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Noninvasive Liquid Biopsy Assays Integrating Tumor and Immune Biomarkers Prove to Be Promising Tools in Immuno-Oncology

Tumor tissue biopsies have been the traditional method of identifying and analyzing cancer cells. But in addition to being invasive and risky, they have many limitations. Tumor accessibility is often problematic as are issues with monitoring the progression of disease. Tissue samples from a single location don’t always provide a full picture of a tumor’s genetic heterogeneity, limiting accuracy.

The more data oncologists have about a patient’s cancer, including genetic information, the more precisely it can be diagnosed, treated, and monitored. Immunotherapy has quickly evolved as a promising treatment modality over the past decade. And, it turns out immune biomarkers can be used to identify specific cancers and their characteristics using liquid biopsies such as non-invasive samples of blood, saliva, urine, cerebrospinal fluid, bile, and other biofluids.

This allows for:

  • Potential earlier detection of cancer, which historically has led to better treatment outcomes.
  • Selection of specific modes of immunotherapy best suited for the patient.
  • Easier sampling, supporting ongoing monitoring prior to, during, and after treatment.

Evolution of Liquid Biopsies

The first liquid biopsy test was approved by the U.S. Food and Drug Administration (FDA) in 2013. Used to monitor metastatic breast, colon, and prostate cancers, the CellSearch® CTC enumeration platform tracked circulating tumor cell (CTC) counts. The FDA approved a cell-free DNA (cfDNA) liquid biopsy test in 2016, which was used along with a diagnostic test that could detect epidermal growth factor receptor (EGFR mutations in circulating tumor DNA (ctDNA) associated with non-small cell lung cancer. In their early development, liquid biopsies had limitations. These included isolating analytes and a lack of knowledge of tumor behavior and shedding dynamics.

From the development of personalized targeted immunotherapies to being able to better predict a treatment’s success, the potential for liquid biopsies continues to grow. Applications in oncology include screening, early diagnosis, patient stratification (often used to monitor drug efficacy in clinical trials), therapeutic guidance, and treatment response monitoring. Liquid biopsies also help monitor residual disease, predict relapse, and provide a more accurate prognosis.

What Is a Biomarker?

The International Program on Chemical Safety, a joint venture between the National Institutes of Health (NIH) and World Health Organization (WHO), defines a biomarker as “any substance, structure, or process that can be measured in the body or its products and that can influence or predict the incidence of outcome or disease”. Recent computational and experimental developments have led to the discovery of biomarkers that could be used for precision oncology. By identifying and monitoring these using liquid biopsy assays, tumors can be closely analyzed without performing an invasive tissue biopsy.

Types of Biomarkers/Analytes

Information on a tumor can be derived from cellular material, DNA, RNA, and proteins as well as metabolites. The types of biomarkers that can be detected using a liquid biopsy assay include:

