Large‐scale evaluation of SLC18A2 in prostate cancer reveals diagnostic and prognostic biomarker potential at three molecular levels

Abstract

Diagnosis Prognosis Biomarkers A B S T R A C TLimitations of current diagnostic and prognostic tools for prostate cancer (PC) have led to over-diagnosis and over-treatment. Here, we investigate the biomarker potential of the SLC18A2 (VMAT2) gene for PC at three molecular levels. Thus, SLC18A2 promoter methylation was analyzed in 767 malignant and 78 benign radical prostatectomy (RP) samples using methylation-specific qPCR and Illumina 450K methylation microarray data. SLC18A2 transcript levels were assessed in 412 malignant and 45 benign RP samples using RNAseq data. SLC18A2 protein was evaluated by immunohistochemistry in 502 malignant and 305 benign RP samples. Cancer-specificity of molecular changes was tested using ManneWhitney U tests and/or receiver operating characteristic (ROC) analyses. Log rank, uni-and multivariate Cox regression tests were used for survival analyses. We found that SLC18A2 promoter hypermethylation was highly cancer-specific (area under the curve (AUC): 0.923e0.976) and associated with biochemical recurrence (BCR) after RP in univariate analyses. SLC18A2 transcript levels were reduced in PC and had independent prognostic value for BCR after RP (multivariate HR 0.13, P < 0.05). Likewise, SLC18A2 protein was

Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients

“…In this study, we obtained comparable AUCs, ranging from 0.79 ( GABRE ) to 0.98 ( SLC18A2 ), from analyses of malignant vs. non-malignant diagnostic prostate needle biopsies, which not only corroborate the previous reports but also highlight the robustness of our qMSP assays, even for low input DNA samples. Moreover, the significant positive association between methylation levels in PC biopsies and D’Amico risk score for all candidate genes, is also in agreement with previous findings from RP specimens, where high methylation levels were generally associated with at least one adverse clinicopathological factor (high PSA, high Gleason score, positive surgical margins, and/or advanced pT-stage)20212343.…”

“…Hence, our results confirm and expand on previous reports of PC-specific hypermethylation of these genes in prostatectomy specimens1820212223. Furthermore, to investigate if epigenetic cancer field effects exist for our eight novel candidate genes, we analysed non-malignant diagnostic needle biopsy samples from 79 patients with/without cancer in other biopsies using qMSP.…”

“…Based on analyses of RP specimens, we and others have previously shown that AOX1, CCDC181, GABRE, GAS6, HAPLN3, KLF8, MOB3B , and SLC18A2 as well as GSTP1 , are common targets (AUC > 0.90) of aberrant promoter hypermethylation in PC tissue samples1820212223. In this study, we obtained comparable AUCs, ranging from 0.79 ( GABRE ) to 0.98 ( SLC18A2 ), from analyses of malignant vs. non-malignant diagnostic prostate needle biopsies, which not only corroborate the previous reports but also highlight the robustness of our qMSP assays, even for low input DNA samples.…”

“…In addition, future biopsy-based studies could include analysis of KLF8 and SLC18A2 for which our previous study in two large patient cohorts showed that a higher methylation level in PC tissue samples from RP specimens was associated with early biochemical recurrence in univariate analyses2023. Finally, we note that G AS6 and MOB3B methylation did not show significant prognostic value in our previous study of two large RP cohorts20, while the potential prognostic value of GSTP1 hypermethylation has been evaluated by multiple research groups, however with conflicting results14.…”

“…To date, several genes have been identified as common targets for aberrant promoter hypermethylation in PC17, including the extensively studied GSTP1 (Glutathione S-Transferase pi 1) gene that is hypermethylated in more than 90% of all PC tissue samples1819. Moreover, we have previously reported similarly high frequencies of cancer-specific promoter hypermethylation for the eight biomarker candidate genes AOX1 (Aldehyde Oxidase 1), CCDC181 (Coiled-Coil Domain Containing 181, also known as C1orf114 ), GABRE (Gamma-Aminobutyric Acid A Receptor Epsilon), GAS6 (Growth Arrest-Specific 6), HAPLN3 (Hyaluronan and Proteoglycan Link Protein 3), KLF8 (Kruppel-like Factor 8), MOB3B (MOB kinase activator 3B), and SLC18A2 (Solute Carrier Family 18 vesicular monoamine Member 2) in malignant tissue samples from radical prostatectomy (RP) specimens20212223. However, while hypermethylation-based cancer field effects have been demonstrated for GSTP1 in several previous studies of PC242526272829, the existence of such epigenetic field effects remains to be investigated for our eight novel candidate methylation marker genes.…”