Rebecca C. Fitzgerald

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.

Population-Based Study Reveals New Risk-Stratification Biomarker Panel for Barrett's Esophagus

BACKGROUND & AIMSnThe risk of progression of Barretts esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues.nnnMETHODSnWe analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia ≥ 6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status.nnnRESULTSnLow-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia.nnnCONCLUSIONSnLow-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Mechanisms and sequelae of E‐cadherin silencing in hereditary diffuse gastric cancer

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. Copyright

Clinical implications of E-cadherin associated hereditary diffuse gastric cancer

Approximately 1–3% of gastric cancers arise as a result of inherited gastric cancer predisposition syndromes. These may be of the diffuse or intestinal type. Linkage analysis has recently implicated E-cadherin mutations in an estimated 25% of families with an autosomal dominant predisposition to diffuse type gastric cancers. This subset of gastric cancer has been termed hereditary diffuse gastric cancer (HDGC).

The combination of autofluorescence endoscopy and molecular biomarkers is a novel diagnostic tool for dysplasia in Barrett's oesophagus

Objective Endoscopic surveillance for Barretts oesophagus (BO) is limited by sampling error and the subjectivity of diagnosing dysplasia. We aimed to compare a biomarker panel on minimal biopsies directed by autofluorescence imaging (AFI) with the standard surveillance protocol to derive an objective tool for dysplasia assessment. Design We performed a cross-sectional prospective study in three tertiary referral centres. Patients with BO underwent high-resolution endoscopy followed by AFI-targeted biopsies. 157 patients completed the biopsy protocol. Aneuploidy/tetraploidy; 9p and 17p loss of heterozygosity; RUNX3, HPP1 and p16 methylation; p53 and cyclin A immunohistochemistry were assessed. Bootstrap resampling was used to select the best diagnostic biomarker panel for high-grade dysplasia (HGD) and early cancer (EC). This panel was validated in an independent cohort of 46 patients. Results Aneuploidy, p53 immunohistochemistry and cyclin A had the strongest association with dysplasia in the per-biopsy analysis and, as a panel, had an area under the receiver operating characteristic curve of 0.97 (95% CI 0.95 to 0.99) for diagnosing HGD/EC. The diagnostic accuracy for HGD/EC of the three-biomarker panel from AFI+ areas was superior to AFI− areas (p<0.001). Compared with the standard protocol, this panel had equal sensitivity for HGD/EC, with a 4.5-fold reduction in the number of biopsies. In an independent cohort of patients, the panel had a sensitivity and specificity for HGD/EC of 100% and 85%, respectively. Conclusions A three-biomarker panel on a small number of AFI-targeted biopsies provides an accurate and objective diagnosis of dysplasia in BO. The clinical implications have to be studied further.

Complex Diseases in Gastroenterology and Hepatology: GERD, Barrett’s, and Esophageal Adenocarcinoma

Gastroesophageal reflux disease, Barretts esophagus, and esophageal adenocarcinoma are related diseases with environmental and genetic determinants. The genetic changes are relevant in 2 distinct ways. First, there are heritable variations in germline DNA that may influence the individual susceptibility to cancer. Second, there is an accumulation of somatic-cell genetic and epigenetic changes within the epithelium during the metaplasia-dysplasia-carcinoma sequence, which may be an important determinant for the likelihood for cancer progression. Esophageal cancer occurring in the context of a familial syndrome is rare and most cases are sporadic. The sporadic cases still may have heritable germline influences, but these are likely to involve multiple, low-penetrance susceptibility genes. To date, the relative contribution and identity of such genes are unknown. However, in the future the identification of susceptibility genes could have important public health implications for patient management. With regard to the epithelium, a map gradually is being created of the frequently occurring alterations. Some of these changes are critical whereas others are bystanders. As well as the identification of abnormalities in target genes, it also is possible to determine the global gene expression profile of these diseases and to correlate this profile with clinical outcome. It is hoped that these complementary approaches will enable patients to be stratified in terms of their cancer risk so that prevention, surveillance, and treatment strategies can be targeted appropriately. At the current time, genetics does not influence routine clinical management of patients with gastroesophageal reflux disease, Barretts esophagus, or esophageal adenocarcinoma.

Genome-wide association studies in oesophageal adenocarcinoma and Barrett's oesophagus: a large-scale meta-analysis

