A cell-based BCR-ABL1 secondary reference panel traceable to the World Health Organization BCR-ABL1 International Genetic Reference Panel – with an additional MR4.5 level – provides easier access to International Scale calibration and can act as a tool for assay optimization, validation, and quality assurance for molecular monitoring in chronic myeloid leukemia patients.

Such monitoring is important to disease management, especially for decision making with respect to treatment cessation. The new secondary reference panel would allow laboratories to circumvent the oft-used and time-consuming sample exchange process with reference laboratories for International Scale (IS) calibration, Nicholas C. P. Cross, PhD, of the University of Southampton (England) and colleagues wrote in an article published in Leukemia (2016 Jun 3;30:1844-52).

“The development of BCR-ABL1 tyrosine kinase inhibitors ... has enabled progressively deeper molecular responses in CML patients undergoing tyrosine kinase inhibitor therapy,” the authors wrote, noting that deeper molecular responses are “important milestones for patients considering treatment cessation,” and that “other landmarks on the IS also represent different treatment decision thresholds and prognostic outcomes.”

For this reason, regular molecular monitoring using real-time reverse-transcription quantitative PCR (RqPCR) is recommended for optimal disease management, they said.

“As treatment decisions are directly impacted by test results, accuracy and precision of BCR-ABL1 assays across the entire measurement range is crucial for patient management, especially in patients with deep molecular responses when considering possible treatment cessation,” they wrote.

Access to the material for the WHO BCR-ABL1 reference panel (MR1-MR4), which was developed in 2010 as a primary standard for BCR-ABL1 assay IS calibration, is limited, particularly for smaller laboratories, the authors said.

The secondary reference panel they developed, however, is “traceable to and faithfully replicates the WHO panel in both raw materials (lyopholized.K562 and HL-60 cell mixes) and manufacturing process, with the addition of a MR4.5 level.” The secondary panel was calibrated to IS using digital PCR against ABL1, BCR, and GUSB as reference genes, and was successfully evaluated by 45 different BCR-ABL1 assays at 44 different clinical laboratories in a multinational evaluation study.

The MR4.5 level was added to allow for more accurate IS calibration “as CML patients reaching this deep molecular response are increasingly being considered for treatment cessation,” the authors noted.

“Quality-control assessments indicated that the secondary panel had minimal residual moisture, excellent vial-to-vial homogeneity and greater than 2.5 years of real-time stability,” they said.

Further, the panel was successfully processed by all of the laboratories, indicating that it is compatible with many different BCR-ABL1 test configurations, they added.

Of note, the number of assays that achieved good precision and sensitivity exceeded the number that achieved good IS accuracy, suggesting an unmet need for “a simple and broadly available calibration mechanism, such as this secondary panel, to ensure IS accuracy is maintained in laboratories over time,” they wrote, concluding that such a panel “can provide easier access to IS calibration, as well as act as a tool for assay optimization, validation and quality assurance.”

In a letter to the editor in regard to the findings by Cross et al., Maria Sol Ruiz of Instituto Alexander Fleming in Buenos Aires, and colleagues wrote about their own development and validation of “secondary reference materials calibrated to the IS through the WHO primary standards in order to facilitate standardization of molecular monitoring in Latin America,” (Leukemia. 2016 Aug 19. doi: 10.1038/leu.2016.197).

In their study, the letter’s authors demonstrated that secondary reference biological calibrators anchored to the WHO primary standards can decrease inter-laboratory variability.

“Our results, together with those recently reported by Cross et al., substantiate the objective initially set during the establishment of the WHO primary standards, that is, to facilitate worldwide diffusion of the IS. For the first time in Latin America, this study provides a platform on which to assess the performance of distinct clinical BCR-ABL1 tests and confirm the utility of secondary reference materials to further improve IS accuracy and inter-laboratory precision,” they wrote, noting that their efforts will continue through provision of secondary reference material to centers involved in their project, as well as to potential new participants.

“Moreover, due to its higher precision and absolute quantification capability, we are evaluating the possibility of including digital PCR as the calibration method for the future,” they said.

The study discussed in the letter to the editor was supported by a grant from Novartis to one of the authors. Novartis also paid speaking fees to some of the authors. The study by Dr. Cross et al. was also funded by Novartis and some authors are employed by Novartis. The remaining authors, including Dr. Cross, reported having no disclosures.

sworcester@frontlinemedcom.com

Secondary reference panel expands access for CML monitoring