Minimal Residual Disease Testing Research Articles

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&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 2417: Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic

Abstract Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation. Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across >1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population. To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting. Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [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 2417.

Abstract 6881: Investigating the role of post-transplant bone marrow immune dysregulation in multiple myeloma

Abstract Multiple myeloma (MM) is a cancer of bone marrow plasma cells (BMPCs), which to date remains incurable. Standard of care for qualifying patients involves high-dose chemotherapy followed by autologous stem cell transplant (ASCT), after which minimal residual disease (MRD) testing is used to assess the degree of tumor clearance. Though strongly associated with better survival, MRD-negativity is an imperfect predictor for patient outcomes and fails to account for the pro-tumorigenic BM dysregulation often found in MM patients. As the post-transplant BM milieu in MM is not well understood, we conducted 3' single-cell RNA sequencing (scRNAseq) of 49 newly diagnosed (NDMM) and 79 day-100 post-ASCT whole-BM aspirates to investigate how immunosuppressive microenvironmental signatures may persist or develop following ASCT to influence patient outcomes. 68 of 79 post-ASCT samples come from uniformly-treated clinical trial enrollees with progression-free survival (PFS) ranging from 10 months to over 7 years, with both MRD- and MRD+ cases at either PFS extreme. We observe that although post-ASCT samples have uniformly lower PC count than NDMM samples, only erythroblasts and B cells appear relatively depleted in NDMM samples to compensate for higher PC proportions. T/NK cells and myeloid lineages, though similar in percentage across samples, exhibit clear subtype bias between the two timepoints. Post-ASCT samples are significantly enriched for GZMH+ CD8+ T and NK cells, HIF1A+ hypoxia-responsive monocytes, and depleted for S100A8/9-high CD14+ monocytes. NDMM samples are also enriched for SPINK2+, CDK6+, and AVP+ neutrophils, which are rare in post-ASCT samples. Notably, higher HIF1A expression in myeloid cells post-ASCT is further associated with longer PFS. Regardless of MRD status, NFKBIA, FOS, and GNLY upregulation and GZMK downregulation in post-ASCT T cells are associated with worse PFS. Conversely, enrichment of CD16+ IFN-responsive monocytes and depletion of ARG1+/FUT4+/CEACAM8+ myeloid-derived suppressor cells (MDSCs) are associated with longer PFS. Beyond such general trends, we observe MRD-dependent PFS-association of several CD8+ T cell subsets. Overall, this study enhances our understanding of how post-transplant BM repopulation and immune interaction may impact treatment response in MM. Citation Format: Julia T. Wang, Mark Fiala, Kazuhito Sato, Julie Fortier, Ravi Vij, Li Ding. Investigating the role of post-transplant bone marrow immune dysregulation in multiple myeloma [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 6881.

Abstract 5022: MEDOCC-CrEATE trial: Feasibility of measuring circulating tumor DNA after surgery to guide adjuvant chemotherapy in stage II colon cancer patients

Abstract Introduction: Patients with stage II colon cancer not classified as high risk (pT4 microsatellite stable) do not receive adjuvant chemotherapy (ACT) according to Dutch guidelines. However, 15-20% of patients with stage II colon cancer experience a disease recurrence, indicating that there is an unmet clinical need to identify patients who could benefit from adjuvant treatment. Observational studies demonstrate that postoperative circulating tumor DNA (ctDNA) is indicative of minimal residual disease (MRD) and a strong prognostic biomarker for disease recurrence. Aim: The MEDOCC-CrEATE trial aims to assess 1) the proportion of stage II colon cancer patients with detectable postoperative ctDNA accepting ACT and 2) whether ctDNA-guided ACT reduces 2-year recurrence rate (RR). Methods: MEDOCC-CrEATE is an interventional trial following the ‘trial within cohorts’ design. Participants of the Prospective Dutch Colorectal Cancer cohort with stage II colon cancer and no indication for ACT randomized to the intervention arm undergo postoperative tumor-informed ctDNA testing using next-generation sequencing of 33 genes through the PGDx elio™ liquid biopsy assay. If ctDNA is detected, they are offered 4 cycles of adjuvant capecitabine plus oxaliplatin. Patients in the intervention arm who test ctDNA negative and patients in the control arm receive standard of care follow-up. For all patients, blood is collected every 6 months for 3 years to monitor disease recurrence. The primary endpoint is the proportion of patients with detectable postoperative ctDNA willing to receive ACT. Secondary endpoints include 2-year RR, disease-free and overall survival, quality of life and cost-effectiveness of ctDNA-guided ACT. The study is powered for 2-year RR, randomizing 660 patients to each study arm in order to treat 30 patients in the intervention arm with detectable postoperative ctDNA, assuming 5% ctDNA detection and 10% noncompliance. Results: Logistics for timely multicenter collection of tumor tissue and blood have been optimized across 28 Dutch hospitals. At present, 240 patients have been randomized. Of the 119 patients in the intervention arm, 98 provided consent for immediate postoperative ctDNA analysis (82%). Of the 92 currently available results, 5 (5 %) had detectable ctDNA. The median time from surgery to blood collection was 15 days (IQR 11-19), with a median turnaround time from surgery to ctDNA result of 47 days (IQR 39-54). Conclusion: Multicenter postoperative tumor-informed ctDNA testing for MRD is operationally and technically feasible within the clinically relevant 8-12 week window to start ACT. The observed ctDNA detection rate is in accordance with expectations and the study design. Upon study finalization, the results will be used for health technology assessment to demonstrate the putative clinical utility of ctDNA-guided ACT in stage II colon cancer. Citation Format: Ingrid Franken, Suzanna Schraa, Karlijn van Rooijen, Dave van der Kruijssen, Carmen Rubio-Alarcón, Steven Ketelaars, Sietske van Nassau, Teunise Snetselaar, Bregje Adriaans, Jillian Phallen, Sam Angiuoli, Amy Greer, Ellen Verner, Veerle Coupé, Helena Verkooijen, Miranda van Dongen, Linda Bosch, Mirthe Lanfermeijer, Daan van den Broek, Gerrit Meijer, Victor Velculescu, Mark Sausen, Miriam Koopman, Remond Fijneman, Geraldine Vink, on behalf of the PLCRC-MEDOCC group. MEDOCC-CrEATE trial: Feasibility of measuring circulating tumor DNA after surgery to guide adjuvant chemotherapy in stage II colon 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 5022.

