EUROPEAN JOURNAL OFCANCER

Transcription

1 Volume 49, No. 5, March 2013 ISSN EJC EUROPEAN JOURNAL OFCANCER Reprinted from Eur J Cancer. 2013;49(5): BRAF inhibitor activity in V600R metastatic melanoma Oliver Klein, Arthur Clements, Alexander M. Menzies, Sandra O Toole, Richard F. Kefford, Georgina V. Long THE OFFICIAL JOURNAL OF

2 European Journal of of Cancer (2013) (2013) 49, 49, Available at journal homepage: BRAF inhibitor activity in V600R metastatic melanoma Oliver Klein a, Arthur Clements a, Alexander M. Menzies a,b, Sandra O Toole c,d,e,f, Richard F. Kefford a,b,d, Georgina V. Long a,b,d, a Westmead Hospital, Sydney, Australia b Melanoma Institute Australia, Sydney, Australia c Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia d University of Sydney, Sydney, Australia e The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia f St. Vincent s Clinical School, University of New South Wales, Sydney, Australia Available online 10 December 2012 KEYWORDS Metastatic melanoma V600E V600R BRAF inhibitor Vemurafenib Dabrafenib Abstract Activating mutations in the BRAF gene occur in approximately 50% of melanomas. More than 70% of BRAF mutations are V600E and 10 30% are V600K. Potent and selective BRAF inhibitors have demonstrated significant clinical benefits in patients with V600E and V600K BRAF-mutated melanoma. V600R mutations constitute approximately 3 7% of all BRAF mutations and the activity of BRAF inhibitors in patients with this mutation is unknown. We have treated 45 patients with V600 mutated melanoma including patients with V600R mutation between July 2011 and October 2012 with the selective BRAF inhibitor dabrafenib (n = 43) or vemurafenib (n = 2) via a compassionate access programme. The overall response rate was 50% for the whole population with a progression-free survival of 5.5 months. Five objective responses were seen in six assessable patients with V600R BRAF mutation (n = 9). Our experience suggests that patients with V600R BRAF mutations can be treated successfully with oral BRAF inhibitors, and molecular diagnostic assays should include detection of this type of mutation. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Cutaneous melanoma is genetically heterogeneous with subsets defined by specific driver mutations. 1 Corresponding author: Address: Melanoma Institute Australia and Westmead Hospital, University of Sydney, 40 Rocklands Road, North Sydney, New South Wales, Australia. Tel.: ; fax: address: georgina.long@sydney.edu.au (G.V. Long). Approximately 50% of melanomas carry mutations in the BRAF oncogene leading to constitutive activation of the MAP kinase pathway. The majority of BRAF mutations occur in exon 15 of the BRAF gene and 74 95% are due to a V600E amino acid substitution. 2 5 More than 99% of cases of V600E are due to the point mutation c.1799t>a, (Catalogue of Somatic Mutations In Cancer (COSMIC) ID 476). 5 V600K (c.1798_1799gt>aa, COSMIC ID 473), the second most common mutation, /$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

