Artificial intelligence to empower diagnosis of myelodysplastic syndromes by multiparametric flow cytometry.

The diagnosis of myelodysplastic syndromes (MDS) might be challenging and relies on the convergence of cytological, cytogenetic, and molecular factors. Multiparametric flow cytometry (MFC) helps diagnose MDS, especially when other features do not contribute to the decision-making process, but its usefulness remains underestimated, mostly due to a lack of standardization of cytometers. We present here an innovative model integrating artificial intelligence (AI) with MFC to improve the diagnosis and the classification of MDS. We develop a machine learning model through an elasticnet algorithm directed on a cohort of 191 patients, only based on flow cytometry parameters selected by the Boruta algorithm, to build a simple but reliable prediction score with five parameters. Our AI-assisted MDS prediction score greatly improves the sensitivity of the Ogata score while keeping an excellent specificity validated on an external cohort of 89 patients with an Area Under the Curve of 0.935. This model allows the diagnosis of both high- and low-risk MDS with 91.8% sensitivity and 92.5% specificity. Interestingly, it highlights a progressive evolution of the score from clonal hematopoiesis of indeterminate potential (CHIP) to highrisk MDS, suggesting a linear evolution between these different stages. By significantly decreasing the overall misclassification of 52% for patients with MDS and of 31.3% for those without MDS (P=0.02), our AI-assisted prediction score outperforms the Ogata score and positions itself as a reliable tool to help diagnose MDS.

Machine learning-based improvement of MDS-CBC score brings platelets into the limelight to optimize smear review in the hematology laboratory.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal hematopoietic diseases of the elderly characterized by chronic cytopenias, ineffective and dysplastic haematopoiesis, recurrent genetic abnormalities and increased risk of progression to acute myeloid leukemia. A challenge of routine laboratory Complete Blood Counts (CBC) is to correctly identify MDS patients while simultaneously avoiding excess smear reviews. To optimize smear review, the latest generations of hematology analyzers provide new cell population data (CPD) parameters with an increased ability to screen MDS, among which the previously described MDS-CBC Score, based on Absolute Neutrophil Count (ANC), structural neutrophil dispersion (Ne-WX) and mean corpuscular volume (MCV). Ne-WX is increased in the presence of hypogranulated/degranulated neutrophils, a hallmark of dysplasia in the context of MDS or chronic myelomonocytic leukemia. Ne-WX and MCV are CPD derived from leukocytes and red blood cells, therefore the MDS-CBC score does not include any platelet-derived CPD. We asked whether this score could be improved by adding the immature platelet fraction (IPF), a CPD used as a surrogate marker of dysplastic thrombopoiesis. METHODS: Here, we studied a cohort of more than 500 individuals with cytopenias, including 168 MDS patients. In a first step, we used Breiman's random forests algorithm, a machine-learning approach, to identify the most relevant parameters for MDS prediction. We then designed Classification And Regression Trees (CART) to evaluate, using resampling, the effect of model tuning parameters on performance and choose the "optimal" model across these parameters. RESULTS: Using random forests algorithm, we identified Ne-WX and IPF as the strongest discriminatory predictors, explaining 37 and 33% of diagnoses respectively. To obtain "simplified" trees, which could be easily implemented into laboratory middlewares, we designed CART combining MDS-CBC score and IPF. Optimal results were obtained using a MDS-CBC score threshold equal to 0.23, and an IPF threshold equal to 3%. CONCLUSIONS: We propose an extended MDS-CBC score, including CPD from the three myeloid lineages, to improve MDS diagnosis on routine laboratory CBCs and optimize smear reviews.

Automated Detection of Dysplasia: Data Mining from Our Hematology Analyzers.

Myelodysplastic syndromes (MDSs) are clonal hematopoietic diseases of the elderly, characterized by chronic cytopenia, ineffective and dysplastic hematopoiesis, recurrent genetic abnormalities and increased risk of progression to acute myeloid leukemia. Diagnosis on a complete blood count (CBC) can be challenging due to numerous other non-neoplastic causes of cytopenias. New generations of hematology analyzers provide cell population data (CPD) that can be exploited to reliably detect MDSs from a routine CBC. In this review, we first describe the different technologies used to obtain CPD. We then give an overview of the currently available data regarding the performance of CPD for each lineage in the diagnostic workup of MDSs. Adequate exploitation of CPD can yield very strong diagnostic performances allowing for faster diagnosis and reduction of time-consuming slide reviews in the hematology laboratory.

