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Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients*[S]

  • Andrei P. Drabovich
    Correspondence
    To whom correspondence may be addressed:
    Affiliations
    ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5T 3L9 Canada

    §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, M5T 3L9 Canada

    ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9 Canada
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  • Punit Saraon
    Affiliations
    ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5T 3L9 Canada
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  • Mikalai Drabovich
    Affiliations
    ‖Independent researcher, Palo Alto, California 94303
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  • Theano D. Karakosta
    Affiliations
    §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, M5T 3L9 Canada
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  • Apostolos Dimitromanolakis
    Affiliations
    §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, M5T 3L9 Canada
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  • M.Eric Hyndman
    Affiliations
    **Department of Surgery, Division of Urology, Southern Alberta Institute of Urology, University of Calgary, Calgary, AB T2V 1P9, Canada
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  • Keith Jarvi
    Correspondence
    To whom correspondence may be addressed:
    Affiliations
    ‡‡Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9 Canada

    §§Department of Surgery, Division of Urology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, M5T 3L9 Canada
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  • Eleftherios P. Diamandis
    Correspondence
    To whom correspondence may be addressed:
    Affiliations
    ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5T 3L9 Canada

    §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, M5T 3L9 Canada

    ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9 Canada

    ‡‡Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9 Canada
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  • Author Footnotes
    * This work was supported by grants from the Canadian Institute of Health Research (#285693) to E.P.D., K.J., and A.P.D, and Prostate Cancer Canada (RS2015-01) to A.P.D.
    [S] This article contains supplemental material.
Open AccessPublished:June 27, 2019DOI:https://doi.org/10.1074/mcp.RA119.001612
      Seminal plasma, because of its proximity to prostate, is a promising fluid for biomarker discovery and noninvasive diagnostics. In this study, we investigated if seminal plasma proteins could increase diagnostic specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. To select 147 most promising biomarker candidates, we combined proteins identified through five independent experimental or data mining approaches: tissue transcriptomics, seminal plasma proteomics, cell line secretomics, tissue specificity, and androgen regulation. A rigorous biomarker development pipeline based on selected reaction monitoring assays was designed to evaluate the most promising candidates. As a result, we qualified 76, and verified 19 proteins in seminal plasma of 67 negative biopsy and 152 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which predicted prostate cancer on biopsy and outperformed age and serum Prostate-Specific Antigen (PSA). A machine-learning approach for data analysis provided improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by an in-house immunoassay, TGM4 protein was upregulated 3.7-fold (p = 0.006) and revealed AUC = 0.66 for detecting prostate cancer on biopsy for patients with serum PSA ≥4 ng/ml and age ≥50. Very low levels of TGM4 (120 pg/ml) were detected in blood serum. Collectively, our study demonstrated rigorous evaluation of one of the remaining and not well-explored prostate-specific proteins within the medium-abundance proteome of seminal plasma. Performance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.

