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Recent Advances in

Histopathology 23
To all downloaders, share as much as you want but just be man enough to give
credit to us.
Massimo Pignatelli MD PhD FRCPath
Dean of Medicine, Nazarbayev University, Astana
Adjunct Professor of Pathology, University of Pittsburgh School of Medicine
Pittsburgh, USA

Patrick Gallagher MD PhD FRCPath

Senior Clinical Lecturer, University of Bristol
Bristol, UK

London Philadelphia Panama City New Delhi

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We are delighted to present Recent Advances in Histopathology 23 after careful selection of the most
important developments within the field.
The practice of pathology is a constantly evolving speciality. To reflect this, one of the main goals
of this edition is to review the rapid development and application of molecular techniques to
complement the morphological assessment of tissue diagnosis, prognosis and response to treatment.
We have commissioned a wide range of chapters, in order to provide a comprehensive update
of key topics in histopathology for those preparing for their pathology postgraduate exams. We
believe that both generalist as well as specialist consultant and academic histopathologists will find
information of relevance to their daily practice and continued professional development.
This volume of Recent Advances in Histopathology continues the series role as a key resource for
pathologists, both in the UK and internationally, who wish to remain up to date with current trends
in modern histopathology. We are especially grateful to the contributors, who responded to our
invitation with enthusiasm and produced well written and interesting chapters in a timely fashion. The
next edition is already under development, and we welcome chapter suggestions from our readers.

Massimo Pignatelli
Patrick Gallagher
March 2014


Chapter 1 Antibody-mediated rejection of solid organ allografts 1

Chapter 2 The maternal death autopsy 17
Chapter 3 Classification and treatment of non-small-cell
lung carcinoma 31
Chapter 4 Pathology of obesity 47
Chapter 5 Stratified medicine for cancer: the role of the
histopathologist 61
Chapter 6 Mucosal pathology of the gastric cardia and
Barretts oesophagus 73
Chapter 7 Pathology of regenerative and neoplastic
hepatocellular nodules 87
Chapter 8 Serrated lesions of colon and rectum 103
Chapter 9 An update on the pathology of chronic
inflammatory bowel disease 117
Chapter 10 Diagnosis and therapy of gastrointestinal MALT
lymphoma 135
Chapter 11 Medical revalidation for histopathologists 149
Chapter 12 Molecular testing for human papilloma virus 159
Chapter 13 Tensins in health and disease 169
Index 181
Chapter 1

Antibody-mediated rejection of
solid organ allografts
Margaret M Burke, Desley AH Neil, Annalisa Angelini

Transplantation is the treatment of choice for end-stage organ failure. Overall survival rates
in the UK [1] (Table1.1) are comparable to those from the international registries. However,
despite improved management of acute cellular rejection (ACR) and of the complications
of immunosuppression, antibody-mediated rejection (AMR) remains a significant cause of
morbidity and mortality early and late after transplantation.
AMR may be asymptomatic or cause allograft dysfunction resistant to therapy for
ACR [2] It affects recipients sensitised with pre-transplant donor-specific antibodies
(DSAs) to human leucocyte antigens (HLAs) or post-transplant de novo HLA or non-HLA
antibodies. Its overall incidence is difficult to estimate as criteria for diagnosis are neither
agreed nor standardised. It may coexist with ACR, the combination being associated with
a worse outcome. Risk factors include younger age, female gender, prior sensitization
to OKT3, cytomegalovirus seropositivity, pregnancy, previous transfusions, surgery and
transplantation, pre-transplant cardiac support with ventricular assist device and pre- and
post-transplant haemodialysis. Treatment options are limited and are associated with
considerable morbidity. Varying combinations of plasmapheresis, immunoadsorption,
intravenous immunoglobulin (Ig) and intensive immunosuppression with drugs such as
mycophenolate mofetil and high-dose cyclophosphamide have limited success. Newer
biological agents such as monoclonal antibodies to CD20 (rituximab), plasma cells
(bortezomib) and complement C5 (eculizumab) are currently under investigation.
Recently significant progress has been made in our understanding of the pathology and
immunology of AMR. In this chapter, we will briefly summarise the role of antibodies in
pathogenesis of AMR and review the current status of pathological diagnosis of AMR in
kidney, heart, liver and lung allografts.

Margaret M Burke, MB, FRCPath, Department of Pathology, Harefield Hospital, Royal Brompton & Harefield NHS
Foundation Trust, London, UK
Email: M.Burke@rbht.nhs.uk (for correspondence)
Desley AH Neil, BMedSc, MBBS, PhD, FRCPath, Department of Cellular Pathology, Queen Elizabeth Hospital
Birmingham, Birmingham, UK
Annalisa Angelini, MD, Heart Transplant Pathology Unit, Department of Cardiac, Thoracic and Vascular Sciences,
University of Padua, Padua, Italy
2 Antibody-mediated rejection of solid organ allografts

Table 1.1 Mean patient survival 1, 5 and 10 years after solid organ transplantation
(UK data 19982010*)
Organ Alive at 1 year (%) Alive at 5 years (%) Alive at 10 years (%)

Adult (DBD) 95.75 88.3 75
Adult (living donor) 98.50 95.5 90
Paediatric (DBD) 99.25 98.3 94
Paediatric (living donor) 98.50 96.6 93
Adult 81.25 71 55
Paediatric 92.50 81.6 59
Adult (DBD) 88.25 74.6 59
Paediatric (DBD) 92.75 86.3 81
Adult 78 53 31
Paediatric NA NA NA

*Figures averaged from NHS Blood and Transplant [1].

DBD, deceased brain donor.

AntIbodIes And pAthogenesIs of AMr

Capillary endothelial cells (ECs) form the boundary between allograft parenchyma and the
recipients blood and are the main target in AMR, mediated by DSAs. Over the last decade,
immunophenotypic and molecular studies of ECs has contributed significantly to our
understanding of their complex interaction with DSAs and its consequences, manifested
in allograft biopsies as microvascular inflammation [3]. All nucleated cells in the body
express Class I HLAs (HLAs A, B and C) on their surface, whilst Class II HLAs (HLAs DP,
DQ and DR) are constitutively expressed on the surface of antigen-presenting cells and ECs
of capillaries, but not larger vessels [4]. Inflammation upregulates Class II HLA expression
by ECs which express receptors on their surface to complement, Fc fragment of Ig and
cytokines such as interferon-gamma. Antibody binding to antigen activates complement
via the classical pathway, generating C4b and C3b which rapidly undergo proteolytic
cleavage to form the stable split degradation products C4d and C3d. These are bound to
ECs and act as indirect tissue markers of complement activation. Immunopathological
detection of capillary C4d deposition in allografts has evolved as a sensitive and specific
diagnostic tool for AMR [5].
Preformed and de novo DSAs are central to pathogenesis of hyperacute and acute
rejection and contribute to late allograft loss [4]. Potential recipients are screened for
panel-reactive antibodies (PRAs) to detect those at risk, and in positive cases complement-
dependent lymphocytotoxicity (CDL) cross-matches a surrogate marker of DSA are
performed against potential donors. Different IgG subclasses of preformed DSAs may
vary in pathogenicity. Recipients with IgG3 DSAs are most at risk of graft loss. IgG1 DSAs
Pathology 3

are the most prevalent, with least risk to the graft [6]. De novo DSAs develop in 2030% of
transplant recipients, usually to Class II or combined Class I and II HLA antigens [4].
De novo non-HLA antibodies may develop, such as anti-vimentin against endothelium,
and mediate graft damage. Anti-MICA (MHC Class I related chain A) may be associated
with AMR and late allograft failure. Other non-HLA antibodies may be markers of previous
graft injury with exposure of self antigens [2]. Transplants have been done successfully
across the ABO blood barrier raising questions about differences in the immune response
to blood group (carbohydrate) antigens versus HLA (glycoprotein) antigens and endothelial
susceptibility to both. Antibodies to glutathione S-transferase T1 (GSTT1) develop in liver
transplant recipients with an allograft genetically mismatched for this protein [7].
CDL is a specific but not sensitive test for detection of anti-HLA antibodies. A recent
important advance in technology is the solid phase assay (Luminex assay) which uses flow
cytometry to detect antibodies to HLAs by the use of single antigens bound to polystyrene
beads. With its greater sensitivity compared with CDL assays, it has led to an increase in the
number of detectable anti-HLA antibodies. Many of these are DSAs, but not all are harmful
to the graft [6]. Recently the addition of labelled C4d or C1q to the Luminex assay has
enabled detection of C4d-fixing or C1q-fixing DSAs which appear to be strongly associated
with poor allograft survival compared with noncomplement-fixing DSAs [6,8]. In a further
refinement of the test single antigen kits to individual HLAs can enable identification of
individual DSAs which can then be quantified by their mean fluorescence intensity (MFI)
level. Increasingly the MFI is used to monitor the impact of DSA-depleting therapies. Whilst
there is considerable interest in these functional assays, there is no agreement as yet on
their potential value in individual recipients or, in the case of MFIs, what level should be set
as the threshold for significance.
Nonetheless these advances raise the possibility of developing systems of risk stratification
providing there is international standardization of methodology and interpretation of results.
Thus, development of personalised immunosuppression and desensitization in high-risk
DSA-positive potential recipients may become feasible in the future [9].

The pathologist is a key member of the clinical team managing allograft recipients. Regular
updates and access to results of biopsy reproducibility studies using digital technology
[10,11] are available in international forums and publications such as those led by the
Banff Foundation for Allograft Pathology (http://www.banfffoundation.org/) and the
International Society for Heart and Lung Transplantation (ISHLT) Annual Scientific
Meetings (http://www.ishlt.org/). This ensures continual refinements of grading systems
for biopsy diagnosis of AMR in the different organs in the light of outcome data from
international multicentre studies.
In 2004, a National Institute of Health conference on diagnosis of AMR in solid
organ allografts proposed a generic classification system based on a multidisciplinary
approach using serology for DSAs, C4d deposition in tissues, histopathological changes
and graft dysfunction [12] (Table 1.2). Recent molecular studies, mainly in kidney, have
highlighted the importance of the histopathological features of AMR in the absence
of C4d positivity, and the combination of C4d and histology without a proven DSA in
diagnosing AMR [3].
4 Antibody-mediated rejection of solid organ allografts

Table 1.2 Modified NIH proposal for working classification of antibody-mediated rejection*
Antibody-mediated rejection:
Allograft dysfunction, histopathological findings, capillary C4d deposition, circulating DSAs (HLA or non-HLA)
Asymptomatic antibody-mediated rejection:
Histopathological findings, capillary C4d deposition, circulating DSAs (HLA or non-HLA)
Silent/latent antibody-mediated response:
Capillary C4d deposition and/or circulating DSAs (HLA or non-HLA)

*Adapted from Takemoto et al. [12].

Molecular studies in kidney suggest that C4d or DSAs alone with positive histopathology may equate with antibody-
mediated rejection [3,17]
DSAs, donor-specific antibodies; HLA, human leucocyte antigens.

In kidney and heart allografts acute AMR is manifested as microvascular inflammation

with capillary endothelial activation, accumulation of intravascular (IV) inflammatory cells
and capillary C4d deposition as a sign of complement activation [1315]. The picture is less
clear in liver and lung allografts [16,17]. Other pathology such as oedema, haemorrhage,
microvascular thrombosis and tissue necrosis may reflect increasing severity of AMR.
Capillary C4d deposition appears as intensely staining granular endothelial deposits. It
is scored by intensity and distribution of deposition in kidney and heart allografts. A scoring
system has not been agreed universally for lung and liver allografts. Changes of long-
standing AMR (chronic AMR) are described in biopsies from renal, but not other allografts,
although AMR contributes, with ACR and nonimmune factors, to the development of
chronic rejection in cardiac and pulmonary allografts, manifested as cardiac allograft
vasculopathy (CAV) and obliterative bronchiolitis (OB), respectively.
The gold standard tissue staining technique for C4d is immunofluorescence (IF) using
a monoclonal antibody. Polyclonal antibodies are available for use on paraffin wax sections
with immunohistochemistry (IHC) which is the method of choice with, or instead of, IF for
many pathologists. However, a small number of studies have compared IHC with IF and, as
exemplified in the heart, show variable sensitivity for IHC [18]. Hence, a low threshold for
reporting positivity is needed when using IHC.
Properly standardised technology is important to avoid pitfalls in interpretation of
C4d, especially using IHC. Controls for C4d should include tissue with known hyperacute
rejection or AMR. C4d deposition in arterial endothelium and artefacts such as nonspecific
staining of arteriolar elastic lamina are useful as internal positive controls. Interstitial
staining may interfere with evaluation of capillary deposition. Intense serum staining
should not be mistaken for a positive result as it lacks the circumferential granular staining
typical for endothelial C4d deposition. Capillary C4d deposition may also occur in lesions
not relevant to diagnosis of AMR such as biopsy site scars in the heart. Other pathologies
such as hyaline membranes in diffuse alveolar damage, necrotic myocytes and necrotic
hepatocytes may be positive.
The pathological features of AMR specific to each organ will be presented in the
following sections. They are best developed and understood in renal allografts.

The presentation of renal AMR may be acute (aAMR) with rapid reduction in renal
function, often associated with proteinuria, or may be more insidious [13]. Ongoing
Pathology 5

untreated, partially treated or multiple episodes of aAMR can result in structural changes
in the allograft. Once these develop the process is called chronic AMR (cAMR) [9]. The
histological features depend on the timing of the biopsy.
By Banff criteria the diagnosis of renal AMR is multidisciplinary and requires DSA
positivity, C4d deposition in peritubular capillaries (PTCs) and histological features of
AMR [3,19]. If only two of the three features are present, or the pathologist is unaware of the
DSA data then it is classified as suspicious of AMR. However these criteria are increasingly
recognised as missing AMR with clinical impact [13,20].

In aAMR microcirculatory changes occur in PTCs and glomerular capillaries (GCs),
termed peritubular capillaritis and transplant glomerulitis, respectively (Figure 1.1a and
b), with accumulation of macrophages and T lymphocytes [13,21]. In severe cases, there is
thrombotic microangiopathy (TMA) with thrombi in arterioles and/or glomeruli [13]. The
other cause of TMA in this setting is acute calcineurin inhibitor (CNI) toxicity. Fibrinoid
necrosis in larger vessels is recognised as a feature of AMR [3] as is transmural and intimal

a b

c d

Figure 1.1 Acute renal antibody-mediated rejection. (a) Glomerulitis: Periodic acid Schiff-stained section of
glomerulus showing segmental capillary loops plugged with inflammatory cells (arrow). Most other capillary loops
are empty. (b) Peritubular capillaritis: Haematoxylin and eosin-stained section of vacuolated tubular epithelium
indicating acute tubular injury, interstitial oedema separating the tubules and peritubular capillaritis (arrows) with
intravascular inflammatory cells. (c) C4d-immunostained section showing deposition on endothelium of glomerular
capillaries and (d) on peritubular capillaries.
6 Antibody-mediated rejection of solid organ allografts

arteritis [13]. Peritubular capillaritis should be assessed only in cortex as it is less specific in
medulla, where it may be seen in ascending infection.
In cAMR there is reduplication of glomerular basement membrane (GBM) and
peritubular capillary basement membranes (PTCBMs) [13,22]. On light microscopy, this
is seen as double contours of GBMs, termed transplant glomerulopathy. Other causes
of double contours must be excluded such as recurrent or de novo immune complex
glomerulonephritis and chronic TMA due to other causes. Chronic vascular rejection,
consisting of fibromuscular intimal thickening, with minimal elastosis, is seen as a
consequence of cAMR, although other factors such as increased cold ischaemia time and
CMV infection may be involved.

C4d deposition is seen in PTCs and/or GCs (Figure 1.1 c and d) [23], but only peritubular
capillary deposition is assessed as part of Banff criteria for diagnosis of AMR. By IF, Banff
criteria state that 50% of PTCs need to show positive staining to be considered positive
[3]. However, recent evidence suggests that any staining has an impact on long-term graft
survival [23]. On IF GC C4d deposition may be difficult to assess because of glomerular
autofluorescence. The presence of glomerular C4d positivity increases the risk of long-term
graft loss. The medulla is the most sensitive area in which to detect C4d deposition in PTCs.
C4d deposition in atrophic areas has been shown to affect long-term graft survival [3]. With
the recent understanding of the role of natural killer (NK) cells in AMR, a marker for NK
cells may help in diagnosis [20].

Electron microscopy
The earliest feature of AMR is swelling of ECs, with increase in organelles of GC and PTC.
Within glomeruli this is associated with or followed by electron-lucent widening and
accumulation of debris on the subendothelial side of the GBM [24,25]. With more severe,
AMR there is necrosis of ECs and accumulation of platelets, both features of TMA.
Untreated glomerular changes progress with deposition of a new layer of GBM just
under the EC layer (Figure 1.2a). There may be interposition of mesangial cells in the
GBM, but there are no electron dense deposits, a feature which allows differentiation from
immune complex-mediated reduplication of the GBM. Parallel changes occur in PTCs with
reduplication of the PTC basement membrane (Figure 1.2b) [13,22]. Three to four layers
are evidence of early cAMR and five or more layers are diagnostic of cAMR [3,24]. The
ultrastructural features of cAMR precede the light microscopic features of double contours.

Molecular studies in renal AMR

Molecular studies on C4d-positive biopsies have helped to further the understanding
of the pathology of AMR. Endothelial transcripts, a marker of endothelial activation, are
upregulated and associated with NK cell transcripts, indicating a potential role of NK cells
in AMR. By looking at AMR transcripts across a broad spectrum of biopsies, the importance
of microvascular inflammation as a marker of AMR has been highlighted and is potentially
more sensitive than C4d staining [3].

The incidence of cardiac AMR is estimated as 1020%. It may occur early or late
after transplantation. By recently established ISHLT criteria, diagnosis is based on
morphological and immunopathological criteria alone [14,15].
Pathology 7

Figure 1.2 Chronic renal antibody-

mediated rejection. (a) Electron
micrograph of glomerular capillary
wall showing endothelial cells
(curved arrows) lining the capillary
loop, and epithelial cells (Epi) lining
the outer aspect of the glomerular
basement membrane (GBM).
There is a new layer of glomerular
basement membrane (straight
arrows) on the subendothelial
GBM aspect, separated from the original
Epi glomerular basement membrane
by an area of electron lucency.
(b) Electron micrograph of part
of a peritubular capillary. There
a b
is reduplication of the basement
membrane with eight to nine layers
(}) beneath the endothelium (arrow).

Several studies over the last two decades have correlated microvascular inflammation and
capillary C4d positivity with DSA positivity, graft dysfunction, CAV and a poor outcome
[26,27]. Diagnostic criteria for AMR were included in the 2005 revision of ISHLTs 1990 working
formulation for biopsy diagnosis of rejection [28]. In 2010, the requirements outlined in 2005
for DSA positivity and graft dysfunction were dropped after acceptance of asymptomatic AMR
as an entity, with the diagnosis of AMR to be made henceforth by pathological criteria alone
[2]. In 2011, a preliminary grading system for AMR was drawn up and published by members
of ISHLTs Pathology Council [14] with publication of the definitive version in 2013 [15]. It
remains subject to review as experience of its utility increases.

Definitions and grading

The pathological features of AMR, or pathologic AMR (pAMR), are graded on the
combination of histopathological and immunopathological findings. pAMR 1 is
characterised by either histopathological changes [pAMR1(h+)] or immunopathological
findings [pAMR1(i+)]. pAMR2 is characterised by both histopathological changes
and immunopathological findings. pAMR3, or severe AMR, is characterised by
histopathological and immunopathological evidence of extensive myocardial damage and
often disruption of the microcirculation. Concomitant ACR is frequently found and should
be graded separately [14,15,26,28].

Diffuse myocardial microvascular inflammation is the hallmark of cardiac AMR (Figure
1.3a) and is associated with capillary C4d deposition and, often, circulating DSAs with/
without cardiac allograft dysfunction [14]. In pAMR1 (h+) and pAMR2 capillaries are
distended, with lumens narrowed by plump activated ECs and plugs of IV macrophages
[26,29] (Figure 1.3b and c). In pAMR3 there is oedema, vasculitis, haemorrhage,
microvascular thrombosis, capillary damage and myocyte necrosis with neutrophil
infiltration and karyorrhectic debris, usually in the clinical setting of profound irretrievable
allograft dysfunction. Occasional IV macrophages may be also seen in the quiescent state,
ACR, ischaemic injury, infection and healing biopsy sites.
8 Antibody-mediated rejection of solid organ allografts

a b

c d

Figure 1.3 Cardiac antibody-mediated rejection. (a) Haematoxylin and eosin-stained section of myocardium which
shows diffuse microvascular inflammation. (b) Capillary endothelial cells are prominent (short arrows) and lumens
contain mononuclear cells (long arrows). (c) The intravascular cells are CD68-positive, thus confirming them as
macrophages. (d) There is diffuse capillary C4d deposition.

Antibodies for biopsy evaluation of AMR are C4d and CD68 using IHC and C3d, C4d and
HLA-DR using IF [14,15]. Some pathologists use either HLA-DR by IF or CD31/CD34 by
IHC to assess capillary integrity which may be lost in pAMR3 or after repeated episodes
of lesser grades of pAMR. In pAMR1 (i+) and pAMR2 a positive C4d result by IHC and IF
is multifocal (>50% of intact myocardium) or diffuse capillary deposition of any intensity
[14,15] (Figure 1.3d). Diffuse weak capillary C4d deposition may reflect previous or
resolving AMR. Focal strong C4d deposition by IHC (>1050% of intact myocardium) may
represent evolving AMR and the pathologist should recommend close monitoring of graft
function and peripheral blood DSA studies. In pAMR3 C4d deposition may be absent, weak
or multifocal because of endothelial damage. All C4d-positive cases should be followed
up by IHC/IF until negative. Perimyocytic C4d deposition is occasionally observed, but its
significance is currently unknown. Capillaries in Quilty lesions, scars and ischaemic injury
may also stain but are not relevant to diagnosis of AMR at this time. Results of IF staining
with the monoclonal antibody to C3d may predict AMR [30]. Results of IHC staining with
the polyclonal antibody to C3d are neither specific nor sensitive for AMR and are not
recommended at this time.
Pathology 9

The potential screening value of the histological features of AMR is poor [31,32], but
when confirmed by IHC, CD68-positive IV macrophages predict myocardial capillary C4d
deposition, circulating DSA and clinical symptoms early, but not late after transplantation
[29]. Although IV macrophages are typical for cardiac AMR, IV T lymphocytes have also
been identified, raising the question of evolving ACR, perhaps potentiating AMR [33] C4d
positivity by IF and/or IHC, without morphological changes, (i.e. pAMR1 (i+)], may predict
AMR [30], correlate with DSA production [27], graft dysfunction and loss and lead to later
accelerated CAV. Prospective detection of C4d and C3d in allograft biopsies using IF also
predicts AMR, allograft vasculopathy and increased mortality [30]. One study using paraffin
section IHC showed, surprisingly, that C3d, but not C4d deposition predicts CAV [34].

Immunopathological surveillance of cardiac AMR

The recommended schedule for serological and immunopathological studies is 2 weeks,
then 1, 3, 6 and 12 months after transplantation, annually and when AMR is clinically
suspected early or late (>1 year) after transplantation [2]. Allograft dysfunction in the
absence of ACR or other causes, the presence of known risk factors for AMR and positive
histopathology should also prompt immunopathological studies. In one study utilizing IF,
intensity of staining began to diminish after 1 week of treatment. Capillary staining of C3d
cleared within 2 weeks to 1 month whilst that for C4d cleared within 12 months [30]. The
experience of clearing of capillary staining with IHC methodology has not been published.

The true incidence and impact of AMR in the liver are unknown due to the lack of large
prospective studies assessing DSAs. This has also held back the interpretation of the
pathology, in particular the interpretation of C4d staining [35]. Its diagnosis and prevalence
are the major topics for discussion at the forthcoming Banff allograft pathology meeting in
August 2013 where further guidelines may be established. Currently, the diagnosis of AMR
in the liver should be based on graft dysfunction, a proven DSA and histological features
suggestive of AMR with a positive C4d stain in portal or sinusoidal microvasculature.

As hyperacute rejection rarely occurred with livers transplanted against a positive cross-
match, the belief that the liver was protected from AMR has long been held despite
early studies showing an increased risk of graft loss in the first year from rejection [16].
Preformed DSAs are associated with worse allograft survival. Their incidence ranges from
10.5% to 22.2% and a positive pretransplant CDL cross-match occurs in about 10% of
recipients [36]. Preformed DSAs are cleared relatively quickly post-transplant (17 days)
in 6585% of patients. Persistent DSA is associated with steroid-resistant rejection, AMR
and the development of chronic rejection. De novo DSAs occur in up to 10% of recipients
in the first year post-transplant and around 50% longer term. They are associated with
increased risk of acute and chronic rejection, particularly in the presence of a high MFI,
with progressive graft fibrosis and the development of cirrhosis [37].
Autoantibodies of the type found in autoimmune hepatitis, such as smooth muscle
actin, but with an atypical staining pattern, are found in up to 75% of paediatric and adult
recipients [37]. Antibodies to GSTT1, a protein expressed in hepatocytes and bile ducts
but lacking in 20% of Caucasians and up to 58% of non-Caucasians, develop when there
is a genetic mismatch between the donor and recipient, the donor having the wild type
10 Antibody-mediated rejection of solid organ allografts

gene and the recipient the null genotype. They develop prior to the development of graft
inflammation, and only with a mismatched graft, suggesting that they are allogeneic
although their histological pattern is different to HLA DSAs [7].

The histological features suspicious of AMR are portal tract changes indicative of duct
obstruction portal oedema and a ductular reaction (Figure 1.4a), often accompanied
by a neutrophilic infiltrate. Sinusoidal inflammation is less clearly defined; however,
microvascular inflammation, with an increase in inflammatory cells, possibly macrophages
or neutrophils, has been found [36,38]. The presence of perivenulitis in ACR should raise
suspicion of coexistent AMR [38]. Features of evolving chronic rejection (dysplastic/sick
bile ducts and progressive paucity of bile ducts often in the setting of unresolving acute
rejection) should also prompt IHC for C4d deposition and serology for DSAs [16,35].
In the GSTT1-related DSA setting plasma cells are prominent, and in recipients with
chronic hepatitis autoantibodies are often detected. HLA DSAs have also been detected
in patients with this pattern, particularly when fibrosis is severe with bridging and
cirrhosis. Areas with unexplained coagulative necrosis may also be an indication of AMR.
In recipients with preformed antibodies and a positive cross-match nonspecific changes
resembling preservation-reperfusion injury may be seen in biopsies taken early post-
transplant, including hepatocyte swelling, cholestasis and platelet microthrombi.

The interpretation of C4d staining is still incompletely understood due to a lack of
correlation with DSAs in most studies [35]. C4d may be deposited in portal and/or
sinusoidal microvasculature (Figure 1.4b and c) [3840]. It has been shown that IF is more
sensitive than IHC but the staining patterns differ, with portal vascular staining dominating
in IHC and sinusoidal staining in IF [36,39]. In the setting of chronic rejection, C4d
deposition often appears to be sinusoidal in areas of central perivenulitis [38].

The true incidence of pulmonary AMR is unknown as definitions and diagnostic criteria are
currently lacking. Recently recommendations for biopsy interpretation have been published
in the first attempt to reach international consensus on pathology [17]. Two multicentre
studies are in progress with updates expected from the forthcoming Banff Foundation Allograft
Pathology Conference in August 2013 and at ISHLTs Annual Scientific Meeting in 2014.
The lung allograft is subject to challenge from both the recipients immune system and
the environment. This is reflected in overlapping patterns of pathology, with many potential
causes. Consequently, the diagnosis of pulmonary AMR is one of exclusion, made in a
multidisciplinary context and based on graft dysfunction, proven circulating DSAs and
histopathological features suggestive of AMR whether or not capillary C4d is detected (the
triple test).

Known patterns of immunologic injury in the lung associated with de novo DSAs
include persistent/recurrent ACR of all grades, lymphocytic bronchiolitis and
Pathology 11


a b

Figure 1.4 Possible mixed hepatic rejection. (a)

Haematoxylin and eosin-stained section of a portal
tract which is inflamed with bile duct (BD) and venous
(v) inflammation features of acute cellular rejection.
Portal oedema and marginal ductular reaction (arrows)
raise the possibility of concurrent antibody-mediated
rejection. (b) C4d-immunostained section of a portal
tract showing staining of portal microvasculature and
(c) (from another patient) of liver parenchyma showing
C4d deposition on sinusoidal endothelium.

bronchiolitis obliterans syndrome(BOS) with its histopathologic correlate of OB [17].

Capillary C4d deposition has been reported in ACR in DSA-positive recipients and
to a lesser extent in DSA-negative recipients. Septal capillary injury or pulmonary
capillaritis has been proposed as a specific histological marker of pulmonary AMR
[41]. Capillaritis has also been reported in ACR in DSA-positive recipients, some
recipients responding to steroids, others to plasmapheresis and IVIg, suggesting an
antibody-mediated component [42].Other patterns seen in DSA-positive recipients
include acute and organizing lung injury without ACR, arterial endothelitis, small
airway inflammation and diffuse alveolar damage [43]. However, in this group C4d and
C3d deposition show little correlation with DSA status or with histology, reflecting the
need for refinement of diagnostic criteria.

Interpretation of lung allograft pathology is on routinely fixed and processed transbronchial
lung biopsies and may be difficult because the biopsies are often small and fragmented
and may not fully inflate despite gentle agitation immediately after immersion in formalin.
Histopathological patterns which should prompt staining for C4d deposition are given
in Table 1.3 [17]. Chief amongst them is neutrophilic capillaritis, defined as patchy or
12 Antibody-mediated rejection of solid organ allografts

diffuse dense neutrophilic septal infiltrates with karyorrhectic debris. There may also be
fibrin platelet thrombi in the microvasculature, alveolar haemorrhage and flooding of
neutrophils into adjacent alveolar spaces. Neutrophilic capillaritis should be distinguished
from neutrophilic margination, i.e. neutrophilic infiltrates in the septae in the absence
of karyorrhexis and fibrin. Both may produce a histopathological picture of diffusely
thickened and cellular alveolar septae which should prompt higher power examination for
the above features (Figure 1.5a).

Although the evidence for the role of capillary C4d deposition in pulmonary AMR is not
robust, it is still advised as part of allograft biopsy evaluation to enable pathologists to
gain more experience in its interpretation (Figure 1.5b). A scoring system analogous
to that for renal and cardiac biopsies (distribution of staining >50% = positive) is
suggested. Nonspecific C4d deposition in elastin in the alveolar walls is a significant
problem interfering with assessment of capillary deposition. Its deposition in hyaline
membranes may reflect other mechanisms of activation such as the nonimmune
mannose-lectin pathway of complement activation, perhaps explaining its occurrence
in infection, sepsis and reperfusion injury in both transplanted and nontransplanted
lungs [43].

Because of overlapping patterns of pathology with many different causes, a definition
and grading system for pulmonary AMR cannot be recommended at this time [17].
However, neutrophilic capillaritis +/ capillary C4d deposition should be reported as

Table 1.3 Indications for C4d immunostaining of lung allograft biopsies*

Histopathological criteria
1. Neutrophilic capillaritis
2. Neutrophilic septal margination
3. High-grade acute cellular rejection (A3)
4. Persistent/recurrent acute cellular rejection (any A Grade)
5. Acute lung injury pattern/diffuse alveolar damage
6. High-grade lymphocytic bronchiolitis (Grade B2R)
7. Persistent low-grade lymphocytic bronchiolitis (Grade B1R)
8. Obliterative bronchiolitis (Grade C1)
9. Arteritis in the absence of infection or cellular rejection
Other criteria
10. Graft dysfunction without morphological explanation
11. Any histological findings in setting of de novo DSA positivity

*Adapted from Berry et al. [17].

DSA, donor-specific antibodies.
Conclusion 13

a b

Figure 1.5 Diagnosing pulmonary antibody-mediated rejection (AMR). (a) Haematoxylin and eosin-stained section
of a transbronchial lung biopsy. The alveolar septae are slightly thickened with increased cellularity due to a sparse
mixed inflammatory infiltrate. The picture is nonspecific; possible causes include early bacterial or viral infection.
AMR cannot be excluded. (b) C4d staining showed diffuse capillary deposition. The recipient had presented with
decreasing lung function despite treatment of acute cellular rejection and was found to be donor-specific antibody-
positive. In the absence of other causes, especially infection, the clinicopathological diagnosis was findings
consistent with pulmonary AMR.

findings suggestive of AMR provided the diagnosis is put in its clinical context and
correlated with the results of contemporaneous DSA results (the triple test). Other
causes such as infection, acute lung injury of any cause and drug-related changes
should be excluded. If serological and clinical data are not available at the time of
reporting, a recommendation for serological testing should be made. Future critical
appraisal of this approach may enable progress towards a definition and an agreed
grading system for pulmonary AMR.

The pathological hallmarks of AMR have been described in several, but not all solid
organ allografts and are still evolving. Currently, microscopic features common to all
are capillary endothelial activation, microvascular inflammation, IV inflammatory cells
(nearly always macrophages) and capillary deposition of C4d. Molecular assessment
of biopsies in renal allograft recipients has revealed new information on aspects of
histopathlogical features of importance but does not, as yet, have a well-defined role in
A standard approach to diagnosis and management of AMR should include correlation
of the biopsy findings with serological evidence of DSAs and assessment of allograft
function. This requires a multidisciplinary team approach by clinicians, immunologists
and pathologists. The advent of technology to quantitate circulating DSAs and to
determine their ability to fix complement and their IgG subtypes suggests that risk
stratification in an individual recipient should be possible, perhaps leading to personalised
immunosuppression in high-risk cases.
14 Antibody-mediated rejection of solid organ allografts

Key points for clinical practice

The pathologist is a core member of the multidisciplinary clinical team ensuring a
standardised approach to diagnosis and management of allograft recipients with AMR.
AMR is a significant cause of morbidity and mortality after solid organ transplantation and
is difficult to treat. It is caused by DSAs against HLA and non-HLA antigens on capillary
endothelium of the allograft. It frequently coexists with ACR.
Classification of AMR into latent, silent, subclinical and clinical categories relies on circulating
DSAs, biopsy appearances, capillary C4d deposition and assessment of allograft function.
Recent molecular research suggests additional entities of C4d negative AMR and DSA-
negative AMR, both with positive histology.
DSAs to HLA antigens may occur prior to transplantation or de novo post-transplantation.
De novo non-HLA antibodies may develop late after transplantation. Recent advances in
serological testing may identify high-risk recipients for whom therapy could be tailored.
Current pathological criteria common to solid organ allografts are capillary endothelial
activation, microvascular inflammation and capillary deposition of C4d. Haemorrhage,
necrosis, microvascular thrombi and neutrophilic infiltration may indicate severe AMR.
Pathology of AMR specific to the different organs forms the basis of international grading
systems in kidney and heart allografts. Grading systems for liver and lung allografts are
being developed.
Technical standardization for C4d immunostaining is essential as C4d detection by IHC is
less sensitive than by IF and can lead to underdiagnosis of a positive result. The pathologist
should be aware of pitfalls in biopsy interpretation of C4d deposition.
Finally before making a diagnosis of AMR think of other potential diagnoses. This is especially
important in liver and lung biopsies. This should be done in a clinicopathological setting.

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5. Feucht HE. Complement C4d in graft capillaries -- the missing link in the recognition of humoral
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18. Chantranuwat C, Qiao JH, Kobashigawa J, et al. Immunoperoxidase staining for C4d on paraffin-embedded
tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence. Appl
Immunohistochem Mol Morphol 2004; 12:166171.
19. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future
directions. Am J Transplant 2008; 8:753760.
20. Farkash EA, Colvin RB. Diagnostic challenges in chronic antibody-mediated rejection. Nat Rev Nephrol
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21. Hidalgo LG, Campbell PM, Sis B, et al. De novo donor-specific antibody at the time of kidney transplant
biopsy associates with microvascular pathology and late graft failure. Am J Transplant 2009; 9:25322541.
22. Papadimitriou JC, Drachenberg CB, Ramos E, et al. Antibody-mediated allograft rejection: morphologic
spectrum and serologic correlations in surveillance and for cause biopsies. Transplantation 2013; 95:128-
23. Batal I, Girnita A, Zeevi A, et al. Clinical significance of the distribution of C4d deposits in different anatomic
compartments of the allograft kidney. Mod Pathol 2008; 21:14901498.
24. Wavamunno MD, OConnell PJ, Vitalone M, et al. Transplant glomerulopathy: ultrastructural abnormalities
occur early in longitudinal analysis of protocol biopsies. Am J Transplant 2007; 7:27572768.
25. Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-mediated
rejection and transplant glomerulopathy. Am J Transplant 2011; 11:21232131.
26. Lones MA, Czer LS, Trento A, et al. Clinical-pathologic features of humoral rejection in cardiac allografts: a
study in 81 consecutive patients. J Heart Lung Transplant 1995; 14:151162.
27. Smith RN, Brousaides N, Grazette L, et al. C4d deposition in cardiac allografts correlates with alloantibody. J
Heart Lung Transplant 2005; 24:12021210.
28. Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization
of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 2005; 24:17101720.
29. Fedrigo M, Feltrin G, Poli F, et al. Intravascular macrophages in cardiac allograft biopsies for diagnosis of
early and late antibody-mediated rejection. J Heart Lung Transplant 2013; 32:404409.
30. Tan CD, Sokos GG, Pidwell DJ, et al. Correlation of donor-specific antibodies, complement and its regulators
with graft dysfunction in cardiac antibody-mediated rejection. Am J Transplant 2009; 9:20752084.
31. Hammond ME, Stehlik J, Snow G, et al. Utility of histologic parameters in screening for antibody-mediated
rejection of the cardiac allograft: a study of 3,170 biopsies. J Heart Lung Transplant 2005; 24:20152021.
32. Fedrigo M, Gambino A, Benazzi E, et al. Role of morphologic parameters on endomyocardial biopsy to
detect sub-clinical antibody-mediated rejection in heart transplantation. J Heart Lung Transplant 2011;
33. Fedrigo M, Leone O, Burke M, et al. Inflammatory cell burden and phenotype in endomyocardial biopsies
from patients with antibody-mediated rejection (AMR) - An AECVP multicenter study. J.Heart Lung
Transplant 2013;32(4S):S19.
34. Moseley EL, Atkinson C, Sharples LD, Wallwork J, Goddard MJ. Deposition of C4d and C3d in cardiac
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applications and pitfalls. Liver Transpl 2011; 17:747750.
16 Antibody-mediated rejection of solid organ allografts

36. Kozlowski T, Andreoni K, Schmitz J, Hayashi PH, Nickeleit V. Sinusoisdal C4d deposits in liver allografts
indicate an antibody-mediated response: diagnostic considerations in the evaluation of liver allografts.
Liver Transpl 2012; 18:641658.
37. Evans HM, Kelly DA, McKiernan PJ, Hubscher S. Progressive histological damage in liver allografts following
pediatric liver transplantation. Hepatology 2006; 43:11091117.
38. Bellamy CO, Herriot MM, Harrison DJ, Bathgate AJ. C4d immunopositivity is uncommon in ABO-compatible
liver allografts, but correlates partially with lymphocytotoxic antibody status. Histopathology 2007;
39. Lunz J, Ruppert KM, Cajaiba MM, Isse K, et al. Re-examination of the lymphocytotoxic crossmatch in liver
transplantation: can C4d stains help in monitoring? Am J Transplant 2012; 12:171182.
40. Musat AI, Agni RM, Wai PY, et al. The significance of donor-specific HLA antibodies in rejection and
ductopenia development in ABO compatible liver transplantation. Am J Transplant 2011; 11:500510.
41. Badesch DB, Zamora M, Fullerton D, et al. Pulmonary capillaritis: a possible histologic form of acute
pulmonary allograft rejection. J Heart Lung Transplant 1998; 17:415422.
42. Astor TL, Galantowicz M, Phillips A, Palafox J, Baker P. Pulmonary capillaritis as a manifestation of acute
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Heart Lung Transplant 2013; 32:326332.
Chapter 2

The maternal death autopsy

Sebastian Lucas

In the UK, the current maternal mortality rate is 11.4/100,000 maternities, resulting in
about 120 deaths a year [1]. About 80% are autopsied by instruction from a coroner or fiscal
(consented autopsies are most unusual). Thus, such autopsies are relatively rare, and the
appropriate trend is for them to be examined in regional centres by specialist pathologists.
This is the ideal since the range of possible causes of death is very wide (Table 2.1); the
evaluation of causation can be complex, requiring much histopathology and thinking time;
and the medical, social and legal consequences of such deaths are profound, prolonged
and expensive.

Classification of maternal deaths

Internationally, maternal deaths are defined as deaths at any time during pregnancy,
delivery and up to 42 days postdelivery. Delivery includes miscarriages and abortions
(spontaneous, legal and unsafe), live and stillbirths, and vaginal and caesarean deliveries.
Deaths after 42 days from delivery are included only if they result from a problem that arose
before that caesura, such as peripartum cardiomyopathy (PPCM) or prolonged survival in
intensive care.
Maternal deaths are subdivided into direct, indirect and coincidental see Table 2.2 for
definitions and examples. This is primarily to quantify and compare the quality of obstetric
practices internationally. In low-income countries, direct deaths outnumber indirect;
whilst in high-income countries with good obstetrics, indirect deaths are relatively more
common. The latest causes of maternal deaths in the UK are published in the triennial
confidential enquiry, along with much useful discussion on clinical pathology [1].

The role of the autopsy

The role of the autopsy is to help work out what actually happened to result in death.
Most maternal deaths are not preventable or predictable: they happen, and are usually
multifactorial in causation. By following a standard protocol (e.g. the RCPath autopsy
guideline [2]), the pathologist leaves the mortuary with all the information and samples
required to resolve this causation, whatever it is. Obtaining the placenta, if possible, is

Sebastian Lucas, FRCP, FRCPath, Department of Histopathology, St Thomas Hospital, London, UK

Email: Sebastian.lucas@kcl.ac.uk (for correspondence)
18 The maternal death autopsy

Table 2.1 Comprehensive list of the causes of maternal death in the UK

Hypertensive disease of a.Subtype of PET: HELLP syndrome in PET (haemolysis, elevated liver
pregnancy [pre-eclampsia, enzymes, low platelets)
Peripartum haemorrhage (PPH) a. Uterine atony
b. Abruption of the placenta
c. Placenta praevia
d. Abnormally adherent placenta
i. Placenta accreta, increta, percreta
e. Genital tract trauma tear, laceration
i. Spontaneous or iatrogenic forceps, episiotomy
f. Retained placental material
g. Rupture of the uterus
i. Spontaneous or iatrogenic
h. Life support for PPH
i. Transfusion-associated lung injury ( TRALI), fluid overload
Peripartum dilated
cardiomyopathy (PPCM)
Amniotic fluid embolism
syndrome (AFES)
Early pregnancy deaths a.Ectopic pregnancy and haemorrhage
b.Spontaneous miscarriage/abortion
c.Legal termination (medical or surgical procedures)
d.Unsafe (criminal) abortion
Sepsis Many categories (see Table 2.4)
Obstetric anaesthesia a. General anaesthesia
i. Cardiac or ventilatory problems
b. Epidural (spinal) anaesthesia
ii. Infection
iii.Dural puncture, cerebrospinal fluid (CSF) leakage and subdural

Air embolism
Choriocarcinoma and
hydatidiform mole
Ovarian hyperstimulation
syndrome (OHSS)
Acute fatty liver of pregnancy
Venous thromboembolism Pulmonary embolism and dural venous thrombosis
Cardiac a.Congenital heart lesion with pulmonary hypertension
b.Inheritable cardiomyopathy, e.g. hypertrophic cardiomyopathy (HOCM),
arrhythmogenic right ventricular cardiomyopathy (ARVCM)
c.Acquired cardiac muscle disease, e.g. ischaemic heart disease, endocardial
fibroelastosis, myocarditis
d.Sudden unexpected arrhythmic cardiac death syndrome (SADS)
e. Obesity and sudden cardiac death
f. Valvular disease, e.g. in IV drug users, rheumatic mitral stenosis

The role of the autopsy 19

Table 2.1 Continued

Systemic hypertension
Idiopathic arterial (primary)
pulmonary hypertension
Pre-existing thrombophilia states,
e.g. antiphospholipid syndrome
Thrombotic thrombocytopaenic
purpura (TTP)
Stroke Subarachnoid haemorrhage , intracerebral haemorrhage, and cerebral
Arterial wall degeneration a.Dissection of aorta
b.Dissection of coronary , splenic and other abdominal arteries
Psychiatric, including suicide
related to pregnancy and delivery
Epilepsy [sudden unexplained
death in epilepsy (SUDEP)]
Tumours Malignant disease worsened by pregnancy (breast, cervix)
Other diseases a.HIV/AIDS, tuberculosis, community-acquired nongenital tract sepsis,
influenza (e.g. epidemic type A - H1N1)
b.Sickle cell disease (HbSS and HbSC)
c.Connective tissue disease - systemic lupus erythematosus (SLE)
d.Diabetes mellitus gestational and pre-existing diabetes; this includes the
hypoglycaemic dead in bed syndrome

Modified from the Guidelines of the Royal College of Pathologists [2])

Table 2.2 Classification of maternal deaths

Direct Indirect Coincidental
Definition Diseases that are directly Diseases that are not directly Disease or event unrelated to
related to pregnancy and/ related to pregnancy or pregnancy and not influenced
or delivery, i.e. only happen delivery, but are exacerbated by pregnancy
through pregnancy by pregnancy and/or delivery.
Examples Pre-eclampsia Dissection of aorta Homicide
Amniotic fluid embolism Sudden cardiac death with a Road collision
Genital tract trauma morphologically normal heart Illicit drug toxicity
Postpartum haemorrhage Congenital heart disease Most cancers
Genital tract sepsis Venous thromboembolism Some suicides

always helpful. Although virtually all maternal autopsies are medicolegal, coroners do not
place obstacles in the way of the investigation, being increasingly only too pleased to have
them sorted out unambiguously.
More attention in this chapter is directed to the direct deaths; indirect and coincidental
deaths encompass the range of pathologies encountered in any women, although there are
aspects where pregnancy accentuates the pathology.
20 The maternal death autopsy

Direct maternal deaths

Amniotic fluid embolism syndrome (AFES)
This, although a common direct cause of maternal death in high-income countries,
is a pathophysiological puzzle. The classic form is sudden cardiorespiratory collapse
resulting in acute hypoxia, occurring during or just after labour or caesarean section. The
clinical triad is hypotension or cardiac arrest, pulmonary vasospasm and coagulopathy
with severe bleeding. There is no in-life diagnostic test, so the case definition requires
exclusion of other diagnoses (Table 2.3) [3]. Mortality is high, and treatment is

The important autopsy pathology is in the lungs. Amniotic fluid, amniotic and fetal
squamous cells and fetal hair embolise to the small vessels of the lungs. They may be
seen easily on haematoxylin and eosin stains, but it is critical to perform supportive
special stains: Alcian blue to show amniotic acid mucin, and high molecular weight
keratin immunohistochemistry (e.g. LP34) to demonstrate the squames. An endothelial
CD31 immunostain completes the panel, to distinguish embolic squames from sloughed
endothelial cells (Figure 2.1).
In the renal glomeruli, fibrin thrombi are usually found in the capillary lumens,
reflecting the common disseminated intravascular coagulation (DIC) that is part of the AFE
syndrome (Figure 2.2). The uterus will usually show much mucosal bleeding, and perhaps
AF material in the mural veins. In principle, it may be possible to demonstrate the location
of entry of AF into the uterine veins, e.g. via a caesarean incision or mucosal split, but in
practice this rarely pertains.
The pathogenesis is debated [4]. Traditionally, the amniotic material embolising into the
lung is thought to trigger an acute anaphylactic response with cardiopulmonary shut down,
whilst also triggering the clotting cascade and consumptive coagulopathy. But some argue
that AFE syndrome is merely an example of systemic inflammatory response syndrome
(SIRS) from inappropriate release of endogenous inflammatory mediators, an abnormal
maternal immune response to fetal antigens.

Table 2.3 Differential diagnosis of sudden collapse in pregnancy, during or after delivery
Venous thromboembolism
Amniotic fluid embolism
Hypovolaemic shock from haemorrhage
Cardiac arrhythmia, e.g. SADS, ischaemic heart disease
Fulminant sepsis
Arterial/aortic rupture
Air embolism
SUDEP sudden unexpected/unexplained death in epilepsy
Direct maternal deaths 21

Figure 2.1 Amniotic fluid embolism. (a) Lung arterioles

contain keratin and mucus (haematoxylin and eosin). (b)
Immunohistochemistal stain against LP34 highlighting
the squames.

Negligence aspects
Medicolegally, AFE is important since it used as a defence against claims of clinical
negligence where there has been fatal peri- or postpartum haemorrhage (PPH); for
whatever the cause of the haemorrhage, AFE would make it inevitably fatal. So it is
important to look for AFE in all cases where it might be a relevant factor, to prove or
exclude it.

