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Myeloid/lymphoid neoplasms with eosinophilia (M/LN-eo) and tyrosine kinase (TK) gene fusions are a rare group of haematopoietic neoplasms with a broad range of clinical and morphological presentations. Paediatric cases have increasingly been recognised. Importantly, not all appear as a chronic myeloid neoplasm and eosinophilia is not always present. In addition, standard cytogenetic and molecular methods may not be sufficient to diagnose M/LN-eo due to cytogenetically cryptic aberrations. Therefore, additional evaluation with fluorescence in-situ hybridisation and other molecular genetic techniques (array-based comparative genomic hybridisation, RNA sequencing) are recommended for the identification of specific TK gene fusions. M/LN-eo with JAK2 and FLT3-rearrangements and ETV6::ABL1 fusion were recently added as a formal member to this category in the International Consensus Classification (ICC) and the 5th edition of the WHO classification (WHO-HAEM5). In addition, other less common defined genetic alterations involving TK genes have been described. This study is an update on M/LN-eo with TK gene fusions with focus on novel entities, as illustrated by cases submitted to the Bone Marrow Workshop, organised by the European Bone Marrow Working Group (EBMWG) within the frame of the 21st European Association for Haematopathology congress (EAHP-SH) in Florence 2022. A literature review was performed including paediatric cases of M/LN-eo with TK gene fusions., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

The World Health Organization classification (WHO-HAEM5) and the International Consensus Classification (ICC 2022) of myeloid neoplasms are based on the integration of clinical, morphologic, immunophenotypic, and genomic data. Flow cytometric immunophenotyping (FCIP) allows the identification, enumeration, and characterization of hematopoietic cells, and is therefore a powerful tool in the diagnosis, classification, and monitoring of hematological neoplasms. The vast majority of flow cytometry (FCM) studies in chronic myeloid neoplasms focus on its role in myelodysplastic neoplasms (MDS). FCM can also be helpful for the assessment of myeloproliferative neoplasms (MPN) and MDS/MPN, including the early detection of evolving myeloid or lymphoid blast crisis and the characterization of monocytic subsets. The classification of acute myeloid leukemia (AML) is primarily based on cytogenetic and molecular findings; however, FCIP is needed for subclassification of AML, not otherwise specified (NOS; ICC)/AML defined by differentiation (WHO-HAEM5). The main role of FCM in AML remains in making a rapid diagnosis and as a tool for measurable residual disease monitoring. Machine learning and artificial intelligence approaches can be used to analyze and classify FCM data. This article, based on an invited lecture at the 106th Annual Meeting of the German Society of Pathology in 2023, reviews the role of FCM in the classification of myeloid neoplasms, including recent publications on the application of artificial intelligence., (© 2023. The Author(s).)

Background: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures., Methods: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group., Results: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies., Conclusions: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice., (© 2021 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34 + CD19 - ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS., (© 2022 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Current guidelines recommend flow cytometric analysis as part of the diagnostic assessment of patients with cytopenia suspected for myelodysplastic syndrome. Herein we describe the complete work-up of six cases using multimodal integrated diagnostics. Flow cytometry assessments are illustrated by plots from conventional and more recent analysis tools. The cases demonstrate the added value of flow cytometry in case of hypocellular, poor quality, or ambiguous bone marrow cytomorphology. Moreover, they demonstrate how immunophenotyping results support clinical decision-making in inconclusive and clinically 'difficult' cases., (© 2022 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Background: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML)., Methods: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML)., Results: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%)., Conclusion: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter., (© 2022 International Clinical Cytometry Society.)

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients., (© 2021 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1., Objectives: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients., Methods: Nine patients were enrolled and received at least two courses of ara-C (1 g/m 2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m 2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m 2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed., Results: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 10 9 /L and to platelet recovery >50 × 10 9 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission., Conclusion: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study., (© 2022 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

