Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.

Shared and distinct mechanisms of UBA1 inactivation across different diseases.

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Serious infections in patients with VEXAS syndrome: data from the French VEXAS registry.

INTRODUCTION: Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is an acquired autoinflammatory monogenic disease with a poor prognosis whose determinants are not well understood. We aimed to describe serious infectious complications and their potential risk factors. METHODS: Retrospective multicentre study including patients with VEXAS syndrome from the French VEXAS Registry. Episodes of serious infections were described, and their risk factors were analysed using multivariable Cox proportional hazards models. RESULTS: Seventy-four patients with 133 serious infections were included. The most common sites of infection were lung (59%), skin (10%) and urinary tract (9%). Microbiological confirmation was obtained in 76%: 52% bacterial, 30% viral, 15% fungal and 3% mycobacterial. Among the pulmonary infections, the main pathogens were SARS-CoV-2 (28%), Legionella pneumophila (21%) and Pneumocystis jirovecii (19%). Sixteen per cent of severe infections occurred without any immunosuppressive treatment and with a daily glucocorticoid dose ≤10 mg. In multivariate analysis, age >75 years (HR (95% CI) 1.81 (1.02 to 3.24)), p.Met41Val mutation (2.29 (1.10 to 5.10)) and arthralgia (2.14 (1.18 to 3.52)) were associated with the risk of serious infections. JAK inhibitors were most associated with serious infections (3.84 (1.89 to 7.81)) compared with biologics and azacitidine. After a median follow-up of 4.4 (2.5-7.7) years, 27 (36%) patients died, including 15 (56%) due to serious infections. CONCLUSION: VEXAS syndrome is associated with a high incidence of serious infections, especially in older patients carrying the p.Met41Val mutation and treated with JAK inhibitors. The high frequency of atypical infections, especially in patients without treatment, may indicate an intrinsic immunodeficiency.

Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study.

DDX41 germline mutations (DDX41MutGL) are the most common genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia (AML). Recent reports suggest that DDX41MutGL myeloid malignancies could be considered as a distinct entity, even if their specific presentation and outcome remain to be defined. We describe here the clinical and biological features of 191 patients with DDX41MutGL AML. Baseline characteristics and outcome of 86 of these patients, treated with intensive chemotherapy in 5 prospective Acute Leukemia French Association/French Innovative Leukemia Organization trials, were compared with those of 1604 patients with DDX41 wild-type (DDX41WT) AML, representing a prevalence of 5%. Patients with DDX41MutGL AML were mostly male (75%), in their seventh decade, and with low leukocyte count (median, 2 × 109/L), low bone marrow blast infiltration (median, 33%), normal cytogenetics (75%), and few additional somatic mutations (median, 2). A second somatic DDX41 mutation (DDX41MutSom) was found in 82% of patients, and clonal architecture inference suggested that it could be the main driver for AML progression. DDX41MutGL patients displayed higher complete remission rates (94% vs 69%; P < .0001) and longer restricted mean overall survival censored at hematopoietic stem cell transplantation (HSCT) than 2017 European LeukemiaNet intermediate/adverse (Int/Adv) DDX41WT patients (5-year difference in restricted mean survival times, 13.6 months; P < .001). Relapse rates censored at HSCT were lower at 1 year in DDX41MutGL patients (15% vs 44%) but later increased to be similar to Int/Adv DDX41WT patients at 3 years (82% vs 75%). HSCT in first complete remission was associated with prolonged relapse-free survival (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88; P = .02) but not with longer overall survival (hazard ratio, 0.77; 95% confidence interval, 0.35-1.68; P = .5).

Involvement of the JAK-STAT pathway in the molecular landscape of tyrosine kinase fusion-negative hypereosinophilic syndromes: A nationwide CEREO study.

