Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria

Background: Mutations in the E-cadherin (CDH1) gene are a well documented cause of hereditary diffuse gastric cancer (HDGC). Development of evidence based guidelines for CDH1 screening for HDGC have been complicated by its rarity, variable penetrance, and lack of founder mutations.

Methods: Forty three new gastric cancer (GC) families were ascertained from multiple sources. In 42 of these families at least one gastric cancer was pathologically confirmed to be a diffuse gastric cancer (DGC); the other family had intestinal type gastric cancers. Screening of the entire coding region of the CDH1 gene and all intron/exon boundaries was performed by bi-directional sequencing.

Results: Novel mutations were found in 13 of the 42 DGC families (31% overall). Twelve of these mutations occur among the 25 families with multiple cases of gastric cancer and with pathologic confirmation of diffuse gastric cancer phenotype in at least one individual under the age of 50 years. The mutations found include small insertions and deletions, splice site mutations, and three non-conservative amino acid substitutions (A298T, W409R, and R732Q). All three missense mutations conferred loss of E-cadherin function in in vitro assays. Multiple cases of breast cancers including pathologically confirmed lobular breast cancers were observed both in mutation positive and negative families.

Conclusion: Germline truncating CDH1 mutations are found in 48% of families with multiple cases of gastric cancer and at least one documented case of DGC in an individual under 50 years of age. We recommend that these criteria be used for selecting families for CDH1 mutational analysis.

     

Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes

Invasive breast cancer shows a wide range of morphological differentiation, associated with differences in prognosis, but as yet, the underlying genetic mechanisms cannot be accounted for. In order to establish a model of the possible progression from the different subtypes of ductal carcinoma in situ (DCIS) to invasive breast cancer, 77 selected cases of invasive breast cancer representing distinct morphological subtypes were investigated by means of comparative genomic hybridization (CGH). There was a high degree of genetic homology between tubular and tubulo-lobular carcinoma and well-differentiated DCIS, and between ductal invasive carcinoma G3 and poorly differentiated DCIS. Highly differentiated invasive breast cancers were characterized by a loss of 16q and a low average number of aberrations per case. In high-grade tumours, losses of this chromosomal region were seen with a much lower frequency in cases with evidence of an aneuploid tumour status. These data demonstrate the close genetic similarity of well-, intermediately, and poorly differentiated DCIS and distinct morphological types of invasive breast carcinoma, providing further evidence that DCIS is a direct precursor lesion of invasive breast cancer and that various evolutionary genetic pathways exist.     

Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands

E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.     

Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer

Inherited mutations in the E-cadherin gene ( CDH1 ) were described recently in three Maori kindreds with familial gastric cancer. Familial gastric cancer is genetically heterogeneous and it is not clear what proportion of gastric cancer susceptibility in non-Maori populations is due to germline CDH1 mutations. Therefore, we screened eight familial gastric cancer kindreds of British and Irish origin for germline CDH1 mutations, by SSCP analysis of all 16 exons and flanking sequences. Each family contained: (i) two cases of gastric cancer in first degree relatives with one affected before age 50 years; or (ii) three or more cases of gastric cancer. Novel germline CDH1 mutations (a nonsense and a splice site) were detected in two families (25%). Both mutations were predicted to truncate the E-cadherin protein in the signal peptide domain. In one family there was evidence of non-penetrance and susceptibility to both gastric and colorectal cancer; thus, in addition to six cases of gastric cancer, a CDH1 mutation carrier developed colorectal cancer at age 30 years. We have confirmed that germline mutations in the CDH1 gene cause familial gastric cancer in non-Maori populations. However, only a minority of familial gastric cancers can be accounted for by CDH1 mutations. Loss of E-cadherin function has been implicated in the pathogenesis of sporadic colorectal and other cancers, and our findings provide evidence that germline CDH1 mutations predispose to early onset colorectal cancer. Thus, CDH1 should be investigated as a cause of inherited susceptibility to both gastric and colorectal cancers.     

