Immunohistochemical Staining for p63 is

Useful in the Diagnosis of Anal Squamous

Cell Carcinomas

Am J Surg Pathol 2007;31:285290

Int

Background
Carcinomas in the anal canal account for about
1.5% of gastrointestinal cancers in the United
States, and approximately 80% of these are
squamous cell carcinomas (SCCs).
SCCs of the anus are frequently related to
chronic infection with human papilloma virus
(HPV)
Background
Usually occur in the sixth to seventh decade of
life, occur in younger patients when they are
immunocompromised
Male:Female=2:1
HIV/AIDS, and the increasing use of
immunosuppressive therapy for solid organ
transplantation, inflammatory bowel disease and
collagen vascular diseases has meant an
increasing incidence of HPV infection and anal
SCC.
Background
The main differential diagnosis of carcinomas in
the anus includes SCC, well-differentiated
neuroendocrine carcinoma/carcinoid tumor
(NEC), and poorly differentiated
adenocarcinoma (ADC).
Background
Morphologic clues can be sought to aid in
diagnosis, including squamous eddy/keratin
pearl formation in SCC and lumen formation in
ADC.
Such carcinomas can look remarkably similar in
small biopsies, and a panel of immunostains is
often used for diagnosis.
Background
In diagnosing primary anal SCC, such panels
rely on negativity for markers of ADC and
neuroendocrine differentiation, along with
positivity for one or more cytokeratins (CK),
suggesting squamous differentiation.
Combination of CK 5 and 6 (CK5/6) is used as
evidence for squamous lineage.
Background
Loss of keratin expression in poorly
differentiated SCC !!
Accurate diagnosis is imperative for the
development of an effective treatment plan
P63 gene !!
Background
The p63 gene resides on the long arm of
chromosome 3 at 3q27-28.
P63 is a member of the p53 gene family and, as
such, participates in epithelial proliferation and
differentiation.
Background
Antibody to p63 is frequently used in the
diagnosis of prostate carcinoma, because
normal prostatic glands are lined by p63-positive
basal cells, whereas carcinomatous glands lack
a basal layer.
Be found to be associated with SCC in a number
of sites, including the head and neck, lung and
uterine cervix.
Background
It is also expressed in some breast carcinomas
(particularly metaplastic carcinoma) and in
urothelial carcinomas.
Given its association with SCC in other sites,
authors elected to investigate the utility of p63
staining in the diagnosis of anal SCC!!
Methods and Materials
All cases were submitted for diagnosis during
the period from January 1, 2004 to December 31,
2005, and originated within the University of
Michigan system.
24 anal squamous carcinomas, 68 colorectal
adenocarcinomas (including a tissue microarray
of 49 ADC), and 32 colorectal neuroendocrine
carcinomas from the archives at the University of
Michigan
Methods and Materials
11 of the SCC cases were deemed poorly
differentiated based on the presence of large
areas consisting of monotonous, immature-
appearing cells and the relative lack of areas
showing squamous eddy or keratin pearl
formation.
Methods and Materials
Colorectal NECs included tumors from
throughout the colon, and the appendix.
ADCs from throughout the colon and rectum
were included.
Both biopsy and resection specimens were used.
Methods and Materials
The squamous carcinoma cases included 11
resection specimens and 13 biopsies. Three
resection specimens containing normal anal
transition zone (ATZ) were also stained to
evaluate the staining pattern in the ATZ.
Methods and Materials
Immunohistochemical stain:
P63 CK5/6 chromogranin A(CGA)
synaptophysin(SYN)
Only nuclear staining for p63 was considered
positive. A section of prostate tissue was used
as a control, with p63 staining the basal cells in
the normal prostate
Methods and Materials
A section of tonsil was used as a positive control
for CK5/6 staining, and a section of adrenal
gland for CGA and SYN staining.
Staining for CK5/6, CGA, and SYN was
considered positive when cytoplasmic staining
was present.
Methods and Materials
Positive predictive values (PPV) and
specificities were calculated for p63
and CK5/6 staining in SCC.
Disease
positive negative
Test positive True Positive(TP) False Positive(FP)
negative False Negative(FN) True Negative(TN)
Positive predictive values(PPV) =TP/TP+FP
Results-P63
All 24 (24/24; 100%) of the SCC were positive
for nuclear p63 staining.
None of the ADCs were positive(0/68; 0%).
Two NEC, both classic appendiceal carcinoids,
showed nuclear positivity with p63(2/32; 6.25%).
Results-P63
Within the SCC, staining was present in both
well and poorly differentiated areas.
SCC cases included 4 carcinomas with basaloid
morphology, a pattern termed cloacogenic
carcinoma in the past. These carcinomas also
stained strongly with p63.
Invasive SCC
stained with p63,
showing strong
nuclear staining
(100X)

Classic appendiceal
carcinoid tumor
showing strong
nuclear staining with
p63 (100X)
Basaloid SCC
(cloacogenic carcinoma)
with nuclear staining for
p63 (100X)
Results-P63
Although none of the ADC and only 2 NEC
showed nuclear staining, there was scattered
nonspecific cytoplasmic staining.
However, the lack of nuclear staining was easily
discerned in these cases.
Invasive ADC stained
with p63, showing lack
of nuclear staining.
Some nonspecific
cytoplasmic staining is
present (100X)

Results-CK5/6
All of the SCCs (24/24; 100%) were also strongly
positive for CK5/6 staining, and negative for
CGA and SYN.
Cytoplasmic staining with CK5/6 was so strong
as to obscure most cellular and nuclear detail in
the carcinomas.
Results-CK5/6
The CK5/6 immunostain was strongly
positive in adnexal structures of the
perianal skin where present on resection
specimens, again obscuring most
cytologic detail.

