ctDNA Status Research Articles

Undetectable pre-radical cystectomy circulating tumour DNA status predicts improved oncological outcomes.

To assess recurrence-free survival (RFS) in patients with undetectable tumour-informed circulating tumour DNA (ctDNA) before radical cystectomy (RC) and evaluate if those who converted from detectable to undetectable ctDNA status after RC have similar RFS outcomes as those with persistently undetectable ctDNA status. Patients who underwent RC had prospectively and longitudinally collected tumour-informed ctDNA analyses during 2021-2023. ctDNA status was informed from the pre-RC specimen. The minimal residual disease (MRD) window was defined as the initial 90 days after RC. RFS was evaluated using the Kaplan-Meier method. Cox regression analysis was performed to find predictors of disease recurrence. The cohort included 135 patients with 647 ctDNA analyses. The median (interquartile range [IQR]) age was 71 (63-77) years. Over a median (IQR) follow-up of 11 (7-18) months, 41 patients (30%) had a recurrence. Pre-RC undetectable ctDNA status was found in 54 patients (40%). The RFS rates at 6, 12, and 21 months were 98%, 93%, and 82%, respectively. Of 77 patients with undetectable ctDNA status at the MRD window available for conversion dynamics analysis, 43 had persistently undetectable ctDNA status (both at pre-RC and MRD window) and 31 converted from pre-RC detectable to MRD undetectable status (conversion group). The persistently undetectable group had significantly better RFS than the conversion group (log-rank, P < 0.001), with 12-month RFS rates of 97% vs 51%, and 18-month RFS rates of 88% vs 51%, respectively. On Cox multivariate analysis, only the conversion group status predicted disease recurrence. Patients with undetectable pre-RC ctDNA status have a favourable prognosis and may be candidates for treatment de-escalation. Those with persistently undetectable ctDNA had superior RFS compared to the conversion group. Pre-RC ctDNA status should be incorporated into trials examining ctDNA use in clinical decision-making.

Discerning the impact of ctDNA detection on patient decision-making in early-stage breast cancer

The impact of knowledge of circulating tumor DNA (ctDNA) status on patient decisions in high-risk triple-negative breast cancer (TNBC) weighing benefit and toxicity is unknown. Here, 286 women with a history of non-metastatic breast cancer who had received chemotherapy completed a survey mimicking scenarios of residual TNBC after chemotherapy and unknown, negative, or positive ctDNA status to determine the shift in the decision to receive adjuvant therapy. Participants were then presented scenarios mimicking possible post-neoadjuvant therapies and rated acceptability. A general linear model with repeated measures determined contributions of risk reduction and toxicity. When the hypothetical risk of recurrence mimicked ctDNA negativity, significantly less participants were accepting of adjuvant capecitabine versus no therapy. When presented with ctDNA positivity and increased recurrence risk, the degree of benefit impacted acceptability more than the toxicity profile. As genomic technology advances and ctDNA assays become commercially available, it is imperative to understand the impact on patient decision-making.

CtDNA SNORD3F Hypermethylation is a Prognostic Indicator in EGFR-TKI-Treated Advanced Non-Small Cell Lung Cancer.

DNA methylation plays a regulatory role in the oncogenesis and tumor progression and is valuable in the diagnosis and prognosis of cancer. While circulating tumor DNA (ctDNA) is widely used in the detection of oncogenic mutations and the guidance of treatment in advanced non-small cell lung cancer (NSCLC), studies of ctDNA methylation remains insufficient. We aim to investigate the methylation profiles of ctDNA in patients with advanced NSCLC undergoing EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy and to discover novel biomarkers with predictive or prognostic value. We recruited 49 patients with EGFR-mutated advanced NSCLC undergoing EGFR-TKI as first-line treatment. Utilizing next-generation sequencing, we examined the somatic mutations and methylation signatures within the tumor-associated genomic regions of ctDNA from pre-treatment blood. Subsequently, we explored the association of these molecular features with the patients' response to therapy and their progression-free survival (PFS). Genomic mutation profiling revealed no significant association of PFS or best overall response (BOR) and ctDNA status. Evaluation of ctDNA methylation showed a negative correlation between the methylation of small nucleolar RNA (snoRNA) genes and PFS (R=-0.31, P=0.043). Furthermore, high-level methylation of SNORD3F was associated with poorer PFS (mPFS 346d vs 243d, HR 0.49, 95% CI 0.24-0.93, P=0.029). Our study explored the prognostic value of ctDNA methylation in patients with advanced NSCLC undergoing targeted therapies and first revealed the predictive role of SNORD3F.

Tumor-informed circulating tumor DNA in urothelial carcinoma: a promising novel biomarker.

