Hematologist-Level Classification of Mature B-Cell Neoplasm Using Deep Learning on Multiparameter Flow Cytometry Data.

The wealth of information captured by multiparameter flow cytometry (MFC) can be analyzed by recent methods of computer vision when represented as a single image file. We therefore transformed MFC raw data into a multicolor 2D image by a self-organizing map and classified this representation using a convolutional neural network. By this means, we built an artificial intelligence that is not only able to distinguish diseased from healthy samples, but it can also differentiate seven subtypes of mature B-cell neoplasm. We trained our model with 18,274 cases including chronic lymphocytic leukemia and its precursor monoclonal B-cell lymphocytosis, marginal zone lymphoma, mantle cell lymphoma, prolymphocytic leukemia, follicular lymphoma, hairy cell leukemia, lymphoplasmacytic lymphoma and achieved a weighted F1 score of 0.94 on a separate test set of 2,348 cases. Furthermore, we estimated the trustworthiness of a classification and could classify 70% of all cases with a confidence of 0.95 and higher. Our performance analyses indicate that particularly for rare subtypes further improvement can be expected when even more samples are available for training. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Expression of CD56 defines a distinct subgroup in childhood T-ALL with inferior outcome. Results of the ALL-BFM 2000 trial.

This study reports the prognostic impact of the expression of the natural killer cell marker CD56 in a large series of risk-adapted paediatric patients with T cell acute lymphoblastic leukaemia (T-ALL; n = 493) treated within the ALL-Berlin-Frankfurt-Münster (BFM) 2000 protocol. The immunophenotype was analysed centrally at diagnosis using flow cytometry and correlated with clinical parameters and outcome. CD56 expression was detected in 7·1% and early T-cell precursor (ETP) phenotype in 6·7% of all T-ALL patients. The percentage of ETP in the CD56+ T-ALL cohort was 4-fold higher than in the whole cohort. CD56+ T-ALL frequently expressed the progenitor marker CD34 and myeloid antigens CD13 and CD33. The 5-year event-free survival (EFS) rates for the European Group for the Immunological classification of Leukaemias/World Health Organization subgroups and the ETP phenotype were not statistically different. By contrast, patients with CD56 expression had a significantly reduced EFS (60 ± 8%) and overall survival (60 ± 8%) at 5 years, with a hazard ratio of 2·46 (P = 0·002) and 2·99 (P < 0·001), respectively. Moreover, CD56 expression in combination with the minimal residual disease (MRD)-based high risk assignment defined a population with a 'very-high' risk probability of relapse in the ALL-BFM 2000 trial. The CD56 marker has the potential to augment MRD-based risk stratification and may serve as a molecular target for antibody-based treatment strategies in childhood T-ALL.

Peripheral blood sCD3⁻ CD4⁺ T cells: a useful diagnostic tool in angioimmunoblastic T cell lymphoma.

Angioimmunoblastic T cell lymphoma (AITL) belongs to the subgroup of mature T cell lymphomas according to the World Health Organization and is one of the common T cell lymphomas in Western countries. Particularly in cases in which histological confirmation cannot be easily achieved, immunophenotyping of peripheral blood can give important information for the differential diagnosis of AITL. sCD3⁻ CD4⁺ T cells are a typical feature of AILT in flow cytometry of peripheral blood. In this retrospective study, the diagnostic value of flow cytometry for the diagnosis 'AITL' was assessed by comparing the frequency of sCD3⁻ CD4⁺ T cells in leukemic AITL patients and in patients with other leukemic CD4⁺ T cell lymphomas. Immunophenotyping of peripheral blood by flow cytometry was performed in a lymphocyte gate using fluorochrome-labelled antibodies against CD3, CD2, CD4, CD5, CD7, CD8, CD10, CD14, CD16, CD19, CD56, CD57 and T cell receptor. In 17/17 leukemic AITL patients, a small but distinct population of sCD3⁻ CD4⁺ T cells was detected (mean percentage of sCD3⁻ CD4⁺ T cells in the lymphocyte gate: 11.9 ± 15.4%, range 0.1-51.8%). In contrast, sCD3⁻ CD4⁺ T cells were found in only 1/40 patients with other leukemic CD4⁺ T cell lymphomas (one patient with mycosis fungoides). sCD3⁻ CD4⁺ T cells have a high positive predictive value (94%) for the diagnosis 'AITL'. Flow cytometry is particularly useful in the differential diagnosis of AITL, even if the aberrant T cell population has a very low frequency. Further biological characterization of this subfraction of lymphoma cells is warranted.

Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification.

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission.

Childhood near-tetraploid acute lymphoblastic leukemia: an EGIL study on 36 cases.

OBJECTIVES: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. METHODS: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. RESULTS: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n=2, T-II n=2, T-III n=3, T-IV n=3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. CONCLUSIONS: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations.

