Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry.

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group "CytHem-LMMC" are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.

[MDS & CMML: Diagnostic and classification]. / SMD & LMMC : diagnostic et classification.

In 2023, a diagnosis process of myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML) is mainly based on morphological results obtained on bone marrow and blood smears which could be completed by cytogenetical analyses. Due to recent finding, flow cytometry data are recognized as useful for the diagnosis of CMML especially. Actual classifications and prognostic scoring systems have changed and nowadays include results of high-throughput sequencing approaches in addition to cytogenetical results. All together, these data allow the medical world to correctly evaluate the prognosis of these patients and to provide some information for targeted therapies. This chapter will provide the most important modifications recently published in the field of diagnosis and prognosis of MDS and CMML.

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.

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.

ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

BACKGROUND: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB. METHODS: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available. RESULTS: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB. CONCLUSIONS: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples.

Evaluation of two new highly multiplexed PCR assays as an alternative to next-generation sequencing for IDH1/2 mutation detection.

IDH1 and IDH2 somatic mutations have been identified in solid tumors and blood malignancies. The development of inhibitors of mutant IDH1 and IDH2 in the past few years has prompted the development of a fast and sensitive assay to detect IDH1R132 , IDH2R140 and IDH2R172 mutations to identify patients eligible for these targeted therapies. This study aimed to compare two new multiplexed PCR assays - an automated quantitative PCR (qPCR) on the PGX platform and a droplet digital PCR (ddPCR) with next-generation sequencing (NGS) for IDH1/2 mutation detection. These assays were evaluated on 102 DNA extracted from patient peripheral blood, bone marrow and formalin-fixed paraffin-embedded tissue samples with mutation allelic frequency ranging from 0.6% to 45.6%. The ddPCR assay had better analytical performances than the PGX assay with 100% specificity, 100% sensitivity and a detection limit down to 0.5% on IDH1R132 , IDH2R140 and IDH2R172 codons, and a high correlation with NGS results. Therefore, the new highly multiplexed ddPCR is a fast and cost-effective assay that meets most clinical needs to identify and follow cancer patients in the era of anti-IDH1/2-targeted therapies.

Fusion Gene Detection and Quantification by Asymmetric Capture Sequencing (aCAP-Seq).

Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, µBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.

Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes.

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.

Machine learning identifies the independent role of dysplasia in the prediction of response to chemotherapy in AML.

The independent prognostic impact of specific dysplastic features in acute myeloid leukemia (AML) remains controversial and may vary between genomic subtypes. We apply a machine learning framework to dissect the relative contribution of centrally reviewed dysplastic features and oncogenetics in 190 patients with de novo AML treated in ALFA clinical trials. One hundred and thirty-five (71%) patients achieved complete response after the first induction course (CR). Dysgranulopoiesis, dyserythropoiesis and dysmegakaryopoiesis were assessable in 84%, 83% and 63% patients, respectively. Multi-lineage dysplasia was present in 27% of assessable patients. Micromegakaryocytes (q = 0.01), hypolobulated megakaryocytes (q = 0.08) and hyposegmented granulocytes (q = 0.08) were associated with higher ELN-2017 risk. Using a supervised learning algorithm, the relative importance of morphological variables (34%) for the prediction of CR was higher than demographic (5%), clinical (2%), cytogenetic (25%), molecular (29%), and treatment (5%) variables. Though dysplasias had limited predictive impact on survival, a multivariate logistic regression identified the presence of hypolobulated megakaryocytes (p = 0.014) and micromegakaryocytes (p = 0.035) as predicting lower CR rates, independently of monosomy 7 (p = 0.013), TP53 (p = 0.004), and NPM1 mutations (p = 0.025). Assessment of these specific dysmegakarypoiesis traits, for which we identify a transcriptomic signature, may thus guide treatment allocation in AML.

Cytokine-like protein 1-induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML.

Mouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56- in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.

Exome sequencing for diagnosis of congenital hemolytic anemia.

BACKGROUND: Congenital hemolytic anemia constitutes a heterogeneous group of rare genetic disorders of red blood cells. Diagnosis is based on clinical data, family history and phenotypic testing, genetic analyses being usually performed as a late step. In this study, we explored 40 patients with congenital hemolytic anemia by whole exome sequencing: 20 patients with hereditary spherocytosis and 20 patients with unexplained hemolysis. RESULTS: A probable genetic cause of disease was identified in 82.5% of the patients (33/40): 100% of those with suspected hereditary spherocytosis (20/20) and 65% of those with unexplained hemolysis (13/20). We found that several patients carried genetic variations in more than one gene (3/20 in the hereditary spherocytosis group, 6/13 fully elucidated patients in the unexplained hemolysis group), giving a more accurate picture of the genetic complexity of congenital hemolytic anemia. In addition, whole exome sequencing allowed us to identify genetic variants in non-congenital hemolytic anemia genes that explained part of the phenotype in 3 patients. CONCLUSION: The rapid development of next generation sequencing has rendered the genetic study of these diseases much easier and cheaper. Whole exome sequencing in congenital hemolytic anemia could provide a more precise and quicker diagnosis, improve patients' healthcare and probably has to be democratized notably for complex cases.

Incorporating flow cytometry and next-generation sequencing in the diagnosis of CMML. Are we ready for prime?

In the last version of the WHO classification of myeloid malignancies, flow cytometry and molecular investigation are listed as potentially useful, yet non-essential diagnostic tools in hard-to-recognize chronic myelomonocytic leukemias (CMML). Flow recognition of CMML was initially based on an increase in the fraction of peripheral blood, CD14+,CD16- classical monocytes ≥94% of total monocytes. An associated inflammatory disease can preclude the detection of classical monocyte fraction increase by inducing accumulation of CD14+,CD16+ intermediate monocytes. In such a situation, decrease in the Slan+,CD14low,CD16+ non-classical monocyte fraction below 1.7% still supports CMML diagnosis. This robust, two-step flow cytometry assay identifies CMML with a very high sensitivity. Otherwise, detection of one or several acquired gene mutations with high variant allele frequency supports the diagnosis of CMML, oligomonocytic CMML or clonal monocytosis of clinical significance. Together, recent investigations support integration of flow cytometry analysis of peripheral blood monocyte subsets and new generation sequencing of a panel of 20-30 recurrently mutated genes in the diagnostic work-up of CMML.