EBV-driven lymphoid neoplasms associated with pediatric ALL maintenance therapy.

The development of a second malignancy after the diagnosis of childhood acute lymphoblastic leukemia (ALL) is a rare event. Certain second malignancies have been linked with specific elements of leukemia therapy, yet the etiology of most second neoplasms remains obscure and their optimal management strategies are unclear. This is a first comprehensive report of non-Hodgkin lymphomas (NHLs) following pediatric ALL therapy, excluding stem-cell transplantation. We analyzed data of patients who developed NHL following ALL diagnosis and were enrolled in 12 collaborative pediatric ALL trials between 1980-2018. Eighty-five patients developed NHL, with mature B-cell lymphoproliferations as the dominant subtype (56 of 85 cases). Forty-six of these 56 cases (82%) occurred during or within 6 months of maintenance therapy. The majority exhibited histopathological characteristics associated with immunodeficiency (65%), predominantly evidence of Epstein-Barr virus-driven lymphoproliferation. We investigated 66 cases of post-ALL immunodeficiency-associated lymphoid neoplasms, 52 from our study and 14 additional cases from a literature search. With a median follow-up of 4.9 years, the 5-year overall survival for the 66 patients with immunodeficiency-associated lymphoid neoplasms was 67.4% (95% confidence interval [CI], 56-81). Five-year cumulative risks of lymphoid neoplasm- and leukemia-related mortality were 20% (95% CI, 10.2-30) and 12.4% (95% CI, 2.7-22), respectively. Concurrent hemophagocytic lymphohistiocytosis was associated with increased mortality (hazard ratio, 7.32; 95% CI, 1.62-32.98; P = .01). A large proportion of post-ALL lymphoid neoplasms are associated with an immunodeficient state, likely precipitated by ALL maintenance therapy. Awareness of this underrecognized entity and pertinent diagnostic tests are crucial for early diagnosis and optimal therapy.

Asciminib stands out as the superior tyrosine kinase inhibitor to combine with anti-CD20 monoclonal antibodies for the treatment of CD20+ Philadelphia-positive B-cell precursor acute lymphoblastic leukemia in preclinical models.

Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCPALL) is a high-risk acute lymphoblastic leukemia subtype characterized by the presence of BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) combined with chemotherapy are established as the first-line treatment. Additionally, rituximab (RTX), an anti-CD20 monoclonal antibody (mAb) is administered in adult BCP-ALL patients with ≥20% of CD20+ blasts. In this study, we observed a marked prevalence of CD20 expression in patients diagnosed with Ph+ BCP-ALL, indicating a potential widespread clinical application of RTX in combination with TKIs. Consequently, we examined the influence of TKIs on the antitumor effectiveness of anti-CD20 mAbs by evaluating CD20 surface levels and conducting in vitro functional assays. All tested TKIs were found to uniformly downregulate CD20 on leukemic cells, diminishing the efficacy of RTX-mediated complement-dependent cytotoxicity. Interestingly, these TKIs displayed varied effects on NK cell-mediated antibody-dependent cytotoxicity and macrophage phagocytic function. While asciminib demonstrated no inhibition of effector cell functions, dasatinib notably suppressed the anti-CD20-mAb-mediated NK cell cytotoxicity and macrophage phagocytosis of BCP-ALL cells. Dasatinib and ponatinib also decreased NK cell degranulation in vitro. Importantly, oral administration of dasatinib, but not asciminib, compromised NK cell activity within patients' blood, determined by ex vivo degranulation assay. Our results indicate that asciminib might be preferred over other TKIs for combination therapy with anti-CD20 mAbs.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Multiomics to investigate the mechanisms contributing to repression of PTPRC and SOCS2 in pediatric T-ALL: Focus on miR-363-3p and promoter methylation.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous and aggressive malignancy arising from T-cell precursors. MiRNAs are implicated in negative regulation of gene expression and when aberrantly expressed contribute to various cancer types, including T-ALL. Previously we demonstrated the oncogenic potential of miR-363-3p overexpression in a subgroup of T-ALL patients. Here, using combined proteomic and transcriptomic approaches, we show that miR-363-3p enhances cell growth of T-ALL in vitro via inhibition of PTPRC and SOCS2, which are implicated in repression of the JAK-STAT pathway. We propose that overexpression of miR-363-3p is a novel mechanism potentially contributing to overactivation of JAK-STAT pathway. Additionally, by combining the transcriptomic and methylation data of T-ALL patients, we show that promoter methylation may also contribute to downregulation of SOCS2 expression and thus potentially to JAK-STAT activation. In conclusion, we highlight aberrant miRNA expression and aberrant promoter methylation as mechanisms, alternative to mutations of JAK-STAT-related genes, which might lead to the upregulation of JAK-dependent signaling in T-ALL.