We investigated using a custom NGS panel of 149 genes the mutational landscape of 64 consecutive adult patients with tyrosine kinase fusion-negative hypereosinophilia (HE)/hypereosinophilic syndrome (HES) harboring features suggestive of myeloid neoplasm. At least one mutation was reported in 50/64 (78%) patients (compared to 8/44 (18%) patients with idiopathic HE/HES/HEUS used as controls; p < .001). Thirty-five patients (54%) had at least one mutation involving the JAK-STAT pathway, including STAT5B (n = 18, among which the hotspot N642H, n = 13), JAK1 (indels in exon 13, n = 5; V658F/L, n = 2), and JAK2 (V617F, n = 6; indels in exon 13, n = 2). Other previously undescribed somatic mutations were also found in JAK2, JAK1, STAT5B, and STAT5A, including three patients who shared the same STAT5A V707fs mutation and features consistent with primary polycythemia. Nearly all JAK-STAT mutations were preceded by (or associated with) myelodysplasia-related gene mutations, especially in RNA-splicing genes or chromatin modifiers. In multivariate analysis, neurologic involvement (hazard ratio [HR] 4.95 [1.87-13.13]; p = .001), anemia (HR 5.50 [2.24-13.49]; p < .001), and the presence of a high-risk mutation (as per the molecular international prognosis scoring system: HR 6.87 [2.39-19.72]; p < .001) were independently associated with impaired overall survival. While corticosteroids were ineffective in all treated JAK-STAT-mutated patients, ruxolitinib showed positive hematological responses including in STAT5A-mutated patients. These findings emphasize the usefulness of NGS for the workup of tyrosine kinase fusion-negative HE/HES patients and support the use of JAK inhibitors in this setting. Updated classifications could consider patients with JAK-STAT mutations and eosinophilia as a new "gene mutated-entity" that could be differentiated from CEL, NOS, and idiopathic HES.

The spectrum of glomerular and vascular kidney pathology associated with myeloproliferative neoplasms.

A high prevalence of chronic kidney disease (CKD) occurs in patients with myeloproliferative neoplasms (MPN). However, MPN-related glomerulopathy (MPN-RG) may not account for the entirety of CKD risk in this population. The systemic vasculopathy encountered in these patients raises the hypothesis that vascular nephrosclerosis may be a common pattern of injury in patients with MPN and with CKD. In an exhaustive, retrospective, multicenter study of MPN kidney biopsies in four different pathology departments, we now describe glomerular and vascular lesions and establish clinicopathologic correlations. Our study encompassed 47 patients with MPN who underwent a kidney biopsy that included 16 patients with chronic myeloid leukemia (CML) and 31 patients with non-CML MPN. Fourteen cases met a proposed definition of MPN-RG based on mesangial sclerosis and hypercellularity, as well as glomerular thrombotic microangiopathy. MPN-RG was significantly associated with both myelofibrosis and poorer kidney survival. Thirty-three patients had moderate-to-severe arteriosclerosis while 39 patients had moderate-to-severe arteriolar hyalinosis. Multivariable models that included 188 adult native kidney biopsies as controls revealed an association between MPN and chronic kidney vascular damage, which was independent of established risk factors such as age, diabetes mellitus and hypertension. Therefore, MPN-RG is associated with myelofibrosis and has a poor kidney prognosis. Thus, our findings suggest that the kidney vasculature is a target during MPN-associated vasculopathy and establish a new link between MPN and CKD. Hence, these results may raise new hypotheses regarding the pathophysiology of vascular nephrosclerosis in the general population.

Hematopoietic differentiation is characterized by a transient peak of entropy at a single-cell level.

BACKGROUND: Mature blood cells arise from hematopoietic stem cells in the bone marrow by a process of differentiation along one of several different lineage trajectories. This is often represented as a series of discrete steps of increasing progenitor cell commitment to a given lineage, but as for differentiation in general, whether the process is instructive or stochastic remains controversial. Here, we examine this question by analyzing single-cell transcriptomic data from human bone marrow cells, assessing cell-to-cell variability along the trajectories of hematopoietic differentiation into four different types of mature blood cells. The instructive model predicts that cells will be following the same sequence of instructions and that there will be minimal variability of gene expression between them throughout the process, while the stochastic model predicts a role for cell-to-cell variability when lineage commitments are being made. RESULTS: Applying Shannon entropy to measure cell-to-cell variability among human hematopoietic bone marrow cells at the same stage of differentiation, we observed a transient peak of gene expression variability occurring at characteristic points in all hematopoietic differentiation pathways. Strikingly, the genes whose cell-to-cell variation of expression fluctuated the most over the course of a given differentiation trajectory are pathway-specific genes, whereas genes which showed the greatest variation of mean expression are common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. CONCLUSIONS: These data suggest that human hematopoietic differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination, and strongly support the stochastic view of differentiation. They also highlight the need to consider the role of stochastic gene expression in complex physiological processes and pathologies such as cancers, paving the way for possible noise-based therapies through epigenetic regulation.

Azacitidine for patients with Vacuoles, E1 Enzyme, X-linked, Autoinflammatory, Somatic syndrome (VEXAS) and myelodysplastic syndrome: data from the French VEXAS registry.

