Prenatal origin of NUTM1 gene rearrangement in infant B-cell precursor acute lymphoblastic leukaemia.

Rearrangement of NUTM1 gene (NUTM1r) is one of the most frequent aberrations occurring in infants (younger than 1 year at diagnosis) with B-cell precursor Acute Lymphoblastic Leukaemia (BCP-ALL). In this study we had the unique opportunity to analyze the umbilical cord blood (UCB) sample from one infant patient with NUTM1r BCP-ALL. Herein we reported for the first time that NUTM1r infant ALL arise prenatally, as both the patient-specific CUX1::NUTM1 fusion gene, as well as two IG/TR leukaemic markers were already present and detectable in the patient's UCB at birth. Our results clearly demonstrate the prenatal origin of NUTM1r infant BCP-ALL.

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.

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

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.

Health technology assessment-based approach to flow cytometric immunophenotyping of acute leukemias: a literature classification.

OBJECTIVE: Acute leukemia (AL) is a broad, heterogeneous group of malignant diseases. The diagnostic workup of AL is based on several clinical and laboratory findings, including flow cytometric immunophenotyping. However, the role of this assay in the diagnosis of AL has not been systematically investigated. The aim of this study was to determine the accuracy and utility of flow cytometric immunophenotyping in the identification, characterization, and staging of AL. METHODS: We performed a systematic selection and classification of the literature since 1980, focused on flow cytometric immunophenotyping of AL. We applied a 6-variables model to cover both the technical capabilities and the clinical value of flow cytometric immunophenotyping in the diagnosis of AL. RESULTS: Using 3 key words (acute leukemia, immunophenotyping, flow cytometry), we screened the literature from January 1985 to April 2015 in PubMed and Embase databases and found 1010 articles. A total of 363 were selected and submitted to the expert panel, which selected a final data set of 248 articles to be analyzed. Of these, 160 were focused on clinical and biological issues, 55 were technical articles, and 31 were reviews. These 248 articles were then analyzed according to the 6-variables model and definitively classified. CONCLUSIONS: We assessed the literature on flow cytometric immunophenotyping of AL over 3 decades as the first step toward an evidence-based analysis of the impact of this technology on the clinical management of patients with AL.

AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.

Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia.

BACKGROUND: Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia. DESIGN AND METHODS: Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial. RESULTS: The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year event-free survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%). CONCLUSIONS: Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

Modulation of antigen expression in B-cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: evidence for a drug-induced regulatory phenomenon. Results of the AIEOP-BFM-ALL-FLOW-MRD-Study Group.

BACKGROUND: Changes of antigen expression on residual blast cells of acute lymphoblastic leukemia (ALL) occur during induction treatment. Many markers used for phenotyping and minimal residual disease (MRD) monitoring are affected. Glucocorticoid (GC)-induced expression modulation has been causally suspected, however, subclone selection may also cause the phenomenon. METHODS: We investigated this by following the phenotypic evolution of leukemic cells with flow cytometry from diagnosis to four time points during and after GC containing chemotherapy in the 20 (of 360 consecutive) B-cell precursor patients with ALL who had persistent MRD throughout. RESULTS: The early expression changes of CD10 and CD34 were reversible after stop of GC containing chemotherapy. Modulation of CD20 and CD45 occurred mostly during the GC phase, whereas CD11a also changed later on. Blast cells at diagnosis falling into gates designed according to "shifted" phenotypes from follow-up did not form clusters and were frequently less numerous than later on. CONCLUSIONS: Our data support the idea that drug-induced modulation rather than selection causes the phenomenon. The good message for MRD assessment is that modulation is transient in at least two (CD10 and CD34) of the five prominent antigens investigated and reverts to initial aberrant patterns after stop of GC therapy, whereas CD20 expression gains new aberrations exploitable for MRD detection.

Risk of relapse of childhood acute lymphoblastic leukemia is predicted by flow cytometric measurement of residual disease on day 15 bone marrow.

