Circulating tumor DNA for predicting complete response to total neoadjuvant therapy in locally advanced rectal cancer: ENSEMBLE-2.
Authors: Watanabe Jun , Kagawa Yoshinori , Ando Koji , Uemura Mamoru , Fujimoto Yoshiaki , Suwa Yusuke , Nishizawa Yujiro , Matsuhashi Nobuhisa , Izawa Naoki , Muto Osamu , Kinjo Tatsuya , Miyo Masaaki , Laliotis George , Nakamura Yoshiaki , Bando Hideaki , Takemasa Ichiro , Oba Koji , Yoshino Takayuki , Oki Eiji 
Published: January 2025

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Background:
Total neoadjuvant therapy (TNT) has dramatically shifted the paradigm in the treatment of locally advanced rectal cancer (LARC), prolonging survival with high rates of pathologic complete response (pCR) and introducing non-operative management (NOM). However, no predictive biomarkers of TNT efficacy, the regrowth of NOM, or prognosis have been developed. Circulating tumor DNA (ctDNA) is a minimally invasive biomarker used to detect molecular residual disease (MRD) and predict recurrence in colon cancer after curative resection. The effectiveness of ctDNA MRD status as a predictive biomarker for TNT was evaluated in the Phase II TNT study, ENSEMBLE-2 (jRCTs071210143), conducted in Japan.
Methods:
Patients with LARC undergoing TNT were enrolled in ENSEMBLE-2. Protocol treatment was defined as total mesorectal excision (TME) following long course chemoradiotherapy (LCCRT: 50.4Gy, capecitabine) plus four cycles of CAPOX. NOM was allowed if a clinical complete response (cCR) was achieved in the evaluation after TNT. ctDNA MRD was measured by Signatera (Natera, Inc.) at the following time points: baseline, after LCCRT, after TNT, post operative 4w, 12w, 24, 36 and 48w in the GALAXY trial (UMIN000039205).
Results:
A total of 28 patients were enrolled in the study. Treatment was discontinued at the patient's request in one case. After completing TNT, TME and NOM were performed in 21 (77.8%) and 6 (21.4%) patients, respectively. ctDNA positivity rates were 96.4. % (27/28) at baseline, 14.8% (4/27) after LCCRT, and 34.6 % (9/26) after TNT. Post-LCCRT ctDNA status was not significantly associated with cCR + near CR (nCR) vs. incomplete clinical response (iCR), pCR vs. non-pCR (p=0.065 and p=0.539, respectively). In contrast, ctDNA status after TNT was significantly associated with cCR + nCR vs. iCR (p=0.028), as well as pCR vs. non-pCR (p=0.038).
Conclusions:
Our study indicates that post TNT ctDNA status may be a predictive biomarker for TNT response in LARC patients. ctDNA negativity upon completion of neoadjuvant treatment may indicate a favorable response.
Clinical trial information: jRCTs071210143
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Correlation with ctDNA, clinical response, treatment after TNT and pathological response.

After LCCRT
p value
After TNT
p value

Clinical ResponsecCR + nCR vs. iCR (ctDNA -/+)
cCR + nCR 18 / 1
0.065
cCR + nCR 15 / 4
0.028

iCR 5 / 3
iCR 2 / 5

Treatment after TNTNOM vs. TME (ctDNA -/+)
TME 17 / 3
0.438
TME 11 / 9
0.063

NOM 6 / 0
NOM 6 / 0

Pathological response after TMEpCR vs. non pCR (ctDNA -/+)
pCR 5 / 0
0.539
pCR 5 / 0
0.038

non pCR 12 / 3
non pCR 6 / 9

Fisher's exact test was used to statistically examine the correlation between ctDNA MRD status and the factors at each timing.