Generic Protocols for Analytical Validation of Tumor-Informed Circulating Tumor DNA Assays for Molecular Residual Disease: The Blood Profiling Atlas in Cancer's Molecular Residual Disease Analytical Validation Working Group Consensus Recommendation

The presence of circulating tumor DNA (ctDNA) in patients indicates post-treatment molecular residual disease (MRD). Given the complexity of ctDNA-based MRD detection tests, consensus on analytical validation (AV) criteria is needed. To address this, the Blood Profiling Atlas in Cancer (BLOODPAC) Consortium's MRD AV Working Group evaluated existing protocols to develop standardized guidance for tumor-informed assays. Protocols pertaining to blood collection tube types, quantitative output, tissue processing, tumor or matched normal sequencing, software, and clinical validation were considered out of scope. After alignment on objectives and assumptions, study designs on the basis of best practices in the field, available assay validation guidance documents, and unique performance challenges for tumor-informed MRD assays were authored. Each protocol contains introduction, experimental design, statistical analysis, and an example data presentation per the US Food and Drug Administration (FDA) Center for Devices and Radiological Health standard format. Biostatisticians were consulted to define minimal test requirements, sample size, and appropriate statistical analyses. The protocols were submitted to the FDA via the presubmission process for formal written feedback followed by a meeting. BLOODPAC's generic protocols for the AV of tumor-informed ctDNA assays for MRD are designed to provide test developers with a core baseline of standardized AV protocols such that methods described can be adapted and applied for any tumor-informed MRD assay irrespective of technology, panel design algorithm, or workflow component. These protocols aim to optimize test developers' presubmission reviews with the FDA, ensuring productive meetings while enabling reviewers to streamline feedback. As always, test developers are encouraged to communicate with FDA directly around their particular AV methods.