Small RNAs in Seminal Plasma as Novel Biomarkers for Germ Cell Tumors

SIMPLE SUMMARY: Testicular cancer is the most common cancer among young men. It is rarely diagnosed at early stages, being only detected with a highly invasive procedure that presents notable side-effects. Circulating small RNAs have recently been identified as testicular tumor markers, but are unable to diagnose testicular cancer at an early pre-invasive stage. So far, studies have been limited to microRNAs, with other small RNAs remaining unexplored as likely biomarkers. By sequencing all small RNAs in semen samples from men with different stages of testicular cancer and healthy men, we identify signatures predictive of cancer, even at an early stage. Thus, our study provides great potential for non-invasive early diagnosis of testicular cancer. Extensive biological variance in small RNA levels across samples, together with small sample sizes, limit the power to detect single small RNA markers. Hence, larger studies are needed to confirm our findings and deduce their full diagnostic capacity. ABSTRACT: Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor lesions, germ cell neoplasia in situ (GCNIS). Small RNAs isolated from SP from men with TGCTs (n = 18), GCNIS-only (n = 5), and controls (n = 25) were sequenced. SP from men with TGCT/GCNIS (n = 37) and controls (n = 22) were used for validation by RT-qPCR. In general, piRNAs were found at lower levels in SP from men with TGCTs. Ten small RNAs were found at significantly (q-value < 0.05) different levels in SP from men with TGCT/GCNIS than controls. Random forests classification identified sets of small RNAs that could detect either TGCT/GCNIS or GCNIS-only with an area under the curve of 0.98 and 1 in ROC analyses, respectively. RT-qPCR validated hsa-miR-6782-5p to be present at 2.3-fold lower levels (p = 0.02) in the SP from men with TGCTs compared with controls. Small RNAs in SP show potential as novel biomarkers for diagnosing men with TGCT/GCNIS but validation in larger cohorts is needed.