1. Research Presentation Jason M. Leibowitz, MD June 25, 2009 Preceptor: Marcia S. Brose, MD PhD Jason M. Leibowitz, MD June 25, 2009 Preceptor: Marcia S. Brose, MD PhD Otorhinolaryngology: Head and Neck Surgery at PENN Excellence in Patient Care, Education and Research since 1870

2. Overview  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions

3. Thyroid Cancer in the United States  Thyroid cancer is the most common endocrine neoplasm.  Thyroid cancer will be diagnosed in 33,550 individuals (8070 men and 25,480 women) this year.  From 1997-2004 incidence of thyroid cancer increased by 6.2% mostly due to increased detection.  From 1985 to 2004 mortality rate increased by 0.3% a year.

4. RAI-Refractory Disease  25-50% of metastatic thyroid cancers lose ability to take up Iodine.  Iodine Uptake inversely correlates with survival.  This is attributed to down regulation of the Na+/I- Symporter (NIS).  Limited treatment options for unresectable thyroid cancer refractory to RAI.  25-50% of metastatic thyroid cancers lose ability to take up Iodine.  Iodine Uptake inversely correlates with survival.  This is attributed to down regulation of the Na+/I- Symporter (NIS).  Limited treatment options for unresectable thyroid cancer refractory to RAI.

5. Molecular Changes in Thyroid Cancer

6. Molecular Pathway involved in Thyroid Cancer  Activation of MAPK pathway  Oncogenic activation of this pathway in 70% of all thyroid cancers.  BRAF is a serine threonine kinase  Activation of MAPK pathway  Oncogenic activation of this pathway in 70% of all thyroid cancers.  BRAF is a serine threonine kinase Xing, 2007.

7. BRAF V600E in Thyroid Cancer 2003: The BRAF V600E mutation is the most common genetic alteration in thyroid cancer, occurring in about 45% of sporadic papillary thyroid cancers (PTCs). V600E

8. BRAF V600E  Point mutation in 40-45% of PTC  Upregulation of MMP, VEGF --> invasion, angiogenesis  Silencing of tumor suppressive genes, genes involved in iodine transport  BRAF mutation associated with multiple negative prognostic indicators.  Point mutation in 40-45% of PTC  Upregulation of MMP, VEGF --> invasion, angiogenesis  Silencing of tumor suppressive genes, genes involved in iodine transport  BRAF mutation associated with multiple negative prognostic indicators.

9. RAS  Family of small G-proteins involved in transduction of cellular signals from the cell membrane.  Mutations in RAS gene lead to inappropriate activation with constitutively activated downstream pathways and also promote chromosomal instability.  20% FTC contain a RAS mutation RAS mutations may correlate with aggressive behavior (tumor dedifferentiation and poorer prognosis).  Family of small G-proteins involved in transduction of cellular signals from the cell membrane.  Mutations in RAS gene lead to inappropriate activation with constitutively activated downstream pathways and also promote chromosomal instability.  20% FTC contain a RAS mutation RAS mutations may correlate with aggressive behavior (tumor dedifferentiation and poorer prognosis).

10. Targeted Therapy in Thyroid cancer  Loss of differentiation (inability to trap RAI), unresectable lesion, leads to poor prognosis  BRAF inhibitors  BAY 43-9006 (Sorafenib)  Multikinase inhibitor  Loss of differentiation (inability to trap RAI), unresectable lesion, leads to poor prognosis  BRAF inhibitors  BAY 43-9006 (Sorafenib)  Multikinase inhibitor

11. Sorafenib  Orally active multikinase inhibitor (study dose 400mg BID).  Monoclonal antibody with multiple targets including BRAF, VEGFR1, VEGFR2.  Blocks tumor cell proliferation and angiogenesis.  FDA approved for treatment of RCC and hepatocellular carcinoma.  Orally active multikinase inhibitor (study dose 400mg BID).  Monoclonal antibody with multiple targets including BRAF, VEGFR1, VEGFR2.  Blocks tumor cell proliferation and angiogenesis.  FDA approved for treatment of RCC and hepatocellular carcinoma.

