Detection Of Molecular Residual Disease Research Articles

Association of ultra-sensitive ctDNA assay to identify actionable variants and response to immune checkpoint inhibitor (ICI) therapy in metastatic melanoma.

9562 Background: Detection of molecular residual disease (MRD) via circulating tumor DNA (ctDNA) can identify therapeutic response/resistance months in advance of imaging, and monitoring clinically actionable variant dynamics in ctDNA may be important for guiding treatment. Despite this, adoption has been slow due to the reduced sensitivity and reproducibility of current approaches. Here, we profile melanoma patients receiving ICI over several years using an ultra-sensitive, tumor-informed ctDNA platform, and correlate the findings with clinical outcome. Methods: Over 150 plasma samples from 23 advanced melanoma patients (stage IV) were collected during ICI treatment (up to 40 months) and profiled with NeXT Personal, a tumor-informed ctDNA assay that leverages whole-genome sequencing of tumor/normal samples to generate personalized liquid biopsy panels. Each bespoke panel consists of up to 1,800 selected variants which enable sensitive MRD detection, and a fixed set of 2,100 known clinically actionable and resistance loci for detection of variants emerging under therapeutic pressure. MRD signal and variant dynamics were then correlated with RECIST assessments and outcome. Results: In this cohort, ctDNA was detected across a broad dynamic range (2.3-100,000 PPM; median limit of detection = 1.97 PPM) with 37% of detections occurring below 100 PPM. Baseline ctDNA was detected in 94% (17/18) of patients, and was significantly correlated with S100B and metastatic burden (Spearman’s rho, 0.73 and 0.5; p, 0.0002 and 0.025). The mutation-rate normalized count of melanoma-specific driver genes detected at baseline predicted reduced overall survival (OS) (p = 0.03). 94% (17/18) of ctDNA+ patients displayed dynamic variant allele frequency (VAF) shifts, impacting 149 loci across 37 ICI-related genes including BRAF, CDKN2A, KIT, MAP2K1, and NRAS. Delta ctDNA from baseline to the first on-treatment time point correlated with PFS (log rank p = 0.004). Treatment elicited greater than 3-fold reduction in average PPM, and the magnitude was significantly correlated with progression (p = 0.0005). VAF increases in known ICI-related genes (JAK1, ALK, etc) predicted shortened OS (p = 0.007) and fewer variants in genes conferring sensitivity to (NRAS, BRAF, etc) ICI were detected on-treatment in progressing patients (p = 0.025). Conclusions: We tracked both tumor-informed MRD and tumor-agnostic evolution of ICI related variants over the course of treatment using a single liquid biopsy platform. We achieved ultra-sensitive ctDNA detection down to 2.3 PPM and dynamics in ctDNA signals were predictive of clinical outcome. Furthermore, we demonstrated that ctDNA measurements and VAF dynamics of clinically actionable and resistance variants correlated with response as well as resistance to ICI.

A phase II single-arm, open-label trial of T-DM1 (ado-trastuzumab emtansine) and neratinib for HER2-positive breast cancer with molecular residual disease (KAN-HER2 MRD).

TPS620 Background: HER2-positive breast cancer is a biologically and clinically aggressive subtype that has historically been associated with poor outcomes, but for which there are now multiple effective targeted therapies. Patients who have residual disease following standard neoadjuvant therapy have an elevated risk of metastatic recurrence. In addition, the detection of circulating tumor DNA (ctDNA) in the adjuvant period is strongly associated with relapse and can further stratify patients with residual disease. The ctDNA-based detection of molecular residual disease (MRD) is an emerging strategy to identify patients for treatment intensification, and may enable the development of novel curative treatment strategies for “recurrence interception”. Preclinical and clinical evidence support the investigation of neratinib, an irreversible inhibitor of the HER2 tyrosine kinase, in combination with standard T-DM1 in the adjuvant setting for patients with HER2-positive breast cancer and evidence of MRD. Methods: KAN-HER2 MRD is a multicentre, investigator-initiated, open label, single arm phase II trial to evaluate the addition of neratinib to standard T-DM1 in patients with detected MRD. Participants with HER2-positive breast cancer and residual disease following neoadjuvant therapy are pre-screened (Part A) for MRD using a tumor-informed assay (NeoGenomics RaDaR). Those with ctDNA detected following 2-6 cycles of adjuvant T-DM1 will proceed to the interventional phase (Part B) where neratinib is added to standard T-DM1 at the previously-determined recommended phase 2 combination dose of 160 mg/day, in continuous cycles. The primary endpoint is the rate of ctDNA clearance at 12 weeks after treatment initiation (ctDNAwk12); the study is designed with a 2-stage approach and has sufficient power to detect a clearance rate of 40%. Secondary endpoints include MRD rates and their clinical correlates, invasive disease free survival (iDFS), and safety. Bio-specimens including diagnostic biopsies, residual disease, and peripheral blood are being collected from all participants for additional correlative studies. Enrolment in Part A was initiated at the first trial site (Princess Margaret Cancer Centre) in December 2022. KAN-HER2 represents the first reported MRD-directed interception trial for HER2-positive breast cancer. Clinical trial information: NCT05388149 .

Longitudinal circulating tumor DNA monitoring for detection of molecular residual disease in patients with surgically resected stage II/III melanoma.