      Graphical Abstract

      Prostate cancer (PCa)
      The abbreviations used are:
      PCa
      prostate cancer
      BH-adjusted t-test
      Benjamini-Hochberg-adjusted t-test
      CV
      coefficient of variation
      FDR
      false discovery rate
      FWHM
      full width at half maximum
      GS
      Gleason score
      ELISA
      enzyme-linked immunosorbent assay
      IQR
      interquartile range
      LFQ
      label-free quantification
      MWU
      Mann Whitney Unpaired t-test
      PSA
      prostate-specific antigen
      ROC AUC
      receiver operating characteristic area under the curve
      S/N
      signal-to-noise
      SP
      seminal plasma
      SRM
      selected reaction monitoring
      TGM4
      protein-glutamine gamma-glutamyltransferase 4
      XGBoost
      eXtreme Gradient Boosting algorithm.
      1The abbreviations used are:PCa
      prostate cancer
      BH-adjusted t-test
      Benjamini-Hochberg-adjusted t-test
      CV
      coefficient of variation
      FDR
      false discovery rate
      FWHM
      full width at half maximum
      GS
      Gleason score
      ELISA
      enzyme-linked immunosorbent assay
      IQR
      interquartile range
      LFQ
      label-free quantification
      MWU
      Mann Whitney Unpaired t-test
      PSA
      prostate-specific antigen
      ROC AUC
      receiver operating characteristic area under the curve
      S/N
      signal-to-noise
      SP
      seminal plasma
      SRM
      selected reaction monitoring
      TGM4
      protein-glutamine gamma-glutamyltransferase 4
      XGBoost
      eXtreme Gradient Boosting algorithm.
      is the most frequently diagnosed neoplasm and the third leading cause of cancer mortality in men. Its incidence rate has continued to increase rapidly during the past two decades, especially in men over the age of 50 years. Worldwide, close to 260,000 men die from PCa every year (
      • Jemal A.
      • Bray F.
      • Center M.M.
      • Ferlay J.
      • Ward E.
      • Forman D.
      Global cancer statistics.
      ). Our best current strategy to help PCa patients is early diagnosis and administration of the most appropriate therapy, including active surveillance only (
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      Radical prostatectomy versus observation for localized prostate cancer.
      ).
      The most commonly used PCa biomarker, prostate-specific antigen (PSA), is secreted by both normal prostate cells and PCa cells. There is no question that the introduction of PSA testing over the last two decades revolutionized the practice of urology. As a result of PSA screening, most men today with PCa are presented with localized disease and serum PSA values <10 ng/ml. However, the widespread use of PSA screening is not without controversy (
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      ,
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      ).
      Although PSA is an excellent biochemical marker, it has a number of important limitations, including lack of specificity and prognostic significance. PSA expression is prostate tissue-specific but not prostate cancer-specific. Serum PSA levels are increased in both PCa and in other non-malignant prostatic diseases, including benign prostatic hyperplasia and prostate inflammation. Because of the above limitations, clinicians currently perform on average four prostatic biopsies in order to detect one prostate cancer. PSA levels also do not predict the clinical significance or aggressiveness of PCa. Most men with PCa are destined to die of another condition before PCa becomes clinically significant (
      • Konety B.R.
      • Bird V.Y.
      • Deorah S.
      • Dahmoush L.
      Comparison of the incidence of latent prostate cancer detected at autopsy before and after the prostate specific antigen era.
      ). Lack of specificity and prognostic significance are two major limitations of PSA and constitute the major unmet needs in the current clinical diagnostics of PCa.
      There have been intense efforts for the identification of novel PCa biomarkers in blood or urine. Prostatic acid phosphatase has been discovered in the 1930s (
      • Gutman A.B.
      • Gutman E.B.
      An “Acid ” phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland.
      ) and for almost 50 years was used to indicate the success of hormonal therapy, whereas its clinical utility for diagnosis was limited. Apart from total PSA, which was characterized in the 1970s (
      • Wang M.C.
      • Valenzuela L.A.
      • Murphy G.P.
      • Chu T.M.
      Purification of a human prostate specific antigen.
      ,
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      • Inoue T.
      • Fukuyama T.
      [Some physico-chemical characteristics of “ -seminoprotein”, an antigenic component specific for human seminal plasma. Forensic immunological study of body fluids and secretion. VII].
      ) and approved in 1986 (
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      • Redwine E.
      Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate.
      ), current U.S. Food and Drug Administration (FDA)-approved tests include only three tests: Prostate Health Index, PCA3 and CellSearch (
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      • Ismail G.
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      ). Prostate Health Index is a multivariate index assay which includes immunoassay measurements of total PSA, free PSA and [-2]proPSA in blood serum, and is intended for diagnosis of PCa in men aged ≥ 50 years with total PSA 4.0–10 ng/ml and negative digital rectal examination (
      • Lughezzani G.
      • Lazzeri M.
      • Haese A.
      • McNicholas T.
      • de la Taille A.
      • Buffi N.M.
      • Fossati N.
      • Lista G.
      • Larcher A.
      • Abrate A.
      • Mistretta A.
      • Bini V.
      • Palou Redorta J.
      • Graefen M.
      • Guazzoni G.
      Multicenter European external validation of a prostate health index-based nomogram for predicting prostate cancer at extended biopsy.
      ). PCA3 test measures the relative amount of a non-coding RNA PCA3 in the post-digital rectal examination urine and is indicated to aid in the decision for repeat biopsy in men aged ≥ 50 years who have had previous negative prostate biopsies (
      • Bussemakers M.J.
      • van Bokhoven A.
      • Verhaegh G.W.
      • Smit F.P.
      • Karthaus H.F.
      • Schalken J.A.
      • Debruyne F.M.
      • Ru N.
      • Isaacs W.B.
      DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.
      ). The CellSearch detects circulating tumor cells of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+) in whole blood and has been only approved for monitoring patients with metastatic PCa (
      • Goldkorn A.
      • Ely B.
      • Quinn D.I.
      • Tangen C.M.
      • Fink L.M.
      • Xu T.
      • Twardowski P.
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      • Datar R.H.
      • Garzotto M.
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      • Lara Jr, P.
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      • Thompson Jr, I.M.
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      • Vogelzang N.J.
      Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer.
      ). Emerging tests yet to be approved for the clinical use include 4Kscore (immunoassay measurements of kallikrein-2 and total, free and intact forms of PSA in serum) (
      • Nordstrom T.
      • Vickers A.
      • Assel M.
      • Lilja H.
      • Grönberg H.
      • Eklund M.
      Comparison between the four-kallikrein panel and prostate health index for predicting prostate cancer.
      ), STHLM3 model (6 serum proteins, 232 single nucleotide polymorphisms and clinical parameters) (
      • Strom P.
      • Nordstrom T.
      • Aly M.
      • Egevad L.
      • Grönberg H.
      • Eklund M.
      The stockholm-3 model for prostate cancer detection: algorithm update, biomarker contribution, and reflex test potential.