Characteristics and treatment results of refractory and relapsed acute myeloid leukaemia in paediatric patients treated in Polish Paediatric Leukaemia/Lymphoma Study Group institutions according to the Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012 and a review of novel treatment possibilities in paediatric acute myeloid leukaemia.

Introduction: This study aimed to present the clinical features and results of treatment of patients diagnosed with refractory or relapsed acute myeloid leukaemia (AML) in Polish Paediatric Leukaemia/Lymphoma Study Group (PPL/LSG) institutions, treated in accordance with the Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012, as their first-line therapy. Material and methods: The outcome data of 10 patients with refractory AML (median age 9.5 years) and 30 with relapsed AML (median age 12 years) were analysed retrospectively. Re-induction was usually based on idarubicin, fludarabine, and cytarabine along with allogeneic haematopoietic stem cell transplant (allo-HSCT) in 5 patients with refractory AML and 7 relapsed AML children. Results: 37.5% (3/8) of refractory AML patients achieved second complete remission second complete remission (CRII). One of ten patients (1/10; 10%) was alive and stayed in complete remission for 34 months after the allo-HSCT. The probability of 3-year event-free survival (pEFS) in this group was 0.125 ±0.11. In the group of relapsed AML patients, the CRII was achieved in 9 patients (34%), and the probability of survival was: pEFS = 0.24 ±0.08; probability overall survival (pOS) = 0.34 ±0.09, with significantly better results achieved in patients who underwent allo-HSCT (pOS = 0.54 ±0.14 vs. 0.08 ±0.08, p < 0.0001). Conclusions: The prognosis of refractory AML and the first AML recurrence in children who were first-line treated in PPL/LSG centres according to Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012 is poor. Failures of re-induction treatment particularly result from difficulties in achieving remission. Allogeneic HSCT improves prognosis in children with refractory and first recurrent AML, under the condition it is performed in complete remission. Novel therapeutic approaches are needed to increase the remission rate and improve the outcomes.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Isolated central nervous system relapses in patients with high-risk neuroblastoma -clinical presentation and prognosis: experience of the Polish Paediatric Solid Tumours Study Group.

Although isolated central nervous system (CNS) relapses are rare, they may become a serious clinical problem in intensively treated patients with high-risk neuroblastoma (NBL). The aim of this study is the presentation and assessment of the incidence and clinical course of isolated CNS relapses. Retrospective analysis involved 848 NBL patients treated from 2001 to 2019 at 8 centres of the Polish Paediatric Solid Tumours Study Group (PPSTSG). Group characteristics at diagnosis, treatment and patterns of relapse were analysed. Observation was completed in December 2020. We analysed 286 high risk patients, including 16 infants. Isolated CNS relapse, defined as the presence of a tumour in brain parenchyma or leptomeningeal involvement, was found in 13 patients (4.5%; 8.4% of all relapses), all of whom were stage 4 at diagnosis. Isolated CNS relapses seem to be more common in young patients with stage 4 MYCN amplified NBL, and in this group they may occur early during first line therapy. The only or the first symptom may be bleeding into the CNS, especially in younger children, even without a clear relapse picture on imaging, or the relapse may be clinically asymptomatic and found during routine screening. Although the incidence of isolated CNS relapses is not statistically significantly higher in patients after immunotherapy, their occurrence should be carefully monitored, especially in intensively treated infants, with potential disruption of the brain-blood barrier.

Prognostic significance of IKZF1 deletions and IKZF1plus profile in children with B-cell precursor acute lymphoblastic leukemia treated according to the ALL-IC BFM 2009 protocol.

The strongest predictors of outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are minimal residual disease (MRD) and specific molecular abnormalities. One unfavorable prognostic factor is the presence of IKZF1 gene aberrations, particularly when co-occurring with high MRD level at the end of induction treatment. The present study determines the predictive value of a recently-defined IKZF1-plus (IKZF1plus ) microdeletion profile in 373 children with BCP-ALL treated according to the ALL-intercontinental Berlin-Frankfurt-Munster protocol 2009 protocol. IKZF1-wild type (IKZF1wt ) patients demonstrated lower leukemic burden parameters than those carrying IKZF1 deletion (IKZF1del [n = 26, 7.0%]) or IKZF1plus pattern (n = 34, 9.1%): (i) median blast percentage at diagnosis (78.0% vs. 86.9% vs. 86.0%; p = 0.021); (ii) median MRD level at day 15 of induction protocol (0.3% vs. 2.1% vs. 0.8%; p = 0.011); (iii) poor steroid response (7.6% vs. 26.5% vs. 12.5%; p = 0.010). Minimal residual disease level at day 33 (MRD33) exceeding 10-4 was more frequently observed in both the IKZF1del and IKZF1plus subgroups than in IKZF1wt patients (n = 9 [36.0%] vs. n = 13 [41.9%] vs. n = 70 [24.0%], p = 0.051). IKZF1plus individuals showed a tendency for a lower MRD reduction between day 15 and 33 compared to IKZF1del patients (p = 0.124). IKZF1del and IKZF1plus patients showed decreased relapse-free survival (HR [95%CI] for IKZF1wt as reference = 2.72 [1.21-6.11] and 2.00 [0.87-4.49], respectively, p = 0.023). Both genetic markers including IKZF1del and IKZF1plus microdeletion profile provide additional predictive value of treatment outcome in childhood BCP-ALL and may contribute to more efficient patient stratification; the same is true in MRD guided protocols, which are based on flow cytometric measurements on day 15 of induction protocol.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.

Expression of CD73 on leukemic blasts increases during follow-up - a promising candidate marker for minimal residual disease detection in pediatric B-cell precursor acute lymphoblastic leukemia.

Flow cytometry (FCM) is a precise and well-established tool to assess the minimal residual disease (MRD) level in childhood acute lymphoblastic leukemia (ALL). It is crucial to distinguish leukemic cells from their normal counterparts; thus new markers should be evaluated, to increase the accuracy of the analysis. The expression of CD73 on blast cells was measured and compared at the day of diagnosis and at days 15 and 33 of treatment. To determine antigen expression levels, a normalized scale based on median fluorescence intensity (nMFI) was used. The study group consisted of 188 patients from the Polish Pediatric Leukemia and Lymphoma Study Group. From 177 patients with positive MRD at day 15 of treatment, in 147 (83.1%) cases an increase of CD73 expression was observed (mean increase of +17 nMFI units). In addition, an increase of CD73 expression was noted in 26 of 31 (83.9%) patients at day 33 of treatment. In turn, a decrease of CD73 expression was observed only in 13/177 (7.3%) and 1/31 (3.2%) cases at days 15 and 33 of treatment, respectively. In 17 (9.6%) patients no change in expression of CD73 between diagnosis and day 15 of treatment was observed. In the great majority of cases the expression of CD73 is not only stable but increases during the early stages of treatment, which makes it a very useful marker to be used for MRD monitoring in childhood B-cell precursor (BCP)-ALL patients.

