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In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×10

To the editor: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood cancer and is molecularly heterogeneous. Identical twins with concordant BCP-ALL have provided a unique and tractable model to delineate the natural history and clonal evolution of the disease. Such studies have led to the 2-hit model for pediatric BCP-ALL, elucidated by Greaves, which provides unambiguous evidence that an initiating alteration, often occurring in utero, generates a preleukemic clone, which eventually gives rise to an overt leukemia upon acquisition of secondary cooperating mutations. Importantly, genome-wide sequencing has revealed a strikingly silent mutational landscape in childhood BCP-ALL, further indicative of a developmental cancer of prenatal origin, with a short window of time to accumulate molecular alterations.

The germline predisposition associated with the autosomal dominant inheritance of the 14q32 duplication implicating ATG2B/GSKIP genes is characterized by a wide clinical spectrum of myeloid neoplasms. We analyzed 12 asymptomatic carriers and 52 patients aged 18-74 years from six families, by targeted sequencing of 41 genes commonly mutated in myeloid malignancies. We found that 75% of healthy carriers displayed early clonal hematopoiesis mainly driven by TET2 mutations. Molecular landscapes of patients revealed two distinct routes of clonal expansion and leukemogenesis. The first route is characterized by the clonal dominance of myeloproliferative neoplasms (MPN)-driver events associated with TET2 mutations in half of cases and mutations affecting splicing and/or the RAS pathway in one-third of cases, leading to the early development of MPN, mostly essential thrombocythemia, with a high risk of transformation (50% after 10 years). The second route is distinguished by the absence of MPN-driver mutations and leads to AML without prior MPN. These patients mostly harbored a genomic landscape specific to acute myeloid leukemia secondary to myelodysplastic syndrome. An unexpected result was the total absence of DNMT3A mutations in this cohort. Our results suggest that the germline duplication constitutively mimics hematopoiesis aging by favoring TET2 clonal hematopoiesis.

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.

International audience; Prophylactic or preemptive treatment strategies are required to prevent human cytomegalovirus (CMV) infections in transplant recipients. However, treatment failure occurs when CMV resistant-associated variants (RAVs) are selected. Although the diversity of CMV is lower than that of RNA viruses, CMV appears to show some genetic instability, with possible minor emerging resistance that may be undetectable by Sanger sequencing. We aimed to examine CMV-resistance mutations over time by ultra-deep sequencing (UDS) and Sanger sequencing in a kidney transplant recipient experiencing CMV infection. This patient showed a transient response to three different antiviral drugs (valganciclovir, foscarnet, and maribavir) and four episodes of CMV resistance over two years. The full-length UL97 (2.3kpb) and partial UL54 (2.4kpb) CMV genes were studied by UDS and Sanger sequencing and linkage mutations calculated to determine RAVs. We detected four major and five minor resistance mutations. Minor resistant variants (2-20%) were detected by UDS, whereas major resistance substitutions (>20%) were identified by both UDS and Sanger method. We detected cross-resistance to three drugs, despite high CMV loads, suggesting that the fitness of the viral mutants was not impaired. In conclusion, CMV showed complex dynamic of resistance under antiviral drug pressure, as described for highly variable viruses. The emergence of successive RAVs constitutes a clinically challenging complication and contributes to the difficulty of therapeutic management of patients.

We report the case of a patient with a history of Epstein-Barr virus-positive large B-cell lymphoma, who relapsed with an angioimmunoblastic T-cell lymphoma (AITL) associated with a chronic myelomonocytic leukaemia (CMML). We performed targeted next-generation sequencing on CMML and AITL DNA, which revealed mutations of TET2, DNMT3A, SRSF2, NRAS and IDH1, thus confirming that the spectrum of AITL mutations share similarities with myeloid disorders. The frequencies of TET2/DNMT3A and SRSF2 variants could support the hypothesis that TET2/DNMT3A mutations occurred in an early progenitor cell, which later progressed to both the AITL and CMML clones. Treatment with 5-azacytidine led to the complete remission of both diseases. Thus, targeting DNA methylation abnormalities in AITL may be an alternative strategy to chemotherapy. Copyright © 2016 John Wiley & Sons, Ltd.