      ) and ConfirmMDx (hypermethylation of GSTP1, APC and RASSF1 genes in biopsies) (
      • Van Neste L.
      • Partin A.W.
      • Stewart G.D.
      • Epstein J.I.
      • Harrison D.J.
      • Van Criekinge W.
      Risk score predicts high-grade prostate cancer in DNA-methylation positive, histopathologically negative biopsies.
      ). TMPRSS2-ERG fusion (
      • Hessels D.
      • Smit F.P.
      • Verhaegh G.W.
      • Witjes J.A.
      • Cornel E.B.
      • Schalken J.A.
      Detection of TMPRSS2-ERG fusion transcripts and prostate cancer antigen 3 in urinary sediments may improve diagnosis of prostate cancer.
      ) and SPINK1 mRNA (
      • Tomlins S.A.
      • Rhodes D.R.
      • Yu J.
      • Varambally S.
      • Mehra R.
      • Perner S.
      • Demichelis F.
      • Helgeson B.E.
      • Laxman B.
      • Morris D.S.
      • Cao Q.
      • Cao X.
      • Andrén O.
      • Fall K.
      • Johnson L.
      • Wei J.T.
      • Shah R.B.
      • Al-Ahmadie H.
      • Eastham J.A.
      • Eggener S.E.
      • Fine S.W.
      • Hotakainen K.
      • Stenman U.H.
      • Tsodikov A.
      • Gerald W.L.
      • Lilja H.
      • Reuter V.E.
      • Kantoff P.W.
      • Scardino P.T.
      • Rubin M.A.
      • Bjartell A.S.
      • Chinnaiyan A.M.
      The role of SPINK1 in ETS rearrangement-negative prostate cancers.
      ) measured in urine are also promising biomarkers. With diagnostic and prognostic AUCs (area under the receiver operating characteristic curve) in the range 0.66–0.70, these novel serum or urine biomarkers do not substantially outperform PSA.
      Although much of the work to identify and characterize PSA was originally carried out in seminal plasma (SP) (
      • Rao A.R.
      • Motiwala H.G.
      • Karim O.M.
      The discovery of prostate-specific antigen.
      ), there are only few comprehensive proteomic studies on identification of novel PCa biomarkers in SP (
      • Karakosta T.D.
      • Soosaipillai A.
      • Diamandis E.P.
      • Batruch I.
      • Drabovich A.P.
      Quantification of human kallikrein-related peptidases in biological fluids by multiplatform targeted mass spectrometry assays.
      ,
      • Neuhaus J.
      • Schiffer E.
      • von Wilcke P.
      • Bauer H.W.
      • Leung H.
      • Siwy J.
      • Ulrici W.
      • Paasch U.
      • Horn L.C.
      • Stolzenburg J.U.
      Seminal plasma as a source of prostate cancer peptide biomarker candidates for detection of indolent and advanced disease.
      ,
      • Flores-Morales A.
      • Iglesias-Gato D.
      Quantitative mass spectrometry-based proteomic profiling for precision medicine in prostate cancer.
      ) or expressed prostatic secretions (
      • Kim Y.
      • Ignatchenko V.
      • Yao C.Q.
      • Kalatskaya I.
      • Nyalwidhe J.O.
      • Lance R.S.
      • Gramolini A.O.
      • Troyer D.A.
      • Stein L.D.
      • Boutros P.C.
      • Medin J.A.
      • Semmes O.J.
      • Drake R.R.
      • Kislinger T.
      Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer.
      ,
      • Kim Y.
      • Jeon J.
      • Mejia S.
      • Yao C.Q.
      • Ignatchenko V.
      • Nyalwidhe J.O.
      • Gramolini A.O.
      • Lance R.S.
      • Troyer D.A.
      • Drake R.R.
      • Boutros P.C.
      • Semmes O.J.
      • Kislinger T.
      Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer.
      ). SP has total protein concentration of 40–60 mg/ml and a dynamic range of at least nine orders of magnitude, with semenogelin-1 (20 mg/ml) and interleukin-12 (10 pg/ml) being one of the most and least abundant proteins, respectively (
      • Drabovich A.P.
      • Saraon P.
      • Jarvi K.
      • Diamandis E.P.
      Seminal plasma as a diagnostic fluid for male reproductive system disorders.
      ). Nearly a quarter of molecular composition of SP is secreted by prostate (
      • Drabovich A.P.
      • Saraon P.
      • Jarvi K.
      • Diamandis E.P.
      Seminal plasma as a diagnostic fluid for male reproductive system disorders.
      ), with the rest produced by seminal vesicles, epididymis, testis, and periurethral glands (
      • Robert M.
      • Gagnon C.
      Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein.
      ,
      • Mann T.
      • Lutwak-Mann C.
      ). We previously completed extensive studies on the SP proteome and identified more than 3,000 proteins in SP of healthy men and patients with infertility (
      • Batruch I.
      • Lecker I.
      • Kagedan D.
      • Smith C.R.
      • Mullen B.J.
      • Grober E.
      • Lo K.C.
      • Diamandis E.P.
      • Jarvi K.A.
      Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system.
      ,
      • Batruch I.
      • Smith C.R.
      • Mullen B.J.
      • Grober E.
      • Lo K.C.
      • Diamandis E.P.
      • Jarvi K.A.
      Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility.
      ). Our work resulted in first-of-a-kind SP biomarkers for the differential diagnosis of male infertility (
      • Drabovich A.P.
      • Dimitromanolakis A.
      • Saraon P.
      • Soosaipillai A.
      • Batruch I.
      • Mullen B.
      • Jarvi K.
      • Diamandis E.P.
      Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.
      ,
      • Drabovich A.P.
      • Jarvi K.
      • Diamandis E.P.
      Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay.
      ,
      • Korbakis D.
      • Schiza C.
      • Brinc D.
      • Soosaipillai A.
      • Karakosta T.D.
      • Légaré C.
      • Sullivan R.
      • Mullen B.
      • Jarvi K.
      • Diamandis E.P.
      • Drabovich A.P.
      Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.
      ). Success with male infertility biomarkers motivated us to apply a similar strategy to PCa. We previously extensively validated by targeted proteomics and immunoassays the prostate-specific kallikrein-4 as a potential PCa biomarker in SP and blood serum (
      • Karakosta T.D.
      • Soosaipillai A.
      • Diamandis E.P.
      • Batruch I.
      • Drabovich A.P.
      Quantification of human kallikrein-related peptidases in biological fluids by multiplatform targeted mass spectrometry assays.
      ).
      In this work, we hypothesized that SP could contain novel PCa biomarkers within the medium-abundance range of concentrations (0.1–100 μg/ml) of the SP proteome. Some of these proteins have never been previously studied in the context of PCa. To select the most promising biomarker candidates, we combined proteins identified through five data mining and experimental -omics approaches, such as transcriptomics, proteomics, secretomics, tissue specificity and androgen regulation. Only those proteins which were previously identified in the SP proteome were considered as candidates and were qualified and verified by mass spectrometry-based selected reaction monitoring (SRM) assays. According to the fit-for-purpose approach to biomarker measurement assays (
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      ,
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      ), Tier 3 exploratory SRM assays were developed for the cost-effective qualification of dozens of candidates, followed by well-validated quantitative Tier 2 SRM assays for verification of a small number of candidates in hundreds of SP samples, followed by a high-precision Tier 1 immunoassay for orthogonal verification of a single biomarker in SP and blood serum samples. Powerful nonlinear machine-learning algorithms (
      • Chen T.
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      XGBoost: A Scalable Tree Boosting System.
      ) were utilized to evaluate potential multi-marker models for PCa diagnosis and prognosis. Our study was designed to simultaneously assess biomarker candidates for the two unmet clinical needs: (1) differentiation between PCa and negative biopsies, and (2) discrimination between low- and high-grade PCa. To our knowledge, this work is one of the largest and the most comprehensive proteomic studies on SP proteins and PCa.

