Characterization of the P373L E-cadherin germline missense mutation and implication for clinical management.

AIM: Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by E-cadherin germline mutations. One-third of these mutations are of the missense type, representing a burden in genetic counselling. A new germline missense mutation (P373L) was recently identified in a HDGC Italian family. The present work aimed at addressing the disease-causative nature of the P373L mutant. METHODS: Assessment of the P373L mutation effect was based on cell aggregation and invasion assays. LOH analysis at the E-cadherin locus, search for somatic E-cadherin mutations and for promoter hypermethylation were performed to identify the mechanism of inactivation of the E-cadherin wild-type allele in the tumour. RESULTS: In vitro the P373L germline mutation impaired the E-cadherin functions. E-cadherin promoter hypermethylation was observed in the tumour of the P373L mutation carrier. CONCLUSION: We conclude that the combination of clinical, in vitro and molecular genetic data is helpful for establishing an accurate analysis of HDGC-associated CDH1 germline missense mutations and subsequently for appropriate clinical management of asymptomatic mutation carriers.

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Distribution of HPV infection and tumour markers in cervical intraepithelial neoplasia from cone biopsies of Mozambican women.

AIMS: To evaluate human papillomavirus (HPV) infection in whole cervical cone specimens with cervical intraepithelial neoplasia (CIN). In addition, to evaluate the relation between the presence of CIN lesions and HPV infection and the expression of Ki-67, p53, cytokeratins, Gp230 glycoprotein, and simple mucin-type carbohydrates. METHODS: Cervical cone specimens from five patients with CIN were studied. For each specimen, serial sections encompassing the whole cone were collected (52 samples). HPV infection and HPV types were detected by the polymerase chain reaction and enzyme immunoassay. The expression of Ki-67, p53, cytokeratins, Gp230, and simple mucin-type carbohydrates was examined immunohistochemically. RESULTS: All cases showed high risk HPV types, namely types 16, 33, 35, and 58. Four of the five patients were infected by multiple viral types. HPV-58 was always seen in CIN III, whereas HPV-35 was more frequent in CIN I. The expression of Ki-67 and p53 was higher in CIN III lesions. The expression of cytokeratins 8 and 17 showed complete or almost complete overlap with CIN III. Altered expression of Gp230, Tn, and sialyl-T was often seen in all grades of CIN. CONCLUSIONS: When whole cervical cone specimens are evaluated the rate of multiple HPV infection is very high. The expression of cytokeratins 8 and 17 is a useful marker of CIN III.

Concurrent hypermethylation of gene promoters is associated with a MSI-H phenotype and diploidy in gastric carcinomas.

Changes in the pattern of DNA methylation are among the most common alterations observed in human cancers, such as gastric carcinomas. We analysed in a series of 51 sporadic gastric carcinomas the methylation status of the promoter regions of the hMLH1, CDH1, MGMT and COX2 genes. We aimed to determine the frequency of CpG island hypermethylation and to find out whether the occurrence of concurrent hypermethylation is related to the clinicopathological features of the gastric carcinomas. Using methylation-sensitive restriction analysis/polymerase chain reaction (PCR) and methylation-specific PCR (MSP) strategies, we searched for the presence of hypermethylation on the promoter region of the 4 selected genes. All showed hypermethylation of their promoter regions with frequencies of 37, 51, 61 and 29% for hMLH1, CDH1, MGMT and COX2, respectively. Concurrent hypermethylation was more frequently observed in MSI-H (P=0.0005) and diploid (P=0.029) tumours. Hypermethylation of hMLH1 was associated with MSI-H tumours (P=0.0001), whereas hypermethylation of MGMT was associated with MSI-H (p=0.021) and diploid tumours (p=0.012). Our results indicate that concurrent hypermethylation is a common event in gastric cancer, suggesting that global methylation changes play an important role in the development of sporadic gastric carcinoma. Moreover, inactivation of different gene promoters by hypermethylation is significantly associated with microsatellite instability (MSI-H) and diploidy: hMLH1 determines MSI-H and MGMT the diploid status of gastric carcinomas.

Detection of N-myc amplification in neuroblastomas using Southern blotting on fine needle aspirates.

OBJECTIVE: To verify the possibility of detecting N-myc amplification in fine needle aspiration (FNA) cytology from neuroblastomas by the Southern blotting technique. STUDY DESIGN: Fifteen neuroblastomas diagnosed by FNA in the Department of Pathology, Hospital de S. João, between 1990 and 1998, were studied for N-myc amplification using the Southern blotting technique in cytologic and histologic material. RESULTS: DNA extraction from the cytologic material was performed in all cases (N = 15). In two cases the quality/quantity of the DNA did not allow the study of N-myc status by the Southern blotting technique. We detected N-myc amplification in 1 of 13 patients (7.6%) from whom material was available for genetic study. CONCLUSION: It is possible to use the Southern blotting technique to demonstrate N-myc amplification in material obtained from FNA of neuroblastomas.

MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile.

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC.

Microsatellite instability in hyperplastic and adenomatous polyps of the stomach.

