Measurable Residual Disease Detection Research Articles

FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia

FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Gilteritinib as Post-Transplant Maintenance for AML With Internal Tandem Duplication Mutation of FLT3.

Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 (FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.

Measurable Residual Disease Detection in Acute Myeloid Leukemia: Current Challenges and Future Directions.

Acute myeloid leukemia (AML) is an aggressive malignant disease with a high relapse rate due to the persistence of chemoresistant cells. To some extent, these residual cells can be traced by sensitive flow cytometry and molecular methods resulting in the establishment of measurable residual disease (MRD). The detection of MRD after therapy represents a significant prognostic factor for predicting patients' individual risk of relapse. However, due to the heterogeneity of the disease, a single sensitive method for MRD detection applicable to all AML patients is lacking. This review will highlight the advantages and limitations of the currently available detection methods-PCR, multiparameter flow cytometry, and next generation sequencing-and will discuss emerging clinical implications of MRD test results in tailoring treatment of AML patients.

Phenotypic Analysis of Hematopoietic Stem and Progenitor Cell Populations in Acute Myeloid Leukemia Based on Spectral Flow Cytometry, a 20-Color Panel, and Unsupervised Learning Algorithms.

The analysis of hematopoietic stem and progenitor cell populations (HSPCs) is fundamental in the understanding of normal hematopoiesis as well as in the management of malignant diseases, such as leukemias, and in their diagnosis and follow-up, particularly the measurement of treatment efficiency with the detection of measurable residual disease (MRD). In this study, I designed a 20-color flow cytometry panel tailored for the comprehensive analysis of HSPCs using a spectral cytometer. My investigation encompassed the examination of forty-six samples derived from both normal human bone marrows (BMs) and patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) along with those subjected to chemotherapy and BM transplantation. By comparing my findings to those obtained through conventional flow cytometric analyses utilizing multiple tubes, I demonstrate that my innovative 20-color approach enables a more in-depth exploration of HSPC subpopulations and the detection of MRD with at least comparable sensitivity. Furthermore, leveraging advanced analytical tools such as t-SNE and FlowSOM learning algorithms, I conduct extensive cross-sample comparisons with two-dimensional gating approaches. My results underscore the efficacy of these two methods as powerful unsupervised alternatives for manual HSPC subpopulation analysis. I expect that in the future, complex multi-dimensional flow cytometric data analyses, such as those employed in this study, will be increasingly used in hematologic diagnostics.

Measurable residual disease detection in acute lymphoblastic leukaemia – comparison between NGS and ASO-qPCR

Measurable residual disease detection in acute lymphoblastic leukaemia – comparison between NGS and ASO-qPCR

Quantification of Measurable Residual Disease Detection by Next-Generation Sequencing–Based Clonality Testing in B-Cell and Plasma Cell Neoplasms

Next generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in post-treatment settings can be crucial for relapse risk stratification in patients with B-cell and plasma cell neoplasms. Prior studies have focused on validation of various technical aspects of the MRD assays, but more studies are warranted to establish the performance characteristics and enable standardization and broad utilization in routine clinical practice. Here, we describe our approach and clinical experience with an NGS-based IGH MRD quantification assay, incorporating a spike-in calibrator for monitoring B-cell and plasma cell neoplasms based on their unique IGH rearrangement status. Comparison of MRD status (positive or undetectable) by NGS and flow cytometry (FC) assays showed high concordance (91%, 471/519 cases) and overall good linear correlation in MRD quantitation, particularly for chronic lymphocytic leukemia and B-lymphoblastic leukemia/lymphoma (R=0.85). Quantitative correlation was lower for plasma cell neoplasms, where underestimation by FC is a known limitation. No significant effects on sequencing efficiency by the spike-in calibrator were observed, with excellent inter- and intra-assay reproducibility within our laboratory, and in comparison to an external laboratory, using the same assay and protocols. Assays performed both at internal and external laboratories showed highly concordant MRD detection (100%) and quantitation (R=0.97). Overall, this NGS-based MRD assay showed highly reproducible results with quantitation that correlated well with FC MRD assessment, particularly for B-cell neoplasms.

A 19-color single-tube full spectrum flow cytometry assay for the detection of measurable residual disease in acute myeloid leukemia.

Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a diagnostic sample into several tubes, restricting the number of cells and the complexity of immunophenotypes that can be analyzed. Full spectrum flow cytometers overcome this limitation by enabling the simultaneous use of up to 40 fluorescent markers. Here, we used this approach to develop a good laboratory practice-conform single-tube 19-color MRD detection assay that complies with recommendations of the European LeukemiaNet Flow-MRD Working Party. We based our assay on clinically-validated antibody clones and evaluated its performance on an IVD-certified full spectrum flow cytometer. We measured MRD and normal bone marrow samples and compared the MRD data to a widely used reference MRD-MFC panel generating highly concordant results. Using our newly developed single-tube panel, we established reference values in healthy bone marrow for 28 consensus leukemia-associated immunophenotypes and introduced a semi-automated dimensionality-reduction, clustering and cell type identification approach that aids the unbiased detection of aberrant cells. In summary, we provide a comprehensive full spectrum MRD-MFC workflow with the potential for rapid implementation for routine diagnostics due to reduced cell requirements and ease of data analysis with increased reproducibility in comparison to conventional FlowMRD routines.

