Minimal Residual Disease Assay Research Articles

Abstract PO3-19-06: MRD Assay evaluates Recurrence and response via a tumor Informed Assessment: MARIA-Breast Observational Trial

Abstract Background: Detectable ctDNA in patients with solid tumors has been associated with disease prognosis pre-treatment, assessing response to therapy in the form of minimal residual disease (MRD), and monitoring for recurrence after curative intent treatment. Utilizing patient-specific genomic mutation profiling of an individual’s cancer from a tissue sample, in conjunction with the patient’s germline DNA, to create a personalized sequencing panel to analyze for a subset of these genetic mutations from ctDNA in blood is a strategy that has high sensitivity for detecting MRD. Studies have shown that pretreatment levels of ctDNA using this approach are a potential early indicator of disease recurrence after surgery, that ctDNA clearance may be an early predictor of favorable outcomes and has been shown to correlate with pathologic complete response (Forde et al. N Engl J Med. 2022, PMID:35403841), and that this approach has high sensitivity for detecting recurrence for patients in advance of the current standard of care (Abbosh et al. Cancer Res (2020) 80 (16_Supplement): CT023). Methods: This is a multi-site, prospective, observational trial in the United States of 200 patients with early stage breast cancer using a patient-specific tumor-informed MRD assay for ctDNA analysis. Participants are asked to provide study specimens prior to initial treatment intervention, after curative intent surgical resection, during adjuvant therapy (as applicable) and pre-recurrence follow-up. ctDNA will be analyzed with an NGS-based, tumor-informed MRD assay that identifies somatic mutations from DNA obtained from the patient’s tumor tissue, subtracts germline variants via NGS-based analysis of the patient’s germline DNA, and detects a selected set of between 18-50 tumor-specific ctDNA in their blood. All primary tumor specimens will undergo full exome sequencing using the Personalized Cancer Monitoring (PCM) assay. Impact of results of this CLIA-approved MRD assay on clinical decision making will be captured. The primary objective is to assess the ability of MRD to predict post-treatment recurrence. Further objectives are to correlate MRD status with pathologic complete response, determine the lead time to detection of recurrence compared to standard of care, and the association of MRD status with overall survival. Active enrollment started in March, 2022. Support: Invitae. Clinical trial information: NCT05219734. Citation Format: Edward Esplin, Kelli Swan, Lee Ifhar, Brandie Heald, Sarah Nielsen, Daniel Pineda-Alvarez, William O'Callaghan, Robert Daber, Darrel Ross, Carlos Vieira, Aadel Chaudhuri, W. Korn. MRD Assay evaluates Recurrence and response via a tumor Informed Assessment: MARIA-Breast Observational Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-19-06.

Minimal Residual Disease using a Plasma-Only Circulating Tumor DNA Assay to Predict Recurrence of Metastatic Colorectal Cancer Following Curative Intent Treatment.

Minimal residual disease (MRD) detection identifies patients with colorectal adenocarcinoma (CRC) likely to recur following definitive treatment. We evaluated a plasma only MRD assay to predict recurrence and survival in metastatic CRC patients undergoing curative intent procedures (surgery and/or radiotherapy), with or without (neo)adjuvant chemotherapy. The primary objective of this study was to assess the correlation of post-procedure tumor cfDNA detection status with radiographic disease recurrence (RFS). Pre- and post-procedure longitudinal samples were collected from 53 patients and analyzed with a multiomic MRD assay detecting circulating tumor DNA (ctDNA) from genomic and epigenomic signals. Pre- and post-procedure ctDNA detection correlated with recurrence-free and overall survival. 230/233 samples from 52 patients were successfully analyzed. At the time of data cutoff, 36 (69.2%) patients recurred with median follow-up of 31 months. 19/42 patients (45.2%) with ctDNA analyzed 3 weeks post-procedure had detectable ctDNA. ctDNA detection 3 weeks post-procedure was associated with shorter median RFS (HR 5.27; 95% CI, 2.31-12.0, p<0.0001) and overall survival (OS) (HR 12.83; 95% CI, 3.6-45.9, p<0.0001). Pre-procedure ctDNA detection status was not associated with RFS but was associated with improved OS (HR 4.65; 95% CI, 1.4-15.2, p=0.0111). Undetectable ctDNA pre-procedure had notable long-term overall survival, >90% 3 years post-procedure. In this cohort of oligometastatic CRC, detection of ctDNA pre- or post-procedure was associated with inferior outcomes even after accounting for prognostic clinicopathologic variables. This suggests ctDNA may enhance current risk stratification methods helping evaluate novel treatments and surveillance strategies toward improving patient outcomes.

Abstract 3675: Design of high-performance tumor-informed minimal residual disease (MRD) panels from low FFPE tumor input

Abstract Background: Minimal residual disease (MRD) testing can detect cancer recurrence months to years earlier than the current standard of care, enabling earlier treatment of recurrence and improved patient outcomes. Tumor-informed MRD assays typically utilize formalin-fixed paraffin-embedded (FFPE) tumor tissue, which is available in limited quantities for some patients, for example, following core needle biopsy (CNB), after neoadjuvant treatment or when patients need multiple tests from the same tumor sample. To determine the lower limit of tissue input, we evaluated our MRD assay performance across a range of extracted tumor volumes. Methods: Resected tumors and CNBs were sectioned, H&amp;E stained, and macro-dissected. Tumor gDNA was extracted, quantified, prepared into libraries and sequenced. Sequenced libraries were aligned and evaluated for depth of coverage, variation of coverage, and duplication rate. Somatic calling was performed on matched tumor and normal samples. Up to 1000 target sites were selected for high-depth targeted sequencing of the tumor and normal gDNA for confirmation of somatic variant calls. The positive predictive value (PPV) was computed as the percent of putative somatic variant sites that were present in the tumor capture library and absent in the normal capture library. Results: Extracted tumor volumes varied by almost two orders of magnitude, from 0.06mm3 (equivalent to needle core or fine needle aspirate biopsies) to 5mm3 (achievable with resected tumor). gDNA amount varied linearly (3.6ng to 1549ng) with tumor tissue input, indicating the low tissue to paraffin ratio did not have an adverse effect on yield. Tumor gDNA inputs into library prep ranged from 2.5ng to 100ng. DNA amounts above 100ng into library prep had no discernable benefit. Below 10 ng, depth of coverage and the coefficient of variation in coverage indicated poor-quality libraries. Samples with ~10 ng of gDNA input into library prep showed depth of coverage comparable to higher inputs and saturated the achievable library complexity. Additionally, PPV of somatic calling was consistent across the range of gDNA inputs from 10-100 ng, demonstrating equivalent assay performance. Conclusion: Tumor-informed MRD assays have immense potential for increasingly sensitive treatment response and recurrence monitoring that can inform better treatment decisions. FFPE tumor tissue is a critical input into MRD assays but is a limited resource. This study supports a minimum DNA input of 10 nanograms for a single attempt at extraction, corresponding to a tissue volume of 0.2mm3 or a single 10µm slide with a 20mm2 area, representing one of the lowest tissue input requirements for an MRD assay. Low FFPE tissue requirements expand the patient population that benefit from MRD testing by utilizing samples that have low tumor content, are post-neoadjuvant therapy or do not meet the tumor volume requirements of competing MRD offerings. Citation Format: Matt LaBella, Ashley Acevedo, Ravi Patel, Kiefer Haug, Sangita Ganesh, Elise Buser, Nafei Xu, Shalee Carlson, Kyle Trettin, Sarah Ratzel, Kieko Hara, Pavlos Msaouel, Kanishka Sircar, Chad Tang, Dale Muzzey, Genevieve Gould. Design of high-performance tumor-informed minimal residual disease (MRD) panels from low FFPE tumor input [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3675.

