Circulating tumor DNA is increasingly being explored as a way to refine response assessment in rectal cancer, particularly when organ preservation is being considered. In early-stage, node-negative disease, where tumor burden is relatively low, whether ctDNA can provide clinically useful information before and after neoadjuvant treatment remains an important question.
A new analysis from the phase II Canadian Cancer Trials Group CO.28 NEO trial examined a tissue-free epigenomic ctDNA assay in patients with node-negative rectal cancer treated with neoadjuvant chemotherapy followed by transanal excision. The study explored whether ctDNA detection could provide additional information alongside established clinical assessments when evaluating response and the need for total mesorectal excision. The study was published online on October 2, 2026, in JCO Precision Oncology.
Title: “Tumor-Free Circulating Tumor DNA in Node-Negative Rectal Cancers Undergoing Neoadjuvant Chemotherapy, Excision, and Observation in the Phase II NEO Trial (CO.28)”
Authors: Jonathan M. Loree, MD, MS, Emma Titmuss, MSc, Carl J. Brown, MD, Thereasa Rich, MS, Kimberly Banks, MS, Vallerie Gordon, MD, Sunil Patel, MD, Marianne Krahn, MD, Antonio Caycedo-Marulanda, MD, Manoj Raval, MD, Dongsheng Tu, PhD, Derek J.Jonker, MD, Christopher J. O’Callaghan, PhD, DVMD, and Hagen F. Kennecke, MD
Organ Preservation in the NEO Trial
Organ preservation is an important consideration in early-stage rectal cancer because TME can affect quality of life through changes in bowel and sexual function and the potential need for temporary or permanent stomas. The phase II CCTG CO.28 NEO trial previously evaluated an organ-preserving strategy in patients with cT1-T3ab, node-negative, well- or moderately differentiated rectal adenocarcinoma. Patients received 3 months of neoadjuvant capecitabine plus oxaliplatin or infusional fluorouracil, leucovorin, and oxaliplatin, without radiotherapy, followed by transanal endoscopic surgery.
One challenge with organ-preservation strategies is determining whether the tumor has responded sufficiently to safely avoid more extensive surgery. Response assessment and surveillance typically rely on MRI, endoscopy, physical examination, and CT imaging. The current analysis explored whether ctDNA could provide additional information alongside these established assessments.
Tissue-Free ctDNA Analysis
Of the 58 patients enrolled in CO.28, 53, or 91.4%, had at least one successfully sequenced ctDNA sample available for analysis. Blood samples were collected before chemotherapy, after chemotherapy and before transanal endoscopic surgery, and during follow-up at 12, 24, and 36 months. Samples at disease progression were also obtained when available.
The investigators used Guardant Reveal, a tissue-free epigenomic assay assessing more than 20,000 regions for ctDNA detection. Prechemotherapy samples were available from 48 patients, while 46 had samples available after chemotherapy and before surgery. Forty-one patients had samples at both time points.
ctDNA Detection Decreased After Chemotherapy
Before chemotherapy, ctDNA was detected in 22 of 48 patients, corresponding to a detection rate of 45.8%. Detection did not significantly differ according to clinical T stage, although numerically higher detection rates were observed in patients with T2 and T3 disease compared with T1 disease.
Following neoadjuvant chemotherapy, ctDNA detection decreased substantially. Only 4 of 46 patients, or 8.7%, had detectable ctDNA after chemotherapy and before transanal excision, compared with 45.8% before treatment (P < .0001). Among the 41 patients with paired samples, 20 had no detectable ctDNA at either time point. Eighteen had a reduction in estimated tumor fraction after chemotherapy, and 17 of these 18 patients completely cleared detectable ctDNA. Three patients had an increase in estimated tumor fraction.
The largest reduction in ctDNA detection was seen among patients with clinical T2 tumors, where detection decreased from 53.1% before chemotherapy to 3.6% after treatment.
Postchemotherapy ctDNA and Recommendation for TME
The association between ctDNA after chemotherapy and subsequent recommendation for TME was one of the main findings of the analysis. Overall, 23 patients were recommended TME because of insufficient response to chemotherapy or high-risk features identified at transanal excision.