Tumor-informed circulating tumor DNA (ctDNA) is an emerging biomarker in urothelial carcinoma. Recent clinical trials have investigated the integration of ctDNA into clinical decision-making in patients with muscle-invasive bladder cancer, their findings suggest that ctDNA may potentially revolutionize the way we stratify patients to different treatment modalities. ctDNA informed from TURBT specimens was found to be prognostic of disease outcomes among patients with localized nonmetastatic bladder cancer. Detectable precystectomy ctDNA status was associated with worse survival outcomes. Additionally, ctDNA status was predictive of adverse disease on radical cystectomy, including the likelihood of disease upstaging, lymph node involvement, and having a locally advanced disease (≥pT3a). In the postcystectomy minimal residual disease (MRD) period, ctDNA status may refine patient selection to adjuvant therapy, and if validated by ongoing clinical trials, patients with undetectable postcystectomy ctDNA status may forgo adjuvant treatment, regardless of pathological stage. On the contrary, patients with pre or postcystectomy detectable ctDNA status may benefit from treatment intensification. The integration of ctDNA in clinical decision-making has the potential to revolutionize the way we manage urothelial carcinoma by refining patient selection to different treatment modalities. This approach could ultimately lead to personalization of oncological care, with the potential to reduce both treatment-related and financial toxicity.

The prognostic significance of circulating tumor DNA in patients with positive lymph node disease after robotic-assisted radical cystectomy: A contemporary analysis

Background and objectiveNeoadjuvant therapy followed by radical cystectomy with lymphadenectomy remains the gold standard of treatment in patients with muscle-invasive bladder cancer. Pathologically positive lymph node (pN+) disease is known to convey a poor prognosis. Tumor-informed circulating tumor DNA (ctDNA) has emerged as a possible novel prognostic biomarker in the field. We seek to assess recurrence-free survival (RFS) for patients undergoing robotic-assisted radical cystectomy (RARC) with extended pelvic lymphadenectomy (ePLND) and to assess whether ctDNA status can be a prognostic marker for RFS outcomes in patients with pN+ disease. MethodsPatients who underwent RARC + ePLND during 2015 to 2023 were included. A sub-group analysis (n = 109) of patients who had prospectively collected serial-longitudinal tumor-informed ctDNA analyses during 2021-2023 was performed. Survival analysis and Cox-regression models were conducted. ResultsIncluded were 458 patients with a median age of 69 (IQR 63–76), and a median follow-up time of 20 months (IQR 10-37). RFS for pN0 (n = 353) and pN+ (n = 105) at 12, 24 and 36 months were 87% vs. 54%, 80% vs. 39%, and 74% vs. 35%, respectively (log-rank, P < 0.0001). On Cox multivariate analysis ≥pT3 disease (Hazzard ratio [HR] = 3.36 [2.18–5.18], P < 0.001), pN+ disease (HR = 2.39 [1.55–3.7], P < 0.001), and recipients of neoadjuvant treatment (HR = 1.61 [1.11–2.34], P = 0.013) were predictive of disease relapse. Patients with pN+ disease and undetectable precystectomy or postcystectomy ctDNA status had similar RFS to patients with pN0 with undetectable ctDNA. On Cox-regression multivariate sub-group analysis, detectable precystectomy ctDNA status (HR = 3.89 [1.32–11.4], P = 0.014), detectable ctDNA status in the minimal residual disease window ([MRD], HR = 2.89 [1.12–7.47], P = 0.028), and having ≥pT3 with pN+ disease (HR = 4.2 [1.43–12.3], P = 0.009) were predictive of disease relapse. ConclusionsPatients with pN+ .after RARC had worse oncological outcomes than patients with pN0 disease. Undetectable ctDNA status was informative of RFS regardless of nodal status at both the precystectomy and the MRD window. Patients with undetectable ctDNA status and pN+ disease may benefit from treatment de-escalation.

1211P IMpower010: ctDNA status and 5y DFS follow up in patients (pts) with resected NSCLC who received adjuvant chemotherapy (chemo) followed by atezolizumab (atezo) or best supportive care (BSC)

1211P IMpower010: ctDNA status and 5y DFS follow up in patients (pts) with resected NSCLC who received adjuvant chemotherapy (chemo) followed by atezolizumab (atezo) or best supportive care (BSC)

Tumor Characteristics Associated With Preoperatively Detectable Tumor-Informed Circulating Tumor DNA in Patients With Renal Masses Suspicious for Renal Cell Carcinoma.

