PITX3 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients after radical prostatectomy

Abstract

Background: Molecular biomarkers that might help to distinguish between more aggressive and clinically insignificant prostate cancers (PCa) are still urgently needed. Aberrant DNA methylation as a common molecular alteration in PCa seems to be a promising source for such biomarkers. In this study, PITX3 DNA methylation (mPITX3) and its potential role as a prognostic biomarker were investigated. Furthermore, mPITX3 was analyzed in combination with the established PCa methylation biomarker PITX2 (mPITX2).

Methods: mPITX3 and mPITX2 were assessed by a quantitative real-time PCR and by means of the Infinium HumanMethylation450 BeadChip. BeadChip data were obtained from The Cancer Genome Atlas (TCGA) Research Network. DNA methylation differences between normal adjacent, benign hyperplastic, and carcinomatous prostate tissues were examined in the TCGA dataset as well as in prostatectomy specimens from the University Hospital Bonn. Retrospective analyses of biochemical recurrence (BCR) were conducted in a training cohort (n = 498) from the TCGA and an independent validation cohort (n = 300) from the University Hospital Bonn. All patients received radical prostatectomy.

Results: In PCa tissue, mPITX3 was increased significantly compared to normal and benign hyperplastic tissue. In univariate Cox proportional hazards analyses, mPITX3 showed a significant prognostic value for BCR (training cohort: hazard ratio (HR) = 1.83 (95 % CI 1.07-3.11), p = 0.027; validation cohort: HR = 2.56 (95 % CI 1.44-4.54), p = 0.001). A combined evaluation with PITX2 methylation further revealed that hypermethylation of a single PITX gene member (either PITX2 or PITX3) identifies an intermediate risk group.

Conclusions: PITX3 DNA methylation alone and in combination with PITX2 is a promising biomarker for the risk stratification of PCa patients and adds relevant prognostic information to common clinically implemented parameters. Further studies are required to determine whether the results are transferable to a biopsy-based patient cohort. Trial registration: Patients for this unregistered study were enrolled retrospectively.

Keywords: DNA methylation; PITX2; PITX3; Prognostic biomarker; Prostate cancer.

Fig. 1

Genomic location, design, and validation of the PITX3 QM Assay. a PITX3 quantitative-methylation (QM) assay located on the reverse strand of chromosome 10. Both PITX3 splice variants PITX3-001 and PITX3-201 are shown. The information was taken from Ensembl Homo sapiens version 82.38 (GRCh38.p3). The two beads of the Illumina Infinium HumanMethylation450 BeadChip (cg12324970 and cg23095743) used from the TCGA dataset are shown. The GC content (%) is shown with the dashed line indicating 50 % GC. b The QM real-time PCR assay was validated on a dilution series of bisulfite-converted artificially methylated and unmethylated DNA. Each sample was measured in duplicate

Fig. 2

PITX3 DNA methylation in prostatectomy specimens. Median methylation is indicated by the gray line. PITX3 DNA methylation is significantly higher in prostate cancer (PCa) tissue compared to corresponding normal adjacent tissue (NAT) and benign prostatic hyperplasia (BPH). a NAT and PCa samples of the training cohort (TCGA). p values refer to Wilcoxon-Mann-Whitney test. b NAT, BPH, and PCa samples from a test study comprising 71 patient samples. Each sample was measured in triplicate. p values refer to Kruskal-Wallis test

Fig. 3

Frequency and prognostic value of mPITX3 and mPITX2 in the training (n = 498) and validation (n = 300) cohorts. PITX3 and PITX2 DNA methylation was analyzed in prostate carcinoma patients from two cohorts. Methylation frequencies (a, c, and e) and Kaplan-Meier analyses of BCR-free survival in patients stratified according to dichotomized mPITX3 and mPITX2 levels are shown (b, d, and f). a mPITX3 analysis in the training cohort revealed a symmetric, bell-shaped distribution covering a broad spectrum of values (22–92 %). An optimal cutoff was elaborated by an iterative approach (68.2 %) stratifying patients into mPITX3 hyper- (mPITX3 _high) and hypomethylated (mPITX3 _low) cases. b Patient survival in the training cohort according to mPITX3 _low and mPITX3 _high status. Patients with mPITX3 _low tumors show a better prognosis. Approximate mean BCR-free survival: 93 months (mPITX3 _low, 95 % CI 85–100 months, n = 301) and 76 months (mPITX3 _high, 95 % CI 63–90 months, n = 117; LR = 5.05; p = 0.025), respectively. c mPITX2 analysis in the training cohort revealed an uneven distribution covering an altogether lower spectrum of values than mPITX3 (5–79 %). An optimal cutoff was elaborated by an iterative approach (34.3 %) stratifying patients into mPITX2 hyper- (mPITX2 _high) and hypomethylated (mPITX2 _low) cases. d Patient survival in the validation cohort according to mPITX2 _low and mPITX2 _high status. Patients with mPITX2 _low tumors show a better prognosis. Approximate mean BCR-free survival: 96 months (mPITX2 _low, 95 % CI 88–105 months, n = 220) and 78 months (mPITX2 _high, 95 % CI 67–89 months, n = 198; LR = 7.95; p = 0.005), respectively. e mPITX3 analysis in the validation cohort revealed a flattened, bell-shaped distribution covering (5–100 %). An optimal cutoff was elaborated by an iterative approach (61.8 %) stratifying patients into mPITX3 hyper- (mPITX3 _high) and hypomethylated (mPITX3 _low) cases. f Patient survival in the validation cohort according to mPITX3 _low and mPITX3 _high status. Patients with mPITX2 _low tumors show a better prognosis. Approximate mean BCR-free survival: 125 months (mPITX3 _low, 95 % CI 118–132 months, n = 145) and 103 months (mPITX3 _high, 95 % CI 91–115 months, n = 105; LR = 11.17; p = 0.001), respectively. Patient survival in the validation cohort according to mPITX2 _low and mPITX2 _high status is reported elsewhere [30]

Fig. 4

Survival according to combined mPITX3 and mPITX2 status. Kaplan-Meier analysis of BCR-free survival in prostate cancer patients stratified according to PITX3 and PITX2 DNA methylation status. Training cohort (n = 498, a): After a homogenous dropout within the first months after prostatectomy in all three groups, patients with low methylation values in PITX2 and PITX3 genes show the lowest number of BCR events (n = 182). Patients with high methylation in PITX2 and PITX3 genes present with the highest rate of BCR events (n = 67). Intermediate numbers of BCR events are observed in patients with low methylation in one PITX gene member and high methylation in the other PITX gene member (n = 169). Validation cohort (n = 300, b): Patients with low methylation values in PITX2 and PITX3 genes show the lowest number of BCR events (n = 136). Patients with high methylation in PITX2 and PITX3 genes present with the earliest BCR events (n = 32). Patients with low methylation in one PITX gene member and high methylation in the other PITX gene member (n = 82) show the highest number of BCR events, however, more protracted than patients with high methylation in both PITX genes