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

The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E-cadherin expression. In the present study we investigated various ways in which loss of function of the E-cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cadherin-negative samples by immunohistochemical analysis. Methylation-specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadherin-negative tumours. All 16 exons of E-cadherin (including intron-exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation-sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the 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 with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.     

The incidence of microsatellite instability and loss of heterozygosity in fibroadenoma of the breast

A majority of studies have shown an increase in the risk of breast cancer among women previously diagnosed with fibroadenoma (FA). At present there is conflicting evidence whether some of the chromosome abnormalities frequently found in breast carcinoma, such as loss of heterozygosity (LOH), are already present in FAs and other types of benign breast disease and, if present, whether such abnormalities are associated with the observed increase in risk. Microsatellite instability (MSI) is also recognised as a marker of genetic damage and is thought to occur when there has been damage to the cell's mismatch repair (MMR) system. We have analysed 39 cases of FA obtained from paraffin-embedded tissue for the presence of MSI and LOH at 11 loci to determine if these types of genetic alterations occur in FA. The incidence of MSI and LOH found were 4 of 395 (1.0%) and 5 of 271 (1.8%) informative loci tested respectively. Approximately 8% of cases were positive for MSI and 10% were positive for LOH, with one specimen having multiple occurences of both MSI and LOH. We conclude that these forms of genetic alteration do occur in FAs but that the incidence is low.     

Germline mutations of the E-cadherin gene in families with inherited invasive lobular breast carcinoma but no diffuse gastric cancer

BACKGROUND:

Present data are highly suggestive but do not unequivocally prove the cosegregation of germ‐line CDH1 mutations with inherited invasive lobular breast cancer (ILBC).

METHODS:

Two Caucasian families with 6 pathologically confirmed ILBC cases but no diffuse gastric cancer (DGC) were identified in our oncogenetics consultations. Screening for mutations of CDH1, BRCA1, and BRCA2 genes was performed on blood samples. When available, loss of heterozygosity (LOH) and immunohistochemistry (IHC) analyses were performed on tumor samples.

RESULTS:

No BRCA1 or BRCA2 mutation was found. Deleterious CDH1 germ‐line mutations c.283C>T and c.1582del were found in all the 4 living women with ILBC in family 1 and family 2, respectively. The mutation c.283C>T was also present in a healthy 71‐year‐old male and 2 obligate carriers in family 1. No DGC was observed in the 2 families. Loss of the wild‐type CDH1 allele in 1 of the breast tumors was confirmed by LOH and IHC studies, in accordance with the “2‐hit” model of tumor suppressor genes.

CONCLUSIONS:

Germline CDH1 mutation can be cosegregated with ILBC in the absence of DGC. Present data do not support recommendation of prophylactic gastrectomy in CDH1 germline mutation carriers with ILBC. Cancer 2011. © 2011 American Cancer Society.     

Infrequent occurrence of microsatellite instability in sporadic and familial breast cancer

Microsatellite instability was analysed in 93 primary breast tumours at 13 chromosomal loci frequently altered in breast cancer. RER (replication errors) were observed at a low (5%) frequency in sporadic, familial and hereditary breast tumours, as well as in breast tumours from patients with multiple primary cancers. Our study suggests that the RER+ phenotype is rare in breast tumours, and that breast cancer is not included in the hereditary non-polyposis colon cancer (HNPCC) syndrome. Moreover, the RER+ tumours revealed an atypical pattern of microsatellite alteration as compared with those usually seen in HNPCC tumours. In agreement with the findings in HNPCC tumours, all RER+ breast tumours were diploid, although having a similar frequency of allelic imbalance as RER— tumours. Thus, mismatch repair deficiency is rare in breast cancer, is most likely caused by somatic mutations, and possibly in a set of DNA repair genes different from that involved in the HNPCC syndrome.     

乳腺癌APC基因突变和杂合缺失及甲基化的研究

目的:探讨结肠腺瘤性息肉病基因(adenomatous polyposis coli, APC)基因在乳腺癌发生发展中的作用.方法: 应用PCR-SSCP、微卫星分析、甲基化特异性PCR和RT-PCR方法检测乳腺单纯性增生、非典型增生和癌组织中APC基因突变密集区突变状况、APC基因杂合性缺失(LOH)、启动子1A区甲基化状态以及mRNA表达.结果: 乳腺癌组织中未发现APC基因突变.乳腺癌组织中APC基因启动子1A区甲基化率为36.8%,甲基化与TNM分期呈正相关,P《0.05.乳腺癌组织中非甲基化组织APC mRNA表达为0.55±0.09,甲基化组织表达为0.21±0.14,两者比较差异有统计学意义,P《0.05.APC基因LOH发生率达32.6%,LOH和甲基化呈正相关,P《0.05.结论: 乳腺癌发生发展过程中APC基因出现异常改变.乳腺癌组织中APC基因失活的机制不是基因突变,而是该基因LOH和启动子1A区甲基化.     

