Genetics & Molecular Markers for Renal Cell Carcinoma

1. Genetics & Molecular Markers for Renal Cell Carcinoma Christopher G. Wood, M. D., FACS Professor and Deputy Chair Department of Urology The University of Texas MD Anderson Cancer Center

2. Outline • Genetics • Overview • Familial Syndromes • Molecular Mechanisms • Molecular Markers • Overview • Specific Molecular Markers • Integrated Staging Systems • Molecular profiling

3. RCC Genetics Family of epithelial tumors Distinctive tumor biology Unique response to treatment Differing prognosis Renal Cell Carcinoma as a “metabolic” disease Familial syndromes von Hippel-Lindau (vHL) Hereditary papillary RCC (HPRCC) Hereditary Leiomyomatosis RCC (HLRCC) Birt-Hogg-Dubé (BHD) Linehan et al. J Urol, 2003

4. Human Renal Epithelial Neoplasms Linehan et al. J Urol, 2003

5. Familial RCC • Genetic linkage analysis • Specific mutations associated with histologic subtypes • Occurs at an early age • Bilateral &/or multifocal tumors • Associated with cutaneous & systemic processes

6. Knudson “two-hit” Hypothesis

7. Genetic Alterations Lam et al. Urology, 2005

8. von-Hippel Lindau (vHL) Clinical Manifestations Retinal angioma Endolymphatic sac tumors (10%) Hemangioblastoma of the spine & cerebellum (60-80%) Pancreatic cysts & islet cell tumors Pheochromocytoma (10-20%) Epididymal cystadenoma Clear cell RCC (24-45%) Renal cysts Linehan et al. J Urol, 2003 Lonser et al. Lancet, 2003

9. Clinical Spectrum of vHL • Type 1 • ccRCC • Hemangioblastoma • Type 2A • Hemangioblastoma • Pheochromocytoma • Type 2B • ccRCC • Hemangioblastoma • Pheochromocytoma • Type 2C • Pheochromocytoma Cohen et al. NEJM, 2005

10. vHL Genetics Autosomal Dominant (90% penetrance) 70% of sporadic ccRCC’s have lost pVHL expression Deletion Silencing Chromosomal alteration in 3p25 Function Fibronectin assembly Microtubule stability Tumor suppressor Linehan et al. J Urol, 2003 Lonser et al. Lancet, 2003

11. vHL Forms a ubiquitin-protein ligase complex Normoxia pVHL bind hydroxylated hypoxia inducible factor (HIF) HIF-a is ubiquitinated & targeted for proteasome-mediated degradation Hypoxia HIF-a dimerizes with HIF-b HIF accumulates & translocates to nucleus … binds HRE Activation of multiple pathways Metabolism (GLUT1, CAIX) Angiogenesis (VEGF, PDGF, EGF, Erythropoetin) ECM remodeling (MMP-2, TGF) Semenza et al. Molecular Cell, 2006 Roe et al. Molecular Cell, 2006

12. vHL Pathway Cohen et al. NEJM, 2005

14. The HIF Story (Part 1) HIF-1a Expressed ubiquitously Activates glycolytic genes Inhibits cell-cycle progression by opposing c-Myc Inhibits tumor growth HIF-2a Differentially expressed in endothelium, kidney, heart, lungs, & small intestine Activates VEGF, TGFa, lysyl oxidase, Oct4, & Cyclin D1 Increased cell proliferation by promoting c-Myc transcription &cell-cycle progression May promote angiogenesis Promotes tumor growth and increases the degree of dysplasia Conclusion: Differential control of cell-cycle progression based on amplitude of HIF-a subunit expression Gordan et al. Cancer Cell, 2007

15. The HIF Story (Part 2) 57 sporadic human ccRCC’s VHL WT with undetectable HIF-a – 12% VHL deficient with both HIF-1a and HIF-2a (H1H2) – 61% VHL deficient with HIF-2a exclusively (H2) – 27% WT Increased mTOR growth factor signaling (ERK & S6) H1H2 tumors More angiogenic than WT tumors Activation of Akt/mTOR and ERK/MAPK1 growth factor signaling pathways Increased markers of DNA damage from replication stress H2 tumors Increased c-Myc activated targets (cyclin D2 and E2F1) Decreased c-Myc repressive targets (p21 and p27) More efficient HR-mediated repair and resolution of replication stress Reduced accumulation of DNA damage Reduced activation of checkpoint responses Further progression of cell cycle Gordan et al. Cancer Cell, 2008

