Circulating tumor cell
Circulating tumor cells (CTCs) are cells that have shed into the vasculature from a primary tumor and circulate in the bloodstream. CTCs thus constitute seeds for subsequent growth of additional tumors (metastasis) in vital distant organs, triggering a mechanism that is responsible for the vast majority of cancer-related deaths.
CTCs were observed for the first time in 1869 in the blood of a man with metastatic cancer by Thomas Ashworth, who postulated that “cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of origin of multiple tumours existing in the same person”. A thorough comparison of the morphology of the circulating cells to tumor cells from different lesions led Ashworth to conclude that “One thing is certain, that if they [CTC] came from an existing cancer structure, they must have passed through the greater part of the circulatory system to have arrived at the internal saphena vein of the sound leg”. Modern cancer research has demonstrated that CTCs derive from clones in the primary tumor, validating Ashworth's remarks. The significant efforts put into understanding the CTCs biological properties have demonstrated the critical role circulating tumor cells play in the metastatic spread of carcinoma. However, only recently it has been demonstrated that these circulating tumor cells reflect molecular features of cells within tumor masses. CTCs thus could be considered a “liquid biopsy” which reveals metastasis in action, providing live information about the patient’s disease status. Analysis of CTCs from blood samples could be an invaluable tool for early stage detection of cancer as well as for neoplastic progression and recurrence monitoring. Blood tests are easy and safe to perform and multiple samples can be taken over time. By contrast, analysis of solid tumors necessitates invasive procedures that might limit patient compliance. The ability to monitor disease progression over time could facilitate appropriate modification to a patient's therapy, potentially improving their prognosis and quality of life. To this end, technologies with the requisite sensitivity and reproducibility to detect CTCs in patients with metastatic disease have recently been developed.
Frequency of CTCs
The detection of CTCs may have important prognostic and therapeutic implications but because their numbers can be very small, these cells are not easily detected. It is estimated that among the cells that have detached from the primary tumor, only 0.01% can form metastases.
Circulating tumor cells are found in frequencies on the order of 1-10 CTC per mL of whole blood in patients with metastatic disease. For comparison, a mL of blood contains a few million white blood cells and a billion red blood cells, see figure 1. This low frequency, associated to difficulty of identifying cancerous cells, means that a key component of understanding CTCs biological properties rely on the development of technologies and approaches capable of isolating them in significant numbers (enrichment step) and preserving CTCs for further molecular, morphological and functional analysis. To date CTCs have been detected in several epithelial cancers (breast, prostate, lung, and colon) and clinical evidences indicate that patients with metastatic lesions are more likely to have CTCs isolated.
CTCs are usually captured from the vasculature by using specific antibodies able to recognize specific tumoral marker (usually EpCAM); however this approach is biased by the need for a sufficient expression of the selected protein on the cell surface, event necessary for the enrichment step. Moreover, since EpCAM and other proteins (e.g. cytokeratins) are not expressed in some tumors and can be down regulated during the epithelial to mesenchymal transition (EMT), new enrichment strategies are required.
First evidence indicates that CTC markers applied in human medicine are conserved in other species. Five of the more common markers including CK19 are also useful to detect CTC in the blood of dogs with malignant mammary tumors.
To date, a variety of research methods have been developed to isolate and enumerate CTC. The only U.S. Food and Drug Administration (FDA) cleared methodology for enumeration of CTC in whole blood is the CellSearch system. Extensive clinical testing done using this method shows that presence of CTC is a strong prognostic factor for overall survival in patients with metastatic breast, colorectal or prostate cancer, see figure 2 
This method is based on the use of iron nano-particles coated with a polymer layer carrying biotin analogues and conjugated with antibodies anti EpCAM for capturing CTCs, and on the use of an analyzer to take images of isolated cells upon their staining with specific fluorescent antibody conjugates. Blood is sampled in an EDTA tube with an added preservative. Upon arrival in the lab, 7.5mL of blood is centrifuged and placed in a preparation system. This system first enriches the tumor cells immunomagnetically by means of ferrofluid nano-particles and a magnet. Subsequently recovered cells are permeabilized and stained with a nuclear stain, a fluorescent antibody conjugate against CD45 (leukocyte marker), and cytokeratin 8, 18 and 19 (CKs). The sample is then scanned on an analyzer which takes images of the nuclear, cytokeratin, and CD45 stains. To be considered a CTC a cell must contain a nucleus, be positive for cytoplasmic expression of cytokeratin as well as negative for the expression of CD45 marker, and have a diameter larger than 5 µm. If the total number of tumor cells found to meet the criteria cited above is 5 or more, a blood sample is positive. In studies done on prostate, breast and colon cancer patients, median survival of metastatic patients with positive samples is about half the median survival of metastatic patients with negative samples. This system is characterized by a recovery capacity of 93% and a detection limit of one CTC per 7.5 mL of whole blood. Despite its sensitivity and reproducibility, the CellSearch Method requires specific equipment to perform the analysis.
Maintrac is a diagnostic platform applying microscopic methods to identify rare cells in body fluids and their molecular characteristics.
