Flow Plex-A tool for unbiased comprehensive flow cytometry data analysis.

INTRODUCTION: The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training. METHODS: We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets. RESULTS: We developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health. CONCLUSIONS: We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.

Is a 500-Cell Count Necessary for Bone Marrow Differentials?: A Proposed Analytical Method for Validating a Lower Cutoff.

OBJECTIVES: By convention, 500 cells are counted for bone marrow aspirate differentials. Evidence supporting such a cutoff is lacking. We hypothesized that 300-cell counts could be sufficient. METHODS: Cell count results from 165 cases, for which values were recorded at 300 and 500 cells, were analyzed. We tested for statistical differences and changes in diagnostic classification between the two cutoffs. RESULTS: Three hundred cell counts did not produce diagnostically different results, particularly for myeloblasts and plasma cells, where cell percentages are critical for disease classification. Method comparison analysis did not reach statistical significance for any cell type when comparing the two methods. Bias plots showed narrow, even spread about the mean bias. Contingency table analysis yielded no significant diagnostic discrepancies. CONCLUSIONS: Performing differential counts on 300 cells would produce clinically and statistically similar results to 500 cells. Reducing the cell number counted has potential cost/labor reductions without affecting quality of care.

Grouped gene selection and multi-classification of acute leukemia via new regularized multinomial regression.

Diagnosing acute leukemia is the necessary prerequisite to treating it. Multi-classification on the gene expression data of acute leukemia is help for diagnosing it which contains B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL) and acute myeloid leukemia (AML). However, selecting cancer-causing genes is a challenging problem in performing multi-classification. In this paper, weighted gene co-expression networks are employed to divide the genes into groups. Based on the dividing groups, a new regularized multinomial regression with overlapping group lasso penalty (MROGL) has been presented to simultaneously perform multi-classification and select gene groups. By implementing this method on three-class acute leukemia data, the grouped genes which work synergistically are identified, and the overlapped genes shared by different groups are also highlighted. Moreover, MROGL outperforms other five methods on multi-classification accuracy.

Myeloid neoplasms in the World Health Organization 2016 classification.

In the 2016 revision of the World Health Organization (WHO) classification, the categories of myeloid neoplasms have not been revised significantly from the 2008 fourth edition. However, recent discovery of molecular abnormalities provides a new perspective regarding the diagnostic and prognostic markers. In myeloproliferative neoplasms, the identification of CALR gene mutation, in addition to the JAK2 and MPL mutations, has impacted the diagnostic criteria. In myelodysplastic syndromes and acute myeloid leukemia, in addition to alterations in the transcription factors and signal transduction pathways, discovery of gene mutations in the epigenetic regulators that are involved in DNA methylation, histone modification, cohesin complex, and RNA splicing, by comprehensive genetic analyses, has improved our understanding of the pathobiology of these diseases. Moreover, recent large-scale sequencing studies have revealed the acquisition of clonal somatic mutations, in the myeloid neoplasm-associated genes of the hematopoietic cells. Such mutations were detected in people with normal blood cell counts, without any apparent disease. Presence of these mutations confers an increased risk for subsequent hematological neoplasms, indicating the concept of clonal hematopoiesis of indeterminate potential. This updated WHO classification incorporates the criteria of new clinical, prognostic, morphologic, immunophenotypic, and genetic findings in myeloid neoplasms.

BCR-ABL-positive acute myeloid leukemia: a new entity? Analysis of clinical and molecular features.

BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks.

Cytogenetic profiles of 2806 patients with acute myeloid leukemia-a retrospective multicenter nationwide study.

The cytogenetic and molecular data is recognized as the most valuable prognostic factor in acute myeloid leukemia (AML). Our aim was to systemically analyze the cytogenetics of Korean AML patients and to compare the cytogenetic profiles of various races to identify possible geographic heterogeneity. We retrospectively reviewed medical records of 2806 AML patients diagnosed at 11 tertiary teaching hospitals in Korea between January 2007 and December 2011. The most common recurrent chromosomal abnormality was t(8;21) (8.8 %, 238/2717), but t(15;17) showed an almost same number (8.6 %,235/2717). Among de novo AML, the most frequent aberrations were t(15;17), observed in 229 (10.7 %). The most common French-American-British (FAB) classification type was M2 (32.2 %), and recurrent cytogenetic abnormalities correlated with the FAB subtypes. Among 283 secondary AML cases, myelodysplastic syndrome was the most common predisposing factor. About 67.1 % of the secondary AML cases were associated with chromosomal aberrations, and chromosome 7 abnormalities (n = 45, 15.9 %) were most common. The incidence of FLT3 internal tandem duplication mutation was relatively low at 15 %. Our study reports certain similarities and differences in comparison to previous reports. Such discrepancies call for extensive epidemiological studies to clarify the role of genetic as well as geographic heterogeneity in the pathogenesis of AML.

