Precision oncology leverages molecular information about cancer to personalize therapy and improve outcomes. The discovery and development of relevant biomarkers is a prerequisite to fulfillment of the promise of precision oncology, but the challenge for researchers is that clinically actionable mutations occur at very low frequency and sourcing biospecimens with specific mutations is extraordinarily difficult.

In this article, we discuss the utility of liquid biopsy in cancer genomics as well as a novel, next-generation sequencing (NGS) initiative designed to support and accelerate liquid biopsy-based diagnostics for precision oncology.

Making the Case for Liquid Biopsy Companion Diagnostics
Tissue biopsy samples are commonly used for tumor characterization but are limited by sampling bias and constraints on sampling frequency. Liquid biopsies offer a minimally invasive alternative, enabling analysis of tumor cells or tumor cell products that have been shed into bodily fluids. In principle, liquid biopsy has the potential to reflect all subclones present at a specific point in time and enable sequential, longitudinal monitoring of a tumor’s evolutionary dynamics.

With advances in technology, it is now possible to analyze the genetic material of circulating tumor cells (CTCs) at the single cell level to evaluate spatial and temporal dynamics in circulation. Liquid biopsy has also expanded to include a variety of matrices beyond CTCs, including circulating tumor DNA (ctDNA), cell-free DNA or RNA (cfDNA or cfRNA), soluble proteins, and even exosomes.

Potential for Clinical Utility of Liquid Biopsy
This modality has a broad range of possible applications, from elucidating mechanism of action to stratifying patients and monitoring response to treatment. Other potential clinical uses include monitoring of radiologically undetectable tumors, predicting recurrence, assessing tumor burden, tracking the clonal evolution of tumors, and evaluating immune biomarkers over time.[1]

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Studies have demonstrated the potential of liquid biopsies for capturing tumor heterogeneity. One study found that prostate cancer CTCs can demonstrate a high degree of phenotypic heterogeneity and that this heterogeneity correlated with poorer outcomes in metastatic castration-resistant prostate cancer (mCRPC).[2] Another study comparing targeted sequencing of ctDNA and matched metastatic tissues in patients with mCRPC showed that gene alterations identified from ctDNA and tissue were highly concordant. In some cases, ctDNA sequencing revealed clinically relevant alterations that were not identified in the tumor biopsy.[3]

A growing body of evidence supports the capability of liquid biopsy to detect residual disease, recurrence, and even early signs of therapeutic resistance.[4] Numerous studies have demonstrated high concordance between the mutations found in cfDNA and matched solid biopsy samples.[5][6] Moreover, plasma cfDNA levels may increase as tumors progress and there is evidence that cfDNA acts as a signaling molecule that induces metastasis.[4] cfDNA has been extensively studied across many cancer types with promising results as a tool for detecting cancer, monitoring tumor mutations, determining treatment eligibility, and even monitoring therapeutic response.[7]

Advantages of Liquid Biopsy
The potential clinical utility of liquid biopsy is accompanied by the following potential advantages:

  • Ease of collection;
  • Low risk of adverse effects;
  • Ability to perform serial testing, making it an attractive option for use in clinical trials, either as a supplement or an alternative to tumor biopsy;
  • Opportunity to assess the whole-body burden of disease and response to therapy;
  • Preservation of cellular contents, allowing for gene expression profiling at the single-cell level and other downstream analyses.

Paradigm-Shifting Approvals
In 2020, the U.S. Food and Drug Administration (FDA) approved two companion diagnostics—the Guardant360 CDx assay and the FoundationOne Liquid CDx test—that combine liquid biopsy and next-generation sequencing, ushering in a new era of multi-gene mutation testing and biomarker profiling. In their efforts to continue moving precision oncology forward, researchers are increasingly relying on genomic profiling to inform the discovery of biomarkers, whether functional, diagnostic, predictive, or prognostic.

Using Genomic Insights to Support Liquid Biopsy Companion Diagnostic Development
Genomic insights have played a critical role in advancing our understanding of cancer biology and driving the development of precision therapeutics. Using tumor genomic alterations to predict therapeutic benefits from targeted therapy has improved clinical outcomes in a range of neoplasms. [8] Next-generation sequencing is a genomic profiling approach that has been widely adopted due to its massively parallel sequencing capability and its compatibility with low-quantity input DNA. Analysis can be limited to targeted gene panels or can involve the whole exome or genome. NGS is the preferred sequencing method when the target mutations are not known. As it enables simultaneous analysis of a spectrum of genomic alterations, including mutations, copy number variations, translocations, and fusions, NGS may lead to a greater yield of actionable findings.