3 1074 O. Klein et al. / European Journal of Cancer 49 (2013) occurs in older patients or those with evidence of chronic sun damage at the site of the primary cutaneous melanoma, 6 and comprises 10 30% of the BRAF mutation-positive melanoma population. 2 4 V600R (c.1798_1799gt>ag, COSMIC ID 474) mutations are the third most common mutation occurring in approximately 5 7% of patients with BRAF-mutant melanoma. 7,8 The selective BRAF inhibitors, vemurafenib and dabrafenib, are active in V600E BRAF mutation-positive metastatic melanoma with Response Evaluation Criteria in Solid Tumours (RECIST) 9 response rates of approximately 50% 10 14, a clinical benefit observed in nearly 90% of patients, and a prolonged progression-free and overall survival compared with dacarbazine chemotherapy. 12,14 Activity of dabrafenib has been observed in prospective studies of patients with V600K mutationpositive melanoma 13,15 and retrospective analyses of clinical trials and case series of vemurafenib 11,12,16 but lower response rates and a shorter progression-free survival have been reported in these patients compared with the more common V600E mutation. 15,17 There remains uncertainty as to whether BRAF inhibitors are effective in patients with non-v600e/k mutations. Although there is limited pre-clinical evidence that selective BRAF inhibitors might be active in V600R melanoma, 18 clinical activity has not been reported. This report describes our clinical experience of treating nine patients with V600R mutated metastatic melanoma with dabrafenib (n = 7) or vemurafenib (n = 2) on compassionate access programmes. 2. Patients and methods 2.1. Patient population Between June 2011 and October patients were enroled in a compassionate access programme to receive the selective BRAF inhibitor dabrafenib and an additional two patients with V600R received compassionate vemurafenib. Eligible patients had histologically confirmed metastatic melanoma with a BRAF V600 mutation, were not eligible for an active trial or expanded access programme by the same sponsor, and had an ECOG performance status of three or less. Patients with brain metastases could be enroled if no immediate local treatment was required. Patients received dabrafenib at a dose of 150 mg orally twice daily and the two patients treated with vemurafenib received 960 mg orally twice daily. Radiological disease assessment included computed tomography (CT) scan, and for those with brain metastases at baseline, magnetic resonance imaging (MRI) of the brain with gadolinium enhancement. Assessments were performed at baseline and every 6 10 weeks. Patients were allowed to continue treatment beyond disease progression if there was ongoing clinical benefit as determined by the treating clinician BRAF mutation testing BRAF mutation testing was performed on sections from archival FFPE tissue blocks at 1 of 3 laboratories; Peter MacCallum Cancer Centre, Department of Diagnostic Molecular Pathology (Melbourne, Australia), Royal Prince Alfred Hospital, Department of Clinical and Molecular Genetics (Sydney, Australia) and Healthscope Pathology (Clayton Australia) using one of the following methods. At Peter MacCallum Cancer Centre, samples were macro-dissected and subjected to high resolution melting (HRM) analysis using primers flanking codon 600 in the BRAF gene. These primers identify variations in exon 15 of the BRAF gene between nucleotides c.1788 and c.1823 in reference sequence NM_ , corresponding to codons All abnormal HRM traces were subjected to bidirectional DNA sequencing. At Royal Prince Alfred Hospital, samples were reviewed by a tissue pathologist, macro-dissected to enrich for tumour and the DNA was extracted from formalin fixed paraffin embedded (FFPE) tissue using NucleoSpin FFPE DNA Kit (Macherey Nagel) according to the manufacturer s instruction with an overnight proteinase digestion. The quality and quantity of the extracted DNA was assessed using NanoDrop Ò ND Spectrophotometer. A minimum of 480 ng of DNA was required for successful mutational analysis. Samples were amplified for 238 variant targets in a 24- multiplex polymerase chain reaction (PCR) using the OncoCarta Panel v1.0 Kit (ABL1, AKT1, AKT2, BRAF, CDK, EGFR, ERBB2, FGFR1, FGFR3, FLT3, JAK2, KIT, MET, HRAS, KRAS, NRAS, PDGFR, PIK3CA, RET) and analysed based on the matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF) technology on the Sequenom MassArray platform. At Healthscope Pathology, haematoxylin and eosinstained sections of FFPE tissue were reviewed by a pathologist, followed by macrodissection to ensure the percentage of tumour cells was enriched to at least 60%. DNA was then extracted using the Qiagen QIAmp mini extraction columns. Exon 15 of the BRAF gene was amplified by PCR. A single-base extension assay was performed using a forward primer that interrogated the nucleotide at position 1798, as well as a reverse primer that interrogated the nucleotide at position The primers were designed using the Sequenom Assay Designer software. Standard protocols based on the Sequenom Typlex kit were then followed according to the manufacturer s instructions. Products of the extension reaction are fired and analysed on a Sequenom Massarray mass spectrometer.

