Assessment of circulating tumor DNA in patients with locally advanced rectal cancer treated with neoadjuvant therapy

Abstract

Post-neoadjuvant therapy (post-NAT) and post-surgical circulating tumor DNA (ctDNA) risk stratification may enhance the management of patients with locally advanced rectal cancer (LARC). In this study, we assessed the prognostic value of ctDNA-based MRD detection in LARC patients using a personalized, tumor-informed ctDNA assay. Plasma samples from LARC patients (N = 30) were analyzed retrospectively using the Signatera™ assay. The neoadjuvant rectal (NAR) score was calculated and compared to ctDNA status to predict recurrence risk and survival outcomes. ctDNA-positive patients post-NAT and post-surgery had worse Disease Free Survival (DFS) (HR: 7.82; p = 0.001, HR: 19.65; p = 0.001) when compared to ctDNA-negative patients. In the post-NAT setting, patients who responded to NAT had superior DFS compared to patients who did not clear their ctDNA or showed no radiological response (HR: 24.7, p = 0.001 and HR: 5.1, p = 0.054, respectively). When ctDNA status is used alongside the NAR score in the post-NAT setting, patients who were ctDNA-positive with an intermediate or high NAR score showed significantly worse DFS (HR: 47.5, p < 0.001) compared to ctDNA-negative patients with either a low or intermediate/high NAR score (HR: 9.8, p = 0.0301). Post-NAT ctDNA status, whether used alone or in combination with the NAR score, may predict NAT response, and improve risk stratification.

Keywords: Biomarker; Circulating tumor DNA; Locally advanced rectal cancer; NAR score; Neoadjuvant therapy.

Fig. 1

(A) Overview plot showing longitudinal ctDNA status, treatment regimen, and clinical outcomes for patients with stage II–III rectal cancer. (B) Heat map showing clinicopathological and ctDNA detection data for patients. ACT adjuvant chemotherapy, ctDNA circulating tumor DNA, NAR neoadjuvant rectal score, NAT neoadjuvant therapy, pCR pathologic complete response.

Fig. 2

(A) Kaplan–Meier estimates of patients with stage II–III rectal cancer representing disease-free survival stratified by ctDNA status (negative or positive) post-NAT at week 11. (B) Kaplan–Meier estimates of disease-free survival stratified by ctDNA status (positive or negative) and radiological response post-NAT, prior to surgery (left panel). Correlation of ctDNA clearance post-NAT with radiological relapse (right panel). (C) Kaplan–Meier estimates of disease-free survival stratified by ctDNA status post-NAT and rNAR score. (D) Kaplan–Meier estimates of disease-free survival stratified by ctDNA status post-NAT and pCR status. The HRs and 95% CIs were calculated using the Cox proportional (Fig. 2A-B-C) and Cox Regression with Firth’s Penalized Likelihood (D) hazard model. The P value was obtained with Fisher’s exact test whereas the OR and 95% CIs with simple logistic regression. ctDNA circulating tumor DNA, Cl confidence interval, DFS disease-free survival, HR hazard ratio, NAT neoadjuvant therapy, pCR pathologic complete response, + positive, − negative.

Fig. 3

(A) Kaplan-Meier estimates of patients with stage II–III rectal cancer representing NAR scores stratified by DFS. (B) Association between post-NAT ctDNA status and NAR score (i) and rates of recurrence based on patient NAR scores (low, intermediate, or high) and ctDNA status post-NAT (ii). The p-value for the association between post-NAT ctDNA and NAR score was assessed with the Wilcoxon rank sum two-sided test (left panel) and Fisher’s exact test (right panel). C. Kaplan-Meier estimates of patients with stage II–III rectal cancer representing disease-free survival stratified by post-NAT NAR scores and ctDNA status. HRs and 95% CIs were calculated using Cox Regression with Firth’s Penalized Likelihood (A,C) hazard model. P values were calculated using the two-sided log-rank test. ctDNA circulating tumor DNA, Cl confidence interval, DFS disease-free survival, HR hazard ratio, NAR neoadjuvant rectal score, NAT neoadjuvant therapy, + positive, − negative.

Fig. 4

Kaplan-Meier estimates of patients with stage II–III rectal cancer representing disease-free survival stratified by (A) ctDNA status at the MRD time point (week 2–12 post-surgery). (B) ctDNA dynamics during the post-NAT to MRD time point. To account for immortal time bias, a landmark analysis was performed 12 weeks after surgery. (C) Multivariate and univariate analysis of prognostic factors and their association with DFS, as indicated by HR, analyzed across the cohort; HR hazard ratio. ctDNA circulating tumor DNA, Cl confidence interval, DFS disease-free survival, HR hazard ratio, MRD molecular residual disease, NAR neoadjuvant rectal score, NAT neoadjuvant therapy.