Postoperative ctDNA Research Articles

Circulating tumor DNA dynamics and response to immunotherapy in colorectal cancer.

Circulating tumor DNA (ctDNA) is increasingly being investigated as a tool to detect minimal residual disease in resected, stage I-III colorectal cancer. Recent ctDNA studies have indicated that detection of ctDNA following surgery for resectable colorectal cancer confers a significantly higher risk of recurrence than those with negative ctDNA postoperatively. In those with postoperative ctDNA positivity, clearance of minimal residual disease with adjuvant chemotherapy is a positive prognostic indicator. Lastly, ctDNA has demonstrated superior sensitivity to the conventional blood tumor marker carcinoembryonic antigen (CEA) and can offer median lead times of up to 11 months for radiographic detection of recurrence during the surveillance of resected, stage I-III colorectal cancer. In metastatic colorectal cancer (mCRC), there is growing evidence to suggest that plasma ctDNA can be used to monitor tumor response to conventional chemotherapy as well. The present case series demonstrated that plasma ctDNA is a predictor of tumor response to immunotherapy in patients with mCRC that are microsatellite stable or microsatellite instability high. Plasma ctDNA could serve as a dynamic marker of immunotherapy response even in colorectal tumors that were CEA non-producers. Overall, these findings add to ongoing efforts to establish the role of plasma ctDNA in monitoring response to immunotherapy in CRC.

Effect of Co-mutation of RAS and TP53 on Postoperative ctDNA Detection and Early Recurrence after Hepatectomy for Colorectal Liver Metastases.

Circulating tumor DNA (ctDNA) is a promising biomarker for patients undergoing hepatectomy for colorectal liver metastases (CLM). We hypothesized that post-hepatectomy ctDNA detection would identify patients at highest risk for early recurrence of CLM. Patients with CLM who underwent curative-intent hepatectomy with ctDNA analysis within 180 days postoperatively (1/2013 and 6/2020) were included. Tissue somatic mutations and ctDNA analyses were performed by next-generation sequencing panels. Survival analyses determined factors associated with clinical recurrence 1 year or earlier after hepatectomy. Patients with primary tumors in situ and without 1-year follow-up were excluded. Median follow-up was 28.3 months. Of 105 patients, 32 (30%) were ctDNA positive (ctDNA+) after curative-intent hepatectomy. Compared with ctDNA-negative patients, ctDNA+ patients had multiple CLM (84% vs 55%, p = 0.002) and co-mutated RAS/TP53 (47% vs 23%, p = 0.018). Multiple CLM (odds ration (OR), 5.43; p = 0.005) and co-mutated RAS/TP53 (OR, 3.30; p = 0.019) were independently associated with post-hepatectomy ctDNA. Although perioperative carcinoembryonic antigen levels were not prognostic, postoperative ctDNA+ (hazard ratio (HR), 2.04; p = 0.011) and extrahepatic disease (HR, 2.45, p = 0.004) were independently associated with worse recurrence-free survival. After adjusting for extrahepatic disease, preoperative chemotherapy, multiple CLM, tumor viability of 50% or greater, and co-mutated RAS/TP53, ctDNA+ within 180 days was the only independent risk factor for recurrence 1 year or earlier after hepatectomy (94% vs 49%; HR, 11.8; p = 0.003). Postoperative ctDNA detection is associated with early recurrence 1 year or earlier after curative-intent hepatectomy for CLM, and RAS/TP53 co-mutations result in a more than 3-fold increased risk for postoperative ctDNA positivity. This highlights the complementary effect of tumor tissue and circulating mutational profiling for patients with CLM.

Prognostic Value of Postoperative Circulating Tumor DNA in Patients With Early- and Intermediate-Stage Hepatocellular Carcinoma.