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 >100 million reads. Somatic mutations were identified by exome sequencing (200x) tumor and blood. Five patients had <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 >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.

MAGIC-DR: An interpretable machine-learning guided approach for acute myeloid leukemia measurable residual disease analysis.

Multiparameter flow cytometry is widely used for acute myeloid leukemia minimal residual disease testing (AML MRD) but is time consuming and demands substantial expertise. Machine learning offers potential advancements in accuracy and efficiency, but has yet to be widely adopted for this application. To explore this, we trained single cell XGBoost classifiers from 98 diagnostic AML cell populations and 30 MRD negative samples. Performance was assessed by cross-validation. Predictions were integrated with UMAP as a heatmap parameter for an augmented/interactive AML MRD analysis framework, which was benchmarked against traditional MRD analysis for 25 test cases. The results showed that XGBoost achieved a median AUC of 0.97, effectively distinguishing diverse AML cell populations from normal cells. When integrated with UMAP, the classifiers highlighted MRD populations against the background of normal events. Our pipeline, MAGIC-DR, incorporated classifier predictions and UMAP into flow cytometry standard (FCS) files. This enabled a human-in-the-loop machine learning guided MRD workflow. Validation against conventional analysis for 25 MRD samples showed 100% concordance in myeloid blast detection, with MAGIC-DR also identifying several immature monocytic populations not readily found by conventional analysis. In conclusion, Integrating a supervised classifier with unsupervised dimension reduction offers a robust method for AML MRD analysis that can be seamlessly integrated into conventional workflows. Our approach can support and augment human analysis by highlighting abnormal populations that can be gated on for quantification and further assessment. This has the potential to speed up MRD analysis, and potentially improve detection sensitivity for certain AML immunophenotypes.

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.

Isoelectric focusing followed by affinity immunoblotting to detect monoclonal free light chains in monoclonal gammopathies: Comparison with immunofixation electrophoresis and free light chain ratio.

Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC). We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis. Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%). The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing.

Development and application of a precision cell-free DNA (cfDNA) minimal residual disease (MRD) test to enable optimized treatment selection in patients with genitourinary (GU) cancers.

