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Circulating Tumor Cells and the CellSearch Assay

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Published: April 2011

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Dr. Halling will provide an overview of the metastatic process in cancer and how circulating tumor cell counts influence prognosis and overall survival in metastatic breast, colorectal, or prostate cancer.

Mr. Campion will discuss the technical aspects of a novel assay for enumerating circulating tumor cells in peripheral blood and cite several clinical trial studies describing the clinical utility of this assay.

Presenters: Kevin Halling, MD, PhD

Presenters: Michael Campion

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenters for this program are Kevin Halling, MD, PhD and Michael Campion from the Division of Laboratory Genetics and Anatomic Pathology at Mayo Clinic. Dr. Halling will provide an overview of the metastatic process in cancer and how circulating tumor cell counts influence prognosis and overall survival in metastatic breast, colorectal, or prostate cancer. Mike Campion will discuss the technical aspects of a novel assay for enumerating circulating tumor cells in peripheral blood and cite several clinical trial studies describing the clinical utility of this assay.

Mike Campion and I are going to discuss the use of the Cell Search assay for the detection of circulating tumor cells. We believe that circulating tumor cell analysis is a valuable new tool for monitoring patients with metastatic cancer and that it will be increasingly used in the clinical management of patients with metastatic cancer.

Circulating Tumor Cell (CTC)

A circulating tumor cell is a cell that has detached from a solid tumor and entered the peripheral bloodstream. A number of studies have now demonstrated that the presence of circulating tumor cells in the peripheral blood is associated with decreased progression-free survival and decreased overall survival in patients with metastatic breast, colorectal, or prostate cancer. Mike will review the findings of some of these studies later in this presentation.

The Metastatic Process

This slide provides an overview of the metastatic process. The metastatic process begins when a cancer cell breaks away from a tumor and enters the lymphatic or peripheral bloodstream. At some point a circulating tumor cell then comes to rest against a vessel wall and extravasates into the surrounding tissue. This circulating tumor cell may then establish a new tumor at a site distant from the original tumor, in other words a metastasis. Tumor angiogenesis helps establish the new tumor.

Needle in a Haystack...

Tumors shed an enormous number of tumor cells. It is estimated that approximately one million circulating tumor cells are shed per gram of tumor tissue. However, only a small proportion of these CTCs survive. Within 24 hours, less than 0.1% of these cells are still viable and less than 0.01% survive to produce metastases. Patients with metastatic cancer have been found to have approximately 1 tumor cell per 100,000 to 10 million mononuclear cells in their peripheral blood.

Circulating Tumor Cells

There has been a great deal of clinical interest in CTCs for decades. Many studies have been conducted to examine the clinical utility of CTC detection. These studies reveal that CTC detection has a number of utilities but currently this type of testing is primarily used to evaluate a patient's prognosis and to assist in the therapeutic management of patients with metastatic cancer. And now Mike will discuss the technical aspects of the CellSearch assay and several clinical trials that have addressed the clinical utility of the assay.

The CellSearch System (Veridex LLC)

Before I get into the details of the system, I would just like to quickly go over a few key points about the system. The CellSearch System is manufactured by Veridex Corporation which is a subsidiary of Johnson and Johnson. The CellSearch assay consists of 2 main components, the Autoprep which isolates the CTCs from the whole blood, and an analyzer that contains a fluorescent microscope which differentiates the CTCs from nonspecific cells and debris. This platform is the only FDA-approved platform for the capture, analysis, and enumeration of circulating tumor cells. The process, which will be explained in detail later, allows for the detection of as few as 1 CTC in 7.5 ml of blood. One thing that makes the CellSearch system stand out from other methods for capturing circulating tumor cells is the fact that the sample preparation has been automated and standardized. That process is made more robust by CTC images that are then reviewed by trained technologists.

Automated Sample Preparation

This slide shows in detail the sample preparation that takes place in the Autoprep. 7.5 mL of blood is drawn into Cellsave tubes that contain a preservative that allows for sample to be stored at room temperature for 96 hours. Seven point five mL of blood is removed and placed in a processing tube and centrifuged at 800g for 10 minutes. That tube is then placed on the Autoprep for processing. The plasma is aspirated and buffer containing ferrofluid compounds that are bound to EpCam antibodies are added.

