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Old 07-02-2008, 08:04 PM
Dross Dross is offline
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Default Circulating tumor cells can reveal genetic signature of dangerous lung cancers

Massachusetts General Hospital (MGH) investigators have shown that an MGH-developed, microchip-based device that detects and analyzes tumor cells in the bloodstream can be used to determine the genetic signature of lung tumors, allowing identification of those appropriate for targeted treatment and monitoring genetic changes that occur during therapy. A pilot study of the device called the CTC-chip will appear in the July 24 New England Journal of Medicine.

"The CTC-chip opens up a whole new field of studying tumors in real time," says Daniel Haber, MD, director of the MGH Cancer Center and the study's senior author. "When the device is ready for larger clinical trials, it should give us new options for measuring treatment response, defining prognostic and predictive measures, and studying the biology of blood-borne metastasis, which is the primary method by which cancer spreads and becomes lethal."

CTCs or circulating tumor cells are living solid-tumor cells found at extremely low levels in the bloodstream. Until the development of the CTC-chip by researchers from the MGH Cancer Center and BioMEMS (BioMicroElectroMechanical Systems) Resource Center, it was not possible to get information from CTCs that would be useful for clinical decision-making. The current study was designed to find whether the device could go beyond detecting CTCs to helping analyze the genetic mutations that can make a tumor sensitive to treatment with targeted therapy drugs.

The researchers tested blood samples from patients with non-small-cell lung cancer (NSCLC), the leading cause of cancer death in the U.S. In 2004, MGH researchers and a team from Dana-Farber Cancer Institute both discovered that mutations in a protein called EGFR determine whether NSCLC tumors respond to a group of drugs called TKIs, which includes Iressa and Tarceva. Although the response of sensitive tumors to those drugs can be swift and dramatic, eventually many tumors become resistant to the drugs and resume growing.

The CTC-chip was used to analyze blood samples from 27 patients, 23 who had EGFR mutations and 4 who did not, and CTCs were identified in samples from all patients. Genetic analysis of CTCs from mutation-positive tumors detected those mutations 92 percent of the time. In addition to the primary mutation that leads to initial tumor development and TKI sensitivity, the CTC-chip also detected a secondary mutation associated with treatment resistance in some participants, including those whose tumors originally responded to treatment but later resumed growing.

"Patients found to have resistance mutations before treatment probably won't benefit as much or as long from single-agent TKI therapy as those without such baseline mutations," says Lecia Sequist, MD, MPH, of the MGH Cancer Center, a co-lead author of the NEJM paper. "For those patients we may need to consider other modes of therapy, including combinations of targeting agents or second-generation TKIs that can overcome the most common resistance mutation."

Blood samples were taken at regular intervals during the course of treatment from four patients with mutation-positive tumors. In all of those patients, levels of CTCs dropped sharply after TKI treatment began and began rising when tumors resumed growing. In one patient, adding additional chemotherapy caused CTC levels to drop again as the tumor continued shrinking.

Throughout the course of therapy, the tumors' genetic makeup continued to evolve. Not only did the most common resistance mutation emerge in tumors where it was not initially present, but new activating mutations, the type that causes a tumor to develop in the first place, appeared in seven patients' tumors, indicating that these cancers are more genetically complex than expected and that continuing to monitor tumor genotype throughout the course of treatment may be crucial.

"If tumor genotypes don't remain static during therapy, it's essential to know exactly what you're treating at the time you are treating it," says Haber. "Biopsy samples taken at the time of diagnosis can never tell us about changes emerging during therapy or genotypic differences that may occur in different sites of the original tumor, but the CTC-chip offers the promise of noninvasive continuous monitoring." Haber is the Kurt J. Isselbacher/Peter D. Schwartz Professor of Medicine at Harvard Medical School.