473 Background: By predicting those most likely to relapse, cfDNA-based MRD testing has potential clinical utility for adjuvant therapy decision making. Ideally, MRD would be paired with validated treatment selection testing to enable precision adjuvant therapy. Herein we report the development and application of a combined personalized MRD and treatment selection test to patients with GU cancer. Methods: Patients with clinically localized solid tumors undergoing definitive therapy were enrolled on a prospective trial (NCT05082701) where precision MRD testing was performed with standard imaging-based disease recurrence monitoring. StrataMRD was performed on 2 tubes of peripheral blood through assessment of 1-12 personalized tracer mutations identified via tumor tissue profiling by Strata Select, which also provides validated genomic profiling, and angiogenesis inhibitor and immunotherapy treatment response scores (Angio TRS and IRS, respectively). Patients with GU cancers and valid StrataMRD results from at least one time point were eligible for this analysis. Tissue biomarkers were compared vs. 792 patients with advanced GU cancers tested by the same tissue platform. Results: A total of 49 patients with GU cancers who underwent definitive surgery and had at least one valid cfDNA based MRD test were eligible (*Table). The median age was 64 years, 92% of patients were male, and 49% of patients were stage I or II (vs. 51% stage III); patients underwent initial MRD testing at a median of 11 weeks after surgery and a median of 3 MRD tests have been performed per patient. Overall, 4/49 (8.2%) patients had an initial MRD+ test, and 8/49 (16.3%) had a MRD+ test at any time point (4 converted from MRD- to MRD+). Patient level MRD sensitivity and specificity in the evaluable cohort (with concurrent or subsequent imaging) was 100% (6 MRD+ / 6 recurrences) and 100% (24 MRD- / 24 not recurred), respectively. In this cohort, 34% and 52% of the renal cell carcinoma (RCC) patients were IRS- and Angio TRS-High, respectively, compared to 29% and 50%, respectively, of patients with advanced RCC undergoing similar tissue testing. Similarly, 15%, and 14% of the patients with bladder or other GU cancers were IRS-High, respectively, compared to 27% and 13% of those with advanced bladder or other GU cancers, respectively. Conclusions: Personalized cfDNA based MRD testing had high sensitivity and specificity vs. routine imaging for detecting disease recurrence in patients with GU cancers. Combined tumor testing with validated angiogenesis inhibitor and immunotherapy treatment selection biomarkers enables individualized adjuvant therapy decision making. Clinical trial information: NCT05082701 . [Table: see text]

Japanese Society of Medical Oncology clinical guidelines: Molecular testing for colorectal cancer treatment, 5th edition.

Molecular testing to determine optimal therapies is essential for managing patients with colorectal cancer (CRC). In October 2022, the Japanese Society of Medical Oncology published the 5th edition of the Molecular Testing Guideline for Colorectal Cancer Treatment. In this guideline, in patients with unresectable CRC, RAS/BRAF V600E mutational and mismatch repair tests are strongly recommended prior to first-line chemotherapy to select optimal first- and second-line therapies. In addition, HER2 testing is strongly recommended because the pertuzumab plus trastuzumab combination is insured after fluoropyrimidine, oxaliplatin, and irinotecan in Japan. Circulating tumor DNA (ctDNA)-based RAS testing is also strongly recommended to assess the indications for the readministration of anti-EGFR antibodies. Both tissue- and ctDNA-based comprehensive genomic profiling tests are strongly recommended to assess the indications for targeted molecular drugs, although they are currently insured in patients with disease progression after receiving standard chemotherapy (or in whom disease progression is expected in the near future). Mutational and mismatch repair testing is strongly recommended for patients with resectable CRC, and RAS/BRAF V600E mutation testing is recommended to estimate the risk of recurrence. Mutational and mismatch repair and BRAF testing are also strongly recommended for screening for Lynch syndrome. Circulating tumor DNA-based minimal residual disease (MRD) testing is strongly recommended for estimating the risk of recurrence based on clinical evidence, although MRD testing was not approved in Japan at the time of the publication of this guideline.

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]

Redefining the prognostic significance of RAS and BRAF V600E mutations on disease free survival in patients with colorectal cancer in the era of ct-DNA defined minimal residual disease: Results from the MD Anderson INTERCEPT Program.

196 Background: In patients (pts) with resected colorectal cancer (CRC), circulating tumor DNA (ctDNA) is an emerging tool for the detection of minimal residual disease (MRD) following the completion of curative intent therapy. The presence of MRD is highly prognostic, with MRD+ pts having worse disease-free survival (DFS). Prior to the advent of ctDNA, the modest negative prognostic implications of BRAF and RAS mutations (mt) on DFS were established in some studies. However, it is unclear if these previously noted prognostic effects of RAS/BRAF mts will be maintained with the information provided with ctDNA testing for MRD. Methods: As part of the INTERCEPT program, pts with Stage II-IV CRC treated with curative intent at MD Anderson Cancer Center were evaluated with a tissue-informed ctDNA assay (Signatera). This ctDNA testing was performed along with routine surveillance visits (post-operatively and Q3 months). A cohort of patients from the INTERCEPT program also had correlative analysis performed including whole exome sequencing called by GATK-Mutect2 to assess for BRAFV600E and RAS mt. Results: Within the INTERCEPT program, 265 pts had whole exome sequencing and were included for this analysis, with RAS mutations in 44% (n=116) and BRAF V600E in 3.8% (n=10). After a median follow up of 16-m, the median was DFS 7.93-m (95% CI 5.52 – 10.33-m) in ctDNA+ vs not reached in ctDNA-ve. There was no significant difference between ctDNA+ rates in RAS mt vs RAS wt pts (χ2 p=0.41); 217 pts were ctDNA-ve with a DFS HR=1.81 for RAS mut vs wt (95% CI 0.57-5.73, p=0.30). The 12-m DFS in this cohort was 95.2% in RAS mt vs 97.9% in RAS wt (OR 1.25, 95% CI 0.4-4.4, p=NS). 48 pts were ctDNA+, with no difference in DFS by RAS mt status with HR=1.42 (95% CI 0.76 – 2.64, p = NS), which was unchanged after excluding those with radiographically evident disease at or shortly after the finding of ctDNA+. In a multivariate model, after controlling for ctDNA status, there was no prognostic impact of RAS on DFS in the larger population with HR 1.51 (95% CI 0.88 – 2.60; p = NS). While the number of pts with BRAF V600E mt was low, there was no evidence of a prognostic effect after controlling for ctDNA status. Conclusions: In CRC pts treated with curative intent, positive ctDNA at any point post-operatively or during surveillance is highly predictive of disease recurrence. In these pts, RAS mt status was not associated with DFS. Moreover, RAS mt pts are not more likely to be ctDNA+ve. Thus, our data suggest that the presence of ctDNA defined MRD has greater prognostic value than the presence or absence of RAS or BRAF V600E mt.

