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Updated: May 13, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients
Donghui Huang1, Dong-Wan Kim, Athanasios Kotsakis
1Pfizer Oncology, San Diego, CA, USA. donghuistephen.huang@pfizer.com
Abstract:
The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10-11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.
Insights
A new deep sequencing method can rapidly detect ALK resistance mutations in non-small cell lung cancer (NSCLC) patients treated with crizotinib. This aids in understanding treatment resistance and guiding future therapies.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Crizotinib is approved for ALK-positive non-small cell lung cancer (NSCLC).
- Tumor resistance to crizotinib develops due to ALK kinase domain mutations in about one-third of patients.
- Understanding resistance mechanisms is crucial for improving patient outcomes.
Purpose of the Study:
- To develop a rapid and sensitive method for detecting ALK resistance mutations.
- To analyze genomic DNA from NSCLC tumor biopsies of relapsed patients.
Main Methods:
- Developed a multiplex deep sequencing assay utilizing semiconductor sequencing technology.
- Applied a novel base-pair specific error-weighted mutation calling algorithm (BASCA).
- Analyzed genomic DNA from thirteen relapsed NSCLC patients.
Main Results:
- Successfully detected three known crizotinib resistance mutations: C1156Y, L1196M, and G1269A.
- The assay demonstrated robust and sensitive detection of ALK kinase mutations.
- Identified specific resistance mutations in the analyzed patient samples.
Conclusions:
- The developed deep sequencing assay is effective for identifying ALK resistance mutations in NSCLC.
- This method aids in the elucidation of clinical resistance mechanisms.
- Facilitates personalized treatment strategies for patients with ALK-positive NSCLC.
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