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Supplementary Data from Circulating Tumor DNA in Stage III Colorectal Cancer, beyond Minimal Residual Disease Detection, toward Assessment of Adjuvant Therapy Efficacy and Clinical Behavior of Recurrences

Purpose:Sensitive methods for risk stratification, monitoring therapeutic efficacy, and early relapse detection may have a major impact on treatment decisions and patient management for stage III colorectal cancer patients. Beyond assessing the predictive power of postoperative ctDNA detection, we explored the added benefits of serial analysis: assessing adjuvant chemotherapy (ACT) efficacy, early relapse detection, and ctDNA growth rates.Experimental Design:We recruited 168 patients with stage III colorectal cancer treated with curative intent at Danish and Spanish hospitals between 2014 and 2019. To quantify ctDNA in plasma samples (n = 1,204), 16 patient-specific somatic single-nucleotide variants were profiled using multiplex-PCR, next-generation sequencing.Results:Detection of ctDNA was a strong recurrence predictor postoperatively [HR = 7.0; 95% confidence interval (CI), 3.7–13.5; P < 0.001] and directly after ACT (HR = 50.76; 95% CI, 15.4–167; P < 0.001). The recurrence rate of postoperative ctDNA-positive patients treated with ACT was 80% (16/20). Only patients who cleared ctDNA permanently during ACT did not relapse. Serial ctDNA assessment after the end of treatment was similarly predictive of recurrence (HR = 50.80; 95% CI, 14.9–172; P < 0.001), and revealed two distinct rates of exponential ctDNA growth, slow (25% ctDNA-increase/month) and fast (143% ctDNA-increase/month; P < 0.001). The ctDNA growth rate was prognostic of survival (HR = 2.7; 95% CI, 1.1–6.7; P = 0.039). Serial ctDNA analysis every 3 months detected recurrence with a median lead-time of 9.8 months compared with standard-of-care computed tomography.Conclusions:Serial postoperative ctDNA analysis has a strong prognostic value and enables tumor growth rate assessment. The novel combination of ctDNA detection and growth rate assessment provides unique opportunities for guiding decision-making.See related commentary by Morris and George, p. 438

Objective: Circulating tumor DNA (ctDNA) is a candidate biomarker of cancer with practice-changing potential in the detection of both early and residual disease. Disease stage and tumor size affect the probability of ctDNA detection, whereas little is known about the influence of other tumor characteristics on ctDNA detection. This study investigates the impact of tumor cell whole-genome doubling (WGD) on the detection of ctDNA in plasma collected preoperatively from newly diagnosed colorectal cancer (CRC) patients.Methods: WGD was estimated from copy numbers derived from whole-exome sequencing (WES) data of matched tumor and normal DNA from 833 Danish CRC patients. To explore if tumor WGD status impacts ctDNA detection, we applied tumor-informed ctDNA analysis to preoperative plasma samples from all patients.Results: Patients with WGD+ tumors had 53% increased odds of being ctDNA positive (OR = 1.53, 95%CI: 1.12-2.09). After stratification for UICC stage, the association persisted for Stage I (OR = 2.44, 95%CI: 1.22-5.03) and Stage II (OR = 1.76, 95%CI: 1.11-2.81) but not for Stage III (OR = 0.83, 95%CI: 0.44-1.53) patients.Conclusion: The presence of WGD significantly increases the probability of detecting ctDNA, particularly for early-stage disease. In patients with more advanced disease, the benefit of WGD on ctDNA detection is less pronounced, consistent with increased DNA shedding from these tumors, making ctDNA detection less dependent on the amount of ctDNA released per tumor cell. Objective: Circulating tumor DNA (ctDNA) is a candidate biomarker of cancer with practice-changing potential in the detection of both early and residual disease. Disease stage and tumor size affect the probability of ctDNA detection, whereas little is known about the influence of other tumor characteristics on ctDNA detection. This study investigates the impact of tumor cell whole-genome doubling (WGD) on the detection of ctDNA in plasma collected preoperatively from newly diagnosed colorectal cancer (CRC) patients. Methods: WGD was estimated from copy numbers derived from whole-exome sequencing (WES) data of matched tumor and normal DNA from 833 Danish CRC patients. To explore if tumor WGD status impacts ctDNA detection, we applied tumor-informed ctDNA analysis to preoperative plasma samples from all patients. Results: Patients with WGD+ tumors had 53% increased odds of being ctDNA positive (OR = 1.53, 95%CI: 1.12–2.09). After stratification for UICC stage, the association persisted for Stage I (OR = 2.44, 95%CI: 1.22–5.03) and Stage II (OR = 1.76, 95%CI: 1.11–2.81) but not for Stage III (OR = 0.83, 95%CI: 0.44–1.53) patients. Conclusion: The presence of WGD significantly increases the probability of detecting ctDNA, particularly for early-stage disease. In patients with more advanced disease, the benefit of WGD on ctDNA detection is less pronounced, consistent with increased DNA shedding from these tumors, making ctDNA detection less dependent on the amount of ctDNA released per tumor cell.