All four patients with detectable ctDNA after chemotherapy were recommended TME, compared with 16 of 42 patients, or 38.1%, without detectable ctDNA (P = .03). Only two of the four ctDNA-positive patients ultimately underwent TME. The other two declined the recommended surgery.
Importantly, baseline ctDNA status did not show the same relationship. TME was recommended in 50.0% of patients with detectable pretreatment ctDNA and 38.5% of patients without detectable pretreatment ctDNA, with no significant difference between the groups. The findings therefore suggest that ctDNA measured after neoadjuvant chemotherapy may warrant further study as a supplemental tool for treatment-response assessment.
What Did ctDNA Show During Follow-Up?
Five patients developed clinical recurrence during follow-up, including two local and three distant recurrences. The study did not demonstrate a statistically significant association between ctDNA detection and recurrence-free survival.
Among patients assessed before chemotherapy, 3-year recurrence-free survival was 90.9% with detectable ctDNA versus 88.5% without detectable ctDNA. The hazard ratio was 0.79, with a 95% confidence interval of 0.13–4.75 (P = .80). At the postchemotherapy time point, 3-year recurrence-free survival was 75.0% versus 90.5% in patients with and without detectable ctDNA, respectively. The hazard ratio was 2.82, with a wide 95% confidence interval of 0.31–25.29 (P = .36).
The small number of recurrences limited the ability to evaluate the prognostic value of ctDNA. Among the two patients with local recurrence who had samples obtained at progression, ctDNA was detected in one. No progression ctDNA samples were available for the three patients who developed distant recurrence. Notably, none of the 90 longitudinal samples collected from patients who remained recurrence-free had detectable ctDNA.
Tissue-Free vs Tumor-Informed ctDNA
The investigators had previously evaluated a tumor-informed ctDNA assay in the same CO.28 population. In that analysis, pretreatment ctDNA was detected in 12 of 49 patients, or 24.5%. In the current study, the tissue-free assay detected ctDNA in 45.8% of patients before chemotherapy.
A tissue-free approach may also have practical advantages in patients with limited diagnostic tissue because it does not require tumor tissue to construct an individualized assay. Approximately 10% of patients in this cohort had previously been found to have insufficient diagnostic tissue for development of a tumor-informed panel.
The authors noted that findings from ctDNA studies should be continually reassessed as assay performance changes, while further studies are needed to establish the clinical utility of tissue-free ctDNA in this setting.
Where Could ctDNA Fit Into Organ Preservation?
The study provides early evidence that tissue-free ctDNA can be detected in node-negative rectal cancers despite the relatively low tumor burden in this population. More importantly, detectable ctDNA after chemotherapy was associated with inadequate treatment response and recommendation for TME.
This does not mean that ctDNA can currently determine whether a patient should proceed with organ preservation or radical surgery. The ctDNA analysis was retrospective and does not establish a ctDNA-guided treatment strategy. Rather, ctDNA could be further studied as a supplemental tool alongside MRI, endoscopy, and other clinical assessments.
Study Limitations
The analysis was limited by its small cohort and low number of recurrence events. Only 53 patients had ctDNA samples available, and five recurrences occurred during follow-up. Longitudinal sampling was also incomplete, including the absence of progression samples in the patients who developed distant recurrence.
These limitations reduced the ability to determine the sensitivity of ctDNA for detecting recurrence or to establish its prognostic value. The authors therefore called for larger studies, ideally prospective interventional trials with longer follow-up, to determine whether ctDNA can improve decision-making in patients being considered for organ preservation.
What Comes Next?
The NEO trial analysis showed that tissue-free ctDNA detection decreased from 45.8% before chemotherapy to 8.7% after treatment, while all four patients with detectable postchemotherapy ctDNA were recommended TME. However, the small cohort and low number of recurrence events limited the ability to determine the prognostic value of ctDNA in this setting.
The findings support further evaluation of tissue-free ctDNA as a supplemental tool alongside MRI, endoscopy, and other clinical assessments when selecting patients for organ-preservation strategies. Larger, ideally prospective interventional studies with longer follow-up are needed to establish its clinical utility in early-stage, node-negative rectal cancer.
The full article is available in JCO Precision Oncology.
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