Multiple ways of silencing E-cadherin gene expression in lobular carcinomaof the breast

The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epit helial cells, in which it mediates adhesion and morphogenesis. Invasive lob ular carcinoma (ILC) characteristically infiltrates diffusely as single cel ls; by immunohistochemistry, many of these tumours lack E-cadherin expressi on. in the present study we investigated various ways in which loss of func tion of the E-cadherin gene could occur in ILCs, namely, promoter methylati on, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cad herin-negative samples by immunohistochemical analysis. Methylation-specifi c polymerase chain reaction (PCR) showed that 17/22 (77%) of these rumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadher in-negative tumours, All 16 exons of E-cadherin (including intron-exon boun daries) were amplified from chromosomal DNA and screened for mutations by c onformation-sensitive gel electrophoresis (CSGE), Bands with altered mobili ty were analysed by direct sequencing. We identified five frameshift mutati ons, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed u sing microsatellite markers, and 9/18 (50%) informative tumours showed LOH, We conclude that most ILCs show genetic or epigenetic changes affecting th e E-cadherin gene and that many of these tumours lack E-cadherin expression . In all cases in which there was loss of expression, this was consistent w ith biallelic inactivation of CDH1 by promoter methylation, mutation or all elic loss in any combination. (C) 2001 Wiley-Liss. Inc.