Purpose: Response to preoperative chemo-radiotherapy (CRT) varies. We assessed whether circulating tumor DNA (ctDNA) might be an early indicator of tumor response or progression to guide therapy adaptation in rectal cancer.Experimental Design: A total of 243 serial plasma samples were analyzed from 47 patients with localized rectal cancer undergoing CRT. Up to three somatic variants were tracked in plasma using droplet digital PCR. RECIST and MRI tumor regression grade (mrTRG) evaluated response. Survival analyses applied Kaplan-Meier method and Cox regression.Results: ctDNA detection rates were: 74% (n ¼ 35/47) pretreatment, 21% (n ¼ 10/47) mid CRT, 21% (n ¼ 10/47) after completing CRT, and 13% (n ¼ 3/23) after surgery. ctDNA status after CRT was associated with primary tumor response by mrTRG (P ¼ 0.03). With a median follow-up of 26.4 months, metastases-free survival was shorter in patients with detectable ctDNA after completing CRT [HR 7.1; 95% confidence interval (CI), 2.4-21.5; P < 0.001], persistently detectable ctDNA pre and mid CRT (HR 3.8; 95% CI, 1.2-11.7; P ¼ 0.02), and pre, mid, and after CRT (HR 11.5; 95% CI, 3.3-40.4; P < 0.001) compared with patients with undetectable or nonpersistent ctDNA. In patients with detectable ctDNA, a fractional abundance threshold of !0.07% mid CRT or !0.13% after completing CRT predicted for metastases with 100% sensitivity and 83.3% specificity for mid CRT and 66.7% for CRT completion. All 3 patients with detectable ctDNA post-surgery relapsed compared with none of the 20 patients with undetectable ctDNA (P ¼ 0.001).Conclusions: ctDNA identified patients at risk of developing metastases during the neoadjuvant period and post-surgery, and could be used to tailor treatment.

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Objective: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. Methods: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of lowfrequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. Results: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89e0.95]) and reproducible (>0.87 [95% CI: 0.87 e0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. Conclusions: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.