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Aug 1, 2014 (Vol. 34, No. 14)

Finer Screens for Circulating Tumor Markers

  • Circulating tumor cells (CTCs) and circulating tumor (cell-free) DNA (ctDNA) are valuable research tools for studying the biology, genetic makeup, and activity of metastatic cancer. 

    They are increasingly being used for diagnostic and prognostic applications to guide patient care and therapeutic decision making. Advances in the detection, isolation, and analysis of CTCs, ctDNA, and exosomes were the focus of several presenters at SelectBio’s “Circulating Biomarkers” conference held recently in Boston, including Robert McCormack, Ph.D., head of technology innovation and strategy at Janssen Diagnostics, a subsidiary of Johnson & Johnson.

    It has become apparent that “CTC’s reflect more the biology of a cancer than the amount of cancer,” said Dr. McCormack, and “therein lies their true value.” 

    CTCs can inform clinical decisions and guide drug development, according to Dr. McCormack, and clinicians are using CTCs to determine therapy effectiveness, elucidate mechanisms underlying therapy failure, and identify therapeutic options for individual patients.

    CellSearch® from Janssen Diagnostics, the only in vitro device for the detection and isolation of CTCs that has been cleared by the FDA, is used in the clinical setting to capture and count CTCs in blood samples to determine the prognosis of patients with metastatic breast, colorectal, or castration-resistant prostate cancer. CellSearch and the majority of currently available methods use an enrichment step, or positive selection to detect CTCs based on antibody binding to epithelial cell markers, most notably epithelial cellular adhesion molecule, or EpCAM, which is found on the surface of most epithelial cell tumors, as well as cytokeratin.

  • Click Image To Enlarge +
    Researchers at the University of Louisville are developing a method of detecting and isolating circulating tumor cells that is designed to capture a comprehensive set of cells, including those that do not express the EpCAM (epithelial cellular adhesion molecule) marker.

    As Lori Millner, Ph.D., a clinical chemistry fellow at the University of Louisville, and her colleagues at PGXL Technologies have pointed out, an estimated 37–39% of patients with metastatic breast cancer have no detectable CTCs using epithelial cell surface markers as the criteria for isolation. “There is definitely no consensus on what are CTCs or how to capture them,” Dr. Millner remarked.

    It is possible for cancer cells to go through epithelial cell to mesenchymal cell transition and to downregulate certain markers, including EpCAM. Furthermore, Dr. Millner continued, there is evidence indicating that those patients with metastatic breast cancer who have no detectable CTCs using currently available methods tend to have more aggressive types of breast cancer.

    Going through the epithelial-to-mesenchymal transition may confer an advantage to CTCs in the circulation, helping them “avoid immune detection and be more robust,” Dr. Millner offered. “Arguably, they are the most dangerous cells.”

    The researchers at Louisville are developing a method of detecting and isolating CTCs designed to capture a more comprehensive set of cells, including those that do not express EpCAM. With funds from a National Cancer Institute SBIR grant, they are completing a Phase I proof-of-concept study of a single-cell analysis method tested on four breast cancer cell lines. Their positive selection method is based on a multi-antigen approach, including the antibodies for EpCAM and HER2.

    CTC isolation is then carried out on a Silicon Biosystems DEP Array system, which sorts and separates cells using dielectrophoresis. The user can instruct the system to sort the cells into defined single-cell populations.

    Using this system, Dr. Millner’s group has demonstrated the capability to perform single-cell Sanger sequencing. Future technology development will focus on using next-generation sequencing (NGS) to do whole-genome and whole-transcriptome analysis, and a proposed Phase II trial would apply the CTC isolation method to patient samples.

  • Next-Generation CTC Technology

    Commercially available since 2004, CellSearch has open-channel capabilities built into the technology that allow users to include additional antibodies for further phenotypic specification, or for targeting a therapy (such as the identification of HER2 receptors). Janssen Diagnostics is collaborating with Massachusetts General Hospital (MGH) on the development of next-generation CTC technology that will isolate CTCs based on both positive and negative selection.

    The recently published results from the SWOG S0500 study demonstrated the predictive value of CTC enumeration in patients with metastatic breast cancer as an indicator of the response to first-line chemotherapy and to guide the decision to change to an alternative chemotherapeutic regimen with the aim of improving overall or progression-free survival (PFS). Patients with elevated CTC counts prior to the start of therapy who subsequently showed low CTC counts three weeks later had a significantly better PFS and overall survival (OS) than did patients whose CTC counts did not decrease after the start of therapy.

    The latter patients, however, did not benefit from an early switch to an alternative chemotherapy. The authors concluded that for these patients, participation in clinical trials and access to experimental therapies should be considered instead of another line of chemotherapy.

    Negative selection of CTCs has particular advantages when analyzing the mRNA content of tumor cells, as “binding of anything to the surface of a membrane initiates signal transduction and starts changing the expression of certain molecules in a cell,” explained Dr. McCormack. Negative-selection methods also allow for single-cell sorting for NGS applications. In addition, Janssen Diagnostics has had success culturing CTCs, which can then be used to test for drug susceptibility or resistance.

    David Miyamoto, M.D., Ph.D., instructor in radiation oncology, Massachusetts General Hospital, described the successive generations of microfluidic devices a team of bioengineers, biologists, and clinicians at MGH have developed to isolate CTCs from blood samples. Through improvements and modifications in design and materials, the chip-based device has evolved to be able to capture single CTCs as well as clusters of CTCs and to provide enhanced adherence across the chip surface.

    MGH has tested its second-generation “herringbone CTC chip” in several pilot studies in various cancers including prostate, breast, and melanoma. Prototypes of a third-generation chip, the CTC iChip (the “i” stands for “inertial focusing device”), is  in development in collaboration with Johnson & Johnson. These prototypes have been engineered to facilitate downstream assays. Instead of the CTCs remaining trapped on the device, as with previous versions of the chip, the CTC iChip releases the CTCs into solution.

    “This allows for a range of applications and enables single-cell analysis,” noted Dr. Miyamoto. “You can isolate single cells” for RNA expression analysis, DNA analysis, or mutation analysis, for example.

    A key advantage of the third-generation device is its use of both positive and negative selection for CTC isolation, according to Dr. Miyamoto. Negative selection is achieved by coating all of the non-CTCs in a sample with magnetic beads, a procedure that targets the non-CTCs for removal, leaving the CTCs unperturbed. Dr. Miyamoto is studying the use of the device as a prognostic tool in prostate cancer, and in particular to isolate CTCs in blood samples from patients with metastatic castration-resistant prostate cancer to assay for androgen receptor signaling prior to treatment with second-generation androgen receptor targeting agents.

    “I think CTC technology is going beyond the enumeration and examination of molecular pathways that are activated or turned off as a result of therapies,” projected Dr. Miyamoto. “[It is becoming] a tool to guide targeted therapies and advance personalized medicine.”

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