The Li-Fraumeni syndrome (LFS): a model for the initiation of p53 signatures in the distal Fallopian tube

Abstract

A candidate early precursor to pelvic serous cancer, the 'p53 signature', is commonly found in the benign mucosa of the distal Fallopian tube and harbours p53 mutations and evidence of DNA damage. We examined tubes from women with pre-existing (germ-line) mutations in p53 [Li-Fraumeni syndrome (LFS)] for evidence of this precursor. Fallopian tubes from two cases of LFS were immunostained for p53, Ki-67 (proliferation) and H2AX (DNA damage response) and analysed for p53 mutations by laser capture microdissection (LCM) and p53 genomic sequencing (exons 2-11). A common single nucleotide repeat (snp) in exon 3 (rs1042522) and deletion sequencing chromatograms in exon 4 were examined in combination to estimate LOH in both LFS tubes and advanced serous carcinomas from the general population. LFS tubal epithelium contained abundant (10-20 per section) p53 signatures with evidence of DNA damage and low proliferative activity. Six of 11 LFS microdissected p53 signatures (55%) and 15 of 21 serous carcinomas (71%) revealed LOH at the p53 locus, relative to background epithelium. The LFS model confirms prior observations that the distal Fallopian tube is particularly prone to focal epithelial p53 gene inactivation-p53 mutation and LOH-in the absence of malignancy or increased epithelial proliferation. The fact that the LFS is not associated with ovarian cancers is consistent with the concept that loss of p53 function must be accompanied by at least one more genotoxic event (including BRCA1/2 functional inactivation) to produce the malignant phenotype. This is in keeping with a general model of carcinogenesis, in which different and often independent risk factors operate at multiple points in the serous carcinogenic spectrum.

1

p53 signatures in a distal fallopian tube segment from two patients with Li-Fraumeni syndrome (LFS) (A&B) are characterized by strong linear nuclear p53 localization in secretory cells. The number of cells demonstrating nuclear DNA proliferation is low (C).

2

A p53 signature at higher power demonstrates normal morphology (A) and strong p53 nuclear staining (B). Punctate nuclear staining with an antibody to H2AX is characteristic of localization of γ-H2AX (C, arrows). Another p53 signature (D) stains strongly for bcl-2, a secretory cell marker (E).

3

Comparison of deletion chromatograms from control epithelia (no LOH, upper) and a p53 signature (LOH, lower). The sequence chromatogram of the heterozygous LFS deletion 482_487delCCATCT is shown in panel A. The position of the deletion is marked by the arrow and both wild type and mutant alleles are clearly present (as demonstrated by the presence of sequence from both alleles superimposed after cDNA position 481). The sequence chromatogram from sample LFS10 is shown in panel B. Only the mutant allele is present demonstrating loss of heterozygosity of the wild type allele.

4

A model for serous carcinogenesis in the distal fallopian tube, genotoxic stress imposed on the secretory cells (ovals) produces a p53 mutation followed by LOH of the non-mutated allele, with accumulation of p53 protein. Capacity of the affected cell for expansion is variable. Functional loss of BRCA1/2 via one or more mechanisms is a necessary component of progression to malignancy and is increased in women with inherited heterozygous BRCA1/2 mutations.