Hypertensive diseases of pregnancy

Pre-eclampsia and eclampsia may develop in the third trimester; but up to a quarter of
cases may happen without warning up to 2 weeks postpartum. Pre-eclampsia is defined
as raised blood pressure, oedema, and proteinuria. Predisposing factors for pre-eclampsia
include essential hypertension, renal disease and obesity. The aetiopathogenesis is not
understood but immune factors and generalised endothelial cell upregulation and damage
are pertinent [5], i.e. a generalised vasculopathy (but not a vasculitis). Eclampsia is defined
as clonic-tonic seizures occurring in a patient with pre-eclampsia, and it has a high
22 The maternal death autopsy

a b

Figure 2.2 Thrombotic microangiopathy in the kidney glomerulus. (a) Disseminated intravascular coagulation
fibrin thrombi in amniotic fluid embolism syndrome (haematoxylin and eosin). (b) Platelet thrombi in thrombotic
thrombocytopaenic purpura immunohistochemical stain against CD61.

mortality untreated. The pre-eclampsia-associated HELLP variant is haemolysis, elevated

liver enzymes, low platelets and also has a high mortality.
The mode of acute death in these hypertensive disorders of pregnancy was considered
to be primarily hypertensive-type intracerebral haemorrhage. However, it is now realised
that deaths occur in mothers with PET but without brain haemorrhage. The pathogenesis
appears to be an encephalopathy caused by vasogenic oedema, i.e. a more severe,
generalised version of the posterior reversible leukoencephalopathy syndrome (PRES),
due to endothelial cell damage [6]. Some deaths appear to be cardiac, although the heart
is usually normal; it is hypothesised that these are similar to the mechanism of death in
sudden unexpected death in epilepsy (SUDEP), where the brain triggers a fatal cardiac
arrhythmia. Another potentially fatal crisis comes with HELLP with liver failure, and
capsular rupture causing intra-abdominal haemorrhage.

In about 60% of fatalities, there is deep intracerebral haemorrhage, without pre-existing
berry aneurysm, or other predisposing lesion. Diffuse cortical petechial haemorrhages
are another version, particularly prominent in the occipital lobes. Otherwise there is brain
swelling and diffuse cerebral oedema.

The lesion of glomerular endotheliosis is characteristic and unique to PET [7]. The endothelial
cells are swollen, making the glomerular capillaries appear bloodless (Figure 2.3). The
glomerulus may also herniate into the proximal tubule. Endothelial cells may be vacuolated
with lipid (best seen with electron microscopy). With silver staining, basement membrane
thickening and remodelling produces a string-of-beads appearance.

Uterus and placenta

If the placenta is available, it will show the effects of reduced arterial blood supply on the
villi, with foci of infarction. The decidua characteristically shows atherosis and fibrinoid
necrosis of the spiral arterioles.
Direct maternal deaths 23

Figure 2.3 Pre-eclampsia. The

kidney glomerulus shows bloodless
capillaries, endothelial cell swelling,
and herniation into the proximal
tubule (haematoxylin and eosin).

Grossly the liver shows blotchy focal or confluent haemorrhagic necrosis [8]. Histologically,
there is periportal fibrin deposition, haemorrhage and hepatocyte necrosis, a lesion unique
to PET.

Peri- and postpartum haemorrhage

There are many causes (Table 2.1). In some cases, there will have been an emergency
hysterectomy, to stop the bleeding; the specimen will be in the histopathology laboratory
and must be examined. The general autopsy features of hypovolaemic shock include
body pallor, pituitary infarction and hypoxic-ischaemic neuronal necrosis in the brain;
but much will depend on the medical interventions and how long the woman lived post-

Uterine atony, placenta praevia, retained placenta

Uterine atony is the commonest cause of PPH, but leaves no definable pathology. Placenta
praevia will have been diagnosed in life, and the uterus will show the low attachment site.
Retained placental material is rarely fatal in the UK, though it has caused haemorrhagic
death following birth at home.

Placental abruption
Abruption of the uterus will have been heralded clinically. It leaves a clot between the
maternal placental surface and uterus which usually indent both. It is often accompanied
by a severe coagulopathy.

Creta syndromes
The placenta creta syndromes commonly follow from previous caesarean section, with
the fibrotic scar rendering the decidua suboptimal. The placental villi then attach direct to
the uterine muscle (accreta), or invade further into the myometrium (increta) and, rarely,
through it (percreta). Torrential bleeding follows when the placenta is detached in the
24 The maternal death autopsy

third stage of labour. Histological examination of the uterus bed confirms the diagnosis
(Figure 2.4).

Genital tract trauma

The most taxing scenario for the pathologist is genital tract trauma. The vagina, cervix
and lower uterus can be torn and lacerated by large babies as well as clumsy assisted
delivery. Arteries and veins in the submucosa are ruptured and haemorrhage is severe.
It is worth removing the entire genital tract en bloc from vagina to uterine fundus by
careful dissection; the piece is fixed, and then serially horizontally sliced, and sampled
histologically to depict the tear dimensions and vessel ruptures.

Uterine rupture
Rupture of the uterus is classically a consequence of big baby, small pelvis and prolonged
labour. Modern obstetrics mandates caesarean section, so this is rarely seen in the UK.
Previous Caesarean section is a risk factor. Drugs that enhance uterine contraction, used
in termination, labour and postpartum, can sometimes result in rupture; these include
misoprostol and oxytocics. The rupture is typically lateral.

Spontaneous abortion or miscarriage (delivery before 24 weeks gestation without
iatrogenic induction) may be septic or aseptic. Causes of death include ascending genital
tract sepsis, uterine haemorrhage and molar pregnancy [1].
Legal, i.e. medically safe, termination of pregnancy has a minimal mortality. But
occasional fatality results from rupture of the uterus due to misoprostol, or comorbidity
such as taking cocaine. Unsafe (criminal) abortion is fortunately rare in the UK, but can
cause death from infection and haemorrhage.

Figure 2.4 Placenta increta.

Thinned uterine wall (from
previous Caesar section) and
invasion of placental villi through it
(haematoxylin and eosin).
Direct maternal deaths 25

Sepsis in pregnancy is complicated because it is not one syndrome but several, with different
pathogeneses [1]. In severe and fatal cases, the end result is bacteraemic septic shock and
multiorgan failure, often with DIC. The syndromes and pathologies are depicted in Table 2.4 [9].
Examining the placenta is critical for sepsis autopsies, ideally with microbiology culture
as well as histopathology. Pre-evisceration maternal blood cultures taken as aseptically as
possible from the neck vessels or heart are necessary, and should be part of the standard
protocol for all maternal autopsies. And check on any predeath cultures done since they
will be even more useful if positive.
In category 4 sepsis, note that there is no inflammation of the genital tract despite the
high bacterial load (Figure 2.5). This form of toxic shock sepsis accounted for one-third of
fatal sepsis in the last UK triennial report [1], and the pregnancy per se may not actually be
relevant to its occurrence in predisposed individuals.
In postdelivery genital tract sepsis, it is often not clear how the infection (most
commonly group A Streptococcus) entered the body. The Semelweis syndrome of
infection from the hands of health care workers directly into the genital tract is rare in
modern obstetrics in high-income countries. Inadvertent contamination by the mothers
hand, from her nasal carriage of community-acquired organisms, is probably frequent.

Table 2.4 Sepsis classification and pathology [1,9]

Category Case definition Typical infection agent Pathology
1. Unsafe abortion Unsafe/illegal termination Clostridium spp Genital tract necrotising
of pregnancy sepsis, septic shock
multiorgan pathology
2. Ruptured membranes Presenting with genital Escherichia coli i.e. Infected and inflamed
(genital tract sepsis) tract infection at or around perineal infection from gut placenta, cord and
time of spontaneous commensals membranes, genital tract
ruptured membranes; sepsis, MOF
typically occurs in second
3. Postdelivery (genital Vaginal or caesarean Group A Streptococcus Genital tract sepsis,
tract sepsis) delivery or termination of pyogenes (GAS) sometimes necrotising,
pregnancy; a well interval with high bacterial load,
of one or more days; with MOF
genital tract infection
evidenced by clinical,
microbiological and
histopathological features
4. Community-acquired Membranes intact, not in GAS, pneumococcus Toxic shock syndrome,
sepsis labour; bacteraemic septic MOF
(toxic) shock
5. Postpartum sepsis Various: e.g. infected Gram-negative and gram- Localised sepsis, leading
related to birth process spinal anaesthesia, Caesar positive organisms to MOF
but genital tract not section wound infection
26 The maternal death autopsy

Figure 2.5 Postpartum sepsis. Uterus and cervix with

fulminant group A Streptococcus pyogenes infection.
a (a) Gross specimen. (b) Gram stain showing vast
numbers of cocci against a necrotic background.

Indirect maternal deaths

Venous thromboembolism (VTE)
This, in the form of massive pulmonary embolism, was previously the commonest UK
cause of death in pregnancy, prior to the introduction of protocols of thromboprophylaxis,
particularly following caesarean sections. Women who collapse and die suddenly and
unexpectedly are suspected to have VTE (see Table 2.3 for differential diagnosis) and may
be thrombolysed. Hence, it is critical to examine the entire length of the pulmonary artery
tree thoroughly to show or exclude massive thromboembolism. Note that emergency
enzymatic thrombolysis does not dissolve the thrombus within hours of administration. In
the brain, thrombosis of dural veins results in haemorrhagic infarction.
VTE is important because pregnancy is a procoagulant state; this is needed to help
prevent severe haemorrhage when the placenta detaches from the decidua. The down-side
is that this confers a 10-fold relative risk of VTE in women, a risk which runs right through
pregnancy to a week or so after delivery.

Cardiovascular disease
In the UK, cardiac and vascular diseases are the commonest category of maternal death
[1]. Weakening of the walls of the aorta and some medium or large arteries (most often
the splenic or coronary artery) result in aneurysm, dissection and rupture usually in the
third trimester. The aetiology appears to be multihit: an inherent predisposition combined
with progesterone-associated weakening of the media. Histologically, there is elastic
degeneration, deposits of mucin and attenuated muscle. The outcome is usually a sudden
unexpected collapse from shock (Table 2.3).

Cardiac disease
Cardiac disease is increasing in prevalence in pregnancy [1]. The causes are listed in
Table 2.1. Whilst ischaemic heart disease is explicable on the grounds of lifestyle, obesity
Indirect maternal deaths 27

and the increasing age of pregnant women, sudden unexpected arrhythmic cardiac death
syndrome is a puzzle. Yet significant numbers of women die suddenly in the third trimester
or after delivery, the autopsy is negative and the heart is morphologically normal. We
postulate that these may represent potentially inheritable cardiac conditions, such as long
QT syndrome. Thus, it is essential in this scenario to exclude all other possible causes of
death, including cocaine and other stimulatory drugs, and to retain a piece of frozen spleen
tissue for later DNA analysis. The blood relatives will be examined in a cardiac genetic clinic
to determine whether there is a recognised genetic disease.
Congenital heart disease (CHD) is increasingly better managed, and thus pregnancy
has become safer. But the large shift of blood volume and changing intravascular pressures
that take place physiologically, just after delivery, still mean that those with CHD and
pulmonary hypertension are at significant risk of cardiac arrest.

Peripartum cardiomyopathy
This is defined as heart failure during the last month of pregnancy and up to 5 months
postdelivery, with all other causes excluded. It is a dilated cardiomyopathy with the usual
nonspecific histology. Aetiologically, the current view is of an oxidative proapoptotic stress
on myocytes, related to prolactin [10]. Technically, PPCM is a direct maternal death.

Thrombotic thrombocytopaenic purpura (TTP)

Pregnancy probably increases the risk of TTP [11], which happens following abnormalities
of von Willebrand factor physiology that promote platelet clustering and adhesion to
the endothelia of the microvasculature. Platelet thrombi block small vessels in the brain,
kidney, heart and elsewhere. Laboratory blood data demonstrate low platelets but normal
clotting factors and fibrin.
The clinical presentation is usually postpartum, with confusion, microangiopathic
anaemia and renal failure. If it causes rapid death, it is because of blockage of arterioles and
venules in the myocardium, with haemorrhagic microinfarction and acute heart failure.

Pregnancy-associated infections
As well as genital tract and other acute bacterial sepsis syndromes (see above), there are
other important associations, but the immunopathology is not well understood. Because
pregnancy is a relative immunodepressed state with regard to cell-mediated immunity,
viral infections (herpes simplex, viral hepatitis, influenza), listeriosis and tuberculosis
may be more aggressive than in the nonpregnant woman. However, there is no proof of a
general immunodepression in pregnancy that predisposes to bacterial infections that are
countered by neutralising antibody responses. Two viral syndromes are discussed below.

Epidemic influenza
The 2009-2010 pandemic of type A/H1N1 influenza demonstrated the impact of pregnancy
upon the clinical manifestations of H1N1 infection. It affected mainly third trimester
pregnant woman, who became severely ill from influenza pneumonitis and acute
lung injury, requiring treatment in intensive care. Many acquired secondary bacterial
pneumonia. Proportionately, pregnancy was the pre-eminent risk factor for death with
H1N1 infection with, roughly, a x100 relative risk of death compared with nonpregnant
women [12].
28 The maternal death autopsy

This is not a significant issue in the UK, where nearly all HIV-positive pregnant women
(about 1500 pa) are identified before delivery, and treated for their benefit as well as
reducing the risk of maternofetal transmission of HIV [1]. But in low-income countries with
high HIV prevalence (e.g. sub-Saharan Africa), it is a major (and preventable) contribution
to mortality, increasing the maternal mortality rates by about 10-fold [13]. The typical
scenario is of late presentation with advanced HIV disease at around the time of delivery,
and death shortly after from tuberculosis or other opportunistic infections; or from sepsis
or complications of abortion.

This account is necessarily selective. It is likely that the rank order of types of death will
change as obstetrics and populations change; for example, increasing maternal age and
obesity [1,14] should impact on indirect maternal death rates. The maternal autopsy will
continue to provide powerful information on such future trends.

Key points for clinical practice

Consider whether you are experienced enough to be doing this autopsy, or should hand it
on to a specialist pathologist.
Obtain as much clinical information and laboratory data as possible before starting the
autopsy; enquire what the clinicians consider to be the main problems.
Before evisceration, take sterile blood cultures; and later, retain a femoral venous blood sample.
Be present throughout the evisceration, and do or supervise the dissection yourself.
Be familiar with the range of clinical pathology that could have resulted in death in this case,
and how to make/exclude the diagnoses.
During dissection, pay particular close attention to the pulmonary artery, the heart and the
genital tract.
Sample all organs systematically for histopathology. Take multiple blocks of organs such as
brain, heart and lung when appropriate.
If the autopsy is negative, retain a piece of spleen in the freezer.
To establish the cause of death, discuss the case openly with the relevant obstetricians,
physicians, anaesthetists and intensivists; seek help from more experienced pathologists if
The autopsy report will be scrutinised by the coroner, involved doctors, the family, lawyers
and a confidential enquiry peer-review panel; it may also become the basis for, or the
refutation of, a clinical negligence claim. Its vital to get it right.

1. Cantwell R, Clutton-Brock T, Cooper G et al. Saving Mothers Lives: Reviewing maternal deaths to make
motherhood safer: 2006-2008. The Eighth Report of the Confidential Enquiries into Maternal Deaths in the
United Kingdom. BJOG 2011;118 (Suppl 1):1-203.
2. Lucas S. Guidelines on autopsy practice. Scenario 5: maternal death, G100. London: The Royal College of
Pathologists, 2010.
References 29

3. Knight M, Tuffnell D, Brocklehurst P, Spark P, Kurinczuk JJ. UK obstetric surveillance system. Incidence and
risk factors for amniotic-fluid embolism. Obstet Gynecol 2010; 115:910917.
4. Clark SL. Amniotic fluid embolism. Clin Obstet Gynecol 2010; 53:322328.
5. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005; 365:785799.
6. Zeeman GG. Neurologic complications of pre-eclampsia. Sem Perinatol 2009; 33:166172.
7. Mirza FG, Cleary KL. Pre-eclampsia and the kidney. Sem Perinatol 2009; 33:173178.
8. Joshi D, James A, Quaglia A, Westbrook RH, Heneghan MA. Liver disease in pregnancy. Lancet 2010;
9. Lucas S. The autopsy pathology of sepsis-related death. In: Fernandez R (ed.), Severe Sepsis and Septic
Shock - Understanding a Serious Killer. Rijeka: InTech, 2012.
10. Hilfiker-Kleiner D, et al. 16-kDa prolactin and bromocriptine in post-partum cardiomyopathy. Curr Heart
Fail Rep 2012; 9:174182.
11. Hunt BJ, Thomas-Dewing RR, Bramham K, Lucas SB. Preventing maternal deaths due to acquired
thrombotic thrombocytopenic purpura. J Obstet Gynaecol Res 2013; 39:347350.
12. Moran NF, Moodley J. The effect of HIV infection on maternal health and mortality. Int J Gynaecol Obstet
2012; 119:S2629.
13. Lucas SB. Predictive clinicopathological features derived from systematic autopsy examination of patients
who died with A/H1N1 influenza infection in the UK 200910 pandemic. Health Technol Assess 2010;
14. Knight M, Kurinczuk JJ, Spark P, Brocklehurst P. UK Obstetric Surveillance System. Extreme obesity in
pregnancy in the United Kingdom. Obstet Gynecol 2010; 115:989997.
Chapter 3
Classification and treatment of
non-small-cell lung carcinoma
Golda Shelley-Fraser, Nidhi Bhatt, Adam Dangoor and Matthew Sephton

Lung cancer mortality remains a major health issue causing over a million deaths worldwide
in 2000 according to WHO data [1]. Cancer trends have been steadily changing in recent
times. Whilst lung cancer in wosmen remains low in developing countries, incidence and
mortality in women have almost doubled in developed countries over a 30-year period.
Latest data from WHO GLOBOCAN 2008 [2] shows that lung cancer has surpassed breast
cancer as the leading cause of cancer death in women. Alternatively, incidence in men
has plateaued in developed countries. Changing lung cancer trends have been related to
increasing incidence of smoking in women, particularly of cigarettes with lower tar and
nicotine that are also reportedly associated with increase in adenocarcinoma incidence.
Investigative studies into vast survival differences in adenocarcinoma subtypes (as in
2004 WHO classification) in last few years have resulted in major advancements in the
management of non-small-cell lung carcinoma (NSCLC).

In 2011, the International Association for the Study of Lung Cancer, American Thoracic
Society and European Respiratory Society (IASLC/ATS/ERS) jointly developed a
multidisciplinary approach to classification and management of adenocarcinoma [3]. In
essence, a diagnostic approach for former bronchioloalveolar carcinoma (BAC) has been
developed (Figure 3.1). There is emphasis on approach to small specimens for diagnosis as
well as to optimise tissue for molecular tests.

Preinvasive neoplasia
Atypical adenomatous hyperplasia (AAH)
This is defined as a 5mm area of atypical pneumocyte proliferation lining centriacinar
alveoli with minimal septal widening. The lesional cells are believed to be type 2

Golda Shelley-Fraser, FRCPath, Histopathologist, University Hospitals Bristol, UK

Email: golda.shelley-fraser@uhbristol.nhs.uk (for correspondence)
Nidhi Bhatt, MD(Path), FRCPath, Histopathologist, University Hospitals Bristol, UK
Adam Dangoor, MRCP, MD Medical Oncologist, Bristol Haematology & Oncology Centre
Matthew Sephton, MD, Medical Oncologist, Yeovil District Hospital, Yeovil, UK
32 Classification and treatment of non-small-cell lung carcinoma

Figure 3.1 Peripheral circles

illustrate newly defined and
more accurate categories in the
Adenocarcinoma 2011 classification which replace
in situ the former confusing term of
bronchioloalveolar carcinoma
Minimally Lepidic
invasive predominant
adenocarcinoma adenocarcinoma

with a lepidic

pneumocytes and/or Clara cells. They have a hobnail appearance and a high nuclear:
cytoplasmic ratio.
AAH can be multifocal and is often an incidental finding in the background of NSCLCs.
It may be difficult to differentiate from type 2 pneumocyte hyperplasia, but AAH is discrete
unlike the ill-defined and patchy nature of the latter.

Adenocarcinoma in situ (AIS)

AIS replaces former BAC. It is morphologically similar to AAH but larger (>5mm). It is
defined as a localised (3cm) neoplastic lesion growing along existing alveolar walls in
a lepidic growth pattern. Alveolar septal widening is usually present. There must be no
evidence of parenchymal, pleural or lymphovascular invasion. Diagnosis should be made
only after the entire lesion has been examined histologically. If strict diagnostic criteria are
used, AIS has a 100% 5-year disease-free survival (DFS).
AIS may be nonmucinous or mucinous most are, however, nonmucinous. Nonmucinous
AIS has a hobnail appearance and discrete borders. Monotonous morphology and diffuse
strong p53 expression differentiates it from type 2 pneumocyte hyperplasia or bronchiolar
metaplasia. Mucinous AIS is composed of columnar mucinous epithelium with foveolar/
goblet cell morphology and may be difficult to differentiate from upper gastrointestinal
metastases as thyroid transcription factor 1 (TTF-1) is often not expressed.

Invasive neoplasia
Minimally invasive adenocarcinoma (MIA)
MIA is a small (3cm) adenocarcinoma with lepidic growth pattern and 1 foci of invasion
measuring 5mm. Invasion is defined by the presence of angulated tumour nests or
acini within a desmoplastic or myofibroblastic stroma. By definition, it lacks pleural and
lymphovascular invasion and shows no evidence of tumour necrosis. As for AIS, diagnosis
Adenocarcinoma 33

should be made only after the entire lesion has been examined pathologically. If strict
diagnostic criteria are used, MIA has a 100% 5-year DFS.

Invasive nonmucinous adenocarcinoma (INA)

As predominant histological subtypes have been shown to predict prognosis and correlate
with molecular data, the 2011 classification recommends recording the percentages of
various subtypes within an adenocarcinoma (Figure 3.2).

Lepidic predominant adenocarcinoma (LPA)

Larger (>3cm) adenocarcinomas that comprise mainly lepidic growth but also display
invasion are classified as LPAs. Also included are 3cm INAs that show tumour necrosis,
lymphovascular invasion or pleural invasion. Stage 1 LPA reportedly shows 90% 5-year DFS.

Acinar predominant adenocarcinoma (APA)

These are invasive adenocarcinomas displaying glandular structures with visible lumina.
They may be closely packed or back-to-back or may infiltrate desmoplastic stroma. APA is
the commonest invasive pattern seen in a MIA. Cribriform growth pattern is also included.

Papillary predominant adenocarcinoma (PPA)

These are invasive adenocarcinomas showing true luminal papillary structures with
fibrovascular cores. Demonstration of desmoplastic stromal reaction is not a prerequisite.
This growth pattern has prognosis similar to acinar subtype.

a b c

d e f

Figure 3.2 Subtypes of invasive nonmucinous adenocarcinoma. (a) Lepidic component of a lepidic predominant
adenocarcinoma seen confined to the alveolar lining with no evidence of stromal invasion. (b) Lepidic
predominant with possible acinar component. (c) Acinar predominant adenocarcinoma showing glandular
profiles invading desmoplastic stroma. (d) Papillary predominant adenocarcinoma showing intra-alveolar tumour
papillae with fibrovascular cores. (e) Micropapillary predominant adenocarcinoma showing intra-alveolar tufts with
no stromal cores. (f ) Solid adenocarcinoma showing no morphological evidence of glandular differentiation.
34 Classification and treatment of non-small-cell lung carcinoma

Micropapillary predominant adenocarcinoma (MPA)

This subtype is characterised by intra-alveolar detached papillae, tufts or rings of
neoplastic cells lacking fibrovascular cores. Tumour groups readily infiltrate the
lung parenchyma with minimal stromal response. Lymphovascular invasion is often
widespread at the time of diagnosis. Micropapillary and solid subtypes have been shown
to have the worst prognosis [4].

Solid predominant adenocarcinoma (SPA)

Solid subtype is characterised by nests and cords of tumour cells that show
minimal morphological evidence of glandular differentiation. Mucin stains and
immunophenotyping are often needed to demonstrate their glandular nature. Pure
forms may be mistaken for nonkeratinising squamous cell carcinoma (SCC) or large cell
carcinoma (LCC).

Invasive mucinous adenocarcinoma (IMA)

This term replaces former mucinous BAC. IMAs are now recognised as being
morphologically, genetically and clinically distinct from nonmucinous adenocarcinomas.
Most common genetic abnormalities are KRAS mutations and EML4-ALK translocation.
They usually present at a higher stage and are often mistaken for metastatic disease or
multifocal consolidation on imaging due to multifocal involvement probably resulting from
aerogenous spread.
IMA comprises mucinous columnar or goblet cell epithelium and shows
intracytoplasmic mucin production. The tumour cells show minimal cytological atypia and
maintain nuclear polarity. They may display architectural patterns similar to INA, but this is
not known to be prognostically significant.
In the 2011 classification, colloid adenocarcinoma and fetal adenocarcinoma subtypes
have been retained but mucinous cystadenocarcinoma, signet ring carcinoma and clear
cell carcinoma have been abolished as their pure forms are rare and overlap with the
existing morphological variants (Figure 3.3).

Colloid adenocarcinoma
This is characterised by extracellular mucin lakes that expand and/or destroy existing
alveolar spaces and contain groups of malignant mucinous epithelium, which are often
difficult to visualise. The mucinous epithelium is of goblet cell or columnar type.

Fetal adenocarcinoma
This is characterised by cribriform and tubular structures composed of columnar cells with
clear cytoplasm that recapitulate fetal lung tubules and resemble early secretory phase
endometrium. Morular metaplasia, as in endometrioid adenocarcinoma, is also present.
Pure variants are usually seen in younger age groups. They are associated with
-catenin mutations and show nuclear (as opposed to membranous) -catenin [5]. In the
authors experience, they are often seen as a minor component in otherwise conventional
adenocarcinomas; these have normal membranous -catenin expression.

Enteric adenocarcinoma
This variant is defined as the presence of enteric differentiation (acinar/cribriform
architecture and intra-acinar necrosis) in at least 50% of the tumour. Presence of other
Large cell carcinoma 35

a b c

d e f

Figure 3.3 Mucinous lung neoplasia. (a) Mucinous atypical adenomatous hyperplasia. (b) Mucinous
adenocarcinoma in situ. (c) Mucinous minimally invasive adenocarcinoma. (d) Invasive mucinous adenocarcinoma.
(e) Enteric adenocarcinoma. (f ) Fetal adenocarcinoma.

subtypes of primary lung adenocarcinomas facilitates distinction from metastatic

colorectal adenocarcinoma.

Squamous cell carcinoma

According to WHO criteria, SCC is an epithelial tumour that shows evidence of
keratinisation and /or intracellular bridges arising from bronchial epithelium. SCC
represents approximately 30% of NSCLCs. They have a strong association with cigarette
smoking but due to changes in smoking behaviour, the incidence is decreasing unlike
adenocarcinoma [1].
SCC usually presents as a central tumour close to the hilum. They tend to produce
symptoms earlier than adenocarcinoma and commonly show direct spread into adjacent
structures, in particular hilar lymph nodes. However, they are late to metastasis compared
with adenocarcinoma. SCC often grows to a large size, becomes necrotic and cavitates; they
are commonly associated with obstructive pneumonitis downstream.
The current WHO classification mentions four subtypes: papillary, clear cell, small cell
and basaloid. SCC expresses high molecular weight keratins, CK5/6 and shows nuclear
expression with p63; very few express CK7 or TTF-1.

Large cell carcinoma

LCCs are undifferentiated carcinomas lacking squamous and glandular differentiation.
They represent approximately 10% of NSCLCs and have a strong association with cigarette
smoking. They commonly present as large peripheral tumours that macroscopically show a
fleshy, necrotic cut surface without cavitation.
36 Classification and treatment of non-small-cell lung carcinoma

This is a diagnosis of exclusion that can only be accurately made on a surgical specimen
rather than biopsy due to the heterogeneity of lung carcinomas. WHO has separated LCC
into subtypes: large cell neuroendocrine carcinoma, large cell undifferentiated carcinoma,
basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma and LCC
with rhabdoid differentiation.
Large cell neuroendocrine carcinomas express neuroendocrine markers including
synaptophysin, chromogranin and CD56, although only expression of one is required
for diagnosis. Large cell undifferentiated carcinoma usually expresses cytokeratins
and approximately half express TTF-1; neuroendocrine markers are negative. Basaloid
carcinoma is rare, resembling its cutaneous counterpart. Lymphoepithelial-like carcinoma
of the lung shows a syncytial growth pattern and a prominent lymphocytic infiltrate;
there is an association with EpsteinBarr virus. Clear cell carcinoma shows prominent
glycogen containing clear cytoplasm and should be distinguished from metastatic renal cell
carcinoma. In LCC with rhabdoid differentiation, at least 10% of tumour cells should show
rhabdoid differentiation.

Adenosquamous carcinoma
Adenosquamous carcinoma is defined as carcinoma showing both squamous and
glandular differentiation, with each component comprising at least 10%, according to WHO
criteria. They represent between 0.4% and 4% of NSCLCs and are associated with cigarette
smoking. They often present as a peripheral mass and can show central scar formation.
Histologically, they must show both squamous and glandular differentiation. The
two components may be separate or may mingle. Diagnosis on biopsy is dependent
on sampling; definitive diagnosis is best made on the surgical specimen. On
immunohistochemistry, the adenocarcinoma component should express TTF-1 and CK7,
whilst the squamous component is usually decorated by p63 and CK5/6.

Sarcomatoid carcinoma
Sarcomatoid carcinomas are a group of poorly differentiated non-small cell carcinomas.
They represent approximately 1% of non-small cell carcinomas and are strongly associated
with tobacco smoking. WHO divides this subtype into 5: pleomorphic carcinoma, spindle
cell carcinoma, giant cell carcinoma, carcinosarcoma and pulmonary blastoma.
They commonly arise centrally, although pleomorphic carcinomas tend to be peripheral
showing a predilection for chest wall involvement. Pleomorphic carcinoma is composed of
non-small cell carcinoma either SCC, adenocarcinoma or LCC admixed with a population
of malignant spindle or giant cells, the latter occupying at least 10% of the total tumour
Spindle cell carcinoma is composed wholly of malignant spindle cells, and likewise
giant cell carcinoma consists solely of malignant giant cells. Carcinosarcoma is a biphasic
tumour composed of both epithelial and sarcomatoid elements; the carcinoma component
is generally SCC. The sarcomatoid component may display chondroid, rhabdoid or
osteoid differentiation. Pulmonary blastoma is a primitive tumour composed of epithelial
and mesenchymal foci. The epithelial component may resemble fetal adenocarcinoma;
squamous morulae may be included. The stromal cell component is undifferentiated and
Staging TNM 7th edition 37

resembles blastema. The epithelial elements usually express cytokeratins and the blastema
cells express vimentin and smooth muscle actin.

Staging TNM 7th edition

Tumour size
TNM 7th edition includes the additional size cut-offs of 2, 5 and 7cm further to 3cm. T1
tumours are classified as T1a if 2cm or T1b if >2 but <3cm. T2 tumours are classified as
T2a if 5cm or as T2b if >5cm but <7cm [6,7].

Multiple tumours
Multiple tumours can present challenges. All tumours must be identified macroscopically,
measured accurately and their relationship to one another established. The tumours
need to be compared microscopically to determine whether they have similar histological
appearances. If similar they are likely to represent intrapulmonary metastases, if not
synchronous primaries are favoured (Table 3.1) [8,9].

Pleural invasion
TNM 7th edition made recommendations for evaluation of visceral pleural invasion. If
there is no suggestion of pleura involvement, the tumour is classified as PL0. If the tumour
invades beyond the outer elastic layer, it is classified as PL1. If tumour extends to the
pleural surface, it is PL2. The tumour is classified as PL3 when there is parietal pleural
involvement. The use of elastic stains is advocated. PL1 or PL2 upstages an otherwise T1
tumour to T2 and PL3 upstages a tumour to T3 (Figure 3.4) [10].

Table 3.1 Comparison of TNM 6th edition classification with the changes in TNM 7th edition
Description 6th edition TNM 7th edition TNM
2cm T1 T1a
>23cm T1 T1b
5cm T2 T2a
>57cm T2 T2b
>7cm T2 T3
Invasion of chest wall, diaphragm, phrenic nerve, parietal pleura, T3 T3
mediastinal pleura, parietal pericardium
Separate tumour nodules in the same lobe T4 T3
Invasion of heart, great vessels, trachea, oesophagus, vertebral T4 T4
body, carina
Separate tumour nodules in an ipsilateral lobe M1 T4
Malignant pleural or pericardial effusion T4 M1a
Separate tumour nodules in a contralateral lobe M1 M1a
Distant metastases M1 M1b
38 Classification and treatment of non-small-cell lung carcinoma

Figure 3.4Demonstration
of visceral pleural invasion. (a)
The pleura appears to be intact
without invasion on haematoxylin
and eosin staining; the elastic
stain (EVG) demonstrates invasion
of tumour beyond the outer
elastic layer (PL1). (b) Tumour
a b extending to the visceral pleural
surface (PL2).

c d

Problems in diagnosis
Interobserver reproducibility in subtypes
Several recent studies have investigated interobserver variability amongst experts in
diagnosing the newly described INA subtypes. Whilst these have reported high level of
agreement (approaching 90%) in identifying a single pattern as the predominant pattern
[11,12], similar agreement is not achievable in identifying all patterns and even less so
in determining the percentage proportion of each subtype (authors experience). The
prognostic significance of the latter is debatable but may be useful in distinguishing
between a metachronous/synchronous primary and a metastasis.

Assessment of invasion
Distinguishing between AIS and LPA is usually straightforward but separating AIS and MIA
& MIA and LPA may be quite challenging even after the entire lesion has been examined
histologically [1]. An elastin stain has been recommended to differentiate alveolar collapse
from alveolar destruction by invasion, but this is often difficult to interpret.

Squamous cell carcinoma

Cross-cut squamous dysplasia which is growing along mucinous structures can be
mistaken for invasion and misdiagnosed as SCC. The other differential diagnoses are solid
adenocarcinoma and the small cell variant of SCC which may mimic small cell carcinoma.
Clarification can be sought with immunostaining a solid adenocarcinoma should express
TTF-1 and be negative for p63 (Figure 3.5). It is worth noting that SCC can show focal
mucin deposition. Small cell carcinoma will express neuroendocrine markers and TTF-1,
whilst a squame will not.
Problems in diagnosis 39

Figure 3.5 Subtypes of non-

small cell lung cancer with similar
morphologies. (a) Squamous cell
carcinoma (with positive nuclear
expression of p63). (b) Solid
subtype adenocarcinoma (with
positive nuclear expression of
thyroid transcription factor 1 (TTF-
a b
1). (c) Large cell neuroendocrine
carcinoma (with positive
expression of CD56). (d) Large cell
carcinoma undifferentiated (with
positive pan cytokeratin staining).

c d

Differentiating between a metastatic SCC in the lung from a primary tumour can be
difficult. Immunohistochemistry is not particularly helpful in this matter, although a
metastasis from a head and neck tumour may express p16 and/or HPV if HPV related.
Otherwise, architectural features such as vascular invasion, a well-circumscribed lesion
and the lack of an in situ component favour a metastasis over a primary lesion.

Large cell carcinoma

Large cell neuroendocrine carcinoma often shows cytological features suggestive of
neuroendocrine differentiation such as fine chromatin without nucleoli. Differentiation
from an atypical carcinoid is based on morphological criteria such as >10 mitoses per
10 high-power field and from small cell carcinoma on cell size, morphology and Ki67
proliferation index.

Adenosquamous carcinoma
Common pitfalls in the diagnosis of adenosquamous carcinoma are entrapped benign
bronchial glands within a SCC and squamous dysplasia in an adenocarcinoma. Diagnosis
is straight forward if squamous differentiation is associated with acinar, papillary or
lepidic growth patterns of adenocarcinoma. The diagnosis is more made difficult when the
adenocarcinoma component shows a solid growth pattern. Mucin droplets can be seen
in SCC and so in this situation more than five mucin droplets per high-power field are
required for a diagnosis [1].
The main differential diagnosis is mucoepidermoid carcinoma, in particular a high-
grade mucoepidermoid carcinoma. Low-grade mucoepidermoid carcinomas are generally
easy to separate from adenosquamous carcinoma as they lack significant cytological atypia.
Also, mucoepidermoid carcinomas tend to be centrally located, whereas adenosquamous
carcinomas are generally peripheral.
40 Classification and treatment of non-small-cell lung carcinoma

Treatment of NSCLC
First-line chemotherapy
The management of metastatic NSCLC has grown in complexity in the past 5 years, guided
by the increasing influence from histological and molecular analyses. In the past, it was
considered sufficient to have obtained histological confirmation of the diagnosis of lung
cancer and having simply differentiated between NSCLC and small cell lung cancer. This
was commonly possible on bronchial washings and brushings. Small cell lung cancer is
traditionally treated with a platinum agent, cisplatin or carboplatin, in combination with
a second agent, such as etoposide. For the first-line treatment of patients with metastatic
NSCLC, the platinum agents have commonly been combined with one other agent such
as gemcitabine, paclitaxel or vinorelbine in a platinum doublet, regardless of histological
These regimens have been shown in phase III studies to have comparable efficacy
with varying side effect profiles [1315]. Until 2008-2009, patients in the United Kingdom
were commonly treated with a platinum and gemcitabine combination for all histological
subtypes of NSCLC (Figure 3.6), with median overall survival and progression-free
survival in the ranges of 9.59.9 months and 4.65.5 months respectively [14]. With respect
to whether cisplatin or carboplatin should be the platinum-containing agent of choice,
the British Thoracic Oncology Group (BTOG) trial BTOG2 showed that carboplatin
and gemcitabine were not inferior to the higher dose of cisplatin (80mg/m2) used in
combination with gemcitabine in one of the trial arms in terms of median overall survival
[16] and therefore carboplatin/gemcitabine remained a popular treatment option for
metastatic NSCLC until 2009.
In 2008, the importance of identifying the histological subtype of NSCLC in guiding the
management was shown. Scagliotti et al. [17] identified that overall survival in patients
with NSCLC receiving cisplatin/pemextrexed was noninferior to cisplatin/gemcitabine
(median survival 10.3 vs. 10.3 months respectively). However, the study identified that
patients with adenocarcinoma subtype had a 1.7 month improved overall survival if
they received cisplatin /pemetrexed, rather than cisplatin/gemcitabine (12.6 vs. 10.9
months respectively). In the case of large-cell carcinoma histology, the improvement in

For palliative chemotherapy

NSCLC confirmed

Figure 3.6 Typical management

First line:
of metastatic non-small cell lung
cancer (NSCLC) in 2009. Once
histopathological confirmation
of the diagnosis had been made,
further pathological or molecular
Histopathological/ analyses played no further role in
molecular analysis
Second line: altering the management. This is a
erlotinib or docetaxel Progressive disease typical situation and is by no means
Treatment of NSCLC 41

overall survival with cisplatin/pemetrexed was even more marked compared to cisplatin/
gemcitabine (10.4 vs. 6.7 months respectively). In contrast, the reverse situation was seen
in patients with squamous cell histology, where there was significantly worse survival with
cisplatin/pemetrexed versus cisplatin/gemcitabine (9.4 vs. 10.8 months respectively).
This was the first prospective phase III study in NSCLC to show survival differences
based on histological subtype. It is thought that the reason for this difference in efficacy of
chemotherapy regimens is based on significantly increased levels of thymidylate synthetase
(TS) in squamous cell histology. Increased levels of TS have been shown in preclinical data
to correlate with reduced sensitivity to pemetrexed. The work from Scagliotti et al. shifted
the need from simply diagnosing NSCLC, to the need to identify the histological subtype,
in order for the optimal chemotherapy regimen to be recommended for the patient.
The National Institute for Health and Clinical Excellence (NICE) in the United Kingdom
approved the use of pemetrexed in combination with cisplatin for the first-line treatment of
patients with locally advanced or metastatic NSCLC with non-squamous histology [18].

Epidermal growth factor receptor tyrosine kinase inhibitors

The epidermal growth factor receptor (EGFR) has been used as a therapeutic target in
NSCLC for a number of years. In early trials, EGFR detection by immunohistochemistry
was used as a marker for EGFR expression, but this did not correlate well with responses
to EGFR tyrosine kinase inhibitors (TKIs). It was the identification of certain activating
mutations in EGFR, present in a proportion of patients with NSCLC, which correlated
better with response to EGFR TKIs [19]. Prior to 2010, EGFR TKIs were commonly used in
predominantly second-line treatment of NSCLC after disease progression despite first-
line cytotoxic chemotherapy. At this time, activating mutations in EGFR were commonly
not assessed. The BR21 trial showed that the median overall survival for patients receiving
erlotinib, used in the second- and third-line settings for NSCLC, regardless for EGFR
mutation status, was 6.7 months compared with 4.7 months for placebo [20]. As a result
of an indirect comparison of trials comparing docetaxel and erlotinib in the second-line
setting for NSCLC, NICE approved the use of erlotinib as an alternative to docetaxel in this
setting in 2008 [21] (Figure 3.6)
Shortly after this, interest of the use of EGFR TKIs in the first-line setting developed
and in 2009 the results of the IressaPan-Asia Study (IPASS) were published [22]. This study
compared gefitinib (Iressa) with carboplatin/paclitaxel in the first-line setting in patients
with advanced pulmonary adenocarcinoma in East Asia, who were nonsmokers or former
light smokers. The 12-month rates of progression-free survival were 24.9% with gefitinib
and 6.7% with carboplatin/paclitaxel, confirming the noninferiority and, in fact, superiority
of gefitinib compared with carboplatin/paclitaxel with respect to progression-free survival
in the intention-to-treat population. In the subgroup of patients who were positive for an
EGFR gene mutation, progression-free survival was significantly longer for those patients
who received gefitinib than those who received carboplatin/paclitaxel (Hazard ratio (HR)
for progression, 0.48; 95% CI, 0.360.64; P<0.001).
Objective response rates were also remarkably high in patients with a mutation
treated with gefitinib, compared to chemotherapy (71.2% vs. 47.3% respectively). In
contrast, in patients who were negative for an EGFR mutation, progression-free survival
was significantly longer amongst those who had received carboplatin/paclitaxel (HR for
progression with gefitinib, 2.85; 95% CI, 2.053.98; P<0.001), and the use of gefitinib in
this subgroup of patients was associated with a significantly lower objective response rate
compared with chemotherapy (1.1% vs. 23.5% with carboplatin/paclitaxel). The IPASS
42 Classification and treatment of non-small-cell lung carcinoma

study was performed in a population known to have high rates of EGFR gene mutations
(Asian patients, non-smokers or former light smokers), but the subsequent EURTAC study
demonstrated similar results in the European population using a different EGFR TKI,
erlotinib (Tarceva), where median progression-free survival was 9.7 months with erlotinib
versus 5.2 months with chemotherapy in EGFR mutation positive patients [23].
In addition to IPASS and EURTAC, other studies have confirmed the additional benefit of
using first-line EGFR TKIs in patients with EGFR mutations [24]. It has also been shown that
this treatment is commonly better tolerated than cytotoxic chemotherapy and therefore
knowing the EGFR mutation status of a patient with metastatic NSCLC is of key importance
in guiding the appropriate treatment, in addition to knowing the histological subtype of
NSCLC. Rates of EGFR mutations are higher in nonsquamous subtypes (approximately
10% in adenocarcinomas [25] versus 3.4% in squamous cell subtypes [26]), and therefore
it is common tumours of the nonsquamous subtype that are sent for EGFR mutation
analysis. The importance of testing appropriate samples for EGFR mutation analysis has
implications for the need of larger sample sizes during diagnostic procedures.

Anaplastic lymphoma kinase pathway

In addition to the EGFR pathway, other potential therapeutic targets have been studied in
NSCLC. In 2007, rearrangements in the gene coding anaplastic lymphoma kinase (ALK),
leading to ALK fusion proteins, were found in patients with NSCLC [27]. These fusion
proteins appeared to play a critical role in tumourigenesis. The most common ALK fusion
protein in NSCLC is the echinoderm microtubule-associated protein-like 4 (EML4-ALK),
although other transforming ALK fusion proteins have been described. Further research
has indicated that ALK-positive tumours account for approximately 35% of NSCLC cases
[28,29], mostly in patients who are nonsmokers (or 10 pack years) of a younger age than
the average for lung cancer, and of adenocarcinoma histology [16].
Preclinical studies of the dual ALK and mesenchymal-epithelial transition factor
TKI, crizotinib (Xalkori), were rapidly undertaken and the promising significant clinical
responses using crizotinib in ALK-positive patients with NSCLC in phase I and phase
II studies led to a prospective, randomised phase III study, PROFILE 1007. This study,
comparing second-line crizotinib with pemetrexed or docetaxel chemotherapy in patients
with ALK-positive NSCLC, who had received previous platinum-based chemotherapy,
showed an improvement in progression-free survival with crizotinib compared to
chemotherapy (7.7 months vs. 3.0 months respectively) and an improvement in objective
response rate (65.3% with crizotinib vs. 19.5% with chemotherapy) [30]. Crizotinib is
therefore indicated for the treatment of previously treated ALK-positive advanced NSCLC
and has been referred for review by NICE. Additional confirmatory randomised phase III
studies, including those in the first-line setting, are underway.
The development of crizotinib, and the speed of progress from early phase trials to
clinical use, illustrates how rapid this process can now be when an appropriate target
and effective agent can be identified. Crizotinib is an example of an agent that has been
developed to provide personalised medicine for patients with ALK-positive NSCLC.
Tailoring a treatment based on histological and molecular analyses is an expanding area
not only in the treatment of lung cancer but also in oncology in general. The Cancer
Research UK Stratified Medicine Programme is an example of this drive, with the aim of
making genetic testing more readily available, therefore improving the access of targeted
therapies available for patients (Table 3.2).
Treatment of NSCLC 43

Table 3.2 Tyrosine kinase inhibitors in clinical use in NSCLC

Tyrosine kinase inhibitor Description
Gefitinib EGFR inhibitor
Used as first-line treatment in EGFR mutation positive patients
Erlotinib EGFR inhibitor
Used as first-line treatment in EGFR mutation positive patients
Used as second-line treatment after progression with chemotherapy
Crizotinib ALK, c-Met and HGFR inhibitor
Used as first-line treatment in patients positive for EML4-ALK fusion protein
Afatinib EGFR, Her2 and Her4 inhibitor
Maybe effective after failure of previous EGFR TKI therapy

NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma kinase; c-Met,
mesenchymal-epithelial transition factor; HGFR, hepatocyte growth factor receptor; EMLA4, echinoderm microtubule-
associated protein-like 4; Her2, human epidermal growth receptor 2; Her4, human epidermal growth factor receptor 4.

Optimising the diagnostic pathway to obtain sufficient tissue for analysis is an

increasingly important focus of the lung cancer multidisciplinary team; whilst the
development of optimal pathological and cytogenetic techniques is required to provide
reliable data on which to base treatment decisions. This is an exciting time in the
development of new diagnostics and treatments of lung cancer (Figure 3.7), which should
ultimately result in more effective treatment of patients.

For systemic therapy

EGFR mutation ALK Non-squamous Squamous

positive positive histology histology

First line: First line:

First line:
gefitinib First line: platinum/gemcitabine
or crizotinib or
erlotinib vinorelbine

Second line:
Histopathological/ docetaxel
molecular analysis
Progressive disease erlotinib

Figure 3.7 Typical management of metastatic non-small cell lung cancer (NSCLC) in 2013. There is increasing
influence of pathological and molecular analyses on the management. This is a typical situation and is by no means
extensive. NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; ALK, anaplastic lymphoma
44 Classification and treatment of non-small-cell lung carcinoma

Key points for clinical practice

The term bronchioloalveolar carcinoma (BAC) is now obsolete and should no longer be
used in pathology reports.
Predominant histological subtype of adenocarcinoma should be recorded as this has been
shown to have prognostic implications. Percentages of secondary subtypes should also be
recorded until further data emerges, especially the presence of subtypes associated with
worse prognosis, i.e. micropapillary and solid subtypes.
The entire lesion should be processed for histology if it has a radiological ground-glass
appearance and is <3cm to enable distinction between AIS and MIA.
Due to sampling error and heterogeneity of non-small cell carcinomas, diagnosis of the rarer
subtypes should be made on the resection specimen rather than on biopsy.
Use of connective tissue stains is recommended for accurate assessment of pleural invasion
by carcinoma.
Pathological and molecular analyses are playing an increasing role in guiding the
management of patients with NSCLC.
The increasing need for larger samples of tissue for the appropriate analyses means that
bronchial washings and brushings are increasing becoming inadequate.
Identifying the histological subtype of NSCLC is important in guiding the initial management
and recommending further analyses, such as EGFR mutation analysis, on tissue samples of
nonsquamous subtype.
Many of the newer therapies, such as TKIs, have varying side effect profiles, which may be
different from cytotoxic chemotherapy and maybe better tolerated by the patient.
Further research is investigating the resistance mechanisms involved when a treatment,
such as with a TKI, ceases to work. The role of repeat biopsying in this situation is uncertain
at present but likely to become more commonly requested.

