1. Question: Should genome sequencing of multiple oncogenes surplant BRAF V600 mutation testing by an FDA approved test? Answer: Yes Jeffrey A Sosman MD Ingram Chair for Cancer Research- Professor of Medicine, Director, Melanoma Program

2. Why Melanoma? 2012- Target therapy –Therapy for BRAF V600E melanoma –Therapy for CKIT mutated melanoma (exon 11 mutations) Other BRAF mutations- V600K,M,R,D,E’ (20% of V600 mutations), L597 mutations Expansion in NRAS melanoma 15-20% of all melanoma- targeted therapy in development –MEK inhibitor+AKT inhibitor, MEK inhibitor+CDK4 inhibitor Expansion into alternate genes- NF1, MEK1, MEK2, HRAS, CRAF (all components of MAP kinase pathway) RAC1, PPP6C, GRIN2A, targets?? Or modulating Other mutations which are activating same genes

3. Melanoma is Comprised of Clinically Relevant Molecular Subsets Curtin et al. NEJM 2005; Curtin et al. JCO 2006 Arising from Skin Without Chronic Sun Damage Arising from Skin With Chronic Sun Damage Arising from Mucosal Surfaces Arising from Acral Surfaces 50% BRAF 20% NRAS 1-2% KIT 10% BRAF 10% NRAS 5% KIT 5% BRAF 15% NRAS 20% KIT 15% BRAF 15% NRAS 15% KIT

4. Goals of the VICC PCMI To establish ‘reflex’ testing of ‘common’ clinically relevant genetic alterations in lung cancers and melanomas. To develop a clinically-applicable high-throughput molecular genotyping facility for ‘rarer’ genetic variants. To develop bioinformatic algorithms to report genetic results in the electronic medical record in ways that are clinically useful for practicing oncologists. –Collaboration among Depts of Medicine, Pathology, BioInformatics, and VICC –Sounds simple, but…requires high level of collaboration/coordination

5. S37 p.S37Fc.110C>T p.S37Yc.110C>A S45 p.S45Pc.133T>C p.S45Fc.134C>T p.S45Yc.134C>A Q209 p.Q209Pc.626A>C p.Q209Lc.626A>T p.Q209Rc.626A>G GNAQ PositionAA mutantNucleotide mutant G12 p.G12Cc.34G>T p.G12Sc.34G>A p.G12Rc.34G>C p.G12Vc.35G>T p.G12Ac.35G>C p.G12Dc.35G>A G13 p.G13Ac.38G>C p.G13Vc.38G>T p.G13Rc.37G>T p.G13Dc.38G>A Q61 p.Q61Ec.181C>G p.Q61Hc. 183A>T p.Q61Hc.183A>C p.Q61Lc.182A>T p.Q61Lc.182_183AA>TG p.Q61Kc.181C>A p.Q61Pc.182A>C p.Q61Rc.182A>G p.Q61Rc.182_183AA>GG NRAS CTNNB1 W557 p.W557Rc.1669T>C p.W557Rc.1669T>A V559 p.V559Ac.1676T>C p.V559Dc.1676T>A L576 p.L576Pc.1727T>C K642 p.K642Ec.1924A>G D816p.D816Hc.2446G>C KIT Q209 p.Q209Pc.626A>C p.Q209Lc.626A>T GNA11 PositionAA mutantNucleotide mutant V600 p.V600Rc.1798_1799GT>AG p.V600Kc.1798_1799GT>AA p.V600Ec.1799T>A p.V600Ec.1799_1800TG>AA p.V600Mc.1798G>A p.V600Gc.1799T>G p.V600Dc.1799_1800TG>AT BRAF 43 Somatic Point Mutations in 6 Genes Relevant to Targeted Therapy in Melanoma

6. BRAF _V600 1799T>A/G NRAS_Q61 182A>T/C/G NRAS_G13 (R) 38 G>A/T/C BRAF_V600 (R) 1800 G>A/T KIT_V559 1676 T>C/A B-CAT_S45 (R) 133 T>C NRAS_G12 35 G>T/C/A BRAF _V600 (R) 1799 T> G/A B-CAT_S37 110C>A/G/T B-CATS45 (R) 134R C>A/T BRAF_V600 1798G>A NRAS_G13 (R) 37G>T/C NRAS_Q61 (R) 183 A>G/T/C NRAS_G12 (R) 34G>A/T/C KIT_K642 1924A>G NRAS_Q61 181C>A/G GNA11_Q209 (R) 626A/T/C KIT_W557 1669T>A/C KIT_L576 1727T>C KIT_D816 2446G>C GNAQ_Q209 626A>T/C/G Fig 1B

