MiR-193b promoter methylation accurately detects prostate cancer in urine sediments and miR-34b/c or miR-129-2 promoter methylation define subsets of clinically aggressive tumors

Prostate cancer (PCa) is the most incident male cancer in western countries, constituting the second most common cause of cancer and the sixth leading cause of death by cancer among men worldwide [1]. For 2012, it was estimated that PCa alone accounted for 420,000 newly diagnosed cancer cases and 101,000 of all cancer-related deaths in European men [2]. PCa is age-related and very heterogeneous, both molecularly and clinically, ranging from relatively indolent to highly aggressive. It is typically asymptomatic at its earliest stages, when adequate treatment is mostly curative, in contrast with its late diagnosis, which usually impairs a curative-intent therapeutic strategy [3]. This led to the widespread use of serum PSA as screening tool for PCa. However, it is now commonly accepted that this entailed overdiagnosis and overtreatment, justifying the strong recommendation against PCa screening and prompting the search for more effective biomarkers [4].

DNA methylation is a chemically stable and easily quantified alteration [5]. We and others have previously reported on the use of quantitative promoter methylation of several protein-coding genes for early diagnosis and prognostication of PCa [6]. Although several gene methylation panels have been then developed [7, 8], both sensitivity and specificity must be perfected to allow for clinical translation.

MicroRNAs, a class of small (19–25 nucleotides) non-coding RNA, are involved in virtually all cellular processes and frequently deregulated in cancer cells [9], although its abrogation due to aberrant promoter methylation has been seldom reported [10]. Because this epigenetic alteration is likely to be highly cancer-specific, it might constitute an effective cancer biomarker. Thus, we aimed to explore the potential of microRNA-coding genes promoter methylation as diagnostic and prognostic biomarkers in PCa. Therefore, after genome-wide screening, a set of putative tumor-suppressor microRNAs (miR-34b/c, miR-129-2, miR-152, miR-193b, miR-663a and miR-1258) with increased promoter methylation levels in PCa compared to normal prostate tissues was identified and further validated in clinical samples.

PCa remains one of the most prevalent neoplasms and a leading cause of morbidity and mortality in men. Although PSA screening has decreased the number of men diagnosed with metastatic PCa, this was accomplished at the cost of overdiagnosis and overtreatment of a sizeable proportion of men carrying indolent/non-life threatening tumors [13]. Thus, a strong recommendation against serum PSA-based PCa screening has been issued [14], prompting the search for more effective biomarkers allowing for better risk stratification of PCa suspects. Herein, we aimed to tackle this clinical quest through discovery and preliminary validation of novel biomarkers for PCa detection and prognostication, using methylation analysis of microRNAs gene promoters.

Owing to our previous experience in DNA methylation analysis of PCa [6, 11], we searched for altered methylation patterns at the promoter regions of microRNAs deregulated in PCa. This information was then used to develop novel biomarkers, instead of microRNA expression levels, as previously attempted by other researchers [15]. Indeed, DNA methylation is easier to assess than microRNA expression, it is more specific and, importantly, more stable. Moreover, because microRNAs downregulation in cancer is more common than upregulation, it seemed likely that aberrant promoter methylation might constitute an underlying mechanism, similar to protein-coding genes [16]. Although several strategies might be used to identify microRNAs putatively downregulated due to promoter hypermethylation, high-throughput technologies such as methylation-array analysis are able to simultaneously pinpoint putative candidates [17] and the reliability of the results might be readily assessed through analysis of well-known hypermethylated loci. Indeed, results of the methylation array experiments confirmed the high prevalence of GSTP1 and APC promoter methylation (data not shown), as we previously demonstrated in PCa [18]. To increase the likelihood of finding robust candidate biomarkers, we used stringent conditions based on high fold-variation of methylation levels between cancerous and non-cancerous tissue samples. From methylation-array analysis, six candidate microRNAs, putatively deregulated by promoter hypermethylation were identified. MiR-1258, miR-193b and miR-34b/c were the most promising candidates, displaying substantial PCa-specificity compared with other urinary tract tumors, an attractive feature for testing in bodily fluids. MiR-129-2 and miR-663a showed modest results and their inability to discriminate PCa from bladder cancer rendered it unsuitable for testing in urine samples.

Association between promoter methylation levels in tumor tissue samples and standard clinicopathological variables was also assessed. Higher miR-129-2 promoter methylation levels associated with higher GS and stage, suggesting prognostic value. MiR-34b/c, miR-663a and miR-1258 methylation levels also associated with pathological stage, but higher diagnostic performance underscores the potential for detecting PCa at early stages instead of prognostication, as we previously reported for EFEMP1 promoter methylation [19]. Nevertheless, in this series of radical prostatectomies (Cohort #1) higher miR-129-2 methylation conveyed independent prognostic information, although only for DSS. Importantly, these results are in line with previous observations concerning the association of higher gene promoter methylation levels with clinicopathological features of more aggressive disease [11, 20].

