Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons.,We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice.,All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers.,Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.,Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway.,Anti-oxidants promoted distant metastasis in NSG mice.,Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice.,Oxidative stress thus limits distant metastasis by melanoma cells in vivo.
The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma.,Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response.,Therefore, the precise identification of the underlying somatic mutations is essential.,Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.,Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples.,DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction.,BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC).,All mutations were independently reassessed by Sanger sequencing.,Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).,There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%).,All mutations down to 6.6% allele frequency could be detected with 100% specificity.,In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity.,The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib.,NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity.,IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.,Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.