Tumour Markers are substances present in the tumour, produced by the tumour or by the host as a response to the presence of the tumour, providing information about biological characteristics of the tumour. these tumour markers may specific for the tissue but often get elevated in neoplastic as well non-neoplastic lesions, further Various analytical platforms available for serum tumour markers lack standardisation. These factors add to interpretative challenges in serum tumour markers

1. Role of Tumour Markers in
Clinical Practice
DR RAJESH V BENDRE
CHIEF PATHOLOGIST
MD(PATH), DNB(PATH), DPB.
MUMBAI

2. Tumour Marker- Introduction
• Tumour Markers are substances present in the
tumour, produced by the tumour or by the host
as a response to the presence of the tumour,
providing information about biological
characteristics of the tumour
• Quantitative determination – in Serum or
Biological fluids, dynamic follow-up
macromolecules whose appearance and changes
in concentration are related to the genesis and
growth of malignant tumours
• Qualitative determination – Histopathologic, in
Tumour tissue
Antigens Hormones
Enzymes
Tissue
Specific
TUMOUR
MARKERS

3. Ideal tumor marker needs to : Be specific of one pathogy , Be sensitive : its cut-off is low;
Deliver a value correlated to the tumor mass; Have a short life, which enables efficient follow-up :
decreasing during treatment, increasing before a relapse.
Based on these points, tumor markers are useful in various cancers, for diagnosis, monitoring or Both.
IDEAL TUMOUR MARKER - DOES IT EXISTS?
TUMOUR MARKER
CHARACTERISTICS
EXPLANATION IDEAL TUMOUR MARKER
Specificity & Positive
Predictive Value of a
Tumour Marker
The percentage of normal persons or persons with benign conditions for whom a negative
result is obtained. The greater the specificity, the fewer the false-positives. target
specificity should be 95% ( 5% false positives). The probability with which a tumour exists
within a control group in the case of positive test results
High specificity – not present in
other diseases, non-tumours and in
healthy subjects Organ Specific
Sensitivity & Negative
Predictive Value of a
Tumour Marker
The percentage of test results which are correctly positive in the presence of a tumour.
The greater the sensitivity, the fewer the false-negatives. The probability with which no
tumour exists within a control group in the case of negative test results
High sensitivity –
Early Diagnosis
Cut-off value The concentration of a tumour marker which differentiates healthy subjects from
diseased subjects.
Correlation with the tumour mass &
prognosis
Standardised Testing
Assay
Challenges due to tumour heterogeneity, variable epitopes with different mutations,
variable host clearance & metabolism.
Minimal Variability between
methods

4. Screening
Early
Detection
Diagnosis
Prognosis
&
Monitor

5. Individual Tumour Markers –
Past & Present

6. Ca - 125
Ca125, glycosylated phosphatidylinositol - linked cell
surface protein. It is an antigen expressed by fetal
amniotic & coelomic epithelium & found in tissues
derived from the coelomic epithelium ( ovarian capsule,
mesothelial cells of pleura, peritoneum ,pericardium)
Ca-125 as a screening test was initiated by the fact that
about 83% of patients with epithelial ovarian cancers
have elevated levels.
Lack of Specificity
Elevated in:
• Benign gynaecology – Endometriosis,
Fibroids, Pelvic Inflammatory Disease
• Other peritoneal inflammation
• Cyclical variations in pre-menopausal age
group
• Other Benign conditions- Urinary retention,
Chronic renal failure, Pancreatitis
• Other malignant disease – gastric and lung
Lack of Sensitivity
• Ca 125 not raised in 30% of
women with ovarian cancer –
Early Stage disease
Ca 125 levels U/mL Sensitivity % Specificity %
65 and greater 79 83
150 69 93
190 63 95

7. Ca-125
The American College of Obstetricians and Gynecologists and Society of
Gynecologic Oncologists guidelines for women with a pelvic mass suggestive of
ovarian cancer - serum CA-125 value higher than 35 U/mL in postmenopausal
women or higher than 200 U/mL in premenopausal women.
Premenopausal
◦ CA125 > 200
◦ Ascites
◦ Evidence of metastasis
◦ Family history of breast
or ovarian cancer
Postmenopausal
◦ CA125 >35
◦ Ascites
◦ Fixed or nodular mass
◦ Evidence of metastasis
◦ Family history of breast or
ovarian cancer
ACOG Practice Bulletin.
Obstet Gynecol. 2007;110:201-213.