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.

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment.

Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow.

Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr-/- cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34+ cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation.

Phenotypic landscape of granulocytes and monocytes by multiparametric flow cytometry: A prospective study of a 1-tube panel strategy for diagnosis and prognosis of patients with MDS.

BACKGROUND: Multiparametric flow cytometry (MFC) was recently reported to be a helpful additional tool in the diagnosis of myelodysplastic syndromes (MDS). However, numerous aberrancies have been reported that makes their evaluation difficult as part of a routine diagnosis. METHODS: Here, we validated a 1-tube panel for the evaluation of granulocytic and monocytic maturation by MFC and correlated our findings with diagnosis and prognosis of MDS. A total of 251 samples with MDS suspicion were prospectively analyzed and compared to an internal reference database leading to the calculation of the Diff score. RESULTS: The associated specificity and sensitivity values of this scoring system were 92.1% and 60.4% in a first learning cohort and 96.7% and 65.2% in a second independent validation cohort. The combination of the Diff score with the concomitantly calculated Ogata score increased the sensitivity to 74.2% and 78.3% in the learning and validation cohorts, respectively. Finally, a normal Diff score in MDS patients was associated with a significant prolonged progression-free survival. CONCLUSIONS: Taken together, the present data indicate that our strategy is a sensitive and specific MFC tool for the diagnosis of MDS-related cytopenia(s) which could be also useful for predicting evolution of these diseases.

A hierarchical approach in the diagnostic workflow of chronic myelomonocytic leukemia: Pivotal role of the "Mono-dysplasia-score" combined with flow cytometric quantification of monocyte subsets.

INTRODUCTION: Monocytosis is a frequent trigger for blood smear review in a routine hematology laboratory whereas chronic myelomonocytic leukemia (CMML) is infrequent and arises mostly in elderly patients. In order to define the best workflow for monocytosis, we studied three diagnostic approaches: the classical morphology approach (blood smear review), the flow cytometry assay (quantification of monocyte subsets as described by Selimoglu-Buet et al in 2015), and the "mono-dysplasia-score" also referred to as "Monoscore (as described by our team in 2018 using the structural parameters of the Sysmex XN™ analyzers). METHODS: Studying a multicentric cohort of 196 nonclonal monocytoses and CMML patients aged over 50 years, we compared the diagnostic performance of the three approaches alone and in combination to propose a diagnostic decision tree. RESULTS: In patients presenting with additional criteria for slide review to monocytosis (37% of our cohort), we propose to sequentially combine morphology, Monoscore, and flow cytometry. On the contrary, for patients with isolated monocytosis (63%), slide review is not mandatory and we suggest performing flow cytometry depending on the Monoscore value. Using the proposed algorithm, 98% of CMML patients would have been correctly identified, slide review rate drastically reduced, and flow cytometry would have been carried out in 44% of patients. CONCLUSION: We have shown that implementation of Monoscore is a useful input filter to significantly reduce slide reviews without losing sensitivity and that flow cytometry is a performant technique in the second step of the diagnostic workup of CMML.

The fraction of CD117/c-KIT-expressing erythroid precursors predicts ESA response in low-risk myelodysplastic syndromes.

BACKGROUND: Compelling evidence has emerged for the relevance of flow cytometry (FC) in the diagnostic work-up of myelodysplastic syndromes (MDS) but due to technical issues, the erythroid lineage has been under investigated, specifically in the therapeutic context. METHODS: Using the "no red cell lysis" method developed to set up the RED-score, we specifically quantified the fraction of CD117/c-KIT-expressing erythroid precursors in a cohort of 144 MDS patients and studied the correlation with response to erythropoiesis-stimulating agents (ESA) in a sub cohort of 63 low-risk MDS patients. RESULTS: We confirmed the previously reported increase in CD117/c-KIT-expressing erythroid precursors in a subset of MDS patients and demonstrated a strong association between a cut off of CD117/c-KIT-expressing erythroid precursors ≥3% and ESA response (P = 0.001), independent of red blood cell requirement. From our observations, we hypothesized that a decrease in CD117/c-KIT-expressing erythroid precursors could be a mechanism of ESA failure. Moreover, the fraction of CD117/c-KIT-expressing erythroid precursors was correlated with progression-free survival in low-risk MDS patients (P = 0.018). In vitro, we demonstrated in an EPO dependent cell line that CD117/c-KIT expression is necessary for cell survival under EPO stimulation. CONCLUSIONS: The quantification of the CD117/c-KIT-expressing erythroid precursors could be proposed as a new theranostic and prognostic marker in MDS treated by ESA. Future studies will be required to determine whether modulating CD117/c-KIT expression and signaling could be used to improve anemia in MDS. © 2019 International Clinical Cytometry Society.

