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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
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Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory
Tindaro Giardina1, Cleo Robinson2, Fabienne Grieu-Iacopetta1
1Anatomical Pathology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA, Australia.
Pathology
|May 13, 2018
Summary
Targeted next-generation sequencing (NGS) provides a reliable method for detecting multiple genetic mutations in cancer specimens, showing high concordance with existing platforms. This approach is suitable for various sample types, including those with limited material or low tumor cell content.
Area of Science:
- Oncology
- Genetics
- Molecular Diagnostics
Background:
- Routine diagnostic use of next-generation sequencing (NGS) is developing, facing challenges with complexity.
- Targeted NGS offers an efficient alternative to single-target assays for identifying multiple cancer-related genetic aberrations.
- Clinical demand for comprehensive genetic testing in cancer specimens is increasing.
Purpose of the Study:
- To validate targeted NGS performance against established mutation detection platforms in a diagnostic laboratory setting.
- To assess the concordance of targeted NGS results with Sanger sequencing, pyrosequencing, CAST PCR, and Cobas assays.
- To evaluate the suitability of targeted NGS for various formalin-fixed, paraffin-embedded (FFPE) cancer sample types.
Main Methods:
- A blinded validation study was conducted on 113 FFPE tumor samples (core biopsies, resections, cytology) from non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM), and gastrointestinal stromal tumor (GIST).
- DNA was extracted, and libraries were prepared using the TruSight Tumor 26 gene panel for targeted NGS on a MiSeq instrument.
- NGS results were compared with those obtained from Sanger sequencing, pyrosequencing, CAST PCR, and Cobas assays.
Main Results:
- Targeted NGS demonstrated high concordance (94.7%) with conventional methods, with 107 out of 113 cases showing agreement.
- False negatives were attributed to sequencing quality failures or mutations falling outside the NGS panel's target range.
- NGS identified 113 additional mutations, including 26 with known clinical importance and 37 with potential clinical significance, using low DNA input (10-20 ng) and samples with <50% tumor cell content.
Conclusions:
- Targeted NGS is highly concordant with established mutation testing platforms and is suitable for routine diagnostic use.
- The method performs well across diverse FFPE sample types, including those with limited material or low tumor cell content.
- This study established quality parameter settings for robust mutation data generation via NGS in a diagnostic laboratory.
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