Ekaterina Olkhov-Mitsel

Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers

DNA methylation, consisting of the addition of a methyl group at the fifth‐position of cytosine in a CpG dinucleotide, is one of the most well‐studied epigenetic mechanisms in mammals with important functions in normal and disease biology. Disease‐specific aberrant DNA methylation is a well‐recognized hallmark of many complex diseases. Accordingly, various studies have focused on characterizing unique DNA methylation marks associated with distinct stages of disease development as they may serve as useful biomarkers for diagnosis, prognosis, prediction of response to therapy, or disease monitoring. Recently, novel CpG dinucleotide modifications with potential regulatory roles such as 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxylcytosine have been described. These potential epigenetic marks cannot be distinguished from 5‐methylcytosine by many current strategies and may potentially compromise assessment and interpretation of methylation data. A large number of strategies have been described for the discovery and validation of DNA methylation‐based biomarkers, each with its own advantages and limitations. These strategies can be classified into three main categories: restriction enzyme digestion, affinity‐based analysis, and bisulfite modification. In general, candidate biomarkers are discovered using large‐scale, genome‐wide, methylation sequencing, and/or microarray‐based profiling strategies. Following discovery, biomarker performance is validated in large independent cohorts using highly targeted locus‐specific assays. There are still many challenges to the effective implementation of DNA methylation‐based biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing these gaps in the field of epigenetics. The development of DNA methylation‐ and hydroxymethylation‐based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical settings.

Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer

DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.

Urinary DNA Methylation Biomarkers for Noninvasive Prediction of Aggressive Disease in Patients with Prostate Cancer on Active Surveillance

Purpose: Patients with prostate cancer on active surveillance are monitored by repeat prostate specific antigen measurements, digital rectal examinations and prostate biopsies. A subset of patients on active surveillance will later reclassify with disease progression, prompting definitive treatment. To minimize the risk of under treating such patients on active surveillance minimally invasive tests are urgently needed incorporating biomarkers to identify patients who will reclassify. Materials and Methods: We assessed post‐digital rectal examination urine samples of patients on active surveillance for select DNA methylation biomarkers that were previously investigated in radical prostatectomy specimens and shown to correlate with an increasing risk of prostate cancer. Post‐digital rectal examination urine samples were prospectively collected from 153 men on active surveillance who were diagnosed with Gleason score 6 disease. Urinary sediment DNA was analyzed for 8 DNA methylation biomarkers by multiplex MethyLight assay. Correlative analyses were performed on gene methylation and clinicopathological variables to test the ability to predict patient risk reclassification. Results: Using backward logistic regression a 4‐gene methylation classifier panel (APC, CRIP3, GSTP1 and HOXD8) was identified. The classifier panel was able to predict patient reclassification (OR 2.559, 95% CI 1.257–5.212). We observed this panel to be an independent and superior predictor compared to current clinical predictors such as prostate specific antigen at diagnosis or the percent of tumor positive cores in the initial biopsy. Conclusion: We report that a urine based classifier panel of 4 methylation biomarkers predicts disease progression in patients on active surveillance. Once validated in independent active surveillance cohorts, these promising biomarkers may help establish a less invasive method to monitor patients on active surveillance programs.

Epigenome-Wide DNA Methylation Profiling Identifies Differential Methylation Biomarkers in High-Grade Bladder Cancer

