Tumor Markers.pptx

Tumor Markers

1. Tumor Markers Dr. Indranil Bhattacharya Consultant Pathologist Dept. of Pathology Jagjivan Ram Hospital Mumbai

2. What is Tumor Markers A tumor marker is a substance found in blood, urine, or body tissue. The term "tumor markers" may refer to proteins that are made by both healthy cells and cancer cells in the body. It may also refer to mutations, changes, or patterns in a tumor's DNA. Tumor markers are also called biomarkers .

3. How do we diagnose cancer today? Physical Examination Blood tests CT scans Biopsy Human Prostate Cancer Normal Blood Smear Chronic Myeloid Leukemia

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 micro-metastases. The level should not have large fluctuations that are independent of changes in tumor size. 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. Analyte Cancer Use CEA Monitor colorectal, breast, lung cancer CA-125 Ovarian cancer monitoring CA15-3, 27. 29 Monitor recurrences of breast cancer AFP Germ cell tumors, liver cancer Total PSA Screen and monitor prostate cancer Free PSA Distinguish prostate cancer from BPH HCG Germ cell and trophoblastic tumors Hormone receptors Breast cancer therapy NMP 22, BTA. FDP Monitor recurrences of bladder cancer COMMON TUMOR MARKERS

6. New cancer cases per year Cancer Site or Type New Cases Prostate 218,000 Lung 222,500 Breast 207,500 Colorectal 149,000 Urinary system 131,500 Skin 68,770 Pancreas 43,100 Ovarian 22,000 Myeloma 20,200 Thyroid 44,700 Germ Cell 9,000

7. Types of Tumor Markers • Hormones (hCG; calcitonin; gastrin; prolactin;) • Enzymes (acid phosphatase; alkaline phosphatase; PSA) • Cancer antigen proteins & glycoproteins (CA125; CA 15.3; CA19.9) • Metabolites (norepinephrine, epinephrine) • Normal proteins (thyroglobulin) • Oncofetal antigens (CEA, AFP) • Receptors (ER, PR, EGFR) • Genetic changes (mutations/translocations, etc.)

8. Characteristics of an ideal tumor marker • Specificity for a single type of cancer • High sensitivity and specificity for cancerous growth • Correlation of marker level with tumor size • Homogeneous (i.e., minimal post-translational modifications)

9. Roles for tumor markers • Determine risk (PSA) • Screen for early cancer (calcitonin, occult blood) • Diagnose a type of cancer (hCG, catecholamines) • Estimate prognosis (CA125) • Predict response to therapy (CA15-3, CA125, PSA, hCG) • Monitor for disease recurrence or progression (most widely used function) • Therapeutic selection (her2/neu, kras)

10. Tumor markers in routine use Marker CA15-3, BR 27.29 CEA, CA 19-9 CA 72.4, CA 19-9, CEA NSE , CY F A 21 . 1 PSA, PAP C A 125 Calcitonin, thyroglobulin hCG CA 19-9, CEA AFP, CA 19-1 BAP, Osteocalcin, NTx Catecholamines, metabolites Fecal occult blood Cancer Breast C o l o r ec t al Gastric Lung Prostate Ovarian Thyroid Trophoblastic Pancreatic Hepatocellular Bone Pheoch r o m oc y t o ma Colon cancer

11. Case report • 38-y M complains of severe headaches, episodic, and uncontrolled by analgesics. • No hx of migranes. In clinic, blood pressure 160/110 mmHg. • 24 hour urine is collected in acid container. Urine is tested for catecholamines.

12. LC-electrochemical detector results standard patient sample 1. Increased catecholamines. 2. Disproportinate increase in epinephrine. Diagnosis: pheo c h r o m o c y t o m a

13. Alpha Fetoprotein • Hepatocellular carcinoma • Germ Cell Tumors – Classifying and staging with hCG • Nonseminomas: both AFP & hCG elevated (90%) • Seminomas: AFP not elevated, hCG elevated 30% • AFP level not directly related to tumor size • Elevated in pregnancy, liver disease (hepatitis, cirrhosis, GI tumors) • AFP Tumor-specific glycoforms may improve specificity of AFP for HCC

14. CEA • CEA 150-300 kDa glycoprotein • Elevated in smokers and elderly • Elevated in breast, pancreatic, GI, and lung cancer – Breast cancer: used for detecting and monitoring metastatic disease • Elevated in benign diseases: cirrhosis, emphysema & rectal polyps • CEA – Not useful for C olorectal C ancer (CRC) Screening • New more specific marker for CRC: TIMP-1 (Tissue inhibitor of metalloprotease)

