Detecting breast cancer’s fingerprint in a droplet of blood

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McGill team develops new technology that can accurately measure protein biomarkers

One in eight women will be diagnosed with breast cancer during her lifetime. The earlier cancer is detected, the better the chance of successful treatment and long-term survival. However, early cancer diagnosis is still challenging as testing by mammography remains cumbersome, costly, and in many cases, cancer can only be detected at an advanced stage. A team based in the Dept. of Biomedical Engineering at McGill University’s Faculty of Medicine has developed a new microfluidics-based microarray that could one day radically change how and when cancer is diagnosed. Their findings are published in the April issue of the journal Molecular & Cellular Proteomics.

For years, scientists have worked to develop blood tests for cancer based on the presence of the Carcinoembryonic Antigen (CEA), a protein biomarker for cancer identified over 40 years ago by McGill’s Dr. Phil Gold. This biomarker, however, is also found in healthy people and its concentration varies from person to person depending on genetic background and lifestyle. As such, it has not been possible to establish a precise cut-off between healthy individuals and those with cancer.

“Attempts have been made to overcome this problem of person-to-person variability by seeking to establish a molecular ‘portrait’ of a person by measuring both the concentration of multiple proteins in the blood and identifying the signature molecules that, taken together, constitute a characteristic ‘fingerprint’ of cancer,” explains Dr. David Juncker, the team’s principal investigator. “However, no reliable set of biomarkers has been found, and no such test is available today. Our goal is to find a way around this.”

Dr. Mateu Pla-Roca, the study’s first author, along with members of Juncker’s team, began by analyzing the most commonly used existing technologies that measure multiple proteins in the blood and developing a model describing their vulnerabilities and limitations. Specifically, they discovered why the number of protein targets that can be measured simultaneously has been limited and why the accuracy and reproducibility of these tests have been so challenging to improve. Armed with a better understanding of these limitations, the team then developed a novel microfluidics-based microarray technology that circumvents these restrictions. Using this new approach, it then became possible to measure as many protein biomarkers as desired while minimizing the possibility of obtaining false results.

Juncker’s biomedical engineering group, together with oncology and bioinformatics teams from McGill’s Goodman Cancer Research Centre, then measured the profile of 32 proteins in the blood of 11 healthy controls and 17 individuals who had a particular subtype of breast cancer (estrogen receptor-positive). The researchers found that a subset of six of these 32 proteins could be used to establish a fingerprint for this cancer and classify each of the patients and healthy controls as having or not having breast cancer.

“While this study needs to be repeated with additional markers and a greater diversity of patients and cancer subsets before such a test can be applied to clinical diagnosis, these results nonetheless underscore the exciting potential of this new technology,” said Juncker.

Looking ahead, Juncker and his collaborators have set as their goal the development of a simple test that can be carried out in a physician’s office using a droplet of blood, thereby reducing dependence on mammography and minimizing attendant exposure to X-rays, discomfort and cost. His lab is currently developing a hand-held version of the test and is working on improving its sensitivity so as to be able to accurately detect breast cancer and ultimately, many other diseases, at the earliest possible stage.

This study was funded by the Canadian Institutes for Health Research (CIHR), Genome Canada; Génome Québec; The Canada Foundation for Innovation (CFI), The Natural Science and Engineering Research Council (NSERC); and the Banque de tissue et de données of the Réseau de la Recherches sur le cancer (RRCancer) of the Fonds de recherche en santé du Québec (FRSQ).

http://mcponline.org/content/11/4/M111.011460.abstract

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Dr. Robert Nagourney
Medical and Laboratory Director
Rational Therapeutics, Inc.
Long Beach, California

A report last year described a novel application of the cell search technology developed by Veridex, LLC (a subsidiary of Johnson & Johnson) that may provide an extremely sensitive tool for the early detection of cancer. Four major cancer centers in the United States were to conduct an analysis to determine the accuracy of this method for early diagnosis.

Over recent years, it has been recognized that cancer patients circulate small numbers of tumor cells in their blood. Using microbead technology, these tumor cells can be isolated from the blood stream and characterized. The original application of the technology was a prognostic marker by which patients with breast, colorectal or prostate cancers and high levels of circulating tumor cells, fell in the “high-risk” groups.

The more recent iteration of this technique will allow investigators to not only identify but also characterize the isolated tumor cells. This provides an exciting new opportunity for early diagnosis.

As we speculate on the ramifications of this discovery, certain questions are raised. The most immediate being: What to do with the data? It has previously been suggested that many cancers arise 20 or 30 years before they are clinically detected. Malignant populations measuring in the hundreds of thousands, millions or even hundreds of millions, may still lie below the radar screen of modern diagnostic tools.

If we have the capacity to identify patients 10 or 20 years before their cancers can be clinically detected, would we then begin therapy decades before clinical disease arises? If so, what treatments will we administer? Will the early detection of cancer cells be associated with the further characterization of tumors, such that targeted agents can be utilized to eliminate these clones at their earliest inception?

We will watch the development of these clinical studies with great interest. It will be even more interesting to see how we answer the questions that arise.