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.

Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Atypical Hemolytic Uremic Syndrome (aHUS) and Adenosine Deaminase (ADA)-Deficient Severe Combined Immunodeficiency (SCID)-Two Diseases That Exacerbate Each Other: Case Report.

Hemolytic uremic syndrome (HUS) is defined by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury (AKI). Atypical HUS (aHUS), distinguished by its etiology, is caused by uncontrolled overactivation of the alternative complement pathway. The correct diagnosis of aHUS is complex and involves various gene mutations. Severe combined immunodeficiency (SCID), characterized by severe T-cell lymphocytopenia and a lack of antigen-specific T-cell and B-cell immune responses, is of seldom occurrence. In 10-15% of pediatric patients, SCID is caused by adenosine deaminase (ADA) deficiency. The authors describe the case of a boy who suffered from both aHUS and ADA-deficient SCID. At the age of 9 months, the patient presented acute kidney injury with anuria and coagulopathy. The diagnosis of aHUS was established on the basis of alternative complement pathway deregulation and disease-associated gene mutations. Further examination revealed immune system failure and, at the age of 13 months, the ADA deficiency was confirmed by genetic tests and the boy was diagnosed with ADA-SCID. ADA SCID has recently been described as a possible triggering factor of aHUS development and progression. However, more research is required in this field. Nevertheless, it is crucial in clinical practice to be aware of these two co-existing life-threatening diseases.

Multicolor flow cytometry immunophenotyping and characterization of aneuploidy in pediatric B-cell precursor acute lymphoblastic leukemia.

The aim of this study was to assess the incidence of DNA aneuploidy in Polish children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and the relationship between aneuploidy and immunological phenotype, age, leukocyte count, S-phase fraction (SPF) and early response to induction chemotherapy assessed by the percentage of residual blast cells in bone marrow aspirates. The study group consisted of 267 patients. DNA content and immunophenotype were assessed in the bone marrow before treatment using multicolor flow cytometry (FC). DNA aneuploidy was detected in 50/267 (19%) patients. High hyperdiploidy was found to be associated with lower leukocyte count (p = 0.006) and common ALL immunophenotype. Flow cytometry analysis revealed that high hyperdiploid BCP-ALL patients showed significantly higher expression of CD9, CD20, CD22, CD58, CD66c, CD86 and CD123 antigens as compared to other groups of ploidy. In contrast, CD45 showed decreased expression. The percentage of leukemic blasts at diagnosis was lower in high hyperdiploid BCP-ALL cases than in diploid (79% vs. 85.7%, p = 0.001). The difference in minimal residual disease (MRD) levels on day 15 and 33 of induction therapy between analyzed groups was not significant. This study showed that high hyperdiploidy is associated with lower WBC count and specific immunological phenotype. Flow cytometric evaluation of expression of selected antigens can be used for fast identification of markers of aneuploidy in pediatric BCP-ALL, before genetic tests results are available. Understanding the biological significance of aneuploidy in leukemia can potentially be exploited therapeutically using targeted therapies against specific blast cell subclones.

Mixed phenotype acute leukemia: Biological profile, clinical characteristic and treatment outcomes: Report of the population-based study.

