Background:Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-γ). We have explored whether the expression of IFNG, the gene encoding IFN-γ, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined.Methods:Total RNA from 17 NSCLC and 21 melanoma patients was analyzed by quantitative reverse transcription PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, IFNG, PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8+ T-cells were also evaluated. Progression-free survival and overall survival were estimated.Results:A total of 17 NSCLC patients received nivolumab and 21 melanoma patients received pembrolizumab. Progression-free survival with nivolumab was significantly longer in NSCLC patients with high versus low IFNG expression (5.1 months versus 2 months, p = 0.0124). Progression-free survival with pembrolizumab was significantly longer in melanoma patients with high versus low IFNG expression (5.0 months versus 1.9 months, p = 0.0099). Significantly longer overall survival was observed for melanoma patients with high versus low IFNG expression (not reached versus 10.2 months p = 0.0183). There was a trend for longer overall survival for NSCLC patients with high versus low IFNG expression.Conclusions:IFN-γ is an important marker for prediction of response to immune checkpoint blockade. Further research is warranted in order to validate whether IFNG is more accurate than PD-L1.

Highlights d T cell-induced IFN-g correlates the best to immune checkpoint blockade (ICB) therapy d Immune signatures increase in on-therapy patients regardless of response to therapy d ICB therapy decreases MYC and WNT signaling in patients with clinical response d IFN-g exposure of melanoma cells leads to a conserved transcriptome signature

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BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5'-nuclease PCR (Taqman) in the presence of a peptide-nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.

Immunotherapy of metastatic melanoma consists of various approaches leading to specific or non-specific immunomodulation. The use of FDA-approved interleukin (IL)-2 alone, in combination with interferon α, and/or with various chemotherapeutic agents (biochemotherapy) is associated with significant toxicity and poor efficacy that does not improve overall survival of 96% of patients. Many studies with allogeneic and autologous vaccines have demonstrated no clinical benefit, and some randomised trials even showed a detrimental effect in the vaccine arm. The ongoing effort to develop melanoma vaccines based on dendritic cells and peptides is driven by advances in understanding antigen presentation and processing, and by new techniques of vaccine preparation, stabilisation and delivery. Several agents that have shown promising activity in metastatic melanoma including IL-21 and monoclonal antibodies targeting cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) or CD137 are discussed. Recent advances of intratumour gene transfer technologies and adoptive immunotherapy, which represents a promising although technically challenging direction, are also discussed.