Genetic instability in lung cancer: concurrent analysis of chromosomal, mini- and microsatellite instability and loss of heterozygosity

To investigate what kind of genetic instability plays important roles in lung carcinogenesis, we analyzed micro- and minisatellite instability, loss of heterozygosity (LOH) and chromosome instability in 55 cases of lung cancer, including, 10 squamous cell, 5 large cell, and 3 small cell carcinomas, and 37 adenocarcinomas. Analysis of minisatellite instability, the mechanism of which is different from microsatellite instability, has not been reported previously. Minisatellite instability was detected in only one case (1/55, 1.8%), and the frequency of microsatellite instability was low, being found only in three cases (3/55, 5.5%). In contrast, LOH, for at least in one locus, was observed in 27 cases (49.1%). In adenocarcinomas, the frequency of LOH was higher in poorly differentiated compared to more differentiated carcinomas. For chromosome instability, a similar correlation between differentiation grade and instability was observed in adenocarcinomas. And instability was more common in large cell and small cell carcinomas than in adenocarcinomas. Our analysis showed that chromosome instability and LOH, rather than mini- and microsatellite instability, play significant roles in the development of lung cancer.     

Crossregulation of NF-kappaB by the APC/GSK-3beta/beta-catenin pathway

Glycogen synthase kinase-3beta (GSK-3beta) and adenomatous polyposis coli (APC) play an important role in the regulation of beta-catenin. Inhibition of or defects in their functions can lead to activation of beta-catenin. beta-catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF-kappaB). However, the regulatory roles of GSK-3beta/APC on the NF-kappaB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK-3beta/APC might regulate NF-kappaB activity through beta-catenin. We found that inhibition of GSK-3beta suppressed NF-kappaB activity, whereas reexpression of APC restored NF-kappaB activity in APC mutated cells. The regulatory effects were through beta-catenin because depletion of beta-catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF-kappaB target TRAF1 was inversely correlated with activated beta-catenin. Thus, APC/GSK-3beta, through beta-catenin, may crossregulate NF-kappaB signaling pathway.     

Detection of microsatellite alterations in nipple discharge accompanied by breast cancer

Nipple discharge in breast cancer cases was examined loss of heterozygosity (LOH). DNA samples were extracted from both supernatant and cell pellet components of the discharge, and examined for LOH at microsatellite markers, D11S1818, D11S2000, D16S402, D16S504, D16S518, D17S520, and D17S786. At least one LOH was found in either the supernatant or cell pellet in seven out of 10 patients (70%). Five of seven samples, which were cytologically negative, were LOH positive, and only one case, which was cytologically positive, showed no LOH on the markers examined. All three samples, which were judged negative by CEA measurement (<400 ng/ml), were LOH positive. This method could be a useful novel diagnostic modality for nonpalpable breast cancer with nipple discharge.     

Characterization and clinical evaluation of microsatellite instability and loss of heterozygosity in tumor-related genes in gastric cancer

Microsatellite instability (MSI) detection is widely used in the diagnosis and prognosis evaluation of colorectal cancer. However, for gastric cancer (GC), there is no standard panel of microsatellites (MSs) used in clinical guidance. The present study aimed to identify useful predictors of the clinical features and for the prognosis of GC, based on an investigation of MSI and loss of heterozygosity (LOH) in tumor-related genes. First, from 20 tumor-related genes which were proven to be important to the development of GC, 91 MSs were identified, and PCR amplification, short tandem repeat scanning analysis and TA clone sequencing were used to analyze MSI and LOH in the first set of 90 GC samples. Subsequently, the same method was used to detect the MSI/LOH of the optimized loci in the second set of 136 GC samples. MSI/LOH in the mismatch repair genes was highly consistent with that in oncogenes and tumor suppressor genes, respectively. The length of the core sequence was a main factor for the MSI/LOH rate. The MSI of 12 single loci was significantly associated with lymph node metastasis. The MSI in TP53-1 and the LOH in MGMT-10 were significantly associated with early stages of tumor infiltration depth. The LOH in MGMT-10, PTN-2 and MCC-17 was significantly associated with TNM stage. The LOH in TP53-1 and ERBB2-12 was associated with adenocarcinoma. The MSI/LOH in 6 single loci of 5 tumor-related genes was associated with poor prognosis of GC. The present study demonstrated that the MSI/LOH of loci in tumor-associated genes was associated with 4 clinicopathological characteristics and outcomes of GC. These results may provide potential specific biomarkers for the clinical prediction and treatment of GC.     

