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Background <p>The emergence of Androgen Receptor variants (AR-Vs) during hormonal treatment is believed to be one of the key mechanisms of resistance. AR-Vs lack the ligand-binding domain and function as constitutively active transcription factors, rendering AR-targeted endocrine-based therapeutics ineffective in tumors that express AR-Vs. The goal of this study was to develop a targeted multiplex assay to identify known and unexplored AR-V proteins and help guide the stratification of PCa patients based on their AR-V protein profiles.</p> Methods <p>A targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC–MS/MS) was developed to quantify total AR proteins using the AR N-terminal domain (AR-Nterm), a well-characterized AR splice variant AR-V7 (AR-V7), a putative, previously identified but uncharacterized protein isoform (AR-V12) and isoforms that code for an exon 3 duplicated junction seen in AR-V2 and AR- full length with two copies of exon 3. Specimens included three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative PCa cell lines (PC3, DU145), and a patient-derived xenograft (PDX) model of CRPC. Endogenous target peptides were quantified according to tier-2 targeted mass spectrometry “fit-for-purpose” guidelines to ensure analytical rigor.</p> Results <p>Analytical performance was characterized across a linearity range of 4.15 fmol to 4000 fmol followed by evaluations of precision, accuracy, selectivity, stability, ion suppression and repeatability studies. The overall coefficient of variation (CV) was &lt; 20% across seven independent validation experiments. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R<sup>2</sup> &gt; 0.95). Four targets, including AR-Nterm and 3 AR-variants, were identified and quantified in PCa cell lines and in a PDX pre-clinical model.</p> Conclusion <p>This study establishes a robust, validated MRM targeted proteomics assay capable of reproducibly detecting and quantifying both known and previously uncharacterized AR-Vs at the protein level  . The assay demonstrated &lt; 20% analytical variation at the lower limit of quantification of 4.25 femtomole (fmol) for AR-Nterm and 8.25 fmol for AR-V7, AR Exon3-Dup/AR-V2 and AR-V12 and, showed robust analytical performance in preclinical models and is ready for further evaluation in clinical specimens. This provides a clinically applicable platform for the comprehensive profiling of AR-V proteins in human subjects, enabling improved stratification of patients with CRPC and supporting precision therapeutic strategies.</p>

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Targeted proteomics for the detection of androgen receptor variants in preclinical models of castration-resistant prostate cancer

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月12日

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  • Zoi E. Sychev,
  • Gabrianne Larson,
  • Megan L. Ludwig,
  • Seiei Shiba,
  • Kiel Tietz,
  • Joshua W. Russo,
  • Steven Balk,
  • Eva Corey,
  • Stephen Plymate,
  • Emmanuel S. Antonarakis,
  • Scott M. Dehm,
  • Jesse C. Seegmiller,
  • Justin M. Drake

摘要

Background

The emergence of Androgen Receptor variants (AR-Vs) during hormonal treatment is believed to be one of the key mechanisms of resistance. AR-Vs lack the ligand-binding domain and function as constitutively active transcription factors, rendering AR-targeted endocrine-based therapeutics ineffective in tumors that express AR-Vs. The goal of this study was to develop a targeted multiplex assay to identify known and unexplored AR-V proteins and help guide the stratification of PCa patients based on their AR-V protein profiles.

Methods

A targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC–MS/MS) was developed to quantify total AR proteins using the AR N-terminal domain (AR-Nterm), a well-characterized AR splice variant AR-V7 (AR-V7), a putative, previously identified but uncharacterized protein isoform (AR-V12) and isoforms that code for an exon 3 duplicated junction seen in AR-V2 and AR- full length with two copies of exon 3. Specimens included three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative PCa cell lines (PC3, DU145), and a patient-derived xenograft (PDX) model of CRPC. Endogenous target peptides were quantified according to tier-2 targeted mass spectrometry “fit-for-purpose” guidelines to ensure analytical rigor.

Results

Analytical performance was characterized across a linearity range of 4.15 fmol to 4000 fmol followed by evaluations of precision, accuracy, selectivity, stability, ion suppression and repeatability studies. The overall coefficient of variation (CV) was < 20% across seven independent validation experiments. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R2 > 0.95). Four targets, including AR-Nterm and 3 AR-variants, were identified and quantified in PCa cell lines and in a PDX pre-clinical model.

Conclusion

This study establishes a robust, validated MRM targeted proteomics assay capable of reproducibly detecting and quantifying both known and previously uncharacterized AR-Vs at the protein level  . The assay demonstrated < 20% analytical variation at the lower limit of quantification of 4.25 femtomole (fmol) for AR-Nterm and 8.25 fmol for AR-V7, AR Exon3-Dup/AR-V2 and AR-V12 and, showed robust analytical performance in preclinical models and is ready for further evaluation in clinical specimens. This provides a clinically applicable platform for the comprehensive profiling of AR-V proteins in human subjects, enabling improved stratification of patients with CRPC and supporting precision therapeutic strategies.