Diverse p53 alterations in ulcerative colitis-associated low-grade dysplasia: full-length gene sequencing in...

In long-standing ulcerative colitis (UC), p53 mutations have been shown to occur by indirect detection methods such as PCR-SSCP. To clarify whether p53 gene mutations are early events in UC-associated neoplasia and to analyse clonality within dysplasia-associated lesions or masses (DALMs), the entire coding region of the p53 gene was analysed in DNA of microdissected single crypts by the polymerase chain reaction (PCR)-direct sequencing method. With a novel microdissection method using serial histological sections, the p53 gene (exons 2-11) was analysed in a total of 11 regenerative crypts and 76 single crypts within seven DALMs selected from three colectomy specimens of long-standing UC patients. Although p53 point mutations were found in at least one crypt in each DALM, heterogeneity in terms of the presence and the type of genetic change was marked, except in one carcinoma. As early events, p53 gene mutations were apparent even in some regenerative crypts (8/12 crypts). Some were of silent type. Altered p53 protein expression was confirmed in only 14/32 mutated crypts and was also evident in 24 other non-mutated examples by immunostaining of serial sections. Polyclonal p53 gene mutations were found in regenerative (REG) crypts and low-grade dysplasia (LGD), but monoclonal changes were noted in high-grade dysplasia (HGD) or carcinoma (Ca) in long-standing UC. At the single crypt level, however, p53 point mutations were not always linked to p53 overexpression, indicating a discrepancy between gene alteration and protein accumulation in LGD.