[Acquired hemophilia A associated with bullous pemphigoid and multiple myeloma: a case report]. / Hémophilie A acquise associée à une pemphigoïde bulleuse et à un myélome multiple : à propos d'un cas.

Acquired hemophilia A (AHA) is a rare and potentially severe bleeding disorder caused by circulating autoantibodies directed against factor (F) VIII. Apart from idiopathic cases, AHA is associated with autoimmune diseases, cancers, use of medications, pregnancy and the post-partum period. We report the case of a 78-year-old a male patient presenting with symptoms of a hematoma after a fall three days previously. He is medically followed for multiple myeloma and bullous pemphigoid. Laboratory investigations revealed isolated and recent increased of activated partial thromboplastin time (80,6/32,1s) and a markedly low FVIII activity (< 1%). The high-titer of FVIII inhibitor (19 Bethesda units/mL) confirmed the diagnosis of AHA diagnosis. The symptoms were noticeably alleviated following bortezomib, cyclophosphamide and dexamethasone therapy. This report describes a rare case of AHA associated with bullous pemphigoid and multiple myeloma. These pathologies induce an immunity modification that can predispose or be associated with the development of anti-FVIII inhibitors. This case illustrates two possible physiopathological hypotheses for the development of AHA, which will be discussed in this case report.

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (P=0.05) and a hepcidin:ferritin ratio <9 (P=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (P=0.0008 and P=0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. ClinicalTrials.gov registration: NCT 03598582.

CD13 expression in B cell malignancies is a hallmark of plasmacytic differentiation.

The diagnosis of Waldenström Macroglobulinaemia (WM)/lymphoplasmacytic lymphoma (LPL) remains one of exclusion because other B-cell lymphoproliferative disorders (B-LPD), such as marginal zone lymphoma (MZL), can fulfil similar criteria, including MYD88 L265P mutation. It has been suggested that expression of the myeloid marker CD13 (also termed ANPEP) is more frequent in LPL than in other B-LPD and has also been described on normal and malignant plasma cells. Here, CD13 expression was tested in a cohort of 1037 B-LPD patients from 3 centres by flow cytometry. The percentage of CD13-expressing cells was found to be variable among B-LPD but significantly higher in WM/LPL (median 31% vs. 0% in non-WM/LPL, P < 0·001). In multivariate linear regression, CD13 expression remained significantly associated with a diagnosis of WM/LPL (P < 0·001). A cut-off value of 2% of CD19+ cells co-expressing CD13 yielded the best diagnostic performance for WM/LPL assertion. This was further improved by association with the presence or absence of IgM paraprotein. Finally, given that previously published transcriptomic data revealed no difference in CD13 (also termed ANPEP) mRNA between normal and pathological B-cells, the hypothesis of some post-transcriptional regulation must be favoured. These results suggest that testing for CD13 expression in routine flow cytometry panels could help to discriminate WM/LPL from other B-LPD.

A new approach for diagnosing chronic myelomonocytic leukemia using structural parameters of Sysmex XNTM analyzers in routine laboratory practice.

According to WHO recommendations, diagnosis of chronic myelomonocytic leukemia (CMML) beforehand requires microscopic examination of peripheral blood to identify dysplasia and/or blasts when monocytes are greater or equal to 1.0 × 109/L and 10% of leucocytes. We analyzed parameters derived from SysmexTM XN analyzers to improve the management of microscopic examination for monocytosis. We analyzed results of the complete blood count and the positioning and dispersion parameters of polymorphonuclear neutrophils and monocytes in 61 patients presenting with CMML and 635 control patients presenting with a reactive monocytosis. We used logistic regression and multivariate analysis to define a score for smear review. Three parameters were selected: neutrophil/monocyte ratio, structural neutrophil dispersion (Ne-WX) and monocyte absolute value. We established an equation in which the threshold of 0.160 guided microscopic examination in the search for CMML abnormalities with a sensitivity of 0.967 and a specificity of 0.978 in the learning cohort (696 samples) and 0.923 and 0.936 in the validation cohort (1809 samples) respectively. We created a score for microscopic smear examination of patients presenting with a monocytosis greater or equal to 1.0 × 109/L and 10% of leucocytes, improving efficiency in laboratory routine practice.