      DISCUSSION

      Prostate cancer biomarkers studied up to date included genetic (
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      • Fraumeni Jr, J.F.
      • Hoover R.N.
      • Chanock S.J.
      • Hayes R.B.
      Variation in KLK genes, prostate-specific antigen and risk of prostate cancer.
      ) and epigenetic markers (
      • Van Neste L.
      • Partin A.W.
      • Stewart G.D.
      • Epstein J.I.
      • Harrison D.J.
      • Van Criekinge W.
      Risk score predicts high-grade prostate cancer in DNA-methylation positive, histopathologically negative biopsies.
      ), molecular markers such as RNA (
      • Bussemakers M.J.
      • van Bokhoven A.
      • Verhaegh G.W.
      • Smit F.P.
      • Karthaus H.F.
      • Schalken J.A.
      • Debruyne F.M.
      • Ru N.
      • Isaacs W.B.
      DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.
      ,
      • Hessels D.
      • Smit F.P.
      • Verhaegh G.W.
      • Witjes J.A.
      • Cornel E.B.
      • Schalken J.A.
      Detection of TMPRSS2-ERG fusion transcripts and prostate cancer antigen 3 in urinary sediments may improve diagnosis of prostate cancer.
      ,
      • Tomlins S.A.
      • Rhodes D.R.
      • Yu J.
      • Varambally S.
      • Mehra R.
      • Perner S.
      • Demichelis F.
      • Helgeson B.E.
      • Laxman B.
      • Morris D.S.
      • Cao Q.
      • Cao X.
      • Andrén O.
      • Fall K.
      • Johnson L.
      • Wei J.T.
      • Shah R.B.
      • Al-Ahmadie H.
      • Eastham J.A.
      • Eggener S.E.
      • Fine S.W.
      • Hotakainen K.
      • Stenman U.H.
      • Tsodikov A.
      • Gerald W.L.
      • Lilja H.
      • Reuter V.E.
      • Kantoff P.W.
      • Scardino P.T.
      • Rubin M.A.
      • Bjartell A.S.
      • Chinnaiyan A.M.
      The role of SPINK1 in ETS rearrangement-negative prostate cancers.
      ), proteins (
      • Lughezzani G.
      • Lazzeri M.
      • Haese A.
      • McNicholas T.
      • de la Taille A.
      • Buffi N.M.
      • Fossati N.
      • Lista G.
      • Larcher A.
      • Abrate A.
      • Mistretta A.
      • Bini V.
      • Palou Redorta J.
      • Graefen M.
      • Guazzoni G.
      Multicenter European external validation of a prostate health index-based nomogram for predicting prostate cancer at extended biopsy.
      ,
      • Nordstrom T.
      • Vickers A.
      • Assel M.
      • Lilja H.
      • Grönberg H.
      • Eklund M.
      Comparison between the four-kallikrein panel and prostate health index for predicting prostate cancer.
      ,
      • Strom P.
      • Nordstrom T.
      • Aly M.
      • Egevad L.
      • Grönberg H.
      • Eklund M.
      The stockholm-3 model for prostate cancer detection: algorithm update, biomarker contribution, and reflex test potential.
      ) and metabolites (
      • Wu C.L.
      • Jordan K.W.
      • Ratai E.M.
      • Sheng J.
      • Adkins C.B.
      • Defeo E.M.
      • Jenkins B.G.
      • Ying L.
      • McDougal W.S.
      • Cheng L.L.
      Metabolomic imaging for human prostate cancer detection.
      ), and circulating tumor cells (
      • Goldkorn A.
      • Ely B.
      • Quinn D.I.
      • Tangen C.M.
      • Fink L.M.
      • Xu T.
      • Twardowski P.
      • Van Veldhuizen P.J.
      • Agarwal N.
      • Carducci M.A.
      • Monk 3rd, J.P.
      • Datar R.H.
      • Garzotto M.
      • Mack P.C.
      • Lara Jr, P.
      • Higano C.S.
      • Hussain M.
      • Thompson Jr, I.M.
      • Cote R.J.
      • Vogelzang N.J.
      Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer.
      ). These markers were studied in serum, urine, prostatic secretions, prostate tissues, and cells found in urine. The reality of PCa diagnostics, however, turned out as very challenging. Novel clinical tests only marginally improved PSA performance. Prostate Health Index (Phi), an FDA-approved blood serum test for three PSA forms, was intended for use before the initial biopsy in men with elevated PSA (>4 ng/ml), and revealed AUC = 0.68 to predict the initial biopsy outcome (
      • Stephan C.
      • Jung K.
      • Semjonow A.
      • Schulze-Forster K.
      • Cammann H.
      • Hu X.
      • Meyer H.A.
      • Bögemann M.
      • Miller K.
      • Friedersdorff F.
      Comparative assessment of urinary prostate cancer antigen 3 and TMPRSS2:ERG gene fusion with the serum [-2]proprostate-specific antigen-based prostate health index for detection of prostate cancer.
      ). An FDA-approved PCA3 test in urine after prostate massage was intended to predict the need for secondary biopsy, and revealed AUC = 0.70 (
      • Stephan C.
      • Jung K.
      • Semjonow A.
      • Schulze-Forster K.
      • Cammann H.
      • Hu X.
      • Meyer H.A.
      • Bögemann M.
      • Miller K.
      • Friedersdorff F.
      Comparative assessment of urinary prostate cancer antigen 3 and TMPRSS2:ERG gene fusion with the serum [-2]proprostate-specific antigen-based prostate health index for detection of prostate cancer.
      ). Emerging 4KScore test predicted PCa on primary biopsies with AUC = 0.69 (
      • Nordstrom T.
      • Vickers A.
      • Assel M.
      • Lilja H.
      • Grönberg H.
      • Eklund M.
      Comparison between the four-kallikrein panel and prostate health index for predicting prostate cancer.
      ). A comprehensive STHLM3 model (6 blood serum proteins, 232 risk SNPs and 5 clinical parameters) facilitated detection of GS ≥7 with the cumulative AUC = 0.76, whereas individual markers had AUCs in the range of 0.59–0.67 (