Risk-adapted treatment in acute lymphoblastic leukemia (ALL) relies on genetic information and measurable residual disease (MRD) monitoring. In this proof of concept study, DNA from diagnostic bone marrow (BM) of six children with ALL, without stratifying genetics or central nervous system (CNS) involvement, underwent whole-genome sequencing (WGS) to identify structural variants (SVs) in the leukemic blasts. Unique sequences generated by SVs were targeted with patient-specific droplet digital PCR (ddPCR) assays. Genomic DNA (gDNA) from BM and cell-free DNA (cfDNA) from plasma and cerebrospinal fluid (CSF) were analyzed longitudinally. WGS with 30× coverage enabled target identification in all cases. Limit of quantifiability (LoQ) and limit of detection (LoD) for the ddPCR assays (n = 15) were up to 10 -5 and 10 -6 , respectively. All targets were readily detectable in a multiplexed ddPCR with minimal DNA input (1 ng of gDNA) at a 10 -1 dilution, and targets for half of the patients were also detectable at a 10 -2 dilution. The level of MRD in BM at end of induction and end of consolidation block 1 was in a comparable range between ddPCR and clinical routine methods for samples with detectable residual disease, although our approach consistently detected higher MRD values for patients with B-cell precursor ALL. Additionally, several samples with undetectable MRD by flow cytometry were MRD-positive by ddPCR. In plasma, the level of leukemic targets decreased in cfDNA over time following the MRD level detected in BM. cfDNA was successfully extracted from all diagnostic CSF samples (n = 6), and leukemic targets were detected in half of these. The results suggest that our approach to design molecular assays, together with ddPCR quantification, is a technically feasible option for accurate MRD quantification and that cfDNA may contribute valuable information regarding MRD and low-grade CNS involvement., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arthur, Rezayee, Mogensen, Saft, Rosenquist, Nordenskjöld, Harila-Saari, Tham and Barbany.)

Objectives: To report the findings of the 2019 Society for Hematopathology/European Association for Haematopathology Workshop within the categories of reactive eosinophilia, hypereosinophilic syndrome (HES), germline disorders with eosinophilia (GDE), and myeloid and lymphoid neoplasms associated with eosinophilia (excluding entities covered by other studies in this series)., Methods: The workshop panel reviewed 109 cases, assigned consensus diagnosis, and created diagnosis-specific sessions., Results: The most frequent diagnosis was reactive eosinophilia (35), followed by acute leukemia (24). Myeloproliferative neoplasms (MPNs) received 17 submissions, including chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Myelodysplastic syndrome (MDS), MDS/MPN, and therapy-related myeloid neoplasms received 11, while GDE and HES received 12 and 11 submissions, respectively., Conclusions: Hypereosinophilia and HES are defined by specific clinical and laboratory criteria. Eosinophilia is commonly reactive. An acute leukemic onset with eosinophilia may suggest core-binding factor acute myeloid leukemia, blast phase of chronic myeloid leukemia, BCR-ABL1-positive leukemia, or t(5;14) B-lymphoblastic leukemia. Eosinophilia is rare in MDS but common in MDS/MPN. CEL, NOS is a clinically aggressive MPN with eosinophilia as the dominant feature. Bone marrow morphology and cytogenetic and/or molecular clonality may distinguish CEL from HES. Molecular testing helps to better subclassify myeloid neoplasms with eosinophilia and to identify patients for targeted treatments., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: The aim of this study was to evaluate the diagnostic and prognostic role of multiparameter flow cytometry (FC) in patients with idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS)., Methods: We performed FC using a standardized panel and two different diagnostic algorithms (Ogata, Wells) in a well-characterized cohort of 79 patients with ICUS/CCUS and compared it with a retrospective blinded morphological evaluation and data from targeted next-generation DNA sequencing of 20 myelodysplastic syndrome (MDS)-related genes., Results: Our data show that FC has low sensitivity in distinguishing CCUS from ICUS patients (40.5% for Ogata score and 59.5% for Wells score). The Wells score was suggestive of dysplasia in ICUS/CCUS patients with concurrent morphological signs of dysplasia in the bone marrow (following re-evaluation by two hematopathologists) and in CCUS patients with a higher mutational burden. Eight patients with ICUS/CCUS from our cohort progressed to another myeloid malignancy (MDS, acute myeloid leukemia, or chronic myelomonocytic leukemia), all showing flow cytometric signs of dysplasia., Conclusion: FC performs poorly in diagnosing CCUS versus ICUS. However, it can potentially provide prognostic information in cytopenic patients by identifying a subgroup of patients with a higher grade of dysplasia, higher mutational burden, and higher risk of progression and, together with mutational screening, also identify a group of patients who might require morphological reassessment of dysplastic changes in their bone marrow., (© 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.)

