IMMUNOHISTOSELECTIVE SEQUENCING (IHSS) OF P53 TUMOR-SUPPRESSOR GENE IN HUMAN ESOPHAGEAL PRECANCEROUS LESIONS

Accumulation of p53 protein occurs in human oesophageal precancerous l esions and even in near-normal oesophageal epithelium, In some instanc es, p53 gene mutations have been detected, In many of the cases of p53 protein accumulation in early lesions, however, p53 mutations were no t detected due to either the lack of mutation or the low abundance of cells with a mutation, In order to enrich p53 immunostain-positive cel ls for single strand conformation polymorphism (SSCP) analysis and DNA sequencing, an immunohisto-selective sequencing (IHSS) method was dev eloped, Anti-p53 antibody-peroxidase stained oesophageal tissue sectio ns were subjected to ultraviolet (UV) irradiation to damage the DNA in p53 immunostain-negative cells, The immunostain protected p53 immunos tain-positive cells from the UV light and thus preserved the DNA in th ose cells for PCR amplification, Comparison of the SSCP results from s ections with and without UV treatment showed that the IHSS method sele ctively enriched p53 immunostain-positive cells. With this method, we could analyse mutations in samples with as few as 30 p53 immunostain-p ositive cells per tissue section, Analysis was carried out on tissues with precancerous lesions from six surgically-resected oesophageal spe cimens and 13 oesophageal biopsies from symptom-free subjects, The res ults of mutation analysis for some of the samples were confirmed by mi crodissection to enrich the p53-positive cells, The mutations in tissu es with precancerous lesions were compared with those in the correspon ding squamous cell carcinomas, The IHSS method is shown to be a simple and effective way to analyse mutations in p53 immunostain-positive ce lls, IHSS may also be a general method for molecular analysis of biolo gical specimens after immunohistochemical staining.