The causes and functional consequences of E-cadherin (E-CD) loss in breast cancer are poorly understood. E-CD loss might act in concert with alterations in the APC/␤-catenin pathway to permit oncogenic ␤-catenin signaling. To test this hypothesis, we have analyzed the presence of genetic and epigenetic alterations affecting E-CD (CDH1), APC and ␤-catenin (CTNNB1) genes and the immunohistochemical expression of E-CD, ␤-and ␥-catenin in a series of 46 infiltrating lobular breast carcinomas (ILCs). Since 80% of ILCs featured complete loss of E-CD expression, we analyzed the molecular alterations responsible for E-CD inactivation in these tumors. We found that 10 of 46 (22%) cases harbored mutations in CDH1, including 1 case with 2 different mutations (1 of which was germline). CDH1 was also inactivated by loss of heterozygosity (LOH; 30/41, 73%) and promoter hypermethylation (19/46, 41%). Interestingly, LOH and mutations were also detected in the corresponding in situ lesions of the ILCs, implying that these alterations are early events in lobular cancer tumorogenesis. Additionally, the presence of a polymorphism in the CDH1 promoter was found to be inversely correlated with CDH1 mutations, but not with E-CD levels. We next examined whether alterations in the APC/␤-catenin pathway also occurred in the same series of ILCs. Although no CTNNB1 or APC mutations were detected, promoter methylation (25/46, 52%) and LOH (7/30, 23%) of APC were found. Moreover, methylation of APC and CDH1 occurred concordantly. However, ␤-and ␥-catenin were severely reduced or absent in 90% of these tumors, implying that alterations in CDH1 and APC genes do not promote ␤-catenin accumulation in ILC. These molecular alterations were not associated with microsatellite instability. In summary, several different mechanisms (mutations, LOH, methylation) are involved in the frequent CDH1 inactivation in invasive and in situ lobular breast cancer. The same tumors also show genetic and epigenetic alterations of APC gene. However, altered CDH1 and APC genes do not promote ␤-catenin accumulation in this tumor type.Key words: lobular breast cancer; E-cadherin; ␤-catenin; APC; mutation; methylation; loss of heterozygosity; microsatellite instability Lobular carcinoma of the breast accounts for 10 -15% of breast cancer cases and has a distinctive histology characterized by a diffuse growth pattern that is due to loss of cell cohesiveness. This weakened intercellular adhesion might be mediated by dysfunction of the E-cadherin/catenin adhesion complex and related molecules. The transmembrane glycoprotein E-cadherin, which mediates calcium-dependent intercellular adhesion in epithelial tissues, plays an important role in embryogenesis, polarization, differentiation and cell migration. 1 E-cadherin-mediated adhesion is essential for maintaining normal epithelium, since its dysfunction is intimately related to malignancy. 2 In fact, E-cadherin gene (CDH1) is considered a tumor suppressor, and its loss has also been demonstrated to promote tumor invasion and...

Cyclin D1 is frequently overexpressed in human neoplasias by gene rearrangement and amplification, but no mutations in the CCND1 gene have so far been reported. However, in vitro mutagenesis of CCND1 has shown that substitutions affecting threonine 286 residue produced cyclin D1 nuclear accumulation, by interfering with protein degradation and induced neoplastic transformation in murine fibroblasts. To test whether similar genetic changes may occur in vivo, we analysed a series of 60 endometrioid endometrial carcinomas (EECs) for cyclin D1 expression and gene amplification by immunohistochemistry and FISH, respectively. Two of 17 carcinomas showing cyclin D1 expression in more than 5% of neoplastic cells, but without gene amplification, were found to harbor single-base substitutions in CCND1 that changed proline 287 into threonine and serine, respectively. Both cases expressed cyclin D1 in more than 50% of neoplastic cells. Additionally, seven tumors with cyclin D1 overexpression of an independent series of 59 EECs were also analysed, and a 12-bp in-frame deletion that eliminated amino acids 289-292 was detected in one case with cylin D1 expression in more than 50% of neoplastic cells. In contrast, no mutations of the CCND1 gene were detected in a set of breast carcinomas with cyclin D1 overexpression without gene amplification. In summary, our data indicate that mutations of CCND1, which probably render the protein insensitive to degradation, represent a previously unreported mechanism of cyclin D1 overexpression in human tumors in vivo.

