Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Establishment of the 1st world health organization international genetic referrence panel for quantitation of BCR-ABL mRNA

The International Consensus Classification of acute myeloid leukemia

The Bcr/Abl plays a central role in Philadelphia chromosome‐positive (Ph+) leukemia because of the constitutively activated Abl tyrosine kinase and its downstream pathways. Currently, the clinical treatment of imatinib‐resistant patients with tyrosine kinase inhibitors is severely limited by drug resistance and adverse effects. Herein, a dual‐targeting proteolysis‐targeting chimera (PROTAC) protein drug, termed PMIBcr/Abl‐R6, is designed by engrafting an MDM2/p53 inhibition peptide sequence onto the Bcr/Abl tetramerization domain. PMIBcr/Abl‐R6, harboring a Bcr/Abl targeting sequence and an MDM2 binding sequence, acts as a PROTAC drug in Ph+ leukemia cells. Its dual‐targeting constitution suggests that PMIBcr/Abl‐R6 designs to target the tetramerization domain instead of the Abl kinase domain, therefore has the potential to overcome drug resistance mutations in the kinase domain. The efficient ability of PMIBcr/Abl‐R6 is demonstrated to simultaneously induce Bcr/Abl degradation and activate the p53 pathway. PMIBcr/Abl‐R6 has the potential to overcome drug resistance in Ph+ leukemias by multiple mechanisms.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/advs.202104850

Kill Two Birds with One Stone: A Multifunctional Dual‐Targeting Protein Drug to Overcome Imatinib Resistance in Philadelphia Chromosome‐Positive Leukemia

La cryoconservation de tissu ovarien peut etre proposee, avant traitements hautement gonadotoxiques, a des patientes afin de preserver leur fertilite. L'autogreffe de tissu ovarien est actuellement la seule methode de reutilisation du tissu ovarien disponible, mais en cas de pathologie neoplasique, elle presente un risque de reintroduction d'eventuelles cellules malignes via le greffon. L'objectif de ce travail a ete de developper une methode pour detecter la maladie residuelle (MRD) dans le tissu ovarien par cytometrie en flux multicouleurs (CMF) en cas de leucemie aigue. Une technique de dissociation de cortex ovarien a ete mise au point a partir de tissu ovarien de reference provenant de resections percoelioscopiques. Un modele experimental de detection de la MRD a ete valide et consistait a ajouter des cellules de leucemie aigue lymphoblastique (LAL) ou myeloide (LAM) a une suspension de cellules ovariennes isolees de reference. La methode a ensuite ete utilisee pour rechercher la MRD dans le tissu ovarien cryoconserve de 11 patientes atteintes de leucemie aigue (7 LAL et 4 LAM).Le modele experimental a permis de valider une sensibilite de 10"4 et une specificite elevee tant pour les LAL que pour les LAM. Lorsqu'un marqueur moleculaire etait disponible pour la recherche de la MRD, nous avons observe une bonne correlation entre la CMF et la PCR quantitative. La detection par CMF de la MRD dans le tissu ovarien des patientes leucemiques etait positive chez 3 des 11 patientes etudiees.Cette technique est essentielle pour evaluer le risque carcinologique avant de proposer la reutilisation du tissu ovarien cryoconserve par technique d'autogreffe.

https://tel.archives-ouvertes.fr/tel-01245803/document

Qualification biologique des greffons de tissu ovarien autoconservé : Contribution à la recherche de maladie résiduelle en cas de pathologie néoplasique

The efforts made in the last decade regarding the molecular landscape of acute myeloid leukemia (AML) have created the possibility of obtaining patients’ personalized treatment. Indeed, the improvement of accurate diagnosis and precise assessment of minimal residual disease (MRD) increased the number of new markers suitable for novel and targeted therapies. This progress was obtained thanks to the development of molecular techniques starting with real-time quantitative PCR (Rt-qPCR) passing through digital droplet PCR (ddPCR) and next-generation sequencing (NGS) up to the new attractive metabolomic approach. The objective of this surge in technological advances is a better delineation of AML clonal heterogeneity, monitoring patients without disease-specific mutation and designing customized post-remission strategies based on MRD assessment. In this context, metabolomics, which pertains to overall small molecules profiling, emerged as relevant access for risk stratification and targeted therapies improvement. In this review, we performed a detailed overview of the most popular modern methods used in hematological laboratories, pointing out their vital importance for MRD monitoring in order to improve overall survival, early detection of possible relapses and treatment efficacy.

/pdf/revealing-the-mysteries-of-acute-myeloid-leukemia-from-cdpi6uat.pdf

Revealing the Mysteries of Acute Myeloid Leukemia: From Quantitative PCR through Next-Generation Sequencing and Systemic Metabolomic Profiling

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35–45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

t(9;22)(q34;q11) with the BCR-ABL mBCR fusion gene transcript - in: "Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program"

A. Navarrete

Papers

t(9;22)(q34;q11) with the BCR-ABL mBCR fusion gene transcript - in: "Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program"