Cancer‐related genetic variants of Helicobacter pylori strains determined using gastric wash‐based whole‐genome analysis with single‐molecule real‐time technology

Helicobacter pylori (H. pylori) are a primary factor in the pathogenesis of gastric cancer (GC); GC ranks third among cancer‐related mortality. A clear understanding of the H. pylori genome factors underlying GC is necessary to develop more effective methods to prevent GC. A single‐molecule real‐time DNA sequencing‐based H. pylori genome‐wide association study analysis was performed using the H. pylori genome present in five early‐stage GC (EGC) and five non‐GC clinical DNA samples recovered from gastric washes. A total of 275 genes with 702 nucleotide variants (NVs) were found to be common to three or more patients with EGC but no non‐GC patients (single‐NV: 654/702, 93.2%; multi‐NV: 40/702, 5.7%; deletion: 3/702, 0.4%; insertion: 3/702, 0.7%). Gene ontology analysis of H. pylori revealed that genes involved in the mitochondrial electron transport system, glycolytic processes and the TCA cycle were highly enriched. Cancer‐related NVs were most frequently found in a member of the Helicobacter outer membrane protein family, hopL. In particular, one of the NVs in hopL was a novel six‐nucleotide insertion (1159095?1159096, TACTTC); this mutant was detected more frequently in a validation set of 50 additional EGC samples (22/50, 44.0%) than in 18 non‐GC samples (3/18, 16.7%, P = .04). These results suggest that the hopL variant is associated with the development of GC and may serve as a genetic biomarker of H. pylori virulence and GC risk. Our assay can serve as a potent tool to expand our understanding of bacteria‐associated tumorigenesis.     

Gastric cancer occurrence in preneoplastic lesions: A long‐term follow‐up in a high‐risk area in Spain

There are no established criteria to classify patients into high or low risk of progressing to gastric cancer (GC). The aim of the study was to identify predictors of GC occurrence among patients with gastric preneoplastic lesions. A prospective and retrospective follow‐up study was carried out in a province in Spain with one of the highest risk of GC. The study included 478 patients who underwent gastric biopsy in 1988–1994 with diagnoses of normal mucosa, nonatrophic gastritis (NAG), non‐metaplastic multifocal atrophic gastritis (MAG) and complete or incomplete intestinal metaplasia (IM) and who accepted to undergo a new biopsy during 2005–2007 or had an event during follow up. Inter‐ and intra‐observer variability of histological diagnosis was assessed. Analysis was done using Cox proportional hazards risk (HR) models. The mean age of the patients was 50 years, 47% were males and the mean follow‐up time was 12.8 years. During follow‐up, 23 GC (4.8%) were diagnosed (21 adenocarcinomas and 2 lymphomas) with an incidence of 3.77 per 1,000 person per year. The incidence rate of GC for those with incomplete IM was 16.5 per 1,000 person years. Out the 21 adenocarcinomas, 16 had an incomplete IM in the baseline diagnosis. Incomplete IM (HR 11.3; 95% CI 3.8–33.9) and a family history of GC (HR 6.1; 95% CI 1.7–22.4) were the strongest risk factors for gastric adenocarcinoma. Subtyping of IM and family history of GC may be useful for the identification of high‐risk patients who need more intensive surveillance.     

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.     

The prognostic value of E‐cadherin in gastric cancer: A meta‐analysis

The prognostic impact of E‐cadherin downregulation in gastric cancer has been assessed for years while the results are controversial and heterogeneous. We thus comprehensively reviewed the evidence for evaluation of E‐cadherin expression in gastric cancer to determine this effect. We searched PubMed and Embase to identify eligible studies, and 26 studies comprising 4,383 gastric cancer patients were included to assess the association between E‐cadherin immunohistochemical expression and overall survival (OS) and clinicopathological characteristics. Summary hazard ratios (HRs) or odds ratios (ORs) with 95% confidence interval (95% CI) were calculated to estimate the effect. We also performed meta‐regression and subgroup analysis according to study location, publication year, number of patients, quality score of studies and cut‐off value. Reduced E‐cadherin expression was significantly correlated with poor OS of gastric cancer patients (HR 1.62, 95% CI 1.34–1.96). Subgroup analysis indicated that E‐cadherin low‐expression had an unfavorable impact on OS in Asian patients (HR 1.87, 95% CI 1.45–2.41). Moreover, downregulation of E‐cadherin was significantly associated with TNM stage (OR 2.52, 95% CI 1.85–3.43), the depth of invasion (OR 2.01, 95% CI 1.39–2.90), lymph node metastasis (OR 2.39, 95% CI 1.68–3.40), distant metastasis (OR 2.23, 95% CI 1.21–4.11), grade of differentiation (OR 2.26, 95% CI 1.60–3.21), vascular invasion (OR 1.86, 95% CI 1.10–3.13) and histological type of gastric cancer (OR 4.22, 95% CI 2.96–6.02). This meta‐analysis revealed that E‐cadherin expression might be a predicative factor of poor prognosis for gastric cancer particularly in Asia.     

