Quantitative Multiplex Measurement of BCR-ABL1 Expression on the International Scale Using the BCR/ABL1 Quant™ RUO Reagents and the LightCycler® 480
Authors: Brown Justin T , Laosinchai-Wolf Walairat , Labourier Emmanuel , Ginsburg Philip J , Ryder Thomas 
Published: October 2019

Abstract 4430

Introduction:

BCR-ABL1 e13a2 or e14a2 fusion transcripts corresponding to the translocation (9;22) are present in >95% of chronic myeloid leukemia (CML) patients. This molecular signature is thus useful to monitor tumor burden and tyrosine kinase inhibitor treatment response in CML. The e1a2 variant is found in about 5% of children with acute lymphoblastic leukemia (ALL) and 20–35% of adult ALL. Growing evidence indicates that the e1a2 variant is associated with inferior outcome and higher risk of relapse in CML. It is therefore also important to study e1a2 expression levels in these individuals. The objective of this study was to evaluate the use of a single multiplex assay (BCR/ABL1 Quant™ RUO) for the quantitative detection of e1a2, e13a2, and e14a2 fusion transcripts. These reagents were initially optimized for use with the ABI 7500 series of instruments (Brown et al., Blood Cancer Journal 2011). Here we investigated their compatibility and performance on the LightCycler® 480 System (LC480).

Method:

Control RNAs containing or lacking BCR-ABL1 transcripts were obtained from commercial sources. The kit’s Calibrator standard curve samples and the ARQ IS Calibrator Panel were both prepared by heat lysis prior to reverse transcription (RT). The RNA was then reverse transcribed and then amplified by multiplex, real-time quantitative PCR on the ABI 7500 Fast Dx or LC480 using target-specific primers for e1a2, e13a2, e14a2, and ABL1 as an endogenous internal control. Runs on the LC480 used a ramp rate of 1.6 degrees/second to mimic that of the ABI 7500. Also, color compensation reagents (positive simplex reactions) were used on the LC480 per manufacturer’s recommendations to generate a color compensation profile to enhance the performance of the multiplex reactions. Four-point standard curves were generated in each run using the included multiplex ARQ Calibrators (each calibrator sample contains each target: BCR/ABL1, ABL1, and BCR/ABL1 Quant Norm, an exogenous internal control). Fusion transcript identity was determined by capillary electrophoresis on an ABI 3130xl series instrument with 36 cm capillaries using POP7. Data were analyzed using either instrument’s software (SDS v1.4 for the ABI 7500 Fast Dx and LightCycler software v1.5 for the LC480) or proprietary Excel-based software developed by Arista Molecular.

Result:

Analytical performance assessed using cell line dilutions showed a lower limit of detection equivalent to at least one translocation-positive cell in 100,000 normal cells. Linearity of >5 logs was observed with these samples. Breakpoints e13a2, e14a2, and e1a2 were all detected with various control materials. Negative specimens did not generate signal. ANOVA of multiple independent runs of controls sample replicates in randomly assigned plate locations on the LC480 showed excellent inter- and intra-run reproducibility and no detectable spatial bias across the plate. Side-by-side runs of the samples on both the 7500 and LC480 showed excellent correspondence of the results. Finally, testing of residual clinical specimens and prototype secondary IS reference materials (ARQ IS Calibrator Panel) linked to the WHO 1st International Genetic Reference Panel for the quantitation of BCR-ABL translocation indicated that the assay has the performance required to report on the International Scale (IS) when performed on the LC480.

Conclusion:

The BCR/ABL1 Quant RUO reagents are compatible with use on the LC480 instrument. They have the advantage of both quantifying and distinguishing e1a2, e13a2, and e14a2 fusion transcripts in a single reaction. Streamlined reagent formulation, multiplex assay format, and inclusion of ARQ calibrators improve the workflow and increase the number of specimens that can be tested per run. Our results suggest that this research assay has the performance required for reporting on the IS. Further evaluation with a larger set of clinical specimens is warranted.

Disclosures:

Brown: Asuragen: Employment. Laosinchai-Wolf:Asuragen: Employment. Labourier:Asuragen: Employment.