7 research outputs found
The Plateau of BCR-ABL Transcript Level â„0.1% May Select CML Patients in Complete Cytogenetic Remission for Mutation Analysis.
P669: ANALYSIS OF FACTORS INFLUENCING THE DECISION TO STOP TKI TREATMENT IN CML PATIENTS IN DEEP MOLECULAR RESPONSE
Results of the European survey on the assessment of deep molecular response in chronic phase CML patients during tyrosine kinase inhibitor therapy (EUREKA registry)
Purpose: the advent of tyrosine kinase inhibitor (TKI) therapies has revolutionized the treatment of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCRâABL1 transcripts by real-time quantitative PCR every three months during TKI treatment. Since a proportion of patients in deep molecular response (DMR: MR4, MR4.5, MR5 ) maintain remission after treatment stop,assessment of DMR is crucial. However, systematically collected molecular data,monitored with sensitive standardized assays, are not available outside clinical trials.
Methods: data were collected on the standardized assessment of molecular response in the context of real life practice. BCR-ABL1 transcript levels after >2 years of TKI therapy were evaluated for DMR. Since standardized molecular monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating laboratories was carried out.
Results: between 2014-2017, 3,377 peripheral blood samples from 1,117 CML patients were shipped to 11 standardized reference laboratories in six European countries. BCRABL1 transcript types were b3a2 (41.63%), b2a2 (29.99%), b2a2/b3a2 (3.58%) and atypical (0.54%). For 23.72% of patients, the initial transcript type had not been reported. Response levels (EUTOS laboratory) were: no MMR, n=197 (6.51%); MMR, n=496 (16.40%); MR4 , n=685 (22.64%); MR4.5, n=937 (30.98%); MR5 , n=710 (23.47%). With a Cohenâs kappa coefficient of 0.708, a substantial agreement between EUTOScertified and local laboratories was shown.
Conclusions: multicenter DMR assessment is feasible in the context of real life clinical practice in Europe. Information on the BCR-ABL1 transcript type at diagnosis is crucial to accurately monitor patientsâ molecular response during or after TKI therap
Analysis of chronic myeloid leukemia during deep molecular response by genomic PCR: a traffic light stratification model with impact on treatment-free remission
This work investigated patient-specific genomic BCR-ABL1 fusions as markers of measurable residual disease (MRD) in chronic myeloid leukaemia, with a focus on relevance to treatment-free remission (TFR) after achievement of deep molecular response (DMR) on tyrosine kinase inhibitor (TKI) therapy. DNA and mRNA BCR-ABL1 measurements by qPCR were compared in 2189 samples (129 patients) and by digital PCR in 1279 sample (62 patients). A high correlation was found at levels of disease above MR4, but there was a poor correlation for samples during DMR. A combination of DNA and RNA MRD measurements resulted in a better prediction of molecular relapse-free survival (MRFS) after TKI stop (nâ=â17) or scheduled interruption (nâ=â25). At 18 months after treatment cessation, patients with stopped or interrupted TKI therapy who were DNA negative/RNA negative during DMR maintenance (green group) had an MRFS of 80% and 100%, respectively, compared with those who were DNA positive/RNA negative (MRFSâ=â57% and 67%, respectively; yellow group) or DNA positive/RNA positive (MRFSâ=â20% for both cohorts; red group). Thus, we propose a âtraffic lightâ stratification as a TFR predictor based on DNA and mRNA BCR-ABL1 measurements during DMR maintenance before TKI cessation
Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy
Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (nâ=â10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (pâ=â0.015), and the amplification efficiency was 2% greater (Pâ=â0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (nâ=â67) compared to e14a2 (nâ=â78) when analysed by RT-qPCR (Pâ=â0.0005) but not ddPCR (Pâ=â0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management
Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study
Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR
4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1
IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.
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A certified plasmid reference material for the standardisation of BCRâABL1 mRNA quantification by real-time quantitative PCR
Serial quantification of BCRâABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCRâABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 Ă 106, 1.08±0.11 Ă 105, 1.03±0.10 Ă 104, 1.02±0.09 Ă 103, 1.04±0.10 Ă 102 and 10.0±1.5 copies/ÎŒl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCRâABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCRâABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)