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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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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). amPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. b Patient survival in the training cohort according to mPITX3low and mPITX3high status. Patients with mPITX3low tumors show a better prognosis. Approximate mean BCR-free survival: 93 months (mPITX3low, 95 % CI 85–100 months, n = 301) and 76 months (mPITX3high, 95 % CI 63–90 months, n = 117; LR = 5.05; p = 0.025), respectively. cmPITX2 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- (mPITX2high) and hypomethylated (mPITX2low) cases. d Patient survival in the validation cohort according to mPITX2low and mPITX2high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 96 months (mPITX2low, 95 % CI 88–105 months, n = 220) and 78 months (mPITX2high, 95 % CI 67–89 months, n = 198; LR = 7.95; p = 0.005), respectively. emPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. f Patient survival in the validation cohort according to mPITX3low and mPITX3high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 125 months (mPITX3low, 95 % CI 118–132 months, n = 145) and 103 months (mPITX3high, 95 % CI 91–115 months, n = 105; LR = 11.17; p = 0.001), respectively. Patient survival in the validation cohort according to mPITX2low and mPITX2high status is reported elsewhere [30]
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Fig3: 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). amPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. b Patient survival in the training cohort according to mPITX3low and mPITX3high status. Patients with mPITX3low tumors show a better prognosis. Approximate mean BCR-free survival: 93 months (mPITX3low, 95 % CI 85–100 months, n = 301) and 76 months (mPITX3high, 95 % CI 63–90 months, n = 117; LR = 5.05; p = 0.025), respectively. cmPITX2 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- (mPITX2high) and hypomethylated (mPITX2low) cases. d Patient survival in the validation cohort according to mPITX2low and mPITX2high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 96 months (mPITX2low, 95 % CI 88–105 months, n = 220) and 78 months (mPITX2high, 95 % CI 67–89 months, n = 198; LR = 7.95; p = 0.005), respectively. emPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. f Patient survival in the validation cohort according to mPITX3low and mPITX3high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 125 months (mPITX3low, 95 % CI 118–132 months, n = 145) and 103 months (mPITX3high, 95 % CI 91–115 months, n = 105; LR = 11.17; p = 0.001), respectively. Patient survival in the validation cohort according to mPITX2low and mPITX2high status is reported elsewhere [30]

Mentions: For the analysis of PITX3 promoter methylation (mPITX3) in the training cohort, results obtained from two Illumina Infinium HumanMethylation450 BeadChip beads from the TCGA dataset (cg12324970 and cg23095743) were used. Both beads were located within the CpG island of PITX3 (Fig. 1a). Firstly, PCa (n = 498) and normal adjacent tissue (NAT, n = 50) samples from the training cohort were analyzed with respect to mPITX3. Patient samples showed a significantly lower level of mPITX3 in NAT compared to PCa samples (p < 0.001, Fig. 2a). A histogram of mPITX3 showed a bell curve with a minor depression at ≈68 % (Fig. 3a). mPITX3 levels as a continuous variable were related to prognostic clinicopathological variables and were found to be significantly correlated with the ISUP Gleason grading group (ρ = 0.112; p = 0.012), pathologic tumor (pT) category (ρ = 0.123; p = 0.006), presurgical PSA (ρ = 0.134; p = 0.003), and the AR activity score (ρ = 0.154; p = 0.005) as obtained from TCGA [27] in the training cohort. In order to analyze the suitability of mPITX3 for the stratification of patients at risk for biochemical recurrence (BCR), mPITX3 was dichotomized by an optimized cutoff (mPITX3low < 68.2 % ≤ mPITX3high; Table 1) which was identified by an iterative approach. In the training cohort, mPITX3high was significantly associated with BCR in the univariate Cox proportional hazards model (hazard ratio (HR) = 1.83 (95 % CI 1.07–3.11); p = 0.027; (Table 2)) and the Kaplan-Meier analysis (likelihood ratio (LR) = 5.05; p = 0.025, Fig. 3b).Fig. 1


