Vemurafenib

Cell viability was determined using the MTT assay kit (Cat 11465007001, Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, 5 × 10⁴ cells were seeded per well in 100 μL DMEM (HyClone; Thermo Scientific, 90 Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, 91 Lawrenceville, GA) and 1% penicillin/streptomycin mixture (Sigma-Aldrich, MO). Melanoma cell lines treated with decitabine were considered MEL-like or MES-like based on the protein expression of E-cadherin versus N-cadherin and ZEB1, respectively, which showed similar results to our patient-derived cell lines. For the experiments, cells were exposed to increasing concentrations of either the BRAF inhibitor vemurafenib (Roche, IN) for 24 h or the demethylating agent decitabine (Sigma-Aldrich, MO) for 72 h, or DMSO vehicle control. At the end of treatment, the cells were incubated with 10 μL/well of the MTT labeling reagent for 4 h, then incubated with 100 μL of the Solubilization solution overnight. Absorbance of each sample was measured on a microplate reader (Molecular Devices, CA) at the wavelength of 560 nm.

Vemurafenib (PLX4032 or RG7204) was kindly donated by Roche for study purpose. Vemurafenib was dissolved in DMSO to a stock concentration of 1 mg/ml. The Vemurafenib stock was freshly diluted before each experiment to 60 μg/ml unless stated otherwise. Dabrafenib and trametinib were obtained from Alsachim (Illkirch-Graffenstaden, France). Both drugs were diluted freshly to 90 and 12 ng/ml, respectively.

Chemicals Reagents (Sigma-Aldrich) (unless otherwise stated), Vemurafenib (Roche, Paris, France) and U0126 SelleckChem (Euromedex, Souffelweyersheim, France).

All animal experiments were performed in accordance with E.U. directive 2010/63 (regional animal ethics committee of Gothenburg approval #287/289-12 and #36-2014). An aliquot of dispersed patient cells was mixed with an equal volume of Matrigel and injected subcutaneously into the flanks of immunocompromised, non-obese severe combined immune deficient interleukin-2 chain receptor γ knockout mice (NOG mice; Taconic, Denmark) to form xenografts. First-passage PDXes were passaged twice until they achieved 80-100 mm³ in the treatment phase. The mice were treated 5 days per week for a minimum of three weeks with 0.3 mg/kg trametinib (Selleck Chem) twice daily, 120 mg/kg vemurafenib (Zelboraf, a Roche product purchased at the hospital pharmacy) twice daily or both trametinib/vemurafenib twice daily. Once a week, tumor sizes were measured with a caliper. Before treatment, ten days after the start of treatment and at the end of the experiment, blood samples were drawn from the hind leg vein (vena saphena). Plasma levels of human S100B were measured with an ELISA kit (Abnova, Taiwan).

One hundred and eighty treatment‐naïve or pretreated patients with BRAF‐mutant, metastatic melanoma of unresectable stage III or IV (American Joint Committee on Cancer [AJCC; 10]) who had progressed on treatment with single‐agent dabrafenib (Novartis, Nürnberg, Germany) or vemurafenib (Roche, Germany, Grenzach‐Wyhlen) from February 2010 to April 2015 were included in our multicenter retrospective data analysis. These consisted of patients with primary BRAFi resistance as well as patients who first responded to treatment and developed secondary resistance. The study was approved by the Ethics Committee, Hannover Medical School, Hannover, Germany.

Cobimetinib (provided by Hoffmann-La Roche) is an oral selective inhibitor of the MEK pathway. Vemurafenib (provided by Hoffmann-La Roche) is a low-molecular-weight inhibitor of BRAF serine–threonine kinase. Atezolizumab (provided by Hoffmann-La Roche) is an anti-PD-L1 antibody.
The study will include three possible treatments: (1) Vemurafenib 960 mg twice daily per os from day 1 to 42; (2) Vemurafenib 720 mg twice daily per os from day 1 to 42; (3) Cobimetinib 60 mg once daily per os from day 1 to 21 and from day 29 to 42; Cobimetinib should not be taken on days 22–28; and (4) Atezolizumab 840 mg intravenous for two cycles (days 22 and 43).
Patients will be randomized to three different arms: A) BRAF-mutated patients will receive (1) + (3) for 6 weeks; B) BRAF-mutated patients will receive (2) + (3) + (4) for 6 weeks; C) BRAF WT patients will receive (3) + (4) for 6 weeks.
All patients will also receive Atezolizumab 1200 mg intravenous every 3 weeks for 17 cycles after surgery and after a second screening period (up to 6 weeks).