A liquid biopsy starts with examining a sample of fluid from the body which may include blood, urine, saliva, and cerebrospinal fluid.Blood contains a mixture of various substances including red blood cells, white blood cells, platelets, exosomes, RNA, DNA, and proteins. The plasma is obtained by centrifuging blood which can further be simplified into serum. The serum refers to the part of the plasma that lacks clotting agents because of its coagulated nature, and thus simplifies the liquid to focus on what is needed for a biopsy. While the majority of liquid biopsies utilize blood, urine can prove extremely useful, particularly for cancers relating to the endocrine system.

Exosomes are vesicles from blood cells that may be used for diagnostic purposes due to the presence of DNA, RNA, and proteins from their original cell.Such nanoparticles have been found to spread cancer throughout the body by travelling in the blood and fusing with healthy cells at distant sites. Therefore, by targeting exosomes directly, further metastases may be prevented. According to  San Lucas  et al., exosomes contain high quality DNA that shows 90-95% of the same genetic markers as the origin tumor, and thus are useful in the diagnosis and tracking of cancer. Exosomes, along with blood, can also be found in urine and used in the diagnosis and tracking of various cancers including prostate, kidney, and colorectal cancer (Nilsson et al.).

As a vital component of liquid biopsies, messenger RNA (mRNA) found in urine may be used in the diagnosis of kidney, bladder, and prostate cancers (Di Meo et al.). RNA has long been hypothesized to have evolved before DNA, but it is primarily DNA that tends to be used in liquid biopsies. While DNA is often found in the cell nucleus and other organelles such as mitochondria, it is not associated with any cells in the blood plasma.

A tumor is a growth of cells that may or may not be cancerous. It is theorized that when cell death occurs, some of the cellular DNA begins circulating in the blood and other fluids as cell-free DNA (cfDNA). Some of the various forms of cfDNA in the blood include cell-free fetal DNA (cffDNA), donor derived cell free DNA (ddcfDNA), and circulating tumor DNA (ctDNA). This ctDNA is often the most important element in a liquid biopsy for cancer due to the ability to track the DNA to the organ of origin and reliability provided for locating solid tumors (Karachaliou et al.). Further research by  Bettegowda et al.  has demonstrated that both metastatic and localized cancers are capable of detection through ctDNA fragments in the blood. All of these various types of DNA work with liquid biopsies due to a process called Next Generation Sequencing, which will be examined later in detail.

Liquid biopsies are useful in diagnoses, treatments, transplants, and prenatal health. The next section will provide insight into some of these applications.

Despite the liquid biopsy initially being considered revolutionary for its early and simple diagnosis of cancer, its versatility extends to many other applications. As a result, cancer is not the only priority for the oncological test.

For example, cffDNA, or DNA that originates from a fetus, can be found in a mother’s body and examined in liquid biopsies (Chitty & Lo). Referred to as Non-Invasive Prenatal Testing (NIPT), the analysis can be used to screen for chromosomal abnormalities such as Down’s Syndrome, Edward’s Syndrome, and Patau Syndrome. If abnormalities are found, a more invasive method (e.g. amniocentesis or chorionic villus sampling) will likely be recommended to form a diagnosis.

The scope of liquid biopsies further extends to organ transplants. As  Hofste et al.  note, transplanted organs release donor-derived cell free DNA (ddcfDNA) in the body. The amount of the ddcfDNA can be compared to the body’s original cfDNA in a liquid biopsy using blood. The results can then help determine whether the body is accepting or rejecting a graft in the early stages of a transplant and even help determine immunosuppression levels that will ensure transplant success.

Oncology has long been the primary field of liquid biopsies with regards to both diagnosis and treatment.

Precision, or personalized, medicine is a subfield in oncology care that focuses on the patient’s genetic profile to target their cancer. Molecular profiling assesses cfDNA to find markers deemed significant which are then used in treatment (Di Meo et al.). Unfortunately, not all cancer is the same, and thus, blanket treatments may cause more harm than good. Utilizing a liquid biopsy can help identify if a treatment is effective and whether or not it should be continued. As mentioned earlier, exosomes may be one of those targets for treatment that may prevent the advance of cancer.

Tumors can be unaffected by treatment using DNA associated with resistance in the past. In fact, Helwick  reports in one study that resistance was successfully identified in 27% of patients using a liquid biopsy. Acquired resistance is also a major obstacle to eradicating cancer with the use of drugs and treatments, such as radiation, that have severe side effects. Using specific genetic mutations from ctDNA, a process called DNA sequencing can decrease associated dangers of such treatment.

T-cell transfer immunotherapy is a treatment in which specific T-cells are collected from a patient, cultured in the lab, then returned back into the patient’s body in increased numbers to fight the cancer.  Xi et al.  demonstrated in their research regarding the procedure that ctDNA could be used to determine whether a treatment was effective within a mere two weeks. The test’s rapid determination of treatment effectiveness will aid patients from further continuing potentially ineffective and painful treatments.

Applications of a Liquid Biopsy in Oncology (Bettegowda et al.)

Workflow for Breast Cancer

The application of liquid biopsies employs Next Generation Sequencing (NGS), a specific type of Whole Genome Sequencing (WGS) which arranges and maps the DNA of an organism’s entire genome within a day (Behjati & Tarpey). Fragments of DNA are processed multiple times to provide an extremely detailed and accurate overview of exomes, which comprise a fraction of the genome. Using NGS, hundreds of different breast cancer tumors have been sequenced. The results revealed previously unknown mutations in DNA that may be associated with breast cancer in potential patients (Desmedt et al.). Such mutations are divided into driver mutations (those that contribute to the development of a tumor) and passenger mutations (those that can change through the course of cancer treatment into resistant clones of a tumor).

Extensive research using 20,592 women with breast cancer has helped map out an entire panel of genes to be tracked through liquid biopsies, as reported by O’Leary et al. These genes include mutations leading to breast cancer, common gynecologic cancers, and other tumors. Abnormal methylation, an epigenetic aberration that may indicate a marker for breast cancer, can further be detected by liquid biopsies in cfDNA during the diagnosis process. The potential of liquid biopsies can be illustrated in this regard by  Petr et al.  through a study which identified 3,506 methylated regions within the examined breast cancer.

After a diagnosis is made, treatments can be decided and initiated using the aforementioned techniques of precision medicine. During the process, liquid biopsies can serve to determine the efficacy and efficiency of the treatment. Even after a patient has reached remission, liquid biopsies prove absolutely vital as a screening tool to track and manage the return of the breast cancer.

A focus on the future of liquid biopsies, due to their large candidate eligibility, continues to make them an increasingly available and accessible screening tool for the general population. While the general population certainly stands to benefit from liquid biopsies, one demographic in particular stands to gain the most: cancer patients.

As  Miller et al.  reported in 2016, there were approximately 15.5 million cancer survivors in the United States alone, a number that is rapidly increasing as technology and treatment methods improve. While the demographic is at an increased risk for relapse, liquid biopsies serve to provide patients and providers with a cheap and non-invasive alternative to regularly test for the return of cancer.

Further populations that are vulnerable also need a method that will not compromise their immune system or extend recovery time. For example,  Ilie et al.  observed that liquid biopsies detected lung cancer early using ctDNA in those with chronic obstructive pulmonary disease (COPD), a condition which prevents a traditional diagnostic method such as tissue biopsy from being used. Similarly,  Mischak  observed that in patients with Chronic Kidney Disease (CKD), liquid biopsies can provide a less intrusive alternative to the usage of kidney tissue to track certain proteins.

Estimated Survivors of Cancer in 2016 and Projected Survivors in 2026 (Miller et al.)

It is important to note that at their current stage, liquid biopsies should be used in conjunction with traditional methods as necessary rather than solely. The next section will discuss how each form of biopsy has its own advantages and disadvantages.

Traditional biopsy methods can be invasive or dangerous for patients, often qualifying as surgery, and can include tissue biopsies (tissue, marrow), imaging scans (x-ray, MRI, ultrasound), and lab tests (blood, urine).

One of the highest priorities for liquid biopsies is increased commercialization to make the tool as cost-effective and widely available as possible.  Volik et al.  report that great progress has been made in commercializing liquid biopsies for cancer diagnoses.

In 2016, the  FDA  approved the first liquid biopsy test for lung cancer. Since then, the field has exploded with innovation: applications from fetal testing and organ transplantation to genome mapping show that liquid biopsies have a scope far beyond oncology. Even within the field of cancer research, multiple ongoing studies continue to demonstrate the superiority of liquid biopsies over traditional methods.

For example, work is being done on a test that spots both DNA and protein biomarkers for pancreatic cancer. While the test is not yet ready for commercialization, it has been claimed that mutations in DNA shed from cancer cells and found in the blood is “exquisitely specific” for cancer. “If cancer-linked DNA can be found in the blood of an individual, it is very likely that person has cancer,” says Vogelstein, co-director at the  Johns Hopkins  Kimmel Cancer Center. Studies by the clinical research center and others have shown more than 85% of patients with advanced cancer have identifiable DNA in their blood. Today, the possibility of identifying such small pieces of DNA in the blood without prior knowledge of the genetic status of the cancers demonstrates how quickly the field is advancing.

Key Trends in the Liquid Biopsy Market Value (Future Market Insights)

A current ongoing research study done by the   Stanford Cancer Institute and the National Cancer Institute  is set to determine the effectiveness of genomic profiling in precision treatments. Further research studies continue to focus on being able to identify markers for diseases identified by a biopsy.

BioChain is proud to offer a range of   specialized  products  designed to extract cfDNA in blood plasma or serum. The magnetic bead-based product can perform with less than 1 mL of a sample despite being able to maximize sensitivity of cfDNA. In fact, BioChain’s kit recovers more cfDNA per mL of plasma when compared to competing products, possesses convenient automation compatibility, and is strictly tested for consistency in results.

Uses of the extraction kit range from NGS to PCR for DNA amplification while the scalable system allows the sample to be conserved and used only as needed. The significantly cost-effective method can be utilized for further applications, from prenatal genetic testing to early cancer diagnoses. Compared to traditional biopsy methods, BioChain’s cfDNA extraction kits can provide a more efficient method of extraction from smaller fragmented chains. Leading at the forefront of biotechnological advancement, BioChain continues to couple premier therapeutic products with competitive pricing to provide an exceptional clinical and research experience.