Tumor markers are biological substances that can be detected in cancer patients that are produced either by the tumor itself or by the body in response to cancer. While ideal tumor markers would be highly specific to cancer, many tumor markers can also be elevated in benign conditions. This can lead to false positives and incorrect interpretation of tumor marker test results. Some conditions that may cause transient or non-transient rises in common tumor markers include cirrhosis, inflammatory bowel disease, pregnancy, and smoking. The lack of specificity of some tumor markers limits their use for screening asymptomatic populations but they can be useful for monitoring known cancer patients. Improved specificity of tumor markers through new technologies may help address this limitation.

2. • Biological substances synthesized and
released by cancer cells themselves or
• Produced by the host in response to the
presence of tumor
• Most tumor markers are proteins
• Detected in a solid tumor, in circulating
tumor cells in peripheral blood, in serum,
lymph nodes, in bone marrow, or in other
body fluid (urine, stool, ascites)

3. PAST
• First tumor marker reported was bence jones
protein in 1847.
• AFP-1963.
• CEA- 1965.

4. IDEAL TUMOR MARKERS
• Be specific to the tumor
• Level should change in response to tumor size
• An abnormal level should be obtained in the presence of
micrometastases
• Levels in healthy individuals are at much lower concentrations
than those found in cancer patients
• Predict recurrences before they are clinically detectable
• Test should be cost effective

5. 1. Screening To identify early cancer risk
2. Diagnosis To corroborate the diagnosis
3. Staging To assess & stratify the risk
4. Prognosis To predict the outcome
5. Localization To locate the primary
6. Therapy To target the therapy
7. Surveillance To detect recurrence in F-Up
8. Monitoring To evaluate response to Rx.

7. • Enzymes (PSA, NSE, VMA)
• Cell membrane receptors (ER, PR)
• Tumor antigens (CEA, AFP)
• Antibodies (IgA, IgG, IgM, IgD)
• CA-specific proteins(CA 19-9, CA 125)
• Gene mutation products (BR CA 1, 2)

8. • Tissue-specific proteins (PSA, hCG)
• Special hormones (b-hCG )
• Catecholamines (VMA, ACTH)
• Cytoplasmic / Nucleic material (DNA)
• Products of cell turn-over (TNF)
• Cellular modulators (ki-62, c-erb-2)

9. • ELISA
• Immuno-histochemistry (IHC)
• Polymerase chain reaction (PCR)
• Fluorescence in situ hybridization (FISH)
• Cluster Kits ( All-in-One Kit)
– Detects profiles
– Patterns
– Prototypes
– Constellations

11. STORAGE OF TEST KITS
Unopened test kits should be stored at 2-8oC upon receipt. The
microtiter plate should be kept in a sealed bag with desiccants,
to minimize exposure to damp air. Opened test kits will remain
stable until the expiration date, provided it is stored as
described above.

12. REAGENT PREPARATION
1.All reagent should be brought to room temperature (18-22oC )
before use.
2. Dilute 1 volume of Wash Buffer Concentrate (50x) with 49
volumes of distilled water. For example,
Dilute 15 ml of Wash Buffer (50x) into distilled water to prepare
750 ml of washing buffer (1x). Mix
well before use.

14. LIMITATIONS OF THE
PROCEDURE
1. Reliable and reproducible results will be obtained when the assay
procedure is carried out with a complete understanding of the package insert
instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor
precision and falsely elevated absorbance readings.
3. Heterophilic antibodies such as human anti-mouse antibodies (HAMA) are
frequently found in the serum of human subjects. Those antibodies can cause
severe interference in many immunodiagnostic procedures .

15. • Prostate Specific Antigen(PSA) is a glycoprotein
• Ideal as a tumor marker, high tissue specificity
• High sensitivity for prostate cancer
• Also elevated in BPH & prostatitis
• Useful in
– Dx. & follow up of prostate Ca, Prognostic factor
– To monitor recurrence & response to treatment
– ? For screening of prostate cancer along with DRE

16. PSA velocity. This is the change in PSA concentrations over time. If the
PSA continues to rise significantly over time (at least 3 samples at least
18 months apart), then it is more likely that prostate cancer is present. If
it climbs rapidly, then the affected person may have a more aggressive
form of cancer.
PSA doubling time. This is another version of the PSA velocity. It
measures how rapidly the PSA concentration doubles.
PSA density. This is a comparison of the PSA concentration and the
volume of the prostate (as measured by ultrasound). Men with larger
prostates tend to produce more PSA, so this factor is an adjustment to
compensate for the size
.
Age-specific PSA ranges. Since PSA levels naturally increase as a
man ages, it has been proposed that normal ranges be tailored to a
man's age.

