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CTC-Chip Dream Team Progress Update

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CTC-Chip Dream Team Progress Update

Bioengineering and Clinical Applications of Circulating Tumor Cell Chip

Funding: $15 million

Leader: Daniel A. Haber, M.D., Ph.D., Director, Massachusetts General Hospital Cancer Center
Co-Leader: Mehmet Toner, Ph.D., Professor, Biomedical Engineering, Harvard Medical School


Fast Facts on Prostate Cancer and Lung Cancer:

  • Circulating tumor cells (CTCs) are extremely rare cancer cells that have detached from the primary tumor and can be found in the blood of cancer patients.
  • In the United States, it is estimated that 228,190 men and women will be diagnosed with and 159,4800 men and women will die of cancer of the lung and bronchus in 2013.
  • Based on rates from 2007-2009, 1 in 14 men and women will be diagnosed with cancer of the lung and bronchus during their lifetime.
  • It is estimated that 238,590 men will be diagnosed with and 29,720 men will die of cancer of the prostate in 2013.
  • Based on rates from 2007-2009, 1 in 6 men will be diagnosed with cancer of the prostate during their lifetime.

 

Project Background

Cancers arise from cells within an organ, such as the breast or prostate, but cause death by spreading—or metastasizing—through the bloodstream to the bone, liver, lungs, or brain.

Cancer cells that detach from the primary tumor can be found in the blood of cancer patients. These cancer cells, called circulating tumor cells (CTCs), are extremely rare—there is one for every one billion normal cells. The ability to detect and analyze CTCs could help physicians detect and treat cancer. It could also help scientists learn how cancers spread.

The technologies that are currently available for detecting CTCs have helped scientists learn about these cells. But they are not sensitive or reliable enough for physicians to use in the clinic to make cancer treatment decisions.

This Dream Team, comprised of clinicians, bioengineers, and molecular biologists, has developed a novel approach to detect and isolate CTCs. They have created a business card-size CTC-Chip that contains 78,000 microscopic columns that have been coated with a special material that is able to catch the CTCs but let normal blood cells flow through. This makes it possible for scientist to study and analyze CTCs. The Team hopes that their work will lead to an expansion of research and a revolution in clinical care through the use of the CTC-Chip to quickly and non-invasively monitor the status of a tumor.

 

Status Update

6 month milestones
The Team initiated a study focused on prostate cancer patients for three reasons: there are unique biomarkers for prostate cancer, metastatic prostate cancer is difficult to biopsy so other approaches are required, and it is difficult to predict whether the cancer will be indolent or aggressive. They developed standard protocols for CTC staining, and showed that CTC levels can be used as a biomarker for tumor response to therapy. They also found that RNA could be collected from CTCs with analysis showing that a common gene fusion present in approximately half of prostate tumors could be identified.

A second pilot study developed a new way to visualize a biomarker for aggressive prostate cancer, and is now being tested on a larger cohort of patients. The Team observed that CTC levels go down after prostate surgery, and is testing a hypothesis that CTC levels can be used to predict the risk for relapse of prostate cancer.

The Team has designed a second-generation CTC-chip that is easier to produce, easier to use, cheaper to manufacture, and more functional than the early version. Standard protocols for increased functionality and high throughput analysis are being developed. The new chips are scheduled to be distributed to SU2C institutions in year two of the grant. 

The Team is also developing technology to understand how the tumor microenvironment influences the growth of CTCs.  An Extracellular Matrix Microarray has been designed, built, and characterized that will allow researchers to test cell lines in 800 different conditions. This technology will allow investigators to test and discover key biological processes that lead to metastases in a variety of cancer types.

12 month milestones
The Team extended the pilot study initiated in the first six months to detect Prostate Specific Antigen-positive CTCs in patients with early-stage cancer before and after their surgery.  The study found that 60% of patients did not have CTCs before their surgery. They also found that, in six of the eight patients that had CTCs before surgery, their CTC levels decline by more than 50% within 24 hours following surgery; the other two had a delay in the decline. By three months following surgery all patients had no detectable CTCs. The Team has now initiated a clinical trial of 200 patients to determine if pre- and post-surgery CTC levels and rate of decline are predictive of relapse. In addition, the Team is continuing to investigate a second gene, Ki67, which is a biomarker for aggressive cell growth that may be correlated to metastatic prostate cancer.