7. First 150 Patients: 20% of BRAF V600 Mutations Would Have Been Missed by Allele-Specific PCR Lovly, Dahlman, Fohn, Su et al ‘12

8. Vanderbilt-Ingram Cancer Center First 150 Patients: 40% of Pts with Mutant Metastatic Disease  Genotype-Driven Treatment Lovly, Dahlman, Fohn, Su et al ‘12

9. Melanoma SNaPshot genotyping in CLIA Lab (652 samples, from Jul 2010 to June 2012) Distribution of BRAF V600 mutations Distribution of all mutations detected

10. Melanoma 7/1/2010-11/1/2012 759 Specimens – 65% specimens with mutation detected – 16 specimens with 2 mutations – 3 specimens with 3 mutations 715 patients – 64% patients with mutation detected

11. 11 Vemurafenib (PLX4032)

12. 100 90 80 70 60 50 40 30 20 10 0 Overall survival (%) 06121824 Vemurafenib (n=337) Median f/u 12.5 months Dacarbazine (n=338) Median f/u 9.5 months 338 337 173 280 79 178 24 44 0101 244 326 111 231 50 109 4747 9.713.6 Overall survival (February 01, 2012 cut-off) censored at crossover Hazard ratio 0.70 (95% CI: 0.57–0.87) p<0.001 (post-hoc) Time (months) No. at risk Dacarbazine Vemurafenib 15.9 BRIM2

13. c-KIT Mutations in Melanoma 4q12 –Selectively amplified in acral/mucosal –Candidate genes → c-Kit amplifications → point mutations C-Kit by Subtype –Acral11% Mt 25% Amp –Mucosal21% Mt 29% Amp –Cutaneous +CSD 1-18% Mt 6% Amp C-Kit: Melanoma vs GIST –Point mutations –↑ Exon 13 & 17 mutations –Amplified wild-type c-KIT –Lack of 2 ndary mutations Woodman, BCP, 2010

14. Phase II Studies of Imatinib 400 mg BID in Advanced Melanoma kit c-abl PDGFR-α PDGFR-β Imatinib Three “large” studies have been embarked upon include both KIT mutated and amplified  Hodi- DFCC central with imatinib, sunitinib, or nilotinib for imatinib fail  Carvajal- MSKCC central with imatinib  Guo-. Peking Univ, Beijing, China- imatinib

15. . Treatment Response Over Time by Melanoma Subtype and Genetic Alteration of KIT Carvajal, R. D. et al. JAMA 2011;305:2327-2334 Copyright restrictions may apply.

16. Kit Inhibition in Melanoma  Kit Inhibitors can produce dramatic effects in patients with melanomas containing a variety of C-kit mutations  Kit mutations are seen in 2% of all melanomas  Role of Kit inhibition in Kit amplified tumors has yet to be established  Multiple studies currently underway  Imatinib, sunitinib, dasatinib, nilotinib  International Phase II trial (nilotinib )- comp[leted  Exciting, but not the answer for the majority of patients with melanoma

17. Vanderbilt-Ingram Cancer Center Index Case: Using NGS to Find Novel Drivers 75 year old male presented with ulcerated right ear melanoma  resected 4 mos later – local recurrence  re-resection and radiation; BRAF V600E and KIT mutations not detected 12 mos later – widespread mets  palliative thyroidectomy; no mutations detected by SNaPshot Whole genome sequencing performed on thyroid metastasis (90% tumor) and matched normal blood Dahlman, Xia, Hutchinson et al ‘12

18. WGS Analysis of “Pan-Negative” Melanoma GAIIx Paired-end SAMtools Pindel CREST FREEC Dahlman, Xia, Hutchinson et al ‘12

19. Melanoma SNaPshot Negative Patient Whole-genome sequencing  BRAF L597R Sensitive to MEK inhibition in vitro SNaPshot Limitation Example: Melanoma Patient with BRAF L597 Mutation Dahlman, Xia, Hutchinson et al, Cancer Discov, 2012) Patient with BRAF L597S, treated with TAK-733

