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New tool reveals molecular signature of cancer and HIV

27.10.2004


’LigAmp’ highly sensitive



Scientists have designed a new molecular tool, dubbed "LigAmp," to pinpoint DNA mutations among thousands of cells, the equivalent of searching for a single typo in an entire library of books. Preliminary studies in a small number of cell lines and body fluids show the ultra-sensitive test may help detect microscopic cancer and HIV drug resistance.
"Other molecular tests make it very difficult to locate a mutation in a particular cell surrounded by thousands of other cells that don’t have the mutation," says James Eshleman, M.D., Ph.D., who led the study with colleagues from the Johns Hopkins Department of Pathology and Kimmel Cancer Center. "LigAmp essentially filters background ’noise’ caused by normal cells and reveals specific mutations."

The researchers say that sensitive tests to locate mutations could identify cancer in patients at high-risk for the disease. Such tests could even help detect a recurrence of cancer by monitoring whether the number of mutations rises above a predetermined threshold value.



In addition to cancer detection, the Hopkins mutation-finder appears able to detect drug-resistant HIV. The team tested it on blood samples from a handful of patients with HIV and located DNA mistakes in the virus itself that make it resistant to certain antiretroviral drugs. Results of analyses of the new test are published in the November issue of Nature Methods.

"We designed LigAmp to improve how we look for extremely subtle variations in viral and cellular DNA," says Eshleman, an associate professor of pathology and oncology and associate director for the DNA Diagnostics Laboratory at Johns Hopkins. "The molecular code of normal cells may look identical to cancerous except for a single rung in the DNA ladder-structure."

The test works by creating a molecular "magnet" with an affinity for the DNA mistake, also known as a point mutation. If the mutation is found, the magnet binds to it and inserts a bacterial gene. The bacterial gene serves as a red flag and produces a fluorescent color visible to powerful computer programs.

In their studies, the Hopkins investigators tested LigAmp on colon cancer cell lines, blood from HIV patients, and fluid from cancer patients’ pancreatic ducts. Single mutations in colon cancer cells and drug-resistant HIV viruses were detected at dilutions of up to 1 in 10,000 molecules. Mutations of the KRAS2 gene were detected in duct fluid samples from three pancreatic cancer patients, which also corresponded to mutations found in their tumors. LigAmp also located a drug-resistance mutation, called K103N, in blood samples from three HIV patients.

Further analysis of LigAmp with larger sample sizes and blinded panels of clinical samples currently is under way. "Some initial studies show that we can simultaneously look for different mutations and quantify the number of mutated molecules present. This may help us build panels of cancer markers for screening and determine low or high levels of mutation."

Funding for this research was provided by the Maryland Cigarette Restitution Fund, the National Cancer Institute, and the National Institute of Allergy and Infectious Diseases.

Johns Hopkins colleagues working with Eshleman are Chanjuan Shi, Susan Eshleman, Dana Jones, Noriyoshi Fukushima, Li Hua, Antony Parker, Charles Yeo, Ralph Hruban, and Michael Goggins.

Vanessa Wasta | EurekAlert!
Further information:
http://www.jhmi.edu

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