9582 Background: Over the past decade, advances in immunotherapy (IO) have dramatically improved overall survival (OS) for patients with metastatic melanoma. Immune checkpoint inhibitors and BRAF/MEK inhibitors also became standard of care for surgically resected stage III melanoma, and most recently pembrolizumab was approved for stage IIB/C patients. Although the phase III trials that led to their respective approvals met their primary endpoints for relapse-free survival, no benefit in OS has been observed. Given that a large proportion of early-stage melanoma patients are cured with surgery alone, the decision to proceed with adjuvant therapy should be weighed against the risk of potential life-altering toxicity. There is a high unmet need to develop biomarkers to accurately select patients who are most likely to benefit from adjuvant IO while sparing a group of low risk patients from unnecessary treatment. In this study, we evaluated the prognostic value of personalized, tumor-informed circulating tumor DNA (ctDNA) testing in patients with resectable stage II/III melanoma. Methods: In this real-world study, longitudinal plasma samples (n = 159) were analyzed in real-time from 45 stage IIA-IIID cutaneous melanoma patients treated at Rush University between 03/30/2021 and 01/12/2023. A personalized, tumor-informed ctDNA assay (Signatera bespoke, mPCR-NGS assay) was used for detection and quantification of ctDNA in plasma samples. Results: Personalized, tumor-informed ctDNA assays were successfully designed for 93% (42/45) of patients using tissue samples from either surgical resection (14/42, 33%) or biopsy (26/42, 67%; 16 shave, 10 excisional, 1 punch, 1 core). ctDNA was detectable at one or more time points in 36% (15/42) of patients. We observed a higher ctDNA-positivity rate in stage III patients (11/20, 55%) compared to stage II patients (4/22, 18%). Twelve of the 15 ctDNA-positive patients received adjuvant IO/treatment escalation after a positive ctDNA result; 6 patients had sustained ctDNA clearance, 2 had transient clearance, and 4 continue to receive adjuvant IO and follow-up ctDNA testing. Radiographic recurrence was observed in 7/42 patients, 6 of whom were ctDNA-positive. Ninety-six percent (26/27) of patients with persistent ctDNA-negativity remain clinically and radiographically progression-free. Conclusions: Our data suggest that molecular residual disease detection using a personalized, tumor-informed ctDNA assay is highly prognostic in melanoma patients undergoing curative resection. Sustained clearance of ctDNA was achieved with adjuvant therapy in a subset of this cohort. Prospective studies evaluating the utility of ctDNA to aid clinical decision-making regarding adjuvant treatment intensification or de-escalation are warranted.

Circulating tumor DNA–based detection of molecular residual disease as a biomarker for recurrence monitoring predict disease recurrence in patients with ovarian cancer.

e17542 Background: Despite recent advances in cancer diagnostics and treatments, nearly 75% ovarian cancer patients relapse within two years of primary therapy and become candidates for treatment of recurrent diseases. A novel approach to post-surgical risk stratification for molecular residual disease (MRD) detection and treatment monitoring is needed to improve patient outcomes. Methods: We analyzed a cohort of 22 stage Ⅰ-Ⅳ ovarian cancer patients which were followed up at median 6 (range,3-15) times between July 2017 and December 2020. Among the 22 patients, stage I, II, III and Ⅳ comprised 9%, 5%, 50% and 36%, respectively. Tumor tissue and blood samples of all subjects were collected at Ruijin Hospital, Shanghai Jiao Tong University.Somatic mutations were identified by whole exome sequencing and assessed from plasma collected during treatment using personalized tumor-informed MRDtect assays with a median of 25（range,17-30） amplicons/assay targeting tumor-specific variants unique to each patient, sequenced at mean 200,000X depth. Plasma samples with ≥2 variants detected were considered MRD-positive and ctDNA levels were reported in mean tumor molecules per mL (MTM/mL) of plasma. Sequencing data were analyzed using PiVAT secondary analysis software and MRD positive was concluded when at least two variants were detected in plasma. Results: CtDNA was preoperatively detectable in 18 (85.7%) patients, and the positive rate became 38.8% (7/18) after surgery. Serial ctDNA analysis during surveillance after definitive treatment of the 22 patients with longitudinal collected plasma samples identified relapse with 100% sensitivity and 100% specificity. The median lead time from ctDNA detection in plasma to relapse detection by standard-of-care computed tomography was 135 days (range,45-387days). Variants from a total of 526 genes were selected from the WES results of these 22 patients. Among these 526 genes, 96% (505 genes) were selected for only one patient, 3.4% (18 genes) were selected for two patients and 0.2% (one gene) were selected for three patients. Compared with tumor-agnostic assay, tumor-informed assay is a more accurate route that is more consistent with the heterogeneity of tumor and can maximize the ability to reflect the real situation of each patient. Conclusions: Sensitive methods for risk stratification, therapeutic efficacy monitoring, and early relapse detection may have a major impact on treatment decisions and patient management for ovarian cancer patients. MRD can be used as an effective biomarker to prompt prognosis and recurrence risk, providing powerful assistance for accurate diagnosis and treatment for patients.

Molecular residual disease (MRD) detection using bespoke circulating tumor DNA (ctDNA) assays in localized soft tissue sarcoma (STS).

11509 Background: Surgery and (neo)adjuvant radiotherapy (RT) are the mainstay curative treatments for localized STS. Despite treatment, up to 50% of STS patients experience metastatic relapse, and routine use of adjuvant systemic therapy (AST) remains controversial. The presence of ctDNA following curative-intent treatment of STS is a potential biomarker for MRD and may identify patients who are likely to benefit from AST. Given the genomic heterogeneity of STS, a histology-agnostic approach to ctDNA detection in this population is desirable. Methods: Patients (pts) with localized, high risk (size ≥ 5cm, grade ≥ 2) STS were enrolled prior to (neo)adjuvant RT and surgery. Pts who received (neo)adjuvant systemic treatment were excluded. Blood samples for ctDNA analysis were collected at diagnosis, post-RT, post-surgery and every 3 months for up to 2 years. Whole exome sequencing (WES) of archival tumor and matched normal were carried out to identify patient-specific, somatic, single nucleotide variants. Personalized and tumor-informed, multiplex PCR next generation sequencing-based ctDNA (Signatera) assays were then developed to track ctDNA in serially collected plasma samples. ctDNA levels were expressed as mean tumor molecules per milliliter (MTM/ml) of plasma. Radiologic surveillance was performed every 3 months following surgery. The primary endpoint was a ctDNA detection rate of >70% at diagnosis. Secondary endpoints included MRD detection after local therapy and correlation of ctDNA levels with disease relapse. Results: A total of 140 plasma samples from 22 pts [18 male and 4 female; median age: 65 years, range: 30 – 84] were obtained. RT was preoperative in 19/22 pts. Of the 22 tumor samples, 20 had adequate tissue quality for WES to enable ctDNA assay design. Tumor histologic subtypes included undifferentiated pleomorphic sarcoma (n = 6), myxofibrosarcoma (n = 5), and liposarcoma (n = 9). A median of 7 plasma samples per patient (range: 2 – 10) were analyzed. ctDNA was detected in 80% of pts (16/20) at diagnosis, with median ctDNA level of 3.4 MTM/mL (range: 0.2 – 1393.9). Of these 16 pts, 15 (94%) became ctDNA negative at the immediate post-surgical timepoint. In addition, ctDNA was detected in 4 pts (80%) prior to or around radiologic relapse with a median lead time of 92 days. Conclusions: Personalized, tumor-informed ctDNA assays can detect MRD after definitive local therapy and/or prior to radiologic recurrence in patients with localized high-risk STS. As such, serial ctDNA monitoring provides prognostic value and may further identify patients that will benefit from AST treatment. Additional studies evaluating ctDNA as a predictive biomarker for AST benefit are ongoing.

