The utility of liquid biopsy tests in cancer diagnostics provide potential and beneficial outcomes and turn the focus of cancer treatments towards it. Currently, tissue biopsy is most frequently used invasive molecular analysis for cancer diagnostics in clinical practice. One main limitation with tissue biopsy approach is that repeated sampling over time is generally not practicable because of invasive method. Hence, monitoring of therapy in patients and detection of tumor location cannot be possible with tissue biopsy. Due to Invasion method in tissue biopsies, causes adverse side effects such as the risks of tumor dissemination, infections and bleeding. To overcome these major issues associated with tissue biopsy, liquid biopsy is now a days in focus as a potential diagnostic technique for the cancer patients. A liquid biopsy which is less invasive technique, also known as fluid biopsy or fluid phase biopsy, is performed by taking bodily fluids like blood, urine, cerebrospinal fluids and stool samples from the patient for diagnosis of cancer.

Liquid biopsies could provide a potential revolution in the field of cancer diagnosis and treatment. Liquid biopsy analysis for the diagnosis of cancer has been based on analytes of biological fluid. The analytes which are released by tumors in bodily fluids and analyzed as biomarker by liquid biopsy for the diagnosis of cancer are circulating tumour cells (CTCs), circulating nucleic acids (circulating tumour DNA (ctDNA), the tumour-derived fraction of cell-free DNA (cfDNA) in the plasma and cell-free RNAs (mRNAs, long non-coding RNAs and microRNAs), extracellular vesicles, tumour-educated platelets, proteins and metabolites.

Liquid biopsy is less invasive and samples are more easily obtainable throughout the management of the disease. It can be easily repeated because of its less invasive nature and helpful for monitoring patients’ responses to therapy during and after treatment to shift it towards personalized medicines. Liquid biopsy provides changes in molecular information of the cancer during and after treatment and hence, it could be a potential tracker for the identification of causes for the development of drug resistance and helpful for the treatment selection. It can be used to monitor the progression of cancer and act as a monitoring method for cancer patients who have completed treatment but are at high risk of recurrence of disease.

The biomarkers for liquid biopsy are-

1. Circulating tumor cells (CTCs)

Circulating tumor cells are carcinoma cells with a half-life of 1-2.4 h which detach from a primary tumor or metastatic lesions and travel in the bloodstream. These cells can be isolated from the blood of cancer patients as single cells or cell clusters but they are low in numbers (< 10 cells/mL of blood) even in metastatic settings and vary between types of tumor. Hence, it requires a large amount of blood sample (8 ml). Only FDA approved immunoaffinity-based CellSearch technology for CTCs detection, separation and counting. In this method, CTCs can be separated using specific antigens which are expressed on the surface of CTCs only.

2. Tumor educated platelets (TEPs)

Tumor educated platelets (TEPs) are another biomarker of liquid biopsy. TEPs are platelets which are formed due to the modification by a tumor. The RNA profile of these platelets is altered by tumor cells and PETs act as a carrier and protector of metastasis. The altered mRNA in TEPs could be used for the diagnosis of cancer. Platelets are anucleate cells originating from megakaryocytes, playing a crucial role in the systemic and local responses to tumor growth. The merits of the use of TEPs over other blood-based biosources include their abundance, easy sequestration and ability to process high quality RNA in response to external signals. Combinatorial analysis of TEPs with other complementary biosources like extracellular vesicles circulating tumor DNA and CTCs, but possibly also imaging and protein markers, warrants consideration as next-generation biomarker troves, thereby seeking optimal diagnostic synergy.

3. Extracellular Vesicles (EVs)

Extracellular vesicles (EVs) are developing as vital intercellular messengers regulating tumor progression and cell metabolism. There are mainly two types of EVs- small EVs (≤200nm) or exosomes and large or medium EVs (≥ 200 nm) including microvesicles, shed bodies, ectosomes and microparticles. Exosomes are small nano-sized EVs which are derived from the late endocytic pathway and carry a large variety of molecules, such as proteins, DNA fragments and RNAs. The plasma membrane which is guided by specific molecular machinery to form outward budding known as EVs. Exosomes have been thoroughly investigated and found them to be a recognized circulating biomarker in cancer diagnosis. Due to lack of standardized methods for the isolation of nano-vesicles, few of them have been used in clinical practice. The limitations in use of small EVs are the requirement of large amount of biological fluids for downstream analyses, high costs and analytical time. However, large EVs are also a circulating diagnostic tools for cancer and advantageous over small EVs due to its particular composition and easy recovery from biological fluids. Large EVs can be isolated by using ultracentrifugation, ultrafiltration, gel filtration, precipitation and immunoaffinity.

