LAUSR

The diagnosis of chronic myelomonocytic leukemia (CMML) relies on both a persistent peripheral blood monocytosis (monocytes ≥1x10/L) and monocytes accounting for ≥10% of the white blood cell (WBC) differential count. We showed that a relative accumulation of classical monocytes (cMo: CD14CD16) ≥94% among the total peripheral blood monocytes measured by flow cytometry could help to distinguish CMML from other causes of monocytosis. Since then, the quantification of circulating monocyte subsets for the diagnosis of this disease has become widespread. The increase in cMo is related to a decrease in the fraction of non-classical monocytes (ncMo: CD14CD16). This flow cytometry criterion can be challenged by the co-occurrence of an inflammatory disease, observed in up to 20% of CMML patients depending on the series studied, or an inflammatory state. Inflammation can provoke an increase in the intermediate subset of monocytes (iMo: CD14CD16) with a concomitant decrease in the relative cMo percentage below the typical 94% threshold, erasing the CMML signature and generating an easily recognizable “bulbous” profile. As the CMML-associated decrease in the ncMo fraction persists in an inflammatory setting, it might be a relevant criterion for differentiating CMML from a reactive monocytosis, pending an accurate delineation between iMo and ncMo subsets. At present, routine use of a decreased ncMo fraction in diagnostics is precluded by the lack of inter-operator reproducibility of its measurement, unlike cMo fraction measurement. Slan, also known as 6-sulfo LacNac, a carbohydrate modification of P-selectin glycoprotein ligand 1, is considered to be a marker of ncMo. To improve the flow cytometry distinction between CMML and reactive monocytosis, we analyzed the utility of measuring the slan-positive (slan) ncMo fraction in the peripheral blood of CMML patients, especially those with a concomitant inflammatory state. We first analyzed, by flow cytometry, slan expression at the surface of circulating blood cells in samples collected from 22 young (mean age: 38±13 years) and 35 elderly (mean age: 76±8 years) healthy donors. The mean absolute monocyte count (AMC) of the young individuals was 0.51±0.19x10/L. while that of the older subjects was 0.6±0.15x10/L). Whole blood samples were stained as described elsewhere with anti-CD45, CD2, CD56, CD24, CD14, CD16 (Beckman-Coulter or BDBiosciences) and anti-slan (Miltenyi-Biotec) antibodies and analyzed with either a Navios (Beckman Coulter) or Fortessa (BDBiosciences) cytometer. Regardless of age, slan was detected only at the surface of monocytes (6.5±3.2%), not being expressed on neutrophils (0.05±0.2%) or lymphocytes (0.3±0.2%), including natural killer cells (0.1±0.2%) (Figure 1A). Following an exclusion gating strategy, monocytes were separated into cMo (CD14CD16), iMo (CD14CD16) and ncMo (CD14/CD16) subsets. The expression of slan was observed to be mostly restricted to ncMo, with 95.7±3.4% of slan cells gathering within this subpopulation (Figure 1B, C). To ensure a reproducible definition of slan cells among monocyte subsets, we chose a slan/CD16 dot plot representation, which allowed an accurate definition of the threshold for slan positivity, determined against the cMo population (Figure 1C). Only a fraction of ncMo (51.7±12.0%) were slan, corresponding to monocytes with the weakest expression of CD14. Slan and slannegative ncMo subpopulations displayed similar morphological features (Figure 1D). These results confirmed that slan is specifically expressed by a fraction of ncMo. Between November 2017 and October 2018, we collected peripheral blood samples or peripheral blood mononucleated cells from a learning cohort of subjects consisting of 26 patients with a reactive monocytosis, 55 patients with newly diagnosed CMML (diagnosis made according to World Health Organization criteria) and 72 healthy donors (37 young and 35 elderly) in two centers (Gustave Roussy and Henri Mondor University Hospitals) following approval by the Ile-de-France 1 (DC2014-2091) and the Groupe Francophone des Myélodysplasies ethical committees (Table 1). The CMML patients had a mean age of 75±9 years, with a typical male predominance (male to female ratio, 2.5:1) and WBC count ranging between 3x10/L and 116x10/L, Eighteen of these patients (33%) were classified as having proliferative CMML (WBC ≥13x10/L). Their AMC ranged from 1.0x10/L to 40.7x10/L with a mean monocyte percentage of 28.4±12.7%. Bone marrow analysis confirmed dysplasia in 96% of the samples (multilineage dysplasia in 81%) and allowed subdivision of these patients into 18 with CMML-0 (33%), 28 with CMML-1 (51%), and 9 with CMML-2 (16%). These patients had slight decreases of both hemoglobin concentration and platelet count. CMML-2 patients showed a significantly deeper thrombocytopenia compared to CMML-1 and CMML-0 patients (P<0.01). Patients with reactive monocytosis were younger (65±14 years) than CMML patients; they had similar mean WBC counts but lower AMC (range: 1x10/L-8.9x10/L). The male to female ratio was 1.9:1 and these patients presented with various causal diseases including systemic autoimmune and/or inflammatory disease, acute infection, metastatic neoplasm, and diffuse lymphoma. We analyzed monocyte subset distribution in this learning cohort. Forty-eight of the 55 CMML patients had cMo ≥94% (Figure 1E), in accordance with previous series. The flow cytometry profile of all the seven CMML patients whose cMo percentage was below 94% had an easily recognized “bulbous” appearance (Figure 1F) that has been associated with an inflammatory state (C-reactive protein level, available for 4 of these 7 patients: 68.3±80.5 mg/L). This particular profile was due to an increase in the iMo fraction (12.4±7.3%), in comparison to the fraction present in the CMML patients without an inflammatory state (2.0±1.0%), combined with the disappearance of the ncMo subset (Table 1). A decrease of the ncMo fraction below 1.13% of total circulating monocytes was recently proposed to identify CMML (Figure 1G). This characteristic feature of the disease was shown to involve the epigenetic deregulation of a miR-150/TET3 pathway that prevents the conversion of cMo into ncMo. The quantification of ncMo requires a robust delineation between iMo and ncMo. This latter subpopulation was first defined by CD14 expression level; subsequently, this strategy was refined using an oblique line to gate the population. Discrepancies between ncMo percentages determined by these two strategies are illustrated in Figure 1H, I, which highlights the difficulty in accurately and reproducibly quantifying the ncMo subset. Using the oblique line strategy, the relative fraction of ncMo was significantly lower in CMML patients (1.6±1.3%) than in controls (11.4±4.9%) or in patients with reactive monocytosis (10.1±5.4%) (Figure