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BloodPAC Publishes Analytical Validation Protocols for NGS Liquid Biopsy Assay Development

NEW YORK – The Blood Profiling Atlas in Cancer Consortium (BloodPAC) has released a series of analytical protocols for validating next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assays, which the consortium believes will help accelerate the development and regulatory reviews of new tests.

Developed by consortium members, the resource may potentially serve as a set of generic analytical protocols and help to define industry standards for developing liquid biopsy assays.

As research groups and molecular diagnostic firms continue to develop accurate ctDNA tests, the liquid biopsy space requires a standardized approach to validate assays to guarantee the legitimacy of information that clinicians will need for their treatment decisions. Uncertainty in the space also exists regarding the reliability of ctDNA testing, as studies have shown conflicting results between ctDNA and tissue genotyping.

BloodPAC's Analytical Variables Working Group (AV WG) published the resource this week in Clinical Chemistry. It contains four standard methods and 11 protocols offering guidance on different aspects of validation studies, including limits of detection, accuracy, and contrived sample functional characterization.

The group aimed to develop a standardized, generic starting point for all analytical validation protocol discussions so that the methods could be adapted for any NGS ctDNA assay, regardless of design, enrichment technology, or any workflow component.

AV WG's recommended protocols address specific parts of the validation process, including the collection of normal human plasma; sample preparation; preparing contrived samples using ctDNA from culture media; and preparing contrived samples using fragmented cell line genomic DNA.

By developing the protocols with input from the US Food and Drug Administration through its official pre-submission review process, the team hopes to minimize the length of pre-submission review, typically about three months.

BloodPAC's team consists of members from industry, academia, not-for-profits, and US government agencies, including firms that sell liquid biopsy assays, companies that use liquid biopsy assays as companion diagnostics, organizations that do research related to liquid biopsy assays, organizations that conduct clinical trials involving liquid biopsies, and agencies that develop policies and procedures related to liquid biopsies.

In addition to helping pre-submission discussions between manufacturers and the FDA, BloodPAC hopes that the standardization protocol for analytical validation will support discussions between assay developers and potential industry partners.

In addition, groups developing laboratory developed tests (LDTs) that use plasma-based NGS assays may also find the best practices informative for their work.

The study authors include employees from Thermo Fisher Scientific, Guardant Health, Personal Genome Diagnostics, Novartis Pharmaceuticals, Sysmex Inostics, and Illumina.

"Liquid biopsy technology holds great promise in improving the outcome of patients with cancer, but without a common 'yardstick' with which the FDA can measure analytical performance, real gains for patients will be slow to materialize," Jim Godsey, VP of assay development at Illumina and co-chair of the BloodPAC's AV WG, said in a statement. "These protocols establish a vital piece of infrastructure that will accelerate the development of new tools to improve cancer care and are the tangible fruit of collaboration across industry, academic researchers, and the FDA."

While the primary focus of the project was to validate ctDNA assays that could help guide targeted therapies and drug response monitoring, the AV WG will also measure assay performance to support new uses for ctDNA analysis in the future.

The FDA has also asked BloodPAC to begin a second phase to develop analytical validation protocols designed to detect and monitor cancer patient status post-treatment.

"Those working to develop assays designed to detect and monitor ctDNA must overcome unique hurdles in order to deliver accurate results," Mickey Williams, director of the molecular characterization laboratory at the Frederick National Laboratory for Cancer Research and consultant for BloodPAC, said in a statement. "The BloodPAC efforts have produced a thoughtful manuscript that ctDNA assay developers should read for helpful advice on assay analytical validation."