Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based Circulating Tumor DNA Assays, Version 2.0.

Abstract

Circulating tumor DNA (ctDNA) is a promising biomarker for cancer detection, treatment selection, and disease monitoring. As a result, an increasing number of diagnostic developers are pursuing liquid biopsy assays using next-generation sequencing (NGS) to detect ctDNA. To support clinical adoption, these assays must undergo rigorous analytical validation (AV) to facilitate regulatory approval, support market entry, and ultimately improve outcomes for patients with cancer. To this end, the BLOODPAC AV Working Group developed and published a set of generic AV protocols in September 2020. These included 11 protocols and 4 standard methods designed as a foundational resource for in vitro diagnostic developers, support validation discussions with regulatory bodies, promote alignment between pharmaceutical and diagnostic partners, and help reduce regulatory review cycles by standardizing expectations for study design. In 2023, after over 2 years of practical application, BLOODPAC launched an open comment period (April-July 2023) to gather user feedback. Nine institutions provided comments, which were reviewed by the Working Group and aggregated with feedback received from the (US) Food and Drug Administration via presubmission process. This input informed the development of the updated version to ensure the protocols remain practical, scientifically robust, and aligned with current technologies. This paper outlines the feedback process, reviews key challenges identified, and summarizes the resulting changes. It presents the second iteration (version 2.0) of the BLOODPAC generic AV protocols, reaffirming the consortium's commitment to maintaining their relevance, usability, and scientific rigor.