A Novel Sequencing Initiative
To address the unmet need for biospecimens with a mutation of interest, the Biospecimen Solutions team at Precision for Medicine developed a collaborative initiative focused on building a searchable database of NGS profiles that could be used to help accelerate the discovery of relevant biomarkers and development of both tissue and liquid biopsy-based diagnostics for precision oncology.

In collaboration with academic and industry partners, Precision for Medicine launched the Precision Oncology Sequencing Initiative (Project P.O.S.I.), an ambitious two-phase NGS initiative to screen samples in its extensive biorepository for key biomarkers across cancer indications. At the core of this initiative are two commercially available assays that analyze variants across known cancer-related genes:

  • The Oncomine Precision Assay profiles DNA or DNA from formalin-fixed, paraffin-embedded (FFPE) tissue or plasma, analyzing curated biomarker content spanning 50 genes. This assay is run on the Ion Torrent™ Genexus™ System, which integrates and automates nucleic acid extraction and purification, library preparation, sequencing, and reporting.
  • The TSO500, run on an Illumina platform, includes pan-cancer biomarker content for a variety of solid tumor types and can also measure microsatellite instability (MSI) and tumor mutational burden).

Phase one of the project, which is ongoing, focuses on NGS screening of FFPE samples with the Oncomine Precision Assay. Phase two, which is also ongoing, involves large-scale screening of liquid biopsies using a variety of NGS techniques and panels, including both the Oncomine Precision Assay and the TSO500.

Developing cfDNA Diagnostics
Recently, Pillar Biosciences (Pillar) partnered with Precision for Medicine as part of its effort to advance development of cfDNA diagnostics. This partnership leverages a global network Precision for Medicine has created for actively and prospectively collecting cfDNA from liquid biopsies obtained from patients with cancer. The specimens are processed and sequenced, and the resulting genomic data is then combined with clinical information and liquid biopsy metadata and analyzed using QuartzBio®, Precision for Medicine’s multi-omic data processing engine.

A critical advantage of Project P.O.S.I. is its use of real clinical samples, rather than contrived specimens. Generating data from clinical samples makes it easier for diagnostic developers to select and optimize an NGS approach in the earliest stages of development. Precision for Medicine’s prospective collection capabilities allow for serial sampling at multiple timepoints and may even allow for the collection of matched tissue specimens. This work is being conducted in Precision for Medicine’s Houston CLIA lab, home of the proprietary ApoStream® platform that isolates CTCs for research use.

Key Takeaways
High-quality, well-characterized biospecimens are building blocks for the exploration of disease drivers, drug targets, and novel biomarkers. Large-scale genomic profiling programs are revolutionizing the field of precision oncology, creating libraries of NGS profiles that can be mined for insights that reveal potential drug targets and enhance diagnosis, prognostication, and prediction of outcomes. As our knowledge base expands, we expect precision medicine will integrate multi-omic tumor characterization and dynamic monitoring of liquid biopsy samples for stratifying treatment and monitoring therapeutic response.

[1] Malone ER, et al. Molecular profiling for precision cancer therapies. Genome Med. 2020;12(1):8.
[2] Scher HI, et al. Phenotypic heterogeneity of circulating tumor cells informs clinical decisions between AR signaling inhibitors and taxanes in metastatic prostate cancer. Cancer Res. 2017;77:5687-5698.
[3] Wyatt AW, et al. Concordance of circulating tumor DNA and matched metastatic tissue biopsy in prostate cancer. J Natl Cancer Inst. 2017;109.
[4] Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 2019;20(2);71-88.
[5] Lebofsky R, et al. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types. Mol Oncol. 2015; 9:783-790.
[6] Janku F, et al. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies. Oncotarget. 2015; 6:12809-12821.
[7] Qvick A, et al. Liquid biopsy as an option for predictive testing and prognosis in patients with lung cancer. Mol Med. 2021;27:68.
[8] Ramalingam SS, et al. Overall survival with osimertinib in untreated, EGFR-mutated advanced NSCLC. N Engl J Med. 2020;382(1):41-50.

Featurted Image: DNA Genotyping and Sequencing. Technician loads robot for genetic studies of the human papillomavirus (HPV) at the Cancer Genomics Research Laboratory, part of the National Cancer Institute’s Division of Cancer Epidemiology and Genetics (DCEG). Courtesey: Photo by National Cancer Institute on Unsplash.

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