Tumour markers by dr narmada

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  • tumour,  also spelled  tumor,  also called  neoplasm ,  a mass of abnormal tissue that arises without obvious cause from preexisting body cells, has no purposeful function, and is characterized by a tendency to independent and unrestrained growth.  Marker, a substance used as an indicator of a biological state
  • Tumour markers by dr narmada

    1. 1. • 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)
    2. 2. PAST• First tumor marker reported was bence jones protein in 1847.• AFP-1963.• CEA- 1965.
    3. 3. 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
    4. 4. 1. Screening To identify early cancer risk2. Diagnosis To corroborate the diagnosis3. Staging To assess & stratify the risk4. Prognosis To predict the outcome5. Localization To locate the primary6. Therapy To target the therapy7. Surveillance To detect recurrence in F-Up8. Monitoring To evaluate response to Rx.
    5. 5. • 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)
    6. 6. • 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)
    7. 7. • ELISA• Immuno-histochemistry (IHC)• Polymerase chain reaction (PCR)• Fluorescence in situ hybridization (FISH)• Cluster Kits ( All-in-One Kit) – Detects profiles – Patterns – Prototypes – Constellations
    8. 8. STORAGE OF TEST KITSUnopened test kits should be stored at 2-8oC upon receipt. Themicrotiter plate should be kept in a sealed bag with desiccants,to minimize exposure to damp air. Opened test kits will remainstable until the expiration date, provided it is stored asdescribed above.
    9. 9. REAGENT PREPARATION1.All reagent should be brought to room temperature (18-22oC )before use.2. Dilute 1 volume of Wash Buffer Concentrate (50x) with 49volumes of distilled water. For example,Dilute 15 ml of Wash Buffer (50x) into distilled water to prepare750 ml of washing buffer (1x). Mixwell before use.
    10. 10. LIMITATIONS OF THEPROCEDURE1. Reliable and reproducible results will be obtained when the assayprocedure is carried out with a complete understanding of the package insertinstructions and with adherence to good laboratory practice.2. The wash procedure is critical. Insufficient washing will result in poorprecision and falsely elevated absorbance readings.3. Heterophilic antibodies such as human anti-mouse antibodies (HAMA) arefrequently found in the serum of human subjects. Those antibodies can causesevere interference in many immunodiagnostic procedures .
    11. 11. • 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
    12. 12. PSA velocity. This is the change in PSA concentrations over time. If thePSA continues to rise significantly over time (at least 3 samples at least18 months apart), then it is more likely that prostate cancer is present. Ifit climbs rapidly, then the affected person may have a more aggressiveform of cancer.PSA doubling time. This is another version of the PSA velocity. Itmeasures how rapidly the PSA concentration doubles.PSA density. This is a comparison of the PSA concentration and thevolume of the prostate (as measured by ultrasound). Men with largerprostates tend to produce more PSA, so this factor is an adjustment tocompensate for the size.Age-specific PSA ranges. Since PSA levels naturally increase as aman ages, it has been proposed that normal ranges be tailored to amans age.
    13. 13. What does the test result mean?Total PSA level greater than 10.0 ng/ml are at an increased risk forprostate cancerLevels between 4.0 ng/ml and 10.0 ng/ml may indicate prostate cancerBPH, or prostatitis. These conditions are more common in the elderly, as isa general increase in PSA levels. Concentrations of total PSA between 4.0ng/ml and 10.0 ng/ml are often referred to as the "gray zone.
    14. 14. 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 (microgramsper milliliter).
    15. 15. 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
    16. 16. • 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
    17. 17. • 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.