  • Circulating Tumor Cells (CTC): Discovered in 1869, CTCs became a focal point in testing and monitoring by the late 1990s and have become a focus of liquid biopsy testing. These cells detach from the tumor and circulate freely in the bloodstream via a process known as epithelial-mesenchymal transition (EMT). The abundance of CTCs in the blood is important in determining the severity of the cancer, while they often retain markers that identify the tumor’s origin. Enrichment methods have been developed to identify surface cell proteins, cell size, electric charge, and other biological properties; in patients with metastatic breast cancer, whole-genome sequencing of CTCs has been achieved.
  • Circulating Cell-free DNA (cfDNA): By looking for cfDNA molecules, assays can find ctDNA. Fragments of DNA circulate in the blood of all individuals, which was first reported in 1948. Its association with the presence of cancer was identified in 1977. The release of cfDNA occurs via cell death and active cell secretion; while an increase in cell-free DNA counts was once thought to be a cancer biomarker, autoimmune diseases, heavy exercise, trauma, and pregnancy can also increase counts. However, profiling ctDNA can help determine tumor burden, metabolism, and origin by analyzing the fraction of these fragments compared to total cfDNA.
  • Epigenetic Modifications: Cellular processes such as DNA transcription, repair, and replication become dysregulated due to changes in epigenomic regulators, leading to mutations associated with many cancer types. Alterations in methylation patterns can be used to differentiate normal and cancer cells and have been identified in ctDNA and tissues of origin. In fact, DNA methylation patterns are tissue specific, so testing for epigenomic biomarkers has become promising in the use of liquid biopsy testing.
  • Transcriptome: By assessing all expressed RNA, including, mRNA, rRNA, tRNA, and other more-recently discovered RNA species such as microRNA (collectively known as the transcriptome), specific changes can be identified. Transcriptional changes have been correlated with carcinogenesis. More specifically, irregularities in mRNA expression have been associated with cancer-related dysregulation. Profiling gene patterns has allowed researchers to identify various cancer subtypes based on molecular analysis, including patient-specific mutations.Non-coding RNA (ncRNA) is of particular interest, as a subclass, called miRNAs regulate tumor suppressors and oncogenes. These serve as biomarkers in all stages of cancer. Since miRNA expression is cancer-specific, it can identify the type and origin of cancer and be used for early detection, prognosis, and treatment selection.
  • Proteome: The entire set of proteins expressed in an organism, the proteome can be measured down to the tissue or cellular level. It changes over time and is influenced by a variety of conditions. A sub-field known as oncoproteomics looks at the molecular mechanisms involved in carcinogenic processes. Numerous circulating biomarkers can be tracked in the proteome, as proteins can have diagnostic and predictive benefits. While there previously have been limitations in using protein biomarkers, newer high-throughput techniques can probe a cancer’s molecular signature and analyze concentrations, modifications, and other aspects of expressed proteins. Mass-spectrometry is often used, as is protein array technology that immobilizes up to thousands of high-density proteins to identify biomarkers.
  • Metabolome: Metabolic reprogramming, known as the Warburg effect, is a major hallmark of cancer cells. Some aspects of cancer metabolism include a higher glycolytic flux and higher lactate production than normal cells. The mechanisms behind metabolic reprogramming have been under investigation. Nevertheless, it can be measured by looking at the number of oncometabolite, or molecules that increase in abundance in relation to enzyme mutations associated with cancer. The idea of searching for metabolomic biomarkers is relatively new, but more promising methods include analyzing metabolite panels or signatures to quantify metabolites or measure metabolic pathways of cancer cells.
  • Exosomes: Released from endosomes of most cells, these lipid-enclosed nanovesicles contain DNA, different types of RNA, proteins, lipids, and a variety of other metabolites. The composition of an exosome can reflect that of its origin cell. Exosomes are thought to play a role in intercellular communication; their potential to promote/restrain tumor growth, progression, angiogenesis, and metastasis is under investigation. They are also being studied as biomarkers and targets for anticancer-therapy as well as possible drug delivery vectors. Exosomes are found in many different body fluids and can be used for genomic, transcriptomic, proteomic, and metabolomic analyses as part of a liquid biopsy assay.
  • T-Cells: Immune systems cells are readily accessible and can be easily isolated and studied. As with studying circulating proteins and cytokines, these can be used to predict a patient’s immune and disease response to a targeted therapy. T-cells are especially viable because they can be isolated in a fully functional state. They can also be used to measure expression of the PD-1 biomarker, while the T-cell receptor (TCR) repertoire is a novel biomarker in immune-oncology that tracks variations in chains the TCR is encoded by.

Limitations & Potential

As of the writing of this article, the FDA has approved at least seven liquid biopsy tests, including Cancer SEEK, a multianalyte test that detects ctDNA mutations and measures protein biomarkers for eight types of cancer. Clinical limitations include the lack of a biomarker panel to define specific types of tumors and their stages, which is in development. Sample preparation, standardization, and acceptance in clinical settings are other current limitations as well.

However, liquid biopsy assays have vast potential as blood analysis is far more advantageous than obtaining tissue samples. Biomarkers can be used to identify and quantify disease as well as monitor therapy success. Being noninvasive, a liquid biopsy can be repeated multiple times and may be used for follow up studies during treatment. And, while not yet common practice, it is rapidly evolving as the technologies behind it continue to advance.

Partner With BioAgilytix

BioAgilytix, specializing in large molecule bioanalysis, provides biomarker, immunogenicity, cell-based assay, and other laboratory services for pharma and biotech companies around the world. We are a leader in immunogenicity testing, an important step in drug development, evaluating anti-drug antibodies prior to treatment, and analyzing drug efficacy in a patient over time.

Leveraging our expertise, we’ve also developed a menu of over 600 biomarkers and are renowned for biomarker assay development, validation, and sample testing. We use a number of innovative platforms for biomarker analysis to help find the optimal solution for each customer’s application.

Contact BioAgilytix today with any questions or requests, or for information on our expertise in tumor/immune biomarkers and liquid biopsy assays.

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