Abstract 961: Detection of minimal residual disease in lymph predicts recurrence in HPV-negative head and neck cancer patients

Abstract Background: Locoregional cancer relapse remains a major cause of failure in head and neck squamous cell carcinoma (HNSCC), particularly for HPV-negative patients whose 2-year locoregional failure rate is up to 50%. There is unmet need for an accurate diagnostic test that predicts risk of recurrence prior to adjuvant therapy selection. We present a novel proximal assay for minimal residual disease (MRD) profiled in lymphatic exudate collected via surgical drains (“lymph”). Methods: Lymph, plasma, and blood were collected from 46 HPV-negative HNSCC patients postoperatively at 24 hours along with resected tumor. Cell-free DNA was extracted from lymph and plasma and sequenced using the TruSeq Oncology 500 panel to a depth of &amp;gt;100 million reads. Somatic mutations were identified by exome sequencing (200x) tumor and blood. Five patients had &amp;lt;2 mutations in tumor and were excluded. Two patients were censored due to lack of clinical data, yielding 18 patients with disease recurrence (REC) and 21 with no evidence of disease (NED) with &amp;gt;1 year of follow-up. Tumor-specific variants were force-called in lymph and plasma using a custom pipeline. Patients were considered MRD positive if the mean variant allele fraction (mVAF) was greater than 0.015% (the estimated limit of detection). The Kaplan-Meier (KM) estimator with log-rank test and Cox proportional-hazards model were used for survival analyses. Logistic regression model was performed with 5-fold cross-validation. Results: KM survival analyses showed lymph accurately predicts recurrence (sensitivity (SN) = 78%, specificity (SP) = 67%; p = 0.003, Hazard ratio (HR) = 4.7), while plasma was not significant (p = 0.92) at this postoperative timepoint. Performance was enhanced for locoregional relapse (SN = 92%, SP = 67%; p = 0.001, HR = 13.9. N=33). We also observed accurate recurrence prediction (SN = 83%, SP = 69%, p = 0.03, HR = 7.7) in the 19 N0 patients (node-negative by pathology). Lymph MRD outperformed individual pathology features (extranodal extension, perineural invasion, lymphovascular invasion, and nodal disease status) for recurrence prediction (HR = 3.4, 0.88, 2.5, 2.4, respectively). A logistic regression model combining lymph MRD with these 4 high-risk pathologic features showed superior performance over lymph alone or pathology alone (SN = 89%, SP = 67%; p = 0.0007, HR = 8.6). Conclusions: ctDNA analysis of lymph from surgical drains represents a novel proximal MRD approach in HPV-negative HNSCC. Postoperative lymph significantly outperforms plasma for prediction of recurrence, particularly in patients with locoregional relapse. Accurate MRD identification in patients with lower risk pathologic features suggests that lymph MRD testing has potential to augment traditional pathology and provide more personalized adjuvant treatment decision-making in patients with HPV-negative HNSCC. Citation Format: Wendy Winckler, Zhuosheng Gu, Damion Whitfield, Noah Earland, Adam Harmon, Megan Long, Peter Harris, Zhongping Xu, Ricardo Ramirez, Sophie Gerndt, Maciej Pacula, Marra S. Francis, Jose P. Zevallos, Aadel A. Chaudhuri. Detection of minimal residual disease in lymph predicts recurrence in HPV-negative head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 961.

Abstract 986: Mathematical modeling of ctDNA dynamics to predict utility of ultrasensitive MRD assays in early stage non-small cell lung cancer (NSCLC)