Intrachromosomal amplification of chromosome 21 (iAMP21) is a cytogenetic subtype associated with relapse and poor prognosis in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). The biology behind the high relapse risk is unknown and the aim of this study was to further characterize the genomic and transcriptional landscape of iAMP21. Using DNA arrays and sequencing, we could identify rearrangements and aberrations characteristic for iAMP21. RNA sequencing revealed that only half of the genes in the minimal region of amplification (20/45) were differentially expressed in iAMP21. Among them were the top overexpressed genes ( p  < 0.001) in iAMP21 vs. BCP ALL without iAMP21 and three candidate genes could be identified, the tyrosine kinase gene DYRK1A and chromatin remodeling genes CHAF1B and SON. While overexpression of DYRK1A and CHAF1B is associated with poor prognosis in malignant diseases including myeloid leukemia, this is the first study to show significant correlation with iAMP21-positive ALL.

The 13th workshop of the European Bone Marrow Working Group in Utrecht, The Netherlands, was devoted to studying myelodysplastic syndromes (MDS) and their boundaries. The panel received 44 cases submitted to the 3 invited categories, which included: reactive cytopenias with dysplasia, idiopathic cytopenia of undetermined significance, clonal haematopoiesis of indeterminate potential, idiopathic dysplasia of uncertain significance and overt MDS. For this summary, we have selected 17 cases that highlight difficulties in separating true MDS from other causes of cytopenia and the intricate relationship between clonal haematopoiesis and true MDS. In addition, cases of overt MDS with challenging features were also selected. All cases were stained for p53 expression. Using instructive submitted cases we discuss the following: (1) cytopenia with clonal haematopoiesis not fulfilling MDS criteria, (2) cytopenia and/or dysplasia with germline mutations and/or familial history suggesting an underlying gene defect, (3) MDS based on a recurrent chromosomal abnormality and (4) overt MDS with diagnostic difficulties due to concurrent treatment or disease. The lively discussion in the open forum of the workshop illustrated the need for better integrative understanding of the evolution of acquired genetic abnormalities in haematopoiesis, and the challenge of diagnosing true MDS in cytopenic patients with genetic abnormalities, either germline or acquired., (© 2018 S. Karger AG, Basel.)

Diagnosis of myelodysplastic syndromes (MDS) is not straightforward when objective data, such as blast excess and abnormal cytogenetics, are lacking. Expert laboratories use flow cytometry (FCM) to help diagnose MDS. However, most of FCM protocols for MDS are complex, requiring a high level of expertise and high cost. We have reported a FCM mini-panel consisting of four FCM parameters (so-called Ogata score), which is simple to conduct and inexpensive. In this paper, to refine this mini-panel, we have introduced a new FCM parameter, which quantifies CD33 expression on CD34+ cells (called Granulocyte/CD34 cell CD33 ratio). Bone marrow cells from MDS without blast excess (low-grade MDS) and controls were stained with CD34, CD45, and CD33 and analyzed for five parameters ("Granulocyte/CD34 cell CD33 ratio" plus four parameters in the Ogata score). By a multivariate logistic regression model, only three parameters, including "Granulocyte/CD34 cell CD33 ratio" had statistically significant power for diagnosing low-grade MDS. Based on the results, we constructed a new scoring system, which showed approximately 50% sensitivity and more than 95% specificity in diagnosing low-grade MDS. Our revised mini-panel is suitable for screening samples suspected for MDS and provides a basis for further improvement in diagnostic FCM protocols for MDS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The mechanisms underlying lenalidomide-resistance of del(5q) MDS stem cells remain to be elucidated and may include cell-intrinsic as well as microenvironmental causes. Abnormal hypolobated megakaryocytes constitute one of the hallmarks of del(5q) MDS. We hypothesized that these cells have potential implications for the regulation of haematopoietic stem cells (HSC) similarly to what has recently been described for megakaryocytes in the murine system. Therefore, we conducted a study to determine the response of abnormal hypolobated megakaryocytes to lenalidomide therapy. We studied lenalidomide-treated patients in the MDS-004 trial as well as a cohort seen at our institution. Morphological evaluation at time of complete cytogenetic remission (CCyR) demonstrated the persistence of hypolobated megakaryocytes in all evaluable patients (n = 9). Furthermore, we provide evidence that the abnormal hypolobated morphology is restricted to del(5q) megakaryocytes, both at diagnosis and during CCyR. Using fluorescence in situ hybridisation analysis on flow-sorted stem- and progenitor populations, we observed a similar degree of clonal involvement in megakaryocyte-erythroid-progenitors as in HSC. Taken together, our findings suggest that megakaryocyte morphology might aid in the evaluation of patients where discontinuation of lenalidomide is considered and offers interesting hypotheses for further investigation of lenalidomide resistance., (© 2018 John Wiley & Sons Ltd.)

Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal ( SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS., (© 2017 by The American Society of Hematology.)

The European LeukemiaNet MDS (EUMDS) registry is collecting data of myelodysplastic syndrome (MDS) patients belonging to the IPSS low or intermediate-1 category, newly diagnosed by local cytologists. The diagnosis of MDS can be challenging, and some data report inter-observer variability with regard to the assessment of the MDS subtype. In order to ensure that correct diagnoses were made by the participating centres, blood and bone marrow slides of 10% of the first 1000 patients were reviewed by an 11-person panel of cytomorphologists. All slides were rated by at least 3 panel members (median 8 panel members; range 3-9). Marrow slides from 98 out of 105 patients were of good quality and therefore could be rated properly according to the WHO 2001 classification, including assessment of dysplastic lineages. The agreement between the reviewers whether the diagnosis was MDS or non-MDS was strong with an intra-class correlation coefficient (ICC) of 0.85. Six cases were detected not to fit the entry criteria of the registry, because they were diagnosed uniformly as CMML or AML by the panel members. The agreement by WHO 2001 classification was strong as well (ICC = 0.83). The concordance of the assessment of dysplastic lineages was substantial for megakaryopoiesis and myelopoiesis and moderate for erythropoiesis. Our data show that in general, the inter-observer agreement was high and a very low percentage of misdiagnosed cases had been entered into the EUMDS registry. Further studies including histomorphology are warranted.

A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53 -mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53 , RUNX1 , and TET2 , respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making., (Copyright© Ferrata Storti Foundation.)

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117 + erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage., (Copyright© Ferrata Storti Foundation.)

Cytopenia is common in the elderly population and etiology may be difficult to assess. Here, we investigated the occurrence of mutations in patients with idiopathic cytopenia of undetermined significance and the usefulness in improving diagnostics. We included 60 patients with persistent cytopenia > 6 months without definite diagnosis of hematological neoplasm after routine assessment. Bone marrow material underwent a blinded morphology review and DNA was sequenced with a targeted 20 gene panel representing the most commonly mutated genes in myelodysplastic syndrome. Thirty seven (62%) patients carried at least one mutation at inclusion, and of these 95% carried a mutation in TET2, ASXL1, SRSF2, or DNMT3A. The most commonly mutated gene was TET2 observed in 43% of all patients. During one to eight years follow-up seven patients progressed to a myeloid neoplasm and six of these had a detectable mutation at study entry. Median time to progression was 53 months (range 10-78), and at time of progression each patient had at least two mutations detected. Mutations in TP53 and NRAS were not present in patients at inclusion, but identified as secondary hits triggering progression. The morphology review was concordant in 68% of all cases, and 93% of the cases reclassified into the group "highly suspicious for MDS" had a mutation. All patients who had a concordant review "highly suspicious for MDS" had at least two mutations detected. Overall, we show that morphology examination is challenging in this heterogeneous group and targeted sequencing helps identify patients at risk of progression. Am. J. Hematol. 91:1234-1238, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The telomerase reverse transcriptase (TERT) gene rs2736100&#95;C allele has recently been shown to be associated with an increased risk for myeloproliferative neoplasms (MPNs) among Caucasians. However, it is unknown if this association is present in other ethnical populations and whether rs2736100 allele frequencies mirror the incidence of MPNs in a population. Here we genotyped TERT rs2736100 variants in 126 Swedish and 101 Chinese MPN patients and their age-, sex-, and ethnically-matched healthy controls. Healthy Chinese adults had a higher frequency of the A allele and lower frequencies of the C allele compared to Swedish counterparts (57.4 vs 47.0 % for A, 42.6 vs 53.0 % for C, P = 0.006). Both Swedish and Chinese patients harbored significantly higher C allele frequency than their controls (62.7 vs 53.0 % and 57.4 vs 42.6 % for Swedish and Chinese, respectively, P = 0.004). Swedes and Chinese bearing the CC genotype had a significantly increased risk of MPN compared to AA carriers (OR = 2.47; 95 % CI: 1.33-4.57, P = 0.003, for Swedes, and OR = 3.45; 95 % CI: 1.52-7.85, P = 0.005, for Chinese). Further analyses showed that rs2736100&#95;CC was associated with robustly enhanced risk in males only (CC vs AA, OR = 5.11; 95 % CI: 2.19-11.92, P < 0.0001). The CC-carrying MPN patients exhibited significantly higher TERT expression than patients with the AC genotype. Collectively, the rs2736100&#95;C is a risk allele for MPNs in Swedish and Chinese males, and the lower incidence of MPNs in the Chinese population is correlated with a lower rs2736100&#95;C risk allele frequency., Competing Interests: Compliance with ethical standards The study was approved by the Shandong University Second Hospital Ethics Committee and Stockholm Regional Ethics Review Board, and informed consent was obtained from all the participants. Conflict of interest The authors declare that they have no conflict of interest.

