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Abstract: EP660

Type: E-Poster Presentation

Session title: Chronic myeloid leukemia - Biology & Translational Research

Background
Monitoring of BCR-ABL1 mRNA levels using RT-qPCR assays standardised to the International Scale (IS) is essential for management of CML patients, especially when cessation of tyrosine kinase inhibitor therapy is intended. Before the development of secondary reference materials, laboratories used an expensive sample exchange process with reference laboratories for IS calibration. Secondary cell-based reference panels are now available that are traceable to and replicate the WHO primary reference material with an additional standard to include MR4.5.

Aims
To develop and evaluate the use of lyophilised cell line panels to derive and monitor stability of laboratory specific conversion factors (CFs) and the ability of laboratories to measure MR4.5.

Methods
From 2016 – 2020 samples were distributed annually to EUTOS references labs who agreed to participate (2016 n= 49, 2017 n=48, 2018 n=50, 2019 n=49). The samples were: three HL60/K562 cell line lysates at approximately 10%, 0.1% and 0.0032%BCR-ABL IS, a cDNA sample with known ABL1 and BCR-ABL1 copy number and secondary reference material panels composed of five vials of lyophilised cells (HL60/K562; LYO panel) supplied by Novartis (2016 -2019) or Thermo Fisher Scientific (Acrometrix^TM, 2020). IS values for both panels were assigned by reference to the WHO primary reference material for both ABL1 and GUSB. All samples tested by RT-qPCR following normal lab protocols. Anonymised control gene transcript copy numbers over a 4 week audit period for 50 local routine samples were also collected.

Results
CFs were determined using the secondary reference lyophilised cell line panels using the method described at https://www.nibsc.org/documents/ifu/09-138.pdf. The validity of lab CFs was determined annually using: Optimal (+/- 1.2 fold): Previous panel CF / New panel CF = 0.83 – 1.2; Satisfactory (+/- 1.6 fold): Previous panel CF / New panel CF = 0.63 – 1.58; Unvalidated: Previous panel CF / New panel CF <0.63 or >1.58. The validity of CFs for all rounds are shown in the Table.

To assess whether labs could detect MR4.5 data were analysed in six categories: median number of CG transcripts reported for cell line lysates, detection of BCR-ABL1 in DMR cell line samples and MR4.5 reference panel samples, CG and BCR-ABL1 transcript number per ul cDNA, %BCR-ABL1/CG for cDNA sample and quality of CG audit data. The scores for each category were weighted, and combined scores calculated. In all rounds >95% of labs were assessed as being able to detect MR4.5 in all or most samples.

Conclusion
The % of labs with CFs validated as optimal or satisfactory increased from 67.5% (2016) to 92.3% (2020) and 36.4 (2016) to 53.9% (2020) for ABL1 and GUSB respectively. The percentage of laboratories able to detect MR4.5 in most samples was high across all years with a median of 98%. The EUTOS molecular standardisation study indicates that secondary reference panels can be used effectively to obtain and validate IS CFs over time, validate MR4.5 detection and monitor additional aspects of quality assurance.

Keyword(s): Chronic myeloid leukemia, MRD