BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients

Wilms’ tumor 1 (WT1) gene alterations in acute myeloid leukemia

Le gène Wilms’ Tumor 1 (WT1), localisé en 11p13, code pour une protéine régulatrice de la transcription. Initialement découvert du fait de son altération dans la tumeur de Wilms, le gène WT1 s’est avéré être aussi impliqué dans les hémopathies malignes, en particulier les leucémies aiguës myéloïdes (LAM). Le rôle de WT1 dans les LAM a été souligné par la présence d’une surexpression de WT1 dans la majorité des LAM et de mutations de WT1 dans environ 10% des cas, mais sa contribution à la leucémogenèse reste à préciser. Evaluer la fréquence, les caractéristiques associées et l’impact pronostique des mutations de WT1 et du SNP (single nucleotide polymorphism) rs16754 de WT1 dans les LAM, et déterminer si la maladie résiduelle (minimal residual disease, MRD) utilisant l’expression de WT1 (MRD-WT1) est prédictive de l’évolution clinique. Méthodes. Les mutations des exons 7 et 9 de WT1, ainsi que le SNP rs16754, ont été recherchés sur des échantillons diagnostiques sanguins ou médullaires de 517 patients présentant une LAM de novo et traités dans les protocoles français ALFA-9801 ou ALFA-9802. L’exon 7 (incluant le SNP rs16754) et l’exon 9 de WT1 ont été amplifiés par PCR sur ADN génomique puis séquencés selon la méthode de Sanger. Les taux de transcrits de WT1 ont été quantifiés par PCR quantitative en temps réel (RQ-PCR) au diagnostic et au cours du suivi thérapeutique chez un total de 221 patients atteints de LAM et traités par chimiothérapie intensive. Résultats. Dans la cohorte du protocole ALFA-9802 (patients âgés de 15 à 50 ans), les mutations de WT1 ont été identifiées chez 14/268 (5%) patients. Les mutations de WT1 étaient associées à un âge plus jeune (p=0.02) et à la présence d’une duplication en tandem de FLT3 (p=0.03). Les patients mutés WT1 présentaient un risque de rechute accru (82% vs 46% à 4 ans, p<0.001) et une survie globale plus courte (22% vs 56% à 4 ans, p=0.01) que les patients non mutés. Au sein des LAM à caryotype normal (LAM-CN) (n=106), la présence d’une mutation de WT1 constituait un facteur défavorable indépendant pour le risque de rechute. Dans la cohorte du protocole ALFA-9801 (patients âgés de 50 à 70 ans), la fréquence des mutations de WT1 était seulement de 6/249 (2.5%). En raison du faible effectif de patients mutés, la valeur pronostique des mutations de WT1 n’a pas pu être étudiée dans cette tranche d’âge. L’allèle minoritaire du SNP rs16754 a été retrouvé chez 141/511 (28%) patients, à l’état hétérozygote (WT1AG) chez 123 (24%) patients et homozygote (WT1GG) chez 18 (4%) patients. Dans la cohorte globale ainsi que dans le sous-groupe de LAM-CN (n=208), le statut du SNP rs16754 n’a pas montré d’impact pronostique significatif sur l’évolution clinique. Au total, 80-90% des échantillons présentaient une surexpression de WT1 au diagnostic. Les taux de transcrits de WT1 au diagnostic étaient plus élevés dans les LAM avec cytogénétique favorable, c’est-à-dire t(15;17), t(8;21), ou inv(16)/t(16;16), et les LAM avec mutations de NPM1, et plus bas dans le sous-type FAB M5 et en cas de réarrangement du gène MLL. Le taux d’expression de WT1 au diagnostic n’était pas corrélé à l’évolution clinique. Après la chimiothérapie d’induction, un taux faible de MRD-WT1 était associé à un risque de rechute inférieur et une survie plus longue (p<0.001). La décroissance de la MRD-WT1 exprimée en log réduction était aussi significativement associée au risque de rechute, même après ajustement sur l’âge, la leucocytose initiale et la cytogénétique (p=0.004). En fin de consolidation, la MRD-WT1 était également prédictive du risque de rechute (p=0.004). Conclusion. Dans cette étude, les mutations de WT1 et la MRD-WT1, mais pas le SNP rs16754, sont significativement