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Unraveling metastasis with circulating tumor cells

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Anne Paxton

May 2014—Some diseases have clear origins and unfold in predictable ways, but cancer isn’t one of them. Despite legions of studies over the decades, cancer tumorigenesis and its deadly sequel, metastasis, essentially remain a riddle, wrapped in a mystery, inside an enigma, as Winston Churchill once described Russia.

But scientists do know that circulating tumor cells (CTCs) are one of the keys to metastasis. Like migrants, invaders, or colonizers from nation-states seeking to expand, CTCs leave the primary tumor, travel through the body’s vasculature, and somehow serve as seeds for cancer’s spread.

It’s been 10 years since seminal research led by Massimo Cristofanilli, MD, reported in the New England Journal of Medicine (2004;351:781–791), showed that the presence of CTCs revealed by a diagnostic assay of the blood is predictive of overall survival in patients with metastatic breast cancer. That study helped lead the Food and Drug Administration to approve CellSearch, by Veridex (now Janssen Diagnostics LLC), the first test to count CTCs in a blood sample to help clinicians with prognosis in breast cancer. FDA approval of CellSearch use in prostate and colorectal cancer followed soon after.

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Circulating tumor cells are “seeds” of fatal metastasis, a process consisting of sequential and rate-limiting steps where rare metastasis-competent CTCs shed from primary tumors are able to survive in the circulation and colonize organs distant from the primary lesion. CTC-induced brain metastasis results from a CTC subpopulation with defined characteristics. Illustration courtesy of Dr. Marchetti.

With the mass of research and new test development completed since then, does it appear that CTCs can shed light on tumorigenesis and metastasis—and more importantly, that their use can help stage and treat patients with metastatic cancer? CAP TODAY asked pathologists and clinicians in the field that question and found that circulating tumor cells are yielding up the secrets of their role in cancer progression reluctantly, but that clinical use of CTCs continues to grow.

As with any biomarker, not all the hoped-for applications have panned out, and widespread clinical use remains several years away. Still, tantalizing research findings, innovative test technologies, and promising clinical applications are re-stoking optimism about CTCs’ future.

“To me and to many of my colleagues, CTC is a very important test right now to help us understand whether or not therapy is working, and if it isn’t, to really make some judgments about what to do next in those patients,” says Richard J. Cote, MD, chair of the Department of Pathology at the University of Miami School of Medicine, chief of pathology at Jackson Memorial Hospital, and director of the Dr. John T. Macdonald Foundation Biomedical Nanotechnology Institute. He predicts an even more central role for CTCs in the future. “In the broader perspective, CTCs will perhaps be the single most important general test that we can perform in cancer, particularly in patients with metastasis.”

That broader perspective may be needed, because recently the field of CTC research was unsettled by disappointing results of a major clinical trial. Hopes that CTCs could be used to guide breast cancer therapy were dealt a setback in December at the San Antonio Breast Cancer Symposium, when investigators reported on results of a phase three clinical trial funded by the National Cancer Institute. The study, known as SWOG S0500, found that when metastatic breast cancer patients were switched from one form of chemotherapy to another based on a continuing elevated CTC count, using the CellSearch test, it made no difference in the patients’ overall survival or time to progression.

Those findings, of course, could relate more to shortcomings of the therapies than to the value of CTCs as a biomarker. The study did validate that patients with elevated CTC counts (five or more cells per 7.5 mL sample), at both baseline and 21 days after starting their first chemotherapy, have a worse prognosis, and SWOG study coordinator Jeffrey B. Smerage, MD, PhD, said this result could indicate that this patient population needs more effective treatment options beyond traditional chemotherapy.

Dr. Marchetti

Dr. Marchetti

But the report was not particularly encouraging news, says Dario Marchetti, PhD, professor of pathology and immunology and director of the CTC Core Facility at Baylor College of Medicine in Houston. Unfortunately, most insurance companies do not consider tests for CTCs to be reimbursable based on the clinical results so far, and this new study will not help the field of CTC research. He sees the findings as affirming the need for strong characterizing studies, looking at CTC properties by marker presence, functionality, and other parameters.

“We need to further our knowledge regarding CTCs and cancer, and we also need to potentially make additional CTC platforms available for clinical testing,” Dr. Marchetti says. He stresses that CellSearch detects only a subset of CTCs, and it’s important not to leap to conclusions about the clinical usefulness of CTCs based on this one study.

