Liquid biopsy is a minimally invasive technology for detection of molecular biomarkers without the need for costly or invasive procedures. It is a simple and non-invasive alternative to surgical biopsies which enables medical doctors to discover a range of information about a disease or a tumour through a simple blood sample. Circulating cancer cells or traces of the cancer’s RNA or DNA in the blood can give clues about which treatments are most likely to work for that patient. Circulating nucleic acids are protected by extracellular micro-vesicles, mainly exosomes.

New dedicated methods enable you to enrich and purify from this liquid biopsy: circulating free DNA (cfDNA), circulating small-RNA, circulating tumour cells (CTCs), extracellular micro-vesicles (including exosomes) containing small-RNA, mRNA and DNA.
A test done on a sample of blood to look for cancer cells from a tumour that are circulating in the blood or for pieces of DNA from tumour cells that are in the blood. A liquid biopsy may be used to help find cancer at an early stage. It may also be used to help plan treatment or to find out how well treatment is working or if cancer has come back. Being able to take multiple samples of blood over time may also help doctors understand what kind of molecular changes are taking place in a tumour.
According to Y. M. DENNIS LO, Annals of the New York Academy of Sciences 2001, Volume 945 Issue CIRCULATING NUCLEIC ACIDS IN PLASMA OR SERUM II, Pages 1 – 7. The recent interest in nucleic acids in plasma and serum has opened up numerous new areas of investigation and new possibilities for molecular diagnosis. In oncology, tumour-derived genetic changes, epigenetic alterations, and viral nucleic acids have been found in the plasma/serum of cancer patients. These findings have important implications for the detection, monitoring, and prognostication of many types of malignancies. In prenatal diagnosis, the discovery of fetal DNA in maternal plasma and serum has provided a non-invasive source of fetal genetic material for analysis. This development has important implications for the realization of non-invasive prenatal diagnosis and has provided new methods for the monitoring of pregnancy-associated disorders. Plasma DNA technology has also found recent applications in the fields of organ transplantation, post trauma monitoring, and infectious agent detection. Future areas of study include circulating RNA in plasma and the elucidation of the biology of release, clearance, and possible functionality of plasma nucleic acids.

Circulating microRNA Biomarkers as Liquid Biopsy for Cancer Patients: Pros and Cons of Current Assays. Ono S, Lam S, Nagahara M, Hoon DS J Clin Med. 2015 4(10): 1890-1907, An increasing number of studies have focused on circulating microRNAs (cmiRNA) in cancer patients' blood for their potential as minimally-invasive biomarkers. Studies have reported the utility of assessing specific miRNAs in blood as diagnostic/prognostic biomarkers; however, the methodologies are not validated or standardized across laboratories. Unfortunately, there is often minimum limited overlap in techniques between results reported even in similar type studies on the same cancer. This hampers interpretation and reliability of cmiRNA as potential cancer biomarkers. Blood collection and processing, cmiRNA extractions, quality and quantity control of assays, defined patient population assessment, reproducibility, and reference standards all affect the cmiRNA assay results. To date, there is no reported definitive method to assess cmiRNAs. Therefore, appropriate and reliable methodologies are highly necessary in order for cmiRNAs to be used in regulated clinical diagnostic laboratories. In this review, we summarize the developments made over the past decade towards cmiRNA detection and discuss the pros and cons of the assays.

Valid biomarker signatures from liquid biopsies – how to standardise NGS:0- Dominik Buschmann, Benedikt Kirchner, Michael W. Pfaff, Drug Target Review, 206(4): 40-44, The advent of Next-Generation Sequencing (NGS) techniques has revolutionized transcriptomics research and opened numerous avenues for scientific and clinical applications. While reverse transcriptase quantitative real-time PCR (RT-qPCR) is still considered the gold standard of gene expression analysis, its high throughput, single-nucleotide resolution and ever-plummeting costs have made NGS an intriguing and increasingly accessible alternative to this classical method. In addition to mere transcript quantification, RNA-Seq offers exciting new insights such as the discovery of novel transcripts and detection of alternative splice variants or chimeric transcripts. While DNA sequencing yields fascinating discoveries about the genomic makeup of target tissues, RNA-Seq might hold even more potential for biomarker research and drug discovery.

Real-time liquid biopsy circulating tumour cells versus circulating tumour DNA. Alix-Panabières C and Pantel K, Ann Transl Med. 2013 1(2): 18, Liquid biopsy is a new diagnostic concept, i.e., analysis of therapeutic targets and drug resistance-conferring gene mutations on circulating tumour cells and cell-free circulating tumour DNA released into the peripheral blood from metastatic deposits. Here, we discuss current challenges and future perspectives of the liquid biopsy concept in clinical oncology. We postulate that this concept will contribute to a better understanding and clinical management of drug resistance in cancer patients.