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.

The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues was last updated in 2008. Since then, there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias, largely derived from gene expression analysis and next-generation sequencing that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists, and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition. The major changes in the classification and their rationale are presented here.

Azacitidine front-line in 339 patients with myelodysplastic syndromes and acute myeloid leukaemia: comparison of French-American-British and World Health Organization classifications.

BACKGROUND: The MDS-IWG and NCCN currently endorse both FAB and WHO classifications of MDS and AML, thus allowing patients with 20-30 % bone marrow blasts (AML20-30, formerly MDS-RAEB-t) to be categorised and treated as either MDS or AML. In addition, an artificial distinction between AML20-30 and AML30+ was made by regulatory agencies by initially restricting approval of azacitidine to AML20-30. Thus, uncertainty prevails regarding the diagnosis, prognosis and optimal treatment timing and strategy for patients with AML20-30. Here, we aim to provide clarification for patients treated with azacitidine front-line. METHODS: The Austrian Azacitidine Registry is a multicentre database (ClinicalTrials.gov: NCT01595295). For this analysis, we selected 339 patients treated with azacitidine front-line. According to the WHO classification 53, 96 and 190 patients had MDS-RAEB-I, MDS-RAEB-II and AML (AML20-30: n = 79; AML30+: n = 111), respectively. According to the FAB classification, 131, 101 and 111 patients had MDS-RAEB, MDS-RAEB-t and AML, respectively. RESULTS: The median ages of patients with MDS and AML were 72 (range 37-87) and 77 (range 23-93) years, respectively. Overall, 80 % of classifiable patients (≤30 % bone marrow blasts) had intermediate-2 or high-risk IPSS scores. Most other baseline, treatment and response characteristics were similar between patients diagnosed with MDS or AML. WHO-classified patients with AML20-30 had significantly worse OS than patients with MDS-RAEB-II (13.1 vs 18.9 months; p = 0.010), but similar OS to patients with AML30+ (10.9 vs 13.1 months; p = 0.238). AML patients that showed MDS-related features did not have worse outcomes compared with patients who did not (13.2 vs 8.9 months; p = 0.104). FAB-classified patients with MDS-RAEB-t had similar survival to patients with AML30+ (12.8 vs 10.9 months; p = 0.376), but significantly worse OS than patients with MDS-RAEB (10.9 vs 24.4 months; p < 0.001). CONCLUSIONS: Our data demonstrate the validity of the WHO classification of MDS and AML, and its superiority over the former FAB classification, for patients treated with azacitidine front-line. Neither bone marrow blast count nor presence of MDS-related features had an adverse prognostic impact on survival. Patients with AML20-30 should therefore be regarded as having 'true AML' and in our opinion treatment should be initiated without delay.

A Ranking Approach for Probe Selection and Classification of Microarray Data with Artificial Neural Networks.

Acute leukemia classification into its myeloid and lymphoblastic subtypes is usually accomplished according to the morphology of the tumor. Nevertheless, the subtypes may have similar histopathological appearance, making screening procedures difficult. In addition, approximately one-third of acute myeloid leukemias are characterized by aberrant cytoplasmic localization of nucleophosmin (NPMc(+)), where the majority has a normal karyotype. This work is based on two DNA microarray datasets, available publicly, to differentiate leukemia subtypes. The datasets were split into training and test sets, and feature selection methods were applied. Artificial neural network classifiers were developed to compare the feature selection methods. For the first dataset, 50 genes selected using the best classifier was able to classify all patients in the test set. For the second dataset, five genes yielded 97.5% accuracy in the test set.

Acute Myeloid Leukemia Genetics: Risk Stratification and Implications for Therapy.