Understanding the specific tumor characteristics associated with detectable circulating tumor DNA (ctDNA) in patients with renal cell carcinoma (RCC) is critical for informing future studies aiming to establish the clinical utility of such testing. We characterized the pathologic and clinical features associated with preoperatively detectable ctDNA in patients with renal masses suspicious for RCC. Consecutive patients who underwent partial or radical nephrectomy for nonmetastatic suspected RCC (cT1b-T3) during 2022-2023 had prospectively collected tumor-informed ctDNA analyses conducted preoperatively and postoperatively. Descriptive statistics and univariate analyses were used to describe the study findings. Sixty-nine patients with a median age of 62 years (IQR, 51-70) and a median follow-up time of 7 months (IQR, 3-11) had 205 ctDNA samples collected for analysis. Thirty-nine (61%) had preoperative detectable ctDNA of 64 patients. Postoperative ctDNA status was available for 47 patients, and three (6%) had detectable ctDNA. Two had inferior vena cava (IVC) involvement, and one developed metastatic disease. Subgroup analysis of solely malignant RCC (n = 65) revealed that patients with preoperative detectable ctDNA had a higher pathologic stage (P = .001), larger tumors (7 v 4.5 cm; P = .001), higher tumor complexity (P = .022), and increased rates of tumor grades 3-4 (P = .038). All patients with gross renal vein or IVC involvement (n = 9) and those with lymphovascular invasion (n = 6) on pathology had detectable preoperative ctDNA. On univariate analysis, high tumor complexity, larger tumors, and tumor grades 3-4 were found to be predictors of preoperatively detectable ctDNA status. Preoperative ctDNA was detectable in 61% of patients with nonmetastatic RCC, and it correlated with clinically relevant features. Clinical trials should consider incorporating both preoperative and postoperative ctDNA analyses to augment prediction of disease recurrence and to refine treatment decision making.

63O Transcriptional features of stage IIT4N0/III colon cancer associated with residual disease and ctDNA status after surgery in the PEGASUS trial

63O Transcriptional features of stage IIT4N0/III colon cancer associated with residual disease and ctDNA status after surgery in the PEGASUS trial

Circulating tumour DNA dynamics predict recurrence in stage III melanoma patients receiving neoadjuvant immunotherapy

BackgroundNeoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy.MethodsPlasma samples were collected pre-neoadjuvant treatment, pre-surgery and/or six weeks post-surgery from 40 patients enrolled in the OpACIN-neo and PRADO clinical trials. Patients received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent unbiased pre-amplification followed by tumour-informed mutation detection using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system.ResultsPre-treatment ctDNA was detectable in 19/40 (48%) patients. Among these, 17/19 (89%) zero-converted within six weeks of surgery and none recurred. Positive ctDNA post-surgery (N = 4), irrespective of pre-treatment ctDNA status, was 100% predictive of recurrence (sensitivity 44%, specificity 100%). Furthermore, ctDNA cleared prior to surgery in 7/9 (78%) patients who did not recur, warranting further investigation into ctDNA-guided surgical management.ConclusionPost-surgery ctDNA positivity and zero-conversion are highly predictive of recurrence, offering a window for personalised modification of adjuvant therapy.

Circulating tumor DNA for predicting radiographic and pathologic response to total neoadjuvant therapy in locally advanced rectal cancer: ENSEMBLE-1.

71 Background: Total neoadjuvant therapy (TNT) has dramatically shifted the treatment paradigm for locally advanced rectal cancer (LARC), prolonging survival with high rates of pathologic complete response (pCR) and introducing opportunities for nonoperative management (NOM). However, there is a need for robust predictive biomarkers of TNT efficacy, local cancer regrowth in NOM, and overall prognosis. Circulating tumor DNA (ctDNA) is a minimally invasive biomarker used to detect molecular residual disease (MRD) and predict recurrence in colorectal cancer after curative resection. The effectiveness of ctDNA MRD status specifically as a predictive biomarker for TNT was evaluated in the Phase II TNT study, ENSEMBLE-1 (jRCTs051200113), conducted in Japan. Methods: Patients with LARC undergoing TNT were enrolled in ENSEMBLE-1. Protocol treatment was defined as short-course radiotherapy (SCRT: 25Gy) followed by six cycles of CAPOX and total mesorectal excision (TME). NOM was allowed if a clinical complete response (cCR) was achieved after TNT. ctDNA was measured by SignateraTM (Natera, Inc.) at the following time points: baseline (pre-treatment), after SCRT, after 4 cycles of CAPOX, before TME, and post operatively at 4w, 12w, 24, 36 and 48w in the GALAXY trial (UMIN000039205). Results: A total of 30 patients were enrolled in the study. After completing TNT, TME and NOM were performed in 20 and 7 patients, respectively. ctDNA was measured in 26 patients (TME: 19 patients, NOM: 7 patients). ctDNA was detected in 100% (25/25) at baseline, 81% (21/26) after SCRT, 31% (8/26) after 4 cycles of CAPOX, and 45% (9/20) after TNT. The attached table shows the results of statistical analyses of the correlation between ctDNA status, clinical response, TME or NOM, and pathologic complete response (pCR). It was found that ctDNA status after SCRT and after 4 cycles of CAPOX was associated with cCR or non cCR (p=0.007 and p=0.007, respectively). Additionally, ctDNA status after TNT was associated with pCR or non pCR (p=0.029). Conclusions: Our study demonstrates that ctDNA-based MRD may predict TNT response in LARC patients. Negative ctDNA during treatment correlates with favorable responses and may be an aid in NOM decision making. Clinical trial information: jRCTs051200113 . [Table: see text]

Circulating tumor DNA analysis in patients with esophageal cancer treated with neoadjuvant chemoradiotherapy followed by surgery.