7517 Background: Clonal hematopoiesis of indeterminate potential (CHIP) has been described in blood samples from large series of patients. Its prevalence and consequences remain questioned because sequencing methods vary and because most studies were performed in cohorts of individuals suffering from non-hematological diseases (solid cancers, diabetes, cardiovascular or psychiatric diseases). CHIP has been described as a risk factor for blood cancer. However, the diagnosis of most blood cancers relies on morphologic and genetic examination of the bone marrow, and clonal hematopoiesis has never been evaluated in bone marrow samples from healthy individuals. Hence, it is not clear whether the current definition of CHIP is clinically relevant. To address this issue, we studied clonal hematopoiesis in paired peripheral blood and bone marrow samples from an unprecedented cohort of rigorously selected healthy volunteers. Methods: Here, we investigated the frequency of clonal hematopoiesis in 82 paired bone marrow and blood samples from carefully selected healthy adult volunteers (HEALTHOX clinical trial, ClinicalTrials.gov Identifier: NCT02789839). This study was approved by the ethics committees (CPP Tours and AFSSAPS identifier ID-RCB: 2011-A00262-39; CPP Ile-de-France III: 2753). Forty-one genes known to be mutated in myeloid malignancies were sequenced with a 1% threshold of detection. All variants were checked using IGV software v2.3. Statistical analyses were performed using Mann-Whitney, chi-squared, Fisher’s exact, Wilcoxon matched-pairs tests or Spearman correlation using GraphPad Prism 6. Results: In bone marrow samples, clones were found in almost 40 % of healthy volunteers over 50 years-old (yo). The most frequent mutations were found in DNMT3A (48%) and TET2 (28%) and the other mutations were found in ASXL1 (8%), JAK2 (8%), RAD21 (4%) and SRSF2 (4%) with one individual carrying three variants. Blood parameters and bone marrow smears were normal with the exception of two individuals with mild macrocytosis or thrombocytosis. Clonal hematopoiesis cases differed from others by age (62.8 vs. 38.6 years, P < .0001) and platelet count (294 vs. 241 G/L, P < .0208). Conclusions: These results confirm that clonal hematopoiesis 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 in the bone marrow.

International audience; AMP activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK 1/ 2 catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.

Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders in which recurrent mutations define clonal hematopoiesis. The origin of the phenotypic diversity of non-del(5q) MDS remains unclear. Here, we investigated the clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment and interrogated dominant clones for MDS-initiating cells. We found that clones mainly accumulate mutations in a linear succession with retention of a dominant subclone. The clone detected in the long-term culture-initiating cell compartment that reconstitutes short-term human hematopoiesis in xenotransplantation models is usually the dominant clone, which gives rise to the myeloid and to a lesser extent to the lymphoid lineage. The pattern of mutations may differ between common myeloid progenitors (CMPs), granulomonocytic progenitors (GMPs), and megakaryocytic-erythroid progenitors (MEPs). Rare STAG2 mutations can amplify at the level of GMPs, from which it may drive the transformation to acute myeloid leukemia. We report that major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knock-down (KD) in normal CD34+ progenitors modifies their granulocytic and erythroid differentiation. Clonal architecture of the HSPC compartment and mutations selected during differentiation contribute to the phenotypic heterogeneity of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation and may guide the search for novel therapeutic strategies.

