Effets fonctionnels des mutations de KDM6A dans la leucémie à tricholeucocytes

Hairy cell leukemia (HCL) and its variant form (HCL-v) are B-cell lymphoproliferative disorders characterized by BRAF and MAP2K1 mutations responsible of abnormal activation of MAPkinases signaling pathway. Additional mutations to these driver anomalies have been identified. We are interested in the KDM6A gene which codes for a lysine demethylase exerting a role of tumor suppressor. Its loss of expression would sensitize tumor cells to inhibitors of EZH2, a methyltransferase. In order to study the functional impact of the inactivation of KDM6A in HCL, we transfected a CRISPR- Cas9 system targeting exon 4 of the gene in the JVM-3 cell line. The loss of expression of KDM6A in this line promotes activation of the MAPK pathway without modifying the proliferative capacities of the cells, nor their sensitivity to BRAF inhibitors. The edited cells show better resistance to nutrient deprivation, consistent with an increase in the expression of survivin. No increased sensitivity of the modified cells to epigenetic therapies including EZH2 inhibitors has been observed. The inactivation of KDM6A thus appears to contribute to the activation of the oncogenic pathway of MAPK in HCL but does little to modify the capacities of the hairy-cells and their response to chemotherapies and targeted therapies.La leucémie à tricholeucocytes (HCL) et sa forme variante (HCL-v) sont des syndromes lymphoprolifératifs B caractérisés par des mutations de BRAF et de MAP2K1 responsables d’une activation anormale de la voie des MAP-kinases. Des mutations additionnelles à ces anomalies drivers ont été identifiées. Nous nous sommes intéressés au gène KDM6A qui code pour une lysine déméthylase exerçant un rôle de suppresseur de tumeur. Sa perte d’expression sensibiliserait les cellules tumorales aux inhibiteurs d’EZH2, une méthyltransférase. Afin d’étudier l’impact fonctionnel de l’inactivation de KDM6A dans la HCL, nous avons transfecté un système CRISPR-Cas9 ciblant l’exon 4 du gène dans la lignée cellulaire JVM-3. La perte d’expression de KDM6A dans cette lignée favorise l’activation de la voie des MAPK sans modifier les capacités prolifératives des cellules, ni leur sensibilité aux inhibiteurs de BRAF. Les cellules éditées présentent une meilleure résistance à la privation en nutriments concordante avec une augmentation de l’expression de la survivine. Il n’a pas été observé de sensibilité accrue des cellules modifiées aux thérapies ciblant l’épigénétique incluant les inhibiteurs d’EZH2. L’inactivation de KDM6A paraît ainsi concourir à l’activation de la voie oncogénique des MAPK dans la HCL mais modifie peu les capacités des tricholeucocytes et leur réponse aux chimiothérapies et thérapies ciblées

Leucémie à tricholeucocytes Correspondance : Points essentiels

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Variant form of hairy cell leukemia

International audienceMature lymphoid B-cell proliferations with hairy cells represent heterogeneous entities where specific diagnosis is difficult but important since it impacts therapeutic management. The clinical cases of variant hairy cell leukemia reported herein illustrate the persistence of a clear interest in the use of splenectomy as a therapeutic alternative. Furthermore, ibrutinib appears to be a promising treatment in patients with relapsed/refractory disease

A population-based study of hairy cell leukemia over a period of 20 years

International audienceThere are limited population-based studies of hairy cell leukemia (HCL), a rare chronic lymphoproliferative disorder of B-cells. We conducted a population-based study that included all patients diagnosed with HCL between 1996 and 2016 in Western Normandy. Recorded data focused on medical history, clinical presentation, biological results, treatment modalities in the first line and in relapsed/refractory patients and the occurrence of secondary malignancies. One hundred and twenty-three HCL patients were registered in the database. HCL represented 0.7% of all malignant hematological disorders and 3.0% of all leukemia. The overall age-standardized incidence ratio (SIR) was 0.39/100,000 inhabitants in men and 0.09/100,000 in women, and it remained stable over the 20-year period analyzed. One hundred and seven patients (88%) received first-line treatment, 33 patients (27%) received at least 2 lines of treatment and 14 patients (11%) received more than 2 lines. Cladribine used as first-line treatment induced a high hematological complete response (HCR) rate of 92%. The median overall survival (OS) was over 15 years, with 5-year and 10-year survival rates of 84% and 70.5%. No significant differences in OS were observed between men and women, between the calendar periods studied or between patients who received a single line treatment with IFN-α or PNA. The risk of relapse was higher with IFN-α treatment, requiring subsequent treatments in that patients. The time to next treatment (TTN) tends to be longer for PNAs compared to IFN-α even if difference is not significant. Secondary cancers were observed in 9/123 patients (7.3%) with solid tumors in 8 patients and hematological malignancy in one patient. Our data confirm in real life that single courses of cladribine administered to patients with HCL induce high response rates, the majority of which are HCR. Relapses seem less frequent than with IFN-α and the administration schedule is less restrictive for the patients. The emergence of chemo-immunotherapy and the development of effective new drugs such as recombinant immunotoxins and BRAF targeting will offer new possibilities in the management of HCL patients

New generation sequencing of targeted genes in the classical and the variant form of hairy cell leukemia highlights mutations in epigenetic regulation genes

International audienceClassical hairy cell leukemia (HCL-c) is a rare lymphoid neoplasm. BRAFV600E mutation, detected in more than 80% of the cases, is described as a driver mutation, but additional genetic abnormalities appear to be necessary for the disease progression. For cases of HCL-c harboring a wild-type BRAF gene, the differential diagnosis of the variant form of HCL (HCL-v) or splenic diffuse red pulp lymphoma (SDRPL) is complex. We selected a panel of 21 relevant genes based on a literature review of whole exome sequencing studies (BRAF, MAP2K1, DUSP2, MAPK15, ARID1A, ARID1B, EZH2, KDM6A, CREBBP, TP53, CDKN1B, XPO1, KLF2, CXCR4, NOTH1, NOTCH2, MYD88, ANXA1, U2AF1, BCOR, and ABCA8). We analyzed 20 HCL-c and 4 HCL-v patients. The analysis of diagnostic samples mutations in BRAF (n = 18), KLF2 (n = 4), MAP2K1 (n = 3), KDM6A (n = 2), CDKN1B (n = 2), ARID1A (n = 2), CREBBP (n = 2) NOTCH1 (n = 1) and ARID1B (n = 1). BRAFV600E was found in 90% (18/20) of HCL-c patients. In HCL-c patients with BRAFV600E , other mutations were found in 33% (6/18) of cases. All 4 HCL-v patients had mutations in epigenetic regulatory genes: KDM6A (n = 2), CREBBP (n = 1) or ARID1A (n = 1). The analysis of sequential samples (at diagnosis and relapse) from 5 patients (2 HCL-c and 3 HCL-v), showed the presence of 2 new subclonal mutations (BCORE1430X and XPO1E571K ) in one patient and variations of the mutated allele frequency in 2 other cases. In the HCL-v disease, we described new mutations targeting KDM6A that encode a lysine demethylase protein. This opens new perspectives for personalized medicine for this group of patients

Tailored Digital PCR Follow-Up of Rare Fusion Transcripts after Initial Detection through RNA Sequencing in Hematological Malignancies

International audienceThe management of hematologic malignancies has entered a new era in which minimal residual disease (MRD) monitoring plays a pivotal role. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-sequencing (RNA-seq) approach (FusionPlex) with a whole-transcriptomic strategy (Advanta RNA-Seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r = 0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection of <0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets

Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies