Liquid biopsies easing diagnosis

June 23, 2021

When a doctor says, “you have a lump,” your heart drops, and your mind races to the worst case scenario. For years, to determine if the lump was cancerous, it might involve a biopsy where a chunk of tissue is cut from your flesh, leaving scars that take a while to heal and can cause emotional issues, such as insecurities about body image. Now, there is the advent of liquid biopsies, a minimally invasive diagnostic tool that does not require an operating room, but relies on laboratory professionals detecting genetic mutations in bodily fluids.

History of oncology biomarkers

While some may not be entirely familiar with the the term liquid biopsy, the search for cancer biomarkers traces back to Henry Bence Jones discovering the Bence Jones Protein in 1848, which was the first major milestone of oncology biomarkers,1 followed by Thomas Ashworth describing circulating tumor cells (CTCs) with microscopic observation of a blood with metastases, leading to metastatic cancer.

It was not until 1965, when Phil Gold, MD, PhD, and Samuel O. Freedman, OC CQ FRSC, discovered Carcinoembryonic antigen (CEA), and in 1970, Richard Albin, PhD, DSc, found the Prostrate-Specific Antigen (PSA) for prostrate cancer. This is the same year that oncogenes reveal the molecular basis of cancer.

In 1981, Robert C. Bast, Jr., discovered the CA 125 antigen to identify ovarian cancer, and CA 19-9, seen in the serum of patients with colon and pancreatic cancer. The human epidermal growth receptor, known as HER2, was found in 1989 by Dennis Slamon, MD, PhD, et al, and in 1991, Curtis Harris, MD, discovered p53 when he was exploring the consequences of un-repaired DNA. Now, more than half of the clinical trials in the States are being conducted with biomarker testing.1

Use of liquid biopsy

“Liquid biopsy has great potential to transform cancer patient testing and is rapidly becoming a compliment to the common tissue biopsy techniques,” said Rajeshwari Samanth, Research Consultant at Fact.MR. Most commonly, liquid biopsy is used for cancer, including lung, ovarian, breast and prostate. “Commonly used liquid biopsy is EGFR Mutation Test v2 to determine the eligibility of non-small cell lung cancer patients to receive certain EGFR tyrosine kinase inhibitors. Liquid biopsies are particularly used in patients receiving targeted therapies and to check disease progression and attainment of resistance mutations,” such as BRCA1 reversions in ovarian cancers receiving platinum- or PARP-based treatment.

Currently, techniques used include Circulating Tumor DNA (ctDNA) or Cell-Free DNA Tests, as well as Circulating Tumor Cell Tests (CTCs). CTCs are rare, such as one cell in 109 blood cells, creating challenges for isolation. More recently, people have been turning their attention to extracellular vesicles (EVs) and ctDNA to potentially create a global tumor genome and transcriptome. EVs are released by dividing and growing new tumor cells, so EVs have cellular proteins on their surface.

Only a third of the cancer biomarker tests currently ordered are liquid, using blood, tears, pleural effusion bronchoalveolar lavage, urine, cerebral spinal fluid (CSF), saliva, ascites peritoneal lavage, seminal fluid, breast ductal fluid, amniotic fluid, vaginal secretions, utero-tubular lavage and more.

Many liquid biopsies search for cancer-specific genetic changes or mutations of DNA. Another approach uses methylation, chemical tags known as methyl groups, on DNA in bodily fluids. These chemical tags do not change the genetic code, but they play a role in activating or deactivating the genes.

DNA methylation patterns can distinguish between cancerous and normal tissues, as well as the type of tissue, be it breast, brain, lung, kidney, or other.2 Daniel De Carvalho, PhD, of Princess Margaret Cancer Centre in Toronto, led a team that developed the DNA methylation-based liquid biopsy technique, citing it may be useful for tumors that do not shed much DNA, such as kidney and brain cancers. In non-epithelial cancers, such as brain gliomas, identifying a tumor specific membrane protein is challenging, as only those CTCs with target protein are isolated.

Though MRI or other imaging methods can detect a tumor, the test cannot tell what type of brain cancer it is, or if it is metastatic, originating from elsewhere and spreading. Combining these imaging methods with liquid biopsies and other testing approaches may be the key to early detection for things like brain cancer.

Recent advances in liquid biopsies

“Recently in 2020, The Food and Drug Administration (FDA) approved new tests to detect DNA circulating in the blood after being released by cancerous tumors,” explained Samanth. “This test is used for multiple cancers and biomarkers. For example, to detect mutations in BRCA1 and BRCA2 genes in patients with ovarian cancer. This is the first approval to combine two technologies — NGS and liquid biopsy — in one diagnostic test. These tests, along with advanced testing technology, help to provide genetic information if cancer drugs may treat them.”