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The cells that slough off from a cancerous tumor into the bloodstream are a genetically diverse bunch, Stanford University School of Medicine researchers have found. Some have genes turned on that give them the potential to lodge themselves in new places, helping a cancer spread between organs. Others have completely different patterns of gene expression and might be more benign, or less likely to survive in a new tissue. Some cells may even express genes that could predict their response to a specific therapy. Even within one patient, the tumor cells that make it into circulating blood vary drastically.

The finding underscores how multiple types of treatment may be required to cure what appears outwardly as a single type of cancer, the researchers say. And it hints that the current cell-line models of human cancers, which showed patterns that differed from the tumor cells shed from human patients, need to be improved upon.

The new study, which was published online in PLoS ONE, is the first to look at so-called circulating tumor cells one by one, rather than taking the average of many of the cells. And it's the first to show the extent of the genetic differences between such cells.

"Within a single blood draw from a single patient, we're seeing heterogeneous populations of circulating tumor cells," said senior study author Stefanie Jeffrey, MD, professor of surgery and chief of surgical oncology research.

For over a century, scientists have known that circulating tumor cells, or CTCs, are shed from tumors and move through the bloodstreams of cancer patients. And over the past five years, there's been a growing sense among many cancer researchers that these cells - accessible by a quick blood draw - could be the key to tracking tumors non-invasively. But separating CTCs from blood cells is hard; there can be as few as one or two CTCs in every milliliter of a person's blood, mixed among billions of other blood cells.

To make their latest discovery, Jeffrey, along with an interdisciplinary team of engineers, quantitative biologists, genome scientists and clinicians, relied on a technology they developed in 2008. Called the MagSweeper, it's a device that lets them isolate live CTCs with very high purity from patient blood samples, based on the presence of a particular protein - EpCAM - that's on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, the team first tested whether they could accurately detect the expression levels of 95 different genes in single cells from seven different cell-line models of breast cancer - a proof of principle since they already knew the genetics of these tumors. These included four cell lines generally used by breast cancer researchers and pharmaceutical scientists worldwide and three cell lines specially generated from patients' primary tumors.

"Most researchers look at just a few genes or proteins at a time in CTCs, usually by adding fluorescent antibodies to their samples consisting of many cells," said Jeffrey. "We wanted to measure the expression of 95 genes at once and didn't want to pool our cells together, so that we could detect differences between individual tumor cells."

So once Jeffrey and her collaborators isolated CTCs using the MagSweeper, they turned to a different kind of technology: real-time PCR microfluidic chips, invented by a Stanford collaborator, Stephen Quake, PhD, professor of bioengineering. They purified genetic material from each CTC and used the high-throughput technology to measure the levels of all 95 genes at once. The results on the cell-line-derived cells were a success; the genes in the CTCs reflected the known properties of the mouse cell-line models. So the team moved on to testing the 95 genes in CTCs from 50 human breast cancer patients - 30 with cancer that had spread to other organs, 20 with only primary breast tumors.

"In the patients, we ended up with 32 of the genes that were most dominantly expressed," said Jeffrey. "And by looking at levels of those genes, we could see at least two distinct groups of circulating tumors cells." Depending on which genes they used to divide the CTCs into groups, there were as many as five groups, she said, each with different combinations of genes turned on and off. And if they'd chosen genes other than the 95 they'd picked, they likely would have seen different patterns of grouping. However, because the same individual CTCs tended to group together in multiple different analyses, these cells likely represent different types of spreading cancer cells.

The diversity, Jeffrey said, means that tumors may contain multiple types of cancer cells that may get into the bloodstream, and a single biopsy from a patient's tumor doesn't necessarily reflect all the molecular changes that are driving a cancer forward and helping it spread. Moreover, different cells may require different therapies. One breast cancer patient studied, for example, had some CTCs positive for the marker HER2 and others lacked the marker. When the patient was treated with a drug designed to target HER2-positive cancers, the CTCs lacking the molecule remained in her bloodstream.

When the team went on to compare the diverse genetic profiles of the breast cancer patients' CTCs with the cells they'd studied from the cell lines, they were in for another surprise: None of the human CTCs had the same gene patterns as any of the cell-line models.

"These models are what people are using for drug discovery and initial drug testing," said Jeffrey, "but our finding suggests that perhaps they're not that helpful as models of spreading cancers." While the human cell-line cells did show diversity between each of the seven cell lines, they didn't fall into any of the same genetic profiles as the CTCs from human blood samples.

These results don't have immediate impacts for cancer patients in the clinic because more work is needed to discover whether different types of CTCs respond to different therapies and whether that will be clinically useful for guiding treatment decisions. But the finding is a step forward in understanding the basic science behind the bits of tumors that circulate in the blood. It's the first time that scientists have used high-throughput gene analysis to study individual CTCs, and opens the door for future experiments that delve even more into the cell diversity. The Stanford team is now working on different methods of using CTCs for drug testing as well as studying the relationship between CTC genetic profiles and cancer treatment outcomes. They've also expanded their work to include primary lung and pancreatic cancers as well as breast tumors.

Source: Stanford University Medical Center