[show abstract][hide abstract] ABSTRACT: Around 25-40% of cases of hereditary diffuse gastric cancer (HDGC) are caused by heterozygous E-cadherin (CDH1) germline mutations. The mechanisms for loss of the second allele still remain unclear. The aims of this study were to elucidate mechanisms for somatic inactivation of the wild-type CDH1 allele and to seek evidence for cadherin switching. Archival tumour material was analysed from 16 patients with CDH1 germline mutations and seven patients fulfilling HDGC criteria without CDH1 germline mutations. The 16 CDH1 exons were sequenced. E-cadherin promoter methylation was analysed by bisulphite sequencing and pyrosequencing and allele specificity was determined using polymorphic loci. Loss of heterozygosity was analysed using microsatellite markers. Cadherin expression levels were determined by real-time RT-PCR and immunohistochemistry. Six of 16 individuals with germline mutations had at least one second hit mechanism. Two exonic mutations (exon 9 truncating, exon 3 missense) and four intronic mutations which may affect splicing were identified. Tumours from 4/16 individuals had promoter hypermethylation that was restricted to the A allele haplotype in three cases. E-cadherin loss (mRNA and protein) generally correlated with identification of a second hit. In cases without germline E-cadherin mutations there was no evidence for somatic mutation or significant promoter methylation. P-cadherin (>25% cells) was expressed in 7/13 (54%) and 4/5 (80%) with and without germline CDH1 mutations, respectively, independent of complete E-cadherin loss. Overall, inactivation of the second CDH1 allele occurs by mutation and methylation events. Methylation is commonly allele-specific and is uncommon without germline mutations. P-cadherin over-expression commonly occurs in individuals with diffuse type gastric cancer.
The Journal of Pathology 09/2008; 216(3):295-306. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Germline mutations in the gene encoding the tumour suppressor E-cadherin (CDH1) are the underlying genetic defect responsible for hereditary diffuse gastric cancer (HDGC). A remarkably high percentage ( approximately 80%) of CDH1 mutations in HDGC patients and carriers generate premature termination codons (PTCs). Here, we examined whether CDH1 transcripts harbouring PTCs are downregulated by nonsense-mediated decay (NMD), an RNA surveillance pathway that degrades PTC-bearing transcripts. Using an allele-specific expression (ASE) assay to differentiate between mutated and wild-type CDH1 alleles, we found that PTC-bearing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several CDH1 mutation carriers. We show that NMD is responsible for this robust downregulation, as CDH1 transcripts harbouring PTCs in the KATO-III gastric tumour cell line were upregulated in response to protein synthesis inhibitors or depletion of the NMD factors UPF1 and eIF4AIII. Analysis of HDGC patients harbouring CDH1 alleles with PTCs at a wide variety of different positions indicates an association of their predicted ability to induce NMD and an earlier age of onset of gastric cancer. This suggests that NMD may be detrimental for HDGC patients and therefore NMD is a potentially useful therapeutic target for CDH1 mutation carriers.
[show abstract][hide abstract] ABSTRACT: BRAF kinase is a downstream target of KRAS and activates the MAPK pathway. These two molecules are prone to mutations in sporadic microsatellite unstable (MSI) colorectal carcinomas (CRC) and BRAF V600E mutations are inversely associated with oncogenic KRAS mutations. The biological significance of BRAF V600E oncogenic activation is not well established in this type of tumour. We aimed to study proliferation and survival effects induced by BRAF inhibition in MSI CRC cell lines harbouring distinct genetic backgrounds (BRAF V600E or KRAS G13D). Suppression of BRAF in BRAF V600E MSI CRC cell lines by RNA interference significantly inhibited proliferation and induced apoptosis, as demonstrated by BrdU incorporation and TUNEL assay, respectively. No significant differences were seen in proliferation and apoptosis, in cell lines harbouring KRAS G13D, after BRAF inhibition. We further analysed proliferation-associated molecules (pERK1/2, cyclin D1, p27 Kip1) and apoptosis-associated molecules (Bcl-2, Bax, pAkt, pBad, XIAP) in all cell lines. After BRAF down-regulation, we found a more pronounced decrease in ERK1/2 phosphorylation and cyclin D1 expression levels in BRAF-mutated cell lines in comparison to KRAS mutated cells. Upon BRAF inhibition, we also found an increase in p27(Kip1) levels and a more pronounced decrease in the levels of anti-apoptotic protein Bcl-2, specifically in cell lines with BRAF V600E. In conclusion, we have shown that MSI KRAS and BRAF mutant CRC cell lines respond differently to BRAF knockdown. This report provides evidence supporting BRAF as a good target for therapeutic intervention in patients with sporadic MSI CRC harbouring activating mutations in BRAF but not in KRAS.