Concerning circulating tumor cells, maintrac is using an approach based on microscopic identification of circulating tumor cells. To prevent damage and loss of the cells during the process, maintrac uses just two steps towards the identification. In contrast to many other methods, maintrac does not purify the cells or enriches them, but identifies them within the context of the other blood compounds. To obtain vital cells and to reduce stress of those cells, blood cells are prepared by only one centrifugation step and erythrocyte lysis. Like CellSearch maintrac uses an EpCAM antibody. It is, however, not used for enrichment but rather as a fluorescent marker to identify those cells. Together with the nuclear staining with propidium iodide the maintrac method can distinguish between dead and living cells. Only vital, propidium excluding EpCAM positive cells are counted as potential tumor cells. Only living cells can grow into tumors, therefore dying EpCAM positive cells can do no harm. The suspension is analysed by fluorescence microscopy, which automatically counts the events. Simultaneous event galleries are recorded to verify whether the software found a true living cell and to differentiate between skin epithelial cells for example. Close validation of the method showed that additional antibodies of cytokeratins or CD45 did not have any advantage.
Unlike other methods maintrac does not use the single cell count as a prognostic marker, rather maintrac utilizes the dynamics of the cell count. Rising tumor cell numbers are an important factor that tumor activity is ongoing. Decreasing cell counts are a sign for a successful therapy.
Therefore maintrac can be used in following situations
Studies showed that under certain circumstances also EpCAM positive cells can be found in the blood. Inflammation diseases like Morbus Crohn or Colitits Ulcerosa also show increased levels of EpCAM positive cells. Patients with severe skin burns can also carry EpCAM positive cells in the blood, which may falsify results. So use of EpCAM positive cells as a tool for early diagnosis is not recommended.
CTCs are pivotal to understanding the biology of metastasis and promise potential as a biomarker to noninvasively evaluate tumor progression and response to treatment. However, isolation and characterization of CTCs represent a major technological challenge, since CTCs make up a minute number of the total cells in circulating blood, 1–10 CTCs per mL of whole blood compared to a few million white blood cells and a billion red blood cells. Therefore the major challenge for CTC researchers is the prevailing difficulty of CTC purification that allows the molecular characterization of CTCs. Several methods have been developed to isolate CTCs in the peripheral blood and essentially fall into two categories: biological methods and physical methods.
Biological methods are separation based on antigen-antibody bindings. Antibodies against tumor specific biomarkers including EpCAM, Her2, PSA are used. The most common technique is magnetic nanoparticle-based separation (immunomagnetic assay) as used in CellSearch or MACS. Other techniques under research include microfluidic separation and combination of immunomagnetic assay and microfluidic separation. Oncolytic viruses such as vacinia viruses are developed to detect and identify CTCs.
Physical methods are often filter-based, enabling the capture of CTCs by size. ScreenCell is a filtration based device that allows sensitive and specific isolation of CTCs from human whole blood in a few minutes. Peripheral blood is drawn and processed within 4 hours with a ScreenCell isolation device to capture CTCs. The captured cells are ready for cell culture or for direct characterization using ViewRNA in situ hybridization assay.
Characterization of CTC
Any useful method for isolation of CTCs must allow (i) their identification and enumeration and (ii) their characterization through immunocytochemistry, fluorescence in situ hybridization (FISH) DNA and RNA assays, and all other relevant molecular techniques using DNA and RNA. When circulating tumor cells are captured from blood using filtration devices (such as ScreenCell isolation device), further morphological and molecular characterization is required to reveal important predictive information and report changes in CTC biology, for example during tumor relapse. ViewRNA assay for CTCs characterization is the only in situ hybridization technology that allows multiplex, single-molecule RNA detection of any RNA target. The exceptional sensitivity and specificity is achieved by using proprietary probe design, simultaneous branched DNA (bDNA) signal amplification and background suppression.
The captured CTCs on the filter membrane of a ScreenCell isolation device, are transferred to a 24-well cell culture plate for enumeration/characterization using ViewRNA ISH Cell Assay. A target-specific probe set containing 20 oligonucleotide pairs hybridizes to the target RNA. An oligo pair hybridization event is essential for support of the signal amplification structure, which is assembled by a series of sequential hybridization steps. Each fully assembled amplification structure is contained within 40−50 bp of target RNA with the capacity for 400-fold signal amplification. Therefore, a typical target-specific probe set (containing 20 oligo pairs) can generate 8,000-fold signal amplification at the location of the target RNA.
Further Characterisation of CTC
Some drugs are particularly effective against cancers which fit certain requirements. For example Herceptin is very effective in patients who are Her2 positive, but much less effective in patients who are Her2 negative. Once the primary tumor is removed, biopsy of the current state of the cancer through traditional tissue typing is not possible anymore. Often tissue sections of the primary tumor, removed years prior, are used to do the typing. Further characterisation of CTC may help determining the current tumor phenotype. FISH assays has been performed on CTC to as well as determination of IGF-1R, Her2, Bcl-2, [ERG (gene)|ERG], PTEN, AR status using immunofluorescence.
Morphological appearance is judged by human operators and is therefore subject to large inter operator variation. Several CTC enumeration methods exist which use morphological appearance to identify CTC, which may also apply different morphological criteria. A recent study in prostate cancer showed that many different morphological definitions of circulating tumor cells have similar prognostic value, even though the absolute number of cells found in patients and normal donors varied by more than a decade between different morphological definitions.
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