    18. 18. CEA Distribution In Healthy Individuals and Patients with Non-Malignant Conditions % Distribution of CEA ng/mL ng/mL ng/mLHealthy Subjects 0-3.0 3.1-10 >10.0Non Smokers 96 4 0Smokers 80 19 1Non-Malignant DiseasesCirrhosis 53 42 5Ulcerative Colitis 65 26 9Rectal polyps 78 19 3Pulmonary 52 39 9Gastrointestinal 76 21 3
    19. 19. CEA Distribution In Healthy Individuals and Patients with Non-Malignant Conditions % Distribution of CEA ng/mL ng/mL ng/mLHealthy Subjects 0-3.0 3.1-10 >10.0Non Smokers 96 4 0Smokers 80 19 1Non-Malignant DiseasesCirrhosis 53 42 5Ulcerative Colitis 65 26 9Rectal polyps 78 19 3Pulmonary 52 39 9Gastrointestinal 76 21 3
    20. 20. CEA Distribution In Patients With Malignant Disease % Distribution of CEA 0-3 3.1-10 >10 ng/mL ng/mL ng/mLColorectal 28 20 52Breast 50 27 23Ovarian 80 16 4Pulmonary 39 29 32
    21. 21. • 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
    22. 22. – 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
    23. 23. • 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
    24. 24. CA-125 Distribution In Patients With Malignant Disease % Distribution of CA-125Cancers <35 35-65>65 u/mL u/mL u/mLOvarian 14 9 77Lung 56 19 25Breast 82 8 10Endometrial 70 8 22Cervical 66 15 19Colorectal 76 11 12
    25. 25. 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
    26. 26. • 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
    27. 27. • 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)
    28. 28. • 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)
    29. 29. • 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
    30. 30. Hormones as T M• ACTH • LUNG• ADH • LUNG,ADRENAL CORTEX, PANCREATIC, • DUODENAL• BOMBESIN • LUNG• CALCITONIN • MEDULLARY CA THYROID • PHEOCHROMOCYTOMA• VASOACTIVE INTESTINAL PEPTIDE
    31. 31. ONCOFETAL ANTIGEN AS T M• AFP • HEPATOCELLULAR, GERM CELL TUMOR• CARCINOFETAL • LIVER FERRITIN• TENNESSEE ANTIGEN • COLON , BLADDER.• TISSUE POLYPEPTIDE • BREAST, ANTIGEN COLORECTAL,OVARIAN, BLADDER
    32. 32. 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.
    33. 33. • 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
    34. 34. PROTEINS AS TM• C- PEPTIDE • INSULINOMA• FERRITIN • LIVER,LUNG ,BREAST,LEUKEMIA• IMMUNOGLOBULIN • MULTIPLE MYELOMA, LYMPHOMA• MELANOMA ASSOCITED ANTIGEN • MELANOMA• PROTHROMBIN PRECURSOR • LIVER
    35. 35. 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.
    36. 36. MUCIN AS T M• CA 125 • OVARIAN, ENDOMETRIAL• CA 15-5 • BREAST, OVARIAN• CA 549 • BREAST, OVARIAN• MCA • BREAST, OVARIAN• CA27.29 • BREAST
    37. 37. • 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
    38. 38. 1. Cancer heterogeneity2. Lack of Specificity – false positives3. Lack of Sensitivity - false negatives4. Benign diseases - positive CA 125 or CEA5. Smokers have raised CEA6. Normal persons also have small amounts7. Higher levels only with large tumor volume8. Some cancers never have higher levels
    39. 39. THANK YOUSPEAKER- DR.NARMADA PRASAD TIWARI
    40. 40. 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 )
    41. 41. • 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)
    42. 42. 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]
    43. 43. ExamplesTumor 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]
    44. 44. 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, lungvimentin carcinoma, lymphoma, leukemia, melanoma[5]
    45. 45. ELISAEnzyme-Linked Immunosorbent Assay (ELISA) is used mainly todetect and quantify proteins, antibodies, peptides, or hormonesin a sample. In ELISA, antigens are immobilized on a solidsupport, either coated directly or through specific captureantibodies. Primary detection antibodies are then applied,forming a complex with the antigen. If conjugated with anenzyme, the detection antibody can directly be used to quantifythe antigen, or can itself be quantified by another enzyme-conjugated secondary antibody. The method chosen depends onwhich target you are investigating.
    46. 46. 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.

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