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Chapter 4

Pathology of obesity
Eve P Fryer, Ian SD Roberts, Clare Verrill

The prevalence of obesity worldwide is increasing at an alarming rate. In the USA, 35.7%
of adults are obese [1] and obesity accounts for approximately 280,000325,000 deaths
annually [2]. Degrees of obesity are also rising; for example in the USA, numbers of
people in body mass index (BMI) categories >40 and >50 have increased by 50% and 75%
respectively between 2000 and 2005 [3]. Pathologists will therefore inevitably encounter
the effects of obesity in their surgical diagnostic work or in their post-mortem practice and
increasingly, pathologists will be asked to perform autopsies in those who have undergone
bariatric surgery.
Post-mortems in the obese are challenging and it is not just the logistical and
physical challenges of dealing with larger body sizes which are important, but also
there are a number of fatal conditions which are effectively exclusive to obesity such as
obstructive sleep apnoea (OSA), obesity hypoventilation syndrome (OHS) and obesity
cardiomyopathy. These conditions are likely being under-recognised by pathologists, who
attribute death to other conditions such as nonsignificant coronary artery atherosclerosis.
In addition, there are a number of conditions for which obesity is a significant, but lesser
known independent risk factor such as venous thromboembolic disease. It is important
that the role obesity plays in these deaths is acknowledged by inclusion in the medical
certificate of cause of death. This, however, is difficult to implement when, e.g., body
height and weight are not being recorded in approximately half of coronial autopsy reports
in the United Kingdom [4].
The concept that adipose tissue is not an inert substance, but an organ which has
multisystemic effects when present in increased amounts in obesity, is gaining acceptance.
As a consequence, obese individuals are therefore not just larger than normal, but
have altered physiology. Adipose tissue has an endocrine role, secreting adipokines
and promoting inflammation by secretion of cytokines, which are implicated in the
development of cancer, atherosclerosis and thrombosis.

Eve P Fryer, FRCPath, Consultant Pathologist, Oxford University Hospitals NHS Trust, Oxford, UK
Ian SD Roberts, FRCPath, Professor of Cellular Pathology, University of Oxford, Consultant Pathologist, Oxford
University Hospitals NHS Trust, Oxford, UK
Clare Verrill, FRCPath, Consultant Pathologist, Oxford University Hospitals NHS Trust, Oxford, UK
Email: Clare.Verrill@OUH.nhs.uk (for correspondence)
48 Pathology of obesity

Obesity is most commonly assessed by BMI which is calculated by weight in kilograms

divided by the square of the height in metres (kg/m2), although waist: hip ratio is probably
a better measure of cardiovascular disease risk. The WHO classification system for BMI
defines obesity as a BMI of >30 with further subcategories (Table 4.1) [5]. Obesity is a
heterogeneous group, and to aid description of more extreme degrees of obesity, further
categories have been proposed (Table 4.1).
This article aims to summarise the effects of obesity which may be encountered by
pathologists in diagnostic or autopsy practice and to provide guidance as to how best to
manage these complex cases. It is beyond the scope of this review to cover every condition
to which obesity is related. For an overview of these conditions, see the review article by RW
Byard [6].

Respiratory system
Obstructive sleep apnoea
OSA is a condition of recurrent episodes of apnoea (interrupted air flow) due to obstruction
of the upper airway during sleep, followed by transient awakening to restore airway patency
[7]. The condition occurs in the obese because of accumulation of fat in the neck region.
The diagnosis is suggested by a history of snoring and observed apnoeas. The diagnosis
is confirmed by polysomnography and it is usually treated by continuous positive airway
pressure (CPAP). The condition is exacerbated by smoking (which increases inflammation
of the upper airways), sedative drugs and alcohol consumption (as they worsen the
apnoeas and arterial de-saturations).
Recurrent apnoeas cause chronic alveolar hypoxia, which can result in pulmonary
artery constriction with subsequent pulmonary hypertension and cor pulmonale. Right
ventricular failure is usually accompanied by left ventricular failure due to the direct effects
of obesity or hypertension. Far from just causing poor quality sleep, OSA has numerous
effects, being an independent risk factor for congestive cardiac failure, hypertension,
myocardial infarction, cardiac arrhythmias and sudden death [7]. It is also associated with
increased rates of car and work-related accidents [8].

Table 4.1 The WHO 2000 classification of obesity and other commonly used terminology
BMI (kg/m2) Alternative, commonly used WHO classification 2000 (WHO
terminology 2000)
<18.5 Underweight Underweight
18.5024.99 Normal Normal range
25.0029.99 Overweight Overweight
30.0034.99 Obese Obese class I
35.0039.99 Obese class II
40.00 Morbid obesity Obese class III
50.00 Superobesity
60.00 Super-superobesity
Respiratory system 49

These deaths are often challenging as autopsy findings may be limited to obesity only, but
death can be attributed to OSA provided a thorough approach is taken. The circumstances
of the death are crucial to the diagnosis and therefore details such as exact body position
must be actively sought from the Coroner. In a textbook case, the pathologist would be
provided with a history of sleep apnoea during life, the body would be found in bed with
the deceased on their back (supine), and they may have ingested alcohol or other sedatives
at a level which alone is insufficient to account for death. There may be right ventricular or
biventricular failure. If there is no history of sleep apnoea, it is still possible to attribute death
due to the condition, but as the definitive test (polysomnography) is performed during life,
this becomes more difficult. Features of the case such as being found deceased in bed in the
supine position and a history from relatives suggestive of sleep apnoea, such as snoring, may
support the diagnosis. A post-mortem where OSA is being considered as the cause of death
requires full histological and toxicological assessment.
The suggested criteria for issuing a cause of death at post-mortem due to OSA are [9,10]
as follows:
1. A diagnosis during life of OSA, even in the absence of respiratory failure. Without an
established diagnosis, an appropriate history, e.g. snoring
2. The circumstances of the death, in particular death always in bed, during sleep in the
supine position, often whilst not using CPAP
3. Absence of specific autopsy findings such as an acute cardiac or cerebral event
4. Evidence of intoxication with alcohol or other sedatives

Obesity hypoventilation syndrome

OHS otherwise known as Pickwickian syndrome is a condition comprising hypoventilation,
daytime hypercapnia and hypoxaemia (PaCO2 >45mmHg and PaO2 <70mmHg) in an obese
patient with sleep-disordered breathing (usually OSA) in the absence of any other causes
of hypoventilation such as chronic obstructive pulmonary disease (COPD) [11]. It is the
presence of chronic daytime hypercapnia in OHS which distinguishes it from OSA and as
a result, patients with OHS are more likely to develop cor pulmonale than those with sleep
apnoea alone. The diagnostic test for OHS is a daytime arterial blood gas.
There are three hypotheses for the pathogenesis of chronic daytime hypoventilation
in OHS: impaired respiratory mechanics because of obesity, leptin resistance leading to
central hypoventilation and impaired compensatory response to acute hypercapnia in
OSA [11]. OHS has a high mortality rate, 46% over 50 months if untreated [11], and unlike
OSA, deaths are not a result of respiratory failure or cardiac arrhythmia due to prolonged
hypoxia, but are usually deaths due to cor pulmonale with the mode of death being acute
decompensation or lethal ventricular arrhythmia. Death is usually a sudden collapse, e.g.
in the street or being found suddenly deceased at home and not necessarily in bed. As in
sleep apnoea, there may be no history of OHS during life. In this situation, the features that
would suggest OHS as the cause of death are prominent right ventricular hypertrophy and/
or failure with a history of sleep-disordered breathing (see Figure 4.1). A post-mortem
where OHS is being considered will often require full histological and toxicological analysis.
The criteria for issuing a cause of death due to OHS are [9] as follows:
1. A history during life of OHS. In the absence of this, a history of OSA or snoring during
life. There may be no prior history
50 Pathology of obesity

Figure 4.1 A midventricular slice

from a heart in a death due to
obesity hypoventilation syndrome
with cor pulmonale in a 71-year-old
man found deceased at home. The
body mass index was 46 and heart
weight 768g with biventricular
hypertrophy, most marked on the

2. Sudden death in the absence of a clear alternative cause of death such as an acute
cardiac or cerebral event
3. Features of pulmonary hypertension and cor pulmonale such as right ventricular
hypertrophy and dilatation. In everyday practice, right ventricular hypertrophy can be
defined by the Fulton technique as an isolated right ventricular weight of >65g or a left:
right ventricular weight ratio of <2.33.3:1.

Obesity cardiomyopathy
Obesity directly causes alterations in the structure and function of the heart even in the
absence of other conditions associated with obesity such as hypertension. The mechanism
responsible is an increase in total blood volume and cardiac output because of the high
metabolic activity of excess adipose tissue [12]. The high cardiac output state results in
ventricular dilatation and left and right ventricular hypertrophy with systolic dysfunction.
The term obesity cardiomyopathy is used when these changes lead to congestive cardiac
If systemic hypertension is also present, this further promotes left ventricular
dilatation and hypertrophy, the effect being additive to that of obesity, not synergistic [12].
Hypertensive heart disease can be differentiated from obesity cardiomyopathy by the left
ventricular hypertrophy being concentric in hypertension versus eccentric in obesity.
There may be a history during life of hypertension and there may be changes suggestive of
hypertension in other organs such as the kidney [9].
Obesity cardiomyopathy typically occurs in those who have severe and long-standing
obesity. The modes of death are either progressive congestive heart failure or sudden
arrhythmic cardiac death. Previously, fat infiltration in the myocardium was proposed as
the cause of obesity cardiomyopathy. This is now largely discredited as fat can be seen in the
myocardium of the right ventricle in normal hearts. The currently accepted model of obesity
cardiomyopathy is multifactorial, involving metabolic disturbances (insulin resistance,
Heart 51

increased free fatty acid levels and increased levels of adipokines), activation of the renin
angiotensinaldosterone and sympathetic nervous systems, myocardial remodelling and
small vessel disease [13].
Obesity cardiomyopathy is most likely to be confused with dilated cardiomyopathy
at post-mortem, and obviously the distinction is important as dilated cardiomyopathy
has genetic implications. Dilated cardiomyopathy shows ventricular dilatation with
an inadequate degree of left ventricular hypertrophy (left ventricle wall thickness
<10mm), whereas obesity cardiomyopathy tends to show left ventricular hypertrophy
and dilatation (wall thickness >10mm) [9]. Microscopically, in dilated cardiomyopathy
there is myocardial fibrosis that is not usually seen in obesity cardiomyopathy. Fat may be
present in the right ventricle in obesity cardiomyopathy, but not usually in the left ventricle
unless it surrounds blood vessels in the outer wall (which is considered normal) [9]. It is
recommended that a sample of heart and spleen is frozen for genetic studies in case of an
inherited cardiomyopathy.
Defining cardiomegaly in obesity is difficult. Typically in obesity cardiomyopathy the
heart weight is markedly above that predicted for body weight and typically >800900g.
However, it may be close to that predicted for body weight, but always greater than if
one accepts a fixed value of an upper limit of normal such as 400g for females and 500g
for males [14]. It is known that heart weight increases with body weight due to left and
right ventricular hypertrophy and tables have been proposed which allow calculation of
predicted heart weight from the body weight [15]. At some point, however, even though the
heart weight appears proportional to the body weight, this must be considered potentially
pathological as the increased ventricular mass predisposes to arrhythmia. In the authors
experience, there is a relatively high proportion of deaths in the obese in which there are few
findings, namely obesity, an apparent arrhythmic death and left ventricular hypertrophy.
Criteria for issuing a cause of death due to obesity cardiomyopathy [9,16] are as follows:
1. Heart weight increased over value predicted for normal body weight
2. Left ventricular or biventricular hypertrophy and dilatation of atria and ventricles. Small
foci of interstitial fibrosis may be present, but not extensive ischaemic fibrosis
3. There may be marked fat in the right ventricle usually in the epicardial surface
and extending in with blood vessels (in the absence of fibrosis which suggests
arrhythmogenic right ventricular cardiomyopathy), often up to the trabeculae. These
changes usually affect the anterior and lateral wall and less so the posterior wall
(Figure 4.2)
4. Exclusion of significant coronary artery disease, myocarditis, myocardial infarction or
other clear alternative cause of death

Sudden death in the obese

Obese subjects have an increased risk of arrhythmias and sudden death, even in the
absence of obvious cardiac disease. The sudden cardiac mortality rate is 40x higher than
the rate of unexplained cardiac arrest in a matched nonobese population [17]. A prolonged
QT interval is seen in a relatively high percentage of obese subjects [17], but the clinical
significance of this remains speculative. It seems plausible that there is a sudden death
in the morbidly obese scenario related to cardiac arrhythmia in those where cardiac
hypertrophy or dilatation is not prominent and that this is a diagnosis of exclusion,
analogous to scenarios such as sudden unexpected death in epilepsy (SUDEP) and sudden
unexpected death in alcohol misuse (SUDAM) [18].
52 Pathology of obesity

Figure 4.2 Fat infiltration in the

right ventricle. This may be a feature
of obesity cardiomyopathy or can
be seen in normal hearts as an age-
related phenomenon. There is an
absence of fibrosis which if present
would suggest arrhythmogenic
right ventricular cardiomyopathy.

Coronary artery disease

Obesity has been shown on meta-analysis to be an independent risk factor for the
development of coronary heart disease [19], and it has been proposed that the
proinflammatory state caused by excess adipose tissue contributes to the development of
atherosclerosis. Implicated adipokines include adiponectin which is associated with insulin
resistance and inflammation, and low levels are associated with coronary heart disease.
Also, leptin which controls satiety, and is increased in obese individuals, is associated with
an increased risk of coronary heart disease [20]. Interestingly, there is an obesity paradox
whereby excess body weight confers a lower risk for death in cardiovascular disease
including heart failure [21].

Obesity causes a spectrum of changes within the liver from simple steatosis through to
nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma.
The majority of patients with morbid obesity will have steatosis (91% in a recent systematic
review). In the same review, the prevalence of NASH was 37%, but this was extremely
variable between individual studies [22]. NASH is associated with increased mortality
compared with the normal population, partly due to an increase in liver-related deaths, but
it has also recently emerged as a predictor of cardiovascular disease [23].
The histological features of NASH are steatosis, ballooning of hepatocytes, lobular
inflammation, Mallory bodies and a pericellular pattern of fibrosis. The NAFLD activity
score can be useful in determining if a given case meets the criteria for a diagnosis of
steatohepatitis. [24] Histological assessment cannot reliably distinguish between alcoholic
steatohepatitis and NASH. However, features said to favour alcohol over nonalcoholic
causes are neutrophils, prominent Mallory bodies and extensive zone 3 fibrosis. Marked
steatosis with less severe steatohepatitis with nuclear vacuolation (possibly suggesting
insulin resistance) favours NASH over alcohol [23].
The mechanism of NASH development remains uncertain, but has been proposed to
involve insulin resistance, iron accumulation, oxidative stress and hepatocyte death with an
Kidney 53

imbalance in anti- and proinflammatory factors [25]. The current model suggests that two
hits are required, with the first hit being peripheral insulin resistance, leading to hepatic
steatosis. These lipid-laden hepatocytes are then vulnerable to a combination of second
hits caused by cytokines, oxidative stress or genetic factors. Adipose tissue is a source of
inflammation and therefore a plausible cause of the second hit.

A high BMI is associated with an increased risk of chronic kidney disease (CKD).
This association is partly explained by the increased frequency of hypertension and
diabetes mellitus in obesity. In addition, high BMI results in an increased renal mass,
glomerulomegaly and hyperfiltration-associated injury [26]. Histologically, this manifests
as an increase in mesangial matrix and secondary focal segmental glomerulosclerosis
(FSGS) with sclerosis and hyalinosis adjacent to the vascular pole of the glomerulus
(Figure 4.3). This pattern of glomerular injury is associated with proteinuria and
progressive interstitial fibrosis and tubular atrophy. The proteinuria may be in the

a b

c d

Figure 4.3 Effects of obesity on the kidney. An obese 42-year-old man (weight 140kg) presented with nephrotic
proteinuria (6g/day). Renal biopsy shows marked glomerulomegaly (a) with focal segmental sclerosis and hyalinosis
adjacent to the vascular pole (b). An obese 71-year-old man with type II diabetes mellitus developed acute renal
failure (serum creatinine rising from 200 to 500mol/L) shortly after starting on orlistat. Renal biopsy shows a
diabetic nephropathy with nodular glomerulosclerosis (c), together with intratubular calcium oxalate crystals,
indicative of an oxalate nephropathy (d).
54 Pathology of obesity

nephrotic range (>3.5g/24 hours) but without features of the nephrotic syndrome. At
electron microscopy, there is effacement of podocyte foot processes that is in proportion
to the severity of proteinuria and is generally less extensive than that seen in primary
FSGS [27].
In addition to the above structural changes, obesity exacerbates injury associated
with many primary renal diseases. For example, in a recent series of patients with IgA
nephropathy, a high BMI was found to be an independent predictor of progressive loss
of renal function [28]. Obesity is also associated with an increased risk of renal stone
disease [29].
Weight loss following bariatric surgery is associated with reduction in proteinuria
and stabilisation of CKD [30]. However, gastric bypass is associated with a risk of enteric
hyperoxaluria and oxalate nephropathy [31]. This complication typically presents as
acute renal failure, often on a background of CKD. Histologically, there are abundant
calcium oxalate crystals within tubules, with associated acute and chronic tubular injury
(Figure 4.3). Prognosis is generally poor, with most patients requiring renal replacement
therapy. Similarly, oxalate nephropathy may result from orlistat therapy for obesity [32].
This drug reduces digestion of dietary fats by inhibiting the production of gastric and
pancreatic lipase. Intestinal absorption of oxalate is normally limited by the formation
of insoluble calcium oxalate in the bowel lumen. Enteric hyperoxaluria develops as a
result of reduced absorption of fats and bile acids. In the presence of fat malabsorption,
free fatty acids within the colonic lumen bind calcium ions, thus increasing oxalate

Other systemic complications of obesity

Metabolic syndrome
Various definitions exist of the metabolic syndrome. The WHO definition of the metabolic
syndrome is of insulin resistance, impaired glucose tolerance or diabetes mellitus together
with at least two of the following: (1) hypertension, (2) obesity, (3) high triglycerides and/or
low high-density cholesterol and (4) microalbuminaemia. The metabolic syndrome confers
an increased risk of cardiovascular disease.

Obesity is associated with increased risk of several cancer types: breast, endometrial,
colorectal, pancreatic, gastric, oesophageal adenocarcinoma (related to Barretts
oesophagus), cholangiocarcinoma, hepatic, melanoma and renal cancer. Insulin resistance,
hyperinsulinaemia, increased insulin-like growth factor (IGF), elevated steroid and peptide
hormones and systemic inflammation as a result of adipocytokine production, e.g. leptin
and tumour necrosis factor alpha (TNF-a), all appear to play a role in the development of
malignancy in the obese.

Production of adipocytokines by excess fat disturbs the balance between adipose tissue and
the immune system by causing dysregulated immune response, impaired chemotaxis and
altered macrophage differentiation [33].
Bariatric surgery 55

Obesity is an established risk factor for surgical site infection, hospital acquired
infections and skin infection. It also predisposes to urinary tract infections [33]. It is not
certain whether obesity affects the risk and outcomes of community acquired infections
such as pneumonia, bacteraemia and sepsis [33].

Venous thromboembolic disease

Obesity is a significant and well-established independent risk factor for development
of venous thromboembolic disease [34]. Despite the fact that the pathogenesis involves
biological markers specific to obesity and is not just a reflection of reduced mobility in
the obese, this risk factor is probably lesser known amongst pathologists and is being
unintentionally omitted from death certificates.
The pathogenesis of increased venous thromboembolic risk in the obese is complex,
potentially involving a number of hormones, cytokines and growth factors secreted by
adipose tissue such as leptin [35]. Venous stasis is also common in obesity, promoting
endothelial activation and accumulation of prothrombotic substances.

Bariatric surgery
The current National Institute for Clinical Excellence (NICE) guidelines on surgery in
obesity state that bariatric surgery should be offered to those with a BMI of 40kg/m2 or
over, and also to those patients with a BMI of 3540kg/m2 who also have obesity-related
complications, such as diabetes mellitus [36]. Bariatric surgery reduces overall mortality
by approximately 40% with reductions in deaths from heart disease, diabetes mellitus
and cancer, together with improvement in cardiac function and reversal of obesity
cardiomyopathy [37]. Mortality from bariatric surgery is low; a meta-analysis of 361 studies
comprising 85,048 patients showed a mortality at <30 days (early) of 0.28% and 30 days to 2
years (late) of 0.35% [38].
Increasingly, pathologists will be asked to perform autopsies following bariatric
surgery. The cause of death may be directly or indirectly related to the surgery, related to
complications of obesity or unrelated. Bariatric surgical options are all usually performed
laparoscopically and are divided into restrictive procedures (laparoscopic gastric banding
or vertical sleeve gastrectomy) or combined restrictive and malabsorption procedures
(most commonly Roux-en-Y gastric bypass) [39]. Laparoscopic gastric banding creates
a small gastric pouch, which restricts calorie intake proximal to a band (Figure 4.4a).
This is connected to a subcutaneous access port and can be adjusted by changing the
volume of saline within the band. Complications of gastric banding include nausea and
vomiting, dehydration with resultant electrolyte disturbances and/or prerenal failure. Rare
complications are gastrointestinal haemorrhage, aspiration, erosions caused by ischaemia
due to pressure on the gastric wall by the band which may result in gastric necrosis (which
requires resection), oesophageal or gastric perforation, ulcers, band slippage or abdominal
Insertion of a gastric balloon (to result in a feeling of fullness) can be performed
endoscopically. The balloons can deflate and rupture with migration and rarely cause
small bowel obstruction. Vertical sleeve gastrectomy involves creating a sleeve of stomach
with a lesser capacity along the greater curvature (Figure 4.4b), and tube stricture is the
main late complication specific to this procedure. Other complications are similar to the
56 Pathology of obesity

Figure 4.4 Commonly performed

Aspiration Oesophagus types of bariatric surgery annotated
Gastric pouch with possible complications. All
Perforation of these procedures are usually
Band access port
Erosion due to performed laparoscopically. Figure
ischaemia 4a shows gastric banding and
Gastric necrosis possible complications. Figure 4b
Band slippage Nausea + shows vertical sleeve gastrectomy.
Ulceration vomiting A late complication specific to
Electrolyte this procedure is tube stricture.
disturbances Other complications are similar
to the technical complications
of Roux-en-Y gastric bypass and
would include anastomotic leak,
haemorrhage and perforation.
Duodenum Figure 4c shows Roux-en-Y gastric
bypass with possible complications
of surgery and the effects of
subsequent malabsorption

Sleeve of stomach

Tube stricture


b Duodenum

Bowel ischaemia
Oesophagus Marginal ulcers
Anastomotic leak
Anastomotic stricture (late)


Duodenum Jejunum

Jejunal ulceration
Nausea + vomiting Intestinal obstruction
Malabsorption secondary to
adhesions or internal
Small intestinal
bacterial overgrowth
Conclusion 57

technical complications of Roux-en-Y gastric bypass and would include anastomotic leak,
haemorrhage and perforation.
Roux-en-Y gastric bypass creates a small gastric pouch. The distal stomach and proximal
small bowel are bypassed by anastomosing the gastric pouch to the jejunum. The other limb
is then anastomosed to the small bowel (see Figure 4.4c). Immediate complications include
anastomotic leak, perforation, intestinal ischaemia, bleeding or pulmonary embolism. Late
complications include intestinal obstruction secondary to adhesions or internal herniation,
as patients with a Roux-en-Y bypass have several mesenteric defects created by the surgery
through which a small bowel loop can pass and become obstructed or strangulated,
anastomotic stricture and marginal ulceration (ulcers which occur at the gastrojejunal
anastomosis of uncertain, but probably multifactorial origin). Autopsy studies have also found
a high incidence of nonischaemic arrhythmic cardiac deaths after bariatric surgery [40].
The National Confidential Enquiry into Patient Outcome and Death (NCEPOD)
published an audit of 29 UK post bariatric surgery deaths in 2012. Two of the deaths
followed gastric balloon insertion; one death due to gastric rupture on removal of the
device, the other a cardiac arrest associated with a large heart. Of the other 27 deaths, the
commonest causes of death were: anastomotic leak and sepsis (10 deaths), pulmonary
thrombo-embolism (6 deaths), intra-abdominal haemorrhage (3 deaths) and malnutrition
from short bowel (2 deaths). Deaths which were either directly or indirectly related to the
surgery occurred at all time points, including many years after surgery [41].
Cholelithiasis (cholesterol gall stone formation) is a frequent consequence of the rapid
weight loss following bariatric surgery. Roux-en-Y surgery can be complicated by dumping
syndrome and Roux-en-Y stasis syndrome (chronic abdominal pain, nausea and vomiting
due to stasis of solids in the stomach), and nutritional deficiencies are common. Small
intestinal bacterial overgrowth (SIBO) syndrome is characterised by an increased number
(usually 105 colony forming units of bacteria per mL of proximal jejunal aspirate) and/or
abnormal type of bacteria in the small bowel. It shows increased incidence in the morbidly
obese when compared with nonobese subjects and can complicate gastric bypass surgery.
SIBO may be clinically asymptomatic or cause diarrhoea or anaemia. In more severe cases,
there are features of malabsorption and malnutrition. The gold standard for diagnosis
during life is microbiological assessment of jejunal aspirate, although the hydrogen breath
test is also commonly used.

Pathologists will increasingly encounter obesity-related pathology in their practice, with
many conditions specific to obesity. The pathology of obesity-related conditions is complex
and often multifactorial, requiring specific knowledge of these conditions and a careful
thorough approach to diagnosis.

Key points for clinical practice

Obese individuals have altered physiology due to excess adipose tissue that secretes
adipocytokines which have been implicated in the development of cancer, coronary
atheroma and venous thromboembolic disease.
There are a number of potentially fatal conditions which are almost exclusive to the obese
and which pathologists will increasingly encounter at post-mortem: obesity cardiomyopathy,
OSA and OHS.
58 Pathology of obesity

Post-mortems in the obese require specific attention, as findings in conditions such as OSA
may be limited and it is the circumstances of the death which are key to the diagnosis.
Obesity directly affects the heart, resulting in left ventricular hypertrophy that may progress
to obesity cardiomyopathy when congestive cardiac failure is present.
Pathologists will increasingly encounter autopsy cases where there has been bariatric
surgery and although mortality for these laparoscopic procedures is low, there are a number
of complications which can occur and could result in death, although death may be a
consequence of obesity itself or be entirely unrelated to the surgery.

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Chapter 5

Stratified medicine for cancer:

the role of the histopathologist
Emily Shaw, Margaret Ashton-Key, Peter WM Johnson

Cellular pathologists have been supporting and refining the process of accurate disease
classification for decades, with methods such as immunohistochemistry and in situ
hybridisation adopted in order to supplement morphological assessment of tumours as
part of the diagnostic process. Stratified cancer medicine involves predictive analysis: the
characterisation of tumours according to the presence or absence of specific molecular
abnormalities that are associated with differential treatment responses, in order to offer
appropriately targeted therapy and avoid unnecessary treatment. This is being applied to
an increasing number of solid tumour types to complement the traditional morphological
organ or tissue of origin-based assessment of tumours. Thanks to recent advances in
genomic technology that have opened up new possibilities for molecular taxonomy in
cancer, this is a rapidly evolving area which is having a direct impact on histopathology
practice. This represents an important part of the wider concept of delivering more
personalised or precision medicine across many different disease areas in the current
postgenomic era, since the elucidation and publication of the first human genome
over a decade ago. This review aims to summarise advances in this area with a focus on
solid tumours in adult patients. A detailed review of the clinical trials in this area and
applications in neuropathology, haematolymphoid or paediatric malignancies are beyond
the scope of this article.
Many cancer genomes have now been sequenced and published, including as part
of the International Cancer Genome Consortium (http://icgc.org). Cancer genome
sequencing projects have generated findings of clinical and therapeutic relevance,
e.g. the identification of the mutated BRAF oncogene as a key driver in just over half of

Emily Shaw, BM(Hons), MRCP(UK), Specialty Registrar in Histopathology, Salisbury Hospital NHS Foundation Trust
and Clinical Lead for the Stratified Medicine Programme, Cancer Research UK, London, UK
Margaret Ashton-Key, DM, FRCPath, Consultant Histopathologist and Honorary Senior Lecturer, University Hospital
Southampton NHS Foundation Trust, Southampton, UK
Peter WM Johnson, MD, FRCP, Chief Clinician for Cancer Research UK, London and Professor of Medical Oncology
at Southampton University Faculty of Medicine and University Hospital Southampton NHS Foundation Trust ,
Southampton, UK
Email: ecshaw@doctors.org.uk (for correspondence)
62 Stratified medicine for cancer: the role of the histopathologist

Table 5.1 Terms used to classify and describe the effects of gene mutations in cancer
Timing Germline (constitutional): a mutation Somatic (acquired): a mutation occurring
present in one parental gamete and during DNA replication and cell division
transferred to all cells resulting from during life and present only in a subset of
subsequent cell divisions cells in a tumour or tissue
Effect on protein coding Synonymous (silent): a nucleotide Nonsynonymous: a nucleotide change
change resulting in the same amino leading to a different amino acid, e.g. the
acid, due to redundancy in nucleotide DNA sequence GTA codes for valine but a
combinations coding for each amino change to GAA would result in glutamic
acid, e.g. coding DNA strands containing acid instead
GTA and GTG would both encode the
amino acid valine
Effect on protein function Activating (gain of function): a change Inactivating: a change leading to a
leading to enhanced effect of a protein, nonfunctional or reduced function
e.g. the codon 600 BRAF V600E mutation protein, e.g. codon 594 mutations in the
leads to increased activity of the BRAF BRAF gene cause loss of kinase activity
protein irrespective of usual regulatory
Functional effect Pathogenic: a mutation that can be Nonpathogenic: a genetic abnormality
experimentally demonstrated or that does not appear to contribute to
predicted to contribute to initiation or initiation or progression of tumours
progression of a tumour
Prediction of treatment Sensitising: a mutation that has been Resistance: a mutation that has been
response shown in clinical trials to be associated shown in clinical trials to be associated
with treatment response, e.g. the L585R with treatment resistance, e.g. the T790M
mutation in the EGFR gene is predictive mutation in the EGFR gene predicts a
of response to EGFR tyrosine kinase lack of response to EGFR tyrosine kinase
inhibitor drugs in patients with non-small inhibitor drugs in patients with non-small
cell lung cancer cell lung cancer. This may be seen as a
primary or secondary phenomenon,
possibly due to clonal selection pressures
in tumours during treatment
Importance in Driver: a mutation that is recurrent Passenger: a mutation that does not
carcinogenesis between different tumours, and can be appear to play a role in the initiation or
functionally linked to carcinogenesis, progression of cancer and is likely to
e.g. KRAS mutations in colorectal cancers have occurred as a bystander effect due
cause increased activity of the mutated to genomic instability. These may be
KRAS protein leading to abnormal cell private to particular tumours and are
proliferation typically not recurrent
Mutation nomenclature
Mutations are conventionally described using nomenclature agreed by the HGVS (Human Genome Variation
Societys) with reference to both the coding and protein changes. The following example is for the most common
mutation in the BRAF gene, a point mutation due to a single nucleotide substitution
Coding (c.): This is the description of the abnormality at DNA nucleotide level, according to the numbered
nucleotide position on the sense DNA strand (5 to 3 direction) of the reference genome:
For example c.1799T>A refers to substitution of adenine for thymine at position 1799
Protein (p.): This describes the abnormality at protein coding, amino acid level, according to the number of the
affected amino acid. The reference amino acid is denoted by its one or three letter code at the start of the sequence
and the mutant amino acid follows the position number at the end of the sequence:
For example p.V600E or Val600Glu refers to coding for glutamate rather than valine at codon 600
Introduction 63

malignant melanomas. The timescale from this discovery to the licensing of the BRAF
inhibitor vemurafenib was encouragingly short in drug development terms. Apart from
the immunomodulator therapy ipilimumab, this represents the first effective treatment
option for patients with advanced melanoma. One of the challenges in making sense
of the deluge of data from genome sequencing studies is in distinguishing key driver
mutations from nonpathogenic bystander or passenger mutations, particularly since
cancer is characterised by genomic instability, with each cancer containing anything
from 1000 to 100,000 different point mutations [1], some of which are private to that
tumour, making assessment of their role in pathogenesis difficult. There is an increasing
requirement for the cellular pathologist to be conversant in the language describing
the effects of genetic abnormalities identified through cancer genome screening, and
some of the terminology is summarised in Table5.1. Recent research into cancer
genomic evolution gives insights into the degree of spatial and temporal heterogeneity
and complexity [2]; challenges to the delivery of personalised cancer medicine through
Histopathologists are accustomed to the use of molecular markers for diagnostic
purposes, e.g. characteristic chromosomal translocations in soft tissue tumours and
haematolymphoid malignancies. An exemplar for the application of newer predictive
molecular markers was the introduction of HER2 testing in breast cancer to identify
patients who may benefit from treatment with trastuzumab (Herceptin, Roche). Table 5.2
summarises the currently licensed therapeutic agents for use in patients with tumours
bearing specific genetic aberrations.

Table 5.2 Cancer medicines active against specific genetic abnormalities and currently
approved for use worldwide.
Drugs Molecular marker predictive of response Disease indication
ATRA (all-trans retinoic acid) t(15;17) translocation Acute promyelocytic leukaemia
Imatinib t(9;22) translocation (Philadelphia Chronic myeloid leukaemia
chromosome); BCR-ABL fusion

KIT/PDGFRA mutation Gastrointestinal stromal tumours

Trastuzumab HER2 gene amplification Breast cancer
Trastuzumab emtansine Metastatic gastric cancer
(antibody-drug conjugate) (trastuzumab only)
Cetuximab KRAS wild-type, i.e. lack of mutation Metastatic colorectal carcinoma
Gefitinib EGFR mutation Non-small cell lung carcinoma
Crizotinib ALK gene rearrangement
Vemurafenib BRAF codon 600 mutation, especially V600E Malignant melanoma
64 Stratified medicine for cancer: the role of the histopathologist

Historical background
In the past, pharmaceutical companies have usually provided initial funding following the
introduction of a new predictive marker, often through a limited number of laboratories.
When this funding has ended, new service and funding agreements have had to be found.
The United Kingdom National External Quality Assurance Scheme (UK NEQAS) for
Molecular Genetics and Immunocytochemistry and in situ hybridisation have been quick
to establish quality assurance schemes for new markers and currently there are established
disease-based schemes encompassing analysis of HER2, EGFR, KRAS, BRAF and KIT.
Testing has either been delivered by histopathology or molecular genetics laboratories, and
increasingly by unified departments of molecular pathology.

Sample requirements and technologies for

mutation detection
Molecular analysis protocols have been developed and optimised for formalin fixed,
paraffin embedded (FFPE) tumour tissue. Material can be submitted as sections on glass
slides or as scrolls and ideally a matched haematoxylin and eosin (H&E) stained section
should also be provided, with the percentage tumour nuclei content assessed and outlined
on the slide by the referring pathologist. Some laboratories perform macrodissection of
slide-mounted sections to isolate and enrich the material for tumour nuclei and avoid
analysis of any adjacent non-neoplastic tissue; however, the workload implications of this
are significant and the development of more sensitive analysis techniques may mean that
this is not required in future. Macrodissection is generally considered mandatory with
current methods if the tumour content of the section is assessed as <20% by the reporting
pathologist. For currently available methods, mutation analysis for mutation hotspots in up
to five genes can be performed on DNA extracted from a single 5 micron paraffin section
with tumour cellularity >50%. Each section can be expected to yield at least 150ng DNA,
with inputs as low as 10ng yielding a meaningful result; however, variability in quality of
DNA due to the effects of formalin fixation may mean that only a small proportion of the
extracted DNA can be amplified and that larger amounts of starting material are required to
compensate for this [3].
As well as assessing tumour content, the pathologist can provide useful information
to the molecular laboratory about other specimen-dependent factors that may influence
the likelihood of success in subsequent analysis, such as necrosis, or melanin pigment
in malignant melanoma, which inhibits the polymerase chain reaction (PCR). Specimen
handling during the preanalytical phase has an important impact on the outcome of
mutation analysis, and there is potential for optimisation by simple changes in practice in
cellular pathology laboratories, such as use of a clean microtome blade for cutting sections
from each new block in order to prevent cross-contamination of DNA and tissue between
Detection of gene amplification or translocations may be performed in thin sections
on the glass slide using in situ hybridisation, whereas detection of gene mutations
or fusion transcripts requires extraction of nucleic acids and analysis using PCR and
sequencing-based methods (Table 5.3). Some screening techniques involve comparison
to a known normal sample, such as high-resolution melt (HRM) analysis and single-strand
conformation polymorphism analysis (SSCP or SSCA) [4,5]. This allows identification of
Sample requirements and technologies for mutation detection 65

Table 5.3 Summary of selected techniques for mutation detection

Method Brief description Comparative Comparative Limit of Main Main dis
of method DNA input sensitivity detection* advantages advantages
Sequencing-based screening methodologies (detection of all variants: known and unknown)
Sanger Amplification Low Lowest 1020% Identification Labour-
(dideoxy-) and sequencing (~100ng) of known and intensive;
sequencing of polymerase novel variants may miss low-
chain reaction level variants
(PCR) products
by selective
incorporation of
during in vitro
DNA replication
Pyrosequencing Sequencing Low High 5% Can also Comparatively
by synthesis: (~100ng) be used for high
detection of the targeted sequencing
luminescence mutation error rate
released when a detection
pyrophosphate- Fast method
labelled nucleotide with real-time
molecule is readout
during DNA
Next- Massively parallel High High 10% Highest Larger input
generation sequencing of (~500ng) (dependent throughput quantities
sequencing thousands-millions on read technology, of DNA
of amplified depth) enabling required and
DNA molecules greater scope complex data
using capture- or of analysis interpretation
amplicon-based up to whole requirements
approaches exome/
Screening methods using comparison of mutated with normal DNA (detection of all variants, known and
High-resolution Screening of Low Low 5% Quick, Nonspecific
DNA melting samples using (~100ng) melting amplification
analysis (HRM) comparison of the products of products
melting curves can be can lead to
of PCR products sequenced to missed calls
against known identify exact
normal samples abnormality
Single-strand Heat-denatured Low High 110%, Established Technical
conformational PCR products are (~100ng) varies by technique, parameters
polymorphism compared with mutation low-cost (e.g.
analysis (SSCP known samples temperature,
or SSCA) using capillary gel
electrophoresis composition)
and analysed must be
according to strictly
electrophoretic controlled
66 Stratified medicine for cancer: the role of the histopathologist

Table 5.3 Continued

Targeted mutation detection methods (genotyping of known mutations only)
Amplification Selective High Highest <18%, Fast and Only detects
refractory amplification (~500ng) varies by sensitive predefined
mutation of sequences mutation technique hotspot
system containing a mutations
defined mutation
over those that
do not
Fragment DNA fragment Low High 12% Fast and Cannot be
length analysis length analysed (~100ng) sensitive used to
against size technique detect point
standards to mutations
detect deletions
and insertions

*% mutant alleles in wild-type background

those samples that are not normal, for further work to characterise the precise abnormality
present if required. Determination of the sequence can be achieved by conventional
Sanger (dideoxy-) sequencing, which is considered to be more labour intensive and have
a lower sensitivity than more modern next-generation sequencing technologies. The limit
of detection for direct sequencing is generally considered to be 20%, i.e. a mutation must
be present in 20% of the DNA within a sample to be confident of picking it up by direct
sequencing analysis. It is currently uncertain what effect if any mutations present at a
low frequency within a tumour have on overall biological behaviour, and therefore whether
there is a threshold of significance. Pyrosequencing is a similar but slightly more sensitive
technique for sequence determination and mutation detection, with an estimated limit of
detection of 5% [6].
Methods for targeted analysis include amplification refractory mutation system (ARMS),
which is a technique in combination with real-time quantitative PCR to selectively amplify
those sequences containing a defined mutation over those that do not, i.e. are wild-type as
well as fragment length analysis. Fragment length analysis can be used to detect insertions
or deletions but not point mutations.
The analytical sensitivity of the mutation detection methods in use currently is between
75% and 90% for sequencing and HRM analysis and >90% for pyrosequencing, SSCP,
fragment length analysis, next-generation sequencing and allele-specific PCR [7]. The
choice of technique involves a trade-off balancing analytical sensitivity (ability to correctly
detect mutation-positive cases) and limit of detection (minimum detectable percentage of
mutant vs. wild-type alleles in a sample) with the specificity of the method.
Following analytical and clinical interpretation, the output of the molecular analysis
is formulated into a report for the requesting clinician or pathologist. The International
Organization for Standardization (ISO), the College of American Pathologists and UK
NEQAS have all issued guidance on the contents of this report (summarised in Table5.4)
[8]. In some centres, the report is sent to the referring clinician only and filed in the
patient record, and in others the report is received by the histopathologist and issued as
a supplementary report or integrated molecular pathology report. Since the mutational
Selected examples of disease applications 67

Table 5.4 Requirements of a molecular pathology report

Dates Of sample receipt and report authorisation
Patient information Three-point identifiers, e.g. patient name, date of birth and reference number
Information about request Nature of sample, tissue and tumour type, percentage content tumour nuclei
as assessed by referring pathologist, clinical indication for analysis, name and
address of referrer
Information about the analysis Technique(s) used, scope of test, sensitivity/limit of detection
Results Presence or absence of abnormality in gene(s) in question expressed using
standard Human Genome Variation Society (HGVS) nomenclature, interpretation
of clinical significance of result (may be unknown)
Contact information For laboratory as well as name/job title of person authorising report

profile is an attribute of the tumour rather than the patient, the latter approach seems more
logical and may allow the molecular results to be further interpreted in the context of the
morphological and immunohistochemical features of the tumour.

Selected examples of disease applications

Lung cancer
Over recent years a number of key driver mutations have been discovered in pulmonary
adenocarcinoma, and those which have been clinically validated so far are EGFR mutations
and ALK translocations. Novel targets also identified and linked to drugs in development
or clinical trials include KIF5B-RET and ROS [9,10]. Progress in pulmonary squamous
cell carcinomas has not been so promising, though occasional rare cases with EGFR and
ALK abnormalities have been described [1012]. This presents a dilemma for treatment
of these patients, since they are often not representative of the population of preapproval
clinical trials. Also this adds complexity to the process of determining optimal testing
strategies, with economics of testing affected by the prevalence of the mutation in question.
Molecular analysis in lung cancer is ahead of other tumour types in that the multiple
tests now required have developed to be used sequentially, reinforced by the US model
of an approved companion diagnostic to accompany each drug. The relative anatomical
inaccessibility of lung cancers and the resulting small tissue samples compound the
problem of limited tissue availability, with current analysis methods consuming significant
amounts of DNA.

EGFR in lung cancer

The EGFR gene (epidermal growth factor receptor gene, also known as ERBB1 or HER1)
encodes the cell membrane-bound epidermal growth factor (EGF) receptor, and mutations
in this gene determine response to tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib
in patients with non-small cell lung cancer (NSCLC). Ninety per cent of EGFR mutations
are located in the tyrosine kinase-binding domain (exons 18-21). The mutant EGFR protein
activates cellular pathways implicated in cancer cell growth, survival and migration. The
most common activating mutations are exon 19 deletions and a codon 858 missense
mutation in exon 21 (L585R). The most common resistance mutation is EGFR T790M, but
68 Stratified medicine for cancer: the role of the histopathologist

other mechanisms of resistance such as amplification or overexpression of MET, PIK3CA

mutations and transformation to small cell lung carcinoma have also been described
[13]. Histological features associated with EGFR mutations are adenocarcinoma of any
growth pattern, especially papillary or micropapillary, but with the exception of mucinous
carcinomas (the latter are associated instead with KRAS mutations). EGFR mutations
appear much less common in neuroendocrine, mucoepidermoid and adenoid cystic
carcinoma [14].
The mutant EGF receptor can be targeted using small molecule inhibitor drugs, which
act inside the cell against the internal tyrosine kinase domain of the receptor, so-called
tyrosine kinase inhibitors, such as erlotinib (Tarceva) or gefitinib (Iressa). These are the
drugs that have demonstrated clinical response in lung cancer patients in clinical trials

EML4-ALK in lung cancer

The EML4-ALK fusion gene is derived from an inversion affecting chromosome 2 and
leading to fusion of the EML4 (echinoderm microtubule-associated protein-like 4) gene
with the ALK (anaplastic lymphoma kinase) gene. This fusion gene is found in 47%
of unselected NSCLCs, predominantly adenocarcinomas, with the translocation very
occasionally detected in squamous cell carcinomas [15,16]. Alternative ALK fusion partners
(e.g. TRK-fused gene TFG, nucleophosmin gene NPM1 and kinesin family member 5B
gene, KIF5B) have been described but are much less common than EML4-ALK. ALK gene
rearrangements generally occur exclusively of EGFR or KRAS mutations, though this may
simply reflect the fact that both are relatively uncommon events and therefore statistically
unlikely to coexist and case reports have documented tumours with combinations of
abnormalities [17]. The fusion gene encodes a fusion protein with overactivity of ALK due
to ligand-independent dimerisation, and ALK signaling leads to cellular proliferation.
Clinical correlates of the presence ALK mutation are never or light cigarette smoking
history, younger age at onset of disease and there is also a strong association with
adenocarcinoma showing a signet ring or acinar growth pattern [15,16]. The ALK/MET
inhibitor crizotinib (Xalkori, Pfizer) is a multitargeted small molecule TKI, administered
orally, which inhibits ALK phosphorylation and signal transduction. Crizotinib was licensed
for use in NSCLC by the US Food and Drug Administration (FDA) in 2011. Unusually, the
FDAs accelerated approval was based not on evidence of survival benefit, but instead on
trial data demonstrating a response rate of up to 57% in patients with a fusion-gene positive
tumour [18]. A phase III trial is studying effect on progression-free survival. At the time
of approving crizotinib, the FDA also licensed a specific break-apart fluorescence in situ
hybridisation probe (Abbott) as the requisite companion diagnostic for detection of the ALK
gene translocation. Crizotinib resistance mutations have been detected following therapy
[19]. Crizotinib is also being investigated as a treatment of aggressive and resistant forms of
anaplastic large cell lymphoma carrying the t(2;5) translocation involving NPM1 and ALK
and neuroblastoma in the paediatric population [20].

Breast cancer
Ascertaining the HER2 status of invasive breast cancer has now been the standard of care
for over a decade, and this is achieved using immunohistochemical assessment of protein
expression in the majority of cases. In situ hybridisation can be used to confirm gene
amplification and is generally reserved for cases with equivocal immunohistochemistry
Challenges of the approach 69

results. In the past few years, there has been interest in using gene expression profiling
in breast cancer to provide risk stratification in addition to traditional histopathological
parameters such as grade, vascular invasion and lymph node involvement. Data from
gene expression profiling tools such as Oncotype DX (21 genes, Genomic Health) and
MammaPrint (70 genes, Agendia) can be used to identify a subset of patients with such
a good prognosis that they can be spared adjuvant chemotherapy since adverse effects
would be likely to outweigh the potential benefits. A recent NICE appraisal of gene
expression arrays in breast cancer approved Oncotype DX as cost-effective for use in the
in certain situations, but recommended further research to establish the utility of the IHC4
panel (immunohistochemistry for oestrogen and progesterone receptors, HER2 and the
proliferation marker Ki67) [21]. In terms of molecular taxonomy, a landmark study using
expression arrays led to classification of breast cancer into five molecular subtypes [22].
These were further expanded into ten subtypes in the METABRIC study published last year
[23], and although this work has led to mechanistic insights and the discovery of new driver
mutations in breast cancer, translation to the clinic is still some way off.

The BRAF gene encodes a serine/threonine kinase, an enzyme activated by
phosphorylation and responsible for transferring phosphate groups to other proteins to
modulate their function that is part of the Raf kinase family. Over 90% of BRAF mutations
are in codon 600, the commonest being V600E. The BRIM3 trial recently provided evidence
that patients with previously untreated, unresectable stage IIIC/IV melanoma with
V600E mutation had improved overall and progression-free survival with vemurafenib
when compared with standard dacarbazine therapy [24]. An unexpected finding was the
increased risk of cutaneous squamous cell carcinoma in patients receiving vemurafenib
therapy, and a possible mechanism for this is paradoxical stimulation of events in a related
cellular pathway in epidermal cells with wild-type BRAF. Ongoing studies are focusing on
improving the durability of response to BRAF inhibitors by trialling them in combination
with other targeted agents acting on related pathways. The MEK pathway also shows
overactivity in melanomas harbouring BRAF mutations and phase III clinical trials of MEK
inhibitors are currently underway [25].

