High-Accurate Third-Generation Sequencing to Comprehensively Decipher BCR::ABL1 TKIs in-Cis Resistant Mutations

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Background Drug-resistant mutations in the ABL1 kinase domain (KD) of BCR::ABL1 are primary mechanisms of resistance to tyrosine kinase inhibitor (TKI) therapy in Ph-positive leukemia. These mutations alter the conformation of ABL1 within BCR::ABL1, impairing TKI and thus diminishing their anti-leukemia efficacy. Our previous studies, presented at the 61st and 62nd ASH Annual Meeting, demonstrated the superiority of next-generation sequencing (NGS) over Sanger sequencing for detecting BCR::ABL1-KD mutations. NGS offers higher sensitivity, accurate mutation frequency determination, and the ability to identify compound or polyclonal mutations within the same amplicon. However, NGS still needs to improve, including its short read length and the disadvantages in identifying in-cis compound mutations, lack of flexibility, and long turnaround time. Leveraging advancements in a novel third-generation sequencing (TGS) technology - characterized by single-molecule sequencing capability, long reads, real-time sequencing, and high accuracy - we have developed an approach for directly full-length in-cis BCR::ABL1-KD mutation screening. Methods The new approach allows for a comprehensive in-cis analysis of resistance mutations in the BCR::ABL1-KD using a novel TGS platform based on single-molecule side synthesis side nanopore sequencing (NSBS), distinct from PacBio or ONT sequencing. We retrospectively analyzed 30 specimens from 30 cases previously tested using NGS, including 15 mutation-positive and 15 mutation-negative specimens under previous investigation. Among them, there were ten single mutations, four double mutations, and one triple mutation, with the variant allele frequency (VAF) ranging from 7.8% to 99.6%. Results The TGS approach can directly analyze in-cis compound BCR::ABL1-KD mutations in a full-length BCR::ABL1-KD sequencing model with high single-base accuracy and superior to the NGS protocol. The novel TGS approach can detect the complete BCR::ABL1 fusion gene sequence (including p190 and p210 types, sequence range 1696~1717bp), which can more accurately obtain the ABL1 sequence in the BCR::ABL1 fusion gene, thus analyzing the veritable resistance mutations in the ABL1 KD of the BCR::ABL1 fusion gene; it has higher detection sensitivity (average sequencing coverage depth of 13086x, range 8226~24152x; Q30 can reach 99.39%; VAF as low as 1%). The TGS approach detected more mutations than NGS (27 vs. 21) and accurately distinguished more complex mutation patterns, including multiple compound or polyclonal mutations. It detected five mutations in the NGS mutation-negative group. The VAF obtained in mutation analysis was inconsistent compared to NGS, indicating that the mutations and mutation frequencies detected by TGS are more in line with the true situation and significant for continuous monitoring. Conclusion Through comparative analysis with the NGS project, the novel TGS approach shows the advantages of longer reads, higher sensitivity and accuracy, and the ability to complete the scheme from library construction to data output within 12 hours. It may provide the clinic with faster and more accurate results of in-cis BCR::ABL1-KD resistance mutation analysis with higher sensitivity and guide the clinic to quickly change treatment plans, thereby improving therapeutic outcomes.

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