4 O. Klein et al. / European Journal of Cancer 49 (2013) Statistical analysis SPSS software was used to calculate the Kaplan Meier median progression-free survival curve with two-sided 95% confidence interval (CI). 3. Results Between July 2011 and October 2012, 45 patients with BRAF V600 mutated metastatic melanoma received compassionate access to the selective BRAF inhibitors dabrafenib (n = 43) or vemurafenib (n = 2). BRAF mutation genotyping revealed a V600E mutation in 23 (51%), a V600K mutation in 13 (29%) and a V600R mutation in nine (20%) patients (Fig. 1). The majority of patients were male with stage M1c disease, 15 (33%) patients had brain metastases prior to commencement of the BRAF inhibitor, and more than half of all patients had an elevated lactate dehydrogenase (LDH) level (Table 1). Only two patients had received prior systemic therapy with chemotherapy or targeted agents (vemurafenib, trametinib), and none had received ipilimumab. Nine patients of the 45 were not included in the efficacy analysis; three patients deceased prior to the first response assessment and six had not been radiographically assessed for response at the time of reporting. According to the clinician s assessment, the overall response rate was 50% (18/36) for the whole population of assessable patients with 17 patients achieving a partial response and one patient a complete response. The median progression-free survival was 5.5 months (95% CI, ) (Fig. 2). According to the BRAF V600 mutation genotype, 11 of 20 (55%) patients with the V600E mutation, 2 of 10 (20%) with the V600K and 5 of 6 (83%) with the V600R mutation achieved a clinical and radiological response (Fig. 2). Most patients with V600R mutated melanoma (n = 9) were elderly males with a history of an ulcerated primary melanoma (Table 2). One patient, the only female, could be not assessed for response as she deceased due to an intracranial haemorrhage 3 days after commencing dabrafenib, and this was considered Fig. 1. Spectrographs showing the c.1798_1799 GT>AG (V600R) BRAF mutation. The Oncocarta v1.0 assay demonstrates additional mutant peaks at the complementary bases (arrows).

5 1076 O. Klein et al. / European Journal of Cancer 49 (2013) Table 1 Clinical characteristics of patients treated with dabrafenib and vemurafenib. Characteristics Patients (N = 45), n (%) Age, median(range), years 61 (17 84) Sex Male 35 (78) Female 10 (22) BRAF genotype V600E 23 (51) V600K 13 (29) V600R 9 (20) Stage AJCC M1a 1 (2) M1b 4 (9) M1c 40 (89) Brain metastases Present 15 (33) Absent 30 (67) Lactate dehydrogenase Elevated 24 (53) Normal 21 (47) unrelated to dabrafenib. Overall, five of six evaluable patients with V600R mutated melanoma treated with selective BRAF inhibitors achieved a clinical and radiological response (e.g. Figs. 3 and 4). Two additional patients achieved rapid clinical responses shortly after commencing treatment, with a rapid reduction in the size of subcutaneous metastases in both patients, and resolution of neurological symptoms (left hemiparesis power 1/5 to 4/5) in one. Both patients are awaiting their first radiological assessment. Given the lack of knowledge about the clinical efficacy of selective BRAF inhibitors in patients with V600R mutated metastatic melanoma, we describe the clinical course of two patients in more detail Case 1 A 74-year-old man presented with an acute onset of severe headaches and a brain CT scan revealed three haemorrhagic tumours involving the right frontal and parietal lobes. Two of the tumours were resected and histological assessment confirmed metastatic melanoma. Further imaging revealed multiple lung metastases (Fig. 3C). The patient had no history of a primary cutaneous melanoma and skin lesions suspicious of melanoma could not be identified at the time of diagnosis. A V600R mutation in exon 15 of the BRAF gene was detected in the tumour sample and he was subsequently commenced with the BRAF inhibitor dabrafenib after enrolment onto the compassionate access programme. A progress CT scan, 8 weeks after commencement of treatment, confirmed a partial response with a significant decrease in size of the pulmonary metastases and stable intracranial disease. Progress imaging at 16 and 24 weeks (Fig. 3D) after commencement of dabrafenib, revealed an ongoing response with a further decrease in the size of the pulmonary metastases and stable intracranial disease. Ten months after commencing treatment, he progressed with multiple new brain metastases and an increase in size of the pre-existing pulmonary metastases. Subsequently, he has undergone whole brain radiotherapy and continues on dabrafenib. The patient is alive 12 months after commencing drug. A B 1.0 Overall response 0.8 Proportion Progression-free PFS=5.5 months (95% CI, ) Months Fig. 2. Overall response rates (A) and Kaplan Meier progression-free survival analysis (B) of patients with V600 mutated melanoma treated with dabrafenib and vemurafenib.

