Circulating+Tumor+Cells+and+Cancer+Survival

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=An Introduction to Circulating Tumor Cells=

Overview of CTCs
One of the most dangerous capabilities of cancer is its ability to metastasize and spread to distant tissues. At some point in the cancer’s development, the primary tumor will launch seed cells that will invade adjacent tissues and start new colonies. Circulating tumor cells, often abbreviated as CTCs, are these cells that have arisen from both the initial and metastatic tumor sites that have been released into the bloodstream. In order to successfully invade and survive in other tissues, these seed cells need to not only gain the capability to detach from the tumor, but also adapt to the new environments which they tether themselves to. However, the development of these adaptations have made these seed cells enormously complex. This is further convoluted by CTC heterogeneity, which can cause CTCs to differ between different cancers. Other than their general function as seed cells, there is no single parameter such as size, cytomorphology, or biomarker that is considered essential for defining a CTC. CTC properties have been shown to differ across cancer type. Currently, the most popular method for discerning CTCs is the CellSearch system, which utilizes epithelial cell adhesion molecules (EpCAM) that are embedded to the membranes of CTCs, but not to those of other cells common to the blood stream. Due to measuring EpCAM expression, CellSearch is only effective on breast, prostate, and colon cancer. This greatly limits CTC studies on other cancer types.Therefore despite the danger that this process poses, metastasis is still incompletely understood. Nonetheless, current research into the subject has brought up some startling implications, the most relevant of which being the possible correlation between their detection and unfavorable clinical outcome. Because cancer deaths are largely attributed to cancer’s metastasizing ability, analysis of circulating tumor cells in order to determine cancer progression has become a prominent area dedicated to monitoring cancer progression. By utilizing the relationship between the number of CTCs in the bloodstream, it may be possible to predict patient response to therapy as well as the overall chance of survival. Furthermore because CTCs can be collected by blood draw, they would provide a noninvasive means to analyze cancer. As cancer continues to be a prevalent issue in today's society, new and more efficient manners of analyzing and monitoring cancer growth are being introduced with the hopes of increasing detection accuracy and minimizing the side effects of more invasive procedures. (van de Stolpe, et al. 2011)

CTC Production
While many studies have found that the large production of CTCs is a strong indicator of cancer fatality, it does not imply the same for tumor size. Nonetheless, there are indications that CTCs can predict some degree of tumor growth. There is little information as to when exactly CTCs are expressed during a tumor’s development. One study in particular purported that in 83% of mice inoculated with orthopedic breast cancer and 35% inoculated with melanoma, the highest CTC rate of production was detected while the primary tumor was still small. Furthermore, this maximum rate appeared to be connected to a subsequent increase in tumor size. After the tumor had grown large, the number of CTCs then appeared to greatly decrease. However in a few tests, a second peak appeared in a few tests even after the tumor had grown large, but this peak was smaller than the earlier one.The results from several of these tests are displayed in Figure 2. The appearance of this phenomenon suggests that the production of CTCs is in some ways an indicator of periods of cancer growth. However, it is not a strictly linear relationship. A large increase in CTC production apparently occurs just before progressive tumor growth. The subsequent smaller peaks would therefore be indicative of continued growth over time. This would make sense as the increase in cell division due to growth would also increase the number of CTCs produced and released. Furthermore, the appearance of these traits in two different tumor types suggests that they are general characters for metastatic disease. The differences in expressiveness from mouse to mouse as well as across different cancer types (35% for melanoma and 83% for breast) suggest the affect of tumor type and host environment on the data. However, this study only been tested these claims in inoculated mice. Endogenous human testing needs to be performed in order to properly verify these claims. (Juratli, et al. 2014)

=CTC Analysis=

Favorable and Unfavorable CTC Levels
Due to the relative novelty of CTC studies, there is a lack of general consensus on what constitutes dangerous levels of CTC concentration in the bloodstream. Therefore in order to properly analyze the effects of CTCs, most studies must first determine at what levels the patient would be most at risk of having a complication. Once determined, this level is labeled the cutoff value. In general, studies must set aside a statistically relevant portion of their samples in order to estimate this value. They must then validate that it is replicable for all other patients through subsequent tests. This cutoff value is estimated by determining at what concentration of CTCs the hazard ratio significantly increases. A hazard ratio is a description of the relative risk of a complication occurring with or without a specific event. In CTC studies the event would refer to CTC concentration while the complication could be death or cancer progression. However, the identity of these variables is the choice of the researchers. Once a cutoff value has been determined, it is then used to mark the change between high and low CTC counts. (More information on hazard ratios can be found here).

