A Comparison of Two Standardized Quantitative RT-PCRs with CE IVD Kit for Digital PCR in CML Patients with Different Level of BCR-ABL1 Transcripts

The detection and quantification of BCR-ABL1 transcripts play a key role in therapy management of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard strategy for monitoring of minimal residual disease (MRD). Recently, digital PCR (dPCR) was introduced as a new quantification method based on determination of absolute target sequence quantity. In connection with the deep molecular response (MR) assessment and the option of TKIs treatment cessation, the dPCR has a potential of high sensitivity, robustness and accuracy of BCR-ABL1 transcripts detection. The aim of present work was to compare the results of two standardized RT-qPCR methods with RT-dPCR detection in clinical samples of CML patients with different BCR-ABL1 fusion transcripts level. In total, 62 peripheral blood samples of CML patients with the level of BCR-ABL1 transcripts (international scale, IS) between 100% and undetectable were enrolled. The samples were analyzed by three methods: (1) CE IVD Xpert BCR-ABL Ultra Kit (GeneXpert, Cepheid), (2) conventional RT-qPCR assay according to ELN guidelines and certified by EUTOS for the level of MR4.5 BCR-ABL1 detection (ABI7300, Applied Biosystems), and (3) RT-dPCR using CE IVD QXDx BCR-ABL %ID Kit (Bio-Rad) on QX200 Droplet Digital PCR System (Bio-Rad). All steps were performed according to manufacturer´s instructions. A linear regression analysis and an overall reporting bias analysis using the Bland-Altman test were used for comparison of the methods. Based on the results of GeneXpert as the determined routine method for BCR-ABL1 transcripts quantification, the samples were divided into two groups: 50/62 samples were scored as BCR-ABL1 positive (the first group), while 12/62 samples were scored as BCR-ABL1 undetectable (the second group). The first group of GeneXpert positive samples was analyzed using both conventional RT-qPCR and RT-dPCR (both in duplicates). The median of the sum of ABL copies per sample was 180,511 copies (range 56,466 - 536,453) by RT-qPCR vs. 39,960 (range 14,360 - 71,690) by RT-dPCR. Both conventional RT-qPCR and RT-dPCR did not detect any BCR-ABL1 transcript in 4/50 samples. In addition, RT-dPCR did not detect any BCR-ABL1 transcript in 5 other samples (Figure 1). Linear regression analysis showed good correlation between both sets of assays: GeneXpert vs. RT-dPCR (R2=0.965, N=41) and conventional RT-qPCR vs.