Long-read RNA sequencing of archival tissues reveals novel genes and transcripts associated with clear cell renal cell carcinoma recurrence and immune evasion

The use of long-read direct RNA sequencing (DRS) and PCR cDNA sequencing (PCS) in clinical oncology remains limited, with no direct comparison between the two methods. We used DRS and PCS to study clear cell renal cell carcinoma (ccRCC), focussing on new transcript and gene discovery. Twelve primary ccRCC archival tumors, six from patients who went on to relapse, were analysed. Results were validated in an independent cohort of twenty patients by qRT-PCR and compared to DRS analysis of RCC4 cells. In archival clinical samples and due to long-term storage, average read length was lower (400-500nt) than that achieved through DRS of RCC4 cells (>1100nt). Still, deconvolution analysis showed a loss of immune infiltrate in primary tumors of patients who relapse as reported by others. Differentially expressed genes in patients who went on to relapse were determined with good overlap between DRS and PCS, identifying LINC04216 and the T cell exhaustion marker TOX as novel candidate recurrence-associated genes. Novel transcript analysis revealed over 10,000 candidate novel transcripts detected by both methods and in ccRCC cells in vitro, including a novel CD274 ( PD-L1 ) transcript encoding for the soluble version of the protein with a longer 3' UTR and lower stability than the annotated transcript. Both methods identified 414 novel genes, also detected in RCC4 cells, including a novel noncoding gene over-expressed in patients who relapse. Overall, we showcase use of PCS and DRS in archival tumor samples to uncover unmapped features of cancer transcriptomes, linked to disease progression and immune evasion.