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Published on: April 11, 2016
Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay
Daniel Stetson1, Ambar Ahmed1, Xing Xu2
1Translational Sciences, Oncology, IMED Biotech Unit, AstraZeneca, Boston, MA.
Purpose:
Discordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored.
Materials And Methods:
To measure these variables, we tested four next-generation sequencing (NGS) gene panel assays for mutations in circulating tumor DNA (ctDNA) using replicate sets of 24 plasma samples and compared the results with matched tumor-normal tissue pairs.
Results:
Our orthogonal approach identified false-negative (FN) and false-positive (FP) variants with high confidence and revealed substantial variability among the ctDNA assays, with a range of sensitivity (38% to 89%) and positive predictive value (36% to 80%). Most discordance in our cross-vendor study was observed below 1% variant allele frequency. FP variants displayed mutational biases and tended to be novel variants not found in somatic databases. Of the 56 unique variants called by all four ctDNA assays, 41 (68%) resulted from technical discordance.
Conclusion:
These findings suggest that most NGS assay discordance is a result of technical variations and, to a lesser extent, biologic factors such as clonal hematopoiesis of indeterminate potential and tumor heterogeneity.
Insights
Technical variables, not just tumor heterogeneity, cause discordance in circulating tumor DNA (ctDNA) variant calling. Most next-generation sequencing (NGS) assay discrepancies stem from technical factors, impacting accuracy in liquid biopsy analysis.
Area of Science:
- Molecular Biology
- Genomics
- Oncology
Background:
- Discordance between plasma and tumor variant detection is often attributed to tumor heterogeneity.
- Technical variables influencing circulating tumor DNA (ctDNA) analysis remain under-investigated.
Purpose of the Study:
- To quantify technical variables impacting next-generation sequencing (NGS) gene panel assays for ctDNA.
- To compare variant calling concordance between plasma ctDNA and matched tumor-normal tissue pairs.
Main Methods:
- Four NGS gene panel assays were tested using replicate plasma samples.
- Results were compared against matched tumor-normal tissue data to identify false positives and negatives.
- Variant allele frequency and assay performance metrics (sensitivity, PPV) were evaluated.
Main Results:
- Significant variability was observed among ctDNA assays, with sensitivity ranging from 38% to 89% and positive predictive value (PPV) from 36% to 80%.
- Most discordance occurred below 1% variant allele frequency.
- 68% of unique variants identified by all four ctDNA assays resulted from technical discordance, with false-positive variants showing mutational biases.
Conclusions:
- Technical variations are the primary driver of NGS assay discordance in ctDNA analysis.
- Biologic factors like clonal hematopoiesis and tumor heterogeneity contribute to a lesser extent.
- These findings highlight the need to optimize technical aspects of ctDNA assays for improved diagnostic accuracy.