Last edited by gdpawel : 04-15-2012 at 12:29 AM. Reason: post full article
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Old 07-04-2008, 04:41 AM
gdpawel gdpawel is offline
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Default Determining Genetic Signatures

The interest in and the knowledge of gene expression profiling in cancer medicine has heighten since the completion of the human genome project (the world's most expensive telephone book*). However, researchers have cautioned the science of gene expression profiling, in which scientists examine the genetic signature of a cell.

The gene chip is a device that measures differences in gene sequence, gene expression or protein expression in biological samples. It may be used to compare gene or protein expression under different conditions, such as cells found in cancer.

Hence the headlong rush to develop tests to identify molecular predisposing mechansims whose presence still does not guarantee that a drug will be effective for an individual patient. Nor can they, for any patient or even large group of patients, discriminate the potential for clinical activity among different agents of the same class.

The challenge is to identify which patients the targeted treatment will be most effective. Tumors can become resistant to a targeted treatment, or the drug no longer works, even if it has previously been effective in shrinking a tumor. Drugs are combined with existing ones to target the tumor more effectively. Most cancers cannot be effectively treated with targeted drugs alone.

What is needed is to measure the net effect of all processes within the cancer, acting with and against each other in real time, and test living cells actually exposed to drugs and drug combinations of interest. The key to understanding the genome is understanding how cells work. How is the cell being killed regardless of the mechanism?

The core understanding is the cell, composed of hundreds of complex molecules that regulate the pathways necessary for vital cellular functions. If a targeted drug could perturb any of these pathways, it is important to examine the effects of drug combinations within the context of the cell. Both genomics and proeomics can identify potential therapeutic targets, but these targets require the determination of cellular endpoints.

Eur J Clin Invest, Volume 37(suppl. 1):60, April 2007
BMJ 2007;334(suppl 1):s18 (6 January), doi:10.1136/bmj.39034.719942.94

* The sequencing of the entire human genome gave us the address and the next door neighbors of every human gene, yet we don't know what they do, how they do it, why they do it, or who they do it with. - Dr. Robert A. Nagourney

Last edited by gdpawel : 04-25-2013 at 10:51 AM. Reason: additional info
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Old 01-22-2011, 11:05 AM
gdpawel gdpawel is offline
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Default Circulating Cells: Helpful for Cancer Patients or Just Interesting?

Emile E. E. Voest
Department of Medical Oncology
UMC Utrecht, the Netherlands


In the era of targeted therapy a multitude of new agents to treat cancer is developed. Unfortunately only 5% of these agents will ultimately be approved for clinical use. One of the reasons for this high failure rate is our inability to select patients for the appropriate therapy. The potential recurrence rate of an individual tumor is relatively well defined by prognostic factors, however, our tools to predict response to therapy are very limited. Developing predictive markers to assess which patients will benefit from treatment are therefore highly needed. This educational session will be devoted to circulating biomarkers. In this presentation I will focus on circulating cells as potential markers for treatment response. Several circulating cell types currently under investigation: circulating tumor cells; circulating (endothelial) progenitor cells (C(E)PC); and circulating endothelial cells (CTC), the value of measuring these cells will be discussed in detail below.


Circulating Tumor Cells

Of all surrogate tumor tissues, CTCs have probably received the greatest attention the last years (1–8). It is becoming increasingly clear that the number, and change in number, of CTCs is prognostic for several types of cancer, including breast, colorectal- and prostate cancer (4–7) In the NIH clinical trials database, currently 298 trials are listed that measure CTC and correlate these measurements with treatment outcome. Few of these trials prospectively uses CTC to make treatment decisions. Now that CTC detection techniques have significantly improved and proper logistics for CTCs have become implemented in trials, a feasible, new goal is to characterize CTCs and to study specific molecular targets on CTCs (8). However, several limitations should be taken into consideration. A substantial percentage of patients have no detectable CTCs. Furthermore, CTCs may serve as surrogate tissue but may not be representative for real tumor tissue. CTCs may represent a subset of tumor cells. Next to this, EpCAM-based CTC detection may cause a bias for cells that have a low or no EpCAM expression.

