Upon tumor cell death (via necrosis or apoptosis) occurring as a consequence of cellular turnover, small fragments of DNA, known as circulating tumor DNA (ctDNA) are released into the bloodstream. Solid tumor masses can also shed cancer cells into the bloodstream. Those cells are known as circulating tumor cells (CTCs). The term Liquid Biopsy refers to body fluid sampling (blood, saliva, urine, etc.) and analysis of ctDNA and/or CTCs. Analysis of ctDNA and CTCs extracted from a peripheral blood sample enables cancer detection.

There is growing evidence that the genetic alterations detected in liquid biopsy reflect in most cases the same mutational pattern identified through traditional tumor biopsy at the primary tumor site. A liquid biopsy can therefore be used to provide an accurate "snapshot" of the tumor genomic profile, in order to monitor the progression of the tumor itself, its response to therapy and the emergence of resistance to therapy. Further applications of liquid biopsies involve early screening for malignant tumors as well as development of personalized therapies.

Thanks to the introduction of molecular diagnostic techniques, cancer has been identified as a heterogeneous clinical entity. Each primary tumor site can comprise several tumor subtypes, each one presenting a specific genetic signature and clinical features. Metastatic lesions can also diverge from the primary tumor type in terms of molecular and clinical features. The DNA released into the bloodstream by apoptotic and necrotic tumor cells can give information related not only to the genetic profile of the primary tumor (and its heterogeneity), as is the case of traditional biopsy (tissue DNA), but also to metastatic lesions.

Solid tumors are often asymptomatic and clinically undetectable until they reach a critical mass (usually 1-2 cm in diameter). However, smaller lesions (e.g. less than 1cm) are thought to be capable of releasing circulating cell-free DNA (cfDNA) into the bloodstream. DNA released by cancer cells into the bloodstream is called circulating tumor DNA (ctDNA). The tumor mass can also shed cancer cells into the bloodstream, which are defined as Circulating Tumor Cells (CTCs). The study of circulating nucleic acids has evolved considerably in the last years. Circulating free DNA is a "naked" DNA that is not contained within a cell membrane, is fragmented, is present also in healthy subjects because of normal tissue turnover and is quantitatively very variable.
ctDNA is the portion of circulating free DNA (cfDNA) resulting from cancer cells. The fragments are on average 150-180bp long and have a high prevalence of mutations linked to cancer. Several studies have demonstrated that ctDNA from plasma can be used as a tumor biomarker since its quantity increases in cancer patients, its length is different from the characteristic fragments of healthy subjects and it can present the same genetic alterations of the primary tumor lesion.

The detection of genetic alterations can give information on cancer evolution and on the response to treatment. State of the art DNA sequencing technologies have led to the discovery of new cancer-associated genetic mutations that may have prognostic value and may guide the design of targeted therapeutic treatments and monitoring programs. ctDNA analysis is of considerable interest for personalized medicine for several reasons: it allows rapid and sensitive genetic diagnosis, it enables monitoring of therapy response and it is based on minimally invasive and repeatable sampling procedures.

Next Generation Sequencing, also known as High-throughput Sequencing, is a set of different modern sequencing techniques that allow DNA sequencing in a much faster and less expensive way compared to the classic method (called Sanger sequencing method). The resulting increase in number of sequences of human genomes available has revolutionized genomics and molecular biology. Not only it is now possible to sequence, in a short time, ctDNA and DNA molecules derived from CTCs, but it is also possible to obtain a large amount of information by their sequencing.

Cancer cells that detach from a solid tumor mass and are detected in the bloodstream are referred to as Circulating Tumor Cells (CTCs). CTCs have the ability to extravasate from blood circulation and seed metastases at distant sites. The detection and analysis of CTCs may constitute a non-invasive procedure to monitor response to therapy, as well as to stratify patients before treatment. CTCs in cancer patients are present in the blood at low frequency, usually from 0 to 10 cells per ml of whole blood.

The DNA contained within the cells bears the hereditary information(i.e. the genes), which controls all cellular functions including growth, cell division and programmed cell death (apoptosis). Cancer cells bear DNA alterations that lead to uncontrolled growth. Initially, cancer cells multiply and proliferate locally, but when the tumor becomes bigger and acquires additional genetic alterations, they might migrate to distant sites leading to disease progression and metastasis formation.

With specialized technology, it is now possible to isolate CTCs from the peripheral blood of cancer patients and determine their genetic profile. This can reveal the insurgence of drug resistance mechanisms and guide the selection of an appropriate therapy. This information may also influence decisions about an early administration of therapy or supportive treatments. The genomic profiling of CTCs may provide guidelines on the most effective drug targets and identify genotypes known as drug-resistant. Features of CTCs:

• CTCs are cells detached from the primary tumor and may cause metastasis
• A high number of CTCs correlates with adverse prognosis and short survival in many cancer types
• Can provide a molecular profile of the tumor of origin
• Immunological profile: EpCAM+, Cytokeratin+, CD45- , intact nucleus

Efficient isolation of extremely rare cells such as CTC requires a technology that satisfy various requirements:

• Maximum recovery of labelled target cells
• Minimum non-specific binding of non-target cells to device surfaces
• Minimum loss of target cells during recovery
• Easy recovery of small numbers of target cells for additional analysis

Bioscience has an automated platform for liquid biopsy analysis which allows the separation and recovery of rare cells starting from whole blood samples. This system is able to recover up to 1 CTC per ml of blood (~ 1 cell in 7x106 white blood cells per ml) with the purity necessary for subsequent molecular characterization.