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Tumor Biomarkers

Tumor Biomarkers. Kentucky Cancer Registry Fall Conference Joe Pulliam, M.D. Tumor Markers. Defined by NCI as substances found in abnormal amounts in patients with cancer Blood Other body fluids Other tissues

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Tumor Biomarkers

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  1. Tumor Biomarkers Kentucky Cancer Registry Fall Conference Joe Pulliam, M.D.

  2. Tumor Markers • Defined by NCI as substances found in abnormal amounts in patients with cancer • Blood • Other body fluids • Other tissues • Conceptually also applies to physical exam findings and radiologic markers (clinical tumor markers)

  3. Tumor Markers • Some markers are unique for a cancer type • Others are found in many types of cancers • Others may also be found in patients without cancer but that have other diseases • Ex. Prostate specific antigen

  4. Tumor Markers • Used for: • Diagnosis • Monitor response to treatment • Prognosis • Predict response to particular treatments • Generally cannot be used alone to diagnose cancer – must be used with other methods such as biopsy

  5. How are Tumor Markers selected? • Basic Research • To show a potential marker exists • Evidence-based Translational Research • To show the markers add value to healthcare • Practice Guidelines Organizations • Review the literature and provide standards • American Society of Clinical Oncology • National Comprehensive Cancer Network • National Academy of Clinical Biochemistry • College of American Pathologists

  6. How are Tumor Markers selected? Basic Research Translational Research and Evidence Based Medicine Observed Changes in Treatment Outcome Clinical Practice Guidelines Changes in Patient Care

  7. How are Tumor Markers selected? • Coordinated efforts of cancer staging and database centers • American Joint Commission on Cancer • International Union for Cancer Control • Prognostic Factors Task Force • NCI Surveillance Epidemiology and End Results • Non-anatomic tumor markers were first added to AJCC Cancer Staging Manual, 6th ed. and expanded in the 7th ed.

  8. New Tumor Markers • Many of the new non-anatomic tumor markers refer to components of tumor signaling pathways • Prognostic significance • Potential therapeutic targets (predictive markers)

  9. Ligand Receptor Intracellular Signaling Molecules Cellular Changes

  10. www.biocarta.com/pathfiles/egfPathway.aspaccessed 9-8-10

  11. New Tumor Biomarkers:Her2 in Breast Cancer • Human Epidermal Growth Factor Receptor 2 • Nomenclature: HUGO designation ERBB2 • aka: NEU, NGL, HER2, TKR1, HER-2, c-erb B2, Her2/neu • Cell membrane tyrosine kinase receptor • Signals after forming homo- or heterodimers with other EGFR family members • Amplified in 18 to 20% of breast cancers

  12. New Tumor Biomarkers:Her2 • Clinical relevance: • Correlates with poor prognosis • Correlates with resistance to endocrine therapy • Her2 positive tumors often have lost expression of ER/PR • Predicts response to anthracyclines through linkage to TOPO2 gene, with which it is often co-amplified • Predicts response to targeted therapy (Trastuzumab/Herceptin)

  13. New Tumor Biomarkers:Her2 • Detection of increased Her2 can be performed in several ways • Immunohistochemistry (IHC) • Fluorescence in situ hybridization (FISH) • Chromogenic in situ hybridization (CISH) • Silver-enhanced in situ hybridization (SISH) • Quantitative reverse-transcriptase polymerase chain reaction (Q RT-PCR)

  14. Her2 Detection • Can use DNA, RNA or protein www.herceptin.com/.../interpreted-results.jspaccessed 9-8-10

  15. Her2 by IHC www.herceptin.com/.../interpreted-results.jspaccessed 9-8-10

  16. Her2 by FISH • This diagram gives the ratio of Her2 signals to a chromosome 17 centromeric probe • FISH can also be done with a single Her2 probe and reported as the number of probe signals visualized www.herceptin.com/.../interpreted-results.jspaccessed 9-8-10

  17. Advantages Quick, cheap, easy Storage for years Tumor morphology visible FDA approved Disadvantages Susceptible to variations in testing protocol Semiquantitative and subjective score interpretation Her2 by IHC AJCP 2009 132:539

  18. Advantages Less susceptible to variations in testing Score interpretation more objective, quantiative Identifies Her2 amplified cases within IHC 2+ cases FDA approved Disadvantages Expensive equipment and probes Signal decays with time Difficult to see tumor morphology (dark field) Her2 by FISH AJCP 2009 132:539