Magnets are then engaged which draws the ferrofluid particles to the side of the tube, leaving any unlabeled cells to be aspirated. The magnets are then removed and target cells are resuspended in buffer that contains permeabilizing and staining reagents. This epithelial-enriched sample is then placed in a cell presentation device called a Magnest that attracts the magnetically labeled epithelial cells to the surface of the cartridge.

Labeling and Identification of CTCs

Besides using the EpCam antibodies for immunomagnetic labeling and capturing of the epithelial cells, the CellSearch System also uses 3 stains to help distinguish true CTCs from contaminating leukocytes. Circulating tumor cells are identified by using antibodies labeled with phycoerythrin (PE) to cytokeratins 8, 18, and 19. Leukocytes are marked using allophycocyanin (APC)-labeled antibodies to CD45. Additionally, DAPI stains the nuclei of cells and helps distinguish viable cells from nonspecific debris.

Labeling and Identification of Cells

This is a graphical representation of the staining pattern. As you can see, the circulating tumor cells are identified by a positive EpCam, positive cytokeratin, and a positive DAPI pattern. In turn, a leukocyte is positive for CD45 and DAPI, but negative for cytokeratin staining. These staining patterns are then used by a trained technologist to interpret images that are presented in a gallery on the Celltracks analyzer.

Cell Analysis

The final step in the identification of a circulating tumor cell is done by a review of images by a technologist. The technologist reviews the gallery of images and selects those images that fit the criteria of an intact tumor cell, which once again is EpCam positive, cytokeratin positive and DAPI positive. Once that is complete, a pathologist reviews the selections and makes any changes that he or she feels is warranted.

Mayo Medical Laboratories FDA-Cleared Assays

At Mayo Clinic we offer all 3 of the FDA approved tests for circulating tumor cells using the CellSearch platform. Test number 89089, is for circulating tumor cells in metastatic beast cancer. Test number 89162, is for circulating tumor cells in metastatic colorectal cancer. And finally, test number 60142, which is for circulating tumor cells in metastatic prostate cancer patients.

Metastatic Breast Cancer (MBC)

In 2010, an estimated 209,000 new cases of breast cancer were diagnosed in the United States and approximately 40,000 deaths from this disease. Of newly diagnosed cases of breast cancer, 5% of patients will have metastatic breast cancer at diagnosis, and another 30% will ultimately develop metastatic disease. With these numbers, it is easy to see how an assay that could monitor the metastatic process would be very useful.

CellSearch Use in MBC Clinical Trial

A multicenter, prospective, longitudinal clinical trial using the CellSearch system was conducted between 2001 and 2003, and the results published in 4 scientific publications. The summary of these results can be seen in this chart. The trial included 177 patients with metastatic disease as defined by standard imaging techniques. Results were used to determine whether the number of circulating tumor cells predicted disease progression and survival in metastatic breast cancer patients. A baseline circulating tumor cell count was determined prior to start of new therapy and an initial follow-up count was determined after the start of therapy. Progression-free survival was determined from the time of the baseline draw to identifiable disease progression based on CT scans or clinical indications. Overall survival was measured from baseline blood draw to the time of death. Lets take a look at these results a little more closely on the next few slides.

Progression-Free Survival in MBC

Kaplan-Meier analyses were performed to determine whether circulating tumor cell counts could predict progression-free survival. Patients were separated into 2 groups based on their circulating tumor cell count. The graph on the left shows the results of baseline circulating tumor cell counts. The favorable group represented patients with a baseline circulating tumor cell count <5, while the unfavorable group represented patients with a baseline count =5. The median progression-free survival was approximately 7.0 months for the favorable group and 2.7 months for the unfavorable group, which was statistically significant.

The graph on the right shows the value of monitoring circulating tumor cell levels during the course of treatment. The most striking results show that patients with =5 circulating tumor cells at both baseline and at the end of treatment had significantly shorter median progression-free survival (1.8 months) than patients in all other groups. Patients with <5 circulating tumor cells at each time point had the longest median progression-free (7.2 months). Interestingly, patients with =5 circulating tumor cells at baseline, which decreased to <5 at the end of treatment had significantly longer median progression-free survival than patients who maintained counts of at least 5 at both time points, 6.1 months versus 1.8 months.