Tumor-informed circulating tumor DNA (TI-ctDNA) based molecular residual disease (MRD) detection and relapse risk for patients with stage II-IV melanoma in surgical remission: A single center experience.

9572 Background: Melanoma is associated with significant risk of relapse after surgery. While the odds of relapse increase with advancing stage, long term relapse free survival (RFS) is possible without adjuvant therapy in a significant subset of patients as shown by AJCC v8 staging long term survival data. TI-ctDNA (Signatera™) is a non-invasive biomarker for MRD detection and functions by sequencing the patient’s own tumor for the creation of a customized panel of PCR probes based upon a tumor’s unique mutational profile. Positive TI-ctDNA testing is significantly associated with relapse after surgical resection in colorectal cancer and may help inform treatment decision-making, allowing for intensification or de-intensification of patient management. This study describes the operating characteristics of TI-ctDNA in monitoring for relapse in patients with resected melanoma. Methods: This is a single center retrospective analysis of patients with stage IIb-IV melanoma in surgical remission. Between January 2021 and January 2023, male and female patients presenting in surgical remission were monitored with TI-ctDNA at 6- or 12-week intervals. Surveillance imaging for relapse was performed according to standard of care guidelines appropriate for tumor stage. Patients were treated with immunotherapy, targeted therapy, or melanoma specific anti-tumor vaccine. Duration of RFS from surgery to detectable TI-ctDNA was recorded, along with duration of RFS from surgery to confirmed melanoma relapse. Results: 45 eligible patients were included in the analysis. 10 patients had detectable TI-ctDNA during the study period. 7 patients had confirmed relapse, 6 of whom were TI-ctDNA positive and 1 who was TI-ctDNA negative. The total relapse rate in TI-ctDNA positive patients (6/10) was significantly higher than the relapse rate in TI-ctDNA negative patients (1/35) [p = 0.0002]. Time to detection of positive TI-ctDNA was variable and ranged from 0 to 664 days (median 73 days). Conclusions: TI-ctDNA testing demonstrated preliminary ability to differentiate melanoma patients at the highest risk of relapse. Prospective evaluation of TI-ctDNA monitoring for determination of patients at high versus low risk of relapse is warranted and may help to inform future clinical trial designs by allowing for patient stratification to low risk (undetectable TI-ctDNA) and high risk (detectable TI-ctDNA) disease states.

Prospective dynamic sampling and molecular residual disease monitoring to predict clinical outcomes for patients with unresectable, locally advanced non-small cell lung cancer undergoing radical radiotherapy.

8549 Background: The role of molecular residual disease (MRD) detection in non-small cell lung cancer (NSCLC) patients after definitive resection has been progressively elaborated. However, the dynamic patterns of circulating tumor DNA (ctDNA) during and after radical radiotherapy (RT) remain unclear but are critical for detecting MRD in this context. Methods: We enrolled 139 patients with locally advanced NSCLC treated with radical RT in this prospective study. And 761 blood samples were successfully detected by ctDNA-MRD assay at different preset timepoints before, during and after RT. Results: There was a declining trend in ctDNA concentrations of “during RT” time point (RT reached 40 Gy) relative to those after RT. Patients with negative ctDNA of “during RT” and “after RT” time points may indicate an early response to RT, and had better progression-free survival (PFS) than those with late response to RT(ctDNA clearance only at “after RT” time point, HR = 0.30, 95%CI: 0.12-0.75, p = 0.004). Moreover, patients with both negative ctDNA of “during RT” and “after RT” time points could not benefit from consolidation therapy (HR = 0.72, 95%CI: 0.29-1.78, p = 0.478), while patients with any positive ctDNA of them could (HR = 0.59, 95%CI: 0.38-0.93, p = 0.023).The sensitivity of longitudinal MRD was 97.8% and only two patients with local progression were overlooked. The 2-year cancer-specific PFS of patients with longitudinal undetectable MRD was 88.4%, which corresponds to the potentially cured population. For patients with only blood-informed MRD detection, the sensitivity and median lead time of longitudinal MRD were 97.7% and 3.9 months, respectively, which indicating the feasibility and efficacy of blood-informed MRD detection strategy. Conclusions: These data shed new light on the ctDNA-MRD detection for localized NSCLC patients who received radical RT.