The clinical value of microsatellite instability and a loss in heterozygosity in sporadic breast cancers

We analyzed loss of heterozygosity (LOH) and microsatellite instability (MI) in 108 cases of sporadic breast cancers using 22 microsatellite markers on 12 chromosomes. LOH was frequently seen in 1p (13%), 6p (18%), 8p (11%), 11p (18%), 13q (21%), 16q (31%), 17p (44%) and 17q (29%). Individual patients were scored according to the degree of LOH at the above eight chromosomal markers. Patients with no LOH were scored as 1, patients with one to three LOH were scored as 2, and patients with four or more LOH were scored as 3. A high LOH score correlated with a high histological grade (p = 0.019) and a poor prognosis (p = 0.0035). Eleven (10.2%) of 108 patients with breast cancer showed MI, with 6 cases showing MI at a single locus and 5 at multiple loci. Of the 11 Mi-positive patients only one had lymph node involvement (p = 0.015), none had histological grade 3 disease, and Mi-positive patients tended to have a better prognosis than Mi-negative ones. These data suggest that MI may be an early event in mammary tumorigenesis, and that LOH occurs at a later stage. The LOH score may be a useful prognostic marker of operable breast cancer.     

Genetic alterations on chromosome 17 in human breast cancer: relationships to clinical features and DNA ploidy

Summary We analyzed DNA from 105 primary breast cancers to assess amplification of the ERBB2 gene and loss of heterozygosity (LOH) on chromosome 17 using 4 polymorphic markers, and investigated the relationships of these genetic alterations to clinicopathological characteristics including DNA ploidy. Amplification of the ERBB2 gene was observed in 28% of the tumors. ERBB2 was amplified in tumors of all clinical stages and amplification was significantly linked to lymph node metastasis. LOH atD17S5 was observed in 28 of 57 informative tumors, while 17 of 62 informative tumors showed allelic loss atTP53. Among the 37 tumors informative for both loci, 32% showed LOH at these loci and 49% retained both alleles, indicating that there was a significant relationship between LOH atD17S5 and atTP53. We also examined LOH at theD17S74 andNME1 loci on chromosome 17q. LOH atD17S74 andNME1 was observed in 20% and 22% of the informative tumors, respectively, but there was no significant association between LOH at these loci. Of the 4 loci tested, LOH atTP53, D17S74, andNME1 was associated with clinical stage. Lymph node metastasis was correlated with LOH atNME1. Moreover, allelic loss was more frequent in aneuploid tumors than in diploid tumors. These results suggest that certain combinations of genetic alterations on chromosome 17 may cooperate in the development and/or progression of breast cancer. Furthermore, it seems likely that analysis of these alterations in breast cancer patients may provide useful prognostic information.     

Detection of microsatellite alterations in bronchial washings in squamous cell lung cancer: the first study from India

BACKGROUND: In recent times, the possibility of detecting lung cancer using microsatellite alterations (microsatellite instability and loss of heterozygosity) in DNA of bronchial washings has been explored. However, no data regarding the presence of microsatellite alterations in lung cancer are available from India, a country which contributes significantly to the lung cancer burden of the world. METHODS: Bronchial washings as well as tumor specimens obtained on bronchoscopy were analyzed for the presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) using the D3S1300 microsatellite marker on chromosome 3p and the TP53 marker on chromosome 17p. RESULTS: The sensitivities of the TP53 and D3S1300 loci in bronchial washings were 35% and 45% (combined 50%), respectively, which was significantly better than conventional cytology (positive for malignant cells in 15%). The presence of these microsatellite alterations was not related to the age, cumulative smoking exposure or smoking status (current or former) of patients. CONCLUSION: Microsatellite alterations, particularly LOH, occur in a significant proportion of Indian patients with squamous cell carcinoma of the lung.     

Mutational analysis of the class I beta-tubulin gene in human breast cancer

Non-small cell lung cancers have a high incidence of somatic mutations of the beta-tubulin (class I) gene, suggesting involvement in the acquisition of resistance to taxanes, which exert their effects through binding to beta-tubulin. Since taxanes are often used in the treatment of breast cancer, we carried out a mutational analysis of the class I beta-tubulin (GenBank accession AF070600) gene in breast cancer. We paid special attention to the primer design so as not to amplify the pseudogenes. We identified 1 somatic mutation, codon 306 [Arg (CGC) to Cys (TGC)], and 2 genetic polymorphisms, codon 217 [Leu (CTG) to Leu (CTA)] and (C to T) at 57 bases downstream from exon 4. Our results suggest that acquisition of resistance to taxanes is unlikely to be explained by somatic mutations of the class I beta-tubulin gene in most breast cancers. In addition, the overestimation of the incidence of somatic mutations of the class I beta-tubulin gene due to the pseudogenes is discussed.