Genomic breakpoint-specific monitoring of measurable residual disease in pediatric non-standard-risk acute myeloid leukemia

Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia : An I-BFM-FLOW-Network Report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.publishersversionPeer reviewe

Expression Patterns of Coagulation Factor XIII Subunit A on Leukemic Lymphoblasts Correlate with Clinical Outcome and Genetic Subtypes in Childhood B-cell Progenitor Acute Lymphoblastic Leukemia

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An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts

UMAP Based Anomaly Detection for Minimal Residual Disease Quantification within Acute Myeloid Leukemia

Leukemia is the most frequent malignancy in children and adolescents, with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as the most common subtypes. Minimal residual disease (MRD) measured by flow cytometry (FCM) has proven to be a strong prognostic factor in ALL as well as in AML. Machine learning techniques have been emerging in the field of automated MRD quantification with the objective of superseding subjective and time-consuming manual analysis of FCM-MRD data. In contrast to ALL, where supervised multi-class classification methods have been successfully deployed for MRD detection, AML poses new challenges: AML is rarer (with fewer available training data) than ALL and much more heterogeneous in its immunophenotypic appearance, where one-class classification (anomaly detection) methods seem more suitable. In this work, a new semi-supervised approach based on the UMAP algorithm for MRD detection utilizing only labels of blast free FCM samples is presented. The method is tested on a newly gathered set of AML FCM samples and results are compared to state-of-the-art methods. We reach a median F1-score of 0.794, while providing a transparent classification pipeline with explainable results that facilitates inter-disciplinary work between medical and technical experts. This work shows that despite several issues yet to overcome, the merits of automated MRD quantification can be fully exploited also in AML

FCM marker importance for MRD assessment in T-Cell Acute Lymphoblastic Leukemia: An AIEOP-BFM-ALL-FLOW Study Group Report

Background: T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15 % of pediatric and about 25 % of adult ALL cases. Minimal/measurable Residual Disease (MRD) assessed by Flow Cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. Methods: We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear Support Vector Machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Results: 91 bone marrow samples of 43 pediatric T-ALL patients from 5 reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of 8 different markers. For all laboratories, CD48 and CD99 were among the top 3 markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and timepoint (diagnosis, day15, day33). Conclusions: Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for minimal residual disease (MRD) monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future. This article is protected by copyright. All rights reserved

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL

Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels