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
Graphical Abstract
Prostate cancer (PCa)
1
The abbreviations used are:
PCa
prostate cancer
BH-adjusted t-testBenjamini-Hochberg-adjusted t-test
CVcoefficient of variation
FDRfalse discovery rate
FWHMfull width at half maximum
GSGleason score
ELISAenzyme-linked immunosorbent assay
IQRinterquartile range
LFQlabel-free quantification
MWUMann Whitney Unpaired t-test
PSAprostate-specific antigen
ROC AUCreceiver operating characteristic area under the curve
S/Nsignal-to-noise
SPseminal plasma
SRMselected reaction monitoring
TGM4protein-glutamine gamma-glutamyltransferase 4
XGBoosteXtreme Gradient Boosting algorithm.
1The abbreviations used are:PCa
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 (prostate cancer
BH-adjusted t-testBenjamini-Hochberg-adjusted t-test
CVcoefficient of variation
FDRfalse discovery rate
FWHMfull width at half maximum
GSGleason score
ELISAenzyme-linked immunosorbent assay
IQRinterquartile range
LFQlabel-free quantification
MWUMann Whitney Unpaired t-test
PSAprostate-specific antigen
ROC AUCreceiver operating characteristic area under the curve
S/Nsignal-to-noise
SPseminal plasma
SRMselected reaction monitoring
TGM4protein-glutamine gamma-glutamyltransferase 4
XGBoosteXtreme Gradient Boosting algorithm.
1
). Our best current strategy to help PCa patients is early diagnosis and administration of the most appropriate therapy, including active surveillance only (2
).- Wilt T.J.
- Brawer M.K.
- Jones K.M.
- Barry M.J.
- Aronson W.J.
- Fox S
- Gingrich J.R.
- Wei J.T.
- Gilhooly P.
- Grob B.M.
- Nsouli I.
- Iyer P.
- Cartagena R.
- Snider G.
- Roehrborn C.
- Sharifi R.
- Blank W.
- Pandya P.
- Andriole G.L.
- Culkin D.
- Wheeler T.
Radical prostatectomy versus observation for localized prostate cancer.
N. Engl. J. Med. 2012; 367: 203-213
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 (
3
, - Schroder F.H.
- Hugosson J.
- Roobol M.J.
- Tammela T.L.
- Ciatto S.
- Nelen V.
- Kwiatkowski M.
- Lujan M.
- Lilja H.
- Zappa M.
- Denis L.J.
- Recker F.
- Berenguer A.
- Määttänen L.
- Bangma C.H.
- Aus G.
- Villers A.
- Rebillard X.
- van der Kwast T.
- Blijenberg B.G.
- Moss S.M.
- de Koning H.J.
- Auvinen A.
ERSPC Investigators Screening and prostate-cancer mortality in a randomized European study.
N. Engl. J. Med. 2009; 360: 1320-1328
4
).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 (
5
). 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 (
6
) 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 (7
, 8
) and approved in 1986 (9
), current U.S. Food and Drug Administration (FDA)-approved tests include only three tests: Prostate Health Index, PCA3 and CellSearch (10
). 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 (11
). 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 (- 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.
Eur. Urol. 2014; 66: 906-912
12
). 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 (13
). 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) (- 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.
J. Clin. Oncol. 2014; 32: 1136-1142
14
), STHLM3 model (6 serum proteins, 232 single nucleotide polymorphisms and clinical parameters) (15
) and ConfirmMDx (hypermethylation of GSTP1, APC and RASSF1 genes in biopsies) (16
). TMPRSS2-ERG fusion (17
) and SPINK1 mRNA (18
) 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.- 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.
Cancer Cell. 2008; 13: 519-528
Although much of the work to identify and characterize PSA was originally carried out in seminal plasma (SP) (
19
), there are only few comprehensive proteomic studies on identification of novel PCa biomarkers in SP (20
, 21
, 22
) or expressed prostatic secretions (23
, - 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.
Mol. Cell. Proteomics. 2012; 11: 1870-1884
24
). 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 (25
). Nearly a quarter of molecular composition of SP is secreted by prostate (25
), with the rest produced by seminal vesicles, epididymis, testis, and periurethral glands (26
, 27
). 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 (28
, - 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.
J. Proteome Res. 2011; 10: 941-953
29
). Our work resulted in first-of-a-kind SP biomarkers for the differential diagnosis of male infertility (30
, 31
, 32
). 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 (20
).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 (
33
, - Lee J.W.
- Devanarayan V.
- Barrett Y.C.
- Weiner R.
- Allinson J.
- Fountain S.
- Keller S.
- Weinryb I.
- Green M.
- Duan L.
- Rogers J.A.
- Millham R.
- O'Brien P.J.
- Sailstad J.
- Khan M.
- Ray C.
- Wagner J.A.
Fit-for-purpose method development and validation for successful biomarker measurement.
Pharm. Res. 2006; 23: 312-328
34
), 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 (- Carr S.A.
- Abbatiello S.E.
- Ackermann B.L.
- Borchers C.
- Domon B.