Background: Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement based on cytomorphological examination of the cerebrospinal fluid (CSF) alone is not as sensitive with low cell counts as flow cytometric immunophenotyping (FCI) of the CSF. However, the importance of low CSF blasts counts at diagnosis is uncertain. We sought to determine the significance of FCI in relation to conventional morphological examination., Procedure: We retrospectively compared FCI of the CSF with cytomorphology at diagnosis or relapse of childhood ALL. All patients were diagnosed 2000-2012 in Stockholm or Umeå, Sweden. Clinical data were collected from medical records and the Nordic leukemia registry. Treatment assignment was based on morphological examination only., Results: The cohort was comprised of 214 patients with ALL. CSF involvement was detected by both methods in 20 patients, in 17 by FCI alone, and in one patient by cytomorphology alone. The relapse rate was higher for patients with negative cytology but positive FCI compared to those without CNS involvement using both methods. The difference was especially marked in the current protocol. However, none of the patients with negative CSF cytology but positive FCI had a CNS relapse., Conclusions: FCI of the CSF increased the detection rate of CNS involvement of ALL approximately two times compared to cytomorphology. Patients with low-level CNS involvement may benefit from additional intensified systemic or CNS-directed therapy, but larger studies are needed., (© 2014 Wiley Periodicals, Inc.)

Objectives: Thrombocytopenia is an independent adverse prognostic factor in patients with Myelodysplastic syndromes (MDS). Azacitidine, first-line treatment for the majority of patients with higher-risk MDS, is associated with aggravated thrombocytopenia during the first cycles. Eltrombopag is a novel thrombopoietin receptor agonist, which also has been shown to inhibit proliferation of leukaemia cell lines in vitro. This phase I clinical trial was designed to explore the safety and tolerability of combining eltrombopag with azacitidine in patients with MDS. In addition, we assessed the potential effects of eltrombopag on hematopoietic stem and progenitor cells (HSPCs) from included patients., Patients and Methods: Previously untreated patients with MDS eligible for treatment with azacitidine and with a platelet count <75 × 10(9) /L were included. Patients received eltrombopag in dose escalation cohorts during three cycles of azacitidine., Results: Twelve patients, with a median age of 74 yr, were included. Severe adverse events included infectious complications, deep vein thrombosis and transient ischaemic attack. The maximal tolerated eltrombopag dose was 200 mg qd. Complete remission or bone marrow remission was achieved in 4 of 12 patients. Platelet counts improved or remained stable in 9 of 12 patients despite azacitidine treatment. No increase in blast count, disease progression, or bone marrow fibrosis related to study medication was reported. Eltrombopag did not induce cycling of HSPCs., Conclusion: The combination of eltrombopag with azacitidine in high-risk MDS patients is feasible and well tolerated. Improvements in platelet counts and the potential antileukaemic effect of eltrombopag should be explored in a randomised study., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The cannabinoid receptors are upregulated in many types of cancers, including mantle cell lymphoma (MCL) and have been suggested to constitute novel therapeutic targets. The expression pattern of the key members of the endocannabinoid system was analyzed in a well-characterized MCL patient cohort and correlated to biological features. 107 tumor tissues were analyzed for the mRNA levels of cannabinoid receptors 1 and 2 (CNR1 and CNR2) and the two main enzymes regulating the endocannabinoid anandamide levels in tissue: NAPEPLD and FAAH (participating in synthesis and degradation, respectively). NAPEPLD, CNR1 and CNR2 were overexpressed while FAAH expression was reduced in MCL compared to non-malignant B-cells. Both low CNR1 and high FAAH levels correlated with lymphocytosis (p=0.016 and p=0.022, respectively) and with leukocytosis (p=0.0018 and p=0.047). Weak to moderate CNR1 levels were a feature of SOX11 negative MCL (p=0.006). Both high CNR2 and high FAAH levels correlated to anemia (p=0.0006 and p=0.038, respectively). In conclusion, the relative expression of the anandamide synthesizing and metabolizing enzymes in MCL is heavily perturbed. This finding, together with high expression of cannabinoid receptors, could favor enhanced anandamide signaling and suggest that targeting the endocannabinoid system might be considered as part of lymphoma therapy.

