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In Circulation

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They are small, just 22 nucleotides or so in length, but they may have a big role to play in diagnosing and determining the course of various cancers.

Many researchers had long thought that microRNAs played a role in cancer development and progression. But in 2008, some noticed that miRNAs could be detected in the blood, possibly paving the way for their use as cancer biomarkers.

"Before 2008, it wasn't even known that circulating microRNAs existed," says Muneesh Tewari, a researcher at the Fred Hutchinson Cancer Research Center in Seattle. He began studying the role of miRNAs in cancer in 2005, when he had "this idea that perhaps microRNAs might be released from the tumor cells and they might be more stable than longer messenger RNAs. And we just tested it out."

The expression of a number of miRNAs has been linked to different cancers and their progression. But the origins of these miRNAs and how they arrive in the blood are not yet fully known. Still, miRNAs as biomarkers of cancer are being validated and are wending their way toward the clinic.

"MicroRNAs are, for several reasons, interesting biomarkers," says Jörg Ellinger, a researcher and clinician at University Hospital Bonn in Germany. "They are functionally important ... and microRNA expression profiles allow [for] distinguishing malignant and non-malignant tissue and allow [for] distinguishing different tumor entities."

MiRNAs have a number of other properties that would make them good biomarkers, researchers say — they are important in gene regulation, they are amplifiable, and they are stable.

In normal tissue, miRNAs regulate genes. The Hutch's Tewari points out that one miRNA can regulate anywhere from a dozen to a hundred messenger RNAs. "MicroRNAs are inherently regulatory molecules, and there is evidence from tissue biomarker studies that microRNAs can be pretty important biomarkers in the tissue," he says, adding that "measuring the microRNA may basically have a lot of information content."

And as compared to proteins — which are also used as biomarkers — miRNAs "are one level higher in the regulatory hierarchy," says Luke Selth, a research fellow at the University of Adelaide in Australia.

There is also a small number of miRNAs. Emily Wang at City of Hope in California says there are only about 800 validated miRNAs, compared to, for example, hundreds of thousands of messenger RNAs. "We only need to analyze about 800 to 1,000 microRNAs to get a global picture of a given tumor," she says. Further, because they are nucleic acids, there are a number of extant tools that can be used to analyze miRNAs, like PCR.

MiRNAs are also uncommonly stable. Adelaide's Selth notes that in his work, he uses blood samples that are up to a decade old. "The microRNAs are totally fine and can be readily quantitated," he says.

Tewari says that miRNAs are also quite stable in newer clinical specimens. "We can take plasma, leave it at the bench for 24 hours, and there's no decline in the microRNAs that we measure. We can freeze and thaw it several times without observing a decline," he adds.

This stability might be due to the miRNAs' small size, but also to the complexes in which they are found in the blood. Shortly after reports came out showing that miRNAs were present in the blood, new research indicated that such circulating miRNAs were often found either in exosomes or microvesicles. "A leading hypothesis in the field at the time was that microRNAs were potentially stable because they were encapsulated in these vesicles," Tewari says.

But, more recently, his group reported in PNAS that most circulating miRNAs are actually associated with Argonaute2, which is part of the RISC silencing complex. "It makes sense that microRNAs might be circulating as a RISC complex since that's how they exist inside the cell," he says.

Linked to cancer

In a 2008 PNAS paper establishing that circulating microRNAs could be used to detect cancer, the Hutch's Tewari and his colleagues also found that serum levels of miR-141 could distinguish prostate cancer patients from healthy controls. Similarly, Ellinger's group has found that miR-1233 circulates in higher concentrations in people with renal cell carcinoma, which it reported in PLOS One in 2011. That miRNA, Ellinger says, was basically unknown prior to that study, but his group suggests that it has diagnostic implications.

Other circulating miRNAs have been linked to patient prognosis. City of Hope's Wang has tied an miRNA to cancer progression. Her team examined serum from 42 breast cancer patients with locally advanced disease and found two miRNAs that correlated with outcomes, as it reported in the Journal of Translational Medicine earlier this year. In a validation set, the prevalence of one — miR-122 — in the blood of early-stage breast cancer patients seemed to predict metastasis.

Origins

But just where circulating microRNAs come from — normal tissue or tumor tissue — and how they are released into body fluids — through cell death or some other process — are mostly unanswered questions, though researchers have a few ideas.