Assessing the pathogenicity of MLH1 missense mutations in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with clinical, genetic and functional features

Assessing the pathogenicity of missense mutations of MLH1 and MSH2 is critical to counsel patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC). Approximately 32% of all MLH1 mutations and 18% of MSH2 mutations are missense variants which often have an uncertain genetic significance. To assess the pathogenicity of four MLH1 missense mutations which were found in five patients with suspected HNPCC, P648S (CCC --> TCC), L559R (CTG --> CGG), K618A (AAG --> GCG), Y646C (TAT --> TGT), we studied their ability to disrupt MLH1 protein function and their relationship with all those clinical, genetic and pathological features which are typical of this syndrome. Our results indicated that the P648S and L559R mutations were probably pathogenic because they disrupted MLH1 protein interaction with its partner PMS2 in vitro and abolished MLH1 expression in HCT116 cells. In addition these variants were associated with features often found in HNPCC patients: in particular high microsatellite instability, occurrence of high grade tumours and, in one case, strong family history. The pathogenicity of the K618A and Y646C mutations was questionable as their correlation with features typical of HNPCC was low and the outcome of the functional analysis was ambiguous. These observations suggested that a clinically usable assessment of the pathogenicity of MLH missense variants can be achieved through the analysis of multiple mutation characteristics among which loss of protein function, occurrence of microsatellite instability and family history seemed to have a predominant role.     

A TP53-truncating germline mutation (E287X) in a family with characteristics of both hereditary diffuse gastric cancer and Li-Fraumeni syndrome

Mutations in CDH1, which encodes E-cadherin, have been associated with hereditary diffuse gastric cancer (HDGC) in Western populations but have not been shown to play a major role in Asians. Recently, a patient with familial gastric cancer (FGC) was shown to harbor a germline mutation in the TP53 gene, which encodes p53 and has been previously associated with Li-Fraumeni Syndrome (LFS). To determine whether mutations in TP53 are associated with FGC in Asians, we screened the entire coding region of TP53 in probands from 23 Korean FGC families. We identified a nonsense (E287X) TP53 germline mutation in a family whose history is compatible with both HDGC and LFS. Two members of this family (SNU-G2) were afflicted with brain tumors, seven with gastric cancers, two with sarcomas, and one with both gastric cancer and a sarcoma. The E287X TP53 mutation segregated with the cancer phenotype in the family members from whom DNA samples were available. To our knowledge, this is the first report of a large family with both HDGC and LFS. Our results suggest that TP53 mutational screening in FGC families should be interpreted with caution because additional TP53 mutation-carrying HDGC families may also show LFS-related phenotypes.     

Sociodemographic,psychosocial and clinical factors associated with uptake of genetic counselling for hereditary cancer: a systematic review

Evidence suggests that a significant proportion of individuals referred to cancer genetic counselling (GC) do not attend, and thus may not be engaged in adequate cancer risk management. We aimed to review the literature to better understand barriers to accessing GC and how they may be overcome. We conducted a systematic literature search for articles examining factors influencing cancer GC uptake as well as motivators and barriers to GC attendance. Factors were categorised as sociodemographic, psychosocial or clinical. The literature search identified 1413 citations, 35 of which met the inclusion criteria. GC uptake ranged from 19% to 88%. With the exceptions of education level, socioeconomic status, cancer‐specific distress, personal cancer diagnosis and actual and perceived risk of cancer, support was lacking for most sociodemographic, clinical and psychosocial factors as predictors of GC uptake. Cost and logistical barriers, emotional concerns, family concerns and low perceived personal relevance were reported as important considerations for those declining GC. We conclude that there is poor understanding of GC and a lack of decision support among those referred to GC. Research into ways of providing education and support to referred individuals will be important as the scope and availability of GC and genetic testing broaden.     

FGFR3 mutations,but not FGFR3 expression and FGFR3 copy-number variations,are associated with favourable non-muscle invasive bladder cancer