Azacitidine can be effective in myelodysplastic syndromes (MDS) associated with inflammatory/autoimmune diseases. Vacuoles, E1 Enzyme, X-linked, Autoinflammatory, Somatic syndrome (VEXAS) is a new monogenic autoinflammatory syndrome caused by somatic ubiquitin-like modifier-activating enzyme 1 (UBA1) mutation, often associated with MDS, whose treatment is difficult and not yet codified. Based on a French nationwide registry of 116 patients with VEXAS, we report the efficacy and safety of azacitidine treatment in 11 patients with VEXAS with MDS. Clinical response of VEXAS to azacitidine was achieved in five patients (46%), during 6, 8+, 12, 21, 27+ months respectively, suggesting that azacitidine can be effective in selected patients with VEXAS and associated MDS.

Myeloproliferative neoplasms and clonal haematopoiesis in patients with giant cell arteritis: a case-control and exploratory study.

OBJECTIVES: GCA is a large vessel vasculitis for which triggering factors remain unknown. Clonal haematopoiesis (CH) was associated with atherosclerosis through the induction of inflammation in myeloid cells, and data suggest that CH expansion and inflammation may support each other to induce a pro-inflammatory loop. Our objective was to describe the impact of JAK2p.V617F-mutated myeloproliferative neoplasms (MPNs) on GCA and to screen MPN-free patients for CH mutations. METHODS: We performed a retrospective case-control study comparing the characteristics of 21 GCA patients with MPN and 42 age- and gender-matched GCA patients without MPN. Also, 18 GCA patients were screened for CH through next-generation sequencing (NGS). RESULTS: The most frequent associated MPN was essential thrombocythaemia (ET; n = 11). Compared with controls, GCA patients with MPN had less-frequent cephalic symptoms (71.4 vs 97.6%; P = 0.004) and higher platelet counts at baseline [485 × 109/l (interquartile range 346-586) vs 346 (296-418); P = 0.02]. There was no difference between groups for other clinical features. Overall survival was significantly shorter in patients with MPN compared with controls [hazard ratio 8.2 (95% CI 1.2, 56.6); P = 0.03]. Finally, screening for CH using NGS in 15 GCA patients without MPN revealed CH in 33%. CONCLUSION: GCA patients with MPN display higher platelet counts and shorter overall survival than controls. This association is not fortuitous, given the possible pathophysiological relationship between the two diseases. CH was found in one-third of GCA patients, which may be higher than the expected prevalence for a similar age, and should be confirmed in a larger cohort.

APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Luspatercept (RAP-536) modulates oxidative stress without affecting mutation burden in myelodysplastic syndromes.

In low-risk myelodysplastic syndrome (LR-MDS), erythropoietin (EPO) is widely used for the treatment of chronic anemia. However, initial response to EPO has time-limited effects. Luspatercept reduces red blood cell transfusion dependence in LR-MDS patients. Here, we investigated the molecular action of luspatercept (RAP-536) in an in vitro model of erythroid differentiation of MDS, and also in a in vivo PDX murine model with primary samples of MDS patients carrying or not SF3B1 mutation. In our in vitro model, RAP-536 promotes erythroid proliferation by increasing the number of cycling cells without any impact on apoptosis rates. RAP-536 promoted late erythroid precursor maturation while decreasing intracellular reactive oxygen species level. RNA sequencing of erythroid progenitors obtained under RAP-536 treatment showed an enrichment of genes implicated in positive regulation of response to oxidative stress and erythroid differentiation. In our PDX model, RAP-536 induces a higher hemoglobin level. RAP-536 did not modify variant allele frequencies in vitro and did not have any effect against leukemic burden in our PDX model. These results suggest that RAP-536 promotes in vivo and in vitro erythroid cell differentiation by decreasing ROS level without any remarkable impact on iron homeostasis and on mutated allele burden.

EZH1/2 function mostly within canonical PRC2 and exhibit proliferation-dependent redundancy that shapes mutational signatures in cancer.

Genetic mutations affecting chromatin modifiers are widespread in cancers. In malignant peripheral nerve sheath tumors (MPNSTs), Polycomb repressive complex 2 (PRC2), which plays a crucial role in gene silencing, is inactivated through recurrent mutations in core subunits embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), but mutations in PRC2's main catalytic subunit enhancer of zeste homolog 2 (EZH2) have never been found. This is in contrast to myeloid and lymphoid malignancies, which harbor frequent loss-of-function mutations in EZH2. Here, we investigated whether the absence of EZH2 mutations in MPNST is due to a PRC2-independent (i.e., noncanonical) function of the enzyme or to redundancy with EZH1. We show that, in the absence of SUZ12, EZH2 remains bound to EED but loses its interaction with all other core and accessory PRC2 subunits. Through genetic and pharmacological analyses, we unambiguously establish that EZH2 is functionally inert in this context, thereby excluding a PRC2-independent function. Instead, we show that EZH1 and EZH2 are functionally redundant in the slowly proliferating MPNST precursors. We provide evidence that the compensatory function of EZH1 is alleviated upon higher proliferation. This work reveals how context-dependent redundancies can shape tumor-type specific mutation patterns in chromatin regulators.

