In the course of patient care, formalin-fixation and paraffin-embedding (FFPE) of biopsy tissue samples are routinely performed, where these samples can be analyzed by histology and archived to link the sample with clinical long-term follow-up. With the development of advanced NGS-based oncology gene panels, it is becoming increasingly important to consider pre-analytic variables when extracting nucleic acids from FFPE-treated samples. This post covers frequently asked questions (FAQs) around the extraction of nucleic acids from FFPE samples for downstream NGS analysis.
Answering frequently asked questions (FAQs)
"The most effective ways to use QC data to monitor the health of your NGS assay."
Previously, we wrote about some of the Quality Control challenges that clinical laboratories performing Next Generation Sequencing (NGS) face towards ensuring their assays are safe and effective for guiding medical management decisions. Reliable access to high quality reference materials is necessary to help overcome these challenges; however, it is not sufficient. Insights that reference materials provide into the health of an NGS assay are only as good as laboratories’ ability to use their QC data effectively.
With limited time and resources to collect, organize, access, and analyze QC metrics, laboratories may frequently rely on reference materials as binary indicators of Pass/Fail: As long as the expected endpoint results are obtained, an assay is considered to be performing well. The drawback of this approach is that it is reactive, rather than proactive: A sufficient number of failures must occur within a given timeframe before a troubleshooting investigation is performed. By the time a problem is recognized, resources have been wasted and turnaround times (TAT) delayed; in some cases, fidelity of patient results may even have been put at risk. Additional time and costs are then incurred as the investigation proceeds.
Specimen analysis by NGS yields a wealth of information in addition to endpoint variant calls that is indicative of assay performance. Data such as nucleic acid quantity and quality at different steps throughout the workflow (PDF) and sequencing library characteristics are generated every time a reference material is tested. However, these data must be carefully tracked and trended to allow use as highly informative QC parameters. For clinical laboratories whose primary focus is on patient testing and reporting, granular QC metrics may not be captured and reviewed as part of routine test monitoring.
“Ensure NGS-based tests for Personalized Medicine are safe and effective for guiding medical management decisions”
Since the introduction of the GS20 in 2005 by 454 Life Sciences, Next Generation Sequencing (NGS) has found many applications in clinical diagnostics. As a result of this transition from the long-held gold standard, Sanger sequencing, the primary challenge for clinical laboratories has shifted from data acquisition to ensuring these tests are safe and effective for guiding medical management decisions.
Many laboratories struggle to gain a thorough understanding of the analytic performance characteristics of their NGS tests. The difficulty arises from the fact that these assays are comprised of highly complex, fragmented workflows, and have many different intended uses. However, across the various practices currently used for NGS assay development, validation, and performance monitoring, there is a common goal: results must be as accurate, precise, and consistent as possible.
The distinction between accuracy and precision.
If you took a university introductory statistics course, you may have learned the distinction between accuracy and precision. It may likely have been presented with an archery analogy, where ‘Accurate’ was represented by arrows loosely clustered around the target’s bull’s-eye, ‘Precise’ was shown as a tight grouping displaced from the center, and ‘Accurate and Precise’ was depicted as what every archer aims for, a tight grouping directly at the bull’s-eye. Suddenly, words that are used interchangeably in everyday conversation took on dramatically different meanings.
Important information for assay development and review by the FDA
The presentations during the FDA-AACR Liquid Biopsies in Oncology Drug and Device Development Workshop on July 19, 2016 included several important pieces of information that will likely guide the development of assays and their review by the FDA.
After 17 months of deliberations since its first open meeting February 20, 2015 on NGS IVD assay oversight, the U.S. Food and Drug Administration (FDA) issued DRAFT guidance for Stakeholders and FDA staff. The document “Use of Standards in FDA Regulatory Oversight of Next Generation Sequencing (NGS)-Based In Vitro Diagnostics (IVDs) Used for Diagnosing Germline Diseases” was published online on July 6, 2016. This document (PDF located here) is for analytical validity and not for clinical validation.
A handy infographic to compare the first-generation (Sanger) and next-generation sequencing (NGS) technologies
It is hard to believe that next-generation sequencing has only been around for a little over ten years, but has had a profound impact on many frontiers of basic and applied genetics. To contrast the first-generation (Sanger sequencing by capillary electrophoresis) and the next-generation sequencing (NGS) approaches, we provide you with this infographic.
Interview with Dr. Greg Tsongalis, Professor of Pathology, Dartmouth-Hitchcock Medical Center
In this video interview Dr. Greg Tsongalis addresses sources of pre-analytic variability when processing formalin-fixed, paraffin-embedded (FFPE) samples in a pathology laboratory setting. By 'pre-analytic', he refers to the kinds of kits used for DNA extraction, and other factors that may have a significant impact on the quality of the results. He also discusses his work on clinical assay development for somatic mutation detection using NGS technology.
Recent publication highlights potential of personalized medicine for young cancer patients
There are many challenges for pediatric patients struggling with recurrent or refractory cancer but a recent report and accompanying editorial published in the Journal of American Medical Association brings new hope for patients and their families.1 2This three year study also demonstrates how the principles of precision medicine can be successfully applied with existing DNA and RNA sequencing technologies for many patients.
Are you wondering why reference materials are needed for precision medicine?
In this video interview, SeraCare’s Chief Scientific Officer Dr. Russell Garlick shares some background about SeraCare’s history of manufacturing controls and reference materials for infectious disease diagnostics, and how advances in Precision Medicine reveal a need for similar materials for the fields of oncology and maternal health.