Understanding differential technologies for detection of MRD and how to incorporate into clinical practice.

Patient- and leukemia-specific factors assessed at diagnosis classify patients with acute myeloid leukemia (AML) in risk categories that are prognostic for outcome. The induction phase with intensive chemotherapy in fit patients aims to reach a complete remission (CR) of less than 5% blasts in bone marrow by morphology. To deepen and sustain the response, induction is followed by consolidation treatment. This postremission treatment of patients with AML is graduated in intensity based on this favorable, intermediate, or adverse risk group classification as defined in the European Leukemia Network (ELN) 2022 recommendations. The increment of evidence that measurable residual disease (MRD) after induction can be superimposed on risk group at diagnosis is instrumental in tailoring further treatment accordingly. Several techniques are applied to detect MRD such as multiparameter flow cytometry (MFC), quantitative (digital) polymerase chain reaction (PCR), and next-generation sequencing. The clinical implementation of MRD and the technique used differ among institutes, leading to the accumulation of a wide range of data, and therefore harmonization is warranted. Currently, evidence for MRD guidance is limited to the time point after induction using MFC or quantitative PCR for NPM1 and core binding factor abnormalities in intermediate-risk patients. The role of MRD in targeted or nonintensive therapies needs to be clarified, although some data show improved survival in patients achieving CR-MRD negativity. Potential application of MRD for selection of conditioning before stem cell transplantation, monitoring after consolidation, and use as an intermediate end point in clinical trials need further evaluation.

Prognostic Significance of Measurable Residual Disease Detection by Flow Cytometry in Autologous Stem Cell Apheresis Products in AML.

Prognostic Significance of Measurable Residual Disease Detection by Flow Cytometry in Autologous Stem Cell Apheresis Products in AML.

A Novel and Cost-Efficient Measurable Residual Disease (MRD) Detection Assay Using a Single Molecule Molecular Inversion Probe-Based Next Generation Sequencing (NGS) Assay Predicts Overall Survival in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction Relapse remains the main reason for poor outcomes of patients (pts) with acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the preferred post-remission treatment in pts at high risk of relapse. The presence of measurable residual disease (MRD) after intensive frontline treatment or before or after HSCT has emerged as an important predictor of relapse. Various methods are used for MRD detection, including flow cytometry and molecular assays detecting individual gene mutations (mut) such as NPM1mut. Next generation sequencing (NGS)-based assays covering multiple genes commonly mutated in AML may allow sensitive MRD detection in a broad range of pts with diverse genetic characteristics. Methods We established a targeted, error corrected NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs). The smMIP protocol uses a hybridization-capture approach to selectively enrich and amplify both DNA strands of 92 regions in 32 genes in a single reaction. Unique molecular identifiers (UMIs) facilitate computational correction of sequencing errors to enable reliable detection of variants ≥0.5% variant allele frequency (VAF), with the additional advantage of relatively low reagent and sequencing costs of &amp;lt; $100 per sample. The smMIP assay was used to evaluate MRD status on pre-HSCT bone marrow samples from 98 AML pts who received an HSCT in first (n=83) or second (n=15) complete remission with or without count recovery. Median age at HSCT was 59 (range 20-75) years, 42% were female; 70% had de novo AML, 19% had an antecedent myeloid neoplasm and 11% had AML post cytotoxic therapy. European LeukemiaNet (ELN)-2022 risk groups at diagnosis were favorable in 11%, intermediate in 33%, adverse in 20% and unknown in 22%. All pts had received intensive induction chemotherapy. Twenty-nine percent received a myeloablative conditioning regimen (MAC), while 23% received reduced intensity conditioning (RIC) and 48% a non-myeloablative (NMA) conditioning regimen. Donors were matched related (18%), haploidentical (6%), or matched (55%) or mismatched (21%) unrelated. The median follow-up for pts alive was 34.8 months. Results Overall, we detected 190 variants in 71 (72%) of 98 pre-HSCT remission samples. The most commonly affected genes were DNMT3A (in 37% of pts), TET2 (21%), PPM1D (12%), IDH2 (10%) and TP53 (5%). Twenty-nine percent of pts had only variants affecting the clonal hematopoiesis-related genes DNMT3A, TET2 or ASXL1 (‘DTA‘), while 44% of patients had ≥1 variant involving a non-DTA gene. MRD-positive pts, defined as those with ≥1 non-DTA variant, had similar baseline and HSCT-related characteristics (includingage, sex, ELN risk group, de novo vs. secondary AML) compared to pts without non-DTA variants. Overall survival (OS) was significantly inferior in NGS MRD-positive pts compared to those with no non-DTA variant (Figure A, 5-year OS, 49% vs. 85%, P=.011). MRD-positive pts also had a non-significant higher cumulative incidence of relapse at 5 years (46% vs. 24%, P=.14), while non-relapse mortality was similar in both groups (P=.72). We next performed multivariable analyses, considering variables associated with OS at an univariable P&amp;lt;.10 (MRD status, patient sex, therapy-related AML, conditioning intensity), and using stepwise backward variable selection to identify factors significantly associated with OS (Figure B). In the final multivariable model, pre-HSCT MRD detection remained associated with inferior OS (hazard ratio 2.51; 95% confidence interval, 1.07 - 5.91; P=.035). Female sex associated with favorable OS, while myeloablative conditioning (MAC) showed a borderline significant association with superior OS. Of note, we did not detect an interaction between conditioning intensity and detection of MRD with regard to OS, i.e. the prognostic association of MRD detection was not different between pts receiving myeloablative or less-intensive conditioning. However, this analysis was limited by the small patient number. Conclusion Detection of MRD by targeted NGS using the smMIPs approach provides prognostic information in pts undergoing allogeneic HSCT, including those without established molecular markers such as NPM1mut. This method is applicable to a large proportion of AML pts and is cost-efficient for broad clinical use. S.M.K. and T.H., and M.J. and K.H.M. contributed equally to this abstract.