associées à l’évolution clinique dans les LAM. Ces résultats suggèrent que les mutations de WT1 et la MRD-WT1 pourraient contribuer à améliorer l’évaluation pronostique et la prise en charge thérapeutique des patients atteints de LAM.Wilms’ tumor 1 (WT1) gene, located at chromosome band 11p13, encodes a transcriptional regulator. Originally named for its role in Wilms’ tumor, a pediatric kidney malignancy, WT1 has since been found to be implicated in several hematological malignancies, particularly in acute myeloid leukemia (AML). The role of WT1 in AML has been underlined by the finding of WT1 overexpression in the majority of AML and somatic WT1 mutations in approximately 10% of AML, but its contribution to leukemogenesis has still not been clarified. To assess the incidence, the main associated features and the prognostic significance of WT1 mutations and WT1 single nucleotide polymorphism (SNP) rs16754 in AML patients, and to determine whether minimal residual disease (MRD) based on WT1 expression (MRD-WT1) is predictive of clinical outcome. Diagnostic bone marrow or peripheral blood samples from 517 patients (15-70 years) with previously untreated primary AML enrolled on the French ALFA-9801 or ALFA-9802 trials were analyzed for the presence of WT1 exons 7 and 9 mutations and WT1 SNP 16754. WT1 exons 7 (including SNP 16754) and 9 were amplified by PCR from genomic DNA and directly sequenced using the Sanger method. (WT1 transcript levels were quantified by real-time quantitative PCR (RQ-PCR) at diagnosis and during follow-up in a total of 221 AML patients treated with intensive chemotherapy. In the ALFA-9802 cohort (patients aged 15-50 years), WT1 mutations were identified in 14/268 (5%) cases and were associated with a younger age (p=0.02) and the presence of a FLT3 internal tandem duplication (p=0.03). Patients with WT1 mutations were found to have a shorter overall survival (4-year OS: 22% vs 56%, p=0.01) and a higher risk of relapse (4-year RR: 82% vs 46%, p=0.0008) compared to wild-type cases. Within the subgroup of patients with cytogenetically normal (CN) AML (n=106), WT1 mutation was found to be an independent adverse prognostic factor for the risk of relapse. In the ALFA-9801 cohort (patients aged 50-70 years), WT1 mutations occurred in only 6/249 (2.5%) cases. Because of the small number of WT1 mutants in this age range, we could not investigate the prognostic significance of WT1 mutations in older patients. The minor allele of WT1 SNP rs16754 was found in 141/511 (28%) patients, at the heterozygous state (WT1AG) in 123 (24%) and homozygous state (WT1GG) in 18 (4%). In the whole patient cohort and within the subset of CN-AML (n=208), WT1 SNP rs16754 status showed no significant impact on clinical outcome. Overall, WT1 overexpression was found in 80-90% of diagnostic samples. WT1 transcript levels at diagnosis were significantly higher in AML with favorable cytogenetics, that is t(15;17), t(8;21), and inv(16)/t(16;16), or with NPM1 mutations, and lower in M5 FAB subtype and in case of MLL rearrangements. Pre-treatment WT1 expression level was not correlated with clinical outcome. After induction chemotherapy, low MRD-WT1 levels were associated with reduced relapse risk and longer overall survival (p<0.001). Greater log reduction in MRD-WT1 after induction also predicted a decreased risk of subsequent relapse, that remained significant when adjusted on age, white blood cell count and cytogenetic risk group (p=0.004). After completion of consolidation, MRD-WT1 level was again predictive of the risk of relapse (p=0.004). We found that WT1 mutations and MRD-WT1, but not WT1 SNP rs16754 status, significantly affect clinical outcome in AML. These results suggest that WT1 mutations and MRD-WT1 may contribute to improve prognostic assessment and therapeutic management of AML patients

