BRAF-Mutations in MelanomasL. Mazzucchelli

Istituto Cantonale di Patologia, Locarno

77. Annual Meeting Swiss Society of Pathology, Lucerne 2011Sponsored by Roche Pharma Switzerland

� Melanoma has increased at an alarming rate during the past 40 years (estimated life time risk of 1 in 75).

� Early melanoma can be cured through surgical excision � Prognosis of metastatic melanoma is dismal. It is not

responsive to current available chemotherapy, immunologic therapy or radiotherapy (3-years OS: 10-15%).

� Recent discoveries in melanoma pathogenesis and genetics have yielded various new molecular targets in the MAPK pathway.

� Novel, small molecule compunds targeting BRAF have already shown great promise in preclinical, early-phase, and phase III clinical trials.

Normal RAS-RAF (MAPK) pathway signaling

� The RAS-RAF pathway is a series of protein kinasesthat transmit signals to control proliferation, differentiation, and survival

� Activated ERK can enter the nucleus and phosphorylate transcription factors such as ETS, which mediate gene transcription, ultimately stimulating cell survival and proliferation

AKT

PTEN

RAF

J Pathol 2011;223:219

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Normal cell proliferation and survival

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Excessive cell proliferation and survival

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Activating BRAF mutations in 50% of melanoma

� BRAFV600E is capable of causing RAS-independent activation of ERK, MEK-dependent cell proliferation, and transformation

1. Can we predict BRAF mutations from clinical and pathological features?

2. Has BRAF mutation a prognostic significance?

Features associated with BRAF mutational status:

� Histological subtype (SSM and NM)

� Presence of mitoses � Single melanoma � Truncal location� Age at diagnosis <50 yr� Lack of evidence of CSD� Increased upward migration and

nest formation of intraepidermalmelanocytes

� Thickening of the involved epidermis, with sharper demarcation to the surrounding skin

� Larger, rounder, and more pigmented tumor cells

� Brain metatstasis. Worse prognosis?

� Associated nevus� Breslow thickness � Ulceration

Features not associated with BRAF mutational status:

Flaherty KT, et al. Cancer 2010;116:4902–13. Viros A, et al. PLoS Med 2008;5:e120Long GV, et al. JCO 2011;29:1239

YES NO

3. Does a single BRAF mutation induce melanoma?

BRAF

Br J Cancer 2011;104:392-398; JCO 2011;29:1239

Frequencies of BRAF mutations in various cancers

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Flaherty K et al. Cancer 2010, 116:4902

Common genetic alterations in melanoma

CSD-Skin

Non-CSD-Skin

c-kit mutations (1%)

4. Which target?

Targeting metastatic melanoma

Flaherty KT. Ann Rev Med 2012;63:21.1-21.13

Phase 3 randomized clinical trial. NEJM 2011, June 5

Overall survival

Progression-free survival

All patients 672Age: <65 yrs

�65 yrs512160

Sex: FemaleMale

293379

ECOG status: 01

457215

Disease stage: IIIcM1aM1bM1c

3374

126439

LDH : NormalElevated

390282

FactorNumber

of patients

0.2 0.4 0.6 1.0 2 4 106Hazard ratio and 95% confidence interval

Favorsvemurafenib

Favorsdacarbazine

20

BRIM3: OS by baseline characteristic

5. What are the side effects?

Selected adverse events (% of patients)Vemurafenib, n= 336 Dacarbazine, n= 282

Adverse events All Grade 3 Grade� 4 All Grade 3 Grade �4

Arthralgia 49 3 - 3 <1 -

Rash 36 8 - 1 - -

Fatigue 33 2 - 31 2 -

Photosensitivity 30 3 - 4 - -

↑LFTs 18 7 <1 5 1 -

Cutaneous SCC 12 12 - <1 <1 -

Keratoacanthoma 8 6 - - - -

Skin papilloma 18 <1 - - - -

Nausea 30 1 - 41 2 -

Neutropenia <1 - <1 11 5 3

Discontinuations due to AE: 6% Vemurafenib; 4% Dacarbazine

V600EBRAF cells

Nissan MH , Solit DB, Curr Oncol Rep 2012 (published online 2011)

Paradoxical hyper-activation of MEK and ERK by PLX4032 in BRAF wild-type cells

Nissan MH , Solit DB, Curr Oncol Rep 2012 (published online 2011)

Paradoxical CRAF activation by BRAF

Heidorn SJ et al. Cell 2010;140:209-220

BRAF wt

BRAF mt

BRAF mt

BRAF mt

BRAF wt

BRAF wt

BRAF wt

N Engl J Med 2011;365:1448

Mechanisms of resistance to BRAF inhibitors and strategies to overcome

resistance

Baseline Days 15 7 months

BRAF and resistance

� Primary (or intrinsic) resistanceAny mechanism that allows all or a subpopulation of cells within a BRAF mutant melanoma to survive the initial weeks or months of therapy

� Secondary (or acquired) resistanceAny mechanisms by which tumors begin to grow again following the initial response to therapy

Fedorenko et al. BiochemicalPharmacology 2011 Article in press

Combined immunotherapy with anti-CTLA4 (ipilimumab)?

Activation of intrinsic survival pathways or exogen growth support from the microenvironment

PI3K inhibitorsPI3K activation bypass BRAF

New drugs able to block RAF-dimers

BRAF mutations able to reactivate MAPK pathway (i.e. dimerization of BRAF)

Secondary Resistance

PI3K inhibitorsCDK4 inhibitorsPI3K inhibitorsMEK inhibitors

Selection of clones with additional mutationsPTEN lossP16 inactivationPI3K activationNRAS/ MEK activation

Combined therapy with MEK inhibitors

Incomplete pathway inhibition (residual pathway activity)

Possible solutionsPrimary Resistance

6. So what?

� Recent advances in molecular biology have identified BRAF, NRAS, and c-KIT mutations as major contributors to melanoma pathophysiology in approximately 70% of all cases.

� BRAF inhibitors demonstrate high response rates exclusively in patients whose melanoma harbors an activating BRAF mutation.

� Although response duration is highly variable, BRAF inhibitors improve survival outcome in the metastatic setting.

� Combination of BRAF inhibitors with immune-enhancing therapy with a CTLA-4 blocking antibody may be very effective.

� Additional pathways appear to contribute to primary resistance of BRAF inhibition therapy.

� The optimal strategy for combined therapy in melanoma patients remains to be determined.

Flaherty KT. Ann Rev Med 2012;63:21.1-21.13

� BRAF mutations can be easily detected by direct sequencing or real-time PCR (cobas® 4800 BRAF V600 Mutation Test).

� NRAS mutations should be investigated in BRAF wild-type melanoma.

� c-KIT mutations should be investigated in BRAF/NRAS wild-type melanoma, and in acral or mucosal melanoma, in vulvar melanoma, or melanomas occurring at chronically sun-damaged skin (role of c-KIT amplification is still unclear).

� Additional pathways appear to contribute to primary or secondary resistance to BRAF inhibitor therapy

� “We are now entering an era of personalized therapy for patients with advanced melanoma”.