Title: Characterising the blood methylome to develop an early detection test for high grade serous carcinomas.
Funder: Cancer Research UK

Abstract: Background: Currently there is no effective screening method for ovarian cancer. USPLCO and UKCTOCS trials in the US and UK, respectively, have shown little benefit in combining ultrasound scanning and serum CA-125 for early diagnosis. Serum CA-125 is most effective as a marker of disease status in patients undergoing chemotherapy treatment for epithelial ovarian cancer (EOC). It is not specific to malignancy, expressed in several benign conditions including endometriosis, pelvic infection, and uterine leiomyomata. In addition, the current CA-125 assay lacks sensitivity and is elevated in only 50% of early stage EOCs. Recent evidence suggests that the most common and most aggressive form of EOC, High Grade Serous Carcinoma (HSGC), arise from the distal fallopian tube and not the ovary, with a proposed precursor lesion being serous tubal intraepithelial carcinoma (STIC). Early detection of STICs would be a major advance in HGSC management since the majority of HGSCs present as advanced (FIGO stage 3/4) disease. We have preliminary data which shows that there are numerous hypermethylation events which occur in STICs and are maintained in the resultant HGSCs, relative to matched normal fallopian tube (NFT) tissues. Indeed we have selected four hypermethylated target regions which show strong discriminatory power between formalin-fixed paraffin-embedded (FFPE) HGSC and NFT tissues. We propose that developing a test capable of measuring these methylation changes in circulating tumour DNA (ctDNA) from blood could be a promising new approach in the early detection and diagnosis of HGSCs. Aims: 1. Development of a robust MS-PCR test for detecting cfDNA in sera 2. MeDIP-sequencing of ctDNA 3. Validation methylation markers in independent clinical samples Methods: 1. Probe-based Methylation-Specific PCR (MS-PCR) and Methylation Specific Restriction Endonuclease PCR (MSRE-PCR) and Digital Droplet PCR (DD-PCR) approaches will be developed for all 4 markers. 2. DNA Methylation (DNAme) immunoprecipitation followed by deep sequencing (meDIP-seq) will be performed with a collaborator on ctDNA derived from ongoing collections at both sites. 3. Prospective collections between the two sites will be organised. Markers identified by meDIP-seq will be pyrosequenced and tested by PCR assays (optimised in Aim 1) on independent ctDNA samples, both as early warning markers and as MRD markers (for longitudinal collections). How results will be used: Data generated from this primer award would form the basis for future project (and possibly programme) grant applications to CRUK (and other funding bodies). Ultimately DNAme markers could be developed as an early detection test for HGSC.

Lay abstract: Background: Currently there is no effective screening method for ovarian cancer. USPLCO and UKCTOCS trials in the US and UK, respectively, have shown little benefit in combining ultrasound scanning and serum CA-125 for early diagnosis. Serum CA-125 is most effective as a marker of disease status in patients undergoing chemotherapy treatment for epithelial ovarian cancer (EOC). It is not specific to malignancy, expressed in several benign conditions including endometriosis, pelvic infection, and uterine leiomyomata. In addition, the current CA-125 assay lacks sensitivity and is elevated in only 50% of early stage EOCs. Recent evidence suggests that the most common and most aggressive form of EOC, High Grade Serous Carcinoma (HSGC), arise from the distal fallopian tube and not the ovary, with a proposed precursor lesion being serous tubal intraepithelial carcinoma (STIC). Early detection of STICs would be a major advance in HGSC management since the majority of HGSCs present as advanced (FIGO stage 3/4) disease. We have preliminary data which shows that there are numerous hypermethylation events which occur in STICs and are maintained in the resultant HGSCs, relative to matched normal fallopian tube (NFT) tissues. Indeed we have selected four hypermethylated target regions which show strong discriminatory power between formalin-fixed paraffin-embedded (FFPE) HGSC and NFT tissues. We propose that developing a test capable of measuring these methylation changes in circulating tumour DNA (ctDNA) from blood could be a promising new approach in the early detection and diagnosis of HGSCs. Aims: 1. Development of a robust MS-PCR test for detecting cfDNA in sera 2. MeDIP-sequencing of ctDNA 3. Validation methylation markers in independent clinical samples Methods: 1. Probe-based Methylation-Specific PCR (MS-PCR) and Methylation Specific Restriction Endonuclease PCR (MSRE-PCR) and Digital Droplet PCR (DD-PCR) approaches will be developed for all 4 markers. 2. DNA Methylation (DNAme) immunoprecipitation followed by deep sequencing (meDIP-seq) will be performed with a collaborator on ctDNA derived from ongoing collections at both sites. 3. Prospective collections between the two sites will be organised. Markers identified by meDIP-seq will be pyrosequenced and tested by PCR assays (optimised in Aim 1) on independent ctDNA samples, both as early warning markers and as MRD markers (for longitudinal collections). How results will be used: Data generated from this primer award would form the basis for future project (and possibly programme) grant applications to CRUK (and other funding bodies). Ultimately DNAme markers could be developed as an early detection test for HGSC.

Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CANCER AND NEOPLASMS