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

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.

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.

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.

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.”

ExoCap™, a new kit available from JSR Life Sciences for research applications, uses a magnetic bead-based isolation method to extract and enrich exosomes for use with cell culture supernatants without the need for ultracentrifugation. The magnetic beads are coupled to antibodies that recognize antigens on the surface of exosomes. The four ExoCap kits include an antibody targeted to capture exosomes with the EpCAM, CD9, CD63, or CD81 surface antigen, and the Composite ExoCap Kit, which combines all four antibodies.

“Binding [of the antibody-bead complexes] should not alter the exosomes,” asserted Kenneth Henry, Ph.D., senior research scientist. The company has shown that the vesicles isolated are characteristic of exosomes. They have a lipid bilayer membrane and a particle size distribution of about 100 nm, and they contain cargo of the sort found in exosomes.

Studies are under way to demonstrate whether these exosomes are biologically active once they are released from the beads. Nucleic acid analysis can be done directly from isolated exosomes that remain bound to the magnetic beads, Dr. Henry explained, or users can add a reagent included in the kit to release the exosomes from the beads for subsequent analysis.

Side-by-side comparison of miRNA recovery efficiency using a standardized sample of ultra-centrifuged exosomes from HT-29 cell culture supernatant spiked into buffer. [JSR Life Sciences]

Genotyping ctDNA

Mark Sausen, Ph.D., director of R&D at Personal Genome Diagnostics (PGDx), identifies three main areas of technology and applications development in the commercialization of noninvasive diagnostic approaches for using NGS in cancer. These include the capture and characterization of CTCs; the isolation and analysis of ctDNA; and exosome-based approaches.

PGDx is focusing on ctDNA, and earlier this year the company introduced the METDetect™ assay for detecting amplifications and structural changes of the MET cancer gene in blood samples from cancer patients. These changes are associated with the response to specific targeted therapies in clinical trials as well as resistance to other targeted therapies.

Furthermore, these structural alterations can serve as markers for monitoring treatment response and of overall prognosis and cancer recurrence. Whereas the mere presence of ctDNA has been used as a prognostic marker—for example, to detect minimal residual disease following surgical resection and as an indication of a greater chance for relapse—more recently, molecular tools such as NGS have been applied to ctDNA to look for a particular genotype.

Detection and NGS of ctDNA “is a very powerful tool to evaluate the genotype of a tumor without a biopsy,” said Dr. Sausen.

Both CTCs and ctDNA have advantages and challenges, and these may depend on the application you are using or clinical question you are asking. Overall, “it seems the levels of ctDNA are higher than the levels of CTCs in a particular blood sample,” Dr. Sausen observed. A challenge in isolating CTCs is not to miss them, as their numbers can be quite low.

With ctDNA, continued Dr. Sausen, “you are looking for somatic mutations that occur at low levels (with a large range)—from 50%—in the presence of a lot of wild-type DNA.” Detecting very low-level mutations in ctDNA is particularly problematic given that the error rates of NGS instruments can be approximately 1%, although experimental and bioinformatics approaches can overcome this limitation.

Norgen Biotek specializes in technology for collecting RNA or DNA from CTCs, exosomes, and other types of samples. Its silicon carbide resin “is much more sensitive than traditional silica-based columns,” according to Bernard Lam, Ph.D., a senior scientist at the company. It can be used to isolate DNA or RNA from single cells and can capture rare copies of transcripts without the need for carrier RNA. “By adjusting the chemistry, you can choose to elute either DNA or RNA, or both,” explained Dr. Lam. The samples are eluted in small volumes “and can go directly into next-generation applications,” he added.

The silicon carbide resin is also applicable for collecting microRNAs. “The technology has no bias,” Dr. Lam asserted, and can capture larger mRNA transcripts and smaller miRNAs without having to skew the chemistry to account for the different GC content in various RNA species.

Personal Genome Diagnostics (PGDx) performs CLIA-validated next-generation sequencing for the identification of MET amplification in plasma using circulating tumor DNA. Here, clinical libraries generated from cell-free DNA for the MET locus are being enriched in preparation for sequencing.

Biostabilization at Ambient Conditions

CTC technologies are among the many advanced molecular diagnostic tools being developed that require a high degree of precision, and “if something goes wrong from [sample] collection to analysis” it can introduce a lot of variability into the sample “and mask the results,” cautioned Rolf Müller, Ph.D., president, CSO, and founder of Biomatrica. Sample destabilization and degradation will affect the outcomes of analytical methods including detection of cell surface markers and cell-free DNA as well as genomic, transcriptomic, and proteomic analyses.

At present, “99% of biostability is done using cold chain management,” noted Dr. Müller. Whether current approaches to cold chain management will remain adequate, however, is unclear. Biostability demands are expected to grow with the rise in omics-based tests that require intact DNA, RNA, proteins, and metabolites. Another relevant trend is the commercial-scale development of diagnostics that rely on isolated CTCs or other live cell samples.

Scaling up the cold chain is costly, complex, and unreliable, according to Dr. Müller, and interruptions in the cold chain can result in freeze-thaw cycles, causing “tremendous cell stress—especially for CTCs—and cell lysis and degradation of nucleated cells.” Furthermore, cell surface markers “are extremely sensitive to freezing and thawing,” which can result in changes in biomarker profiles.

Biomatrica has developed a technology platform for the preservation of biomaterials at ambient temperatures that includes the stabilization of DNA, RNA, and proteins; blood and other patient samples; diagnostic assays; and live mammalian cells. It provides an alternative to cold chain storage and transport and cryopreservation.

Different types of biological samples and different applications have varying requirements for biostability. Accordingly, Biomatrica not only offers off-the-shelf biostabilization reagents, it also uses its proprietary screening technology and library of biostabilizers to optimize the collection of a particular sample type and develop a custom stabilization approach for a specific assay.

Earlier this year, Biomatrica and American Type Culture Collection (ATCC) signed a licensing agreement. As per the agreement, Biomatrica will supply ATCC with its DNAstable® and RNAstable® reagents for the stabilization of DNA and RNA standards at room temperature.

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