The fibroblast growth factor receptor 3 (FGFR3) is a tyrosine kinase receptor frequently activated by point mutations in bladder cancer (BC). These mutations are associated with genetically stable, Ta and low-grade BC, representing the favourable BC pathway. Conversely, FGFR3 over-expression was recently found in 40 % of muscle invasive BC. We examined FGFR3 mutation status and protein expression in patients originally diagnosed as T1. We also investigated copy-number variations in FGFR3 as a possible alternative mechanism to activate FGFR3. We included 84 patients with T1 BC as their initial diagnosis. A uropathologist reviewed the slides for grade and (sub)stage. The FGFR3 mutation status was examined by PCR-SNaPshot and FGFR3 protein expression by standard immuno-histochemistry (FGFR3-B9). Copy-number status was determined in 69/84 cases with nine probes covering nine exons of the FGFR3 gene (MLPA). Of 27 BC with FGFR3 mutations, 26 (96 %) showed FGFR3 over-expression. Of the 57 wild-type BC, 27 (47 %) BC showed over-expression. Pathological parameters significantly differed (p?<?0.01) between mutant and wild-type tumours with the FGFR3 mutation pointing to more favourable BC. However, if the BC exhibited wild-type FGFR3, FGFR3 protein status had no influence on grade and (sub)stage. We found six tumours with more than or equal to three copies of FGFR3. Only 1 of 22 wild-type tumours with over-expression of FGFR3 had more than or equal to three gene copies. In initially diagnosed T1 BC, only the FGFR3 mutation was significantly associated with favourable BC disease characteristics. In addition to almost all FGFR3 mutant BC, 47 % of wild-type BC displayed FGFR3 over-expression, suggesting an alternative mechanism to activate FGFR3. Increased FGFR3 copy number was a rare event and did not account for this mechanism. Nevertheless, FGFR3 wild-type tumours with over-expression of the protein may still represent a subset that might potentially benefit from FGFR3-targeted therapy.     

Expression of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex correlates with the macroscopic appearance of early gastric cancer

E-cadherin and its associated cytoplasmic proteins, alpha-, beta-, and gamma-catenins, play an essential role in the control of epithelial differentiation. We have previously shown that loss or down-regulation of E-cadherin/catenin correlates with poor survival in advanced gastric adenocarcinoma. The aim of this study was to assess the expression of E-cadherin and catenins in early gastric cancers (EGCs). Immunohistochemical staining for E-cadherin and alpha-, beta-, and gamma-catenins was performed on 41 paraffin-embedded gastrectomy specimens of EGC using an indirect immunoperoxidase technique. The pattern of expression and cellular localization of the E-cadherin/catenin complex in tumour cells were correlated with the macroscopic appearance of the tumour according to the Japanese Endoscopic Society classification. The tumours were classified as follows: three type I (protruding) and 38 type II (superficial), of which ten were type IIa (elevated), one was type IIb (flat), and 27 were type IIc (depressed). E-cadherin and alpha-, beta-, and gamma-catenins were expressed at the cell-cell junctions in normal mucosa. Forty out of 41 tumours showed abnormal expression (loss of membranous immunoreactivity and/or nuclear staining) of at least one component of the E-cadherin catenin complex. Loss of E-cadherin immunoreactivity was more frequently seen in type IIb (1/1, 100%) and type IIc (27/27, 100%) than in type I (1/3, 33%) and type IIa (1/10, 10%) (p<0.01). Abnormal expression of E-cadherin and alpha-catenin was more frequently seen in diffuse-type than in intestinal type tumours (p<0.05). Abnormal immunoreactivity of beta- and gamma-catenin, including nuclear localization, was observed in 34% and 7.3% of tumours, respectively, but there was no significant correlation with tumour type or endoscopic appearance. In conclusion, abnormal expression of the E-cadherin/catenin complex occurs in EGC and seems to correlate with macroscopic appearances.     

Late-onset familial Mediterranean fever (FMF): A subset with distinct clinical,demographic, and molecular genetic characteristics

To determine the prevalence and characterize demographic, clinical, and genetic features of familial Mediterranean fever (FMF) of late onset, all patients experiencing their first FMF attack at age 40 years or more were identified using the computerized registry of our FMF clinic, and then thoroughly interviewed and examined. The control group consisted of 40 consecutive FMF patients, who arrived at the FMF clinic for their regular follow-up visit and were 40 years of age or older at the time of the examination. The severity of the disease in patients and controls was determined using a modified score, developed previously. Mutational analysis in the FMF gene was performed using a commercial kit. Only 20 of 4000 (0.5%) patients had late-onset FMF. These patients were mostly men, of non-North African origin, P < 0.05 compared to controls. All had abdominal attacks and in most these were the only manifestation of their disease, P < 0.001. None had chronic or prolonged manifestations of FMF, for example, amyloidosis, chronic arthritis, or protracted myalgia, P < 0.001. The response to treatment was good despite using low colchicine dose, P < 0.05. The overall severity score indicated a mild disease, P < 0.001. Mutational analysis revealed absence of M694V homozygosity, P < 0.01, compared to our regular FMF population. We conclude that the onset of FMF in a late age defines a milder form of disease with typical clinical, demographic, and molecular genetic characteristics. Am. J. Med. Genet. 87:30–35, 1999. © 1999 Wiley-Liss, Inc.     