PURPOSE: Speed of blast clearance is an indicator of outcome in childhood acute lymphoblastic leukemia (ALL). Availability of measurement of minimal residual disease (MRD) at an early time point with a reduced-cost method is of clinical relevance. In the AIEOP-BFM-ALL (Associazione Italiana Ematologia Oncologia Pediatrica and Berlin-Frankfurt-Münster Study Group) 2000 trial, patients were stratified by levels of polymerase chain reaction (PCR) MRD at day +33 and +78. AIEOP studied the prognostic impact of MRD measured by flow cytometry (FCM) at day 15 of induction therapy. PATIENTS AND METHODS: Bone marrow samples from 830 Italian patients were collected on day 15, after 14 days of steroids, and one dose of intrathecal methotrexate, vincristine, daunorubicine, and asparaginase. Cells were analyzed by four-color FCM for detection of leukemia-associated immunophenotypes. RESULTS: Three patient risk groups were identified by FCM: standard (< 0.1% blast cells; 42% of the total), intermediate (0.1 to < 10%; 47%), and high (> or = 10%; 11%). Their 5-year cumulative incidences of relapse were 7.5% (SE, 1.5), 17.5% (SE, 2.1), and 47.2% (SE, 5.9), respectively. In multivariate analysis, FCM was the most important prognostic factor among those available by day 15, with two-fold and five-fold increase in the risk of relapse compared with patients with less than 0.1%. PCR MRD, when added to the model, had significant prognostic impact; yet high levels of FCM MRD retained an independent ability to detect a significantly higher risk of relapse. CONCLUSION: Measurement of FCM MRD in day 15 bone marrow was the most powerful early predictor of relapse, applicable to virtually all patients; it may complement PCR MRD-based stratification including later time points, thus allowing additional treatment tailoring.

Standardization of flow cytometric minimal residual disease evaluation in acute lymphoblastic leukemia: Multicentric assessment is feasible.

BACKGROUND: Single-laboratory experience showed that flow cytometric (FCM) assessment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) is feasible in most patients and gives independent prognostic information. It is, however, not known whether FCM analysis can reliably be standardized for multicentric application. METHODS: An extensive standardization program was installed in four collaborating laboratories, which study FCM-MRD in children treated with the AIEOP-BFM-ALL 2000 protocol. This included methodological alignment, continuous quality monitoring, as well as personnel education by exchange and performance feed-back. RESULTS: Blinded inter-laboratory tests of list-mode data interpretation concordance (n = 202 blood and bone marrow samples from follow-up during induction of 31 randomly selected patients of a total series of n = 395) showed a very high degree of inter-rater agreement among the four centers despite differences in cytometers and software usage (intraclass correlation coefficient [ICC] 0.979 based on n= 800 single values). Lower concordance was reached with amounts of MRD below 0.1%. Comparing data from sample exchange experiments (n = 42 samples; ICC 0.98) and from independent patient cohorts from the four centers (regarding positive samples per time-point of follow-up as well as risk estimates) concordance was also good. CONCLUSION: MRD-evaluation by FCM in ALL can be standardized for reliable multicentric assessment in large trials.

Prednisone induces immunophenotypic modulation of CD10 and CD34 in nonapoptotic B-cell precursor acute lymphoblastic leukemia cells.

BACKGROUND: Immunophenotypic modulation is induced by steroids in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients during remission induction therapy. METHODS: We cultured BCP-ALL blasts from diagnostic bone marrow (BM) samples (n = 20) in the presence of prednisone on stroma layer obtained from BM-derived mesenchymal cells to maintain viability. Antigen expression was assessed by multiparametric flow cytometry. RESULTS: Leukemia samples that sustained the treatment in vitro with prednisone, showed significative reduction of CD10 and CD34 expression compared with control, and it was comparable with that observed in residual leukemic cells of the same patients in BM at day 15 of treatment. Modulated cells were viable as determined by Annexin V staining and preserved light scattering properties. Of note, the extent of antigen modulation in vitro correlated with response to prednisone in vivo. CONCLUSIONS: The prednisone-induced immunophenotypic modulation can be reproduced in vitro and this phenomenon may reflect sensitivity to chemotherapy.

Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation.