CTCs have a clear potential as pharmacodynamic biomarker in early oncology trials. Potential applications of measuring actual target modulation are, for example, to provide proof of mechanism of action of the drug and to study the biologically active dose range. With the availability of pharmacodynamic assays for growth hormone receptors on CTCs, opportunities arise in monitoring of activating- or resistance-conferring mutations and measuring change in activity of down-stream signaling molecules intracellularly that can indicate the level of inhibitory activity of the drug. The development of new techniques that improve CTC detection sensitivity allows for increasing sensitivity in subsequent characterization. These advanced techniques may enable further specified CTC analysis which could lead to a more personalized therapy for the patient in the future. In summary, there are many interesting and encouraging developments in the field of CTC detection and their characterization that may lead to further development and incorporation of CTCs as pharmacodynamic biomarker in early clinical trials of targeted anti-cancer therapy.

Circulating Endothelial (Progenitor) Cells

In addition to CTC, circulating normal cells may also predict tumor progression or host responses to treatment. The best studied cells are circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC). The relevance of EPCs in cancer growth suggests that EPCs might be used as a surrogate marker for angiogenic activity (9–12). Both circulating mature endothelial cells (CECs) and endothelial progenitor cells (EPCs) are increased in the blood of cancer patients and correlate with angiogenesis and tumor volume. Therefore these cells might serve as a biomarker to determine prognosis, response to therapy and the optimal biological dose (OBD) of anti-angiogenic agents.

CEC levels correlate with progressive disease, as patients with growing tumors have higher CEC levels compared to patients with stable disease. Conversely, CEC levels return to normal after successful treatment. This suggests that CECs correlate with the presence and the activity of a tumor and indicates that CECs hold the potential to measure changes in disease activity and therefore response to therapy. Clinically this has been investigated in patients with metastatic breast cancer treated with low dose metronomic chemotherapy. In these patients the CEC count after 2 months of continuous therapy could predict both disease-free and overall-survival after a prolonged follow-up of more than 2 years. Others showed that high baseline levels of CECs predicted response to metronomic chemotherapy combined with bevacizumab. We showed that CEC and EPC were increased in the blood of cancer patients after treatment with various chemotherapeutic regimens. The increase in CEC and EPC is seemingly unrelated to the presence of a tumour since adjuvant chemotherapy showed similar kinetics. This suggests that EPC and CEC release after chemotherapy is part of a reactive host response independent of tumor type and chemotherapy regimen. This response may very well be an important factor in determining the outcome of patients, as EPC and CEC have been found to stimulate tumour growth, metastasis formation and limit chemotherapeutic efficacy by prevention of necrosis. The magnitude of the increase of CEC and EPC after chemotherapy was associated not only with response to chemotherapy after 3 cycles but also with PFS and OS. This correlation between CEC/EPC and prognosis of patients is supported by other studies (13, 14). There are several limitations to take into account. EPC and CEC detection techniques are labor intensive, time consuming, often require fresh samples and the number of circulating cells are commonly very low.

In summary, circulating EPC and CEC are biologically interesting but presently the detection techniques and inter- and intrapatient variability prohibit wide spread use of these cells in routine clinical care.

Future Directions: Can We Use Circulating Cells in Clinical Decision Making?

The above described studies have greatly contributed to our understanding of the biology of cancer. Measurement of these cells has clearly prognostic value. It furthermore indicates avenues to further refine specific assays to use circulating cells as biomarkers. However, the data are presently insufficient to consider circulating cells to predict outcome of treatment in such a manner that anti-cancer treatment can be started or even more important stopped. Given the response rates of current anti-cancer treatment and the willingness of patients to undergo treatment even for relatively low success percentages imposes high sensitivity and specificity requirements on potential predictive tests. Presently, none of these circulating cell assays fulfil these requirements but the enormous potential of these circulating cells as pharmacodynamic markers deserves prospective clinical trials to further assess their value.


