Liquid biopsy technologies to study, diagnose, and track cancer hold immense potential, particularly if they can help detect cancer early. Technological advances that enable us to probe deeper into liquid-biopsy biomarker sources such as circulating tumor DNA and RNA (ctDNA and ctRNA), extracellular vesicles (EVs), tumor cells, and tumor-educated immune cells are paving the way for a new era of cancer precision medicine.
Can Liquid Biopsy Deliver on the Promise of Precision Medicine?
Cancer is notorious for its heterogeneity. For many cancers, the inability to capture this heterogeneity is considered a leading cause of treatment failures. (1) With cancer precision medicine, we aim to understand this diversity at a deeper level and use the information to personalize treatments. Liquid biopsy is already enabling us to realize a part of the precision medicine vision, with well-developed technologies already in clinical use for cancer therapy monitoring and disease relapse. For instance, in non‐small‐cell lung cancer (NSCLC), cell-free DNA (cfDNA) testing is recommended for guiding treatment in specific clinical circumstances, such as when patients are medically unfit for invasive tissue sampling. Several liquid biopsy platforms have received approval from the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) as companion diagnostic kits to guide the treatment of NSCLC patients with EGFR Tyrosine kinase inhibitors (TKIs). (2) Despite these triumphs, several challenges need to be tackled before we can achieve its full potential across a patient’s journey – from screening and early detection to assessing the patient’s condition after diagnosis and relapse and evaluating treatment effectiveness.
Cancer Precision Diagnostics
Why Are We Not There Yet?
Detection of ctDNA and Other Liquid Biopsy Biomarker Sources is a Challenge
Circulating tumor DNA (ctDNA) are DNA fragments derived from tumor cells that circulate in the blood along with cfDNA from other sources. cfDNA obtained from body fluids such as blood, lymph, urine, and semen are used for biomarker testing, not only because it’s less invasive than tissue biopsy but also because it can provide better tumor heterogeneity data. (3) However, detection of ctDNA and other liquid biopsy biomarker sources such as cfRNA, EVs, and others remains a challenge due to various reasons:
- Limited amounts. ctDNA represents only a small fraction of the total circulating cfDNA, particularly in early-stage cancers. This limits the sensitivity of ctDNA analysis, often leading to unreliable results. (4)
- Variable amounts. Biomarkers are often not just low but variable in amounts. Hence, different blood samples from the same individual could yield different results. (4)
- Sensitivity versus specificity: Low and variable biomarker amounts call for tests that can offer high sensitivity. However, high sensitivity can affect the specificity of the test. For example, a highly sensitive test could pick up benign mutations giving rise to false-positive results. (4)
- Lack of pathophysiologic information: Liquid biopsy has the potential to detect early lesions. However, a robust understanding of the biological nature of these lesions is lacking. Such data could be invaluable to plan optimal treatment strategies. (4)
Technologies Pushing Diagnostic Boundaries
The advent of highly precise and sensitive liquid genotyping technologies is slowly changing the cancer diagnostic landscape. Recent guidelines issued by the ESMO Precision Medicine Working Group on the use of ctDNA assays in clinical practice discuss limitations and considerations of ctDNA testing as well as the need for advanced techniques capable of reliably capturing intra-patient spatial and temporal tumor heterogeneity. (5)
Sensitive PCR-Based Technologies
Sensitive PCR-based technologies such as digital PCR (dPCR), real-time PCR (qPCR) along with next-generation (NGS) technologies are leading the way here. Several studies comparing ctDNA testing to tissue-based PCR and NGS testing in advanced cancers have shown excellent specificity and positive predictive values between the two. (5)
- Highly sensitive nanofluidic digital PCR can be particularly valuable for detecting variants that occur at a low frequency relative to wild-type background DNA, which is often the case in liquid biopsy samples. A study evaluating the correlation between liquid biopsy and tissue biopsy using samples from TKI-treated advanced-NSCLC patients, and using three different technologies (TaqMan digital PCR assays, amplification-refractory mutation system PCR, and peptide nucleic acid (PNA)-mediated PCR clamping), showed that digital PCR yielded the highest sensitivity and best correlation. (6)
- RT-qPCR remains a favored technique for the discovery of biomarkers from miRNA, which like ctDNA is a valuable source of cancer biomarkers. For example, using TaqMan™ microRNA assays and RT-qPCR in Bevacizumab-treated metastatic colorectal cancer (mCRC) patient samples, researchers showed that expression levels for multiple miRNAs correlated with disease progression. (7)
- Next-generation sequencing (NGS) trumps PCR-based methods in many aspects, including speed and throughput. NGS can detect mutant allele frequencies (MAFs) < 1%. Moreover, techniques such as unique molecular identifiers or unique barcodes can help to increase sensitivity and reduce false negatives. (8) For example, one study demonstrated incorporating molecular barcodes into DNA fragments before sequencing using the Ion PGM Dx system reduced background errors to detect rare mutations in ctDNA in patients with esophageal cancer. (9)
In this guide, you will find several use cases from peer-reviewed publications where researchers have successfully employed qPCR, dPCR and NGS technologies to solve cancer research challenges.