17. What does the test result mean?
Total PSA level greater than 10.0 ng/ml are at an increased risk for
prostate cancer
Levels between 4.0 ng/ml and 10.0 ng/ml may indicate prostate cancer
BPH, or prostatitis. These conditions are more common in the elderly, as is
a general increase in PSA levels. Concentrations of total PSA between 4.0
ng/ml and 10.0 ng/ml are often referred to as the "gray zone.

18. Neuron-specific enolase
(NSE)
• A neuronal isoenzyme of cytoplasmic enzyme enolase, in
neuroendocrine cells
• As a prognostic factor in neuroblastoma
• Occurs in neuroendocrine tumors: medullary carcinoma of the
thyroid, pheochromocytoma, carcinoid tumors; immature
teratoma, small cell carcinoma of lung, non-small-cell cancer,
melanoma. Correlate with stage and bulk of disease
• Abnormal levels are usually higher than 9 ug/mL (micrograms
per milliliter).

19. Estrogen Receptor (ER)
– 2 isoforms ： ERa and ERb
– ERa → better prognosis, predictor of relapse
– useful when deciding on adjuvant hormone
treatment
– As diagnostic marker when it is a primary
unknown tumor
– ERb → Good prognostic factor, correlates with
low grade and negative axillary LN status

20. • HER-2/neu oncogene (using monoclonal
antibody) - over expression related to poor
prognosis in breast cancer
• Oncogene c-erbB-2 gene ： over expressed in
30% of breast cancers, correlation between c-
erbB-2 gene positivity, positive axillary node
status, reduced time to relapse and reduced
overall survival
• BRCA1 gene on chromosome 17q ： familial
breast-ovarian cancer syndrome, and breast
cancer in early-onset breast cancer families →
high risk screening

21. • Complex glycoprotein that is associated with the plasma
membrane of tumor cells, from which it may be
released in to the blood.
• Elevated specially in Colon cancer, Adeno. Ca uterus.
• Also in Pancreatic, Gastric, Lung, breast & Ovarian Ca.
• Also in cirrhosis, inflammatory bowel disease, chronic
lung disease, pancreatitis, fibrocystic breast disease.
• 19% of smokers, 3% of healthy population
• Not satisfactory for screening for a healthy population
• Good for monitoring recurrence & to monitor Rx.

22. CEA Distribution In Healthy Individuals and
Patients with Non-Malignant Conditions
% Distribution of CEA
ng/mL ng/mL ng/mL
Healthy Subjects 0-3.0 3.1-10 >10.0
Non Smokers 96 4 0
Smokers 80 19 1
Non-Malignant Diseases
Cirrhosis 53 42 5
Ulcerative Colitis 65 26 9
Rectal polyps 78 19 3
Pulmonary 52 39 9
Gastrointestinal 76 21 3

23. CEA Distribution In Healthy Individuals and
Patients with Non-Malignant Conditions
% Distribution of CEA
ng/mL ng/mL ng/mL
Healthy Subjects 0-3.0 3.1-10 >10.0
Non Smokers 96 4 0
Smokers 80 19 1
Non-Malignant Diseases
Cirrhosis 53 42 5
Ulcerative Colitis 65 26 9
Rectal polyps 78 19 3
Pulmonary 52 39 9
Gastrointestinal 76 21 3

24. CEA Distribution In Patients
With Malignant Disease
% Distribution of CEA
0-3 3.1-10 >10
ng/mL ng/mL ng/mL
Colorectal 28 20 52
Breast 50 27 23
Ovarian 80 16 4
Pulmonary 39 29 32

25. • Alfa Feto Protein is a serum fetal protein synthesized by the
liver, yolk sac, gastrointestinal tract – a glycoprotein
• In Hepatocellular Cancer: It is diagnostic (>500) and also
useful for screening of high risk population (HBV, HCV)
• Benign conditions: hepatic parenchymal inflammation,
hepatic necrosis, pregnancy, primary biliary cirrhosis, extra
hepatic biliary obstruction give positive test.
• Testicular germ cell tumor (embrional or endodermal):
• Diagnosis, Prognosis, to monitor recurrence & response
• The absolute AFP level correlates with tumor bulk
• Cancers of pancreas, colon, stomach & bronchogenic Ca

26. – Cell surface glycoprotein, present during embryonic
development of coelomic epithelium & is present in
adult structures derived from it
– For follow up, an increase may predict recurrent
disease, precedes clinical recurrence by months
– Correlates with tumor bulk,
– ↑ levels also found in PID, 1st trimester