1. Stebbing J, Jiao LR. Circulating tumour cells as more than prognostic markers. Lancet Oncol 2009; 10: 1138–9.

2. Mostert B, Sleijfer S, Foekens JA, Gratama JW. Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer. Cancer Treat Rev 2009; 35: 463–74.

3. Dotan E, Cohen SJ, Alpaugh KR, Meropol NJ. Circulating tumor cells: evolving evidence and future challenges. Oncologist 2009; 14: 1070–82.

4. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004; 351: 781–91.

5. Hayes DF, Cristofanilli M, Budd GT, et al. Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 2006; 12: 4218–24.

6. Cohen SJ, Punt CJ, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26: 3213–21.

7. de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 2008; 14: 6302–9.

8. Lianidou ES, Mavroudis D, Sotiropoulou G, Agelaki S, Pantel K. What's new on circulating tumor cells? A meeting report. Breast Cancer Res 2010; 12: 307.

9. Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science 2008; 319: 195.

10. Kaplan RN, Riba RD, Zacharoulis S, et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 2005; 438: 820.

11. Shaked Y, Ciarrocchi A, Franco M, et al. Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors. Science 2006; 313: 1785.

12. Shaked Y, Henke E, Roodhart JM, et al. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 2008; 14: 263.

13. Roodhart JM, Langenberg MH, Vermaat JS, et al. Late release of circulating endothelial cells and endothelial progenitor cells after chemotherapy predicts response and survival in cancer patients. Neoplasia 2010; 12: 87–94.

14. Roodhart JM, Langenberg MH, Daenen LG, Voest EE. Translating preclincal findings of (endothelial) progenitor cell mobilization into the clinic; from bedside to bench and back. BBA–Reviews on Cancer, 2009; 1796: 41–9.

Gregory D. Pawelski

Last edited by gdpawel : 01-20-2013 at 11:37 PM. Reason: corrected url address
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Old 02-19-2011, 09:46 AM
gdpawel gdpawel is offline
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Default Heterogeneous populations of circulating tumor cells

The cells that slough off from a cancerous tumor into the bloodstream are a genetically diverse bunch, Stanford University School of Medicine researchers have found. Some have genes turned on that give them the potential to lodge themselves in new places, helping a cancer spread between organs. Others have completely different patterns of gene expression and might be more benign, or less likely to survive in a new tissue. Some cells may even express genes that could predict their response to a specific therapy. Even within one patient, the tumor cells that make it into circulating blood vary drastically.

The finding underscores how multiple types of treatment may be required to cure what appears outwardly as a single type of cancer, the researchers say. And it hints that the current cell-line models of human cancers, which showed patterns that differed from the tumor cells shed from human patients, need to be improved upon.

The new study, which was published online in PLoS ONE, is the first to look at so-called circulating tumor cells one by one, rather than taking the average of many of the cells. And it's the first to show the extent of the genetic differences between such cells.

"Within a single blood draw from a single patient, we're seeing heterogeneous populations of circulating tumor cells," said senior study author Stefanie Jeffrey, MD, professor of surgery and chief of surgical oncology research.

For over a century, scientists have known that circulating tumor cells, or CTCs, are shed from tumors and move through the bloodstreams of cancer patients. And over the past five years, there's been a growing sense among many cancer researchers that these cells - accessible by a quick blood draw - could be the key to tracking tumors non-invasively. But separating CTCs from blood cells is hard; there can be as few as one or two CTCs in every milliliter of a person's blood, mixed among billions of other blood cells.

