Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing
Chih-Jian Lih1, Han Si1, Biswajit Das1
1Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
The Journal of Molecular Diagnostics : JMD
|July 27, 2016
Summary
The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 aids in evaluating HER2 gene amplification accuracy. This genomic DNA reference material helps improve confidence in clinical reporting for breast cancer diagnostics.
Area of Science:
- Genomics
- Molecular Diagnostics
- Biotechnology
Background:
- Accurate quantification of HER2 gene copy number is critical for breast cancer diagnosis and treatment.
- Standard Reference Materials are essential for validating molecular diagnostic assays.
Purpose of the Study:
- To evaluate the performance of next-generation sequencing (NGS) methods using NIST Standard Reference Materials 2373.
- To assess laboratory and bioinformatic approaches for determining HER2 gene amplification ratios.
Main Methods:
- Utilized NIST Standard Reference Materials 2373, genomic DNA from five breast cancer cell lines.
- Employed real-time quantitative PCR, digital PCR, targeted-amplicon, whole-exome, and whole-genome sequencing.
- Compared laboratory and bioinformatic performance across different amplification ratios.
Main Results:
- High reproducibility was observed across all tested NGS methods.
- Slight variations in HER2 copy number were detected, attributed to platform-specific biases.
- Demonstrated the utility of SRMs 2373 for assay validation and performance evaluation.
Conclusions:
- NIST Standard Reference Materials 2373 serve as valuable calibrators for HER2 amplification assays.
- The reference material enhances confidence in reporting HER2 amplification for clinical applications.
- Identified platform-specific biases in NGS methods, highlighting the need for careful assay validation.