15. CA 15-3/ CA27.29 • High molecular weight glycoprotein (Polymorphic Epithelial Mucin) • Breast cancer marker – Correlate with stage and tumor size – Prognosis & predict response to chemotherapy – Detect residual disease following initial therapy – Detect recurrence, correlates with disease progression or regression – NOT sensitive enough for early detection • Elevated in benign diseases of liver & breast • Elevated in other cancers: pancreatic, lung, ovarian, colorectal & liver

16. CA 125 • 200-2000 kDa glycoprotein • Increased in benign diseases: pregnancy, endometriosis, ovarian cysts, PID, cirrhosis, hepatitis, pericarditis • Increased in other cancers: lung, breast, GI, endometrial, & pancreatic

17. Cytokeratin fragment 21-1 • Cytokeratins are intermediate filament structural proteins found in cytoskeleton of epithelial cells. • Increased CYFRA 21-1 seen in all histologic types of lung cancer but especially non-small cell lung cancer. • CYFRA 21-1 is used for diagnosis, prognosis, and monitoring after chemotherapy. • May be increased in benign respiratory disease, urological, gastrointestinal and gynecological cancers.

18. Thyroglobulin

19. Thyroglobulin as a tumor marker Monitoring of the recurrence or metastasis of differentiated thyroid cancer Papillary cancer Anaplastic cancer Follicular cancer Differentiated

20. Prostate specific antigen • PSA Forms / Measurements: – 55-95% PSA complexed with antichymotrypsin (PSA- ACT) – 5-45% free PSA (fPSA) – Total PSA = fPSA + PSA-ACT • Total PSA ranges: – 0-4 ng/mL = Low risk of PCA (22% positive) – 4-10 ng/mL = diagnostic gray zone (PCA & BPH) – >10 ng/mL = 40-50% with PCA

21. Prostate specific antigen • Enhancing Differential Diagnosis PCA – PSA velocity – increases over time – % fPSA – PSA density – tPSA/prostatic volume – Age-race- adjusted reference ranges

22. Free PSA (fPSA) • Unbound portion of PSA is inversely related to probability of prostatic carcinoma • Differentiation from carcinoma and BPH – When the total PSA is between 4 and 10 ng/mL: % Free PSA Probability of carcinoma 56% 28% 20% 16% 5% - 10 10 - 15 15 - 20 20 - 25 > 25

23. Prostate specific antigen clinical applications PSA >10 ng/mL with +DRE = Biopsy PSA 4-10 ng/mL and –DRE = Biopsy • Early detection in conjunction with DRE • Determine success of radical prostatectomy • Recurrence following treatment • Monitoring hormonal treatment * DRE: Digital Rectal Examination

24. Genetic tumor markers and disease • Oncogenes: – N-ras: leukemia – K-ras: colon/ gastric – C-erB-2: Breast/gastric – N-myc: Breast/Neuro – c-abl/bcr: CML – bcl-2: leukemia/lymp – HER-2/INT2/ATM/ H-ras: Breast – MCC: colon • Tumor Suppressors: – p53: Breast/colon/lung – RB1: Retinoblastoma – WT1& 2: Renal – BRCA1& 2: Breast/ pancreatic/Ovarian – BRCA1:prostate/stom. – APC: Colorectal – MTS1: Melanoma – DCC: colon/gastric

25. Estrogen and progesterone receptors • ER pos itive have more favorable prognosis within first 5 y ear after diagnosis • Hormone therapy blocks binding of estrogen to estrogen receptors: – Block receptor using tamoxifen or aromatase inhibitors – 60% of patients with primary tumors with ER/PR respond to hormone therapy • ER/PR measured in tumor tissue by immunohistochemistry or ELISA (tumor tissues)

26. HER-2/neu (c-erbB-2) • 185 kDa tyrosine kinase growth factor receptor • Gene amplification/overexpession occurs in 30% patients & correlates with aggressive disease & shortened survival • Moderate negative predictive factor for response to endocrine therapy or alkylating agents • Strong predictive factor for response to trastuzumab (Herceptin) • Methods approved by FDA: FISH and IHC

27. Immunohistochemistry for her-2/neu Ne g a t i v e 3+

28. Fluorescence in situ hybridization testing

29. Immunohistochemistry vs. FISH for HER - 2/neu testing

30. ER/PR and her-2/neu status and survival Onitilo et al. Clin Med Res 2009;7:4-13.

31. Other companion diagnostic tests Barrett et al. Clin Chem 2013;59:198-201. Biomarker Drug Cancer H e r 2 . neu KRAS BRAF ALK Fusion EGFR BCL-ABL translocation Trastuzumab Cetuximab, panitumumab Vemurafenib Crizotinib Gefitinib, erlotinib Imatinib, dasatinib, nilotinib Breast ca. C o l o r e c t al Melanoma Non-small cell lung ca. Non-small cell lung ca. Chronic myeloid leukemia