8. CA – 125 & HE 4 : ROMA
HE4 - Human epididymis protein 4 (HE4), a relatively new marker for ovarian carcinoma, is the product of the
WFDC2 (WAP type four disulphide core 2) gene that is overexpressed in patients with ovarian carcinoma.
Many studies have shown that HE4 and ROMA improve the detection of ovarian cancer.
◦ Moore et al have shown that at 75% specificity, ROMA exhibits sensitivities of 76.5% and 81.3% for
premenopausal women in a gynecological oncologist setting and a primary care setting, respectively. In
postmenopausal women, the sensitivities at 75% specificity were 92.3% and 90.2% for women in a
gynecological oncologist setting and a primary care setting, respectively.
◦ Kim et al & Ruggeri et al concluded that the ROMA algorithm showed a good accuracy for discriminating
women at high risk for epithelial ovarian cancer.
◦ Molina et al & Lenhard et al showed that ROMA had the highest sensitivity at 95% specificity compared to
CA125 and HE4, especially in early stage disease.

9. CA – 125 & HE 4 : ROMA
Combination of HE4 and CA125 has 76.4% sensitivity and 95% specificity, making the combination more
accurate than either test alone especially in Stage I, or early ovarian cancer .
Computing Risk of Ovarian Malignancy Algorithm (ROMA) classifies patients as being low or high risk for
epithelial ovarian cancer using the following algorithms:
Premenopausal: predictive index (PI)
= -12.0 + (2.38 x LN(HE4)) + (0.0626 x LN(CA125))
Postmenopausal: PI
= - 8.09 + (1.04 x LN(HE4)) + [0.732 x LN(CA125))
Predicted probability: (PP) = 100 x exp(PI)/(1 + exp(PI))
Premenopausal
ROMA > 13% = High Risk for epithelial ovarian cancer
ROMA < 13% = Low Risk for epithelial ovarian cancer
Postmenopausal
ROMA > 28% = High Risk for epithelial ovarian cancer
ROMA < 28% = Low Risk for epithelial ovarian cancer

10. Produced by prostatic alveolar and ductal epithelial cells, a
serine protease with t1/2-2~3 days & partially responsible for
semen liquefaction.
In serum various molecular forms exists because of complex
formation with protease inhibitors. Major immunoreactive form
is PSA complexed with a1-antichymotrypsin (PSA-ACT).
Others such as linked to a1-antitrypsin (trace quantity) and a2-
macroglobulin (undetectable by current immunoassays). A non-
complexed free form (fPSA) represents 5 to 40% of the “total”
PSA ( fPSA + a1-antichymotrypsin complex).
Free PSA： used to enhance the specificity of total PSA in
detecting prostate cancer, especially when total PSA between 4-
10 ng/ml.
Age Serum PSA (ng/ml)
40~50 0~2.5
50~60 0~3.5
60~70 0~4.5
70~80 0~6.5
PSA
Effects of urological manipulations on PSA levels-
It is advisable to perform PSA measurement before rather than
after any of these manipulations.
- DRE May cause minor increases
- Prostate massage May cause minor elevations
- Ejaculation may increase PSA levels.
- TURP & Needle Biopsy -Increases PSA levels significantly. It
is recommended to wait at least 6 weeks before drawing blood
for PSA assay.
- Ultrasound Increases PSA levels in a minority of subjects.
- Cystoscopy Flexible cystoscopy does not appear to increase
PSA levels but rigid cystoscopy may increase levels.

11. Use of Ratio- free/total PSA
(cut off as 0.11)
An ↑in ratio is associated with increased
probability of prostate cancer
97% specific for this disease, 96% sensitivity in detecting
disease
• Post Surgery- PSA is expected to be undetectable (below 0.2
ng/ml) by 6-8 weeks after the radical prostatectomy,
persistent elevated level indicate residual disease or distant
metastases
• Biochemical recurrence as an initial PSA value 0.2 ng/mL
followed by a subsequent, persistent repeat value. However,
a cut-point of 0.4 ng/mL predict the risk of metastatic
relapse.
PSA

12. Alpha-Fetoprotein
Glycoprotein, found in fetal liver, yolk sac, GI tract, biochemically
related to albumin in adults half-life：4~6 days
Increased in 70% HCC, 20~70% germ cell tumors (yolk sac tumors,
embryonal cell carcinoma) of testis and ovary, except dysgerminoma
The absolute AFP level correlates with tumor bulk
Benign：conditions that cause hepatic parenchymal inflammation,
hepatic necrosis and hepatic regeneration, ex. hepatitis, pregnancy,
primary biliary cirrhosis, extrahepatic biliary obstruction. For Hbs Ag
(+) chronic hepatitis/cirrhosis screening, further correlation with USG
12~15th gestational week 30~40 ng/ml
At birth 30 ng/ml
1 years old <20 ng/ml
Adult 5 ng/ml