Introduction Myelodysplastic syndromes (MDS) are phenotypically and genotypically heterogeneous diseases with several driver mutations, which are closely related to patient prognosis. Dynamic evolution of mutations reflects the selection of subclones during disease evolution until transformation in secondary acute myeloid leukemia. Thanks to increasing knowledge in gene functions, target drugs are now available in therapeutic. However, questions remain on the impact of such treatments on malignant cells. We have previously investigated the effects of lenalidomide on clonal evolution, by monitoring variant allele frequencies (VAF) using next generation sequencing (NGS) in non-del5q MDS patients (Chesnais et al, Blood 2015). Here, we present a rapid and ultra-sensitive method using picoliter-droplet digital PCR for mutation detection in MDS with ring sideroblasts (RS). Materials and Methods Bone marrow aspirates were obtained from MDS patients included at diagnosis in a multicentric observational trial (PHRC MDS-04, NCT02619565). Three cell lines (HL60, OCI-AML3, UKE-1) were also used to establish the specificity and the sensitivity of assays. Both frozen living cells and extracted DNA were used. Selected samples were screened for mutations in 39 genes by an NGS approach using a Personal Genome Machine® (PGM, ThermoFisher Scientific, Waltham, MA, USA). Primers and probes were designed for Taqman assays based on allelic discrimination of recurrent mutations found in DNMT3A, SF3B1, JAK2 and NRAS genes. For the detection of SF3B1 p.K700E mutation, 3 locked nucleic acids were notably added to the probes to improve specificity. Picoliter-droplet digital PCR was performed on RainDrop® Digital PCR System (RainDance™ Technologies). Results Allelic discrimination assays were validated on genomic DNA extracted from cell lines and patient samples harboring or not targeted mutations using the RainDance system. About 5.106 droplets were generated using RainDrop Source. Wild-type (WT) DNA was tested in order to assess false positive signals for each design, characterized by λFP (mean number of false positive signals), limit of blanck (LOB) and limit of detection (LOD) for all experiments. The limit of blanck (LOB) defined here the highest number of droplets corresponding to apparent droplets containing mutated amplicons while testing wild type DNA. The limit of detection (LOD) was the lower number of droplets which can be distinguish from LOB while testing DNA with very low concentration of mutant genome. All the designed assays were also strongly approved for linearity using mixtures of mutated and WT DNA from cell lines (0.01% to 100% mutated allele frequency). Specificity, linearity and sensibility of the selected assays were validated on genomic DNA. Later on, we investigate genomic DNA of 3 MDS patients with RS and harboring JAK2 and SF3B1 mutations. For these patients, we obtained comparable results using both NGS and picoliter-droplet digital PCR in term of mutant allele burden quantification. Moreover, a triplex assay allowing mutant allele discrimination in JAK2 and SF3B1 genes was established on these patients. Further analyses were conducted on living cells harboring JAK2 or NRAS mutations. This approach was first conducted using a "home made" microfluidic system based on the detection of fluorescent probes in living cells encapsulated into agarose beeds. We obtained specific fluorescent signals corresponding to the genotypes. In parallel, an alternative method based on the QX100™Droplet Digital™PCR system (Biorad) also demonstrated the feasibility of allelic discrimination in living cells. Experiments based on frozen cells of MDS patients are currently under investigation. Conclusion This study is the first application of multi-target digital PCR used to detect and quantify somatic mutations recurrently found in MDS. Analyses of the clonal architecture determined on living cells and its evolution upon treatment in MDS patients with RS by this approach will help us to investigate the monitoring of the therapeutic response. Our study supports a proof of principle for further large-scale analyses of MDS patients at diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