A phase II trial of TAS-102 in patients with colorectal cancer with ctDNA-defined minimal residual disease post-adjuvant therapy: Results from the MD Anderson INTERCEPT Program.

136 Background: Circulating tumor DNA (ctDNA) has emerged as a novel marker with high specificity and positive predictive value for identifying radiographically occult microscopic or minimal residual disease (MRD) in colorectal cancer (CRC) patients (pts). An additional widely acknowledged unmet need is the development of therapeutic strategies to eradicate MRD and increase the cure rate of the disease. Ongoing trials are evaluating MRD as a biomarker for determining the intensity of post-operative adjuvant therapy. However, the feasibility and efficacy of therapy in pts who are MRD+ during surveillance (after all curative intent treatments including adjuvant chemotherapy) is unknown. Methods: In this proof-of-concept phase II study, pts with stages II-IV CRC noted to be MRD+ during surveillance (ctDNA+ but no radiographic evidence of disease on imaging in last 30 days) received 6 months (m) of TAS-102. Pts were identified through the MD Anderson INTERCEPT program that integrates MRD testing with a tissue informed assay (Signatera) into standard of care for CRC pts. The primary endpoint was 6-m ctDNA clearance and secondary objectives included 3-m ctDNA clearance, and disease-free survival (DFS). Assuming H0 = 5% spontaneous, HA = 30% ctDNA clearance with TAS 102 &amp; 1-sided α = 0.05, 15 patients are required for β = 85%. Results: Between 7/22/22 and 9/7/23,15 pts with MRD were enrolled. Baseline characteristics included a median age of 62 years (range: 37-73), Caucasian (77%), male (77%), resected Stage IV (92%) and median ctDNA level of 1.55 MTM/ml (range 0.04-28.72). At data cutoff, 11 pts had 3-m ctDNA levels and 10 had a decrease in their ctDNA level (91%) including 5 with undetectable levels (45%). At 6-m, 60% of patients had ctDNA levels at or below prior levels; three maintained undetectable ctDNA at 6-m: of those, two pts had subsequent radiographic progression following therapy completion. Median DFS was 9.4-m in the full cohort. Conclusions: We demonstrate the feasibility of identifying and enrolling MRD+ pts through screening during surveillance onto therapeutic trials and show the ability of an experimental therapy to convert patients to MRD negative status, a necessary but not sufficient step for disease eradication. Preliminary results suggest that treatment with TAS-102 is effective in inducing ctDNA clearance in the short term in most pts, but with no evidence of persistent clearance. Updated results including 6-m ctDNA levels and survival data will be presented. Clinical trial information: NCT05343013 .

Clinical application of tumour-in-normal contamination assessment from whole genome sequencing

The unexpected contamination of normal samples with tumour cells reduces variant detection sensitivity, compromising downstream analyses in canonical tumour-normal analyses. Leveraging whole-genome sequencing data available at Genomics England, we develop a tool for normal sample contamination assessment, which we validate in silico and against minimal residual disease testing. From a systematic review of 771\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$771$$\\end{document} patients with haematological malignancies and sarcomas, we find contamination across a range of cancer clinical indications and DNA sources, with highest prevalence in saliva samples from acute myeloid leukaemia patients, and sorted CD3+ T-cells from myeloproliferative neoplasms. Further exploration reveals 108 hotspot mutations in genes associated with haematological cancers at risk of being subtracted by standard variant calling pipelines. Our work highlights the importance of contamination assessment for accurate somatic variants detection in research and clinical settings, especially with large-scale sequencing projects being utilised to deliver accurate data from which to make clinical decisions for patient care.