4. Circulating nucleic acids

4.1 Circulating tumor DNA (ctDNA)

The DNA which are released by the tumor cell in bloodstream of cancer patients through apoptosis, necrosis or active release are called ctDNA. It is typically cleared from the blood within hours by nuclease activity, by the liver and spleen, and excretion through the kidneys. The tumor-specific mutations in ctDNA sequence can provide a new way for the screening of cancer patients on large scale from a group of healthy individuals. The concentration of ctDNA in plasma can be correlate with tumor size and stage of cancer. Patients having cancer at stage I had less than 10 copies/5 ml of tumor mutations and these number increased 10 to 100 times in plasma among late-stage cancer patients. It can more precisely detect late stages of cancers as concentrations of ctDNA are increased. However, for the early stage diagnosis, ctDNA test should be highly sensitive. There are basically PCR-based and NGS based sequencing techniques are available for the analysis of ctDNA. PCR-based sequencing for ctDNA analysis can be used for targeted panel and single-locus or multiplexed assays. It is fast and inexpensive compared with NGS sequencing technique. It cannot be used for daily clinical usage as cost of analysis will increase and it can only screen known variants. In addition, it can only detect mutant allele fraction which is ≥ 10%. However, NGS-based sequencing can be applied to any size of panel and screen unknown variants too. It is able to detect mutant allele fractions < 1%. NGS can applied to the targeted panel for specific and highly sensitive identification of targeted ctDNA mutations. The Tagged-Amplicon deep sequencing (TAm-seq), Safe-Sequencing System (Safe-SeqS), CAncer Personalized Profiling by deep sequencing (CAPP-Seq), and Ion Torrent are the methods which can be applied NGS to target panel for detection of ctDNA.

The ctDNA has shown many encouraging results for cancer classification, monitoring, prognosis, and treatment selection for patients. The major challenge with ctDNA is its low concentration in the blood. However, some NGS-based sequencings improve the sensitivity of ctDNA assays. The other main concern with clinical use of ctDNA is cost of the sensitive processes. The ctDNA should be combined to increase sensitivity and specificity with other biomarkers of liquid biopsy. If the cost for sequencing continues to decrease, using liquid biopsy for cancer prevention and management hold promise in the future.

4.2 miRNAs

These are most abundant endogenous and small non-coding RNAs (21–25 bps), circulating nucleic acids in the blood. They can act as oncogenes and carried by TEPs and exosomes. They have important role in tumor growth and development of treatment resistance. In case of solid tumors, the concentration and composition of exosomal miRNAs differ between cancer patients and healthy subjects and it may be used as novel potential diagnostic, prognostic and predictive biomarkers for several types of cancers.

4.3 Circulating free DNA (cfDNA)

Circulating free DNA (cfDNA) are degraded fragments of DNA (50 – 200 bp) released to the blood plasma in cancer patients. The concentrations of cfDNAs increase with the stages of cancer and has been found higher in cancer patients as compared to healthy individuals. The main issue with its use as diagnostic cancer biomarker in liquid biopsy is availability of its concentration in pregnancy, injury and some non-pathological cancer diseases like diabetes, inflammation and infections. This problem with cfDNA shifted the focus of research towards its tumor fraction ctDNA.

Proteins biomarkers such as CA125, CEA, CA19-9 and S100 are clinically diagnosed cancer with liquid biopsy test in patients, but many have limited specificity and use.

5. FDA Approved liquid biopsy tests

In 2016, FDA approved a liquid biopsy test, called the cobas EGFR Mutation Test for the detection of EGFR gene mutations in ctDNA of patient’s plasma with lung cancer. Recently the FDA approved the first two NGS-based liquid biopsy solutions: Guardant360 in August, 2020 and Foundation One CDx in November 2020.

An impressive literatures on liquid biopsy have recently highlighting the potentials of it in the field of oncology research and promote its clinical applications in cancer treatment. To achieve efficient clinical usage of liquid biopsies, standardization of both pre-analytical (blood collection tubes specifications, time and procedure to obtain plasma from the blood and procedure for the isolation of biomarkers of liquid biopsy from plasma) and analytical procedures (focusing to maximization of yield of biomarkers from the plasma) is still in process to performed and generalized them for all liquid biopsies components. Cancer-ID in Europe (https://www.cancer-id.eu/the-project/overview/) network contain large amount of relevant information regarding the liquid biopsy.

References

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2. Velt SGJGI & Wurdinger T. 2019. Tumor-educated platelets. Blood. 2019; 133(22): 2359-2364.

3. Chen M, Zhao H. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics. 2019 Aug 1;13(1):34.

4. Andersson D et al.2020. A Liquid biopsy analysis in cancer diagnostics. Mol Aspects Med. 72:100839.

5. Poulet G et al. 2019. Liquid Biopsy: General Concepts. Acta Cytol. 63(6):449-455.