Overall Survival in MBC

Kaplan-Meier analyses were also performed to determine whether circulating tumor cell counts could predict overall survival. The graph on the left shows that when comparing the favorable and unfavorable groups, the median overall survival was shown to be significantly longer in the favorable group than in the unfavorable group (21.9 versus 10.9 months).

The graph on the right shows that metastatic breast cancer patients who had =5 CTCs at all timepoints after the initiation of therapy had a much shorter overall survival of 4.1 months. Whereas those patients that were less than 5 CTCs at all time points had the longest overall survival of 22.6 months. Interestingly, those patients who have = 5 CTCs at baseline but eventually decreased to less than 5 CTCs after the start of therapy have approximately the same risk of death as those who never exceed the 5 circulating tumor cells threshold.

This led to the FDA approval of the CellSearch system for use in metastatic breast cancer patients in 2004.

Metastatic Colorectal Cancer (MCRC)

Now let's discuss the use of the CellSearch Assay for metastatic colorectal cancer.

In 2010, an estimated 142,000 new cases of colorectal cancer were diagnosed in the United States with approximately 51,000 deaths due to colorectal cancer. Of the new cases, nearly 20% had metastatic colorectal cancer when diagnosed and nearly one-third will develop metastases during clinical followup. Therefore, approximately 75,000 new patients are being treated for metastatic colorectal cancer every year.

CellSearch Use in MCRC Clinical Trial

A multicenter, prospective clinical trial was conducted between 2004 and 2006 to determine whether circulating tumor cell enumeration could predict colorectal cancer disease progression and survival. Prior to initiation of a new line of therapy, 430 patients with measurable metastatic colorectal cancer were enrolled. Measurable disease was defined as a minimum of one 2-cm lesion to a maximum of 10 lesions. Imaging methods were determined for each patient as per the current standard of care defined by the patient's physician. All lesions seen at baseline were followed using the same method for all successive studies, either CT or MRI of the chest, abdomen, and pelvis.

A baseline circulating tumor cell count was established prior to the start of therapy and follow-up counts were determined every 3 to 4 weeks. Progression-free survival was measured from the time of baseline draws until the time of detectable disease progression based on CT or MRI scans and clinical findings. Overall survival was determined from the time of baseline blood draws to the time of death. Finally, unlike the metastatic breast cancer trial, which used a cutoff of greater than or equal to 5 circulating tumor cells, the metastatic colorectal trial used a cutoff of greater than or equal to 3 circulating tumor cells.

Progression-Free Survival in MCRC

The chart on the left is a Kaplan-Meier curve that was generated to evaluate progression-free survival between 2 patient cohorts based on baseline circulating tumor cell counts. The favorable group which consists of those patients with less than 3 circulating tumor cells had an n value of 305, while the unfavorable group which was those patients with greater than or equal to 3 circulating tumor cells had an n value of 108. The results of this study show that the favorable group had a median progression-free survival of 7.9 months compared to the unfavorable group's median progression-free survival of 4.5 months.

The chart on the right shows that metastatic colorectal cancer patients with =3 CTC at all time points had the shortest median progression-free survival, which was significantly different compared to the median progression-free survival of the other groups. One of the take home messages from this chart is that the difference in the median progression-free survival between those patients who showed a CTC reduction after the initiation of therapy was significantly longer compared to those patients who showed a CTC increase.

Overall Survival in MCRC

Kaplan-Meier analyses were also performed to determine whether circulating tumor cell counts could predict overall survival. The graph on the left shows that when comparing the favorable and unfavorable groups, the median overall survival was shown to be significantly longer in the favorable group than in the unfavorable group (18.5 versus 9.4 months).

The graph on the right shows that metastatic colorectal cancer patients who exceed the threshold of 3 CTC at any point after the initiation of therapy had a much higher likelihood of dying sooner. Patients with =3 CTC at all time points had the shortest median overall survival. Patients with <3 CTC at all time points had the longest median overall survival. This chart also shows that patients who showed a decrease in CTC count had a significantly lower risk of death compared to those patients with an increase in CTC count.

This clinical trial led to FDA approval of the CellSearch system for use in metastatic colorectal cancer in 2007.