Tumor-Informed Circulating Tumor DNA for Minimal Residual Disease Detection in the Management of Colorectal Cancer.

Recurrence after curative-intent treatment occurs in 20%-50% of patients with stage II-IV colorectal cancer (CRC), underscoring the need for early detection of minimal residual disease (MRD) using circulating tumor DNA (ctDNA). Here, we examined the pattern of use of a tumor-informed ctDNA assay in CRC MRD monitoring in routine clinical practice at Mayo Clinic, Rochester. We conducted a retrospective analysis of health records of patients with CRC who had at least one tumor-informed ctDNA assay from May 2019 through July 1, 2022. Recurrence was defined as radiographic evidence of disease. Descriptive characteristics of the cohort, ctDNA results, and subsequent interventions were recorded. Of the 120 patients included, the median age at diagnosis was 67 years, 46% were female, and 94% were White. At diagnosis, 10 patients had stage I, 23 stage II, 60 stage III, and 25 stage IV disease. Of 476 ctDNA assays performed, 70% were performed in patients who had recurrent disease most commonly to monitor the effectiveness of therapeutic interventions and 16% resulted in a change in clinical decision making. There were 110 recurrences identified in 62 patients, as some patients experienced more than one recurrence over time. Compared with serum carcinoembryonic antigen levels, ctDNA results correlated better with radiologic imaging. Routine ctDNA monitoring for MRD detection has been adopted in clinical practice; however, 84% of ctDNA assays performed did not result in a change in clinical management. This suggests the need for further clinical research data to guide routine clinical use of ctDNA MRD testing in CRC.

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.

Minimal Residual Disease (MRD) Testing By Next Generation Sequencing (NGS) after Two Cycles (CY) of Non-Intensive Chemoimmunotherapy Is Predictive of Remission Duration and Need for Maintenance Therapy (MT) in Previously Untreated Mantle Cell Lymphoma (MCL): A Wisconsin Oncology Network Study

Introduction: Despite the variable course of MCL, limited prospective markers exist to identify more indolent disease. MCL is often diagnosed in older patients (pts) less able to tolerate intensive approaches. Bendamustine/rituximab (BR) is a standard non-intensive regimen for MCL, and use of MT remains controversial. Emerging data in MCL suggest a correlation between achieving MRD negativity (MRD-) and progression-free survival (PFS). The GALLIUM study (Marcus R, et al, N Engl J Med 2017) previously showed improved PFS with a bendamustine/obinutuzumab (BO) induction compared with BR in follicular lymphoma, and we postulated this benefit may be observed in MCL. Using MRD testing, we predicted that MT may be omitted in pts achieving MRD- status after induction BO chemoimmunotherapy for MCL. Methods: Adult pts ≥18 years (yrs) with previously untreated MCL who were ineligible for more intensive therapies (including transplant) were enrolled. Pts received 4-6 CY of BO induction intravenously (IV) every 28 days (D) with B 90 mg/m2 IV D1 &amp; 2 every 28D for 6 CY concurrent with O IV (cycle 1 = 100 mg D1, 900 mg D2, and 1000 mg D8 &amp; 15; then 1000 mg D1, CY 2-6). Peripheral blood (PB) MRD was obtained using NGS (ClonoSEQ®) after cycle 2 BO as an exploratory endpoint. Consolidation O (CO) 1000 mg IV weekly for 4 doses was initiated ≤12 weeks after the final dose of BO. Restaging and MRD testing of PB and bone marrow aspirate (BMA) was done ≤30 days after CO completion. For pts with complete response (CR) radiographically and MRD- in PB/BMA, maintenance O (MO) was omitted. For pts without CR or persistent MRD positivity (MRD+) in the PB or BMA, MO was given with 1000 mg IV D1 of a 56-day cycle for 8 CY. Pts were followed for ≥2 years from therapy completion. The primary endpoint is PFS from D1 of BO induction. Secondary endpoints are MRD status, concordance rate of PB/BMA in predicting MRD status, response rates and overall survival (OS). Results: Twenty-one pts (of planned 32) enrolled beginning 2/2018, with follow-up through 6/1/2023. The study closed prematurely due to slow enrollment. Median age is 70 yrs with 3 pts &amp;gt;80 yrs; majority were men (76%). Median MIPI score is 6.11 and median ECOG performance status 0. Twenty pts (95%) have stage IV disease. Twenty pts completed 4 (n=1) or 6 CY BO. Ten pts received MO; 10 did not receive MO per protocol due to radiographic CR and MRD- PB/BMA post-CO. Six pts did not complete MO for reasons of progressive disease (n=4), infection (n=1) and new carcinoma (n=1). After cycle 2 BO, PB analysis was MRD+ in 7 pts and MRD- in 13. Concordance rates between post-CO MRD testing in PB and BMA was 70% (14/20). Of non-concordant values, all had PB MRD- but BMA MRD+. Best responses in 20 assessable pts are: CR 75% (15/20), partial response 20% (4/20), and stable disease 5% (1/20). Most common grade 3/4 toxicities were: neutropenia (10/21), leukopenia (6/21), infections (5/21) and infusion reactions (2/21). One pt had grade 5 cardiac arrest with sepsis during BO. Most common grade 3 infections were abdominal/colitis (n=3) and pneumonia (n=2). Most common grade 1/2 toxicities were nausea (14/21), diarrhea (10/21), constipation (8/21), dysgeusia (6/21), fatigue (8/21) and dry skin/rash (12/21). After a median duration of follow-up of 43.9 months (mos) (95% confidence interval [CI] 28.3-60.1), PFS at 2-yrs and 3-yrs is 66.0% (95% CI 41.6-82.2) and 58.7% (95% CI 33.2-77.3), respectively; and median PFS is 46.5 mos (95% CI 16.4-∞). MRD- status after the exploratory endpoint post-C2 BO was predictive of 2-year PFS (Figure 1a), 83.3% (95% CI 48.2-95.6) for MRD- vs. 42.9% (95% CI 9.78-73.4) for MRD+, with hazard ratio [HR] 0.34 (95% CI 0.09-1.39), p value 0.133. Evaluation of PFS by MRD status post-CO showed similar outcomes regardless of receiving MO (Figure 1b): 2-year PFS is 77.8% (95% CI 36.5-93.9) in MRD- pts post-CO vs 60.0% (95% CI 25.3-82.7) in MRD+ pts; HR 0.45 (95% CI 0.10-1.91, p value 0.278. Median OS is 46.5 mos (95% CI 35.1-∞). Conclusion: Omission of MO in pts achieving MRD- status after induction/CO did not result in worsening PFS. Observed PFS and toxicities with BO induction and CO appears comparable with other data sets utilizing a BR-based induction +/- MT in older, less fit MCL pts. Correlation between PFS and PB MRD testing after C2 induction therapy represents a potential new prognostic marker of MCL behavior, and may be an important early disease indicator in development of risk-adapted therapies.

