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Patients who discontinued therapy before best response were also excluded.Seven non-BRAF patients were selected to serve as controls.Expression of mutated protein BRAF as measured by intensity, per cent immunoreactive cells, or IRS did not correlate with RECIST response, TTBR, PFS or OS, including on multivariate analysis.Heterogeneity of staining was seen in 22% of cases and did not correlate with outcome.Clinical outcome was assessed using time to best response (TTBR), PFS and OS from commencement of BRAF inhibitor.

Briefly, sections were stained with undiluted hybridoma supernatant of BRAF-specific clone VE1 (provided by Andreas von Deimling, now commercially available at Spring Bioscience, Pleasanton, CA, USA) on a Ventana Bench Mark XT immunostainer (Ventana Medical Systems, Tucson, AZ, USA).All immunostained slides were evaluated twice by two independent observers (JSW and RAS) blinded to all clinical, histopathological and mutation data.The BRAF VE1 antibody staining was scored for the percentage of immunoreactive cells.The presence of intra-tumoural heterogeneity for BRAF expression was correlated with RECIST response, time to best response (TTBR), progression-free survival (PFS) and overall survival (OS).Expression was generally high (median IRS 28 (range 5–30)) and homogeneous (78%).We have previously shown that immunohistochemistry (IHC) with the BRAF mutation status (Long et al, 2013).Detection of the target protein of the BRAF inhibitor using IHC has a number of advantages over other molecular mutation testing techniques.Clinical and follow-up details were collected and analysed on all patients as approved by the Westmead and Royal Prince Alfred Hospital Research Ethics Committees.All patients treated with vemurafenib received 960 mg twice daily.The selection criteria for the current study included all patients diagnosed with a BRAF mutation via somatic mutation testing, who received a BRAF inhibitor on the abovementioned clinical trials, and had sufficient archival tissue to perform IHC.Patients who did not receive the recommended phase 2 doses (RP2D) of vemurafenib (960 mg BD) or dabrafenib (total daily dose of 300 mg) were excluded.

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