Liquid biopsy is a non-invasive sampling approach also used to predict the progression and growth dynamics of cancer and provide guidance for therapy monitoring. This blog covers various features and issues associated with liquid biopsy as exemplified by tumor markers.

Liquid biopsy is a minimally invasive technology for the detection of molecular biomarkers in a patient’s biological fluids, such as blood. 

A liquid biopsy helps the treating oncologist to gain precise information about the clinical state of a particular tumor. Bodily fluids including peripheral blood, cerebrospinal fluid (CSF), urine, or saliva can be easily collected and assessed as liquid biopsies, Liquid biopsy sampling is a simple, reasonably low-cost alternative to surgical biopsies.

Liquid biopsies have generated considerable attention within applications pertinent to many diseases and health conditions, like non-invasive prenatal diagnostics, organ grafting, and particularly in oncology. Liquid biopsies have developed rapidly as a diagnostic and monitoring tool to detect, characterize, and monitor circulating cancer cells and provide an approach to tumor molecular profiling.

The benefits of liquid biopsy

In current cancer diagnostics, histopathological analysis of a tissue biopsy is the "golden standard”, although invasive sampling may put the patient at risk. Furthermore, specific regions, such as the brain or the lungs, are very difficult to access to obtain surgical biopsy samples.

Some cancers are asymptomatic at an early stage. Early diagnosis by using liquid biopsy is hence of crucial importance since it may provide faster access to therapeutic options: think of an example of liver transplantation in patients affected by hepatocellular carcinoma.

Liquid biopsies have many advantages over tissue biopsies, like the non-invasive types, allowing for multiple time points of monitoring, the ability to identify over 20 types of cancer, and the identification of mechanisms of resistance.

Keeping in mind that Positron Emission Tomography (PET)-scanning cannot visualize solid tumors with less than 6 million cells per location, liquid biopsies might identify tumor progression long before the tumor lesions are large enough to become amenable for diagnostic imaging.

A tissue biopsy is an invasive procedure that only captures a snapshot of a tumor. By contrast, liquid biopsies being non-invasive, facilitate serial sampling and the ability to monitor potential dynamic changes in the tumor throughout the course of the anti-cancer therapy. 

Blood: the game-changer in cancer detection

The blood harbors a diversity of tumor cells and tumor cell-derived biomarkers that can be readily monitored by molecular analysis. The two most well-developed biological analytes detected by liquid biopsy are circulating tumor cells (CTCs) and circulating cell-free “naked” tumor DNA (ctDNA). These, and other biomarkers like extracellular vesicles (also called exosomes), the non-coding DNA (erroneously known as junk-DNA), and microRNAs, provide valuable complementary biomarkers to standardized tumor risk-stratification methods, as well as for therapeutic monitoring and analysis of disease progression.

The plasma level of CTCs and ctDNA positive correlates with tumor volume and clinical stage of cancer. Circulating cancer cells or vestiges of tumor-derived DNA in collected blood can hence give important clues about the prognosis and the stage of the disease and help clinicians find the particular therapies most likely to work for that individual patient.

Circulating biomarkers: the tumor signature in the blood

Tumor biomarkers are often increased in the blood of patients with malignant tumors, usually at stage III and IV, like the prostate, colorectal, liver, stomach, lung, and breast tumors. CTC or circulating tumor biomarkers may indicate and reflect ongoing metastasis allowing insights into the molecular makeup of cancer in any patient. 

ctDNA is more abundant in the circulation among metastatic cancers than in early-stage disease, and the prevalence of ctDNA detected in patients with no radiographic evidence of metastasis varies between 49–78%, compared to 86–100% in metastatic disease (Speicher et al., 2014).

An example of ctDNA with a predictive biomarker potential is the measurement of O6-methyl-guanine-methyltransferase (MGMT) promoter methylation from ctDNA in glioblastoma multiforme (GBM) patients (Balaña C. et al. 2003).

The European PROLIPSY project focuses on the early detection of prostate cancer and combines analysis of CTCs, circulating cell-free DNA (cfDNA), and exosomes in people with high serum levels of the protein prostate-specific antigen (PSA). The aim is to first identify people with prostate cancer and then to distinguish those with aggressive cancer from those with non-aggressive disease.

Another example, reported by Sundaresan et al. is the use of ctDNA, complemented by mutation analyses of CTCs and tumor biopsies for improving the detection rate of T790M EGFR resistant mutation to molecular targeted therapy of non-small cell lung cancers.

Not yet a standard clinical tool, why?

