Assessment of tumor burden and response to therapy in patients with colorectal cancer using a quantitative ctDNA test for methylated BCAT1/IKZF1

Abstract

Failure of colorectal cancer (CRC) treatment is due to residual disease, and its timely identification is critical for patient survival. Detecting CRC-associated mutations in patient circulating cell-free DNA is confounded by tumor mutation heterogeneity, requiring primary tumor sequencing to identify relevant mutations. In this study, we assessed BCAT1 and IKZF1 methylation levels to quantify circulating tumor DNA (ctDNA) and investigated whether this method can be used to assess tumor burden and efficacy of therapy. In 175 patients with CRC who were ctDNA-positive pretreatment, ctDNA levels were higher with advancing stage (P < 0.05) and correlated with tumor diameter (r = 0.35, P < 0.001) and volume (r = 0.58, P < 0.01). After completion of treatment (median of 70 days [IQR 49-109] after surgery, +/- radiotherapy, +/- chemotherapy), ctDNA levels were reduced in 98% (47/48) and were undetectable in 88% (42/48) of patients tested. For those with incomplete adjuvant chemotherapy after surgery, roughly half remained ctDNA-positive (11/21, 52.4%). The presence of ctDNA after treatment was associated with disease progression (HR 9.7, 95%CI 2.5-37.6) compared to no ctDNA. Assaying blood for ctDNA methylated in BCAT1/IKZF1 has the potential for identifying residual disease due to treatment failure, informing a potential need for therapy adjustment in advanced disease.

Keywords: BCAT1; IKZF1; circulating tumor DNA; colorectal cancer; efficacy; methylation.

Fig. 1

Disposition of cases and how they were selected for the main analyses. cfDNA: cell‐free DNA; ctDNA: circulating tumor DNA; MRI: magnetic resonance imaging; CT: computed tomography.

Fig. 2

Relationship between the amount of methylated ctDNA in circulation and (A) maximum tumor diameter in patients with CRC stages I and II (n = 66, r = 0.359, P = 0.003), (B) estimated tumor volume in patients with CRC stages I and II (n = 50, r = 0.586, P < 0.001), (C) sum of maximum tumor diameters (primary and evident metastatic sites) in patients with CRC stages III and IV (n = 85, r = 0.319, P = 0.003), and (D) sum of maximum tumor diameters (primary and evident metastatic sites) in all patients (n = 152, r = 0.321, P < 0.001). Statistical correlations were performed with Pearson correlation analysis.

Fig. 3

Levels of methylated BCAT1/IKZF1 (% methylation—see Methods) in patients with CRC before and after cessation of treatment with (A) surgery (n = 30); (B) neoadjuvant therapy, surgery, and adjuvant chemotherapy (n = 6); (C) surgery and adjuvant chemotherapy (n = 12); and (D) incomplete adjuvant chemotherapy (n = 21). Each marker with the joining line represents the methylation levels before and after treatment. The pink markers indicate patients that had detectable ctDNA levels pre‐ and post‐treatment.

Fig. 4

Kaplan–Meier curves for progression‐free survival stratified according to post‐treatment detection of ctDNA (methylated BCAT1/IKZF1); positive ctDNA, n = 13, negative ctDNA, n = 42. Hazard ratio for disease progression for those with a post‐treatment positive ctDNA was 9.69, 95% confidence interval 2.50‐37.59. Statistical analysis was with Cox regression analysis.