Abstract LB-223: Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples
The increased reliance on liquid biopsy next generation sequencing assays for oncology clinical decisions has highlighted the importance of understanding the factors that impact the analytical validation and clinical utility of these tests. Although recent studies have been published examining the c...
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| Published in: | Cancer research (Chicago, Ill.) Vol. 78; no. 13_Supplement; p. LB-223 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
01-07-2018
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| Online Access: | Get full text |
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| Summary: | The increased reliance on liquid biopsy next generation sequencing assays for oncology clinical decisions has highlighted the importance of understanding the factors that impact the analytical validation and clinical utility of these tests. Although recent studies have been published examining the concordance between tumor and plasma sequencing data from the same patient, there has been a lack of insight into the intra-assay variability within these tests. We have previously performed a replicate study across multiple ctDNA sequencing vendors and found a high degree of variability among assays particularly at low allele fractions (AF<1%). To further extend our analysis, we examined the variability of ctDNA sequencing results from replicate patient plasma samples with the same vendor assay. The cohort consisted of 25 total samples: seven cancer patient plasma samples, representing triplicate 2 ml baseline plasma samples, plus four healthy normal samples, representing two different pools of six subjects in duplicate. The plasma names were blinded and shipped to Guardant Health for Guardant360 panel testing and both the raw data and reported variant calls were returned to AstraZeneca for comparative analysis. A total of 17 unique variants were called in this study, of which four variants were concordant for all three replicate plasmas and 13 variants were reported in only one or two replicates. Examining the raw data for all samples, the majority of the discordant variants were able to be identified at low levels. Plasmas had low amounts of ctDNA, and two patients had low unique molecule counts, with allele fractions ranging from 0.10-1.13%. Interestingly, the reported yield of the extracted DNA did not correlate with the ability to detect low frequency somatic variants. Data will be presented showing the unblinded comparisons between the replicate samples for both the cancer patient plasma data and the pooled normal plasmas. This study highlights the importance of reproducibility studies for ctDNA samples with variants at low allele fractions to discern between false negative and false positive finding.
Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Barrett Nuttall, Tristan J. Lubinski, Fakhera Afshar, Amelia Raymond, Carl Barrett. Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-223. |
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| ISSN: | 0008-5472 1538-7445 |
| DOI: | 10.1158/1538-7445.AM2018-LB-223 |