Continual Improvement of the Reliability of Next-Generation Sequencing-Based ctDNA Analysis: A Long-Term Comparison of ctDNA Detection in China

Abstract

Background: Since circulating tumor DNA (ctDNA) sequencing is increasingly being applied in clinical management of patients with cancer, its testing accuracy has become a matter of serious concern. To address this issue, a long-term ctDNA analysis proficiency testing (PT) scheme for next-generation sequencing (NGS) was launched in China in 2018, serving as an educational tool for assessing and improving the testing quality of NGS-based ctDNA detection.

Methods: Feedback from participating laboratories across 23 different PT samples containing different variants with varying variant allele frequency was collected between 2018 and 2021. To further show the landscape of changing conditions in accuracy and reliability of NGS-based ctDNA testing, performance was analyzed by evaluating the cfDNA extraction kits, testing panels, target enrichment strategies, and sequencing platforms.

Results: During the 4 years, 2745 results reported from 504 laboratories were evaluated. Only 66.3% of results from laboratories were entirely in concordance with the expected results. Nonetheless, along with an increasing number of participating laboratories, the number of errors occurring in laboratories, and the proportion of laboratories that experienced errors both showed a significant downward trend. No obvious differences in the error rates were found regarding the kit manufacturers or sequencing platform. Moreover, the individual performances of the laboratories improved when they participated in more PT scheme rounds.

Conclusions: These data demonstrated that the performance of individual Chinese laboratories for NGS-based ctDNA analysis continuously improved over time with participation in PT schemes. However, further care must also be taken in standardized operations and validations.

Keywords: circulating tumor DNA; external quality assessment; next-generation sequencing; proficiency testing; standardization.