Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

Journal of Translational Medicine

Alagarsamy Srinivasan³  Albert Dobi³  Gyorgy Petrovics³  Shiv Srivastava³  David G McLeod⁴  Sudhir Srivastava⁵  Jacob Kagan⁵  Sreedatta Banerjee³  Wei Huang³  Ahmed A Mohamed³  Wusheng Yan³  Shyh-Han Tan³  Anshu Rastogi³  David G Camp²  Tao Liu²  Karin D Rodland²  Wei-Jun Qian²  Richard D Smith²  Yuqian Gao²  Thomas L Fillmore¹  Chaochao Wu²  Tujin Shi²  Athena A Schepmoes²  Jintang He² 

[1] Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA;Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA;Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA;Urology Service, Walter Reed National Military Medical Center, Bethesda, MD, USA;Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, USA

关键词: Diagnosis;    Prostate cancer;    ELISA;    Mass spectrometry;    MRM;    PRISM-SRM;    Biomarker;    Quantification;    ERG;   

Others  :  1137567
DOI  :  10.1186/s12967-015-0418-z

 received in 2014-11-21, accepted in 2015-01-23,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 He et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Albertson DG, Collins C, McCormick F, Gray JW: Chromosome aberrations in solid tumors. Nat Genet 2003, 34(4):369-76.
• [2]Rao VN, Papas TS, Reddy ES: Erg, a human ets-related gene on chromosome 21: alternative splicing, polyadenylation, and translation. Science 1987, 237(4815):635-9.
• [3]Reddy ES, Rao VN, Papas TS: The erg gene: a human gene related to the ets oncogene. Proc Natl Acad Sci U S A 1987, 84(17):6131-5.
• [4]Lin B, Ferguson C, White JT, Wang S, Vessella R, True LD, et al.: Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2. Cancer Res 1999, 59(17):4180-4.
• [5]Ernst T, Hergenhahn M, Kenzelmann M, Cohen CD, Bonrouhi M, Weninger A, et al.: Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue. Am J Pathol 2002, 160(6):2169-80.
• [6]Kumar-Sinha C, Tomlins SA, Chinnaiyan AM: Recurrent gene fusions in prostate cancer. Nat Rev Cancer 2008, 8(7):497-511.
• [7]Mani RS, Iyer MK, Cao Q, Brenner JC, Wang L, Ghosh A, et al.: TMPRSS2-ERG-mediated feed-forward regulation of wild-type ERG in human prostate cancers. Cancer Res 2011, 71(16):5387-92.
• [8]Petrovics G, Liu A, Shaheduzzaman S, Furusato B, Sun C, Chen Y, et al.: Frequent overexpression of ETS-related gene-1 (ERG1) in prostate cancer transcriptome. Oncogene 2005, 24(23):3847-52.
• [9]Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, et al.: Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005, 310(5748):644-8.
• [10]Vanaja DK, Cheville JC, Iturria SJ, Young CY: Transcriptional silencing of zinc finger protein 185 identified by expression profiling is associated with prostate cancer progression. Cancer Res 2003, 63(14):3877-82.
• [11]Zammarchi F, Boutsalis G, Cartegni L: 5' UTR control of native ERG and of Tmprss2:ERG variants activity in prostate cancer. PLoS One 2013, 8(3):e49721.
• [12]Rubin MA, Maher CA, Chinnaiyan AM: Common gene rearrangements in prostate cancer. J Clin Oncol 2011, 29(27):3659-68.
• [13]Clark J, Merson S, Jhavar S, Flohr P, Edwards S, Foster CS, et al.: Diversity of TMPRSS2-ERG fusion transcripts in the human prostate. Oncogene 2007, 26(18):2667-73.
• [14]Sementchenko VI, Schweinfest CW, Papas TS, Watson DK: ETS2 function is required to maintain the transformed state of human prostate cancer cells. Oncogene 1998, 17(22):2883-8.
• [15]Rahim S, Uren A: Emergence of ETS transcription factors as diagnostic tools and therapeutic targets in prostate cancer. Am J Transl Res 2013, 5(3):254-68.
• [16]Prensner JR, Rubin MA, Wei JT, Chinnaiyan AM: Beyond PSA: the next generation of prostate cancer biomarkers. Sci Transl Med 2012, 4(127):127rv123.
• [17]Tomlins SA, Aubin SM, Siddiqui J, Lonigro RJ, Sefton-Miller L, Miick S, et al.: Urine TMPRSS2:ERG fusion transcript stratifies prostate cancer risk in men with elevated serum PSA. Sci Transl Med 2011, 3(94):94ra72.