Combined targeting of high‐mobility group box‐1 and interleukin‐8 to control micrometastasis potential in gastric cancer

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial‐to‐mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT‐promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high‐mobility group box‐1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin‐8 (IL‐8) at early‐stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV‐SPORT6‐HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU‐1 and KATOIII), N87‐xenograft BALB/c nude mice and human paired serum‐tissue GC samples. HMGB1‐induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL‐8 were evaluated in N87‐xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1‐induced IL‐8 overexpression contributed the HMGB1‐induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL‐8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL‐8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL‐8 as a promising diagnostic and therapeutic target to control GC micrometastasis.     

Increased T‐helper 17 cell differentiation mediated by exosome‐mediated microRNA‐451 redistribution in gastric cancer infiltrated T cells

MicroRNA (miR)‐451 is a cell metabolism‐related miRNA that can mediate cell energy‐consuming models by several targets. As miR‐451 can promote mechanistic target of rapamycin (mTOR) activity, and increased mTOR activity is related to increased differentiation of T‐helper 17 (Th17) cells, we sought to investigate whether miR‐451 can redistribute from cancer cells to infiltrated T cells and enhance the distribution of Th17 cells through mTOR. Real‐time PCR was used for detecting expression of miR‐451 in gastric cancer, tumor infiltrated T cells and exosomes, and distribution of Th17 was evaluated by both flow cytometry and immunohistochemistry (IHC). Immunofluorescence staining was used in monitoring the exosome‐enveloped miR‐451 from cancer cells to T cells with different treatments, and signaling pathway change was analyzed by western blot. miR‐451 decreased significantly in gastric cancer (GC) tissues but increased in infiltrated T cells and exosomes; tumor miR‐451 was negatively related to infiltrated T cells and exosome miR‐451. Exosome miR‐451 can not only serve as an indicator for poor prognosis of post‐operation GC patients but is also related to increased Th17 distribution in gastric cancer. miR‐451 can redistribute from cancer cells to T cells with low glucose treatment. Decreased 5′ AMP‐activated protein kinase (AMPK) and increased mTOR activity was investigated in miR‐451 redistributed T cells and the Th17 polarized differentiation of these T cells were also increased. Exosome miR‐451 derived from tumor tissues can serve as an indicator for poor prognosis and redistribution of miR‐451 from cancer cells to infiltrated T cells in low glucose treatment can enhance Th17 differentiation by enhancing mTOR activity.     

Changes in aberrant DNA methylation after Helicobacter pylori eradication: A long‐term follow‐up study

Changes of DNA methylation in gastric mucosae after eradication of Helicobacter pylori have not been clarified yet. From this background, we investigated time course of DNA methylation following H. pylori eradication in 221 successfully H. pylori eradicated subjects with endoscopic follow‐up at least for 6 months, including 114 controls, 53 subjects with gastric dysplasia and 54 patients with early gastric cancer. All dysplasia and gastric cancer patients underwent endoscopic resection at the time of enrollment. The methylation levels in LOX, APC and MOS genes from noncancerous gastric mucosae using quantitative methylation‐specific PCR, as well as the histologic findings of gastric mucosae, were compared before and after eradication. Average follow‐up duration was 26.0 months (range: 6 to 76 months). H. pylori eradication decreased methylation levels in LOX (p‐value for slope < 0.001) but not in APC. In MOS, decrease of its methylation level following H. pylori eradication was significant among controls without intestinal metaplasia (IM) (p‐value for slope < 0.05); however, it was not observed among patients with IM or those with dysplasia or gastric cancer. After H. pylori eradication, methylation level in MOS persistently increased in patients with dysplasia or gastric cancer (p < 0.01). In conclusion, H. pylori eradication decreases aberrant DNA methylation with gene‐specific manner. Methylation level in MOS is associated with IM and may be used as a surrogate marker for gastric cancer risk, regardless of H. pylori eradication history.     

Detection of gastric carcinoma‐associated MG7‐Ag by serum immuno‐PCR assay in a high‐risk Chinese population,with implication for screening

To evaluate gastric carcinoma‐associated antigen, MG7‐Ag, for detection of gastric cancer in a high‐risk population, a population‐based screening of gastric cancer was conducted in Linqu County, Shandong Province, China. In 2002 and 2003, a total of 2,710 participants aged 35–65 years received an endoscopic examination with 5 biopsies taken from standard sites with pathological diagnosis, and serum samples were collected to detect MG7‐Ag by serum‐based Immunopolymerase chain reaction (PCR) assay. The sensitivity and specificity of MG7‐Ag Immuno‐PCR assay in detecting of gastric cancer were assessed. Of 2,710 participants, 148 (5.46%) were determined to be MG7‐Ag positive. The sensitivity of MG7‐Ag Immuno‐PCR assay for the detection of gastric cancer was 77.5% (31 of 40 gastric cancer cases), the specificity was 95.62% (2,553 of 2,670 nongastric cancer subjects) and the accuracy was 73.12%. A total of 24 gastric cancer cases were in Stage I or II, of which 17 (70.8%) were MG7‐Ag positive. However, the proportion of MG7‐Ag positivity in subjects with superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, indefinite dysplasia or dysplasia was ranged from 3.00% to 5.61% in comparison with 77.5% in those with gastric cancer. Our findings suggest that MG7‐Ag was a sensitive and specific serum biomarker and may have a potential for gastric cancer screening in the high‐risk population.     

