Circulating tumor-derived DNA (ctDNA) clearance in patients with locally advanced rectal cancer treated with multimodal treatment

The management of locally advanced rectal cancer (LARC) relies on a multimodal approach, including chemotherapy, radiation, and surgery. Neither instrumental work-up nor molecular biomarkers are currently available to identify a risk-adapted strategy. Circulating tumor DNA (ctDNA) is a fast-growing and promising tool potentially improving patient stratification and prognosis. We aim to investigate the role of ctDNA and its clearance at different timepoints during chemo-radiotherapy (CT-RT) and correlate with clinical outcomes.

We conducted a prospective observational study enrolling patients with LARC managed with neoadjuvant CT-RT (capecitabine 1650 mg/m2 concomitant with 50,4 Gy pelvic long course radiotherapy), surgery at IEO between November 2014 and November 2019. Blood samples for ctDNA were obtained at pre-planned timepoints: baseline (T0), end of chemoradiation (T1), after surgery (T2), end of adjuvant chemotherapy (T3). ctDNA was extracted from plasma and mutations detected on tumor biopsy (KRAS, NRAS, BRAF, PIK3CA) were evaluated on ctDNA. The primary endpoint was ctDNA clearance, defined as 100% decrease of variant allele frequency (VAF) in a target mutation, assessed at T1, T2 and T3 (clearance 1 [C1], C2 and C3, respectively); it was analyzed for correlation with pathologic complete response (pCR) down-staging, event-free survival (EFS), and overall survival (OS). Survival was calculated using Kaplan–Meier method and log-rank tests. Statistical analyses were performed using SPSS software (version 28.0.1.0).

We enrolled 115 patients. Sixty (52%) ones had tumoral mutations on tissue and underwent liquid biopsy. Rectal primary site was distal in 28 (47%), middle in 23 (38%) and proximal in 9 (15%). Fifty-six (93%) had a T3 tumor and 51(85%) had clinical node-positive disease. Twelve (20%) patients obtained pCR and 48 (80%) down-staging. Moreover, thirty-four (57%) received adjuvant chemotherapy. After a median follow-up of 82 months, median EFS was 91.8 months (95% CI, 61.2-137.6) and median OS was not reached; the 3-year EFS and OS rates were of 83% and 87%, respectively. Liquid biopsy at T0, T1, T2 and T3 was available for 60 (100%), 57 (95%), 28 (47%) and 9 (15%) patients, respectively. The median ctDNA value at T0 was 0.11 ng/2ml (range: 0.00-17.40). C1, C2 and C3 were observed in 16/57 (28%), 9/48 (19%) and 2/29 (7%) patients. Twenty-five patients (42%) obtained ctDNA clearance at least in one timepoint. No statistically significant associations emerged between ctDNA clearance and pCR, down-staging, EFS and OS. The 3-year EFS rate was of 92% for patients with at least one ctDNA clearance and 79% for patients with no clearance (Hazard Ratio [HR]: 0.5; 95% CI: 0.1-2.4); considering any single timepoint the HR for EFS was 1.1 (95% CI, 0.3-3.1) for C1, 0.6 (95% CI, 0.1-2.9) for C2, and 1.1 (95% CI, 0.1-9.1) for C3.

In patients with ctDNA clearance at any timepoint the 3-year EFS rate was longer compared with patients without clearance, although not statistically significant. Its determination after surgery may represent the most informative timepoint for survival assessment. Larger prospective translational studies using deeper molecular techniques are required to define the role of ctDNA analysis to guide the multimodal treatment in LARC.

The authors.

Has not received any funding.

D. Ciardiello: Travel / Accommodation / Expenses: Sanofi, BMS, Mrck Serono. G. Curigliano: Advisory / Consultancy: Roche, BMS, Pfizer, Seagen, Lilly, Novartis, Astra Zeneca, Daichii Sankyo, Gilead, Ellipsis.; Speaker Bureau / Expert testimony: Pfizer; Leadership role: ESMO Clinical Practice Guidelines Chair; Research grant / Funding (self): Merck. All other authors have declared no conflicts of interest.