Pre-Transplant Measurable Residual Disease (MRD) Detection of KMT2A-rearranged Acute Myeloid Leukemia Is Strongly Associated with Inferior Post-Transplant Outcome

Introduction KMT2A-rearranged ( KMT2Ar ) acute myeloid leukemia (AML) is associated with inferior survival and allogeneic hematopoietic cell transplant (HCT) in first remission (CR1) is an important post-remission objective (Issa et al, Blood Cancer Journal 2021). Despite HCT, however, the relapse risk is ~50% (Menghrajani et al, Blood Adv. 2022). Several small cohort studies have described the prognostic utility of pre-HCT MRD assessment using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR), but currently, this is not standard practice (Scholl et al, Haematologica 2005; Huang et al, Med Sci Monitor 2016; Liu et al, Biol Blood Marrow Transplant 2014; Heuser et al, Blood 2021). A significant barrier to widespread implementation of KMT2A::X MRD monitoring is the presence of diverse fusion-partner genes and varied breakpoint regions. The potential to detect and pre-emptively target pre-HCT KMT2A::X MRD has relevance with the development of menin inhibitors, which have been shown to be active in the clinic (Issa et al, Nature 2023). In the current work, we report the prognostic impact of pre-HCT KMT2A::X MRD detection by RT-qPCR and the relationship with post-HCT outcome in adults with KMT2Ar AML. We also describe a novel 3 common primer/probe KMT2A::X reverse transcription digital PCR (RT-dPCR) MRD assay as a simplified dPCR alternative to MRD monitoring. Methods Archived pre-HCT cDNA were available from 54 patients treated in the UK (including UK NCRI AML17 and AML19) and Australia. Samples were analyzed by RT-qPCR using previously described methods in 45 patients (Abildgaard et al, Eur J Haematol. 2013), and RT-dPCR in 9 patients (Ivey, unpublished 2023). The RT-dPCR method, which uses a common 3 primer/probe set to capture breakpoints within KMT2A obviating the need for fusion-gene specific plasmid standards, showed high concordance with RT-qPCR across 20 samples (R 2=0.9915) . Copy numbers were expressed per 100 ABL copies. Assay sensitivity was ~0.001%. Survival outcomes were estimated using the Kaplan-Meier method for relapse-free survival (RFS, date of HCT to relapse or death from any cause) and overall survival (OS, date of HCT to death or last follow-up). Cox proportional hazards was used to assess variables impacting RFS and OS. Categorical variables were calculated using Fisher exact test and continuous variables using Mann-Whitney-U test. The study was approved by human research ethics committees (AUS 21/26; UK 14/WA/1056, NC10.13). Results A total of 54 patients with KMT2Ar AML underwent HCT between 2010-2022. Median age was 43 years (range, 17-70), 63% were female, 94% had de novo AML, all had received prior intensive chemotherapy and 76% were transplanted in CR1, 50% with myeloablative conditioning. KMT2A::X subtypes included t(9;11)/ KMT2A::MLLT3 in 35%, t(6;11)/ KMT2A::AFDN in 26%, t(11;19)/ KMT2A::ELL in 13% and KMT2A::MLLT1 in 5%, t(10;11)/ KMT2A::MLLT10 in 17% and other rare KMT2A::X variants in 4%. Pre-HCT MRD was detected (MRD-pos) in 46% of patients (25 of 54) at a median level of 0.3 copies/100ABL (range, 0.006-35.0394). The frequency of post-HCT relapse was significantly higher in patients with RT-qPCR/RT-dPCR levels ≥0.01% pre-HCT (68% relapsed vs 24% if MRD-neg, p=0.002) (Figure 1). Median time to relapse was 4.7 months (range, 0.8-22.5), with 100% of patients (22 of 22 with available molecular status at relapse) retaining the KMT2A::X variant at relapse. With median follow-up time of 37 months, pre-HCT KMT2A::X MRD-pos was associated with significantly inferior 2-year RFS (19% vs. 63%, p=0.0013) (Figure 2) and 2-year OS (47% vs. 69%, p=0.031). On univariate analysis, variables associated with inferior RFS were pre-HCT MRD-pos status (hazard ratio (HR) 3.2 [95% confidence interval (CI) 1.5-6.9], p=0.002) and transplant in second remission vs. CR1 (HR 2.6 [95% CI 1.2-5.5], p=0.01), however, pre-HCT MRD-pos status was the only variable associated with inferior RFS on multivariate analysis (HR 2.8 [95% CI 1.3-6.1], p=0.01). Conclusions Pre-HCT KMT2A::X MRD detection by RT-qPCR and RT-dPCR is a robust prognostic indicator of post-transplant relapse and inferior survival outcome. Targeting patients with KMT2A::X MRD positive disease pre-HCT using novel menin inhibitors is currently the subject of an enrolling clinical trial (AMLM26 INTERCEPT) (ACTRN12621000439842).