Epigenetic changes, including CpG island hypermethylation, occur frequently in bladder cancer (BC) and may be exploited for BC detection and distinction between high-grade (HG) and low-grade (LG) disease. Genome-wide methylation analysis was performed using Agilent Human CpG Island Microarrays to determine epigenetic differences between LG and HG cases. Pathway enrichment analysis and functional annotation determined that the most frequently methylated pathways in HG BC were enriched for anterior/posterior pattern specification, embryonic skeletal system development, neuron fate commitment, DNA binding, and transcription factor activity. We identified 990 probes comprising a 32-gene panel that completely distinguished LG from HG based on methylation. Selected genes from this panel, EOMES, GP5, PAX6, TCF4, and ZSCAN12, were selected for quantitative polymerase chain reaction–based validation by MethyLight in an independent series (n = 84) of normal bladder samples and LG and HG cases. GP5 and ZSCAN12, two novel methylated genes in BC, were significantly hypermethylated in HG versus LG BC (P ≤ .03). We validated our data in a second independent cohort of LG and HG BC cases (n = 42) from The Cancer Genome Atlas (TCGA). Probes representing our 32-gene panel were significantly differentially methylated in LG versus HG tumors (P ≤ .04). These results indicate the ability to distinguish normal tissue from cancer, as well as LG from HG, based on methylation and reveal important pathways dysregulated in HG BC. Our findings were corroborated using publicly available data sets from TCGA. Ultimately, the creation of a methylation panel, including GP5 and ZSCAN12, able to distinguish between disease phenotypes will improve disease management and patient outcomes.

Germline mutations in the kallikrein 6 region and predisposition for aggressive prostate cancer

Background There is a need for markers that can specifically identify individuals at increased risk of harboring aggressive forms of prostate cancer (PCa). Methods We surveyed the Kallikrein ( KLK ) region ( KLK 1-15) for single-nucleotide polymorphisms (SNPs) associated with aggressive PCa (Gleason Score ≥ 8) in 1858 PCa patients. Discovery cohorts (Swiss arm of the European Randomized Study of Screening for PCa, n = 379; Toronto, Canada, n = 540) and a validation cohort (Prostate, Lung, Colorectal and Ovarian [PLCO] screening trial, n = 939) were analyzed. Fine-mapping within the KLK region was carried out by genotyping and imputation in the discovery cohort, whereas PLCO data were provided through database of Genotypes and Phenotypes ( dbGaP ). The influence of SNPs of interest on biochemical-free survival was evaluated in a cohort of localized PCa from the International Cancer Genome Consortium (ICGC; n = 130) analyzed with next-generation sequencing. Single- and multi-SNP association studies, as well as haplotype analyses, were performed. All statistical tests were two-sided. Results Several SNPs in very strong linkage disequilibrium in the KLK 6 region and located within the same haplotype (rs113640578, rs79324425, rs11666929, rs28384475, rs3810287), identified individuals at increased risk of aggressive PCa in both discovery (odds ratio [OR] = 3.51-3.64, 95% confidence interval [CI] = 2.01 to 6.36, P = 1.0x10 -5 -8.4x10 -6 ) and validation (OR = 1.89-1.96, 95% CI = 0.99 to 3.71, P = .04-.05) cohorts. The overall test of haplotype association was highly statistically significant in each cohort ( P = 3.5x10 -4 and .006, respectively) and in the three data sets combined ( P = 2.3x10 -5 ). These germline SNPs independently predicted relapse in the ICGC cohort (hazard ratio = 3.15, 95% CI = 1.57 to 6.34, P = .001). Conclusions Our fine-mapping study has identified novel loci in the KLK 6 region strongly associated with aggressive PCa.

PD65-01 GERMLINE MUTATIONS IN THE KALLIKREIN 6 REGION AND PREDISPOSITION FOR AGGRESSIVE PROSTATE CANCER

Laurent Briollais, Hilmi Ozcelik, Jingxiong Xu, Toronto, Canada; Maciej Kwiatkowski, Aarau, Switzerland; Emilie Lalonde, Dorota H Sendorek, Neil E Fleshner, Toronto, Canada; Franz Recker, Aarau, Switzerland; Cynthia Kuk, Ekaterina Olkhov-Mitsel, Toronto, Canada; Sevtap Savas, St. John’s, Canada; Sally Hanna, Tristan Juvet, Geoffrey A Hunter, Matt Friedlander, Hong Li, Karen Chadwick, Ioannis Prassas, Antoninus Soosaipillai, Toronto, Canada; Marco Randazzo, Aarau, Switzerland; John Trachtenberg, Ants Toi, Yu-Jia Shiah, Michael Fraser, Theodorus van der Kwast, Robert G Bristow, Bharati Bapat, Eleftherios P. Diamandis, Paul C Boutros, Alexandre R Zlotta*, Toronto, Canada