Abstract Background: ctDNA assay use for minimal residual disease (MRD) detection in early stage NSCLC has been limited by suboptimal sensitivity of first generation assays. It is unclear how improving assay limit of detection (LOD) would translate to improvements in clinical sensitivity. Here, we performed mathematical modeling of ctDNA dynamics after curative-intent treatment in early stage NSCLC to predict the clinical impact of MRD assays with improved analytical sensitivity. Methods: We analyzed a recently reported dataset of post-surgical ctDNA MRD surveillance in early stage NSCLC (Abbosh et al., Nature 2023) to investigate ctDNA growth dynamics. Of 70 relapsed patients in this cohort, 23 had 3 consecutive ctDNA-positive samples with no intervening therapy. We used these to generate patient-specific mathematical models for ctDNA variant allele fractions (VAF) over time, initially assuming exponential growth. We assessed this assumption by creating log-linear models for ctDNA increase in these cases. Using slopes of the log-linear models, we calculated patient-specific doubling times and assessed their relation to stage, histology, and outcome. We used the log-linear models to simulate a VAF distribution each day after surgery, enabling projection of clinical sensitivities and estimation of lead times for recurrence detection at varying LODs. Results: For the 23 cases with 3+ consecutive ctDNA positive samples, we developed log-linear models for ctDNA VAF growth, which were strongly correlated (r &amp;gt; 0.5) in 18/23 (78%) patients (median r = 0.89), supporting that ctDNA can be modeled as exponential growth. We calculated ctDNA doubling times for the 18 patients with exponential ctDNA kinetics and 10 patients with 2 consecutive ctDNA positive samples. Doubling times were remarkably consistent with median 51 days (IQR 40-79). Doubling times were longer for stage I tumors compared to stage II/III (p = 0.04) but were not different between NSCLC subtypes. Doubling time correlated with time to clinical relapse, with patients having more rapid ctDNA growth relapsing earlier (r = 0.52, p=0.03). Finally, we used patient-specific log-linear models to project ctDNA VAFs at a landmark 30 days after surgery. By improving the LOD for ctDNA detection from 10−4 to 10−6, the projected clinical sensitivity doubled (from 43% to 86%). Simulations predicted that this improvement in sensitivity would increase median lead time from 183 days for LOD=10−4 to 349 days for LOD=10−6 (p&amp;lt;0.0001). Conclusion: For most relapsed NSCLC patients, the natural history of ctDNA MRD growth can be reliably modeled with exponential growth kinetics. ctDNA doubling time is associated with stage and clinical outcome. Ultrasensitive MRD assays with LOD of 10−6 are predicted to meaningfully improve clinical sensitivity at the post-surgical landmark and to increase the lead time for MRD detection before relapse. Citation Format: Jordan S. Goldstein, Christopher Abbosh, Ash A. Alizadeh, Maximilian Diehn, David M. Kurtz. Mathematical modeling of ctDNA dynamics to predict utility of ultrasensitive MRD assays in early stage non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 986.

Minimal residual disease assessment and progress in multiple myeloma

With the rapid iteration of multiple myeloma therapeutics over the last two decades, as well as increasing remission rates and depth of remission in patients, traditional methods for monitoring disease response are insufficient to meet the clinical needs of new drugs. Minimal residual disease (MRD) is a more sensitive test for determining the depth of response, and data from multiple clinical trials and meta-analyses show that a negative MRD correlates with a better prognosis than a traditional complete response. MM is at the forefront of MRD evaluation and treatment. MRD detection methods have been continuously updated. The current MRD assessment has three dimensions: bone marrow-based MRD testing, MRD testing based on images of residual metabolic of focal lesions, and peripheral blood-based MRD testing. The various MRD assessment methods complement one another. The goal of this article is to discuss the currently used MRD assays, the progress, and challenges of MRD in MM, and to provide a reference for clinicians to better use the techniques.

Prognostic value of minimal residual disease profiling in resectable hepatocellular carcinoma.

530 Background: Detecting post-surgical residual disease is a critical clinical requirement in resectable hepatocellular carcinoma (HCC). Previous studies focused on specific genomic regions have indicated that ctDNA holds promise as a tool for prognostic assessment. Nevertheless, these methods exhibited limited sensitivity and failed to meet the minimal residual disease (MRD) threshold. Here we report the prognostic value of MRD profiling in HCC patients who have undergone hepatectomy. Methods: This retrospective study involved 88 HCC patients who underwent surgical resections in China, from January to May in 2016. During surgery, tumor and normal tissue specimens were collected. Plasma samples were obtained 7 days post-surgery. PredicineBEACON, a baseline tissue- or blood-informed MRD assay, was used for MRD profiling. This entailed identifying tumor-specific mutations through whole exon sequencing of tissue samples. Subsequently, a personalized MRD panel, selecting up to 50 mutations via bioinformatics pipelines merged with a fixed panel featuring 500 tumor actionable hotspots, was created for each patient. To detect MRD in post-surgery plasma samples, we employed ultra-deep sequencing at a coverage of 100,000X, utilizing the designed panel. Results: In the cohort of 88 patients, the distribution based on Barcelona Clinic Liver Cancer (BCLC) staging was as follows: 79.5% stage A (N=70), 12.5% stage B (N=11), and 8.0% stage C (N=7,). The MRD assay identified 36 patients as MRD+ and 52 patients as MRD-. We observed significant correlations between MRD status and both relapse-free survival (RFS) and overall survival (OS). For MRD+ patients, the median RFS was 17.1 months, while it was not reached for MRD- patients (p=0.0013). Likewise, the median OS for MRD+ patients was 40.1 months, in contrast to it not being reached for MRD- patients (p=0.0012). The MRD positivity rate stood at 100% in stage C patients, a significantly higher percentage compared to the rates observed in stage B (36.4%, p=0.0256) and stage A (36.2%, p=0.0042). Significantly, MRD status emerged as a pivotal prognostic differentiator among clinically low-risk patients in stage A: among MRD+ patients, the median RFS was 23.3 months, whereas it was not reached for MRD- patients (p=0.05). Additionally, the median OS of both MRD+ and MRD- was not reached but their survival curves showed significant differences (p=0.038). Conclusions: This study demonstrated the clinical utility of ctDNA MRD assay in patients with resectable HCC, as notably evident in the robust detection of MRD using plasma samples collected 7 days after surgery. Furthermore, the assessment of post-surgical MRD status provided valuable prognostic insights into both patient survival and the risk of disease relapse. ctDNA MRD status played a pivotal role as a prognostic differentiator, particularly among clinically low-risk patients.

A randomized controlled trial of CAPEOX vs observation in patients with early-stage colorectal cancer with positive MRD after curative surgery (CAREME).

TPS225 Background: Most patients (pts) with early-stage colorectal cancer (CRC) have a good prognosis, but some still relapse shortly after surgery. Effective methods to assess the risk of recurrence in these pts and guide adjuvant chemotherapy (ACT) decision-making are lacking. Increasing studies have shown that circulating tumor DNA (ctDNA) can detect minimal residual disease (MRD) and identify pts with a high risk of recurrence. ACT could improve survival of MRD-positive pts with stage Ⅱ CRC suggested by recent studies, whether it is appropriate for those with stage Ⅰ or clinically low-risk stage Ⅱ CRC remains unknown. Methods: CAREME is a multicenter randomized controlled clinical trial aimed at investigating the benefit of chemotherapy for MRD-positive pts with early-stage CRC. Pts with resected stage Ⅰ or Ⅱ CRC will be evaluated by oncologists. Those who did not receive neoadjuvant therapy and are deemed suitable for active surveillance (i.e., ACT is not needed) will undergo a postoperative ctDNA test using MinerVa MRD assay (a tumor-informed assay covering 769 cancer-related genes). Pts with mismatch repair-deficient tumors will be excluded and stage Ⅱ pts should have no traditional high-risk features according to clinical practice guidelines. Pts with positive ctDNA (N = 38) will be equally randomized into two groups: treatment group (Arm A) or surveillance group (Arm B). Arm A will receive 8 cycles of CAPEOX therapy while pts in Arm B will not receive any ACT; both groups will undergo ctDNA tests at 6 months after randomization and be followed up every 3 months. The primary endpoint is 18-month recurrence-free survival, and a key secondary endpoint is ctDNA clearance at 6 months after randomization. Tumor samples from the pts will be collected for MRD assay and exploratory research. Accrual started in February 2023. Support: Genecast Biotechnology Company. Clinical trial information: NCT05699746 .