Urine is a key sample to evaluate DNA methylation biomarkers for PCa, as it is readily collected and biomarkers are diluted to a smaller extent than in plasma, providing higher sensitivity[21]. Nevertheless, the amount of DNA potentially deriving from prostatic cells is variable, usually low, entailing the use of a panel with limited number of biomarkers. Thus, only miR-34b/c, miR-193b and mir-1258, were tested in urine samples (Cohort #3). From these, Mir-193b was previously shown to be aberrantly methylated in PCa cell lines as well as in primary tumors, but no data is available regarding its performance as PCa detection biomarker [22, 23]. Indeed, Mir-193b performed best, with high AUC, sensitivity, specificity and PPV, whereas miR-34b/c performance was more modest.

Intriguingly, miR-1258, which showed the best performance in tissue samples (Cohort #1), displayed a strikingly different result in urines as its methylation levels were higher in controls than in PCa patients. The reason for this discrepant result is not immediately apparent, but it might be due to high miR-1258 promoter methylation in non-epithelial cells, such as leucocytes, which are relatively more abundant in urine than in tumor tissue samples. Moreover, median miR-1258 promoter methylation levels in urines from PCa were substantially inferior to those of miR-193b, impairing the robustness of the assay. It should be recalled that, contrarily to other studies, the urine samples we used were not collected following DRE or prostatic massage, which are usually employed in an attempt to increase sensitivity. Studies dealing with PCa biomarkers in urine vary in the method of urine collection and the real impact of prostatic massage has never been evaluated [24]. It could be argued that the distance from the peripheral zone to the urinary tract flow may render urinary based tests less sensitive, which would be an important issue since most malignancies arise from this zone. Nevertheless, studies on PCA3 did not find a difference in the levels of this biomarker between patients with peripheral versus transitional zone PCa [25, 26].

Currently, the performance of serum PSA and urinary PCA3, the only biomarkers approved for clinical use is rather limited. The reported performance of serum PSA as PCa biomarker is somewhat modest, with AUC ranging from 0.54 to 0.70 [27, 28]. Even other serum PSA-derived measurements, like PSA-density, free PSA percentage and PSA-velocity have not significantly improved performance [28]. Nonetheless, PCA3, which was reported to perform better than serum PSA both in urine and ejaculates but has not been approved for population-based screening, displays AUCs varying from 0.66 to 0.79 [27‐30]. Additionally, although miRs’ expression has been extensively investigated in liquid biopsies, available data for urine samples is rather limited. Nevertheless, an AUC of 0.74 was reported for miR-107 [31] and simultaneous quantification of miR-107 and miR-574-3p in urine showed an AUC of 0.83, for PCa cancer detection [32]. We should emphasize that in our dataset, urinary miR-193b promoter methylation (AUC = 0.96) outperformed not the only currently approved clinical biomarkers, but also the previously mentioned miRs, constituting a promising tool for non-invasive PCa detection.

Because a major goal of this study was to discriminate clinically aggressive from indolent PCa, it was critical to test the prognostic value of microRNAs in a pre-therapeutic setting, which was accomplished in series of prospectively collected prostate biopsies (Cohort #3). In univariable analysis, most standard clinicopathological parameters associated with DFS and DSS, clinically validating this dataset. The same was demonstrated for higher miR-129-2 and miR-34b/c promoter methylation levels. The CAPRA score, however, only associated with DSS but not DFS. This was unexpected as its determination at diagnosis associated with DFS in patients with clinically localized disease submitted to RP [12]. Notwithstanding, our prostate biopsy series included PCa at diverse clinical stages, submitted to different therapeutic modalities (RP, radiotherapy, androgen-deprivation therapy), which might explain the apparent flaw of CAPRA score. In multivariable analysis, only clinical stage, amongst all clinicopathological parameters, retained independent prognostic value, both for DFS and DSS. Remarkably, high miR-34b/c promoter methylation levels also predicted shorter DFS and DSS, suggesting that it might constitute a useful PCa prognostic biomarker. These results suggest that high miR-34b/c promoter methylation levels identify clinically aggressive PCa, irrespective of disease extent at diagnosis.

It should be acknowledged that in spite of the excellent diagnostic performance of miR-193b promoter methylation in urine, additional patient sets must be tested. Furthermore, a larger cohort of patients submitted to biopsy and subjected to different therapies is required to further validate our observations. Ultimately, we plan to develop a multiplex assay to simultaneously assess miR-193b, miR-129-2 and miR34b/c promoter methylation, allowing for diagnostic and prognostic assessment of PCa suspects in a single analysis.

Through genome-wide screening, a set of methylation-based PCa biomarkers was identified and validated. MiR-193b demonstrated high sensitivity and specificity for detection of PCa, both in tissue and urine, whereas high miR-129-2 and miR-34b/c methylation levels independently predicted for shorter DSS and DFS or DSS, respectively. If confirmed in larger and independent datasets, quantitative promoter methylation of selected miRs might provide useful tools for clinical management of PCa patients. The authors would like to extend their appreciation to all the patients and control subjects that kindly provided the biological samples for this study.