RHCG and TCAF1 promoter hypermethylation predicts biochemical recurrence in prostate cancer patients treated by radical prostatectomy

Abstract

Purpose: The lack of biomarkers that can distinguish aggressive from indolent prostate cancer has caused substantial overtreatment of clinically insignificant disease. Here, by genome-wide DNA methylome profiling, we sought to identify new biomarkers to improve the accuracy of prostate cancer diagnosis and prognosis.

Experimental design: Eight novel candidate markers, COL4A6, CYBA, TCAF1 (FAM115A), HLF, LINC01341 (LOC149134), LRRC4, PROM1, and RHCG, were selected from Illumina Infinium HumanMethylation450 BeadChip analysis of 21 tumor (T) and 21 non-malignant (NM) prostate specimens. Diagnostic potential was further investigated by methylation-specific qPCR analysis of 80 NM vs. 228 T tissue samples. Prognostic potential was assessed by Kaplan-Meier, uni- and multivariate Cox regression analysis in 203 Danish radical prostatectomy (RP) patients (cohort 1), and validated in an independent cohort of 286 RP patients from Switzerland and the U.S. (cohort 2).

Results: Hypermethylation of the 8 candidates was highly cancer-specific (area under the curves: 0.79-1.00). Furthermore, high methylation of the 2-gene panel RHCG-TCAF1 was predictive of biochemical recurrence (BCR) in cohort 1, independent of the established clinicopathological parameters Gleason score, pathological tumor stage, and pre-operative PSA (HR (95% confidence interval (CI)): 2.09 (1.26 - 3.46); P = 0.004), and this was successfully validated in cohort 2 (HR (95% CI): 1.81 (1.05 - 3.12); P = 0.032).

Conclusion: Methylation of the RHCG-TCAF1 panel adds significant independent prognostic value to established prognostic parameters for prostate cancer and thus may help to guide treatment decisions in the future. Further investigation in large independent cohorts is necessary before translation into clinical utility.

Keywords: DNA methylation; biomarker; diagnosis; prognosis; prostate cancer.

Figure 1. Differential methylation (T

vs. NM) according to Illumina 450K array. A. Multi-dimensional scaling plot of samples included in 450K analysis, based on the 10,000 most variable CpG sites across all samples. T samples (N = 21): Triangles; AN samples (N = 12): Circles; N samples (N = 9): Crosses. B.-E. Distribution of differential methylation (T vs. NM) according to the 450K array. Differentially methylated CpG sites (DMCs) were defined as CpG sites with Δβ ≥ |0.2 | and adj. P < 0.05. B. Differential methylation across all probes. C. Distribution of DMCs. D. Distribution of hypermethylated DMCs. E. Distribution of hypomethylated DMCs.

Figure 2. Selection of biomarker candidates

A: Candidate selection process. Of the 324 top candidate significant DMCs, 259 were associated to a total of 163 different genes. Eight genes were selected for further validation. B: Mean methylation of representative DMCs for each selected candidate in T and NM samples according to the discovery 450K dataset (T, N=21; NM, N=21). C: Mean methylation of the same DMCs as in (B) for each candidate in T and NM samples according to the TCGA 450K dataset for prostate cancer (T, N=297; NM, N=34).” .

Figure 3. Diagnostic potential of candidate methylation markers in T, AN and BPH samples (cohort 1)

ROC analysis of NM samples (AN and BPH, N = 30) vs. T samples (N = 203). Left: Box plots of methylation levels in NM and T samples. (**) P < 0.001, rank-sum test. Right: ROC curves of data displayed in box plots. A. COL4A6. B. CYBA. C. HLF. D. LINC01341. E. LRRC4. F. PROM1. G. RHCG. H. TCAF1.

Figure 4. Kaplan-Meier analysis of association between methylation levels of candidate biomarkers and time to BCR after RP

Patients were divided into high- or low methylation groups based on ROC analysis of BCR status (36 months after RP, cohort 1). A, B: RHCG analyzed in cohort 1 (A) and cohort 2 (B). C, D: TCAF1 analyzed in cohort 1 (C) and cohort 2 (D). P-values from log-rank test, adjusted according to the Hochberg procedure.

Figure 5. Kaplan-Meier analysis of the association between methylation levels of the marker panel d

RHCG-TCAF1 and time to BCR after RP. Patients in cohort 1 (A) and cohort 2 (B) divided into high-and low methylation groups: High: High methylation of both candidates. Low: High methylation of only one, or low methylation of both candidates. RT: RHCG-TCAF1 panel. P-values from log-rank test.

Figure 6. Kaplan-Meier analysis of the association between methylation levels of the marker panel t

RHCG-TCAF1 and time to BCR after RP. Patients in cohort 1 (A) and cohort 2 (B) divided into high-, low- and intermediate methylation groups. High: High methylation of both candidates. Low: Low methylation of both candidates. Intermediate: High methylation of one candidate only. RT: RHCG-TCAF1 panel. P-values from log-rank test.