Successful nelarabine and venetoclax treatment of a relapsed/refractory mediastinal myeloid sarcoma with clonal TCR rearrangement.

Myeloid sarcomas represent a heterogeneous group of diseases with a tumoral presentation of acute myeloid leukemia. The clinical presentation of these hematologic cancers is typically aggressive and thus rapidly fatal in the absence of treatment, which relies on intensive chemotherapy that is sometimes followed by allogeneic hematopoietic stem-cell transplant (AHSCT). However, the global treatment strategy for these lesions is currently not well established. We report the case of a patient presenting with a highly refractory mediastinal myeloid sarcoma with uncommon morphologic and phenotypic characteristics and a clonal TCR rearrangement. The patient's disease was progressive despite multiple courses of intensive chemotherapy and a combination of nelarabine and venetoclax finally led to a complete metabolic response consolidated by an AHSCT. This treatment regimen, which has never been reported before, was very well tolerated especially on the neurologic and hematologic levels. This case underlines the clinical, histologic and molecular heterogeneity of what is called myeloid sarcoma and the importance of next-generation sequencing analysis of the tumor mass with both myeloid and lymphoid panels to better classify this rare entity and identify therapeutic targets.

Mastocytosis onset in a patient with treated hairy cell leukemia: Just a coincidence?

Mastocytosis is a mast cell disease caused by functionally defective infiltrating mast cells and CD34+ mast cell precursors. The heterogeneous group of mast cell disorders is categorized into five variants in the updated 2017 World Health Organization (WHO) classification among those systemic mastocytosis with an associated neoplasm (SM-AHN). Except for myeloid neoplasia, lymphoproliferative disorders associated to SM-AHN are more scarce. Here, we report the second case ever described of associated mastocytosis and hairy-cell disease. A 38-year-old female patient without any specific medical history was diagnosed a hairy cell leukemia and BRAFV600E mutation was found in hairy cells. Since purine-analogs were avoided to prevent prolonged myelosuppression, she was treated with vemurafenib and rituximab. Despite early discontinuation due to vemurafenib-induced agranulocytosis, a partial response was observed. Strikingly, bone marrow biopsy performed one month after vemurafenib discontinuation revealed a nodular infiltration by 30% tumoral mastocytes. Along with elevated tryptase level, KITD816V mutation on mastocytes and clinical exam, the patient was diagnosed with systemic mastocytosis with an associated hematological neoplasm (SM-AHN). No BRAFV600E mutation was found on mastocytes. The physiopathology of this association is not known and might be only a coincidence or a common genetic driver mutation enhancing mast and hairy cells.

Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.

OBJECTIVES: Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up-regulation of MCL-1 expression. We thus tested the anti-leukemic activity of S63845, a specific MCL-1 inhibitor. METHODS: Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co-cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL-2, MCL-1, and BCL-XL determined by Western Blot and mass spectrometry-based proteomics. RESULTS: We observed that even if MCL-1 expression is weak compared to BCL-2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax-mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition. CONCLUSION: These results are therefore of great relevance for clinicians as they provide the rational for combining BCL-2 and MCL-1 inhibition in AML.

Impact of genotype in relapsed and refractory acute myeloid leukaemia patients treated with clofarabine and cytarabine: a retrospective study.

The treatment of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains a challenge. Among salvage chemotherapy regimens, the clofarabine and cytarabine (CLARA) combination has been widely evaluated and has a favourable safety/efficacy balance. Predictive factors of efficacy in patients with R/R AML are unclear, particularly the impact of AML-related gene mutations. We report our single-centre experience on 34 R/R AML patients treated with CLARA, with a focus on the genetic characterization of our cohort. CLARA yielded a 47% response rate among this poor-prognosis AML population, while two patients (5·8%) died due to treatment-related toxicity. The two-year progression-free survival and overall survival rates were 29·4% and 35·3%, respectively. Nine patients (26%) had long-term response with a median follow-up of 39·5 months among the responders, of whom six underwent haematopoietic stem cell transplantation. Adverse karyotype did not correlate with response or survival, and secondary AML were more frequent among responders to CLARA, suggesting that this combination may successfully salvage R/R AML patients regardless of adverse prognostic markers. We also observed that a low mutational burden and absence of splice mutations correlated with prolonged survival after CLARA, suggesting that extensive genotyping may have prognostic implications in R/R AML.

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.