Dig deeper into the technologies that enabled some of the most exciting discoveries in cancer research.
In this guide, you will find several area-and technology-specific examples, which might be just what you need to solve your current cancer research challenge.
- Applied Biosystems technologies and how they could be used for cancer research
- Molecular mechanisms of malignancy; genetic and epigenetic mechanisms
- New approaches to cancer detection; solid tumor biopsy, liquid biopsy and genetic tests
- Cancer biomarker discovery; development of vaccines and therapeutics
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- Hernández Martínez A, Madurga R, García-Romero N, Ayuso-Sacido Á. Unravelling glioblastoma heterogeneity by means of single-cell RNA sequencing. Cancer Lett. 2022;527:66-79. Published 2022 Feb 28. doi: 10.1016/j.canlet.2021.12.008.
- Romero A, Jantus-Lewintre E, García-Peláez B, et al. Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing: results of the RING observational trial. Mol Oncol. 2021;15(1):43-56. Published 2021 Jan. doi:10.1002/1878-0261.12832
- Macías M, Alegre E, Díaz-Lagares A, et al. Liquid biopsy: from basic research to clinical practice. Adv Clin Chem. 2018;83:73-119. Published 2018. doi:10.1016/bs.acc.2017.10.003
- Crosby D. Delivering on the promise of early detection with liquid biopsies. Br J Cancer. 2022;126(3):313-315. Published 2022 Feb 1. doi:10.1038/s41416-021-01646-w
- Pascual J, Attard G, Bidard FC, et al. ESMO recommendations on the use of circulating tumour DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group Ann Oncol. 2022;S0923-7534(22)01721-5. Published (online) 2022 Jul 06. doi:10.1016/j.annonc.2022.05.520
- Siggillino A, Ulivi P, Pasini L, et al. Detection of EGFR mutations in plasma cell-free tumor DNA of TKI-treated advanced-NSCLC patients by three methodologies: Scorpion-ARMS, PNAClamp, and digital PCR. Diagnostics (Basel). 2020;10(12):1062. Published 2020 Dec 7. doi:10.3390/diagnostics10121062
- de Miguel Pérez D, Rodriguez Martínez A, Ortigosa Palomo A, et al. Extracellular vesicle-miRNAs as liquid biopsy biomarkers for disease identification and prognosis in metastatic colorectal cancer patients. Sci Rep. 2020;10(1):3974. Published 2020 Mar 4. doi:10.1038/s41598-020-60212-1
- Chen M, Zhao H. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics. 2019;13(1):34. Published 2019 Aug 1. doi:10.1186/s40246-019-0220-8
- Hagi T, Kurokawa Y, Takahashi T, et al. Molecular Barcode Sequencing for Highly Sensitive Detection of Circulating Tumor DNA in Patients with Esophageal Squamous Cell Carcinoma. Oncology. 2020;98(4):222-229. doi:10.1159/000504808