Impact des cellules NK dans le traitement de la Leucémie Lymphoïde Chronique et de la maladie de Waldenström par anticorps anti-CD20 optimisés

L introduction de l anticorps monoclonal anti-CD20 rituximab a transformé le traitement de deux hémopathies du sujet âgé, la leucémie lymphoïde chronique (LLC) et la maladie de Waldenström (MW), mais ne permet pas l obtention d une guérison. Des anti-CD20 optimisés, dont le LFB-R603, ont été développés pour augmenter leur affinité pour les récepteurs Fc RIIIa et induire une plus forte cytotoxicité médiée par un anticorps (ADCC). Etant donné le rôle des cellules NK dans l ADCC, nous avons réalisé leur expertise phénotypique et fonctionnelle. Les cellules NK des sujets âgés préservent la majorité de leurs caractéristiques phénotypiques et fonctionnelles avec des signes de maturité accentués. Leur fonction ADCC est cependant parfaitement préservée. Dans la LLC, les cellules NK présentent une expression normale des récepteurs inhibiteurs et activateurs. Bien qu elles soient peu activées, leurs principales fonctions sont préservées, incluant la cytotoxicité naturelle et l ADCC induite par le rituximab contre des cellules Raji. Les activités ADCC in vitro montrent que le LFB-R603 est plus efficace que le rituximab à faibles concentrations, aussi bien contre les Raji que les cellules B de LLC. Chez les patients atteints de LLC avec délétion 17p (del17p), incluant TP53, nous observons une diminution de l ADCC in vitro, probablement en raison d une résistance des cellules de LLC avec del17p. Cependant, de faibles réponses ADCC restent détectables avec le LFB-R603. Enfin, une étude phénotypique et fonctionnelle des cellules NK a été réalisée dans la MW. Cette étude montre que des anti-CD20 optimisés peuvent avoir leur place dans l arsenal thérapeutique de la LLC et de la MWThe introduction of rituximab, an anti-CD20 monoclonal antibody (mAb), has transformed the treatment of two hematological diseases of the elderly, chronic lymphocytic leukemia (CLL) and Waldenström s macroglobulinemia (WM), but it doesn t allow obtaining a recovery. Optimized anti-CD20 mAbs, including LFB-R603, have been developed to increase their affinity to Fc RIIIa and thus induce a higher mAb-mediated cytotoxicity (ADCC). Given the role of NK cells in ADCC, we performed a phenotypic and functional expertise of these cells. NK cells from older subjects preserved most of their phenotypic and functional characteristics with accentuated maturity features. It is important to note that their ADCC function is well preserved. In patients with CLL, NK cells show normal expression of inhibitory and activating receptors. Although most of NK cells are poorly activated, their main functions are preserved, including cytotoxicity against K562 target and rituximab induced ADCC against Raji cells. ADCC activity in vitro shows that LFB-R603 is more efficient than rituximab in inducing ADCC at low doses against Raji cells and CLL cells. For patients with 17p deletion (del17p), including TP53, an anti-CD20 ADCC efficiency decrease was observed, probably linked to a potential resistance of CLL B cells with del17p. However, low ADCC responses were observed with LFB-R603. Finally, we have evaluated the phenotypic and functional capacities of NK cells in MW. In conclusion, these findings highlight that optimized anti-CD20 mAb, including LFB-R603, may participate to the control of CLL and WM, and prompt further investigations for clinical applications in combination with chemotherapiesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Evaluation of paroxysmal nocturnal hemoglobinuria screening by flow cytometry through multicentric interlaboratory comparison in four countries.

OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is currently diagnosed by flow cytometry; although highly sensitive, its interpretation and reporting appear as critical as its technique. Thus, we developed a quality control scheme for the French-speaking region based on the international recommendations for PNH screening. METHODS: After a topical workshop, we proposed a 1-year, two-step survey program to any volunteering French-speaking clinical laboratory. The first survey consisted of sending raw data files to evaluate gating and the interpretation strategy of each center. The second stipulated sending fresh whole-blood samples to evaluate the whole process and its practice. RESULTS: Forty-nine participants from voluntary centers returned results for each of the two successive surveys. On virtual survey, 27% reported false-positive PNH created by immature granulocytes, whereas the minor PNH clone was not detected by 9%. On fresh survey, 63% of centers used at least the same six-color combination (CD24, CD14, CD33, CD15, CD45, and fluorescent aerolysin), and nearly 70% of participants were able to perform a sensitivity test less than 0.1% on neutrophils. All participants detected the major PNH clone, yet 16% returned false-positive results for the non-PNH clone case. CONCLUSIONS: We succeeded in rallying numerous French-speaking clinical laboratories for both surveys and in harmonizing the technical practice by highlighting common pitfalls