MP68-04 A FIVE—GENE DNA—METHYLATION BIOMARKER PANEL SENSITIVELY DETECTS BLADDER CANCER AND DISCRIMINATES BETWEEN HIGH—GRADE AND LOW—GRADE DISEASE IN VOIDED URINE

RESULTS: Our series of patients included 39 BCG-successful and 20 BCG-failure patients. The expression levels of OCT3/4 in the BCG non-responders were significantly higher than those of the BCG responders. In vitro study UM-UC3 showed a strong resistance to BCG treatment than T24. The expression levels of CSLCs-related genes (OCT3/4, Nanog, and SOX2) and EMT-related genes (Snail) increased after BCG treatment in UM-UC3 whereas T24 showed the contrary results. In sphere formation assay UM-UC3 showed a higher colonization ability than T24 after BCG treatment. CONCLUSIONS: Higher expression levels of CSLCsand EMT-related genes reflected BCG failure. The population of CSLCs in the primary CIS may be one of the predictors for BCG failure.

PD46-07 URINARY DNA METHYLATION BIOMARKERS: A NON-INVASIVE METHOD FOR PROSTATE CANCER MONITORING

INTRODUCTION AND OBJECTIVES: The majority of men with prostate cancer (PCa) are diagnosed with low-risk disease given effective PSA screening. To avoid over-treatment, men with low-risk PCa are encouraged to be followed on an Active Surveillance (AS) protocol in which repeated blood tests, digital rectal examinations (DRE) and prostate biopsies are utilized to monitor disease progression. Aberrant DNA methylation is a common feature in a variety of tumours. Tumour-specific gene methylation would be an ideal biomarker since only minute amounts of DNA are required which could be extracted from post-DRE urine samples, thus being a much less invasive method for PCa screening as compared to frequent biopsies. Our group previously identified and validated a panel of DNA methylation biomarkers that predicted the presence of high risk disease and/or post treatment biochemical recurrence in radical prostatectomy samples. In the current study, we investigated if this same panel of biomarkers can be detected in post-DRE urine samples of PCa patients participating in an AS program, and whether methylation of these biomarkers could have prognostic value in predicting development of high-risk PCa. METHODS: We collected post DRE urine samples from PCa patients with lowand intermediate-risk PCa patients followed in an AS program. DNA extracted from urine cell pellets was analyzed for selected methylation biomarkers by multiplex MethyLight, a highly sensitive, methylation specific qPCR assay. Patients were stratified using Chi-square analysis by the percent of methylated reference (PMR). Correlative studies were performed on methylation data and clinical variables such as Gleason score and clinical stage. RESULTS: DNA extracted from DRE urine cell pellets of 142 patients was analyzed for the presence of 9 DNA methylation biomarkers. DNA methylation in one or more biomarkers was seen in 73% of patients. Methylation of 3 independent biomarkers was able to distinguish between low(GS6) and intermediate-risk (GS7) PCa (p<0.05). Multigene analysis showed that methylation in a combination of any 3 genes was able to stratify between GS6 and GS7 patients (p<0.05). CONCLUSIONS: We have demonstrated that our panel of DNA methylation biomarkers can be detected in DRE urine samples of PCa patients. These markers have prognostic value in distinguishing low-risk vs intermediate risk PCa. The promising results from this study may help establish a less invasive method for PCa monitoring for patients in AS programs, resulting in better management and treatment for PCa patients.

625 A non-invasive urine-based epigenetic urinary biomarker panel for the detection of bladder cancer and the discrimination of high-grade and low-grade disease

1Princess Margaret Cancer Centre, Dept. of Surgical Oncology, Division of Urology, University of Toronto, Toronto, Canada, 2Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Canada, 3University Health Network, Dept. of Surgery, Division of Urology, University of Toronto, Toronto, Canada, 4Mount Sinai Hospital, Dept. of Surgery, Division of Urology, Toronto, Canada, 5Dalhousie University, Dept. of Urology, Halifax, Canada, 6University of Liege, Dept. of Urology, Liege, Belgium, 7University Health Network, Dept. of Pathology, University of Toronto, Toronto, Canada