Challenges of the approach

There are technical challenges involved with molecular analysis of FFPE material. Formalin
fixation leads to cross-linking and degradation of DNA into fragments typically <200 base
pairs in length; however, overfixation and underfixation of specimens could potentially
both cause problems. There is a need for optimal, standardised sample handling protocols
in the preanalytical phase to maximise the potential for obtaining diagnostically useful
information from DNA extracted from FFPE tumour samples. Advances in interventional
techniques such as endobronchial ultrasound-guided sampling (EBUS) fine needle
aspiration of lung or mediastinal lymph node lesions have enabled combined cytological
diagnosis and staging, but contribute to a trend for smaller samples.
Most novel cancer drugs such as kinase inhibitors act to reduce or halt tumour growth
rather than killing existing cells, in a situation analogous to bacteriostatic rather than
bactericidal antimicrobial drugs. This means that patients tend to experience a temporary
response to therapy only, with subsequent development of resistance and the need to
70 Stratified medicine for cancer: the role of the histopathologist

move to a different treatment strategy. As a consequence of tumour heterogeneity and

temporal evolution, there is an increasing requirement to track the mutational profile using
longitudinal tissue sampling, at different timepoints in a patients illness, and to assess the
mutation status of multiple genes at the same time rather than in a consecutive manner.
This approach is supported by the results of a recent retrospective study of pre- and
post-treatment biopsies in EGFR mutation positive NSCLC patients. This demonstrated a
range of new molecular abnormalities conferring resistance in the post-treatment biopsy
samples, including the emergence of the most common resistance mutation, T790M, in
nearly half of the 63 patients in the series [26]. Current clinical trials are exploring the use
novel agents in combination, such as BRAF and MEK inhibitors in advanced melanoma, to
try and produce a more sustained response without unacceptable toxicity.
Pathology departments must be adequately resourced for sample preparation,
macrodissection and any testing performed within the cellular pathology department,
particularly if the demand continues to grow as we suspect it will. Equally important is the
reliability of the output of testing, and robust quality assurance schemes should continue
to evolve and cover the end-to-end process from sample preparation through testing to
interpretation and reporting, wherever this is performed. The entire process is complicated
by the constantly changing landscape of analysis required, as well as the technology and
platforms available to deliver it, with most molecular genetics laboratories now venturing
into the introduction of next-generation sequencing to diagnostic work. Whatever
technology is used, it is important that accurate results can be delivered within a clinically
relevant turnaround time that is appropriate for the particular analysis required and
circumstances of the patient.

Progress towards routine delivery of predictive

molecular analysis in solid tumours
DNA-based predictive analysis of solid tumours is currently carried out on individual genes
in a sequential manner for particular licensed therapies. The request is usually initiated by
an oncologist rather than as a reflex test from the pathologist. A number of initiatives are
underway around the world to perform broader molecular profiling closer to the point of
Since 2011, the Cancer Research UK Stratified Medicine Programme has been underway
at a number of clinical and laboratory sites in England, Wales and Scotland. Phase One of
the initiative has been piloting the routine delivery of mutational analysis of four to five
key genes of interest in different solid tumour types (breast, colorectal, lung, ovarian and
prostate cancer as well as malignant melanoma). Nine thousand tumour samples have
undergone analysis and the efforts have generated a wealth of insights into numerous
aspects of this approach. Phase Two of the initiative commenced in summer 2013 and will
focus on lung cancer, with profiling of a far wider panel of genetic abnormalities using next-
generation sequencing technology. This should yield greater opportunities for patients to
enter clinical trials and access novel treatments based on the results.

The characterisation of tumour genomes to identify oncogenic drivers and therapeutic
targets is beginning to have a significant impact on the management of people with cancer,
References 71

with important implications for histopathologists. As well as mutation testing performed on

nucleic acids extracted from paraffin sections of the tumour, recent work has focused on the
potential for minimally invasive techniques for treatment response and disease monitoring
through so-called liquid biopsies, using circulating tumour cells or free circulating tumour
DNA fragments isolated from plasma [27,28]. We anticipate a need to move from the
requirement for a single gene mutation test to the simultaneous assessment of a panel of
inter-related biomarkers, a change that is already evident in pulmonary adenocarcinoma
where assessment of EGFR and EML4-ALK provides information about two different
licensed treatment options. Since histopathology is the gold standard for classification of
disease, predictive molecular analysis must in due course become fully integrated into the
process of cancer diagnosis and prognostication in histopathology. This will require sharing
of expertise and close working between histopathologists and molecular geneticists. There
is an urgent need to prepare current and future biomedical/clinical scientists and medically
qualified pathologists to deliver integrated histopathology, incorporating predictive
molecular analyses as the next level of refinement of comprehensive cancer reporting and

Key points for clinical practice

The characterisation of tumour genomes to identify oncogenic drivers and therapeutic
targets is beginning to have a significant impact on the management of cancer, with
implications for histopathologists.
DNA-based tests linked to currently licensed treatments in the UK are as follows: HER2
amplification in breast cancer, KRAS mutations in metastatic colorectal cancer, BRAF
mutations in advanced malignant melanoma, EGFR mutations and ALK translocations in
pulmonary adenocarcinoma.
Molecular analysis can be performed in FFPE tumour tissue.
Detection of gene amplification or translocations may be performed in thin sections on
the glass slide using in situ hybridisation, whereas detection of gene mutations or fusion
transcripts requires extraction of nucleic acids and analysis using PCR and sequencing-
based methods.
Major challenges of the stratified medicine approach include tumour heterogeneity and
treatment resistance.
Higher throughput next-generation sequencing technologies are enabling a broader scope
of analysis but bring new levels of complexity to data interpretation.

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Chapter 6

Mucosal pathology of the gastric

cardia and Barretts oesophagus
James J Going

Barretts columnar-lined oesophagus has a long history [13], but only since Norman
Barrett, Philip Allison and others drew attention to it in the mid-twentieth century [46] did
it attract increasing notice, further stimulated by the emergence of a strong association with
oesophageal adenocarcinoma [7].
Adenocarcinomas arising from mucosa of Barretts oesophagus and the gastric cardia
(junctional cancers) present late, and are often inoperable. As surgery, chemotherapy and
radiotherapy are only moderately effective, prognosis is poor and lethality high. This state
of affairs is a challenge to do better.
As the dietary austerities of the Second World War and after receded into history, the
incidence of oesophageal and junctional adenocarcinoma, previously rare, began to
increase in Western societies [8]. This increase has been absolute, with increasing obesity
(identified by epidemiological studies as an important risk factor; [9]) leading to reflux
of acid and bile into the distal oesophagus, injuring the native oesophageal mucosa, and
in some people stimulating glandular (columnar) metaplasia. Barretts oesophagus
is however only identified in a minority of people with reflux, and conversely may be
present in people who have never experienced troublesome reflux symptoms. Continuing
reflux with ongoing mucosal injury and inflammation are likely to further promote
The increase in oesophageal adenocarcinoma and junctional gastric cancer has also
been inversely related to distal gastric cancer associated with Helicobacter pylori infection,
as improved social conditions reduced the number of people infected with H. pylori during
The response of the medical profession to the challenge of oesophageal and junctional
adenocarcinoma has focused on endoscopic and biopsy surveillance in the small minority
of people known to have Barretts columnar-lined oesophagus, the only definitely known
precursor of oesophageal adenocarcinoma apart from obesity and reflux.

James J Going, BSc, PhD, MRCP, FRCPath Senior Lecturer in Pathology and Honorary Consultant Pathologist,
University of Glasgow and Southern General Hospital, and Department of Pathology, Southern General Hospital,
Glasgow, UK
Email: going@udcf.gla.ac.uk (for correspondence).
74 Mucosal pathology of the gastric cardia and Barretts oesophagus

This seems to present a tantalising opportunity for early detection and prevention of
these unpleasant cancers, but unfortunately, as most of them occur in people not known
to have had Barretts oesophagus, this can never have much impact on mortality from
these diseases. Conversely, not only will most people with Barretts oesophagus not die of
oesophageal adenocarcinoma, most will never even know they had it. At least as many will
die of colon cancer.
There are many other challenges. Even the definition of Barretts oesophagus is
contested, and the level of cancer risk associated with it, the diagnosis of dysplasia can be
problematical, and the role of biomarkers in risk stratification remains unclear. Much of the
available evidence is of poor quality [10].
Some junctional cancers arise in a biological context similar to Barretts oesophagus,
in patients with normal gastric mucosa, whilst others develop on a background of chronic
gastritis, intestinal metaplasia and glandular atrophy [11] resembling in these respects
distal gastric cancer of intestinal type. This review is chiefly concerned with Barretts
oesophagus and junctional cancers arising on a comparable background. The Siewert
classification is not easily related to biological context and will not be considered further.
This review will examine: (1) Mucosal phenotypes of Barretts columnar-lined
oesophagus and the gastric cardia. (2) Diagnosis of Barretts oesophagus. (3) Is there a
case for population screening for Barretts oesophagus? (4) How dangerous is Barretts
oesophagus? (5) The case for postascertainment endoscopic and biopsy surveillance of
Barretts oesophagus. (6) The challenge of dysplasia diagnosis in Barretts oesophagus. (7)
Advanced imaging and detection of Barretts dysplasia. (8) The potential of biomarkers to
improve risk stratification in Barretts oesophagus. (9) Endoscopic treatment of the high-
risk oesophagus and cardia.

Mucosal phenotype of Barretts oesophagus and

the gastrIc cardIa
Barretts metaplasia establishes several differentiated mucosal phenotypes with three
common patterns: cardiac-like, fundic-like (cardio-oxyntic) and intestinal (with goblet
cells) (Figure6.1). This creates a challenge of interpretation of biopsies from a potential
Barretts oesophagus and requires circumspection from the reporting pathologist: a label
of Barretts oesophagus is troublesome, potentially expensive, and may induce anxiety, so it
should not be applied lightly.
Biopsy features diagnostic of an oesophageal origin include full-thickness
nonkeratinising stratified squamous epithelium native to the oesophagus; islands
of immature squamous epithelium, often present around the ostia of oesophageal
submucosal gland ducts, to which they may draw attention; these ducts; or the submucosal
glands themselves. It is not clear that so-called pancreatic metaplasia is specifically
indicative of an oesophageal origin, but this has been claimed for transitional multilayered
epithelium with a mixed squamous and glandular phenotype (Figure 6.2); [12], which is
also regarded as a marker of gastro-oesophageal reflux [13]
A biopsy with definite oesophageal features does not establish that another biopsy in the
same specimen container is necessarily from the oesophagus. The classical definition of
metaplasia (replacement of one differentiated tissue type by another) does not necessarily
mean that the new tissue is identical to a mature tissue found in some other location. In
particular, Barretts mucosa of intestinal phenotype would rarely be mistaken for normal
Mucosal phenotype of Barretts columnar-lined oesophagus and the gastric cardia 75

Figure 6.1 Two characteristic

mucosal phenotypes of Barretts
oesophagus: cardiac (left) and
intestinal (right, with goblet
cells). Cancer risk of these two
phenotypes is a matter of debate.
As intestinal metaplasia is present
in most long-segment cases, the
distinction is usually academic.
In short and ultra-short Barretts
oesophagus, intestinal metaplasia
may be a less constant feature, and
the distinction potentially more

Figure 6.2 A clue to an

oesophageal origin of a mucosal
biopsy: multilayered epithelium
with a squamoid appearance at
the base, but a clearly glandular
differentiation on the surface.
This mucosal type is most often
seen near the gastro-oesophageal

small or large intestinal mucosa. Although Barretts mucosa may be indistinguishable

from cardiac mucosa, there is controversy about whether cardiac mucosa is itself usually
metaplastic in character. Mucosa from an oesophageal location may also resemble gastric
body or antral mucosa.
All three mucosal phenotypes of Barretts oesophagus may occur at any distance from
the oesophagogastric junction, but parietal cells in oxyntic and cardio-oxyntic mucosae
are more prevalent in the distal few centimetres of the oesophagus than proximally
[14]. Cardiac- and intestinal-type mucosa with goblet cells may be found at any level.
The mucosa is a mosaic of phenotypes: cardiac and oxyntic mucosae are often seen
side-by-side in the same biopsy, likewise cardiac and intestinal type mucosa. Oxyntic
and intestinal mucosae colocate less often in the same biopsy. The intestinal phenotype
is almost invariably incomplete: a well-developed crypt-and-villus architecture and
76 Mucosal pathology of the gastric cardia and Barretts oesophagus

a well-developed brush border are almost never seen, and Paneth cells are seldom

dIagnosIs of Barretts oesophagus

Arriving at a new diagnosis of Barretts oesophagus is a joint venture between endoscopist
and pathologist. In some cases, the diagnosis is obvious with circumferential replacement
of the pale oesophageal squamous mucosa by tongues or patches of salmon-pink mucosa
extending well proximal to the most proximal extent of the gastric mucosal folds or the
most distal extent of oesophageal submucosal palisade blood vessels, an anatomical
landmark favoured by Japanese endoscopists, but histological confirmation of a compatible
mucosal phenotype is usually required.
Often the diagnosis is less obvious. Oesophagitis with erythema may be mistaken for
Barretts mucosa. Biopsy will usually correct this misidentification. More challenging
are biopsies which could have been derived from the anatomical oesophagus (any one
of the possible Barretts mucosal phenotypes, including intestinal metaplasia). Without
histological proof of an oesophageal origin, or certainty on the part of the endoscopist that
all the biopsies submitted in that specimen container were from the oesophagus, there
must be ambiguity.
This difficulty is pertinent to some recent literature which claims that the long-term
cancer risk of Barretts oesophagus is significantly less than has been thought. These
population based studies ascertain Barretts oesophagus on the basis of biopsies stated to
be oesophageal in origin and the presence in at least some of those biopsies of glandular
mucosa consistent with a diagnosis of columnar-lined oesophagus. Whilst these studies
probably avoid referral bias, there is a possibility, given that determining the exact origin
of biopsies is not always straightforward (and these biopsies are likely to be taken by
endoscopists with varying degree of experience and expertise in identifying anatomical
landmarks around the distal oesophagus), that some in fact derive from the proximal
stomach and may not represent Barretts oesophagus at all. This will be discussed below.

can populatIon screenIng for Barretts

oesophagus Be justIfIed?
Screening cannot be justified without clear evidence of benefit. This does not exist for
Barretts oesophagus and there is little prospect of it doing so soon. Also, many people
with Barretts oesophagus do not have obvious gastro-oesophageal reflux disease (GORD)
symptoms, so one would probably have to screen the entire adult population above a
certain age (which might be greater for women than men). Such an exercise would be
costly and of doubtful value. More oesophageal adenocarcinoma might be prevented by
spending the money on obesity prevention and smoking cessation.

exactly how dangerous Is Barretts oesophagus?

It is widely believed that cancer risk in Barretts oesophagus is associated with intestinal
metaplasia, characterised by goblet cells. This is difficult to test: it is hard to prove there
are no goblet cells in a particular oesophagus, as there could always be some in the
Postascertainment surveillance 77

next biopsy. Also, there is almost no spatiotemporal data relating intestinal metaplasia
in Barretts oesophagus with dysplasia and its progression. The relationship between
intestinal metaplasia and dysplasia has been questioned [15], and the occurrence of
genetic and epigenetic abnormalities similar to those in cancers in nonintestinal glandular
mucosa also calls in question the idea that intestinal type mucosa only is at increased risk
of malignancy [16].
Accurate assessment of patient-specific cancer risk in Barretts oesophagus will require
better knowledge of controls of mucosal phenotype (cell fate specification) and the
evolution of dysplastic clones than is presently available. This is unlikely to be reducible to
whether intestinal metaplasia is present or absent, so requiring intestinal metaplasia as a
necessary precondition for Barretts oesophagus is too rigid to justify on present evidence.
It may also hinder understanding of carcinogenesis in metaplastic oesophageal mucosa
and at the oesophagogastric junction, and its use to define Barretts columnar-lined
oesophagus is illogical.
Published studies of adenocarcinoma risk in Barretts oesophagus yield a wide variety of
estimates. Provenzale et al. [17] demonstrated publication bias and proposed an incidence
of symptomatic adenocarcinoma in Barretts oesophagus about 0.5% per annum, a figure
widely accepted since then, but even this lower figure has been challenged more recently
by several allegedly population-based studies.
Reported incidence of adenocarcinoma in Barretts oesophagus is between 0% and 3.5%
per annum [18,19]. Risk estimates in some studies were probably overestimated (small
size, short follow-up, variable definitions, inclusion of prevalent cancers and high-grade
dysplasia, HGD) [20]. Current surveillance guidelines are based on an adenocarcinoma
risk 0.5% per annum estimated from a meta-analysis of available data to the year 2000 [21].
A more recent meta-analysis by Desai et al. [22] excluding prevalent cancers and HGD
reported an incidence of adenocarcinoma of 0.33% per annum. These figures are important
because estimates of early adenocarcinomas (EAC) incidence influence the degree to which
endoscopic and biopsy surveillance are likely to be beneficial in Barretts oesophagus.
Bhat et al. [23] reported cancer risk estimates for an unselected cohort of Barretts
oesophagus (not all with intestinal metaplasia, ascertained from pathology reports) in
Northern Ireland, with mean follow-up of 7 years. After excluding baseline HGD and EAC,
combined EAC, HGD and cancer of the gastric cardia risk estimate was 0.13% per annum
(CI 0.100.16). When the analysis was restricted to cases with intestinal metaplasia and
visible Barretts mucosa, the incidence of EAC was 0.18% per annum and the combined
incidence of HGD, EAC and gastric cardia cancer was 0.33% per annum, identical to the
estimate of Desai et al. [22].
The risk of adenocarcinoma amongst Barretts oesophagus patients in a population-
based study from Denmark [24] was the same at 0.12% per annum. The relative risk of
adenocarcinoma amongst Barretts oesophagus patients compared with the general
population was 11.3%. These findings have been said to call into question current screening
and surveillance regimens. However, de Jongh et al. [25] found a progression rate more
typical of previous estimates at about 0.4% per annum in a Dutch population study.

postascertaInMent surveIllance
For patients known to have Barretts oesophagus, current guidelines continue to recommend
surveillance endoscopy and biopsy every 35 years for patients without dysplasia; every
78 Mucosal pathology of the gastric cardia and Barretts oesophagus

612 months for low-grade dysplasia (LGD) and quarterly for HGD [26,27] in the plausible,
but unproven expectation that HGD and low-stage adenocarcinoma (pT1a/b) will be more
amenable to treatment. There are however no controlled trials of surveillance efficacy, so the
extent to which current strategies are optimal (or even justified) is unknown.
If the risk of progression to adenocarcinoma is really much lower than previously
reported, the value of surveillance is called into question, particularly in Barrett patients
without dysplasia, in whom even quinquennial surveillance may not be cost-effective [28].
The wide variation in reported EAC progression rate in Barretts oesophagus [20] increases
doubt about effectiveness and cost-effectiveness of current surveillance strategies [17,29].
Most studies of cost-effectiveness of Barretts surveillance have assumed an EAC incidence
of 0.5% per annum, which as mentioned above may be an overestimate [3033]. Continuing
reflux, age >50 years, maleness, Caucasian ethnicity, elevated body mass index (BMI) and
especially dysplasia are additional adenocarcinoma risk factors. Diagnostic accuracy of
dysplasia also needs to be considered.

dysplasIa dIagnosIs In Barretts oesophagus

This remains a significant challenge. There have been many studies showing that
agreement on dysplasia grading between pathologists is not good, and no evidence of
any great progress in the last 25 years towards doing better. In particular, overdiagnosis
of LGD is not uncommon, but if that diagnosis stands up to review by competent experts,
progression risk is actually comparable with the risk associated with some published series
of HGD [34,35].
The challenge is of course twofold: in a perfect world one would neither overdiagnose
regenerative and reactive changes as dysplastic nor underdiagnose true dysplasia as
reactive. The hazards of dysplasia diagnosis in the presence of florid active inflammation
are well known. If in doubt, rebiopsy following a period of proton pump inhibitors (PPI)
therapy on a sufficient dosage to allow significant healing should be undertaken.
Practical difficulties encountered in routine practice include biopsy trauma (Figure 6.3),
variable section thickness, variable staining, vagaries of biopsy orientation and background
inflammation (Figures 6.4 and 6.5). A prudent histopathologist has a low threshold for
seeking the opinion of an experienced colleague or colleagues if there is any difficulty
in deciding whether dysplasia is present, and if so whether low grade or high grade. The
diagnosis indefinite for dysplasia is not helpful to the clinician who is seeking to develop
a follow-up strategy for an individual patient but on some occasions it cannot be avoided.
Abrupt transitions between dysplastic and nondysplastic epithelial clones (Figure 6.6) and
abnormal mitoses, suggesting aneuploidy (Figure 6.7), are useful clues.
Not only must the pathologist recognise dysplasia, the endoscopist must take biopsies
including dysplastic mucosa in the first place. The necessity for some kind of systematic
approach to the diagnosis led to the implementation of the Seattle protocol (four biopsies
every 2cm of Barretts mucosa; four biopsies every centimetre if dysplasia is suspected).
There have been relatively few studies of the efficacy of systematic versus nonsystematic
biopsy strategies. Abela et al. [36] did however show that careful systematic biopsy
following a new diagnosis of long-segment Barretts oesophagus yielded a relatively high
prevalence of dysplasia followed by a very low incidence of new dysplasia, in contrast to
nonsystematic biopsy which yielded a much lower prevalence of dysplasia identified within
6 months of the index endoscopy, but thereafter a considerably higher rate of incident
Advanced imaging and detection of Barretts dysplasia 79

Figure 6.3 Low-grade dysplasia, missed in routine practice. Left panel: No dysplasia. Right panel: fragmented
dysplastic surface epithelium and keratin debris. Reported as no dysplasia. Disruption of mucosal architecture
probably made this dysplastic focus harder to recognise. Subsequent biopsies confirmed persistent low-grade

dysplasia diagnosis, which in reality was probably pseudoincident, in the sense that it had
probably been present all along, but not been detected by an inefficient biopsy protocol.
Careful implementation of Seattle biopsy protocol is demanding for patient,
endoscopist, histology laboratory and interpreting pathologist. It remains to be shown that
alternative approaches are better. Unfortunately, in white light endoscopy, flat dysplasia is
hard to see but if reliable endoscopic visualisation of dysplastic Barretts mucosa could be
achieved, considerable efficiency might be achieved, with improved predictive value and

advanced IMagIng and detectIon of

Barretts dysplasIa
Autofluorescence imaging (AFI), narrow band imaging (NBI) and confocal laser
endomicroscopy (CLE) may improve detection of dysplasia and early cancer.
NBI visualises submucosal vascular changes and detects more and higher grades of
dysplasia in fewer biopsies than white light endoscopy [37]. A meta-analysis of NBI in
upper GI endoscopy has claimed sensitivity 97%, specificity 94%, and overall accuracy 96%
for NBI in differentiating dysplasia from nondysplastic Barretts mucosa [38].
Curvers et al. [39] performed a multicentre randomised study of trimodal endoscopy
(AFI, NBI and high-definition white light endoscopy, HD-WLE) with improved detection
80 Mucosal pathology of the gastric cardia and Barretts oesophagus

Figure 6.4 High-grade dysplasia,

missed in routine practice. This
panel shows a markedly villous
mucosal biopsy with disordered
glandular architecture. High-
power details emphasise severity
of cytological and architectural
atypia. This biopsy was reported
as showing nondysplastic
inflammatory atypia.

Figure 6.5 Genuine nondysplastic inflammatory atypia in Barretts oesophagus.

Advanced imaging and detection of Barretts dysplasia 81

Figure 6.6 A useful clue to the

presence of dysplasia. An abrupt
morphological transition between
cells associated with adjacent
crypts emphasises the atypia of
the dysplastic cells on the right in
comparison with the more orderly
epithelium on the left.

Figure 6.7 Subtle low-grade

dysplasia with maturation on the
mucosal surface. Some maturation
on the mucosal surface is usual in
dysplastic Barretts mucosa. The
presence of abnormal mitoses
(centre right) is a useful pointer to

of HGD and early cancer in Barretts oesophagus. In another multicenter randomised

controlled trial, Sharma et al. [40] compared CLE, HD-WLE and NBI separately and
together. CLE with HD-WLE was significantly better than white light endoscopy alone in
detecting HGD and early cancer in Barretts oesophagus.
These studies offer some promise of more cost-effective Barretts oesophagus
endoscopic surveillance. More reliable detection of HGD and intramucosal
82 Mucosal pathology of the gastric cardia and Barretts oesophagus

adenocarcinoma and nonsurgical treatments, such as radio frequency ablation (RFA)

and endoscopic mucosal resection, could favourably affect the cost-benefit balance
of Barretts oesophagus surveillance [41]. Careful appraisal of diagnostic accuracy of
advanced imaging techniques in Barretts surveillance is needed as they become more
widely available.

BIoMarkers and rIsk stratIfIcatIon In

Barretts oesophagus
Risk stratification is at the heart of Barretts oesophagus management and effective risk
biomarkers would be of great value [42]. More intense surveillance of high-risk patients and
less intense surveillance of low-risk patients would be practical and cost-effective. Genetic
and molecular biomarkers continue under investigation because of a conviction they ought
to be the key to cancer prevention and surveillance of high-risk individuals. Biomarkers
might also decrease interobserver variability in pathologists interpretation of dysplasia.
Progress towards these goals has been disappointingly slow.

Abnormal cellular DNA content (aneuploidy) is associated with increased risk of
adenocarcinoma. Reid et al. [43] showed that patients without aneuploid cells have a low
risk of progression, whilst patients in whom baseline biopsies demonstrated aneuploidy,
tetraploidy, or HGD progressed to cancer over 5 years in 43% and 59% of cases. In some
specialist centres, flow cytometry to assess ploidy is undertaken in the assessment
of Barretts mucosa, but it is not widely used probably for reasons including cost,
reimbursement issues and technical challenges. Flow cytometry divorces DNA content
measurements from morphology. Image cytometry of nuclei from thick sections, however,
allows some histological control, and image cytometry on histological sections gives best
correlation of DNA content with morphology, but introduces its own problems with nuclear
truncation and overlapping.
Molecular markers. TP53 is usually wild type in nondysplastic Barretts mucosa [44].
One TP53 allele may be inactivated by mutation and loss of heterozygosity at TP53 is
common in adenocarcinomas [44,45]. Overexpression of p53 (and sometimes circulating
p53 antibodies) is associated with progression to adenocarcinoma [46], and patients with
TP53 loss of heterozygosity (LOH) are more likely to progress to aneuploidy, HGD and
adenocarcinoma [47]: Thirty-seven per cent of patients with baseline p53 LOH progressed
to adenocarcinoma, against 3% without. Prevalence of LOH increases from 6% in
nondysplastic Barretts mucosa to 57% in HGD.
Aberrant tumour suppressor gene (TSG) promoter methylation is also an
adenocarcinoma risk biomarker [48]. In a multicenter study of 200 patients by Jin et al.
[49], an eight-biomarker panel predicted about half of HGDs and adenocarcinomas, with
reasonable specificity. Although these results appear encouraging, nonstandardised
methodology and an almost complete lack of prospective trials are problematical. Without
Endoscopic treatment of the high-risk oesophagus and cardia 83

robust validation in large-scale multicenter trials, uptake of molecular risk stratification in

endoscopic surveillance is unlikely.

endoscopIc treatMent of the hIgh-rIsk

oesophagus and cardIa
At one time, the only definitive treatment of oesophageal neoplasia was oesophagectomy.
Even in good surgical centres, this is risky and with significant morbidity. Newer,
endoscopic treatments include argon beam coagulation, photodynamic therapy,
endoscopic mucosal resection and RFA. RFA is currently approved for the treatment of
HGD. In the long run its greatest value may however turn out to be in the treatment of LGD.
The intention of treatment is complete ablation of potentially dysplastic Barretts mucosa.
Up to three treatments may be required to achieve this and even then some patients are
refractory to treatment (Figures 6.8 and 6.9). Data from the SURF trial of RFA in Barretts
oesophagus with LGD presented in May 2013 [50] suggest that it may be very effective in
preventing progression to HGD and oesophageal adenocarcinoma; if longer follow-up
confirms these early findings, removing the necessity for intensive long-term surveillance
may be cost-effective.

Figure 6.8 Oesophagectomy may still be required in

some high-risk patients in whom endoscopic treatment
has not eradicated neoplastic changes. This patient
had persistent high-grade dysplasia/intramucosal
adenocarcinoma following several cycles of endoscopic
mucosal resection and radio frequency ablation (RFA).
This unusual long (M10) but nowhere-circumferential
(C0) Barretts segment is a post-treatment appearance
in a patient partially refractory to RFA. The specimen has
been opened and pinned out to optimise fixation.
84 Mucosal pathology of the gastric cardia and Barretts oesophagus

Figure 6.9 This dysplastic long segment Barretts

oesophagus was also refractory to radio frequency
ablation. The markedly villous mucosal architecture may
have contributed to this state of affairs.

Key points for clinical practice

A new diagnosis of Barrett's oesophagus should not to be undertaken lightly. Oesophageal
biopsies with an appropriate glandular phenotype may be described as 'consistent with',
even 'typical of' Barrett's oesophagus.
Oesophageal submucosal glands or ducts, native squamous epithelium and multilayered
epithelium are useful pointers to an oesophageal biopsy origin.
Reactive changes are easily mistaken for dysplasia if the oesophagus is heavily inflamed.
Rebiopsy after a period of acid suppression may help.
Dysplastic changes may also be mistakenly regarded as reactive. A prudent pathologist has
a low threshold for seeking a second or third opinion if in doubt.
Confirmation in particular of a new diagnosis of dysplasia by an experienced colleague is
good practice.
Abrupt transitions between morphologically different populations of epithelial cells should
raise a suspicion of dysplasia.
Although failure of maturation on the mucosal surface is a useful pointer to the presence of
dysplasia, this feature is not always present, and even if present, it may not be seen in the
plane of section. Deeper levels may help.
Good-quality histology is essential. Sections which are too thick and overstaining make
diagnosis significantly more difficult. Immunohistochemistry has little to offer.
Diagnostic and therapeutic endoscopic mucosal resections should be processed in their
entirety in parallel slices 23mm thick. Diathermy artifact is often a challenge.
If in doubt, consult.
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progression prediction in Barretts esophagus. Cancer Res 2009; 69:41124115.
50. Phoa KYN, van Vilsteren FG, Pouw RE, et al. Radiofrequency ablation in Barretts oesophagus with
confirmed low-grade dysplasia: Interim results of a European multicenter randomised controlled trial
(SURF). Gastroenterology 2013; 155:s187.
Chapter 7

Pathology of regenerative and

neoplastic hepatocellular nodules
Alberto Quaglia

Primary liver tumours are currently classified into epithelial, mixed/uncertain origin,
mesenchymal, germ cell and lymphomas [1]. Epithelial tumours are further subdivided
into hepatocellular and biliary, based on their resemblance to their normal epithelial
counterparts. This does not imply exclusive derivation from mature hepatocytes
or cholangiocytes. The liver contains a reserve regenerative compartment made of
hepatic progenitor cells believed to reside in the canal of Hering [2]. This progenitor cell
compartment is capable of activating, proliferating and differentiating into both hepatocyte
and cholangiocyte lineages, particularly when mature epithelial cells are damaged or
their replication is inhibited [3]. Hepatic progenitors are activated in many chronic
liver disorders [4] and may be targeted by carcinogenesis. Neoplastic transformation of
progenitor cells leads to neoplastic progenies which maintain the ability to differentiate
into both lineages . This in turn explains the observation of hepatic progenitor cell features
in hepatocellular neoplasms at an early stage (dysplastic nodules) or advanced tumours
with mixed hepatocellular and cholangiocellular phenotypes [3,5,6]. Concepts such
as tumour acquisition of progenitor cell features by de-differentiation, bone marrow
derivation of a proportion of hepatic progenitor/stem cells, cell fusion and cancer
cooperation add a further layer to the intricate pathogenesis and phenotypic complexity of
liver tumours [3,711].
The 4th edition of the WHO classification of tumours of the digestive system [1]
subdivides hepatocellular epithelial tumours into benign tumours [hepatocellular
adenoma (HCA), focal nodular hyperplasia (FNH)], malignancy-associated and
premalignant lesions (large cell change, small cell change, dysplastic nodules) and
malignant tumours [hepatocellular carcinoma (HCC) and variants, hepatoblastoma,
undifferentiated]. These categories partly overlap and partly divert from previous
classifications, in particular the widely adopted Working Party 1995 [12] consensus based
on the classification of hepatocellular nodules into regenerative lesions [monoacinar (e.g.
nodular regenerative hyperplasia), multiacinar, lobar/segmental hyperplasia, cirrhotic
nodule, FNH] and dysplastic or neoplastic lesions (HCA, dysplastic focus, low-grade/high-
grade dysplastic nodule) and HCC. Considerations on the general definition of terms such
as tumour, nodule, regenerative, neoplastic, benign, dysplastic and malignant are beyond

Alberto Quaglia, MD, PhD, FRCPath, Institute of Liver Studies, Kings College Hospital, London, UK
Email: alberto.quaglia@nhs.net (for correspondence)
88 Pathology of regenerative and neoplastic hepatocellular nodules

the purpose of this review, which for sake of simplicity and authors personal preference
maintains the time honoured distinction of regenerative versus neoplastic lesions.
This review also focuses on the more practical issues of differentiating histologically
between these various entities, in adult patients. The reader is referred to textbooks
[1315] or topical reviews [1618] for more comprehensive systematic descriptions of the
histopathology of hepatocellular lesions, and the most recent advances in their molecular
biology aspects [19,20].
The diagnosis of liver nodules rests on proper clinicopathological correlation. A full
clinical history, close correlation with imaging findings and ideally an adequate sample
of lesional tissue along with a separate sample of nonlesional tissue to assess the status of
background liver should be the basis for their histological interpretation.

Regenerative hepatocellular nodules

The term regenerative hepatocellular nodule, regenerative nodule or regenerative lesion is
used when hepatic plates expand and assume a nodular configuration. This can occur in
response to necrosis, altered vascular supply or other stimuli [12]. The size of regenerative
nodules can vary from an area corresponding to approximately the size of a single acinus
or lobule to multiple ones, including many portal tracts and at times reaching even several
centimetres in diameter (monoacinar and multiacinar regenerative nodules, respectively).
Their boundaries consist usually of fibrous septa, collapsed reticulin strands, multilobular
areas of parenchymal collapse, atrophic hepatic plates, other nodules or a combination
of the above. The hepatocytes of regenerative nodules are usually bland, and do not show
any of the atypical features of dysplastic nodules (see below). The hepatic plates may be
two-cell thick. The reticulin framework is preserved, without the rarefaction or loss noted
usually in HCC.
Regenerative nodules may form in response to acute severe hepatocellular necrosis
as observed, for example, in cases of acetaminophen overdose. This is usually a diffuse
injury, affecting the whole organ in a relatively homogenous distribution, without much
difference between different lobes or segments [21]. At microscopy, the centro-mid lobular
hepatocytes are typically affected. The remaining periportal hepatocytes enter the cell
cycle and proliferate. This may lead to reconstitution of the lobular mass within few days/
weeks as shown in serial biopsies of livers from survivors, including recipients of auxiliary
grafts [22,23]. In the early stage of this process, the hepatic plates show a micronodular
configuration due to the combination of centro-mid lobular collapse, inflammation, fibrous
scarring and regeneration (Figure 7.1). Matrix reabsorption, and plate remodelling result
eventually in the reconstitution of the lobular architecture.
Acute liver injury caused by aetiologies other than acetaminophen overdose, and many
cases of subacute liver injury, are characterised by large areas of parenchymal loss spanning
over many lobules (multilobular or multiacinar necrosis, massive hepatic necrosis when
about three-fourths or more of the organ are affected). In some cases, the injury is extreme
to the point that no hepatic plates are found even after close examination of the whole
organ. Often, however, interspersed amongst areas of multilobular collapse are nodules of
parenchyma of variable size, ranging from <1mm to several centimetres, in a map-like
pattern, considered to be the regenerative response to parenchymal injury [21,22].
Regenerative hepatocellular nodules 89

a b

c d

e f

Figure 7.1 Reticulin stain (Gordon and Sweet). (ac) Acute liver failure due to acetaminophen overdose. Serial
biopsies from native liver during its recovery phase following auxiliary transplantation (donor right lobe attached to
recipients left lobe). (a) 29 days, (b) 146 days, and (c) 350 days. Acute necrosis with sparing of periportal hepatocytes
results in an initial phase of hepatocellular proliferation, acute inflammation and reticulin collapse with parenchymal
nodular transformation. Resolution of inflammation, matrix reabsorption and hepatic plates remodelling
and growth lead eventually to full reconstitution of the liver mass, involution of the graft and withdrawal of
immunosuppression. (d) Regenerative nodule in nodular regenerative hyperplasia. The nodular area is flanked by
atrophic plates with condensation of reticulin fibres but no bridging fibrosis. (e) Regenerative nodule in advanced
stage chronic hepatitis C. The nodular area is surrounded by bridging fibrous septa. (f ) Primary biliary cirrhosis.
Bridging fibrosis linking up portal tracts generate a jigsaw puzzle pattern rather than round nodules.
90 Pathology of regenerative and neoplastic hepatocellular nodules

In advanced chronic liver disease, fibrosis progression leads to the formation of fibrous
septa, which encase regions of hepatic parenchyma to form nodules (cirrhotic nodules),
the size and shape of which depend essentially on the underlying disease. For example, in
chronic biliary disorders the regenerative nodules are typically in a jig-saw puzzle pattern,
the fibrous septa connecting portal tracts reciprocally, and sparing the hepatic venules
(Figure 7.1). In chronic hepatitis C, the nodules are of small size due to porto-portal and
porto-central bridging with loss of the vascular anatomical relationships (Figure 7.1). Two-
cell-thick hepatic plates are often present, and irrespective of the aetiology there may be
deposition of granules of copper-binding protein at the interface with the fibrous septa. This
indirect sign of advanced fibrosis may be helpful in the interpretation of core needle biopsy
specimens, in which nodule formation is not readily apparent due to the small size of the
sample or the large size of the nodules or both.
Some cirrhotic nodules may enlarge, and become dominant compared with the adjacent
regenerative nodules, to the point that they stand out at macroscopic examination of a
surgical specimen or may even be identified on imaging (large regenerative nodules,
macroregenerative nodules in older terminology). Sinusoidal capillarisation and individual
arteries may be present but are usually focal. In conditions such as BuddChiari syndrome
and primary sclerosing cholangitis, large areas or segments with preserved vascular outflow,
biliary drainage or portal vein supply in combination may become hyperplastic, particularly
in comparison with adjacent underperfused or poorly drained areas. This may cause, in
some cases, severe distortion of the lobar anatomy (Figure 7.2).
Nodular regenerative hyperplasia is the term used to describe thickened hepatic plates
arranged in a nodular configuration, often irrespective of specific lobular zonal boundaries
and with intervening atrophic hepatic plates, dilated sinusoids to a variable extent but no
bridging fibrosis. It typically occurs when small portal veins are obliterated, in the context,
for example, of systemic microvascular disease. The nodular hepatic plates are probably
those with preserved portal perfusion, whereas the atrophic ones are those affected by the
obliteration of their small portal vein tributaries (Figure 7.1). Areas of nodular regenerative
hyperplasia can become dominant and form regenerative masses, which can be identified

Figure 7.2 Primary sclerosing

cholangitis. Marked segmental
hypertrophy and lateral atrophy
lead to severe distortion of the
lobar anatomy.
Regenerative hepatocellular nodules 91

on imaging, causing difficulties in the differential diagnosis with HCC. Terms such as
adenomatous hyperplasia or partial nodular transformation have been used in the past to
name such lesions. These masses resemble FNH and probably share a similar pathogenesis
FNH is considered to be the typical example of a localised regenerative process, leading
to the formation of a mass identifiable on imaging and rarely even symptomatic. It is
usually solitary, but multiple FNH can occur in the context of vascular malformations
and coexist with intracranial tumour. It usually develops in noncirrhotic livers of women
3040 years of age, but FNH-like lesions have been described in cirrhosis [26,27]. It is
considered a benign hyperplastic regenerative process secondary to a local abnormality
of blood flow due to a vascular malformation or obliteration and arterialisation. This is
supported by its polyclonality, increased ANGPT1/ANGPT2 ratio, and association with
vascular disorders such as BuddChiari syndrome, hereditary haemorrhagic telangiectasia,
portal vein thrombosis, portal vein atresia and congenital portosystemic shunt [28,29].
Macroscopically, FNH can range in size from a few millimetres to several centimetres.
It usually shows a pale, micronodular and firm parenchymal surface, with or without
a central scar. There is usually no capsule, the periphery of the lesion merging with the
adjacent liver. Microscopically, it consists of nodules of benign-looking hepatocytes
separated by fibrous septa containing a ductular reaction, a lymphoid infiltrate and
aberrant vascular structures. Bioulac-Sage et al. [30] have recently shown a fairly
characteristic pattern of glutamine synthetase expression, typically patchy, highlighting the
hepatocytes away from the fibrous septa (Figure 7.3b). There may be a degree of sinusoidal
capillarisation by CD34 immunostaining. Associated features include steatosis, siderosis
and cholate stasis.
The differential diagnosis of regenerative nodules depends on the clinical context. In
cirrhotic patients, large regenerative nodules need to be differentiated from dysplastic
nodules and HCC. Dominant, mass-forming areas of nodular regenerative hyperplasia
and FNH may mimic radiologically HCA, HCC or nonhepatocellular lesions. These specific
differential diagnoses are discussed below.

a B

Figure 7.3 (a) A 32-year-old woman transplanted for glycogen storage disease type 1. A 10-mm hepatocellular
adenoma showed diffuse staining for glutamine synthetase. No nuclear or cytoplasmic staining for -catenin was
identified. Please note background liver on the right hand side showing normal glutamine synthetase expression by
perivenular hepatocytes. (b) 52-year-old woman. Liver resection for focal nodular hyperplasia. In comparison with
picture (a), the pattern of glutamine synthetase is typically map-like.
92 Pathology of regenerative and neoplastic hepatocellular nodules

An important practical point is the identification of a regenerative nodule in a core

needle biopsy specimen, as the changes can be very subtle and easily overlooked. Lack
of portal tracts over a large span of parenchyma, or portal tracts irregularly distributed,
with or without a local hepatic venule could imply that the sample is from a large
regenerative nodule. Two-cell-thick hepatic plates in biopsy cores from adult patients, thin
hepatic plates sometimes in a parallel configuration, sinusoidal dilatation without other
accompanying features of venous outflow blockage or even a few focal deposits of copper
binding protein on the orcein or Victoria blue stain without other signs of biliary pathology
could all represent signs that the needle has hit a regenerative area. Even in advanced
chronic liver disease, fibrous septa may not be evident if a large regenerative nodule is
sampled. On the contrary, FNH in a core needle biopsy can be indistinguishable from
cirrhosis, particularly if the histopathologist is not given sufficient clinical information,
is not aware that the biopsy was targeted and there is no available sample from the
background liver well away from the lesion for comparison. Additional stains, which
can be of help in identifying the porto-lobular relationship, are immunohistochemistry
for cytokeratins (e.g. Ker 7) and glutamine synthetase to highlight portal regions and
centrilobular venules, respectively.

Neoplastic hepatocellular nodules

Dysplastic nodules
This term is usually applied to nodules of relatively small size, up to about 1015mm,
identified in cirrhotic livers, and showing atypical histological features without fulfilling
the criteria of HCC. These nodules are thought to be HCC precursors, based mainly on the
following observations: (1) their occurrence in livers affected elsewhere by overt HCC [31],
(2) the identification of small nodular lesions with atypical features and containing foci of
overt HCC (nodule-in-nodule pattern) [32], (3) similarities at molecular level with HCC
[33], (4) clinicopathological studies based on biopsy and clinical follow-up suggesting their
transformation into HCC [34,35], and (5) change in their vascular supply.
This latter point is particularly important. Progression from cirrhosis to HCC is
characterised by a switch from a dual portalarterial to a predominantly arterial supply,
due to neoangiogenesis. Histologically, this is associated with the presence of unpaired
arteries [36] and a change in the characteristics of sinusoidal endothelial cells (sinusoidal
capillarisation) marked by the immunohistochemical staining pattern with markers such
as CD34 [37]. Arterialisation is exploited radiologically by demonstrating contrast uptake in
arterial phase and rapid washout in the venous phase [38] to the point that demonstration
of arterialisation by one or two imaging modalities (for lesion greater or smaller than 2cm,
respectively) is considered to be sufficient for the diagnosis of HCC without the need of
histological confirmation. The use of liver biopsy is therefore confined to lesions with
atypical features on imaging. Of note, a small proportion of HCC, may be hypovascular,
and a small proportion of nodules identified by imaging in cirrhotic patients may not be
hepatocellular [39].
Macroscopically, dysplastic nodules are distinct from the background liver in terms of
their size, appearance, colour, texture and/or bulging cut surface. Microscopically, they
may show a range of changes, which deviate from the background regenerative nodules.
Particularly helpful features are those suggestive of clonal expansion, such as well-defined
areas (nodule-in-nodule pattern, Figure 7.4) characterised by presence or absence, or
Neoplastic hepatocellular nodules 93

Figure 7.4 Dysplastic nodule in

cirrhotic liver showing a nodule-in-
nodule pattern. The line of triangles
marks the boundary of the nodule
against the background liver. The
arrow indicates an area of increased
cell density.

significant increase or decrease in some changes in comparison with the adjacent tissue.
These changes include siderosis, copper/copper-binding protein deposits, steatosis, clear
cell change, MalloryDenk bodies, increased trabecular thickness, increased cell density,
pseudoglandular structures, nuclear hyperchromasia or irregularity of the nuclear contour,
cytoplasmic basophilia, increased proliferative rate within areas of a nodule or compared
with background liver and loss of the reticulin stroma. Large cell change, originally called
liver cell dysplasia [40], describes the presence, usually in the context of chronic liver
disease, of more or less demarcated areas of liver parenchyma in which hepatocytes show
large atypical nuclei with preserved nuclearcytoplasmic ratio. It has been shown to be
associated with a risk of development of HCC, particularly in patients with viral hepatitis,
although its association with cholestasis and senescence suggests that this change may
have more than one pathogenesis [41]. Small cell change refers to the presence of areas
of increased cell density due to a reduction in hepatocyte size and increased nuclear
cytoplasmic ratio, often associated with nuclear hyperchromasia and irregularity of the
nuclear contour. A relationship between small cell change and progenitor cells has been
proposed on the basis of a similar immunohistochemical profile [42]. Small cell change is
considered to be a true precursor of HCC. In some cases, nodules fulfilling the criteria of
dysplastic nodules harbour foci of overt HCC in a nodule-in-nodule pattern.
According to a recent consensus paper [43], dysplastic nodules are further subdivided
into low-grade and high-grade lesions. Low-grade dysplastic nodules may be
indistinguishable from large regenerative nodules, differing only by the focal presence of
some of the dysplastic changes described above. High-grade dysplastic nodules may be
indistinguishable from early HCC probably because they represent part of a continuum.
Recent studies have shown that identification of stromal invasion, i.e. the presence
of tumour cells inside portal tracts or fibrous septa, may be of help in differentiating
between dysplastic nodules and HCC, but in the authors opinion, its interpretation is not
straightforward, even with the aid of immunohistochemistry [44].
Recent molecular studies [45] have led to the introduction of an immunohistochemical
panels including heat-shock protein 70, glypican 3 and glutamine synthetase, deemed to
be useful in differentiating dysplastic nodules from overt HCC [46]. Lack of staining with all
94 Pathology of regenerative and neoplastic hepatocellular nodules

three markers supports a high-grade dysplastic nodule, staining for one marker supports
well-differentiated HCC but does not exclude a high-grade dysplastic nodule, and staining
for two or three markers favours a well-differentiated HCC over a high-grade dysplastic
nodule. Clathrin heavy chain [47] and annexin-A2 [48] have been shown to improve the
performance of this panel.
To summarise, rather than separating categorically dysplastic nodules from HCC,
the currently accepted view is that HCC development and the transition between
dysplasia and HCC can be defined by three phases [43]: (1) high-grade dysplastic nodule
characterised by absence of stromal invasion, iso or hypovascularised with individual
arteries and portal perfusion; (2) well-differentiated (early) HCC, vaguely nodular with
indistinct margins, individual arteries, residual portal perfusion and iso-hypovascularity
on imaging, with or without stromal invasion; (3) moderately differentiated (progressed)
distinctly nodular HCC, with or without stromal invasion, with individual arteries, no portal
tracts and characteristic imaging features of hypervascularity in the arterial phase, and
hypovascularity in the venous phase. Vascular invasion may or may not be present.
Despite widespread interest in the concept of dysplastic nodule, the practical
diagnostic implications are largely dependent on local clinical practice. In view of the
current clinicoradiological criteria, small nodular lesions in cirrhotic patients are rarely
biopsied, or may be biopsied during local ablative procedure. The histological differential
diagnosis between large regenerative nodule, dysplastic nodule and HCC in this
setting may be challenging, severely limited by sampling error and essentially based on
clinicopathological correlation. The practical issues on the diagnosis of dysplastic nodules
in livers removed at transplantation are tempered, as the clinical emphasis in this context is
on the identification of histological predictors of tumour recurrence after transplantation,
which usually applies to overt HCC. Identification of incidental dysplastic nodule in
resection specimens of HCC from patients with compensated cirrhosis may confirm field
change and risk of malignant transformation in the remaining liver.
Small cell change and large cell change are part of a constellation of features which are
occasionally seen incidentally, either in a biopsy specimen not targeted radiologically to
a specific lesion or in sections from surgical specimens outside the boundaries of lesions
identified macroscopically. These changes include the iron-free foci in livers of patients
with genetic hemochromatosis described by Deugnier et al. in 1994 [49], the areas of
severe irregular regeneration of hepatocytes described by Shibata et al. in 1998 [50] or the
dysplastic foci characterised by any of the changes characteristic of dysplastic nodules,
in areas not >1mm. The general view is that these changes should at least be mentioned
in a histology report as they may indicate an increased risk of HCC. How this risk can be
quantified or whether and how it should dictate further clinical management is not clear.
Gene expression profiling has recently produced a prognostic gene expression signature to
predict the development of HCC [51].