69 Background: The aim of this prospectively study is to investigate circulating tumor DNA (ctDNA) as a prognostic marker for survival in locally advanced esophageal squamous cell cancer (ESCC) patients treated with neoadjuvant chemoradiotherapy (nCRT) and surgery. Methods: Serial plasma samples were collected from 68 ESCC patients treated with neoadjuvant chemoradiotherapy before surgery. ctDNA was detected before treatment, after neoadjuvant chemoradiotherapy and after surgery by personalized, next-generation sequencing assay. Cox regression analyses were used to evaluate the prognostic significance of ctDNA for recurrence-free survival (RFS) and overall survival (OS). Results: ctDNA was detectable in 89.7%, 17.2%, and 6.9% of pretreatment, post-neoadjuvant chemoradiotherapy and post-surgery plasma samples, respectively. The conversion of ctDNA status from positive at baseline to negative at 4-6 weeks after neoadjuvant chemoradiotherapy was significantly associated with pathological complete response (pCR) ( P = 0.02). Significant worse RFS was related to detectable ctDNA after neoadjuvant chemoradiotherapy ( P = 0.008) or after surgery ( P = 0.001). Detectable ctDNA at baseline ( P = 0.01) and after surgery ( P = 0.008) were associated with worse OS. In multivariate analysis, detectable postoperative ctDNA was significantly associated with RFS ( P = 0.012) and OS ( P = 0.025) after adjusting for other clinicopathological risk factors. Conclusions: ctDNA status is a strong prognostic factor of recurrence in locally advanced ESCC patients. Consequently, ctDNA could potentially improve pre- and post-treatment risk assessment and facilitate individual therapy for ESCC patients.

New Biomarkers to Define A Biological Borderline Situation for Pancreatic Adenocarcinoma - Results of An Ancillary Study of the PANACHE01-PRODIGE48 Trial.

To investigate in patients treated for a resectable pancreatic ductal adenocarcinoma (PA), the prognostic value of baseline CA19-9 and circulating tumour DNA (ctDNA) for overall survival (OS), to improve death risk stratification, based on a planned ancillary study from PANACHE01-PRODIGE 48 trial. Biological borderline situation that was first used by the MD Anderson, became a standard practice following the international consensus conference in 2016 to manage PA. Regarding the risk of systemic disease especially in the setting of "markedly elevated" CA19-9, neoadjuvant therapy is advised to avoid unnecessary surgery, with risk of early recurrence. To best define biological borderline situations, new biomarkers are needed. Characteristics at diagnosis and OS were compared between patients with or without ctDNA status available. OS was estimated with Kaplan Meier method and compared with log-rank test. Restricted cubic spline approach was used to identify optimal threshold for biological parameters for death risk stratification. Univariate and multivariate Cox proportional hazard models were estimated to assess the association of ctDNA status and other parameters with OS. Among the 132 patients from the primary population for analysis in the PANACHE01 -PRODIGE 48 trial, 92(71%) were available for ctDNA status at diagnosis. No selection bias was identified between patients with or without ctDNA status. 14 patients (15%) were ctDNA+ and exhibited a higher risk for death (P=0,0188; HR95% CI: 2.28 (1.12-4.63). In the 92 patients with ctDNA status available among the others parameter analysed only CA19-9 was statically associated with OS in univariate analysis. Patients with log of CA19-9 equal or superior to 4.4 that corresponds to a CA19-9 of 80 UI/mL were identified at higher risk for death (P=0,0143; HR95% CI: 2.2 (1.15-4.19). In multivariate analysis CA19-19 remained independently associated with OS (p-value=0.0323). When combining the two biomarkers, median OS was of 19.4 (IC 95% 3.8-Not reached) months, 30.2 (IC 95% 17.1-NR) months and not reached (IC 95% 39.3-NR) for "CA19-9 high and ctDNA+ group", "CA19-9 high or ctDNA+ group", and "CA19-9 low and ctDNA- group", respectively (logrank P=0,0069). Progress in the management of potentially operable PA remains limited, relying solely on strategies to optimize the sequence of complete treatment, based on modern multidrug chemotherapy (FOLFIRINOX, GemNabPaclitaxel) and surgical resection. The identification of risk criteria, such as the existence of systemic disease, is an important issue, currently referred to as "biologic borderline disease". Few data, particularly from prospective studies, allow us to identify biomarkers other than CA19-9. Combining ctDNA to CA19-9 could be of interest to best define biological borderline situations in PA.