      • Strom P.
      • Nordstrom T.
      • Aly M.
      • Egevad L.
      • Grönberg H.
      • Eklund M.
      The stockholm-3 model for prostate cancer detection: algorithm update, biomarker contribution, and reflex test potential.
      ). Most of these tests re-grouped different PSA proteoforms in combination with additional clinical characteristics, whereas novel unique protein biomarkers demonstrated only marginal performance (AUC = 0.59, 0.60 and 0.59 for KLK2, MSMB and MIC1, respectively). Non-protein biomarkers, such as hypermethylation of GSTP1, APC and RASSF1 genes in tissue biopsies (ConfirmMDx) revealed AUC = 0.66 to predict GS≥7 (
      • Van Neste L.
      • Partin A.W.
      • Stewart G.D.
      • Epstein J.I.
      • Harrison D.J.
      • Van Criekinge W.
      Risk score predicts high-grade prostate cancer in DNA-methylation positive, histopathologically negative biopsies.
      ).
      There were only few previous studies on PCa biomarkers in SP (
      • Karakosta T.D.
      • Soosaipillai A.
      • Diamandis E.P.
      • Batruch I.
      • Drabovich A.P.
      Quantification of human kallikrein-related peptidases in biological fluids by multiplatform targeted mass spectrometry assays.
      ,
      • Neuhaus J.
      • Schiffer E.
      • von Wilcke P.
      • Bauer H.W.
      • Leung H.
      • Siwy J.
      • Ulrici W.
      • Paasch U.
      • Horn L.C.
      • Stolzenburg J.U.
      Seminal plasma as a source of prostate cancer peptide biomarker candidates for detection of indolent and advanced disease.
      ,
      • Flores-Morales A.
      • Iglesias-Gato D.
      Quantitative mass spectrometry-based proteomic profiling for precision medicine in prostate cancer.
      ) or expressed prostatic secretions (
      • Kim Y.
      • Ignatchenko V.
      • Yao C.Q.
      • Kalatskaya I.
      • Nyalwidhe J.O.
      • Lance R.S.
      • Gramolini A.O.
      • Troyer D.A.
      • Stein L.D.
      • Boutros P.C.
      • Medin J.A.
      • Semmes O.J.
      • Drake R.R.
      • Kislinger T.
      Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer.
      ,
      • Kim Y.
      • Jeon J.
      • Mejia S.
      • Yao C.Q.
      • Ignatchenko V.
      • Nyalwidhe J.O.
      • Gramolini A.O.
      • Lance R.S.
      • Troyer D.A.
      • Drake R.R.
      • Boutros P.C.
      • Semmes O.J.
      • Kislinger T.
      Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer.
      ). Overall, SP is a highly relevant biological fluid to search for biomarkers because prostate-secreted proteins are found at much higher concentrations in SP than in serum or urine (
      • Drabovich A.P.
      • Saraon P.
      • Jarvi K.
      • Diamandis E.P.
      Seminal plasma as a diagnostic fluid for male reproductive system disorders.
      ,
      • Bieniek J.M.
      • Drabovich A.P.
      • Lo K.C.
      Seminal biomarkers for the evaluation of male infertility.
      ), and might be more easily identified and quantified by mass spectrometry. No doubt that semen and SP are unconventional fluids for PCa diagnostics, and that some older patients may have difficulty in providing SP for analysis. However, discussions of our urologists with patients (50 to 75 y.o.) indicated that most of them were willing and able to provide SP for diagnostic testing, if such test would replace invasive biopsies.
      We previously completed extensive studies on SP proteome, identified more than 3,000 proteins in SP of healthy men and patients with infertility (
      • Batruch I.
      • Lecker I.
      • Kagedan D.
      • Smith C.R.
      • Mullen B.J.
      • Grober E.
      • Lo K.C.
      • Diamandis E.P.
      • Jarvi K.A.
      Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system.
      ,
      • Batruch I.
      • Smith C.R.
      • Mullen B.J.
      • Grober E.
      • Lo K.C.
      • Diamandis E.P.
      • Jarvi K.A.
      Analysis of seminal plasma from patients with non-obstructive azoospermia and identification of candidate biomarkers of male infertility.
      ), and developed a simple 2-biomarker algorithm for the differential diagnosis of male infertility (
      • Drabovich A.P.
      • Dimitromanolakis A.
      • Saraon P.
      • Soosaipillai A.
      • Batruch I.
      • Mullen B.
      • Jarvi K.
      • Diamandis E.P.
      Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.
      ,
      • Korbakis D.
      • Brinc D.
      • Schiza C.
      • Soosaipillai A.
      • Jarvi K.
      • Drabovich A.P.
      • Diamandis E.P.
      Immunocapture-selected reaction monitoring screening facilitates the development of ELISA for the measurement of native TEX101 in biological fluids.
      ). In this study, we designed a rigorous biomarker development pipeline with discovery, qualification and verification phases (
      • Drabovich A.P.
      • Martinez-Morillo E.
      • Diamandis E.P.
      Toward an integrated pipeline for protein biomarker development.