A 9-gene signature in circulating tumor DNA based on molecular residual disease detection to predict recurrence of resectable non–small-cell lung cancer.

e20579 Background: Molecular residual disease (MRD) status is associated with the recurrence of non-small cell lung cancer (NSCLC) after complete resection. In clinical practice, MRD-positive patients are still uncertain of the specific recurrence time, and may even not relapse. Besides, it remains unclear whether the mutational signatures of circulating tumor DNA (ctDNA) based on MRD detection (ctDNA-MRD) may help predict recurrence. Therefore, we performed this analysis to explore whether the ctDNA-MRD mutational signatures could help predict recurrence, especially in MRD-positive population. Methods: Two investigators independently searched public databases for articles related with MRD in lung cancer published up to July 31, 2022. After screening, raw data from two published studies on resectable NSCLC were analyzed. Gene clusters were established based on the clearance of ctDNA after surgery. Kaplan-Meier and multivariate analyses were performed to find genes (Monitor genes) and clinical variables associated with recurrence-free survival (RFS). Lasso method was used to find recurrence-related genes based on perioperative ctDNA (preoperative and longitudinal ctDNA). Monitor genes and recurrence-related genes were defined as 9-gene signature. The mutational status of Monitor genes and 9-gene signature in perioperative ctDNA, as well as clinical variables, were used to establish prognostic models. The power of the models in distinguishing recurrence was shown as the area under the curve (AUC). Results: Data from 168 patients with at least one somatic mutation detected in tumors were included in the analysis. DNMT3A, EGFR, TP53, KEAP1, NAV3 (Monitor genes), and clinical variables (stage, adjuvant therapy) were factors associated with RFS. The AUC of the model based on Monitor gene mutations in perioperative ctDNA was 0.774, and the AUC reached 0.807 when clinical variables were included. The AUC of the models based on clinical variables, 9-gene signature (Monitor genes, KRAS, NTRK3, RPL5, and SOX2) in perioperative ctDNA for predicting the recurrence in the overall population and MRD-positive population were 0.840 and 0.832, respectively. Conclusions: This analysis reveals that perioperative ctDNA mutational signatures are helpful to predict postoperative recurrence, so it is necessary to pay attention to the specific gene mutations in ctDNA-MRD. [Table: see text]

Large healthy donor dataset demonstrates high analytical specificity and repeatability for the NeXT Personal molecular residual disease assay.

e15031 Background: Residual and recurrent cancer detection using circulating cell-free DNA (cfDNA) in liquid biopsy samples has proven promising in the past decade, with emerging technologies driving increasingly lower limits of detection (LOD). At ultra-high detection sensitivity levels, it is critical to warrant a high specificity to ensure that the detected signals are true measurements of the residual disease. Methods: The molecular residual disease (MRD) detection application of the NeXT Personal platform relies on up to 1,800 personalized somatic variants identified by whole genome sequencing (WGS) of the patient-specific tumor compared with the matched normal sample. Under the assumption that individuals with undetectable cancer should have negative MRD detection, we evaluated the MRD analytical specificity of the assay by measuring background noise using over 200 patient-specific panel designs to a large cohort of healthy donors that should display no tumor signals at any MRD target loci. These panels were designed and manufactured based on a pan-cancer cohort of tumor-normal paired samples whole genome data. Then, those designed panels were evaluated on cfDNA extracted from the plasma of over 200 unique healthy donors. 30-50 ng (median 50 ng) of cfDNA was used for the assay for each sample. Furthermore, six of the 200 patient panels were used to measure assay repeatability by hybridizing to at least 28 different healthy donors per panel. Results: The pan-cancer cohort used for panel design was composed of predominantly stage II ~ IV patients from 12 distinct tumor types. Across all patient samples, no MRD was detected, resulting in 100% specificity (95% confidence interval: 98.23-100%). In addition to the 100% specificity observed, the median anticipated limit of detection (LOD) as calculated by the number of targets and the number of molecules at each target was 2.6 PPM, with the lowest LOD being 1.4 PPM. The average observed unexpected signal was nearly one order of magnitude lower than the average LOD of each panel. Lower LODs were associated with larger panels (up to 1,800 mutation targets) and higher tumor purity. No correlation was observed between noise and tumor type of the designed panels or healthy plasma donor age. Further, we observed 100% repeatability across the six panels that were each applied to at least 28 unique plasma donors, with MRD not being detected in any of the replicates. Conclusions: The large healthy donor screening study demonstrated that our assay maintains excellent specificity and repeatability while still achieving ultra-sensitive detection. In all cases, the observed error-rates in the panels were very low and did not affect detection specificity. Overall these landmark specificity and repeatability results lend further confidence to the robustness of the analytical performance of NeXT Personal.

Cost-effectiveness analysis of a circulating tumor DNA-based molecular residual disease assay to guide adjuvant chemotherapy decisions in patients with resectable early stage colorectal cancer.

e18929 Background: An ongoing prospective study in patients with stage II or III colorectal cancer (CRC) has demonstrated ctDNA testing to be prognostic of recurrence risk and predictive of adjuvant chemotherapy (ACT) benefit. From the perspective of a US payer, we aimed to investigate the cost effectiveness of incorporating ctDNA analysis using the commercially available molecular residual disease (MRD) assay (Signatera™) into standard practice to aid in adjuvant chemotherapy decision-making, where traditional high-risk clinicopathological (CP) features have not shown a significant benefit of ACT in Stage II CRC. Methods: We developed a decision model to project lifetime clinical and economic consequences of different adjuvant treatment-guiding strategies. The model was parameterized using a follow-up of up to 2-years from a recently published data from GALAXY study and the cost data from the US literature. GALAXY is an observational arm of the ongoing CIRCULATE-Japan study (UMIN000039205) that analysed pre-surgical and post-surgical ctDNA in patients with stage II-IV resectable CRC. Costs are presented in 2023 US dollars. Future costs and benefits were discounted at 3%. Results: Compared to the CP predictors alone-based strategy, ctDNA analysis using the MRD assay in combination with CP predictors-based strategy led to an increase of 0.8 life years and 0.7 quality adjusted-Life Years (QALY) and cost savings of $9,771 per patient. Conclusions: The addition of MRD detection by ctDNA to traditional CP predictors aids in adjuvant chemotherapy decision-making in patients with stage II or III resectable CRC and is likely to be cost effective in the US healthcare system. ctDNA testing should therefore be considered for adoption in this disease setting.

