Application of a colorectal cancer ctDNA methylation test in endocrine cancers.

Abstract

e15533 Background: Assays for circulating tumor DNA (ctDNA) have shown utility for cancer detection and management. It is important to demonstrate cancer specificity of targeted ctDNA biomarkers. Detection of methylated BCAT1 and IKZF1 ctDNA has shown high sensitivity for colorectal cancer (CRC). The aim of this study was to investigate whether hypermethylation of these two biomarkers is also present in prostate or breast adenocarcinoma (PCa or BCa) tissues, and if they are detectable as ctDNA from such cancer patients. Methods: A subset of TCGA data comprising matched tumor and normal samples from PRAD (n = 50), BRCA (n = 90) and COADREAD (n = 44) cohorts were accessed to interrogate DNA methylation (450K) at CpG probes covered by the BCAT1/ IKZF1 ctDNA assay (cg10764357 and cg07589773/cg18607529, respectively). Linear regression of BCAT1/ IKZF1 methylation and tumor staging was performed for cases with AJCC staging data (PRAD n = 503; BRCA n = 788; COADREAD n = 284). Blood was collected from BCa (n = 32) or PCa (n = 101) patients prior to starting treatment at Flinders Medical Centre (South Australia). DNA isolated from plasma was bisulphite-converted and assayed for BCAT1/ IKZF1 methylation. Age-adjusted comparisons of ctDNA positivity were made with blood results from N = 310 pre-treatment CRC patients (60% male), as well as male (N = 344) and female (N = 383) controls with a clear colonoscopy and no history of cancer. Results: BCAT1 and IKZF1 were significantly hypermethylated in PCa and CRC tumors compared to matched normal tissues (Table). Conversely, BCAT1/ IKZF1 CpG probes were hypomethylated in breast tumors compared to matched normal samples. There was no correlation between hypermethylation in IKZF1/ BCAT1 and stage of PCa or CRC. In contrast to PCa in silico findings of hypermethylation, BCAT1/IKZF1 ctDNA methylation was only positive in 7/101 PCa blood samples (6.9%; 95%CI 3.2-13.9%). Three of 32 BCa blood samples (9.4%; 95%CI 2.5-25%) were positive for BCAT1/IKZF1 methylation. These positivity rates were not significantly different to controls at 8.5% for females (p = 0.80 vs BCa) and 8.2% for males (p = 0.73 vs PCa), but all were significantly lower than that observed in CRC cases (59.0%, 183/310; p < 0.001). Conclusions: While interrogation of TCGA datasets revealed IKZF1 and BCAT1 hypermethylation in prostate tumors, detection rate of these biomarkers in blood from PCa patients was not higher than that of non-cancer controls. Likewise, the BCAT1/IKZF1 ctDNA assay positivity rate in BCa patients was also no different to non-cancer controls. These findings highlight the specificity of methylated BCAT1 and IKZF1 ctDNA for detection of CRC.[Table: see text]