Search

Clonal hematopoiesis (CH) of indeterminate potential has been described in blood samples from large series of patients. Its prevalence and consequences are still not well understood because sequencing methods vary and because most studies were performed in cohorts comprising individuals with nonhematologic diseases. Here, we investigated the frequency of CH in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers. Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. In bone marrow samples, clones were found in almost 40% of healthy volunteers more than 50 years old. The most frequent mutations were found in DNMT3A and TET2, with 1 individual carrying 3 variants. Variant allele frequencies were highly concordant between blood and bone marrow samples. Blood parameters were normal except for those in 2 individuals: 1 had a mild macrocytosis and 1 had a mild thrombocytosis. Furthermore, no morphologic abnormalities or dysplasia were detected when bone marrow smears were carefully evaluated. Individuals with CH differed from others by age (62.8 vs 38.6 years; P < .0001) and platelet count (294 vs 241 ×109/L; P = .0208), the latter being no more significant when removing the 2 individuals who carried the JAK2 p.V617F mutation. These results confirm that CH is a very common condition in healthy adults over 50 years old. Consequently, the detection of driver myeloid mutations should be interpreted with caution in the absence of cytologic abnormalities in the blood and/or the bone marrow.

INTRODUCTION Cytokines are involved in many processes, including hematopoiesis and inflammation. Aging is associated with the onset of clonal hematopoiesis (CH) of indeterminate potential, putatively associated with a higher risk of progression to hematological malignancies such as myelodysplastic syndromes or acute myeloid leukemia. Moreover, CH may participate to create a pro-inflammatory environment contributing to the pathogenesis of age-related diseases, such as cardiovascular diseases. This is likely to be driven by or translated in changes in bone marrow (BM) and/or peripheral blood (PB) soluble factors for which reference values still remain unclear, because BM cytokines levels have never been determined in strictly selected healthy people. Indeed, control BM samples classically used in studies are from subjects undergoing surgeries for non-hematologic causes, such as total hip replacement or cardiac surgery, patients suffering from immune thrombocytopenic purpura, brain death patients or allogeneic BM donors. In this study, the BM and PB plasma concentrations of 49 hematopoietic and inflammatory cytokines were measured in a representative panel of 94 healthy adult volunteers and the results were analyzed considering their age and presence of CH. METHOD Ninety-four healthy donors aged from 18.6 to 80.1 years old (yo), including 58 women were recruited for this study (HEALTHOX protocol, CPP Tours, AFSSAPS identifier ID-RCB: 2016-A00571-50 and ClinicalTrials.gov # NCT02789839). The presence or absence of CH (>1% of variant allele frequency) in this cohort is already known (Guermouche H, Ravalet N et al, Blood Adv 2020;4(15):3550-3557). BM samples were obtained through sternal aspiration using a classical procedure in France, and PB sampling was performed at the same time by venipuncture. Samples were collected on sodium heparin or EDTA, centrifuged twice (1200 g, 10 min, 20 C°), aliquoted and stored at -80°C. A 48-plex human cytokine panel assay was used to quantify 48 human cytokines in PB and BM plasmas [beta-NGF, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL27, CLEC11A, CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, FGF-2, G-CSF, GM-CSF, HGF, IFN-alpha-2, IFN-gamma, IL-1 alpha, IL-1 beta, IL-1ra, IL-2, IL-2-RA, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-12B, IL-13, IL-15, IL-16, IL-17A, IL-18, KITLG, LIF, LT-alpha, M-CSF, MIF, PDGF subunit B, TNF, TNFSF10, VEGF-A]. MIF and FLT3L quantification were performed by ELISA. Regarding CH, the controls were subjects older than 50 yo without CH. Statistical analyses were performed with the R (3.6.3) and Rstudio version 1.2.5042 (www.rstudio.org) software. Comparisons were computed by Wilcoxon and Kruskal-Wallis tests. All pairwise multiple comparisons were performed using Dunn's-test for multiple comparisons of independent samples (PMCMR package). Correlations between cytokine levels in PB and BM were tested with Pearson and Spearman methods. Correlation matrices were plotted using the "corrplot" package. RESULTS CH was detected in 16 volunteers, mostly in individuals over 50 yo. BM and PB plasma samples were studied in 3 age-groups: 18-40, 40-60 and 60-80 yo. With aging, variations were observed for 18 BM cytokine levels, with 7 increasing (FLT3L, CXCL9, HGF, FGF-2, CCL27, IL-16, IL-18) and 8 decreasing (G-CSF, TNF, IL-2, IL-15, IL-17a, IL-4, LT-alpha, IL-1 alpha). In PB, 10 cytokines significantly increased with age (CXCL9, FLT3L, CCL27, CXCL10, HGF, CCL11, IL-16, IL-6, IL-1 beta, CCL2). CH was associated with significantly higher BM levels of MIF and IL-1 beta, lower BM levels of IL-9 and IL-5 and higher PB levels of IL-15, VEGF-A, IL-2, CXCL8, CXCL1 and G-CSF (Table). CONCLUSION In this study and for the first time, we concomitantly analyzed BM and PB concentrations of a panel of hematopoietic and inflammatory cytokines in a cohort of strictly selected healthy volunteers. In addition to the establishment of reference values, useful for various biological studies, and correlations between blood and BM levels, we identify variations in the BM of key cytokines according to age and CH. The differences in these cytokine concentrations, either as causes or as consequences, may shape both the BM microenvironment and hematopoietic processes, eventually leading to the beginning of age-related myeloid malignancies or inflammatory conditions. Figure 1 Figure 1. Disclosures Hirsch: Daiichi Sankyo Oncology: Consultancy; Novartis Pharma: Consultancy. Suner: Sanofi - Genzyme: Consultancy. Delhommeau: Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy.

