Development of Xpert® BCR-ABL Ultra, an Automated and Standardized Multiplex Assay with Required Performance Characteristics for BCR-ABL1 Quantitative Measurement on an International Reporting Scale
Authors: Day Gwo-Jen , Lockwood Christina , Payton Jacqueline E. , Uy Geoffrey L. , Schiffer Charles , Feldman Gerald , Bossler Aaron , Woolworth Julie , Mims Alice S. , Levitas Alba , Shridhar Krupa , Huang Sandy , Xiao Vivian , Wu Natalie , Bates Michael P. , Wong Wendy 
Published: October 2019

Abstract
Monitoring BCR-ABL transcript levels in patients on TKI therapy using real-time quantitative PCR is standard of care in the management of CML, and achieving sensitivity to 4.5 logs below baseline is important in patients being considered for TKI discontinuation. In order to accurately assess treatment response, it is essential to ensure that variability in testing methodologies is tightly controlled. This has resulted in substantial efforts to standardize BCR-ABL molecular measurement across laboratories by introducing an International Scale (IS). Here we describe the development and analytical evaluation of Xpert® BCR-ABL Ultra, an automated cartridge-based BCR-ABL monitoring assay incorporating lot-specific standardization to the International Scale as defined by WHO.
The BCR-ABL Ultra cartridge simplifies the generation of highly accurate and reproducible assay results by integrating nucleic acid extraction, target amplification, and quantification of the BCR-ABL b3a2 or b2a2 transcripts relative to ABL in peripheral blood specimens. The assay turn-around-time is approximately 2.5 hours and has a %BCR-ABL/ABL(IS) dynamic range of ~6 logs, including two key milestones for the assessment of clinical response at ~10% (IS) (SD=~0.08Log) and ~0.0033% (IS) (SD=~0.25Log) (CMR4.5). Lot-to-lot standardization is achieved using a secondary standard panel of controls derived from and aligned to the first WHO international genetic reference panel. The secondary standards are produced at Cepheid as part of the assay kit manufacturing process and applied to every production lot of cartridges in order to improve assay reliability and reporting comparability. Analytical sensitivity studies using spiked-in CML cell lines demonstrated a limit of detection (LoD) and limit of quantification (LoQ) of 0.003% BCR-ABL/ABL (IS) with a limit of blank (LoB) of 0.00017%. In an analytical specificity study, assay results were negative in 100% of 70 known CML-negative samples, including 20 AML or ALL patients and 50 healthy donors. A study was further conducted to estimate the lower limit of clinical sensitivity using blood from 12 CML patients on TKI therapy who had achieved and maintained MMR with reporting below 0.05% (IS). Eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (SD=~0.17Log) to 0.0011% (IS) (SD=~0.4Log).
In conclusion, the Xpert BCR-ABL Ultra assay demonstrates sensitivity in the range necessary to make clinical decisions regarding the continuation of TKI therapy in patients achieving CMR4.5, and every production lot of cartridges is assigned a unique conversion factor guaranteeing alignment to the WHO International Scale.

Disclosures
Uy: Novartis: Research Funding.