Majority of patients with resected early- and intermediate-stage liver cancer will experience postoperative recurrence. This study aimed to investigate the application of ctDNA sequencing in the postoperative period of hepatocellular carcinoma. A total of 96 patients with liver cancer were enrolled in this study. Postoperative peripheral blood samples were collected from all patients after surgery and analyzed using hybridization capture-based next-generation sequencing. Identification of at least one somatic mutation in the peripheral blood was defined as ctDNA+. Five genetic features in tumor tissues were associated with disease-free survival (DFS) using Lasso-Cox model. The area under the receiver operating characteristic curve was 0.813 and 0.882 in training and validation cohorts, respectively. The recurrence rate in ctDNA+ and ctDNA- groups was 60.9% and 27.8%, respectively. Multivariate Cox regression analysis showed that the postoperative ctDNA was an independent prognostic predictor of DFS (HR [hazard ratio]: 6.074, 95% Cl [confidence interval]: 2.648-13.929, P<0.001) and overall survival (OS) (HR: 4.829, 95% CI: 1.508-15.466, P=0.008). Combined ctDNA with AFP improved prediction performance. The median DFS was 2.0, and 8.0 months in ctDNA+/AFP-H and ctDNA+/AFP-L groups, respectively; while ctDNA-/AFP-H and ctDNA-/AFP-L groups had not reached the median time statistically (Log-rank test, P < 0.0001). Furthermore, ctDNA- patients had better prognosis than ctDNA+ patients irrespective of tumor stage. Postoperative ctDNA sequencing has great prognostic value in patients with liver cancer. Patients with positive ctDNA should receive more intensive disease monitoring and more aggressive treatment strategies to improve the survival time.

Prognostic utility of preoperative and postoperative circulating tumor DNA (ctDNA) in resected pancreatic ductal adenocarcinoma: A systematic review and meta-analysis.

595 Background: Surgical resection offers the only chance of cure for localized pancreatic ductal adenocarcinoma (PDAC). Despite surgical resection, 80% of patients experience disease recurrence. There is growing evidence that support the prognostic role of perioperative KRAS-mutated circulating tumor DNA (ctDNA). We conducted a systematic review and meta-analysis to investigate the prognostic utility of preoperative and postoperative KRAS-mutated ctDNA testing in resected PDAC. Methods: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a comprehensive search of PubMed/MEDLINE, Embase, and Cochrane Central Register of Controlled Trials databases was performed in September 2021. We included studies that reported on the effects of preoperative and postoperative KRAS-mutated ctDNA on overall survival (OS) and/or relapse free survival (RFS) in resected PDAC. The random-effects model was used to calculate pooled OS and RFS hazard ratios (HRs) and 95% confidence intervals (CIs). Publication bias was assessed by visual inspection of a funnel plot of the included studies. Results: We identified 6,986 studies, and 13 studies were eligible for analysis. A total of 954 patients were included for the final evaluation. In the preoperative setting, positive ctDNA correlated with worse RFS in 8 studies (HR, 2.067; 95% CI, 1.346-3.174; P &lt; 0.001) and worse OS in 10 studies (HR, 2.170; 95% CI, 1.451-3.245; P &lt; 0.001) compared to negative ctDNA. In the postoperative setting, positive ctDNA correlated with worse RFS across 7 studies (HR, 2.986; 95% CI, 1.897-4.699; P &lt;.001), and worse OS in 5 studies (HR, 5.812; 95% CI, 1.757-19.228; P = 0.004) compared to negative ctDNA. There was visible symmetry in the funnel plot of the studies included, suggesting no publication bias. Conclusions: In resected PDAC, preoperative and postoperative KRAS-mutated ctDNA positivity may be useful markers of poor prognosis in terms of RFS and OS. Clinically, KRAS-mutated ctDNA testing may also have implications when considering the aggressiveness and duration of adjuvant therapy in PDAC, although prospective trials are needed to assess this utility.

NRG-GI008: Colon adjuvant chemotherapy based on evaluation of residual disease (CIRCULATE-US).

