Enzymatic mutation detection method evaluated for detection of p53 mutations in cDNA from breast cancers.
BACKGROUND
Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMD(TM)) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53.


METHODS
The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products 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 specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47).


CONCLUSIONS
EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.
Norberg
T
T
Cancer Centre Karolinska, Radiumhemmet, Karolinska Institute & Hospital, SE-171 76 Stockholm, Sweden. torbjorn.norberg@cck.ki.se
Klaar
S
S
Lindqvist
L
L
Lindahl
T
T
Ahlgren
J
J
Bergh
J
J
D001943:Breast Neoplasms / Q000737:chemistry*; D018076:DNA, Complementary / Q000737:chemistry*; D004706:Endodeoxyribonucleases; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D011933:Reagent Kits, Diagnostic; D016159:Tumor Suppressor Protein p53; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII