Determination of BCR-ABL Overexpression by Correlating qRT-PCR and IP-FISH in Imatinib Treated CML Patients.
Authors: Bianchini Michele , Gargallo Patricia , Alú Fernanda , Barreyro Paula , Rosales Belen , Bengió Raquel , De Brasi Carlos , Larripa Irene 
Published: October 2019

Abstract
Imatinib mesylate (IM) has proven to be the most effective treatment in Chronic Myeloid Leukemia (CML). Nevertheless, IM resistance and molecular evidence of persistent disease have raised concern and prompted interest in additional strategies to achieve disease eradication. Resistance to Imatinib in the treatment of CML is mainly associated to 3 mechanisms: acquired mutations in the kinase domain of BCR-ABL protein, genetic amplification and transcript overexpression of BCR-ABL rearrangement. Therefore an accurate assesment of molecular resistance is particularly important to improve strategies to overcome IM resistance. Amplification of BCR-ABL gene can be determined by interphase fluorescence in situ hybridization (IP-FISH) while BCR-ABL transcript expression can be determined by quantitative real time PCR (qRT-PCR) and normalized by the simultaneous quantification of ABL housekeeping gene [MR (Molecular Ratio): BCR-ABL/ABL]. With the aim to determine overexpression of BCR-ABL, we propose a method that correlates qRT-PCR and FISH data from the same peripheral blood sample. Informed consent was obtained from all cases. The ratio between both methodologies (MR)/(IP-FISH) permit to calculate the Expression Index (EI) for each patient. The last ratio allow to estimate the level of BCR-ABL transcripts expressed per tumor cell and to characterize the overexpression. The EI was determined in 93 CML patients (53 males and 40 females) in different disease phases (chronic 55%, accelerated 32% and blast crisis 13%). The median EI value including all cases was 0.25 (range 8×10−6- 86). Percentile analysis was performed to stratify the patients in different groups; those cases included in percentile 90 showed an increment of EI above 1 Log respect to the median value, that we defined as cases with overexpression of BCR-ABL transcripts (10/93, 10.7%). We also evaluated the EIs using ROC curve, which permit an analysis of the tradeoff between sensitivity and specificity at variable cut points. The ROC curve was constructed using EI values from two groups of CML patients defined as: test group (n=18) with discordant data (FISH < 5%, MR > 0.01%) and control group including responder patients (FISH < 5%, MR < 0.01%) and not responder patients (FISH > 5%, MR > 0.01% or MR < 0.01%) (total n=75). The area under the ROC curve (AUC) quantifies the overall ability of the test to discriminate between those individuals with or without BCR-ABL overexpression. We obtained a significant (p < 0.0001) AUC value of 0.85 (95% CI, 0.74 to 0.95); choosing an EI cutoff of 1.84 we obtained a sensitivity of 96% (95% CI, 88.7% to 99.2%) and a specificity of 61% (95% CI, 35.7% to 82.7%). This cutoff is slightly lower than the value we obtained by the percentile stratification; therefore, more patients were included in the overexpressing group (n=14). Finally, several point mutations in the ABL tyrosine kinase domain were detected in 17 out of 93 patients (18.3%) at the time of resistance. All cases (n=14) included in the overexpressing group were resistant to imatinib treatment and any other cause of resistance such as, point mutations, amplification and clonal evolution, could be described within this group. These results represent the characterization of a methodology to determine BCR-ABL overexpression in CML patients; in conclusion our findings show that EI cutoff value in a screening population is a potential valuable marker for BCR-ABL overexpression detection.