27. • Elevated in Ovarian, Endometrial, Pancreatic, Lung,
Breast, Colon cancers and also in
• Menstruation, Pregnancy, Endometriosis and other
gynecological and non gynec conditions.
• Useful in monitoring ovarian Ca recurrence & Rx.
• Screening of high risk population (BRCA1-2
Carriers); Not useful for routine screening

28. CA-125 Distribution In Patients With
Malignant Disease
% Distribution of CA-125
Cancers <35 35-65>65
u/mL u/mL u/mL
Ovarian 14 9 77
Lung 56 19 25
Breast 82 8 10
Endometrial 70 8 22
Cervical 66 15 19
Colorectal 76 11 12

29. CA 19-9 is elevated in
• In 21-42% patients of gastric Ca
• In 20-40% patients of colonic Ca
• In 71-93% patients of pancreatic Ca

30. • Human chorionic gonodotropin (βHCG)
– Glycoprotein synthesized by syncytio trophoblastic
cells of normal placenta
– Serum and urine HCG ↑ in early gestation and peak
in the first trimester (60~90 days)
– Elevated in Gestational trophoblastic disease (a
progressive rise in after 90 days of gestation → highly
suggestive), choriocarcinoma
– Elevated in testicular cancer, βHCG after surgery
– Monitor treatment response, relapse & recurrence

31. • Tyrosinase
– Use RT-PCR to detect hematogenous spread of
melanoma cells from a solid tumor in peripheral
blood
• S100B protein
– For confirmation of amelanotic malignant
melanoma by immunohistology
– ↑in 70% with stage IV metastasized melanoma
• MIA (melanoma inhibitory activity)

32. • Thyroglobulin
– Tissue-specific, glycoprotein produced by thyroid
follicular cells
– Also increased in breast or lung cancer
• Thyrocalcitonin
– From thyroid C cells & increased in medullary thyroid
cancer
– Effective to screen patients with 1st degree relatives
affected by medullary thyroid cancer and multiple
endocrine neoplasia type 2 (MEN2)

33. • Burkitt’s type lymphoma and leukemia
– T (8;14) due to juxtaposition and activation of the c-myc
gene
• CD 25 most sensitive serum marker for tumor burden
• CD 44 high concentration indicates poor prognosis
• Lactate dehydrogenase (LDH)
– Normal: 100~250 IU/L
– High-grade lymphomas, blood levels correlate closely with
disease activity and response to therapy

34. Hormones as T M
• ACTH • LUNG
• ADH • LUNG,ADRENAL
CORTEX, PANCREATIC,
• DUODENAL
• BOMBESIN • LUNG
• CALCITONIN • MEDULLARY CA
THYROID
• PHEOCHROMOCYTOMA
• VASOACTIVE
INTESTINAL PEPTIDE

35. ONCOFETAL ANTIGEN AS T M
• AFP • HEPATOCELLULAR,
GERM CELL TUMOR
• CARCINOFETAL • LIVER
FERRITIN
• TENNESSEE ANTIGEN • COLON , BLADDER.
• TISSUE POLYPEPTIDE • BREAST,
ANTIGEN COLORECTAL,OVARIAN,
BLADDER

36. GENETIC MARKERS
• SIS • ASTROCYTOMA,
OSTEOSARCOMA
• RET • MEDULLARY CA
THYROID, MEN 2
• K-RAS • COLON, LUNG
,PANCREATIC CA.
• H-RAS
• BLADDER, KIDNEY
• N-RAS
• MELANOMAS,HEMATOL
OGIC MALIGNANCY.

37. • ABL • CML,ALL
• C-MYC • BURKITT LYMPHOMA
• N-MYC • NEUROBLASTOMA,
• SMALL CELL LUNG CA
• L- MYC • SMALL CELL CA LUNG
• CYCLIN-D • MANTLE CELL
LYMPHOMA
• CDK4 • GLIOBLASTOMA,
• MELNOMA, SARCOMA

38. PROTEINS AS TM
• C- PEPTIDE • INSULINOMA
• FERRITIN • LIVER,LUNG
,BREAST,LEUKEMIA
• IMMUNOGLOBULIN • MULTIPLE MYELOMA,
LYMPHOMA
• MELANOMA ASSOCITED
ANTIGEN • MELANOMA
• PROTHROMBIN
PRECURSOR • LIVER

39. BLOOD GROUP ANTIGEN AS TM
• CA 19-9 • PANCREATIC,
GIT,HEPATIC
• CA 19-5 • GIT,PANCREATIC,
• OVARIAN
• CA 50 • PANCREATIC, GIT
• CA72-4 • OVARIAN,BREAST,GIT
• CA242 • GIT,PANCREATIC.