Acute myeloid leukemia is a category of diseases with a common aggressive clinical presentation but with a prognosis and management that is dependent upon the underlying genetic characteristics of the neoplasm. The purpose of this brief review is to update the practicing pathologist on the current standard of care in the genetic evaluation of acute myeloid leukemia and to highlight future directions in the classification, genetic assessment, and management of these devastating diseases.

Acute myeloid leukemia: advances in diagnosis and classification.

Acute myeloid leukemia is an aggressive myeloid neoplasm characterized by ≥20% myeloblasts in the blood or bone marrow. Current treatment strategies for acute myeloid leukemia are based on both patient-related parameters such as age and performance status as well as the intrinsic characteristics of particular disease subtypes. Subtyping of acute myeloid leukemia requires an integration of information from the patient's clinical history (such as any prior preleukemic myeloid neoplasm or cytotoxic potentially leukemogenic therapy), the leukemia morphology, cytogenetic findings, and the mutation status of particular genes (NPM1, FLT3, and CEBPA). In recent years, a barrage of information has become available regarding gene mutations that occur in acute myeloid leukemia and their influence on prognosis. Future therapies for acute myeloid leukemia will increasingly rely on the genetic signatures of individual leukemias and will adjust therapy to the predicted disease aggressiveness as well as employ therapies targeted against particular deregulated genetic pathways. This article reviews current standards for diagnosing and classifying acute myeloid leukemia according to the 2008 WHO Classification. Data that have subsequently accumulated regarding newly characterized gene mutations are also presented. It is anticipated that future leukemia classifications will employ a combination of karyotypic features and the gene mutation pattern to stratify patients to increasingly tailored treatment plans.

MicroRNAs distinguish cytogenetic subgroups in pediatric AML and contribute to complex regulatory networks in AML-relevant pathways.

BACKGROUND: The role of microRNAs (miRNAs), important post-transcriptional regulators, in the pathogenesis of acute myeloid leukemia (AML) is just emerging and has been mainly studied in adults. First studies in children investigate single selected miRNAs, however, a comprehensive overview of miRNA expression and function in children and young adults is missing so far. METHODOLOGY/PRINCIPAL FINDINGS: We here globally identified differentially expressed miRNAs between AML subtypes in a survey of 102 children and adolescent. Pediatric samples with core-binding factor AML and promyelocytic leukemia could be distinguished from each other and from MLL-rearranged AML subtypes by differentially expressed miRNAs including miR-126, -146a, -181a/b, -100, and miR-125b. Subsequently, we established a newly devised immunoprecipitation assay followed by rapid microarray detection for the isolation of Argonaute proteins, the hallmark of miRNA targeting complexes, from cell line models resembling core-binding factor and promyelocytic leukemia. Applying this method, we were able to identify Ago-associated miRNAs and their targeted mRNAs. CONCLUSIONS/SIGNIFICANCE: miRNAs as well as their mRNA-targets showed binding preferences for the different Argonaute proteins in a cell context-dependent manner. Bioinformatically-derived pathway analysis suggested a concerted action of all four Argonaute complexes in the regulation of AML-relevant pathways. For the first time, to our knowledge, a complete AML data set resulting from carefully devised biochemical isolation experiments and analysis of Ago-associated miRNAs and their target-mRNAs is now available.

[State of medical care of patients with hematological malignancies in Kyïv].

In the article data are presented about morbidity by oncogematologic pathology - one of the most meaningful of social-economic problems. In Ukraine annually diagnose the to 8 thousand new cases of haemoblastosis. Indexes of morbidity on a 100 thousand population are 5,2; at illness of Hodgkin's lymphoma - 2,5, at plural myeloma - 1,6; at leukemia - 8,1. Morbidity by haematological pathology in Kyiv long time remains high: annually 250 expose patients with malignant lymphnoma, 57 - with myeloma, 190 - with leukemia, from them at 55 % is a sharp form and at 40 % - chronic. The anxiety of doctors causes circumstance that the special treatment is overcome 58,1 % patients by leukemia, 68,6 % - plural myeloma and 77,8 % patients with malignant lymphoma. World experience shows that application of complex methods of therapy allows to prolong life-span 80-90 % patients with Hodgkin's malignant lymphoma on 10, and at 95 % patients by a lymphogranulomatosis - to attain nonrecurrence survival to 5 years.