Liquid biopsy:-  monitoring cancer-genetics in the blood. Emily Crowley, Federica Di Nicolantonio, Fotios Loupakis & Alberto Bardelli, Nature Reviews Clinical Oncology 10, 472-484, Cancer is associated with mutated genes, and analysis of tumour-linked genetic alterations is increasingly used for diagnostic, prognostic and treatment purposes. The genetic profile of solid tumours is currently obtained from surgical or biopsy specimens; however, the latter procedure cannot always be performed routinely owing to its invasive nature. Information acquired from a single biopsy provides a spatially and temporally limited snap-shot of a tumour and might fail to reflect its heterogeneity. Tumour cells release circulating free DNA (cfDNA) into the blood, but the majority of circulating DNA is often not of cancerous origin, and detection of cancer-associated alleles in the blood has long been impossible to achieve. Technological advances have overcome these restrictions, making it possible to identify both genetic and epigenetic aberrations. A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions (primary and metastases) as well as offering the opportunity to systematically track genomic evolution. This Review will explore how tumour-associated mutations detectable in the blood can be used in the clinic after diagnosis, including the assessment of prognosis, early detection of disease recurrence, and as surrogates for traditional biopsies with the purpose of predicting response to treatments and the development of acquired resistance.

Circulating Cancer Biomarkers:- The Macro-revolution of the Micro-RNA. Montani F and Bianchi F EBioMedicine. 2016 Feb 28;5: 4-6 e Collection 2016, MicroRNAs (miRNAs) are small non-coding RNAs that act as master regulators of many cellular processes. The expression of miRNAs is often deregulated in human tumours, causing the alteration of molecular mechanisms relevant for cancer progression. Importantly, miRNAs are detectable in the blood and their quantity fluctuations are the hallmark of pathogenic conditions, including cancer. Several groups reported the identification of circulating cell-free miRNAs (cf-miRNAs) in the human serum and plasma and demonstrated their diagnostic and prognostic utility. Other studies also shown that it may be feasible to apply such cf-miRNA signatures within screening programs in order to improve cancer early detection. Circulating cf-miRNAs therefore appear to be excellent candidates for blood-borne cancer biomarkers.

Current Status of CTCs as Liquid Biopsy in Lung Cancer and Future Directions:- Zhang Z, Ramnath N, Nagrath S, Front Oncol. 2015 Sep 30;5: 209, Circulating tumour cells (CTCs) have garnered a lot of attention in the past few decades. Isolation of these rare cells from the billions of blood cells has been a challenge until recent times. With the advent of new sensitive technologies that permit live cell isolation and downstream genomic analysis, the existing paradigm of CTC research has evolved to explore clinical utility of these cells. CTCs have been identified as prognostic and pharmacodynamics biomarkers in many solid tumours, including lung cancer. As a means of liquid biopsy, CTCs could play a major role in the development of personalized medicine and targeted therapies. This review discusses the state of various isolation strategies, cell separation techniques and key studies that illustrate the application of liquid biopsy to lung cancer.
Bio fluids Guidelines -- Liquid Biopsy Research, by Roche Molecular Diagnostics, a liquid biopsy is a simple and non-invasive alternative to surgical biopsies which enables doctors to discover a range of information about a tumour through a simple blood sample. Traces of the cancer’s DNA in the blood can give clues about which treatments are most likely to work for that patient. Bio fluids Guidelines -- Analysing microRNAs in liquid biopsies, blood serum, plasma, urine, CSF and exosomes, microRNA sequencing and qPCR analysis.

By Exiqon MicroRNAs in liquid biopsies hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. These short regulating RNAs have wide-ranging biological potential, are limited in number and are relatively stable in clinical samples such as serum / plasma, urine and other bio fluids. However, microRNA profiling in bio fluid samples is challenging in many ways. Bio fluids contain low levels of RNA, high levels of inhibitors and are susceptible to many preanalytical variables. To address these challenges, we have focused on developing highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive QC procedures. Exiqon offers a range of products and services for highly sensitive, specific and robust detection of microRNAs in bio fluids. The techniques we have developed are based on many years’ experience with both microRNA profiling and biomarker discovery and validation in bio fluid samples. Next Generation Sequencing (NGS) is a powerful tool for the discovery of novel microRNAs. We have optimized microRNA sequencing specifically for serum / plasma, to enable reliable results using Exiqon’s NGS Services. Our rigorously validated LNA™- enhanced qPCR assays offer sensitive and specific analysis of both known and novel microRNAs. These guidelines focus on setting up microRNA profiling experiments from blood serum and plasma, and urine, but contain useful information for microRNA experiments using other bio fluids as well. The guidelines provide important information and tips to ensure successful microRNA profiling using either Next Generation Sequencing or the miRCURY LNA™ Universal RT microRNA PCR System.

Pioneering the Liquid Biopsy Revolution:- Enrich. Purify. Discover by Qiagen, QIAGEN’s liquid biopsy solutions empower you to sensitively, specifically and rapidly analyse circulating nucleic acids, giving you the first step towards uncovering valuable biomarkers in your samples.
Liquid biopsy is a new, minimally invasive technology for detection of disease biomarkers without the need for costly or invasive procedures. The dedicated kits enable you to enrich and purify circulating free DNA (cfDNA), as well as nucleic acids from circulating tumour cells (CTCs) and extracellular vesicles. Liquid biopsy solutions deliver: Unbiased, specific enrichment of desired nucleic acids, Flexibility in sample volume for increased sensitivity, High performance and confidence in your results.

Liquid Biopsy Research by Thermo Fischer Scientific, Significant progress has been made in the development of new molecular methods to unlock the potential of peripheral monitoring using circulating tumour cells (CTCs) and cell-free DNA (cfDNA) from liquid biopsy samples. Advanced molecular profiling methods using next-generation sequencing (NGS) and digital PCR are enabling clinical researchers to accurately profile mutations of interest in blood samples. These methods may potentially impact our approach to monitoring therapy and recurrence in the future.