Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Abstract

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 MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS 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.

Patient Versus Physician Perspective in the Management of Chronic Myeloid Leukemia During Treatment with Tyrosine Kinase Inhibitors

“…It has been demonstrated that patient adherence to guideline recommendations on molecular monitoring contributes to better clinical outcomes [ 2 , 15 ]. In our study, a significant proportion of patients (76.7%) reported achieving response milestones at 3, 6, and 12 months.…”

European LeukemiaNet laboratory recommendations for the diagnosis and management of chronic myeloid leukemia

“…Testing laboratories using RT-qPCR or RT-dPCR generally express results on the IS by using (i) laboratory-specific conversion factors (CFs) to the IS derived by sample exchange with an established reference laboratory [ 85 , 87 , 88 ], (ii) laboratory-specific CFs derived from secondary reference reagents calibrated to the 1st World Health Organisation (WHO) International Genetic Reference Panel for the quantitation of BCR::ABL1 [ 84 , 89 ] or (iii) validated diagnostic kits or devices calibrated to the WHO panel. Technical guidelines have defined in detail how to derive IS values from raw RT-qPCR (or RT-dPCR) data, including sample quality criteria, how to score replicate reactions and what to do if BCR::ABL1 mRNA is not detected [ 90 , 91 ]. The frequency of monitoring should follow, resources permitting, that specified by the current version of the European LeukemiaNet (ELN) clinical guidelines.…”

“…To enable assessment of DMR, it is important that laboratories use optimised tests that are able to detect MR 4.5 in most clinical samples. This requires the ability to detect small numbers of BCR::ABL1 mRNA targets as well as good quality RNA/cDNA and consistent criteria to distinguish between low-level positive and undetectable disease, as described in detail elsewhere [ 90 , 91 ]. Determination of the LoD must be performed with clinical samples; estimates of sensitivity derived solely from cell line mixtures are meaningless and should not be used or quoted on clinical reports.…”

“…Determination of the LoD must be performed with clinical samples; estimates of sensitivity derived solely from cell line mixtures are meaningless and should not be used or quoted on clinical reports. Optimization of test performance to achieve the desired LoD may be necessary, but attention should also be paid to the limit of blank (LoB), i.e., the background false positive rate, which may compromise accurate determination of DMR [ 91 ]. Use of automated, sensitive RT-qPCR systems for measuring BCR::ABL1 on the IS, such as the Cepheid GeneXpert Ultra, or IS-calibrated RT-qPCR/RT-dPCR kits validated to detect DMR, e.g.…”

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2V617F Mutation

“… 14 15 16 However, it has been widely recognized that real-time qPCR results exhibit huge variability due to its susceptibility to sample quality and operator experiences and extensive efforts are required to enhance the standardization and assay quality. 17 Droplet digital PCR (ddPCR) is an innovative PCR-based technology that partitions the nucleic acid samples into thousands of nanoliter-sized droplets, and PCR amplification is carried out within each droplet, which enables absolute quantification of DNA copies. Sample partitioning mitigates the effects of target competition, making PCR amplification less sensitive to inhibition and greatly improving the discriminatory capacity of assays that differ by only single nucleotide.…”