  19. Testing Algorithm for Her2 IHC/FISH Arch Pathol Lab Med 2007, 131:18

  20. Her2 by CISH or SISH AJCP 2009 132:539

  21. Advantages Bright field – conventional microscopy Stable signal over time Can visualize tumor morphology DNA more stable analyte than protein Disadvantages New technique, therefore little experience Some discordance with FISH for low level amplification Her2 by CISH or SISH AJCP 2009 132:539

  22. Scarff-Bloom-Richardson (SBR) Grade in Breast Cancer • Histologic grading system for breast cancer • Correlates well with prognosis • Adopted by World Health Organization in 1968 • Also referred to as Nottingham modification, or Bloom-Richardson grading

  23. Scarff-Bloom-Richardson (SBR) Grade • Tubule Formation • Majority of tumor (>75%) – 1 point • Moderate degree (10-75%) – 2 points • Little or none (<10%) – 3 points • Nuclear pleomorphism (compare to adjacent normal epithelium) • Small, regular uniform cells – 1 point • Moderately increased size and variability – 2 points • Marked variation – 3 points • Mitotic Count (must adjust for microscope field) • Low – 1 point • Moderate – 2 points • High – 3 points • Overall Grade is Sum of Scores • Grade 1 – well differentiated – 3 to 5 points • Grade 2 – moderately differentiated – 6 to 7 points • Grade 3 – poorly differentiated – 8 to 9 points

  24. Scarff-Bloom-Richardson (SBR) Grade Grade 2 Grade 3 Grade 1 Tubule Formation Nuclear Pleomorphism Mitotic Count Adapted from www.breastpathology.info/Special%20Types.html9-8-10

  25. Proliferation as a Tumor Marker • The definition of significantly “proliferative” varies with the type of cancer • In some tumors, proliferation may be used as a marker for diagnosis • Burkitt non-Hodgkin lymphoma • In other tumors, proliferation may be correlated with prognosis

  26. Proliferation as a Tumor Marker • How to measure proliferation • Mitotic count – simplest • Ex. SBR grade for Breast Cancer • Mitosis-Karyorrhexis Index • Neuroblastoma • Recognizes difficulty distinguishing mitotic cells from apoptotic cells • Immunohistochemistry • Special proteins expressed during cell cycle (usually with DNA synthesis) • PCNA – Proliferating Cell Nuclear Antigen • Mib1 • Ki-67 • Flow cytometry or Image analysis • The amount of DNA per cell correlates with phase of cell cycle • Formerly used in breast cancer

  27. Mib-1 in Burkitt Lymphoma

  28. Multigene Signature Methods • Most commonly used in Breast cancer • Will likely become important in other tumors • Gene expression microarray • Include several pathways of significance for each type of cancer • Correlate with distinct biological subtypes • Methods • Mammaprint – 70 gene • Oncotype Dx – 21 gene • others

  29. Multigene Signature Methods • Derived from basic research and translational research using tumor biospecimens correlated with tumor registry outcome data • Potential tumor markers validated in subsequent testing NEJM 2009, 360:790

  30. Multigene Signature Methods • 4 molecular breast cancer subtypes • Basal-like • Luminal A • Luminal B • Her2-like • Good correlation with standard pathological variables • Potential insight into treatment failures or new therapeutic interventions NEJM 2009, 360:790

  31. Mammaprint • Gene signature derived from selected retrospective review • 78 node negative breast cancer patients not treated with adjuvant therapy • Supervised top-down approach • Two outcomes – “Low Risk” or “High Risk” of disease recurrence without adjuvant therapy • Uses fresh or frozen tumor, not formalin-fixed paraffin-embedded • 70 gene cDNA microarray • FDA approved

  32. www.nature.com/.../v17/n7/fig_tab/2402974f1.html

  33. Oncotype Dx • Uses a “candidate-gene” approach to measure gene expression by Q-RT-PCR http://www.oncotypedx.com/en/Breast/HealthcareProfessional/Underlying.aspxaccessed 9-8-10

  34. Oncotype Dx • Uses formalin-fixed, paraffin-embedded tissue • Normalized gene ratios by comparing 16 cancer genes to 5 reference genes • this also corrects for variable RNA quality between samples Nature Protocols 1, 1559 - 1582 (2006)

