Background: Resistance to BRAF inhibitors is an emerging problem in the melanoma field. Strategies to prevent and overcome resistance are urgently required. Methods: The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot. The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture. Results: BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling. Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy. A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance. Conclusion: Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF -V600E-mutated melanomas.

Purpose: To determine safety and feasibility of adjuvant ipilimumab following resection of high-risk melanoma and to identify surrogate markers for benefit.Experimental Design: In this phase II trial, 75 patients with resected stage IIIc/IV melanoma received the CTLA-4 antibody ipilimumab every 6 to 8 weeks for 1 year. Eligible patients received further maintenance treatments. The first 25 patients received 3 mg/kg of ipilimumab, and an additional 50 patients received 10 mg/kg. HLA-A*0201þ patients received multipeptide immunizations in combination with ipilimumab. Leukapheresis was performed prior to and 6 months after initiation of treatment.Results: Median overall and relapse-free survivals were not reached after a median follow-up of 29.5 months. Significant immune-related adverse events were observed in 28 of 75 patients and were positively associated with longer relapse-free survival. Antigen-specific T cell responses to vaccine were variable, and vaccine combination was not associated with additional benefit. No effects on T regulatory cells were observed. Higher changes in Th-17 inducible frequency were a surrogate marker of freedom from relapse (P ¼ 0.047), and higher baseline C-reactive protein (CRP) levels were associated with freedom from relapse (P ¼ 0.035).Conclusions: Adjuvant ipilimumab following resection of melanoma at high risk for relapse appeared to be associated with improved outcome compared to historical reports. Significant immune-related adverse events were generally reversible and appeared to be associated with improved relapse-free survival. Although vaccination failed to induce a consistent in vitro measurable response, a higher change in Th-17 inducible cells and higher baseline CRP levels were positively associated with freedom from relapse.

BackgroundWe evaluated whether tumor infiltrating lymphocytes (TIL) could be expanded from surgically resected tumors from pancreatic cancer patients.MethodsTumors were resected from pancreatic cancer patients. Tumors were minced into fragments and cultured in media containing high dose interleukin-2 (IL-2) for up to 6 weeks. T cell phenotype, activation markers, and reactivity were measured.ResultsTIL expansion was measured in 19 patient samples. The majority of these TIL were CD4+ T cells and were highly activated. Purified CD8+ T cells produced IFN-γ in response to HLA-matched pancreatic tumor targets. PD-1 blockade and 4-1BB stimulation were demonstrated as effective strategies to improve effective TIL yield, including the production of tumor-reactive pancreatic TIL.ConclusionsTIL expanded from pancreatic tumors are functional and able to respond to pancreatic tumor associated antigens. PD-1 blockade, 41BB stimulation, and CD8+ T cell enrichment are effective strategies to improve TIL yield and tumor reactivity. These results support the development of adoptive cell therapy strategies using TIL for the treatment of pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-016-0164-7) contains supplementary material, which is available to authorized users.

BackgroundIpilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4+ and CD8+ T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.MethodsMicroarray analysis of flow-cytometry purified CD4+ and CD8+ T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.ResultsIpilimumab up-regulated Ki67 and ICOS on CD4+ and CD8+ cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8+ at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4+ and CD8+ cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES+CD8+, GranzymeB+EOMES+CD8+ and decreased Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67+CD8+ T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67+EOMES+CD8+ T cells were associated with relapse (p ≤ 0.001), and low Ki67+EOMES+CD4+ T cells were associated with irAE (p ≤ 0.008).ConclusionsUp-regulation of proliferation and activation signals in CD4+ and CD8+ T cells were pharmacodynamic markers for ipilimumab. Ki67+EOMES+CD8+ and Ki67+EOMES+CD4+T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.