OBJECTIVES: The aim of this population-based, retrospective study was to analyze biological and clinical features and treatment results in children diagnosed with MPAL in all Polish pediatric oncology centers between 2007 and 2018. METHODS: Among 2893 children and adolescents diagnosed and treated for acute leukemia, 39 (1.35%) patients fulfilled the WHO criteria of MPAL. The T/myeloid phenotype was most prevalent. RESULTS: Cytogenetics findings were seen in 2 (5.1%), while chromosomal abnormalities were found in 14 (35.9%) patients. Thirty-two patients achieved CR-1, including 23 (92.0%) treated with ALL-directed chemotherapy and 9 (64.3%) treated with AML-type induction regimens. Within these patients, 4 (12.5%) died due to treatment-related complications and 11 (34.4%) relapsed. Nineteen (63.3%) patients underwent allo-HSCT in CR-1 and 14 (73.7%) of them have been in CR-1. In total, 17 (43.6%) patients remain in CR-1 for 1-12 years, including 14 (58.3%) with T/myeloid MPAL. The 5-year pOS and pEFS were 51.8% and 44.2%, respectively. The overall survival for ALL-directed therapy was significantly better than the one for AML-type chemotherapy (P = .001). It was also better for patients who underwent HSCT in CR-1 (P = .001). CONCLUSIONS: The prognosis of MPAL is unsatisfactory, but initial treatment with ALL-directed chemotherapy consolidated with allo-HSCT improves the outcomes in MPAL.

GATA3 germline variant is associated with CRLF2 expression and predicts outcome in pediatric B-cell precursor acute lymphoblastic leukemia.

The germline variant at rs3824662 in GATA3 is a risk locus for Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), the biological subtype of B-cell precursor (BCP)-ALL defined by a distinct gene expression profile and the presence of specific somatic aberrations including rearrangements of CRLF2. In this study, we investigated whether rs3824662 in GATA3 associates with CRLF2 expression in leukemic cells and predicts prognosis in pediatric BCP-ALL patients treated according to the ALL Intercontinental Berlin-Frankfurt-Münster (IC BFM) 2009 (n = 645) and the ALL IC BFM 2002 (n = 216) protocols. High expression of CRLF2 was observed at both protein and mRNA levels (fourfold higher in AA than in CA + CC) among GATA3 AA variant carriers, independent of the presence of P2RY8-CRLF2 fusion. Additionally, the AA variant at rs3824662 was a significant factor affecting minimal residual disease level at the end of induction phase and overall survival regardless of the risk group and the protocol. The germline variant at rs3824662 in GATA3 is a prognostic factor which associates with CRLF2 expression in leukemic cells supporting the hypothesis that GATA3 may have a regulatory effect on the CRLF2 pathway in pediatric BCP-ALL.

The influence of fixation of biological samples on cell count and marker expression stability in flow cytometric analyses.

The most common applications of flow cytometry (FC) include diagnostics of haemato-oncological disorders, based on analysis of bone marrow, peripheral blood (PB), or cerebrospinal fluid (CSF) samples. A proper diagnostic process requires standardisation in setting the optimal time frame between material collection and the assay. Unfortunately, this might be difficult to achieve in daily practice due to unintended shipment delays, which might compromise large-scale multicentre studies. Thus, material fixation should be considered as a solution. The most widely used fixative agents are: paraformaldehyde, TransFix®, Cyto-Chex®, and serum-containing media. In this review, we attempted to summarise the literature data on the influence of sample storage under different temperatures and times combined with different fixation conditions on the cell count and marker expression levels. Based on the findings of several extensive studies employing fixed PB samples, it can be concluded that the performance of particular fixative greatly depends on the analysed marker and specific PB cell population expressing a given antigen. Preservation of absolute cell count was usually better in Cyto-Chex®-fixed PB samples, whereas TransFix® tended to better stabilise marker expression levels. CSF-based studies reveal that both serum-containing media and TransFix® can prevent cellular loss and enhance FC-based detection of leptomeningeal localisations of haematological malignancies, the latter being more available and having longer shelf-life. As both cell count and marker expression level are the main determinants of quality of biological samples dedicated to FC analyses, it remains to be addressed by the investigators which is the fixative of choice for their specific research aims.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection.

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34high pre-B-I cell immunophenotype. These CD34-dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34-dim pre-B-I cells. Our results indicate that newly identified CD34-dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34-dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.