Circulating tumor DNA methylation marker-based minimal residual diseases detection and recurrence monitoring of cancer.

e15172 Background: Circulating tumor DNA (ctDNA) has been heralded as a promising tool to detect residual disease, relapse, and monitor treatment response by detecting molecular residual disease (MRD) in solid tumors. Numerous previous studies have demonstrated the molecular characteristics of ctDNA, such as the mutational landscape, single-nucleotide variations, copy number variations, and methylation landscape. Based on the methylation characteristics of ctDNA, we developed a novel MRD evaluation system (PcMM-qMSP) with ctDNA methylation markers, and evaluated the association of changes in ctDNA level with clinical outcome. Methods: We used sequencing data from public databases for data mining by clinical Natural Language Processing (NLP) to screen the pan-cancer ctDNA methylation markers (PcMM). A PcMM-based MRD detection method was established by quantitative methylation-specific PCR (qMSP). Moreover, 99 clinical blood samples [lung cancer (41), colorectal cancer (18), gastric cancer (5), pancreatic adenocarcinoma (16), liver cancer (6), cervical cancer (3), ovarian cancer (4), breast cancer (3), and unknown (3)] were collected to evaluate the consistency of ctDNA level between PcMM-qMSP and next-generation sequencing (NGS). Results: We evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility. PcMM-qMSP showed coefficients of variation (CVs) of the intra- and inter-batch assay of 0.42-0.65% and 0.05-0.27%, the accuracies were 100%. Best-in-class assays demonstrated a limit of detection (LoD) of approximately 30 copies of methylated DNA, with lower LoD at 10 copies. The evaluation results of 99 pan-solid tumor blood samples showed that the consistency of PcMM-qMSP and NGS in ctDNA level assessment was 0.82. The specific data are shown in the Table. Conclusions: PcMM is a pan-tumor specific methylation biomarker. The results demonstrated that PcMM-qMSP and NGS were highly functionally consistent for estimating ctDNA level. PcMM-qMSP detection time is less than 24 hours and costs USD 300 as compared to NGS, which takes seven days and costs USD 900. Hence, PcMM-qMSP is economical, rapid, accurate and highly sensitive for detecting pan-solid tumors. [Table: see text]

Diagnostic methods for peritoneal molecular residual disease in gastric cancer

Peritoneal metastasis of gastric cancer serving as the most frequent form of metastasis, is one of the leading causes of death. A portion of surgically treated patients often suffer from small peritoneal residual metastasis, which will lead to recurrence and metastasis of gastric cancer patients after surgery. Given these, the prevention and treatment of peritoneal metastasis of gastric cancer deserves more attention. Molecular residual disease (MRD) refers to the molecular abnormalities of tumor origin that cannot be found by traditional imaging or other laboratory methods after treatment, but can be found by liquid biopsy, representing the possibility of tumor persistence or clinical progress. In recent years, the detection of MRD based on ctDNA has gradually become a research hotspot in the prevention and treatment of peritoneal metastasis. Our team established a new method for MRD molecular diagnosis of gastric cancer, and reviewed the research achievements in this field.

Early Detection of Molecular Residual Disease and Risk Stratification for Stage I to III Colorectal Cancer via Circulating Tumor DNA Methylation

Detection of molecular residual disease and risk stratification as early as possible may improve the treatment of patients with cancer. Efficient pragmatic tests are therefore required. To measure circulating tumor DNA (ctDNA) with 6 DNA methylation markers in blood samples and to evaluate the association of the presence of ctDNA with colorectal cancer (CRC) recurrence throughout the disease course. In this multicenter prospective longitudinal cohort study performed from December 12, 2019, to February 28, 2022, 350 patients with stage I to III CRC were recruited from 2 hospitals for collection of blood samples before and after surgery, during and after adjuvant chemotherapy, and every 3 months for up to 2 years. A multiplex, ctDNA methylation, quantitative polymerase chain reaction assay was used to detect ctDNA in plasma samples. A total of 299 patients with stage I to III CRC were evaluated. Of 296 patients with preoperative samples, 232 (78.4%) tested positive for any of the 6 ctDNA methylation markers. A total of 186 patients (62.2%) were male, and the mean (SD) age was 60.1 (10.3) years. At postoperative month 1, ctDNA-positive patients were 17.5 times more likely to relapse than were ctDNA-negative patients (hazard ratio [HR], 17.5; 95% CI, 8.9-34.4; P < .001). The integration of ctDNA and carcinoembryonic antigen tests showed risk stratification for recurrence with an HR of 19.0 (95% CI, 8.9-40.7; P < .001). Furthermore, ctDNA status at postoperative month 1 was strongly associated with prognosis in patients treated with adjuvant chemotherapy of different durations and intensities. After adjuvant chemotherapy, ctDNA-positive patients had a significantly shorter recurrence-free survival than did the ctDNA-negative patients (HR, 13.8; 95% CI, 5.9-32.1; P < .001). Longitudinal ctDNA analysis after the postdefinitive treatment showed a discriminating effect in that ctDNA-positive patients had poorer recurrence-free survival than did the ctDNA-negative patients (HR, 20.6; 95% CI, 9.5-44.9; P < .001). The discriminating effect was enhanced (HR, 68.8; 95% CI, 18.4-257.7; P < .001) when ctDNA status was maintained longitudinally. Postdefinitive treatment analysis detected CRC recurrence earlier than radiologically confirmed recurrence, with a median lead time of 3.3 months (IQR, 0.5-6.5 months). The findings of this cohort study suggest that longitudinal assessment of ctDNA methylation may enable the early detection of recurrence, potentially optimizing risk stratification and postoperative treatment of patients with CRC.

Investigate the application of postoperative ctDNA-based molecular residual disease detection in monitoring tumor recurrence in patients with non-small cell lung cancer--A retrospective study of ctDNA.

