The impact of <i>PTEN</i> deletion, <i>TMPRSS2</i> : <i>ERG</i> fusion, and androgen receptor variant 7 (AR-V7) on resistance to novel hormone therapies (NHTs) in castrate-resistant prostate cancer.

e17036 Background: Despite improved treatment outcomes of prostate cancer (PC) from NHTs, acquired resistance via AR-dependent and/or AR-independent pathways remains a challenge. Here, we examined the role of AR-V7 expression, PTEN loss, and TMPRSS2:ERG fusion in PC progression in NHT-treated castrate-resistant prostate cancer (CRPC) patients. Methods: Whole exome sequencing and RNA-seq data were analysed for 48 CRPC patients with available treatment history who received the BostonGene TumorPortrait test in 2022–2024. Among them, 25 received long-term (&gt; 3 months) NHT (NHT+) and 23 did not (NHT-). Normalized AR-V7 levels expressed in splice junctions per million (SJPM; determined by STAR aligner) were used to stratify patients into the AR-V7+ and AR-V7- subgroups using a threshold of 0.05 SJPM. Tumor microenvironment (TME)-related gene signatures were analysed based on ssGSEA scores (Bagaev et al., 2021). Pathway activity assessment was performed with PROGENy. Mutation frequencies and copy number alterations (CNAs) were identified using strelka2 and sequenza, respectively. Results: We observed significantly higher (p=0.01, Mann–Whitney U test) AR-V7 expression in the NHT+ group. Namely, 68% (17/25) of NHT+ samples classified as AR-V7+. Among these AR-V7+ samples, 64% (11/17) presented with AR amplification, compared to 12.5% (1/8) of AR-V7- samples. TMPRSS2:ERG fusion was observed exclusively in AR-V7+ patients. Genomic events that activate the PI3K/Akt/mTOR pathway, such as PTEN loss and alterations in AKT1 and PIK3CA , prevailed in AR-V7- samples. Notably, while PTEN loss was detected in both AR-V7- and + samples (4 vs 6, respectively), it co-occurred with the TMPRSS2:ERG fusion in 5/6 of AR-V7+ samples. Thus, restoration of the AR transcriptome mediated by TMPRSS2:ERG in the absence of PTEN , previously observed only in murine models (Chen et al., 2014), was confirmed in human patients. Within the NHT+ group, 4/8 AR-V7- samples and 3/17 AR-V7+ samples exhibited the Immune-Enriched Non-Fibrotic TME, though this difference in TME composition did not reach statistical significance. Pathway analysis revealed PI3K/Akt/mTOR to be significantly upregulated in AR-V7- samples (p=0.002, t -test), while AR signaling was activated in AR-V7+ samples (p=0.013, t -test). Conclusions: Analysis of CRPC NHT+ patients confirmed two mechanisms of NHT resistance in CRPC. First is an AR-dependent pathway mediated by AR-V7 expression. Second is an AR-independent pathway driven by PI3k/Akt/mTOR signaling. Notably, while PTEN loss occurred in both mechanisms, the presence of TMPRSS2 : ERG fusion likely skewed the preference towards the AR-dependent pathway. Our findings highlight the importance of comprehensive biomarker evaluation for precise post-NHT treatment selection (e.g., PI3k/Akt inhibitors) for CRPC patients.