      ). Quantitative multiplex SRM assays were a foundation of our pipeline and allowed simultaneous verification of dozens of candidates in hundreds of SP samples within a realistic timeline (near 30-day continuous SRM data acquisition). We qualified 76 candidates and verified 19 candidates in SP, whereas our top candidate TGM4 protein was also evaluated in blood serum. Majority of our candidate proteins have never been previously quantified in SP or investigated in the context of PCa, and the molecular function of some proteins (olfactomedin-4, twisted gastrulation protein homolog 1, etc.) may not be well known. We demonstrated that levels of most prostate-specific proteins previously thoroughly characterized in blood (prostatic acid phosphatase, kallikreins 2 and 3, prostate-specific membrane antigen, beta-microseminoprotein, neuropeptide Y, transmembrane protease serine 2, and others) remained unchanged in SP of PCa versus negative biopsy patients. In addition, no difference was found for the levels of androgen-regulated proteins, except for TGM4. Multi-variable machine learning analysis provided a unique combination of TGM4 with a pregnancy-associated endometrial alpha-2 globulin (PAEP). Such 2-marker combination improved detection of PCa on biopsy (AUC = 0.76) and could be further investigated in detail.
      Previous studies on TGM4 revealed it as a key regulator of invasiveness (
      • Davies G.
      • Ablin R.J.
      • Mason M.D.
      • Jiang W.G.
      Expression of the prostate transglutaminase (TGase-4) in prostate cancer cells and its impact on the invasiveness of prostate cancer.
      ) and cell adhesion (
      • Jiang W.G.
      • Ye L.
      • Sanders A.J.
      • Ruge F.
      • Kynaston H.G.
      • Ablin R.J.
      • Mason M.D.
      Prostate transglutaminase (TGase-4, TGaseP) enhances the adhesion of prostate cancer cells to extracellular matrix, the potential role of TGase-core domain.
      ), and demonstrated association of TGM4 with the epithelial-mesenchymal transition and interaction between cancer and vascular endothelial cells (
      • Jiang W.G.
      • Ablin R.J.
      • Kynaston H.G.
      • Mason M.D.
      The prostate transglutaminase (TGase-4, TGaseP) regulates the interaction of prostate cancer and vascular endothelial cells, a potential role for the ROCK pathway.
      ). TGM4 was previously suggested as a PCa biomarker, but results were inconsistent and revealed either significant over-expression (
      • Cao Z.
      • Wang Y.
      • Liu Z.Y.
      • Zhang Z.S.
      • Ren S.C.
      • Yu Y.W.
      • Qiao M.
      • Zhai B.B.
      • Sun Y.H.
      Overexpression of transglutaminase 4 and prostate cancer progression: a potential predictor of less favourable outcomes.
      ) or under-expression (
      • Kim Y.
      • Jeon J.
      • Mejia S.
      • Yao C.Q.
      • Ignatchenko V.
      • Nyalwidhe J.O.
      • Gramolini A.O.
      • Lance R.S.
      • Troyer D.A.
      • Drake R.R.
      • Boutros P.C.
      • Semmes O.J.
      • Kislinger T.
      Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer.
      ,
      • Sequeiros T.
      • Rigau M.
      • Chiva C.
      • Montes M.
      • Garcia-Grau I.
      • Garcia M.
      • Diaz S.
      • Celma A.
      • Bijnsdorp I.
      • Campos A.
      • Di Mauro P.
      • Borrós S.
      • Reventós J.
      • Doll A.
      • Paciucci R.
      • Pegtel M.
      • de Torres I.
      • Sabidó E.
      • Morote J.
      • Olivan M.
      Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer.
      ,
      • Cho S.Y.
      • Choi K.
      • Jeon J.H.
      • Kim C.W.
      • Shin D.M.
      • Lee J.B.
      • Lee S.E.
      • Kim C.S.
      • Park J.S.
      • Jeong E.M.
      • Jang G.Y.
      • Song K.Y.
      • Kim I.G.
      Differential alternative splicing of human transglutaminase 4 in benign prostate hyperplasia and prostate cancer.
      ) of TGM4 in PCa versus benign disease, or slightly opposite directions based on different assays (
      • Kim Y.
      • Ignatchenko V.
      • Yao C.Q.
      • Kalatskaya I.
      • Nyalwidhe J.O.
      • Lance R.S.
      • Gramolini A.O.
      • Troyer D.A.
      • Stein L.D.
      • Boutros P.C.
      • Medin J.A.
      • Semmes O.J.
      • Drake R.R.
      • Kislinger T.
      Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer.
      ). TGM4 was found downregulated 1.7-fold in urinary extracellular vesicles of PCa and had AUC 0.58 to diagnose PCa on biopsy (
      • Sequeiros T.
      • Rigau M.
      • Chiva C.
      • Montes M.
      • Garcia-Grau I.
      • Garcia M.
      • Diaz S.
      • Celma A.
      • Bijnsdorp I.
      • Campos A.
      • Di Mauro P.
      • Borrós S.
      • Reventós J.
      • Doll A.
      • Paciucci R.
      • Pegtel M.
      • de Torres I.
      • Sabidó E.
      • Morote J.
      • Olivan M.
      Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer.
      ). Immunohistochemistry with tissue microarrays revealed under-expression of TGM4 in prostate tissues (p < 0.001) and AUC of 0.81 to detect PCa versus benign disease (
      • Sequeiros T.
      • Rigau M.
      • Chiva C.
      • Montes M.
      • Garcia-Grau I.
      • Garcia M.
      • Diaz S.
      • Celma A.
      • Bijnsdorp I.
      • Campos A.
      • Di Mauro P.
      • Borrós S.
      • Reventós J.
      • Doll A.
      • Paciucci R.
      • Pegtel M.
      • de Torres I.
      • Sabidó E.
      • Morote J.
      • Olivan M.
      Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer.
      ). TGM4 in the urinary extracellular vesicles also differentiated between low- and high-grade PCa with high sensitivity and specificity (p < 0.001; AUC 0.82). Mining of recent genomic and transcriptomic data sets revealed that TGM4 gene was amplified in 23% of patients with neuroendocrine PCa (
      • Beltran H.
      • Prandi D.
      • Mosquera J.M.
      • Benelli M.
      • Puca L.
      • Cyrta J.
      • Marotz C.
      • Giannopoulou E.
      • Chakravarthi B.V.
      • Varambally S.
      • Tomlins S.A.
      • Nanus D.M.