Real-World Patterns of MRD Testing and Impact on Treatment for Chronic Lymphocytic Leukemia Patients

INTRODUCTION Therapeutic options for Chronic Lymphocytic Leukemia (CLL) have advanced significantly over time, resulting in a heterogeneous treatment landscape. Insights into the depth of treatment response can be gained through highly-sensitive detection of residual cancer cells using measurable (also known as minimal) residual disease (MRD) testing. Initially, MRD testing was limited to prognostic use within clinical trials. However, in recent years it has expanded into various clinical settings, although the extent of real-world use is not well established. METHODS 766 individuals with CLL enrolled in the Leukemia and Lymphoma Society (LLS) registry had their medical records collected and data extracted via the Invitae Ciitizen® platform, a patient-centric platform that leverages HIPAA right-of-access to organize clinical data into a standardized, research-ready format, using a combination of machine learning and human clinician review. Proprietary logic-based algorithms were used to characterize lines of therapy (LOTs), MRD testing methods, outcomes and duration of therapy. Descriptive findings are reported below. RESULTS Of the 766 CLL participants, 117 (15%) received a total of 311 MRD tests (mean=3 tests/person, median=2, max=16). 67 individuals had one or more MRD negative results (134 negative results total) and 78 individuals had one or more MRD positive results (177 positive results total). 56/117 (47.9%) patients had one or more MRD tests as part of a clinical trial, for a total of 132/311 (42.2%) MRD tests performed during a trial. Total MRD tests included 260 (83.6%) evaluated by flow cytometry, 8 (2.6%) by next generation sequencing (NGS), with the remainder not having documented methods (43; 13.8%). Since 2014, when MRD testing was integrated as a prognostic indicator in clinical trials, there has been increasing use of MRD testing ( Figure 1) within this cohort, as assessed by percentage of individuals receiving one or more tests per year. Interestingly, there is a peak of percentage tested in a clinical trial setting in 2017, after which a continued increase in those tested outside of a clinical trial setting shifts the overall proportion down. We also note the appearance of NGS testing in 2018. The 117 individuals who were MRD tested received 40 unique LOTs (e.g., “obinutuzumab, venetoclax”). We then quantified the MRD outcomes as either “achieved MRD-negative” or “did not achieve MRD-negative” for the most commonly-used therapies (n≥5) ( Table 1). Ibrutinib and acalabrutinib monotherapies had the lowest proportion of MRD-negative outcomes (1/15 and 0/6, respectively), whereas obinutuzumab-venetoclax +/- ibrutinib combination therapies had the highest proportion of MRD-negative outcomes (4/5 and 17/17, respectively). In order to assess the impact of MRD outcome on treatment patterns, the duration of therapy following a MRD-positive and negative test were evaluated. Therapy continued a mean duration of 691 days (n=126, median=488) after a MRD-positive test and 419 days (n=66, median=230) after a MRD negative test. CONCLUSION In this real-world dataset of 766 individuals with CLL, we detect an increasing rate of MRD testing since 2014. Notably, this usage is not limited to the clinical trial setting, underscoring the growing significance of MRD testing as a more general tool for evaluating disease control. Exploration of which LOTs achieve proportionally more MRD-negative statuses highlights that venetoclax-containing fixed-duration regimens as well as venetoclax monotherapy have higher rates of MRD-negativity. Conversely, BTK monotherapies ibrutinib and acalabrutinib were associated with the lowest percentage of MRD-negative outcomes. Finally, we find that the duration of therapy from the time of MRD testing is shorter when the result is MRD-negative, suggesting that MRD testing as a component of outcomes assessment used in the real-world setting may be useful in deciding when to discontinue therapy. This aligns with ongoing clinical trials investigating fixed duration therapies and the use of MRD-negative status as an outcome measure to assess depth of remission. Future studies on larger datasets will explore how MRD testing is integrated into clinical practice and its impact on treatment decisions.