16. The HIF Story (Conclusion) VHL-deficient ccRCC’s can be distinguished on the basis of differential HIF-a expression c-Myc drives proliferation in H2 tumors WT and H1H2 tumors utilize growth factor signaling pathway The combination of VHL genotype & HIF-a expression allows the stratification of ccRCC’s into biologically distinct groups Subclassification of ccRCC’s into subgroups provides a framework for targeted therapy. Growth factor driven ccRCC’s (WT and H1H2) are more likely to respond to TKI’s Expression of HIF-2a alone may mark a subset of RCC’s that are uniquely resistant to the current targeted therapies. Gordan et al. Cancer Cell, 2008

17. Hereditary Papillary RCC Clinical Manifestations Rarest form of hereditary kidney cancer Onset within the 4th- 6th decade of life Type 1 papillary RCC Bilateral & multiple tumors Less aggressive & fewer less metastases Once metastatic … worst prognosis No extra-renal findings

18. Hereditary Papillary RCC Genetics Autosomal Dominant Chromosomal alterations 7q31 Trisomy 7 & 17 Loss of Y c-MET mutation Missense mutation in tyrosine kinase domain Constitutive activation of the MET oncogene (HGF-R)

19. MET Pathway

20. Birt-Hogg-Dubé (BHD) Clinical Manifestation Hereditary oncocytomas (familial renal oncocytosis) Multiple, bilateral RCC Commonly chromophobe Fibrofolliculomas of the head & neck Pulmonary cysts & spontaneous pneumothorax (25%) Colonic polyps (possible increased cancer risk) Other Nevi, PTH adenomas, lipomas, oral mucosal papules

21. Fibrofolliculomas

22. Birt-Hogg-Dubé (BHD) Genetics Autosomal Dominant Chromosomal alteration in 17p11.2 Folliculin (FLCN) Binds AMPK & negatively regulates mTOR Tumor suppressor

23. Hereditary Leiomyomatosis RCC (HLRCC) Clinical Manifestations Type 2 papillary RCC Solitary, multiple, or bilateral Aggressive behavior & poor prognosis Painful cutaneous leiomyomas Uterine leiomyomas (fibroids) Multiple Painful Early age

24. HLRCC Genetics Chromosomal alteration in 1q42 Fumarate hydratase (FH) Kreb’s cycle (TCA) enzyme Catalyzes malate to fumarate ATP production through the mitochondrial respiratory chain Impairs apoptosis Tumor suppressor

25. FH Pathway

26. Tuberous Sclerosis Complex (TSC) Clinical Manifestations … Neurocutaneous syndrome Benign hamartomas (tuber) of cerebral cortex MR Seizures Sebaceum adenomas of face “Ash-leaf” spots Angiomyolipomas Subependymal giant astrocytoma Increased risk of RCC ? Lendvay et al. J Urol, 2003

27. Adenoma Sebaceum

28. Benign Tubers

29. Tuberous Sclerosis Complex (TSC) Genetics TSC1 gene (9q34) … tumor suppressor Encodes for hamartin Altered cell adhesion Altered actin organization TSC2 gene(16p13) … tumor suppressor Encodes for tuberin Regulates cytoplasmic vesicle transport Overexpression inhibits cell growth Energy deprivation AMPK activates TSC mTOR is decreased Crino et al. NEJM, 2006 Lendvay et al. J Urol, 2003 Henske et al. Curr Mol Med, 2004

30. TSC Pathway

31. Inter-related Molecular Pathways • Cell signaling • PI3K & Akt • mTOR • Cellular Organization • TSC • Angiogenesis • HIF-a • VEGF • Cell Metabolism • AMPK • FH • SDH

34. Importance of Molecular Markers Stratify risk of disease recurrence/progression Estimate Disease Free Survival Molecular staging Identify candidates for adjuvant therapy Predict response to treatment Immunotherapy Small molecule inhibitors

35. Model for Molecular Markers

36. Molecular Markers Hypoxia Inducible CAIX CXCR’s HIF-a & VEGF-R IGF-1 Proliferation Ki-67 PCNA Ag-NORs (Argyrophilic nucleolar organizer region) Cell Cycle Regulation p53 PTEN CDK’s mTOR STAT (signal transducer & activator of transcription) P21 Immunogens TATI (tumor associated trypsin inhibitor) TPS (tissue polypeptide specific) antigen Apoptosis Based BCL-2 CD Based CD10 (CALLA) CD154 Cell Adhesion EpCAM (epithelial cell adhesion molecule) EMA (epithelial membrane antigen) E-cadherin b-catenin P-selectin Cytoskeleton Laminin Collagen IV Fibronectin Vimentin Enzymatic Gamma-Enolase Pyruvate Kinase Type M2 iNOS Receptor Tyrosine Kinase-EphA2 TS (thymidylate synthase) Glycoproteins Vinculin Tissue Based Gp200 Miscellaneous CA-125 Caveolin-1 Erythropoetin Ferritin NMP-22 Neopterin MMP’s

