Genomic analyses of bilateral testicular germ cell cancers (BITCOIN study) from the Italian Germ Cell Cancer Group (IGG).

617 Background: Despite Testicular Germ Cell Tumors (TGCT) being the most common malignancy in young men aged 15 to 44 years, bilateral TGCT (bTGCT) accounts for 2–5% of TGCT cases. To date there are only small case reports exploring genomic aberrations in bTGCT, with no shared single nucleotide variants or structural chromosomal rearrangements identified in paired TGCTs, suggesting they develop independently. Methods: BITCOIN is an ambispective translational study that recruited 53 patients with bTGCT within 6 Italian Canters of Reference for TGCTs. We collected FFPE tissue specimens of both tumors (29 pts), or from one of those (14 pts) along with a blood sample when feasible (43 pts). Illumina Stranded Total RNA sequencing was performed to generate 100 bp-paired reads that were aligned to hg38 human genome using HISAT2. Raw gene-level counts, obtained with FeatureCounts algorithm, were then filtered for low expression, and normalized using TMM method for downstream differential expression analysis (DGE) via edgeR. Visualization of differentially expressed genes (FDR 1.5) and gene set enrichment (GSE) analysis were carried out with ggplot2 and clusterProfiler, respectively. Results: Overall, in this preliminary analysis, 57 samples were sequenced from 30 unique pts. Of these, 27/57 (47%, representing 19 unique pts) reached at least 30 million reads and >50% of reads assigned to features. Notably, mean age of these specimens was 8 years. We identified among the bTGCT most expressed genes, embryonic/stem-like TGCT signatures (i.e. NR6A1 and BCN2 ) together with epigenetic regulators ( KMT2C, ARID1B ) and lncRNA drivers ( MALAT1 , NEAT1 , FTX , XACT ). DGE analysis was performed on 8 pts with paired tumors and 11 pts for whom only one tumor sample was available, highlighting differentially expressed genes according to the site (right R vs left L). Among these, APOB was the main marker of L localization. Finally, GSE using the Hallmark gene sets revealed a mild enrichment of epithelial-mesenchymal transition and TNFA signaling via NFKB (NES > 0.5 and FDR < 0.01 for both) in the L side. Conclusions: To date this represents the largest study on bTGCT. In this preliminary analysis, we demonstrate the feasibility of RNAseq on archival FFPE tissues identifying possible genomic characteristics of bilateral cancers.