Two Thai families with Norrie disease (ND): association of two novel missense mutations with severe ND phenotype, seizures, and a manifesting carrier

We describe two Thai families with Norrie disease (ND) in three generations, including 10 affected males and one manifesting female. All affected males in each family had severely defective eye development with complete loss of vision. In addition, three male patients (one from family 1 and two from family 2) suffered from epilepsy, and one female carrier from one family manifested blindness with phthisis bulbi in her right eye. Mutation analysis of the ND gene (NDP) revealed two different novel missense mutations (L16P and S75P) that co-segregated with ND in each family, suggesting that the newly appearing proline at codon 16 or codon 75 alters the conformation of the ND protein and contributes to the severe phenotype of ND in each family. Other studies suggest that epileptic seizures or growth retardation that is associated with ND is the consequence of loss of contiguous genes, because most such patients had deletions extending beyond the Norrie locus. Our finding that the three affected males in the two families with the missense mutations had epilepsy does not support a contiguous gene effect, but favors the pleiotropism of NDP, at least as far as the epileptic manifestation is concerned. The unilateral blindness in the female carrier may have been due to non-random X-inactivation.     

Late-onset familial Mediterranean fever (FMF): a subset with distinct clinical, demographic, and molecular genetic characteristics.

To determine the prevalence and characterize demographic, clinical, and genetic features of familial Mediterranean fever (FMF) of late onset, all patients experiencing their first FMF attack at age 40 years or more were identified using the computerized registry of our FMF clinic, and then thoroughly interviewed and examined. The control group consisted of 40 consecutive FMF patients, who arrived at the FMF clinic for their regular follow-up visit and were 40 years of age or older at the time of the examination. The severity of the disease in patients and controls was determined using a modified score, developed previously. Mutational analysis in the FMF gene was performed using a commercial kit. Only 20 of 4000 (0.5%) patients had late-onset FMF. These patients were mostly men, of non-North African origin, P < 0.05 compared to controls. All had abdominal attacks and in most these were the only manifestation of their disease, P < 0.001. None had chronic or prolonged manifestations of FMF, for example, amyloidosis, chronic arthritis, or protracted myalgia, P < 0.001. The response to treatment was good despite using low colchicine dose, P < 0.05. The overall severity score indicated a mild disease, P < 0.001. Mutational analysis revealed absence of M694V homozygosity, P < 0.01, compared to our regular FMF population. We conclude that the onset of FMF in a late age defines a milder form of disease with typical clinical, demographic, and molecular genetic characteristics.     

Multifocal PEComa (PEComatosis) of the female genital tract associated with endometriosis, diffuse adenomyosis, and endometrial atypical hyperplasia

The authors describe a case of multifocal perivascular epithelioid cell tumor (PEComa) arising in the pelvis of a 39-year-old woman affected by tuberous sclerosis. The tumor presented in the form of multiple fascicular, focally cystic nodules involving the uterine corpus, both ovaries, and the omentum. Microscopically, the nodules were composed of foci of adenomyosis and endometriosis (with focal atypical complex hyperplasia) associated with a stromal spindle cell population immunoreactive for HMB-45, smooth muscle actin, and estrogen and progesterone receptors. We interpret these foci as the result of a widespread proliferation of perivascular epithelioid cells (PEC). Because of the diffuse quality of the process, the designation of PEComatosis seems warranted.     

Genomic structure of human mismatch repair gene, hMLH1, and its mutation analysis in patients with hereditary non-polyposis colorectal cancer (HNPCC)

Mutation of hMLH1, a gene involved in DNA mismatch repair, isresponsible for some families carrying the hereditary non-polypoticcolorectal cancer (HNPCC) syndrome. To establish a basis forpresymptomatic diagnosis of HNPCC patients who carry germlinemutations in this gene, we determined the exon—intronorganization of hMLH1. The results indicated that hMLH1 consistsof 19 coding exons spanning approximately 100 kb, and that exons1–7 contain a region that is highly conserved in the MLH1and PMS1 genes of yeast. We used PCR—SSCP analysis andDNA sequencing to examine the entire coding region of the MLH1gene in DNAs of 34 unrelated cancer patients who belong to HNPCCpedigrees. Germline mutations were detectable in eight (24%)of these patients; four of them were missense mutations, onehad occurred in an Intron where it would affect splicing, andthe remaining three were frameshift mutations resulting in truncationof the gene product downstream of the mutation site.