Next-Generation Sequencing (NGS) for Residual Disease (MRD) Monitoring in T-Lymphoblastic Leukemia (T-ALL)

Background: Flow Cytometry is the current standard for detection of measurable residual disease (MRD) in T-lymphoblastic leukemia (T-ALL) for North America but relies on subjective interpretation of data by a highly trained interpreter, which raises concerns for reproducibility between laboratories. Nevertheless, MRD &amp;gt; 0.01% at End of Induction (EOI) and End of Consolidation (EOC) have been associated with poorer outcome in T-ALL. MRD detection by next-generation sequencing (NGS) offers the potential for reduced subjectivity and increased sensitivity, as has already been demonstrated in B-lymphoblastic leukemia. This study assesses the performance characteristics and potential contribution of NGS for the detection of T-ALL MRD in pediatric patients. Method: Residual patient samples from COG AALL1231 concurrently tested for T-ALL MRD by flow cytometry were tested for the presence of leukemia-associated clonal TCRB sequences at the University of Washington using the immunoSEQ kit from Adaptive Biotechnologies. The sensitivity of the assay for these samples is estimated at 0.002%. The samples included 615 pretreatment samples, 298 EOI samples, and 72 EOC samples. The EOI samples were negative for MRD by flow cytometry (MRD&amp;lt;0.01%). The EOC samples were from patients positive for MRD at EOI, as per the treatment protocol. 111 samples lacking a pretreatment clonal TCRB sequence for which residual material was available were submitted to Adaptive Biotechnologies for TCRG sequencing. ETP status was determined on pre-treatment samples by flow cytometry. A pretreatment clonal sequence representing &amp;gt; 19% of the total T cell sequences was considered leukemia-associated and the presence any leukemia-associated clonal sequence post-treatment was considered MRD positive. The MRD results were correlated with clinical outcome. Results: Clonal TCRB leukemia-associated sequences were identified in 68.5% of pretreatment samples and correlated with ETP status (ETP 3.6% (N=56) , Near-ETP 44.8% (N=96), Not-ETP 82.4% (N=449), unknown 50% (N=14)). Clonal TCRG leukemia-associated sequences were identified in an additional 44.1% of pretreatment samples lacking a clonal TCRB sequence (N=111). EOI MRD was identified in 71.0% of samples assayed for TCRB (ETP not evaluable (N=0), Near-ETP 76.2% (N=21), Not-ETP 70.3% (N=263), Unknown 77.8% (N=9)). Additional EOI samples not informative for TCRB and assayed for TCRG (N=29) identified 89.7% as MRD positive. EOC MRD was identified in 63.9% of samples assayed for TCRB (ETP 66.7% (N=3), Near-ETP 45.5% (N=11), Not-ETP 68.4% (N=57), Unknown 0% (N=1)). Additional EOC samples not informative for TCRB and assayed for TCRG (N=35) identified as 68.6% MRD positive. Correlation of MRD status with clinical outcome data revealed no difference in disease-free survival (DFS) at either EOI or EOC, although all relapses but one at each timepoint (N=15 EOI, N=15 EOC) occurred in the MRD positive groups. Conclusion: A significant subset of T-ALL samples do not contain clonal leukemia-associated TCRB or TCRG sequences (31.5% for TCRB, 17.6% using both TCRB or TCRG) and cannot be evaluated for MRD using this NGS approach. Samples lacking a clonal leukemia-associated sequence are associated with an immature immunophenotype (ETP or Near-ETP), although 17.6% of Not-ETP samples also lack a clonal sequence, suggesting that flow cytometric classification of maturational stage is likely imperfect. A high frequency of MRD positivity is present at both EOI and EOC that does not reflect the excellent outcome that most patients experience, thus raising concerns about the specificity of these clonal sequences at very low levels of detection. Evaluation of sequence specificity across the cohort and use of higher MRD thresholds to optimize the correlation with clinical outcome is underway. A negative MRD result by NGS has a high negative predictive value for relapse/death at either EOI (95.2%) or EOC (92.9%) but a poor positive predictive value (6.8% at both EOI and EOC).

