Tumor markers

Dr WT Poon

Definition
A broad spectrum of molecules, with
widely varying characteristics,
produced or induced by the tumor
cell and which reflect its growth
and/or activity and allow the
presence, development or
therapeutic response of a malignant
tumor to be known

Specificity
TMs are not specific to cancer, as the
majority of them are also synthesized
and released by normal cells
Various benign pathologies can cause
an increase in serum levels of TMs,
giving rise to false positives

Sensitivity
Serum TM levels varies according to different factors
The greater the number of tumor cells (size), the greater will
be the concentration of the TM
The greater the ease with which TM reach the circulation
(vascularization, intracellular location, release mechanism),
the greater are the concentrations of TMs
Small hepatic metastatic nodules (highly vascularized tissue)
can produce large increases in TMs, whilst other metastasis
(skin) are associated with normal values or only slight
increases
A well differentiated tumor is one which resembles the cells
from which it comes, maintaining its principal characteristics,
including synthesis of TM, which can disappear in the case of
undifferentiated tumors

 The greater the TM concentration

detected in patient, the greater the
likelihood of presence of a malignant
tumor
Take CEA as example

CEA example
CEA in patients with localized cancers are
normal or slightly elevated, between 10 20
ng/mL; similar figures to these are usually
found in patients with liver cirrhosis, renal
failure or chronic obstructive pulmonary
disease
On the other hand, in patients with
metastasis, the concentrations of CEA may
be above 50 ng/mL, and levels such as this
are seldom detected in the absence of tumor

Exclusion of benign
pathology
When there is an increase in a TM, the
existence of certain benign disorders
which can cause an increase has to be
ruled out
The majority of TMs are catabolized in the
liver and excreted via the renal route
Majority of TMs show moderate increases
(2 to 4 times the upper limit of normal) in
patients with liver cirrhosis or renal failure

 Some TMs can reach serum

concentrations in patients with renal
failure similar to those found with
malignant diseases and can therefore
not be used in renal failure patients

 The changes in tissues which

produce a particular TM can also
cause moderate increases, examples
include
PSA in prostatitis or benign prostatic
hypertrophy
CEA in ulcerative colitis or Crohns
disese

Sequential study of TMs

An isolated finding of high levels of any TM is
of limited value
2 or 3 sequential measurements should be
carried out at intervals of more than its plasma
half-life (15-20 days for the majority of TMs)
If the TM values show a continuous increase
(greater than the analytical imprecision and
intra-individual biological variation), it can be
concluded with a high level of probability that
it is of malignant origin

Reference Change Value

Differences in serial results occur due to analytical
and intrinsic within-subject biological variation
The analytical imprecision for measuring a TM
would range from 5-8% depending on method
The within-subject biological variation for a TM e.g.
CEA is ~13%
In order to decide whether a sequential TM trend is
genuine, the Critical Difference, or better termed,
the Reference Change Value (RCV) that is due to
the inherent sources of error must be exceeded

 This can be calculated as: RCV = Z *

2* (CVA2+ CVI2)
For 95% probability Z is 1.96, and for
99% probability Z is 2.58
CVA: analytical imprecision
CVI: within-subject biological variation
For CEA, a rise or fall of >40% would
be required for significance

Technical Issues
Results for a TM obtained by
commercial methods are not always
similar to one another, and this can
cause considerable discrepancies, in
particular in the case of CA19.9
Reasons for this include use of
antibodies of differing specificity, crossreactions with other molecules or the
presence of heterophile antibodies etc

TM for health check?

Positive & negative predictive

value
PPV: The probability that a person with
a positive test result has disease = a/
(a+b)
NPV: The probability that a person with
a negative test result has no disease
= d/(c+d)
Value depend on the disease
prevalence

 The effect of
disease prevalence
on the positive
predictive value
(PPV) for a test
with sensitivity and
specificity of 95%
is shown in the
table:

Prevalence

PPV

0.10%

1.90%

1%

16%

10%

68%

20%

83%

50%

95%

 Even tumor markers that are highly

sensitive and specific will have low
positive predictive values (PPV) given
the low prevalence of cancer
(<0.1%) in health check subjects

Principal TMs in current use

Alpha-fetoprotein (AFP)
Human chorionic gonadotropin (hCG)
Carcinoembryonic Antigen (CEA)
CA 125
CA 19-9
CA 15-3
Prostate specific antigen (PSA)

