Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC’s Analytical Variables Working Group

Abstract

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC’s Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors’ presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency.

Establishing the Clinical Utility of ctDNA Analysis for Diagnosis, Prognosis, and Treatment Monitoring of Retinoblastoma: The Aqueous Humor Liquid Biopsy

“…We characterize the details of ctDNA in the AH, including diagnostic and prognostic genomic biomarkers, evaluated at diagnosis and throughout treatment longitudinally with at least 12 months in follow-up. For the purpose of establishing clinical utility, we review our past publications to demonstrate the assay’s fit for purpose, focusing on the pre-analytical, analytical, and clinical validity that have empowered us to begin establishing clinical utility, prospectively, for RB [ 20 ].…”

Clinical Utility of Plasma KRAS, NRAS and BRAF Mutational Analysis with Real Time PCR in Metastatic Colorectal Cancer Patients—The Importance of Tissue/Plasma Discordant Cases

“…The most probable explanation of this finding is the higher false negativity rate of liquid biopsy testing observed when a specific mutation present in the tumor tissue is analysed in the plasma of patients with low tumor burden. Modern techniques with improved sensitivity would probably detect circulating mutations also in case of low mutant allele fraction (MAF) [ 21 , 22 , 23 ].…”

Single-copy detection of somatic variants from solid and liquid biopsy

“…To estimate the limit of detection, we performed probit regression on log(µ), as recommended by BloodPAC 28 . We also compare our assay to an ideal assay that always generates a positive signal in presence of one, or more, amplifiable variant molecule(s).…”