• [18]Furusato B, Tan SH, Young D, Dobi A, Sun C, Mohamed AA, et al.: ERG oncoprotein expression in prostate cancer: clonal progression of ERG-positive tumor cells and potential for ERG-based stratification. Prostate Cancer Prostatic Dis 2010, 13(3):228-37.
• [19]Park K, Tomlins SA, Mudaliar KM, Chiu YL, Esgueva R, Mehra R, et al.: Antibody-based detection of ERG rearrangement-positive prostate cancer. Neoplasia 2010, 12(7):590-8.
• [20]Rosen P, Pfister D, Young D, Petrovics G, Chen Y, Cullen J, et al.: Differences in frequency of ERG oncoprotein expression between index tumors of Caucasian and African American patients with prostate cancer. Urology 2012, 80(4):749-53.
• [21]Rastogi A, Tan SH, Banerjee S, Sharad S, Kagan J, Srivastava S, et al.: ERG monoclonal antibody in the diagnosis and biological stratification of prostate cancer: delineation of minimal epitope, critical residues for binding, and molecular basis of specificity. Monoclon Antib Immunodiagn Immunother 2014, 33(4):201-8.
• [22]Braun M, Goltz D, Shaikhibrahim Z, Vogel W, Bohm D, Scheble V, et al.: ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer–a comparative study of two monoclonal antibodies. Prostate Cancer Prostatic Dis 2012, 15(2):165-9.
• [23]Farrell J, Young D, Chen Y, Cullen J, Rosner IL, Kagan J, et al.: Predominance of ERG-negative high-grade prostate cancers in African American men. Mol Clin Oncol 2014, 2(6):982-6.
• [24]Magi-Galluzzi C, Tsusuki T, Elson P, Simmerman K, LaFargue C, Esgueva R, et al.: TMPRSS2-ERG gene fusion prevalence and class are significantly different in prostate cancer of Caucasian. Afr Am Japan Patients Prostate 2011, 71(5):489-97.
• [25]Rawal S, Young D, Williams M, Colombo M, Krishnappa R, Petrovics G, et al.: Low frequency of the ERG oncogene alterations in prostate cancer patients from India. J Cancer 2013, 4(6):468-72.
• [26]Mao X, Yu Y, Boyd LK, Ren G, Lin D, Chaplin T, et al.: Distinct genomic alterations in prostate cancers in Chinese and Western populations suggest alternative pathways of prostate carcinogenesis. Cancer Res 2010, 70(13):5207-12.
• [27]Shi T, Su D, Liu T, Tang K, Camp DG 2nd, Qian WJ, et al.: Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics. Proteomics 2012, 12(8):1074-92.
• [28]Lange V, Picotti P, Domon B, Aebersold R: Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 2008, 4:222.
• [29]Picotti P, Aebersold R: Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat Meth 2012, 9(6):555-66.
• [30]Shi T, Fillmore TL, Sun X, Zhao R, Schepmoes AA, Hossain M, et al.: Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum. Proc Natl Acad Sci U S A 2012, 109(38):15395-400.
• [31]Shi T, Qian WJ: Antibody-free PRISM-SRM for multiplexed protein quantification: is this the new competition for immunoassays in bioanalysis? Bioanalysis 2013, 5(3):267-9.
• [32]Shi T, Sun X, Gao Y, Fillmore TL, Schepmoes AA, Zhao R, et al.: Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion. J Proteome Res 2013, 12(7):3353-61.
• [33]He J, Sun X, Shi T, Schepmoes AA, Fillmore TL, Petyuk VA, et al.: Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer. Mol Oncol 2014, 8(7):1169-80.
• [34]MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al.: Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 2010, 26(7):966-8.
• [35]Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, et al.: Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 2008, 26(3):317-25.
• [36]Reis PP, Waldron L, Goswami RS, Xu W, Xuan Y, Perez-Ordonez B, et al.: mRNA transcript quantification in archival samples using multiplexed, color-coded probes. BMC Biotechnol 2011, 11:46. BioMed Central Full Text
• [37]Rigau M, Olivan M, Garcia M, Sequeiros T, Montes M, Colas E, et al.: The present and future of prostate cancer urine biomarkers. Int J Mol Sci 2013, 14(6):12620-49.
• [38]Leyten GH, Hessels D, Jannink SA, Smit FP, de Jong H, Cornel EB, et al.: Prospective multicentre evaluation of PCA3 and TMPRSS2-ERG gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer. Eur Urol 2014, 65(3):534-42.