AFP
Glycoprotein (69000 Da)
Protein composition very similar to albumin,
differs mainly in the N terminal segment
AFP synthesis starts early in the fetus, first
in yolk sac and then fetal liver
AFP conc. during pregnancy are very high,
reaching levels 25-30 times the normal
value in adults
Also very high in neonates

 Increase AFP up to 4000 times normal

have been reported in cord blood,
and return to normal adult values
during the first few months (<10
ng/mL)
Like albumin, its function involves the
transport of different substances
(non-esterified fatty acids, steroid
hormones, zinc, copper, bilirubin etc)

 The most common cause of AFP false

positives are benign, acute or chronic
liver disease (cirrhosis), viral or toxic
hepatitis, hepatic abscess, biliary
atresia etc
Increases are usually moderate <100
ng/mL
>1000 ng/mL can be reached in
hereditary tyrosinemia and ataxia
telangiectasia

 Main use of AFP as TM is in

hepatocellular cancer (HCC),
testicular tumor and endodermal
sinus neoplasms
Moderate increases are also detected
in 15% of GI cancers, mainly gastric,
which are associated with a worse
prognosis

Early diagnosis in high risk

group
AFP is used as a method of early
diagnosis in places where HCC is
endemic
HCC is one of the most common
cancers in Hong Kong. About 8% of
our population is chronic carrier of
the hepatitis B virus (HBV) and they
are at risk of developing cirrhosis and
HCC

 Clinical guidelines suggest the

combined use of AFP, with a cut-off
point of 20 ng/mL, and hepatic
ultrasound every 3-6 months in at-risk
groups (cirrhosis, Asian males >= 40
years, Asian women >= 50 years etc)
Using these methods, AFP is positive
(>20 ng/mL) is 50-70% of cases of
HCC, with a specificity of ~85%

Diagnostic aid
Apart from screening, serum AFP
levels are useful in the evaluation of
patients who present with liver mass
In patients with a hypervascularised
mass >2 cm, AFP > 200 ng/mL may
be sufficient for diagnosis without the
need for a biopsy

Prognosis
A positive AFP result is an
independent risk factor for
recurrence and mortality
Patients with AFP > 1000 ng/mL had
a greater incidence of vascular
invasion (61% vs 32%)

Follow-up
In patients who have undergone surgical
resection, a rapid decrease is observed, with a
half life of between 3-4 days
Extension of the half life of AFP is a sign of poor
prognosis
Presence of high level indicates residual tumor,
but a negative result does not exclude this
In patients treated with radiotherapy,
monitoring of AFP is better, as imaging methods
do not distinguish fibrosis caused by radiation

AFP L3 fraction
The glycosylation of AFP in HCC is
different
The AFP in patients with HCC binds to
Lectin A, but the normal AFP does not
Commercial techniques are available
which can distinguish AFP isoforms
specific to HCC
Their use improves diagnostic
sensitivity in patients with moderate
increases in AFP (20 -1 100 ng/mL)

Case
Abdominal mass in a two-month-old
baby girl
CT scan showed an intrinsic mass
within the left lobe of the liver
hepatoblastoma
Initial laboratory testing showed AFP
level of 1600 g/L (age related
reference value: 16 2000 g/L)

Interpretation
The AFP result was normal within
the age-related reference value for
infants
AFP is higher in infants than in adults

 As hepatoblastoma is expected to
have a much higher AFP level, the
apparently normal level was not
compatible with the clinical picture
The lab reanalyzed the original
sample with 100 fold dilutions and
revealed the true AFP level of
1,000,000 g/L

Hook effect
The initial normal AFP level was an
analytical artifact caused by high
dose hook effect

Human chorionic gonadotropin

(hCG)
Glycoprotein hormone secreted by the
syncytiotrophoblastic cells of the placenta
Its function is to maintain both the secretion of
progesterone and the function of corpus luteum
during the initial phases of pregnancy
hCG is formed by two subunits, alpha and beta,
with a molecular weight of 15000 and 22000 Da
respectively
Alpha subunit: FSH / LH / TSH
Beta subunit: specific to hCG
Normal values < 5 IU/L

 hCG synthesis starts from the 8th day following

ovulation, the level doubling every 2-4 days, to
reach a peak between 10th and 12th weeks of
gestation, and then falls
Tumors can produce several variants of hCG
i.e. hyper-glycosylated forms, variants without
the beta subunit C-terminal segment etc
These forms are detected with varying degrees
of sensitivity depending on the method used