INTERCEPT Program of circulating tumor DNA (ctDNA) testing for minimal residual disease (MRD) in colorectal cancer (CRC): Results from a prospective clinical cohort.

27 Background: Minimal residual disease, defined by ctDNA+ in the absence of macroscopic of disease, represents a new and clinically poorly defined subset of patients (pts). While observational cohorts have been reported on the prognosis of MRD, there is little information on the impact of returning and acting on ctDNA results on the subsequent ability to detect radiographic disease. Here we provide real-world evidence form a large prospective cohort where ctDNA testing was routinely performed and returned to pts and providers, providing opportunities to understand the prognostic performance of ctDNA in practice, including the rate of false negatives defined as radiographic recurrence without detectable ctDNA. Methods: The INTERCEPT program enrolled pts undergoing curative intent surgery for stages II-IV CRC at MD Anderson Cancer Center. A tumor-informed MRD assay (Signatera) was drawn post-operatively and every 3 months as per established reimbursement guidelines. Pts enrolled between Jan-Dec 2022 were included with follow up through Aug, 2023. Post-op timepoint was defined as any test within 6.5 months of surgery to include adjuvant therapy. A false negative result was defined as a negative test but with radiographic detection of recurrence within the subsequent 4 months. Results: A total of 1,140 pts were included, with 44.7% female and 39% with advanced disease. At least one ctDNA+ result was obtained in 338 patients (29.6%). When limited to pts with a post-operative/post-adjuvant timepoint (n= 520) and a median follow up of 10.4m, the 12m DFS was 94.2% for ctDNA-, and 54.3% for pts with at least 1 ctDNA+ test (median DFS of 14.9m); HR 17.67 (95% CI: 10.08 – 30.99), p&lt;0.0001. When segregated by stage, ctDNA remained the most important predictor of outcomes [Table], such that pts with stage IV ctDNA- had better prognosis than pts with stage II ctDNA+ (38.9% vs 87.7% 12mDFS, p=.002). For 15 pts, we detected a false negative ctDNA, representing a rate of 1.3% (15/1140) corresponding to a negative predictive value (NPV) of 98.1%. Conclusions: In this first prospective real-world ctDNA program, MRD testing had a high NPV (98.1%) with very few false negative cases. The prognostic importance of ctDNA was confirmed and exceeded the risk prediction of stage alone, underlying and supporting the importance of introducing the test in practice and the need to investigate interventional studies, as are ongoing in the INTERCEPT program (crcmrd.org). [Table: see text]

Predicting recurrence using a tumor-uninformed ctDNA assay detecting MRD in patients with resected stage II or III colorectal cancer: Subset analysis from the GALAXY study in CIRCULATE-Japan.

21 Background: The presence of circulating tumor DNA (ctDNA) after curative-intent surgery can identify patients with minimal residual disease (MRD) who are at risk of recurrence and aid with postsurgical risk stratification. ~80% of stage II and only 50% of stage III colorectal cancer (CRC) patients are cured by surgery alone. A tumor-uninformed plasma-only approach for MRD assessment accelerates the turnaround time, enabling rapid adjuvant chemotherapy (ACT) treatment decision making, optimizing the impact on patient outcomes. Methods: A subset of samples from GALAXY, an observational arm of the CIRCULATE-Japan study (UMIN000039205), were analyzed for postsurgical ctDNA detection at a landmark time point (LMT) defined as 4 weeks after curative surgery in pathological stage II or III CRC patients. In a prospective lead-in to clinical validation, 80 patients meeting pre-specified eligibility criteria were randomly selected with enrichment for recurrence to 50% while maintaining the stage II:III recurrent (R)/nonrecurrent (NR) ratio to that observed in GALAXY. Residual plasma samples were analyzed with the Tempus xM assay (xM), a tumor-uninformed ctDNA MRD assay that integrates methylation and genomic variant MRD classifiers to deliver a binary MRD call. All calls were blinded to clinical outcomes. The methylation-based classifier detects fragments with CRC methylation signatures in differentially methylated regions trained by sequencing CRC and presumed-healthy samples on a 6Mbp panel. The variant-based MRD classifier detects highly prevalent CRC variants. Artifacts, CHIP, and germline variant filtering algorithms were also applied. Results: Of the 80 patients (pts), 70 [Stage II n= 29 (41%), Stage III n= 41 (59%)] were evaluable (36 R and 34 NR). At the LMT and with at least 1-year follow-up, 18/36 R pts had detectable ctDNA (xM MRD+) and 30/34 NR pts had undetectable ctDNA (xM MRD-), providing a sensitivity of 50% and specificity of 88%. Additionally, xM MRD+/- status strongly correlated with disease-free survival (DFS) with a hazard ratio (HR) of 5.09, adjusted to account for a 24% recurrence rate in GALAXY. In an updated, ongoing longitudinal analysis of the 4 false positive pts, 2 were Stage III, MRD+ from both the methylation and genomic classifiers, received ACT, and have likely cleared their ctDNA without any sign of clinical recurrence. An adjusted sensitivity and specificity analysis resulted in xM clinical performance of 53% (20/38 R) and 94% (30/32 NR), respectively. Conclusions: xM is a novel tumor-uninformed assay that demonstrated remarkable performance to detect clinical recurrence at a LMT, and a clinically meaningful correlation with DFS. A larger clinical validation study including longitudinal analysis, correlation with ACT, and outcomes from the GALAXY study is currently underway.

Association of pathologic nodal status with minimal residual disease after neoadjuvant treatment and resection of locally advanced rectal cancer.