An unusual case of acute leukemia

International audienceWe report the case of a 31 year-old man diagnosed with an atypical acute leukemia difficult to characterize cytologically. The immunophenotyping identified a blastic population co-expressing myeloid, lymphoidBand lymphoid T markers suggesting the diagnosis of either a mixed phenotype acute leukemia (MPAL) or an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Because of the poor prognosis linked to these leukemias, the patient benefited from chemotherapy targeting both myeloid and lymphoid components, followed by allogeneic hematopoietic stem cell transplantation. DNA-based techniques analyzing B and T-cell clonality identified partial rearrangements in immunoglobulin and TCR genes, allowing the monitoring of minimal residual disease. This observation highlights the difficulty to classify some atypical cases of acute leukemias. It emphasizes on the complementarity of cytomorphology, immunophenotyping by flow cytometry and molecular techniques in order to promptly characterize and treat these leukemias

ISOLD: A New Highly Sensitive Interleukin Score for Intraocular Lymphoma Diagnosis

International audienc

Primary vitreoretinal lymphoma: short review of the literature, results of a European survey and French guidelines of the LOC network for diagnosis, treatment and follow-up

International audiencePurpose of review. The aim of this study was to highlight the diagnostic and management challenges of primary vitreoretinal lymphoma (PVRL) through a review of the literature and a European survey on real-life practices for PVRL.Recent findings. The care of PVRL patients is heterogeneous between specialists and countries. Upfront systemic treatment based on highdose methotrexate chemotherapy, with or without local treatment, might reduce or delay the risk of brain relapse. Ibrutinib, lenalidomide with or without rituximab, and temozolomide are effective for patients with relapsed/refractory PVRL and should be tested as first-line treatments.Summary. The prognosis of PVRL remains dismal. No firm conclusion regarding optimal treatment can yet be drawn. The risk of brain relapse remains high. Diagnostic procedures and assessment of therapeutic responses need to be homogenized. Collaboration between specialists involved in PVRL and multicentric prospective therapeutic studies are strongly needed. The recommendations of the French group for primary oculocerebral lymphoma (LOC network) are provided, as a basis for further European collaborative work

Persistence of PNH Clones over Time: Insights from the Mid-Term Analysis of the French Nation-Wide Multicenter Prospective Observational Study

