Comprehensive Quantitative RT-PCR Detection of Multiple BCR-ABL1 mRNA Species for Diagnosis and Monitoring of Leukemias

Introduction:

The BCR-ABL1 genomic fusion event in chronic myelogenous and B-lymphoblastic leukemias (CML, ALL) commonly produces the major chimeric transcript variants e13 (or e14)-a2 or e1-a2, which are translated respectively to p210 and p190 bcr-abl1 oncoproteins. However, substantial transcript heterogeneity is present including alternative splice forms in a given leukemia, as well as rare alternative chimeric transcript types. Relatively little is known regarding the molecular response of alternative transcript variants of BCR-ABL1 during tyrosine kinase inhibition (TKI) therapy, in part because of the lack of effective disease burden monitoring methods. We present an approach to detection of the various BCR-ABL1 mRNA types using a multiplex quantitative RT-PCR (QRT-PCR) methodology. This technique enables diagnosis and molecular tracking of unusual BCR-ABL1 variants and can improve our understanding of how these uncommon leukemias behave in response to TKI therapies. Furthermore, the method is more generally applicable for simultaneous QRT-PCR detection and measurement of different mRNA species.

Methods:

Patient samples for diagnostic evaluation were obtained and total RNA extraction was performed using the Qiagen RNeasy Mini Kit (Qiagen USA, Valencia, CA). Reverse transcription was done using Multiscribe Reverse Transcriptase (Life Technologies, Grand Island, NY). Eleven fluorescent primer and probe mixes were designed (e1a2-FAM, e6a2-Cyc 5, e13a2-FAM, e14a2-FAM, e19a2-Cyc 5, e1a3-FAM, e12a3-Cyc 5, e13a3-FAM, e14a3-FAM, e19a3-Cyc 5, and Gusb reference control-HEX). 10 uL reactions were performed (95 C X 10 min, 45 cycles of 95 C X 10 sec and 60 C X 30 sec) using four multiplex wells per sample. Double strand oligonucleotides (oligos) for each target type were synthesized (IDT, Coralville IA) and diluted in a range of 10⁶, 10⁵, 10⁴, 10³, 100, 10, 1 and 0.1 copies/uL. Results of the new multiplex QRT-PCR assay were compared against our standard method of qualitative multiplex RT-PCR and fluorescent probe hybridization.

Results:

Results for the target BCR-ABL1 oligo controls are summarized in the table. The data show a wide dynamic range of detection with reproducible copy number detection at very low level. All PCR targets had excellent correlation coefficients (R²) and good to excellent PCR efficiency. Specificity was excellent, except for some cross-reactivity of A3 templates. This minor interference did not detract from assay performance or interpretation. Thirty-two patient samples were evaluated using the multiplex QRT-PCR assay and the current lab method. For 13 known BCR-ABL1 negative and 19 positive patient samples, concordance/accuracy and specificity were 100%. Furthermore, relative quantification of transcript abundance was obtained by the multiplex QRT-PCR approach, enabling distinction between diagnostic and post-treatment mRNA levels. Alternative splicing events producing additional transcripts (e1-a2) were resolved in the same specimen and always quantified well below the primary transcript level.

Conclusions:

We demonstrate a powerful and relatively simple method for concurrent detection and quantitative measurement of multiple common and unusual alternative BCR-ABL1 mRNA transcripts. The results of this study provide a basis for more detailed monitoring of molecular disease levels during TKI therapy in leukemic patients with rare BCR-ABL1 types, and in turn, potentially enhance the ability to more optimally individualize clinical management for these patients. The platform is flexible and novel BCR-ABL1 transcript types can also be incorporated relatively easily. A similar strategy is applicable to the detection of chimeric mRNA species in other types of leukemias. Our approach can also be used to evaluate targeted RNA expression profiles of other prognostic or theranostic tumor markers in individual patients.