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FEATURE ARTICLE

Salivary Diagnostics

Amazing as it might seem, doctors can detect and monitor diseases using molecules found in a sample of spit

David T. Wong

RNA and Cancer

Members of my laboratory discovered a few years ago that, much to everyone's astonishment, many RNA molecules, or transcripts, as they're often called, are present in the cell-free portion of human saliva. These transcripts include messenger RNA or "mRNA," the kind of RNA that cells use to convey the instructions carried in DNA for the manufacture of proteins. This finding was surprising because RNA tends to be quite fragile outside the confines of the cell. Indeed, we don't know for certain where this RNA comes from. Cells do not ordinarily secrete this type of molecule, so we suspect that it comes from leaky or broken cells. The makeup of the RNA population doesn't suggest that it originates from a single type of tissue, which leads us to conclude, for now, that such leaks or breaks may not be unusual considering the quantity of cells in the human body—a number that most experts estimate to be in the trillions. In addition to proteins, the collection of RNA molecules constitutes a second saliva-based diagnostic alphabet for disease diagnosis.

In a 2004 publication, we noted that saliva contains approximately 3,000 mRNAs, of which 180 were common between samples from 10 healthy people. This slight degree of overlap (17 percent) might be considered low for cells, all of which require a common set of "housekeeping genes" to provide their basic building blocks and to enable routine metabolism. However, because we studied the cell-free portion of saliva, we weren't particularly surprised by the results. Nonetheless, expecting the variation and explaining it are quite different propositions, and we cannot say why the large majority of RNAs from our study subjects were different. We suspect that this aspect of salivary function, similar to so many other physiological differences between one person and the next, will prove to be a product of both genes and environment.

Having established an RNA baseline for normal saliva, we began searching for RNA biomarkers that could help diagnose disease. Our first target was oral cancer, the most common form of cancer in developing countries. In India, for example, oral cancer accounts for 40 percent of all malignancies. The incidence is lower in the United States, where oral cancer makes up less than 3 percent of the total. Nevertheless, more than 28,000 new cases were diagnosed in this country in 2004. Tobacco use, whether it is smoked, chewed or held in the mouth, represents the primary risk factor for cancers of the mouth and pharynx. Drinking alcohol also raises the risk in a dose-dependent fashion, but to a much lesser extent than does tobacco. Additionally, some studies implicate certain human papilloma viruses in the genesis of these tumors. However, more than a quarter of oral cancer victims neither smoke nor drink and have no other lifestyle-related risk factors.

Oral cancer typically begins as a lump within the mouth, but finding it is not always straightforward. In the early stages, such growths are hidden more often than not, making visual inspection an unreliable means of diagnosis. Many of these abnormal tissues, visible or invisible, will never become cancerous. Furthermore, it can be difficult, even for experts, to distinguish between benign tumors and early-stage malignant ones. Of course, the most reliable means of diagnosis, a surgical biopsy, is also the most invasive and is unsuited for use as a screening technique. As a result, several other means of identifying this type of cancer have emerged in recent years. Some, such as illumination with certain wavelengths of light (autofluorescence) or application of the dye toluidine blue, detect higher concentrations of RNA and DNA in clusters of cancerous cells, which are often crowded together compared with normal cells. Suspicious areas can then be examined further with a biopsy. (Autofluorescence has already entered clinical practice in the United States; toluidine blue is in phase-III clinical trials and has been approved for use in Europe.) Another new technique uses a combination of vinegar, dye and special lights to heighten the ability to see lesions in the mouth. It's also possible for scientists to search under the microscope for cancer cells in tissue from a scrape of the cheek.

Figure%206.%20Micrograph%20showing%20a%20small%20tumorClick to Enlarge ImageThese techniques are generally effective at identifying malignancies. However, they have some disadvantages. Principally, they are all limited by the need to examine the external surfaces of the mouth, which must miss some proportion of early-stage cancers. In addition, and perhaps more important from a public-health standpoint, none of these technologies promises to become inexpensive enough to screen people in the general population, because they all require expert medical personnel to conduct the test and judge the outcome.

I hope that a saliva-based test will one day provide an improvement over any of these methods. The trick, of course, is to identify the telltale signs of oral cancer in saliva. In a search for RNA biomarkers, my colleagues and I compared the transcripts found in the saliva of people with early-stage oral cancer with those of people without cancer. This work involved a technique called microarray analysis, a type of test that allows the simultaneous measurement of thousands of RNAs.

In a microarray experiment, scientists attach a kind of molecular beacon to all the RNAs in a particular sample and then see what DNA sequences they attach to. (An RNA molecule and its mirror image, or complement, DNA bind tightly to each other.) The microarrays are palm-sized quartz wafers on which millions of DNA molecules are laid out in a grid, or array. There are about 22,000 different sequences among these short pieces of DNA, each corresponding to a different RNA transcript. The same sequence is present at many locations on the grid. This redundancy helps eliminate false signals and provides an internal check. After the tagged RNAs find their DNA complements, a laser "reads" each position on the array and indicates whether those RNAs are present in the sample and in what quantities.

Using this approach, we examined the salivary RNA from 32 patients with early stage oral cancer and 32 controls and found that four of the 180 common transcripts showed consistently different levels in cancer patients. Among them, 91 percent had the distinctive changes in these four RNAs, confirming that the markers are reliable indicators of cancer. When we searched a larger group of people, we found that very few people had these four biomarkers. But among those that did, more than 9 out of 10 had oral cancer. The next step will be to conduct a similar study on a wider scale. To date, we've confirmed the findings in more than 300 oral-cancer patients, and the National Cancer Institute's Early Disease Research Network has validated our results.








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