205 Background: Studies have shown that minimal residual disease (MRD) identified by detection of circulating tumor DNA (ctDNA) is associated with recurrence after surgical resection of colorectal cancer. Though multiple trials are evaluating the use of ctDNA to guide adjuvant therapy, data on the utility of ctDNA after neoadjuvant therapy is limited. In this study, we evaluated the associations between ctDNA and recurrence after neoadjuvant treatment and resection of rectal cancer (LARC). Methods: Consecutive patients with primary rectal cancer treated with neoadjuvant systemic therapy and/or chemoradiotherapy followed by resection between 12/2020-4/2023 were identified. Patients were tested for ctDNA via the MD Anderson INTERCEPT platform using a high-sensitivity patient informed assay. Patients with metastases or a first ctDNA test more than 3 months after resection were excluded. MRI-determined stage, extramural vascular invasion (mrEMVI), pelvic sidewall adenopathy (PSW), pathologic lymphovascular invasion (LVI), perineural invasion (PNI) and tumor regression grade (TRG) were collected. Associations between these factors and ctDNA status were analyzed with Fischer’s exact test. Recurrence free survival (RFS) was analyzed with the log-rank test. Results: Sixty-seven patients (3 treated with chemoradiotherapy alone, 15 with systemic therapy alone and 49 with total neoadjuvant therapy) were identified. Positive ctDNA was identified in 6 (8.9%) within 3 months after resection, all of whom received total neoadjuvant therapy, and 3/6 recurred within 12 months. Clinical T status (p=0.146), N status (p=0.842), mrEMVI (p=0.475) and PSW (p=0.318) were not associated with MRD. TRG was not associated with MRD (TRG0-1: 4% vs TRG2-3: 11.3%, p=0.547). Pathologic N status was associated with MRD (ypN0: 4.4% vs ypN+: 18%, p=0.049). MRD was associated with a median RFS of 3.2 months (HR 39, 95% CI: 4.1-380, p=0.0015). 1-year RFS was 98% for patients without MRD. Conclusions: Minimal residual disease after multi-modality treatment of LARC is associated with early metastatic recurrence. The patient informed MRD assay appears both sensitive and specific for detection of relapse. Advanced clinical stage and high-risk radiologic features were not associated with MRD, while post-treatment pathologic N status was. Given the high relapse rate for MRD+ patients, MRD detection after resection warrants additional investigation for metastases and/or enrollment on MRD treatment clinical trial. Methods to predict MRD prior to resection are urgently needed.

Immunoglobulin High Throughput Sequencing (Ig-HTS) Minimal Residual Disease (MRD) Analysis is an Effective Surveillance Tool in Patients With Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) accounts for 4% to 6% of B-cell non-Hodgkin lymphoma with historically poor outcomes. With the advent of intensive first-line, targeted, and cellular therapies, outcomes have improved, and initial remission can be 8 to 10 years or longer. As patients experience longer remissions, this raises the question of the optimal surveillance modality. Peripheral blood minimal residual disease (MRD) analysis offers a potential alternative to surveillance imaging that is sensitive, less costly, and eliminates the risk of radiation exposure. Materials and MethodsThe clonoSEQ assay (Adaptive Biotechnologies) is an FDA-cleared commercially available Ig-HTS MRD assay with a sensitivity of 1 cell in 1,000,000. We performed a retrospective analysis of 34 patients from 2015 to 2021, who underwent MRD testing after achieving remission with first-line therapy. ResultsWith a median follow-up of 6.5 years, 10-year progression free survival (PFS) was 60% and 10-year overall survival was 92% of the entire cohort. Among 12 patients who sustained a radiographic relapse, peripheral blood became MRD+ either at or prior to the time of relapse in 11 patients (92%). The first MRD+ test had a lead time of 0 to 24 months (median 34 days) prior to radiographic relapse. Only 1 patient had a MRD− result while being found to have progressive disease on imaging. Among 22 patients who sustained continuous clinical remission, 21 have remained MRD−. Several patients were able to enjoy 2 to 4-year intervals without surveillance imaging. ConclusionsOur data suggest that the clonoSEQ MRD assay is an effective surveillance tool for MCL patients following first-line therapy and is predictive of relapse prior to imaging.

Optimizing Circulating Tumor DNA Limits of Detection for DLBCL during First Line Therapy