The Team investigated whether the CTC Chip might be beneficial in the treatment of non-small cell lung cancer. They studied blood samples from 32 patients to determine which mutations can be identified using CTCs. They also conducted studies to develop tumor molecular profiles to predict drug resistance. They initiated a study to identify mutations associated with drug resistance that will profile tumor mutations in CTCs in patients before they start treatment, after treatment ends, and at the time of disease progression.  The Team collaborated with researchers conducting a phase II clinical trial for 89 patients with hard-to-treat tumors that showed CTCs decreased or remained the same in 89% of the patients whose tumors responded to the study drug, and that high CTC levels were associated with worse overall survival.

The Team previously reported that they had identified a common gene amplification (HER2) in metastatic breast cancer. They conducted two studies designed to follow-up on these preliminary findings, which found that 98% of the patients with HER2-positive tumors had HER2-positive CTCs and 66% of the patients with HER2-negative tumors had HER2-negative CTCs.

18 month milestones
The Team successfully scaled up it efforts to produce the second generation CTC-Chip. Each collaborator site has been or will soon be trained in the use of the CTC-Chips and the instruments that process and stain them. The Team also started enrollment of patients in a clinical study of 200 men to determine if preoperative CTCs and postoperative CTC rate of decline can predict which noninvasive early prostate cancer will go on to become invasive disease. In a separate study, they investigated markers of androgen signaling that can be used to guide treatment decisions.

The Team began using the second generation CTC-Chip along with multi-color imaging to monitor how tumors respond to targeted therapies used to treat non-small cell lung tumors that have an EGFR or an ALK genetic mutation. They have developed assays to detect cancer-specific mutations in DNA taken from the plasma of patients with stage IV disease. They detected mutations in 22 of the 33 samples.

The Team collaborated with researchers conducting a Phase II clinical trial of the drug temozolomide (brand name Temodar) in patients with hard-to-treat small cell lung cancer. The study showed that, after the first drug cycle, CTCs decreased or remained the same in 92% of the patients that showed a response to treatment. High CTC levels were associated with worse overall survival.

The Team also worked to identify capture antibodies that can be put on the CTC-Chip so that it can be used to study CTCs in patients with melanoma and glioblastoma, developed a precise way to measure the physical properties of individual CTCs, developed a new protocol for determining the glycan structures on a cell’s surface, and began to develop strategies to modify the surface of the CTC-Chip to make it easier to detach the CTCs.

24 month milestones
The Team successfully scaled up it efforts to produce the CTC-Chip. Each collaborator site has been trained in the use of the CTC-Chips and the instruments that process and stain them.  The Team has started a study to determine a baseline of CTCs found in healthy patients and has accrued 45 of 200 patients. The Team is developing a new array of antibodies to use in the CTC-Chip to enhance CTC capture.

The Team has accrued 53 of 200 patients in a clinical study determine if preoperative CTCs and postoperative CTC rate of decline can predict which noninvasive early prostate cancer will go on to become invasive disease. The Team previously reported that they had identified a common gene amplification (HER2) in metastatic breast cancer CTCs. They have followed up on this finding by conducting a study designed to compare the HER2 status of the CTCs with the HER2 status of the primary tumor. This study enrolled 65 patients. In patients with HER2-negative tumors, 35% of CTCs were HER2 amplified.  To determine if HER2 status changed over time, 33 patients had a repeat CTC assay performed 3-6 weeks after initial analysis. 10 of 12 HER2 amplified patients remained so after retesting. The two patients that became HER2 negative received trastuzumab (Herceptin) based therapy in the meantime. A total of 17 of 21 HER2 negative patients remained so after retesting.

30 month milestones
The Team has developed the use of triple antibody cell capture (antibody to EpCAM, EGFR, HER2), which provides improved capture of lung CTCs, since not all cells express high levels of EpCAM. They have also developed a refined method to perform single molecule RNA sequencing from nucleic acids extracted from CTCs. This method was applied successfully to pancreatic CTCs and identified an altered signaling pathway in a mouse model of pancreatic cancer. Extension to human pancreatic CTCs confirmed the abnormal signaling is evident in CTCs from approximately 50% of cases.

