Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics   diagnosis   MeSH
• Gene Rearrangement * MeSH
• Genes, Immunoglobulin MeSH
• Real-Time Polymerase Chain Reaction methods   standards   MeSH
• Humans MeSH
• Neoplasm, Residual * genetics   diagnosis   MeSH
• Practice Guidelines as Topic standards   MeSH
• Quality Assurance, Health Care MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * blood   diagnosis   genetics   therapy   MeSH
• Child MeSH
• False Positive Reactions MeSH
• Humans MeSH
• Adolescent MeSH
• Polymerase Chain Reaction * MeSH
• Child, Preschool MeSH
• Prognosis MeSH
• Neoplasm, Residual MeSH
• Hematopoietic Stem Cell Transplantation * MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial MeSH
• Multicenter Study MeSH

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis   MeSH
• Humans MeSH
• Polymerase Chain Reaction methods   MeSH
• Predictive Value of Tests MeSH
• Recurrence MeSH
• Neoplasm, Residual diagnosis   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

• MeSH
• CD2 Antigens immunology   MeSH
• Cell Lineage MeSH
• Child MeSH
• Immunophenotyping MeSH
• Cohort Studies MeSH
• Humans MeSH
• Adolescent MeSH
• Monocytes pathology   MeSH
• Multiplex Polymerase Chain Reaction MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma immunology   pathology   MeSH
• Child, Preschool MeSH
• Prognosis MeSH
• Neoplasm, Residual MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH
• Names of Substances
• CD2 Antigens MeSH

BACKGROUND: Most minimal residual disease-directed treatment interventions in current treatment protocols for acute lymphoblastic leukemia are based on bone marrow testing, which is a consequence of previous studies showing the superiority of bone marrow over peripheral blood as an investigational material. Those studies typically did not explore the prognostic impact of peripheral blood involvement and lacked samples from very early time points of induction. DESIGN AND METHODS: In this study, we employed real-time quantitative polymerase chain reaction analysis to examine minimal residual disease in 398 pairs of blood and bone marrow follow-up samples taken from 95 children with B-cell precursor acute lymphoblastic leukemia treated with the ALL IC-BFM 2002 protocol. RESULTS: We confirmed the previously published poor correlation between minimal residual disease in blood and marrow at early treatment time points, with levels in bone marrow being higher than in blood in most samples (median 7.9-fold, range 0.04-8,293-fold). A greater involvement of peripheral blood at diagnosis was associated with a higher white blood cell count at diagnosis (P=0.003) and with enlargement of the spleen (P=0.0004) and liver (P=0.05). At day 15, a level of minimal residual disease in blood lower than 10(-4) was associated with an excellent 5-year relapse-free survival in 78 investigated patients (100% versus 69 ± 7%; P=0.0003). Subgroups defined by the level of minimal residual disease in blood at day 15 (high-risk: ≥ 10(-2), intermediate-risk: <10(-2) and ≥ 10(-4), standard-risk: <10(-4)) partially correlated with bone marrow-based stratification described previously, but the risk groups did not match completely. No other time point analyses were predictive of outcome in peripheral blood, except for a weak association at day 8. CONCLUSIONS: Minimal residual disease in peripheral blood at day 15 identified a large group of patients with an excellent prognosis and added prognostic information to the risk stratification based on minimal residual disease at day 33 and week 12.

• MeSH
• Time Factors MeSH
• Child MeSH
• Infant MeSH
• Bone Marrow metabolism   pathology   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Adolescent MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma blood   diagnosis   drug therapy   pathology   MeSH
• Child, Preschool MeSH
• Prognosis MeSH
• Antineoplastic Combined Chemotherapy Protocols administration & dosage   MeSH
• Neoplasm, Residual MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH