This is Part 3 in a 3-part Q&A blog series with a panel of liquid biopsy experts addressing many of the issues faced in developing and deploying NGS-based liquid biopsy assays for clinical applications in oncology. At a 2020 liquid biopsy webinar, Dr. Vollbrecht shared a molecular pathologist’s perspective on the current state of liquid biopsy. Laboratory processing and analysis of cfDNA samples is a multi-step process that requires a high degree of precision to achieve consistent results. Her presentation focused on pre-analytics variables, which are often left out of discussions and tend to focus on biochemical manipulation of isolated nucleic acids. Seemingly simple factors at the point of sample collection such as problems with blood test tube filling, storage and labelling are able to affect the cfDNA stability, abundance, and confound the reliability of final interpretation. Variation in sample treatment during laboratory processing, including but not limited to, cfDNA quantification and QC methodology are also amongst the challenges for liquid biopsy.

  This is Part 2 in a 3-part Q&A with a panel of liquid biopsy experts addressing many of the issues faced in developing and deploying NGS-based liquid biopsy assays for clinical applications in oncology. At a 2020 liquid biopsy webinar, Professor Schuuring discussed the plethora of options available to detect low copy number mutations in plasma cfDNA of lung cancer patients. His research laboratory combines NGS, ddPCR, qPCR and mass spectrometry approaches to address three main applications: (1) primary diagnosis by detection of predictive mutations, (2) monitoring of treatment response based on changes in plasma mutant levels, and (3) detection of therapy resistance mechanisms.

  This is Part 1 in a 3- series deep-dive Q&A with expert panelists addressing many of the issues faced in developing and deploying NGS-based liquid biopsy assays for clinical applications in oncology. At the 2020 liquid biopsy webinar, Professor Sandi Deans highlighted a recent EQA scheme aimed at evaluating the standard of cfDNA testing in NSCLS and CRC patients. It was driven by demand from participants themselves, as well as pharmaceutical companies, IVD manufacturers and IQNPath (International Quality Network for Pathology).

This is the introduction to a 3-part Q&A with a panel of liquid biopsy experts addressing some of the issues faced in developing and deploying NGS-based liquid biopsy assays for clinical applications in oncology. In this 3-part blog series, we will share highlights from a recent Liquid Biopsy Expert Panel webinar sponsored by LGC Seracare and facilitated by GenomeWeb, which brought together academic research and clinical experts in liquid biopsy technologies to discuss the benefits, shortcomings, challenges and recommendations for liquid biopsy adoption in the context of cancer disease management. The webinar drew a lot of interest and sparked in-depth questions from attendees, which required a post webinar follow-up response from all 3 panelists.

  This is part 2 of 2 of the MRD blog post. (Click here for part 1). In this section, we will discuss how to overcome some of the most common challenges of MRD testing. Overcoming the Challenges In order to mitigate sequencing errors, methods using Molecular Barcodes (MBCs), Unique Molecular Identifiers (UMIs), etc. (which are essentially all the same) may be used, where each starting molecule is sequenced many times. The MBCs are then used to generate consensus sequences from sequences that were likely obtained from the same starting molecule. The assumption is that errors appear due to somewhat stochastic processes and that the consensus sequences will likely be correct. This requires many observations of the same starting molecule, so it will be recommended to generate 10-fold more sequences than there are molecules. Therefore, with 8,000 genomic equivalents, we might want to target a sequencing depth of 80,000. This is a reason why using 10-fold more input ccfDNA may not necessarily be a good thing (in addition to having to obtain a 10-fold larger liquid biopsy) since we may have to increase sequencing depth accordingly to 800,000, which could increase the cost of sequencing 10-fold, which could reduce the likelihood for payment and running the assay profitably.

Part 1 of 2   Background Measurable Residual Disease (MRD) monitoring – for purposes of this blog – will be the act of looking for somatic variants in a liquid biopsy sample by analyzing circulating cell-free DNA (ccfDNA). This is done to monitor the disappearance of a metastatic solid tumor during treatment and to follow any future reemergence of that cancer. Analyzing ccfDNA assumes that circulating tumor DNA (ctDNA) will be present, and the median ctDNA frequency in patient samples across cancers seems to be around 0.5 to 1 %. Thus, the median variant allele frequencies (VAFs) of the somatic variants will start around this range, and the goal of MRD monitoring is to be able to detect them at much lower VAFs. This can be challenging and if we are going to design an assay for MRD monitoring, then we need to be aware of them and overcome them.

Cancer research is purposely methodical and measured. So – somewhat paradoxically – it can be difficult to keep up with the steady stream of discoveries in the literature and presented at conferences like AACR. As a developer and manufacturer of platform-agnostic NGS reference standards, we’re in a unique position to collaborate with cancer genomics assay developers, laboratories, pharmaceutical companies, and other organizations invested in more precise and robust cancer tests.

I am pleased to share findings from a newly published peer-reviewed study with foundational circulating tumor DNA (ctDNA) pre-analytical and analytical testing in multiple technologies and assay chemistries. The study, “Multi-laboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements,” was just published in The Journal of Molecular Diagnostics (He, Stein et al. 2019).

At the recently-concluded 2018 AMP Meeting, researchers at the New York Presbyterian Hospital (NYPH) and Weill Cornell Medical Center (WCMC) presented a poster1 on the validation of an Oncomine™ cell-free DNA Lung Assay using ctDNA NGS standards developed by SeraCare (Seraseq® ctDNA v2 Reference Materials),2

“So as everyone here is aware, I’m sure, detection of circulating tumor DNA is challenging. There’s very little of it, to start with.” Hardly a revolutionary statement by Tony Godfrey, PhD, (Associate Chair, Surgical Research and Associate Professor of Surgery, Boston University School of Medicine) but an important acknowledgement from a leading expert of the difficulty faced by laboratorians