TPS212 Background: Currently, there are no biomarkers validated prospectively in randomized studies for resected colon cancer (CC) to determine need for adjuvant chemotherapy (AC). However, circulating tumor DNA (ctDNA) shed into the bloodstream represents a highly specific and sensitive approach (especially with serial monitoring) for identifying microscopic or residual tumor cells in CC patients (pts) and may outperform traditional clinical and pathological features in prognosticating risk for recurrence. CC pts who do not have detectable ctDNA (ctDNA-) are at a much lower risk of recurrence and may not need AC. Furthermore, for CC pts with detectable ctDNA (ctDNA+) who are at a very high risk of recurrence, the optimal AC regimen has not been established. We hypothesize that for pts whose colon cancer has been resected, ctDNA status may be used to risk stratify for making decisions about AC. Methods: In this prospective phase II/III trial, up to 1,912 pts with resected stage III A, B (all pts) and stage II, IIIC (ctDNA+ only) CC will be enrolled. Based on the post-operative ctDNA status using Natera’s Signatera assay, those who are ctDNA- (Cohort A) will be randomized to immediate AC with fluoropyrimidine (FP) + oxaliplatin (Ox) for 3-6 mos per established guidelines vs serial ctDNA monitoring. Patients who are ctDNA+ post-operatively or with serial monitoring (Cohort B) will be randomized to FP + Ox vs more intensive AC with addition of irinotecan (I) for 6 mos. The primary objectives for Cohort A are time to ctDNA+ status (phase II) and disease-free survival (DFS) in phase III in the immediate vs delayed AC arms. The primary objective for Cohort B is DFS in the FP + Ox vs FP + Ox + I arms for both phase II and phase III portions of the trial. Secondary objectives include prevalence of detectable ctDNA post-operatively, time-to event outcomes (overall survival &amp; time to recurrence) by ctDNA status, and the assessment of compliance to adjuvant therapy. Biospecimens including archival tumor tissue, post-operative and serial matched/ normal blood samples will be collected for exploratory correlative research. Study will activate in early 2022 across the NCTN. NCT#: Pending. Support: U10-CA-180868, -180822; UG1CA-189867; Natera.

Plasma-informed minimal residual disease (MRD) assay: A multicenter prospective study in Japanese patients with stage II colorectal cancer.

161 Background: ̃80% of stage II colorectal cancer (CRC) can be cured by surgery alone. However, adjuvant chemotherapy is recommended for patients with high risk features such as bowel obstruction, &lt; 12 lymph nodes examined and T4 tumors. Traditional pathological grading and biomarkers such as carcinoembryonic antigen has limited sensitivity. Several reports indicated circulating tumor DNA (ctDNA) may represent a promising prognostic factor to assess MRD as a factor for prediction of recurrence after surgery. Here, we present a proof-of-concept study for the development of a novel plasma-based highly sensitive Next Generation Sequencing (NGS) panel using SafeSEQ technology in operable CRC Japanese patients. Methods: This multicenter prospective study recruited patients diagnosed as operable clinical stage II CRC (n = 46) with pre- and post- (4̃6 weeks) operative plasma samples collected between Nov, 2019 and Jan, 2021. ctDNA were extracted and a 14-gene NGS panel was used to analyze single nucleotide variants (SNVs) and Indels covered by gene-specific amplicons. MRD variant was defined as same variant detected in both pre- and post- operative plasma samples. Tissue NGS by a 500-gene panel was also performed in a small number of tissue samples (n = 5) to compare the concordance of plasma and tissue variants. Results: Pre- and post-operative ctDNA status of 46 patients were analyzed. ctDNA positive was observed in 69.6% (32/46, 95%CI 55.2, 81.0) pre- and 34.8% (16/46, 95%CI 22.7, 49.3) post- samples. AKT1, CTNNB1, NRAS, POLE and PPP2R1A mutation were not detected in this study. TP53 mutation was most frequently detected in both pre- (22/46) and post- (11/46) samples, whereas APC mutation was ranked 2nd in pre- (15/46) but none in post- samples. A combined 96 variants were detected in all samples, in which 76 of them were &lt; 0.5% mutant allele frequency (MAF). MRD variants were detected in 17.4% (8/46, 95%CI 8.82, 30.99) post- samples. Evaluation of positive percentage agreement between tissue and pre- plasma samples in three patients show that a total 7 variants detected in plasma, and 3 of them were detected in tissue samples. Conclusions: This study assesses the feasibility of a plasma-informed NGS panel by evaluating pre- and post- operative plasma samples. The presence of variants with &lt; 0.5% MAF detected in this study indicate a highly sensitive method is required for accurate MRD detection. Further observation is required to explore the relationship between MRD variant and clinical outcome such as 2-year progression-free survival.