Metastatic Prostate Cancer (MPC)

Now let's discuss the use of the CellSearch Assay for metastatic prostate cancer.

In the United States in 2010, there were approximately 217,000 new cases of prostate cancer with 32,000 deaths. Of the new cases, nearly 4% had evidence of metastatic disease at diagnosis.

CellSearch Use in MPC Clinical Trial

A multicenter, prospective clinical trial was conducted between 2004 and 2006 to determine whether the number of circulating tumor cells predicted disease progression and overall survival. The patient sample in this trial consisted of patients with hormone resistant, androgen-independent, or castration resistant tumors. A patient with a castration-resistant tumor is defined as a prostate cancer patient who has had 2 consecutive increases in prostate-specific antigen values despite hormone treatment.

The clinical trial enrolled 231 patients with metastatic prostate cancer who, despite treatment, had an increase in PSA level. Baseline circulating tumor cell counts were determined prior to initiation of a new line of therapy. Most patients (221 of 231) had 1 or more blood draws to monitor circulating tumor cell counts and PSA levels every 4 to 6 weeks after baseline. Progression-free survival was determined by identifying continued increases in PSA values, radiologic imaging evidence of disease progression, or other clinical signs. Overall survival was determined from baseline draw until time of death or until last contact with the patient.

Progression-Free Survival in MPC

Kaplan-Meier analyses were performed to determine whether circulating tumor cell counts could predict progression-free survival. Patients were separated into 2 groups based on their circulating tumor cell count. The graph on the left shows the results of baseline circulating tumor cell counts. The favorable group (n = 94) represented patients with a baseline circulating tumor cell count >5, while the unfavorable group (n = 125) represented patients with a baseline count =5. The median progression-free survival was approximately 5.8 months for the favorable group and 4.2 months for the unfavorable group.

The graph on the right shows that monitoring circulating tumor cells levels during the course of treatment provides additional prognostic information. A reduction of circulating tumor cells to below 5 after the start of therapy predicted longer progression-free survival in metastatic prostate cancer patients. Patients who had <5 circulating tumor cells for all time points had the longest median progression-free survival (6.5 months) of all groups. Patients who had =5 circulating tumor cells for all time points had the worst prognosis and had the shortest median progression-free survival (2.5 months) than the other 3 groups. Interestingly, patients with =5 circulating tumor cells at baseline who had a reduction in circulating tumor cells to <5 at the last draw showed significantly longer progression-free survival (7.3 months) than the group that had an increase of circulating tumor cells from <5 circulating tumor cells at baseline to =5 at last draw with a median progression-free survival time of 4.2 months.

Overall Survival in MPC

Kaplan-Meier analyses were also performed to determine whether circulating tumor cell counts could predict overall survival. The graph on the left shows that when comparing the favorable and unfavorable groups, the median overall survival was shown to be significantly longer in the favorable group than in the unfavorable group (21.7 months versus 11.5 months).

The graph on the right shows that those patients with =5 CTC at any point after the initiation of therapy had a much higher likelihood of dying sooner. Patients with =5 CTC at all time points had the shortest median overall survival, which was significantly different compared to the median overall survival for the group that a rising CTC count. Finally, this graph also demonstrates that patients who showed a decrease in CTC count improve their survival chances and had a median overall survival similar to those patients with favorable CTC count at all draws. The figure also shows that unfavorable CTC levels after the initiation of therapy significantly decreased overall survival.

The Future of CTC

We hope that we have demonstrated the important role of CTC analysis in the management of patients with metastatic breast, colorectal, and prostate cancer. Additional studies are being performed to examine the clinical utility of the CellSearch assay for other tumors such as urothelial cancer, lung cancer, and melanoma. Possibly most importantly are additional studies to determine the utility of this technology for guiding proper patient treatment. For example, a study by the Southwest Oncology Group, is investigating a strategy of changing or maintaining therapy based on circulating tumor cell levels in patients with metastatic breast cancer. This trial might answer the question of whether changing treatment based on an elevated or increasing circulating tumor cell count alone is warranted.

In closing, this concludes our presentation on the use of the CellSearch System for the detection of circulating tumor cells in patients with metastatic cancer. We hope that this information has been useful. Thank you.

 


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