Development and Clinical Validation of a Real-Time PCR Assay for PITX2 DNA Methylation to Predict Prostate-Specific Antigen Recurrence in Prostate Cancer Patients Following Radical Prostatectomy

Abstract

Prostate cancer is the most common cancer among men. The prospective discrimination of aggressive and clinically insignificant tumors still poses a significant and, as yet, unsolved problem. PITX2 DNA methylation is a strong prognostic biomarker in prostate cancer. Recently, a diagnostic microarray for prostate cancer prognosis based on PITX2 methylation has been developed and validated. Because this microarray requires nonstandard laboratory equipment, its use in a diagnostic setting is limited. This study aimed to develop and validate an alternative quantitative real-time PCR assay for measuring PITX2 methylation that can easily be established in clinical laboratories, thereby facilitating the implementation of this biomarker in clinical practice. A methylation cut-off for patient stratification was established in a training cohort (n Z 157) and validated in an independent test set (n Z 523) of men treated with radical prostatectomy. In univariate Cox proportional hazards analysis, PITX2 hypermethylation was a significant predictor for biochemical recurrence (P < 0.001, hazard ratio Z 2.614). Moreover, PITX2 hypermethylation added significant prognostic information (P Z 0.003, hazard ratio Z 1.814) to the Gleason score, pathological T stage, prostate-specific antigen, and surgical margins in a multivariate analysis. The clinical performance was particularly high in patients at intermediate risk (Gleason score of 7) and in samples containing high tumor cell content. This assay might aid in risk stratification and support the decision-making process when determining whether a patient might benefit from adjuvant treatment after radical prostatectomy. (J Mol Diagn 2013, 15: 270e279; http://dx.doi.org/10.1016/j.jmoldx.2012 In the western world, prostate cancer is by far the most common cancer and also one of the leading causes of cancer-related deaths among men.1 However, because of the relatively good prognosis of prostate cancer, only a relatively low percentage of men diagnosed as having localized prostate cancer will die of it. Prostate-specific antigen (PSA) screening for prostate cancer allows for early detection of prostate cancer and, regardless of the limitations of PSA and the problem of overdiagnosis, saves lives.

A DNA Hypermethylation Profile Independently Predicts Biochemical Recurrence Following Radical Prostatectomy

“…In recent years, other variable DNA methylation alterations in neoplastic tissue samples have been also associated with the prediction of PSA recurrence after RP [10,17,18,19,20,21,22,23,24]. These studies have identified new potential tumor markers including methylation of PITX2 , HOXD3 , APC , TGFβ2 , RASSF1A , ANEP , PCDH10 , ZNF132 , TDRD1 , AOX1 , C1orf114 , GAS6 , HAPLN3 , KLF8 , MOB3B , CCND2 , PTGS2 , RARB , SRD5A2 and CYP11A1 , thus emerging as new predictors of BCR particularly in high-risk clinically localized disease.…”

DNA Methylation of PITX2 and PANCR Is Prognostic for Overall Survival in Patients with Resected Adenocarcinomas of the Biliary Tract

“…The assays were sensitive down to 0.8% methylated DNA (equivalent to 40 pg) in a total of 5 ng bisulfite-converted DNA (Fig 2). The analytical performance of the PITX2ab-assay has been described in detail earlier [35]. …”

“…In spite of the presented possible biomarker capabilities, it becomes apparent that aberrant DNA methylation of PITX2 variants is not as prognostic for BTCs as it is for breast or prostate cancer [17,35]. In the latter, these markers may identify patients with an adverse clinical courses who might actually benefit from a radical treatment, while patients with a better prognosis might benefit from a more conservative treatment with fewer side effects.…”

“…The PITX2ab-assay was conducted as previously described [35]. The PITX2c- and PANCR-assay were performed in 20-μl reactions containing 0.25 mM of each dNTP, 1 x PCR buffer [35 mM Tris (pH 8.4), 50 mM KCl, 6 mM MgCl 2 , 4% Glycerol], 3 U FastStart Taq DNA polymerase (Roche), 0.006 μl ROX solution (prepared as previously described in ref.…”

Aberrant hypomethylation-mediated CD147 overexpression promotes aggressive tumor progression in human prostate cancer

“…Several genes, including those that encode glutathione S-transferase π 1 (GSTP1), adenomatous polyposis coli (APC), Ras association domain-containing protein 1 (RASSF1A) and prostaglandinendoperoxide synthase 2 (PTGS2), have been demonstrated to be hypermethylated in PCa, but not in the normal prostate tissues (6,13,14). Similarly, many hypomethylated genes have been identified and demonstrated to be associated with the recurrent status of PCa patients (15)(16)(17). These findings suggest that the DNA methylation of these loci may improve the diagnostic efficiency for PCa.…”