The methodology utilized to study CTCs is exemplified in Figure 3. A study was designed with about 163 breast cancer patients. The CellSearch and CellSpotter Systems were used to identify, isolate, and enumerate CTCs from patient blood draws, which were taken at the first follow-up visit after the initiation of a new line of therapy. The experimenters utilized 95 patients of these patients, labeled as the training set, in order to determine the cutoff value. The ultimate purpose of this cutoff value was to demonstrate that at greater concentrations of CTCs, there would be a significant decrease in the percentage of patient survival. The remaining 68 patients were grouped into the validation set in order to show that the results from the training set are replicable. The experimenters found that the two data sets had similar mean probabilities for progression-free survival and that they also had similar mean probabilities for overall survival. Furthermore, the shapes of each curve were similar for each corresponding graph. Therefore, they concluded that the two sets of data were nearly identical. With this conclusion, they combined the two data sets into one full set. This full set represented the total sum of patients from which they would draw their conclusions concerning the nature of the CTC. (Cristofanilli, et al. 2004)

The cutoff value is variable to the unique conditions attributed to each test. For example, the malleability in cutoff value due to the change in cancer type is exemplified by the study shown in Figure 4. While the study shows that both breast and prostate cancer have a cutoff value of 5 CTCs per 7.5 mL of blood, it also displays that colorectal cancer has a cutoff value of 3 CTCs per 7.5 mL of blood. Thus the value for severe CTC concentration is different for different cancer types. Nonetheless, the cutoff value could change for the same cancer as long as it is across different tests. Despite the similarities shown in Figures 3 and 4 for breast cancer cutoff values, the tests used to obtain these values are different due to being in different studies. Therefore, it is not assumed that 5 CTCs per 7.5 mL of blood is a universal cutoff value for breast cancer. Instead this value is only true for the studies it was calculated for.

CTCs and Patient Survivability
One of the most discussed topics of CTC research is the hypothesized negative correlation between CTC number and patient survivability. As displayed in Figures 3, 4 and 5, this relationship has been noted across numerous cancer types and appears not only in overall survival, but also in progression free survival. Patients that present favorable levels of CTCs are shown to be significantly more likely to survive than patients with unfavorable levels. This is also apparent after treatment, such that a shift in CTC number can illustrate the effectiveness of the treatment as illustrated by the [|study]displayed in Figures 4 and 5. In this particular study, blood samples were taken from 177 patients with metastatic breast cancer, 413 patients with metastatic colorectal cancer, and 218 patients with metastatic prostate cancer. A sample was taken both before treatment and on the follow up visit after treatment. For all three cancer types, the median probability for overall survival before treatment was shown to be twice as high for patients with favorable CTC levels than it was for patients with unfavorable levels. After treatment, the patients were divided into four groups: CTCs that remained favorable, CTCs that changed from favorable to unfavorable, CTCs that changed from unfavorable to favorable, and CTCs that remained unfavorable. Patients that remained at favorable levels or that had converted to favorable levels both expressed higher probabilities of overall survival than patients who had either remained unfavorable or had converted to unfavorable levels. Therefore it would appear that survival is not only negatively correlated to the CTC level, but that it can be used to detect treatment effectiveness. (Miller, et al. 2010)



Factors which Distort CTC Measurement
It is important to realize that there multiple biological and physical factors within the organism that can lead to inaccurate representations of CTC concentration the bloodstream. Because these factors can affect CTC count, they could also cause a misrepresentation of the patient's state. Vicki Plaks' article "Circulating Tumor Cells" in particular discusses these factors. The most pertinent of these issues are filtration, clustering, cloaking, benign circulating epithelial cells, heterogeneity, presence of circulating stem cells, epithelial-mesenchymal transitions, and the seeding potential. Other issues involve general detection limitation. Because there are very few CTCs present in the bloodstream and their study, the current detection methods, such as Cell Search can be somewhat limited and inaccurate. This is especially true on CTCs that do not carry EpCAM markers. This is somewhat mitigated by the research into other identifiers, such as genomic markers. However these new methods are also riddled with measurement inaccuracies because of their novelty.

