Molecular analyses from a phase I trial of vemurafenib to study mechanism of action (MOA) and resistance in repeated biopsies from BRAF mutation–positive metastatic melanoma patients (pts).

8502 Background: Vemurafenib (PLX4032), an oral, selective inhibitor of oncogenic V600E mutant BRAF, was evaluated for safety and pharmacokinetics in the Phase I dose escalation (DE) study PLX06-02 (Flaherty, et al. NEJM, 2010). Exploratory analyses included the investigation of MOA, pathway inhibition, and primary or acquired drug resistance from pts participating in the DE and melanoma extension phase. Methods: Biopsies were taken at baseline (BL), on vemurafenib therapy Day 15 (D15), and at progressive disease (PD). Protein expression (PTEN, Cyclin D1, p27, and Ki67), phosphorylation (pERK, pMEK and pAKT), apoptosis (TUNEL), gene sequence (MEK1), and mutations in oncogenes and tumour suppressor genes (404 mutations incl. BRAF, H-, K-, N-RAS, and PIK3CA using Sequenom MassArray) were analyzed. Results: Tumor tissue of 5 pts from the DE and 17 pts from the extension cohort was obtained (total samples: 18 BL, 12 D15, 20 PD). In 7/7 pts with paired BL/D15 samples, high levels of pERK were observed at BL followed by a considerable reduction at D15. A similar pattern was observed for pMEK. The percent of cells with nuclear staining for Cyclin D1 and Ki67 was markedly reduced at D15 compared with BL. At PD, a broad distribution of pERK levels was observed across the samples. BRAF V600E mutation was present at both BL and DP in all samples analyzed. Genetic analysis performed on 32 samples (16 BL, 1 D15, 15 DP) revealed 1 MEK P124S mutation (at D15; confirmed in archival tissue) and 1 N-RAS G12R mutation (at PD; no pre-treatment sample available). Conclusions: The strong downregulation of MEK/ERK phosphorylation observed in pts treated with vemurafenib is consistent with preclinical studies and confirms the proposed MOA of vemurafenib (blockade of BRAF V600E-driven signalling). This blockade is associated with reduction in Cyclin D1 expression and downregulation of proliferation. That the BRAF mutation persisted in all PD samples and in some cases ERK phosphorylation was reduced suggests that some patients may continue to respond to inhibition of the BRAF V600E-driven signaling pathway although simultaneously requiring inhibition of other activated signaling pathways.