      • Tagawa S.T.
      • Van Allen E.M.
      • Elemento O.
      • Sboner A.
      • Garraway L.A.
      • Rubin M.A.
      • Demichelis F.
      Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer.
      ), TGM4 mRNA was significantly over-expressed (p = 0.00001) in the Cambridge cohort of 125 men with primary PCa versus matched benign tissues (
      • Dunning M.J.
      • Vowler S.L.
      • Lalonde E.
      • Ross-Adams H.
      • Boutros P.
      • Mills I.G.
      • Lynch A.G.
      • Lamb A.D.
      Mining human prostate cancer datasets: The “camcAPP” Shiny App.
      ), and TGM4 mRNA was 14-fold over-expressed in primary PCa versus benign prostatic hyperplasia (
      • Bhowal A.
      • Majumder S.
      • Ghosh S.
      • Basu S.
      • Sen D.
      • Roychowdhury S.
      • Sengupta S.
      • Chatterji U.
      Pathway-based expression profiling of benign prostatic hyperplasia and prostate cancer delineates an immunophilin molecule associated with cancer progression.
      ). Our data suggested that TGM4 protein levels in blood and SP might decrease with age, whereas TGM4 levels in SP were elevated in PCa versus benign disease. The complex interplay of these factors (age-dependence, androgen regulation and intra-individual variability) could explain previous inconsistency regarding the levels of TGM4 in prostate tissues and urine of PCa patients. Overall, TGM4 has all characteristics of a promising biomarker, such as exclusive prostate tissue specificity, secretion into SP and androgen regulation. Even though demonstrated performance of TGM4 as a single biomarker will unlikely result in its immediate use in the clinic, TGM4 needs to be investigated in future as a biomarker of distinct genomic subtypes, or as a protein to be included into emerging multi-marker panels.
      PCa heterogeneity revealed in the recent large-scale genomic studies (
      Cancer Genome Atlas Research, N.The molecular taxonomy of primary prostate cancer.
      ) could obstruct identification of biomarkers with high diagnostic sensitivity. However, next generation sequencing may facilitate stratification of genomic subtypes and identification of “exceptional responder” biomarkers, e.g. high diagnostic specificity-biomarkers which perform only in distinct cancer subtypes (
      • Saner F.A.M.
      • Herschtal A.
      • Nelson B.H.
      • deFazio A.
      • Goode E.L.
      • Ramus S.J.
      • Pandey A.
      • Beach J.A.
      • Fereday S.
      • Berchuck A.
      • Lheureux S.
      • Pearce C.L.
      • Pharoah P.D.
      • Pike M.C.
      • Garsed D.W.
      • Bowtell D.D.L.
      Going to extremes: determinants of extraordinary response and survival in patients with cancer.
      ). For example, more than 70% of primary PCa cases are driven by elevated ETS transcription factors, either through their over-expression because of the androgen-responsive gene fusions (
      Cancer Genome Atlas Research, N.The molecular taxonomy of primary prostate cancer.
      ) or lack of degradation in SPOP-mutated subtypes (
      • Gan W.
      • Dai X.
      • Lunardi A.
      • Li Z.
      • Inuzuka H.
      • Liu P.
      • Varmeh S.
      • Zhang J.
      • Cheng L
      • Sun Y.
      • Asara J.M.
      • Beck A.H.
      • Huang J.
      • Pandolfi P.P.
      • Wei W.
      SPOP promotes ubiquitination and degradation of the ERG oncoprotein to suppress prostate cancer progression.
      ). A rarer and more unique subtype of PCa (8% of primary PCa cases (
      • Armenia J.
      • Wankowicz S.A.M.
      • Liu D.
      • Gao J.
      • Kundra R.
      • Reznik E.
      • Chatila W.K.
      • Chakravarty D.
      • Han G.C.
      • Coleman I.
      • Montgomery B.
      • Pritchard C.
      • Morrissey C.
      • Barbieri C.E.
      • Beltran H.
      • Sboner A.
      • Zafeiriou Z.
      • Miranda S.
      • Bielski C.M.
      • Penson A.V.
      • Tolonen C.
      • Huang F.W.
      • Robinson D.
      • Wu Y.M.
      • Lonigro R.
      • Garraway L.A.
      • Demichelis F.
      • Kantoff P.W.
      • Taplin M.E.
      • Abida W.
      • Taylor B.S.
      • Scher H.I.
      • Nelson P.S.
      • de Bono J.S.
      • Rubin M.A.
      • Sawyers C.L.
      • Chinnaiyan A.M.
      • PCF/SU2C International Prostate Cancer Dream Team
      • Schultz N.
      • Van Allen E.M.
      The long tail of oncogenic drivers in prostate cancer.
      )) is driven by the mutated transcription factor FOXA1, which alters selectivity of androgen receptor binding and changes the pattern of expression of androgen-regulated genes (
      • Robinson J.L.
      • Holmes K.A.
      • Carroll J.S.
      FOXA1 mutations in hormone-dependent cancers.
      ). In our data set (Fig. 5C, ELISA data), we had 11 patients (12%) with very high TGM4 levels >6.3 μg/ml (all patients in the negative biopsy group had TGM4 < 6.3 μg/ml). These estimates may warrant detailed investigation of TGM4 levels in SP of patients with FOXA1 mutations.
      Review of our data on the abundance of SP versus blood serum proteins made us to hypothesize that sensitivity of protein assays (1 pg/ml for ultrasensitive immunoassays) could be insufficient to validate novel PCa biomarkers in blood serum. Indeed, assuming a five order concentration gradient between SP and serum for high- and medium-abundance prostate-specific proteins (KLK3, KLK2, and TGM4), and taking into account the relative abundance of top 300 SP proteins measured by LFQ in this study, it can be estimated that potential serum concentration of the unexplored low-abundance prostate-specific proteins would be much lower than 1 pg/ml, and thus be undetectable by standard immunoassays. Validation of novel biomarkers of primary PCa in blood serum may thus require development of a new generation of protein assays with fg/ml or lower analytical sensitivity.