The Team has also completed accrual of a Phase II trial for patients with relapse sensitive or refractory Small Cell Lung Carincoma. The Team found that at baseline the number of CTCs was not predictive of response to treatment or associated with progression free and overall survival. However, at the end of cycle 1, an increase in the number of CTCs compared to baseline values was correlated with a response to treatment.

36 month milestones
The Team has initiated the joint phase I clinical trial as stated in the original proposal. CTCs will be tested for EGFR mutations and compared with the results obtained from the tumor biopsy in patients with non- small cell lung cancer who are undergoing a re-biopsy for clinically defined resistance to EGFR inhibitors. 40 patients will be accrued across all institutions for this study. Two aims are planned for this clinical study: Demonstrate the feasibility of testing for EGFR mutations from captured CTCs using the latest chip version and next generation sequencing techniques; and describe the agreement of EGFR mutations between the repeat tumor tissue biopsy and the CTC-chip-derived DNA/RNA.

To explore its role in human cancer, the Dream Team investigated epithelial-mesenchymal transition (EMT) in circulating tumor cells (CTCs) from breast cancer patients. EMT is a biologic process that allows an epithelial cell, which normally attaches to surrounding tissue proteins, to enhance its migratory capacity, invasiveness, and resistance to cell death. The Team found that cells rarely expressed mesenchymal and epithelial markers simultaneously in primary tumors, but in CTCs mesenchymal markers were found in a higher percentage of cells and, in some multicellular clusters, all CTCs expressed mesenchymal markers. CTCs were monitored in eleven patients and suggested a possible link between mesenchymal CTCs and disease progression. Additionally, mesenchymal CTCs occurred as both single cells and multicellular clusters, while also expressing genes known to regulate EMT. These data support a role for EMT in breast cancer metastasis, as a potential biomarker of therapeutic resistance, and as a potential drug target in breast cancer.

42 month milestones
The Dream Team has continued the joint phase I clinical trial that compares genetic analyses in CTCs to those of direct tumor biopsies. The SU2C CTC-Chip protocols are fully implemented at Massachusetts General Hospital Cancer Center, Dana Farber Cancer Institute, the University of Texas MD Anderson Cancer Center, and Memorial Sloan Kettering Cancer Center, and the trial has been approved by Institutional Review Boards and opened at all institutions. To date, 38 of 40 patients have been accrued across all institutions. Analysis of the samples to demonstrate the feasibility of testing for EGFR mutations from captured CTCs with next-generation sequencing techniques has begun. Preliminary evidence indicates an agreement of EGFR mutations between the repeat tumor tissue biopsy and the DNA/RNA isolated from CTCs captured using the CTC-Chip.

The Dream Team has also continued to develop its third-generation device called the iChip. Using a size-based method to eliminate red blood cells, inertial focusing to line up the remaining cells in single file, and magnetic antibodies to isolate CTCs, this device will allow scientists to study CTCs in a variety of new ways. Additionally, the Dream Team has developed a new technology to concentrate the CTCs in a much smaller volume of solution that keeps them alive longer than before. Last, the Dream Team has developed a new technology that isolates single CTCs in oil droplets. This will allow scientists to sort the CTCs based on different cell characteristics and analyze single cells.

48 month milestones
The Dream Team has completed enrollment for their joint phase I clinical trial that compares genetic analyses in CTCs to those of direct tumor biopsies. Analysis of the samples includes testing for EGFR mutations from captured CTCs with next-generation sequencing techniques. The results are preliminary but so far point to significant disparity between tumor biopsy results and CTC-derived genotypes.

The Dream Team has also continued to develop its third-generation device called the iChip. Using a size- based method to eliminate red blood cells, inertial focusing to line up the remaining cells in single file, and magnetic antibodies to isolate CTCs, this device will allow scientists to study CTCs in a variety of new ways. The CTC-iChip is not yet ready for routine operation, or wide distribution. Nonetheless, the Dream Team is using CTCs isolated with this new Chip to (i) perform single-cell sequencing, and (ii) establish cultures of CTC-derived cell lines.


Fast Facts on Prevention Tips:

  • The best way to reduce your risk of lung cancer is not to smoke and to avoid second-hand smoke.
  • While a much smaller risk compared to smoking, radon is still an important cause of lung cancer. You can reduce your exposure to radon by having your home tested and treated, if needed.
  • Studies have found that men who get regular physical activity have a slightly lower risk of prostate cancer. Always exercise safely.


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