20. Vanderbilt-Ingram Cancer Center Of 49: 2 L597, 1 D594, 1 K601 (8%) 8% of “Pan-Negative” Samples Harbor non-V600E BRAF Exon 15 Mutations Melanoma Panel: 538 Samples 7/1/10-12/31/11 Dahlman, Xia, Hutchinson et al ‘12 Cosmic: 0.1% of BRAF mutations

21. Mutations in the BRAF gene PRESENTED BY:

22. MEK 162: Best percentage change from baseline and best overall response (NRAS mut) *Patients with missing best % change from baseline and unknown overall response are not included. N=28* Progressive Disease (PD) Stable Disease (SD) Partial Response (PR) Unconfirmed PR 45 mg NRAS Ongoing pts Ascierto, Berking, Agarwala et al. ASCO 2012 Response rate: 21% (6 of 28 pts) Disease control rate: 68%

23. Actionable Mutations- MAPKinase Pathway

25. Vanderbilt-Ingram Cancer Center Summary Routine multiplex mutational profiling of melanoma with a disease-specific panel –Identifies patients with clinically relevant driver mutations –Enables genetically-informed cancer medicine in the clinic –Facilitates clinical trial enrollment –Allows for rapid discovery of potentially targetable novel drivers in ‘pan-negative’ cases BRAF L597 mutations and MEK inhibitors

27. Vanderbilt-Ingram Cancer Center 16 Cancers ALL ALCL Basal Cell Carcinoma Breast Colorectal Gastric GIST IMT Lung Medulloblastoma Melanoma Neuroblastoma Ovarian Rhabdomyosarcoma Thymic Thyroid 24 Genes 271 Disease-Gene- Variant Relationships 16 Cancers ALL ALCL Basal Cell Carcinoma Breast Colorectal Gastric GIST IMT Lung Medulloblastoma Melanoma Neuroblastoma Ovarian Rhabdomyosarcoma Thymic Thyroid 24 Genes 271 Disease-Gene- Variant Relationships

28. Vanderbilt-Ingram Cancer Center

29. More Comprehensive Profiling with Illumina MiSeq Illumina.com Target Enrichment by Probe Hybridization: Amplicon Target Enrichment 1000x read coverage Automated Alignment & Analysis

30. “Vanderbilt Cancer Panel” for MiSeq Panel 1 = 34 genes Targets = 594 (exons) Target bp = 195838 bp # Amplicons = 1494 (max 1536) Coverage = 95% Low-Scoring Targets = 13 AKT1IDH1NF1 ALKIDH2NF2 BRAFKITNRAS CDK4KRASPDGFRA DDR2MAP2K1PIK3CA EGFRMAP2K2PTEN ERBB2METRICTOR FGFR1MLH1RPTOR FGFR2MLH3SMO FGFR3MSH2TSC1 GNA11MTORTSC2 GNAQ Panel 2 = 32 genes Targets = 457 (exons) Target bp = 210570 bp # Amplicons = 1448 (max 1536) Coverage = 93% Low-Scoring Targets = 13 AKT2JAK3NTRK3 AKT3KDRPTCH1 ARAFMCL1PTCH2 BCL2MYCRAF1 BCL2L1MYCL1RB1 ERBB3MYCNRET ERBB4NOTCH1SMAD4 FGFR4NOTCH2STK11 HRASNOTCH3TP53 JAK1NTRK1IGF1R JAK2NTRK2 Design: Illumina Design Studio Targets: All exons of 66 genes

31. Validate with samples with known mutations: –FFPE Patient Tissue –Frozen Patient Tissue –Cell Lines Expand to SNaPshot-negatives/unknowns Design a capture method/panel for fusion genes Now seeking interesting clinical samples! Please contact me (katie.hutchinson@vanderbilt.edu) Implement into Clinical Molecular Diagnostics Lab??? (Cindy Vnencak-Jones) Vanderbilt Cancer Panel Plans

32. The major issues critical to personalized cancer care in melanoma Acquired resistance to BRAF inhibitors- –mechanisms and overcoming resistance Targeting other mutations (NRAS) effectively with new or old drugs Defining new genetic mutations, amplifications, or translocations Need for both clinical and translational collaboration to speed up the discoveries needed for clinical progress Transmitting genetic information to the oncologist in a clinically relevant language