Tumour-agnostic circulating tumour DNA analysis for improved recurrence surveillance after resection of colorectal liver metastases: A prospective cohort study

PurposeNearly 50% of patients recur within two years after curatively intended resection of colorectal cancer liver metastasis (CRLM). The optimal surveillance strategy is unknown due to the lack of evidence. Here, we explored the potential for improving postoperative CRLM surveillance by performing serial circulating tumour DNA (ctDNA) assessments parallel to standard-of-care surveillance. Experimental design499 prospectively collected serial plasma samples from 96 patients undergoing CRLM resection were analysed using the tumour-agnostic methylation multiplex droplet-digital PCR test ‘TriMeth’. ResultsPatients with ctDNA postoperatively or post adjuvant chemotherapy experienced a significant lower recurrence-free survival than patients without ctDNA (hazard ratio (HR) 4.5; P < 0.0001 and HR 8.4, P < 0.0001). ctDNA status was a stronger predictor of recurrence than standard clinical risk factors and carcinoembryonic antigen. Serial TriMeth analysis detected ctDNA before radiological recurrence in 55.6% of ctDNA-positive patients, with up to 10.6 months lead-time (median 3.1 months). During surveillance, 24% of patients had inconclusive CT scans, which was associated with a significant delay in recurrence diagnosis (median 3.5 months versus 1.0 month, P < 0.0001). Uniquely, ctDNA status at the time of inconclusive CT scans predicted recurrence with positive and negative predictive values of 100%, and 75% (P = 0.0003). Serial TriMeth analysis allowed ctDNA growth rate assessment and revealed that fast ctDNA growth was associated with poor overall survival (HR: 1.6, P = 0.0052). ConclusionsSerial postoperative ctDNA analysis has a strong prognostic value and is more sensitive for recurrence detection than standard-of-care CRLM surveillance tools. Altogether, TriMeth provides several opportunities for improving postoperative surveillance of CRLM patients.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0‐19.7; P < .0001; and HR, 4.3; 95% CI, 2.3‐8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision‐making in case of indeterminate CT findings, reducing time‐to‐intervention.

Sensitive Quantification of Cell-Free Tumor DNA for Early Detection of Recurrence in Colorectal Cancer.

The detection of plasma cell–free tumor DNA (ctDNA) is prognostic in colorectal cancer (CRC) and has potential for early prediction of disease recurrence. In clinical routine, ctDNA-based diagnostics are limited by the low concentration of ctDNA and error rates of standard next-generation sequencing (NGS) approaches. We evaluated the potential to increase the stability and yield of plasma cell–free DNA (cfDNA) for routine diagnostic purposes using different blood collection tubes and various manual or automated cfDNA extraction protocols. Sensitivity for low-level ctDNA was measured in KRAS-mutant cfDNA using an error-reduced NGS procedure. To test the applicability of rapid evaluation of ctDNA persistence in clinical routine, we prospectively analyzed postoperative samples of 67 CRC (stage II) patients. ctDNA detection was linear between 0.0045 and 45%, with high sensitivity (94%) and specificity (100%) for mutations at 0.1% VAF. The stability and yield of cfDNA were superior when using Streck BCT tubes and a protocol by Zymo Research. Sensitivity for ctDNA increased 1.5-fold by the integration of variant reads from triplicate PCRs and with PCR template concentration. In clinical samples, ctDNA persistence was found in ∼9% of samples, drawn 2 weeks after surgery. Moreover, in a retrospective analysis of 14 CRC patients with relapse during adjuvant therapy, we successfully detected ctDNA (median 0.38% VAF; range 0.18–5.04% VAF) in 92.85% of patients significantly prior (median 112 days) to imaging-based surveillance. Using optimized pre-analytical conditions, the detection of postoperative ctDNA is feasible with excellent sensitivity and allows the prediction of CRC recurrence in routine oncology testing.

Utility of circulating tumor DNA for predicting prognosis in the management of resectable pancreatic cancer

Background: The measurement of circulating tumor DNA (ctDNA) has been studied in several malignancies, including metastatic pancreatic cancer, but less is known about its utility in monitoring treatment response and recurrence in resectable pancreatic cancer. Methods: We conducted a systematic review of the literature examining the association of ctDNA with overall survival (OS) and disease-free survival. Results: Five articles met our exclusion criteria. Baseline and/or postoperative ctDNA was found to be associated with decreased OS and recurrence-free survival. Discussion: ctDNA has the potential to be used as a prognostic biomarker and to guide therapy in resectable pancreatic cancer.

Results and Molecular Correlates from a Pilot Study of Neoadjuvant Induction FOLFIRINOX Followed by Chemoradiation and Surgery for Gastroesophageal Adenocarcinomas.

