Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR

SIMPLE SUMMARY: Reverse transcriptases (RT) play a crucial role in BCR::ABL1 fusion transcript monitoring of chronic myeloid leukemia (CML). RT enzyme and reaction conditions may contribute to impairment of stoichiometry in cDNA synthesis potentially biasing in qRT-PCR data. We have comparatively investigated the performance of the MLV-RT and SuperScript IV with random-hexamer vs. target-specific priming by means of the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens. Our experiments identified priming type and RT type as major factors for diagnostic data variation. Variation was mainly due to different efficacies of RT enzymes to process low- (<50) and high-copy targets. The impairment of the high-performing SuperScript IV in processing low- (BCR::ABL1) and high-copy-number (GUSB or ABL1) RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. For improving BCR::ABL1 assay sensitivity with a faithful representation of diagnostic targets, increased RNA/cDNA amounts and distinct RT/priming combinations are highly recommended. ABSTRACT: Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.