 The immunoassays currently used

measure both the total HCG and beta
fraction, generally using either
monoclonal or polyclonal antibodies
directed to the beta subunit
It is possible to obtain divergent
results depending on whether they
are better or worse at detecting the
different molecular forms of hCG

 The detection of increases in hCG in nonpregnant women should lead to suspicion of

a malignant tumor
TM in trophoblastic tumors and germ cell
ovarian and testicular cancer
The diagnosis of trophoblastic tumor is based
on two factors: ultrasound exam & hCG conc.
False positives due to presence of
heterophile antibodies should always be
ruled out

Case
A 25 year old woman was incidentally found to
have a positive blood hcG pregnancy test
However, ultrasound failed to reveal a fetal sac,
laparoscopy did not reveal an ectopic pregnancy,
and dilation and curettage revealed no recent
history of pregnancy
The positive hCG persisted and a presumptive
diagnosis of trophoblastic malignancy was made
She was treated with chemotherapy

Interpretation
Phantom hCG
False positive hCG results in patients who were
treated for assumed trophoblastic malignancy
with surgery and/or chemotherapy
The false immunoreactivity was usually due to
serum heterophile antibody that interferes with
hCG assay
Such heterophile antibody is present due to
previous exposure to animals or monoclonal
antibody treatments

Heterophile antibody
interference
To detect heterophile antibody
interference:
Repeat with another immunoassay
method
Add blocking reagent
Measure urine hCG as the heterophile
antibody does not cross the glomerular
membranes into the urine

 Increases in hCG have been

described in 24% of patients with
systemic lupus erythematosis, due to
possible interference with various
ovarian or endometrial antibodies
Renal failure can also cause
moderate increases (up to 10 times
the normal value)
Slight increases during menopause

Pituitary hCG
Apart from malignancy, persistently
low levels of hCG can be produced by
the pituitary gland in perimenopausal or post-menopausal
women
The pituitary hCG production can be
suppressed by a 3-week treatment
with high progesterone oral
contraceptive pills

Carcinoembryonic Antigen
(CEA)
High molecular weight glycoprotein (180000 Da)
Its natural function is unknown, could be related
to cell recognition mechanisms or adhesion
mechanisms
Normal value < 5 ng/mL
Small increases (<10 ng/mL) may be detected in
5 10 % of smokers
CEA can be detected in a large number of
epithelial tumors: colorectal, lung, breast, head
and neck tumors, etc

 Some 15.6% of patients had CEA > 5

ng/mL
Increase in various benign
conditions, such as liver cirrhosis,
renal failure, pulmonary disorders
(COPD, pneumonia, tuberculosis), GI
disorders (ulcerative colitis,
diverticulitis, Crohns disease,
pancreatitis), ovarian cysts or
hyperthyroidism

 None of the patients with renal

failure or liver disease had increases
> 25 ng/mL
Only small increases can be
observed in the absence of
malignancy
Whenever there is doubt, progression
must be monitored

 The marker has no role for screening

of colorectal cancer; its sensitivity
and specificity are not high enough,
particularly for early stages of
disease
No clinical guidelines advise its use
as a method of screening

 CEA is of more use in determining

prognosis and detecting recurrence
Studies have shown that CEA levels
can start to rise up to 10 months in
advance of clinical features of
disease
Early detection of recurring disease
may have survival benefits

CA 125
High molecular glycoprotein
Present in Fallopian tubes and
mesothelial cells (pleura, pericardium,
peritonium)
Normal value < 35 U/mL
TM of choice in ovarian carcinomas
High number of false positives
Menstruation, endometriosis, nephrotic
syndrome, pregnancy: minor increase
<100 U/mL
Renal failure, liver disease: moderate
increase <300 U/mL
Effusion: major increase <1000 U/mL

 The likelihood that elevated CA 125

levels are associated with
malignancy increases in postmenopausal women
The majority of screening studies
using CA 125 are carried out in postmenopausal women

UK NICE recommendation

Women who have ovarian cancer have a greater chance of

surviving the disease if it is caught earlier
CA 125 should be offered to woman (especially if they are
aged 50 or over) who reports any of the following
symptoms, either persistently or frequently (particularly
more than 12 times in a month):
persistent bloated feeling in the abdomen (abdominal
distension)
loss of appetite or feeling full quickly (early satiety)
pain in the abdomen or pelvic area
needing to pass urine urgently or more often than usual

