Circulating Tumor DNA for Prediction of Complete Pathological Response to Neoadjuvant Radiochemotherapy in Locally Advanced Rectal Cancer (NEORECT Trial)

Abstract

Background/objectives: Locally advanced rectal cancer is treated with neoadjuvant chemoradiotherapy (nCRT) followed by total mesorectal excision (TME). As this approach achieves complete pathologic remissions (pCR) in approximately 30% of patients, it raises the question of whether surgery is always necessary. Non-surgical strategies, such as "watch and wait" (W&W), have shown similarly promising outcomes. However, there is an unmet need for reliable biomarkers predicting pCR. Analysis of circulating tumor DNA (ctDNA) has shown potential for monitoring treatment response and detecting minimal residual disease. We hypothesized that monitoring ctDNA changes during nCRT might facilitate the identification of individuals who achieve pCR.

Methods: In the prospective single-center NEORECT trial, the plasma of forty rectal cancer patients was collected before, during, and after nCRT and before TME. Informative somatic mutations were identified in tissue biopsies by NGS and subsequently used for ctDNA quantification by dPCR.

Results: The results identified three distinct ctDNA patterns: increase, decrease, and absence. Remarkably, undetectable DNA was observed in good responders, while a tenfold ctDNA increase was associated with the emergence of new metastases. Despite these insights, ctDNA alone demonstrated low specificity, with no significant correlation to pCR or long-term prognosis. A multimodal approach incorporating routinely available clinical parameters remains inadequate for accurately predicting pCR prior to TME.

Conclusions: In conclusion, the NEORECT trial establishes the feasibility of ctDNA-based personalized monitoring for rectal cancer patients undergoing nCRT. However, the utility of ctDNA in enhancing pCR prediction for a W&W strategy warrants further investigation. Larger studies integrating multi-gene analyses and expanded clinical datasets are essential in the future.

Keywords: circulating tumor DNA; complete pathological response; neoadjuvant radiochemotherapy; rectal cancer; response prediction.

Figure 1

Patient disposition in the NEORECT trial. Of the 40 patients intended to be treated, 26 had a complete dataset after surgery. Due to technical limitations, 18 individuals were eligible for ctDNA tracing in plasma samples.

Figure 2

CtDNA and cfDNA dynamics during treatment of the 18 patients of the NEORECT trial. Dot lines depict ctDNA dynamics as genome equivalents per milliliter plasma (GE/mL plasma, left y-axis) and gray bars show cfDNA as GE/mL plasma (right y-axis). Individual headlines describe the patient ID of the NEORECT trial and its mutated gene and hotspot traced by dPCR as well as the VAFs detected by NGS from the initial biopsy in brackets. Three groups can be defined based on ctDNA dynamics: Undetectable ctDNA at any timepoint (A), increment of ctDNA towards V4 compared at any timepoint during nCRT (B), and overall ctDNA decreasing over the course of therapy (C).

Figure 3

Multimodal disease evaluation before and after surgery based on different clinical and pathological parameters and LBx (n = 18) (A) Yellow: pathological assessment after surgery; green: clinical assessment before surgery; purple: LBx assessment; pCR: pathological complete remission; SR: subtotal remission; CEA: carcinoembryonic antigen. (B) pairwise Pearson correlation of all parameters. Blue depicts a positive correlation and red a negative correlation, respectively. No significant coefficients are shown as blank.

Figure 4

Composite score of clinical diagnostic modalities and ctDNA before surgery (V4). (A) Forming a pre-surgical scoring based on clinical parameters assessed in the context of clinical routine diagnostics and ctDNA status at V4 (NEORECT scoring). CEA: carcinoembryonic antigen; n.a: not assessable/not available; preOP: before surgery. (B) NEORECT scoring of individual participants. Coloring is dependent on pathologically classified Dworak scoring after surgery as depicted for Dworak 4 to 1.

Figure 5

Dynamics of LBx (ct and cfDNA) in a patient with metastatic recurrence during nCRT and a liver-first approach. Both ctDNA (dotted line) and cfDNA (gray bars) are described as genome equivalents per milliliter plasma (GE/mL plasma). The headline describes the patient ID and the mutated gene and hotspot traced by dPCR as well as the VAFs detected by NGS in the respective tissue in brackets (primary tissue/liver metastasis/resected specimen). nCRT: neoadjuvant chemoradio therapy; L. ex: excision of liver metastases; TME: total mesorectal excision.

Figure 6

Disease-free survival (DFS) stratified by histological response and ctDNA measurements. Patients were stratified (A) by histopathological response status, (B) by ctDNA dynamics, (C) depending on V1 ctDNA (even distribution), and (D) depending on V4 ctDNA. Statistical significance was tested based on the log-rank method. p-Values under 0.05 are defined as statistically significant.