Long-read RNA sequencing redefines the clear cell renal cell carcinoma transcriptome and reveals novel genes and transcripts associated with disease recurrence and immune evasion

Background Long-read direct RNA sequencing (DRS) and PCR cDNA sequencing (PCS) of tumour samples could lead to discovery of novel transcript isoforms, novel genes, and transcriptomic co-dependencies missed by conventional short-read sequencing. However, only a handful of reports using DRS or PCS in cancer exist with no direct comparison between the two methods. Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Following primary tumour resection approximately 30% of patients experience disease recurrence. Long-read RNA sequencing has not been applied to kidney cancer. Methods 12 primary ccRCC archival tumours (discovery cohort), 6 from patients who went on to relapse, were analysed by Oxford Nanopore DRS and PCS. Results were validated in an independent cohort of 20 patients and compared to DRS analysis of RCC4 cells. Results DRS and PCS were successfully performed achieving high read length, with PCS achieving higher sequencing depth. Differentially expressed gene sets in patients who went on to relapse were determined with good overlap between DRS and PCS. Deconvolution analysis showed a loss of immune infiltrate in primary tumours of patients who relapse and revealed the CD8+ T cell exhaustion marker TOX as a novel recurrence-associated gene. Notably, novel transcript analysis revealed more than 10,000 uncharacterised candidate novel transcripts detected by both methods and in a ccRCC cell line in vitro . This allowed the definition of the full exonic structure of ccRCC-associated splice variants, including variants of MVK and HPCAL1 . Remarkably, this also revealed a novel s PD-L1 transcript encoding for the soluble version of the protein with a longer 3’UTR and lower stability in ccRCC cells than the annotated transcript. Levels of sPD-L1 transcripts are unchanged in primary tumours that go on to relapse, whereas membrane PD-L1 shows a trend towards down-regulation. Finally, both methods identified 414 novel genes, also detected in ccRCC cells in vitro , including a novel non-coding gene over-expressed in patients who relapse. Conclusions PCS and DRS can be used in tumour samples to uncover substantial yet unmapped features underpinning the plasticity and instability of cancer transcriptomes which are linked to disease progression and immune evasion. ### Competing Interest Statement EAS and DJT are employees of and stock option holders in Oxford Nanopore Technologies. NSV has received grants, speaker honoraria and/or advisory fees from Bristol Myers Squibb, Ipsen, EUSA pharma, Eisai and Pfizer, all outside the submitted work. The remaining authors declare no competing interests. ### Funding Statement This work was funded the Biotechnology and Biological Sciences Research Council (BBSRC) White Rose doctoral training partnership (BB/J014443/1) through an Industrial Cooperative Awards in Science and Engineering (iCASE) studentship supported by Oxford Nanopore Technologies. Additional support was provided by the Hull York Medical School and Oxford Nanopore Technologies. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Yorkshire & The Humber Leeds East Research Ethics Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Material: The sequencing results are available as raw reads and the gene counts tables are available at gene expression omnibus as below: PCS GSE242204, DRS GSE241932, RCC4 DRS: GSE242084. Code: The workflows for reads alignment are available at and . Workflow for differential gene expression identification is available at ().