MRD Assessment in Patients with Newly Diagnosed Multiple Myeloma Using Tokenized Real World Data Sources

Introduction The depth of response in multiple myeloma (MM) after therapy is closely related to the prognosis of the disease. Robust data supports minimal residual disease (MRD) as prognostic after induction, auto-SCT, consolidation; during maintenance therapy; and in the R/R treatment setting. MRD is also prognostic despite unfavorable cytogenetics or transplant eligibility. Accordingly, clinical practice guidelines recommend the assessment of MRD after each phase of treatment, and MRD negativity is increasingly considered an important treatment goal. However, understanding how MRD is currently utilized in routine clinical practice across institutional and community settings is challenging. We have adopted an existing claims-based algorithm and linked MRD test results to explore assessment patterns and attainment of MRD negativity across lines of treatment and patient transplant status in the real-world setting. Methods The study uses Adaptive's internal MRD data linked with HealthVerity closed payer claims data through privacy-preserving record linkage (PPRL) tokenization. PPRL allows for private patient records across different data sources to be linked and shared without transmitting protected health information (PHI). The data source used is Source 20 from the Healthverity Marketplace, representing the largest available source of closed medical and pharmacy claims from health insurers. The data covers 120 million total individuals from over 120 unique payers including commercial, Medicare, and Medicaid, providing a comprehensive treatment journey of the patients. Newly diagnosed MM patients (NDMM) were identified as adults with a diagnosis of MM between January 2015 and December 2022 (the first observed MM diagnosis as Index Diagnosis), with continuous Medical and Pharmacy coverage at least 6 months during pre-index and 12 months during post-index periods. Patients were further required to have initiated MM treatment within 60 days of Index Diagnosis, had at least one MRD assessment using NGS MRD (clonoSEQ® Assay - Adaptive Biotechnologies), and no enrollment in clinical trials nor prior MM treatment pre-index. Lines of therapy were identified following an established algorithm (Fonseca, 2020). We describe patient characteristics, treatment journey, and assessment of MRD test results at a sensitivity of 10 -5 in accordance with IMWG guidance. Results The study identified 244 NDMM patients meeting the above criteria, of which 79 received transplant. Median age was 63 for transplant patients and 65 for non-transplant patients, 60% of all patients were male, and 80% were diagnosed before 2021. The percentage moving to next treatment was greater for transplant patients compared to non-transplant patients (2L: 46%, 3+ line: 19% vs. 2L: 35%, 3+ line: 12%). In both cohorts, front-line therapy usually consisted of an IMiD + dex doublet. For transplant patients, 62% and 46% achieved MRD- at some point during treatment at 10 -5 and 10 -6 thresholds, respectively. Similar MRD- attainment was seen in the non-transplant group (70% and 50%). A total of 562 MRD assessments were performed for these cohorts with a mean of ~2 per patient. The number of patients with &amp;gt;=2 MRD was 50 (63.3%) for transplant, and 91 (55.2%) for non-transplant. Among transplant patients, 88.8% of the MRD tests were performed post-transplant. Among non-transplant patients, 92.9% of the MRD tests were performed on 2L or later. Conclusion This analysis allows us to understand attainment of MRD negativity by transplant status and line of treatment in the real-world setting. Additional data sources are required to confirm outcomes. This study represents the first effort to enrich MRD test results with external claims data sources though tokenized linkage and provides additional patient treatment journey information as identified by the claims data.