      Our study might be one of the largest and most comprehensive studies on PCa biomarkers in SP. Following our previous validation of the prostate-specific kallikrein-4 in SP and blood serum (
      • Karakosta T.D.
      • Soosaipillai A.
      • Diamandis E.P.
      • Batruch I.
      • Drabovich A.P.
      Quantification of human kallikrein-related peptidases in biological fluids by multiplatform targeted mass spectrometry assays.
      ), we evaluated here one of the last and not well-studied prostate-specific proteins within the medium-abundance proteome of SP. However, our study was not without limitations, which included: (1) evaluation of only medium-to-high abundance protein candidates (100 ng/ml - 1 mg/ml) measurable by SRM in the unfractionated digest of SP; (2) some patients with negative biopsy might have had a missed PCa. We also recognize that the only real ground truth for PCa prognosis is a 20-year survival, whereas other clinical parameters (Gleason score, localization, staging etc.) have limitations. It is known that Gleason score correlates with PCa progression, with the 20-year survival rate >70% for GS≤6 and <30% for GS≥8 (
      • Albertsen P.C.
      • Hanley J.A.
      • Fine J.
      20-year outcomes following conservative management of clinically localized prostate cancer.
      ). In our study, Gleason score served to facilitate the easier execution of this project because the 20-year survival data was not available. Our work was primarily indented to demonstrate that our biomarker development pipeline empowered by SRM assays was suitable to search for PCa biomarkers in SP, and to demonstrate that SP could have a value as a clinical sample for PCa diagnostics. We recognize that our set of 152 PCa samples is relatively small and may not represent the true clinical heterogeneity and all distinct genomic subtypes. The only way to validate the relevance of TGM4 would be its measurement in much larger sets of prospectively collected SP samples with known genomic subtypes.
      Even though extensive genomic studies on PCa did not reveal substantial correlations between genomic alterations and PCa aggressiveness (
      Cancer Genome Atlas Research, N.The molecular taxonomy of primary prostate cancer.
      ,
      • Fraser M.
      • Sabelnykova V.Y.
      • Yamaguchi T.N.
      • Heisler L.E.
      • Livingstone J.
      • Huang V.
      • Shiah Y.J.
      • Yousif F.
      • Lin X.
      • Masella A.P.
      • Fox N.S.
      • Xie M.
      • Prokopec S.D.
      • Berlin A.
      • Lalonde E.
      • Ahmed M.
      • Trudel D.
      • Luo X.
      • Beck T.A.
      • Meng A.
      • Zhang J.
      • D'Costa A.
      • Denroche R.E.
      • Kong H.
      • Espiritu S.M.
      • Chua M.L.
      • Wong A.
      • Chong T.
      • Sam M.
      • Johns J.
      • Timms L.
      • Buchner N.B.
      • Orain M.
      • Picard V.
      • Hovington H.
      • Murison A.
      • Kron K.
      • Harding N.J.
      • P'ng C.
      • Houlahan K.E.
      • Chu K.C.
      • Lo B.
      • Nguyen F.
      • Li C.H.
      • Sun R.X.
      • de Borja R.
      • Cooper C.I.
      • Hopkins J.F.
      • Govind S.K.
      • Fung C.
      • Waggott D.
      • Green J.
      • Haider S.
      • Chan-Seng-Yue M.A.
      • Jung E.
      • Wang Z.
      • Bergeron A.
      • Dal Pra A.
      • Lacombe L.
      • Collins C.C.
      • Sahinalp C.
      • Lupien M.
      • Fleshner N.E.
      • He H.H.
      • Fradet Y.
      • Tetu B.
      • van der Kwast T.
      • McPherson J.D.
      • Bristow R.G.
      • Boutros P.C.
      Genomic hallmarks of localized, non-indolent prostate cancer.
      ), future proteomic studies on PCa should consider distinct genomic subtypes of PCa (
      Cancer Genome Atlas Research, N.The molecular taxonomy of primary prostate cancer.
      ). This may facilitate identification of proteomic signatures which correlate with progression of PCa and provide true biomarkers of aggressiveness within each unique genomic subtype of PCa.

      Data Availability

      Raw mass spectrometry shotgun data and MaxQuant output files were deposited to the ProteomeXchange Consortium via PRIDE (www.ebi.ac.uk/pride/archive/login) with the dataset identifier PXD007657. Selected annotated spectra for the single-peptide identification candidate proteins DMBT1, PVRL2, TGFB1 and TFF1 are presented in supplemental Fig. S1. Annotated spectra for single-peptide identification proteins can be viewed with MS-Viewer at the following permanent link http://msviewer.ucsf.edu/prospector/cgi-bin/mssearch.cgi?report_title=MS-Viewer&search_key=enahbvtldi&search_name=msviewer. SRM raw data and processed Pinpoint files for qualification and verification phases were deposited to the Peptide Atlas (www.peptideatlas.org/PASS/PASS00989) with the data set identifier PASS00989. Alternative link is ftp://PASS00989:[email protected].

      Acknowledgments

      We thank Antoninus Soosaipillai for suggestions on ELISA development, Susan Lau for coordinating collection and storage of clinical samples, and Ihor Batruch for assistance with mass spectrometry.

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