- Deutsch E.W.
- Grant R.P.
- Hoofnagle A.N.
- Hüttenhain R.
- Koomen J.M.
- Liebler D.C.
- Liu T.
- MacLean B.
- Mani D.R.
- Mansfield E.
- Neubert H.
- Paulovich A.G.
- Reiter L.
- Vitek O.
- Aebersold R.
- Anderson L.
- Bethem R.
- Blonder J.
- Boja E.
- Botelho J.
- Boyne M.
- Bradshaw R.A.
- Burlingame A.L.
- Chan D.
- Keshishian H.
- Kuhn E.
- Kinsinger C.
- Lee J.S.
- Lee S.W.
- Moritz R.
- Oses-Prieto J.
- Rifai N.
- Ritchie J.
- Rodriguez H.
- Srinivas P.R.
- Townsend R.R.
- Van Eyk J.
- Whiteley G.
- Wiita A.
- Weintraub S.
Targeted peptide measurements in biology and medicine: best practices for mass spectrometry-based assay development using a fit-for-purpose approach.
Mol. Cell. Proteomics. 2014; 13: 907-917
35
) 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 (
60
) and epigenetic markers (- Ahn J.
- Berndt S.I.
- Wacholder S.
- Kraft P.
- Kibel A.S.
- Yeager M.
- Albanes D.
- Giovannucci E.
- Stampfer M.J.
- Virtamo J.
- Thun M.J.
- Feigelson H.S.
- Cancel-Tassin G.
- Cussenot O.
- Thomas G.
- Hunter D.J.
- 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.
Nat. Genet. 2008; 40 (author reply 1035–1036): 1032-1034
16
), molecular markers such as RNA (12
, 17
, 18
), proteins (- 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.
Cancer Cell. 2008; 13: 519-528
11
, - 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.
Eur. Urol. 2014; 66: 906-912
14
, 15
) and metabolites (61
), and circulating tumor cells (13
). 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 (- 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.
J. Clin. Oncol. 2014; 32: 1136-1142
62
). 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.
Clin. Chem. 2013; 59: 280-288
62
). Emerging 4KScore test predicted PCa on primary biopsies with AUC = 0.69 (- 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.
Clin. Chem. 2013; 59: 280-288
14
). 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 (15
). 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 (16
).There were only few previous studies on PCa biomarkers in SP (
20
, 21
, 22
) or expressed prostatic secretions (23
, - 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.
Mol. Cell. Proteomics. 2012; 11: 1870-1884
24
). 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 (25
, 63
), 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 (
28
, - 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.
J. Proteome Res. 2011; 10: 941-953
29
), and developed a simple 2-biomarker algorithm for the differential diagnosis of male infertility (30
, 64
). In this study, we designed a rigorous biomarker development pipeline with discovery, qualification and verification phases (65
). 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 (
66
) and cell adhesion (67
), and demonstrated association of TGM4 with the epithelial-mesenchymal transition and interaction between cancer and vascular endothelial cells (68
). TGM4 was previously suggested as a PCa biomarker, but results were inconsistent and revealed either significant over-expression (59
) or under-expression (24
, 57
, - 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.
Oncotarget. 2017; 8: 4960-4976
58
) of TGM4 in PCa versus benign disease, or slightly opposite directions based on different assays (23
). TGM4 was found downregulated 1.7-fold in urinary extracellular vesicles of PCa and had AUC 0.58 to diagnose PCa on biopsy (- 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.
Mol. Cell. Proteomics. 2012; 11: 1870-1884
57
). 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.
Oncotarget. 2017; 8: 4960-4976
57
). 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 (- 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.
Oncotarget. 2017; 8: 4960-4976
69
), TGM4 mRNA was significantly over-expressed (p = 0.00001) in the Cambridge cohort of 125 men with primary PCa versus matched benign tissues (- 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.
Nat. Med. 2016; 22: 298-305
70
), and TGM4 mRNA was 14-fold over-expressed in primary PCa versus benign prostatic hyperplasia (71
). 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 (
72
) 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 (73
). 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 (- 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.
Nat. Rev. Cancer. 2019; 19: 339-348
72
) or lack of degradation in SPOP-mutated subtypes (74
). A rarer and more unique subtype of PCa (8% of primary PCa cases (75
)) 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 (- 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.
Nat. Genet. 2018; 50: 645-651
76
). 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 (
20
), 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 (42
). 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 (
72
, 77
), future proteomic studies on PCa should consider distinct genomic subtypes of PCa (- 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.
Nature. 2017; 541: 359-364
72
). 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.
Supplementary Material
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Article Info
Publication History
Published online: June 27, 2019
Received:
June 4,
2019
Footnotes
Author contributions: A.P.D., K.J., and E.P.D. designed research; A.P.D., P.S., and T.K. performed research; A.P.D. contributed new reagents/analytic tools; A.P.D., M.D., and A.D. analyzed data; A.P.D. wrote the paper; M.E.H. and K.J. provided clinical samples and clinical expertise.
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Copyright
© 2019 Drabovich et al.
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