32. RT-PCR for circulating tumor cells • Prostate Cancer – PSA, PSMA • Breast Cancer – Cytokeratin 19, CEA, MUC1, hMAM • Melanoma – Tyrosinase, MART1, MAGE3, GAGE

33. Mechanism for circulating tumor cells Metastatic Cascade Cells grow as benign tumor Cells break through the basement membrane Travel through the blood Adhere to capillary wall Escape from blood vessel (extravasation) Proliferate to form metastases

34. mRNA Microarrays • Large mRNA and DNA arrays (Affymetrix, Illumina) enable unfocused genomic signature analysis. • Oncotype DX  and Mamaprint  enable prediction of therapeutic success in breast cancer. • Tumor of Origin  enables identification of the tissue origin of metastasis.

35. Comparative G enomic H ybridization (CGH) • A method of comparing differences in DNA copy number between tests (e.g. tumor) and reference samples • Can use paraffin-embedded tissues • Good method for identifying gene amplifications or deletions by scanning the whole genome.

36. Oncotype Dx Paik et al. N Engl J Med. 2004;351: 2817-2826 16 Cancer and 5 Reference Genes P ROL I F E R A T I ON Ki-67 STK15 Survivin Cyclin B1 MYBL2 ES T R OG EN ER PR Bcl2 S C U BE2 INVASION Stromelysin 3 Cathepsin L2 H E R 2 G R B7 H E R 2 BAG1 G S T M 1 REFERENCE B e t a - a c t i n GAPDH RPLPO GUS TFRC CD68

37. Oncotype Dx Paik et al. N Engl J Med. 2004;351: 2817-2826 Category RS (0-100) Low risk RS <18 Int risk RS ≥18 and <31 High risk RS ≥31 Calculation of the Recurrence Score Result Coefficient x Expression Level RS = + 0.47 x HER2 Group Score - 0.34 x ER Group Score + 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score + 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1

38. Microarray test for tumor of origin indications over biopsy • The cancer is found in an unexpected location or multiple locations, indicating metastatic disease • Tumor is poorly differentiated or undifferentiated • Unresolved differential diagnosis of ≥2 cancer types • The patient has a history of multiple cancers • IHC are inconclusive or conflicting • The specimen is small, constraining the diagnostic work up • Clinical history and histology differ on the dx • There is an atypical distribution of metastases • The diagnosis is questioned when the pt fails to respond to tx

39. Cancer genomics examples Cancer Associated gene Inheritance mode Breast and ovarian cancer BRCA1, BRCA2 Dominant Wilms’ tumor WT1 Dominant Familial retinoblastoma RB1 Dominant Huntington’s disease Huntingtin Dominant Hereditary colorectal cancer MLH1, MSH2,6, PMS1,2 Recessive Skin cancer Xeroderma pigmentosum Recessive XPB, XPD, XPA

40. Self assessment questions Which technique is most useful for detecting gene duplications and deletions? A. Immunohistochemistry B. Comparative genomic hybridization C. Fluorescence in situ hybridization D. Real-time polymerase chain reaction E. Chemilluminescence immunoassay Answer: B. CGH arrays are performed on microchips.

41. Self assessment questions Tumor markers that are glycosylated proteins: A. Are identical between tumors B. Can cause falsely high and low results by immunoassays C. The extent of glycosylation is indicative of disease severity D. Assay inaccuracies can be corrected by standardization E. Are detected by genomic microarrays Answer: B. Variation in tumor marker expression result in discordance between commercial immunoassays.

42. Self assessment questions High sensitivity PSA assays are useful for: A. Early detection of disease recurrence after prostatectomy B. Differentiation between benign prostatic hypertrophy and prostate cancer C. Differentiation between aggressive vs. non- aggressive disease D. Improved screening for prostate cancer E. Selection of hormone vs. chemotherapy Answer: A. As much as 2 years can be gained in some studies

43. Some limits to tumor marker tests : A condition or disease that is not cancer can raise tumor marker levels. Patient without cancer can have high tumor marker levels. Tumor marker levels can change over time. The tests may not get the same result every time. Tumor marker levels may not go up until cancer gets worse. This does not help find cancer early, or in patient at high risk. It also does not help find a recurrence. Some cancers do not make tumor markers that are found in the blood. And, some types of cancer have no known tumor markers. T umor marker levels might not go up, even if type of cancer usually makes tumor markers

44. Conclusions: Tumor marker tests are not perfect. They are often not specific for cancer and may not be sensitive enough to pick up a cancer recurrence. The presence of tumor markers alone is not enough to diagnose cancer.

45. Thank You