13. Hepatocellular Carcinoma (HCC)- Serum Markers
Alpha fetoprotein (AFP), total and L3 percent & Des-gamma-carboxy prothrombin (DCP)
- Surveillance in conjunction with abdominal ultrasound for early detection of HCC in high-risk groups
- Post-treatment monitoring when pretreatment concentration was elevated
AFP-L3
- AFP has 3 isoforms − L1, L2, L3
- L3 isoform is expressed by malignant hepatocytes
- L3 isoform has highest affinity for lectin from
Lens culinaris, which makes it possible to
differentiate L3 from other isoforms
- N.R-10% of total AFP
DCP
- Also referred to as PIVKA-II (protein induced by
vitamin K absence or antagonist II)
- Nonfunctional prothrombin
- Results from lack of carboxylation of 10 glutamic
acid residues
- Vitamin K dependent carboxylase, which
catalyzes this reaction in many HCCs, is absent

14. Glycoprotein synthesized by syncytiotrophoblastic cells
of normal placenta, never in males!
Serum and urine HCG ↑in early gestation and peak in
the first trimester (60~90 days) T ½: 1.25 days, ~30
hours
Elevated in：gestational trophoblastic disease (a
progressive rise in after 90 days of gestation → highly
suggestive), choriocarcinoma
◦ Detection & Monitor treatment response：
production of BHCG ceases on commencement of
tx, rising or persistently elevated levels are
diagnostic of resistance to chemotherapy
◦ evaluate radicality of the surgery: In testicular
cancer, the presence of β-HCG after orchiectomy →
residual cancer and needs further treatment
◦ Monitor relapse (reliable indicator of CR)
βHCG
• The disappearance of hCG is usually
achieved within 8 weeks in ∼40% of
patients, within 9 to 22 weeks in ∼55% of
cases, and in >22 weeks in 5% of
patients. In some cases, hCG
concentrations remain stable or increase,
suggesting the presence of persistent
trophoblastic disease (molar retention,
invasive mole, or choriocarcinoma).
• Unpredictable transient rises in
hCG/hCGβ concentrations after
chemotherapy may occur as a result of
tumor lysis with a subsequent release of a
given marker; consequently, comparisons
of t1/2 calculated from marker values
before treatment and after the second
cycle of chemotherapy are often
unreliable.

15. Monitor treatment and to detect recurrence for breast cancer
◦ Normal：<31 U/ml
◦ ↑in 20% with localized breast cancer,
◦ ~80% with metastatic disease, esp. if with bone involvement
◦ Specificity of 86%, sensitivity of 30%
◦ Also increased in gastric, pancreatic, cervical lung cancer
CA15-3

16. ◦ Fetal glycoprotein found on cell surfaces, produced by fetal GI tract, liver, and pancreas
◦ Normal serum and tissue fluid value：<3.0 ng/ml
◦ Circulating half-life：1 to 5 days
◦ Detect early relapse of colorectal cancer and prognostic indicator
◦ Normal pretherapy CEA：lower metastasis incidence
◦ High initial CEA：higher metastasis incidence
◦ In 2/3 of patients an elevated CEA may be the 1st indication of relapse
Found also in 30~50% of breast cancer, small cell lung cancer, mucinous cystadenocarcinoma of ovary,
adenocarcinoma of cervix
Elevation (<10 ng/ml) in smokers, COPD, inflammatory or peptic bowel disease, liver inflammation or
cirrhosis, renal failure, fibrocystic breast disease
Carcinoembryonic antigen (CEA)

17. A mucin which reacts with monoclonal antibody 111 6 NS 19-9.
Useful for differentiated benign from malignant disease
◦ Benign- Acute and chronic pancreatitis, hepatocellular jaundice, cirrhosis, acute
cholangitis and cystic fibrosis.
◦ Malignant- 71-93% elevated in pancreatic
21-42% elevated in gastric ca
20-40% elevated in colonic ca
As a diagnostic aid for pancreatic carcinoma. Though with inadequate sensitivity
and specificity limit the use of CA 19-9 in the early diagnosis of pancreatic cancer.
However, in non-jaundiced patients, CA 19-9 may complement other diagnostic
procedures
CA- 19.9

