External Quality Assurance of Current Technology for the Testing of Cancer-Associated Circulating Free DNA Variants

Abstract

Liquid biopsy testing is rapidly emerging as a diagnostic means of identifying circulating free DNA (cfDNA) disease-associated variants. However, the reporting of cfDNA variants remains inconsistent due in part to the application of multiple testing pipelines which raise uncertainty about current cfDNA detection efficiency. External quality assurance (EQA) programs are required to monitor, evaluate and help improve laboratory performance for cfDNA variant detection and in clinical interpretation. This study therefore evaluated the performance of diagnostic laboratories currently performing cfDNA testing in China, Australia and New Zealand. A total of 89 laboratories participated in this EQA program. Reference testing material comprised of cfDNA manufactured to contain six different genotypes in four different genes (EGFR, KRAS, BRAF, NRAS). The predicted genotypic variant allelic frequencies ranged between 0.5% - 2.5%. Proficiency testing used a z-score on the laboratory consensus allelic frequency data to compare laboratory performance for the detection of the different genotypes. Allelic frequency genotyping data were received from 88 of the 89 laboratories. Next generation sequencing and digital PCR testing platforms were primarily used by participants in this pilot EQA. The average consensus data for each cfDNA genotype identified allelic frequencies ranging between 0.39% - 4.4%. Z-score proficiency testing found that >92% of clinical laboratories were concordant for detecting the cfDNA variants. The data from this pilot study suggest that current cfDNA testing platforms can detect cfDNA allelic frequency variants from 0.39% and above with high levels of confidence. In addition, these data highlight the importance of laboratories enrolling on EQA programs so that proficiency in cfDNA diagnostic testing can be determined and potential sources of error identified and addressed.

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Funding

This work was supported by the Beijing Natural Science Foundation (No. 7174345; R.Z.), the National Nature Science Foundation of China (No. 81601848; R.Z.), and the Quality Use of Pathology Program (QUPP) funding, Australia.

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Contributions

SYC acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

RP co-developed the EQA material used in this study, acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

RZ co-developed the EQA material used in this study, acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

LZ co-developed the EQA material used in this study, acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

NP acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

KHT acquired and performed data analysis, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

TB performed interpretation of data, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

JL co-developed the EQA material used in this study, performed interpretation of data, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

MPH developed initial manuscript, performed interpretation of data, assisted in the drafting and in revisions of the updated document, approved final version and agreed to be accountable for all aspects of the work performed.

Corresponding authors

Correspondence to Jinming Li or Martin P. Horan.

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No human or animal was used in this study. All reference testing material were synthetically derived using commercially available cells.

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The authors declare no conflict of interest.

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Chai, S.Y., Peng, R., Zhang, R. et al. External Quality Assurance of Current Technology for the Testing of Cancer-Associated Circulating Free DNA Variants. Pathol. Oncol. Res. 26, 1595–1603 (2020). https://doi.org/10.1007/s12253-019-00744-8

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Keywords

  • Circulating free DNA (cfDNA)
  • Next-generation sequencing
  • Digital PCR
  • External quality assurance
  • Liquid biopsy