We performed a NCI-sponsored, prospective study of neoadjuvant FOLFIRINOX followed by chemoradiation with carboplatin/paclitaxel followed by surgery in patients with locally advanced gastric or gastroesophageal cancer. The primary objective was to determine completion rate of neoadjuvant FOLFIRINOX × 8 followed by chemoradiation. Secondary endpoints were toxicity and pathologic complete response (pCR) rate. Exploratory analysis was performed of circulating tumor DNA (ctDNA) to treatment response. From October 2017 to June 2018, 25 patients were enrolled. All patients started FOLFIRINOX, 92% completed all eight planned cycles, and 88% completed chemoradiation. Twenty (80%) patients underwent surgical resection, and 7 had a pCR (35% in resected cohort, 28% intention to treat). Tumor-specific mutations were identified in 21 (84%) patients, of whom 4 and 17 patients had undetectable and detectable ctDNA at baseline, respectively. Presence of detectable post-chemoradiation ctDNA (P = 0.004) and/or postoperative ctDNA (P = 0.045) were associated with disease recurrence. Here we show neoadjuvant FOLFIRINOX followed by chemoradiation for locally advanced gastroesophageal cancer is feasible and yields a high rate of pCR. ctDNA appears to be a promising predictor of postoperative recurrence.See related commentary by Catenacci, p. 6281.

A systematic review and meta-analysis of the association between circulating tumor DNA (ctDNA) and prognosis in pancreatic cancer.

Pancreatic cancer is a deadly disease with limited therapeutic options. Several strategies are being investigated to improve disease management, including the early diagnosis of recurrences and treatment tailoring by better prognosis estimation. Circulating tumor DNA (ctDNA) could be a promising tool in this regard, although the data is limited. Therefore, we conducted a systemical review and meta-analysis of the published studies on the association of ctDNA and survival outcomes in pancreatic cancer. In the pooled analysis, positive preoperative or postoperative ctDNA was associated with lower RFS/PFS (HR: 2.27, 95 % CI: 1.59-3.24, p < 0.001) and OS (HR: 2.04, 95 % CI: 1.29-3.21, p = 0.002) in localized pancreatic cancer. Similarly, positive baseline ctDNA was associated with lower RFS/PFS (HR: 2.61, 95 % CI: 1.94-3.51, p < 0.001) and OS (HR: 2.41, 95 % CI: 1.74-3.34, p < 0.001) in advanced pancreatic cancer. In conclusion, ctDNA could be a promising tool to individualize treatment planning and to improve outcomes in pancreatic cancer.

Circulating Tumor DNA Predicts Pathologic and Clinical Outcomes Following Neoadjuvant Chemoradiation and Surgery for Patients With Locally Advanced Rectal Cancer.

This study was designed to assess the ability of perioperative circulating tumor DNA (ctDNA) to predict surgical outcome and recurrence following neoadjuvant chemoradiation for locally advanced rectal cancer (LARC). Twenty-nine patients with newly diagnosed LARC treated between January 2014 and February 2018 were enrolled. Patients received long-course neoadjuvant chemoradiation prior to surgery. Plasma ctDNA was collected at baseline, preoperatively, and postoperatively. Next-generation sequencing was used to identify mutations in the primary tumor, and mutation-specific droplet digital polymerase chain reaction was used to assess mutation fraction in ctDNA. The median age was 54 years. The overall margin-negative, node-negative resection rate was 73% and was significantly higher among patients with undetectable preoperative ctDNA (n = 17, 88%) versus patients with detectable preoperative ctDNA (n = 9, 44%; P = .028). Undetectable ctDNA was also associated with more favorable neoadjuvant rectal scores (univariate linear regression, P = .029). Recurrence-free survival (RFS) was calculated for the subset (n = 19) who both underwent surgery and had postoperative ctDNA available. At a median follow-up of 20 months, patients with detectable postoperative ctDNA experienced poorer RFS (hazard ratio, 11.56; P = .007). All patients (4 of 4) with detectable postoperative ctDNA recurred (positive predictive value = 100%), whereas only 2 of 15 patients with undetectable ctDNA recurred (negative predictive value = 87%). Among patients treated with neoadjuvant chemoradiation for LARC, patients with undetectable preoperative ctDNA were more likely to have a favorable surgical outcome as measured by the rate of margin-negative, node-negative resections and neoadjuvant rectal score. Furthermore, we have confirmed prior reports indicating that detectable postoperative ctDNA is associated with worse RFS. Future prospective study is needed to assess the potential for ctDNA to assist with personalizing treatment for LARC.

