The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAFV600E metastatic melanoma.,However, resistance to such compounds represents a formidable problem.,Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases.,Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway.,In addition to three independent amplifications and an aberrant form of BRAFV600E, we identified a new activating insertion in MEK1.,This MEK1T55delinsRT mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas.,Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis.,This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient.,In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting.,Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient’s melanoma.
Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer.,However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells.,Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation.,Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II-restricted recognition of tumors by tumor-reactive CD4+ T cells.,Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma.,These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation.