Question: Should genome sequencing of multiple oncogenes surplant BRAF V600

mutation testing by an FDA approved test?

Answer: Yes

Jeffrey A Sosman MDIngram Chair for Cancer Research- Professor of Medicine,

Director, Melanoma Program

Why Melanoma? • 2012- Target therapy

– Therapy for BRAFV600E 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

Melanoma is Comprised of Clinically Relevant Molecular Subsets

Curtin et al. NEJM 2005; Curtin et al. JCO 2006

Arising from SkinWithout Chronic

Sun Damage

Arising from SkinWith Chronic Sun Damage

Arising from MucosalSurfaces

Arising fromAcral

Surfaces

50% BRAF20% NRAS

1-2% KIT

10% BRAF10% NRAS

5% KIT

5% BRAF15% NRAS

20% KIT

15% BRAF15% NRAS

15%KIT

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

S37p.S37F c.110C>Tp.S37Y c.110C>A

S45p.S45P c.133T>Cp.S45F c.134C>Tp.S45Y c.134C>A

Q209p.Q209P c.626A>Cp.Q209L c.626A>Tp.Q209R c.626A>G

GNAQ

Position AA mutant Nucleotide mutant

G12

p.G12C c.34G>Tp.G12S c.34G>Ap.G12R c.34G>Cp.G12V c.35G>Tp.G12A c.35G>Cp.G12D c.35G>A

G13

p.G13A c.38G>Cp.G13V c.38G>Tp.G13R c.37G>Tp.G13D c.38G>A

Q61

p.Q61E c.181C>Gp.Q61H c. 183A>Tp.Q61H c.183A>Cp.Q61L c.182A>Tp.Q61L c.182_183AA>TGp.Q61K c.181C>Ap.Q61P c.182A>Cp.Q61R c.182A>Gp.Q61R c.182_183AA>GG

NRAS

CTNNB1

W557p.W557R c.1669T>Cp.W557R c.1669T>A

V559p.V559A c.1676T>Cp.V559D c.1676T>A

L576 p.L576P c.1727T>CK642 p.K642E c.1924A>GD816 p.D816H c.2446G>C

KIT

Q209p.Q209P c.626A>Cp.Q209L c.626A>T

GNA11

Position AA mutant Nucleotide mutant

V600

p.V600R c.1798_1799GT>AGp.V600K c.1798_1799GT>AAp.V600E c.1799T>Ap.V600E c.1799_1800TG>AAp.V600M c.1798G>Ap.V600G c.1799T>Gp.V600D c.1799_1800TG>AT

BRAF

43 Somatic Point Mutations in 6 Genes Relevant to Targeted Therapy in Melanoma

43 Somatic Point Mutations in 6 Genes Relevant to Targeted Therapy in Melanoma

BRAF _V6001799T>A/G

NRAS_Q61182A>T/C/G

NRAS_G13 (R)38 G>A/T/C

BRAF_V600 (R)1800 G>A/T

KIT_V5591676 T>C/A

B-CAT_S45 (R)133 T>C

NRAS_G1235 G>T/C/A

BRAF _V600 (R)1799 T> G/A

B-CAT_S37110C>A/G/T

B-CATS45 (R)134R C>A/T

BRAF_V6001798G>A

NRAS_G13 (R)37G>T/C

NRAS_Q61 (R)183 A>G/T/C

NRAS_G12 (R)34G>A/T/C

KIT_K6421924A>G

NRAS_Q61181C>A/G GNA11_Q209 (R)

626A/T/C

KIT_W5571669T>A/C

KIT_L5761727T>C

KIT_D8162446G>C

GNAQ_Q209626A>T/C/G

Fig 1B

First 150 Patients: 20% of BRAF V600 Mutations Would Have Been Missed by Allele-Specific PCR

Lovly, Dahlman, Fohn, Su et al ‘12

Vanderbilt-Ingram Cancer Center

Gene # of metastatic cases # patients placed on a

genotype-driven clinical trial (%) BRAF 32 12 (38%)

CTNNB1 1* 1 (100%)

GNAQ/GNA11 6 3 (50%)

KIT 1 1 (100%)

NRAS 15 4 (27%)

No mutation detected 28 N/A

Total cases 82 21/54 (39%)

* This CTNNB1 mutation (CTNNB1 S45P) occurred concurrently with an NRAS Q61L mutation.