Circulating Tumor DNA as a Potential Prognostic Marker in Patients with Borderline-Resectable Pancreatic Cancer Undergoing Neoadjuvant Chemotherapy Followed by Pancreatectomy.

Circulating tumor DNA (ctDNA) might be a promising biomarker for pancreatic cancer in liquid biopsy. This study aimed to evaluate the usefulness of liquid biopsy for patients with borderline-resectable pancreatic cancer (BR-PC). Patients with BR-PC according to the National Comprehensive Cancer Network guidelines (2017) and eligible for neoadjuvant chemotherapy (NAC) followed by pancreatectomy were recruited at Wakayama Medical University Hospital (UMIN000026647) between March 2017 and April 2020. The study enrolled 55 patients with locally advanced PC, and each patient consented to inclusion in the study. The study investigated the relationship between KRAS status in ctDNA and clinicopathologic features, analyzing ctDNA at three time points: pretreatment, post-NAC, and post-operation. Of the 55 enrolled patients with a diagnosis of BR-PC, 34 were scheduled to undergo pancreatectomy. From 27 patients with resected BR-PC, 81 blood samples were analyzed in triplicate for ctDNA. The patients with positive pretreatment and post-NAC ctDNA status had no significant decrease in median relapse-free survival (RFS) or overall survival (OS). However, the patients with positive postoperation ctDNA status had a significantly shorter median OS (723 days) than the patients with negative ctDNA results (not reached; P = 0.0148). A combined analysis of postoperative ctDNA and CA19-9 values showed the cumulative effect on both RFS (P = 0.0066) and OS (P = 0.0046). The adjusted hazard ratio for risk of survival computed for the patients carrying risk factors (either detectable ctDNA or CA19-9 > 37U/ml) increased from 4.13-fold to 17.71-fold (both P = 0.0055) compared with the patients who had no risk factors. Positive ctDNA predicts poor survival for patients with BR-PC who undergo NAC followed by pancreatectomy.

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

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. 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. 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. 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.

Postoperative Circulating Tumor DNA Can Predict High Risk Patients with Colorectal Cancer Based on Next-Generation Sequencing

Simple SummaryCirculating tumor DNA (ctDNA) is a minimally invasive biomarker useful for monitoring minimum residual disease, recurrence, and treatment response in colorectal cancer (CRC). We analyzed circulating tumor DNA from patients with CRC to evaluate analytical and clinical performances using next-generation sequencing (NGS). It is clear that postoperative circulating tumor DNA detection provides valuable information to determine whether a patient might at high risk of disease recurrence or have a persistent tumor lesion. The NGS assay not only showed excellent analytical performance, but also shows a state-of-art diagnostic option in patient-oriented precision medicine.The objective of this study was to characterize circulating tumor DNA (ctDNA) mutations in colorectal cancer (CRC) patients and evaluate their prognostic values during treatment. Forty-nine patients with CRC planned for operation were enrolled. A total of 115 plasma samples were collected pre-operation, post-operation, and post-chemotherapy. ctDNA analysis was performed using next-generation sequencing (NGS) including 14 genes. In 22 (44.9%) out of 49 patients, at least one mutation (40 total mutations) was detected in the initial plasma sample. The median sum of variant allele frequency was 0.74% (range: 0.10–29.57%). TP53 mutations were the most frequent (17 of 49 patients, 34.7%), followed by APC (18.4%), KRAS (12.2%), FBXW7 (8.2%), NRAS (2.0%), PIK3CA (2.0%), and SMAD4 (2.0%). After surgery, five (14.3%) out of 35 patients harbored ctDNA mutation. All five patients experienced relapse or metastasis during follow-up. It was noteworthy that all three patients with persistent ctDNA relapsed after R0 resection. After chemotherapy, ctDNA analysis was performed for 31 patients, all of which were ctDNA-negative. Analytical and clinical performances of NGS to utilize ctDNA in CRC were determined. Results revealed that postoperative ctDNA might serve as a marker for identifying risk of recurrence, thus contributing to patient-oriented treatment strategies.