40. MUCIN AS T M
• CA 125 • OVARIAN,
ENDOMETRIAL
• CA 15-5 • BREAST, OVARIAN
• CA 549 • BREAST, OVARIAN
• MCA • BREAST, OVARIAN
• CA27.29 • BREAST

41. • Genomics – Gene structure
• Proteonomics – Protein structure
• Pharmacogenomics
– Gene-based drugs structuring and delivery
• G-scan – Human genome mapping
• New treatment modalities
• Individualised treatment modalities
• Early detection of malignant change
• Greater sensitivity and specificity
• Better monitoring and follow-up care

43. 1. Cancer heterogeneity
2. Lack of Specificity – false positives
3. Lack of Sensitivity - false negatives
4. Benign diseases - positive CA 125 or CEA
5. Smokers have raised CEA
6. Normal persons also have small amounts
7. Higher levels only with large tumor volume
8. Some cancers never have higher levels

44. THANK YOU
SPEAKER- DR.NARMADA PRASAD TIWARI

45. Benign conditions that may cause rises (some transient) in serum
tumour marker levels that may lead to incorrect interpretation
• Acute cholangitis (CA19-9) • Endometriosis (CA125)
• Acute hepatitis (CA125, CA15-3) • Heart failure (CA125)
• Acute and/or chronic pancreatitis (CA125, CA19- • Irritable bowel syndrome (CA125, CA19-9, CEA)
9) • Jaundice (CA19-9, CEA)
• Acute urinary retention (CA125, PSA ) • Leiomyoma (CA125)
• Arthritis/osteoarthritis/rheumatoid arthritis • Liver regeneration (α fetoprotein)
(CA125) • Menopause (human chorionic gonadotrophin)
• Benign prostatic hyperplasia (PSA) • Menstruation (CA125)
• Cholestasis (CA19-9) • Non-malignant ascites (CA125)
• Chronic liver diseases such as cirrhosis, chronic • Ovarian hyperstimulation (CA125)
active hepatitis (CA125, CA15-3,CA19-9,
carcinoembryonic antigen (CEA)) • Pancreatitis (CA125, CA19-9)
• Chronic renal failure (CA125, CA15-3, CEA, • Pericarditis (CA125)
human chorionic gonadotrophin) Colitis (CA125, • Peritoneal inflammation (CA125)
CA15-3, CEA) • Pregnancy (α fetoprotein, CA125, human
• Congestive heart failure (CA125) chorionic gonadotrophin) Prostatitis (PSA)
• Cystic fibrosis (CA125) • Recurrent ischaemic strokes in patients with
• Dermatological conditions (CA15-3) metastatic cancer (CA125) Respiratory diseases
such as pleural inflammation, pneumonia
• Diabetes (CA125, CA19-9) (CA125, CEA) Sarcoidosis (CA125)
• Diverticulitis (CA125, CEA) • Systemic lupus erythematosus (CA125)
• Urinary tract infection (PSA )

46. • Tumour-Associated Proteins (TAP)
• Cell membrane receptors
• Hormones
• Immunoglobulins / Cellular antigens
• Polyamines
• Protein clusters and fragments
• Chromosomal material
• Genes (single, clusters)
• Genetic material (DNA, RNA, mRNA)
• Cell modulators (transducers / suppressors)

47. gastrointestinal stromal tumor, mastocytosis, seminoma[5]
Chromogranin neuroendocrine tumor[5]
Cytokeratin (various types) Many types of carcinoma, some types of sarcoma[5]
smooth muscle sarcoma, skeletal muscle sarcoma,
Desmin
endometrial stromal sarcoma[5]
Epithelial membrane protein (EMA) many types of carcinoma, meningioma, some types of sarcoma[5]
Factor VIII, CD31 FL1 vascular sarcoma[5]
Glial fibrillary acidic protein (GFAP) glioma (astrocytoma, ependymoma)[5]
Gross cystic disease fluid protein(GCDFP-15) breast cancer, ovarian cancer, salivary gland cancer[5]
melanoma, PEComa (for example angiomyolipoma), clear cell carcinoma,
HMB-45
adrenocortical carcinoma[5]
Human chorionic gonadotropin (hCG) gestational trophoblastic disease, germ cell tumor, choriocarcinoma[5]
immunoglobulin lymphoma, leukemia[5]
sex cord-gonadal stromal tumour, adrenocortical carcinoma,
inhibin
hemangioblastoma[5]
keratin (various types) carcinoma, some types of sarcoma[5]
PTPRC (CD45) lymphoma, leukemia, histiocytic tumor[5]
lymphocyte marker (various types lymphoma, leukemia[5]