We prospectively evaluated the reconstitution of lymphocyte subpopulations in nine children with lysosomal diseases who underwent 11 allogeneic haematopoietic cell transplants (HCTs) following CD34(+) immunomagnetic enrichment, limited T-cell addback and in vivo B-cell depletion. Absolute lymphocyte count recovery was slow to cross the 5th percentile, occurring at a median of 10 months after HCT in patients with full chimaerism. Natural killer cells represented up to 90% of the total lymphoid population during the first 3 months. CD4(+) lymphocyte recovery occurred 9-18 months after HCT. In most patients, CD8(+) lymphocyte recovery was slow and comparable with that of CD4(+) lymphocytes. The CD4(+)/CD8(+) ratio normalised by 3-7 months after HCT in 50% of the patients. CD8(+) lymphocyte recovery was enhanced in patients with viral reactivation. Reconstitution of B-lymphocytes was particularly delayed in patients treated with rituximab. Declining chimaerism, rejection and viral reactivation were the most common problems in our series. Because of the unique graft manipulation, the pace of lymphocyte reconstitution was particularly slow, suggesting that these patients are at a significantly increased risk of infections for up to 2 years after HCT.

Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: CD58, a member of the Ig superfamily, is expressed by hematopoietic and non- hematopoietic cells. It has been demonstrated to be over-expressed in precursor-B acute lymphoblastic leukemia (ALL) blasts when compared to in their normal counterparts, suggesting its potential use in the detection of minimal residual disease (MRD) by flow cytometry (FC). To assess the reliability and accuracy of CD58 for this purpose, we studied its expression in a large series of normal and ALL bone marrow (BM) samples using quantitative FC. DESIGN AND METHODS: We studied 180 precursor-B ALL BM samples at diagnosis (8 pro-B, 164 early-B, 8 mature-B ALL) and 123 follow-up BM samples (n=54 at day +15 and n=69 at day +78), as well as 51 normal BM samples and 7 regenerating BM samples from patients with T-ALL at week 12. We used four-color quantitative FC, focusing analysis on CD58 expression. In follow-up samples from day +78, the MRD level was simultaneously evaluated by real time quantitative polymerase chain reaction (RQ-PCR) amplification of antigen receptor genes. RESULTS: CD58 expression was significantly higher in ALL blasts than in normal B lymphocytes, while no significant differences between regenerating and normal B lymphocytes were observed. CD58 was expressed in 99.4% of the precursor-B ALL cases and 93.5% of these showed over-expression compared to normal. No significant modulation of CD58 expression during remission induction therapy was noted. Finally, 66 (95.6%) of 69 BM samples simultaneously analyzed using both FC and RQ-PCR at day +78 showed concordant results regarding MRD. INTERPRETATION AND CONCLUSIONS: Our results confirm and further evidence the role of CD58 in the diagnosis and monitoring of precursor-B ALL. In particular, we demonstrated its stability and accuracy in MRD detection at clinically relevant time points. These findings indicate that CD58 is a powerful tool for MRD detection in precursor-B ALL.

Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells.

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cells that express CD34 are heterogeneous in their lineage affiliation and degree of maturation. Expression of CD13 and CD33 antigens indicates myeloid lineage association, but the precise sequence of expression of these two markers during differentiation is unclear. We noted the presence of CD34+ cells expressing CD13 but lacking CD33, a subset of cells not yet well characterized. In this report we describe the prevalence and the immunophenotype of this cell subset. DESIGN AND METHODS: We studied the immunophenotype of immature myeloid cells in human bone marrow samples from 11 healthy transplantation donors and in 4 cord blood samples. We used four-color flow cytometry and a large panel of monoclonal antibodies directed against lineage and differentiation-associated antigens. Three additional bone marrow samples were analyzed after immunomagnetic sorting of CD34+ cells. We focused our analysis on the subset of cells defined by the expression of CD34 and CD13 and the lack of CD33. RESULTS: We found CD34+, CD13+, CD33- cells in all 11 bone marrow and 4 cord blood samples studied. These cells represented 0.5 0.5% (mean SD) and 0.8 1.2% of mononucleated cells, respectively. CD34+, CD13+, CD33- cells appeared to be more immature than those expressing CD33 because of their light scatter characteristics (smaller size and lower granularity), the expression of markers associated with early hematopoietic cells (CD90, CD133 and CD117), and the absence of lineage-associated markers. INTERPRETATION AND CONCLUSIONS: These findings suggest that the expression of CD13 precedes that of CD33 during myeloid differentiation, and that CD34+, CD13+, CD33- cells are at an early stage of human myeloid cell differentiation.