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

The Prognostic Impact of Measurable Residual Disease Dynamics in Mantle Cell Lymphoma

Introduction Measurable residual disease (MRD) technologies have greatly enhanced our ability to guide treatment duration and predict outcome in various hematologic malignancies. However, drawing definitive conclusions on the benefits of achieving MRD-negative status in patients with mantle cell lymphoma (MCL) has been challenging due to the limited availability of large-scale MRD detection series. To address this, our study aimed to determine the optimal timing for MRD detection and explore the prognostic value of MRD dynamics in MCL. Methods In present study, we included 102 patients diagnosed with advanced MCL demonstrating clonal B cell involvement in bone marrow (BM) detectable by multiparametric flow cytometry (MFC). 60/102 (58.8%) patients received high dose cytarabine-based intensive immunochemotherapy. BM aspirates were collected and MRD assessment was conducted at specific time points, including the initiation of cycle 3, cycle 5, at the end of induction, and every 6 months during maintenance therapy. Results 77/102 (75.4%) patients reached MRD negativity throughout induction treatment. Of those with MRD − vs MRD + status , 23.4% vs 68.0% were aged &amp;gt;65 years respectively, which was likely a consequence of the greater MRD − rates observed in patients who received intensive immunochemotherapy and auto-transplantation. Besides, elevated lactate dehydrogenase (LDH) level, high-risk MIPI, blastoid/pleomorphic pathology type, and non-cytarabine containing regimen were associated with a lower rate of MRD negativity. Follow-up data revealed that MRD-negative patients had significantly better survival (P&amp;lt;0.001). In subgroup analyses, MRD negativity was prognostic in almost all subgroups. MRD detection serves as a valuable complement to the traditional response evaluation system, particularly benefiting patients who achieve partial remission (PR). This indicated the importance of assessing MRD during induction therapy for PR patients. However, for patients who achieve complete remission (CR), the presence or absence of MRD has minimal impact on prognosis (P=0.921 for progression-free survival (PFS), and P=0.399 for overall survival (OS). Then we evaluated the optimum timing of MRD detection and impact of rapidity of MRD negativity. 31 of the 77 patients (40.3%) had a rapid reduction of tumor burden and achieved MRD negativity after two cycles of treatment. 35 patients (45.5%) were MRD negative after four cycles of treatment. In 14.2% of the patients, MRD turned negative only at the evaluation after six cycles of induction therapy or during maintance duration. Comparing MRD status at different time points, the MRD status after four cycles of therapy was the strongest predictor of survival prognosis (HR=5.37, C-index=0.673). However, the speed of reaching MRD negativity was not significantly correlated with survival prognosis. Patients with high-risk features, such as high-risk MIPI, elevated LDH levels and complex karyotype, tend to achieve MRD negativity at a faster speed, often within two treatment cycles. However, faster rate of achieving MRD negativity did not confer any prognostic benefit to these patients. The overall MRD negativity rate was relatively low, and the prognosis for these patients was worse compared to other patients. In the high dose cytarabine-based intensive immunochemotherapy group, patients who achieved a rapid MRD negativity within two cycles of treatment showed a slightly longer PFS compared to those who achieved MRD negativity after 4-6 cycles (P=0.017). However, there was no significant difference in OS between the two groups. Conclusions In summary,the optimal timing for MRD testing is after four treatment cycles, as this time point demonstrated the best predictive ability for survival prognosis. However, the speed of achieving MRD negativity was not found to be linked with survival prognosis. Although certain high-risk features may lead to faster achievement of MRD negativity, this rapidity did not confer any additional prognostic benefit to these patients. Importantly, our results suggest that the value of MRD negativity as a prognostic marker may be limited in patients who have achieved complete remission and those with blastoid/pleomorphic morphologies. These findings underscore the need for tailored approaches to MRD testing and interpretation in MCL, in alignment with individual patient characteristics and treatment responses.

Comparison of Two Therapeutic Approaches (Pediatric vs. Adult) for Treating Acute Lymphoblastic Leukemia. Experience of Two Centers in Mexico City

Background: Chemotherapy is the most frequently used strategy in treating adult acute lymphoblastic leukemia (ALL) in conjunction with monoclonal antibodies and tyrosine kinase inhibitors. One of the primary considerations is the type of treatment based on pediatric inspiration regimens (CALGB10403, BFM, St Jude XIII) or regimens for adult patients (HyperCVAD), with pediatric regimens being those that have improved the prognosis in the young adult population. Along with this scheme, strict monitoring through measurable residual disease (MRD) is the main dynamic prognostic factor since its positivity is associated with a greater risk of relapse. We aimed to compare the effect on the clinical prognosis of two treatment regimens based on a pediatric regimen versus an adult regimen in patients with ALL. Material and methods: A retrospective, observational, descriptive study of 5 years of follow-up was carried out in patients with ALL at the Hospital General de México and the Hospital Regional de Alta Especialidad de Ixtapaluca, classifying the treatment into two types of regimens (adult regimen vs. pediatric inspiration) evaluating the impact on survival as well as the association of different factors both to diagnosis and measurable residual disease (MRD). Results: A total of 450 patients were studied, 50.4% (n=227) were male, and 49.6% (n=223) were female. 21.1% (n=95) received a pediatric-inspired protocol (CALGB-10403), and 78.9% (n=355) received an adult protocol (Hyper-CVAD). 71.8% (n=323) were considered high risk, and 8.4% (n=38) were identified with Philadelphia positive ALL. 94.4% had a B-cell precursor phenotype ALL, and 8.7% had a nervous system infiltration at diagnosis. Overall, 60.4% integrated complete remission (n=272), with 31.3% (n=141) post-induction MRD negative. The percentage of negative MRD was higher in the pediatric protocol (38.5% vs 29.6%, p=0.000). Relapses occurred in 34% (n=153), and 21.3% (n=96) had a relapse to the central nervous system. Survival at 1000 days of follow-up was 32%, with significant differences in favor of the pediatric protocol (Log Rank 0.000) with 42% at 1000 days (Figure 1), the use of MRD as an impact tool with the prognosis (0.000) (Figure 2), the presence of a post-induction MRD+ (0.000) and high risk (Log-Rank 0.004) were the significant variables. Conclusions: The prognosis of a patient with ALL depends on the type of treatment. The prognosis is favorable when treated with a pediatric protocol. The detection of MRD by flow cytometry is the best follow-up tool, and its positivity decreases survival in adults with ALL.

Recent Advancements in Acute Myeloid Leukemia Have Led to Incremental Improvement in Overall Survival

Background: Treatment approaches in acute myeloid leukemia are rapidly evolving due to a dynamic treatment landscape, routine molecular profiling, and the detection of measurable residual disease. Recently, there has been a surge in FDA approvals for novel therapies (e.g., midostaurin, gilteritinib, CPX-351, enasidenib, ivosidenib, and venetoclax), as well as a departure from a “one-size-fits-all” treatment approach. It is unclear, however, what effect these changes have had on overall survival over time. Since 2017, we have witnessed a significant introduction of novel therapeutics. We hypothesized that patients diagnosed after 2017 may have improved survival due to recent therapeutic advances. Methods: We retrospectively analyzed 627 consecutive patients in the Project ERIS database aged &amp;gt; 18 years with AML treated at VCU Massey Comprehensive Cancer Center from 2015 to 2022. We limited the analysis to January 1, 2015 to December 31, 2019 to avoid confounding from the COVID-19 pandemic. We obtained cytogenetic and molecular characteristics, induction regimens, allogeneic stem cell transplant status, and survival data. The median overall survival (mOS) was compared across calendar years using the Kaplan-Meier method. Categorical variables were analyzed using Fisher's exact test. Results: The median age at AML diagnosis was 62.9 years (range, 19.6-93.0), and the median ECOG performance status was 1 (range, 0-4). The median Charlson Comorbidity Index score was 5 (range, 0-14). There were 139 (22.2%) patients with ELN 2022 favorable-risk disease, 199 (31.7%) with intermediate risk, and 260 (41.5%) with adverse risk. The median OS of all patients diagnosed with AML in 2015 was 8.8 months. In 2016, the mOS was 14.5 months. There was a statistically significant improvement in mOS in 2016 compared to 2015 (HR 0.62 [95% CI 0.40-0.95], p = 0.027). The mOS in 2017, 2018, and 2019 was 12.9 months, 11.1 months, and 19.7 months, respectively. A significant improvement in OS was noted between 2015 and 2019 (HR 0.55 [95% CI 0.36-0.85], p = 0.003) in the entire cohort (Figure 1). When stratified by ELN 2022 genetic risk, clinically relevant improvement in OS was noted across all risk categories from 2015 to 2019, which reached statistical significance in the adverse risk group - favorable risk mOS: 16.4 months vs not reached (p = 0.141); intermediate risk: 13 months vs 18.7 months (p = 0.064); adverse risk: 7.2 months vs 13 months (p = 0.02). We observed expected changes in front-line treatment in this timeframe with decreased single-agent hypomethylating agent (HMA) usage in favor of HMA + venetoclax and increased intensive induction therapies outside of 7+3 (Figure 2). Strikingly, increased procession to allogeneic stem cell transplantation (HCT) was noted in the same timeframe (18.9% in 2015 vs 35.1% in 2019, p = 0.049). Conclusions : Our data demonstrate an emerging trend of clinically significant improvement in overall survival in patients with AML. The introduction of a multitude of therapeutic options may be associated with significantly improved survival over time. In addition to the advent of novel therapeutics leading to improved outcomes, the refinement of SCT conditioning regimens has likely led to the improved accessibility of HCT. In summary, we present an encouraging and significant trend of improved survival and access to HCT, particularly for patients with adverse-risk disease.