Hepatocellular adenoma
HCA is considered to be a benign neoplasm of hepatocytes, although the term benign
contrasts the concept that this lesion carries a risk of malignant transformation. The main
risk factor for developing HCA is exposure to oestrogens or androgens. Outside the context
of hormonal stimulation, HCA is associated with conditions such as glycogen storage
disorder [52,53], and familial adenomatous polyposis. HCA can be single or multifocal, in
Hepatocellular adenoma 95

which case the term adenomatosis may apply. HCAs vary in size and are usually fairly well
circumscribed, with a soft cut surface often similar to background liver, although in many
cases areas of haemorrhage, necrosis or fibrosis may be present. Microscopically, HCA
are composed of hepatic plates similar to those in the background liver and intervening
individual arteries, but usually no portal tracts. Areas of necrosis, haemorrhage or fibrosis
are variably present. Some adenomas are rich in Dubin-Johnson-like pigment, possibly due
to a defect in excretion of organic anions [54]. More specific changes are associated with
the subtypes of HCA recently described on the basis of a correlation between molecular,
histological and clinical features [55,56] (Table 7.1). The subtype associated with mutations
of HNF1a is characteristically steatotic and lacks expression of fatty-acid binding protein.
Small microadenomas with similar characteristics are often found in the adjacent liver.
The inflammatory subtype, previously considered a variant of FNH [57], may be associated
with mutations of the IL6ST gene and tends to occur in associations with obesity, alcohol
consumption and systemic inflammatory signs and symptoms. It is characterised by
sinusoidal ectasia (telangiectasia), an intralesional inflammatory cell infiltrate often related
to intralesional arteries, a ductular reaction and strong expression of serum amyloid A
(SAA) and C-reactive protein (CRP), particularly in comparison with the background liver.
Steatosis may or may not be present. A small percentage of inflammatory adenomas may
harbour mutations of the b-catenin gene, which characterise a third subtype of HCA. The
histological features of this b-catenin-activated variant include pseudogland formation,
nuclear atypia, variable, often very focal nuclear and cytoplasmic staining for b-catenin
and diffuse expression of glutamine synthetase (Figure 7.3). This subtype of HCA occurs
usually in men, is considered to be at risk of malignant transformation and may be very
difficult to differentiate from well-differentiated HCC. The fourth category of HCA includes
all adenomas which do not fit into any of the three categories above.
As in the case of regenerative nodules and FNH, HCA and in particular its inflammatory
subtype may be overlooked in a biopsy sample, particularly if clinical details are lacking
and there is no sample of background liver for comparison. Particular attention needs to
be paid to the presence of individual arteries, and the spots of connective tissue containing
arteries and ductular reaction typical of the inflammatory subtype, which may be easily
mistaken for portal tracts, or FNH septa. Immunohistochemistry for serum amyloid A or
C-reactive protein is usually helpful, as they are both strongly expressed in inflammatory
adenoma, in contrast to FNH or background liver which are negative. Staining for
glutamine synthetase is also effective as, in background liver, it decorates the perivenular

Table 7.1 Hepatocellular adenoma subclassification

Category Histological features Clinical features
-catenin mutated Pseudacinus formation, atypia Malignant transformation, bleeding
HNF1 mutated Steatosis, FABP deficient Bleeding
Inflammatory adenoma (formerly Intralesional lymphocytosis, ductules Bleeding, a percentage bears
telangiectatic FNH) (ck7+ve) and sinusoidal dilation -catenin mutation. Systemic
(telangiectasia). SAA and CRP syndrome. Associated with alcohol,
positive metabolic syndrome
Unclassified Nonspecific features Variable

FABP, fatty-acid binding protein; FNH, focal nodular hyperplasia.

96 Pathology of regenerative and neoplastic hepatocellular nodules

hepatocytes, and in FNH those away from the septa (antiseptal), generating a fairly typical
map-like pattern (Figure 7.3) [30]. The differential diagnosis between HCA and HCC is
described below.

Hepatocellular carcinoma
HCC is considered to be the most common type of primary malignant liver tumour in
adults, although its incidence shows marked geographical variation, depending essentially
on the distribution of its risk factors. HCC is more common in males. The main risk factor
is cirrhosis and the main association is with hepatitis B virus (HBV) and hepatitis C virus
(HCV) infection, aflatoxin and alcohol, but any inherited or chronic liver condition increases
the risk of developing HCC. HCC can affect children with inherited metabolic conditions
such as tyrosinaemia, hypercytrullinaemia, biliary atresia, Bylers disease, bile salt export
pump (BSEP) deficiency and a-1-antitrypsin deficiency. Regression of fibrosis does not
eliminate the risk of HCC. HCC can occur in chronic viral hepatitis at a precirrhotic stage.
This is more frequent in HBV than HCV infection [15]. HCC does occur in noncirrhotic,
sometimes elderly patients. A good proportion of these patients have signs or history of
iron overload, past exposure to HBV or alcohol to excess. Diabetes, obesity and in more
general terms the metabolic syndrome are considered to be significant risk factors for the
development of HCC in livers without significant fibrosis [58]. Some patients, however,
may not have any risk factor or sign of liver injury [59,60]. HCC can develop in patients
taking anabolic steroids, but may show regression after hormonal withdrawal [61]. The
fibrolamellar variant of HCC is a well-recognised entity, which affects young adults, without
underlying history of liver disease. This variant of HCC has peculiar radiological and
histological characteristics, spread and behaviour, and its pathogenesis remains obscure.
HCC tends to be larger in noncirrhotic patients than in cirrhotic ones, partly due to
surveillance programmes in patients with chronic liver disease, leading to its identification
at a relatively early stage. HCC can grow into three main patterns. The nodular pattern
is the one usually observed in cirrhotic liver and consists of an expanding mass well-
demarcated from the surrounding tissue often by interposition of a capsule and with
a multilobulated cut surface. The transition between dysplastic nodules and HCC,
along with the concept of early vaguely nodular and progressed distinctly nodular
HCC, has already been discussed. The term satellite is commonly used to describe the
presence of small tumoural nodules in the vicinity of the main mass. Whether multiple
nodular masses represent multifocal disease (either synchronous or metachronous) or
intrahepatic spread may be difficult to establish. A well-differentiated tumour, or presence
at its periphery of well-differentiated areas or even areas suggestive of origin from a
dysplastic nodule (e.g. nodule-in-nodule pattern) supports a multicentric origin in early
stage tumours [15]. The infiltrative pattern is usually observed in noncirrhotic livers and
consists of a large mass which occupies a good proportion of a lobe or more than one
lobe. The term massive also applies to tumours of this size. There is often involvement of
large portal vein branches. HCC in a diffuse pattern is rare, usually observed in cirrhotic
livers and consists of multiple nodules which may mimic the cirrhotic nodules, may not be
visible on imaging and difficult to identify macroscopically. The term cirrhotomimetic is
often used to describe this pattern [62].
Microscopically, HCC is defined by its resemblance to normal hepatocytes, according
broadly to the traditional Edmondson and Steiner criteria [63] . HCC, however, can
Hepatocellular carcinoma 97

be very heterogeneous and characterised by areas with different growth patterns and
degree of differentiation. Tumour cells are often arranged in a trabecular, pseudoacinar
or solid pattern. Well-differentiated HCC resembles normal hepatocytes with a similar
nuclearcytoplasmic ratio, similar nucleolated nuclei, well-demarcated cell borders
and eosinophilic cytoplasm. The cytoplasm may show steatosis or clarification, and
sometimes accumulation of MalloryDenk bodies, or eosinophilic globular inclusions.
Formation of canalicular bile plugs is a diagnostic feature of HCC, although nonlesional
cholestatic hepatocellular rosettes may become entrapped within the tumour and
mistaken for a tumour component. Staining for CEA using a polyclonal antibody, or
CD10, is the most commonly used method to demonstrate canaliculus formation, but
its expression is not hepatocellular specific; poorly differentiated tumours may not form
canaliculi, and distinction between a canalicular and membranous pattern is based on
subjective interpretation and may not be straightforward. In this respect, the usage of
more hepatocellular-specific canalicular markers appears promising. Two recent studies
have shown excellent specificity of BSEP expression [64,65] in distinguishing between
HCC and its extrahepatic mimics. Outside canalicular expression, arginase-1 [66] has been
noted to have superior sensitivity and specificity than Hep-Par-1 (hepatocyte paraffin
1). Cytokeratins panels (8/18, 19, 7 and 20) are of limited use due to frequent aberrant
HCC expression and considerable overlap with extrahepatic tumours. Alpha-fetoprotein
immunohistochemical detection is often focal and its sensitivity is too low. Glypican-3
may complement Hep-Par-1 in the diagnosis of poorly differentiated HCC [67], but it is
expressed in non-neoplastic liver parenchyma [68] and extrahepatic tumours [67], and
should not be used in isolation. The histological diagnosis of poorly differentiated HCC may
depend ultimately on the identification of better-differentiated areas in resected tumours.
Vascular invasion remains an important prognostic factor. Expression of stem cell markers,
biliary cytokeratins and a gene expression profile similar to hepatoblast identifies a subtype
of HCC with poor prognosis [69]. HCC can grow inside large bile ducts [70].
Fibrolamellar carcinoma is a well-known variant of HCC. It affects usually young adults
and is not associated with underlying chronic liver disease. It is characterised by fibrous
stroma and hepatoid tumour cells of large size, with eosinophilic cytoplasm, cytoplasmic
inclusions and scanty mitotic activity. A recent series has shown that fibrolamellar
carcinoma does not have a better survival than conventional HCC in children [71]. Other
variants include lymphocyte-rich, clear cell, scirrhous and sclerosing HCC. The term
mixed or combined hepatocholangiocellular carcinoma refers to tumours with mixed
hepatocholangiocellular phenotype, or tumours with progenitor cell features.
The differential diagnosis depends on the clinical context. In cirrhotic patients, most
nodular lesions are hepatocellular, but there are exceptions. Caturelli et al [39] showed that
a small proportion of small nodules identified by imaging in cirrhotic patients and suspect
for dysplastic nodules or HCC, were of other nature (e.g. lymphomas, haemangiomas).
Of note cholangiocarcinoma has been described in non-biliary cirrhosis [72]. The issues
regarding the diagnosis of dysplastic nodules and early HCC are described above.
In non-cirrhotic liver the differential diagnosis is usually with other primary epithelial
or non-epithelial tumours (focal nodular hyperplasia, hepatocellular adenoma,
cholangiocarcinoma, angiomyolipoma) or with metastases. FNH is usually the easiest
to differentiate, due to its architecture and bland appearance of lesional hepatocytes. In
contrast, the differential diagnosis with HCA can be challenging for the following reasons:
1) HCAs are considered to be at risk of malignant transformation, particularly those
98 Pathology of regenerative and neoplastic hepatocellular nodules

showing aberrant nuclear/cytoplasmic b-catenin expression, diffuse glutamine synthetase

expression and/or mutation of the b-catenin gene [55]. This risk has been extrapolated
from: a) the observation that some HCCs have well differentiated areas resembling HCA; b)
the description of cases in which b-catenin mutated HCA co-existed with frank HCC in the
same liver; c) the occurrence of HCC in patients with glycogen-storage disease, a condition
characteristically associated with HCA. It remains to be elucidated, however, whether
these observations are evidence of malignant transformation of HCA, or indicate separate
biological entities with a more aggressive behaviour from the start. 2) The histological
appearance of HCA and well-differentiated HCC can overlap considerably. Of note, well-
differentiated HCC can appear very bland, and HCA secondary to hormonal stimulation
can show marked atypia, but regresses following hormonal withdrawal. To complicate the
issue further, there are reports in the literature of complete regression of lesions diagnosed
histologically as overt HCC, following interruption of the hormonal stimulus [61]. 3) There
are no individual diagnostic histological features or immunohistochemical markers that
can be used reliably and in isolation to distinguish between HCA and well-differentiated
HCC. 4) HCC can be very heterogenous, and sampling error needs always to be considered
when examining core needle biopsies, as absence of frank features of malignancy does not
rule out HCC.
Intrahepatic (peripheral) cholangiocarcinoma, is often characterised by the presence of
areas composed of cords or sheets of large cells with eosinophilic cytoplasm and an overall
hepatoid appearance. Lack of bile, no demonstrable canaliculus formation or Hep-Par 1
expression by immunohistochemistry, in the presence of mucus production, cytoplasmic
staining for CEA, CA19-9 and biliary cytokeratins favour cholangiocarcinoma. The presence
of adenocarcinoma with a typical tubulo-glandular pattern, in a core needle biopsy
from a lesion clinically suspected as HCC, raises the possibility of a mixed (combined)
hepatocellular-cholangiocarcinoma. Cholangiocarcinoma can occur in non-biliary
cirrhosis, and simulate clinically HCC [72].
Angiomyolipoma can mimic histologically hepatocellular carcinoma. The diagnosis
is usually based on positive immunohistochemistry for markers such as HMB 45. Many
extrahepatic primary tumours infiltrating the liver can simulate hepatocellular carcinoma
histologically and in particular adrenal cortical carcinoma, neuroendocrine tumours, renal
cell carcinoma, melanoma and hepatoid adenocarcinoma (e.g. gastric origin). Clinical
evidence of an extrahepatic primary, and immunohistochemistry are often sufficient
for the diagnosis, but in some cases, the extrahepatic primary is too small to be detected
clinically (e.g. neuroendocrine tumours), or the tumour is poorly differentiated and does
not fit with any specific immunohistochemical profile. Poorly differentatiated tumours
of intestinal origin, and germ cell tumours may be associated with high serum levels of
alpha-fetoprotein. Immunohistochemical algorithms are provided in various textbooks
[1,13,14,73] to which the reader is referred.

Future directions
The classification of HCA based on molecular, clinical and histological correlation [18]
has shown how the approach to the diagnosis of hepatocellular tumour is changing.
Recent studies using high-throughput molecular techniques have classified HCC into
subcategories associated with specific aetiologies and specific molecular pathways. A
recent meta-analysis of gene expression profiles worldwide identified three robust HCC
Future directions 99

subclasses with strong clinicopathological correlates highlighting the potential role of

molecular taxonomy [74]. The application of genomic profiling to the investigation of HCC
associated with rare conditions such as BSEP deficiency may lead to the identification
of alternative carcinogenic pathways [75]. Proteomics profiling of liver tumours appears
promising in identifying biomarkers, which can be exploited at diagnostic histological
level or clinically for noninvasive diagnosis or to direct treatment [76]. Like in other
areas of medicine, proper histological interpretation should always be part of the clinical
diagnostic and therapeutic process, the design and targeting of molecular techniques (e.g.
laser microdissection), the validation of markers derived from molecular studies and the
classification of disorders in general.

Key points for clinical practice

Primary epithelial liver tumours are subdivided into hepatocellular and biliary, based on
their resemblance to their normal epithelial counterparts. Their pathogenesis however is
complex as it involves mature as well as progenitor cells, which explains the occurrence of
mixed phenotypes.
A full clinical history, close correlation with imaging findings and ideally an adequate sample
of lesional tissue along with a separate sample of non-lesional tissue to assess the status of
background liver should be the basis for the histological interpretation of liver nodules.
Regenerative nodules can occur in response to necrosis, altered vascular supply or other
stimuli. Their changes can be subtle, easily overlooked in biopsy specimens. FNH can be
indistinguishable from cirrhosis.
Dysplastic nodules are thought to be HCC precursors in the context of chronic liver disease.
Progression to HCC occurs through a continuum including high-grade dysplastic nodules,
well-differentiated (early) HCC, vaguely nodular with indistinct margins and moderately
differentiated (progressed) distinctly nodular HCC.
The diagnosis of HCC in cirrhosis is now clinical in most cases; the role of histology in the
diagnosis of dysplastic nodule and early HCC depends on local clinical practice.
Small cell change and large cell change are part of a constellation of histological changes
which may indicate an increased risk of HCC.
HCAs are now subclassified into four categories [steatotic/HNF1a mutated, -catenin
mutated, inflammatory (telangiectatic), unclassified]. Immunohistochemistry is essential.
Features of HCA may be overlooked in biopsy specimens. The inflammatory/telangiectatic
variant may need to be differentiated from FNH. The distinction between HCA and HCC may
be challenging.
Cirrhosis is the main risk factor for HCC, but HCC can arise in noncirrhotic livers. This may
occur in the context of the metabolic syndrome, or other factors (iron overload, past exposure
to HBV, androgens), and occasionally without any signs of liver disease.

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Chapter 8

Serrated lesions of
colon and rectum
Ashraf EK Ibrahim, Mark J Arends

The adenoma is the most frequent precursor of colorectal cancer (CRC) and it is high-risk
adenomas of large size, with villous architecture and high-grade dysplasia that are more
likely to progress to invasive adenocarcinoma. This conventional adenomacarcinoma
sequence is the most common pathway of colorectal neoplastic progression, accounting
for around 7080% of CRC development and it is associated with accumulation of
mutations, DNA deletions/losses, DNA gains and amplifications, with aneuploidy due to
chromosomal instability (CIN), as well as other genetic and epigenetic changes, in a wide
range of cancer-related genes [13]. Molecular genetic analyses have identified many, but
not all, of these molecular changes, including the most frequent genetic and epigenetic
alterations, such as those affecting APC (~80%), KRAS (~40%), TP53 (5060%), SMAD4
(4060%) and others [1,4,5].
In addition to the conventional adenomacarcinoma pathway with underlying
CIN as the main type of genomic instability, there are other pathways of neoplastic
progression (Figure 8.1), such as lesions displaying microsatellite instability (MSI) or
aberrant methylation of CpG islands within gene promoters the CpG island methylator
phenotype (CIMP) [6]. The morphologically serrated pathways represent alternative
routes of colorectal neoplastic progression. The right-sided sessile serrated neoplasia
pathway involves formation of hyperplastic polyps, sessile serrated lesions (SSLs) and
serrated adenomas (of usual type), which may progress to serrated adenocarcinoma,
whereas the left-sided traditional serrated neoplasia pathway includes hyperplastic
polyps and traditional serrated adenomas (TSAs) which may evolve into invasive serrated
adenocarcinomas. The serrated pathway lesions are also characterised by particular genetic
and molecular characteristics, including MSI and CIMP in some, although these are not
as fully characterised at the molecular level as those found in the conventional adenoma
carcinoma pathway (Table 8.1).

Ashraf EK Ibrahim, MBChB (Hons), MSc, PhD, FRCS, FRCPath, University of Cambridge, Department of Pathology,
Addenbrookes Hospital, UK
Email: aeki2@cam.ac.uk (for correspondence)
Mark J Arends, MBChB (Hons), BSc (Hons), PhD, FRCPath, MA, University of Edinburgh, Department of Pathology,
Institute of Genetics & Molecular Medicine, Western General Hospital Campus, Edinburgh, UK
104 Serrated lesions of colon and rectum

Table 8.1 WHO classification [7] and their commonly used synonyms in histopathological
interpretation of serrated polyps and serrated polyposis
WHO classification Synonyms commonly used in histopathological
Microvesicular hyperplastic polyp (MVHP) Hyperplastic polyp
Goblet-cell-rich hyperplastic polyp (GCHP)
Mucin-poor hyperplastic polyp (MPHP)
Sessile serrated adenoma/polyp Sessile serrated lesion (SSL)
Sessile serrated adenoma/polyp with cytological atypia Mixed hyperplastic/adenomatous polyp
Mixed SSL/serrated adenoma (usual type)
Traditional serrated adenoma Traditional serrated adenoma
Serrated polyposis syndrome Hyperplastic polyposis syndrome
Serrated polyposis syndrome

Benign serrated polyps

Hyperplastic polyps
Hyperplastic polyps, formerly called metaplastic polyps, are more common on the left
than the right side of the large bowel, and more common in males than females. They
form small (often <10mm) sessile polyps on the mucosal surface of the large bowel
and are composed of elongated or hyperplastic crypts that have a sawtooth or serrated
architecture. This serrated architecture usually extends down from the surface to involve
half to two-thirds of the crypt, with a relatively straight part of the lower crypt. They often
show in-folded epithelial tufts, with microvesicular cells, and sometimes have infrequent
enlarged goblet cells in the upper zone of the crypts (Figure 8.2). In some, there may be
prominent endocrine cells or there may be a thickened subepithelial collagen plate. Some
have suggested subclassifying hyperplastic polyps into microvesicular types, goblet cell
rich types and mucin-poor types (WHO classification, Table 8.1), and that microvesicular
hyperplastic polyps are more common on the right side of the bowel with goblet cell rich
hyperplastic polyps more common on the left side [8], but this subclassification is not in
common use. Importantly, there is no genuine nuclear dysplasia only some minor nuclear
enlargement with reactive changes (Figure 8.2).
Hyperplastic polyps increase in frequency with age and are 17 times more common
in colons bearing carcinomas. They are associated with similar dietary and lifestyle risk
factors to those for CRC. Molecular analysis has shown that KRAS mutations are common
in hyperplastic polyps and that they are clonal, starting as monocryptal lesions [9].

Hyperplastic polyposis syndrome

The concept of a syndrome of hyperplastic polyposis was put forward following
the recognition of patterns of familial clustering of multiple and sometimes large,
hyperplastic polyps, often in a predominantly proximal location in the bowel. However,
the causative genetic mutation(s) is/are not currently known. This syndrome is usually
asymptomatic and is associated with an increased risk (approximately 25%) of CRC
formation with MSI [10,11].
Benign serrated polyps 105

Figure 8.1 Pathways of

The serrated neoplastic pathways development of serrated
neoplasms of the large bowel.
The left-sided traditional serrated
neoplasia pathway involves distal
Left colon Right colon hyperplastic polyps; sometimes
Traditional serrated Sessile serrated evolving to traditional serrated
neoplasia pathway neoplasia pathway adenomas (TSAs), some of
which may progress to serrated
KRAS mutation BRAF mutation adenocarcinoma. KRAS mutations
occur early and are found in
approximately 80% of TSAs.
The transition to malignancy is
Hyperplastic Proximal associated with low-frequency
polyp hyperplastic polyp CpG island methylator
phenotype - low (CIMP-L), but
with no or only partial MLH1
promoter hypermethylation with
Traditional Sessile no high-frequency microsatellite
serrated adenoma serrated lesion instability (MSI-H). The CIMP-L
status is associated with silencing
of other tumour suppressor
MLH1 hypermethylation
genes. The right-sided sessile
serrated neoplasia pathway
Silencing of tumour starts with proximal hyperplastic
suppressor genes Serrated adenoma polyps, which may progress to
MSS (CIMP-L) sessile serrated lesions, serrated
adenomas (usual type) and
serrated adenocarcinomas.
MSS / CIMP positive, MSI / CIMP positive, BRAF mutations occur early
non-HNPCC, serrated non-HNPCC, serrated and are found in 7582%
adenocarcinoma adenocarcinoma serrated adenomas (usual type).
Serrated adenoma formation or
progression is associated with
hypermethylation of the MLH1
promoter, as part of CIMP-H, causing MSI-H and a markedly increased mutation rate that promotes progression to
invasive serrated adenocarcinoma. The CIMP-H status may also contribute to progression via silencing of cancer-
related genes.

Figure 8.2 Hyperplastic polyp.

The crypts are elongated with a
sawtooth or serrated architecture
seen in the upper two thirds
and a more straight architecture
in the lower third of the crypts
(magnification x100).
106 Serrated lesions of colon and rectum

The current definition of hyperplastic polyposis syndrome (HPS), also called

serrated polyposis syndrome [7,8], includes the findings of either: (1) >5 hyperplastic
polyps proximal to the sigmoid colon, of which two are >10mm in diameter; or (2) >30
hyperplastic polyps of any size, proximal to the sigmoid colon; or (3) any number of
hyperplastic polyps with a first-degree relative with known HPS.
In HPS, the hyperplastic polyps may sometimes be large, villous, show a complex
architecture with polyp branching, may sometimes resemble atypical juvenile polyps, as
well as demonstrating the usual small, sessile hyperplastic polyps. In addition, there may be
some serrated adenomas, some conventional (non-serrated) adenomas, and some mixed
adenoma/hyperplastic polyps, as well as CRC in approximately 25% of cases at diagnosis
[12]. The syndrome is most likely to be diagnosed in patients in their 40s to 60s, although
it has been found in children as young as 11 years of age. It has recently been suggested
that the UK Bowel Cancer Screening Programme is more likely to detect this syndrome.
Other syndromic forms of polyposis should be excluded, including familial adenomatous
polyposis (FAP) and its attenuated variant (AFAP), MUTYH-associated polyposis, Peutz
Jeghers syndrome, juvenile polyposis syndrome and Cowdens syndrome.

Mixed polyps
Mixed polyps contain a mixture of foci of hyperplastic polyp and foci of dysplastic adenoma,
either conventional adenoma or serrated adenoma (Figure 8.3). They were originally thought
to represent collision lesions between a hyperplastic polyp and a nearby adenoma. However,
currently they are regarded as displaying the development of a focus of dysplasia within a pre-
existing hyperplastic polyp [13]. As such, they represent links between hyperplastic polyps
and either serrated adenomas, of usual type, or TSAs in the serrated neoplasia pathways.

Sessile serrated lesions

SSLs, also called sessile serrated polyps (SSP), have been the subject of considerably
confused nomenclature, sometimes with heated disagreements between experts. They
were previously called sessile serrated adenomas, despite the lack of genuine conventional
dysplasia, and are still known by this term in the USA and some European countries.

Figure 8.3 Mixed hyperplastic

polyp/adenoma. The lower left part
is composed of typical hyperplastic
polyp with serrated architecture but
no dysplasia, whereas the adjacent
upper right part shows the features
of a conventional tubulovillous
adenoma with low-grade dysplasia
(magnification x25).
Benign serrated polyps 107

However, following the publication of European guidelines in 2011 [14], there is now
agreement in the UK and participating European countries to use the term Sessile
Serrated Lesion as the preferred term for this lesion. SSLs have some similarities to larger
hyperplastic polyps in that they show a hyperplastic or serrated polyp-like appearance
with some unusual architectural features, of which the key feature is the presence of
horizontal orientation of the deep part of the crypts (just above the muscularis mucosae),
often forming L-shapes (or boot shapes), or inverted T-shapes, or anchor shapes (Figures
8.48.6). However, in hyperplastic polyps the pattern of serration extends around half
way to two-thirds of the way down the crypts (Figure 8.2), in SSLs the pattern of serration
extends down to or nearly to the base of the crypts (Figures 8.58.6). Another key feature
is the lack of conventional or genuine nuclear dysplasia, although the lesional cells may
have some mild nuclear enlargement and nuclear atypia that does not amount to true
dysplasia. Some of the crypts may also show dilatation, sometimes with the most dilated
segment near to the crypt base (Figures 8.48.6). SSLs are more frequent in the right colon

Figure 8.4 Sessile serrated lesion

(SSL). This has elongated crypts
with serrated architecture, but the
basal parts of two crypts show
a horizontal growth pattern just
above the muscularis mucosae.
One horizontal crypt runs straight
along the muscularis mucosae,
whereas the other horizontal crypt
displays a more complex S-shape
(magnification x100).

Figure 8.5 Sessile serrated lesion.

There are elongated crypts with
the serrated architecture extending
down to the basal aspect of the
crypts. The basal part of one crypt
shows a horizontal growth pattern
displaying an anchor-like shape
just above the muscularis mucosae
(magnification x100).
108 Serrated lesions of colon and rectum

Figure 8.6 Sessile serrated lesion

(SSL). This SSL image shows the
basal parts of two crypts with
horizontal growth patterns just
above the muscularis mucosae.
One horizontal crypt displays an
extended horizontal run with
branching, whereas the other
horizontal crypt displays a boot
shape (magnification x100).

and more common in females than males. Sometimes they may be large, sessile, yellow in
colour endoscopically or poorly defined [15].
SSLs may show evidence of abnormal proliferation on MIB-1/Ki-67 immunostaining,
but have a normal subepithelial collagen plate. In some SSLs, there may be evidence of
loss of MLH1 expression by immunohistochemistry. They may express MUC5AC or MUC6
by immunohistochemistry. SSLs may sometimes be found in HPS [16]. The distribution of
SSLs in the large bowel shows a right-sided predominance with involvement of the caecum
and ascending colon in 51% of SSL cases, transverse colon in 16%, descending colon in
11%, sigmoid colon in 12% and rectum in 10% [17].
SSLs may sometimes have foci of dysplasia, representing links in the sessile serrated
neoplasia pathway from SSL to serrated adenoma of usual type. In the WHO (2010)
classification [7] SSPs with cytological atypia replaced the designation mixed polyps and
may show either low-grade or high-grade dysplasia (Table 8.1). There is some uncertainty
about the frequency of SSLs relative to other colorectal polyps and this reaches 7% in some
series, although observer reproducibility is an issue, due to lack of familiarity with the
diagnostic criteria, as well as the nomenclatural confusion leading to mislabelling of SSLs
as adenomas [15,16].

Traditional serrated adenomas

TSAs tend to occur in older adults particularly females and are the least common of
the serrated polyps comprising <6% of serrated polyps and <1% of all colorectal polyps
[13,18,19]. Macroscopically, these lesions are pedunculated and are almost exclusively
limited to the left side of the colon and in the rectum. Microscopically, these lesions
usually show prominent serration and diffuse low-grade dysplasia with approximately 10%
showing high-grade dysplasia [20]. The dysplastic epithelium shows luminal infoldings
aligned perpendicular to the main axis of the crypt, termed ectopic crypt formation or short
ectopic budding crypts [19]. Cellular features include prominent eosinophilic cytoplasm
with no maturation towards the surface, stratification and nuclear hyperchromasia with
elongated nuclei (pencil-like nuclei) (Figure 8.7). An uncommon subtype of the TSA is the
Pathways of pathogenesis 109

Figure 8.7 Traditional serrated

adenoma (TSA). This shows
the typical appearances of
dysplastic epithelium with luminal
infoldings aligned perpendicular
to the main axis of the crypt, in
ectopic crypt formations or short
ectopic budding crypts. There is
prominent eosinophilic cytoplasm,
elongated (pencil-like) nuclei with
hyperchromasia and low-grade
dysplasia (magnification x100).

filiform TSA that shows multiple and prominent thin, elongated projections of TSA-like
dysplastic epithelium with serrated contours [21].

Serrated adenocarcinoma
Serrated adenocarcinomas have been reported to account for approximately 7.5% of all
CRCs, but 17.5% of right-sided colonic adenocarcinomas, although in our experience
and practice the proportions are lower than those reported in the literature. Serrated
adenocarcinomas represent the end-points of the serrated pathways: the majority of
left-sided serrated colorectal carcinomas are microsatellite stable (MSS) or show only
low-frequency MSI (MSI-L) and arise from TSAs [22,23], whereas right-sided serrated
adenocarcinomas appear to arise from SSLs/serrated adenomas (Figure 8.8) and show
a high-frequency MSI (MSI-H) with 30% microsatellite markers displaying changes in
length [22, 2426]. Serrated adenocarcinomas are usually considered to be more prevalent
within the right colon, although in one series over 50% were observed in the distal colon
[22]. Morphologically, serrated carcinomas show serrated architecture often in irregularly
elongated crypt-like structures, but little tubular or papillary growth pattern, with abundant
eosinophilic cytoplasm and are commonly associated with a mucinous component [22]
(Figures 8.9 and 8.10).

Pathways of pathogenesis
Serrated colorectal neoplasia pathways
Overall, it has been estimated that around 2030% CRCs arise via the serrated neoplasia
pathways. The pathways of development of serrated neoplasms can be divided into
two distinct pathways, one that mostly occurs on the right side of the large bowel and
results in sessile serrated neoplastic lesions and the other on the left side or distal bowel
and generates TSAs and associated carcinomas [27,28] (Figure 8.1). In the right-sided
sessile serrated neoplasia pathway, the initial lesion is a hyperplastic polyp (often of
110 Serrated lesions of colon and rectum

Figure 8.8 Serrated adenocarcinoma

ex-serrated adenoma. On the left,
there is a serrated adenoma of usual
type (elsewhere there was a sessile
serrated lesion adjacent to this),
with some low-grade dysplasia on
the left and high-grade dysplasia
centrally. On the right, appearing to
arise from the high-grade dysplastic
epithelium there is an early invasive
adenocarcinoma, just invading
through the muscularis mucosae
with a mucinous component in the
submucosa. This case illustrates the
progression from sessile serrated
lesion (SSL) to serrated adenoma
to serrated adenocarcinoma with a
mucinous component (magnification

Figure 8.9Serrated
adenocarcinoma. This serrated
adenocarcinoma shows invasion
of the muscularis propria by
malignant epithelium forming
markedly elongated, serrated crypt-
like structures, along with some
foci of mucinous differentiation
(magnification x25).

Figure 8.10Serrated
adenocarcinoma. There is invasion
by malignant epithelium forming
irregularly branching, elongated,
serrated crypt-like structures. At the
upper right of the picture, there
is evidence of extracellular mucin
secretion indicative of a mucinous
component (magnification x50).
Molecular abnormalities 111

microvesicular type), which may expand and develop into a SSL. Within this, there may
evolve a focus of dysplasia forming a serrated adenoma (of usual type) and a subset of
these may progress to a proximal serrated carcinoma (50% of which are predominantly
mucinous) and these usually show MSI-H, with silencing of MLH1 expression by promoter
hypermethylation as a part of the CIMP-H phenotype (see below), occurring in the
sporadic or hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome (LS)
setting [28,29].
The left-sided traditional serrated neoplasia pathway starts with distal hyperplastic
polyps, which may progress in some cases to formation of TSAs, some of which may evolve
into distal serrated carcinomas that are MSS or MSI-L with a low-frequency CpG island
methylation phenotype low (CIMP-L), without methylation of the MLH1 promoter, found
in the sporadic or non-HNPCC/LS context [28,29].

Molecular abnormalities
Some of the most frequent molecular alterations in colorectal tumourigenesis include
those affecting APC which is mutated and sometimes deleted in around 80% conventional
(non-serrated) adenomas and carcinomas [30]. This occurs as a very early event in
association with normal epithelium changing to monocryptal or oligocryptal adenomas, as
seen in FAP coli, an inherited syndrome of bowel adenoma and carcinoma susceptibility
due to germline APC mutation [31]. KRAS mutations are found in around 40% colorectal
adenomas and carcinomas, and they occur mostly during the early stages of adenoma
growth and can synergise either with APC mutations or defective mismatch repair (MMR)
to accelerate colorectal tumour formation [32,33]. In contrast, TP53 mutations are observed
in approximately 5060% cancers, often occurring as a late event around the time of
transition from conventional adenoma to carcinoma [34,35]. Many other genetic and
epigenetic changes can occur during this adenoma-carcinoma sequence, including MMR
abnormalities, usually due to acquired MLH1 promoter hypermethylation in 15% sporadic
CRC, as well as in 24% CRC due to inherited mutations in MMR genes in HNPCC/LS, in
addition there is loss of a large part of chromosome 18, containing the SMAD4 and SMAD2
genes, occurring in around 60% CRC, as well as low- or very low-frequency mutations to
a wide range of cancer-associated genes [3638]. Other specific changes to SSLs and TSAs
have been described such as loss of expression and promoter methylation of SLIT2, which
contributes to malignant transformation by E-cadherin degradation [39].

Lynch syndrome and DNA mismatch repair

LS, also known as HNPCC syndrome, is a syndrome of inherited susceptibility to cancers of
the large bowel as well as at a range of other sites including endometrium, ovary, stomach,
small intestine, ureter, renal pelvis, skin (sebaceous tumours) and brain (gliomas) [40].
Carcinogenesis involves the acquisition of defective DNA MMR in tumour cells. The
syndrome is due to dominant inheritance of a mutant MMR gene (mostly either MLH1 or
MSH2), with all somatic cells possessing one mutated and one wild-type MMR allele. During
tumour formation there is inactivation of the second MMR allele, such that the tumour
cell has both MMR alleles inactivated and this can be detected by immunohistochemical
demonstration of abnormal expression or loss of expression of MMR proteins only in the
tumour cells, but not in adjacent stromal or lymphoid cells: loss of MLH1 protein expression
is usually accompanied by loss of its heterodimeric binding partner PMS2, whereas loss
of MSH2 protein expression is usually accompanied by loss of its heterodimeric binding
112 Serrated lesions of colon and rectum

partner MSH6 [41,42]. This leads to defective MMR which appears to be selected due to a
reduction in susceptibility to DNA-damage-induced apoptosis [43,44].
MSH2 and MLH1 are the two most frequently mutated genes in LS, accounting for
4045% LS families each, and minor ones being MSH6 and PMS2 (<10% LS families
each), with rare LS families having other affected genes [45]. The resulting failure to
repair DNA replication-associated errors in these tumours produces a high frequency of
mutations, either as single base mismatches or in regions of short tandem DNA repeats
(the repeat units often being 1-4bp in length), known as microsatellites, such as repetitive
polynucleotide tracts often of the form (A)n or (CA)n. Thus, DNA extracted from such
tumours shows variation in length (both shorter and longer) of a significant proportion of
microsatellites, a phenomenon known as MSI [45].
MSI-H with 30% abnormal microsatellite markers is the hallmark of cancers in LS and
is also observed in 15% of sporadic CRCs, due to somatically acquired MLH1 promoter
hypermethylation, resulting in transcriptional silencing of both copies of MLH1 [11]. The
majority of sporadic CRC exhibiting MSI-H develops in the right side of the large bowel
(caecum, ascending colon and transverse colon) [46]. Deficiency in MMR produces an
increase in the mutation rate (100x 1000x the normal mutation rate), sometimes called
a mutator phenotype, which leads to mutations accumulating in genes that play key
roles in the regulation of growth of normal colonic epithelia and tumour development,
and these are selected during carcinogenesis as part of a progressive deregulation of
growth control [3].

CpG island methylator phenotype and serrated neoplasia

The type of sporadic DNA hypermethylation leading to MLH1 silencing usually occurs
predominantly within a distinct subset of CRC and adenomas designated as showing CpG
island methylator phenotype positivity (CIMP+), in which there is evidence of increased
methylation of the promoters of a range of susceptible genes (e.g. MLH1, p16, MGMT, IGF2,
RUNX3, SOCS1, and NEUROG1) and these CRCs are usually right-sided with an older average
age [6,4648]. CIMP status has been defined using MethyLight quantitative DNA methylation
technology as >10% methylation of 2 out of a panel of 5 gene promoters (CACNA1G, IGF2,
NEUROG1, RUNX3 and SOCS1) and using this definition 18% CRCs can be characterised as
CIMP+ [47]. It has been proposed that CRCs can be subclassified according to whether they
show CIMP-H or low-moderate levels of promoter methylation phenotype - low (CIMP-L) [47].
In the right-sided sessile serrated neoplasia pathway, there is the development of
MSI-H, often around the time of serrated adenoma formation, as a result of silencing of
expression of the MLH1 gene due to promoter hypermethylation, usually as part of CIMP-H
tumour status. These lesions also have a high frequency of BRAF mutations (~7080%).
There is almost always a specific mutation (valine to glutamate) in BRAF at codon 600
(V600E), although other very much less common V600 mutations to other amino acids are
sometimes found, and these V600 BRAF mutations can be identified in a high proportion
(~7080%) of sporadic MSI-H CRCs, but rarely in CRCs due to LS [47].
A study of the prevalence of BRAF mutations in a range of colorectal lesions indicates the
prevalence as conventional (non-serrated) adenomas 0%, typical hyperplastic polyps 19%,
serrated adenomas (usual type) 7582%, TSAs 2030%, mixed polyps 5789%, HNPCC/LS
cancers 0%, all CRCs 15%, MSI-H non-HNPCC/LS cancers 7080% [48].
In the left-sided traditional serrated neoplasia pathway, the TSAs and serrated
carcinomas are usually MSS or show MSI-L, with retention of normal MLH1 expression, with
Clinical implications and predictive factors 113

partial or low-level patterns of DNA promoter methylation (CIMP-L), no BRAF mutations,

but KRAS mutations are found in approximately 80% of TSAs [28,49,50] (Figure 8.1).

The role of immunohistochemistry in the diagnosis of serrated

Ki-67 immunostaining patterns can be helpful in distinguishing HPs, SSLs and TSAs [19].
Other studies have shown differences in the proliferative activity as assessed by Ki-67
immunostaining between the lower, middle and upper thirds of the crypts in these lesions
and adenomas. In HPs, the Ki-67 staining is predominantly limited to the lower third or so
of the crypts. SSLs showed a similar pattern to HPs but with only slightly increased Ki-67
staining within the middle third of the crypts. In contrast, TSAs have more uniform Ki-67
staining throughout the length of the crypts. Similarly, adenomas have more uniform Ki-67
staining, but with an even more widespread labelling towards the upper third of the crypts
[5153]. Other immunohistochemical markers such as cytokeratin 20, MGMT, MLH1,
CDX2, b-catenin and P53 were found not to be helpful in subtyping serrated lesions [19,21].
Most of the studies that examined the use of immunohistochemistry in serrated lesions
were limited to a small number of cases and therefore more work is required to validate
these findings and extend the analysis to other immunostaining markers (such as SLIT2).

Clinical implications and predictive factors

Definitive data on the potential malignant transformation of serrated polyps are
lacking. However, the clinical and molecular data so far suggest that serrated lesions
are biomarkers of future risk of malignancy, as well as having the potential to be part
of serrated lesion-serrated adenomacarcinoma pathways of progression, with the
exception of diminutive distal hyperplastic polyps (unless part of hyperplastic/serrated
polyposis syndrome) [27,5456]. The recently published guidelines issued for the
recommended surveillance intervals after endoscopic resection of serrated polyps are
shown in Table 8.2 [7,57,58].

Table 8.2 American Gastroenterological Association guidelines for endoscopic surveillance

intervals after endoscopic resection of any number of serrated polyps, based on type and
size (not including serrated lesion cases with a diagnosis of hyperplastic/serrated polyposis
syndrome, for which 1 yearly surveillance is recommended)
Serrated lesion Lesion size Recommended surveillance
interval (years)
Hyperplastic polyp <10mm 10
Sessile serrated lesion (SSL) <10mm 5
SSL >10mm 3
Mixed hyperplastic/adenomatous polyp Any size 1
Mixed SSL/serrated adenoma
Traditional serrated adenoma Any size 1
Hyperplastic/serrated polyposis syndrome Any size 1
114 Serrated lesions of colon and rectum

Key points for clinical practice

Serrated lesions of the large bowel include hyperplastic polyps, mixed polyps, SSLs, serrated
adenomas of two types and serrated adenocarcinomas.
The diagnostic accuracy of serrated lesions is important as the different types carry different
levels of risk and require different endoscopic surveillance intervals.
The diagnosis is largely dependent on morphological assessment with limited utility of
immunohistochemical and molecular tests in refining the diagnosis.
There has been nomenclatural confusion regarding SSLs precluding standardisation of
diagnosis and hindering large-scale studies.
The diagnosis of serrated/HPS is important for other family members and should always be
considered when there is history or endoscopic evidence of multiple serrated lesions.
There are two main molecular pathways: the mostly right-sided, sessile serrated neoplasia
pathway and the mostly left-sided, traditional serrated neoplasia pathway.
The sessile serrated neoplasia pathway is associated with acquired MMR deficiency and MSI,
due to MLH1 promoter hypermethylation
The traditional serrated neoplasia pathway produces MSS neoplasms with high-frequency
KRAS mutations.
Both pathways generate serrated adenocarcinomas, often with a mucinous component.

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Chapter 9
An update on the pathology of
chronic inflammatory
bowel disease
Roger M Feakins, Neil A Shepherd

Chronic inflammatory bowel disease (CIBD), in the context of this chapter at least,
represents the range of pathology with classical chronic ulcerative colitis (UC) at one end
and classical Crohns disease at the other. Pathologically, there is very much a spectrum
and there may well be equivocal pathology. In biopsy material, the latter is now termed
inflammatory bowel disease unclassified (IBDU) [1], whereas the term indeterminate
colitis is reserved for equivocal cases of CIBD in resection material [2]. The latter remains
a controversial diagnosis. Many do not like the term but it can be a useful category, when
strictly restricted to resection material, because there is good evidence that the natural
history is like that of UC in the majority of cases. Thus, whilst one can expect increased
complications compared with UC, particularly pelvic sepsis, when ileal pouch construction
is undertaken, there are distinctly dissimilar results for this category compared with
Crohns disease.
Here, we concentrate on diagnostic and management issues of CIBD pathology.
Histology is generally regarded as the gold standard for the diagnosis of CIBD and for the
distinction between UC and Crohns disease. However, clinicians have many other data
on which to make a clinical assessment of such cases. This includes clinical, endoscopic,
radiological and microbiological evidence. So, on the one hand, a biopsy report may simply
provide corroborative evidence for a diagnosis made from many sources of information. On
the other hand, pathologists should understand that a strongly worded pathological report
will often mean that a diagnosis of CIBD or of one of the main types of CIBD is irrevocably
appended to a patient and that this may be inappropriate. Perhaps this is most apposite to
mimics of CIBD, as the microscopic appearances in many conditions, perhaps particularly
infective, can appear very similar to those of CIBD.

Roger M Feakins, MD, FRCPI, FRCPath, Pathology and Pharmacy Building, Royal London Hospital, London, E1
Neil A Shepherd, DM, FRCPath, Gloucestershire Cellular Pathology Laboratory, Cheltenham General Hospital,
Cheltenham, UK
Email: neil.shepherd@glos.nhs.uk (for correspondence)
118 An update on the pathology of chronic inflammatory bowel disease

Accordingly, pathologists should understand that colonoscopic biopsies are often just
a small piece of the evidence that the clinician has for the diagnosis and management of
CIBD patients. Also, it is very important that pathologists avoid inappropriate terminology.
Diagnostic summaries of unqualified colitis can be distinctly misleading. Admittedly, many
gastroenterologists equate the term colitis with ulcerative colitis, but there are many other
causes. The pathologists primary role, in biopsy material, is to make a diagnosis of CIBD
and not colitis. They should then determine whether the features favour UC or Crohns
disease. If they are equivocal, a designation of IBDU is appropriate. Once again, it must be
emphasised that pathologists should be diligent to exclude the many mimics of CIBD and
thus, as ever in gastrointestinal pathology, the context of the pathological appearances is
all-important. This underpins the importance of the pathologist receiving accurate clinical,
endoscopic and imaging information to ensure that an inappropriate diagnosis of CIBD is
not made in cases where there is strong mimicry of the pathological changes of CIBD.

CIBD versus infection in biopsy material

CIBD, at first presentation, needs to be distinguished from other causes of colorectal
inflammation. Clinically, the main differential diagnosis is infective colitis, i.e. an acute
diarrhoeal illness secondary to, or presumed to be secondary to, a common enteroinfective
agent. Despite considerable recent advances in endoscopy and imaging techniques,
mucosal biopsy remains very useful for distinguishing CIBD from infective colitis when the
clinical presentation is not typical [3].
Basal plasmacytosis (with loss of the normal mucosal plasma cell gradient) is one of the
most reliable histological predictors of CIBD in biopsies. Architectural changes, including
crypt distortion, crypt atrophy and a villous mucosal surface, also strongly favour CIBD
over infective colitis. However, distinction is not always straightforward. Crypt changes are
typically diffuse in UC and focal in Crohns disease and accordingly may be absent from
biopsies of Crohns disease if the sample size is small. Also, the timing of investigation
influences the histology. For example, crypt changes are consistently absent before 2 weeks
of symptoms have elapsed and are confined to a minority of biopsies until about 4 months
have passed [47]. In contrast, there are basal plasma cells in over 50% of CIBD biopsies
in the first 2 weeks and in >80% by 30 days. Basal plasmacytosis and architectural changes
are not diagnostic of CIBD and can also occur in other settings such as diverticular colitis.
Rarely, they are seen in infective colitis.
Other histological changes may also be helpful. Granulomas, if unrelated to crypt rupture,
are more common in CIBD than infection (though they tend to be confined to Crohns
disease within the CIBD group)(vide infra). Paneth cell metaplasia is now regarded as a
marker of chronic colorectal inflammation rather than of CIBD. Certain patterns of acute
inflammation may favour CIBD, e.g. high numbers of intraepithelial neutrophil polymorphs
or deep (rather than superficial) crypt abscesses. Changes favouring infective colitis over
CIBD are less well defined and an absence of CIBD-like abnormalities is probably the most
helpful [4,6,7]. Table 9.1 summarises the discriminant features and their frequency and
reproducibility. However, the overall picture should be taken into account. Combinations
of histological features, ideally in biopsies from multiple sites, are considerably more
informative than any individual histological changes. Also, accurate interpretation is
impossible in the absence of a clinical history and comprehensive endoscopic information.
CIBD versus infection in biopsy material 119

Table 9.1 Histological features that help discriminate between IC and CIBD
Histological Reliability for Favours Frequency Frequency Reproducibility Comments
feature discriminating in IC (%) in CIBD
CIBD from IC (%)
Basal High CIBD 06 69100 Moderate/good Best predictor of
plasmacytosis CIBD
Normal in caecum.
Equivalent to loss
of the plasma cell
gradient and to
transmucosal CI
Normal High IC 85 8 Moderate
Crypt distortion High CIBD 030 28100 Variable Loss of parallelism
Caution next to
crypt abscess or
lymphoid follicle
Crypt branching High CIBD 10 79 Variable

Crypt atrophy High CIBD 015 1033 Variable Crypt shortening

and/or wide crypt
Villiform mucosal High CIBD 07 1127 Variable

Granulomas Moderate CIBD 02 627 Good Exclude crypt

Does not
between UC
and IC
Basal lymphoid Fair CIBD 07 1835 Moderate Difficult to
aggregates distinguish from
normal aggregates
Basal giant cells Fair CIBD 02 116 Moderate/good Uncommon;
limited data
Paneth cell Doubtful NA 0 415 Variable Normal in right
metaplasia colon
Probably a feature
of longstanding
Crypt abscesses None NA 4754 7590 Moderate May favour CIBD if
deep or numerous;
limited evidence
Mucin depletion None NA 70 92 Variable

IC, infective colitis; CI, chronic inflammation; CIBD, chronic inflammatory bowel disease; UC, ulcerative colitis.
120 An update on the pathology of chronic inflammatory bowel disease

Specific colorectal infections that can resemble CIBD

Chronic proctocolitis secondary to lymphogranuloma venereum and syphilis may
resemble CIBD clinically and histologically. The diagnosis is often not suspected at
endoscopy. Histological changes include chronic inflammation of the lamina propria,
fibrosis, mild acute inflammation and relatively little crypt distortion or basal plasmacytosis
[8]. Chronic or recurrent infection with Clostridium difficile is another known cause of
crypt architectural changes, although formal comparisons with CIBD histology are sparse.
Amoebic colitis can mimic CIBD and can cause crypt distortion and basal plasmacytosis,
especially if disease is prolonged (Figure 9.1). It can also complicate existing CIBD.
Tuberculosis (TB) elicits granulomas, may cause crypt distortion and is a well-
recognised mimic of Crohns disease. Caseous necrosis and demonstrable acid-fast bacilli
are virtually diagnostic of TB but are usually absent. Confluent granulomas, numerous
granulomas, Langhans giant cells, a lymphoid cuff around granulomas and granulomas
more than 400m in diameter favour TB over CIBD [911]. Another cause of intestinal
granulomatous inflammation is Yersinia infection, which is favoured over CIBD if
granulomas have central necrosis or a lymphoid cuff or if there are stellate abscesses [9,10].
Again, the clinical picture should always be taken into account.

