Migrating to Long-Read Sequencing for Clinical Routine BCR-ABL1 TKI Resistance Mutation Screening

Area of Science:

• Genomics
• Molecular Diagnostics
• Oncology

Background:

• Chronic myeloid leukemia (CML) treatment relies on tyrosine kinase inhibitors (TKIs).
• Emergence of BCR-ABL1 mutations confers TKI resistance, necessitating sensitive monitoring.
• Current mutation screening methods have limitations in sensitivity and detection of low-frequency mutations.

Purpose of the Study:

• To implement long-read sequencing for BCR-ABL1 TKI resistance mutation screening in a clinical setting.
• To establish robust processes and an automated analysis pipeline for clinical molecular diagnostics.
• To validate long-read sequencing against Sanger sequencing for detecting resistance mutations.

Main Methods:

• Development of sample handling and transfer protocols from clinic to sequencing facility.
• Implementation of an automated analysis pipeline for long-read sequence data.
• Clinical validation using parallel Sanger and long-read sequencing for BCR-ABL1 mutations.
• Open-source software (clamp) developed for data management, analysis, and visualization.

Main Results:

• Long-read sequencing demonstrated higher sensitivity, detecting all mutations found by Sanger sequencing, plus 16 additional low-frequency mutations.
• Automated analysis resolved clonal distributions of co-existing mutations.
• Clinical laboratory transitioned from Sanger to long-read sequencing for routine BCR-ABL1 mutation screening post-validation.

Conclusions:

• Long-read sequencing provides superior sensitivity for detecting BCR-ABL1 TKI resistance mutations, enabling earlier identification of emerging resistance.
• The developed workflow and open-source tools facilitate adoption and potential extension to other clinical applications.
• This advancement improves monitoring of CML patients and guides personalized TKI therapy.