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Updated: Jul 16, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and
Maxim Ivanov1,2,3, Konstantin Laktionov4, Valery Breder4
1Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow Region, 141700, Russia.
Background:
Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved.
Methods:
DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines.
Results:
Libraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation -0.81; p < 1e-10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed.
Conclusions:
Reporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.
Insights
Standardizing next-generation sequencing (NGS) in oncology is crucial for precision medicine. This study addresses common NGS diagnostic challenges, offering solutions for reliable cancer gene variant detection in clinical settings.
Area of Science:
- Genomics
- Oncology
- Bioinformatics
Background:
- Next-generation sequencing (NGS) holds promise for precision oncology.
- Lack of standardization hinders clinical adoption of NGS diagnostics.
- This study identifies and proposes solutions for common NGS diagnostic challenges in oncology.
Purpose of the Study:
- To dissect common problems in NGS diagnostics for oncology.
- To propose solutions for enhancing the reliability of NGS in clinical settings.
- To evaluate the impact of standardization on variant detection.
Main Methods:
- DNA extraction from 26 FFPE specimens.
- Targeted sequencing using the TrueSeq Amplicon Cancer Panel (Illumina).
- Comparative analysis of sequencing data using multiple bioinformatics pipelines.
Main Results:
- Sufficient library coverage for low-prevalent mutation detection.
- FFPE artifacts correlate with library concentration, impacting variant cut-offs.
- Identified false negatives/positives in EGFR mutation calls; 33 unique mutations found in 17 genes (TP53 most frequent).
Conclusions:
- Standardization of NGS protocols and data analysis is essential.
- Customization of NGS assays for specific diseases and samples is required.
- Clinical guidelines for reporting somatic mutations are needed for accurate interpretation.
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