• 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)

PAST

• First tumor marker reported was bence jones protein in 1847.

• AFP-1963.• CEA- 1965.

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

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.

• 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)

• 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)

• ELISA • Immuno-histochemistry (IHC)• Polymerase chain reaction (PCR)• Fluorescence in situ hybridization (FISH)• Cluster Kits ( All-in-One Kit)

– Detects profiles– Patterns– Prototypes– Constellations

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.

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.

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.

• 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

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.

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.

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).

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

• 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

• 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.

CEA Distribution In Healthy Individuals and Patients with Non-Malignant Conditions

% Distribution of CEAng/mL ng/mL ng/mL

Healthy Subjects 0-3.0 3.1-10 >10.0

Non Smokers 96 4 0Smokers 80 19 1

Non-Malignant DiseasesCirrhosis 53 42 5Ulcerative Colitis 65 26 9Rectal polyps 78 19 3Pulmonary 52 39 9Gastrointestinal 76 21 3

CEA Distribution In Healthy Individuals and Patients with Non-Malignant Conditions

% Distribution of CEAng/mL ng/mL ng/mL

Healthy Subjects 0-3.0 3.1-10 >10.0

Non Smokers 96 4 0Smokers 80 19 1

Non-Malignant DiseasesCirrhosis 53 42 5Ulcerative Colitis 65 26 9Rectal polyps 78 19 3Pulmonary 52 39 9Gastrointestinal 76 21 3

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

• 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

– 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

• 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

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

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

• 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

• 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)

• 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)

• 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

Hormones as T M

• ACTH• ADH

• BOMBESIN• CALCITONIN

• VASOACTIVE INTESTINAL PEPTIDE

• LUNG• LUNG,ADRENAL

CORTEX, PANCREATIC,• DUODENAL• LUNG• MEDULLARY CA

THYROID• PHEOCHROMOCYTOMA

ONCOFETAL ANTIGEN AS T M

• AFP

• CARCINOFETAL FERRITIN

• TENNESSEE ANTIGEN

• TISSUE POLYPEPTIDE ANTIGEN

• HEPATOCELLULAR, GERM CELL TUMOR

• LIVER

• COLON , BLADDER.

• BREAST, COLORECTAL,OVARIAN,BLADDER

GENETIC MARKERS

• SIS

• RET

• K-RAS

• H-RAS• N-RAS

• ASTROCYTOMA, OSTEOSARCOMA

• MEDULLARY CA THYROID, MEN 2

• COLON, LUNG ,PANCREATIC CA.

• BLADDER, KIDNEY• MELANOMAS,HEMATOLO

GIC MALIGNANCY.

• ABL• C-MYC• N-MYC

• L- MYC

• CYCLIN-D

• CDK4

• CML,ALL• BURKITT LYMPHOMA• NEUROBLASTOMA,• SMALL CELL LUNG CA• SMALL CELL CA LUNG

• MANTLE CELL LYMPHOMA• GLIOBLASTOMA,• MELNOMA, SARCOMA

PROTEINS AS TM

• C- PEPTIDE• FERRITIN

• IMMUNOGLOBULIN

• MELANOMA ASSOCITED ANTIGEN

• PROTHROMBIN PRECURSOR

• INSULINOMA• LIVER,LUNG ,BREAST,LE

UKEMIA• MULTIPLE MYELOMA,

LYMPHOMA

• MELANOMA

• LIVER

BLOOD GROUP ANTIGEN AS TM

• CA 19-9

• CA 19-5

• CA 50• CA72-4• CA242

• PANCREATIC, GIT,HEPATIC

• GIT,PANCREATIC,• OVARIAN• PANCREATIC, GIT• OVARIAN,BREAST,GIT• GIT,PANCREATIC.

MUCIN AS T M

• CA 125

• CA 15-5• CA 549• MCA• CA27.29

• OVARIAN, ENDOMETRIAL

• BREAST, OVARIAN• BREAST, OVARIAN• BREAST, OVARIAN• BREAST

• 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

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

THANK YOU

SPEAKER- DR.NARMADA PRASAD TIWARI

Benign conditions that may cause rises (some transient) in serum tumour marker levels that may lead to incorrect interpretation

• Acute cholangitis (CA19-9) • Acute hepatitis (CA125, CA15-3) • Acute and/or chronic pancreatitis (CA125, CA19-

9) • Acute urinary retention (CA125, PSA ) • Arthritis/osteoarthritis/rheumatoid arthritis