Acute leukaemias of ambiguous lineage (ALAL) represent a rare type of leukaemia, expressing both myeloid and lymphoid markers. This study retrospectively analyzed data from 92 children (biphenotypic n = 78, bilineal n = 6, lineage switch n = 8) with ALAL registered in the Berlin-Frankfürt-Münster (BFM) acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) studies between 1998 and 2006 (2.4% of all cases with acute leukaemia). Our cohort of ALAL patients was characterized by comparatively high median age (8.9 years), high median white blood cell count (14.9 x 10(9)/l), as well as frequent hyperleucocytosis (18.5%) and central nervous system involvement (24.1%). The most frequent cytogenetic abnormalities were ETV6/RUNX1 fusion (16%) and trisomy 8 (14.6%). Complete remission rate was significantly lower than in ALL-BFM patients (91.8% vs. 99.1%, P < 0.001), but comparable to AML-BFM patients (87.9%). Event-free survival (EFS) and overall survival (OS) of ALAL patients were low, at 62 +/- 5%. 5-year probability of EFS was significantly worse than in ALL patients (80 +/- 1%, P < 0.001), but better than for AML patients (49 +/- 2%, P = 0.027). Our data suggest that an intensive therapy regimen including stem cell transplantation may be favourable for bilineal or lineage switch cases, whereas patients with ETV6/RUNX1 fusion, lymphoid morphology and patients with expression of cyCD22 and cyCD79a should be treated with an ALL-directed therapy.

Differential expression of the carcinoembryonic antigen-related cell adhesion molecules panCD66, CD66a, CD66c and of sialyl-Lewis x (CD15s) on blast cells of acute leukemias.

Expression of CD66 has been reported to occur on blast cells from children with acute lymphoblastic leukemia (ALL), but little is known about the differential expression pattern of panCD66 and other members of the CD66 family on blast cells from patients with acute myeloid leukemia (AML). We have performed flow cytometry immunophenotyping on blast cells from 28 patients with acute myeloid leukemia (AML), 13 patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and 7 patients with T-ALL using monoclonal antibodies (mAbs) against panCD66 (clone D14HD11), CD66a (clone 4.3.17), CD66c (clone 9A6) and CD15s. Expression of the panCD66 mAb was found to be positive in 13 of 28 patients with AML (46%) and in 6 of 13 patients with BCP-ALL (46%) but negative for all the seven patients with T-ALL. In AML, panCD66, CD66a and CD66c were more frequently coexpressed with CD65, CD15 and CD64 than with CD13, CD33 or the two progenitor markers CD34 and CD117. In contrast to CD15, the expression of the sialylated Lewis X (CD15s) was associated with CD117 positivity in the majority of AML cases (64 vs. 85%, P = 0.043). Radioimmunotherapeutic strategies targeting CD66 antigens should consider the heterogeneous expression pattern of CD66 molecules in acute leukemias especially in AML where expression is correlated with mature granulomonocytic cells but not with CD34 and CD117 positive progenitor cells.

SDF-1 (CXCL12) in haematopoiesis and leukaemia: impact of DPP IV/CD26.

DPP IV/CD26 contributes to cell signalling by various mechanisms: as a receptor or co-receptor, as a component of a membrane-associated signal transduction complex and by virtue of its exopeptidase activity. The presence of enzymatically active DPP IV in a variety of tissues and in plasma makes it challenging to review clinical consequences of chemokine turnover by DPP IV activity, even more so in times when the concept of DPP IV inhibition for therapeutic purposes has reached the stage of clinical application. Among the known substrates of DPP IV/CD26, Stromal cell-derived factor 1 (SDF-1, CXCL12) has gained attention in recent years as a critical mediator of chemotaxis and tissue invasion, especially in the context of malignant disease. SDF-1 and its receptor, CXCR4, differ from other chemokines and their respective receptors by a lack of redundancy and pleiotropism. Therapeutic intervention using CXCR4 antagonists has been proposed. It seems appropriate to review the role of SDF-1 in haematopoiesis and its malignant counterpart, leukaemia, and to assess the interference of DPP IV/CD26 with the SDF-1/CXCR4 axis as well as possible risks and benefits of DPP IV inhibition.

The prognostic significance of antigen expression in leukaemia.

Numerous immunophenotypic features have been examined for their potential prognostic significance in predicting treatment outcome in leukaemias. These include immunophenotypic subgroups of acute lymphoblastic leukaemia (ALL) and immature acute myeloid leukaemia, expression of individual surface antigens or combined immunophenotypic features, and more recently, molecules mediating the multidrug resistance phenotype or being involved in the regulation of drug-induced apoptosis. Most previous studies investigating the prognostic significance of antigen expression in leukaemia have not used the information provided by multiparameter flow cytometry and have chosen rather arbitrary cut-off points for marker positivity. Moreover, given significant associations between immunophenotypic features and genetic abnormalities in leukaemic cells, immunophenotyping as an independent predictor of treatment outcome has been questioned. Thus, except for lineage assignment of leukaemic blasts and definition of maturational status in ALL, information provided by immunophenotyping is currently not applied to risk-classification systems or used for planning patient treatment in leukaemia. We review some of the recent findings regarding the prognostic impact of distinct immunophenotypic features in acute leukaemias and myelodysplastic syndrome.