Resequencing Array-based Assays
Joseph A. Califano Jr., M.D., otolaryngologist, surgeon, and associate professor at Johns Hopkins University School of Medicine, has had nearly a decade of experience in mutation detection and has recently devised a resequencing array-based assay for detecting tumor-specific mitochondrial mutations in head-and-neck cancers.
Based on small quantities (less than 50 ng) of DNA from frozen tissue samples, the method employs the Affymetrix (www.affymetrix.com) MitoChip v 2.0 mitochondrial genome resequencing array, “which sequences the entire 16-kb mitochondrial genome at least twice,” according to Dr. Califano.
“We developed a simple algorithm to look at the secondary signal, representing hybridization signals from minor populations of mutant DNA. We have also demonstrated proof-of-principle in mixed samples of head-and-neck (tumor) cell lines and have found we can reliably detect mutations in a 1:200 solution.” In the real world, this translates to the much-desired ability to detect cancers in salivary rinses in lieu of invasive biopsy.
Thus far, Dr. Califano’s technique has been shown to detect malignancies in 10 of 13 known cancer patients, “including tumor-specific mutations. The advantage of this technique is the detection of rare mutant signals. There are seven or eight assays out there that perform the same task, but they must be optimized for individual mutations, require research-grade conditions, and incorporate finicky fluorescence-based assays that can take weeks to complete. This technique takes advantage of standard off-the-shelf arrays, standardized array cores, standard software, and a simple algorithm.
“It is widely applicable, easy to use, requires no special expertise, and is objective and quantitative, in contrast to the subjective interpretation of gels or shifts. In addition, mitochondrial mutations, found in virtually all solid tumors, are often many, thus an individual tumor may yield multiple opportunities for detection.”
The technique also has a potential application for monitoring patients after cancer surgery and applicability for detecting, in bodily fluids, mutations specific to other tumor types, Dr. Califano notes.