Holger Vogelsang

Familial gastric cancer: overview and guidelines for management*

Families with autosomal dominant inherited predisposition to gastric cancer have been described. More recently, germlineE-cadherin/CDH1mutations 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 germlineE-cadherin/CDH1mutations 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.

Diffuse type gastric and lobular breast carcinoma in a familial gastric cancer patient with an E-cadherin germline mutation.

E-Cadherin alterations have been reported frequently in sporadic diffuse type gastric and lobular breast carcinomas. Germline mutations of this gene have been identified recently in several gastric cancer families. We analyzed seven patients with a family history of the disease who had diffuse type gastric cancer diagnosed before the age of 45 for germline mutations in CDH1, the gene encoding the E-cadherin protein. We identified a frameshift mutation in exon 3 in one patient with a strong family history of gastric cancer. The same germline mutation was found in the patients mother, who had metachronous development of lobular breast and diffuse type gastric carcinomas. Immunohistochemistry for E-cadherin protein expression revealed an abnormal staining pattern in both of these tumors, suggesting complete inactivation of the cell adhesion molecule. Thus, our finding suggests that besides diffuse type gastric cancer, lobular breast carcinomas may be associated with germline CDH1 mutations.

Germline CDH1 deletions in hereditary diffuse gastric cancer families

Germline CDH1 point or small frameshift mutations can be identified in 30–50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT–PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is ∼46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

Genotype-Phenotype Comparison of German MLH1 and MSH2 Mutation Carriers Clinically Affected With Lynch Syndrome: A Report by the German HNPCC Consortium

PURPOSE Lynch syndrome is linked to germline mutations in mismatch repair genes. We analyzed the genotype-phenotype correlations in the largest cohort so far reported. PATIENTS AND METHODS Following standard algorithms, we identified 281 of 574 unrelated families with deleterious germline mutations in MLH1 (n = 124) or MSH2 (n = 157). A total of 988 patients with 1,381 cancers were included in this analysis. RESULTS We identified 181 and 259 individuals with proven or obligatory and 254 and 294 with assumed MLH1 and MSH2 mutations, respectively. Age at diagnosis was younger both in regard to first cancer (40 v 43 years; P < .009) and to first colorectal cancer (CRC; 41 v 44 years; P = .004) in MLH1 (n = 435) versus MSH2 (n = 553) mutation carriers. In both groups, rectal cancers were remarkably frequent, and the time span between first and second CRC was smaller if the first primary occurred left sided. Gastric cancer was the third most frequent malignancy occurring without a similarly affected relative in most cases. All prostate cancers occurred in MSH2 mutation carriers. CONCLUSION The proportion of rectal cancers and shorter time span to metachronous cancers indicates the need for a defined treatment strategy for primary rectal cancers in hereditary nonpolyposis colorectal cancer patients. Male MLH1 mutation carriers require earlier colonoscopy beginning at age 20 years. We propose regular gastric surveillance starting at age 35 years, regardless of the familial occurrence of this cancer. The association of prostate cancer with MSH2 mutations should be taken into consideration both for clinical and genetic counseling practice.

Efficacy of Annual Colonoscopic Surveillance in Individuals With Hereditary Nonpolyposis Colorectal Cancer

BACKGROUND & AIMS Individuals with hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome) have a high risk for developing colorectal cancer (CRC). We evaluated the efficacy of annual surveillance colonoscopies to detect adenomas and CRCs. METHODS In a prospective, multicenter cohort study, 1126 individuals underwent 3474 colonoscopies. We considered individuals from 3 groups of HNPCC families: those with a pathogenic germline mutation in a mismatch repair gene (MUT group), those without a mutation but with microsatellite instability (MSI group), and those who fulfilled the Amsterdam criteria without microsatellite instability (MSS group). RESULTS Compliance to annual intervals was good, with 81% of colonoscopies completed within 15 months. Ninety-nine CRC events were observed in 90 patients. Seventeen CRCs (17%) were detected through symptoms (8 before baseline colonoscopy, 8 at intervals >15 months to the preceding colonoscopy, and 1 interval cancer). Only 2 of 43 CRCs detected by follow-up colonoscopy were regionally advanced. Tumor stages were significantly lower among CRCs detected by follow-up colonoscopies compared with CRCs detected by symptoms (P = .01). Cumulative CRC risk at the age of 60 years was similar in the MUT and MSI groups (23.0% combined; 95% confidence interval [CI], 14.8%-31.2%) but considerably lower in the MSS group (1.8%; 95% CI, 0.0%-5.1%). Adenomas at baseline colonoscopy predicted an earlier occurrence of subsequent adenoma (hazard ratio, 2.6; 95% CI, 1.7-4.0) and CRC (hazard ratio, 3.9; 95% CI, 1.7-8.5), providing information about interindividual heterogeneity of adenomas and kinetics of CRC formation. CONCLUSIONS Annual colonoscopic surveillance is recommended for individuals with HNPCC. Less intense surveillance might be appropriate for MSS families.