One possibility, the Hutch's Tewari says, is that cell death could cause miRNAs to be released into the blood or other body fluids. Another is that cells might selectively release miRNAs in vesicles or exosomes.

In healthy tissue, Adelaide's Selth adds, there is evidence that cells release miRNAs, both in vesicles and in protein complexes. Those miRNAs can then act as intercellular signaling molecules. "If they get taken up by a recipient cell, they can obviously modulate gene expression of that recipient cell," he says.

Tumor tissue cells may act similarly. They could, Selth says, release miRNAs that promote a microenvironment that helps the tumor. That, he adds, could give tumors a selective advantage. "In cancer and other diseases, I think we'll be finding out that certain of these microRNAs are selectively released by the diseased tissue and are acting to promote the disease," he says.

Overall, he adds, "I think we have a long way to go to determine just how or what the balance is between passive release by various ways or release that is programmed within the cell."

Additionally, Wang at City of Hope says that host cells, like immune cells, might also be sources of miRNAs. "I think that they can come from, in a cancer patient, either tumor cells or host cells," she says.

Tewari's lab has found that some miRNAs linked to cancer may come from blood cells. As he and his team reported in Cancer Prevention Research in 2011, many circulating miRNAs linked to solid tumors are also expressed in blood cells themselves. Tewari says that this is not "a major concern," but it is a factor that researchers need to consider in their analyses.

As part of cancer signatures, however, Wang points out that it often does not matter where miRNAs come from, so long as they are well-validated markers. "Some microRNAs are directly from the tumors, some are not, but the source of the microRNA in the blood would not affect their application as biomarkers," she says.

Development

While much is still unknown about the nature of circulating microRNAs, many of them are trumpeted as possible tests for diagnosing cancer or determining its progression. But before they really catch on as a clinical tool, a number of challenges need to be met.

One such challenge is to establish standardized protocols for extracting and quantifying circulating miRNAs. Adelaide's Selth says that researchers in the field are working toward developing such standards, but it has been difficult to do so as the technology keeps changing and improving. "I think that in the next five to 10 years, we'll have worked out the best way to quantitate microRNAs in blood and also in other body fluids," he says.

The Hutch's Tewari adds that a common way of analyzing miRNA levels — real-time PCR — relies on standard curves or independent synthetic standards. That works well for research studies, he says, but "ultimately, for clinical translation, I think it has a lot of limitations." His group is looking into using digital PCR to work around that drawback of traditional PCR.

Researchers also need large patient cohorts to draw on to thoroughly validate miRNAs they discover in small populations — first for retro-spective studies but then also for large-scale prospective studies. "I think that we definitely require a well-balanced, large cohort," City of Hope's Wang says.

Both she and Selth are planning their own individual retrospective studies and are gathering cohorts for those, to validate markers they have each identified. "And then the next step would be to do prospective studies," Wang says.

Still, tests are making their way toward the clinic. Dave Hoon at the John Wayne Cancer Institute in California has developed a serum-based assay to detect levels of circulating miR-21. He and his colleagues reported in Clinical Chemistry in 2011 that their real-time qPCR-based test can detect miR-21 and can be used to distinguish various stages of breast cancer from one another. "Especially in breast cancer, you want minimally invasive assays at very early stage to detect changes and many blood assays can't do that sensitively," he says.
He and his team are working on a further refinement of their assay that can use smaller amounts of starting material and detect multiple markers.

And in the future, researchers imagine that many miRNAs will be used in conjunction to gauge the status of a patient's cancer. "We know that some of these microRNAs are not specific for one type of cancer," Selth says. He notes that an miRNA that he and his colleagues are interested in, miR-141, occurs at higher levels not only in prostate cancer, but in other cancers as well. "I think they will definitely need to be applied as panels and certainly we know from genes and from proteins that that improves the utility of biomarkers," he adds.

It is already possible to detect such a signature of multiple miRNAs. "It's very easy to do multiplex PCR and look for, for example, five or six microRNAs," Wang says.

Such signatures, though, might not be quite ready for use in the clinic. "I think we've made very good progress," Selth says, referring to circulating miRNA tests for prostate cancer. "I would unfortunately say it's still a ways away."

"I think that for the field as a whole to have a major impact," Tewari adds, "there's a lot more work, really some groundwork that needs to be done."

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