6 O. Klein et al. / European Journal of Cancer 49 (2013) Case 2 Table 2 Characteristics and treatment responses of patients with V600R BRAF-mutated. Overall survival a Progressionfree survival Best response BRAF inhibitor (BRAFi) Lactate dehydrogenase (LDH) Brain metastases Ulceration Stage AJCC Patient Age Gender Site of primary melanoma 1 75 Male Unknown N/A M1c + Normal Dabrafenib PR 10.4 Alive 2 73 Male Upper limb M1c Elevated Dabrafenib PD Male Scalp + M1b Normal Dabrafenib PR 3.5 Alive 4 54 Male Upper limb + M1c + Normal Dabrafenib PR 3.7+ Alive 5 61 Male Trunk + M1a Elevated Dabrafenib PR 3.2+ Alive 6 64 Male Scalp M1c + Normal Dabrafenib b b Alive 7 63 Female Upper limb + M1c + Normal Dabrafenib c c c 8 81 Male Scalp + M1c Elevated Vemurafenib PR Male Scalp + M1a + Normal Vemurafenib b b Alive AJCC: American Joint Committee on Cancer; PR: partial remission; PD: progressive disease. a Time from commencing BRAF inhibitor. b Clinical response, no radiological assessment performed yet. c Patient deceased few days after commencing treatment. An 81-year-old man was diagnosed with a 3.6 mm, ulcerated primary cutaneous melanoma of the occiput and was treated with wide local excision in March Within 6 months the melanoma recurred at the occiput and the left neck. Both recurrences were resected, including a neck dissection which was followed by radiotherapy. Eight months later in May 2011 disease recurred in the contralateral neck and was treated similarly. Within 4 months extensive unresectable subcutaneous melanoma metastases developed involving the scalp and bilateral peri-auricular regions (Fig. 4A). The patient required regular opiate analgesia, and had exhausted local therapies for disease control at the scalp. A staging CT scan showed an additional 7 mm metastasis in the right lung and a 9 mm metastasis in the liver. Testing of a left neck nodal metastasis revealed a V600R mutation in BRAF exon 15. He was commenced on vemurafenib via compassionate use access, had a marked reduction in the size of the subcutaneous lesions within 1 week, and ceased opiate analgesia. Staging at week 8 showed a RECIST 9 partial response (75%) with a marked reduction of the scalp disease (Fig. 4B). The patient progressed in the scalp 19 weeks after commencing vemurafenib, with continued response in the lung and liver. He ceased vemurafenib at 22 weeks and commenced lenvatinib (E7080) and achieved a partial response. The patient died 9.5 months after commencing vemurafenib due to complications from chronic obstructive pulmonary disease. 4. Discussion Vemurafenib was co-developed clinically with the cobas Ò 4800 BRAF V600 diagnostic test, 19 which while highly sensitive and specific for the exon T>A BRAF gene mutation (V600E), lacks similar sensitivity for non-v600e BRAF mutations. 20 Dabrafenib was prospectively tested in patients with a range of BRAF mutations including V600K and K601E. 13,21 No clinical trials of either drug have included patients with V600R BRAF mutation-positive melanoma, the third most common BRAF mutation. 7 Over a period of 15 months we treated 45 patients with V600 BRAF-mutated metastatic melanoma via compassionate access to the selective BRAF inhibitor dabrafenib or vemurafenib (not the expanded access programme). These patients were not eligible for clinical trials and the population was therefore enriched for patients with non-v600e mutations. The observed overall response rate and the progression-free survival in our patient population is in keeping with the outcome previously reported in clinical trials of BRAF inhibitors In this dataset, there appeared to be a lower response

7 1078 O. Klein et al. / European Journal of Cancer 49 (2013) Fig. 3. Chest computed tomography (CT) scan of patients with metastatic V600R mutated melanoma. (A) Bulky axillary lymph nodes at baseline (top) and week 8 (bottom) after treatment with dabrafenib. (B) Lung metastases at baseline (top) and week 16 (bottom) after treatment with dabrafenib. Circles and arrows indicate areas of response. Fig. 4. Subcutaneous scalp metastases of a patient with V600R mutated melanoma at baseline (A) and at week 8 (B) after treatment with vemurafenib. rate in patients with V600K mutated melanoma as previously demonstrated in clinical trials. 15,17 Our screening experience for BRAF mutations in patients with metastatic melanoma confirms that V600R mutations are the third most common BRAF mutation in keeping with data published in the United States and European population. 7,8 We have previously demonstrated specific clinicopathological features associated with V600K mutation-positive metastatic melanoma. 6 The V600R genotype is common in males and those with ulcerated primary melanomas, and like patients with V600K melanoma, it is common in older