Although available clinical data suggest that liquid biopsies can abrogate the shortcomings of tissue biopsies and become ideally suited for cancer identification, these tests are not yet eligible for use in early-stage settings.

Blood tests for tumor markers may be positive indicators of the presence of cancer, but it is not advisable to use them as a stand-alone tumor diagnostic tool. Liquid biopsies should at the current stage considered merely as a valuable complement to tissue biopsies.

Several challenges are associated with promoting liquid biopsy as a standard test in monitoring clinical diagnosis and treatment.

One challenge is the fact that many tumors may become unnoticed or hard to define due to their diminutive size, diffuseness, and localization size. 

The low levels of ctDNA in the blood pose another challenge, despite their increase in number according to clinical stage and tumor size. The total percentage of ctDNA in the blood is hence low and below detection levels. Some scientists recently combined their data on ctDNA mutations with other biomarker parameters like protein occurrence or methylation status to improve the overall sensitivity. At the present time, the liquid biopsy approach does not show optimal sensitivity for all cancer types.

Another challenge is that circulating tumor cells are fragile. It is hence very challenging to isolate ctDNA from the blood for quantitation, not least since the small fragments are easily lost or are being unstable.

More intervention studies are needed

Liquid biopsies and the quantification of circulating biomarkers undoubtedly have a bright future in tumor medicine. The interest in developing assays for liquid biopsies is gaining increasing attention from an ever-increasing number of companies that develop diagnostic kits for the clinical stratification of cancer patients. However, to put liquid biopsy in the hands of the clinicians, the major focus is to prove their case as tumor biomarker assays.

The good news is that this research is well underway. Since the first commercially available liquid biopsy assay became introduced, a very rapid and impressive improvement in throughput and sensitivity of subsequent liquid biopsy assays has occurred, mainly because of advances in DNA and RNA sequencing technologies and the bioinformatics revolution.

Readings

Balaña C. et al., O6-methyl-guanine-DNA methyltransferase methylation in serum and tumor DNA predicts response to 1,3-bis(2-chloroethyl)-1-nitrosourea but not to temozolomide plus cisplatin in glioblastoma multiforme. Clin Cancer Res. 2003;9(4):1461-8

Bi F, et al., Circulating tumor DNA in colorectal cancer: opportunities and challenges. Review. Am J Transl Res. 2020; 5;12(3):1044-1055

Chen VL, et al., Utility of Liquid Biopsy Analysis in Detection of Hepatocellular Carcinoma, Determination of Prognosis, and Disease Monitoring: a Systematic Review. Clin Gastroenterol Hepatol. 2020; 11

Danila DC, et al., Circulating tumor cells as biomarkers in prostate cancer. Clin Cancer Res. 2011; 17(12):3903-12

Ding Y, et al., Perspectives of the Application of Liquid Biopsy in Colorectal Cancer. Review Biomed Res Int. 2020; 6843180

Karachaliou N. et al., Possible application of circulating free tumor DNA in non-small cell lung cancer patients. J Thorac Dis. 2017; 9(Suppl 13): S1364–S1372

Kwapisz D. et al., The first liquid biopsy test approved. is it a new era of mutation testing for non-small cell lung cancer? Ann Transl Med. 2017;5(3):46

Li Y. Biofluid-Based Circulating Tumor Molecules as Diagnostic Tools for Use in Personalized Medicine. J Mol Biomark Diagn 2013, 5:1

Lim M. et al., Liquid Biopsy in Lung Cancer: Clinical Applications of Circulating Biomarkers (CTCs and ctDNA). Review. Micromachines 2018;9,100

Matsutani A, et al., Liquid biopsy for the detection of clinical biomarkers in early breast cancer: new insights and challenges. Pharmacogenomics. 2020; 21(5):359-367

Muller, C. et al. Hematogenous dissemination of glioblastoma multiforme. Sci. Transl. Med. 2014; 6, 247ra101

Rapisuwon S. et al., Circulating biomarkers to monitor cancer progression and treatment. Comput Struct Biotechnol J. 2016;14:211-222

Silva R, et al., Evaluating liquid biopsies for methylomic profiling of prostate cancer. Epigenetics. 2020:1-13

Speicher MR. et al., Tumor signatures in the blood, Nat Biotechnol 2014; 32, 441–443

Sundaresan T.K. et al., Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses. Clin Cancer Res. 2015;22:1103–1110

Wu X. et al., Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma. Int J Biol Sci. 2020;16(9):1551–1562