APG101 efficiently rescues erythropoiesis in lower risk myelodysplastic syndromes with severe impairment of hematopoiesis.

CD95, a member of the death receptor family initiates a caspase-dependent apoptosis, when activated by its ligand CD95L, thought to negatively regulate erythrocyte production in the bone marrow. We have previously shown that CD95 is overexpressed in two thirds of patients with a lower risk myelodysplastic syndrome (MDS) and that resistance to erythropoiesis-stimulating agents (ESA) is linked to poor residual erythropoiesis. In the present study, we show that CD95 overexpression and previous transfusion are independent predictive factors of ESA resistance. To investigate an alternative therapeutic strategy of anemia in ESA-resistant patients, we have conducted a preclinical study of the effects of APG101, a fusion protein consisting of the extracellular domain of human CD95 and the Fc region of human IgG1 on MDS erythropoiesis in vitro. APG101 increases the number of burst-forming unit-erythroid (BFU-E) progenitors derived from CD34+ progenitors in liquid culture and improves overall proliferation rate of erythroid precursors by inhibiting apoptosis. APG101 rescues BFU-E growth in MDS patients presenting with attrition of erythroid progenitors at baseline, independently of CD95 or CD95L expression level. Our data show that overexpression of CD95 at diagnosis is a hallmark of ESA resistance and that severe impairment of erythropoiesis is predictive of erythroid response to APG101 in vitro. These data provide a rationale for further clinical investigation of APG101 in an attempt to treat anemia in lower risk MDS patients.

Presepsin (sCD14-ST) secretion and kinetics by peripheral blood mononuclear cells and monocytic THP-1 cell line.

Presepsin could help for early diagnosis of systemic infection. Little is known regarding its kinetics. We studied presepsin concentration after challenge with bacterial agonist lipopolysaccharide (LPS) stimulation in peripheral mononuclear cells (PMNC) collected from 5 healthy volunteers and in a human cell line of monocytic cells (THP1). In PMNC, an exposure to LPS (100 ng/mL) induced an increase of median presepsin levels as early as hour 1 (+31%, p=0.007), concomitantly to IL-6 synthesis. In THP1 cells, presepsin was detected at 1 hour after LPS exposure, and peaked at 3 hours, in THP1 cells. In conclusion, we report here that presepsin, a surrogate marker of the host response to bacteria, increases early in PMNC and in a monocytic cell lineage. Our findings might confirm the potential usefulness of presepsin bedside as an early marker of infectious diseases.

Characteristic repartition of monocyte subsets as a diagnostic signature of chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/ myeloproliferative neoplasm whose diagnosis is currently based on the elevation of peripheral blood monocytes to >1 × 10(9)/L, measured for ≥3 months. Diagnosis can be ambiguous; for example, with prefibrotic myelofibrosis or reactive monocytosis. We set up a multiparameter flow cytometry assay to distinguish CD14(+)/CD16(-) classical from CD14(+)/CD16(+) intermediate and CD14(low)/CD16(+) nonclassical monocyte subsets in peripheral blood mononucleated cells and in total blood samples. Compared with healthy donors and patients with reactive monocytosis or another hematologic malignancy, CMML patients demonstrate a characteristic increase in the fraction of CD14(+)/CD16(-) cells (cutoff value, 94.0%). The associated specificity and sensitivity values were 95.1% and 90.6% in the learning cohort (175 samples) and 94.1% and 91.9% in the validation cohort (307 samples), respectively. The accumulation of classical monocytes, which demonstrate a distinct gene expression pattern, is independent of the mutational background. Importantly, this increase disappears in patients who respond to hypomethylating agents. We conclude that an increase in the fraction of classical monocytes to >94.0% of total monocytes is a highly sensitive and specific diagnostic marker that rapidly and accurately distinguishes CMML from confounding diagnoses.

Multicentric study underlining the interest of adding CD5, CD7 and CD56 expression assessment to the flow cytometric Ogata score in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative.