Normal RAS-RAF (MAPK) pathway signaling

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1 BRAF-Mutations in Melanomas L. Mazzucchelli Istituto Cantonale di Patologia, Locarno 77. Annual Meeting Swiss Society of Pathology, Lucerne 2011 Sponsored 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 Activating BRAF mutations in 50% of melanoma The RAS-RAF pathway is a series of protein kinases that 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 RAF PTEN AKT Normal cell proliferation and survival Excessive cell proliferation and survival BRAF V600E is capable of causing RAS-independent activation of ERK, MEK-dependent cell proliferation, and transformation J Pathol 2011;223: 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 intraepidermal melanocytes Thickening of the involved epidermis, with sharper demarcation to the surrounding skin Larger, rounder, and more pigmented tumor cells Brain metatstasis. Worse prognosis? Features not associated with BRAF mutational status: Associated nevus Breslow thickness Ulceration YES NO Flaherty KT, et al. Cancer 2010;116: Viros A, et al. PLoS Med 2008;5:e120 Long GV, et al. JCO 2011;29:1239

2 BRAF 3. Does a single BRAF mutation induce melanoma? Br J Cancer 2011;104: ; JCO 2011;29:1239 Frequencies of BRAF mutations in various cancers Common genetic alterations in melanoma c-kit mutations (1%) CSD-Skin Non-CSD-Skin Flaherty K et al. Cancer 2010, 116:4902 Targeting metastatic melanoma 4. Which target? Flaherty KT. Ann Rev Med 2012;63:

3 Overall survival Progression-free survival Phase 3 randomized clinical trial. NEJM 2011, June 5 BRIM3: OS by baseline characteristic Factor All patients 672 Age: <65 yrs 65 yrs Sex: Female Male ECOG status: Disease stage: IIIc M1a M1b M1c LDH : Normal Elevated Number of patients Favors vemurafenib Favors dacarbazine Hazard ratio and 95% confidence interval Selected adverse events (% of patients) Vemurafenib, n= 336 Dacarbazine, n= 282 Adverse events All Grade 3 Grade 4 All Grade 3 Grade 4 5. What are the side effects? Arthralgia <1 - Rash Fatigue Photosensitivity LFTs 18 7 < Cutaneous SCC <1 <1 - Keratoacanthoma Skin papilloma 18 < Nausea Neutropenia <1 - < Discontinuations due to AE: 6% Vemurafenib; 4% Dacarbazine

4 V600E BRAF cells 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) Nissan MH, Solit DB, Curr Oncol Rep 2012 (published online 2011) Paradoxical CRAF activation by BRAF Heidorn SJ et al. Cell 2010;140: N Engl J Med 2011;365:1448 Mechanisms of resistance to BRAF inhibitors and strategies to overcome resistance BRAF and resistance Primary (or intrinsic) resistance Any 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) resistance Any mechanisms by which tumors begin to grow again following the initial response to therapy Baseline Days 15 7 months

5 Fedorenko et al. BiochemicalPharmacology 2011 Article in press Primary Resistance Incomplete pathway inhibition (residual pathway activity) Selection of clones with additional mutations PTEN loss P16 inactivation PI3K activation NRAS/ MEK activation Activation of intrinsic survival pathways or exogen growth support from the microenvironment Secondary Resistance BRAF mutations able to reactivate MAPK pathway (i.e. dimerization of BRAF) PI3K activation bypass BRAF Possible solutions Combined therapy with MEK inhibitors CDK4 inhibitors MEK inhibitors Combined immunotherapy with anti- CTLA4 (ipilimumab)? New drugs able to block RAF-dimers 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: 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.