Guardant360 CDx uses NGS to seek specific EGFRmutations in metastatic non-small cell lung cancer, and FoundationOne Liquid CDx searches 324 genes for mutations, including EGFR, that are implicated in cancer.

A far-field nanoplasmon-enhanced scattering (FF-nPES) can be used as an assay for isolation-free characterization of EVs in serum. This may eventually be used for analysis of EV epithelial cell adhesion molecule (EpCAM) expression to detect pancreatic ductal adenocarcinoma patients.3

Serum EV-miRNA-21, -92a, and -222 are potential biomarkers of bevacizumab-treated metastatic colorectal cancer. Pancreas-associated pathologies have identified KRAS and TP53 mutations as being of interest for future studies.

According to the Department of Health and Human Services (HHS), one study demonstrated as much as 90% of prostate cancer mutations could be detected in blood samples with exome sequencing of immunopurified CTC patients with metastatic prostate cancer.3 In CSF, cell-free DNA and specific mutations in histone H3 could be dectected by ctDNA in brain tumor patients, while glioma patients showed lower levels of Alu methylation, and another study revealed all patients with intracranial tumors adjacent to a cisternal space, as well as those with high grade glima, had detectable ctDNA in CSF.

However, there is a reluctance to give a patient a lumbar puncture when they have an intracranial mass, not wanting to induce a herniation syndrome. The BRAF V600E mutation, which lies within exon 15 of chromosome 7, has also been identified with brain tumors, but this can be detected at both the DNA and mRNA levels, being detected in plasma using a novel plasma-based ddPCR assay.

Samanth shared that liquid biopsies do have limitations. For example, there is a need for an initial histologic analysis that is done by tissue biopsy, and at present, liquid biopsy is not used as a replacement for the tissue biopsy. The big elephant in the room is the lack of valid data for use of liquid biopsies in clinical trials, and test sensitivity can often be challenging when CTCs are minimal in a sample.

Still, liquid biopsies only appear to be gaining momentum, as Labcorp recently announced that it will begin liquid biopsy testing for lung cancer.4 In 2018, Quest had been one of the companies to help fund Clinical Genomics in their creation of COLVERA,5 a liquid biopsy now available in all 50 states that test for aberrant methylation in GCAT1 and IKZF1, which is found at high frequency in colorectal cancer tumor tissue.

Tips for lab professionals

While liquid biopsies are non-invasive or minimally invasive, painless, precise, using a real-time approach, which could potentially reduce cost and diagnostic time, while overcoming the heterogeneity of tumors,6 they are not completely replacing tissue biopsies quite yet.

Samanth warned, “The presence of sample analyte is often low, leading to higher false negative results, and this requires significantly greater technical efforts to interpret the data and conclude the results.”

Storing can also create issues, as well as freezing and thawing protocols, so kits and other consumables should be checked from the manufacturer and stored at recommended conditions only. Macromolecule yield can also be influenced by collection method, processing time, level of hemolysis, and preservation agent exposure. With EVs, the method of vesicle counting can also produce different results.3

Samanth added, “Sample labelling also plays an important role for proper patient identification with all other details and history of disease,” so be sure to take the time for proper identification of samples.


  1. Oates-Zalesky M, Simpson M. Oncology biomarker testing awareness, drivers, and the future. InCrowd. Published May 21, 2021. Accessed June 2, 2021.
  2. Liquid biopsy for brain cancer, early-stage kidney cancer. National Cancer Institute. Published July 23, 2020. Accessed June 2, 2021.
  3. Shankar GM, Balaj L, Stott SL, Nahed B, Carter BS. Liquid biopsy for brain tumors. Expert Review of Molecular Diagnostics. 2017;17(10):943-947. doi:10.1080/14737159.2017.1374854.
  4. Labcorp to introduce combined oncology offering at 2021 ASCO Annual Meeting. Laboratory Corporation of America Holdings. Accessed June 4, 2021.
  5. Clinical Genomics announces nationwide expansion of patient access to COLVERA. Clinical Genomics. Published January 11, 2021. Accessed June 4, 2021.
  6. Liskova A, Samec M, Koklesova L, Giordano FA, Kubatka P, Golubnitschaja O. Liquid biopsy is instrumental for 3PM Dimensional Solutions in cancer management. Journal of Clinical Medicine. 2020;9(9):2749. doi:10.3390/jcm9092749.