Immunophenotypic pattern of myeloid populations by flow cytometry analysis.

We present our experience with immunophenotypic characteristics of benign and malignant myeloid populations, with emphasis on differential diagnosis especially between eosinophils, dysplastic granulocytes, neoplastic promyelocytes, and monocytes. Eosinophils are characterized by bright CD45, high side scatter (SSC), very low forward scatter (FSC), positive CD11b, CD11c, CD13, CD15, and CD33. They are negative for CD10, CD14, CD16, CD56, CD64, and HLA-DR. Mature monocytes are positive for CD11b, CD11c, CD13, CD14, CD33, and CD64, and may express CD2 and CD4. Blasts in acute myeloid leukemias (AML) with minimal differentiation have low SSC and moderate CD45 expression and are positive for CD34, CD117, CD13, HLA-DR, and CD33 and may be positive for TdT, CD4, and CD11c. In acute promyelocytic leukemia (APL), four FC patterns can be recognized. The majority of cases represented classical (hypergranular) APL and were characterized by high SSC, positive CD117, usually negative CD34, heterogeneous CD13, and bright CD33 (pattern 1). The second most common type, corresponding to hypogranular (microgranular) variant of APL, differed from classical APL by low SSC and frequent coexpression of CD2 and CD34 (pattern 2). Rare cases of APL (pattern3) showed mixture of neoplastic cells (SSC(low)/CD2(+)/CD13(+)/CD33(+)/CD34(+)/CD117(+)) and prominent population of benign granulocytes/maturing myeloid precursors (SSC(high)/CD10(+/-)/CD16(+/()/CD117(()). One case showed two APL populations, one with hypogranular and one with hypergranular characteristics (pattern 4). Detailed phenotypic characteristics of neoplastic monocytes and dysplastic granulocytes with their differential diagnosis are also presented.

[microRNA expression in childhood acute granulocytic leukemia and its subtypes].

OBJECTIVE: Recent studies have suggested that there is a close relation between microRNA and acute leukemia (AL). The aim of this study was to investigate and better understand the classification and diagnosis of AL as well as pathogenesis and prognosis of this disease. METHODS: A total of 93 children with AL and and 12 cases of idiopathic thrombocytopenic purpura (as control group) were enrolled in this study. Microarray chip analysis of their bone marrow samples was conducted to evaluate the microRNA profiles. Quantitative real-time PCR was performed for validating the abnormal expression of microRNA. RESULTS: The microRNA expression profiles were different between acute granulocytic leukemia and acute lymphoblastic leukemia and also between the three subtypes (M1, M2 and M3) of acute granulocytic leukemia according to FAB classification based on leukemic cell differentiation. These three subtypes of leukemia could be identified by unsupervised hierarchical cluster analysis of microRNA expression and had specific up-regulation of miR-335, miR-126 and miR-125b, respectively. However, in the M2 and M3 subtypes with positive AML1-ETO and PML-RARα, respectively, which have a better prognosis, the expressions of miR-126 and miR-125b were significantly higher than those with negative AML1-ETO and PML-RARα. Further more, miR-335 and miR-146 were up-regulated in acute granulocytic leukemia observed in this study, which are different from those reported for adult patients. CONCLUSIONS: microRNA cascade may serve as new biomarkers for the classification and diagnosis of pediatric AL. It is also suggested that there might be different pathogenesis and prognosis between AL types related to specific expression and regulation of microRNA.

Eosinophilic myeloid disorders: new classification and novel therapeutic strategies.

PURPOSE OF REVIEW: The aim of this brief review is to evaluate recent developments in the classification and treatment of eosinophilic myeloid disorders in the context of reactive, lymphocyte-variant, and idiopathic eosinophilias. RECENT FINDINGS: The revised 2008 WHO classification recognizes both molecularly defined ('myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1') and undefined (chronic eosinophilic leukemia, not otherwise specified) eosinophilic myeloid disorders. An increasingly sophisticated understanding of the molecular underpinnings of eosinophilia has translated into rational use of biologically targeted therapies such as imatinib mesylate. Conventional cytotoxics and interferon-alpha still have an established role in treating these diseases. Although studied in idiopathic hypereosinophilic syndrome, the therapeutic niche of anti-interleukin-5 (mepolizumab) and anti-CD52 (alemtuzumab) antibody therapy in eosinophilic myeloid diseases has yet to be established. SUMMARY: Molecular/genetic analysis is now mandatory for the diagnosis, classification, and treatment of eosinophilic myeloid disorders. The finding of rearranged, constitutively activated PDGFRA/B identifies patients who are eminently treatable with tyrosine kinase inhibitors.

