Detection of methylated BCAT1 and IKZF1 in circulating tumor DNA is specific for colorectal cancer but not for breast or prostate cancer

Background and Aim: Assays for circulating tumor DNA (ctDNA) biomarkers have shown utility for cancer detection and management. Methylated BCAT1 and IKZF1 are ctDNA biomarkers that have high sensitivity for colorectal cancer (CRC), but it is not clear if these biomarkers are also associated with other common cancers. The aim of this study was to investigate the sensitivity and utility of detecting methylated BCAT1 and IKZF1 in the ctDNA of patients with breast or prostate cancer.

Methods: Blood was collected from patients diagnosed with colorectal, breast, or prostate cancer, before receiving any treatment, at Flinders Medical Centre, South Australia. In addition, blood was collected from healthy controls who had been confirmed to have no evidence of CRC at colonoscopy. Circulating cell-free DNA isolated from plasma was bisulfite converted and assayed for methylation in BCAT1 and IKZF1. Logistic regression was used to assess ctDNA test positivity for BCAT1 and IKZF1 DNA methylation compared with healthy controls. Associations between assay positivity and tumor staging were also assessed by Poisson or logistical regression. To further explore the BCAT1 and IKZF1 DNA methylation levels in these cancers, 450 K DNA methylation array data from The Cancer Genome Atlas (TCGA) were assessed for matched tumor and normal samples from the PRAD (n = 50), BRCA (n = 90), and COADREAD (n = 44) cohorts. DNA methylation was interrogated at three out of 20 CpG sites encompassed in the BCAT1/IKZF1 ctDNA quantitative polymerase chain reaction assay (cg10764357 and cg07589773/cg18607529, respectively). Logistical regression of BCAT1/IKZF1 methylation for the different cancer types and tumor staging was also performed for all tumor cases with American Joint Committee on Cancer staging data (PRAD, 503; BRCA, 788; and COADREAD, 284).

Results: The study included 290 blood samples from patients diagnosed with CRC, 32 with breast cancer, 101 with prostate cancer, and 727 healthy controls. Only 7/101 prostate cancer (6.9%) and 3/32 breast cancer (9.4%) blood samples were positive for BCAT1/IKZF1 DNA methylation. The assay positivity rate was not significantly different to that of healthy controls (8.0% for females vs breast cancer; odds ratio [OR], 0.84; 95% CI, 0.23–3.1; and 7.6% for males vs prostate cancer; OR, 0.86; 95% CI, 0.65–2.1). In patients with CRC, the assay had a 60.3% positivity rate (175/290), which was significantly higher than that in healthy controls (7.7%, 56/727; OR, 18.2; 95% CI, 11.1–29.0). A higher assay positivity rate was observed in more advanced CRC disease, with overall stage IV cases having an assay positivity of 90.7% (39/43) compared with 23.7% (14/59) for stage I (OR, 21.9; 95% CI, 9.4–103.5). TCGA data showed that BCAT1 and IKZF1 were significantly hypermethylated in prostate and colorectal tumors compared with matched normal tissues (P < 0.0001) but were no different for breast tumors versus normal tissue (P = 0.854 for BCAT1 and P = 0.172 for IKZF1). There was no correlation between cancer staging and level of tissue DNA methylation in IKZF1 and BCAT1 for breast, prostate, or colorectal cases in the TCGA dataset.

Conclusion: Circulating tumor DNA methylated in BCAT1 and IKZF1 is specific for CRC and is not sensitive for detection of breast or prostate cancer, despite a subset of the targeted CpG sites in these genes being hypermethylated in prostate tumors. This supports the use of methylated BCAT1 and IKZF1 for CRC detection and monitoring.