Two groups at The University of Texas M.D. Anderson Cancer Center also work on CTCs, but focus on breast cancer. Researchers led by James Reuben, M.D., associate professor, and Massimo Cristofanilli, M.D., an associate professor in the department of breast medical oncology, are working to establish the clinical relevance of heterogeneity. This requires studying a large number of clinical specimens of circulating tumor cells in breast cancer patients. “Using AmpliGrid, we plan to genetically fingerprint subpopulations of tumor cells with a convenient workflow,” says Dr. Reuben.
Using the CellTracks system by Veridex, Dr. Cristofanilli and colleagues were the first to report that in metastatic breast cancer more than five CTCs per sample are associated with a worse prognosis than those samples with fewer than five cells. More recently, in collaboration with Marianna Alunni-Fabbroni, Ph.D., and colleagues at Advalytix in Germany, Drs. Reuben and Cristofanilli have successfully sorted breast cancer cell lines onto an AmpliGrid slide based on a number of surface antigens (EpCAM, CD44, and CD24), the intracellular marker ALDH1, or side population using high-throughput flow cytometry. This was followed by qPCR to identify as many as six genes of interest in a single cell. This technology will be used to identify targets of interest on tumor cells and breast cancer-initiating stem cells, which can be used in the development of targeted therapies for breast cancer.
The M.D. Anderson clinical chemistry diagnostic lab, led by Herbert Fritsche, M.D., has been pioneering the diagnostic use of CTCs, along with Drs. Cristofanilli and Reuben. Using the CellTracks system, which the group had helped validate, they enumerate the number of CTCs for nearly 100 patients per month.
The CellTracks system flags patients’ cells from blood samples as CTCs when they express the EpCAM surface protein and cytokeratin, markers for epithelial cells. Pathologists then evaluate an immunohistochemical image of those cells to count the number of CTCs per sample. Dr. Fritsche and his team have observed that the images of these CTCs are anything but homogeneous: they differ significantly in size, morphology, and fluorescent intensity.
Now, using AmpliGrid single-cell PCR, Dr. Fritsche wants to test his hypothesis that morphologically defined subpopulations correspond to differences in gene expression similar to genetic heterogeneity observed in breast cancer cell lines. The workflow proposed for these studies includes enrichment and identification of EpCAM positive cells on the CellTracks platform, sorting cells onto AmpliGrid slides via flow cytometry, running a multiplex PCR assay on each cell, and finally analyzing the results on one of the genetic analysis platforms at M.D. Anderson, such as an Agilent Bioanalyzer or Beckman Coulter GeXP.
The M.D. Anderson teams expect that the single-cell sensitivity of the AmpliGrid platform will produce robust data to measure heterogeneity by comparing the genetic profiles of individual tumor cells. From a clinical perspective, AmpliGrid meets these throughput, automation, and cost requirements with a simple workflow that seamlessly integrates the steps needed for each clinical specimen, namely: cell sorting, sample preparation, and PCR.
“The ultimate goal is to develop powerful tools to monitor the response to treatment, improve early detection, and personalize treatment for our patients,” says Dr. Cristofanilli.