The importance of macroscopic pathology in CIBD

As in so many branches of pathology, we would strongly emphasise the importance of
an accurate macroscopic assessment of intestinal resection specimens. For instance,
endoscopic assessment is useful for the diagnosis and differential diagnosis of CIBD so it is
only logical that macroscopic pathological assessment should similarly have discriminant
value (Figure 9.2). Indeed, there are certain features that are either pathognomonic or,
at least, highly characteristic of the two major types of CIBD and some of these are only
really assessable at the time of macroscopic pathological assessment. Also, good-quality
photographs are a very important part of the assessment of resection specimens.
In terms of the macroscopic differences in the pathology of UC and Crohns disease,
many of these are recognised and Table 9.2 identifies the characteristic macroscopic

Figure 9.1 Large bowel mucosal

biopsy from a patient with known
amoebic colitis. There is mild
crypt distortion with a few basal
plasma cells. These changes
could cause confusion with
inflammatory bowel disease (CIBD).
Accurate interpretation by the
histopathologist is facilitated by the
provision of a full clinical history
and endoscopic findings.
The importance of macroscopic pathology in CIBD 121

features of the two diseases. Above all else, UC is characteristically continuous, whereas
Crohns disease is notably patchy or discontinuous. Evidence of small bowel involvement
strongly favours Crohns disease, unless the features suggest backwash ileitis. The
thickness of the bowel wall can also be easily appreciated macroscopically.
Although UC is classically continuous, it is important to understand that there may
be discontinuous disease in UC. The caecal patch lesion is now well recognised [12] and
the appendiceal skip lesion is also a characteristic feature of more extensive UC [13].
Furthermore, treatment can have a profound effect on the distribution of disease in UC.
This is seen both macroscopically and microscopically. More powerful immunosuppressive
agents and monoclonal antibodies can produce a notable discontinuity of active disease in
UC and this may readily be apparent macroscopically.

Figure 9.2 A total colectomy

specimen with chronic
inflammatory bowel disease.
There is continuous disease from
the distal margin (at right) to the
transverse colon with a normal
right colon. This is fulminant
ulcerative colitis.

Table 9.2 Macroscopic differences in the pathology of ulcerative colitis and

Crohns disease in the large intestine [12]
Ulcerative colitis Crohns disease
Disease in continuity Disease usually discontinuous
Rectum almost always involved Rectum normal in 50%
Terminal ileum involved in 10% Terminal ileum involved in 30%
Granular and ulcerated mucosa (no fissuring) Discretely ulcerated mucosa; cobblestone appearance;
Often intensely vascular Vascularity seldom
Normal serosa (except in acute fulminating colitis) Serositis common
Muscular shortening of colon; fibrous strictures very rare Shortening due to fibrosis; fibrous strictures common
Never internal spontaneous fistulae Enterocutaneous or intestinal fistulae in 10%
Inflammatory polyposis common and extensive Inflammatory polyposis less prominent and less
Dysplasia and malignant change well recognised Malignant change less common
Anal lesions in <25%; acute fissures, excoriation and Anal lesions in 75%; anal fistulae (often multiple); anal
oedematous anal tags less common ulceration
122 An update on the pathology of chronic inflammatory bowel disease

Table 9.3 Ileal histology in chronic inflammatory bowel disease

Resection (R) Prevalence in ileum Comment Refs
or biopsy (B)
Ileal features favouring CD
Granulomas (noncryptolytic) B and R 33% inflamed CD bxs Specific (unless [1517]
0% UC resections cryptolytic).
However there are
other causes, e.g.
Giant cells B 5% inflamed CD bxs Quite specific, but [15]
Focal isolated ileal erosions with B Indicated CD in [17]
mild active inflammation and mildly one study
active caecal colitis
Patchy LP oedema with crypt disarray B Suggested CD in [17]
and mild inflammation in a small area one study
Transmural lymphoid aggregates in R 0% UC resections [16]
the absence of deep ulcers
Transmural inflammation with R Regarded as [17]
granulomas favouring CD
Submucosal inflammation R 0% UC resections Favours CD [16]
Fissure ulcers R 0% UC resections Favours CD [16, 17]
Extensive ileal disease R Favours CD [17]
Proximal active ileitis separated by R Favours CD [17]
skip regions of uninflamed distal
More inflammatory activity and R Favours CD [17]
injury in ileum than caecum
Neural hyperplasia Favours CD [17]
Ileal features favouring UC
Mild ileal mucosal injury with B Characteristic of [17]
moderate/markedly active caecal ileal UC in one
colitis study
Active inflammation and oedema B Characteristic of [17]
in villus tips; no LP oedema or crypt ileal UC in one
disarray study
Other ileal features observed in CIBD
Ulcer-associated cell lineage/pyloric Both 2% of inflamed CD bxs Not specific; a [15,16]
metaplasia/mucoid gland metaplasia 1% UC resections sign of chronic
Disturbed villous architecture B 84% inflamed CD bxs Architectural [15]
Disturbed crypt architecture B 36% inflamed CD bxs
distinguish Crohns
disease from
infection but not
from neoplasia,
Increased goblet cells in villi B 70% inflamed CD bxs [15]
The upper GI tract and small intestine in CIBD 123

Table 9.3 Continued

Jejunalisation B 34% inflamed CD bxs Associated with [15]
decreased goblet
Increased eosinophils B 17% inflamed CD bxs [15]
Neutrophil polymorphs B 84% inflamed CD bxs [15]
Cryptitis B 41% inflamed CD bxs [15]
Patchy cryptitis and crypt abscesses R 10.5% UC resections [16]
Focal inflammation B 90% inflamed CD bxs [15]
Increased LP inflammatory cells R 3% UC resections [16]
Erosions NOS R 2% UC resections Does not predict [16]
Villous atrophy R 1.5% UC resections [16]

CD, Crohns disease; bx, biopsy; UC, ulcerative colitis; LP, lamina propria; NOS, not otherwise specified; CIBD, chronic
inflammatory bowel disease.

We would particularly identify the importance of the connective tissue changes in Crohns
disease. For instance, fat wrapping is now regarded as pathognomonic of Crohns disease.
It is very common in small intestinal Crohns disease, but it is less common and more
difficult to identify in colonic Crohns disease [14]. Particularly in the small intestine, other
connective tissue changes are well recognised macroscopically. The bowel wall thickening
is caused by both fibrosis and proliferation of smooth muscle in the submucosa (obliterative
muscularisation of the submucosa, OMUS) and neuronal and vascular changes may also be
evident macroscopically [14].

The upper GI tract and small intestine in CIBD

Crohns disease is an established cause of inflammation of the upper GI tract and ileum.
There is also increasing recognition that UC can also involve these sites. Such involvement,
both endoscopically and histologically determined, may provide powerful evidence
towards a diagnosis of CIBD and one of its main subtypes.

Ileal involvement by CIBD is considerably more common in Crohns disease than in
UC in biopsies and strongly favours the former. However, ileal UC may be more easily
detected in resections than in biopsies: there was a prevalence of 17% in one report. The
term backwash ileitis for ileal UC has been questioned, as the evidence for a backwash
mechanism is weak. For example, some patients do not have contiguous caecal and
proximal colonic UC. As regards specific histological features, the presence of non-
cryptolytic granulomas favours Crohns disease over UC but ileal microscopic changes are
otherwise not usually very informative. Table 9.3 lists some of the features seen in ileal
mucosa that may have discriminant value between UC and Crohns disease, often based
on limited published evidence or the opinions of experts [1517]. Initial suggestions that
ileal involvement predicts an increased risk of pouchitis and of carcinoma in patients with
UC have not been confirmed by subsequent studies [15,16,18,19].
124 An update on the pathology of chronic inflammatory bowel disease

Pouchitis and prepouch ileitis

Pouchitis, complicating ileal pouch anal anastomosis (IPAA) and perhaps better known
as chronic relapsing pouchitis to distinguish it from other inflammatory pathologies in
the ileal pouch, is regarded by many authors as an example of small bowel involvement
by UC. A further type of small bowel inflammation that may represent CIBD is prepouch
ileitis, characterised in one report by diffuse inflammation extending from the pouch into
the neoterminal ileum [20] and showing various pathological changes including chronic
inflammation, ulceration, fistulae, submucosal fibrosis and (rarely) cryptolytic granulomas.
There is an almost unique relationship between UC and chronic relapsing pouchitis,
suggesting that the two may be aetiologically linked. However, the response of chronic
relapsing pouchitis to antibiotics and probiotic agents argues for a bacteriological or
chronic infective aetiology.

Upper gastrointestinal (GI) tract

Upper GI involvement by CIBD is more common in Crohns disease than UC overall, is
probably more prevalent in earlier than later disease and is more often encountered in
children than in adults [15,2123]. Reported rates of upper GI involvement by CIBD vary
considerably [24]. This partly reflects difficulties distinguishing CIBD from other causes
such as gastro-oesophageal reflux and inflammation induced by Helicobacter pylori [6,25].
However, published prevalences of other, more specific, features, such as granulomas, also
have a wide range.
Granulomas are the most reliable pointer towards CIBD in the upper GI tract. Not
infrequently, they occur in the absence of previous ileal or colorectal granulomas. In
the setting of CIBD, they suggest involvement by CIBD rather than other causes and
they strongly favour Crohns disease over UC [23]. Other features are less discriminatory
between diagnoses but a few characteristic patterns have emerged.
In known Crohns disease, involvement of the oesophagus is likely if granulomas
are a component of the picture, whilst oesophageal involvement by UC is more
difficult to diagnose and is probably very rare. Recent attention has focused on
lymphocytic oesophagitis (LE), a variably defined and controversial entity with marked
lymphocytosis accompanied by few neutrophil or eosinophil polymorphs (see Table 9.4
for published definitions) (Figure 9.3) [2527]. Almost 40% of children with LE had CIBD
in one report but subsequent studies showed weak or absent associations. In another
study, LE was significantly more common in children with Crohns disease than in those
with UC or in controls, suggesting that the pattern might represent Crohns oesophagitis
in this setting. However, LE occurs in other circumstances and could be a non-specific
reaction [25,26].

Table 9.4 Definitions of lymphocytic oesophagitis

Marked oesophageal lymphocytosis with no or only rare intraepithelial granulocytes (IEG): >50 intraepithelial
lymphocytes (IEL)/hpf and a ratio of >50:1 IEL to intraepithelial granulocytes [25]
Oesophageal inflammation with >20 lymphocytes and sparse neutrophil and eosinophil polymorphs [26]
Dense peripapillary lymphocytic infiltrates and peripapillary spongiosis involving the lower two thirds of the
epithelium [27]
The appendix and CIBD 125

Figure 9.3 Oesophageal mucosa

showing lymphocytic oesophagitis.
The lymphocyte intra-epithelial
count is high and there are
relatively few neutrophils or other
inflammatory cells. Associations
between this entity and chronic
inflammatory bowel disease have
been described, but at present
it is variably defined and of
questionable specificity.

Focally enhanced gastritis (FEG) is characterised by focal perifoveolar or periglandular

inflammation (lymphocytes, plasma cells, macrophages, neutrophil polymorphs) with
associated epithelial damage and a normal background mucosa. It was initially described
as a feature of Crohns disease but has subsequently been demonstrated in many other
conditions. It may be more specific in children than in adults: in one report it was highly
predictive for a diagnosis of CIBD, though not helpful for subclassification [24]. In a detailed
study of gastric histology in UC, three characteristic patterns were observed: focal
gastritis (equivalent to FEG); patchy mixed basal inflammation with loose collections of
lymphocytes, eosinophils, mast cells and plasma cells in the deep lamina propria; and
superficial plasmacytosis [22].
In the duodenum, granulomas are seen in a minority of those with Crohns disease
about 11% of children and 1.5% of adults. Other inflammatory patterns that might be
helpful include duodenal cryptitis, which is more common in Crohns disease than in
controls or in UC, and intraepithelial lymphocytosis, which seems to occur more frequently
in UC than in Crohns disease or controls [21,23]. An apparently unique pattern of diffuse
chronic duodenitis has been described in UC, affecting 10% of those who underwent
duodenal biopsy in one report and characterised by diffuse chronic inflammation, variable
activity and crypt changes reminiscent of the colorectal abnormalities. All patients had
undergone colectomy and the pattern seems to be associated with the development of
(chronic relapsing) pouchitis [22,28].
Overall, a new diagnosis of CIBD is rarely made on the basis of upper GI histology but
might be suggested if there are granulomas or, in children, FEG. Similarly, classification
of known CIBD as UC or Crohns disease is rarely helped by examination of upper GI
histology, in adults, unless there are non-cryptolytic granulomas. Upper GI biopsies may
be of greater value in children and are usually undertaken at the time of colonoscopic
assessment in this group.

The appendix and CIBD

There is no doubt that the assessment of the appendix is becoming increasingly important
in the diagnosis and differential diagnosis of CIBD. Firstly, and intriguingly, there is
126 An update on the pathology of chronic inflammatory bowel disease

now considerable evidence that previous appendicectomy protects patients against the
subsequent occurrence of UC [29]. Many have argued that the reason for this is that the
removal of immunocompetent tissue of the appendix reduces the likelihood of subsequent
UC. We wonder if this is more likely to be because of the influence of faecal stasis on
the generation of mucin changes that lead to UC. We note the association of UC with
sites of faecal stasis notably the rectum, diverticular disease in the sigmoid colon and
the appendix and have postulated that this could be a factor in the protection of the
appendicectomised patient against UC.
The pathological assessment of the appendix is also important in the differential
diagnosis of CIBD. The appendix is now well recognised to be one of the skip lesions
of UC [13]. Indeed, the pathology of the involved appendix in UC is very characteristic.
Just like that in the affected colon and rectum, there is diffuse mucosal pathology with
no semblance of transmural inflammation (Figure 9.4). The pathological assessment of
such an appendix provides powerful evidence towards a diagnosis of UC in colectomy
Florid transmural inflammation in the form of lymphoid aggregates with focal active
inflammation, with or without ulceration, might suggest Crohns disease when seen in an
appendicectomy specimen. However, caution is appropriate in this situation. Chronic or
grumbling acute appendicitis can lead to very similar pathological appearances. We have
seen many cases suggestive of involvement of the appendix by Crohns disease only to find
that the clinicians favour a grumbling appendix and subsequent follow-up has not shown
any evidence of Crohns disease elsewhere. The diligent pathologist requires to be cautious
in this situation and should only suggest a diagnosis of Crohns disease when observing
such changes in an appendicectomy specimen. We believe that this appendiceal scenario
is similar to that seen in resection specimens of sigmoid colon afflicted by diverticular
disease. In this situation, also, there can be profound mimicry of Crohns disease but most
cases appear only to represent mimicry of Crohns disease by complicated diverticular
disease rather than true multifocal Crohns disease [3033].
In a similar vein, caution is appropriate with granulomatous appendicitis. Whilst the
presence of well-formed epithelioid cell granulomata, especially with other features of

Figure 9.4 Histology of the

appendix in ulcerative colitis. There
are the typical changes in the
mucosa with active inflammation,
crypt distortion and mucin
depletion but the disease is notably
restricted to the mucosa with no
transmural inflammation.
Granulomas and CIBD 127

Crohns disease such as focal ulceration and transmural inflammation, might well indicate
Crohns disease, it has been shown that granulomatous appendicitis, per se, is more likely
to represent a chronic acute appendicitis rather than true Crohns disease [34]. So, in
this situation, one should give a guarded report and advise clinicians to seek evidence of
Crohns disease elsewhere.

Granulomas and CIBD

More than 10 years ago, one of us wrote Mention granuloma, certainly to the average
gastroenterologist and probably to many pathologists, in the context of intestinal pathology,
and the diagnosis of Crohns disease is established irrevocably, it seems, often to the
detriment of the patient [35]. Clearly, granulomas are important for the diagnosis of
Crohns disease but we now recognise that the presence of granulomas is not specific to
Crohns disease and can be seen in many other inflammatory pathologies of the intestines.
In particular, specific types of granulomas, especially in the mucosa, are recognised in
many other forms of inflammatory pathology and are certainly not pathognomonic for
Crohns disease. Firstly, it is appropriate to define what is meant by a granuloma and
also define the subtypes. In general, a granuloma is described as a well-circumscribed
collection of epithelioid histiocytes with or without multinucleate giant cells and/or
necrosis. In the intestinal mucosa, smaller collections are known as microgranulomas and
a highly characteristic feature of inflamed intestinal mucosa is the cryptolytic granuloma.
These are well-circumscribed collections of epithelioid histiocytes associated with an
inflamed and disrupted crypt (abscess).
There is no doubt that location of granulomas is important for diagnosis. For instance,
well-formed granulomas present basally in the mucosa or deeper in the bowel wall,
especially in the submucosa, are much more likely to represent Crohns disease than
its many mimics. Furthermore, in Crohns disease, granulomas become more common
as one descends down the GI tract. Thus, in the oesophagus and stomach, for instance,
granulomas are unusual when involved in Crohns disease. In contradistinction, anal
Crohns disease is characterised by a higher likelihood of granulomas [36].
Only about 40% of patients with classical Crohns disease have granulomas and they
are more common in the younger patient rather than the older patient [37]. Furthermore,
they are more strongly associated with a short history of Crohns disease rather than a long
history and long-term disease is associated with effete granulomas [37]. Defunctioned
Crohns disease is especially associated with effete granulomas with Schaumann bodies. In
fact, often the calcified bodies, only, remain and these are the tombstones of granulomas in
the defunctioned intestine [37].
Whilst basal granulomas in the mucosa are characteristic of Crohns disease, granulomas
are also seen in a transmural distribution and are often subserosal and closely associated
with the lymphocytic rosary at the outer aspect of the muscularis propria. They are usually
strongly related to inflammation and ulceration but may also be isolated. A characteristic
feature of granulomas in Crohns disease is that they are adjacent to, and sometimes
within, lymphatic vessels [37]. They are less commonly related to blood vessels, although
a granulomatous vasculitis is a highly characteristic feature of Crohns disease. In about
25% of patients, there are granulomas in lymph nodes, but it is a truism of Crohns disease
that the presence of granulomas only in lymph nodes and not in the intestinal wall is
exceptional [12,37].
128 An update on the pathology of chronic inflammatory bowel disease

Over the years, there has been considerable controversy as to whether the presence
of pathological changes of Crohns disease, at resection margins, is of influence in
determining whether or not that patient is likely to suffer recurrence of the disease. In fact,
there is now substantial evidence that most features of Crohns disease, such as granulomas
and active inflammation, do not have any influence over the recurrence or prognosis
when demonstrated at or close to an excision margin [12,37]. We would therefore suggest
that histological sections taken of resection margins are of little value for determining
likely recurrence or prognosis of Crohns disease. Nevertheless, there is a small amount of
evidence to suggest that Crohns disease-associated myenteric plexitis may be predictive of
recurrence [38,39].
It appears that granulomas cause particular diagnostic problems when they are present
in pathological specimens where there has been previous surgery. For instance, they
are a characteristic feature of diversion proctocolitis, whether the initial indication for
surgery was UC or Crohns disease, being especially seen in the defunctioned rectum after
colectomy for UC [40]. Indeed, well-circumscribed granulomas are almost a normal
feature in the pelvic ileal reservoir mucosa, especially within Peyers patches and lymphoid
aggregates. A pathologist should be very cautious before making a diagnosis of Crohns
disease in pathological specimens where there has been previous surgery.
We have already emphasised the striking mimicry of Crohns disease that can occur
in complicated diverticular disease. Indeed, well-formed epithelioid cell granulomata
certainly occur in this situation. Furthermore, rather characteristic transmural

Figure 9.5 A classical cryptolytic

granuloma is juxtaposed to a more
classical basally sited granuloma.
This is actually a patient with
Crohns disease but isolated
cryptolytic granulomas are not
unique to Crohns disease.
Neoplasia and CIBD 129

inflammation, not necessarily in the form of rosaries but rather migrating radially away
from inflamed diverticula, is seen in complicated diverticular disease. It can be seen that
the combination of granulomas, focal active inflammation, transmural inflammation
and even fissuring ulceration and fistulae, in complicated diverticular disease, can cause
profound mimicry of Crohns disease. As with granulomatous appendicitis, the diligent
clinician and pathologist should seek evidence of Crohns disease elsewhere.
In contradistinction to well circumscribed basal and submucosal granulomas,
cryptolytic granulomas are certainly not specific to Crohns disease (Figure 9.5). These have
been recognised in infective enterocolitis, diversion proctocolitis, diverticular colitis, the
pouch mucosa and pouchitis and secondary colitis. Furthermore, cryptolytic granulomas
are increasingly recognised in active UC [12,41].

Neoplasia and CIBD

Colorectal carcinoma (CRC)
The risk of CRC is higher in patients with CIBD than in those without CIBD. The magnitude
of the increased risk of CRC in UC and Crohns disease is similar, whilst the risk of small
bowel cancer in Crohns disease is greatly increased. Risk factors for CRC in CIBD patients
include duration of disease, extent of disease, severity of histological inflammation, severity
of endoscopic inflammation, the presence of dysplasia, the presence of primary sclerosing
cholangitis (PSC) and a family history of CRC [19,42,43]. Most of the information on CIBD-
related CRC derives from studies of UC and there is a notable lack of knowledge about the
neoplastic risk, and how to manage it, in Crohns disease. Sporadic CRC develops along
various pathways, characterised by chromosomal instability or microsatellite instability
(MSI). MSI may reflect mismatch repair gene mutation or other mechanisms such as gene
promoter methylation. Tumours are often classified as MSI or MSS (microsatellite unstable
or stable) whilst the MSI category is sometimes subdivided into MSI-H (high) or MSI-L
(low) [42,44].
Clinically, CRC in CIBD tends to occur at a younger age than non-IBD CRC (often in
the fifth decade) and is more likely than non-IBD CRC to be multiple. Therefore, it shares
features with CRC arising in the setting of hereditary MSI, i.e. Lynch syndrome (LS).
Furthermore, CRC in CIBD has pathological features in common with both LS-related CRC
and sporadic MSI CRC, including mucinous differentiation, signet ring differentiation, a
Crohns-like peritumoral reaction and a lack of necrosis (Table 9.5) (Figure 9.6) [42,44,45].
Compared with sporadic CRC, APC gene mutation in CIBD-related CRC occurs less
frequently and later, whilst p53 mutation is an earlier event [42,46]. The clinical and
pathological similarities between CIBD-related CRC and non-IBD MSI CRC have raised
the possibility that MSI and the associated serrated pathway are important for the
pathogenesis of CIBD-related CRC. Supporting this suggestion are the observations that
some dysplastic lesions in CIBD are serrated and that BRAF mutations, a feature of the
serrated pathway, occur in a minority of UC-related CRCs. Furthermore, methylation of
MGMT, hMLH1 and p16 has been demonstrated in chronically inflamed CIBD mucosa in
the absence of neoplasia. In fact, MSI does occur in CIBD-related CRC, usually as a result of
promoter hypermethylation. However, it is consistently confined to a minority of cases (less
than one third). Also, detailed studies have shown that the striking pathological and clinical
similarities between CIBD-related CRC and MSI CRC are largely independent of MSI status
(Table 9.5) [42,44]. Therefore, alternative explanations for the similarities are awaited.
130 An update on the pathology of chronic inflammatory bowel disease

Table 9.5 Features of CIBD-related colorectal carcinoma (CRC) and other CRC (%) [42,44,45].
Category of CRC Category of CIBD-related CRC
Sporadic Sporadic Lynch CIBD CIBD- CIBD- UC Crohns
MSS MSI syndrome related related related related related
(hereditary MSS MSI
Clinical feature
Mean age <55 No No Yes Yes Yes Yes
Multiplicity of Rare 010 >10 >10 >10 >10 >10 Rare
Right-sided <50 >50 >50 <50 <50 Rare
Lack of necrosis <50 >50 >50 >50 >50 >50
Crohns-like Rare >50 >50 >50 2550 2550 >50 2550
Mucinous/ <50 >50 4156 >50 29 >50 >50 46
presence of mucin
Well differentiated Rare >40 432 >40 >40 >40 >40 >40
Signet ring cells Rare >10 >10 >10 >10 >10
Medullary Rare >20 >20 Rare Rare Rare

MSS, microsatellite stable; MSI, microsatellite unstable.

Figure 9.6 An adenocarcinoma

arising in the rectum of a patient
with ulcerative colitis (following
ileorectal anastomosis). Mucin-rich
areas are amongst the features
that are seen more often in
inflammatory bowel disease-related
colorectal carcinoma (CRC), and
in microsatellite unstable CRC,
than in sporadic microsatellite
stable CRC. Other such features
include a Crohns-like peritumoral
inflammatory reaction, lack of
necrosis and signet ring cells.
Neoplasia and CIBD 131

Dysplasia in the colorectum

The detection of dysplasia is the basis for CIBD surveillance programmes, whose aim
is to prevent cancer or to treat cancer before it reaches an advanced stage [43]. Their
effectiveness is the subject of controversy. British Society of Gastroenterology (BSG)
guidelines advise initial colonoscopy 10 years after onset of symptoms, with subsequent
screening every 5, 3 or 1 year(s), according to three defined risk categories [47]. Pancolonic
dye spraying and targeted biopsy of endoscopically abnormal areas are now recommended
routinely as there is a considerably higher rate of detection of dysplasia with this method
than with random colorectal biopsies. Other new techniques allowing targeted biopsy
include narrow band imaging and fluorescence-based modalities [43,47].
Dysplastic lesions in CIBD are usually categorised endoscopically as raised or flat.
Those outside the area of colitis are managed in the same way as sporadic lesions. If a
raised dysplastic lesion in the area of colitis is endoscopically resectable and there is no
dysplasia in perilesional biopsies, colectomy is unnecessary. In contrast, unresectable
raised lesions have a significant risk of underlying carcinoma and require colectomy. The
management of flat low-grade dysplasia (LGD) is more controversial; after confirmation
of LGD by a second GI pathologist, options include colectomy or (depending on
circumstances) annual surveillance. For flat high-grade dysplasia, colectomy is usually
recommended [43,47].
Histologically, many lesions resemble adenomas. Formerly, there was considerable
pressure on pathologists to distinguish adenomatous dysplasia from that complicating UC.
However, with the advent of newer endoscopic detection techniques and the ability of any
detectable lesion to be removed by endoscopic mucosal resection (EMR) or endoscopic
submucosal dissection (ESD), there is now much less requirement for a rigid pathological
classification on biopsy material. Instead the pathologist can assess the whole lesion
after EMR or ESD. Some dysplastic lesions in UC are serrated and these may resemble
hyperplastic polyps, sessile serrated polyps or traditional serrated adenomas. Rarely,
dysplasia occurs within an inflammatory polyp.

Pouch cancer
IPAA may be offered to patients with UC who have had a colectomy. It is associated with
a reduced risk of CRC. Carcinoma in the pouch itself is rare and is more likely if there was
preoperative colorectal neoplasia or if there is PSC. Pouch cancer is most likely to arise
in the columnar cuff at the pouchanal junction and thereby likely represents neoplasia
arising in the small segment of lower rectum that is retained at the lower aspect of the
ileal pouch. So, when compared with other UC-associated CRCs, pouch carcinoma is very
similar but is more likely to have a Crohns-like peritumoral reaction [46,47].

Suggestions that the risk of intestinal and extraintestinal lymphoma is increased in long-
standing CIBD are not supported by population studies. However, there is an increased
132 An update on the pathology of chronic inflammatory bowel disease

frequency in CIBD patients on immunosuppressive therapy [48,49], possibly related to

EpsteinBarr virus infection. GI lymphomas that occur in CIBD are more common in men
than in women, are most often of large B cell type and are more likely to be in the large
bowel than the small bowel or other sites (stomach, duodenum and ileal pouch). Despite
the absence of a demonstrably increased risk, the possibility of lymphoma should be
considered when assessing CIBD biopsies, especially if the mucosa appears to be heavily

Appendiceal neoplasia
Appendiceal involvement by CIBD is common. As in the large bowel, chronic mucosal
inflammation may be a risk factor for the development of neoplasia. Indeed, case reports
have suggested an association between CIBD and appendiceal epithelial neoplasms. A
larger study showed that patients with CIBD and synchronous colorectal neoplasia had
a 15-fold increase in the prevalence of appendiceal cystadenoma, compared with those
without CIBD, and an 8-fold increase compared with those with uncomplicated CIBD.
However, the risk in CIBD overall was not increased significantly compared with non-CIBD
patients. Appendiceal neuroendocrine tumours have the same prevalence in CIBD patients
as in the general population [50,51].

Key points for clinical practice

The microscopic features favouring CIBD over infective colitis in biopsies include basal
plasmacytosis and mucosal architectural changes. Reliable interpretation of histology also
requires knowledge of the clinical setting.
Macroscopic assessment of CIBD resections often provides as much information as
histological assessment and some macroscopic pathological features are very specific,
indeed pathognomonic, for each type of CIBD.
Ileal inflammation favours Crohns disease over UC in biopsies, although it may occur in a
significant minority of UC resections. Ileal granulomas strongly favour Crohns disease, whilst
other features are less discriminatory.
Both Crohns disease and UC can involve the upper GI tract. Other causes of inflammation
require exclusion. In the setting of CIBD, granulomas suggest involvement by CIBD and
strongly favour Crohns disease.
LE (lymphocytic oesophagitis), FEG (focally enhanced gastritis) (especially in children),
duodenal cryptitis and UC-like duodenal changes might suggest CIBD but new CIBD is rarely
diagnosed based on upper GI tract changes alone.
The pathological assessment of the appendix is increasingly useful for the differentiation of
UC from Crohns disease in resection specimens.
Granulomas are heavily beholden to context in the diagnosis of CIBD. They are helpful to
diagnose Crohns disease, especially in children and in the upper GI tract, but are more
commonly seen in the large intestinal mucosa where their specificity for Crohns disease is
CIBD-related CRC shares clinical and pathological features with hereditary and sporadic MSI
tumours. However, the similarities appear to be independent of MSI status.
Dysplastic lesions in CIBD are initially managed on the basis of their resectability rather than
grade. Perilesional biopsies help to confirm resectability.
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Chapter 10
Diagnosis and therapy of
gastrointestinal MALT lymphoma
Andrew C Wotherspoon, Larissa Sena T Mendes

In 1983, Isaacson and Wright first reported the existence of a group of indolent B-cell
lymphomas that formed a specific clinicopathological entity and occurred specifically at
extranodal sites [1,2]. These lymphomas structurally showed many of the features of native
lymphoid tissue found distributed along the small intestine, appendix and colorectum.
The most prominent concentration of native mucosa-associated lymphoid tissue (MALT)
is seen in the terminal ileum in the form of Peyers patches, and for this reason the term
lymphomas of MALT applied to this group of lymphoma. A mucosa is not a prerequisite
for these lymphomas as the disease defining features are organisation, cell morphology,
immunophenotype and molecular characteristics [35].
Since that first description, many studies have unravelled the aetiopathogenesis, clinical
behaviour, histological features and molecular mechanisms associated with this group of
tumours. The recognition that some of these lymphomas may respond to antibiotic therapy
means that accurate diagnosis is essential if one is to avoid under treatment of other
lymphoma types. Furthermore, strategies are needed to assess response and determine if
and when more conventional therapies should be applied to cases diagnosed as resistant to
eradication therapies [3,6].

Lymphoma of mucosa-assocIated LymphoId tIssue

and the maLt concept
Extranodal marginal zone lymphoma of MALT lymphoma is the fourth commonest
B-cell non-Hodgkins lymphoma (NHL) worldwide accounting for about 9% of all B NHL
cases. MALT lymphoma has been described at almost every extranodal site, but is most
commonly encountered in the gastrointestinal (GI) tract with the stomach the area most
frequently involved. MALT lymphoma is, however, very rarely seen in the terminal ileum
where native MALT is most abundant [68].

Andrew C Wotherspoon, MB, BCh, FRCPath, Department of Histopathology, Royal Marsden Hospital, London, UK
Email: andrew.wotherspoon@rmh.nhs.uk (for correspondence)
Larissa Sena T Mendes, MD, Department of Histopathology, Royal Marsden Hospital, London, UK
136 Diagnosis and therapy of gastrointestinal MALT lymphoma

The lymphoid tissue in the terminal ileum is the prototype of MALT. There are three
intimately related components to this tissue: the B-cell and T-cell areas in the mucosa and
the draining mesenteric lymph nodes. The Peyers patches in the terminal ileum consist
of lymphoid follicles that occupy the full thickness of the mucosa with the base facing
the muscularis mucosae (Figure 10.1a). The germinal centre has a lower dark zone and
luminal light zone, which shares the organisational characteristics of secondary follicles in
lymph nodes (Figure 10.1bc). The mantle surrounds the germinal centre and is broader
in the subepithelial area. Outside the mantle zone is a further layer of B cells (Figure
10.1bc) that is not seen in peripheral lymph nodes and is characteristically only seen in
the spleen, mesenteric lymph nodes and extranodal lymphoid tissue. This is the marginal
zone which is composed of cells that are slightly larger than mantle zone cells with more
abundant pale cytoplasm. The marginal zone is also broader at the luminal aspect, and
marginal zone B cells are seen to enter the epithelium over the dome of the follicle to form
a lymphoepithelium (Figure 10.1bc). The epithelium in this area contains a population
of specially adapted cells, M cells, which facilitate the transport of large molecules across
the epithelial barrier to enhance and maintain the integrity of intestinal immunity. The
area around the follicle contains T cells, plasma cells and accessory cells. Scattered
intraepithelial T cells are present in the gut epithelium throughout the intestine. MALT
lymphomas recapitulate this structural organisation [4,5].

a b c

figure 10.1 (a) Peyers patches in the terminal ileum. (b) Germinal centre
surrounded by a thin layer of small lymphocytes with scant cytoplasm
forming the mantle zone. Outlining this layer, there is the marginal zone
with slightly larger cells and more abundant cytoplasm. (c) Marginal zone
cells enter the overlying epithelium forming a lymphoepithelium. (d)
Acquired mucosa-associated lymphoid tissue: presence of a lymphoid
follicle and reactive lymphoid infiltrate surrounding and entering the
gastric epithelium.
Clinical features and pathology of GI malt lymphoma of classical type 137

As MALT lymphoma typically arises in areas that are devoid of constitutive organised
lymphoid tissue, acquisition of this is the first step along the path of lymphomagenesis.
Organised lymphoid tissue is usually acquired in the context of either infection or
autoimmune disease. In the stomach organised lymphoid tissue of MALT-type is acquired
most frequently in association with infection by Helicobacter pylori (Hp) but can be seen
with infection by Helicobacter heilmannii and in patients with Sjogrens syndrome. The
acquired lymphoid tissue shares all the features of native MALT with a reactive germinal
centre, mantle and thin marginal zone (Figure 10.1d). There may be infiltration of the
epithelium by marginal zone B cells to form a lymphoepithelium, but the specialised M
cells are not seen [911].

cLInIcaL features and pathoLogy of gI maLt

Lymphoma of cLassIcaL type
GI MALT lymphoma is a disease of the late middle aged/elderly, usually over 50 years
with a median age of about 61 years but can be seen at any age with a slight female
predominance. There is geographical variation with higher incidences in areas of Europe
that have high rates of infection with Hp. However, in underdeveloped areas of the world
with high Hp infection rates MALT lymphoma remains rare [1,3,12,13].
Gastric MALT lymphoma usually presents with vague upper abdominal symptoms
including dyspepsia, nausea and vomiting symptoms that may be attributable to the
associated Hp infection. Lower intestinal MALT lymphoma may present with disordered
bowel habit, vague discomfort or rectal bleeding. Emergency presentation is rare [14].
Endoscopically the appearance of GI MALT lymphoma is variable with some cases
having minimal changes, erythema, thickened folds or erosions. There may be polyps or
occasionally a more solid mass lesion with or without ulceration [7,14].
The histological features of MALT lymphoma in the early stages closely resemble the
appearances of normal MALT. There is expansion of the marginal zone around reactive
follicles that may have intact mantles (Figure 10.2a). The cells in the marginal zone
show cytological atypia and may have a variety of appearances including small regular
lymphocytes, cells with scanty cytoplasm and indented nuclei resembling germinal centre
centrocytes (centrocyte-like cells), cells with marginal zone/monocytoid cell appearances
with abundant pale cytoplasm and well-defined cellular borders and cells with
plasmacytoid/cytic features (Figure 10.2bf). Dutcher bodies may be seen. A mixture of
these cell morphologies is frequently seen in any single case. Plasma cell differentiation is
seen in about a third of cases. Scattered transformed cells are ubiquitous but these should
not be present in sheets [3,7,15,16].
The neoplastic cells extend away from the confines of the follicular structure infiltrating
into the lamina propria around the glands/crypts. Recapitulating the lymphoepithelium of
normal MALT, the neoplastic lymphocytes infiltrate gland/crypt epithelium with destruction
of the architecture resulting in lymphoepithelial lesions (Figure 10.2g). In the earliest stages,
these can be identified by clusters of three or more cytologically atypical neoplastic cells in
the epithelium. With time, there is a sublethal and then lethal effect on the epithelial cells
which become progressively enlarged with eosinophilia of the epithelium and eventually are
destroyed. Neuroendocrine cells appear more resilient and can often be seen as single cells
in an otherwise diffuse sheet of MALT lymphoma cells [1618].
Lymphoid follicles are ubiquitous to MALT lymphoma. In the early stages, they are
intact, but as the disease progresses they frequently become overrun by lymphoma and
138 Diagnosis and therapy of gastrointestinal MALT lymphoma

a b c

d e f

figure 10.2 (a) Gastric mucosa-

associated lymphoid tissue
lymphoma neoplastic cells
infiltrate the lamina propria,
extend around reactive follicles
and reach the epithelium. (b)
Centrocyte-like cells: small-to-
g h medium-sized nuclei and dispersed
chromatin; inconspicuous
nucleoli. (c) Increased amount
of pale cytoplasm giving a
monocytoid appearance. (d) Neoplastic cells showing plasmacytic differentiation. (e) Neoplastic cells resembling
small lymphocytes. (f ) Follicular colonization by plasma cells. (g) Spectrum of lymphoepithelial lesions showing
architectural distortion of the glands (top right) and eosinophilic degeneration (centre). (h) Microlymphoma small
lesion distant from the main one showing CCL cells infiltrating the perifollicular area and ascending towards the

their presence can only be demonstrated by immunostaining of the follicular dendritic

cells (FDC). In some cases, there is specific colonisation of the follicles by neoplastic cells.
The intrafollicular component may resemble the extrafollicular infiltrate, but in some
cases the colonising cells may appear larger and more activated. Cases with plasma cell
differentiation may show infiltration of neoplastic plasma cells in the follicles (Figure
10.2f). Such colonisation recapitulates the behaviour of normal marginal zone B cells
following exposure to antigen [1,17].
Examination of entire gastrectomy specimens from cases of gastric MALT lymphoma
has shown that the mucosa contains multifocal deposits of neoplastic lymphocytes [15,19].
These may be of variable size with the smallest infiltrate being composed of neoplastic
cells around a single follicle forming a microlymphoma (Figure 10.2h). In these cases,
the infiltrates from the multiple lesions have been shown by molecular techniques to be
identical to each other and to the neoplastic clone of the main lymphomatous lesion. It is
assumed that multifocal microlymphomas are a feature of MALT lymphomas at all sites [19].
Immunophenotypically the neoplastic cells express pan-B-cell markers CD19, CD20,
CD22, CD79a and PAX5 (Figure 10.3a). They express surface immunoglobulin which is
Clinical features and pathology of GI malt lymphoma of classical type 139

usually either IgM or IgA and rarely IgG. The cells are typically negative for CD5, CD23,
cyclinD1, CD10 and bcl-6 (Figure 10.3bc). Very occasional cases with expression of
CD5 have been described. There is expression of bcl-2 protein. Expression of CD43 is
seen in approximately 50% of cases (Figure 10.3d). Staining of FDC can be achieved with
antibodies to CD21 and/or CD23 (Figure 10.3c) and staining for cytokeratin may help to
highlight lymphoepithelial lesions (Figure 10.3ef). Staining with antibodies to bcl-6 and
CD10 can highlight residual germinal centres [3,7]. There is light chain restriction (kappa
more frequent than lambda) (Figure 10.3gh).
The presence of a significant number of large cells in clusters or sheets (generally 20 or
more large cells) should provoke a diagnosis of diffuse large B cell lymphoma. It is crucial
to distinguish such clusters from residual naked aggregates of follicle centre cells. This can
be confidently achieved by demonstrating the presence of CD10 and bcl-6 with lack of bc-2
staining characteristic of residual follicle centre cells [20,12].
Immunoglobulin heavy chain and light chain genes show clonal rearrangement and
there is a high load of somatic hypermutation [6]. MALT lymphomas are characterised by
several recurrent chromosomal translocations, which involve the nuclear factor-kappa B
(NF-kB) signalling pathway [6,21]. Of these, the most frequent is the translocation t(11;18)
(q21;q21) which creates a novel functioning fusion product by translocating the amino
terminus region of apoptosis inhibitor 1 (API1) gene to the carboxyl terminus of MALT1.

a b c

d e f

figure 10.3 (a) Neoplastic

cells expressing CD20. (b) CD5
expression in T cells, but is negative
in the lymphomatous infiltrate. (c)
CD23-negative staining. Residual
follicular dendritic cell meshworks
can be observed (top left). (d)
g h CD43 strongly positive. (e and f ).
Cytokeratin staining (AE1/AE3)
highlights the lymphoepithelial
lesions. Immunostaining for kappa (g) and lambda (h) light chains. Kappa light chain restriction in (g) and lambda
(h) light chains showing kappa light chain restriction in (g).
140 Diagnosis and therapy of gastrointestinal MALT lymphoma

The resulting fusion product has the ability to activate the canonical and noncanonical
NF-kB pathways. This translocation appears specific to MALT lymphoma and is present
in about 25% of gastric MALT lymphomas, but the frequency in nongastric GI MALT
lymphoma is less certain. When the t(11;18)(q21;q21) is present, it is almost always the sole
genetic abnormality [22,23].
Other chromosomal translocations have been described including t(1;14)(p22;q32),
found in up to 4% of gastric MALT lymphomas, translocating the BCL-10 gene into to
come under the influence of the promoter of the immunoglobulin heavy chain gene. The
resulting protein also leads to activation of NF-kB pathway. When this translocation is
present there are often further genetic changes particularly trisomies of chromosomes
3, 12 and 18 [9,21,24]. The architectural organisation, immunophenotype and presence
of somatic hypermutation (indicating antigen selection) all point to an origin of MALT
lymphoma from postgerminal centre marginal zone B cells [5,7,10].

ImmunoproLIferatIve smaLL IntestIne dIsease

Immunoproliferative small intestinal disease (IPSID) is a subtype of MALT lymphoma
that has a characteristic geographic distribution, clinical presentation and histological
appearance. IPSID is most frequently encountered in the Middle East and countries
bordering the Mediterranean Sea, the Cape region of South Africa and some parts of the
Indian subcontinent. It may present at any age but is usually seen in young adults who
are commonly of low socioeconomic background [25]. The macroscopic appearances
seen at endoscopy depend on the degree of infiltration at the time of diagnosis and may
vary from minimal mucosal distortion, polyps to general thickening of the wall and more
circumscribed areas [25,26].
Histologically, IPSID is characterised by the presence of sheets of plasma cells with
scanty small lymphocytes. There is widening of the villi. The small lymphocytes generally
resemble the centrocyte-like cells of classical MALT lymphoma. Immunostaining of the
small B cells with antibodies to pan B-cell antigens may facilitate the identification of the
lymphocytic component which is present around epithelial structures with formation of
lymphoepithelial lesions. The immunophenotype of the centrocyte-like cells is similar to
that of classical MALT lymphoma. The plasma cells show synthesis of IgA (usually IgA1),
but light chain production is usually absent. Clinically raised IgA levels can usually be
demonstrated in serum and duodenal juice. Genetically, the translocations seen in classical
MALT lymphoma at other sites have not been demonstrated in IPSID [3,26].

dIfferentIaL dIagnosIs
With advances in the understanding of NHL, the underlying genetic abnormalities and the
increasing development of more subtype-specific treatment options, the accurate diagnosis
of these entities has become more important.

MALT lymphoma versus reactive lymphoid infiltrates

In the early phases, MALT lymphoma very closely resembles non-neoplastic acquired
lymphoid tissue. One of the earliest features seen in MALT lymphoma is expansion of the
marginal zone with extension of small B cells away from the immediate confines of the follicle,
extending into the mucosa around epithelial structures. The presence of moderate cytological
Therapy of GI malt lymphoma 141

atypia, Dutcher bodies and formation of genuine lymphoepithelial lesions have been shown to
be highly associated with MALT lymphoma rather than reactive infiltrates [16,27,28].
Special techniques may be helpful in such cases. Immunophenotypic demonstration
of light chain restriction is characteristic of a neoplastic infiltrate. When expression of
CD43 is demonstrated on the B-cell population, it can be used as a marker of a neoplastic
population. Molecular studies may be useful. Although some studies have suggested that
clonal populations can be demonstrated in reactive infiltrates, adoption of the European
BIOMED-2 primer/protocol has made detection of clonal population more specific and
accurate. Optimised protocol/heteroduplex analysis has made false positive results from
biopsies with reactive infiltrates very rare if the analysis is conducted appropriately.
Detection of a clonal population in a suspicious lymphoid infiltrate should be considered
highly indicative for the diagnosis of MALT lymphoma [15,29].