First 150 Patients: 40% of Pts with Mutant Metastatic Disease Genotype-Driven Treatment

Lovly, Dahlman, Fohn, Su et al ‘12

Melanoma SNaPshot genotyping in CLIA Lab (652 samples, from Jul 2010 to June 2012)

Distribution of BRAF V600 mutations

Distribution of all mutations detected

Melanoma7/1/2010-11/1/2012

• 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

Vemurafenib(PLX4032)

100

90

80

70

60

50

40

30

20

10

0

Ove

rall

surv

ival

(%

)

0 6 12 18 24

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

0

1

244

326

111

231

50

109

4

7

9.7 13.6

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 riskDacarbazine

Vemurafenib

15.9

BRIM2

c-KIT Mutations in Melanoma• 4q12

– Selectively amplified in acral/mucosal– Candidate genes → c-Kit amplifications

→ point mutations

• C-Kit by Subtype– Acral 11% Mt 25% Amp– Mucosal 21% 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 2ndary mutations

Woodman, BCP, 2010

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

. 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.

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

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

WGS Analysis of “Pan-Negative” Melanoma

GAIIxPaired-endSAMtoolsPindelCRESTFREEC

Dahlman, Xia, Hutchinson et al ‘12

• 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

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

Mutations in the BRAF gene

PRESENTED BY:

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%

Actionable Mutations- MAPKinase Pathway

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

Vanderbilt-Ingram Cancer Center

16 CancersALL

ALCLBasal Cell Carcinoma

BreastColorectal

GastricGISTIMTLung

MedulloblastomaMelanoma

NeuroblastomaOvarian

RhabdomyosarcomaThymicThyroid

24 Genes

271 Disease-Gene-Variant Relationships

16 CancersALL

ALCLBasal Cell Carcinoma

BreastColorectal

GastricGISTIMTLung

MedulloblastomaMelanoma

NeuroblastomaOvarian

RhabdomyosarcomaThymicThyroid

24 Genes

271 Disease-Gene-Variant Relationships

Vanderbilt-Ingram Cancer Center

More Comprehensive Profiling with Illumina MiSeq

Illumina.com

Amplicon Target Enrichment

1000x read coverageAutomated Alignment & Analysis

“Vanderbilt Cancer Panel” for MiSeq

Panel 1 = 34 genesTargets = 594 (exons)Target bp = 195838 bp# Amplicons = 1494 (max 1536)Coverage = 95%Low-Scoring Targets = 13

AKT1 IDH1 NF1ALK IDH2 NF2BRAF KIT NRASCDK4 KRAS PDGFRADDR2 MAP2K1 PIK3CAEGFR MAP2K2 PTENERBB2 MET RICTORFGFR1 MLH1 RPTORFGFR2 MLH3 SMOFGFR3 MSH2 TSC1GNA11 MTOR TSC2GNAQ

Panel 2 = 32 genesTargets = 457 (exons)Target bp = 210570 bp# Amplicons = 1448 (max 1536)Coverage = 93%Low-Scoring Targets = 13

AKT2 JAK3 NTRK3AKT3 KDR PTCH1ARAF MCL1 PTCH2BCL2 MYC RAF1BCL2L1 MYCL1 RB1ERBB3 MYCN RETERBB4 NOTCH1 SMAD4FGFR4 NOTCH2 STK11HRAS NOTCH3 TP53

JAK1 NTRK1 IGF1RJAK2 NTRK2

• Design: Illumina Design Studio• Targets: All exons of 66 genes

• 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

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