The Journal of Pathology 03/2008; 214(3):320-7. · 7.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gastric cancer is one of the major causes of cancer-related death worldwide. Familial clustering is observed in about 10% of cases; 1-3% of cases are hereditary. In the latter group, a syndrome which has been well characterised is hereditary diffuse gastric cancer; this is specifically associated with CDH1 (E-cadherin) germline mutations in about 30% of families. In this article, the state of the art of familial gastric cancer regarding the clinical, molecular and pathology features is reviewed, as well as the practical aspects for a correct diagnosis and clinical management.
Journal of clinical pathology 02/2008; 61(1):25-30. · 2.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serous borderline ovarian tumors (SBOTs) are differentiated, slow growing, noninvasive, and have a better prognosis than their invasive counterparts, but recurrence and progression to invasive carcinomas are common, and unlike high-grade serous carcinomas, they tend to be nonresponsive to chemotherapy. However, due to a lack of culture systems and animal models, information about the properties of SBOT and their changes with neoplastic progression is extremely limited. Our objective was to establish a cell culture model for SBOTs and to characterize their phenotype and genotype. We compared cultures derived from two SBOTs, one of which was a short-term culture containing a BRAF mutation but few other cytogenetic changes while the other culture developed into a spontaneously immortalized permanent cell line and had numerical and structural chromosomal abnormalities but lacked RAS/BRAF mutations. Both cultures formed whorl-like epithelial colonies and resembled low-grade invasive carcinomas by their secretion of CA125 and oviduct-specific glycoprotein, production of matrix metalloproteinases, E-cadherin expression, and telomerase activity. Other characteristics associated with neoplastic transformation, including invasiveness, anchorage-independent growth, and tumorigenicity, were not observed. Importantly, cell motility was reduced in both lines, likely contributing to the lack of invasiveness. The results reveal a striking phenotypic similarity between the two cell lines, regardless of their cytogenetic diversity, which suggests that their characteristic phenotype is regulated to a large degree by epigenetic and environmental factors. In conclusion, we have established the first permanent SBOT cell line, which provides a new model to elucidate the undefined relationship of SBOTs to invasive ovarian carcinomas.
International Journal of Gynecological Cancer 02/2008; 18(6):1234-47. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 12/2007; 33(9):1061-7. · 2.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS–BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS–BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.Keywords: KRAS, BRAF, colorectal carcinoma, instability, MAP kinase, lymph node metastases
[show abstract][hide abstract] ABSTRACT: We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.
Journal of Medical Genetics 03/2006; 43(2):138-42. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
European Journal of Cancer 07/2003; 39(9):1222-7. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interleukin (IL)-1 gene cluster proinflammatory polymorphisms have been associated with development of gastric atrophy and with increased risk of gastric carcinoma. We aimed to determine the association between IL-1 loci polymorphisms and increased risk of gastric carcinoma in samples from a Portuguese population, and to find whether there was any relationship with the histologic types of gastric carcinoma.
In a case-control study including 220 controls and 152 patients with gastric carcinoma (intestinal, 76; diffuse, 37; and atypical, 39), both the IL-1B-511 biallelic polymorphism and the IL-1RN penta-allelic variable number of tandem repeats were genotyped.
We found a significant association between the IL-1 polymorphisms and increased risk for tumor development in patients with intestinal-type gastric carcinoma. A trend towards an increased risk of tumor development was also observed in patients with diffuse-type gastric carcinoma. No significant relationship was observed in patients with atypical carcinoma. Carriers of IL-1B-511T and IL-1RN*2 homozygotes had increased risk for developing intestinal-type gastric carcinoma with odds ratios of 2.7 (95% confidence interval [CI], 1.5-4.9) and 3.1 (95% CI, 1.5-6.5), respectively. Statistical analysis showed an interaction between the 2 loci with the risk conferred by the IL-1B-511T allele substantially increased (odds ratio, 9.0; 95% CI, 3.5-23.0) in individuals homozygous for the IL-1RN*2 allele.