To evaluate whether postoperative circulating tumor DNA (ctDNA) in plasma of patients with non-small cell lung cancer (NSCLC) can be used as a biomarker for early detection of molecular residual disease (MRD) and prediction of postoperative recurrence. This study subjects were evaluated patients with surgical resected non-small cell lung cancer. All eligible patients underwent radical surgery operation followed by adjuvant therapy. Tumor tissue samples collected during operation were used to detect tumor mutation genes, and blood samples collected from peripheral veins after operation were used to collect ctDNA. Molecular residue disease (MRD) positive was defined as at least 1 true shared mutation identified in both the tumor sample and a plasma sample from the same patient was. Positive postoperatively ctDNA was associated with lower recurrence-free survival (RFS).The presence of MRD was a strong predictor of disease recurrence. The relative contribution of ctDNA-based MRD to the prediction of RFS is higher than all other clinicopathological variables, even higher than traditional TNM staging. In addition, MRD-positive patients who received adjuvant therapy had improved RFS compared to those who did not, the RFS of MRD-negative patients receiving adjuvant therapy was lower than that of patients not receiving adjuvant therapy. Post-operative ctDNA analysis is an effective method for recurrence risk stratification of NSCLC, which is beneficial to the management of patients with NSCLC.

Abstract 3359: Sensitive detection of locoregional MRD after head and neck cancer surgery

Abstract Background: Plasma cell-free DNA (cfDNA) has low sensitivity to detect locoregional molecular residual disease (MRD) immediately after surgery for limited-stage head and neck cancer. To enhance locoregional MRD sensitivity, we utilize a novel biofluid proximal to the primary tumor: the serosanguinous fluid collected by surgical drains inserted into the wound bed following regional lymph node dissection. We demonstrate that cancer-associated cfDNA in the surgical drain fluid (SDF) is a sensitive biomarker of locoregional MRD detection, risk stratification, and prognostication. Methods: We extracted DNA from 168 SDFs, 48 paired plasmas, and 44 tumor specimens collected 24 hours after surgery from 90 patients: 74 HPV (+) oropharyngeal cancers (OPC), 8 HPV (-) oral cancers, 2 thyroid cancers, and 6 benign neck pathologies. SDFs were also collected at 0, 6, 12, and 24 hours after surgery from 11 HPV (+) patients. We applied TaqMan qPCR to our specimens to quantify cell-free HPV (cf-HPV), a proxy for cancer-associated cfDNA in HPV (+) malignancies. We then measured the association between cf-HPV and extranodal extension (ENE), AJCC 8 staging, and established clinical and pathological risk criteria from 4 HPV (+) OPC clinical trials. We validated our findings using HPV next-generation sequencing. We also compared the predictive value of high-risk pathology with cf-HPV MRD for HPV (+) OPC recurrences. Lastly, we developed a second assay for cell-free BRAF V600E (cf-V600E) in thyroid cancer SDF. Results: Serially collected SDF revealed cf-HPV decreased continuously from 0 to 24 hours postoperative, and that the 24-hour timepoint stratified detectable versus undetectable persistent residual disease. At 24 hours, SDF was significantly more enriched with cf-HPV compared to plasma (P &amp;lt; 0.0001). In a subset of 9 patients, HPV genotype and detection in SDF were 100% concordant between NGS and PCR. Strikingly, SDF cf-HPV was 11-fold higher in pN2 patients versus pN1/N0 (P = 0.02) and 15-fold higher in cases with ENE (P = 0.003), suggesting SDF reflects aggressive nodal pathology and captures locoregional MRD. When we classified our patients according to established clinical and pathological risk criteria, we found significantly higher SDF cf-HPV among high-risk patients (AUC = 0.77). Crucially, in HPV (+) patients treated with surgery alone, SDF MRD detected 100% of locoregional recurrences, while plasma MRD and high-risk pathology detected 0%. Lastly, as proof of concept, we demonstrated SDF cf-V600E detection in the context of high-risk thyroid cancer. Conclusions: Surgical drain fluid cancer-associated cfDNA is a novel minimally invasive biomarker. Our SDF assay was strongly associated with high-risk criteria and detected locoregional relapse with 100% sensitivity compared to 0% in plasma. Our data suggest that SDF liquid biopsy is ultra-sensitive and has the potential to inform postoperative molecular-based risk stratification. Citation Format: Noah Earland, Ricardo J. Ramirez, Sophie P. Gerndt, Peter K. Harris, Andrew I. Hearn, Matthew Inkman, Jeffrey J. Szymanski, Nick Semenkovich, Benjamin M. Wahle, Zhongping Xu, Kevin Chen, Jin Zhang, Jose P. Zevallos, Aadel A. Chaudhuri. Sensitive detection of locoregional MRD after head and neck cancer surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3359.

Abstract 3381: Analytical performance of an ultra-sensitive, tumor-informed liquid biopsy platform for molecular residual disease detection and clinical guidance

Abstract Most circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection methods leverage a limited genomic footprint, restricting detection sensitivity to 10-4 ~ 10-5 tumor fraction and thus their utility in many clinical settings. For example, early-stage, low tumor mutational burden (TMB) cancers may lack sufficient variants in these limited footprints to produce detectable signals. Further, insights into tumor evolution, including actionable mutations may be missed.Here we report a performance update of the NeXT Personal™️ platform. Utilizing whole genome sequencing of tumor and normal DNA to guide design of bespoke MRD assays, we select up to 1800 high signal, low noise MRD targets and up to 400 exonic variants. Along with proprietary algorithms, this achieves high sensitivity with a limit of detection of 1 ~ 3 parts per million, and targets high specificity (99.99%). Specificity was demonstrated with healthy donor plasma samples and &amp;gt; 200 cancer patient panels from a broad range of solid tumors. Sensitivity and linearity of MRD measurements were determined using dilution series from cell lines and clinical samples, which were orthogonally confirmed (R2 = 0.909, PPA = 100%, NPA = 100%) by digital droplet PCR (ddPCR).In addition, NeXT Personal simultaneously surveys tumor-agnostic content in the same workflow, providing detection of actionable mutations, markers of drug resistance, and mechanisms of tumor evolution from a curated set of variants in 90 clinically-relevant genes. Patient plasma (1 ~ 8 mL; 2 ~ 50 ng of cfDNA) can be rapidly queried to provide mutation-level information about tumor biology, longitudinal trajectory, and clinical actionability. The specificity of NeXT Personal variant detection is &amp;gt; 99.99% with 100% PPV, while individual variant content demonstrates high sensitivity at allele fractions of 0.1% and above, with high accuracy and signal linearity as confirmed by ddPCR (R2 = 0.998).To explore the utility of NeXT Personal in a clinical setting, a retrospective analysis was undertaken in an advanced liver cancer (low TMB) cohort of 11 patients undergoing immunotherapy. Our results demonstrated that changes in ctDNA levels during therapy correlated highly with disease status (6wk vs. baseline, p = 0.017; 9wk vs. baseline, p = 0.004), and were detected prior to clinical response confirmation by RECIST 1.1. Examination of the clinical content provided useful insights into the potential of the assay.Our data demonstrate high analytical performance of the NeXT Personal platform in both MRD and individual variant detection. The assay is sufficiently sensitive for early stage, low shedding, and low TMB cancers, early time points or samples with limited input. The addition of clinically relevant tumor-agnostic actionable content makes NeXT Personal unique in its ability to detect MRD and to ultimately help guide clinicians. Citation Format: Rui Chen, Gábor Bartha, John Lyle, Jason Harris, Sean M. Boyle, Josette Northcott, Dan Norton, Rachel M. Pyke, Fábio C. Navarro, Charles W. Abbott, Christian Haudenschild, Rose Santiago, Darren Nichols, Stephanie Huang, Christopher Nelson, Manju Chinnappa, Yi Chen, Yuker Wang, Laurie Goodman, Qi Zhang, Manqing Hong, Xiaoji Chen, Erin Ayash, Nitin Udar, Sebastian Saldivar, Jian Yan, John West, Richard O. Chen. Analytical performance of an ultra-sensitive, tumor-informed liquid biopsy platform for molecular residual disease detection and clinical guidance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3381.