18. Chromogranin A – Circulating Neuroendocrine Marker
• Chromogranin A (CgA) is an acidic glycoprotein that is widely expressed by neuroendocrine cells and
constitutes one of the most abundant components of secretory granules.
• Reference range considered is upto 100 U/L. Values above 200 U/L are highly indicative of
neuroendocrine lesion.
• In pheochromocytoma and functioning-carcinoids, serum CgA shows a comparable, and even better,
sensitivity with urinary reference markers
• It can also be used in tumours of non-neuroendocrine origin as small cell carcinoma lung, other tumours
with neuroendocrine differentiation.
Limitations-
Renal failure, Achlorhydria- atrophic chronic gastritis and patients treated by proton pump inhibitors (as
omeprazole) or H2-receptors antagonists (as ranitidine), steroid therapy can give falsely high readings

19. Cyfra 21-1 - Marker for Non-small cell lung cancer
• CYFRA 21-1 is a cytokeratin 19 fragment that is soluble in serum. Although
expressed in all body fluids, its major occurrence is in the lung.
• Levels rise significantly in patients with epithelial cell-associated carcinomas. CYFRA 21-1 can be
elevated in approx. 70 % of all patients with a Squamous Cell Carcinoma of the lung.
• CYFRA 21-1 can be used for monitoring as well as a predictive and prognostic marker in lung cancer
treatment.
• Cut off level of 3.2 ng/ml is suggested to discreminate benign versus malignant disease.

20. Thyroglobulin And Thyrocalcitonin
Thyroglobulin：Tissue-specific, glycoprotein produced by thyroid follicular cells
• normal: <60 ug/L
• Also increased in breast or lung cancer
• Serum Tg measurement is a technically challenging assay for a marker of choice
during the follow-up of differentiated thyroid cancer. The main challenges being
the hook effect it is careful either to use a technique of serial dilution of the
serum in suspected cases, interferences from Heterophile antibodies &
associated Tg antibodies (TgAb) can give falsely low results.
Thyrocalcitonin：
Produced by thyroid C cells and medullary thyroid cancer
normal: <100 ng/L or <29 p mole/L
Effective in screen patients with 1st degree relatives affected by medullary
thyroid cancer and multiple endocrine neoplasia type 2

21. FDP DR-70) is a quantitative test measuring fibrin and
fibrinogen degradation products (FDP) formed due to a
proteolytic enzyme activator over-production in cancer
tumor cells, used for screening & monitoring.
Cancer causes a dramatic increase in protease activator
molecules in the cell, such as urokinase plasminogen
activator (u-PA) and others which stimulate the protease
(protein cleaving) enzymes to become activated and
ready to breakdown extracellular molecules. These
proteases, such as plasmin, cleave fibrinogen and the
slightly modified form of fibrinogen known as fibrin to
form FDP.
LIMITATIONS: Elevated levels of DR-70 may be seen in
Non-malignant conditions like acute infections,
autoimmune disorders, thrombotic diseases, DIC, DVT,
trauma, etc.
TYPE OF
CANCER
EFFECTIVENESS
SENSITIVITY (%) SPECIFICITY
(%)
Liver 96.5 96.7
Stomach 92 95
Ovarian 84 100
Pancreatic 80 95
Colorectal 75.6 93.4
Tongue 73 92.5
Lung 70.5 92
Breast 65.2 95
FDP-DR 70- Cancer Screening Marker

22. Take Home Messages
Preanalytical factors
• Use recommendations for
usage of tumour markers
Analytical factors
• Knowledge of the assay
method is important in
interpretation of either an
abnormal value or a serial
change in tumor marker
values. Various methods of
detection have their own
specific cut off values and
sensitivities
• Consider Hook Effect
Post- Analytical factors
• Never rely on a single test
result. Follow up testing
from the same laboratory.
• Consider half-life of the
tumor when interpreting
the result
• Consider how the Tumor
Marker is metabolized

23. The New Cancer Diagnostics
DNA
mRNA
Protein

24. Imaging
Multiparametric/miniature testing of serum on a
protein array
Mass spectrometric serum/urine proteomic pattern
generation
Whole genome SNP analysis
General Population
Predisposition to certain disease
.Screen-positive patients
Prevention; Effective Therapy
Asymptomatic individuals
Near Future . Ex. Ca Breast

25. Near Future . Ex. Ca Breast
Cancer patient
Cancerous tissue / body fluid
Tumour fingerprint- DNA/RNA
Individualized treatment
 Surgery / Biopsy
 Liquid biopsy
 Array analysis tissue