BACKGROUND: Few studies have focused on the presence and significance of microsatellite instability (MSI) in gastric polyps, and the results on record are conflicting. The aim of the current study was to address this issue, taking into consideration the 2 main types of gastric polyps, the coexistence of foci of malignant transformation, and the expression of p53 and ERBB-2. METHODS: Six hyperplastic polyps, 10 adenomatous polyps, and 4 adenomatous polyps displaying foci of malignant transformation (intestinal-type carcinoma) were studied for MSI. The authors analyzed a mononucleotide repeat microsatellite (BAT-26) and 5 dinucleotide repeats in microdissected formalin fixed, paraffin embedded tissue sections that were representative of the lesions. Expression of p53 and ERBB-2 were evaluated by immunohistochemistry. RESULTS: BAT-26 positivity was detected in 1 of 6 hyperplastic polyps (16.7%) and in 2 of 10 adenomas (20%) without malignant transformation. In the 4 adenomatous polyps with carcinomatous foci, BAT-26 positivity was detected in 2 cases (50%) in both (adenomatous and carcinomatous) components of the lesions. p53 immunoreactivity was observed in 6 adenomatous polyps, 2 of them with malignant transformation. Overexpression of the ERBB-2 protein was detected in 1 adenomatous polyp with malignant transformation. CONCLUSIONS: Replication error (RER+) phenotype occurs in both hyperplastic and adenomatous polyps of the stomach. The highest frequency is observed in adenomatous polyps with carcinomatous foci, suggesting that MSI may play a role in the process of malignant transformation in this setting. No significant association was observed between RER+ phenotype and overexpression of p53 or ERBB-2 proteins.

Microsatellite instability in medullary breast carcinomas.

Microsatellite instability (MSI) has been reported to occur in a wide variety of sporadic tumours, such as colorectal and gastric cancers. MSI positivity has been associated with a particular clinico-pathologic profile, including the presence of abundant lymphoid infiltration, poor differentiation and a relatively good outcome for the patients. Since medullary breast carcinomas (MBCs) share these clinico-pathologic features with the MSI-positive tumours described above, we evaluated MSI in this particular histologic type of breast cancer. DNA of 24 MBC cases was extracted from formalin-fixed, paraffin-embedded tissue. The presence of MSI was analysed using BAT-26. We also searched mutations in 2 target genes: TGF-beta RII and BAX. Five cases of the series were also analysed for 1 (CA) dinucleotide tandem repeat sequence (D1S158), 8 tetranucleotide repeat sequences (D3S1358, D5S818, D7S820, D8S1179, D13S317, D21S11, FGA and VWA) and 1 pentanucleotide repeat (dAAAAT), localized in intron 1 of p53 gene. We found 2 carcinomas (8.3%) with BAT-26 instability. None of the cases had mutations in the "target genes", TGF-beta RII and BAX, including the 2 cases with BAT-26 instability. No MSI was observed using the panel of tetra- and pentanucleotide markers. Loss of heterozygosity was found in some loci. No significant difference in mean MIB-1 index according to RER status was observed. The low frequency of MSI in MBC is similar to that of other histologic types of breast cancer. Although MBCs share some clinico-pathologic features with colorectal and gastric carcinomas, which exhibit a high frequency of MSI, the underlying genetic events leading to this breast tumour are different from those leading to tumours of the digestive tract.

PCR amplification of DNA obtained from archived hematoxylin-eosin--and giemsa-stained breast cancer aspirates.

The study of DNA abnormalities in fine-needle aspiration (FNA) specimens for breast cancer could be helpful in improving the capacity of diagnosis and specially to obtain prognostic or predictive information. The aim of the present study was to verify whether it is possible to perform molecular analysis in slides of breast cancer FNA specimens. previously stained by hematoxylineosin (H&E) and Giemsa, stored at least for 3 years. For this purpose, 10 cases of FNA obtained from breast cancer patients diagnosed between 1993 and 1994 in our institute were used. Five cytologic smears were alcohol-fixed and stained with H&E. The other five were air-fixed and Giemsa stained. DNA was isolated from the cytologic smears and amplified by using radioactive polymerase chain reaction (PCR) aimed at BAT-26 marker. Our results demonstrate that archived stained smears prepared for cytologic examinations can be used for molecular analyses by using a PCR amplification method. DNA could be isolated and PCR amplified independently of the prior fixation and staining procedures. So, we conclude that the application of molecular biology techniques to the existing archival smears may become a valuable tool to detect genetic changes in samples from breast cancer aspirates.

P53 in breast carcinomas: association between presence of mutation and immunohistochemical expression using a semiquantitative approach.

The aim of this study was to evaluate the concordance between the presence of p53 mutations in breast carcinomas expressing the protein by immunohistochemistry. A series of 60 breast carcinomas was evaluated by immunohistochemistry using monoclonal antibodies against p53 protein (DO 7 and PAb 1801). Twenty cases classified as being positive for p53 according to the current approach (if 5% or more of neoplastic cells contained reaction product in the nucleus) were used for molecular studies. These cases were re-assessed semi-quantitatively using a scoring system based on intensity and percentage of stained cells. DNA was phenol-chloroform extracted from microdissected normal and tumour cells obtained from formalin-fixed, paraffin-embedded tissue sections. Mutations in the p53 gene were analysed by SSCP (single strand conformational polymorphism) with primers covering exons 2-3 to 11. Ten out of the 20 p53-positive cases presented mutations detected by SSCP analysis. Mutations have been found in several exons ranging from exon 4 to exon 10. We observed a positive relationship between the presence of mutations and immunohistochemical evaluation of p53 protein expression using a semiquantitative scoring system. All cases with more than 2/3 stained tumour cells and strong intensity of staining exhibited p53 mutations. At variance, no p53 mutations were found in cases with less than 1/3 stained tumour cells and moderate intensity of staining. Therefore, only the identification of positivity for p53 detected by immunohistochemistry did not always reflect the detection of p53 mutations in breast cancer, however the use of a semi-quantitative approach seems to be useful as an indicator of the presence of mutation.