Kent Man Chu

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis

We analyzed gene expression patterns in human gastric cancers by using cDNA microarrays representing ≈30,300 genes. Expression of PLA2G2A, a gene previously implicated as a modifier of the ApcMin/+ (multiple intestinal neoplasia 1) mutant phenotype in the mouse, was significantly correlated with patient survival. We confirmed this observation in an independent set of patient samples by using quantitative RT-PCR. Beyond its potential diagnostic and prognostic significance, this result suggests the intriguing possibility that the activity of PLA2G2A may suppress progression or metastasis of human gastric cancer.

Gastrointestinal stromal tumors (GISTs) with KIT and PDGFRA mutations have distinct gene expression profiles

Most GISTs require oncogenic activation of the KIT or PDGFRA receptor tyrosine kinase proteins, and the genomic mechanisms of oncogene activation are heterogeneous. Notably, the kinase mutation type correlates with both tumor biology and imatinib response. For example, GISTs with KIT exon 11 mutations are typically gastric and have excellent imatinib response, whereas those with KIT exon 9 mutations generally arise in the small bowel and are less responsive to imatinib. To identify genes that might contribute to these biological differences, we carried out gene expression profiling of 26 GISTs with known KIT and PDGFRA mutational status. Expression differences were then evaluated further by RNA in situ hybridization, immunohistochemistry, and immunoblotting. Unsupervised hierarchical clustering grouped tumors with similar mutations together, but the distinction between the different groups was not absolute. Differentially expressed genes included ezrin, p70S6K, and PKCs, which are known to have key roles in KIT or PDGFRA signaling, and which might therefore contribute to the distinctive clinicopathological features in GISTs with different mutation types. These gene products could serve as highly selective therapeutic targets in GISTs containing the KIT or PDGFRA mutational types with which they are associated.

Self-expanding metallic stent in the treatment of colonic obstruction caused by advanced malignancies

INTRODUCTION: The treatment of malignant obstruction of the left colon or rectum usually requires emergency surgery on poor-risk patients, and the creation of a stoma is usually inevitable. With the use of self-expanding metallic stents, the prompt relief of large-bowel obstruction without surgery has become possible. This report describes our results in the use of self-expanding metallic stents in the treatment of left-sided colonic obstruction resulting from advanced malignancies. METHODS: From November 1997 to March 1999, insertion of self-expanding metallic stents was attempted in 24 patients with acute left-sided colonic obstruction caused by primary or recurrent malignancies. All the procedures were performed by colorectal surgeons. The guidewire was inserted through the channel of the endoscope, and its position was confirmed with fluoroscopy. Uncovered Wallstent® esophageal endoprostheses were used in all except the first case. The insertion and deployment of the stents were under both endoscopic and fluoroscopic guidance. RESULTS: There were 24 patients (15 males) with a mean age of 63.6 (range, 36–98) years. Thirteen patients had primary colorectal cancer and 11 had recurrent cancers (colorectal cancer, 5; gastric cancer, 5; other, 1). In the treatment of primary colorectal cancer, seven procedures were palliative, and no subsequent surgery was planned because of extensive liver metastasis or poor medical risk. The other six patients underwent elective resection after mechanical bowel preparation. There was no mortality related to the procedure. Stenting was successful in the relief of obstruction in 23 patients. Perforation of the colon occurred in one patient, and an emergency Hartmanns operation was performed. Migration of the stents occurred in three patients. Only 3 of the 18 patients in the palliation group required the subsequent creation of stomas. CONCLUSION: The use of the self-expanding metallic stents can achieve rapid and effective nonsurgical means to relieve left-sided colonic obstruction. It provides good palliation for unresectable advanced tumors that cause colonic obstruction. It may also have a role in the temporary relief of obstruction so that subsequent colonic resection can be performed under elective conditions.

Downregulation of ID4 by promoter hypermethylation in gastric adenocarcinoma

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5′ CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix–loop–helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

Comprehensive analysis of the gene expression profiles in human gastric cancer cell lines.

Gastric adenocarcinoma is one of the major malignancies worldwide. Gastric cell lines have been widely used as the model to study the genetics, pharmacology and biochemistry of gastric cancers. Here we describe a comprehensive survey of the gene expression profiles of 12 gastric carcinoma cell lines, using cDNA microarray with 43 000 clones. For comparison, we also explored the gene expression patterns of 15 cell lines derived from lymphoid, endothelial, stromal and other epithelial cancers. Expression levels of specific genes were validated through comparison to protein expression by immunohistochemistry using cell block arrays. We found sets of genes whose expression corresponds to the molecular signature of each cell type. In the gastric cancer cell lines, apart from genes that are highly expressed corresponding to their common epithelial origin from the gastrointestinal tract, we found marked heterogeneity among the gene expression patterns of these cell lines. Some of the heterogeneity may reflect their underlying molecular characteristics or specific differentiation program. Two putative gastric carcinoma cell lines were found to be B-cell lymphoma, and another one had no epithelial specific gene expression and hence was of doubtful epithelial origin. These cell lines should no longer be used in gastric carcinoma research. In conclusion, our gene expression database can serve as a powerful resource for the study of gastric cancer using these cell lines.

Choice of palliation for malignant hilar biliary obstruction

Clinical data from 50 consecutive patients with unresectable hilar tumors situated at or proximal to the common hepatic duct were retrospectively analyzed to aid in the selection of appropriate palliative measures. Thirty-four patients had cholangioenteric bypass (CEB) to either left (28 patients), right (3 patients), or both (3 patients) intrahepatic ductal systems. Sixteen patients had nonoperative drainage (NOD) established either endoscopically (4 patients), percutaneously (9 patients), or using a combined endoscopic-percutaneous approach (3 patients). When compared with patients with CEB, patients with NOD had more frequent medical problems (p less than 0.03) and lower serum albumin levels on admission (p less than 0.03). While comparable postprocedural complications (13 CEB patients versus 4 NOD patients) were observed, patients with NOD had a significantly higher hospital mortality (9 CEB patients versus 9 NOD patients, p less than 0.05). Excluding the 12 patients (6 CEB patients versus 6 NOD patients) who died within 30 days after drainage, the quality of survival of the remaining 38 patients was analyzed with reference to 6 objective parameters. Although patients with NOD had significantly more frequent admissions relating to their catheters (p less than 0.02), there was no qualitative difference in the survival rate between the two groups of patients. For selected high-risk patients with limited life expectancy, NOD should be offered. However, additional prospective studies are required to decide the best choice of palliation for patients who are not at such high risk.

Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

Background Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets.

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AIMS Stress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved. MAIN METHODS The effect of adrenaline on HT-29 cell proliferation was determined by [(3)H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E(2) (PGE(2)) release were determined by zymography and enzyme immunoassay, respectively. KEY FINDINGS Adrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE(2) release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β(1)- and β(2)-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE(2) release in HT-29 cells. SIGNIFICANCE These results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β(1)- and β(2)-adrenoceptors by a COX-2 dependent pathway.