Quality Assessment of a Large Multi-Center Flow Cytometric Dataset of Acute Myeloid Leukemia Patients-A EuroFlow Study.

Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.

Immunophenotypic Analysis of Acute Megakaryoblastic Leukemia: A EuroFlow Study.

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

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.

Robust FCS Parsing: Exploring 211,359 Public Files.

When it comes to data storage, the field of flow cytometry is fairly standardized, thanks to the flow cytometry standard (FCS) file format. The structure of FCS files is described in the FCS specification. Software that strictly complies with the FCS specification is guaranteed to be interoperable (in terms of exchange via FCS files). Nowadays, software interoperability is crucial for eco system, as FCS files are frequently shared, and workflows rely on more than one piece of software (e.g., acquisition and analysis software). Ideally, software developers strictly follow the FCS specification. Unfortunately, this is not always the case, which resulted in various nonconformant FCS files being generated over time. Therefore, robust FCS parsers must be developed, which can handle a wide variety of nonconformant FCS files, from different resources. Development of robust FCS parsers would greatly benefit from a fully fledged set of testing files. In this study, readability of 211,359 public FCS files was evaluated. Each FCS file was checked for conformance with the FCS specification. For each data set, within each FCS file, validated parse results were obtained for the TEXT segment. Highly space efficient testing files were generated. FlowCore was benchmarked in depth, by using the validated parse results, the generated testing files, and the original FCS files. Robustness of FlowCore (as measured by testing against 211,359 files) was improved by re-implementing the TEXT segment parser. Altogether, this study provides a comprehensive resource for FCS parser development, an in-depth benchmark of FlowCore, and a concrete proposal for improving FlowCore. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo.

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. METHODS: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. RESULTS: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. CONCLUSIONS: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.

Lossless Compression of Cytometric Data.

Nowadays, most cytometrists apply lossless compression by storing their FCS files in ZIP archives. Unfortunately, ZIP only achieves modest space savings in cytometric data, due to DEFLATE being used as the underlying lossless compression algorithm (LCA). Presumably, other modern LCA can outperform DEFLATE, especially in terms of space savings. Twenty-one codecs (programs implementing LCA) were evaluated in 167,131 publicly available FCS files. Within floating-point data, as produced by modern instruments, most favorable compression ratios (CRs) were achieved by ZPAQ (median 0.469), BCM (median 0.523), and LZMA (median 0.545). In comparison, the DEFLATE-based codecs only achieved median CR of 0.728 under the most optimal conditions. By default, ZIP offers nine compression level (CL) settings, where lower ZIP-CL optimizes for time efficiency, while higher ZIP-CL optimizes for space efficiency. Interestingly, the third ZIP-CL already resulted in near optimal CR in 90% of the files with floating-point data, as produced by digital cytometers. LZMA is well established, widely supported, and actively maintained (in sharp contrast to ZPAQ and BCM) and therefore arguably the most attractive alternative for ZIP. Within floating-point data, by shifting from ZIP (under optimal conditions) to LZMA (at default settings), the median CR can be improved by 25%. Based on our results, cytometrists can benefit from state-of-the-art compression by choosing the appropriate codec for their situation. Our results are likely to speed-up the adaptation of modern codecs, as CR around 0.5 were beyond all expectations, and such space savings will benefit the field of cytometry. © 2019 International Society for Advancement of Cytometry.

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping.

BACKGROUND: While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies. METHODS: CD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP-ALL, 69 adult BCP-ALL, 101 pediatric T-ALL, and 28 adult T-ALL patients. Paired diagnosis-relapse samples were available for 57 AML and 19 BCP-ALL patients. Leukemic stem cell (LSC) data was available for 32 pediatric AML patients. CD123 expression was evaluated based on mean fluorescence intensity, median fluorescence intensity, and percentage CD123 positive cells. RESULTS: EuroFlow panels were stable over time and between laboratories. CD123 was expressed in the majority of AML and BCP-ALL patients, but absent in most T-ALL patients. Within AML, CD123 expression was lower in erythroid/megakaryocytic leukemia, higher in NPM1 mutated and FLT3-ITD mutated leukemia, and comparable between LSC and leukemic blasts. Within BCP-ALL, CD123 expression was higher in patients with (high) hyperdiploid karyotypes and the BCR-ABL fusion gene. Interestingly, CD123 expression was increased in BCP-ALL relapses while highly variable in AML relapses (compared to CD123 expression at diagnosis). CONCLUSIONS: Authors evaluated CD123 expression in a large cohort of acute leukemia patients, based on standardized and reproducible methodology. Our results may facilitate stratification of patients most likely to respond to CD123 targeted therapies and serve as reference for CD123 expression (in health and disease). © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Usability of femoral head bone marrow to verify reference ranges for the assessment of myelodysplasia by flow cytometry.

BACKGROUND: A flow cytometry score (FCS) based on four parameters has been proposed for analysis of myelodysplastic syndromes patients with <5% blasts. We evaluated whether bone marrow aspirate samples isolated from femoral heads could be used for verification of cutoff values of the individual parameters score (IPS), contributing to the FCS and compared the applicability of FCS parameters in two centers. STUDY DESIGN AND METHODS: Bone marrow cells were obtained from femoral heads of patients who underwent hip replacement surgery. The score of the 4 individual parameters of the cell samples were obtained independently in two centers, using their own facilities and methods. Flow cytometry data files were subsequently exchanged and reanalyzed in the other center. The resulting four data sets were compared to assess reproducibility and outcomes in both centers. RESULTS: Twenty-nine of 40 bone marrow samples contained sufficient cells for analysis. Proposed cutoff values for 3 of 4 individual parameters were appropriate. All 29 samples showed a positive individual parameters score (IPS:1) in 1 of the 4 obtained data sets. Most differences in IPS scores were a result of reanalyzing the data file in the other center, rather than data acquisition. FCS: ≥2 were observed in 11 samples. CONCLUSION: Bone marrow samples from femoral heads are appropriate for verification of the proposed reference cutoff values of the individual parameters. Proposed reference cutoff values needed to be adjusted for reliable interpretation. Standardization of both data acquisition and data analysis is necessary for obtaining uniform results.

No significant prognostic value of normal precursor B-cell regeneration in paediatric acute myeloid leukaemia after induction treatment.

B-cell precursors (BCP) regeneration in bone marrow (BM) after induction chemotherapy is prognostic for good treatment response in adult acute myeloid leukaemia (AML). We detected BCP regeneration in 81% of 59 paediatric AML patients at first complete remission; this compared to 46% in an adult study. BCP regeneration did not correlate with outcome or minimal residual disease levels. In 36 healthy BM controls, BCP levels were significantly higher in children as compared to adults. Therefore, BCP regeneration does not reflect good response to treatment in paediatric AML, possibly due to the relatively high base-line levels of BCP in children.