Anya N. A. Milne

Nature meets nurture: molecular genetics of gastric cancer

The immensity of genes and molecules implicated in gastric carcinogenesis is overwhelming and the relevant importance of some of these molecules is too often unclear. This review serves to bring us up-to-date with the latest findings as well as to look at the larger picture in terms of how to tackle the problem of solving this multi-piece puzzle. In this review, the environmental nurturing of intestinal cancer is discussed, beginning with epidemiology (known causative factors for inducing molecular change), an update of H. pylori research, including the role of inflammation and stem cells in premalignant lesions. The role of E-cadherin in the nature (genotype) of diffuse gastric cancer is highlighted, and finally the ever growing discipline of SNP analysis (including IL1B) is discussed.

Early-onset gastric cancers have a different molecular expression profile than conventional gastric cancers.

Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (≤45 years old) and conventional gastric cancers. Expression of cycloxygenase-2 (COX-2) is elevated in gastric adenocarcinomas compared to non-neoplastic mucosa, and in light of studies showing reduced risk of gastric cancer in nonsteroidal anti-inflammatory drug users, we have chosen to investigate the expression of COX-2 and related molecules in 113 early-onset gastric cancers and compare it with 91 conventional gastric cancers, using tissue microarrays. These markers include molecules known to be important in conventional gastric carcinogenesis, such as E-Cadherin, p53, COX-2, Trefoil Factor-1 (TFF1), β-catenin, p16 and c-myc; as well as molecules not yet described as being important in gastric cancer, such as the transcription factor c-jun, the COX-2 mRNA stabilizer HuR, and C/EBP-β, a transcription factor for COX-2. All markers showed a statistically significant difference between early-onset gastric cancers and conventional gastric cancers, using a χ2 test. In particular, early-onset gastric cancers displayed a COX-2 Low, TFF1-expressing phenotype, whereas COX-2 overexpression and loss of TFF1 was found in conventional cancers, and this difference between early-onset gastric cancers and conventional cancers remained statistically significant when adjusted for location and histology (P<0.0001 and P=0.002 respectively). We found that COX-2 overexpression correlates significantly with loss of TFF1 (P=0.001), overexpression of C/EBP-β (P<0.001) and cytoplasmic HuR (P=0.016). COX-2 was significantly associated with p53 positivity (P=0.003). Abnormalities in E-Cadherin correlated significantly with diffuse phenotype, whereas high expression of COX-2, loss of TFF1 and overexpression of C/EBP-β correlated with the intestinal phenotype. Our results provide further evidence that early-onset gastric cancer exhibits a distinctive expression profile that may have practical implications.

Early Onset Gastric Cancer: On the road to unraveling gastric carcinogenesis

Gastric cancer is thought to result from a combination of environmental factors and the accumulation of specific genetic alterations due to increasing genetic instability, and consequently affects mainly older patients. Less than 10% of patients present with the disease before 45 years of age (early onset gastric carcinoma) and these patients are believed to develop gastric carcinomas with a molecular genetic profile differing from that of sporadic carcinomas occurring at a later age. In young patients, the role of genetics is presumably greater than in older patients, with less of an impact from environmental carcinogens. As a result, hereditary gastric cancers and early onset gastric cancers can provide vital information about molecular genetic pathways in sporadic cancers and may aid in the unraveling of gastric carcinogenesis. This review focuses on the molecular genetics of gastric cancer and also focuses on early onset gastric cancers as well as familial gastric cancers such as hereditary diffuse gastric cancer. An overview of the various pathways of importance in gastric cancer, as discovered through in-vitro, primary cancer and mouse model studies, is presented and the clinical importance of CDH1 mutations is discussed.

Early-onset gastric carcinomas display molecular characteristics distinct from gastric carcinomas occurring at a later age.

Gastric cancer is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently mainly affects older patients (>50 years of age). Fewer than 10% of patients present with the disease before 45 years of age and these young patients are thought to develop carcinomas with a different molecular genetic profile from that of sporadic carcinomas occurring at a later age. Forty early‐onset gastric carcinoma resection specimens were characterized for microsatellite instability (MSI) and loss of heterozygosity status using 22 polymorphic microsatellite markers. Twenty‐four biopsies were additionally evaluated for the presence of MSI. No MSI was observed in any of the cases analysed. Losses were infrequent, but were most common for the D1S234 (26.1%) and D1S1676 (17.4%) markers, flanking the RUNX3 gene; for the p53ALU (23.1%) and TP53 (15.4%) markers, near the TP53 gene; and for the D16S2624 (17.2%) marker, near the E‐cadherin (CDH1) gene. All cases with loss of CDH1, as well as 6/7 cases with loss of TP53, displayed aberrant staining of the corresponding proteins, pointing to a functional role for these proteins in early‐onset gastric carcinogenesis. No germline CDH1, TP53 or RUNX3 mutations were detected in any of the cases analysed. No correlation was observed between non‐functional E‐cadherin and the histological type of the tumours analysed. Finally, Epstein–Barr virus was not detected in any of the cases analysed. On the basis of these results, early‐onset gastric carcinomas appear to have characteristics distinct from gastric carcinomas occurring at a later age. Copyright

