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.

Getting pregnant is not always simple and with an increase in the age of first-time mothers, there is an increased need for in vitro fertilization (IVF). IVF allows the generation of embryos in the lab and subsequent embryo transfer to the patient. Successful implantation of the embryo depends on many factors with chromosomal aneuploidy being one of the important factors in a failed IVF cycle. There are several ways the odds of success can be increased and risks of miscarriage are reduced.

  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.

Overview Tumor mutational burden (TMB) is a measurement of the number of mutations carried by a tumor cell genome. By comparing DNA sequences from a patient’s healthy tissue and tumor cells, and using a number of complex algorithms, scientists can determine the number of acquired somatic mutations present in tumors but not in normal tissues.1 NGS is the primary method employed to measure TMB, either through targeted panels or whole exome sequencing (WES);2 the latter is considered the gold-standard measurement today.

The plans for this year’s NIPT and PGT conferences, like many others, were quickly derailed by the COVID-19 pandemic. The ESHG and ESHRE Annual Meetings were moved to virtual space and even though the format was a new experience, the science did not disappoint. Here we will share some of the highlights from the sessions.

    Tumor profiling assay workflows start from a tissue biopsy sample that is formalin fixed and paraffin embedded (FFPE), a process that introduces various kinds of damage in the nucleic acids in the tissue specimen. Reference materials that closely mimic the damage profile of patient FFPE samples are lacking. Depurination, depyrimidination, deamination, oxidation, nicks, and double strand breaks may be found in DNA extracted from FFPE tissue, despite the use of extraction kits that attempt to repair some of this damage. We have developed a formalin-damaged, multiplexed biosynthetic reference material, Seraseq® Compromised FFPE Tumor DNA, as well as a companion wild-type (WT) material, Seraseq Compromised FFPE WT, to mimic the damage found in patient samples that can be used to assess the entire tumor profiling workflow. We compared the performance of these reference materials in downstream assays to that of FFPE material with minimal DNA damage such as the Seraseq FFPE WT (DNA/RNA) RM product.