Enzymatic mutation detection method evaluated for detection of p53 mutations in cDNA from breast cancers

Background: Rapid, reproducible, and easily run methods with high sensitivi ty and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMD (TM)) method by analysi s of archival cDNA from 203 breast canter patients and comparison with resu lts of cDNA-based sequencing of the tumor suppressor gene p53. Methods: The EMD technology uses the T4 endonuclease VII, which cleaves dou ble-stranded DNA at sites where a DNA mismatch is present because of mispai ring or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were anal yzed separately. After PCR amplification, the fragments were hybridized wit h wild-type p53 and subsequently exposed to the ER ID enzyme. Cleavage prod ucts were analyzed and scored using an ALF (TM) automated DNA sequencer and ALFwin Fragment Analyzer software (Ver. 1.02). Results: The EMD technique had sensitivities of 45% and 64% and specificiti es of 83% and 84% for the two fragments, respectively. Patients with EMD-po sitive, wild-type p53 tumors had a survival similar to that of patients wit h EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutat ed tumors according to sequencing had a statistically significantly worse o verall survival than those with p53 wild-type tumors (P = 0.01.6), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.97).