In fact, he believes CTCs are fundamental in combating metastases, in particular those targeting the brain, which have been his main interest since his father died of brain cancer many years ago. Several studies have shown that CellSearch does not detect the CTCs that invade and colonize the brain. His center was the first to bring this to the forefront, Dr. Marchetti says.

Of several hundred patients he has studied who were clinically diagnosed with brain cancer metastasis, CTCs in the vast majority—65 percent to 70 percent—were not detectable by the CellSearch platform, which uses the epithelial cell adhesion molecule, or EpCAM. Last year in Science Translational Medicine, his center reported that four different proteins, HER2+, EGFR, heparanase, and Notch1, spell out the signature of CTCs that colonize the brain (Zhang L, et al. 2013;5 [180]:180ra48).

Representative image of breast cancer brain metastasis induced by human CTCs following injection into mice. The work published by Dr. Marchetti’s laboratory in Science Translational Medicine (2013) provides the first-time evidence of CTC isolation from patients’ blood, and the characterization of CTCs possessing metastatic competence in experimental animals. Photo courtesy of Dr. Marchetti.

Representative image of breast cancer brain metastasis induced by human CTCs following injection into mice. The work published by Dr. Marchetti’s laboratory in Science Translational Medicine (2013) provides the first-time evidence of CTC isolation from patients’ blood, and the characterization of CTCs possessing metastatic competence in experimental animals. Photo courtesy of Dr. Marchetti.

Dr. Marchetti emphasizes that he has great respect for the CellSearch platform and for the investigators who developed what he calls a “marvelous piece of precision engineering.” But, he adds, the observation about the CTCs that CellSearch did not detect “stimulated our desire to look into alternative methodologies, like flow cytometry, microfiltration, and dielectrophoretic-based CTC platforms. The whole field is a puzzle, a work in progress.”

Since CTCs are the seeds of further metastases, the CTC Core Facility at Baylor is focusing on predicting metastases of the brain as well as other organs, and how to adjust therapy according to biomarkers and progress over time. “By understanding more about the biology of these cells, we have the ultimate goal to develop an assay that can be a clinically useful tool to interrogate and monitor the progression of cancer within the patient in real time,” Dr. Marchetti says. “Taking a blood aliquot is certainly less painful than a biopsy.” And through a real-time “liquid biopsy,” “we can monitor the course of the disease longitudinally in relation to the properties of the CTCs characteristic of these metastases.”

His objective would be one day to put into the clinic the first diagnostic test to predict breast cancer brain metastases. Right now, though, that’s a faraway goal. To develop clinically useful tools for oncologists, Dr. Marchetti stresses the need to learn more about the properties of the CTCs and CTC subsets, especially which cells are able to invade or colonize an organ, versus those that remain dormant or quiescent, or die. “We need to know far more about the molecular characteristics at the base of CTC heterogeneity. This investigation will help towards our goal to monitor therapy effectiveness in patients and adjust it according to the presence or absence of defined biomarkers.”

CTCs are not the only culprit in cancer progression, he cautions, pointing to circulating DNA and exosomes as other possible factors. “But within the complex microenvironment of the blood, CTCs can be one important aspect. Certainly improving our understanding of these cells will be very important, if not essential, in better understanding the biology of cancer metastases. We are convinced, and we are not the only ones who are convinced, that CTCs will be a fundamental piece of the entire puzzle of metastasis.”

Aside from CellSearch, at least five other methods of capturing CTCs are in development or undergoing analytical validation: flow cytometry, which sorts cells by size and surface antigen expression; microchips to capture CTCs as blood flows past EpCAM-coated microposts; filters with pore size that retains CTCs but permits smaller cells to pass; imaging techniques relying on Fiber-Optic Array Scanning Technology (FAST) to use fluorescent labels to identify CTCs; and negative enrichment that eliminates all cells from blood samples except CTCs.