Clinical Utility of Tumor-Naïve Presurgical Circulating Tumor DNA Detection in Early-Stage NSCLC

ObjectivesThe use of tumor-informed circulating tumor DNA (ctDNA) testing in patients with early-stage disease before surgery is limited, mainly owing to restricted tissue access and extended turnaround times. This study aimed to evaluate the clinical value of a tumor-naïve, methylation-based cell-free DNA assay in a large cohort of patients with resected NSCLC. MethodWe analyzed presurgical plasma samples from 895 patients with EGFR and anaplastic lymphoma kinase-wild-type, clinical stage I or II NSCLC. The ctDNA status was evaluated for its prognostic significance in relation to tumor volume, metabolic activity, histologic diagnosis, histologic subtypes, and clinical-to-pathologic TNM upstaging. ResultsPresurgical ctDNA detection was observed in 55 of 414 patients (13%) with clinical stage I lung adenocarcinoma (LUAD) and was associated with poor recurrence-free survival (2-year recurrence-free survival 69% versus 91%; log-rank p < 0.001), approaching that of clinical stage II LUAD. Presurgical ctDNA detection was not prognostic in patients with clinical stage II LUAD or non-LUAD. Within LUAD, tumor volume and positron emission tomography avidity interacted to predict presurgical ctDNA detection. Moreover, presurgical ctDNA detection was predictive of the postsurgical discovery of International Association for the Study of Lung Cancer grade 3 tumors (p < 0.001) and pathologic TNM upstaging (p < 0.001). Notably, presurgical ctDNA detection strongly correlated with higher programmed death-ligand 1 expression in tumors (positive rates 28% versus 55%, p < 0.001), identifying a subgroup likely to benefit from anti–programmed death-ligand 1 therapies. ConclusionThese findings support the integration of ctDNA testing into routine diagnostic workflows in early-stage NSCLC without the need for tumor tissue profiling. Furthermore, it is clinically useful in identifying patients at high risk who might benefit from innovative treatments, including neoadjuvant immune checkpoint inhibitors.

Development of a prediction model for lung metastasis by using postoperative circulating tumor DNA binding clinicopathological risk factors in patients with colorectal cancer.

e15035 Background: Lung metastasis is common in patients with colorectal cancer (CRC). However, diagnosing lung metastasis in CRC is challenging due to slow-growing pulmonary lesions. Circulating tumor DNA (ctDNA) testing provides a direct indication of the presence of minimal residual disease (MRD) which contributing to cancer recurrence. Yet, there are no reports on the potential indications of ctDNA for the site of metastasis. The objective of this study is to develop a predictive model for lung metastasis in CRC patients who have received adjuvant therapy by integrating ctDNA with clinicopathological factors. Methods: Two independent cohorts' series were evaluated including 299 patients from China as training cohort and 134 patients from Spain as validation cohort from 2012-2020. We specifically focused on the detection of ctDNA in these patients. We employed LASSO regression for optimal feature selection and built a predictive model by multivariable logistic regression. Results: In training cohort, 59/299(19.7%) patients have detectable ctDNA. In validation cohort, 24/134 (18.6%) patients have detectable ctDNA. Patients who tested ctDNA-positive had a significantly higher incidence of lung metastasis (33.9% vs. 11.2%, P&lt;0.001) compared to ctDNA-negative patients. A predictive model for lung metastasis was developed by multivariable logistic regression analysis. The model incorporated factors such as ctDNA status, preoperative carcinoembryonic antigen (CEA) level, presence of cancerous nodes, and the mutation status of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) in plasma. The lung metastasis prediction nomogram demonstrated a favorable ability to distinguish with a concordance index of 0.775 (95% CI: 0.706-0.775) during internal validation. The Hosmer-Lemeshow test, with p-value of 0.967, indicated that prediction nomogram model had a good fit. In addition, the prediction model of validation cohort demonstrated a discriminative performance with a concordance index of 0.724(95% CI: 0.698-0.724). Furthermore, the DCA (Decision Curve Analysis) and CIC (Clinical Impact Curve) in both cohorts indicated that the lung metastasis nomogram exhibited a favorable clinical net benefit. Finally, through multi-model comparison, it was found that the predictive performance of the model, which includes ctDNA and blood PIK3CA mutation status, was significantly superior to all clinical prediction models. Conclusions: A lung metastasis prediction nomogram was developed by integrating postoperative ctDNA and clinicopathological risk factors. The purpose of this nomogram is to assist clinicians in predicting the probability of lung metastasis in patients who have undergone surgery for CRC, with the aim to facilitate prompt intervention.

Preliminary results correlating post-operative ctDNA status with disease-free survival in patients with stage II (high risk)/III colorectal cancer in the BNT000-001 epidemiology study.

