Sensitive detection of p53 mutation: analysis by direct sequencing and multisequence analysis.

In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We encountered some differences between the previously published reference p53 sequence and all our sequences and identified polymorphism in six regions.

Sequencing analysis of RNA and DNA of exons 1 through 11 shows p53 gene alterations to be present in almost 100% of head and neck squamous cell cancers.

Data on p53 alterations in human cancers are mainly based on studies restricted to the core domain (exons 5-9), because mutations outside this region are assumed to be rare. To test this assumption, we studied 25 consecutive patients with primary, untreated head and neck squamous cell carcinoma (HNSCC) with a p53 mutation analysis strategy that consists of sequencing all 11 p53 exons and the complete p53 mRNA. With this method, we encountered p53 mutations in 91% of patients; 33% of these were located outside the core domain. Overexpression of the p53 protein was assessed with staining with antibody Bp 53-12-1. Protein overexpression was found in 64%. In one case, p53 overexpression occurred without p53 gene mutations. Analysis of tumor tissue from two autopsied patients with multiple lesions in the head and neck and at distant sites allowed analysis of the clonal relationship of the different tumor foci. In one patient, the head and neck lesion had a mutation different from the one observed at the distant sites, suggesting two different primary tumors, one of them leading to widespread metastastic disease. In all lesions from the second patient, the same mutation was found, suggesting one primary that had metastatized. It appears that sequencing of all exons of the p53 gene is vital for assessment of the real incidence of p53 mutations in HNSCC, because 33% of all mutations are located outside the core domain, leading to a mutation frequency of almost 100% in HNSCC. In 96% of cases, either presence or absence of p53 protein expression could be explained by the type of p53 gene mutation. When only analyzing the p53 core domain, the incidence of p53 mutations in HNSCC is underestimated.

Ki-67 and p53 in T2 laryngeal cancer.

OBJECTIVE: To study the relationship between the proliferative capacity, represented by the immunohistochemical labeling index (LI) of proliferation marker Ki-67, and the p53 status, as in theory an intact p53 cell cycle checkpoint system should result in a lower proliferative capacity. STUDY DESIGN: From a group of 128 patients with a T2 laryngeal carcinoma, presented from 1989 to 1993 at the University Hospital Utrecht, 20 patients with recurrent disease and 16 patients without recurrent disease were randomly selected. All patients received primary irradiation. METHODS: Denaturing gradient gel electrophoresis and immunohistochemistry determined the p53 status. MIB-1 staining was used to determine the Ki-67 LI. RESULTS: In 36% of specimens we found a p53 mutation with overexpression (LI, 31%). In 8% a p53 mutation without p53 overexpression was found (LI, 18%). Forty-two percent showed no mutation but, nevertheless, overexpression (LI, 35%). Neither mutation nor overexpression was found in 14% (LI, 38%). No correlation exists between p53 status and proliferative capacity of tumors (analysis of variance [ANOVA]; P = .104). The proliferation rate as established with Ki-67 LI positively correlates with response to radiotherapy (P = .006). CONCLUSIONS: 1. Overexpression of wild-type p53 protein does not result in cell cycle arrest measurable by a lower Ki-67 LI in comparison with cases overexpressing mutant type p53 protein. 2. A high Ki-67 LI correlates with a favorable response to radiotherapy.

Discordance of p53 status in matched primary tumours and metastases in head and neck squamous cell carcinoma patients.

To study the use of p53 as a diagnostic tool in head and neck squamous cell carcinoma (HNSCC), we analysed 15 primary tumours (PT) and matched lymph node metastases (LNM) for overexpression and mutations of p53. The primary goal was to study whether differentiation between primary and metastatic disease through their p53 status would be possible. Immunohistochemistry for p53 protein (antibody BP 53-12-1) was performed. Mutations of the p53 gene were detected by exon-specific amplification of DNA (exons 4-9), followed by exon analysis using denaturing gradient gel electrophoresis (DGGE). Mutant exons were sequenced. p53 overexpression was detected in seven (47%) of the PT and in seven (47%) of the LNM. 6 patients (40%) exhibited p53 protein overexpression in both PT and LNM. 2 patients had a different p53 protein expression in each sample. Mutations in the p53 gene were detected in 6 patients (40%) in the PT and in 7 patients (47%) in the LNM. In 2 patients (13%), the same mutation was found in the PT and in the LNM. 9 patients (60%) had a different mutation in each sample. We conclude that a poor correlation exists between p53 protein overexpression and p53 gene mutation in HNSCC. Also, a poor correlation for both detection techniques exists, when PT and LNM are compared. The p53 status may seem to differ between PT and LNM because of polyclonality in the PT. More sensitive detection techniques could be promising.

Radioresistance and p53 status of T2 laryngeal carcinoma. Analysis by immunohistochemistry and denaturing gradient gel electrophoresis.

BACKGROUND: Animal experiments and tumor cell line studies have shown that p53 alterations can cause radioresistance. This has not yet been demonstrated in patient groups. METHODS: We report p53 status in 20 patients with T2 laryngeal carcinoma and recurrent disease after curative therapy. The control group consisted of 16 patients with T2 laryngeal carcinoma without recurrent disease. The p53 gene (exons 5 to 9) was analyzed by Denaturing Gradient Gel Electrophoresis (DGGE). Expression of p53 in biopsy material was visualized by immunohistochemistry (monoclonal antibody BP 53-12-1). RESULTS: The group with recurrent disease showed a mutation in 9 cases (45%) and overexpression in 14 cases (70%). In 17 cases (85%) either mutation or overexpression was found. The control group showed a mutation in 7 cases (44%) and overexpression in 14 cases (88%). In 14 cases (88%) either mutation or overexpression was found. Adding up both groups a discordance of 50% was found between both detection techniques. The same mutated exon was found in 6 patients (66%) in both primary and recurrent tumors. CONCLUSIONS: A discordance between immunohistochemistry and DGGE exists in 50% of the cases. Assuming that both p53 mutation and p53 overexpression are indicative of a disturbed p53 checkpoint system, 31 cases (86%) in both groups show an alteration of the p53 system. No significant difference in p53 status in patients with or without recurrent disease exists. Analysis of the p53 status is not of prognostic significance for irradiation as primary treatment.

Unilateral iliofemoral occlusive disease: long-term results of the semi-closed endarterectomy with the ring-stripper.

Nowadays, fewer endarterectomies are performed for treatment of occlusive arterial disease; more often a bypass procedure is done. This study investigates whether the results of the semiclosed endarterectomy for unilateral iliofemoral occlusive disease indeed indicate a wider use of bypass procedures for such short obstructions. Ninety-four patients with an obstructed external iliac and common femoral artery, but with patent ipsilateral common iliac and contralateral iliac arteries, underwent 101 operations. Seven of these patients were operated on at a later stage for occlusive disease on the contralateral side. Ninety-three endarterectomies were performed, and an iliofemoral bypass graft was inserted eight times because an endarterectomy was not feasible. Sixty-two operations were performed for disabling claudication, and 39 operations were performed for limb-threatening ischemia. Eighty-five percent of the patients who underwent an endarterectomy for disabling claudication became asymptomatic. Eighty percent of the patients who underwent an endarterectomy for limb-threatening ischemia became asymptomatic or improved to claudication. After endarterectomy no deaths, false aneurysms, or infections occurred. The patency rates at 1, 5, and 10 years were 94%, 83%, and 65%, respectively. We conclude that the semiclosed endarterectomy with the ringstripper of a unilateral obstruction of one external iliac and common femoral artery can be performed with a low morbidity and without deaths and gives good long-term results.