10/26/04 -- 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.
ther 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
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
"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
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.
Source: Johns Hopkins Medical Institutions