Purpose: Sensitive methods for risk stratification, monitoring therapeutic efficacy, and early relapse detection may have a major impact on treatment decisions and patient management for stage III colorectal cancer patients. Beyond assessing the predictive power of postoperative ctDNA detection, we explored the added benefits of serial analysis: assessing adjuvant chemotherapy (ACT) efficacy, early relapse detection, and ctDNA growth rates. Experimental Design: We recruited 168 patients with stage III colorectal cancer treated with curative intent at Danish and Spanish hospitals between 2014 and 2019. To quantify ctDNA in plasma samples (n = 1,204), 16 patient-specific somatic single-nucleotide variants were profiled using multiplex-PCR, next-generation sequencing. Results: Detection of ctDNA was a strong recurrence predictor postoperatively [HR = 7.0; 95% confidence interval (CI), 3.7–13.5; P < 0.001] and directly after ACT (HR = 50.76; 95% CI, 15.4–167; P < 0.001). The recurrence rate of postoperative ctDNA-positive patients treated with ACT was 80% (16/20). Only patients who cleared ctDNA permanently during ACT did not relapse. Serial ctDNA assessment after the end of treatment was similarly predictive of recurrence (HR = 50.80; 95% CI, 14.9–172; P < 0.001), and revealed two distinct rates of exponential ctDNA growth, slow (25% ctDNA-increase/month) and fast (143% ctDNA-increase/month; P < 0.001). The ctDNA growth rate was prognostic of survival (HR = 2.7; 95% CI, 1.1–6.7; P = 0.039). Serial ctDNA analysis every 3 months detected recurrence with a median lead-time of 9.8 months compared with standard-of-care computed tomography. Conclusions: Serial postoperative ctDNA analysis has a strong prognostic value and enables tumor growth rate assessment. The novel combination of ctDNA detection and growth rate assessment provides unique opportunities for guiding decision-making. See related commentary by Morris and George, p. 438

Background: While detection of circulating tumor DNA (ctDNA) is associated with poor cancer prognosis, the clinical utility for guiding treatment decisions is unresolved. Patients with minimal residual disease (MRD) often have less than one genome equivalent of ctDNA per 10 mL blood. Consequently, it is stochastic whether a 10 mL sample contains ctDNA from a particular genomic locus. Consequently, the sensitivity of ctDNA detection methods targeting a limited number of tumor loci is heavily affected by sampling bias. To overcome this challenge, we developed MRDetect; a whole genome sequencing (WGS) approach, which detects ctDNA using the patient-specific cumulative signal from tens of thousands of mutations throughout the genome. Recently, we showed how MRDetect found ctDNA fractions down to 10-4. Here, we performed a validation study to confirm the prognostic impact of MRDetect. Aim: Validation of MRDetect for sensitive ctDNA detection to monitor residual disease in stage III colorectal cancer (CRC) patients treated with curative intent. Methods: From a large, uniform cohort of stage III CRC patients n = 146), we had plasma samples collected every third month (n = 938, median = 9 per patient) and a median follow-up of 34 months. For each patient, a genome-wide mutational signature was established by WGS of tumor and matched normal DNA. Enhanced by an AI-based error suppression model, this signature was used to detect ctDNA in 1-2 mL plasma samples using WGS (20x coverage). We used de-novo point mutation and copy number variation analysis to investigate cancer evolution after treatment. To evaluate the reproducibility of MRDetect, aliquot samples (n = 2x190 samples) from 5 recurrence and 10 non-recurrence patients were processed and sequenced at two independent laboratories. Outcome measures: ctDNA status, tumor fraction, false positive rate, Time To ctDNA Recurrence (TTcR), and Time To radiological Recurrence (TTrR). Results: Analysis of paired samples showed great reproducibility with high agreement between both ctDNA status calls (Cohens Kappa = 0.81) and the estimated tumor fractions (r2 = 0.99). MRDetect revealed post-operative ctDNA in all recurrence patients (5/5) with detected tumor fractions down to 2 x 10-4. Median TTcR was 0.9 month (range 0.5 - 7.3 months) while median TTrR was 12.8 months (range 11.3 - 31.1 months). The false positive rate was 1% (1/100), assessed in longitudinal samples from the 10 non-relapsing patients. Tumor evolution dynamics in plasma samples revealed novel amplification and deletions, which were absent in the primary tissue but confirmed in metachronous metastases. We will present results from the full cohort at AACR 2022. Conclusion: MRDetect detects ctDNA with high sensitivity and specificity and enables effective postoperative assessment of MRD, cancer evolution dynamics and early relapse detection. Citation Format: Amanda Frydendahl, Thomas Reinert, Jesper Nors, Sunil Deochand, Dillon Maloney, Noah Friedman, Tomer Lauterman, Danielle Afterman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Ravi Kandasamy, Iman Tavassoly, Jonathan Rosenfeld, Anders Husted Andersen, Uffe S. Løve, Per V. Andersen, Ole Thorlacius-Ussing, Lene Hjerrild Iversen, Kåre Andersson Gotschalck, Boris Oklander, Asaf Zviran, Claus Lindbjerg Andersen. Sensitive detection of circulating tumor DNA by whole genome sequencing: Validation of MRDetect using serial blood samples from stage III colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1959.

3540 Background: Challenges in the postoperative management of stage III colorectal cancer include: 1) selection of high-risk patients for adjuvant chemotherapy (ACT), 2) lack of markers to assess ACT efficacy, 3) assessment of recurrence risk after ACT, and 4) lack of markers to guide treatment decisions for high-risk patients e.g. additional therapy or intensified surveillance. Circulating tumor DNA (ctDNA) is a promising marker with potential to mitigate the challenges. Here we used serial ctDNA measurements to assess the correlation between recurrence and ctDNA detection: postoperative, during and after ACT, and during surveillance; and to assess growth rates of metachronous metastases. Uniquely, we also used concurrent CT scans and ctDNA measurements to compare the sensitivity for detecting recurrence. Methods: Stage III CRC patients treated with curative intent at Danish and Spanish hospitals in 2014-2019 were recruited (n = 166). Blood samples (n = 1227) were collected prior to and immediately after surgery, and every third month for up to 36 months. Per patient 16 personal mutations were used to quantify plasma ctDNA (Signatera, bespoke mPCR NGS assay). Results: Detection of ctDNA was a strong recurrence predictor, both postoperatively (HR 7.2, 95% CI 3.8-13.8, P< 0.001), directly after ACT (HR = 18.2, 95% CI 7.1-46, P < 0.001), and when measured serially after end of treatment (HR = 41, 95% CI 16-100, P < 0.001). The recurrence rate of postoperative ctDNA positive patients treated with ACT was 80% (16/20). Patients who stayed ctDNA positive during ACT all recurred. Serial post-treatment ctDNA measurements revealed exponential growth for all recurrence patients following either a SLOW (26%-increase/month) or a FAST (126%-increase/month) pattern (P < 0.001). From