Analytical Platform Evaluation For Quantification Of Erg Oncoprotein In Prostate Cancer Using Protein And Mrna Detection Methods

Current molecular tests that aid in the diagnosis of prostate cancer (PCa) are based on the detection of PSA and PSA isoforms (blood), overexpression of PCA3 (urine), and overexpression of AMACR (tissue) as biomarkers. The discovery of cancer specific fusion transcripts, which are detected in over 50% of prostate cancers, revolutionized the field. As such, the cancer specific TMPRSS2-ERG gene fusion drives the overexpression of the ERG oncoprotein. Based on this, we hypothesized that ERG protein may serve as a valuable biomarker and for which quantification methods can be applied for the diagnosis of prostate cancer. An antibody-free assay for ERG protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment) for evaluation. Recombinant ERG protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine, presumably due to more effective loading of ERG protein and the reduced sample complexity. In comparison the detection limit of the in-house enzyme-linked immunosorbent assay was 30 pg of recombinant ERG protein and 10,000 VCaP cells, while Western blot analysis demonstrated the detection of 195 pg of recombinant ERG protein and 10,000 VCaP cells in these two sample types, respectively. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 250 VCaP cells spiked into female urine, in comparison to NanoString methodologies which detected 10,000 VCaP cells in the same sample. In summary the data presented here suggest that qRT-PCR and PRISM-SRM platforms are highly sensitive in detecting TMPRSS2-ERG transcripts and proteins, respectively. Compared to other RNA (without preamplifcation step) and protein detection technologies, the PRISM-SRM assay has several significant advantages: it is not affected by RNA stability, it does not rely on a specific antibody, and it is ideal for isoform-specific detection with high multiplexing capability. Therefore, PRISM-SRM assays can be adapted to detect ERG protein in cells present in clinical specimens (e.g., tissue and urine), and cell-free ERG protein present in the blood sera, providing an opportunity for their use in the clinical setting for detection of ERG in prostate cancer patients and for defining appropriate treatment strategies. Citation Format: Jintang He, Athena Schepmoes, Anshu Rastogi, Shyh-Han Tan, Wusheng Yan, Wei Huang, Sreedatta Banerjee, Tujin Shi, Chaochao Wu, Thomas Fillmore, Yuqian Gao, Jacob Kagan, Sudhir Srivastava, Richard Smith, Wei-Jun Qian, David McLeod, Gyorgy Petrovics, Albert Dobi, Alagarsamy Srinivasan, Shiv Srivastava, Karin Rodland, Tao Liu, David Camp. Analytical platform evaluation for quantification of ERG oncoprotein in prostate cancer using protein and mRNA detection methods. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1818. doi:10.1158/1538-7445.AM2015-1818