Our results provide further support to the association between IL-1 gene cluster proinflammatory polymorphisms and increased risk of gastric carcinoma. Furthermore, we found evidence pointing to the existence of a synergistic interaction between the IL-1B and IL-1RN polymorphisms.
[show abstract][hide abstract] ABSTRACT: In diffuse gastric carcinoma, despite common E-cadherin gene (CDH1) mutations, tumors show absence of CDH1 loss of heterozigosity (LOH) in most cases. This observation challenges the classical two-hit model of tumor suppressor gene inactivation. In order to investigate whether or not CDH1 promoter methylation may function as the second hit we analysed a series of 23 sporadic gastric carcinomas for the presence of CDH1 mutations, CDH1 promoter methylation, LOH and E-cadherin expression. CDH1 mutations were detected in nine of the 16 (56.3%) diffuse gastric carcinomas and in none of the seven intestinal gastric carcinomas. In diffuse gastric carcinomas harboring CDH1 mutations, LOH was observed in a single case. Loss of plasma membrane E-cadherin expression was consistently found in all nine cases with CDH1 mutation, suggesting that tumors inactivated the remaining CDH1 allele via a different mechanism. CDH1 promoter methylation was observed in nine of the 16 (56.3%) diffuse-type gastric carcinoma cases, including six of the nine cases (66.7%) harboring CDH1 mutations. CDH1 promoter methylation was also seen in two (28.6%) intestinal-type cases. Our results show that CDH1 promoter methylation is the second hit in more than half of the sporadic diffuse gastric carcinoma cases harboring CDH1 mutations.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Families with autosomal dominant inherited predisposition to gastric cancer have been described. More recently, germline E-cadherin/CDH1 mutations have been identified in hereditary diffuse gastric cancer kindred. The need to have protocols to manage and counsel these families in the clinic led a group of geneticists, gastroenterologists, surgeons, oncologists, pathologists, and molecular biologists to convene a workshop to produce consensus statements and guidelines for familial gastric cancer. Review of the available cancer pathology from people belonging to families with documented germline E-cadherin/CDH1 mutations confirmed that the gastric cancers were all of the diffuse type. Criteria to define the different types of familial gastric cancer syndromes were agreed. Foremost among these criteria was that review of histopathology should be part of the evaluation of any family with aggregation of gastric cancer cases. Guidelines for genetic testing and counselling in hereditary diffuse gastric cancer were produced. Finally, a proposed strategy for clinical management in families with high penetrance autosomal dominant predisposition to gastric cancer was defined.
Journal of Medical Genetics 01/2000; 36(12):873-80. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gastric carcinomas with DNA replication errors (RER phenotype) display a particular clinicopathologic profile and carry a putative favorable prognosis. The RER phenotype has been identified as microsatellite instability in noncoding regions, as well as in repeat sequences within exons of several "target genes": TGFbeta RII, IGFII R, and BAX. In an attempt to find out whether the RER status is a significant prognostic factor in gastric carcinoma in a multivariate analysis and whether the clinicopathological features of the RER+ tumors are associated with mutations in the "target genes," we evaluated a series of 152 cases of sporadic gastric carcinoma. Five or six microsatellite loci and/or BAT 26, a poly(A) tract, were analyzed in each case using polymerase chain reaction and electrophoresis. Thirty-five cases (23.0%) were RER+. The RER phenotype was closely associated with a low pTNM stage and carried a significantly better prognosis. The repeat sequences of the target genes were screened for mutations in 28 RER+ and 13 RER-tumors. Mutations in TGFbeta RII occurred in 67.9% of the RER+ tumors and were significantly associated with the glandular histotype. IGFII R and BAX mutations occurred, respectively, in 25.0% and 32.1% of the cases; there was a trend toward an association between mutations in these genes and decreased nodal metastization and wall invasiveness, respectively. We conclude that the RER status is a significant prognostic indicator in gastric carcinoma and that such prognostic influence may be mediated by mutations in TGFbeta RII, IGFII R, and BAX genes.
American Journal Of Pathology 11/1998; 153(4):1211-9. · 4.52 Impact Factor