Reverse transcription can critically impact the diagnostic outcome of BCR::ABL1 quantitative real-time RT-PCR

• Verfasst von: Spiess, Birgit [VerfasserIn]
• Verfasst von: Kleiner, Helga [VerfasserIn]
• Verfasst von: Tarnopolscaia, Irina [VerfasserIn]
• Verfasst von: Naumann, Nicole [VerfasserIn]
• Verfasst von: Fabarius, Alice [VerfasserIn]
• Verfasst von: Hofmann, Wolf-Karsten [VerfasserIn]
• Verfasst von: Saußele, Susanne [VerfasserIn]
• Verfasst von: Seifarth, Wolfgang [VerfasserIn]
• Titel: Reverse transcription can critically impact the diagnostic outcome of BCR::ABL1 quantitative real-time RT-PCR
• Verf.angabe: Birgit Spiess, Helga Kleiner, Irina Tarnopolscaia, Nicole Naumann, Alice Fabarius, Wolf-Karsten Hofmann, Susanne Saussele and Wolfgang Seifarth
• Jahr: 2023
• Umfang: 18 S.
• Fussnoten: Veröffentlicht: 1. August 2023 ; Gesehen am 25.09.2023
• Titel Quelle: Enthalten in: Cancers
• Ort Quelle: Basel : MDPI, 2009
• Jahr Quelle: 2023
• Band/Heft Quelle: 15(2023), 15, Artikel-ID 3914, Seite 1-18
• ISSN Quelle: 2072-6694
• DOI: doi:10.3390/cancers15153914
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: BCR::ABL1 monitoring
• Sach-SW: cDNA synthesis
• Sach-SW: chronic myeloid leukemia (CML)
• Sach-SW: minimal residual disease
• Sach-SW: random hexamer priming
• Sach-SW: reverse transcriptase
• Sach-SW: specific priming
• Sach-SW: strand displacement synthesis activity
• K10plus-PPN: 1860230334

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.