  35. Oncotype Dx • To be used in estrogen/progesterone receptor positive patients • Informative for node positive patients • Not FDA approved • Endorsed by ASCO and NCCN Clinical Practice Guidelines • Recurrence Score – scale from 0 to 100 • Could be thought of as “will patient benefit from traditional chemotherapy” score • Low – 0-18 – low chance of recurrence and no benefit from traditional chemotherapy • High – 31 or more – high chance of recurrence and favorable response to traditional chemotherapy

  36. Multigene Signature Methods • Both Mammaprint and Oncotype Dx are undergoing prospective clinical trials • Mammaprint – MINDACT • will also assess utility in patients with up to 3 positive lymph nodes • Oncotype Dx – TAILORx • designed to further assess utility of intermediate risk scores for treatment decisions

  37. New Tumor Biomarkers:KRAS in Colorectal Cancer • Intracellular second messenger signaling molecule • Relays messages from receptor tyrosine kinases • Other family members include NRAS and HRAS • One of the most frequently mutated oncogenes in a variety of cancers • Activating mutations in 35-45% of colorectal carcinoma • Confer resistance to EGFR-inhibitor drugs

  38. Progression free survival Overall survival

  39. Spectrum of RAS mutation in Colorectal Cancer • Most are activating mutations in codons 12 and 13 (35% of CRC cases) • Initial trials showing resistance to EGFR-inhibitors looked at only these codons • Other mutations (5-10% of CRC cases) are also activating and confer resistance to EGFR-inhibitors • Codons 14, 17, 61, 146 • Activating NRAS mutations (3% of CRC cases also confer resistance

  40. EGFR-RAS-PI3K Signaling Pathways in Colorectal Cancer World J Gastroenterol 2010, 16:1177 accessed 9-8-10

  41. Future Tumor Markers in Colorectal Cancer? • KRAS and BRAF now • PIK3CA, and PTEN in future (Quadruple Negative CRC)? • Also NRAS (Lancet Oncol, 2010 Aug, 11:753) J Clin Oncol 2010, 28:1254 accessed 6-4-10

  42. New Tumor Biomarkers:KIT in Gastrointestinal Stromal Sarcoma (GIST) • GIST – uncommon tumor thought to arise from interstitial cells of Cajal (cells of the autonomic nervous system) • Most GIST tumors have an activating in a receptor tyrosine kinase • KIT mutation in 50-85% of GIST • KIT also known as CD117 or stem cell factor receptor • Nearly all KIT mutations in GIST are in exon 11, some in exon 9 or 13, rarely in exon 17 • PDGFR-alpha mutation in 10-20% of GIST

  43. Fortunately, nearly all KIT and PDGFR-alpha mutations in GIST respond to treatment with Gleevec (a tyrosine kinase inhibitor) Rare exon 17 mutations Detection by IHC sufficient Activating KIT mutations also present in melanoma, mastocytosis, acute myeloid leukemia, myeloproliferative neoplasms However, these frequently have exon 17 mutations resistant to Gleevec, so mutation analysis required New Tumor Biomarkers:KIT in Gastrointestinal Stromal Sarcoma (GIST) PET images before and after 8 weeks of Imatinib for GIST Oncologist, 2006, 11:9

  44. Activating KIT mutations http://atlasgeneticsoncology.org//Genes/KITID127.htmlaccessed 9-8-10

  45. KIT Immunohistochemistry commons.wikimedia.org/wiki/File:Gastric_GIST_...accessed 9-8-10

  46. New Tumor Biomarkers:JAK2 in Myeloproliferative Neoplasms • Myeloproliferative Neoplasms • Class of hematologic cancers in which there is a neoplastic proliferation of maturing blood and marrow cells • Frequently associated with marrow fibrosis • Often characterized by specific mutations • Knowledge of these mutations led World Health Organization to revise it’s diagnostic criteria for myeloproliferative neoplasms • Hopefully this knowledge will lead to targeted therapies

  47. New Tumor Biomarkers:JAK2 in Myeloproliferative Neoplasms • Diagnostic mutations in myeloproliferative neoplasms • JAK2 • Polycythemia Vera – erythroid cells • 90% of P. vera have JAK2 V617F mutations, with other JAK2 mutations identified by sequencing • JAK2 or MPL • Essential Thrombocythemia – platelets and megakaryocytes • Primary Myelofibrosis • Both have JAK2 V617F mutation in 50-60% of cases, MPL W151L/K mutations in 10-15% • KIT • Mastocytosis – mast cells • BCR-ABL • Chronic myelogenous leukemia – myeloid cells • PDGFR-alpha, PDGFR-beta, FGFR1 • Myeloid neoplasms with eosinophilia

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