Independent prognostic factors for AML outcome.

Over the last three decades there have been dramatic advances in deciphering the cytogenetic and molecular lesions underlying the pathogenesis of acute myeloid leukemia (AML). These have not only afforded greater insights into disease biology, but also provided useful information predicting the likelihood of any given patient achieving and maintaining remission following conventional chemotherapy, leading to the development of risk-stratified treatment approaches. However, it is becoming increasingly apparent that AML is highly heterogeneous at the molecular level. Defining the individual genetic abnormalities or combinations of markers that provide significant independent prognostic information and establishing their respective relationships to other pre-treatment characteristics that impact on outcome, such as age and presenting white blood cell count, presents a major ongoing challenge. Moreover, there is increasing evidence that risk of relapse and overall survival can be predicted by assessment of kinetics and depth of response following front-line therapy and monitoring of the leukemic burden using molecular or immunological approaches to minimal residual disease (MRD) detection. These advances present the exciting prospect that panels of pre-treatment parameters affording independent prognostic information can be integrated with precise measurement of treatment response using MRD technologies to provide greater refinement in risk-adapted management of AML. This could lead to further improvements in outcome and serve to identify in a more reliable fashion those patients most likely to benefit from allogeneic transplant in first remission.

An updated approach to the diagnosis of myeloid leukemia cutis.

The diagnosis of myeloid leukemia cutis can be difficult, particularly in the context of an initial skin biopsy with a malignant hematopoietic neoplasm. We studied the immunohistochemical characteristics of 33 cases of myeloid leukemia cutis diagnosed at Stanford University Medical Center, Stanford, CA, 1996-2007, and compared them with the corresponding bone marrow blast immunophenotype and World Health Organization classification (2008). In the skin, CD43 marked 97% of cases (32/33), myeloperoxidase marked 42% (14/33), CD68 marked 94% (31/33), CD163 marked 25% (7/28), and CD56 marked 47% (14/30). CD34 and CD117 were predominantly negative. In 19 cases in which myeloperoxidase was negative, all marked with CD68 and CD43. The flow cytometric immunophenotype of the leukemic blasts in the bone marrow was discordant with the immunohistochemical profile in the skin in all cases, showing loss or gain of at least 1 antigen. Given the immunophenotypic differences between skin and bone marrow blasts, we provide an updated immunohistochemical approach to the diagnosis of myeloid leukemia cutis.

[Classification of myeloid leukemias].

Myeloid leukemia in this series corresponds to the myeloid neoplasms of the 4th WHO classification of pathology and genetics of tumor of haematopoietic and lymphoid tissue. The myeloid neoplasms are composed of six categories, which are 1) myeloproliferative neoplasms (MPN), a new category of 2) myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1, 3) myelodysplastic syndrome (MDS)/MPN, 4) MDS, 5) acute myeloid leukemia (AML) and related precursor neoplasms, and 6) acute leukemias of ambiguous lineage. In MPNs without chronic myelogenous leukemia, the genetic marker of JAK2 V617F is added to the diagnostic criteria for polycythemia vera, essential thrombocythemia and primary myelofibrosis. MDS has the new subtype of refractory cytopenia with unilineage dysplasia composed of refractory anemia, refractory neutropenia and refractory thrombocytopenia. AML with t(9; 11) (p22;q23); MLLT3-MLL, AML with t(6;9) (p23; q34); DEK-NUP214, AML with inv(3) (q21q26.2) or t(3; 3) (q21 ; q26.2); RPN1-EVI1 and AML (megakaryoblastic) with t(1; 22) (p13; q13); RBM15-MKL1 are added to the subtype of AML with recurrent genetic abnormalities, and AML with gene mutations of NPM1 and CEBPA are also added as provisional entities of it. The myeloid neoplasms of the 4th WHO classification are comprehensive and seem to be dynamic by incorporating the results of leukemia researches.