12. Targeted Therapy and Genotype  K-RAS gene mutation and metastatic colorectal carcinoma.  Recent results from Phase II & III clinical trials demonstrate that patients with metastatic colorectal cancer benefit from anti-EGFR therapy.  Patients with K-RAS mutation in codon 12 & 13 should not receive anti-EGFR therapy since they do not receive any benefit.  EGFR and non-small cell lung cancer:  Epithelial growth factor receptor  10% mutated in NSCLC  EGFR mutations are predictors of TKIs responsiveness and may show a long lasting response to TKIs  EXON 19 Deletion respond better to TKIs.  K-RAS gene mutation and metastatic colorectal carcinoma.  Recent results from Phase II & III clinical trials demonstrate that patients with metastatic colorectal cancer benefit from anti-EGFR therapy.  Patients with K-RAS mutation in codon 12 & 13 should not receive anti-EGFR therapy since they do not receive any benefit.  EGFR and non-small cell lung cancer:  Epithelial growth factor receptor  10% mutated in NSCLC  EGFR mutations are predictors of TKIs responsiveness and may show a long lasting response to TKIs  EXON 19 Deletion respond better to TKIs.

13. Prior Data 84 weeks N= 52 N=43 WDTC

14. Papillary vs. Follicular P<0.095 FTC = 19 PTC= 24

15. Prior Data  Conclusions from prior data:  Improved PFS with Sorafenib.  Improved PFS of FTC treated with Sorafenib when compared to PTC.  Conclusions from prior data:  Improved PFS with Sorafenib.  Improved PFS of FTC treated with Sorafenib when compared to PTC.

16. Overview  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions

17. Hypothesis  There are specific genotypes (i.e. BRAF V600E, RAS mutations) that predict favorable response to targeted therapy (Sorafenib).

18. Null Hypothesis  Specific genetic mutations do not predict response to targeted therapy in thyroid cancer.

19. Overview  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions

20. Research Plan  Tissue samples collected from patients with treatment-resistant thyroid cancer with long term follow-up (approximately 30 patients).  All patients received targeted therapy (Sorafenib).  Samples with WDTC analyzed for mutations in BRAF and RAS genes when available:  BRAF - V600E  RAS - Exon 12, 13, 61  Tissue samples collected from patients with treatment-resistant thyroid cancer with long term follow-up (approximately 30 patients).  All patients received targeted therapy (Sorafenib).  Samples with WDTC analyzed for mutations in BRAF and RAS genes when available:  BRAF - V600E  RAS - Exon 12, 13, 61

21. RESULTS

22. Sequence Output  Computer program interprets data and produces an electropherogram, (aka trace)  Each peak represents a base:  A = Adenosine  T = Thymine  C = Cytosine  G = Guanine  N = Reading cannot be determined

23. Overview  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions

24. Results of Stage 1 Analysis N= 30 M = F = 15 PTC=17, FTC= 9, Other (ATC/PD, MTC): 4 Samples analyzed for BRAF mutation: 23/30 (76.6%): samples analyzed for BRAF mutation 4/30 (13%): definite genotype but questioned due to phenotype (ATC/PD, MTC) 2/30 (6%): unable to amplify DNA despite multiple PCR attempts 1/30 (3%): pending analysis 18/30 samples analyzed for RAS mutation, all WT copies of the gene N= 30 M = F = 15 PTC=17, FTC= 9, Other (ATC/PD, MTC): 4 Samples analyzed for BRAF mutation: 23/30 (76.6%): samples analyzed for BRAF mutation 4/30 (13%): definite genotype but questioned due to phenotype (ATC/PD, MTC) 2/30 (6%): unable to amplify DNA despite multiple PCR attempts 1/30 (3%): pending analysis 18/30 samples analyzed for RAS mutation, all WT copies of the gene

25. Results of Stage 1 Analysis  N=22 (interim analysis)  13 WT BRAF  9 BRAF V600E  16 PTC  9 WT BRAF, 7 V600E  6 FTC  4 WT BRAF, 2 V600E  N=22 (interim analysis)  13 WT BRAF  9 BRAF V600E  16 PTC  9 WT BRAF, 7 V600E  6 FTC  4 WT BRAF, 2 V600E

26. BRAF V600E P<0.02 N=13 ( WT=8, V600E=5 )

27. Updated genetics  In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.  We are further investigating BRAF copy number in these patients  In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.  We are further investigating BRAF copy number in these patients p=NS N =22 WT = 13 BRAF V600E = 9

28. Overview  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions  Background  Hypothesis  Methods  Results  Discussion  Conclusions & Future Directions

29. BRAFV600E Correlates with worse Survival Elisei et. al, J Clin Endocrinol Metab, October 2008, 93(10):3943–3949

30. BRAFV600E Correlates with worse Survival State of the mutation in PTC, 10/2008

31. THE BRAF connection Ciampi et al. 2005

32. Updated genetics  In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.  We are further investigating BRAF copy number in these patients  In our expanded analysis to 22 pts with WDTC, the effect is no longer significant but the trend exists.  We are further investigating BRAF copy number in these patients p=NS N =22 WT = 13 BRAF V600E = 9