To make their latest discovery, Jeffrey, along with an interdisciplinary team of engineers, quantitative biologists, genome scientists and clinicians, relied on a technology they developed in 2008. Called the MagSweeper, it's a device that lets them isolate live CTCs with very high purity from patient blood samples, based on the presence of a particular protein - EpCAM - that's on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, the team first tested whether they could accurately detect the expression levels of 95 different genes in single cells from seven different cell-line models of breast cancer - a proof of principle since they already knew the genetics of these tumors. These included four cell lines generally used by breast cancer researchers and pharmaceutical scientists worldwide and three cell lines specially generated from patients' primary tumors.

"Most researchers look at just a few genes or proteins at a time in CTCs, usually by adding fluorescent antibodies to their samples consisting of many cells," said Jeffrey. "We wanted to measure the expression of 95 genes at once and didn't want to pool our cells together, so that we could detect differences between individual tumor cells."

So once Jeffrey and her collaborators isolated CTCs using the MagSweeper, they turned to a different kind of technology: real-time PCR microfluidic chips, invented by a Stanford collaborator, Stephen Quake, PhD, professor of bioengineering. They purified genetic material from each CTC and used the high-throughput technology to measure the levels of all 95 genes at once. The results on the cell-line-derived cells were a success; the genes in the CTCs reflected the known properties of the mouse cell-line models. So the team moved on to testing the 95 genes in CTCs from 50 human breast cancer patients - 30 with cancer that had spread to other organs, 20 with only primary breast tumors.

"In the patients, we ended up with 32 of the genes that were most dominantly expressed," said Jeffrey. "And by looking at levels of those genes, we could see at least two distinct groups of circulating tumors cells." Depending on which genes they used to divide the CTCs into groups, there were as many as five groups, she said, each with different combinations of genes turned on and off. And if they'd chosen genes other than the 95 they'd picked, they likely would have seen different patterns of grouping. However, because the same individual CTCs tended to group together in multiple different analyses, these cells likely represent different types of spreading cancer cells.

The diversity, Jeffrey said, means that tumors may contain multiple types of cancer cells that may get into the bloodstream, and a single biopsy from a patient's tumor doesn't necessarily reflect all the molecular changes that are driving a cancer forward and helping it spread. Moreover, different cells may require different therapies. One breast cancer patient studied, for example, had some CTCs positive for the marker HER2 and others lacked the marker. When the patient was treated with a drug designed to target HER2-positive cancers, the CTCs lacking the molecule remained in her bloodstream.

When the team went on to compare the diverse genetic profiles of the breast cancer patients' CTCs with the cells they'd studied from the cell lines, they were in for another surprise: None of the human CTCs had the same gene patterns as any of the cell-line models.

"These models are what people are using for drug discovery and initial drug testing," said Jeffrey, "but our finding suggests that perhaps they're not that helpful as models of spreading cancers." While the human cell-line cells did show diversity between each of the seven cell lines, they didn't fall into any of the same genetic profiles as the CTCs from human blood samples.

These results don't have immediate impacts for cancer patients in the clinic because more work is needed to discover whether different types of CTCs respond to different therapies and whether that will be clinically useful for guiding treatment decisions. But the finding is a step forward in understanding the basic science behind the bits of tumors that circulate in the blood. It's the first time that scientists have used high-throughput gene analysis to study individual CTCs, and opens the door for future experiments that delve even more into the cell diversity. The Stanford team is now working on different methods of using CTCs for drug testing as well as studying the relationship between CTC genetic profiles and cancer treatment outcomes. They've also expanded their work to include primary lung and pancreatic cancers as well as breast tumors.

Source: Stanford University Medical Center
Gregory D. Pawelski

Last edited by gdpawel : 05-09-2012 at 01:14 AM.
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Old 04-25-2013, 10:50 AM
gdpawel gdpawel is offline
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Default CTC technology has great potential

CTC technology has great potential - for drug selection - ten or twenty years down the road, and they should continue to try and make strides. However, there is a problem with growing or manipulating tumor cells in any way. When looking for cell-death-related events, which mirror the effect of drugs on living tumors, cells are generally not grown or amplified in any way. The object is occurrence of programmed cell death in cells that come into contact with therapeutic agents.

