P53 tumor suppressor gene mutations in laryngeal cancer and in recurrent disease following radiation therapy.

In this study we performed p53 sequencing based mutation analysis in laryngeal cancers and matched recurrent disease following irradiation. The question is if irradiation affects the DNA and introduces or deletes mutations so that p53 cannot be used as a clonal marker anymore. P53 mutations were identified in fresh-frozen laryngectomy specimens with either primary laryngeal cancers, treated by surgery and irradiation post-operative with local failure during follow-up, or with recurrent laryngeal cancers following primary irradiation. In 21 tumors the p53 status was analyzed by direct sequencing full-length mRNA through RT-PCR. DNA sequencing analysis of exons 2 through 11 was performed when RNA isolation could not be performed. The marker mutation identified in this way was detected by DNA sequencing of the corresponding exon in formalin-fixed deparaffinized tumor biopsy samples in respectively matched recurrent disease following surgery and irradiation or primary tumor before irradiation. DNA sequencing analysis of the corresponding exon of peripheral blood leukocytes excluded the presence of germline mutations or polymorphisms. In 16 out of 21 tumors (71%), a mutation was identified. Fifteen of these marker mutations were detected in the matched tumor biopsy sample (94%). The only case lacking the marker mutation probably was a second primary tumor. We conclude that we find no direct evidence for induction or loss of p53 mutations following irradiation. Consequently, p53 may be used as a diagnostic tool when histological examination fails, for example in discriminating between the presence of a second primary tumor in the same area versus recurrent disease.

p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases.

In order to define the diagnostic value of p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma (HNSCC), we investigated p53 mutations in primary tumors (PT) and matched lymph node metastases (LNM); the underlying question being whether differentiation between metastatic disease of a known PT or (a metastasis of) a synchronous or metachronous second tumor is possible by means of p53 sequencing-based mutation analysis. In 15 PT, the p53 status was analyzed, following RNA isolation, cDNA synthesis and polymerase chain reaction amplification, by direct sequencing full-length mRNA. Mutations thus found were confirmed by DNA sequencing analysis of the corresponding exon in the PT. When RNA isolation was defective, DNA sequencing analysis of exons 1 through 11 was performed. In the matched LNM, DNA analysis of the corresponding exon was performed to prove the presence of the same p53 mutation. In the event of small clones not detectable by direct sequencing, an oligo ligation assay was developed to detect a specific mutation. The presence of germline mutations was excluded by DNA sequencing analysis of the corresponding exon of peripheral blood leucocytes. In 14 PT (94%), a mutation was identified. In one PT, no p53 mutation could be identified either after full-length mRNA sequencing or after sequencing exons 1 through 11. In all cases of PT and matched LNM, the mutations proved to be identical. We conclude that p53 mutations develop in carcinogenesis before metastases occur and are maintained during metastasis. Consequently, p53 may serve as a clonal marker not susceptible to change during tumor metastasis. This merits further exploration of the application of p53 mutation analysis in differentiating between metastatic disease from a known PT versus a metastasis of another second PT.

Production of lipid mediators in experimental keratitis of rabbit eye.

The inhibitors of prostaglandin (PG) or leukotriene (LT) synthesis and antagonists of platelet-activating factor (PAF) or LTs are inhibitory in experimental keratitis and clinical symptoms of keratitis are reproduced by application of these lipid mediators. This suggests that PGE2, LTB4, LTD4, and PAF are involved in experimental immunogenic and toxic keratitis. The objective of the present study is the measurement of the concentrations of lipid mediators in the aqueous humour and their release by the cornea and iris during keratitis. In both inflammatory models the concentrations of PGE2, LTB4, LTD4, and PAF in the aqueous humour were significantly increased as compared to their controls. The release of PGE2, LTB4 and LTD4 from the cornea, and of PGE2, LTB4, and PAF from the iris was significantly increased compared to that from control tissues. The results are consistent with a role for these lipid mediators in the inflammatory models. Combined therapeutic use of synthesis inhibitors or antagonists of these mediators in eye inflammation seems possible and may serve as an alternative to topical corticosteroid therapy.

d-Ibuprofen in ocular inflammation induced by paracentesis of the rabbit eye.

The anti-inflammatory compound d-ibuprofen has been investigated for anti-inflammatory and cyclooxygenase inhibitory activity in ocular inflammation induced by paracentesis of eyes of living rabbits. d-Ibuprofen is the dextro-rotary isomer of ibuprofen, a potent non-steroidal anti-inflammatory agent. Eyes pretreated topically with d-ibuprofen 0.8% showed a significant inhibition of aqueous protein (73.0%) and prostaglandin E2 (96.4%) increase after paracentesis as compared to paracentesized untreated fellow eyes and control eyes. In aqueous humor no significant correlation between the increase in prostaglandin E2 and protein could be established after paracentesis. These results indicate that d-ibuprofen could be a useful ocular anti-inflammatory agent as cyclooxygenase inhibitor.