Germline mutations of the E-cadherin(CDH1) and TP53 genes, rather than of RUNX3 and HPP1, contribute to genetic predisposition in German gastric cancer patients

Germline mutations of the cell adhesion molecule E-cadherin have been shown for the first time to underlie a hereditary diffuse type gastric cancer syndrome (HDGC) in Maori families,1 and subsequently have been reported in HDGC patients of various ethnic origins.2–9 Gastric cancer may also be associated with other hereditary tumour syndromes, which are mainly characterised by carcinomas of other organs. One of these syndromes is the HNPCC syndrome (hereditary nonpolyposis colorectal cancer syndrome), which is caused by germline mutations in DNA mismatch repair genes.10 Furthermore, gastric cancer has been observed in the context of the Li Fraumeni syndrome, a rare cancer syndrome due to germline mutations of the TP53 tumour suppressor gene,11–14 as well as in association with the FAP and Peutz–Jeghers syndrome.15 Despite known molecular genetic causes, contributing to a genetic predisposition to gastric cancer, a considerable number of familial cases have been reported that were not attributable to one of this hereditary syndromes, suggesting that unknown susceptibility genes for gastric cancer might exist. Putative tumour suppressor genes that are commonly inactivated in sporadic gastric cancers, such as the RUNX3 or HPP1 genes, are potential candidate susceptibility genes.16,17 In a previous study, we identified one E-cadherin germline mutation among seven diffuse type familial gastric cancer patients, indicating that in addition to E-cadherin , other genes might be involved in genetic predisposition to the disease in this patient group.4 There were three goals of the present study. First, we aimed to extend our analysis of E-cadherin germline mutations to a higher number of patients, to evaluate the contribution of germline mutations in this gene to German familial and early onset gastric cancer patients and to characterise identified missense mutations for their functional relevance. Secondly, we wished to analyse the role …

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2.

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.

Hereditary non-polyposis colorectal cancer: clinical and molecular evidence for a new entity of hereditary colorectal cancer

Background: Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumours. Methods: By thorough molecular and clinical evaluation of 41 families, two different groups were characterised: group 1, 25 families with truncating mutations in MLH1 or MSH2 (12 novel mutations); and group 2, 16 Amsterdam positive families without mutations in these genes and without microsatellite instability in their corresponding tumours. Results: Significant clinical differences between these two groups were found. Firstly, earlier age of onset for all colorectal cancers (median 41 v 55 years; p<0.001) and all tumours (median 43 v 56 years; p = 0.022) was observed, comparing groups 1 and 2. Secondly, 68% of the index colorectal cancers were localised proximally of the splenic flexure in group 1 compared with 14% in group 2 (p<0.010). Thirdly, more synchronous and metachronous colorectal (p = 0.017) and extracolorectal tumours (p<0.001) were found in group 1. Fourthly, a higher colorectal adenoma/carcinoma ratio (p = 0.030) and a tendency towards more synchronous or metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carcinomas. As three mutation negative tumours revealed chromosomal instability after comparative genomic hybridisation, these tumours may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway. Conclusion: These data show that HNPCC includes at least two entities with clinical and molecular differences. This will have implications for surveillance programmes and for cancer research.

Desmoglein 2 is expressed abnormally rather than mutated in familial and sporadic gastric cancer

Alterations of the cell adhesion molecule E‐cadherin have been demonstrated in sporadic and hereditary gastric carcinomas. A cell adhesion molecule with functional similarity to E‐cadherin is desmoglein 2 (Dsg2), a major component of the desmosomes. In this study, we investigated whether alterations of Dsg2 are involved in gastric carcinogenesis and whether germline mutations contribute to a genetic predisposition in familial gastric cancer patients with no germline mutations in the E‐cadherin gene. Seventy‐five formalin‐fixed, paraffin‐embedded tissues from 37 familial and 38 sporadic gastric carcinomas were analysed for Dsg2 expression by immunohistochemistry. DNA from 31 familial gastric cancer patients was analysed for germline mutations and five sporadic tumours were analysed for somatic mutations by DHPLC. Of the 75 tumours, 25 (33%) demonstrated abnormal (reduced and/or non‐membrane‐associated) Dsg2 expression. There was a trend towards more frequent abnormal expression in diffuse type (42%) than in intestinal type tumours (18%) (p = 0.066). One germline missense variant leading to a non‐conservative amino acid change (c. 2810 C > A, Thr 937 Asn) was found in a familial gastric cancer patient with a diffuse type tumour. No somatic mutations were identified. The observed abnormal expression of Dsg2 protein suggests that this molecule is involved in the carcinogenesis of a subset of gastric carcinomas, in particular of the diffuse type. Somatic mutations in the gene do not seem to be a very frequent inactivation event and the finding of no clear pathogenic germline mutation rules out Dsg2 as a major gastric cancer predisposition gene. Copyright

Combined GADD45A and Thymidine Phosphorylase Expression Levels Predict Response and Survival of Neoadjuvant-Treated Gastric Cancer Patients

Purpose: We evaluated the expression of seven therapy-related genes to predict the clinical outcome of advanced gastric cancer patients treated with a neoadjuvant chemotherapeutic protocol. Experimental Design: Pretherapeutic, formalin-fixed, and paraffin-embedded biopsies of 61 patients, who received a 5-fluorouracil (5-FU)– and cisplatin-based chemotherapy were studied. The expressions of the 5-FU–related genes TS, DPD, and TP and of the cisplatin-related genes ERCC1, ERCC4, KU80, and GADD45A were analyzed by quantitative real-time PCR. The expression levels of single genes and of various combinations were tested for an association with response and overall survival. Results: High DPD levels were more frequently found in nonresponding patients and were associated with worse survival. GADD45A and TP levels showed weak associations with response, but GADD45A expression correlated with survival. There was no association with response for TS expression, but tumors with a high TS level were associated with worse survival. The combination of GADD45A and TP revealed the strongest predictive effect. High expression values of TP and/or GADD45A were exclusively found in nonresponding patients (P = 0.002) and were associated with a significantly poorer survival (P = 0.04). Conclusions: Combined gene expression levels of TP and GADD45A represent a new variable to predict the clinical outcome after neoadjuvant chemotherapy in gastric cancer. The association of DPD expression with response and survival underlines a predominant role of DPD to predict 5-FU sensitivity. The association of TS expression levels with survival but not with response suggests an importance of this gene for tumor progression.