CK5/6 staining of
dermal adnexal
structures in
perianal skin (200X)
Results-CK5/6
Five ADC (5/19; 26%) showed strong staining
with CK5/6 and 6 NEC (6/32; 19%) showed a
unique, punctate cytoplasmic staining with
CK5/6.
These results lead to the calculation of a PPV of
69% and a specificity of 78% for CK5/6 staining
in SCC in our series.
CK5/6 staining in
an ADC (200X)
CK5/6
staining in a
NEC (200X)
punctate
cytoplasmic
staining

Results-P63 & CK5/6
Staining with p63 was found in the basal layers
of normal squamous epithelium, and in
dysplastic cells in areas of AIN(I~III).
As the degree and thickness of dysplasia
increased, so did the amount of p63 staining.
In contrast, the normal ATZ showed staining
restricted to the basal layer of cells
Results-P63 & CK5/6
With the CK5/6 antibody, all of the
overlying squamous epithelium,
whether normal or dysplastic,
manifested full-thickness staining.
High-grade
AIN III with
full-thickness
p63 staining
(200X)
ATZ, showing
basal layer
staining with
p63 (400X)
Full-thickness staining
of squamous
epithelium with CK5/6
(200X)
Discussion
Current diagnostic algorithms for anal SCC rely
on negative staining with markers of ADC,
including CDX-2 and CK20, and NEC, including
chromogranin and SYN.
Combined with putative evidence of squamous
differentiation by staining with one or more CK,
most often CK5 and/or CK6.
Discussion
Specificity of the CK5/6 combination for SCC is
not perfect, and these keratins can stain other
types of carcinoma, including up to 30% of
colorectal ADC.
In our series, 26% of ADC were positive for
CK5/6 and 19% of NEC had a punctate,
cytoplasmic positivity for CK5/6.
Discussion-p63
We found that immunohistochemical staining for
p63 is a highly specific and useful tool in the
diagnosis of SCCs of the anal canal.
Our series reveals a specificity of 98% and a
PPV of 92% for squamous differentiation in
invasive carcinomas.
Discussion-p63
In addition, basaloid anal squamous carcinomas,
the type formerly designated cloacogenic
carcinoma, also stained with p63 antibody.
Given their dark, homogeneous nuclear
chromatin and their nested growth pattern, these
carcinomas are easily confused with other types
of carcinoma, particularly neuroendocrine tumors
(different treatment protocol.)
Discussion-p63
The p63 immunostain should prove especially
useful in the small, often poorly preserved
biopsies from which the diagnosis of anal
carcinoma is usually made.
Widespread staining of dysplastic squamous
epithelium in AIN precludes its use for diagnosis
of SCC.(to distinguish from the normal ATZ)
Discussion-p63
The product of the p63 gene has been shown to
be related to SCC in many sites, including the
head and neck, the lung, and the uterine cervix.
And is expressed by a handful of other
carcinomas as well, including metaplastic breast
carcinoma and urothelial carcinoma.
Discussion-p63
Although positive staining with p63 is very
specific for anal SCC, it will not distinguish
carcinomas primary in the anal canal from those
directly extending or metastasis!!

Discussion-p63
The staining of 2 cases of NEC from our series,
both classic appendiceal carcinoid tumors??
The NEC in our series came from throughout the
colon and rectum, but only the carcinoid tumors
from the appendix showed nuclear positivity.
Discussion-p63
The appendix is a product of the embryonic
midgut, which also includes the distal duodenum,
the small intestine, the right colon, and the
proximal three-quarters of the transverse colon.
It is possible that p63 staining varies among
NEC from different parts of the embryologic gut,
and future studies could address this by staining
more NEC, from throughout the gastrointestinal
tract.
Discussion-CK5/6
In our series, no additional utility was gained in
the diagnosis of SCC by adding the CK5/6
immunostain.
On the basis of our study, staining with CK5/6
can be eliminated from any
immunohistochemical panel for anal carcinomas.
Discussion-CK5/6
It provides no added utility to the diagnosis of
SCC, is potentially confusing in its staining of
perianal dermal adnexal structures and normal
squamous epithelium, has a lower specificity and
PPV than p63 for SCC, and provides no utility in
highlighting squamous dysplasia in the overlying
epithelium.
Conclusion
The p63 antibody should prove
especially useful in small biopsies of
the type commonly sent to the
pathology laboratory for the diagnosis
of anal carcinomas.