3526 Background: Despite standard-of-care surgical resection and adjuvant chemotherapy (ACT), disease recurrence in patients (pts) with stage II or III colorectal cancer (CRC) remains high. Circulating tumor DNA (ctDNA) analysis post-surgery with curative intent may help to identify pts with minimal residual disease at risk for recurrence. We report preliminary results of the BNT000-001 study exploring the prevalence and prognostic value of ctDNA positivity post-surgery in pts with stage II (high-risk) / III CRC. Methods: The BNT000-001 (NCT04813627) epidemiological study is enrolling pts in US, Germany (including screening through the Colopredict registry study), Spain, and Belgium with resected high-risk (per NCCN guidelines) stage II / III CRC for longitudinal follow up of their ctDNA status post-surgery and ACT. The primary objective of the study is to estimate the prevalence of ctDNA positivity after surgery. Other objectives include evaluation of the prognostic value of ctDNA and identification of ctDNA positive pts who might be candidates for the interventional clinical trial BNT122-01 (NCT04486378). Disease-free survival (DFS) was measured from the post-surgery visit to disease recurrence/death and analyzed for all eligible pts who remain on study. ctDNA was analyzed using the laboratory developed AVENIO Oncology Assays (AOA) Surveillance test and reported as detected (“positive”), not detected (“negative”), or no result (“non-evaluable”). Results: 1094 pts have been screened, of which 874 pts were analyzed for post-surgery ctDNA status (data cutoff 15 Dec 2023). 142 pts were non-evaluable, resulting in 732 pts with defined ctDNA status. Prevalence of post-operative ctDNA positivity was 12.3% (90*/732). 87.7% (642/732) of pts were ctDNA negative. 826 pts were followed for the prognostic value of post-surgery ctDNA-positivity, excluding 48 ctDNA positive pts who transferred to the BNT122-01 treatment study. At a median follow-up time of 25.9 months, the median DFS of ctDNA positive pts was 12.16 months vs. median DFS not reached for ctDNA negative pts. The 12-month DFS rate was 55% for ctDNA positive vs. 92% for ctDNA negative pts. Of note, our analyses support the identification of ctDNA negative T4N2 pts as a high-risk group for recurrence. Post-surgery ctDNA positivity was associated with significantly increased risk for disease recurrence or death, with a hazard ratio of 10.2. Conclusions: This study supports existing evidence of the prognostic value of post-operative ctDNA in CRC pts. In ctDNA positive pts, the 12-month DFS rate was almost halved (55% vs. 92% in ctDNA negative). Clinical trial information: NCT04813627 . [Table: see text]

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

TPS3641 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) represents a highly specific and sensitive approach (especially with serial monitoring) for identifying minimal/molecular residual disease (MRD) post-surgery 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 be spared the toxicities associated with 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 CC 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 IIB, IIC, and III CC will be enrolled. Based on the post-operative ctDNA status using personalized and tumor-informed assay (Signatera, bespoke 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 vserial ctDNA monitoring. Patients who are ctDNA+ post-operatively, or with serial monitoring (Cohort B), will be randomized to FP+Ox v more intensive AC with addition of irinotecan (I) for 6 mos. One cycle of chemotherapy is allowed while awaiting ctDNA testing results for cohort assignment. The primary endpoints for Cohort A are time to ctDNA+ status (phase II) and disease-free survival (DFS) (phase III) in the immediate v delayed AC arms. The primary endpoint for Cohort B is DFS in the FP+Ox v FP+Ox+I arms for both phase II and phase III portions of the trial. Secondary endpoints include prevalence of detectable ctDNA post-operatively, time-to-event outcomes (overall survival and time to recurrence) by ctDNA status, and the assessment of compliance to adjuvant therapy. Biospecimens including archival tumor tissue, as well as post-operative plus serial matched/normal blood samples, will be collected for exploratory correlative research. Active enrollment across the NCTN started in June 2022 with CCTG sites joining in August 2023. Current accrual (as of 02-02-2024) is 240/1,912. NCT05174169. Support: U10-CA-180868, -180822; UG1CA-189867; Natera, Inc. Clinical trial information: NCT05174169 .

CtDNA monitoring for breast cancer at high risk of recurrence: Interim analysis of JCOG1204A1.

