Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity.,The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy.,Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab).,Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS).,Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type.,Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies.,We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy.,Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease.,Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.
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.