HER-2/neu Testing and Therapy in Gastroesophageal Adenocarcinoma

Despite ongoing advances in the treatment of gastroesophageal cancer, prognosis remains poor. The best promise to improve this poor survival is provided by new targeted agents. Of these, human epidermal growth factor receptor 2 (HER2) is currently in the spotlight. In this review, we provide an overview of recent developments in HER2 testing and results of clinical trials targeting HER2 in gastroesophageal adenocarcinoma. Based on the encouraging ToGA trial findings it is now expected that routine HER2 testing will be included in the diagnostic work-up of patients with advanced gastric cancer. With regard to this testing, overexpression of the HER2 protein seems to possess the best predictive properties. However, HER2 immunohistochemistry (IHC) is subject to assay and interobserver variability, so standardization and internal and external proficiency testing is an absolute prerequisite, especially as the IHC scoring system in gastric cancer is different from that of breast cancer. Further study is needed to investigate the clinical meaning of the significant heterogeneity observed in both gene amplification and protein overexpression in gastroesophageal cancer. Highly effective therapies for gastroesophageal cancer can only be accomplished by a multi-targeted approach, considering crosstalk between pathways and continuing to optimize chemotherapy.

Exclusion of RUNX3 as a tumour-suppressor gene in early-onset gastric carcinomas

Recent studies claim a critical role for RUNX3 in gastric epithelial homeostasis. However, conflicting results exist regarding RUNX3 expression in the stomach and its potential role as a tumour-suppressor gene (TSG) in gastric carcinogenesis. Our aim was to evaluate the role of RUNX3 in early-onset gastric carcinomas (EOGCs). We analysed 41 EOGCs for RUNX3 aberrations using loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analyses. LOH of markers flanking RUNX3 was relatively common, indicating that loss of the gene may play a role in gastric carcinogenesis. However, FISH analysis of selected cases and a panel of 14 gastric carcinoma-derived cell lines showed widespread presence of multiple copies of centromere 1. While RUNX3 copy numbers were generally equal to or fewer than those of centromere 1, at least two copies were present in almost all cells analysed. Accordingly, a subpopulation of tumour cells in 12/37 cases showed RUNX3 protein expression. However, expression was not detected in the adjacent nontumorous mucosa of any case. Together, these observations indicate that chromosome 1 aberrations occur frequently in EOGCs and are reflected in the LOH and IHC patterns found. Our findings refute a role for RUNX3 as a TSG in EOGCs.

A novel region of amplification at 11p12‐13 in gastric cancer, revealed by representational difference analysis, is associated with overexpression of CD44v6, especially in early‐onset gastric carcinomas

Diffuse‐type gastric carcinomas (GCs) are often difficult to characterize because of contamination of tumor samples by surrounding normal tissue. As such, information regarding chromosomal aberrations in this subtype of GCs is limited. In this study, we used representational difference analysis to pinpoint genomic amplifications occurring in diffuse‐type GCs. We found nine differential products from two novel regions of amplification in two tumors: one product mapped to 19p13.1 and eight mapped to a 1.8‐Mb region in chromosomal segment 11p12‐13. These amplifications were confirmed using Southern blot analysis and occurred in 3/16 and 6/15 diffuse‐type GCs, respectively. CD44, a well characterized cellular adhesion molecule involved in several human malignancies, is encoded by a gene located within 200 kb of the 11p12‐13 amplification fragments. We confirmed that overexpression of isoform CD44v6 was correlated with amplification at 11p12‐13 in 11/12 diffuse‐type GCs. Since diffuse‐type GCs occur more frequently in early‐onset gastric carcinomas (EOGCs, presented at 45 years of age or younger) than in “conventional” GCs, and the tumors carrying the original amplifications were EOGCs, we investigated overexpression of CD44v6 in 107 EOGCs and 88 conventional GCs using tissue microarrays. We found frequent CD44v6 overexpression in both tumor groups (76% and 57% respectively) and, interestingly, significantly more cases with overexpression of CD44v6 in EOGCs than in conventional GCs (P = 0.005), irrespective of histology. These findings provide further evidence for both the relevance of CD44 in GC and for distinct molecular characteristics of EOGCs when compared with those of GCs occurring at a later age.