Recommendations on Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Treated with Combination of All- Trans Retinoic Acid and Arsenic Trioxide

Background All- trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination therapy significantly reduces acute promyelocytic leukemia (APL) relapse compared to ATRA combined with chemotherapy, leading to a cure rate of 80-90% in APL patients. The ideal frequency and duration of minimal residual disease (MRD) monitoring needs to be identified in the era of ATO. Methods A total of 1072 APL patients were enrolled in this study, comprising 855 from the APL2012 trial (NCT01987297) and 217 from our center between January 2018 to June 2022 (APL2018 study). The APL2018 protocol was adjusted for consolidation and maintenance therapy based on APL2012 (Chen et. al. PNAS 2021): In consolidation therapy, low-, intermediate-, and high-risk patients were performed following the ATO-group scheme of corresponding risk stratification in the APL2012 study. From December 2019, chemotherapy was no longer administered to high-risk group during consolidation phase. In maintenance treatment, ATO was replaced with oral arsenic realgar-indigo naturalis formula, and methotrexate was removed. RT-PCR or RQ-PCR was routinely used for MRD monitoring, detecting PML::RARA fusion genes in bone marrow before each consolidation and maintenance therapy. MRD testing was conducted every 3 months within the first year, every 6 months within the second year, and annually from 3 to 5 years after treatment. Results A total of 993 patients achieved complete remission (CR) after induction therapy. The negative MRD rates were observed in 61.7% of patients before 1 st consolidation , 95.4% before 2 nd consolidation, 99.3% before 3 rd consolidation, and 99.5% before maintenance therapy , respectively. The median time to achieve negative MRD was 57 (22-301) days with no significant difference between relapsed and non-relapsed patients (p=0.166). A total of 34 patients (30 in the APL2012 trial and 4 in the APL2018 study) relapsed, including 5 at low risk, 9 at intermediate risk, and 20 at high risk. The median recurrence time was 13.5 (3.5-49.0) months after achieving CR, with 94% (32/34) relapsing within 3 years of achieving CR. After induction remission, the risk of relapse decreased with time (44% occurred within the first year, 29% within the second year, 21% within the third year, and only 6% after three years, Figure 1). The median follow-up for the 179 evaluable patients after achieving CR was 39.7 (3.5 -64.6) months in the APL2018 group. The estimated 5-year disease-free survival (DFS) rates were 95.8% (95% CI 92.8-98.9). Prior to the 1 st, 2 nd, and 3 rd consolidation treatment, and before maintenance therapy, the estimated 5-year DFS rates for MRD positive and negative patients were 92.4% (95% CI 86.1-99.2) versus 97.8% (95% CI 94.8-100), p=0.100; 57.1% (95% CI 30.1-100) versus 97.3% (95% CI 94.7-100), p&amp;lt;0.001; 0 versus 96.5% (95% CI 93.1-100), p&amp;lt;0.001; 0 versus 96.8% (95% CI 94.0-99.6), p&amp;lt;0.001, respectively. The results of the APL2012 study were similar to those of APL2018. In the APL2018 study, the estimated 5-year cumulative incidence of relapse (CIR) was 2.51% (95% CI 2.48-2.54), which was 0%, 1.16% (95% CI 1.13-1.19), and 7.83% (95% CI 7.44-8.22) in the low-risk, intermediate-risk, and high-risk patients, respectively (p=0.057). The estimated 5-year CIR for the low to intermediate risk group was 0.74% (95% CI 0.73-0.75), which was statistically different from high-risk patients (p=0.019). The estimated 5-year CIR for MRD positive and negative patients prior to the 1 st and 2 nd consolidation treatments was 4.92% (95% CI 4.76-5.08) versus 1.10% (95% CI 1.08-1.12), p=0.194; 14.29% (95% CI 10.30-18.28) versus 2.08% (95% CI 2.05-2.11), p=0.010, respectively. Conclusion Our results show that MRD after one course of consolidation treatment, rather than before, is a crucial predictive factor for APL relapse. Patients with positive MRD after 1 st consolidation therapy have a high risk of relapse. ATRA-ATO based treatment has significantly reduced APL relapse, so the value of frequent MRD monitoring may be limited. It is recommended to implement an optimized MRD monitoring for risk stratification. For low to intermediate-risk patients and those with negative MRD after a consolidation treatment, the frequency and duration of MRD testing can be minimized. For high-risk patients with sustained MRD negativity for more than 3 years, MRD testing can be considered for discontinuation.