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), focusing on new transcript and gene discovery. Twelve primary ccRCC archival tumors, six from patients who went on to relapse, were analyzed. Results were validated in an independent cohort of 20 patients by qRT-PCR and compared to DRS analysis of RCC4 cells. In archival clinical samples and due to the long-term storage, the average read length was lower (400–500 nt) than that achieved through DRS of RCC4 cells (>1100 nt). 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 overexpressed in patients who relapse. Overall, we showcase the use of PCS and DRS in archival tumor samples to uncover unmapped features of cancer transcriptomes, linked to disease progression and immune evasion.

中文翻译：

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档案组织的长读长 RNA 测序揭示了与透明细胞肾细胞癌复发和免疫逃逸相关的新基因和转录本


长读长直接 RNA 测序 （DRS） 和 PCR cDNA 测序 （PCS） 在临床肿瘤学中的使用仍然有限，两种方法之间没有直接比较。我们使用 DRS 和 PCS 研究透明细胞肾细胞癌 （ccRCC），重点是新的转录本和基因发现。分析了 12 例原发性 ccRCC 存档肿瘤，其中 6 例来自继续复发的患者。通过 qRT-PCR 在 20 名患者的独立队列中验证了结果，并与 RCC4 细胞的 DRS 分析进行了比较。在存档临床样本中，由于长期储存，平均读长 （400–500 nt） 低于通过 RCC4 细胞的 DRS 实现的读长 （>1100 nt）。尽管如此，反卷积分析显示，正如其他人报道的那样，复发患者的原发性肿瘤免疫浸润丢失。确定继续复发的患者的差异表达基因在 DRS 和 PCS 之间具有良好的重叠，将 LINC04216 和 T 细胞耗竭标志物 TOX 确定为新的候选复发相关基因。新的转录本分析揭示了两种方法和体外 ccRCC 细胞中检测到的超过 10,000 个候选新转录本，包括一种编码蛋白质可溶性版本的新型 CD274 （PD-L1） 转录本，具有更长的 3′ UTR 和比注释转录本更低的稳定性。两种方法都鉴定了 414 个新基因，也在 RCC4 细胞中检测到，包括一个在复发患者中过表达的新型非编码基因。总体而言，我们展示了在存档肿瘤样本中使用 PCS 和 DRS 来揭示癌症转录组的未映射特征，这些特征与疾病进展和免疫逃逸有关。