33. BRAF (red) x 3 7 centromere (green) x 3

34. BRAF x4 7 centromere x4

35. 4 copies each 3 copies each

36. THE BRAF connection! Positive Predictor! Ciampi et al, 2005.

37. Summary  Good progression free survival in patients treated with Sorafenib.  BRAF V600E appears to predict for improved outcome in patients treated with sorafenib.  BRAF copy number gain may explain improved outcome of patients with FTC over patients with PTC  Good progression free survival in patients treated with Sorafenib.  BRAF V600E appears to predict for improved outcome in patients treated with sorafenib.  BRAF copy number gain may explain improved outcome of patients with FTC over patients with PTC

38. Future Directions  Completion of genotyping analysis of all patients  Evaluation of copy number gains in WDTC  Hypothesis: Copy number gain accounts for improved survival in FTC treated with Sorafenib  Null: Copy number gain does not influence survival in FTC  Completion of genotyping analysis of all patients  Evaluation of copy number gains in WDTC  Hypothesis: Copy number gain accounts for improved survival in FTC treated with Sorafenib  Null: Copy number gain does not influence survival in FTC

39. Selected Sources  Ciampi R, Zhu Z, Nikiforov YE. BRAF copy number gain in thyroid tumors detected by fluorescence in situ hybridization. Endocrine Pathology 2005; 16(2): 99-105.  Ciampi R, Nikiforov YE. Alterations of the BRAF gene in thyroid tumors. Endocrine Pathology 2005; 16:3): 163-171.  Gupta-Abramson V, Troxel AB, Nellore A, et al. Phase II Trial of Sorafenib in Advanced Thyroid Cancer. Journal Clin Onc 2008; 26 (29): 4714-4719.  Kundra P, Burman KD. Thyroid Cancer Molecular Signaling Pathways and Use of Targeted Therapy. Endoc Metab Clin N Am 2007;36: 839-853  Murer B. Targeted Therapy in Non-Small Cell Lung Cancer. Arch Path Lab Med. 2008; 132: 1573-1575.  Nikiforov YE. Thyroid Carcinoma: Molecular Pathways and Therapeutic targets. Modern Pathology 2008; 21: S37-S43.  Vasko V, Ferrand M, Cristofaro JD et al. Specific Pattern of RAS Oncogene Mutations in Follicular Thyroid Tumors. J. Clin Endocrin. & Metab. 2003; 88(6):2745-2752.  Xing M. BRAF Mutation in Papillary Thyroid Cancer: Pathogenic Role, Molecular Basis, and Clinical Implication. End Rev 2007; 28(7): 742-762.  Ciampi R, Zhu Z, Nikiforov YE. BRAF copy number gain in thyroid tumors detected by fluorescence in situ hybridization. Endocrine Pathology 2005; 16(2): 99-105.  Ciampi R, Nikiforov YE. Alterations of the BRAF gene in thyroid tumors. Endocrine Pathology 2005; 16:3): 163-171.  Gupta-Abramson V, Troxel AB, Nellore A, et al. Phase II Trial of Sorafenib in Advanced Thyroid Cancer. Journal Clin Onc 2008; 26 (29): 4714-4719.  Kundra P, Burman KD. Thyroid Cancer Molecular Signaling Pathways and Use of Targeted Therapy. Endoc Metab Clin N Am 2007;36: 839-853  Murer B. Targeted Therapy in Non-Small Cell Lung Cancer. Arch Path Lab Med. 2008; 132: 1573-1575.  Nikiforov YE. Thyroid Carcinoma: Molecular Pathways and Therapeutic targets. Modern Pathology 2008; 21: S37-S43.  Vasko V, Ferrand M, Cristofaro JD et al. Specific Pattern of RAS Oncogene Mutations in Follicular Thyroid Tumors. J. Clin Endocrin. & Metab. 2003; 88(6):2745-2752.  Xing M. BRAF Mutation in Papillary Thyroid Cancer: Pathogenic Role, Molecular Basis, and Clinical Implication. End Rev 2007; 28(7): 742-762.

40. Thanks  Marcia Brose, MD PhD  Cathy Ma MD, PhD  Kanchan Puttaswamy, MS  Marcia Brose, MD PhD  Cathy Ma MD, PhD  Kanchan Puttaswamy, MS