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Detecting Mutations With Liquid Biopsies: A Novel Approach to Cancer Diagnostics

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Presenting highlights from the webinar “Detection of mutations from liquid biopsies”, hosted by Monika Seidel Ph.D., which is now available to view on-demand here!   

Liquid biopsies can be used to identify the presence of specific analytes in bodily fluids—such as, blood, urine, and cerebrospinal fluid. These tests are used to diagnose disease by detecting biomarkers for cancer, autoimmune disease or diabetes and can be used as early detection tools for organ rejection in transplant patients. This sample method is considerably less invasive compared to more traditional tissue biopsy methods, and is therefore, becoming more widely used. So, let us explore the use of liquid biopsies as tools for cancer diagnosis and research.

Meet Monika

Monika Seidel is a senior development scientist in genomics and cellular research at Cytiva and has a B.Sc. in organic chemistry, M.Sc. in biotechnology and a Ph.D. in medical science from the University of Cardiff, UK. At Cytiva, Monika works on new product development, focusing on numerous activities, from conception and feasibility to manufacturing handover. She has an extensive understanding of liquid biopsy-based diagnostic applications and expertise in molecular biology, biochemistry, immunology-related techniques, mass spectrometry and stem cell biology.

The importance of liquid biopsies

A liquid biopsy sample can contain different types of tumor-derived material, however, most tests work by 
capturing and analyzing a specific biomarker—circulating tumor DNA (ctDNA). ctDNA is a specific type of cell-free DNA (cfDNA) that is released into the bloodstream when a tumor cell dies and is therefore a valuable tool for cancer detection and disease monitoring.

Liquid biopsies can be used to overcome the heterogeneity of tumors, identifying markers that indicate sub-types of cancer (interpatient heterogeneity) and subpopulations of cells within a tumor (intratumor heterogeneity). By gaining a more comprehensive genetic understanding of tumors over time, scientists can look for changes in these markers to monitor treatment response, identify cases of drug resistance and guide future treatment decisions.

In 2020, the US Food and Drug Administration (FDA) approved two cancer liquid biopsy tests. The first is a 
companion diagnostic test that combines two technologies—liquid biopsy and next-generation sequencing (NGS). This test is designed to guide treatment decisions for patients with specific types of mutations of the epidermal growth factor receptor (EGFR) gene in a specific form of metastatic non-small cell lung cancer (NSCLC). The second test detects specific gene mutations found in cfDNA isolated from whole blood plasma specimens and is designed to identify patients diagnosed with prostate cancer and NSCLC who may benefit from targeted therapy. Both approaches are recommended when a tissue biopsy is not feasible, such as when a tumor has already been surgically removed or is particularly difficult to access. Yet, it is worth noting that whilst they are FDA-approved, both tests have low detection sensitivity in early-stage cancer patients and a high risk of false negatives.

Detecting cancer using liquid biopsies

cfDNA is already employed in clinical practice as a tool for tumor profiling and prenatal testing and is the basis for several FDA-approved cancer screening tests. In healthy individuals the fragment length of cfDNA is approximately 170 bp and it is released into the blood via both passive mechanisms (e.g., necrosis and apoptosis) and active mechanisms. The quantity of cfDNA can increase due to several factors, including pregnancy, autoimmune disorders, advanced cancer, and strenuous exercise.

Tests that use cfDNA for cancer mutation detection can have low sensitivity, this can be due to the end-point detection assay for biomarker profiling or specific tumor-derived cfDNA characteristics. In addition, during plasma processing, genomic DNA (gDNA) from white blood cells can contaminate the sample and dilute the tumor fraction. 
cfDNA circulates in many forms, for example as fragments, which are short (> 400 bp), highly degraded and/or (partially) single-stranded. This can cause issues during NGS analysis, particularly if fragments are eliminated during sample processing and therefore are undetected. Tumor-derived cfDNA (ctDNA), is typically even more fragmented than cfDNA (< 100 bp in size) and produces a low yield, 0.01% in early-stage disease and 30% in advanced cancer.

How can cfDNA analysis be improved?

There are numerous ways to 
improve cfDNA analysis. The first is to use a specific collection tube which limits the gDNA contamination and increases the amount of cfDNA. Secondly, you can use an extraction method that is designed to recover small fragments of cfDNA. The third way is to choose the right library preparation protocol for NGS, as some single-strand library preparations can capture degraded or single-stranded fragments of cfDNA. Finally, bioinformatic tools can be used to eliminate sequencing errors and unique molecule identifiers can be implemented to eliminate errors that occur during polymerase chain reaction (PCR).

Sera-XtractaTM Cell-Free DNA kit is a magnetic silica bead-based kit that enables the rapid extraction and purification of cfDNA from blood plasma and it is compatible with downstream molecular biology techniques, including NGS, quantitative PCR (qPCR) and digital droplet PCR (ddPCR). The kit is designed for maximum recovery of small fragment cfDNA whilst minimizing any co-purification of higher molecular weight genomic DNA.

In conclusion, liquid biopsies are a non-invasive alternative to tissue biopsies that can reveal in-depth information about the genetic profile of a tumor. However, downstream analysis of these samples can produce low sensitivity results. A robust extraction method—such as, Sera-Xtracta Cell-Free DNA kit—is therefore key to maximizing the accuracy of results. Listen to the webinar for more information about this kit and to hear about the scientific trials that have been conducted using this solution. 

Ask the Expert

Q: Is the 
Sera-XtractaTM Cell-Free DNA kit compatible with serum samples?

A: Yes, it is compatible and a recent study has demonstrated that the Sera-Xtracta Cell-Free DNA Kit efficiently extracts cfDNA from serum with superior extraction efficiency when compared to an alternative bead-based commercial product and in samples where a significant amount of gDNA is present (notorious with serum samples), the Sera-Xtracta cfDNA kit effectively reduces gDNA carry-over, ensuring maximum enrichment in cfDNA.  

Q: What is the preferable sample type for clinical applications, serum or plasma?

The consensus in the field, is that plasma is the preferred sample in clinical application. This is based on numerous reports showing that mutation allele frequency (MAF) and the detection rate of cancer associated biomarkers is much higher in plasma samples compared to equivalent serum samples. However, it is important that we do not dismiss serum samples completely, as they are very important in retrospective studies for biomarker discovery in large cohorts of patients in biobank samples.

Q: What is the plasma input volume for the Sera-Xtracta
TM Cell-Free DNA kit and how does is correspond to the blood needed?

A: Typically, you can get 4 mL of plasma from 10 mL of blood. The kit has been validated for a plasma input of 0.5-4 mL.

If you missed the webinar, you can view the on-demand video