TY - JOUR
T1 - Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods
AU - He, Jintang
AU - Schepmoes, Athena A.
AU - Shi, Tujin
AU - Wu, Chaochao
AU - Fillmore, Thomas L.
AU - Gao, Yuqian
AU - Smith, Richard D.
AU - Qian, Wei Jun
AU - Rodland, Karin D.
AU - Liu, Tao
AU - Camp, David G.
AU - Rastogi, Anshu
AU - Tan, Shyh Han
AU - Yan, Wusheng
AU - Mohamed, Ahmed A.
AU - Huang, Wei
AU - Banerjee, Sreedatta
AU - Kagan, Jacob
AU - Srivastava, Sudhir
AU - McLeod, David G.
AU - Srivastava, Shiv
AU - Petrovics, Gyorgy
AU - Dobi, Albert
AU - Srinivasan, Alagarsamy
N1 - Publisher Copyright:
© 2015 He et al.
PY - 2015/2/12
Y1 - 2015/2/12
N2 - Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: An antibody-free assay for ERG3 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). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples. Results: Recombinant ERG3 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. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostic and prognostic assays for prostate cancer given their sensitivity, specificity, and reproducibility.
AB - Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: An antibody-free assay for ERG3 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). Enzyme-linked immunosorbent assay (ELISA), western blot, NanoString, and qRT-PCR were also used in the analysis of these samples. Results: Recombinant ERG3 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. The sensitivity of the in-house ELISA was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostic and prognostic assays for prostate cancer given their sensitivity, specificity, and reproducibility.
KW - Biomarker
KW - Diagnosis
KW - ELISA
KW - ERG
KW - MRM
KW - Mass spectrometry
KW - PRISM-SRM
KW - Prostate cancer
KW - Quantification
UR - http://www.scopus.com/inward/record.url?scp=84924328732&partnerID=8YFLogxK
U2 - 10.1186/s12967-015-0418-z
DO - 10.1186/s12967-015-0418-z
M3 - Article
C2 - 25889691
AN - SCOPUS:84924328732
SN - 1479-5876
VL - 13
JO - Journal of Translational Medicine
JF - Journal of Translational Medicine
IS - 1
M1 - 54
ER -