Background: Circulating tumor DNA (ctDNA) assessment is effective in diffuse large B-cell lymphoma (DLBCL) monitoring and risk stratification, with prognostic utility throughout first-line (1L) therapy. DLBCL ctDNA assays vary in analytical sensitivity, or limit of detection (LOD), which range from parts per thousand to below 1 part per million (1 in 10 6). With differing assays and time-points assessed in DLBCL studies, the relationship between analytical and clinical sensitivity for outcome prediction remains unclear. We assessed the prognostic ability of ctDNA at various LODs and used modeling strategies to project the efficacy of assays for minimal residual disease (MRD) detection. Methods: We previously reported a DLBCL dataset assessing plasma ctDNA before, during and after curative-intent 1L anthracycline-based treatment from multiple prospective studies, including RCHOP or EP[O]CH with acalabrutinib, lenalidomide, obinutuzumab, and polatuzumab (Roschewski et al. ASH 2022). In this study, ctDNA was evaluated by Phased Variant Enrichment Detection and Sequencing (PhasED-Seq, Foresight Diagnostics), a ctDNA MRD assay with LOD below 1 in 10 6. Samples from this study and a prospective DLBCL cohort treated with standard 1L therapy at Samsung Medical Center were considered in this analysis. We assessed the prognostic ability of ctDNA detection, considering LODs from 1 in 100 (10 2)to 1 in 10 6 to predict progression free survival (PFS) before, during and after 1L treatment. We assessed the desired sensitivity for ctDNA MRD during treatment by generating patient-specific log-linear models assuming exponential decay. We projected the distribution of variant allele frequencies (VAFs) during therapy from cycles 2 to 5 for patients who progressed to determine the minimal acceptable analytical sensitivity. Results: We included 230 patients consisting of 588 ctDNA samples, with 201 before therapy, 71 at C2D1, 101 at C3D1, 70 at C4D1 and 145 at end of therapy (EOT). Median follow-up was 17.5 months and 62 patients (27%) progressed. To evaluate the impact of LOD at treatment milestones, we applied a threshold for ctDNA positivity ranging from 1 in 10 2 to 1 in 10 6. Increased LOD had no effect on the performance of ctDNA before therapy (Figure A &amp; B). At C2D1, if the LOD was at least 1 in 10 4, there was no difference in MRD prognostic performance. Starting at C3D1, improving the LOD for ctDNA positivity down to 1 in 10 6 showed superior predictive power for PFS at 24 months. The power of ctDNA to predict PFS at 24 months improved later in therapy, with area under the receiver operator curves (AUROCs) for PFS24 of 0.68, 0.73, 0.77, 0.88, and 0.86, at pretreatment, C2D1, C3D1, C4D1, and EOT time-points respectively (Figure A). To further understand the desired LOD for ctDNA MRD, we developed personalized models of ctDNA VAFs. Exponential models fit the data well in 43/44 cases with ≥ 3 MRD-detectable samples through C4D1, with a median correlation of 0.91, confirming their utility for modeling ctDNA. We generated log-linear models for 106 patients with ≥ 2 MRD-detectable samples, and used log-fold change in VAF per cycle (i.e. the slope) to compare patients by progression. Median log-fold change in VAF per cycle was worse for patients who progressed at -1.1 (IQR -1.4, -0.7) compared to those who remained disease-free with median -1.5 (IQR -2.0, -1.1) (p&amp;lt;0.0001). Patients with primary refractory DLBCL on EOT imaging had less robust log-fold change per cycle with median of -0.9 (IQR -1.3, -0.5) compared to those who relapsed after CR with median -1.3 (IQR -1.7, -0.9) (p=0.0004). We used the distribution of slopes to project VAFs for cycles 2 to 5 for patients who progressed to determine the LODs that provide acceptable clinical sensitivity. We found the analytical sensitivity for detecting DLBCL extended lower for each cycle, demonstrating the need for a LOD at least 1 in 10 6 for robust MRD detection at late time-points during 1L treatment. Conclusion: When using an ultrasensitive assay, MRD assessment at later timepoints better predicts PFS than at early timepoints. While the technical LOD does not affect disease burden assessment before therapy, using more sensitive assays during and after therapy improves disease detection and outcome prediction. Utilizing the most sensitive ctDNA MRD assays in 1L DLBCL therapy will maximize the efficacy of MRD-driven therapeutic strategies and MRD as a surrogate endpoint in future trials.

Prognostic Utility of Minimal Residual Disease (MRD) after Curative Intent Induction Therapy for DLBCL: A Prospective Real-World Ctdna Study

Background: The prognostic value of MRD using ultra-sensitive ctDNA assays after 1L DLBCL immunochemotherapy was recently reported (Roschewski, ASH 2022). However, real-world evidence (RWE) for the utility of ctDNA MRD assays to predict survival outcomes after standard-of-care (SOC) 1L treatment for DLBCL remains less clear. Here, we report longitudinally measured ctDNA levels, without use of tumor biopsies, in a real-world population of DLBCL patients treated with curative intent using current anthracycline-based induction regimens. Methods: We prospectively enrolled patients with DLBCLs from Samsung Medical Center undergoing 1L therapy for ctDNA profiling at 3 pre-defined milestones (Baseline, Interim after 3 cycles (C4D1), and end of treatment [EOT]). A total of 99 patients had baseline pretreatment plasma available with sufficient tumor burden for inclusion in this study. 364 samples were profiled in a blinded manner by Phased Variant Enrichment &amp; Detection Sequencing (PhasED-Seq, Foresight Diagnostics). Tumor-derived Phased Variants (PVs) were identified directly from baseline plasma (median 4 mL) without the use of tumor tissue, with matched constitutional DNA from paired PBMCs used to censor germline variants and CHIP. PVs were used to longitudinally assess ctDNA-MRD after therapy, including at the C4D1 and EOT timepoints. MRD levels were compared to standardized responses by PET/CT (Lugano 2014), PFS, and OS. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1:10 6 cfDNA molecules) corresponding to 98% specificity. Results: Among the 99 evaluable patients, the median age was 58 years and 38% were women. 67% had stage III-IV disease, 53% had IPI scores ≥3, and 6% had MYC and BCL2 translocations. All patients were treated with anthracycline based therapy, with 94% receiving RCHOP and 6% receiving DA-EPOCH-R. The median follow-up was 44 months. We tested ctDNA performance at each landmark for predicting outcomes. ctDNA-MRD levels by PhasED-Seq at each landmark were prognostic for both PFS and OS. Specifically, when dividing pretreatment ctDNA levels at the median, ctDNA significantly predicted outcomes (PFS: P=0.004, HR=2.7; OS: P=0.0004, HR=5.1). At the C4D1 and EOT landmarks, we divided patients into those with detectable versus undetectable ctDNA-MRD. Both timepoints significantly stratified patients for outcomes including at C4D1 (PFS: P=0.0002, HR=5.0; OS: P=0.0046, HR=4.1) and at EOT (PFS: P&amp;lt;0.0001, HR=7.8; OS: P&amp;lt;0.0001, HR=10.8). As the current standard for assessing response in DLBCL is imaging, we also examined the prognostic value of PET/CT scans at the same time-point as ctDNA. While interim PET/CT scans in our cohort were prognostic for PFS, they were not significantly prognostic for OS (PFS: P=0.02, HR=2.5; OS: P=0.06, HR=2.4). At EOT, PET/CT scans were prognostic for both PFS and OS (PFS: P=0.011, HR=3.4; OS: P=0.011, HR=4.0). At both milestones, PET/CT appeared less discriminatory than ctDNA-MRD. Exemplifying this, the overall survival of patients at 24 months was better separated at EOT by ctDNA-MRD than it was by PET/CT scan (OS24 ctDNA-MRD+: 37%, ctDNA-MRD-: 94%, compared with PET/CT PR/SD/PD: 50%, PET/CT CR: 86%). Finally, to further compare the prognostic value of ctDNA-MRD in the context of the current standard PET/CT scan, we performed a Cox proportional hazards model to predict PFS and OS, using both PET/CT and ctDNA-MRD as univariate predictors, as well as a multivariate model considering both PET/CT and ctDNA. As expected, interim and EOT ctDNA-MRD and PET/CT scans were predictive of outcomes when considered in univariate analysis. However, when considered in combination, while the prognostic value of ctDNA-MRD for OS remained at both interim and EOT timepoints, PET/CT scan was no longer significantly prognostic of outcomes (Fig 1). Conclusions: These data both demonstrate the feasibility and the prognostic utility of ctDNA-MRD during and after SOC induction therapy for DLBCL in a real-world population using an ultrasensitive ctDNA-MRD assay. The higher predictive value and accuracy of detectable ctDNA-MRD as compared with PET/CT suggest opportunities for integration of such assays in lymphoma response criteria, to potentially inform future clinical decision making.