Dendritic cells (DC) are antigen-presenting cells that play a pivotal role in coordinating functions of the immune system. Previous studies suggest that bone marrow (BM) failure in myelodysplastic syndromes (MDS) may be in part immune-mediated, and that the high propensity for relapse may reflect decreased immune surveillance. This study aimed to assess the frequency of DC in BM samples from well-annotated untreated MDS patients by using 4-colour flow cytometry. DC levels were markedly reduced in all subtypes of MDS. The clinical impact of this finding on therapy response and relapse after, e.g. allogeneic stem cell transplantation warrants further investigation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: To determine the frequency of TP53 mutations and the level of p53 protein expression by immunohistochemistry (IHC) in low-risk myelodysplastic syndromes (MDS) with del(5q) and to assess their impact on disease progression., Patients and Methods: Pre- and postprogression bone marrow (BM) samples from 55 consecutive patients with International Prognostic Scoring System low risk (n = 32) or intermediate-1 risk (n = 23) were studied by next-generation sequencing of TP53. IHC for p53 was performed on 148 sequential BM samples., Results: TP53 mutations with a median clone size of 11% (range, 1% to 54%) were detected in 10 patients (18%) already at an early phase of the disease. Mutations were equally common in low-risk and intermediate-1-risk patients and were associated with evolution to acute myeloid leukemia (5 of 10 v 7 of 45; P = .045). Nine of 10 patients carrying mutations showed more than 2% BM progenitors with strong p53 staining. The probability of a complete cytogenetic response to lenalidomide was lower in mutated patients (0 of 7 v 12 of 24; P = .024)., Conclusion: By using sensitive deep-sequencing technology, we demonstrated that TP53 mutated populations may occur at an early disease stage in almost a fifth of low-risk MDS patients with del(5q). Importantly, mutations were present years before disease progression and were associated with an increased risk of leukemic evolution. TP53 mutations could not be predicted by common clinical features but were associated with p53 overexpression. Our findings indicate a previously unrecognized heterogeneity of the disease which may significantly affect clinical decision making.

Background: The size of colorectal polyps is important in the clinical management of these lesions., Aim: To audit the accuracy in calculating the size of "polyps" by various specialists., Materials and Methods: Eighteen pathologists and four surgeons measured, with a conventional millimetre ruler, the largest diameter of 12 polyp phantoms. The results of two independent measurements (two weeks apart) were compared with the gold standard-size assessed at The Royal Institute of Technology, Sweden., Results: Thirty-one percent (83/264-trial 1) and 33% (88/264-trial 2) of the measurements underestimated or overestimated the gold standard size by >1 mm. Of the 22 experienced participants, 95% (21/22-trial 1) and 91% (20/22-trial 2) misjudged by >1 mm the size of one or more polyps. Values given by 13 participants (4.9%) in trial 1 and by 15 participants (5.7%) in trial 2, differed by > or = +/-4 mm from the gold standard size. In addition, a big difference between the highest and the lowest values was recorded in some polyps (up to 11.4 mm). Those disparate values were regarded as a human error in reading the scale on the ruler., Conclusion: Using a conventional ruler (the tool of pathologists worldwide) unacceptably high intra-observer and inter-observer variations in assessing the size of polyp-phantoms was found. The volume and the shape of devices, as well as human error in reading the scale of the ruler were confounding factors in size assessment. In praxis, the size is crucial in the management of colorectal polyps. Considering the clinical implications of the results obtained, the possibility of developing a method that will allow assessment of the true size of removed clinical polyps is being explored.