If blood levels of CA125 are 35 IU/ml or greater, arrange

ultrasound scan of the abdomen and pelvis

 Differential diagnosis of abdominal

masses, with postmenopausal
women > 35 U/mL and premenopausal women > 200 U/mL to
be at risk

CA 19-9
A tumor-associated carbohydrate antigen
located on the sialylated Lewis A blood group
antigen
Individuals with Lewis a-, comprise 5% of the
population and cannot synthesize CA19-9
Normal value < 37 U/mL
Used principally in GI tumors and TM of
choice for pancreatic carcinomas
Renal failure cause slight increase <150 U/mL

 The main source of false positives for

CA 19-9 is liver disease, mainly with
jaundice and pancreatitis, when
levels > 1000 U/mL can be detected
It is very difficult to achieve a CA19-9
concentration which is indicative of
tumor with absolute certainty
Follow up is necessary in cases of
doubt

 The majority of clinical guidelines do not advise

the use of CA 19-9 in the diagnosis of pancreatic
cancer
It is of little diagnostic value, especially in the
early stages, but may be of interest as an
adjunct to radiological methods, mainly in cases
without jaundice
False negatives can occur in Lewis A negative
genotype patients
False positives can occur in patients with
jaundice

 Serial determination of CA 19-9 can

be used together with imaging
techniques to assess response,
especially in the case of palliative
treatment
Detection of significant increases in
CA 19-9 indicates progression

CA 15-3
Mucin-like antigen
Normal value < 35 U/mL
TM of choice in breast cancer, but it
is not specific
Major increases may be observed in
other tumors: ovarian carcinomas,
endometrial tumors and pulmonary
carcinomas, principally NSCLC

 Minor increase < 100 U/mL in hepatic

and renal diseases, ovarian cyst,
infectious pulmonary diseases,
autoimmune diseases
Increase up to 10 times the normal
value can be detected in
megaloblastic anemia

 In a review, a mean sensitivity of 20%

was obtained with CA 15-3 for breast
cancer
The low sensitivity of CA 15-3 in
locoregional breast cancer means that
their use is of little value for diagnosis
Can be an adjunct to tumor staging:
High levels (>50 U/mL) in patients with
localized disease suggests the presence of
undetected metastasis

Prostate Specific Antigen

(PSA)
Glandular kallikrein enzyme with a
molecular weight of 33000 Da
Synthesized by the prostate and
secreted in the seminal fluid where it
has a liquefying function associated
with its enzyme activity
PSA is regarded as prostate-specific
TM
Normal value < 4 ng/mL

 Concentrations of PSA in prostatitis

can be very high (10 times baseline)
PSA can be increased as a result of
various procedures involving the
prostate e.g. biopsy
Increases in PSA are also detected in
25-50% of patients with BPH,
especially in patients with acute
urinary retention or urinary tract
infection

 PSA level increase with age

Age (years)
Concentration
( g/L) 40-49
0 to 2.5
50-59
0 to 3.5
60-69
0 to 4.5
70-79
0 to 6.5

Some androgenic treatments used in

the treatment of BPH, such as 5alpha reductase inhibitor, can reduce
PSA levels by 40-50%

 Controversy about the use of PSA in

screening for prostate cancer
There is no conclusive evidence that
early diagnosis can improve survival
Screening may result in overdiagnosis and over-treatment of
indolent prostate cancer with little
threat to life

 Various lines of research have been

developed to increase the specificity
of PSA:
PSA velocity (rate of elevation of PSA)
PSA density (PSA divided by prostate
volume)
%free PSA
Prostate health index (PHI)

 PSA circulates bound to protease inhibitor

proteins ( alpha-1-antichymotrypsin, alpha-2
macroglobulin), with a small fraction remaining
in the free state
The %free PSA varies according to the prostatic
disease and is lower in patients with prostate
cancer than in normal individuals or those with
benign disease
For PSA between 4-10, %fPSA <25% can detect
95% of prostate cancer whilst reduce 20% of
unnecessary biopsies

 [-2]proPSA: truncated and inactive

free PSA
Prostate health index: combination of
total PSA, free PSA and [-2]proPSA
High index is associated with
increased prostate cancer risk

END