One Tube 23 Color Full Spectral Flow Cytometry Panel in Detecting Minimal Residual Disease in Pediatric B-Cell Acute Lymphoblastic Leukemia

Introduction Minimal residual disease (MRD) is a normalized tool for risk stratification in the treatment of pediatric acute lymphoblastic leukemia (ALL) and has been introduced into many standardized chemotherapy regimens. Flow cytometry (FC) plays an important role in the monitoring of MRD. However, FC-MRD can't reliably detect residual leukemic cells below level of 0.01% in real clinical scenarios and some patients with negative MRD relapsed, suggesting the current used set of markers are not sensitive enough or specific enough. Published studies has shown that more markers with more reliable combination of antibodies may help improve the sensitivity and specificity of FC-MRD. Spectral multicolor flow cytometry (SMFC) has shown an advantage over traditional FC that more fluorescent markers could be applied simultaneously and more complex marker combinations could be exploited to distinguish leukemic blasts from normal B cell precursors with high specificity. Here we designed a SMFC-based panel of 26 markers with 23 fluorescent colors in one tube to monitor B-ALL MRD. Method The one-tube 23-color panels was designed according to previously published studies and our clinical experience based on standardized EuroFlow 8-color MRD panel. All the leukemia-associated immunophenotypes (LAIPs) were defined based on its deviation from hematogones. For CD10, CD19, CD22, CD34, CD58, CD73, CD86, CD123, CD200and CD304, the overexpression was considered as LAIP, while the under-expression was considered as LAIP for CD24, CD38, CD45 and CD81. Asynchronous co-expression of CD34 and CD20, CD34 and CD21 were also included. The aberrant cross-lineage expression was taken as LAIP for CD2, CD56, CD65, CD15, CD13, CD33, CD66c, NG2 and CD133. For CD44, under-expression or overexpression can both be considered as LAIP. To verify the effectiveness of the panel, 317 bone marrow samples from 317 patients with B-ALL diagnosed between 2009 to 2014 at the Shanghai Children's Medical Center (SCMC) were included. These samples were collected at day 19 to 283 (median: day 35), with 213 (67.19%) collected at around day 35, which was a regular time point to monitor MRD and to readjust the risk stratification. The results were compared with those of conventional 8-color FC-MRD. Results The limit of blank (LOB), limit of detection (LOD), and lower limit of quantitation (LLOQ) were firstly determined, the results showed reproducible sensitivity up to 0.001% (1-in-10 5) after acquisition of 4.8 million cells for 23-color panel. The 8-color and 23-color MRD panels showed high correlation of the quantitation of MRD (r 2=0.80, p&amp;lt;0.0001). Among cases of discordant between 8-color MRD negative (with cutoff &amp;lt;0.01%) and 23-color MRD negative (with cutoff &amp;lt;0.001%), relapse only occurred in patients with 23-color MRD positive and 8-color MRD negative, rather than patients with 23-color MRD negative and 8-color MRD positive, suggesting a higher sensitivity and specificity for 23-color MRD assay (See Figure). Patients with detectable MRD by either 8-color or 23-color had a significantly higher incidence of relapse (CIR, p&amp;lt;0.05) and shorter event free survival (EFS, p&amp;lt;0.05). Positive 23-color MRD seemed to be an even stronger factor impacting CIR and EFS. Compared to patients with 8-color MRD negative and 23-color MRD positive, patients with 8-color MRD positive and 23-color MRD negative had a significantly lower CIR (p&amp;lt;0.05) and a better EFS (p=0.068). Patients with positive 23-color MRD lower than 0.01% showed a trend towards poorer EFS and OS (p=0.083% and p=0.061%, respectively) compared to patients with 23-color MRD negative. Conclusion Based on these evidences, SMFC-based one-tube 23-color MRD panel for B-ALL was convenient and maneuverable with higher sensitivity and specificity compared to conventional 8-color FC-MRD panel. It would be a promising method to improve the risk stratification.

587P Short lead time and high rates of concomitant radiographic recurrences for ctDNA-based minimal residual disease assays colorectal cancer (CRC) during surveillance: Results from the MD Anderson INTERCEPT program

587P Short lead time and high rates of concomitant radiographic recurrences for ctDNA-based minimal residual disease assays colorectal cancer (CRC) during surveillance: Results from the MD Anderson INTERCEPT program

Abstract PR09: Detection of minimal residual disease in post-surgical drain fluid can predict locoregional recurrence in HPV-negative head and neck cancer patients

