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Murray Primary circulating prostate cells

Circulating tumor cells are larger than circulating blood enrichment methods. [72]
cells; filtration methods are based on the physical
properties of these cells and allow enrichment by Negative enrichment methods that deplete normal
size. Isolation of circulating tumor cells was first blood cells using the pan-leukocyte antigen CD45 after
reported in 1964. The filters use pores measuring red cell lysis have also been used. [73]
[65]
between 7.5-8.0 µm in diameter, thus capturing 85-
100% of circulating tumor cells while retaining only Detection of circulating tumor cells
0.1% of circulating blood cells. Three commercially For the detection of enriched circulating tumor cells,
[66]
available filters are available: Screencell Cyto, ISET , two methods have been used: immunocytochemistry
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and Metacell . After filtration the filter membrane is and reverse transcriptase-polymerase chain reaction
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removed and circulating tumor cells are identified (RT-PCR).
by immunocytochemistry. Isolation of tumor cells by
size is fast, simple, and reliable and does not require Immunocytochemistry
high-cost instrumentation. One drawback, though, is The advantage of methods using immunocytochemistry
the need to process samples within four hours. The is the morphological analysis of the detected cells.
system does not detect the rare cells that are smaller The International Society of Hematotherapy and
[74]
than 8 µm; however, it will detect tumor cell clusters. Graft Engineering criteria for circulating tumor cell
The ISET system detects one tumor cell in 1 mL of identification are an object with the appearance of cell
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peripheral blood and permits the evaluation of tumor with a nucleus. Most methods use a combination of
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cells based on morphological criteria. False positivity markers; the CellSearch system defines a circulating
occurs due to the lack of specificity of the enrichment tumor cell as one positive for cytokeratin, negative
technique. Normal epithelial or endothelial cells may for the pan-leukocyte antigen CD45, and expressing
be present due to coring by the sampling needle, and DAPI (4´, 6-diamidino-2-phenylindole) nuclear
staining. The ISET and Metacell systems use anti-
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circulating cells have been described in samples taken cytokeratin staining, while the CTC membrane micro-
from patients with benign conditions. [67,68]
filter, Rosettesep and Nanovelcro CTC Chip , use
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immunofluorescence with a cocktail of anti-EpCAM,
Immunomagnetic selection methods use the specificity anti-cytokeratin, and CD45. All these methods in
of antibody-antigen interactions combined with the essence detect circulating epithelial cells and are not
physical properties of magnetic beads to separate tissue specific. Using basic cell density methods, some
tumor cells from blood cells due to the different authors have attempted to use more specific markers
expression of surface antigens in the differing cell to detect circulating tumor cells, anti-PSA for prostate
populations. This is the basis of enrichment in the cancer, anti-mammoglobin for breast cancer.
[75]
[76]
CellSearch system, the only FDA-approved method As such, these methods are not able to differentiate
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of detecting circulating tumor cells. In the CellSearch between benign and malignant circulating “epithelial”
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system, iron particles are coated with the epithelial cell cells. In patients with benign colonic diseases, up to 29%
surface marker EpCAM, an epithelial marker that is of patients were positive for the Epispot assay, and up
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overexpressed in some cancers but not in normal blood to 19% of patients were positive for the CellSearch
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cells. However, EpCAM positive cells have been assay. One group has used the combination anti-
[69]
[70]
reported in patients with benign colon disease, and PSA and anti-P504S to address this problem. The
[70]
in the original report of Allard et al., women without expression of P504S has been used to differentiate
[69]
evidence of breast cancer had “circulating tumor cells” between benign and malignant prostate tissues in
detected in between 5 and 7% of cases, 1 cell/7.5 mL biopsy samples. P504S is expressed in prostate cancer
blood sample. In addition, the epithelial phenotype cells and those of prostate intra-epithelial neoplasia,
of circulating tumor cells changes, as a result of the but not in benign prostatic tissue. [77,78] The authors
epithelial to mesenchyme transition the expression of report that PSA positive cells can be detected in men
EpCAM decreases and thus there may be failure of with benign prostatic disease, especially prostatitis,
enrichment and as a result circulating tumor cells are but these cells are P504S negative, whereas men
not detected. This applies also to microchip devices with prostate cancer had PSA positive cells which also
that incorporate microposts labeled with anti-EpCAM expressed P504S. [79]
(CTC Chip), using EpCAM coated beads (Dynabeads
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Epithelial enriched)(MACS/auto MACS )(AdnaTest ) In reference to circulating cell clusters, the identification
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or using microvortices in a herringbone pattern to of CTC clusters (defined as ≥ 2 CTCs) has been related
increase the number of interactions between the o poor outcome in stage III-IV breast cancer using the
EpCAM-coated chip surface and circulating tumor CellSearch system, whereas Paoletti et al. defined
[81]
[80]
cells. The same can be said for cytokeratin-based CTC clusters as ≥ 3 CTCs in the CellSearch gallery
[71]
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ December 16, 2016 457