Abstract 6610: Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients

Abstract Breast cancer (BC) is the most frequent neoplasia affecting women worldwide normally detected at early stages. In this regard, early diagnosis drastically decreases mortality, however, around 20% of these patients will later relapse. This is mainly caused by undetectable molecular residual disease (MRD) not eliminated by standard primary treatments. Therefore, it is crucial to detect the after-treatment MRD to stratify the patients by their risk of relapse. Liquid biopsies have emerged as non-invasive method to obtain information about tumors and improve clinical cancer management. Regarding this, much has been hypothesized about utilizing high blood volumes to overcome the necessity of complex and resource-intensive next generation sequencing (NGS) methodologies to detect highly diluted blood tumor components in localized cancers. Herein, we employed a combined analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) together with high blood volumes and single-assay droplet digital PCR (ddPCR) to detect MRD with ultra-high sensitivity. We prospectively assayed 124 samples extracted at baseline, post-neoadjuvant therapy (NAT), post-surgery and a follow-up on a six-monthly basis. A median of 76.40 mL of blood to detect CTCs and 40 mL of plasma to detect ctDNA per patient from 19 BC women were used in this study. ddPCR assays were performed with a median of 14 partitions per determination to detect ctDNA and 12 partitions for CTCs. Overall, ctDNA, CTCs and ctDNA and/or CTCs were detected in 84.21%, 66.66% and 89.47% respectively in the pre-treatment blood samples. MRD (ctDNA and/or CTCs) was detected in 73.68% of the after NAT blood samples. On the other hand, it was detected in 46.66% and 70.00% of the post-surgery and follow-up samples respectively. Post-NAT MRD was detected in 57.14% (4/7) and 83.33% (10/12) of patients with and without pathological complete response pCR respectively. To note, the discordant patients achieving pCR in tissue with detectable MRD in blood were high-risk BC. Importantly, in one of the two patients without pCR and no MRD detected, not enough sample were available to complete the analysis. The other discordant patient presented a localized disease with residual cancer burden value of 1 and no lymph nodes affected. In 1 out of 19 (5.26%) patient clinically relapsed with a positive MRD detection 6 months earlier. Applying this methodology, we observed a sensitivity of 0.004% in ctDNA detection and 0.224 CTCs per mL of blood. Overall, this novel methodology greatly improves sensitivity for ctDNA and CTCs detection in treatment-naïve early BC. In addition, MRD was successfully detected in post-treatment samples antedating clinical relapse by 6 months in one patient. This prospective study is potentially demonstrating that using high blood volumes and a single-assay ddPCR is a cost-effective strategy to monitor localized BC and predict relapses. Citation Format: Alfonso Alba-Bernal, Ana Godoy-Ortiz, María Emilia Domínguez-Recio, Begoña Jimenez-Rodriguez, María Elena Quirós-Ortega, Esperanza López-López, Guillermo Carbajosa-Antona, Jesús Peralta-Linero, Luis Vicioso, Estefanía Bellagarza-García, Guadalupe Dolores Garrido-Ruiz, Cynthia Robles-Podadera, Alicia Garrido-Aranda, María Dunia Roldán-Díaz, Jesús Velasco-Suelto, Rocío Lavado-Valenzuela, Martina Álvarez, Nuria Ribelles, Javier Pascual, Emilio Alba, Iñaki Comino-Méndez. Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6610.

Abstract 1037: Circulating tumor DNA-based molecular residual disease predicts relapse in patients with resectable gastric cancer