518 Background: Monitoring ctDNA after definitive surgery has emerged as an accurate predictor of breast cancer (BC) recurrence. However, further data is needed to determine the proportion and dynamics of ctDNA positivity in different BC subtypes. Here we present ctDNA status and dynamics in patients from an ancillary study (A1) of the prospective randomized phase III JCOG1204 trial (UMIN000012429; Registration: November 2013 - January 2020, N = 1034) in which intensive post-operative follow-up is compared to standard follow-up in BC patients at high risk of recurrence. Methods: Eligible patients enrolled in JCOG1204 were required to have either (1) ≥4 lymph node metastases for estrogen receptor (ER)+ BC without neoadjuvant therapy, (2) ≥1 lymph node metastases for ER- without neoadjuvant therapy, or (3) ≥1 lymph node metastases after neoadjuvant therapy. Additional inclusion criteria for ancillary study JCOG1204A1 include no more than 4 years after surgery and relapse-free at the enrollment. A personalized, tumor-informed assay (Signatera, Natera Inc.) was used for ctDNA surveillance every 3 - 6 months for 2 years for ER- or 5 years for ER+ BC. ctDNA status, time of ctDNA detection and association with subtype were examined. The JCOG DSMC approved the publication of this interim analysis. Results: A total of 248 patients were enrolled (July 2020-October 2023) in JCOG1204A1. Personalized assays were successfully designed for 88% patients, the main reason for failure was insufficient tissue (83%; 25/30). A total of 1440 plasma ctDNA samples were collected from 211 patients. The median age at the time of surgery was 50 years. The most prevalent subtype was ER+ HER2- (Luminal; n = 130), followed by ER- HER2- (TN; n = 38), ER- HER2+ (HER2; n = 24), and ER+ HER2+ (Luminal-HER2; n = 18). 25 patients (11.8%) had at least one positive ctDNA result, with overall sample-level positivity proportion of 4.5% (65 / 1440); 76% (19/25) became detectable with longitudinal testing. Surveillance in TN [the first time point median 31.5 months (12.6-55.8) from surgery] did not result in ctDNA detection. Of the 130 patients with Luminal subtype [the first time point median 36.6 months (12-57) from surgery], 21 (16.2%) had ctDNA first detected at a median 43.5 months (18-72). Additionally, 5 patients (2.4%) had ctDNA detection for the first time after 5 years of surgery. The proportion of ctDNA positivity increased by ~2% annually in Luminal subtype; varied rates of ctDNA level increase have been observed. Conclusions: Monitoring ctDNA in high-risk BC reveals a detection proportion of 11.8% at the patient level (TNBC: 0%; Luminal: 16.2%) Longitudinal testing revealed ctDNA detection in patients with luminal subtype beyond 5 years from surgery, suggesting a correlation with late recurrence. Subsequent reports will focus on investigating the association of ctDNA status with relapse and overall survival.

Circulating tumor DNA (ctDNA) to predict response to neoadjuvant chemotherapy (NAC) and prognosis in patients (pts) with early breast cancer (BC).

e12642 Background: pCR to NAC is an important predictor of clinical recurrence. Up to 16% of patients with pCR may experience disease recurrence, 45% of patients with RD do not. Longitudinal ctDNA testing offers a minimally invasive approach for monitoring treatment response EBC patients receiving NAC. We evaluated whether ctDNA status and dynamics can predict the tumor response to NAC and the risk of recurrence in patients with EBC. Methods: We conducted an observational, retrospective, single-center study to investigate whether ctDNA can predict tumor response to NAC and prognosis in BC patients. 74 pts received standard of care NAC;g 4 patients were treated in the adjuvant setting. Blood samples were collected at diagnosis from 44 pts (baseline) and longitudinally during treatment from 34 patients. Tumor response and progression outcomes were reported. A clinically validated, tumor-informed ctDNA assay (Signatera). ctDNA status and dynamics were correlated with pathologic and radiologic outcomes. Results: 29 TNBC pts were ctDNA+ at baseline, 6 were ctDNA- Of the 29 pts, early ctDNA clearance by week 6 of NAC was observed in 10 pts, weeks 7-16 in 9 pts, clearance post-surgery in 1, 3 pts remained ctDNA+ after surgery, serial ctDNA testing is underway for the remaining 6 pts. All TNBC patients who had radiological complete response (CR) had early clearance of ctDNA . All pts with persistent ctDNA positivity levels after completion of NAC had radiological partial response (PR). Of the 9 TNBC pts with undetectable baseline ctDNA, 33% achieved CR on MRI and 43% achieved pathologic CR. Of the 19 TNBC pts with ctDNA clearance in response to NAC, 89% (17/19) remained ctDNA- and with no evidence of disease (NED) (length of follow up). 1 pt initially cleared their ctDNA on NAC, turned ctDNA+ 4 weeks prior to radiological recurrence. 1 pt with ctDNA clearance on NAC remained ctDNA post-surgery but experienced recurrence. All (n=3) TNBC pts with persistent ctDNA-positivity levels after curative treatment (NAC and surgery) had disease recurrence. The remaining 6 pts ctDNA are ongoing NAC. Of the 6 pts with undetectable baseline ctDNA, 6/6 continued to be ctDNA- regardless of their disease status. At diagnosis, 8/9 ER+ ps were ctDNA+. 3/8 (38%) ER+ pts with positive baseline ctDNA had early clearance of ctDNA after NAC. All 3 had residual disease on pathology. 2/8 (25%) remained ctDNA+ after surgery and neither achieved radiologic CR or pCR. The remaining 3 pts are ongoing NAC. Conclusions: In early TNBC, ctDNA monitoring can be used to predict tumor response to NAC and tailor therapeutic regimens. ctDNA persistence can predict early recurrence and helps identify patients in which treatment escalation or enrollment on clinical trials is necessary. In ER positive breast cancer, and despite the small number of patients, ctDNA clearance does not predict response to NAC.