=Conclusions=

As expected, there appears to be a connection between the concentration of CTCs in the bloodstream and the survivability of the patient, a correlation that appears to persist even throughout treatment. Multiple studies have found that once a patient passes a certain threshold for CTC concentration, his/her chances of survival severely decrease. Furthermore, this has also been observed to persist after treatment, such that the change in the number of CTCs can be used to evaluate the effectiveness of the treatment. If the CTC concentration rises above the established cutoff value, then the treatment is ineffective and if the CTC concentration decreases below this value, the treatment is effective. However there are several notable issues with the study of CTCs. The presence of a spike in CTC production during the earlier stages of tumor growth somewhat implies a connection between growth and cancer survivability. Because CTCs experience their greatest period of production immediately before progressive tumor growth and because the number of CTCs is also negatively correlated to the chances of survival, it can be hypothesized that there may be a connection between continued tumor growth and survivability as well. This would make sense as the increase in tumor growth may signify greater tumor intensity. Furthermore, the increase in tumor size would mean a greater degree of cellular division, which in turn would imply the greater production of CTCs. Because more CTCs are being produced, there is a greater opportunity for the cancer to metastasize, which would decrease the chances of survival. However, while a rise in CTC production has been noted to reflect an increase in tumor growth, the reverse is not necessarily true. Therefore, more research is needed in order to verify this conclusion. On another note, it is important to regard that there is still so much that is unknown about CTCs as well as factors which can meddle with our observations of them. Nonetheless, the relatively consistent data indicates that CTC analysis is a promising area that could lead to easier methods in monitoring cancer. Furthermore, CTCs remain relevant because relatively cheap and easy to measure in that that they do not use a largely invasive procedure.

=Glossary=

=Sources=

1.) Cristofanilli, Massimo, G. Thomas Budd, Matthew J. Ellis, Alison Stopeck, Jeri Matera, M. Craig Miller, James M. Reuben, Gerald V. Doyle, W. Jeffrey Allard, Leon W.M.M. Terstappen, and F. Hayes. "Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer." New England Journal of Medicine 351.8 (2004): 781-791. Web. 18 May 2014.

2.) Jin, Hongchuan, Yanning Ma, Qi Shen and Xian Wang (2012). Circulating Methylated DNA as Biomarkers for Cancer Detection, Methylation - From DNA, RNA and Histones to Diseases and Treatment, Prof. Anica Dricu (Ed.)

3.) Juratli, Mazen A., Mustafa Sarimollaoglu, Dmitry A. Nedosekin, Alexander V. Melerzanov, Vladimir P. Zharov, and Ekaterina I. Galanzha. “Dynamic Fluctuation of Circulating Tumor Cells during Cancer Progression.” Cancers. 6.1 (2014): 128-142. Web. 27 May 2014.

4.) Kalluri, Raghu, and Robert A. Weinberg. "The Basics of Epithelial-mesenchymal Transition." //Journal of Clinical Investigation// 120.5 (2010): 1786. Web. 4 June 2014.

5.) Miller, M. Craig, Gerald V. Doyle, and Leon W. M. M. Terstappen, “Significance of Circulating Tumor Cells Detected by the CellSearch System in Patients with Metastatic Breast Colorectal and Prostate Cancer,” Journal of Oncology, (2010):1-8, May 2014.

6.) Plaks, V., C. D. Koopman, and Z. Werb. "Circulating Tumor Cells." Science 341.6151 (2013): 1186-188. 13 Sept. 2013. Web. 19 May 2014.

7.) van de Stolpe, AnjaKlaus Pantel, Stefan Sleijfer, Leon W. Terstappen, and Jaap M.J. den Toonder. “Circulating Tumor Cell Isolation and Diagnostics: Toward Routine Clinical Use”. Cancer Research 71.18 (2011): 5955–5960. Web.