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BRAF inhibitor activity in V600R metastatic melanoma
Eur J Cancer. 2013;49(5):1073-9
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 BRAFmutations 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 BRAFmutation (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.
Keywords: Metastatic melanoma, V600E, V600R, BRAF inhibitor, Vemurafenib, Dabrafenib.
Cutaneous melanoma is genetically heterogeneous with subsets defined by specific driver mutations. 1 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, 3, 4, and 5More 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, 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 theBRAFmutation-positive melanoma population.2, 3, and 4V600R (c.1798_1799GT>AG, COSMIC ID 474) mutations are the third most common mutation occurring in approximately 5–7% of patients withBRAF-mutant melanoma.7 and 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, 11, 12, 13, and 14, a clinical benefit observed in nearly 90% of patients, and a prolonged progression-free and overall survival compared with dacarbazine chemotherapy.12 and 14Activity of dabrafenib has been observed in prospective studies of patients with V600K mutation-positive melanoma13 and 15and retrospective analyses of clinical trials and case series of vemurafenib11, 12, and 16but lower response rates and a shorter progression-free survival have been reported in these patients compared with the more common V600E mutation.15 and 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 2012 43 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.
2.2. BRAF mutation testing
BRAFmutation 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 theBRAFgene. These primers identify variations in exon 15 of theBRAFgene between nucleotides c.1788 and c.1823 in reference sequence NM_004333.4 , corresponding to codons 597–607. 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® ND-1000 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 eosin-stained 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 1799. 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.
2.3. Statistical analysis
SPSS software was used to calculate the Kaplan–Meier median progression-free survival curve with two-sided 95% confidence interval (CI).
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).BRAFmutation 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.
|Characteristics||Patients (N = 45), n (%)|
|Age, median(range), years||61 (17–84)|
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, 3.4–7.6) ( Fig. 2 ). According to theBRAFV600 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 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.Fig 3 and Fig 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.
|Patient||Age||Gender||Site of primary melanoma||Ulceration||Stage AJCC||Brain metastases||Lactate dehydrogenase (LDH)||BRAF inhibitor (BRAFi)||Best response||Progression-free survival||Overall survival a|
|6||64||Male||Scalp||−||M1c||+||Normal||Dabrafenib||– b||– b||Alive|
|7||63||Female||Upper limb||+||M1c||+||Normal||Dabrafenib||– c||– c||– c|
|9||84||Male||Scalp||+||M1a||+||Normal||Vemurafenib||– b||– b||Alive|
a Time from commencing BRAF inhibitor.
b Clinical response, no radiological assessment performed yet.
c Patient deceased few days after commencing treatment.
AJCC: American Joint Committee on Cancer; PR: partial remission; PD: progressive disease.
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.
3.1. 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. 3 C). 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 theBRAFinhibitor 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. 3 D) 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.
3.2. Case 2
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 2010. 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. 4 A). 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. 4 B). 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.
Vemurafenib was co-developed clinically with the cobas® 4800 BRAF V600 diagnostic test, 19 which while highly sensitive and specific for the exon 15 1799T>ABRAFgene mutation (V600E), lacks similar sensitivity for non-V600E BRAF mutations. 20 Dabrafenib was prospectively tested in patients with a range ofBRAFmutations including V600K and K601E.13 and 21No 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.11, 12, 13, and 14In this dataset, there appeared to be a lower response rate in patients with V600K mutated melanoma as previously demonstrated in clinical trials.15 and 17
Our screening experience for BRAF mutations in patients with metastatic melanoma confirms that V600R mutations are the third most commonBRAFmutation in keeping with data published in the United States and European population.7 and 8We 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 individuals. These observations require confirmation in a larger cohort of patients with V600R mutated melanoma.
Five of six evaluable patients with V600R BRAF-mutant 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 diagnosticBRAFmutation 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 GlaxoSmithKline 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.
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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
Corresponding author: Address: Melanoma Institute Australia and Westmead Hospital, University of Sydney, 40 Rocklands Road, North Sydney, New South Wales, Australia. Tel.: +61 2 9911 7200; fax: +61 2 9954 9290.
© 2012 Elsevier Ltd, All rights reserved.
© 2012 Elsevier Ltd. All rights reserved.
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