(CA125) • Benign prostatic hyperplasia (PSA) • Cholestasis (CA19-9) • Chronic liver diseases such as cirrhosis, chronic

active hepatitis (CA125, CA15-3,CA19-9, carcinoembryonic antigen (CEA))

• Chronic renal failure (CA125, CA15-3, CEA, human chorionic gonadotrophin) Colitis (CA125, CA15-3, CEA)

• Congestive heart failure (CA125) • Cystic fibrosis (CA125) • Dermatological conditions (CA15-3) • Diabetes (CA125, CA19-9) • Diverticulitis (CA125, CEA)

• Endometriosis (CA125) • Heart failure (CA125) • Irritable bowel syndrome (CA125, CA19-9, CEA) • Jaundice (CA19-9, CEA) • Leiomyoma (CA125) • Liver regeneration (α fetoprotein) • Menopause (human chorionic gonadotrophin) • Menstruation (CA125) • Non-malignant ascites (CA125) • Ovarian hyperstimulation (CA125) • Pancreatitis (CA125, CA19-9) • Pericarditis (CA125) • Peritoneal inflammation (CA125) • Pregnancy (α fetoprotein, CA125, human chorionic

gonadotrophin) Prostatitis (PSA) • Recurrent ischaemic strokes in patients with

metastatic cancer (CA125) Respiratory diseases such as pleural inflammation, pneumonia (CA125, CEA) Sarcoidosis (CA125)

• Systemic lupus erythematosus (CA125) • Urinary tract infection (PSA )

• 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)

gastrointestinal stromal tumor, mastocytosis, seminoma[5]

Chromogranin neuroendocrine tumor[5]

Cytokeratin (various types) Many types of carcinoma, some types of sarcoma[5]

Desmin smooth muscle sarcoma, skeletal muscle sarcoma, 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]

HMB-45 melanoma, PEComa (for example angiomyolipoma), clear cell carcinoma, adrenocortical carcinoma[5]

Human chorionic gonadotropin (hCG) gestational trophoblastic disease, germ cell tumor, choriocarcinoma[5]

immunoglobulin lymphoma, leukemia[5]

inhibin sex cord-gonadal stromal tumour, adrenocortical carcinoma, 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]

Tumor marker Associated tumor types

•Alpha fetoprotein (AFP) •germ cell tumor, hepatocellular carcinoma[5]

•CA15-3 •breast cancer[6]

•CA27-29 •breast cancer

•CA19-9•Mainly pancreatic cancer, but also colorectal cancer and other types of gastrointestinal cancer.[7]

•CA-125

•Mainly ovarian cancer,[8] but may also be elevated in for example endometrial cancer, fallopian tube cancer, lung cancer, breast cancer and gastrointestinal cancer.[9] May also increase in endometriosis.[10]

•Calretinin•mesothelioma, sex cord-gonadal stromal tumour, adrenocortical carcinoma, synovial sarcoma[5]

•Carcinoembryonic antigen•gastrointestinal cancer, cervix cancer, lung cancer, ovarian cancer, breast cancer, urinary tract cancer[5]

•CD34•hemangiopericytoma/solitary fibrous tumor, pleomorphic lipoma, gastrointestinal stromal tumor, dermatofibrosarcoma protuberans[5]

•CD99•Ewing sarcoma, primitive neuroectodermal tumor, hemangiopericytoma/solitary fibrous tumor, synovial sarcoma, lymphoma, leukemia, sex cord-gonadal stromal tumour[5]

•CD117 •gastrointestinal stromal tumor, mastocytosis, seminoma[5]

Examples

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]

neuron-specific enolase (NSE) neuroendocrine tumor, small-cell carcinoma of the lung, breast cancer[5]

placental alkaline phosphatase (PLAP) seminoma, dysgerminoma, embryonal carcinoma[5]

prostate-specific antigen prostate[5]

S100 protein

melanoma, sarcoma (neurosarcoma, lipoma, chondrosarcoma), astrocytoma, gastrointestinal stromal tumor, salivary gland cancer, 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]

Tumor M2-PKcolorectal cancer,[11] Breast cancer,[12][13] renal cell carcinoma[14][15] Lung cancer,[16][17] Pancreatic cancer,[18] Esophageal Cancer,[19]

Stomach Cancer,[19]Cervical Cancer,[20]Ovarian Cancer,[21]

vimentin sarcoma, renal cell carcinoma, endometrial cancer, lung carcinoma, lymphoma, leukemia, melanoma[5]

ELISAEnzyme-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.

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.