Introduction Moderate anemia affects a substantial fraction of the elderly population and blood smear analysis can help to guide towards a diagnosis of myelodysplastic syndrome (MDS). Nevertheless, in medical laboratories, review of blood smear is realized only if there is quantitative or qualitative flag on the complete blood count (CBC). Consequently, the suspicion of MDS could be delayed because of the absence of systematic blood smear triggering, this smear being crucial to induce the diagnosis process by cytological analysis of bone marrow aspiration. The DxH 800 Hematology Analyzer measures and calculates 126 cytological parameters based on photometry, impedance or light scatter. These parameters regroup 23 standard CBC parameters and 103 unexploited "research parameters". The goal of our study was to create an original mathematical model using "research parameters" to determine a Suspicion Score of MDS which triggers blood smear examination and consequently the process of identification of subclinical patients suffering from MDS. Methods This study compared the 126 parameters measured by the Beckman-Coulter DxH 800 Hematology Analyzer from peripheral blood samples of MDS patients and healthy volunteers. MDS cohort was composed of 101 patients of the university hospital of Tours (9 MDS-SLD, 28 MDS-MLD, 8 MDS-RS-SLD, 8 MDS-RS-MLD, 20 MDS-EB-1, 19 MDS-EB-2, 9 5q-). The cohort of healthy volunteers included 88 aged-matched subjects from HEALTHOX and PLASMYC protocols (ClinicalTrials.gov # NCT02789839 and # NCT02809222, respectively). The "research parameters" contained Research Use Only (RUO) parameters and Cell Population Data (CPD). CPD were composed of 98 parameters measured by Volume Conductivity Scatter (VCS) module. The parameters derived from reticulocytes, which require a specific prescription, were excluded. Statistical analyses were performed using Rstudio version 1.0.153. The normal distribution of values was studied by Shapiro-Wilk tests (p-value0.05). Means comparisons were performed by Wilcoxon and Student tests. Principal component analysis (PCA) was performed using FactoMineR. The logarithmic logistic regression was done thanks to the glm() function of the stats package. The general linear model was obtained by using logistic regression with weighted parameters and was validated by split-sample strategy (10,000 repeats). Samples were divided into two groups ("learning" and "test" groups) randomly mixing MDS patients and healthy volunteers. The "learning" group (n=130) allowed to build the model and the "test" group (n=59) was used to validate it. Results After mono-parametric and multi-parametric comparisons, 10 parameters were selected to establish the model of interest according to the best contributions at the first axe of PCA, the best correlations at two first axes of PCA and the most significant p-values (parameters described in the Table: MN.LALS.NNRBC, MN.LMALS.NNRBC, MN.UMALS.NNRBC, SD.MALS.NE, SD.UMALS.NE, SD.V.NE, MN.MALS.NE, SD.AL2.NE, SD.AL2.MO, SD.V.MO ). In order to determine the Suspicion Score of MDS (SS-MDS), the 10 selected parameters were weighted during split sampling strategy by logistic regression. After repeat of 10,000 split-samples, the SS-MDS determined using weighted parameters was validated with average efficiency of 92.3%. For each parameter, the median of weighting coefficients was used in the mathematical model: "SS-MDS = ΣCi.Pi + intercept" with "Ci: weighting coefficient of parameter i" and "Pi: parameter i value". On the whole cohort, SS-MDS induced "MDS" flag for 11 MDS patients (red points in the Figures) for which conventional CBC algorithms failed to generate flag spreading peripheral blood smear. By fixing the threshold at -1.5, the sensitivity and specificity of SS-MDS were 100% and 92%, respectively. Moreover, the positive and negative predictive values were 93.5% and 100%, respectively. Conclusions The incidence of MDS increases with aging and the early diagnosis enables optimal care of these diseases. The Beckman-Coulter DxH 800 HematologyAnalyzer is widely used over the world. We propose in this study the clinical use of 10 unexploited "research parameters" to early detect subclinical MDS by selective triggering of blood smear examination. A prospective/multicentric study will allow the optimization of SS-MDS. Figure. Figure. Disclosures No relevant conflicts of interest to declare.