Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study.

PURPOSE: Approximately 10-30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence. EXPERIMENTAL DESIGN: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind. RESULTS: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9-56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions. CONCLUSIONS: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.

Mutated p53 as a molecular marker for the diagnosis of head and neck cancer.

In total, 10-30% of patients with head and neck squamous cell carcinoma (HNSCC) develop local recurrences despite seemingly adequate tumour resection. This may result from minimal residual cancer (MRC): small numbers of tumour cells left behind in the surgical margins, undetectable by routine histopathology. In recent studies, p53 mutations have been considered as selective and sensitive DNA markers of cancer cells. There are two potential problems in using mutated-p53 DNA as a marker. Firstly, p53 mutations occur early in progression and might therefore detect unresected precursor lesions besides tumour cells. Secondly, DNA is a very stable biomolecule that might lead to false-positive results. These two potential problems have been evaluated in this study. Fifty patients with a radical tumour resection were included, of whom 30 showed a p53 mutation in the primary tumour. Histopathologically tumour-free surgical margins were quantitatively analysed for mutated p53 by molecular diagnosis (plaque assay) and subsequent (immuno)histopathology. p53 mutated DNA was detected in the surgical margins of 19/30 patients. Immunohistochemistry confirmed the presence of small tumour foci in 2/19 mutated p53-positive cases. In 7/19 cases, the tumour-specific p53 mutation was found in unresected dysplastic mucosal precursor lesions. Moreover, in a number of cases small p53-immunostained patches were detected, but the mutations found were never tumour-related. By screening contralateral exfoliated cells and plaque assays on RNA it was shown that detection of mutated-p53 DNA is prone to false-positive results. In conclusion, using p53 mutations as a marker, both MRC and unresected mutated p53-positive mucosal precursor lesions are detected within surgical margins. Molecular assessment of surgical margins using p53 mutations enables the selection of HNSCC patients at high risk for tumour recurrence, but tumour RNA seems at present to be a more specific biomolecule for analysis than tumour DNA.

Discordance of genetic alterations between primary head and neck tumors and corresponding metastases associated with mutational status of the TP53 gene.

Ample molecular data are available on the progression from normal mucosa to invasive head and neck squamous cell carcinoma (HNSCC), but information on further genetic progression to metastatic disease is scarce. To obtain insight into the metastatic process, we compared 23 primary HNSCCs with 25 corresponding lymph node metastases (LNMs) and 10 corresponding distant metastases (DMs) with respect to TP53 mutations and patterns of loss of heterozygosity (LOH) based on 26 microsatellite markers on six chromosome arms (3p, 9p, 17p, 13q, 8p, and 18q). In 18 of the 23 patients, a TP53 mutation was detected in the primary tumor, and in all cases the same TP53 mutation was present in the corresponding LNM or DM. In nine of 20 patients with LNMs and three of seven patients with DMs, the LOH pattern of metastasis differed from that of the corresponding primary tumor by at least one marker. Microsatellite markers located on chromosome arms 13q, 8p, and 18q were most frequently discordant, providing evidence that alterations at these chromosomes occur late in HNSCC carcinogenesis. Moreover, evidence was found that DMs had developed directly from the primary tumor and not from LNMs. Remarkably, we observed that the mutational status of the TP53 gene is associated significantly with the degree of genetic differences between primary HNSCCs and corresponding metastases. All patients with TP53 wild-type primary tumors showed significantly more discordant LOH patterns in the corresponding LNMs and DMs than patients with TP53-mutated tumors. The percentages were 100% versus 27% (LNMs) and 100% versus 0% (DMs), respectively (P = 0.008 and P = 0.029; two-sided Fisher exact test). This finding suggests that TP53-mutated tumors need fewer additional genetic alterations to develop metastases compared with TP53 wild-type primary tumors.