Do collision tumors of the gastroesophageal junction exist? A molecular analysis.

Collision tumors are thought to arise from the accidental meeting of two independent tumors. Here we present five gastroesophageal junction tumors consisting of two collision tumors and three composite tumors (characterized by two divergent lineages originating from the same neoplastic clonal proliferation), as diagnosed on histology. In an attempt to prove this distinction at a genetic level, we performed TP53 sequence analysis and p53 immunohistochemistry. In addition, loss of heterozygosity (LOH) analysis using 10 microsatellite markers was carried out. An identical TP53 mutation and a similar pattern of retention and LOH were found in both neoplastic components of the presumed collision tumors, suggesting that both components are derived from a single precursor cell that undergoes divergent differentiation in the evolution of the tumor. In the composite group, 1 case had a genetic basis for the possible diagnosis of a collision tumor, with a TP53 mutation in the adenocarcinoma component only, and a different pattern of retention and loss of heterozygosity. These findings imply that it is not possible to recognize true collision tumors from immunohistologic appearance alone and suggest that the long-standing histologic criteria for the diagnosis of these neoplasms have no molecular basis.

Molecular alterations in gastric cancer with special reference to the early-onset subtype.

Currently, gastric cancer (GC) is one of the most frequently diagnosed neoplasms, with a global burden of 723000 deaths in 2012. It is the third leading cause of cancer-related death worldwide. There are numerous possible factors that stimulate the pro-carcinogenic activity of important genes. These factors include genetic susceptibility expressed in a single-nucleotide polymorphism, various acquired mutations (chromosomal instability, microsatellite instability, somatic gene mutations, epigenetic alterations) and environmental circumstances (e.g., Helicobcter pylori infection, EBV infection, diet, and smoking). Most of the aforementioned pathways overlap, and authors agree that a clear-cut pathway for GC may not exist. Thus, the categorization of carcinogenic events is complicated. Lately, it has been claimed that research on early-onset gastric carcinoma (EOGC) and hereditary GC may contribute towards unravelling some part of the mystery of the GC molecular pattern because young patients are less exposed to environmental carcinogens and because carcinogenesis in this setting may be more dependent on genetic factors. The comparison of various aspects that differ and coexist in EOGCs and conventional GCs might enable scientists to: distinguish which features in the pathway of gastric carcinogenesis are modifiable, discover specific GC markers and identify a specific target. This review provides a summary of the data published thus far concerning the molecular characteristics of GC and highlights the outstanding features of EOGC.

Diagnosis of T1 colorectal cancer in pedunculated polyps in daily clinical practice : a multicenter study

T1 colorectal cancer can be mimicked by pseudo-invasion in pedunculated polyps. British guidelines are currently one of the few which recommend diagnostic confirmation of T1 colorectal cancer by a second pathologist. The aim of this study was to provide insights into the accuracy of histological diagnosis of pedunculated T1 colorectal cancer in daily clinical practice. A sample of 128 cases diagnosed as pedunculated T1 colorectal cancer between 2000 and 2014 from 10 Dutch hospitals was selected for histological review. Firstly, two Dutch expert gastrointestinal pathologists reviewed all hematoxylin-eosin stained slides. In 20 cases the diagnosis T1 colorectal cancer was not confirmed (20/128; 16%). The discordant cases were subsequently discussed with a third Dutch gastrointestinal pathologist and a consensus diagnosis was agreed. The revised diagnoses were pseudo-invasion in 10 cases (10/128; 8%), high-grade dysplasia in 4 cases (4/128; 3%), and equivocal in 6 cases (6/128; 5%). To further validate the consensus diagnosis, the discordant cases were reviewed by an independent expert pathologist from the United Kingdom. A total of 39 cases were reviewed blindly including the 20 cases with a revised diagnosis and 19 control cases where the Dutch expert panel agreed with the original reporting pathologists diagnosis. In 19 of the 20 cases with a revised diagnosis the British pathologist agreed that T1 colorectal cancer could not be confirmed. Additionally, amongst the 19 control cases the British pathologist was unable to confirm T1 colorectal cancer in a further 4 cases and was equivocal in 3 cases. In conclusion, both generalist and expert pathologists experience diagnostic difficulty distinguishing pseudo-invasion and high-grade dysplasia from T1 colorectal cancer. In order to prevent overtreatment, review of the histology of pedunculated T1 colorectal cancers by a second pathologist should be considered with discussion of these cases at a multidisciplinary meeting.