Measurable Residual IDH2 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) in AML patients during remission is associated with adverse clinical outcomes after allogeneic hematopoietic cell transplant (alloHCT), including recent strong evidence from our group regarding residual NPM1 and/or FLT3-ITD variants (PMID: 36881031). Large studies examining the significance of other potential MRD targets, including isocitrate dehydrogenase isoform 2 ( IDH2), are needed. IDH2, which is mutated in approximately 12% of AML patients, is of particular interest due to the possibility of targeted clinical intervention. Methods: Patients over the age of 18 in first complete remission (CR1) with IDH2 mutated AML undergoing alloHCT transplant at a CIBMTR site between 2013-2019 with remission blood samples within 100 days of alloHCT and available clinical data were included in this study. Residual variants in IDH2, NPM1, and FLT3genes were detected by ultrasensitive error-corrected next-generation sequencing (NGS-MRD) on genomic DNA from pre-conditioning blood and validated by digital droplet PCR (ddPCR). The primary outcomes were overall survival (OS) and cumulative incidence of relapse with non-relapse mortality as a competing risk. The day of transplant was considered as time 0, and the median follow-up time was calculated for censored patients. Kaplan-Meier estimation (log-rank tests), Cox proportional hazards models, and Fine-Gray regression models were fitted to examine the clinical outcomes. Results: Among the 257 IDH2-mutated AML patients in this study, 57 (23%) experienced relapse, with 41 events occurring within 12 months after alloHCT. The median follow-up time was 25 months. Among 247 patients with pre-transplant remission flow cytometry results reported, 25% were positive, but no difference in rates of relapse or OS was observed between positive and negative patients. NGS MRD analysis detected residual IDH2 variants in remission samples of 130 patients (51%) with variant allele fraction (VAF) ranging from 0.05-56% (median: 3%), and 100% validation by ddPCR. NGS MRD IDH2 positive patients had increased rates of relapse (p = 0.03; 3yr: +11%, 95% CI: 0.2% to 22%) and decreased OS (p &amp;lt; 0.001; 3yr: -25%, 95% CI: -13% to -38%) compared to those who tested negative (Figure 1A). These OS differences were still observed between NGS MRD IDH2 positive and negative patients, regardless of age stratification (under 60yr p &amp;lt; 0.001, 3yr: -28%, 95% CI: -12% to -43%; over 60yr p = 0.04, 3yr: -19%, 95% CI: -1% to -40%); however, only patients over 60yrs had increased rates of relapse (p = 0.007; 3yr: +21, 95% CI: 6% to 36%). The impact of co-mutation status was examined by subdividing the cohort based on the presence of NPM1 or FLT3-ITD mutations at baseline. Residual IDH2 variants in patients without baseline NPM1 or FLT3-ITD co-mutations (n = 151) were associated with decreased OS and increased relapse ( IDH2+ vs IDH2-; OS p = 0.005, 3yr: -23%, 95% CI: -6% to -40%; Relapse p = 0.01, 3yr: +19, 95% CI: 6% to 32%; Figure 1B). In contrast, residual IDH2 variants were not predictive of relapse in co-mutated patients (n=106, p = 0.5). In these patients, the only prognostic marker for relapse was NPM1 and/or FLT3-ITD MRD, with similar relapse risk between IDH2 only positive and MRD negative groups (p &amp;lt; 0.001; 3yr: 83% vs. 11% vs. 15%; Figure 1B). In multivariable analysis, IDH2 MRD positivity was associated with decreased OS (HR 2.5, 95% CI: 1.5-4.2, p &amp;lt; 0.001) and increased rates of relapse (HR 2.1, 95% CI: 1.4-5.7, p = 0.003) after adjusting for other characteristics. Conclusion: In this study, we demonstrate that adult patients with IDH2-mutated AML who have IDH2 variants detected in CR1 prior to first alloHCT experienced increased relapse rates and decreased overall survival. However, this association is significant only in patients without baseline NPM1 and FLT3-ITD mutations. In co-mutated patients, NPM1 and/or FLT3-ITD MRD serve as superior prognostic markers. Subgroup analyses show the potential influence of factors such as conditioning intensity and age on clinical outcomes. Nevertheless, larger studies with balanced subgroups and more comprehensive baseline mutational information, including IDH2 mutation subtype, are needed to better define the clinical relevance of IDH2 MRD in this context.

