High sensitivity of detection of TP53 somatic mutations by fluorescence-assisted mismatch analysis

We analyzed exons 5-9 of the TP53 gene in 41 breast cancers using direct sequencing, PCR-SSCP (single-strand conformation polymorphism), and fluorescence-assisted mismatch analysis (FAMA), to test the level of specificity and sensitivity of each method. A major issue for the correct detection of TP53 somatic mutations in primary tumors is often represented by the large amount of normal DNA, which can cause excessive dilution of the mutant allele, with consequent possible false-negative results. High sensitivity upon dilution of the mutant allele has been demonstrated for FAMA, a method based on the chemical cleavage of a mismatch within heteroduplex DNA molecules. Exons 5-9 of the TP53 gene were analyzed by FAMA using only two long bifluorescent fragments. Differences in sensitivity, accuracy, and specificity were observed among the above-mentioned procedures. Thirteen of the 41 samples (31.7%) revealed TP53 genetic alterations by automated sequencing, 19 samples (46.3%) were positive for SSCP, whereas 14 samples (34%) showed variants detectable by FAMA. Seven samples were positive in SSCP, but negative in both FAMA and sequencing assays; however, 2 SSCP-negative samples revealed evident signals by FAMA, indicating the presence of TP53 mutations. One of the latter samples showed the alteration by sequence analysis, whereas the other failed to reveal the mutation signal even by sequencing, as a consequence of the very small amount of the mutant allele resulting from the excess of contaminating normal DNA. Our results show that FAMA may represent a suitable and accurate assay for the routine diagnosis of TP53 somatic mutations in DNAs from solid tumor biopsies.