Abstract Background: Circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection has been shown to predict postoperative relapse in several cancers. Currently, fixed and personalized panel-based tumor-informed (TI) ctDNA testing strategies are available for MRD assessment. Here, we aim to evaluate the prognostic value of MRD in resectable gastric cancer (GC) and assess MRD with fixed and personalized panels for comparison. Methods: We prospectively enrolled patients with stage I-III GC who underwent R0 resection at Ruijin Hospital. Blood samples were collected on the day of surgery preoperatively and during the second-fourth week postoperatively. Tissue samples were collected shortly thereafter at the time of curative-intent resection. Both the Patient-specific pROgnostic and Potential tHErapeutic marker Tracking (brPROPHET, a personalized panel-based TI assay, Burning Rock Biotech) and a 168-gene panel-based TI assay (Burning Rock Biotech) were used to quantify ctDNA. Results: A total of 55 patients (19 females and 36 males) with a median age of 65 years (range: 35-87) were enrolled. Precise 5 and 41 patients were treated with neoadjuvant therapy and adjuvant therapy, respectively. The median duration of follow-up was 784 days (range: 582-853). All patients underwent the fixed panel-based TI assay. Of which, 19 patients were collected with postoperative blood samples. The MRD status of postoperative blood by the fixed panel showed a significant association with recurrence-free survival (RFS, MRD-positive [MRD+] vs. MRD-negative [MRD-]: 484 days vs. not reached [NR], hazard ratio [HR] = 8.0, p = 0.02). Of the 55 enrolled patients, 19 patients underwent both personalized and fixed panel-based TI assays. For the personalized panel, preoperative ctDNA was detected in 94.7% (18/19) of the patients with 100% (4/4), 66.7% (2/3), and 91.7% (11/12) in stage I, II, and III, respectively. The numbers were 36.8% (7/19), 0% (0/4), 0% (0/3), and 58.3% (7/12) with the fixed panel, respectively. Among the 19 patients, three of the 13 patients who had postoperative blood samples developed tumor recurrence during the follow-up visit. The sensitivity of the personalized panel for predicting tumor recurrence achieved 100% (3/3) with 90% (9/10) specificity. The sensitivity of the fixed panel was 33.3% (1/3) with 100% (10/10) specificity. In addition, patients with MRD+ by the personalized panel had a significantly shorter RFS than those with MRD- (566 days vs. NR, HR &amp;gt; 100, p = 0.003). Conclusion: Our study indicates that MRD+ by either the personalized or fixed panel was associated with an unfavorable survival outcome. ctDNA-based MRD detection by the personalized panel-based TI assay might be a powerful prognostic biomarker to identify GC patients who underwent radical resection at a higher risk of relapse or with an unfavorable survival outcome. Citation Format: Pei Xue, Yanfei Shao, Xueliang Zhou, Haiyan Li, Yang Wang, Chenyang Wang, Hao Zhang, Bing Li, Shuo Shi, Haiwei Du, Jing Sun. Circulating tumor DNA-based molecular residual disease predicts relapse in patients with resectable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1037.

Abstract 6681: XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening

Abstract The proprietary XNA (xeno nucleic acid) technology has wide applications in sensitive detection of rare mutations in different genomic platforms. These target-specific DNA oligo analogs increase assay sensitivity by blocking the polymerase-dependent amplification of wildtype sequence so only amplification of the mutant sequence occurs. Here we show that the XNA technology helps enrich rare somatic mutations in different human specimen types such as FFPE and plasma samples, using technology platforms such as Sanger sequencing, TaqMan qPCR, and NGS. XNA increases assay sensitivity to 0.1% for Sanger sequencing and qPCR. For NGS, XNA allows detection of the mutant sequence at a lower sequencing depth, thus combining XNA technology with NGS for MRD (molecular residual disease) detection could improve cancer management. In addition, We also show CRISPR mutant screening using a target-specific XNA in a SYBR Green qPCR assay. XNA can be used for rapid CRISPR mutant screening for DNA from pool or individual clones generated from gene editing experiments. In summary, XNA technology is a powerful tool when combined with existing genomic mutation detection platforms to screen for rare somatic mutations and for rapid CRISPR mutant screening. The technology is customizable as a service for easy integration with current technology platforms and applications. Citation Format: Wei Liu, Robert Brown, Andrew Fu, Shuo Shen, Michael Sha, Aiguo Zhang. XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6681.

Perioperative ctDNA-Based MRD Detection in NSCLC-Letter.

We read with great interest the article by Xia and colleagues reporting circulating tumor DNA (ctDNA) as biomarker for early detection of molecular residual disease (MRD) and predictor for relapse after surgery in patients with non–small cell lung cancer in a prospective multicenter cohort named LUNGCA-1 (1).LUNGCA-1 was designed to predict prognosis by perioperative ctDNA detection, including preoperative, 3 days postoperative and 1 month postoperative ctDNA. The number of patients in prognosis analysis is 330 (Fig. 1), whereas Supplementary Table S5 shows only 329 patients with at least one postoperative ctDNA sample. In LUNGCA-1, some patients tested postoperative ctDNA before or without adjuvant therapies, while others tested postoperative ctDNA after adjuvant therapies. We questioned the trial design because the authors mixed different aims: MRD detection at a specific postoperative timepoint for recurrence prediction; MRD detection at both preadjuvant/postadjuvant therapies timepoints for evaluation of the outcome of adjuvant therapies. The authors could separate different aims into different trials or do statistics separately in preadjuvant/postadjuvant groups.The authors defined “MRD positive” for patients with detectable ctDNA at 3 days and/or 1 month postoperatively. We noticed that 5 of 26 patients with MRD positive (19.2%) had ctDNA positive only in the detection at postoperative 3 days and their ctDNA detection was negative before and 1 month after surgery. Therefore, we consider the issue of timepoint setting for postoperative blood collection. Diehl and colleagues reported that ctDNA increased after surgery, most likely due to release of ctDNA from damaged tissue (2). Thus, it may be more reasonable to collect blood at a later timepoint after surgery from 10 days to 16 weeks (3). As reported by Chaudhuri and colleagues (4) and Moding and colleagues (5), blood sample should be collected within 4 months for ctDNA detection after surgery or completing radiation and chemotherapy. In this article, the cell-free DNA concentration at 3 days after surgery is also significantly higher than before and 1 month after surgery (Supplementary Table S1), suggesting a more reasonable manner to collect blood for a longer period after surgery. Also, whether the 5 patients with ctDNA positive for only 3 days after surgery relapsed or not was not mentioned.Chaudhuri and colleagues (4) reported that concentration of ctDNA should be correlated with stage and tumor volume, which the authors did not mention. Furthermore, how to determine ctDNA status is positive or negative needs to be clarified. In addition, the criteria for judging the patient's disease relapse were not mentioned in the text or Supplementary Materials and Methods.See the Response, p. 1156No disclosures were disclosed.We thank the grant from the Fundamental Research Funds for the Central Universities in China (4206-413100049).