Abstract Introduction: Locoregional cancer relapse remains a major cause of failure in head and neck squamous cell carcinoma (HNSCC), particularly for HPV-negative patients whose 3-year locoregional failure rate is 32.5%. There is a major unmet need for an accurate diagnostic test that predicts risk of locoregional recurrence prior to adjuvant therapy selection. We present a novel proximal assay for minimal residual disease (MRD) profiled in lymphatic exudate collected via surgical drains (“lymph”). Methods: Lymph, plasma, and peripheral blood were collected from 22 HPV-negative HNSCC patients postoperatively at 24 hours along with resected tumor. Cell-free DNA was extracted from lymph and plasma and sequenced using the TruSeq Oncology 500 panel to a depth of &amp;gt;100 million reads. One plasma sample failed due to inadequate coverage. Two patients were censored due to lack of clinical data, yielding 9 patients with disease recurrence (REC) and 11 with no evidence of disease (NED) with &amp;gt;1 year of follow-up. Somatic mutations were identified from exome sequencing (200x) in tumor with matched blood. Tumor-specific variants were force-called in lymph and plasma using a custom bioinformatic pipeline. Mutation calls were filtered by a base-specific error model to eliminate artifacts. Student’s t-test was used for group comparisons. The Kaplan-Meier (KM) estimator with log-rank test and Cox proportional-hazards model were used for survival analyses. Results: ctDNA allelic fraction was 1.5x higher in lymph than in plasma (lymph = 0.11% ± 0.16%; plasma = 0.076% ± 0.12%. p = 0.018, N = 100 mutations). Significantly more mutations were detected in REC lymph compared to NED (p = 0.009), but not in plasma REC vs. NED (p = 0.16). We classified patients as positive (&amp;gt;1) or negative (£1) for detected mutations in each analyte and performed a KM survival analysis, showing lymph could accurately predict recurrence (sensitivity = 89%, specificity = 82%; p &amp;lt; 0.005) while plasma could not (sensitivity = 67%, specificity = 40%; p = 0.59). The hazard ratio in lymph was 12.52 (95% CI 1.54-101.61). We stratified REC patients by locoregional or locoregional + distant relapse and observed significantly more mutations detected in lymph (p = 0.01) from locoregional relapse while lymph and plasma performed similarly for locoregional + distant relapse (p = 1.0). We compared lymph MRD outcomes to extranodal extension (ENE), a high-risk pathologic feature. Lymph was concordant with ENE in 12/20 patients and identified an additional 6 ENE-negative relapse cases. Conclusion: Postoperative ctDNA analysis of lymph from surgical drains represents a novel MRD approach in HPV-negative HNSCC. Lymph significantly outperforms plasma for prediction of recurrence, particularly in patients with locoregional relapse. Accurate MRD identification in patients with lower risk pathologic features suggests that postoperative lymph MRD testing has the potential to significantly augment traditional pathology and provide more personalized adjuvant treatment decision-making in patients with HPV-negative HNSCC. Citation Format: Aadel A. Chaudhuri, Zhuosheng Gu, Damion Whitfield, Noah Earland, Adam Harmon, Megan Long, Peter Harris, Zhongping Xu, Ricardo Ramirez, Sophie Gerndt, Maciej Pacula, Marra S. Francis, Wendy Winckler, Jose P. Zevallos. Detection of minimal residual disease in post-surgical drain fluid can predict locoregional recurrence in HPV-negative head and neck cancer patients [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR09.

Personalized K-Track assay to detect actionable mutations and minimal residual disease in solid tumors.

110 Background: Minimal residual disease (MRD) refers to a small number of cancer cells that remain in the body after curative-intent treatment, causing relapse and metastasis. Current tumor biomarkers and imaging methods have limited sensitivity and specificity to monitor MRD for solid tumors. Circulating tumor DNA (ctDNA) in liquid biopsy has emerged as a promising biomarker to detect MRD but current ctDNA assays remain mostly inaccessible and unaffordable for developing countries. Methods: In this prospective multi-center study, 441 Vietnamese patients diagnosed with breast, colorectal, gastric, lung, liver and ovarian cancers, eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived genomic alterations including actionable mutations in 95 cancer-associated genes. Our bioinformatic algorithm then ranked top alterations unique for individual patient, which were then used to monitor ctDNA presence in serial plasma samples collected before surgery and at scheduled visits after surgery by multiplex PCR and ultra-deep sequencing. Results: The assay had limit of detection for somatic mutations down to at least 0.05% variant allele frequency in liquid biopsy.The sensitivity to detect ctDNA in the pre-operative plasma was 91%, 95%, 70%, 77%, 69% and 47% for colorectal, liver, lung, ovarian, high-risk breast cancer and gastric cancer respectively; the specificity was &gt;99%. After 18-month follow-up, our interim analysis revealed a promising 89% detection rate of post-operative ctDNA in all patients that had metastasis and recurrence (31/35), with the lead time of 2.5-13 months ahead of clinical diagnosis. Conclusions: The K-Track assay is streamlined and affordable with dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies. The assay could lay foundation to empower precision cancer medicine in Vietnam and developing countries.

Personalised circulating tumour DNA assay with large-scale mutation coverage for sensitive minimal residual disease detection in colorectal cancer

BackgroundPostoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay.MethodsPersonalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC).ResultsIn 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49–20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001).ConclusionTumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

MRD assay to evaluate recurrence and response via a tumor-informed assessment: MARIA-Breast observational trial.

TPS622 Background: The recognition that cancers release nucleic acids into the peripheral circulation of the patient has enabled a new paradigm for disease detection and surveillance. Detectable ctDNA in patients with solid tumors has been associated with disease prognosis pre-treatment, assessing response to therapy in the form of minimal residual disease (MRD), and monitoring for recurrence after curative intent treatment. Utilizing patient-specific genomic mutation profiling of an individual’s cancer from a tissue sample, in conjunction with the patient’s germline DNA, to create a personalized sequencing panel to then systematically analyze for a subset of these genetic mutations from ctDNA in blood is a strategy that has high sensitivity in detection of MRD. Studies have shown that pretreatment levels of ctDNA using this approach are a potential early indicator of disease recurrence after surgery, that ctDNA clearance may be an early predictor of favorable outcomes and have been shown to correlate with pathologic complete response (Forde et al. N Engl J Med. 2022, PMID:35403841), and that this approach has high sensitivity in its ability to detect recurrence for patients in advance of the current standard of care (Abbosh et al. Cancer Res (2020) 80 (16_Supplement): CT023). Methods: This is a multi-site, prospective, observational trial in the United States of 200 patients with early stage breast cancer undergoing curative intent treatment, who have FFPE tissue available from resection sufficient for a patient-specific bespoke MRD assay who are willing to provide serial whole blood specimens for ctDNA analysis. Participants are asked to provide study specimens prior to initial treatment intervention, after curative intent surgical resection, during adjuvant therapy (as applicable) and pre-recurrence follow-up. ctDNA will be analyzed with an NGS-based, tumor-informed MRD assay that identifies somatic mutations from DNA obtained from the patient’s tumor tissue, subtracts germline variants via NGS-based analysis of the patients germline DNA, and detects a selected set of between 18-50 tumor-specific ctDNA in their blood. All primary tumor specimens will undergo full exome sequencing using the Personalized Cancer Monitoring (PCM) assay. Impact of results of this CLIA-approved MRD assay on clinical decision making will be captured. The primary objective is to assess the ability of MRD to predict post-treatment recurrence. Further objectives are to correlate MRD status with pathologic complete response, determine the lead time to detection of recurrence compared to standard of care, and the association of MRD status with overall survival. Active enrollment started in March, 2022. Clinical trial information: NCT05219734 .