p53 mutations in deep tissues are more strongly associated with recurrence than mutation-positive mucosal margins.

PURPOSE: Application of ultrasensitive diagnostics has shown that small numbers of p53 mutation-positive cells may signify the presence of residual tumor in histologically normal tissues after resection of squamous cell carcinomas arising in the head and neck area. To date, most studies in this area have focused on analysis of tissues at the mucosal aspect of the resection and highlighted the importance of molecular changes in the field with respect to the risk of recurrence. EXPERIMENTAL DESIGN: In the present investigation, we analyzed normal tissues from mucosal and deep surgical margins, referred to as "molecular margins," for the presence of the signature p53 mutation identified for each tumor. RESULTS: The p53 mutation status of these carcinomas did not correlate with clinical or histopathologic variables, but these mutations provided an excellent target for ultrasensitive analysis of margin status. We found that 11 of 16 (68%) of cases with histologically tumor-free (including 9 without dysplasia), but with p53 mutation-positive molecular margins, developed recurrence. The probability of developing local recurrence was significantly higher for the group with p53 mutation-positive margins when compared with the group with clear margins (P = 0.048) and more strongly associated with p53 mutation-positive deep molecular margins than mutation-positive mucosal molecular margins or positivity at both sites (P = 0.009). CONCLUSIONS: This shows that although persistent mucosal fields may contribute to recurrence, clonal p53 mutations in deep tissues are an important cause of treatment failure, and molecular margins from both sites should be analyzed in future prospective series.

Molecular analysis of paired tumours: time to start treating the field.

Molecular analysis of paired tumours highlights the limitations of the current clinical criteria for identifying second primary tumours. At present the finding of identical novel microsatellite alleles in paired lesions provides a "gold standard" marker for establishing clonal origin. However, these aberrations occur at low frequency and other methods for determining clonality have been proposed. In the present study we have applied 3 molecular tests to establish whether it is possible to combine the results obtained with the different approaches to provide information about the likely origin of a second tumour when novel alleles are not found. Our findings provide substantive molecular evidence that a proportion of second tumours are recurrences of an index lesion and suggest that the finding of concordant allelic imbalance at two or more loci at two different chromosome arms together with concordant p53 mutations might provide a useful surrogate. We briefly review other published reports and emphasis the need to plan treatment to eliminate precursor lesions in the field rather than focusing on the visible primary lesion and the 1-2 cm of surrounding mucosa traditionally considered to be "at risk".

Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene.

BACKGROUND: Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations. MATERIALS AND METHODS: We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor. RESULTS: We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences. CONCLUSIONS: The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.