Détermination de la fraction mutée de NPM1 par PCR digitale dans les leucémies aiguës myéloïdes

The mutation in the NPM1 (Nucleophosmin) gene represents one of the most frequently acquired mutations in acute myeloid leukemias (AML). In particular among AMLs with normal karyotype. Among the many types of NPM1 mutants, the type A mutation represents 80% of the mutants. The expression of mutants NPM1 constitutes a reliable molecular marker signifying the presence of residual leukemia cells. Residual disease monitoring is currently based on quantification of real-time quantitative PCR (RT-qPCR) mutated NPM1 transcripts. The results are expressed as "mutated / normal" ratio of mutants NPM1, and require the use of a reference gene. Digital PCR is a recent technology that allows the absolute quantification of a rare genetic event. It is based on the principle of micro-compartmentalisation of the sample, involving a method of dilution of the sample, followed by a conventional end-point PCR. Such an approach consists of the creation of thousands of microdroplets (ddPCR). The advantages of digital PCR are numerous: the reference gene is identical to the desired target, the sensitivity is almost identical to RQ-PCR and no use of plasmid range is required (essential in RQ-PCR). In this work, we have developed a digital droplet PCR (ddPCR), showing its feasibility on both genomic DNA and complementary DNA. At the same time, we were able to validate the applicability of the probe designed for the detection of NPM1 mutation variant A. ddPCR has proven to be a feasible and attractive method for longitudinal monitoring of mutated NPM1 AML patients.La mutation du gène NPM1 (Nucleophosmine) représente d’une des mutations acquises les plus fréquemment observées dans les leucémies aiguës myéloïdes (LAM). En particulier parmi les LAM à caryotype normal. Parmi les nombreux types de mutants NPM, la mutation de type A, représente 80% des mutants. L’expression des mutants NPM1 constitue un marqueur moléculaire fiable signant la présence de cellules leucémiques résiduelles. Le monitoring de la maladie résiduelle s’appuie actuellement sur la quantification des transcrits NPM1 mutés par PCR quantitative en temps réel (RT-qPCR). Les résultats sont exprimés en ratio « muté/normal » de mutants NPM1, et nécessite l’utilisation d’un gène de référence. La PCR digitale, est une technologie récente qui permet la quantification absolue d’un évènement génétique rare. Elle repose sur le principe de micro-compartimentation de l’échantillon, par une méthode de dilution de l’échantillon, suivie d’une PCR classique en point final. L’une de ces approches consiste en la création de milliers de microgouttelettes (ddPCR). Les points forts de la PCR digitale sont nombreux : gène de référence identique à la cible recherchée, sensibilité quasi identique à la RQ-PCR, absence d’utilisation de gamme plasmidique (indispensable en RQ-PCR). Dans ce travail, nous avons mis au point une PCR digitale en gouttelettes (ddPCR), en montrant sa faisabilité à la fois sur l’ADN génomique et l’ADN complémentaire. Parallèlement, nous avons pu valider l’applicabilité de la sonde conçue pour la détection du variant A de mutation NPM1. La ddPCR s’est avérée comme une méthode faisable et attractive dans le suivi longitudinal des patients LAM NPM1 muté

Whole Lung Lavage of Nine Children with Pulmonary Alveolar Proteinosis: Experience in a Tertiary Lung Center

Background: Pulmonary alveolar proteinosis (PAP) is a rare disease in children, characterized by intraalveolar accumulation of large amounts of surfactant proteins, which severely reduce gas exchange. Whole lung lavage (WLL) is the preferred technique for the treatment of severe PAP. Case Presentation: This report presents nine pediatric cases with advanced PAP who underwent WLL under general anesthesia during a 9 year period. One patient was treated with multiple unilateral WLL without employing cardiopulmonary bypass (CPB) and eight cases were treated by simultaneous lavage of both lungs using partial CPB. Conclusion: Our experience suggested that partial CPB was useful to support oxygenation during WLL in small children with severe PAP in whom lung separation and selective lavaging of each lung were impracticable

Reduced Venetoclax Exposition to Seven Days of Azacitidine Is Efficient in Treatment-Naïve Patients with Acute Myeloid Leukemia

International audienc

Next-Generation Sequencing on Circulating Tumor DNA in Advanced Solid Cancer: Swiss Army Knife for the Molecular Tumor Board? A Review of the Literature Focused on FDA Approved Test

FDA-approved next-generation sequencing assays based on cell-free DNA offers new opportunities in a molecular-tumor-board context thanks to the noninvasiveness of liquid biopsy, the diversity of analyzed parameters and the short turnaround time. It gives the opportunity to study the heterogeneity of the tumor, to elucidate complex resistance mechanisms and to adapt treatment strategies. However, lowering the limit of detection and increasing the panels’ size raise new questions in terms of detection of incidental germline alterations, occult malignancies and clonal hematopoiesis of indeterminate potential mutations. In this review, after a technological discussion and description of the common problematics encountered, we establish recommendations in properly using these FDA-approved tests in a molecular-tumor-board context