Serial circulating tumor DNA analysis to assess recurrence risk, benefit of adjuvant therapy, growth rate and early relapse detection in stage III colorectal cancer patients.

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&lt; 0.001), directly after ACT (HR = 18.2, 95% CI 7.1-46, P &lt; 0.001), and when measured serially after end of treatment (HR = 41, 95% CI 16-100, P &lt; 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 &lt; 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 &lt; 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.

Minimal residual disease (MRD) in patients with resected stage I NSCLC: Results of the prospective adjuvant IFCT-0703 trial.

8526 Background: MRD aims to detect circulating biomarkers of micrometastatic disease and ultimately predict recurrences. The IFCT-0703 randomized phase II trial failed to show a benefit of 6 months adjuvant pazopanib (P) vs. placebo after resection of stage I NSCLC (7th TNM edition). The outcome of pts based on their MRD status has been evaluated. Methods: Blood samples were collected in EDTA tubes (Becton Dickinson Company) after surgery (T0), after 3 months (T3) of P or placebo and at the end of treatment (T6). Plasmas were obtained after double centrifugation of total blood. Total nucleic acid was extracted using the Maxwell RSC LV plasma kit (Promega) according to the manufacturer’s protocol. Samples were quantified using the QuBit dsDNA HS Assay kit on a QuBit 3.0 flurometer (Thermo Fisher Scientific). Molecular analysis was performed by next generation sequencing using the Oncomine Lung cfDNA Assay (ThermoFisher Scientific). Two MRD definitions were tested : 1) high level of DNA in the blood or 2) any mutation detected by the standard bioinformatic pipeline was considered present, whatever the allelic fraction. Results: 143 pts were randomized in 29 centers between March 2009 and August 2012, 71 and 72 in the placebo and P arms respectively. Among the 119 pts with evaluable T0 samples, 27 pts recurred and 14 died. Median DNA concentration ([DNA]) was 6.6 ng/ml and an increase of [DNA] of 10 ng/ml was found prognostic of poor DFS and OS, HR=1.4, 95%CI [1.14-1.72], p=0.0016 and HR=1.62, 95%CI [1.15-2.30], p=0.0057 respectively. In 81 pts with available T0-T6 samples, [DNA] variation had no different impact on DFS and OS, in the P arm and the placebo arm. ctDNA mutations (ctDNA+) were detected in 31/119 pts. ctDNA+ were more frequent in samples with high DNA quantity (p=0.0002). Genes mutated at T0 were TP53 in 16, NRAS in 6, MAP2K1 in 2, KRAS in 1, EGFR in 5, BRAF in 1, ALK in 2. 29 pts had 1 mutation, 2 had 2 mutations. DFS and OS were similar between pts with or without ctDNA+ : HR= 1.038 (95%CI 0.438-2.456, p=0.93) and 1.193 (95% CI 0.367-3.882, p=0.77) respectively. Among 27 pts with ctDNA+ at T0 and available sample at T6, 23 had no more mutations at T6. Two pts had a ctDNA+ only at T6 (not at T3), one of them had a recurrence at 7 months. Conclusions: Post-operative ctDNA mutations are found in 26.0% of the pts but their positivity had no impact on DFS or OS. In contrast, DFS and OS were poorer in pts with increased plasma DNA concentration. ctDNA mutations status do not recapitulate the complexity of MRD characterization. NGS will be performed on matched tissues in order to refine MRD definition. Clinical trial information: NCT00775307.

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study.

BackgroundIn patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study.Methods and findingsWe prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing.ConclusionsWe confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM.Trial registrationACTRN12612000345886.

77TiP From liquid biopsy to cure: Using CtDNA detection of minimal residual disease to identify patients for curative therapy after non-small cell lung cancer (NSCLC) resection

Background: For resectable Stage IA2 to IIB (T1b-T3N0) NSCLC, the current practice is surgery followed by observation for Stage IA disease. Adjuvant platinum-based doublet chemotherapy is offered to patients (pts) with stage IB disease with high-risk pathological and radiological features and is recommended for those with Stage II NSCLC. In pts with resected lung cancer, post-operative ctDNA predicts and precedes radiographic recurrences. The survival benefit seen with 1st-line chemo-immunotherapy (chemo-IO) for advanced NSCLC provided the rationale to launch this trial offering concurrent immunotherapy with adjuvant chemo to selected pts.