Profil d'expression de protéines impliquées dans le transport et le métabolisme des xénobiotiques dans le poumon humain sain ou tumoral

LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Comparison of TP53 mutations screening by functional assay of separated allele in yeast and next-generation sequencing in myelodysplastic syndromes

International audienceTP53 mutations are major prognostic factors in many hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Next-generation sequencing (NGS) has improved the detection of such mutations by identifying small mutated clones but functional method like FASAY (functional assay of separated allele in yeast) may prove interesting. We compared the detection of TP53 mutations by FASAY and NGS in 91 patients with AML or MDS. By FASAY, 91% of assays were evaluable and 47 patients (57%) had a functional and 36 (43%) a non-functional p53 protein. FASAY could not conclude in 8 cases (9%), mainly because of poor RNA quality. No TP53 mutation was found using NGS in 50 cases (55%), and at least one mutation was detected in 41 cases (45%). The p53 status was concordant between FASAY and NGS in 95% (79/83) of cases. The four discordances included mutations detected by FASAY only in two cases, and by NGS only in two cases. Mutations not detected by NGS consisted of insertions in intronic regions, which were not analyzed by this assay. Mutations not detected by FASAY were mutations for which the percentage of mutated allele was less than 10%, including one mutation reported as non-deleterious in the IARC database. Overall, our data suggest that FASAY is an effective and reliable method to detect TP53 mutations in AML and MDS, which allows the assessment of the protein functionality, contrary to a sequencing approach

Rare and private spliceosomal gene mutations drive partial, complete, and dual phenocopies of hotspot alterations

Genes encoding the RNA splicing factors SF3B1, SRSF2, and U2AF1 are subject to frequent missense mutations in clonal hematopoiesis and diverse neoplastic diseases. Most "spliceosomal" mutations affect specific hotspot residues, resulting in splicing changes that promote disease pathophysiology. However, a subset of patients carries spliceosomal mutations that affect non-hotspot residues, whose potential functional contributions to disease are unstudied. Here, we undertook a systematic characterization of diverse rare and private spliceosomal mutations to infer their likely disease relevance. We used isogenic cell lines and primary patient materials to discover that 11 of 14 studied rare and private mutations in SRSF2 and U2AF1 induced distinct splicing alterations, including partially or completely phenocopying the alterations in exon and splice site recognition induced by hotspot mutations or driving "dual" phenocopies that mimicked 2 co-occurring hotspot mutations. Our data suggest that many rare and private spliceosomal mutations contribute to disease pathogenesis and illustrate the utility of molecular assays to inform precision medicine by inferring the potential disease relevance of newly discovered mutations

GENETIC TYPING OF CBL, ASXL1, RUNX1, TET2, AND JAK2 IN JUVENILE MYELOMONOCYTIC LEUKEMIA (JMML) REVEALS A GENETIC PROFILE DISTINCT FROM CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML).

International audienceJMML and CMML are rare myelodysplastic/myeloproliferative neoplasms occurring at both ends of life. To investigate relationships between JMML and CMML, genes recently involved in CMML were studied in 68 JMML patients. Mutations in TET2, RUNX1, and JAK2V617F are involved in myelodysplastic and/or myeloproliferative syndromes, and more specifically in CMML but were not found in JMML. Pangenomic analysis by SNP-array showed no abnormality at these loci. Three frameshift mutations of ASXL1 leading to a truncated protein were found in 3 patients (4%) with late onset JMML displaying also RAS activating mutations. Homozygous mutations of CBL with 11q loss of heterozygosity were found in 5 (7%) JMML. CBL substitutions were different from those reported in CMML, exclusive from other RAS activating mutations, and were germline in all patients. Overall, the pattern of genetic lesions observed in JMML differed from that of CMML. Although signalling deregulation is involved in CMML, transcriptional deregulation seems to play a pivotal role, with mutation of RUNX1, ASXL1 or TET2. Conversely, none of these genes involved in transcription or chromatin remodelling was found to be significantly altered in JMML, while CBL mutations confirm the central role of RAS and growth factor signalling deregulation in JMML