Radiation enhances melanoma response to immunotherapeutic and synergizes with benzodiazepines to promote improved anti-tumor activity

The RNA-Seq data of 17 brain metastatic samples short-read sequences were aligned to the hg19 human reference genome using STAR (v 2.4.1a). featureCounts was used to count the reads of the mapped bam files. SAMseq was used to conduct differential expression analysis among the two treatment groups. SAMseq was utilized as it accounts for potential correlation in expression among genes and its permutation-based testing method was deemed more appropriate for a smaller sample size. After identifying the differentially expressed genes (FDR cut-off, 0.05), expression levels were normalized with the samr R package before being log2 transformed. This data was utilized to generate a heatmap with the patients sorted with supervised clustering based upon their immunotherapy/radiation groups and the gene dendrogram was created with Pearson's Correlation Coefficient and with the Ward D2 linkage method. Differentially expressed genes (FDR<0.05[KD1] ) were subjected to pathway analysis using MetaCore. Overall design: Differential expression analysis across two different patient groups based upon their timing of immunotherapy and radiation therapy.