Several molecular alterations, most commonly PTEN mutations but also K-ras mutations, microsatellite instability, and beta-catenin mutations have been detected in endometrioid endometrial carcinoma (EEC). Specifically, mutations in the beta-catenin gene are seen in 15% to 20% of EECs, whereas immunohistochemical expression of beta-catenin ranges from 13% to 44%, nuclear staining being concentrated in areas of immature squamous metaplasia (squamous morules). Complex endometrial hyperplasia with atypia (CEH-A) is a well-known precursor of EEC, which can also show immature squamous metaplasia. In this study, we compared the immunohistochemical and molecular profiles of 13 CEH-A with and 11 CEH-A without squamous morules (SM) for mutations of beta-catenin, PTEN, K-ras, and microsatellite instability (MSI). In all cases of CEH-A with SM, beta-catenin immunostaining showed strong and diffuse nuclear expression in areas of SM and weak to moderate nuclear expression in the glandular component. Six different beta-catenin mutations were found in 7 of 13 cases (54%) (G34E, G34V, S33C, D32Y, S33F, D32A); however, no mutations of the PTEN or K-ras genes were identified. beta-Catenin immunostaining showed focal nuclear staining in only 2 cases of CEH-A without SM. Only 1 case had a beta-catenin mutation (S45A), which was associated with a K-ras mutation (G12D). Another 3 cases had both PTEN and K-ras mutations (C296Stop Ex 8 and G12V, 244del C Ex 7 and G12D, 251ins TGAT Ex 7 and G13D), and one had a PTEN mutation (G230E Ex 7) only. Of all 24 cases, only 1 case of CEH-A without SM showed MSI. In conclusion, marked differences in the molecular profiles regarding beta-catenin, PTEN, and K-ras mutations were observed between CEH-A with and without SM. beta-catenin mutations might represent a signaling pathway leading to a distinctive morphology in hyperplastic/neoplastic endometrium with SM. Other molecular events such as K-ras or PTEN mutations are likely to occur in CEH-A independently from beta-catenin. Furthermore, morphologic differences between CEH-A with SM and CEH-A without SM seem to correlate, at least to some extent, with the clinical course of the disease. In our series, cases of CEH-A with SM and beta-catenin alterations appeared to have a less aggressive behavior when compared with CEH-A without SM and with K-ras and PTEN mutations.

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It has been recently suggested that ovarian serous carcinoma follows a dualistic pathway with low-grade carcinomas arising from borderline tumors and high-grade carcinomas originating de novo. Similarly, our group has shown that based on their molecular profile endometrioid borderline tumors could predate low-grade endometrioid ovarian carcinomas (EOC). It is not clearly understood if low-grade EOC is in turn related to high-grade EOC, or if high-grade EOC may also arise de novo. The aim of our study was to compare the molecular profile of grade 1, 2, and 3 EOCs. Twenty-nine EOCs were selected including 10 grade 1 (G1), 11 grade 2 (G2), and 8 grade 3 (G3). Selected blocks were immunostained with beta-catenin and p53, and also microdissected, DNA extracted and amplified by polymerase chain reaction with primers for exon 3 of the beta-catenin gene, codons 12 and 13 of KRAS and codons 1 to 9 of PTEN. The length of BAT-26 and BAT-25 was analyzed to determine microsatellite instability (MSI). Patients with G1 EOC ranged from 21 to 71 (mean 52) years, those with G2 tumors ranged from 43 to 66 (mean 56) years, and patients with G3 EOC ranged from 41 to 67 (mean 57) years. Immunohistochemical analysis for beta-catenin showed nuclear staining in 14 cases (7 G1, 5 G2, and 2 G3 tumors), whereas the rest showed membranous staining. Beta-catenin mutations were found in only 3 G1 tumors. KRAS mutations were seen in 5 EOCs (2 G1 and 3 G2). MSI and mutations of PTEN were both detected in 1 G1 and 1 G2 tumor, respectively. There was no overlapping expression of MSI, beta-catenin, PTEN, or KRAS mutations. Finally, p53 overexpression was present in 6 EOCs (5 G3 and 1 G2), all G3 p53 positive tumors being negative for all other markers, whereas the G2 tumor also showed a KRAS mutation. In conclusion, beta-catenin and KRAS mutations, and MSI were strongly associated with low-grade EOC. In contrast, p53 overexpression characterized high-grade EOC, with no other molecular alterations present in the vast majority of these tumors. On the basis of these results, we suggest that there may also be a dual pathogenetic pathway for EOC.