In 1991, Johns Hopkins molecular geneticist Bert Vogelstein, M.D., approached his postdoc David Sidransky, M.D., with a startling proposal: Would Sidransky be willing to look for cancerous DNA mutationsin human feces? "This project sounded almost insane to me at the time," recalled Sidransky at the 2004 annual meeting of the American Association for Cancer Research. No one knew in 1991 whether colon cancer DNA could even survive in stool, much less be detected, so Vogelsteins idea sounded futile as well as revolting. "There werent that many people in the lab who wanted to work with stool," Sidransky said. "It made me wonder at the time about how much I wanted to do science."
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But the Exact test does not look for every possible mutation in its four target colon cancer genes, including K-ras. Even with limited coverage, it is still so expensive$795that it cannot effectively compete with colonoscopy for general use as a screening tool. Now, two new studies have found mutations unique to colon cancer that may lead to improvements in the ability to diagnose colorectal cancer at the molecular level.
In the April 23 issue of Science, Victor Velculescu, M.D., Ph.D., of Johns Hopkins University in Baltimore, reported the discovery of mutations in a PI3 kinase gene, and in the April 17 issue of The Lancet, a group led by Martin Midschwendter, M.D., of the Medical University Innsbruck in Austria, showed that methylation of a single gene could, with high sensitivity and specificity, identify colorectal cancer patients. Together with new mutation detection techniques Vogelsteins laboratory has developed, the twin gene findings give DNA cancer diagnostics a boost. No DNA diagnostic test has yet entered widespread use for cancer, but the new discoveries may move the field closer to that goal.
Obstructions to Progress
Colorectal cancer is one of a handful of cancers for which screening has shown the "gold standard" benefita reduction in mortality from the disease in randomized clinical trials. "Cancers can almost always be cured by simple, classical surgical techniques, if theyre detected early," said Vogelstein. With surgery and chemotherapy, doctors can cure 90% of colorectal cancer patients whose cancer is caught early, but only 10% of those with late-stage disease.
Some of the early detection methods in use for colorectal cancer leave a lot to be desired. Fecal occult blood testing, which tests for blood in stool, is cheap but has low sensitivity (ability to detect cancers among people who have the disease). Colonoscopy, while very effective at picking up colorectal cancer, may be ill-suited for population-wide screening because it is an invasive procedure that many people avoid. And even if the demand existed, there still are not enough trained surgeons to perform the test. Flexible sigmoidoscopy is also invasive, and it looks only at the lower colon. CT colonography, also known as virtual colonoscopy, looks promising but so far has performed unevenly in the hands of community radiologists.
A DNA stool test is noninvasive and does not require a vast number of specialists. But it must have high sensitivity and specificity (the ability to rule out disease among people who do not have the disease). Luckily, by the time Sidranksy found K-ras mutations in stool, colorectal cancer had been well-dissected at the genetic level. Mutations in the APC gene, cloned in 1991, are generally associated with early carcinogenesis. Mutations in K-ras and p53 come later. In theory, a panel of mutations selected from all three genes would cast a wide enough net to identify colorectal cancer with enough sensitivity and specificity to serve as a population-wide screening tool.
For the past dozen years the theory has confronted two kinds of obstacles: biological and technical. The biological problems stem from the fact that no single gene mutation was a perfect indicator of cancer. K-ras mutations also occur in hyperplastic polyps and in certain inflammatory conditions. APC mutations occur so early in the process that their presence may not always predict progression to invasive disease. And it was impossible to test for all the possible p53 mutations, because they are spread throughout the gene. Testing for all of them would send the cost of PCR primers and reagents spiraling out of control. So Exacts test, called PreGen-Plus, is a compromise, consisting of 21 different mutations in the three genes, plus a microsatellite instability marker. It also includes a "DNA integrity assay," which tests for differences in DNA length thought to be specific to cancer.
Because of the limited number of markers, PreGen-Plus is far from perfect. In a 2003 study comparing advanced-cancer patients with controls, the test demonstrated 63% sensitivity and 95% specificity. But those results only compared patients with known cancer with healthy controls. Last October, Exact disclosed results of a trial comparing PreGen-Plus with fecal occult blood testing in asymptomatic individualsthe first time the test had been tried on the population it is designed for. PreGen-Plus demonstrated 52% sensitivity overall for cancers and 57% for earlier stage cancers. Despite 95% specificity, this sensitivity is too low for population-wide screening.
Exacts stock price plunged at this news, because it seemed to suggest the test performed worse than before. But Exact Chief Technology Officer Tony Shuber pointed out that the trials statistical power was lowonly 30 cancers among the 5,000 patientsand that it solidly demonstrated that the test worked in asymptomatic people as well as in patients already diagnosed with cancer. Still, Shuber admitted, theres plenty of room for improvement. Shuber would like to see the sensitivity near or above 80%the level already achieved by colonoscopy.
Relief Ahead?
The new gene discovery reported in Science could conceivably help improve the ability of tests such as PreGen-Plus to diagnose cancer at the molecular level. First cloned in 1991, the PI3 kinases have been solidly implicated in carcinogenesis, mainly because the PTEN tumor suppressor gene works by reversing PI3 kinasemediated phosphorylation. But mutations in PI3 kinases were previously unknown in cancer. When Velculescus laboratory set out to systematically sequence the 15 PI3 kinase genes, he found PIK3CA to be the only one with mutations92 in all. "It was totally surprising," said Vogelstein. "When we first saw [the mutations], we said, Wow, its amazing that no one ever found this before." Even more exciting was the fact that most of the genes were clustered in two narrow regions. That meant that only a small fragment of two exons needed to be amplified to get at these mutationsa relatively inexpensive prospect. As a result, "its clearly going to be included in the next panel of markers," Vogelstein predicted.
Other cancer tests may also benefit. Almost one-third of colon cancers were found to have PIK3CA mutations, and such mutations were also found in glioblastomas, gastric cancers, and breast and lung cancers. "Im sure weve just scratched the surface," said Vogelstein.
The Challenges
Although they have yet to be detected in stool, the new mutations could go a long way toward solving the biological problem of the stool test: the need to assemble a panel of genes that will detect upwards of 80% of colon cancers, with few false positives. Vogelsteins laboratory is also attacking the biggest technical problem: how to detect individual DNA mutations in the vast sea of normal DNA in stool. Normal DNA is, on average, a thousand times more abundant than tumor DNA. This problem is a big reason sensitivity for PreGen-Plus is so lowmany mutations go undetected. The development earlier this decade of so-called "digital PCR" and "digital protein truncation" methods by Ie-Ming Shih, M.D., Ph.D., and Givoanni Traverso, Ph.D., among others, in Vogelsteins laboratory suggests a solution. The digital tests amplify small very small pools of DNAonly a few moleculesensuring that any mutant DNA gets detected. "By analyzing individual DNA molecules, you increase the signal-to-noise [ratio] many orders of magnitude," explained Vogelstein. The process is costly and labor-intensive, but Vogelsteins lab is working on improving its efficiency.
The test must compete not only against colonoscopy but also against flexible sigmoidoscopy, virtual colonoscopy, and fecal occult blood testing. "We have tests already that are working well," said Bernard Levin, M.D., vice president of cancer prevention at the University of Texas M. D. Anderson Cancer Center, Houston. "If any one of them were applied you could reduce [cancer] mortality." A noninvasive, highly accurate test would be superior, Levin agreed, if it were cheap enough. "The technology will improve," he said. "Theyre headed in the right direction. The question is, how high can you push the sensitivity and how low can you push the cost?"
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