37. Carbonic Anhydrase (CAIX) G250 renal cancer antigen Function Transmembrane protein Catalyzes reversible reaction of C02 & H20 to HC03- Regulates intracellular & extracellular pH Responds to tumor hypoxia (anaerobic metabolism) Molecular Pathway Target of HIF Acidification of ECM Induce expression of angiogenic factors Inhibit cellular immunity Promotes tumor aggressiveness Loss of contact inhibition & anchorage dependence of tumor cells

38. Importance of CAIX • Useful Molecular Marker • Prognostic • Predicts treatment response • Diagnostic • Therapeutic target

39. CAIX - Prognostic • Absent in most normal tissue • Highly expressed in ccRCC related to vHL • 94% tumor specific • High levels associated with favorable prognosis • Low staining independent poor prognostic factor Bui et al. Clin Cancer Res, 2003

40. CAIX is Prognostic of DSS

41. Importance in Metastatic Disease

42. CAIX Predicts Response to Treatment Complete responders to IL-2 >85% CA IX staining Response to IL-2 Twice as likely in those with high CA IX staining Failed to be a significant predictor of outcome prospectively in the “SELECT” Trial Bui et al. Clin Cancer Res, 2003 Atkins et al. Clin Cancer Res, 2005

43. CAIX may be Diagnostic REDECTANE® Trial PET/CT vs CT Pre-op characterization of renal masses 124I labeled moAb to G250 (Girentuximab) Multicenter study of over 200 patients Outcome 94% sensitive & 100% specific 90% NPV & 100% PPV

44. Primary tumour left kidney Clavicular metastasis Scapula metastasis Phase III Multicenter Comparison of Redectane vs CT (Uzzo, Russo et al.) Courtesy of Michael Yu, Fox Chase Cancer Center • CT: histology of primary and mets unknown • PET/CT: ccRCC primary and mets • Central Pathology: ccRCC

45. CAIX may be Therapeutic Radioimmunotherapy (RIT) Trials Radiolabeled murine G250 Ab 131I or 177Lu Stabilization of disease but no PR or CR Limited by human anti-mouse Ab (HAMA) ARISER Phase III Trial Adjuvant RENCAREX®Immunotherapy trial to Study Efficacy in non-metastatic RCC moAb to chimeric G250 vs placebo High risk ccRCC patients 864 patients accrued from 2004 – 2008 Divgi et al. Clin Cancer Res, 1998

46. Pitfalls of Molecular Markers Tissue preservation &handling is difficult Mixing all pathologic variants pollutes the data Heterogeneity of analyzed material Artificial activation of pathways (false positive) Tumor necrosis Hypoxia Etc. Differing analytic techniques makes interpretation difficult No standardization Cost

47. Integrated Staging Systems UISS (UCLA Integrated Staging System) TNM + ECOG status + Fuhrman grade MISS (Molecular Integrated Staging System) UISS + molecular markers Better prognostic model than TNM stage, grade, or UISS alone Concordance Index of 0.79 vs 0.65, 0.73, 0.76 Significant predictors p53 Gelsolin CAIX Kim et al. Clin Cancer Res, 2004

48. MISS Nomogram

49. Molecular Profiling • Genomics • CGH (comparative genomic hybridization) • SNP (single nucleotide polymorphisms) • Multi-color FISH • High throughput sequencing • High throughput analysis of methylation • Spectral karyotyping • Transciptomics • Microarray (mRNA, miRNA) • SAGE (serial analysis of gene expression) • EST (expressed sequence tags) • DDD (digital differentiation display) • Quantitative RT-PCR • Proteomics • MS (mass spectrometry) • Protein microarray • Chromatography Arsanious et al. Mol Cancer, 2009

50. Summary Renal cell carcinoma is not a single disease Inherited syndromes have led to the discovery of tumor suppressor genes & proto-oncogenes The study of these genes have, in turn, have led to the elucidation of molecular mechanisms in tumorigenesis The discovery of molecular markers has followed We now have powerful tools to help identify disease at a molecular level, prognosticate outcome, and predict response to therapy. We are now able to deliver tumor-specific therapy which feature the use of targeted molecular therapies