Abstract 2266: Detection of androgen receptor splice variants from clinical sequencing
Authors: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun
Journal: Cancer Research (2024)

Abstract Background: Androgen receptor splice variant-7 (AR-v7) has been widely studied as a biomarker of resistance to AR-targeted therapy in castration-resistant prostate cancer (CRPC). Various methods have been used to detect AR-v7 in CRPC specimens, including next generation sequencing (NGS) technology. However, NGS technologies applied in clinical sequencing have focused on using blood or fresh-frozen CRPC tissue specimens. There is not yet an AR-v7 detection system tailored to formalin-fixed paraffin-embedded (FFPE) tissue specimens commonly used for clinical sequencing in hospitals. Methods: In this respect, we propose a novel approach to identify AR-v7 in targeted RNA sequencing (RNA-seq) data derived from FFPE specimens. This approach can identify, in addition to AR-v7, all constitutively active AR splice variants (AR-Vs) that are co-expressed with AR-v7. In short, our two-step approach first gathers soft-clipped or divided reads that are adjacent to the splicing sites of AR-Vs and then yields the number of splitting reads that support the existence of AR-Vs. Next, the algorithm selects the paired-end reads whose one side and the other side are mapped to the preceding and following exons, respectively. The final number of spanning reads are calculated following to the removal of low-quality reads. Results: We validated the proposed approach using two large-scale, independent RNA-seq datasets of prostate cancer (PC) samples: 111 samples from Seoul National University Hospital (SNUH) and 558 samples from The Cancer Genome Atlas (TCGA) dataset. The majority of both datasets were localized PC tumors, especially in the SNUH dataset, where localized PC accounted for 91% of the total. Overall, our approach successfully identified samples with putative AR-Vs, including AR-v7, AR-v3, and AR-v9. Statistical analyses of 111 SNUH PC patients suggested potential detection threshold in clinical sequencing settings (at least 200 split reads). In the SNUH dataset, the AR-v7 positive group, which included 5 patients (5%) with values above the threshold, shared the following characteristics: higher AR-v7/full length AR expression levels, AR amplification and co-expression with AR-v3/AR-v9. AR-v7 positivity was experimentally validated by Sanger sequencing. The similar pattern was also found in the analysis of TCGA dataset. Conclusions: These results demonstrate that the detection system can successfully identify AR-v7 positivity not only for metastatic PC but also for patients with localized PC through clinical sequencing. We expect that the further in-depth analyses including larger samples and clinical outcomes can discover clinical applicability of AR-v7. Citation Format: Suhyun Hwangbo, Sungyoung Lee, Sheehyun Kim, Hongseok Yun. Detection of androgen receptor splice variants from clinical sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2266.