Measurable Residual IDH1 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) prior to allogeneic hematopoietic cell transplant (alloHCT) in patients with acute myeloid leukemia (AML) in remission is associated with increased relapse and inferior survival after transplant. We recently reported that next-generation sequencing (NGS) detection of mutated NPM1 or FLT3-ITD in first complete remission (CR1) blood prior to alloHCT is strongly associated with increased relapse and death (PMID: 36881031). However, the prognostic implications of detecting persistence of other common AML-associated mutations at this treatment landmark remain incompletely defined. Around 20% of patients diagnosed with AML have variants in isocitrate dehydrogenase ( IDH) genes (~7% for IDH1) and clinical outcomes are known to differ due to co-occurrence of other mutations, IDH subtypes, treatment strategies, and patient factors. Studies have examined the relationship between residual IDH1 variants ( IDH1m) in CR and subsequent clinical outcomes, but definitive standardized evidence to inform decision-making for the clinical utility of IDH1m as a target for AML MRD testing was not previously available. Methods: Adult patients undergoing alloHCT for IDH1 mutated AML in CR1 at CIBMTR sites in the USA between 2013-2019 were eligible for the study if remission blood sample collected within 100 days before alloHCT and outcome data were available. Ultrasensitive error-corrected NGS (NGS-MRD) targeting IDH1, NPM1, and FLT3 genes was performed on DNA from pre-conditioning blood to identify residual variants. Detected IDH1m were validated by digital droplet PCR (ddPCR). The day of transplant was considered as day 0. Median follow-up time was calculated for censored patients. Overall survival (OS) was estimated using Kaplan-Meier (log-rank tests) and Cox proportional hazards models (forward selection); cumulative incidence of relapse was examined using Fine-Gray regression models with non-relapse mortality as a competing risk. Results: A total of 148 patients were included in the study, with a median follow-up time of 24 months. Relapse was observed in 37 (25%) patients, with the majority occurring within 1 year after alloHCT (n = 28, 76%). Pre-transplant flow cytometry results were reported by the transplant centers for 97% of patients (n = 144), but no statistically significant differences in clinical outcomes were observed between the positive and negative groups (OS: p = 0.3; relapse: p = 0.07). 53 patients (36%) tested positive for IDH1m persistence in CR1, with a 100% validation rate by ddPCR for variants with an orthogonal assay available. Logistic regression indicated that IDH1m persistence in remission was more likely to occur in patients older than 40 years (OR: 1.02 with every 1-year increase after 40, p = 0.04). Clinical outcomes showed no statistically significant difference between the pre-alloHCT IDH1m positive and negative groups (Figure 1A), and this remained true after further subgroup analysis by age. The variant allele fraction (VAF) of the residual IDH1m ranged from 0.09% to 48.5% (median: 1.5%), and no differences in clinical outcomes were observed when stratifying by high (≥2.5%) or low VAF (&amp;lt;2.5%). Among patients with co-mutated NPM1 and/or FLT3-ITD at baseline (n=69), those with residual mutated NPM1 and/or FLT3-ITD in pre-transplant remission had significantly higher rates of relapse compared to those with residual IDH1m only or NGS-negative groups (p = 0.01, Figure 1B). The cohort excluding patients with co-mutated NPM1 and/or FLT3-ITD did not show any significant clinical outcome differences based on IDH1m persistence. Conditioning intensity did not show an association with clinical outcomes, and multivariable analysis for overall survival and relapse did not identify residual IDH1m as an important marker. Conclusion: Detection of persistent IDH1 variants in the blood of adult patients with AML in CR1 prior to first alloHCT is common, but not associated with increased relapse or death after transplant compared with those testing negative. For those patients with IDH1 mutated AML co-mutated with either NPM1 and/or FLT3-ITD, detection of persistent NPM1 and/or FLT3-ITD was associated with higher rates of relapse. These findings, from the largest study to date, do not support the detection of isolated IDH1 variants in CR1 blood prior to alloHCT as evidence of AML MRD or increased post-transplant relapse risk.