Chronic Myeloid Leukemia (CML) is a disease that falls under the category of myeloproliferative neoplasms. CML is identified by the presence of the Philadelphia (Ph) chromosome, which results from a translocation between chromosomes 9q34 and 22q11. This translocation gives rise to BCR-ABL fusion gene, which encodes the BCR-ABL chimeric protein, a non-receptor tyrosine kinase constitutively active, disrupting downstream cellular pathways and establishing CML. At present there are six tyrosine kinase inhibitors (TKIs) approved as therapy for CML in Brazil: imatinib, dasatinib, nilotinib, bosutinib ponatinib and asciminib. In general, about 20 to 30% of patients will not obtain or keep with the expected responses to TKIs. The major cause of treatment failure is the rise of resistant clones with point mutations in kinase domain (KD) of BCR-ABL. These mutations usually disturbs the competitive inhibition at ATP-binding site of BCR-ABL1 oncoprotein. Up today over 50 point mutations have been listed in the literature and the identification of these mutations is essential to guide new treatment options. For example, the mutations A344P, P465S, and G671R are related to resistance to asciminib and T315I is related to resistance to all TKI except ponatinib and asciminib, however, when TKIs are not available, the patient is referred to hematopoietic stem cell transplantation. Even today, the gold standard technique used to identify mutations in KD of BCR-ABL is still Sanger sequencing technique. This assay detects not only point mutations related to resistance to TKIs, but also alternative splicing variants such as the deletion of exon 7 (Δexon7), analyzed in this study, and the insertion of 35 base pairs between exons 8 and 9. The Δexon7 presents a potential challenge in the analysis and detection of point mutations that occur between aminoacids 360 to 505, which might be related to resistance, such as the P465S mutation. Our aim is to propose a new workflow to eliminate the impairment of Δexon7 in BCR-ABL point mutation analysis.

We employed PCR followed by fragment analysis to detect patients with Δexon7 of BCR-ABL. Subsequently, we performed Sanger sequencing using primers specifically designed to analyze the entire KD region in patients with and without the Δexon7 in the laboratory's diagnostic routine.

We presented a new workflow designed to identify samples with Δexon7 in BCR-ABL gene and eliminate the interference of this variant in sequencing analysis of KD point mutations to effectively provide a complete analysis of point mutations associated with resistance to TKIs.

Although the presence of Δexon7 in the BCR-ABL gene is probably not related with resistance to TKIs, this deletion can be found in around 30% of patients and may mislead the analysis of Sanger sequencing, which is used to detect the presence of point mutations in BCR-ABL gene. In some cases, the analysis of sequences between amino acids 360 to 505 is impaired due to a double sequence in this region corresponding to the region after exon 6 of ABL gene. Some relevant point mutations are described in that region, especially now with the use of inhibitors targeted to myristoyl pocket and as compound mutation. Therefore, the analysis of that region is important to be reported in cases of resistance to TKI.