PITX3 promoter methylation is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients after radical prostatectomy
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). amPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. b Patient survival in the training cohort according to mPITX3low and mPITX3high status. Patients with mPITX3low tumors show a better prognosis. Approximate mean BCR-free survival: 93 months (mPITX3low, 95 % CI 85–100 months, n = 301) and 76 months (mPITX3high, 95 % CI 63–90 months, n = 117; LR = 5.05; p = 0.025), respectively. cmPITX2 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- (mPITX2high) and hypomethylated (mPITX2low) cases. d Patient survival in the validation cohort according to mPITX2low and mPITX2high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 96 months (mPITX2low, 95 % CI 88–105 months, n = 220) and 78 months (mPITX2high, 95 % CI 67–89 months, n = 198; LR = 7.95; p = 0.005), respectively. emPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. f Patient survival in the validation cohort according to mPITX3low and mPITX3high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 125 months (mPITX3low, 95 % CI 118–132 months, n = 145) and 103 months (mPITX3high, 95 % CI 91–115 months, n = 105; LR = 11.17; p = 0.001), respectively. Patient survival in the validation cohort according to mPITX2low and mPITX2high status is reported elsewhere [30]
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Fig3: 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). amPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. b Patient survival in the training cohort according to mPITX3low and mPITX3high status. Patients with mPITX3low tumors show a better prognosis. Approximate mean BCR-free survival: 93 months (mPITX3low, 95 % CI 85–100 months, n = 301) and 76 months (mPITX3high, 95 % CI 63–90 months, n = 117; LR = 5.05; p = 0.025), respectively. cmPITX2 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- (mPITX2high) and hypomethylated (mPITX2low) cases. d Patient survival in the validation cohort according to mPITX2low and mPITX2high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 96 months (mPITX2low, 95 % CI 88–105 months, n = 220) and 78 months (mPITX2high, 95 % CI 67–89 months, n = 198; LR = 7.95; p = 0.005), respectively. emPITX3 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- (mPITX3high) and hypomethylated (mPITX3low) cases. f Patient survival in the validation cohort according to mPITX3low and mPITX3high status. Patients with mPITX2low tumors show a better prognosis. Approximate mean BCR-free survival: 125 months (mPITX3low, 95 % CI 118–132 months, n = 145) and 103 months (mPITX3high, 95 % CI 91–115 months, n = 105; LR = 11.17; p = 0.001), respectively. Patient survival in the validation cohort according to mPITX2low and mPITX2high status is reported elsewhere [30]
Mentions: For the analysis of PITX3 promoter methylation (mPITX3) in the training cohort, results obtained from two Illumina Infinium HumanMethylation450 BeadChip beads from the TCGA dataset (cg12324970 and cg23095743) were used. Both beads were located within the CpG island of PITX3 (Fig. 1a). Firstly, PCa (n = 498) and normal adjacent tissue (NAT, n = 50) samples from the training cohort were analyzed with respect to mPITX3. Patient samples showed a significantly lower level of mPITX3 in NAT compared to PCa samples (p < 0.001, Fig. 2a). A histogram of mPITX3 showed a bell curve with a minor depression at ≈68 % (Fig. 3a). mPITX3 levels as a continuous variable were related to prognostic clinicopathological variables and were found to be significantly correlated with the ISUP Gleason grading group (ρ = 0.112; p = 0.012), pathologic tumor (pT) category (ρ = 0.123; p = 0.006), presurgical PSA (ρ = 0.134; p = 0.003), and the AR activity score (ρ = 0.154; p = 0.005) as obtained from TCGA [27] in the training cohort. In order to analyze the suitability of mPITX3 for the stratification of patients at risk for biochemical recurrence (BCR), mPITX3 was dichotomized by an optimized cutoff (mPITX3low < 68.2 % ≤ mPITX3high; Table 1) which was identified by an iterative approach. In the training cohort, mPITX3high was significantly associated with BCR in the univariate Cox proportional hazards model (hazard ratio (HR) = 1.83 (95 % CI 1.07–3.11); p = 0.027; (Table 2)) and the Kaplan-Meier analysis (likelihood ratio (LR) = 5.05; p = 0.025, Fig. 3b).Fig. 1

View Article: PubMed Central - PubMed

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&thinsp;=&thinsp;498) from the TCGA and an independent validation cohort (n&thinsp;=&thinsp;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)&thinsp;=&thinsp;1.83 (95&nbsp;% CI 1.07&ndash;3.11), p&thinsp;=&thinsp;0.027; validation cohort: HR&thinsp;=&thinsp;2.56 (95&nbsp;% CI 1.44&ndash;4.54), p&thinsp;=&thinsp;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.

No MeSH data available.


Related in: MedlinePlus