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Inside the Lab Liquid biopsy, Oncology, Precision medicine, Screening and monitoring

Liquid Biopsy Insights into Cancer

sponsored by Illumina

The amount of time between the arrival of a new technology and its deployment in the diagnostic laboratory is shrinking. Take immuno-oncology (IO), for example – this powerful new approach harnesses the body’s own immune system and has quickly become one of the most promising approaches to cancer treatment. Yet given the growing number of guideline and clinical trial biomarkers that can be explored, tissue sample availability is becoming a limiting factor. Liquid biopsy provides a noninvasive alternative, not subject to tissue limitations, to gain access to the mutational landscape of tumors.

As molecular pathology assays aim to cover a growing number of guideline-recommended and emerging biomarkers, existing assays that interrogate a small number of variants can now be consolidated onto a single panel, facilitating comprehensive genomic profiling (CGP) of multiple actionable biomarkers and key investigational IO biomarkers through next-generation sequencing (NGS). Covering these needs with a combination of revolutionary sample collection and consolidated testing is key, especially in current times with infectious threats adding another layer of challenge for oncology patients.

Enabling CGP from liquid biopsy sounds like the perfect answer; the approach is noninvasive and enables repeat sampling. However, it raises questions about the feasibility of analyzing a variety of tumor variants and genomic signatures that are circulating in the blood with high sensitivity. We spoke to two experts working in molecular pathology and translational genomics to explore the merit of liquid biopsy to identify tumor insights at a very low limit of detection and learn about the early success of Illumina’s comprehensive RUO assay, TruSight™ Oncology 500 (TSO500) ctDNA.

Multiple markers

“When it comes to sequencing and beyond, the days of the single-focus assay are numbered. There are now hardly any tumor entities in which we only look for one marker,” says Wilko Weichert, Professor of Pathology and Chairman of the Institute of Pathology at the Technical University of Munich, Germany. Larger panels, with a variety of markers enabling the identification of multiple potential driver alterations provide a more detailed prediction of therapy response and will allow clinicians to select the most appropriate therapy according to individual tumor characteristics. “The choice between targeted panels and comprehensive assays depends on intended use – but for anything from exploratory biomarker discovery to composite biomarker testing, it’s important to have multiple markers consolidated into a single assay,” says Stephanie Hastings, Manager in Assay Development, Translational Genomics at Q2 Solutions, USA.

The TSO500 ctDNA assay is [a] highly multiplexed assay that involves the detection of small variants, copy number variations (CNV), fusions, and key genomic signature biomarkers.

The TSO500 ctDNA assay is one such highly multiplexed assay that involves the detection of small variants, copy number variations (CNV), fusions, and key genomic signature biomarkers, including microsatellite instability (MSI) and tumor mutational burden (TMB). MSI is a unique pan-cancer biomarker resulting from defective DNA mismatch repair, which indicates predisposition to mutations. TMB measures the number of mutations within the coding sequence of the tumor genome. Combining multiple genomic aberrations provides a highly personalized assessment.

However, pathologists routinely work with solid tumor tissue samples – so why would they use liquid biopsy? “There are two main advantages of using liquid samples. Blood is readily available, and you can collect it via a minimally invasive procedure, repeating several times if necessary,” explains Weichert. “It’s also accessible in almost every patient – even those in whom you can’t reach the lesion by traditional biopsy due to an increased risk of side effects, such as lung cancer patients with emphysema.”

Another factor to consider is the biology of the two different sample types. Although solid tumor samples might correlate well with histology and cellular phenotypes, they represent only a small, localized primary tumor profile. In contrast, multiple metastatic lesions might all shed DNA into the bloodstream. The subsequent liquid biopsy sample provides a comprehensive patient tumor profile that could be more predictive of therapy response than information from a single tumor site.

The potential value of liquid biopsy is clear – and Weichert sees several scenarios where a liquid sample could prove beneficial. “I believe we will see liquid biopsy used to follow patients, particularly because it’s easier to obtain blood sequentially than tissue. For example, you can measure ctDNA in the blood to check for cancer recurrence or monitor changes in molecular profiles to detect whether resistance mutations have occurred.”

Effective capture

“The real challenge of working with a liquid biopsy sample is its potential for low stability; lysis of white blood cells can cause genomic DNA to spill into the plasma fraction,” says Hastings – and such contamination could potentially conceal the targets of interest in the blood sample.

Therefore, appropriate collection tubes, preanalytical considerations, and using the most effective workflow are key to capturing low-frequency molecular alterations present in cell-free DNA (cfDNA). “When it comes to sample quality for liquid biopsy, the way that blood is collected is crucial – for example, the tube type used can affect the level of stability,” says Hastings. “Once you’ve isolated the ctDNA, the way in which you quantify the amount of material you have is also important for assay performance.” It is critical to use an electrophoretic quantification method, such as Fragment Analyzer or TapeStation, that can specifically measure the cfDNA fraction and exclude high-molecular-weight DNA contamination. Fluorometric methods are not recommended because they quantify all species of DNA sizes contained in the sample, which could potentially overestimate the amount of cfDNA and fail to create a robust library (1).

Hybrid capture is more versatile than amplicon-based chemistry and can be used to detect SNVs, indels, CNVs, fusions, and other structural changes with higher accuracy.

From a library preparation perspective, using hybrid capture-based target enrichment chemistry enables users to generate results from liquid biopsy samples with very high sensitivity. Hybridization probes have tolerance to capture targets even when mutations exist in the hybridized regions and can cover the span of the entire gene sequence. In comparison, amplicon-based chemistries only amplify a subset of the fragmented DNA due to the possibility of break points between primer binding sites. This also makes amplification of novel fusions challenging. Hybrid capture is more versatile than amplicon-based chemistry and can be used to detect SNVs, indels, CNVs, fusions, and other structural changes with higher accuracy. “A hybrid-capture approach is therefore more robust than amplicon because of this feature; you will gain higher sensitivity by having a purer sample for analysis.”

Working in collaboration with Illumina since 2018, Hastings has been able to evaluate the assay chemistry of TSO500 ctDNA that has enabled the detection of low allele frequency variants such as EGFR L858R, MYC indels, and NTRK2 fusions. “Early access to the pre-released version has allowed us to comprehensively evaluate its performance – and, to date, we have analyzed over 1,000 samples using the TSO500 ctDNA assay.”

Liquid biopsy’s future

Liquid biopsy is still very much an evolving application – and its increased use in clinical trials could accelerate adoption. To that end, Weichert believes that a combination of both tissue and liquid samples should be used wherever possible in these scenarios; “We need even more directly comparable data that indicate whether liquid samples are more predictive than tissue samples, as we envisage they might be.”

Whether in the diagnostic laboratory or in clinical trials, liquid biopsy is a fast-moving field that directly feeds into the future of precision medicine. TSO500 ctDNA is proving to be an effective research tool for early-access users, harnessing easily accessible, reproductible tumor content to deliver comprehensive information across 523 cancer-related genes. Hastings certainly believes in the potential of the platform, given that Q2 recently performed analytical validation of TSO500 ctDNA.

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  1. Illumina, “Accurate quantification of cfDNA for use in Trusight™ Oncology 500 ctDNA” (2020). Available at:
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