Dr. Linden

Dr. Linden

But for the time being, CellSearch is the only FDA-approved test, and the CellSearch instrument is finding a home in more and more laboratories. When Michael A. Linden, MD, PhD, was a hematopathology fellow at the University of Washington a few years ago, the laboratory there was one of the early ones to bring the CellSearch instrument online. The University of Minnesota, where Dr. Linden is now assistant professor and hematopathologist in the Department of Laboratory Medicine and Pathology, has acquired CellSearch instrumentation, is conducting clinical validation, and plans to offer the test in-house early this summer.

Interestingly, purchase of the instrument was enabled in part because a patient heard about the test and, considering it to be important, donated a significant amount of money to the institution for its purchase. (The purchase ultimately was made possible by contributions from the private donor, the university, and Fairview Health Services.) “Our initial plan is to offer the test for clinical use, but we’ll also have it as an opportunity for researchers who want to study CTCs as biomarkers in clinical trials, as well as use the platform to investigate new diagnostic assays,” he says.

He considers the CellSearch test remarkable, given that the standard diagnostic cutoff in a 7.5 mL sample of blood is only three to five CTCs amid the billions of red and white blood cells in the sample. “The technology is very powerful,” Dr. Linden says. “But as the test is designed to enumerate very rare events, there is some degree of imprecision at low concentrations of CTCs. This is an important consideration, especially when the measured concentration of CTCs is near the diagnostic cutoff.” In addition, the stains used to detect CTCs in patients with metastatic carcinoma can also detect non-neoplastic epithelial cells, he says. “We don’t normally have detectable epithelial cells circulating in our blood. However, if a single CTC is detected in a patient with a low pretest probability of metastatic carcinoma, the data should not be overinterpreted.”

The kits most commonly used with CellSearch are validated to detect CTCs or carcinoma cells only in breast, prostate, and colorectal cancer, but Dr. Linden is confident that the technology can be adapted to find other cell types. “You’d just have to modify the reagents used to include antibodies that recognize antigens on the surface of your CTC of interest.” Some institutions are already doing studies of that possibility, he says. “I think the technology will be really informative in learning about the biology of metastatic neoplasia.”

While his institution plans to focus on enumeration for now, characterization is becoming a priority for many in the field. “Researchers are interested in elucidating the immunophenotypic and genotypic differences between a primary neoplasm and the CTCs that are shed. Do they express different surface antigens or have different genetic properties that make them capable of circulating rather than remaining localized to the main neoplasm? There are still unanswered questions about the biology of metastatic neoplasia, and characterizing the CTCs that seed metastases may lead to greater understanding of the processes, with implications for new diagnostic and therapeutic strategies.”

Dr. Linden would like to encourage pathologists to take ownership of and be leaders in recommending clinical tests to their clinician partners. “At a certain point, clinicians treating cancer patients are going to order a CTC test and may look to their pathologist for help. Pathologists play a vital role, even if they do not perform the test in-house. We as pathologists can help clinicians understand the data supporting test clinical utility, interpretation of test results, and the analytical limitations of the test.”

Minetta Liu, MD, of the Department of Medical Oncology and the Department of Laboratory Medicine and Pathology at Mayo Clinic Rochester, has already incorporated the use of CTC enumeration in her clinical practice. “The prognostic value of CTC enumeration is repeatedly demonstrated with each reported clinical trial in metastatic breast or prostate cancer,” she says. “Those patients with elevated CTCs are much more likely to have inferior outcomes compared to patients with undetectable levels or low numbers. The questions are: How can we take advantage of this information, convert those patients with unfavorable to favorable CTC counts, and improve overall survival?”

In patients with metastatic breast cancer, she explains, clinicians typically obtain restaging scans at nine- to 12-week intervals to assess for responsive, stable, or progressive disease. However, “inter- and intrareader concordance in determining progression versus no progression by imaging studies alone is not 100 percent,” she says. “We need to improve our ability to identify the proper point in a patient’s disease course to change therapy. I don’t want to waste time and expose patients to toxicity if the drug is no longer helping, but I also don’t want to abandon an intervention if it is still providing benefit.”

CTCs are reflective of underlying tumor biology, she says. “In my clinical practice, I use serial CTC enumeration by the FDA-cleared technology as an adjunct to routine bloodwork, clinical evaluations, and imaging studies. In my laboratory research efforts, we are focused on the molecular characterization of CTCs by various platforms, with the goal of establishing CTC analyses as reliable predictors of treatment benefit to specific agents,” Dr. Liu says.

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