Clinical validation of a novel circulating tumor DNA methylation assay for predicting recurrence of colorectal cancer in patients with curative-intent surgery by monitoring minimal residual disease.

3620 Background: Circulating tumor DNA (ctDNA)-based liquid biopsy has emerged as a promising tool for early detection and monitoring of cancer. In this study, we developed COLO eDX, a digital-PCR based colorectal cancer (CRC) diagnostic assay using a single CRC-specific methylation biomarker. This study aimed to evaluate the prognostic value as the universal assay for recurrence monitoring in CRC patients with curative-intent surgery. Methods: A total of 116 CRC patients who underwent surgery at Seoul National University Boramae Medical Center were enrolled in the study from Mar 2017 to January 2020. For each patient, postoperative plasma specimens were collected at 7 days and at each regular clinical follow-up visit up to 2 years. The enrolled patients were followed up with radiological and clinical measures for maximally 5 years after surgery. The primary outcome was recurrence-free survival (RFS) according to ctDNA status. Results: The majority of samples collected at 7 days after surgery were ctDNA positive by COLO eDX (65/83, 78.3%) and the ctDNA status was not significantly associated with RFS, suggesting that cancer signatures can still be detected in most cancer patients at least 1 week after surgery. However, ctDNA positivity at 1 month or later was associated significantly worse RFS. In plasma taken between 1- and 9-months after surgery, ctDNA positive group had a significantly higher recurrence rate than ctDNA negative group (hazard ratio [HR], 11.4, 95% confidence interval [CI], 3.8-34.4; P&lt;0.0001). Incorporating serial longitudinal samples, ctDNA status was significantly associated with recurrence (HR, 13.4, 95% CI, 4.3-40.6; P&lt;0.0001). Notably, ctDNA analysis by COLO eDX was a more powerful predictor for predicting recurrence than carcinoembryonic antigen (CEA) assessment (HR, 4.0, 95% CI, 1.4-11.8; P=0.0107 and HR, 1.5, 95% CI, 0.3-8.2; P=0.5949). Conclusions: Postoperative ctDNA monitoring using a CRC-specific epigenetic biomarker-based digital PCR assay is a promising strategy for recurrence prediction and personalized clinical management of CRC through early detection of minimal residual disease (MRD).

Association of circulating DNA with recurrence in CRS-HIPEC patients with colorectal and appendiceal peritoneal carcinomatosis: Insights from a phase II randomized trial.

3598 Background: There is a need for biomarkers to guide patient selection for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) in cases of peritoneal carcinomatosis from colorectal and appendiceal cancer. SigNatera is a blood-based quantitative test to detect colorectal cancer recurrence by indicating the presence of at least two altered colorectal cancer-associated genes in Circulating tumor DNA (ctDNA). We performed a correlative study to help determine if using SigNatera ctDNA would be a predictive and prognostic biomarker of recurrent cancer in patients who underwent CRS-HIPEC on a Phase II Randomized Trial Comparing Morbidity and Mortality of CRS-HIPEC using Mitomycin-C versus Melphalan for Colorectal and Appendiceal Peritoneal Carcinomatosis. Methods: 30 patients on the phase II trial had the option for consenting for the ctDNA correlative study utilizing SigNatera sponsored by Natera. Pre-op samples were collected prior to CRS-HIPEC on Day 0 and post-op samples ~2 weeks post-hospital discharge, followed by every 3 months x 2 years. The exploratory objective of the prognostic impact of ctDNA is evaluated by correlating pre-op ctDNA status and Disease-Free Survival (DFS) Results: All 30 pts that underwent ctDNA testing were treated with neoadjuvant chemotherapy prior to CRS-HIPEC. Pre-op ctDNA was negative in 20 pts (8 recurrences, median DFS = 15.9 months) and positive in 10 pts (7 recurrences, median DFS = 39.9 months), p =0.19. Of the 20 pre-op ctDNA(-) pts, 18 remained ctDNA(-) in post-op (12 recurrences, median DFS = 15.6 months) and 2 turned ctDNA(+) (0 recurrences). Of the 10 pre-op ctDNA(+) pts, 2 remained ctDNA(+) post-op (1 recurrence, median DFS = 9.5 months) and 8 turned ctDNA(-) (2 recurrences, median DFS = 39.9 months), p=0.1. Conclusions: The unexpected numerically favorable DFS in the pre-op ctDNA(+) group, in comparison to the pre-op ctDNA(-) group, challenges the utility of preop ctDNA status in guiding CRS + HIPEC decision-making. Though limited by small sample size, this finding may be likely due to the impact of post-op therapy which notably converted 10 preop ctDNA(+) pts to post-op ctDNA(-) status. Our findings warrant validation in a larger study to unravel the multifaceted factors influencing ctDNA’s prognostic value. Clinical trial information: NCT03073694 . [Table: see text]