61st Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Orlando, FL, DEC 07-10, 2019International audienceIntroduction: Since the publication of the International Guidelines (Borowitz, 2010; Illingworth, 2018), no study has assessed the long-term evolution of paroxysmal nocturnal hemoglobinuria (PNH) clones using high-resolution flow cytometry. The sole evaluation, performed by Sugimori et al, using a 2-color flow cytometry test, showed the disappearance of PNH clones in 24% of patients with bone marrow (BM) failure over 5 years (Sugimori, 2009). A diagnostic practice harmonization using high-resolution flow cytometry has spread in France since 2013 through an on-going inter-laboratory comparison program (Debliquis, 2015). Thus, our HPNAFC group has been able to initiate a French nation-wide multicenter prospective observational study.Objective: We aimed to assess the evolution of PNH clones over a long term period using mostly high sensitivity test, which is required for minor clone assessment, with validated flow cytometry data.Methods: All patients of any age with a PNH clone or GPI-deficient cells ≥0.01%, newly- or previously-diagnosed, detected in France from February 29th 2016, could be included in this Observatory, provided that the center had validated a PNH flow cytometry quality control. For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. Referent cytometrist of each center included patients in the e-CRF available on the HPNAFC website providing clinical and biological information as well as flow cytometry raw data files. This study was approved by the national research ethics board.Results: As of July 15th 2019, 48 participating flow cytometry laboratories across France have enrolled 356 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. All cases have been carefully reviewed by the 2 principal investigators, who both thoroughly re-examined flow cytometry data and the e-CRF filling that led to the update of roughly one third of the submitted files. This enabled the validation of 200 patients at diagnosis, the remaining 156 being ongoing. One hundred and three of the 200 validated patients displayed at least one follow-up point (more than 3 months apart from the diagnosis) with a clone size determined at diagnosis (see flow chart figure 1A). For 8/103 patients, exchanges with centers are still ongoing. Thus, we were able to assess the evolution of PNH clones of 95 patients with 2 [range: 1-8] follow-up points over a period of 4.1 [0.3-14.2] years, corresponding to 200 validated follow-up points. The patient median age at diagnosis was 40 years old [10-85] with 3 pediatric cases (<18y) and a M/F sex ratio of 0.86. Diagnoses were made between 2003 and 2018 with clinical information available in 97% of cases: 19 patients (20%) had hemolytic anemia and most patients (n=73, 77%) displayed BM failure including aplastic anemia (n=62), myelodysplastic syndrome (n=7) and unexplained cytopenia(s) (n=4). No case of thrombosis was included. All patients with hemolytic anemia showed an increasing clone size over time including the two who were not treated with eculizumab (Figure 1B; median size at diagnosis on neutrophils: 81.3% vs median size over 5 years: 96.3%). The median clone size at diagnosis for patients with BM failure was 1.5% on neutrophils with a very wide range [0.01-97.87], almost half of them being less than 1%. When comparing the diagnosis point with the latest follow-up point, PNH clone size increased in 37 patients, decreased in 16 of them and remained stable in 20 cases (Figure 1C, D). Nine of the 37 patients reached a PNH clone size above 50% and 4 of them received a treatment by eculizumab in a median delay of 5 years [1.5-6.0]. Interestingly, no patient showed spontaneous disappearance of PNH clones, pending the use of a high-resolution flow cytometry test. The only five patients with undetectable PNH clones (Figure 1D, red lines) were those who underwent BM transplantation.Conclusion: This multicenter study based on robust flow cytometry analysis showed no disappearance of PNH clones, including minor ones, over a long period of time, regardless of the clinical manifestations, except for patients who underwent BM transplantation. Moreover, PNH clone size increased in half of patients with BM failure, justifying a long term PNH clone size monitoring, even in these patients

Identification of 2 DNA methylation subtypes of Waldenström macroglobulinemia with plasma and memory B-cell features

Epigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients

Sofosbuvir plus ribavirin for hepatitis C virus-associated cryoglobulinaemia vasculitis: VASCUVALDIC study

International audienceBackgroundHepatitis C virus (HCV) is the aetiological agent for most cases of cryoglobulinaemia vasculitis. Interferon-containing regimens are associated with important side effects and may exacerbate the vasculitis.ObjectiveTo evaluate safety and efficacy of an oral interferon-free regimen, sofosbuvir plus ribavirin, in HCV-cryoglobulinaemia vasculitis.Patients and methodsWe enrolled 24 consecutive patients (median age of 56.5 years and 46% of women) with HCV-cryoglobulinaemia vasculitis. Sofosbuvir (400 mg/day) was associated with ribavirin (200-1400 mg/day), for 24 weeks. The primary efficacy end point was a complete clinical response of the vasculitis at the end of treatment (week 24).ResultsMain features of HCV-cryoglobulinaemia vasculitis included purpura and peripheral neuropathy (67%), arthralgia (58%), glomerulonephritis (21%) and skin ulcers (12%). Twenty-one patients (87.5%) were complete clinical response at week 24. Complete clinical response was achieved in six (25%) patients at week 4, four (16.6%) at week 8, seven (29.2%) at week 12, three (12.5%) at week 16 and one (4.2%) at week 20. The cryoglobulin level decreased from 0.35 (0.16-0.83) at baseline to 0.15 (0.05-0.45) g/L at week 24. The C4 serum level increased from 0.10 (0.07-0.19) to 0.17 (0.09-0.23) g/L at week 24. Seventy-four per cent of patients had a sustained virological response at week 12 post treatment. The most common side effects were fatigue, insomnia and anaemia. Two serious adverse events were observed.ConclusionsSofosbuvir plus ribavirin combination was associated with a high rate of complete clinical response and a low rate of serious adverse events in HCV-cryoglobulinaemia vasculitis