8 O. Klein et al. / European Journal of Cancer 49 (2013) individuals. These observations require confirmation in a larger cohort of patients with V600R mutated melanoma. Five of six evaluable patients with V600R BRAFmutant metastatic melanoma reported here achieved a partial response by RECIST criteria, including a reduction in the size of lung metastases, liver metastases, stabilisation of brain metastases and a marked reduction in subcutaneous metastases to the scalp due to treatment with a BRAF inhibitor. All nine patients had improved symptoms and performance status within days of commencing treatment, including a patient who had RECIST progression by week 8. Two patients are awaiting radiological confirmation of the clinical response and reduction in subcutaneous metastases observed within the first 2 weeks of treatment. The two patients with the longest follow up had durable responses with one progressing in isolated areas at 19 weeks and the other after 44 weeks of therapy. The rapid clinical responses of patients with V600R BRAF mutation-positive melanoma mirror those observed in patients with V600E and V600K mutations, indicating that selective BRAF inhibitors are active against melanoma with this genotype. Our observations suggest that diagnostic BRAF mutation analysis of metastatic melanoma should test for the rarer non-v600e mutations, in addition to V600E. The clinical benefit of BRAF inhibitors in this subset of patients should be formally elucidated in clinical trials, including trials of combined therapies. Our experience demonstrates that patients with V600R mutation-positive metastatic melanoma can be treated effectively with the BRAF inhibitors vemurafenib and dabrafenib. Conflict of interest statement G.V.L. has received honoraria from Roche for lectures; consultancy fees for advisory board membership and support for congress attendance from Glaxo- SmithKline and Roche. The Melanoma Institute Australia received educational grants from Roche. R.F.K. has received institutional reimbursement for Advisory Board membership and support for travel from GlaxoSmithKline, and institutional reimbursement for lectures and Advisory Board membership from Roche. S.A.O T has consultancy fees for advisory board membership and support for conference attendance from Roche. A.M.M. received travel support for conference attendance from Roche and GSK. References 1. Flaherty KT, Hodi FS, Fisher DE. From genes to drugs: targeted strategies for melanoma. Nat Rev Cancer 2012;12: Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol 2011;29: Jakob JA, Bassett Jr RL, Ng CS, et al. NRAS mutation status is an independent prognostic factor in metastatic melanoma. Cancer 2011;118: Thomas NE, Edmiston SN, Alexander A, et al. Number of nevi and early-life ambient UV exposure are associated with BRAFmutant melanoma. Cancer Epidemiol Biomarkers Prev 2007;16: Catalogue of Somatic Mutations in Cancer (COSMIC) Website. Wellcome Trust Sanger Institute. Available from: 6. Menzies AM, Haydu LE, Visintin L, et al. Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma. Clin Cancer Res 2012;18: Lovly CM, Dahlman KB, Fohn LE, et al. Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials. PLoS One 2012;7:e Houben R, Becker JC, Kappel A, et al. Constitutive activation of the Ras Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J Carcinog 2004;3:6. 9. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45: Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363: Sosman JA, Kim KB, Schuchter L, et al. Survival in BRAF V600- mutant advanced melanoma treated with vemurafenib. N Engl J Med 2012;366: Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011;364: Falchook GS, Long GV, Kurzrock R, et al. Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet 2012;379: Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAFmutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 2012;380: Long GV, Trefzer U, Davies MA, et al. Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB) a multicentre, open-label, phase 2 trial. Lancet Oncol 2012;13: Rubinstein JC, Sznol M, Pavlick AC, et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med 2010;8: Trefzer U, Minor D, Ribas A, et al. BREAK-2: a phase IIA trial of the selective BRAF kinase inhibitor GSK in patients with BRAF mutation-positive (V600E/K) metastatic melanoma Society of Melanoma Research Congress. Pigment Cell Melanoma Res 2011; Yang H, Higgins B, Kolinsky K, et al. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res 2010;70: Cheng S, Koch WH, Wu L. Co-development of a companion diagnostic for targeted cancer therapy. N Biotechnol 2012;29: Halait H, Demartin K, Shah S, et al. Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma. Diagn Mol Pathol 2012;21: Smalley KS, Aplin AE, Flaherty KT, et al. Meeting report from the 2011 International Melanoma Congress, Tampa, Florida. Pigment Cell Melanoma Res 2012;25:E1 E11.

9 2012 Elsevier Ltd. All rights reserved. No responsibility is assumed by Elsevier, its licensors or associates for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. Edited for: Elsevier España, S.L.U. (A member of Elsevier) Av. Josep Tarradellas, Barcelona Tel.: Fax: