BackgroundProstate cancer (PC) can be stratified into distinct molecular subtypes based on TMPRSS2-ERG gene fusion status, but its potential prognostic value remains controversial. Likewise, routine clinicopathological features cannot clearly distinguish aggressive from indolent tumors at the time of diagnosis; thus, new prognostic biomarkers are urgently needed. The DNA methylation variant 5-hydroxymethylcytosine (5hmC, an oxidized derivative of 5-methylcytosine) has recently emerged as a new diagnostic and/or prognostic biomarker candidate for several human malignancies. However, this remains to be systematically investigated for PC. In this study, we determined 5hmC levels in 311 PC (stratified by ERG status) and 228 adjacent non-malignant (NM) prostate tissue specimens by immunohistochemical analysis of a tissue microarray, representing a large radical prostatectomy (RP) cohort with long clinical follow-up. We investigated possible correlations between 5hmC and routine clinicopathological variables and assessed the prognostic potential of 5hmC by Kaplan-Meier and uni- and multivariate Cox regression analyses in ERG+ (n = 178) vs. ERG− (n = 133) PCs using biochemical recurrence (BCR) as endpoint.ResultsWe observed a borderline significant (p = 0.06) reduction in 5hmC levels in PC compared to NM tissue samples, which was explained by a highly significant (p < 0.001) loss of 5hmC in ERG− PCs. ERG status was not predictive of BCR in this cohort (p = 0.73), and no significant association was found between BCR and 5hmC levels in ERG+ PCs (p = 0.98). In contrast, high 5hmC immunoreactivity was a significant adverse predictor of BCR after RP in ERG− PCs, independent of Gleason score, pathological tumor stage, surgical margin status, and pre-operative prostate-specific antigen (PSA) level (hazard ratio (HR) (95 % confidence interval (CI)): 1.62 (1.15–2.28), p = 0.006).ConclusionsThis is the first study to demonstrate a prognostic potential for 5hmC in PC. Our findings highlight the importance of ERG stratification in PC biomarker studies and suggest that epigenetic mechanisms involving 5hmC are important for the development and/or progression of ERG− PC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0146-5) contains supplementary material, which is available to authorized users.

BackgroundPrognostic tools for prostate cancer (PC) are inadequate and new molecular biomarkers may improve risk stratification. The epigenetic mark 5-hydroxymethylcytosine (5hmC) has recently been proposed as a novel candidate prognostic biomarker in several malignancies including PC. 5hmC is an oxidized derivative of 5-methylcytosine (5mC) and can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The present study is the first to investigate the biomarker potential in PC for all four DNA methylation marks in parallel. Thus, we determined 5mC, 5hmC, 5fC, and 5caC levels in non-malignant (NM) and PC tissue samples from a large radical prostatectomy (RP) patient cohort (n = 546) by immunohistochemical (IHC) analysis of serial sections of a tissue microarray. Possible associations between methylation marks, routine clinicopathological parameters, ERG status, and biochemical recurrence (BCR) after RP were investigated.Results5mC and 5hmC levels were significantly reduced in PC compared to NM prostate tissue samples (p ≤ 0.027) due to a global loss of both marks specifically in ERG− PCs. 5fC levels were significantly increased in ERG+ PCs (p = 0.004), whereas 5caC levels were elevated in both ERG− and ERG+ PCs compared with NM prostate tissue samples (p ≤ 0.019). Positive correlations were observed between 5mC, 5fC, and 5caC levels in both NM and PC tissues (p < 0.001), while 5hmC levels were only weakly positively correlated to 5mC in the PC subset (p = 0.030). There were no significant associations between 5mC, 5fC, or ERG status and time to BCR in this RP cohort. In contrast, high 5hmC levels were associated with BCR in ERG− PCs (p = 0.043), while high 5caC levels were associated with favorable prognosis in ERG+ PCs (p = 0.011) and were borderline significantly associated with worse prognosis in ERG− PCs (p = 0.058). Moreover, a combined high-5hmC/high-5caC score was a significant adverse predictor of post-operative BCR beyond routine clinicopathological variables in ERG− PCs (hazard ratio 3.18 (1.54–6.56), p = 0.002, multivariate Cox regression).ConclusionsThis is the first comprehensive study of 5mC, 5hmC, 5fC, and 5caC levels in PC and the first report of a significant prognostic potential for 5caC in PC.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0540-x) contains supplementary material, which is available to authorized users.

Diagnosis Prognosis Biomarkers A B S T R A C TLimitations of current diagnostic and prognostic tools for prostate cancer (PC) have led to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using ManneWhitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni-and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923e0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P < 0.05). Likewise, SLC18A2 protein was

Limitations of current diagnostic and prognostic tools for prostate cancer (PC) contribute to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using Mann-Whitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni- and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923-0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P&lt;0.05). Likewise, SLC18A2 protein was down-regulated in PC (AUC 0.898) and had independent prognostic value for BCR (multivariate HR 0.51, P&lt;0.05). Reduced SLC18A2 protein expression was also associated with poor overall survival in univariate analysis (HR 0.29, P&lt;0.05). Our results highlight SLC18A2 methylation as a new promising biomarker candidate for PC diagnosis. Furthermore, both SLC18A2 RNA and protein expression showed promising prognostic potential beyond routine clinicopathological variables. Thus, novel SLC18A2-based molecular tests could have useful future applications for PC detection and identification of high-risk patients. Citation Format: Christa Haldrup, Anne-Sofie Lynnerup, Tine M. Storebjerg, Søren Vang, Peter Wild, Tapio Visakorpi, Christian Arsov, Wolfgang A. Schulz, Johan Lindberg, Henrik Grönberg, Lars Egevad, Michael Borre, Torben F. Ørntoft, Søren Høyer, Karina D. Sørensen. Large-scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 451.

Background: Prostate cancer (PC) can be stratified into distinct molecular subtypes based on TMPRSS2-ERG gene fusion status, but its potential prognostic value remains controversial. Likewise, routine clinicopathological features cannot clearly distinguish aggressive from indolent tumors at the time of diagnosis, thus new prognostic biomarkers are urgently needed. The DNA methylation variant 5-hydroxymethylcytosine (5hmC, an oxidized derivative of 5-methylcytosine) has recently emerged as a new diagnostic and/or prognostic biomarker candidate for several human malignancies. However, this remains to be systematically investigated for PC. In this study, we determined 5hmC levels in 311 PC (stratified by ERG status) and 228 adjacent non-malignant (NM) prostate tissue specimens by immunohistochemical analysis of a tissue microarray, representing a large radical prostatectomy (RP) cohort with long clinical follow-up. We investigated possible correlations between 5hmC and routine clinicopathological variables and assessed the prognostic potential of 5hmC by Kaplan-Meier, uni- and multivariate Cox regression analyses in ERG+ (n = 178) vs. ERG- (n = 133) PCs using biochemical recurrence (BCR) as endpoint. Results: We observed a borderline significant (p = 0.06) reduction in 5hmC levels in PC compared to NM tissue samples, which was explained by a highly significant (p&lt;0.001) loss of 5hmC in ERG- PCs. ERG status was not predictive of BCR in this cohort (p = 0.73) and no significant association was found between BCR and 5hmC levels in ERG+ PCs (p = 0.98). In contrast, high 5hmC immunoreactivity was a significant adverse predictor of BCR after RP in ERG- PCs, independent of Gleason score, pathological tumor stage, surgical margin status, and pre-operative PSA level (HR (95% CI): 1.62 (1.15-2.28), p = 0.006). Conclusions: This is the first study to demonstrate a prognostic potential for 5hmC in PC. Our findings highlight the importance of ERG stratification in PC biomarker studies, and suggest that epigenetic mechanisms involving 5hmC are important for the development and/or progression of ERG- PC. Citation Format: Siri H. Strand, Soren Hoyer, Anne-Sofie Lynnerup, Christa Haldrup, Tine M. Storebjerg, Michael Borre, Torben F. Orntoft, Karina D. Sorensen. High levels of 5-hydroxymethylcytosine (5hmC) predict biochemical recurrence after prostatectomy in ERG negative prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2774.