Effect of chemoradiation treatment on methylated <scp> <i>BCAT1</i> </scp> and <scp> <i>IKZF1</i> </scp> in rectal cancer

A circulating tumor DNA (ctDNA) assay for methylated BCAT1/IKZF1 can detect rectal adenocarcinoma, but its efficacy after neoadjuvant chemoradiotherapy-commonly used for patients with rectal cancer-is unknown. We determined the effect of radiation treatment on BCAT1/IKZF1 methylation in colorectal cancer (CRC) cell lines and rectal cancer tissues, and determined the value of a ctDNA assay measuring methylated BCAT1/IKZF1 for accurately assessing response to neoadjuvant chemoradiotherapy in patients with rectal cancer. Proof-of-principle experiments assessed CRC cell lines for BCAT1/IKZF1 methylation after irradiation and 5-fluorouracil treatment. Tissue and pre-surgical blood samples were collected from 34 patients diagnosed with rectal cancer, following or without any neoadjuvant treatment, and assayed for BCAT1/IKZF1 DNA methylation using multiplex qPCR. Tissues were scored for histological response, with BCAT1/IKZF1 DNA methylation compared between untreated patients, poor responders, and good responders. Radiation treatment did not affect BCAT1/IKZF1 methylation of CRC cells in vitro. BCAT1/IKZF1 methylation reduced in rectal adenocarcinoma tissues in response to neoadjuvant treatment. Tumor tissue from good responders contained low levels of methylated BCAT1/IKZF1, equivalent to matched normal tissue. The methylated BCAT1/IKZF1 assay had 87% (13/15) sensitivity for rectal cancer in untreated patients. Reduced methylated BCAT1/IKZF1 ctDNA levels were correlated with neoadjuvant therapy-related tumor regression. Radiation does not affect BCAT1/IKZF1 methylation in cancer cells, independent of treatment response. Reduced BCAT1/IKZF1 methylation in rectal tumors and plasma ctDNA reflects reductions in tumor cellularity. These data support future investigations to use methylated BCAT1/IKZF1 ctDNA for monitoring response to neoadjuvant therapy in patients with rectal cancer.