The role of flow cytometry in the classification of myeloid disorders.

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.

Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes: A workshop report with focus on novel entities and a literature review including paediatric cases.

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.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

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.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

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.

A series of case studies illustrating the role of flow cytometry in the diagnostic work-up of myelodysplastic syndromes.

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.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

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.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

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.

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

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/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 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 × 109 /L and to platelet recovery >50 × 109 /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.

Patient-Specific Assays Based on Whole-Genome Sequencing Data to Measure Residual Disease in Children With Acute Lymphoblastic Leukemia: A Proof of Concept Study.

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.

Intracerebral manifestation of iatrogenic, immunodeficiency-associated polymorphic B-LPD with morphology mimicking Hodgkin lymphoma: a case report and literature review.

Iatrogenic immunodeficiency-associated lymphoproliferative disorders (IA-LPD) may arise in patients treated with immunosuppressive drugs for autoimmune disease or other conditions. Polymorphic EBV-positive B-lymphoproliferations often have features mimicking Hodgkin lymphoma and typically a self-limited, indolent course. We present an unusual case with isolated, intracerebral manifestation of polymorphic B-LPD with features of classic Hodgkin-lymphoma in an immunosuppressed patient treated with methotrexate and infliximab, including clinical-radiological features and a detailed description of morphological findings, together with a literature review on reported cases  of primary CNS manifestation of cHL and IA-LPD with Hodgkin-like morphology. The patient achieved complete remission following neurosurgery with gross total tumor resection and drug withdrawal without any additional treatment. Post-operative staging revealed no evidence for focal relapse or systemic disease during the 18 months follow-up period. Among the previously reported 24 cases of primary, isolated Hodgkin lymphoma in the central nervous system, three similar cases of iatrogenic, IA-LPDs were identified and are discussed here. Polymorphic B-LPD are destructive lesions with a range of morphologic features and disease manifestations. It is clinically important to recognize the spectrum of proliferations with features of classic Hodgkin lymphoma in immunodeficiency, iatrogenic settings, because they are likely to impact the choice of treatment strategies. Supplementary Information: The online version contains supplementary material available at 10.1007/s12308-021-00478-0.

Case Report: Whole genome sequencing identifies CCDC88C as a novel JAK2 fusion partner in pediatric T-cell acute lymphoblastic leukemia.

In the present report, we applied whole genome sequencing (WGS) to genetically characterize a case of pediatric T-cell acute lymphoblastic leukemia (ALL) refractory to standard therapy. WGS identified a novel JAK2 fusion, with CCDC88C as a partner. CCDC88C encodes a protein part of the Wnt signaling pathway and has previously been described in hematological malignancies as fusion partner to FLT3 and PDGFRB. The novel CCDC88C::JAK2 fusion gene results in a fusion transcript, predicted to produce a hybrid protein, which retains the kinase domain of JAK2 and is expected to respond to JAK2 inhibitors. This report illustrates the potential of WGS in the diagnostic setting of ALL.

Eosinophilia/Hypereosinophilia in the Setting of Reactive and Idiopathic Causes, Well-Defined Myeloid or Lymphoid Leukemias, or Germline Disorders.

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.

The diagnostic and prognostic role of flow cytometry in idiopathic and clonal cytopenia of undetermined significance (ICUS/CCUS): A single-center analysis of 79 patients.

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.

Overexpression of chromatin remodeling and tyrosine kinase genes in iAMP21-positive acute lymphoblastic leukemia.

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.

Challenges in Diagnosing Myelodysplastic Syndromes in the Era of Genetic Testing: Proceedings of the 13th Workshop of the European Bone Marrow Working Group.

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.

Revising flow cytometric mini-panel for diagnosing low-grade myelodysplastic syndromes: Introducing a parameter quantifying CD33 expression on CD34+ cells.

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.

Megakaryocytes harbour the del(5q) abnormality despite complete clinical and cytogenetic remission induced by lenalidomide treatment.

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.