Summary Background Oesophageal adenocarcinoma represents one of the fastest rising cancers in high-income countries. Barretts oesophagus is the premalignant precursor of oesophageal adenocarcinoma. However, only a few patients with Barretts oesophagus develop adenocarcinoma, which complicates clinical management in the absence of valid predictors. Within an international consortium investigating the genetics of Barretts oesophagus and oesophageal adenocarcinoma, we aimed to identify novel genetic risk variants for the development of Barretts oesophagus and oesophageal adenocarcinoma. Methods We did a meta-analysis of all genome-wide association studies of Barretts oesophagus and oesophageal adenocarcinoma available in PubMed up to Feb 29, 2016; all patients were of European ancestry and disease was confirmed histopathologically. All participants were from four separate studies within Europe, North America, and Australia and were genotyped on high-density single nucleotide polymorphism (SNP) arrays. Meta-analysis was done with a fixed-effects inverse variance-weighting approach and with a standard genome-wide significance threshold (p<5u2008×u200810−8). We also did an association analysis after reweighting of loci with an approach that investigates annotation enrichment among genome-wide significant loci. Furthermore, the entire dataset was analysed with bioinformatics approaches—including functional annotation databases and gene-based and pathway-based methods—to identify pathophysiologically relevant cellular mechanisms. Findings Our sample comprised 6167 patients with Barretts oesophagus and 4112 individuals with oesophageal adenocarcinoma, in addition to 17u2008159 representative controls from four genome-wide association studies in Europe, North America, and Australia. We identified eight new risk loci associated with either Barretts oesophagus or oesophageal adenocarcinoma, within or near the genes CFTR (rs17451754; p=4·8u2008×u200810−10), MSRA (rs17749155; p=5·2u2008×u200810−10), LINC00208 and BLK (rs10108511; p=2·1u2008×u200810−9), KHDRBS2 (rs62423175; p=3·0u2008×u200810−9), TPPP and CEP72 (rs9918259; p=3·2u2008×u200810−9), TMOD1 (rs7852462; p=1·5u2008×u200810−8), SATB2 (rs139606545; p=2·0u2008×u200810−8), and HTR3C and ABCC5 (rs9823696; p=1·6u2008×u200810−8). The locus identified near HTR3C and ABCC5 (rs9823696) was associated specifically with oesophageal adenocarcinoma (p=1·6u2008×u200810−8) and was independent of Barretts oesophagus development (p=0·45). A ninth novel risk locus was identified within the gene LPA (rs12207195; posterior probability 0·925) after reweighting with significantly enriched annotations. The strongest disease pathways identified (p<10−6) belonged to muscle cell differentiation and to mesenchyme development and differentiation. Interpretation Our meta-analysis of genome-wide association studies doubled the number of known risk loci for Barretts oesophagus and oesophageal adenocarcinoma and revealed new insights into causes of these diseases. Furthermore, the specific association between oesophageal adenocarcinoma and the locus near HTR3C and ABCC5 might constitute a novel genetic marker for prediction of the transition from Barretts oesophagus to oesophageal adenocarcinoma. Fine-mapping and functional studies of new risk loci could lead to identification of key molecules in the development of Barretts oesophagus and oesophageal adenocarcinoma, which might encourage development of advanced prevention and intervention strategies. Funding US National Cancer Institute, US National Institutes of Health, National Health and Medical Research Council of Australia, Swedish Cancer Society, Medical Research Council UK, Cambridge NIHR Biomedical Research Centre, Cambridge Experimental Cancer Medicine Centre, Else Kröner Fresenius Stiftung, Wellcome Trust, Cancer Research UK, AstraZeneca UK, University Hospitals of Leicester, University of Oxford, Australian Research Council.

E-cadherin mutations and hereditary gastric cancer: prevention by resection?

Background: A small percentage of gastric cancers (1–3%) arise as a result of clearly identified inherited gastric cancer predisposition syndromes. Recently, linkage analysis has implicated E-cadherin (CDH1) mutations in a subset of dominantly inherited gastric cancers termed hereditary diffuse gastric cancer (HDGC). Heterozygotes with the CDH1 mutation have a 70–80% chance of developing gastric cancer and once patients have invasive, symptomatic disease there is a high associated morbidity and mortality. As a result prophylactic gastrectomy has been advocated for affected family members identified by genetic screening. There are many issues to be considered in order to determine whether prophylactic gastrectomy is the optimal management strategy for these patients. Methods: A MEDLINE literature search was performed and combined with our own experience in order to identify relevant background information on hereditary gastric cancer. In particular we extracted information about prophylactic gastrectomy in HDGC patients. Results: Once the decision has been undertaken to perform genetic testing, the therapeutic options are either endoscopic screening with the aim of identifying an early, curable lesion or prophylactic surgery. Two groups have published their data on prophylactically resected stomachs in asymptomatic patients from affected families. In these series all of the post-gastrectomy specimens had microscopic evidence of diffuse gastric cancer confined within the mucosal layer. These early cancers had not been identified by endoscopic surveillance. Technical issues surrounding the age at which to operate, the best operation to reduce the risk of post-operative complications and the risk of other cancers developing in other organs in these patients are still controversial. Long-term follow-up studies in specialist centres are required to resolve these issues. Conclusions: From the limited experience so far the estimated risk reduction of gastric cancer by gastrectomy is significant.

Pregnancy after prophylactic total gastrectomy.

Hereditary diffuse gastric cancer is an autosomal dominant inherited cancer predisposition syndrome characterized by susceptibility to diffuse gastric and lobular breast cancers. Since current screening options for diffuse gastric cancer are ineffective, prophylactic total gastrectomy (PTG) is a recommended option for unaffected germline CDH1 mutation carriers. It is unknown whether pregnancy after surgery is possible or advisable due to potential maternal nutritional deficiencies. In this report we describe the pregnancy outcomes in three CDH1 mutation positive women after PTG and in a CDH1 mutation negative woman after total gastrectomy for early gastric cancer.

The Evolving Genomic Landscape of Barrett’s Esophagus and Esophageal Adenocarcinoma

We have recently gained unprecedented insight into genetic factors that determine risk for Barretts esophagus (BE) and progression to esophageal adenocarcinoma (EA). Next-generation sequencing technologies have allowed us to identify somatic mutations that initiate BE and track genetic changes during development of tumors and invasive cancer. These technologies led to identification of mechanisms of tumorigenesis that challenge the current multistep model of progression to EA. Newer, cost-effective technologies create opportunities to rapidly translate the analysis of DNA into tools that can identify patients with BE at high risk for cancer, detect dysplastic lesions more reliably, and uncover mechanisms of carcinogenesis.