How do you aggregate a sufficient number of cancer cells to make accurate determinations. Detectable tumor cells in the peripheral blood are present only in extremely small numbers. This precludes allowing a sufficient number of cells to incubate for a few days in the presence of chemotherapeutic agents. Analysis of a relatively small number of isolated cancer cells cannot yield the same quality information as subjecting living cells to chemotherapeutic agents, begging the question of whether or not it can accurately predict which drugs will work and which will not.

CTCs are free-floating cancer cells that can remain in isolation from a tumor for over twenty years. What is the relationship of such long-lasting cells to the tumor cells that need to be attacked through tested substances?

Then there is the question of heterogeneity. Tumors in the body are genetically variable. What is the relationship between CTCs and primary tumors or their already established metastases? It has already been established that the gene expression profile of a metastatic lesion can be different compared to that of the primary. The status of the marker Her2/neu in CTCs sometimes differs from that of the original primary tumor, which would point to different prescriptions for Herceptin.

The number of cells discovered in the CTC technique has turned out to be a good prognosticator of how well empiric treatments are working, but less certain in the ability to use it for drug selection. The level of documentation of the sensitivity and specificity of the drug recommendations that are generated by this procedure has never been the subject of a single peer-reviewed article.

The "problem" is in isolating and analyzing single cancer cells. The supposition is that common cancers can be detected and cured through analysis at a genetic level of a small number of cells or even a single wayward cell.

Genetic or IHC testing examines dead tissue that is preserved in paraffin or formalin. How is that going to be predictive to the behavior of living cells in spontaneously formed colonies or microspheres? Can it describe the complex behavior of living cancer cells in response to the injury they receive from different forms of chemotherapy? There is a big difference between living and dead tissue.

Some molecular tests do utilize living cells, but generally of individual cancer cells in suspension, sometimes derived from tumors and sometimes derived from CTCs. Don't forget, this was tried with the human clonogenic assay, which had been discredited long ago.

Basically, CTC labs use "negative selection" to isolate alleged circulating tumor cells. What that means is methods to "selectively" remove circulating normal cells, such as monocytes, lymphocytes, neutrophils, circulating endothelial cells, etc. The problem is that these normal cells outnumber circulating tumor cells by a factor of a million to one, and no "negative selection" procedure (or combination of procedures) can possibly strip away all the normal cells, leaving behind a relatively pure population of tumor cells.

What you have to do is to use a "positive selection" procedure, meaning selectively extracting the tumor cells out of the vastly larger milieu of normal cells. The problem is, when you do this, there is only a teeny tiny yield of tumor cells:

Here's from Wikipedia:

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.

So, from a typical 7 ml blood draw into a purple top tube, you are going to get, on average, 7 to 70 tumor cells -- total. This may be sufficient for certain molecular type tests (although the degree to which this tiny sample of cells is representative may be questioned), but it isn't nearly sufficient to test even a single drug in a cell culture assay, where one requires millions of cells for quality testing, including requirements for negative and positive controls.

Regardless of all of this, most of the cells that leave home don't survive the journey in the blood or lymph systems and many cancerous cells that eventually do lodge in a distant organ simply remain dormant, leaving it up to the immune system to take care of them.

Full-blown metastasis is an extremely challenging trade and the great majority of cancer cells are not up to the task. Even those malignant characters that manage to slither their way into the blood or lymph system, usually fail to do anything further.

Most tumor cells lack the streamlined form of the blood and immune cells that are designed for cross-body trafficking, shear forces in the smaller vessels may rip the intruders apart. These free-floating cancer cells can remain in isolation from a tumor for over twenty years (Gupta, G.P., and J. Massague. 2006. Cancer metastasis: building a framework. Cell. 127:679-95).
Gregory D. Pawelski
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