48. Examples
Tumor marker Associated tumor types
•Alpha fetoprotein (AFP) •germ cell tumor, hepatocellular carcinoma[5]
•CA15-3 •breast cancer[6]
•CA27-29 •breast cancer
•Mainly pancreatic cancer, but also colorectal cancer and other
•CA19-9
types of gastrointestinal cancer.[7]
•Mainly ovarian cancer,[8] but may also be elevated in for example
endometrial cancer, fallopian tube cancer, lung cancer,
•CA-125
breast cancer and gastrointestinal cancer.[9] May also increase in
endometriosis.[10]
•mesothelioma, sex cord-gonadal stromal tumour,
•Calretinin
adrenocortical carcinoma, synovial sarcoma[5]
•gastrointestinal cancer, cervix cancer, lung cancer, ovarian cancer
•Carcinoembryonic antigen
, breast cancer, urinary tract cancer[5]
•hemangiopericytoma/solitary fibrous tumor, pleomorphic lipoma
•CD34 , gastrointestinal stromal tumor,
dermatofibrosarcoma protuberans[5]
•Ewing sarcoma, primitive neuroectodermal tumor,
•CD99 hemangiopericytoma/solitary fibrous tumor, synovial sarcoma,
lymphoma, leukemia, sex cord-gonadal stromal tumour[5]
•CD117 •gastrointestinal stromal tumor, mastocytosis, seminoma[5]

49. Myo D1 rhabdomyosarcoma, small, round, blue cell tumour[5]
muscle-specific actin (MSA) myosarcoma (leiomyosarcoma, rhabdomyosarcoma)[5]
neurofilament neuroendocrine tumor, small-cell carcinoma of the lung[5]
neuroendocrine tumor, small-cell carcinoma of the lung,
neuron-specific enolase (NSE)
breast cancer[5]
placental alkaline phosphatase (PLAP) seminoma, dysgerminoma, embryonal carcinoma[5]
prostate-specific antigen prostate[5]
melanoma, sarcoma (neurosarcoma, lipoma, chondrosarcoma),
astrocytoma, gastrointestinal stromal tumor, salivary gland cancer,
S100 protein
some types of adenocarcinoma, histiocytic tumor(dendritic cell,
macrophage)[5]
smooth muscle actin (SMA) gastrointestinal stromal tumor, leiomyosarcoma, PEComa[5]
synaptophysin neuroendocrine tumor[5]
thyroglobulin thyroid cancer (but not in medullary thyroid cancer)[5]
thyroid transcription factor-1 all types of thyroid cancer, lung cancer[5]
colorectal cancer,[11] Breast cancer,[12][13] renal cell carcinoma[14][15]
Tumor M2-PK Lung cancer,[16][17] Pancreatic cancer,[18] Esophageal Cancer,[19]
Stomach Cancer,[19]Cervical Cancer,[20]Ovarian Cancer,[21]
sarcoma, renal cell carcinoma, endometrial cancer, lung
vimentin
carcinoma, lymphoma, leukemia, melanoma[5]

50. ELISA
Enzyme-Linked Immunosorbent Assay (ELISA) is used mainly to
detect and quantify proteins, antibodies, peptides, or hormones
in a sample. In ELISA, antigens are immobilized on a solid
support, either coated directly or through specific capture
antibodies. Primary detection antibodies are then applied,
forming a complex with the antigen. If conjugated with an
enzyme, the detection antibody can directly be used to quantify
the antigen, or can itself be quantified by another enzyme-
conjugated secondary antibody. The method chosen depends on
which target you are investigating.

51. Types of ELISA Methods:
• Direct ELISA: Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or
quantify antigen concentration. The specificity of the primary antibody is very important.
o PROS: minimum procedure; avoids cross-reactivity from secondary antibody.
o CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling.
• Indirect ELISA: Primary antibodies are not labeled, but detected instead with enzyme-conjugated
secondary antibodies that recognize the primary antibodies.
o PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies
can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity.
o CONS: cross-reactivity may occur.
• Sandwich ELISA: The antigen to be measured is bound between a layer of capture antibodies and a layer
of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or
competition of binding sites.
o PROS: sensitive, high specificity, antigen does not need to be purified prior to use.
o CONS: antigens must contain at least two antibody binding sites.
• Competitive ELISA: The antigen of interest from the sample and purified immobilized antigen compete for
binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen
alone indicates the presence of antigens in the sample.
o PROS: crude or impure samples may be used, high reproducibility.
o CONS: lower overall sensitivity and specificity.