Diagnostic Precision

A SeraCare blog focused on precision medicine and advanced clinical diagnostics

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The Promise of Liquid Biopsy: A Q&A with Dr. Claudia Vollbrecht

Category: NGS, ctDNA, cfDNA, reference materials, ccfDNA

Posted by Andrew Anfora, PhD on Mar 24, 2021
  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.
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The Promise of Liquid Biopsy: A Q&A with Professor Ed Schuuring

Category: NGS, ctDNA, cfDNA, reference materials, ccfDNA

Posted by Krystyna Nahlik, PhD on Mar 10, 2021
  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.
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The Promise of Liquid Biopsy: A Q&A with Professor Sandi Deans

Category: NGS, ctDNA, cfDNA, reference materials, ccfDNA

Posted by Krystyna Nahlik, PhD on Mar 4, 2021
  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).
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The Promise of Liquid Biopsy for Cancer Diagnostics and Therapeutic Monitoring: Are We There Yet?

Category: NGS, ctDNA, cfDNA, reference materials, ccfDNA

Posted by Krystyna Nahlik, PhD on Feb 25, 2021
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.
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How to resolve the challenges of MRD?

Category: NGS, ctDNA, cfDNA, reference materials, MRD

Posted by Yves Konigshofer, PhD on Oct 7, 2020
  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.
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So, you want to monitor Measurable Residual Disease? What are the challenges?

Category: NGS, ctDNA, cfDNA, reference materials, MRD, Minimal Residual Disease

Posted by Yves Konigshofer, PhD on Oct 1, 2020
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.
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Assessing RNA Extraction with FFPE Fusion RNA Reference Materials

Category: NGS, reference materials, RNA fusion

Posted by Dan Brudzewsky on Mar 11, 2020
This is a third blog in a series on RNA fusions, this time focusing on how the FFPE Fusion RNA materials are used as RNA extraction controls.
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Evolution of non-invasive prenatal testing (NIPT) testing

Category: NGS, NIPT, #Quality, New Reference Material, reference materials, trisomy, Reproductive Health, Non-invasive Prenatal Testing

Posted by Agnes Caruso,PhD on Feb 5, 2020
Prenatal screening for aneuploidy has changed dramatically since the 1970s. Non-invasive methods developed in the 1980s and 1990s, combined measurements of maternal serum analytes and ultrasonography. The problem with those methods was not just a high false-negative rate of 12% to 23%, a high positive rate of 5% and a poor sensitivity, ranging from 50% to 95% 1. Uncertain results frequently led to invasive procedures such as amniocentesis or chorionic villi sampling to perform karyotyping on fetal samples. Both of those procedures carry a risk of miscarriage.
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Multi-Lab Study of Fusion RNA Reference Standards for Targeted NGS

Category: NGS, reference materials, AACR, NTRK, RNA fusion

Posted by Andrew Anfora, PhD on Jan 28, 2020
Sourcing assay validation samples as positive run controls or workflow controls in targeted NGS RNA fusion assays remains a challenge today. This is further exacerbated with clinical labs looking to provide validated NGS assays for patient stratification in a host of new drugs in clinical trials or newly approved targeting fusion genes, such as NTRK genes (Larotrectinib, Loxo/Bayer) and Entrectinib (Genentech/Roche) for rare cancers in adult and pediatric patients, and RET (Loxo/Lilly) for lung cancer. SeraCare produces several RNA fusion reference materials. This article describes the development and multi-laboratory evaluation of a pan-cancer multiplexed Fusion RNA reference standard for analysis of clinically relevant fusion genes in solid tumors. The evaluation was conducted at 5 different laboratories on different NGS platforms (amplicon- and hybridization capture-based) as well as at different RNA inputs within a platform. Results highlight the utility of this Fusion RNA reference material to support clinical NGS assays as positive controls in solid tumor cancer patient stratification for many of these fusion-based targeted therapies.
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What is Non-Invasive Prenatal Testing (NIPT)?

Category: NIPT, #Quality, New Reference Material, reference materials, trisomy, Reproductive Health

Posted by SeraCare Team on Jan 20, 2020
Fetal aneuploidy affects about 9 in 1,000 live births. The definition of aneuploidy is an abnormal number of chromosomes ; with 23 pairs of chromosomes in humans, 46 is the normal number, while aneuploidy individuals will have 45 or 47.  In trisomy, there is one additional chromosome, typically chr21, 18 or 13 (it is not a coincidence that these are the smallest chromosomes in humans).  Historically, the invasive methods amniocentesis and chorionic villus sampling (CVS) were used with risk to the pregnancy, with about a 1% chance of miscarriage due to the procedure. Non-invasive methods based upon ultrasound and serum biomarkers are useful screening tests, but were of limited reliability as they were indirect measures of chromosomal abnormalities1.   Photograph courtesy of Flickr user Can H.
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