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Introduction: Chronic Myeloid Leukemia (CML) represents a paradigm in oncology, characterized by the hallmark Philadelphia chromosome resulting from the t(9;22) reciprocal translocation. This abnormal chromosome gives rise to the BCR::ABL1 fusion gene, which encodes a constitutively active tyrosine kinase protein driving leukemogenesis. The use of tyrosine kinase inhibitors (TKI) have revolutionized CML treatment, demonstrating remarkable efficacy. However, TKI resistance, primarily attributed to point mutations within the BCR::ABL1 kinase domain (KD) remains a significant clinical challenge, affecting approximately 20-30% of patients. The gold standard technique for monitoring mensurable residual disease in CML is the quantitative PCR (qPCR), while Sanger sequencing is utilized to identify KD mutations upon relapse. Objective: This study aims to address the specific challenges posed by two BCR::ABL1 splicing variants — the deletion of exon 7 (Δ7) and the insertion of 35 base pairs between exons 8 and 9 (INS35) — in the molecular monitoring of CML patients. These splicing events occur frequently in CML, observed in approximately 20-25% of cases. Concurrent occurrence of alternative splicing variants with point mutations complicates the detection of low-frequency mutations, potentially leading to mislead or misinterpret results. Additionally, splicing variants are detected by qPCR and incorporated into the Molecular Response (MR) calculation, which may result in poorer MR outcomes, as literature suggests these variants exhibit reduced sensitivity to TKIs. Methods: To address these challenges in CML management, we developed a PCR-based technique coupled with fragment analysis to accurately identify these variants prior to sequencing. We validated this test with 30 samples with Δ7 and/or INS35, prior identified by the Sanger sequence. Moreover, we designed a specific qPCR assay to quantify splicing variants exclusively. Results and conclusion: We performed in silico modeling of the wild-type versus Δ7 and INS35 BCR::ABL1 isoforms, showing the KD disruption caused by the early stop codon resulting from the frameshift caused by Δ7 and INS35. Additionally, our findings indicate that this molecular approach enables highly accurate and sensitive identification of splicing variants, even at very low levels. We observed that these events are independent of CML status, also occurring in healthy donor samples. Furthermore, Δ7 and INS35 are present throughout the longitudinal patient follow-up and are not associated with TKI failure. However, Δ7 with a variant allele frequency (VAF) greater than 0.15 is more prevalent in samples with higher BCR::ABL1 transcript counts, whether at diagnosis, failure, or relapse. This study highlights how Δ7 and INS35 can confound mutational testing results and demonstrates how to overcome these challenges for a more precise analysis of point mutations in CML patients carrying splicing variants. |