ctDNA detection to radiologic recurrence, ctDNA levels of FAST patients increased by a median 117-fold, and up to 554-fold. The 3-year overall survival was 43% for FAST patients and 100% for SLOW and non-recurrence patients (HR = 41.3, 95% CI 7.5-228, P < 0.001). Coinciding CT scans and ctDNA measurements (n = 113 patients, 235 coinciding events, median 2 per patient) showed a high agreement (92%) and ctDNA either detected residual disease before the CT scan (n = 7 patients) or at the same time (n = 14 patients). The median lead-time was 7.5 months. Conclusions: The study confirmed the prognostic power of serial postoperative ctDNA analysis. Moreover, it provided novel analyses demonstrating that ctDNA is more sensitive for recurrence detection than CT scans and can be used for tumor growth rate assessments. The difference between FAST and SLOW growing tumors suggest that growth rates could guide whom to start on systemic therapy rapidly and whom to send for diagnostic imaging. Altogether, the study highlights many potential utilities of ctDNA in guiding clinical decision-making.

BACKGROUND: Postoperative ileus and anastomotic leakage severely impair recovery after colorectal resection. We investigated the effect of perioperative lipid-enriched enteral nutrition versus standard care on the risk of postoperative ileus, anastomotic leakage, and other clinical outcomes.METHODS: We did an international, multicentre, double-blind, randomised, controlled trial of patients (≥18 years) undergoing elective colorectal surgery with primary anastomosis at six clinical centres in the Netherlands and Denmark. Patients were randomly assigned (1:1), stratified by location (colonic and rectal) and type of surgery (laparoscopic and open), via online randomisation software, with block sizes of six, to receive either continuous lipid-enriched enteral tube feeding from 3 h before until 6 h after surgery (intervention) or no perioperative nutrition (control). Surgeons, patients, and researchers were masked to treatment allocation for the entire study period. The primary outcome was postoperative ileus. Secondary outcomes included anastomotic leakage, pneumonia, preoperative gastric volumes, time to functional recovery, length of hospital stay, the need for additional interventions, intensive care unit admission, postoperative inflammatory response, and surgical complications. Analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT02175979, and trialregister.nl, number NTR4670.FINDINGS: Between July 28, 2014, and February 20, 2017, 280 patients were randomly assigned, 15 of whom were excluded after random allocation because they fulfilled one or more exclusion criteria. 265 patients received perioperative nutrition (n=132) or standard care (n=133) and were included in the analyses. A postoperative ileus occurred in 37 (28%) patients in the intervention group versus 29 (22%) in the control group (risk ratio [RR] 1·09, 95% CI 0·95-1·25; p=0·24). Anastomotic leakage occurred in 12 (9%) patients in the intervention group versus 11 (8%) in the control group (RR 1·01, 95% CI 0·94-1·09; p=0·81). Pneumonia occurred in ten (8%) patients in the intervention group versus three (2%) in the control group (RR 1·06, 95% CI 1·00-1·12; p=0·051). All other secondary outcomes were similar between groups (all p>0·05).INTERPRETATION: Perioperative lipid-enriched enteral nutrition in patients undergoing elective colorectal surgery has no advantage over standard care in terms of postoperative complications.FUNDING: Netherlands Organisation for Health Research and Development (ZonMW), Fonds NutsOhra, and Danone Research.