MALT lymphoma versus other small B cell lymphomas

MALT lymphoma with follicular colonisation can closely resemble follicular lymphoma.
Morphological clues for a diagnosis of MALT lymphoma include the presence of a
significant extrafollicular component (usually less pronounced in follicular lymphoma).
Although lymphoepithelial lesions are seen in most cases of MALT lymphoma,
identical features are occasionally seen in other small B cell lymphomas and cannot
be considered a MALT lymphoma specific feature. Immunophenotyping is useful with
staining for CD10 and bcl-6 with characteristic aberrant expression of bcl-2 protein in
follicular lymphoma [7,30].
Distinction between MALT lymphoma and mantle cell lymphoma is important as their
treatment is very significantly different. Morphologically the cells of these lymphomas can
be very similar and the pattern of infiltration, with perifollicular growth and a tendency
to overrun pre-existing follicles may be present in both. However, the immunophenotype
clearly distinguishes the two entities. Expression of CD5 is rarely seen in MALT lymphoma
and the presence of staining for cyclinD1, characteristic of mantle cell lymphoma, is not
present in MALT lymphoma [3,7].
Infiltration of extranodal sites in patients with chronic lymphocytic leukaemia (CLL)
is common, but usually the presence of CLL has been diagnosed prior to diagnostic GI
procedures. Primary small lymphocytic lymphoma (SLL) of the GI tract is exceptionally
rare. Although MALT lymphoma may rarely express CD5 or CD23, the presence of the
characteristic combined immunophenotype CD5+/CD23+ seen in CLL/SLL is not
encountered in MALT lymphoma [30,31].

therapy of gI maLt Lymphoma

Many studies have shown that the presence of gastric MALT lymphoma is highly associated
with mucosal colonisation by Hp, although there may be some geographic variation in
the frequency of Hp-positive lymphomas. In vitro studies have demonstrated that MALT
lymphoma cells proliferate in the presence of Helicobacter organisms in culture systems
that include tumour infiltrating T cells. This T-cell regulated proliferative drive is contact
dependent. The association between the lymphoma and the results of these in vitro studies led
to the hypothesis that gastric MALT lymphoma may respond to Hp eradication therapy [32,33].
Many studies have now shown that approximately 70% of gastric MALT lymphomas
will respond to Hp eradication therapy alone with enduring remission. The choice of
142 Diagnosis and therapy of gastrointestinal MALT lymphoma

eradication therapy and the strategy for managing cases where there is antibiotic resistance
in the organism should be determined by current international guidelines [3436]. There
has been a suggestion that, in a similar way to gastric MALT lymphoma, IPSID may be
associated with infection by Campylobacter jejuni. Whilst definitive evidence is still lacking,
it is certainly true that a proportion of cases of IPSID in early stages will respond to broad
spectrum antibiotic therapy [7,26].
No specific relationship of extragastric classical GI lymphoma to infective organisms
has been shown. Reports of response of MALT lymphoma in the colorectum to antibiotic
therapy exist. In some cases, there is associated gastric Hp infection, but it is unlikely that the
Hp and lymphoma are causally related and the response is likely to be serendipitous [37].
Prediction of gastric MALT lymphoma cases that will respond to Hp eradication alone
would clearly be important to the management of these patients. In reality no clinical,
pathological or molecular feature will absolutely predict those cases that will not respond to
antibiotic based regimes alone. The time to response is also very variable with some patients
showing complete response at the time of first control endoscopy to assess Hp status, whilst
others only regress after months or even years following successful eradication. Success of Hp
eradication should be confirmed by current clinical guidelines but could include a urea breath
test as the most accurate determinant of presence of small residual Hp colonies [35,36].
Absence of Hp infection is likely to be associated with a lack of response, although
there are exceptions and these may be due to the presence of other, as yet undetermined,
infective organisms [15]. The determination of Hp status at diagnosis is therefore crucial to
patient management. Whilst histological evaluation, culture and stool polymerase chain
reaction (PCR)-based studies are all effective detection methods for Hp infection, there
may be cases where previous partial or complete eradication therapy may give a negative
result in patients previously Hp positive. It has been shown that more accurate assessment
of Hp status in the context of MALT lymphomagenesis is achieved by serological based
studies as circulating antibodies may be present up to 2 years following eradication [15,35].
In general, cases with deeper infiltration of the gastric wall by lymphoma with or without
local lymph node involvement are less likely to respond to eradication therapy. This feature
is best determined using endoscopic ultrasound [15,38,39].
Whilst no specific immunophenotypic feature has been shown to predict response to
Hp eradication therapy alone, molecular studies have a role. Whilst some cases of response
have been recorded, the presence of the t(11;18)(q21;q21) is generally a specific predictor
that response to Hp eradication alone is unlikely [40]. This translocation is present in only
approximately 3% of cases responding to Hp eradication therapy alone, whilst it is detected
in about 50% of cases in stage IE and in about 70% of cases in stage IIE or above that do not
respond to this approach [15,39].
The timing when the translocation should be investigated remains controversial,
either at diagnosis or when more conventional treatment is proposed. Assessment is best
performed using interphase fluorescence in situ hybridisation with MALT1 dual-colour
break-apart and API2-MALT1 dual-colour fusion probes or by reverse transcription-
polymerase chain reaction (RTPCR) of the API2-MALT1 fusion transcript [15,31].
When eradication therapy is deemed to have failed or delayed response is considered
unlikely, more conventional standard antilymphoma therapeutic approaches are
indicated [15]. Surgery is no longer considered an option due to the multifocal nature
of the disease [41]. Radiotherapy has been shown to be a highly effective therapy in
gastric MALT lymphomas. Standard chemotherapy using several regimes with or without
immunotherapy has been shown to be effective in the treatment of MALT lymphoma. Of
Assessment of posteradication biopsies 143

note, is the fact that the presence of t(11;18)(q21;q21) is associated with high frequency
of treatment failure in patients given single oral alkylating agents (chlorambucil or
cyclophosphamide) or thalidomide [15,18].
High-grade transformation may occur in MALT lymphoma, although this is very rare in
cases with t(11;18)(q21;q21) [3,7]. Whilst progression to diffuse large B-cell lymphoma has
generally been considered an indication for combination chemotherapy, there is increasing
evidence that these lymphomas may respond to Hp eradication alone. This remains to be
confirmed and such an approach is only recommended in patients unfit for other therapies
or in the context of an appropriate clinical trial [20,42].

assessment of posteradIcatIon bIopsIes

Clearly assessment of post-therapy biopsies is crucial to determining therapeutic strategy
and when pure eradication therapy should be abandoned and more conventional therapy
considered. The duration of follow-up before instigating further therapy remains uncertain
and is highly variable between treating centres [15].
The variable time to disease regression makes assessment of biopsy material crucial
as an indicator of potential response. In many cases, symptoms disappear following
resolution of the acute inflammation induced by Hp-associated gastritis. The GELA group
(Group dEtude des Lymphomes de lAdulte) has developed a scheme for assessment of
posteradication biopsies which is highly reproducible between pathologists (Table 10.1).
This scoring system assesses the presence of the lymphoid infiltrate in the mucosa, the
degree of regression associated fibrosis and the presence of lymphoepithelial lesions and
divides the findings into four groups. In a proportion of cases, there will be no lymphoid
infiltrate detected in the biopsies and these are considered to be complete histological
regression (Figure 10.4a). Other cases will show no change between the appearances in
the follow-up biopsies and those seen at diagnosis (no change group; Figure 10.4d). This

Table 10.1 GELA scoring system for assessment of posteradication therapy

gastric biopsies and clinical recommendations
GELA category Morphological features Recommendation
CR complete histological response Empty appearance of lamina propria
with fibrosis, few glands, small No need of additional therapy
lymphocytes and plasma cells; no
pMRD probable minimal residual Base of lamina propria and/or
disease submucosa with small lymphoid No need of additional therapy
nodules and fibrosis; no LEL
rRD responding residual disease Presence of lymphomatous infiltrate Evaluation of clinical progression
in a diffuse or nodular pattern; some should delineate additional therapy
degree of stromal change (thin areas
of fibrosis); focal or no LEL
NC no change Dense lymphomatous infiltrate Oncological treatment should be
(diffuse or nodular) similar to proposed if infiltrate persists over
diagnostic biopsy; LEL present sequential examinations

GELA, Group dEtude des Lymphomes de lAdulte; LEL, lymphoepithelial lesion.

144 Diagnosis and therapy of gastrointestinal MALT lymphoma

latter group would represent patients in whom, if the absence of change is prolonged,
radiotherapy/chemotherapy may be used at an earlier stage [33,43].
Some biopsies show persistent lymphoid infiltrate, but with reduced density compared
with diagnostic biopsy and with the development of fine fibrosis in the lamina propria with
loss of glands associated with destruction following lymphoepithelial lesion formation. This
is the group considered to be showing responding residual disease (Figure 10.4c). Whilst
this appearance persists, a watchful waiting approach can be continued [18,43].
In many cases, there will be near complete absence of lymphoid infiltrate with only
the presence of occasional lymphoid aggregates within the lamina propria, mainly at the
base of the mucosa (Figure 10.4b). It has been shown that in the majority of cases these
aggregates contain a small population of neoplastic B cells that are derived from the
lymphoma clone. This group has been labelled probable minimal residual disease (pMRD).
Follow-up studies of cases with this residual infiltrate have indicated that this has no
clinical significance in terms of disease progression, and further therapy in these cases is
not indicated [6,15,44].
Sequential follow-up biopsies are indicated in all cases of gastric MALT lymphoma.
Occasional relapses can be seen and these may be associated with reinfection/regrowth of
Hp. In these cases, repeat eradication therapy has proven effective. In some cases, there is

a b c

Figure 10.4 (a) CR complete histological response: empty appearance

of lamina propria, areas of fibrosis and few small lymphoid cells. (b) pMRD
probable minimal residual disease: presence of a lymphoid nodule
(bottom left) in the muscularis mucosa and few areas of fibrosis. (c) rRD
responding residual disease: dense neoplastic infiltrate in the lamina
propria and focal area of fibrosis. (d) NC no change: diffuse infiltrate and
lymphoepithelial lesion with no signs of response to therapy.
Conclusion 145

transient regrowth of lymphoma seen in biopsies in the absence of endoscopic evidence

of relapse and, some of these spontaneously regress in subsequent biopsies. In addition,
there is an associated risk for the development of gastric adenocarcinoma in these patients,
and follow up with repeated biopsies may result in early detection of carcinoma with the
potential for curative therapy [15,31,45].

MALT lymphoma represents a specific lymphoma entity with characteristic clinical,
pathologic and molecular features. The GI tract is the area most frequently involved by
MALT lymphoma with the majority of cases occurring in the stomach. In the stomach, the
greater numbers of cases are associated with Hp infection and eradication of the organism
results in lymphoma regression for most of them, although time to regression can be highly
variable. Certain characteristics, including depth of gastric wall involvement, local lymph
node involvement and presence of the t(11;18)(q21;q21), predict for lack of response to
eradication therapy, in which case radiotherapy or chemotherapy but not surgery is highly
effective in inducing lymphoma regression. Histological assessment of post eradication
gastric biopsies should be performed within the context of the GELA assessment scheme
to provide information about the tempo and direction of travel of potential lymphoma
regression. For extragastric GI MALT lymphoma, antibiotic-based therapy may be effective
in a proportion of cases but generally chemotherapeutic strategies are frequently required.

Key points for clinical practice

MALT lymphomas are a distinct clinicopathological entity with treatment strategies that are
distinct from other NHLs. IPSID is a subtype of MALT lymphoma.
The earliest distinguishing feature in the differential diagnosis between reactive infiltrates
and MALT lymphoma is the extension of atypical small B cells away from follicles into the
superficial mucosa.
Immunohistochemistry can be used to distinguish MALT lymphoma (CD5-/CD23-/CD10-/
bcl-6-/cyclinD1-) from other small B-cell lymphomas.
Lymphoepithelial lesions, whilst characteristically seen in MALT lymphoma, are not essential
for the diagnosis and may be seen in other small B-cell lymphomas.
The majority of gastric MALT lymphomas are associated with infection by Hp.
Helicobacter eradication should be the initial treatment of choice for all gastric MALT
Posteradication biopsy specimens are best assessed within the context of the GELA scoring
The presence of t(11;18)(q21;q21) and increased depth of gastric wall invasion predicts for a
likely lack of response in gastric MALT lymphoma treated with Helicobacter eradication therapy
The time for determining lack of response to Helicobacter eradication therapy in gastric
MALT lymphoma is variable and unpredictable. Some cases may respond only after a year or
more following therapy.
Extragastric GI MALT lymphoma may respond to antibiotic-based therapy but this is less
common than in gastric cases.
146 Diagnosis and therapy of gastrointestinal MALT lymphoma

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Chapter 11
Medical revalidation for
Peter Furness

Introduction how we got to where we are now?

One could never reasonably expect that passing one set of examinations at the beginning
of a professional lifetime would provide a guarantee that a doctor remains fit to practise
for life. Yet this was rarely considered until the latter part of the 20th century, when the
accelerating pace of medical developments made it increasingly important (and time-
consuming) for doctors to keep their practice up to date. The problem was also highlighted
in the public consciousness by the huge media interest in the small number of doctors
whose performance fell well short of the standard that the public could reasonably expect.
In the UK, the most dramatic example of such bad doctors was Dr Harold Shipman, who is
now believed to have murdered several hundred of his patients, but pathologists have also
had their share of adverse media attention.
Solutions to the problem of maintaining and confirming the competence of doctors
have taken different forms and different names in different countries. In some, the problem
has not yet been addressed. In some countries doctors are required to prove that they are
participating in an acceptable amount of postgraduate medical education (usually referred
to as continuing professional development or CPD) thereby making a bold assumption
that such CPD actually has the desired effect. Some countries demand that doctors sit
professional examinations every few years thereby missing the point that bad doctors
may have a problem in their behaviour rather than their knowledge. It is relevant to the
discussion, though rarely acknowledged, that most of these bad doctors, notably including
Shipman, were bad because of the way they behaved, rather than because they had failed to
keep up to date with their specialty.
The present account will focus on medical revalidation in the UK, where the system
recently introduced is arguably more comprehensive (some would say more onerous) than
is in place in any other country in the world. This is in very large part a consequence of the
crimes of Harold Shipman; although, as we shall see, it can be questioned whether our new
system of medical revalidation would have identified Harold Shipman any earlier.
The General Medical Council (GMC: the regulatory authority for doctors in the UK)
has been discussing how to implement a system to check on the performance of doctors

Peter Furness, BM, BCh, PhD, FRCPath, Consultant Histopathologist, University Hospitals of Leicester; Revalidation
Lead, University Hospitals of Leicester, Leicester, Leicestershire, UK. Revalidation Lead, East Midlands; Former vice
Chair and Revalidation Lead, the Academy of Medical Royal Colleges, London, UK
Email: pnf1@le.ac.uk
150 Medical revalidation for histopathologists

since the early 1990s. Proposals based mainly on a requirement to undertake CPD were
well advanced, but had not been implemented, at the time when Harold Shipman was
convicted. It became obvious that the system would be expected to catch the next Harold
Shipman so the GMCs plans were put on hold pending an investigation of the background
of Shipmans crimes by Dame Janet Smith.
Dame Janets report identified, as expected, the need for a system to check up on
the performance of doctors every few years [1]. But she was highly critical of the GMCs
proposals. As a consequence, those plans were abandoned.
The next major development was the publication, by the then Chief Medical Officer Sir
Liam Donaldson, of a White Paper entitled Trust, Assurance and Safety: The Regulation of
Health Professionals in the 21st Century [2]. This was a very broad-ranging document, but
it included proposals for regular checks on the performance of doctors. The whole process
was called revalidation, but it was initially split into two components. Relicensing was to
be a check that a doctor remained competent at the level of basic registration with the GMC
as a medical practitioner. Recertification was to be a check that specialists were operating
at an appropriate level for their specialty. The Medical Royal Colleges, which provide
postgraduate curricula and examinations in all the medical specialties in the UK, were
asked to deliver recertification. This seemed reasonable, as the Medical Royal Colleges are
the bodies that set the standard for postgraduate specialist medical practice in the UK.
However, the Medical Royal Colleges regarded this as a poisoned chalice. The
opportunity for the profession to set and test against its own standards was appealing; but
the risks and logistic requirements were substantial. The White Paper did not specify how
doctors would be evaluated. There appeared to be an assumption that doctors would have
to sit examinations comparable to those which the Colleges already delivered; but this
ignored the high level of medical specialisation in the UK. In histopathology alone, it would
have been necessary to deliver a separate examination for every organ system, every year.
Other medical specialties had the same problem. The Colleges had already suffered an
increasing level of regulatory bureaucracy in relation to their postgraduate curricula and
examinations. Legal challenges by unsuccessful candidates were becoming increasingly
common and expensive. For the Colleges, examinations had become increasingly complex
and expensive to run. And the Colleges are professional membership organisations,
maintained largely by subscriptions from their Fellows. It is one thing for an organisation
funded by its Fellows to tell an aspiring young doctor that he or she has not quite reached
the required standard; it is quite another to tell established Fellows who for many years
have paid their College subscription fees that they are no longer fit to work. The Colleges
declined to do as they were asked.
In the White Paper and in subsequent discussions a consensus emerged that, largely
as a result of the problem of extreme medical specialisation, doctors should be expected
to prove their worth only in relation to their own individual scope of practice. It was soon
recognised that the huge diversity of individual medical practice excluded the option of
sitting examinations at intervals. Furthermore, examinations could not cover the crucial
aspect of what doctors actually do, rather than what they are capable of doing under
examination conditions. The idea that revalidation should be based on an annual review
of the whole of a doctors individual practice gained ground. This would be based on the
annual appraisal which was already demanded by most NHS contracts of employment; but
the process of medical appraisal would have to be enhanced and made more formal if it
was to satisfy the requirements of the GMC.
Appraisal and revalidation 151

As a result, the GMC took the lead in defining how annual appraisals might satisfy this
role. The Medical Royal Colleges became sources of advice on how the GMCs general
requirements should be interpreted in the context of a specific specialty [3]. It was
recognised that if revalidation was to be based on appraisals of a doctors actual practice, the
division into relicensing and recertification was unhelpful, so those terms were dropped.
To deliver this, appraisals would have to fulfil two tasks that, in an educational context,
are usually kept separate; the summative question (Is this doctor fit to practise?) and the
formative question (Can this competent doctor be helped to improve his/her performance
even more?). A decision was made that medical appraisal should be made to answer both
questions, but this has led to some confusion. Some individuals and organisations have
pursued their laudable aims of improving overall standards by demanding more and more
from doctors, using the implied but inappropriate threat of removal of the doctors licence
to practise medicine.
So it is important to start a discussion of the UKs new revalidation processes by making
this summative/formative distinction absolutely clear. Everyone including the GMC,
Royal Colleges and patients wants every doctor to work to the highest level they can. That
is the formative element of appraisal, and for most doctors it should be the major part of the
process. The question of whether or not a doctor keeps a licence to practise is ultimately set
by the GMCs Fitness to Practise Panels [4,5] not by the appraiser or by the Royal Colleges,
although the Colleges may give advice to the GMC. That is the summative question. Those
who wish to use medical appraisal to raise the bar of medical practice should recognise
that if appraisals are calibrated to deliver an opinion that is significantly higher than the
level set by the GMC, we will have a system where appraisal suggests that a doctor is not
fit to practice, but then the GMC overturns that opinion. Then what happens? The doctor
returns to work with a grievance. No one benefits.

Appraisal and revalidation

When the system is established, revalidation will be required every 5 years. We are currently
in an interim phase of implementation, where doctors are being asked to deliver the GMCs
requirements in a rather shorter time frame[6]. However, the core requirement is that each
year every doctor either must have an appraisal to GMC standards, or must have a valid
reason for not having such an appraisal (e.g. prolonged sickness or maternity leave). The
requirements for each appraisal, as applied to pathologists, will be discussed below. Every
doctor now has a Prescribed Connection to a Designated Body which will provide a
Responsible Officer (RO). The connection is Prescribed by the relevant legislation; [7]
doctors are not allowed to choose their own. For most pathologists the Designated Body
will be the hospital where they are employed and the RO its Medical Director; for trainees
the RO will be the Postgraduate Dean responsible for their training. For trainees, the
Annual Review of Competence Progression will take the place of annual appraisal and
award of the Certificate of Completion of Training will, in effect, represent revalidation
at that point in time. But many other routes are defined, e.g. for forensic pathologists and
for those working exclusively in the private sector. The relevant regulations are couched in
somewhat impenetrable legal jargon, but the GMC has provided an interactive system on
its website to allow doctors to identify their Designated Body and RO [8].
ROs have a considerable number of responsibilities beyond that of making revalidation
recommendations to the GMC [7]. Most of these relate to ensuring the quality of medical
152 Medical revalidation for histopathologists

care, including monitoring and responding to adverse clinical incidents and complaints.
So it should be recognised that the outcome of appraisals is only one of the factors that ROs
are expected to consider when making a recommendation.
ROs only have three options when making a recommendation to the GMC [4].
1. Recommend revalidation. This is clearly the option preferred by all concerned, but the
doctor must have contributed to the appraisal process all the information that the GMC
demands. Note that the RO merely recommends; the GMC makes the final decision
2. Recommend deferral. This is appropriate when, for some legitimate reason, there is not
yet enough information on which to base a revalidation recommendation. There must
be a plan in place to obtain that information, as the deferral recommendation must
include an indication of when the information will be available and the delay must be
no >1 year.
3. Failure to engage. There is a degree of vagueness here, because there is no option called
Engaging, but not really trying hard enough. It is yes or no. However, if an RO feels that
a doctor is not engaging with the process, the RO should discuss this with the GMCs
local Employment Liaison Advisor (ELA) even if the problem is identified early in the
revalidation cycle. If the ELA is convinced that there is a problem, the GMC will contact
the doctor concerned directly to warn of the consequences. One possible consequence
is that the doctors revalidation date will be brought forward, potentially to only a few
months away. If the doctor still does not comply with the requirements of revalidation,
the next step would be GMC action for failure to engage, an administrative process
which can result in the removal of the doctors licence to practise medicine without
the involvement of any formal assessment of competence by the GMC. So doctors who
think they need not worry about revalidation in the early years of the cycle may find
their revalidation date brought forward dramatically.
There is no option of making a formal do not revalidate recommendation. That is because
any RO who is unable to make any of the above three recommendations should already
have brought their concerns in relation to the doctors standard of practice to the attention
of the GMC. If the GMC is investigating a doctors performance, the revalidation process is

The appraisal process and its requirements for

The required process for medical appraisal for revalidation differs slightly in the various
parts of the UK. The requirements for doctors in England and Wales have been set out by
the Department of Healths Revalidation Support Team, working in collaboration with the
GMC [9,10]. The information that doctors must provide to facilitate an appraisal to GMC
standards has been set out by the GMC [11]. There is abundant detail available from the
websites of those organisations [9,12], so the requirements will only be summarised here,
with emphasis on how they should be interpreted by pathologists.
Initially, the GMC wanted doctors to provide evidence to demonstrate their compliance
with the GMC publication Good Medical Practice (GMP) [13,14] by mapping every item of
information against the four Domains and twelve Attributes of a good doctor, as defined in
GMP. However, when the process was piloted in 10 sites around the UK this mapping was
found to be very laborious [15]. It was therefore replaced by a standardised set of types of
information. The Academy of Medical Royal Colleges demonstrated that this simpler approach
Quality improvement activities 153

could cover all the GMCs requirements. The resultant list of required information is described
in the GMCs publication on Supporting Information for Medical Appraisal and Revalidation
[11]. Guidance on how these general requirements should be interpreted by pathologists has
been published by the Royal College of Pathologists [16] and is only summarised here.

The scope of work

This is not the same as a doctors job plan. The scope of work sets out everything the doctor
does as a doctor. It is not limited to work done for one organisation. Nor is the scope of work
really concerned with when or how much each type of work is done (unless a low workload
might compromise competence or an excessive workload might compromise patient
safety). Even unpaid work should be included; a pathologist who acts as team doctor
to a local sports club each Saturday must declare such work and provide evidence that
appropriate skills are being maintained. The level of responsibility is important; trainees
and noncareer grade doctors will not be expected to demonstrate performance at the level
of a consultant and the weekend volunteer doctor at the side of the playing field will not be
expected to demonstrate the competence of a consultant in emergency medicine or sports
medicine. The scope of work must provide sufficient detail for an appraiser (who might not
be from the same specialty) to assess the relevance of all the other information provided,
and to identify any important omissions.
Delivering a complete scope of work is important, not only because the GMC demands
it but also because in the future it might be argued that a doctor does not have a right to
practice medicine outside that scope of work. This does not mean that extending ones
scope of practice becomes impossible; but it does mean that any changes before the next
appraisal must be accompanied by a documented plan, such as appropriate extra training
or supervision and subsequent audit, to ensure that safe patient care is maintained.

Documentation from previous appraisals

This is important to facilitate continuity of the process across the whole revalidation cycle.
The Personal Development Plan (PDP) agreed at last years appraisal must be reviewed.
This is a particularly important role of the appraisal, to ensure that progress is being made.
For example, a minor problem identified at one appraisal might be dealt with by inserting
an item in the PDP. If, 12 months later, no progress has been made, then that minor
problem would deserve much more serious consideration.

Continuing professional development

It is not sufficient to confirm that the minimum acceptable number of CPD points has been
delivered. The appraisers task includes checking that the CPD activities undertaken cover the
whole spread of a doctors practice. If not, an item should be inserted in the PDP to correct this.

Quality improvement activities

For most specialties, this heading primarily means audit and outcome data. Both are easier
to deliver in some specialties than others. In histopathology, measuring patient outcomes is
problematic; but participation in appropriate external quality assessment (EQA) schemes is
154 Medical revalidation for histopathologists

essential. It is arguable that the detailed reports from EQA schemes should be discussed in
confidence with the appraiser, as low scores on specific cases may be important guides to
focus on CPD activity. But such schemes test what pathologists can do, in a rather artificial
setting; they do not test what pathologists actually do, which is what matters to patients.
So audit remains a requirement. A variety of other sources of information may be relevant,
including turnaround times, rates of seeking second opinions, numbers of supplementary
reports issued, reflective review of the investigation of individual difficult cases and
more. All must be interpreted with caution, and if possible compared against relevant

Colleague and patient feedback

The GMC has defined in some detail the acceptable methods for collecting feedback
about a doctors performance from colleagues and patients [17]. Questionnaires must be
carefully constructed, validated and administered by a third party, to allow responses to be
confidential. Responses should be from a spread of those with whom a doctor works. Such
surveys must be undertaken once in each revalidation cycle, not every year.
Spontaneous compliments and complaints, whether from patients or colleagues,
should also be considered at appraisal. In this process, the doctor will be expected to
discuss how the complaint was handled, and how future practice might be modified to
make similar complaints less likely. If handled well, complaints can be turned into a very
positive contribution to demonstrating compliance with GMP as long as they are not too
numerous or serious, of course.
Patient feedback poses an obvious difficulty for histopathologists and the GMC accepts
that there are circumstances where this may be omitted. But the appraiser will be expected
to challenge the omission; e.g. a histopathologist who sees patients in a fine-needle
aspiration clinic (or who tends to damaged sportsmen at weekends.) has an opportunity to
deliver patient feedback, and should use it.

Significant events
Any serious mishap in the previous year must be discussed. For a histopathologist, this
could be a wrong diagnosis, a misplaced specimen, a seriously delayed report, an accident
in the laboratory and many more. But the number and magnitude of such incidents will
very rarely be a point of major concern. As with complaints, the GMC is rather more
interested in knowing that when things do go wrong, doctors react appropriately and take
action to minimise damage to patients and to prevent a recurrence. As a result, in medical
appraisal the negative implications of something going wrong can be turned into a very
positive affirmation that the doctor handled the problem well.

Statement of health
All doctors are expected to ensure that their own health does not compromise the care of other
patients. The GMC expects a standard statement to be signed to confirm that this is happening.
After the appraisal the documentation 155

Statement of probity
This is another standard GMC statement. Superficially, it seems a little absurd; if someone
formally declares they are telling the truth, how can we know they are not lying? But the
power of this statement lies in the context. If any of the other information provided at
an appraisal is found to be deliberately incorrect, or in some respects even if it is merely
incomplete, this probity statement is demonstrably false. The consequence could be a very
rapid referral to the GMCs disciplinary procedures.

Information relevant to other activities

Supporting Information has to relate to everything a doctor does as a doctor. Clinical
academics will be familiar with the guidance in the Follett Report relating to joint academic
and clinical appraisal; [18] these new GMC requirements do not alter that. Doctors who
are not clinical academics but nevertheless undertake some research will be expected to
provide information relevant to that role if only to confirm that ethics and governance
requirements have been satisfied. Similarly, those who are involved in teaching or
management will be expected to provide information on those roles that is proportionate to
their level of involvement.

After the appraisal the documentation

As with so much in modern medicine, if it is not recorded, it is assumed not to have
happened. At the end of the appraisal, the appraiser and appraisee must agree a PDP
for the coming year, with personal objectives that are SMART (i.e. Specific, Measurable,
Achievable, Relevant and Time-limited). Then the appraiser must complete a summary
of the appraisal. The content of this summary is quite tightly specified, as it is expected
to demonstrate that all the requirements set out in the GMC document Good Medical
Practice have been considered. The appraiser is then asked to make a series of yes/no
statements for the benefit of the RO. These are set out in the Table 11.1. If any of these
questions are answered with a negative, the appraiser is expected to explain the problem.
The problem might be quite trivial; it may be that formal colleague feedback has not yet

Table 11.1 Statements the appraiser is asked to confirm to the Responsible Officer. If any of
these statements cannot be made, the appraiser is asked to explain why
An appraisal has taken place that reflects the whole of a doctors scope of work and addresses the principles and
values set out in Good Medical Practice
Appropriate supporting information has been presented in accordance with the Good Medical Practice Framework
for Appraisal and Revalidation and this reflects the nature and scope of the doctors work
A review that demonstrates appropriate progress against last years personal development plan has taken place
An agreement has been reached with the doctor about a new personal development plan and any associated
actions for the coming year
No information has been presented or discussed in the appraisal that raises a concern about the doctors fitness to
156 Medical revalidation for histopathologists

been delivered, but is planned for next year. But any serious concerns should be escalated
by the appraiser to the RO.

After the documentation the recommendation

ROs are entitled to review, in confidence, the whole of a doctors appraisal record; but in
practice, if no problems have been highlighted by the appraisers, they are not likely to do so
in any detail. The revalidation recommendation is made largely on the appraisers responses
to the five questions in Table 11.1, coupled with the absence of any concerns raised by the
appraiser or identified through other clinical governance channels. There is no right of
appeal at this stage if an RO refuses to make a positive recommendation. The legal reason for
this is complex, but hinges on it being merely a recommendation. The GMC takes the actual
decision, if necessary after having held a full and separate Fitness to Practise hearing. The
final decision is based on that process, not the revalidation recommendation, so any formal
appeal would not be considered until that hearing was complete.

Would this catch the next Harold Shipman?

Probably not. It is reported that Harold Shipman was intelligent, up to date and well liked
by his patients. He was evil but he was not incompetent. It is possible that confidential
colleague feedback might have raised concerns about unusual patterns of mortality amongst
his patients, but it is far from certain. Reform of the UKs archaic death certification process
is far more likely to be successful in early detection of the next Harold Shipman [20]. But
the new system of revalidation does force every doctor to stop and consider, at least once
each year, whether there could be ways in which they can deliver even better care for their
patients. Indeed, preparation for appraisal forces doctors to consider this throughout the
year. It provides reassurance to the public that their doctors are competent and are keeping
up to date. These are developments that are worth some extra effort.

Key points for clinical practice

Revalidation is not optional; if you do not comply with the GMCs requirements you will lose
your licence to practise medicine in the UK, even if you are a perfectly competent doctor.
Revalidation covers everything you do as a doctor, not just your work for one employer.
The GMCs guidance on Supporting Information is essential reading for all UK doctors. But
histopathologists, surgeons and psychiatrists may satisfy those requirements in different
ways; the Medical Royal Colleges provide specialty-specific guidance.
If you collect together the information all year round, a doctor who is properly keeping up
to date, undertaking appropriate EQA and audit should have relatively little extra work to do
to prepare for annual appraisal.
If you leave everything until the last minute, the GMCs demands will seem extremely
Histopathologists can usually be excused from providing patient feedback. But you must be
prepared to justify that omission on the basis of your whole scope of work.
Items in your PDP must be SMART. If you have not made progress in relation to the items in
your PDP at the time of your next appraisal, then that will be a significant cause for concern.
There are mechanisms to make allowances for prolonged absence from work, travel abroad
etc. But you must plan ahead and make sure the right mechanisms are agreed for you.
References 157

1. Smith J. The Shipman inquiry, third report. Death certification and the investigation of deaths by coroners.
London, Home Department and Secretary of State for Health, 2003.
2. Department of Health. The White Paper, Trust Assurance and Safety: the regulation of health professionals.
London: Department of Health, 2007.
3. Academy of Medical Royal Colleges. Revalidation. In: Review 2012-2013. London: Academy of Medical
Royal Colleges, 2013.
4. General Medical Council. Ready for revalidation. Making revalidation recommendations: the GMC
responsible officer protocol. Guide for responsible officers. Manchester: General Medical Council, 2012.
5. General Medical Council. The investigation process. http://www.gmc-uk.org/concerns/the_investigation_
process.asp. (Last accessed 22 March 2013.)
6. General Medical Council. Ready for revalidation. Meeting the GMCs requirements for revalidation.
Manchester: General Medical Council, 2013.
7. Health Care and Associated Professions, Doctors. The medical profession (responsible officers)
amendment) regulations 2013. Statutory Instruments 2013: 391.
8. General Medical Council. My designated body. http://www.gmc-uk.org/doctors/revalidation/12387.asp.
(Last accessed 22 March 2013.)
9. NHS Revalidation Support Team (RST). http://www.revalidationsupport.nhs.uk/index.php. (Last accessed
22 March 2013.)
10. NHS Revalidation Support Team. Medical appraisal guide. A guide to medical appraisal for revalidation in
England. London: NHS Revalidation Support Team, 2013.
11. General Medical Council. Ready for revalidation. Supporting information for appraisal and revalidation.
Manchester: General Medical Council, 2012.
12. General Medical Council. Revalidation. http://www.gmc-uk.org/doctors/revalidation.asp. (Last accessed 22
March 2013.)
13. General Medical Council. Ready for revalidation. The Good medical practice framework for appraisal and
revalidation. Manchester: General Medical Council, 2012.
14. General Medical Council. Good medical practice. http://www.gmc-uk.org/guidance/good_medical_
practice.asp. (Last accessed 22 March 2013.)
15. NHS Revalidation Support Team. Testing and piloting. http://www.revalidationsupport.nhs.uk/events/
about_the_rst/our_performance/Testingandpiloting.php. (Last accessed 22 March 2013.)
16. Royal College of Pathologists. Supporting information for appraisal and revalidation: guidance for
pathologists. London: Royal College of Pathologists, 2012.
17. General Medical Council. Colleague and patient feedback for revalidation. http://www.gmc-uk.org/
doctors/revalidation/colleague_patient_feedback_intro.asp. (Last accessed 22 March 2013.)
18. Follett B, Paulson-Ellis M. A review of appraisal, disciplinary and reporting arrangements for senior NHS and
university staff with academic and clinical duties. London: Department of Education, 2001.
19. Coroners and Justice Act 2009. Notification, certification and registration of deaths. Chapter 2. In: Coroners
and Justice Act 2009, Chapter 25, Part 1. London: Coroners and Justice Act, 2009.
Chapter 12
Molecular testing for human
papilloma virus
Karin Denton

Human papilloma virus (HPV) is an ancient DNA virus infection with which has been
known for many years to be an essential step in the development of cervical cancer [1,2].
The molecular biology of HPV infection and disease progression is understood in detail
and this has allowed the development of numerous testing strategies. There are now over
140 recognised types of HPV and more than 80 of these have been fully sequenced. Typing
is based on variance demonstrated in DNA sequencing, >10% being required to define
a new type. Types are numbered in relation to order of discovery; there is no relation to
In terms of oncogenesis, HPV 16 is the type involved in most cases of cervical cancer
(5362%), with HPV 18 present in 1315%. The frequency of different HPV types present
in cases of cervical cancer varies in different countries and also probably varies on a much
more local level [3,4]. Other HPV types are causative in fewer cases. In total 14 types are
recognised as being associated with cervical carcinogenesis, these are 16, 18, 31, 33, 35, 39,
45, 51, 52, 56, 58, 59, 68 and 66. These are collectively referred to as high-risk HPV.

Cytology screening strategies

Since the publication of George Papanicolaous original article [5] on morphological
changes in cervical cells sampled from the cervix, cervical cytology has been the mainstay
of cervical cancer prevention. However, implementation even in the developed world
is perhaps surprisingly patchy. A true screening programme involves computerised call
and recall from a population registry, invitations and reminders, sample taking by trained
sample takers, quality assured cytology reporting, referral to colposcopy and integration of
the whole into an organised, monitored, quality assured programme. Very few countries are
able to claim that they have achieved this. The four UK screening programmes are the best

Dr Karin Denton, MBChB FRCPath, Director, Cancer Screening Quality Assurance (SW), Public Health England, UK
Email: karin.denton@phe.gov.uk (for correspondence)
160 Molecular testing for human papilloma virus

Cervical screening in the UK

Cervical screening in the UK is often thought of as a single programme but in fact the four
countries have differences in policy. However, many fundamentals, such as the training and
qualification of screening staff and cytopathologists, use of terminology, and colposcopy
training and accreditation are constant.
The English screening programme currently uses cytology as the primary screening
modality with an HPV test to triage borderline and low-grade abnormalities [6]. This is a
new policy, rolled out during 2012, and replaces cytology screening only. The decision to
implement HPV triage was made after an evaluation by the National Institute for Clinical
Excellence (NICE) and a subsequent pilot, known as the Sentinel sites project [7]. HPV
testing is also used for test of cure to allow routine screening intervals after treatment of
premalignant change in the cervix.
Screening intervals in England are 3 yearly from age 25 to 49 and 5 yearly from age 50 to
64. These features have also been implemented in Northern Ireland. In Wales, all patients
are screened at 3 yearly intervals from age 20 to 64 and there is currently no HPV testing in
use, though HPV test of cure may be implemented soon. There is extensive implementation
of automated screening on cytology material. In Scotland, the intervals are 3 yearly from
age 20 to 59 and HPV is only used for test of cure. Automation of cytology reporting has also
been implemented.

Quality assurance and training

In England, quality assurance is highly structured, and includes the many organisations
which contribute to the commissioning, specification and delivery of programme. Trusts
and individuals are covered by professional requirements and broader quality, health and
safety and other regulation. What distinguishes cancer screening programmes from other
forms of care is the presence of quality assurance as part of the national programme. From
1st April 2013 this function now resides within Public Health England, which is part of the
Department of Health. In order to be commissioned to provide cervical screening services,
providers must meet nationally specified quality standards and these cover a wide range of
both process and outcome measurements.

All four UK countries, in common with elsewhere in the world, have implemented a
programme of HPV immunisation. From 2008 HPV immunisation was offered to girls
aged 1213 through to 18. The only country to implement a national schools-based
immunisation programme earlier was Australia in 2007. Initially the UK procured a
bivalent vaccine against HPV 16 and 18 only, but in 2012 a quadrivalent vaccine including
also HPV 6 and 11, which are responsible for most cases of genital warts, was nationally
commissioned. Coverage in 1213 years olds, delivered to girls through their schools,
has been very high (around 80% in the areas with highest coverage) but the catch up
programme to older girls has never enjoyed such high coverage (around 3035%).
All data suggest that these vaccines are highly effective in preventing infection with HPV
type 16 and 18 in a naive population, and immunisation will definitely have an impact on
the prevalence of HPV-related disease in the future. However, it is important to note that
whilst immunised women have started to be screened in Scotland and Wales; in England
this has not yet occurred as they have not reached the age of 25. Furthermore, the effect will
Potential uses of HPV technology 161

be limited initially due to low coverage in this cohort, and high prevalence of active HPV
infections at the time of immunisation in 1618 years olds. Women immunised at age 1213
will not reach screening age in England until 2020.

Strengths and weaknesses of existing programmes

In the English screening programme, coverage currently stands at 79% overall, though
only at 65% for women aged 2529 [8]. This is a major concern as it is clear that the key to
success in cervical cancer prevention lies in maintaining a high coverage.
The existing UK cytology screening programmes are successful. It is estimated that
up to 80% of cases of cervical cancer which would otherwise occur are prevented by the
programmes [9]. An audit of cases of cervical cancer [10] reveals that not being screened
is a significant risk factor for development of the disease. Since the advent of national
quality assurance structures and detailed programme specification, the programme is more
uniform than ever before and it is reasonable to assume that the sensitivity of cytology,
already probably the highest in the world [11,12], is close to being maximised.
And yet, women who have been screened continue to be diagnosed with, and die from,
cervical cancer. Sometimes on review of the cytology, missed abnormalities are found, but
on other occasions, cervical cytology samples appear to be of adequate quality but with no
abnormal cells present [13].
So sensitivity of the test may be a weakness, but the specificity of the cytology test is
very good. In England, around 80% of women with a high-grade cytology abnormality
have cervical intraepithelial neoplasia 2 (CIN2) or worse at colposcopy with a further
approximately 10% having CIN1. Very few women with high-grade cytology do not have an
abnormality and this indicates the very high specificity of the test.
But the great strength is the organised nature of screening. Even in countries where there
is often quoted to be good organised screening, e.g. Finland, a large number of samples are
taken outside the screening programme. In the United States and Australia, most screened
women have health insurance. Overall population coverage may be lower and because this
is a service provided in private laboratories, detailed specification and quality assurance
are much more difficult to achieve. In an organised programme, it is possible to introduce
changes in an equitable and controlled way. This is a huge strength of the NHS cervical
screening programme which has been used to implement HPV testing.

Potential uses of HPV technology

HPV testing can be used in the following models of provision:
1. Triage of low-grade cytology abnormalities
2. Test of cure checking complete reversion to negative after treatment of CIN
3. HPV as a primary screening test

HPV testing in triage and test of cure have been extensively evaluated [7]. The previous
model for the treatment of low-grade abnormalities was to repeat cytology in 6 months.
The rationale for this was to detect HPV persistence, but it had the disadvantage of poor
compliance with follow-up, delay and inconvenience for women, and also low detection rate
of high-grade disease in women eventually referred to colposcopy. Triage allows women
who are HPV negative to revert to routine recall as their risk of having significant disease is
162 Molecular testing for human papilloma virus

extremely low, and allows selection of the higher risk women for immediate colposcopy,
reducing problems with attendance. However, the detection rate of CIN2+ at colposcopy is
still low at around 15% [7]. Triage is cost-effective and is popular with women.

Test of cure
Previously, women having treatment of high-grade CIN would have annual cytology tests
for 10 years, a significant imposition. Although the detection rate of residual disease was
low, it was always felt that these women were at higher risk than average and needed
enhanced surveillance. Around 85% of women have negative results for both cytology and
HPV at their first follow-up visit, and the facility to return these women to routine recall is
hugely beneficial.

Primary screening
HPV testing has been evaluated in primary screening in many countries for many years.
Several large randomised controlled trials have been undertaken to assess whether this
type of screening gives better outcomes than cytology screening [12,14,15]. Most trials have
used the oldest and best-established HPV test, Hybrid Capture II (Quiagen). It has always
been apparent that the sensitivity of HPV testing for CIN with this technology is very high,
but the specificity is low. HPV infection in the absence of CIN2+ is extremely common with
50% of 1820 years olds testing positive at cytology [16] Even by age 25 this figure is unlikely
to drop below 30%.

Strengths and limitations of HPV testing as a primary screening test

The strength of HPV primary screening is its sensitivity, and the fact that this can be
achieved more easily than with a cytology-based programme, which requires a huge
training and quality assurance effort to get similar sensitivity. Of all the randomised
controlled trials, the only one which approached equality was ARTISTIC, the English
trial. Distinguishing factors were that this was the only one which used liquid-based
cytology (LBC) and the only one which took place in the setting of a highly quality assured
programme, which probably accounts for the better performance of cytology. Even so,
by the third round it was apparent that HPV primary screening gave a longer duration of
protection than a negative cytology result [12,16].
The limitation of HPV testing is always its specificity. Clinically, a positive HPV test
result can only prioritise for further investigation, whereas a negative HPV result is very
useful and can provide reassurance. HPV testing may assist in management of women with
difficult to interpret cytology, especially after treatment. UK guidance is that this off label
testing should only be undertaken after discussion by the multidisciplinary team.
HPV testing also offers a possible route to self-testing. This is not possible with cytology,
but early results suggest a self-administered HPV test is fairly reliable (though not as good
as a professionally taken sample). This may offer a route to reach women who do not
respond to an invitation to be screened.

The genetics of HPV

HPV contains six early and two late genes. E1 is involved in viral replication, and E2 in
transcription regulation. There is no E3 (numbering was originally devised in bovine papilloma
Which HPV test? 163

viruses which do have an E3). E4 disrupts cytokeratins; E5 allows interaction with growth
factor receptors. The two genes of interest in oncogenesis are E6 which is involved in p53
degradation and E7 which binds the retinoblastoma gene, Rb. The L1 and L2 genes code for
capsid proteins. The L1 capsid protein is used in vaccines to generate an immune response.

Which HPV test?

Commercially, there has been much interest in developing HPV testing technology and
it is believed that over 100 systems are commercially available. However, the testing and
reliability for these are very variable. Even amongst those with robust analytic testing, very
few have been evaluated for clinical rather than analytic performance. This is an extremely
important distinction because unusually amongst virology investigations, testing is not for
the presence of the virus itself, but it uses the virus as a surrogate marker of disease. In this
context, extreme sensitivity may not be an advantage.
All the large trials of HPV in a screening setting used Hybrid Capture II as the original
and longest established test. In the UK other platforms have been compared and found to
have comparable sensitivity and equal or improved specificity. The five tests approved for
use in the UK are Hybrid Capture II (Digene) using ThinPrep or SurePath LBC samples with
2 relative light unit (RLU) cut-off, Abbott Real Time high-risk HPV on ThinPrep or SurePath
LBC samples, Roche Cobas 4800 HPV system on ThinPrep or SurePath LBC samples,
Genprobe Aptima HPV assay using Tigris or Panther platforms for ThinPrep LBC or the Tigris
for SurePath LBC samples, and Hologic Cervista HPV test using ThinPrep LBC samples.

Hybrid Capture II
In this test, RNA probes react to DNA targets for 13 high-risk HPV types. The RNADNA
hybrids are detected with a monoclonal antibody to the hybrids and this is then subject
to chemiluminescent detection. There are manual and automated versions available,
but there is still a significant operator requirement. Results are very reproducible and the
system is robust and reliable.
Original evaluations were performed using a cut-off of 1 RLU, but following evaluation in
the ARTISTIC trial [16] a cut-off of 2 RLU is used in England. This is only a semi-quantitative
method, and again the key is to correlate RLU score not with presence of high-risk HPV,
but with presence of CIN2+. It should be noted that in fact quantitation of HPV testing on
cervical cytology samples is illusory, because the samples themselves vary enormously in

Abbott Real Time and Roche Cobas

These two systems utilise real-time polymerase chain reaction (PCR) to detect HPV. Both
can be used to identify HPV types and give results for HPV 16, HPV 18 and HPV high-risk
non-HPV 16HPV 18. This feature may have programme utility which is discussed below.
Both platforms are also used extensively for other diagnostic tests, mainly within virology,
and this has also influenced choice of platform.

Genprobe Aptima
This is the only approved method which detects RNA rather than DNA. The significance
of this is that it should be more specific. HPV infection in its early stages does not lead to
164 Molecular testing for human papilloma virus

integration with host DNA. Whilst other HPV tests detecting viral DNA will be positive,
until E6/E7 has been integrated, this test will remain negative. Integration is the first key
step in oncogenesis. Genprobe uses a transcription-mediated amplification methodology.
The system is highly automated and capable of high-volume testing. Genprobe is also used
for other virological investigations including hepatitis, HIV and Chlamydia, and again
these platforms have been widely implemented in laboratories.

Hologic Cervista
This utilises a novel detection method, invader probe chemistry. This achieves a 110
million fold amplification of HPV DNA. HPV 16 and 18 typing can also be performed as a
reflex using genotype-specific probes.

Significance of HPV 16/18 genotyping

A large US study [17] showed that testing positive for type16/18 HPV conferred about
the same risk as a cytology sample showing ASCUS (equivalent to Borderline change
in UK terminology) for presence of a CIN2+ lesion, whereas the risk of non-HPV 16/18
high-risk types was much lower. This evaluation though is sensitive to variations in
quality of cytology and it remains to be seen whether this result will be replicated in
the UK.

Are all HPV tests the same?

In the English screening programme, a comprehensive evaluation was performed
comparing these five tests with different LBC samples in a triage setting. Results were
surprisingly consistent. As expected, the Genprobe system showed a higher specificity
but the differences were not great. However since this evaluation was carried out, work
elsewhere has demonstrated that there may be greater variation in performance when used
in different settings
Cuzick et al. [18] showed a variation between 10% and 16% positivity in a primary
screening setting and the Danish Horizon studies [19] are raising questions about
reproducibility, again in a screening setting.

Worldwide applications
The five tests described above are only applicable to organised testing in rich countries. Not
only are the tests relatively expensive (though price is reducing with market forces), they
require reliable power sources, mains water and a relatively clean environment. The testing
equipment is large, fragile and not easy to move.
In resource poor settings, which have by far the greatest incidence of cervical cancer,
other tests may be more appropriate. There is extensive experience that introducing
cytology-based screening in these settings is difficult, but limited HPV testing is feasible.
There are at least two HPV tests under development which may well be better suited to
these settings. These are the Care HPV test which uses Hybrid Capture II chemistry and
the lateral flow test. Development of both is being supported by the Bill and Melinda Gates
Foundation. Both require no electricity, running water or refrigeration and give a fast
Practical implications of implementing HPV testing in
cervical screening in the UK and elsewhere

Because of the poor specificity of a positive HPV test result for CIN2+, attention has turned
to indirect markers to see if these could be used to refine the result. Of course, cytology
could be included as an indirect marker and in fact retains the highest specificity of any test
of CIN2+. This is the reason that in the UK HPV primary screening pilots which commenced
in 2013, cytology is being used to triage those samples which test positive for HPV.
Another indirect test which has attracted much attention is P16, a cell cycle protein
which is over expressed when E7 binds to pRB, and which can be detected with
immunohistochemistry. Detection of P16 in histology samples is a useful means to confirm
CIN in difficult cases, and to assist with grading. Dectection in cytology samples, either
on its own or in combination with a proliferation marker, Ki67, looks very promising as a
way of improving the specificity of a HPV positive, cytology low-grade sample [20,21]. An
evaluation in the NHS is due to start in 2013. A similar concept underlies ProExC (Beckton
Dixon), a composite marker comprising identifiction of mini chromosome maintenance
(MCM) protein and topoisomerase II (TOP2A) a marker of cell proliferation.