Introduction: Clonal B-cell lymphocytosis with marginal zone features (CBL-MZ) is a non-CLL type B-cell monoclonal lymphocytosis that has been recently recognized in the 2016 revision of the World Health Organization (WHO) classification of lymphoid neoplasms. Whether CBL-MZ corresponds to the early stage of splenic marginal zone lymphoma (SMZL) is not clearly defined. The aim of the study was to compare CBL-MZ and SMZL in patients diagnosed during the same period in two French university hospitals. Methods: We performed a retrospective study including all consecutive patients with a diagnosis of CBL-MZ or SMZL between 2010 and 2014. For the purpose of the present study, all CBL-MZ and SMZL were reclassified according to the 2016 WHO classification. Patient and disease characteristics comprised clinical data, immunophenotyping of lymphocytes by flow cytometry, cytogenetic studies (G-banding and TP53 FISH) and targeted gene sequencing (TP53, NOTCH2 and MYD88). We assessed progression-free survival (PFS), lymphoma-specific survival (LSS), overall survival (OS) and cumulative incidence of histologic transformation. Results: Forty-one patients were diagnosed with CBL-MZ (n=15) or SMZL (n=26) on consensus review. None had HCV infection. Median age was 74 years (range 43-93). At diagnosis, B symptoms (7% vs 46%, p=0.009), elevated lactate dehydrogenase (13% vs 50%, p=0.023) and elevated β2-microglobulin (50% vs 93%, p=0.039) were significantly more frequent in SMZL patients. HPLL score A was more frequent in patient with CBL-MZ (93% vs 58%, p=0.003). On lymphocyte immunophenotyping, CD23 was expressed only in SMZL (0% vs 38%, p=0.015). Aberrant karyotype was displayed in 5/7 CBL-MZ and 15/18 of SMZL (p=0.591). Fourteen patients (56%; 2 CBL-MZ and 12 SMZL, p=0.177) had at least 3 chromosomal abnormalities and were considered to have a complex karyotype. There was no significant difference between CBL-MZ and SMZL for the frequency of detection of +3, +12, +18, del(7q) or 14q alterations. A chromosome 17p abnormality or monosomy 17 was seen in 12/25 patients (48%) without significant difference between CBL-MZ and SMZL, and was confirmed in 11/11 cases by TP53 FISH analysis (one patient had an isochromosome 17q and TP53 FISH was not done). In 11 other patients, no TP53 deletion was detected by FISH. In the 2 remaining patients (one normal karyotype and one abnormal karyotype without abnormal 17p) there was no more material for FISH analysis. TP53 mutation was present in 1/15 (7%) CBL-MZ and 6/22 (27%) SMZL (p=0.161), NOTCH2 mutation in 1/15 (7%) CBL-MZ and 3/22 (14%) SMZL (p=0,632) and MYD88 mutation in 1/15 (7%) CBL-MZ and 3/22 (14%) SMZL (p=0.632). After a median follow-up of 47 months (CBL-MZ, 52 months; SMZL, 46 months, p=0.771), no CBL-MZ patients had event (progression, histologic transformation or death) or started treatment, whereas 24/26 (92%) SMZL patients started a first-line treatment (chemotherapy in 62%, rituximab in 42% and/or splenectomy in 39%). Twelve SMZL patients (50% of treated patients) relapsed or progressed after the first-line treatment. Among them, 6 had histologic transformation, either in diffuse large B-cell lymphoma (DLBCL, n=5) or Hodgkin lymphoma (HL, n=1). Finally, 5 SMZL patients died (3 of DLBCL, 1 of HL, 1 of SMZL). Overall, 4-year PFS was 100% and 31% (95% CI, 13% to 49%) for CBL-MZ and SMZL, respectively (p=0.002); 4-year LSS was 100% and 77% (95% CI, 61% to 93%) for CBL-MZ and SMZL, respectively (p=0.26); and 4-year OS was 100% and 71% (95% CI, 54% to 88%) for CBL-MZ and SMZL, respectively (p=0.25). Clinical, laboratory, cytogenetic and molecular features were subjected to univariate analyses to evaluate their impact on PFS, LSS, OS and histologic transformation in SMZL patients. In univariate analysis for PFS, only NOTCH2 mutations and TP53 inactivation (i.e. del(17p) or TP53 mutations) predicted poorer PFS, but in multivariate analysis NOTCH2 mutations were the sole factor significantly affecting PFS rates (p=0.041). For LSS, OS and histologic transformation, no prognostic factor was identified in univariate analysis. Conclusions: Our data suggest that CBL-MZ and SMZL share a similar cytogenetic and mutational profile. These findings suggest the possible involvement of a common oncogenic mechanism in the development of these lymphoid neoplasms. Disclosures Hermine: Novartis: Research Funding; Celgene: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau; Alexion: Research Funding.

Purpose: Therapy targeting CTLA-4 immune checkpoint provides increased survival in patients with advanced melanoma. However, immunotherapy is frequently associated with delayed and heterogeneous clinical responses and it is important to identify prognostic immunological correlates of clinical endpoints. Experimental design: 77 patients with stage III/IV melanoma were treated with ipilimumab alone every 3 weeks, during 9 weeks. Blood samples were collected at the baseline and before each dose for in depth immune monitoring. Results: The median follow-up was 28 mo with a median survival of 7 mo. Survival and clinical benefit were significantly improved when absolute lymphocyte count at the baseline was above 1 × 109/L. Notably, ipilimumab had a global effect on memory T cells, with an early increase of central and effector subsets in patients with disease control. By contrast, percentages of stem cell memory T cells (TSCM) gradually decreased despite stable absolute counts and sustained proliferation, suggesting a process of differentiation. Higher proportions of eomes+ and Ki-67+ T cells were observed, with enhanced skin homing potential and induction of cytotoxic markers. Conclusion: These results suggest that CTLA-4 blockade is able to reshape the memory subset with the potential involvement of Eomes and memory subsets including TSCM.