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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
Published on: March 29, 2017
Highly sensitive and accurate detection of ALK-TKI resistance mutations by oligoribonucleotide interference-PCR
Chiori Tabe1, Toshitsugu Fujita2, Kageaki Taima1
1Department of Respiratory Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562 Aomori, Japan.
Background:
Patients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) are treated with ALK tyrosine kinase inhibitors (TKIs). Although most patients benefit from ALK-TKIs, the development of resistance mutations is common and results in NSCLC recurrence. To identify ALK-TKI-resistant NSCLC at the early recurrent phase, highly sensitive and accurate methods for the detection of mutations are essential.
Objective:
The aim of this study was to establish highly sensitive, accurate, cost-effective, and clinically practical methods for the detection of two frequent ALK-TKI resistance mutations, ALK G1202R and L1196M, by liquid biopsy.
Methods:
The efficacy of oligoribonucleotide interference-PCR (ORNi-PCR) was examined by first optimizing experimental conditions to specifically amplify the ALK-TKI resistance mutant DNA corresponding to ALK G1202R and L1196M mutations. ORNi-PCR was then combined with droplet digital PCR (ddPCR) or real-time PCR to detect these mutations in cell-free DNA (cfDNA) extracted from NSCLC patients.
Results:
ORNi-PCR followed by ddPCR/real-time PCR detected 1-10 copy(s) of G1202R and L1196M DNA in model cfDNA. These mutations in patients' cfDNA were identified using ORNi-PCR-based methods, whereas conventional ddPCR failed to detect them.
Conclusion:
ORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.
Insights
This study introduces a highly sensitive liquid biopsy method using oligoribonucleotide interference-PCR (ORNi-PCR) to detect anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor resistance mutations in non-small cell lung cancer (NSCLC). This approach aids in early identification of NSCLC recurrence.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) treatment relies on ALK tyrosine kinase inhibitors (TKIs).
- Resistance mutations to ALK-TKIs are a common cause of NSCLC recurrence.
- Early detection of these resistance mutations is crucial for timely intervention.
Purpose of the Study:
- To develop highly sensitive, accurate, and clinically practical liquid biopsy methods.
- To detect specific ALK-TKI resistance mutations: ALK G1202R and L1196M.
Main Methods:
- Optimization of oligoribonucleotide interference-PCR (ORNi-PCR) for specific amplification of ALK resistance mutant DNA.
- Combination of ORNi-PCR with droplet digital PCR (ddPCR) or real-time PCR.
- Detection of target mutations in cell-free DNA (cfDNA) from NSCLC patients.
Main Results:
- ORNi-PCR combined with ddPCR/real-time PCR successfully detected low copy numbers (1-10) of G1202R and L1196M DNA in model cfDNA.
- These ALK resistance mutations were identified in patient cfDNA using the ORNi-PCR-based methods.
- Conventional ddPCR assays failed to detect these mutations, highlighting the enhanced sensitivity of the new method.
Conclusions:
- ORNi-PCR followed by ddPCR/real-time PCR offers a highly sensitive and accurate liquid biopsy approach for ALK mutation detection.
- These methods show potential for identifying ALK-TKI-resistant NSCLC in its early recurrent phase.
- Further clinical validation is warranted, but the technique shows promise for personalized NSCLC management.
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