Prediction of localized rectal cancer outcomes with ctDNA analysis using a novel ultrasensitive structural variant (SV)-based method.

3619 Background: Rectal cancer has a high risk of recurrence and incidence is rising. Treatment for rectal cancer has moved towards individualization and organ sparing approaches. Selection for treatment is based on radiology, CEA and pathological risk factors, but stronger biomarkers are needed to improve outcomes. Circulating tumor DNA (ctDNA) has shown high prognostic impact on outcome in localized colorectal cancer (CRC), but few patients with rectal cancer have been included in these trials. In this study, we aim to investigate the role of ctDNA in predicting rectal cancer outcomes. Methods: The multicenter CITCCA study included patients with stage I-III CRC planned for curative treatment and investigated prospective, longitudinal ctDNA testing. Neoadjuvant (NAT) and/or adjuvant (ACT) treatment was administered according to local guidelines. All patients underwent surgery. Follow-up was according to Swedish standard-of-care with CT and CEA at 1 and 3 years. Patients were recruited in Sweden, 2020–2024. Sampling of ctDNA occurred before and after surgery at 4-6 weeks, 3 and 6 months,1 and 2 years and was analysed with Pathlight, an ultrasensitive SV-based assay. The primary outcome was recurrence-free interval (RFI). Results: The study included patients with rectal cancer with a median age of 67 years (range 31-81). Ninety-four patients (65%) received upfront surgery and 51 patients (35%) NAT, of which 36 patients (71%) received short-course radiotherapy and 15 (29%) had total neoadjuvant treatment. Fifty-two patients (36%) were (y)pTNM stage I, 45 patients (31%) stage II and 48 patients (33%) stage III. ACT was given to 38 patients (26%). The success rate for assay generation was 96%. At a median follow-up of 35 months (range 2-46), 23 patients recurred or died of rectal cancer. Before surgery, ctDNA was detected in 136 patients (94%). Notably, 92% (55/60) of patients had ctDNA detected prior to surgery, despite NAT. All patients who recurred had positive ctDNA before surgery, regardless of NAT. Postoperatively, at the 4-6 week clinical landmark (CLM), 18 patients (16%) had detectable ctDNA. At the first test after treatment termination, 17 patients (15%) were ctDNA positive and ctDNA detected recurrence ahead of radiology in 13 patients, including 4/4 patients with stage I disease. Positive ctDNA was strongly associated with recurrence (HR: 94; 95% CI: 20-440) and 3-year RFI at CLM was 22% (95% CI: 9%-56%) for ctDNA positive and 95% (95% CI: 90%-100%) for ctDNA negative patients. Conclusions: CtDNA has strong prognostic potential in stage I-III rectal cancer. Using an ultrasensitive SV-based method shows great promise for individualization of treatment in rectal cancer and should be investigated prospectively for organ-sparing selection. Clinical trial information: NCT04726800 .