Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Abstract

word count: 199 Abstract Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear.This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph+ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10 -3 and 36/67 (53%) and 53/67 (79%) at 10 -4 BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph+ALL.

Quantification of minimal disseminated disease by quantitative polymerase chain reaction and digital polymerase chain reaction for NPM-ALK as a prognostic factor in children with anaplastic large cell lymphoma

“… 19 , 35 – 37 In Philadelphia-positive acute leukemia the optimization and standardization process for RQ-PCR-based measurement of m- BCR- ABL1 transcripts underscores the importance of standardization of all steps for quantitative PCR, including data interpretation and quality controls. 38 In the standardization process of MRD assessment of m- BCR-ABL1 fusion gene transcripts organized by the Euro MRD consortium, the usage of a common primer and probe set as well as a centrally distributed plasmid standard curve had the greatest impact on overcoming inter-laboratory variability. 38 …”

“… 38 In the standardization process of MRD assessment of m- BCR-ABL1 fusion gene transcripts organized by the Euro MRD consortium, the usage of a common primer and probe set as well as a centrally distributed plasmid standard curve had the greatest impact on overcoming inter-laboratory variability. 38 …”

Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

“…Fusion genes are good PCR targets because they are related to the oncogenic process and are durable elements of mitosis that are stable throughout the course of the disease. Fusion transcripts also serve as templates for MRD identification, and mRNA can be reverse transcribed to create a template strand for RT-PCR cycling [61]. mRNA can be identified with a limited set of primers and is related to oncogenesis since it is a translocation fusion product directly linked to a translocation genotype.…”

“…Standardized use of EAC primer/probe sets and centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. Stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting was essential [61]. The use of a cell-based secondary reference panel for BCR-ABL1 quantification for MRD analysis in chronic myeloid leukemia was recently published [58].…”

Advances in Fusion Gene Research and Fusion Gene Families in Hematological Malignancies

“…As the most common FG in hematological malignancies, the quantitative standardization of BCR-ABL1 has been carried out for more than ten years, but there are still many problems to be solved. 38,39 Every FG-FM may consist of numerous but individually fairly rare FGs. How to effectively design suitable detection methods and how to promote the standardization of quantitative testing need further optimized solutions.…”