11 Background: Timely detection of recurrence, as well as identification of patients at high risk of recurrence after surgery and after completion of adjuvant therapy, are major challenges in the treatment of colorectal cancer (CRC). Postsurgical circulating tumor DNA (ctDNA) analysis is a promising tool for the identification of patients with minimal residual disease (MRD) and a high risk of recurrence. The objective of this prospective, multicenter study was to determine whether serial postsurgical ctDNA analysis could identify the patients at high risk of recurrence, provide an assessment of adjuvant therapy efficacy and detect relapse earlier than standard-of-care radiological imaging. Methods: The cohort comprises 265 stage I-III CRC patients, the to-date largest cohort assessed for ctDNA. All patients had the tumor resected and a subset of 62.6% (166 /265) was additionally treated with ACT. Plasma samples (n = 1503) were collected at various time points for a median follow-up of 28.4 months (range: 1.2-51.0 months). Individual tumors and matched germline DNA were whole-exome sequenced and somatic single nucleotide variants (SNVs) were identified. Personalized multiplex PCR assays were designed to track tumor-specific SNVs (Signatera, bespoke mPCR NGS assay) in each patient’s plasma sample. Results: Postoperative ctDNA status prior to ACT was assessed in 218 patients, of which 9.17% (20/218) were identified to be MRD-positive and 75% (15/20) eventually relapsed. The remaining 25% (5/20) of MRD-positive patients that did not relapse, received ACT. In contrast, only 13.6% (27/198) of MRD-negative cases relapsed (HR: 11: 95% CI: 5.7-20; p < 0.001). Longitudinal ctDNA-positive status, post-definitive therapy (n = 202) was associated with a HR of 36 (95% CI: 16-81; p < 0.001). For a subset of 155 patients postoperative CEA and ctDNA measurements were compared, wherein, ctDNA-positive status was found to be significantly associated with RFS (HR, 7.1; 95% CI, 3.4-15; P < 0.001) compared to CEA (HR, 1.2; 95% CI, 0.46-3.1; P = 0.73). Serial ctDNA analysis detected MRD up to a median of 8 months (0.56 - 21.6 months) ahead of radiologic relapse. Conclusion: Postoperative ctDNA positive status was associated with markedly reduced RFS compared to CEA. The study also shows that effective therapy can be curative in a portion of MRD-positive patients. In a longitudinal setting, ctDNA analysis predicted the risk of recurrence and is a more reliable biomarker for treatment response monitoring.

BACKGROUND: The use of the vertical rectus abdominis myocutaneous flap in reconstruction after abdominoperineal resection or pelvic exenteration for neoplasia is well documented. However, functional outcomes after vaginal reconstruction, including sexual function, are poorly described. OBJECTIVE: This study aimed to examine sexual function in women following extensive pelvic surgery with colpectomy and vaginal reconstruction with the use of a vertical rectus abdominis myocutaneous flap. DESIGN: This study is a retrospective review of medical records in combination with patient questionnaires. Nonresponders were followed up with a second contact. SETTINGS: This study was performed at a tertiary care university medical center (Colorectal Section, Department of Surgery P, Aarhus University Hospital, Denmark) PATIENTS All women undergoing pelvic surgery and simultaneous vaginal reconstruction with the use of a vertical rectus abdominis myocutaneous flap between 2004 and 2010 at our department were identified from a patient database. Thirty women who were alive at the time of identification were included in the study. MAIN OUTCOME MEASURES: Sexual function before and after surgery was evaluated by the use of the Sexual function Vaginal changes Questionnaire. The main outcome end point was whether the patient was sexually active after vaginal reconstruction. RESULTS: Twenty-six participants (87%) answered the questionnaire. Fifty percent of patients reported an active sex life before surgery. In general, patients reported an unchanged desire for both physical and sexual contact after surgery. However, only 2 patients (14%) reported being sexually active after surgery. LIMITATIONS: This was a retrospective study with a heterogeneous cohort involving several types of cancers and surgical procedures. Factors other than vertical rectus abdominis myocutaneous flap reconstruction itself may interfere with the sexual function. CONCLUSION: Extensive pelvic surgery with colpectomy leads to sexual dysfunction even when the vagina is reconstructed with a vertical rectus abdominis myocutaneous flap. This knowledge may improve the quality of information given to this group of patients before surgery.