Practical implications of implementing HPV testing

in cervical screening in the UK and elsewhere
The move to HPV triage of low-grade cytology and test of cure has involved some
reconfiguration of laboratories to achieve a cost-effective outcome, but in reality, these
reconfigurations may have occurred anyway as small cytology laboratories came under
commissioning scrutiny. However, any move to HPV primary screening would result in a
very significant fall in the number of cytology samples processed, probably in the order of
80%, and this would inevitably lead to further centralisation of HPV testing and cytology
into a small number of high volume expert centres.
The pilot of HPV primary screening will demonstrate what the practical implications are
in terms of cytology, colposcopy and in fact the whole screening programme. No part of
screening is unaffected by this, the most major change ever contemplated. The implications
stretch from information technology, to communication with women, to sample taker
training and to colposcopy. Such a major re-engineering of what is already a successful
programme represents a huge challenge. This is probably the reason why no other national
screening programme has implemented this change, though advisors in several countries
have recommended that they should do so.
It also means that cervical cytology, long regarded by some as a somewhat unfashionable
branch of pathology, is at the cutting edge of molecular diagnostics. A cohort of scientists
crossing the divide between morphology and molecular techniques will be needed to
implement the change and cervical screening, with potentially 4 million samples per year in
England alone, will become one of the largest users of molecular technology in pathology.
In the context of an already successful programme of cervical cancer reduction this is
exciting, but the implications of worldwide implementation of at least some of the benefits
of HPV testing may lead to even greater gains. However, there is need for caution, because
getting a positive HPV test result in a resource poor setting is perhaps the easiest part of the
process. Without colposcopy, histology and treatment of early cervical cancers, all of which
are absent in many of these settings, women will not benefit.
166 Molecular testing for human papilloma virus

Key points for clinical practice

Cervical cancer screening with cytology, as practiced in the UK, is highly effective but may
be reaching a point where further improvements are difficult to achieve.
Changes can be evaluated and implemented in the UK screening programme in a controlled,
quality assured and equitable way.
Primary screening for HPV offers improved sensitivity but lower specificity compared with
Further investigations including cytology and molecular markers are required to improve
Implementation of HPV vaccine necessitates a change in approach to screening.
Many different HPV tests are available; five are approved for use in the UK.
Although performance in a triage setting appears to be very similar, HPV tests may behave
differently in a primary screening setting.
The greatest burden of cervical cancer is in low resource settings, and novel technological
solutions will be required for use in these settings.

1. zur Hausen H. Papillomaviruses in human cancer. Appl Pathol 1987; 5:1924.
2. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive
cervical cancer worldwide. J Pathol 1999; 189:129.
3. Kirschner B, Junge J, Holl K, et al. HPV- genotypes in invasive cervical cancer in Danish women. Acta Obstet
Gynecol Scand 2013 Jun 13. doi: 10.1111/aogs.12197
4. Wheeler CM, Hunt WC, Joste NE, et al. Human papillomavirus genotype distributions: implications for
vaccination and cancer screening in the United States. J Natl Cancer Inst 2009; 101:475487.
5. Papanicoloaou G, Traut H. Diagnosis of uterine cancer by the vaginal smear. 1943.
6. Achievable standards, benchmarks for reporting and criteria for evaluating cervical cytology. NHS CSP
publication 1, 3rd edition, 2012.
7. Kelly RS, Patnick J, Kitchener HC, Moss SM; NHSCSP HPV Special Interest Group. HPV testing as a triage for
borderline or mild dyskaryosis on cervical cytology: results from the Sentinel Sites study. Br J Cancer 2011;
8. http://www.cancerscreening.nhs.uk/cervical/publications/cervical-annual-review-2012.pdf
9. Peto J, Gilham C, Fletcher O, Matthews FE. The cervical cancer epidemic that screening has prevented in
the UK. Lancet 2004 36:249256.
10. Castanon A, Leung VM, Landy R, Lim AW, Sasieni P. Characteristics and screening history of women
diagnosed with cervical cancer aged 20-29 years. Br J Cancer 2013 Jul 2. doi: 10.1038/bjc.2013.322.
11. Cuzick J, Szarewski A, Cubie H, et al. Management of women who test positive for high-risk types of human
papillomavirus: the HART study. Lancet 2003; 362:18711876.
12. Kitchener HC, Gilham C, Sargent A, et al. A comparison of HPV DNA testing and liquid based cytology over
three rounds of primary cervical screening: extended follow up in the ARTISTIC trial. Eur J Cancer 2011;
13. Castanon A, Ferryman S, Patnick J, Sasieni P. Review of cytology and histopathology as part of the NHS
Cervical Screening Programme audit of invasive cervical cancers. Cytopathology 2012; 23:1322.
14. Leinonen MK, Nieminen P, Lnnberg S, et al. Detection rates of precancerous and cancerous cervical
lesions within one screening round of primary human papillomavirus DNA testing: prospective
randomised trial in Finland. Br Med J 2012; 345:e7789.
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Cancer 2012; 107:19171924
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16. Kitchener HC, Almonte M, Thomson C, et al. HPV testing in combination with liquid-based cytology in
primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol 2009; 10:672682
17. Stoler MH, Wright TC, Sharma A, et al. High-risk human papillomavirus testing in women with ASC-US
cytology: results from the ATHENA HPV study. Am J Clin Pathol 2011; 135:468475.
18. Cuzick J, Cadman L, Mesher D, Austin J, Ashdown-Barr L, Comparing the performance of six human
papillomavirus tests in a screening population. Br J Cancer 2013; 108:908913.
19. Preisler S, Rebolj M, Untermann A, et al. HPV detection in Sure Path samples using Roche Cobas real-time
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20. Schmidt D, Bergeron C, Denton KJ, Ridder R; European CINtec Cytology Study Group. p16/ki-67 dual-stain
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LSIL pap cytology results. Am J Clin Pathol 2010; 134:1221.
Chapter 13

Tensins in health and disease

Maham Akhlaq, Hannah Thorpe, Mohammad Ilyas

The organs of multicellular organisms execute specialist functions and usually consist of a
mixture of cells embedded in extracellular matrix (ECM) and arranged in a complex three-
dimensional structure. During organogenesis, cells need to migrate to the appropriate
topographical location and stay there in order to maintain tissue integrity (although
some further movement may be required during organ turn-over). Following injury, cells
may need to migrate into damaged areas and then undergo remodelling into the normal
structure as part of tissue repair. The processes of organogenesis, organ homeostasis and
repair require modulation of cell adhesion to enable cell motility and maintain cell stasis.
Cell adhesion can be viewed as intercellular adhesion (i.e. adhesions between cells) or
cell-to-matrix adhesion. The former is maintained through intercellular junctions, whilst
the latter is maintained predominantly through focal adhesions. In both cases, these
represent points of contact between cells or between the cell and the ECM. They consist of
multimolecular complexes located at the cell membrane which are linked internally to the
cytoskeleton. In response to environmental cues, these complexes can be assembled or
disassembled thereby allowing the cells to become attached/detached from each other and
the ECM which in turn regulates cell motility. An in-depth description of the intercellular
junctions is beyond the scope of this review, but they are briefly described and shown
figuratively in Table 13.1 and Figure 13.1.

The structure and function of focal adhesions

Focal adhesions are central to cellular attachment of cells to the ECM. Critical to the
formation of focal adhesions are integrins, these cell membrane proteins consist
of a heterodimeric combination of one of 18 different -subunits and 8 different

Maham Akhlaq, MBBS, MPhil, Division of Pathology, School of Molecular Medical Sciences, University of
Nottingham, Queens Medical Centre, Nottingham, UK
Hannah Thorpe, BSc, MSc, Division of Pathology, School of Molecular Medical Sciences, University of Nottingham,
Queens Medical Centre, Nottingham, UK
Mohammad Ilyas, BSc, MBChB, PhD, FRCPath Division of Pathology, School of Molecular Medical Sciences,
University of Nottingham, Queens Medical Centre, Nottingham, UK
Email: mohammad.ilyas@nottingham.ac.uk (for correspondence)
170 Tensins in health and disease

Table 13.1 Intercellular junctions and adhesions acting to regulate cellular adhesion and motility
Cell junction Structure and function
Tight junctions Attachment regions formed below the apical surface; composed of transmembrane
occludins, claudins and also zonula occludens proteins which link to the cytoskeleton. These
junctions act to regulate the transport of small molecules and ions, preventing their passage
between cells, and to prevent movement of receptors between apical and basolateral
surfaces thereby maintaining correct function
Adherens junctions Areas of strong mechanical attachment composed of cadherins. Intracellularly these connect
to , and p120 catenins which link to the actin cytoskeleton and contribute to its regulation
Gap junctions Composed of connexin proteins which form a channel structure between neighbouring cells.
Clusters of channels in specialised cellular regions permits passage of small molecules and
ions between adjacent cells, regulating numerous physiological events
Desmosomes Areas of strong cell attachment linked to intermediate filaments. Primarily composed of
membrane desmosomal cadherins and internally, armadillo and plankin proteins together
conferring stability to cell types under frequent mechanical stress

Tight junctions :
ZO proteins

Desmosomes :
Intermediate filaments

Actin cytoskeleton
Gap junctions :

Adherens junctions : Figure 13.1 Protein complexes

Cadherins form intercellular junctions and
-catenin allow cellcell adhesion, a process
-actinin critical for maintenance of tissue
Actin filaments structure and regulation of
physiological events.

-subunits. There are at least 24 different combinations seen and, depending on the
combination, the integrins act as receptors for a variety of different ECM proteins.
Integrins are transmembrane molecules which are attached to the actin cytoskeleton
[1]. This attachment is mediated by a network of proteins clustered on the cytoplasmic
side of the focal adhesions and includes proteins which have structural and signalling
roles (Figure13.2). These include kinases, phosphatases and adaptor molecules and
demonstrate that, in addition to mediating a physical connection from the cell interior to
The structure and function of focal adhesions 171

Extracellular matrix

Paxillin Tensin
p130 cas
-actin Figure 13.2 A typical focal
in adhesion; - and -integrin
Zyxin molecules externally attach
to the extracellular matrix and
Cell intracellular, cytoplasmic tails
Actin fibres connect to a network of proteins
and the actin cytoskeleton.

exterior, focal adhesions also serve roles as signalling platforms, both of which properties
play a pivotal role in regulating cell adhesion.
The life-cycle of focal adhesions can be viewed as consisting of assembly, maturation
and disassembly. Initial binding of integrin heterodimers with the appropriate extracellular
ligand will activate the integrins. Some integrins, such as 1, occur in a high affinity form,
whilst others, such as 3, may require conformational change before activation (known as
outside-in activation). Either way, activation will result in recruitment of adaptor proteins
and cytoplasmic changes resulting in activation of other integrin receptors (known as
inside-out activation) and clustering of integrin receptors around the site of cellECM
contact [2]. The clustering and inside-out activation seem to be mediated by molecules
such as talin and may be metalion dependent [3]. It results in a positive feedback loop
and the formation of early focal adhesions, also known as nascent adhesions, where the
ECM and the cellular cytoskeleton are first linked [2]. The nascent adhesions may undergo
dissolution or, following Rac-dependent actomyosin contraction, may expand to form focal
complexes. Rapid formation and dissolution of nascent adhesions and focal complexes is
a feature of migratory cells but, in the appropriate cellular context, they may progress to
mature focal adhesions which are characterised by contraction of myosin and activation
and recruitment of molecules such as vinculin [4]. This exerts tension on the focal
adhesions and stabilizes the connection with the actin cytoskeleton. In stable nonmotile
cells, the maturation of the focal adhesions is completed by the formation of large actin
stress fibres and the recruitment of Tensins and, in this state, they are sometimes referred
to as fibrillar adhesions [5]. Disassembly of focal adhesions can be mediated through
ubiquitination and proteasomal degradation of talin or degradation of the ECM thereby
removing the signals for integrin activation.
Cell adhesion is critical for cell motility with constant cell attachment at the leading
edge of the cell and detachment at the posterior edge assisting the cyclic process of
cellular protrusion, attachment and traction. The first stage of cell migration involves the
formation of lamellipodia at the leading edge of the cell which protrudes and attaches to
the substratum via focal adhesions. Contact regions at the rear of the cell detach due to
disassembly of focal adhesions and the bulk of the cell follows to relieve tension giving
172 Tensins in health and disease

a shift in cell position [6]. Focal adhesion-mediated cell migration is enabled in part by
activation of Ras homology family member A (RhoA) and subsequently Rho-associated
protein kinase (ROCK), leading to phosphorylation of downstream substrates including
Myosin2. RhoA promotes stress fibre formation contributing to actomyosin contractility
mechanisms which together with activation of mDia1 (also via RhoA) promotes the
formation of thick, parallel actin stress fibres extending from focal adhesion regions.
Together in concert with other signalling axes, continuous cycles of stress fibre attachment
and detachment drive cell migratory processes.
Tensin proteins are localised at focal adhesions and are amongst the molecules acting
as molecular bridges between integrins and the actin cytoskeleton. The Tensin family
members play pivotal roles in key cellular processes including adhesion, migration,
proliferation, differentiation and apoptosis [7]. Recent years have seen an accumulating
interest in Tensin biology and this is the subject of the remainder of this review.

The Tensin gene family

The Tensin gene family consists of four members: Tensin 1 (TNS1, OMIM 600076), Tensin
2 (TENC1, OMIM 607717), Tensin 3 (TNS3, OMIM 606825) and Tensin 4, commonly called
C-terminal Tensin-like, Cten (TNS4, OMIM 608385). These genes encode multidomained
cytoplasmic proteins which, with the exception of Cten, have extensive homology sharing
several functional N- and C-terminus domains yet with divergent central regions. Cten, as
the name suggests, has homology with the other proteins at the C-terminus but is lacking
N-terminus domains which are found in the other members of the family (Figure 13.3).
Tensin 1 maps to chromosome 2q 35-36 and comprises 33 exons. It encodes a 1735
amino acid protein with a predicted molecular mass of 185kDa. Tensin 1 is present in
most tissues with highest levels found in the human heart, skeletal muscles, kidney and
lung when examined by northern blot. Tensin 2 maps to chromosome 12q13, has 29 exons
and encodes a 1285 amino acids with a molecular weight of 170kDa [8]. It was identified
as a protein related to Tensin 1 through homologous N- and C-terminal regions with 60%
and 67% sequence similarity respectively. Tensin 2 shows a similar tissue distribution to
Tensin 1 and is present in most tissues investigated, but it is particularly abundant in heart,
skeletal muscle, liver and kidney [8]. The Tensin 3 gene is localised at 7p12.3. It encodes a
1445 amino acid protein and has a molecular mass of approximately 155kDa. Northern
blot analysis has revealed its expression in most tissues, but it is particularly prevalent in
the placenta and kidney with the spleen, lung, skeletal muscle and heart showing lower
expression [9]. The Cten gene maps to chromosome 17q21.2 and encodes a 715 amino acid
protein with a mass of 77kDa. It is a smaller protein than Tensins 13 due to the absence
of the N-terminus domains (see below) and, in contrast to the other Tensins, expression is
restricted to placenta and prostate tissues [7].

The structure of Tensin proteins

Tensins 13 have highly homologous N- and C-termini but are divergent in their central
regions. In general, the N-terminus comprises actin-binding domains (ABD), whilst the
C-terminus contains a Src-homolgy 2 (SH2) domain and a phosphotyrosine-binding
domain (PTB) (Figure 13.3).
The structure of tensin proteins 173

Binds to sides of F-actin Binds to NPXY motif of -integnin

fibres Binds PP1 tail at focal adhesions (FAB-C)

1 263 463 888 989 1735 aa

Tensin 1
Binds to focal adhesions Weakly caps barbed ends Bind DLC1
(FAB-N) of F-actin filaments 1410 aa
(N-Terminus) Tensin 2 (C-Terminus)
(C1ten) Bind DLC1
Protein Kinase C1 1445 aa

Tensin 3
715 aa

Bind DLC1

Figure 13.3 A detailed diagrammatic representation of the Tensin family protein structure and its binding partners.
Cten lacks the N-terminal common region, which contains the actin-binding activity, one of the two focal adhesion-
targeting regions and the phosphatase-like domain.

Tensin 1 has 60% homology with chick Tensin and contains SH2 and ABD regions
similar to the tumour suppressor phosphatase and Tensin homolog (PTEN) [10]. Tensin 1
has been shown to have actin-binding capabilities through two distinct binding sites in the
ABD at residues 1263 and 263463, although an extra actin-binding site is also present in
the central region at residues 889989. The latter region shares high sequence homology
to insertin (an actin capping protein which retards globular actin polymerisation) and has
therefore been proposed to function similarly. However, later data have contradicted this
suggesting that a barbed end capping mechanism is responsible for actin polymerisation
and depolymerisation through this region [11]. Although PTB domains are known for
binding phosphorylated tyrosines, it is through this domain that Tensin 1 (and the other
Tensins) binds to the NPXY motif on the cytoplasmic tail of -integrins independently
of phosphorylation. The SH2 domain, also located at the C terminus, enables Tensin 1 to
bind phosphorylated tyrosine residues on proteins such as focal adhesion kinase (FAK),
deleted in liver cancer (DLC) 1 and phosphoinositide 3 (PI3) kinase. Tensin 1 also contains
phosphorylated tyrosine residues thereby linking an SH2 containing cytoskeletal protein
with signal transduction to the actin cytoskeleton. At both the N- and C-termini, there
are focal adhesion-binding (FAB) domains which are required for localisation to focal
The structure of Tensins 2 and 3 are similar to that of Tensin 1. Neither contains a central
insertisn region and therefore may not fulfil an actin-capping role as seen in Tensin 1. The
central region of Tensin 2 is found to be proline rich and could potentially act as a binding
site for proteins containing Src homology 3 (SH3) or WW domains [12]. Tensin 2 is the
174 Tensins in health and disease

only member of the Tensin family that has a protein kinase C domain at the N-terminus.
Tensin 3 contains 32 tyrosine residues, 13 of which are predicted to be potential sites of
phosphorylation and possible candidates for signal transduction [9].
Cten shows a high degree of homology at the C-terminus and is thereby included in the
Tensin family of proteins albeit as a more distant and more recently evolved relative. In
contrast to Tensins 13, it does not possess the N-terminus ABD domains which suggest
that Cten still contains the signalling component of other Tensins but lacks the actin-
binding capability and thus may play a novel role in cellular processes [7]. Although focal
adhesion localisation is common amongst all the identified Tensin family members, Cten
has also been detected in the nucleus [13].
As the general structure of the Tensin family of proteins comprises both domains for
binding to cytoskeletal proteins and signal transduction components, they fascinatingly
provide the first link between these two cellular modules. The Tensins have each been
linked to multiple upstream and downstream signalling factors and have also been shown
to differentially interact with such components to aid in mediating their response. Lack of
embryonic lethality in murine knockout models suggests functional redundancy of Tensins,
but the divergent regions also suggest distinct roles.

Functional activity of Tensins

Cell adhesion and motility
Although the exact roles of the individual Tensins are debated and in some circumstances
could be tissue dependent, they are important in both stabilising cell adhesion and
regulating cell motility. Recruitment of Tensins to the focal adhesions is dependent on the
state of maturation of the focal adhesions. Thus, Tensin 2 is recruited to focal complexes
(early stage focal adhesions) and, through its PTB domain, it can bind DLC1. This is
responsible for localisation of DLC1 to focal complexes and it is thought to inhibit DLC1
function [14]. DLC1 functions to stimulate Rho-GTPase activating (RhoGAP) proteins and
thereby inhibits RhoA activity, and thus, when localised to focal complexes, Tensin 2 may
promote cell motility [15]. In contrast, Tensin 1 and Tensin 3 are found in the mature form
of focal adhesions known as fibrillar adhesions [15] (Figure 13.4). Tensin 1 has DLC1-
binding sites in the SH2 domain, whilst Tensin 3, in contrast to the other Tensins, has
DLC1-binding sites located in the ABD. The interaction between Tensin 3 and DLC1 causes
a conformational change in the latter, resulting in an active Rho-GAP activation function.
This leads to increased RhoA-GTP hydrolysis converting it to the inactive GDP-bound state
and inhibiting cell motility [16].
The role of Cten, in direct contrast to that of Tensins 1 and 3, is to stimulate cell motility
and this is executed through two mechanisms. Firstly, it has been shown that following
activation of epidermal growth factor receptor (EGFR) signalling, Tensin 3 levels decrease
whilst cellular Cten levels simultaneously increase (without seemingly affecting the other
Tensin proteins). The Cten displaces Tensin 3 from the focal adhesions and this is known as
the Tensin Switch. As Cten lacks the N-terminus ABD, it is unable to bind the actin stress
fibres, resulting in detachment of focal adhesions from the actin cytoskeleton. This switch
gives rise to actin remodelling and further cytoskeletal rearrangements to provide optimal
conditions to allow for cell migration. Secondly, Cten, through its SH2 domain, can also
bind to DLC1 and there is effectively another switch, whereby Cten replaces Tensin 3 to
Functional activity of tensins 175

Focal adhesions 5
Tensin 1 and Tensin 2 1
Paxillin and Tallin
Tyrosine phosphorylated
proteins increased
Tensin 2
Leading edge Paxillin

Direction of movement Focal adhesions:

Tensin 2
Actomyosin mediated
contractility DLC1
Matrix remodelling
Actin polymerisation Rno A



Retraction Fibrillar adhesions:

Tensin1 and Tensin 3
Tyrosine phosphorylated
proteins decreased
Stress fibers

Actin filaments
Focal adhesions
Fibrillar adhesions

Figure 13.4 In a moving fibroblast Tensin 2 is located towards the leading cell edge causing matrix remodelling,
whilst Tensin 1 and Tensin 3 are present in the cell body in the fibrillar adhesions.

form a complex with DLC1. This results in an inhibition of DLC1 which no longer promotes
Rho-GAP activity, leading to increased RhoA-GTP-mediated signal transduction via ROCK
and subsequent motility [16] (Figure 13.5).
In addition to DLC1, the Tensin proteins can activate and are targets of a variety of other
signalling molecules. FAK has been found in complex with Tensin 1 as well as vinculin,
suggesting that Tensin 1 might mediate signal transduction through this interaction [17].
Conversely, growth hormone stimulation has been shown to cause FAK-mediated tyrosine
phosphorylation of Tensin 1 [18]. Forced expression of Cten in epithelial cell lines has been
shown to cause upregulation and stabilisation of both FAK and integrin-linked kinase (ILK)
together with an associated stimulation of cell motility [19]. Knockdown of FAK and ILK
when Cten has been overexpressed and has resulted in loss of cell motility suggesting that
part of the functional effect of Cten is mediated through these proteins [19,20].
The induction of cell motility by forced expression of Cten is associated with epithelial
mesenchymal transition. This is accompanied by downregulation of E-cadherin and
demonstrates that there is crosstalk between adherens junctions and focal adhesions,
although the signalling pathways involved in this interaction have not been clarified [21].
Obvious candidates would be the ILK and FAK signalling pathways, but, either way, it
demonstrates how activity at the different adhesion junctions can be coordinated to allow
cell adhesion and migration in accordance with cellular requirements.
176 Tensins in health and disease

Steady state G G EGF stimulation



-catenin R -catenin

Tallin Tallin -catenin

Paxillin Paxillin Cten SH2 PTB
ABD Tensin 3 SH2 PTB

Actin filaments

Rho A Rho A

Actin cytoskeleton : Attached Actin cytoskeleton : Detached

Cell rest Cell migration
Rho Gap (DLC1) : Active Rho Gap (DLC1) : Inactive

Figure 13.5 Epidermal growth factor stimulation causes a switch from Tensin 3 to Cten and promotes cell
migration by the Rho A-activated Rho-associated protein kinase pathway. Cten also increases motility by
detachment of cellcell E-cadherin junctions.

Cell survival and apoptosis

Tensins engage with signalling pathways many of which involve the regulation of multiple
biological processes including cell adhesion. For example, the PI3 kinase/Akt signalling
pathways support cell survival through phosphatidylinositol-(3,4,5)-trisphosphate (PIP3)
and PDK1 activation. Furthermore, Cten has been shown to locate to the nucleus where it
is found in complex with -catenin, a member of the Wnt signalling pathway. It would not
be surprising therefore if modulation of Tensin proteins also affected biological activities.
Indeed, forced expression of Cten in both colonic and pancreatic cancer cell lines has been
shown to stimulate anchorage-independent colony formation thereby providing a link
between cell adhesion and properties of stemness [13,20].
Tensins can also be targeted by caspases as part of the process of apoptosis. Tensin 1 is
cleaved by caspase 3 at amino acid 1226 through the DYPD1226G motif and this separates
the SH2/PTB domains from the ABD. The loss of the SH2 domain results in loss of a PI3
kinase-mediated cell survival signal. In addition, the integrins become detached from the
actin cytoskeleton and the focal adhesions become disrupted. This is considered to be an
important step in the cellular changes which characterise the process of apoptosis [22].
Cten has also been shown to be targeted for cleavage by caspase 3 at the DSTD570S motif,
thereby releasing a fragment, containing just the PTB domain. This produces a feed-
forward loop whereby the detached fragment is able to induce apoptosis by competing for
binding sites in the cytoplasmic tails of -integrin, disrupting the links between integrins
and cortical actin fibres [23].
A role for tensins in carcinogenesis 177

Regulation of Tensin proteins

Little is known about the upstream pathways regulating Tensin protein expression, and
most studies have focused on two main pathways the EGFR signalling pathway and the
signal transducer and activator of transcription 3 (Stat3) signalling pathway. Activation of
EGFR signalling through EGF stimulation or c-ErbB2 activity leads to the Tensin switch
with a decrease in Tensin 3 levels accompanied by an increase in Cten (Figure 13.5), whilst
the other Tensins remain unaffected [24]. The signalling pathway between the EGFR and
Tensins 3 and Cten most probably involves the KRAS/BRAF/mitogen activated protein
kinase (MAPK) pathway, as mutational activation of KRAS and BRAF has been shown to be
followed by upregulation of Cten [25]. However, an alternative mechanism has also been
proposed whereby activation of EGFR signalling leads to dephosphorylation of p130cas
and FAK and their consequent dissociation from the Tensin 3-p130cas-FAK multi-protein
complex. This in turn may lead to direct Tensin 3EGFR interaction via phosphorylated
residues in the SH2 and PTB domains of Tensin 3 [9].
Stat3 signalling has been implicated as one of the upstream pathways regulating
expression of Cten. Stat3-dependent overexpression of Cten has been shown to disrupt
cell adhesion and induce motility, whilst this activity was abolished by Cten inhibition.
Furthermore, the cytokine interleukin 6 (IL6) has been shown to induce Cten via Stat3
[26]. However, other studies have suggested that in fact Cten, through its SH2 domain, is
a regulator of Stat3 activity and, in its normal state it is seen to inhibit activation of Stat3
[27]. The precise relationship between Stat3 and Cten still remains to be elucidated and
it is uncertain whether Stat3 is an upstream regulator or a downstream target of Cten or
whether Cten has a feedback mechanism to control the activity of Stat3.
In addition to EGFR and Stat3 signalling, numerous other factors have been reported to
regulate the Tensin proteins, including the ECM, platelet-derived growth factor, thrombin,
angiotensin, Bcr/Abl, p38MAPK and Src. Such effects are suggested to be mediated through
phosphorylation of Tensin residues [28].

A role for Tensins in carcinogenesis

As the Tensins are concerned with cell motility, their involvement in carcinogenesis,
particularly the promotion of metastasis, has been extensively explored. A role for Tensins
in cancer has been most rigorously investigated for Tensin 3 and Cten but evidence clearly
implicates each family member in disease progression.
In investigating the expression of the Tensins in tumours, Tensin 1 was found to
be downregulated in those of the prostate, breast, kidney and skin [2931]. Similarly
Tensin 2 has previously been reported to be downregulated in cancers of the kidney and
lung thereby, indicating a tumour suppressor activity as with Tensin 1 [31]. However,
further investigation in hepatocellular carcinoma revealed Tensin 2 splice variant 3 to
be overexpressed in 46% of tumours in comparison to normal liver tissue exemplifying
contrasting behaviour [32].
Similarly to Tensins 1 and 2, Tensin 3 generally appears to have a tumour suppressive
role and reduced expression has been reported in tumours of the thyroid, kidney and breast
[16,31,33]. Forced expression of Tensin 3 led to decreased colony formation supporting
its role as a tumour suppressor which was consistent with Tensin 3s ability to decrease
cell migration in normal mammary epithelial cells [16]. Conversely, other studies in
breast, melanoma and non-small cell lung carcinoma have found Tensin 3 knockdown
178 Tensins in health and disease

reduced colony formation, thereby implying an oncogenic function [34]. Furthermore,

a transgenic mouse model of breast cancer revealed an increase in Tensin 3 expression
and phosphorylation in comparison to normal mammary tissue [34]. Tensin 3 clearly has
conflicting roles in tumourigenesis and these may be tissue specific, although opposing
views have been observed in cells derived from breast tissue.
Cten was originally identified as a tumour suppressor gene in prostate cancer and is also
downregulated in tumours of the kidney yet, as with Tensin 3, its role in carcinogenesis is
complex [7,31]. Cten is not normally present in most tissues and elevated levels have been
discovered in tumours of the lung, thymus, colon, breast and pancreas, suggesting that in
these tumours it is acting as an oncogene and, in general, greater levels of expression are
associated with advanced disease stage and metastasis [20,21,35,36].
In breast cancer, Cten expression has been shown to stimulate cell motility.
Immunohistochemical analysis of breast carcinomas revealed no correlation between
Cten expression and tumour size, yet there was a significant association with HER2/ErbB2
positive tumours, reduction in oestrogen receptor expression, lymph node metastasis
and tumour grade [24]. A larger study investigated Cten expression in 1409 invasive breast
tumours also using immunohistochemistry. Cten was shown to be associated with tumour
size, grade, nodal involvement and poor Nottingham prognostic index. Patients with high
tumour expression of Cten had a poorer prognosis in comparison to those expressing low
levels and also had increased likelihood of developing metastasis [36]. Cten expression
was also investigated in 462 tumour samples and Cten expression was shown to correlate
with advanced Dukes stage, poor prognosis and distant metastasis in colorectal cancer.
Consistent with this, in vitro investigations have shown Cten to induce cell motility and
promote colony formation and in vivo mouse models further support an oncogenic role of
Cten in colorectal tumours [13,19,25].
Although conflicting reports regarding the role of Tensins in carcinogenesis exist, they
are still considered to have potential as targets for therapeutic agents. Tensin 1 induction
is associated with anticancer properties in epithelioid cancers and leukaemia cells using
a polyphenolic compound resveratrol [37]. Similarly Tensin 2 has also been considered a
novel therapeutic target in myleproliferative disorders [38]. The accumulating evidence
placing Cten under the regulation of the EGFR signalling pathway suggests that targeting
this molecule may have potential in those colorectal tumours resistant to anti-EGFR
therapies due to downstream BRAF and KRAS mutations [25].

Tensins are emerging at the forefront as regulators of cell migration. Their ability to both
bind the actin cytoskeleton and mediate signal transduction events at focal adhesion regions
conveys their importance in regulating cell adhesion and migratory processes. The Tensin
familys role in regulation of cell migration in turn extends to their involvement in cancer
metastasis; however, differential activity in different tissues exemplifies the complexity of
such signalling cascades. Further investigations are warranted to help define the place of
this gene family in the realms of cell migration and tumorigenicity. The targeting of the cell
migratory machinery by using the Tensin family of genes presents an interesting therapeutic
target for anticancer therapies and with further characterisation may provide alternative
targets in areas of need including EGFR inhibitor resistant tumours and possibly overcome
problems associated with toxic anti-Src therapies under development.
References 179

Key points for clinical practice

The tensin family of proteins shows high homology and evolutionary conservation.
They are involved primarily in cell adhesion although they also have roles in regulating
apoptosis and conferring a stem cell phenotype.
They are deregulated in cancer although their function is tissue dependent. Cten is the most
extensively studied tensin molecule and this acts as a tumour suppressor in the prostate but
as an oncogene in colon, breast and lung neoplasia.
Cten is generally a marker of poor prognosis and it may be important in the development
of metastasis.

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Note: Page numbers in bold or italic refer to tables or figures, respectively.

A Barretts oesophagus, 7374

Abortion, 24 adenocarcinomas in, 73, 77
Acetaminophen overdose, and liver failure, 88, 89 biomarkers and risk stratification in, 8283
Acinar predominant adenocarcinoma (APA), 33, 33 cancer risk in, 7677
Acute cellular rejection (ACR), 1 see also Antibody- diagnosis of, 76
mediated rejection (AMR); Transplantation dysplasia diagnosis in, 7879, 7981
Adenocarcinoma, 31, 32 endoscopic treatments, 83, 83, 84
adenocarcinoma in situ, 32 imaging and detection of dysplasia, 79, 8182
atypical adenomatous hyperplasia, 3132 mucosal phenotypes of, 7476, 75
fetal, 34, 35 population screening for, 76
in Barretts oesophagus, 73, 77 (see also Barretts postascertainment surveillance, 7778
oesophagus) Breast cancer, 6869, 178
invasive mucinous, 3435, 35 Bronchiolitis obliterans syndrome (BOS), 11
invasive nonmucinous, 33, 3334 Bronchioloalveolar carcinoma (BAC), 31, 32
minimally invasive, 32 C
Adenosquamous carcinoma, 36, 39
Campylobacter jejuni, 142
Adiponectin, 52
Afatinib, 43
in Barretts oesophagus, risk of, 7677
AFES see Amniotic fluid embolism syndrome (AFES)
gene mutations in, 6163 (see also Gene
Amniotic fluid embolism syndrome (AFES), 20, 2021,
mutations, in cancer)
21, 22
genome screening, 6163, 62
Amoebic colitis, 120, 120
lung (see Lung cancer)
Amplification refractory mutation system (ARMS),
obesity and, 54
66, 66
Tensin proteins in, role of, 177178
AMR see Antibody-mediated rejection (AMR)
Cancer Research UK Stratified Medicine Programme,
Anaplastic lymphoma kinase (ALK), 4243
Antibody-mediated rejection (AMR), 1
Carcinosarcoma, 36
and acute cellular rejection, 1
Cardiac allograft vasculopathy (CAV), 4
antibodies in pathogenesis of, role of, 23
Cardiac antibody-mediated rejection, 69
capillary endothelial cells in, 2
capillary endothelial cells and lumen, 7, 8
cardiac, 69, 8
diffuse capillary deposition, 8, 8
classification of, 3, 4
diffuse microvascular inflammation in, 7, 8
in liver, 910, 11
grading, 7
pathological diagnosis of, 34
ISHLT criteria, 6, 7
pulmonary, 1013, 12, 13
Cell adhesion, 169, 170, 170 see also Focal adhesions;
renal, 46, 5, 7
Tensin proteins
severity of, 4
Cervical cancer, 159 see also Human papilloma virus
ATRA (all-trans retinoic acid), 63
Atypical adenomatous hyperplasia (AAH), 3132
Cetuximab, 63
Autofluorescence imaging (AFI), 79
Chemotherapy, for treatment of non-small-cell lung
B carcinoma, 4041
Bariatric surgery, 5557, 56 Chronic inflammatory bowel disease (CIBD), 117118
and cholelithiasis, 57 amoebic colitis and, 120, 120
deaths after, 55, 57 appendiceal neoplasia in, 132
laparoscopic gastric banding, 55, 56 appendix and, 125127, 126
Roux-en-Y gastric bypass, 56, 57 basal plasmacytosis in, 118
vertical sleeve gastrectomy, 55, 56, 57 colorectal carcinoma in, 129, 130, 130
182 Index

dysplasia in, detection of, 131 EGFR mutations, 6768

granulomas and, 127129, 128 EML4-ALK fusion gene, 68
ileal histology in, 122123, 123 Endothelial transcripts, 6
and infective colitis, 118, 119 Enteric adenocarcinoma, 3435, 35
lymphoma in, 131132 Epidermal growth factor receptor (EGFR) signalling
macroscopic pathological assessment in, 120123, pathway, 176, 177
121, 121 Epidermal growth factor receptor tyrosine kinase
pouch cancer in, 131 inhibitors, in NSCLC treatment, 4142, 43
pouchitis and prepouch ileitis in, 124 Erlotinib, 43, 63, 68
tuberculosis and, 120
upper GI tract in, 124125 F
Yersinia infection and, 120 Fetal adenocarcinoma, 34, 35
Chronic kidney disease (CKD), obesity and, 53, 5354 Fibrillar adhesions, 172, 174
CIBD see Chronic inflammatory bowel disease (CIBD) Fibrolamellar carcinoma, 97
Cirrhotic nodules, 90 Flow cytometry, 82
Clostridium difficile, 120 Focal adhesions, 169172, 171 see also Tensin
Colloid adenocarcinoma, 34 proteins
Colorectal carcinoma, in chronic inflammatory bowel fibrillar adhesions, 172
disease, 129, 130, 130 function of, 171172
Colorectal neoplastic progression life-cycle of, 171
adenomacarcinoma pathway of, 103 nascent adhesions, 172
serrated neoplastic pathways of, 103, 105 (see also structure of, 169170, 171
Serrated pathway lesions) Focally enhanced gastritis (FEG), 125
Complement-dependent lymphocytotoxicity (CDL), Focal nodular hyperplasia (FNH), 91, 91
2, 3 Formalin fixed, paraffin embedded (FFPE) tumour
Confocal laser endomicroscopy (CLE), 79 tissue, 64, 69
Congenital heart disease (CHD), 27 Fragment length analysis, 66
Continuing professional development (CPD), 149, 153
see also Revalidation, medical
Coronary artery disease, obesity and, 52 Gastric cardia, 7476 see also Barretts oesophagus
CpG island methylator phenotype (CIMP), 103 see Gastrointestinal MALT lymphoma, 137140
also Serrated pathway lesions chemotherapy for, 142143
Crizotinib, 42, 43, 63, 68 Helicobacter pylori eradication therapy for,
Crohns disease, 117 see also Chronic inflammatory 141142
bowel disease (CIBD) posteradication biopsies, assessment of, 143,
C-terminal Tensin-like (Cten), 172, 173 see also Tensin 143145
proteins radiotherapy for, 142
Gefitinib, 41, 43, 63, 68
D GELA scoring system, for posteradication biopsies
Dilated cardiomyopathy, 51 assessment, 143, 143
Disseminated intravascular coagulation (DIC), 20, 22 Gene mutations, in cancer, 6163
Donor-specific antibodies (DSAs), 1 breast cancer and, 6869
Dumping syndrome, 57 detection of, techniques for, 6467, 6566, 70
Dysplasia lung cancer and, 6768
in Barretts oesophagus, 7879, 7981 melanoma and, 69
in chronic inflammatory bowel disease, 131 molecular analysis, technical challenges in, 6970
Dysplastic nodules, 9294, 93 molecular pathology report, requirements of, 67
as HCC precursors, 92 terminology related to, 62
high-grade, 9394 General Medical Council (GMC), 149151 see also
large cell change, 93 Revalidation, medical
low-grade, 93 Genital tract trauma, 24
small cell change, 93 Giant cell carcinoma, 36
Glutathione S-transferase T1 (GSTT1), 3, 9
E Granulomatous appendicitis, 126127, 129
Echinoderm microtubule-associated protein-like 4
(EML4-ALK), 42 H
Eclampsia, 21 see also Hypertensive diseases of HCA see Hepatocellular adenoma (HCA)
pregnancy HCC see Hepatocellular carcinoma (HCC)
Index 183

Helicobacter heilmannii, 137 Influenza, 27

Helicobacter pylori, 124 Integrins, 169170
and gastric cancer, 73 Intrahepatic cholangiocarcinoma, 98
MALT lymphoma and, 137 Invasive mucinous adenocarcinoma (IMA), 3435, 35
Hepatic antibody-mediated rejection, 910, 11 Invasive nonmucinous adenocarcinoma (INA), 33,
Hepatic progenitor cells, 87 3334
Hepatocellular adenoma (HCA), 9496, 95
Hepatocellular carcinoma (HCC), 9699 K
and angiomyolipoma, 98 KRAS mutations, in colorectal adenomas, 111
fibrolamellar variant of, 96, 97
growth pattern, 96
histological features, 98 Large cell carcinoma (LCC), 3536, 39
risk factor for, 96 Lepidic predominant adenocarcinoma (LPA), 33, 33
tumour cells in, 97 Leptin, 52
Hepatocellular necrosis, and regenerative nodules, 88 Luminex assay, 3
Hepatocellular nodules, 8788 Lung cancer, 31 see also Non-small-cell lung
neoplastic, 9294 carcinoma (NSCLC)
regenerative, 8892, 8991 molecular analysis in, 6768
Hep-Par-1 (hepatocyte paraffin 1), 97, 98 Lymphocytic oesophagitis (LE), 124, 124, 125
Hereditary non-polyposis colorectal cancer (HNPCC) Lynch syndrome (LS), 111112
see Lynch syndrome (LS)
High-resolution DNA melting analysis (HRM), 65
HIV/AIDS, 28 MALT lymphoma, 135137, 136
HPV see Human papilloma virus (HPV) gastrointestinal, 137140
Human leucocyte antigens (HLAs), 1, 2 and reactive lymphoid infiltrates, 140141
Human papilloma virus (HPV), 159 and small B cell lymphomas, 141
Abbott Real Time and Roche Cobas test, 164 Maternal deaths, 17
biomarkers for, 165 abortion and, 24
and cervical screening programme, 159160 amniotic fluid embolism syndrome and, 20,
genetics of, 162163 2021, 21, 22
Genprobe Aptima HPV assay, 163164 autopsy in, role of, 17, 19
Hologic Cervista HPV test, 164 cardiovascular disease and, 2627
HPV 16/18 genotyping, 164 causes of, 1819
Hybrid Capture II test, 163 classification of, 19
immunisation programme, 160161 coincidental, 17, 19
primary screening test, 162, 165 definition of, 17
testing, 163164 direct, 17, 19, 2026
testing in triage, 161162 epidemic influenza and, 27
test of cure, 162 HIV/AIDS and, 28
types of, and cervical cancer, 159 hypertensive diseases of pregnancy and, 2123,
Hyperplastic polyposis syndrome (HPS), 104, 106 23
Hyperplastic polyps, 104 see also Serrated pathway indirect, 17, 19, 2628
lesions peri- and postpartum haemorrhage and, 2324,
Hypertensive diseases of pregnancy, 2123 24
brain and, 22 sepsis and, 25, 25, 26
kidney and, 22, 23 thrombotic thrombocytopaenic purpura and, 27
liver and, 23 venous thromboembolism and, 26
maternal death in, 22 Mean fluorescence intensity (MFI), 3
uterus and placenta and, 22 Melanoma, 69
Metabolic syndrome, obesity and, 54
I Micropapillary predominant adenocarcinoma (MPA),
Imatinib, 63 33, 34
Immunofluorescence (IF), 4 Microsatellite instability (MSI), 103 see also Serrated
Immunohistochemistry (IHC), 4 pathway lesions
Immunoproliferative small intestinal disease (IPSID), Minimally invasive adenocarcinoma (MIA), 32
140 Miscarriage, 24
Infection, obesity and, 5455 Mucoepidermoid carcinoma, 39
184 Index

Mucosa-associated lymphoid tissue (MALT), 135 Peripartum cardiomyopathy (PPCM), 17, 27

Mutator phenotype, 112 Pertuzumab, 63
Pickwickian syndrome see Obesity hypoventilation
N syndrome (OHS)
Narrow band imaging (NBI), 79 PI3 kinase/Akt signalling pathway, 176
Neutrophilic capillaritis, 1112 Placenta creta syndromes, 2324, 24
and neutrophilic margination, 12 Placental abruption, 23
Next-generation sequencing, 65 Placenta praevia, 23
Nodular regenerative hyperplasia, 89, 9091 Pleomorphic carcinoma, 36
Nonalcoholic steatohepatitis (NASH), 5253 Polymerase chain reaction (PCR), 64
Non-small-cell lung carcinoma (NSCLC), 31 Polysomnography, 48, 49
adenocarcinoma, 3135 Posterior reversible leukoencephalopathy syndrome
adenosquamous carcinoma, 36 (PRES), 22
diagnosis of, problems in, 3839 Pouch cancer, 131
large cell carcinoma, 3536 Pre-eclampsia, 21 see also Hypertensive diseases of
sarcomatoid carcinomas, 3637 pregnancy
squamous cell carcinoma, 35 Primary sclerosing cholangitis, 90, 90
staging of, 37, 37, 38 ProExC (Beckton Dixon), 165
treatment of, 40, 4043, 43, 43 Proteomics profiling, of liver tumours, 99
NSCLC see Non-small-cell lung carcinoma (NSCLC) Proton pump inhibitors (PPI), 78
Pulmonary antibody-mediated rejection, 1013, 12,
O 13
Obesity, 47 Pulmonary blastoma, 36
adipose tissue in, 47 Pyrosequencing, 65, 66
bariatric surgery in, 5557, 56
body mass index and, 47, 48 R
cancer and, 54 Radio frequency ablation (RFA), 82, 83
degrees of, 47 Ras homology family member A (RhoA), 172
fatal conditions with, 47 Regenerative nodules, 8892, 8991
heart, effect on, 5052, 52 acetaminophen overdose and, 88, 89
infection and, 5455 in chronic biliary disorders, 89, 90
kidney, effect on, 53, 5354 in core needle biopsy specimen, 92
liver, effect on, 5253 differential diagnosis of, 9192
metabolic syndrome and, 54 liver injury and, 88, 89
and mortality rate, 47 in nodular regenerative hyperplasia, 89, 9091
respiratory system, effect on, 4850 Renal antibody-mediated rejection, 46
venous thromboembolic disease and, 5455 acute, 4, 56
WHO classification of, 48, 48 Banff criteria, 5, 6
Obesity cardiomyopathy, 5051, 52 chronic, 5, 6, 7
Obesity hypoventilation syndrome (OHS), 4950, 50 glomerulitis, 5, 5
Obliterative bronchiolitis (OB), 4 peritubular capillaritis, 5, 56
Obstructive sleep apnoea (OSA), 4849 transplant glomerulopathy, 6
Oncotype DX, 69 Revalidation, medical, 149151
appraisal and, 151152
P colleague and patient feedback, 154
Panel-reactive antibodies (PRAs), 2 continuing professional development, 153
Panitumumab, 63 documentation from previous appraisals, 153
Papillary predominant adenocarcinoma (PPA), 33, 33 documentation in, 155, 155156
P16, detection of, 165 General Medical Council (GMC) on, 149151
Peri- and postpartum haemorrhage, 18, 2324, 24 historical perspective on, 149150
creta syndromes and, 2324, 24 impact of, 156
genital tract trauma and, 24 process for medical appraisal for, 152153
placental abruption and, 23 quality improvement activities, 153154
placenta praevia and, 23 recommendation, 156
retained placenta and, 23 relicensing and revalidation, 150
uterine atony and, 23 Responsible Officer (RO), 151152
uterine rupture and, 24 scope of work, 153
Index 185

significant events and, 154 Squamous cell carcinoma (SCC), 35, 38, 39
statement of health in, 154 SSLs see Sessile serrated lesions (SSLs)
statement of probity in, 155 Stat3 signalling, 177
summative question/formative question, 151 Stratified cancer medicine, 6170 see also Gene
supporting information in, 155 mutations, in cancer
Rho-associated protein kinase (ROCK), 172 Sudden death in obesity, 51
Roux-en-Y stasis syndrome, 57 Sudden unexpected death in epilepsy (SUDEP), 22
Systemic inflammatory response syndrome (SIRS), 20
Sanger (dideoxy-) sequencing method, 65, 66 T
Sarcomatoid carcinomas, 3637 Tensin proteins, 172 see also Focal adhesions
Seattle biopsy protocol, 78 in carcinogenesis, role of, 177178
Sentinel sites project, 160 cell adhesion and motility, role in, 174175, 175,
Sepsis, 25 176
classification and pathology, 25 cell survival and apoptosis and, 176
postpartum, 26 members of Tensin gene family, 172, 173
in pregnancy, 25 regulation of, 176, 177
Serrated pathway lesions, 103, 104, 105 structure of, 172174, 173
hyperplastic polyps, 104106, 105 Thrombotic thrombocytopaenic purpura (TTP), 27
immunohistochemistry in, diagnosis of, 113 Thymidylate synthetase (TS), 41
mixed polyps, 106, 106 TP53, 82
molecular abnormalities, 111113 Traditional serrated adenomas (TSAs), 103, 108109,
serrated adenocarcinomas, 109, 110 109
sessile serrated polyps, 106108, 107108 Transplantation, 1, 2 see also Antibody-mediated
surveillance intervals after, 113, 113 rejection (AMR)
traditional serrated adenomas, 108109, 109 Trastuzumab, 63
WHO classification, 104 Trastuzumab emtansine, 63
Serrated polyposis syndrome see Hyperplastic Tyrosine kinase inhibitors, in clinical use in NSCLC,
polyposis syndrome (HPS) 4142, 43
Sessile serrated lesions (SSLs), 106108, 107108
Single-strand conformational polymorphism analysis U
(SSCP/SSCA), 6466, 65 Ulcerative colitis (UC), 117 see also Chronic
Sjgrens syndrome, 137 inflammatory bowel disease (CIBD)
Small cell lung cancer, 40 Uterine atony, 23
Small intestinal bacterial overgrowth (SIBO) Uterine rupture, 24
syndrome, 57
Solid predominant adenocarcinoma (SPA), 33, 34
Spindle cell carcinoma, 36 Vemurafenib, 63, 63
Spontaneous abortion, 24 Venous thromboembolism (VTE), 18, 26
obesity and, 55