Antitumor activity of pan‐HER inhibitors in HER2‐positive gastric cancer

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti‐human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti‐HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2‐positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan‐HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2‐amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin‐like growth factor‐1 receptor (IGF‐1R), were less sensitive to pan‐HER inhibitors. A combination therapy consisting of pan‐HER inhibitors and an IGF‐1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2‐amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF‐1R‐targeting therapy can overcome drug insensitiveness in HER2‐amplified gastric cancer.     

Peroxisome proliferator‐activated receptor γ upregulates galectin‐9 and predicts prognosis in intestinal‐type gastric cancer

The importance of PPARγ (peroxisome proliferator‐activated receptor γ) in gastric cancer (GC) is unclear. We investigated the role of PPARγ in GC cell lines and an animal model, and its prognostic significance of PPARγ in GC patients. We controlled PPARγ and galectin‐9 expression by using siRNAs and lentiviral constructs. Interaction between PPARγ and galectin‐9 was evaluated using luciferase and chromatin immunoprecipitation assays. PPARγ expression in GCs was determined by immunohistochemical staining of tissue microarrays and survival analysis was done. Overexpression of PPARγ was accompanied by increased galectin‐9. Enhanced PPARγ or galectin‐9 expression increased E‐cadherin expression; decreased expression of N‐cadherin, fibronectin, snail, twist and slug and reduced cell invasion and migration. PPARγ bound to the galectin‐9 promoter region. Galectin‐9 activity increased in PPARγ‐overexpressing cells but decreased in PPARγ siRNA‐treated cells. In a zebrafish xenograft model, the number of migrated cancer cells and number of fish with AGS cells in the tail vein were reduced in PPARγ‐overexpressing GC cells. PPARγ was expressed in 462 of the 688 patients (69.2%) with GC. In 306 patients with intestinal‐type GC, those with PPARγ‐positive tumors had lower overall and cancer‐specific mortalities than those with PPARγ‐negative tumors. PPARγ expression was an independent prognostic factor for overall and GC‐specific mortality in patients with intestinal‐type GC (adjusted hazard ratio, 0.42; 95% CI, 0.22–0.81). PPARγ inhibits cell invasion, migration and epithelial–mesenchymal transition through upregulation of galectin‐9 in vitro and in vivo.     

Blood leukocyte DNA hypomethylation and gastric cancer risk in a high‐risk Polish population

Global hypomethylation has been shown to increase genome instability potentially leading to increased cancer risk. We determined whether global methylation in blood leukocyte DNA was associated with gastric cancer in a population‐based study on 302 gastric cancer cases and 421 age‐ and sex‐matched controls in Warsaw, Poland, between 1994 and 1996. Using PCR‐pyrosequencing, we analyzed methylation levels of Alu and LINE‐1, 2 CG‐rich repetitive elements, to measure global methylation levels. Gastric cancer risk was highest among those with lowest level of methylation in either Alu (OR = 1.3, 95% CI = 0.9–1.9) or LINE‐1 (OR = 1.4, 95% CI = 0.9–2.0) relative to those with the highest levels, although the trends were not statistically significant. For Alu, the association was stronger among those aged 70 or older (OR = 2.6, 95% CI = 1.3–5.5, p for interaction = 0.02). We did not observe meaningful differences in the associations by other risk factors and polymorphisms examined. For LINE‐1, the association tended to be stronger among individuals with a family history of cancer (OR = 3.1, 95% CI = 1.4–7.0, p for interaction = 0.01), current alcohol drinkers (OR = 1.9, 95% CI = 1.0–3.6, p for interaction = 0.05), current smokers (OR = 2.3, 95% CI = 1.1–4.6, p for interaction = 0.02), those who rarely or never consumed fruit (OR = 3.1, 95% CI = 1.2–8.1, p for interaction = 0.03), CC carriers for the MTRR Ex5+123C>T polymorphism (OR = 2.3, 95% CI = 1.2–4.4, p for interaction = 0.01) and TT carriers for the MTRR Ex15+572T>C polymorphism (OR = 1.7, 95% CI = 1.0–2.8, p for interaction = 0.06). The association was not different by sex, Helicobacter pylori infection, intake of folate, vitamin B6 and total protein and the remaining polymorphisms examined. Our results indicate that interactions between blood leukocyte DNA hypomethylation and host characteristics may determine gastric cancer risk.     

Anti‐angiogenic therapies for gastric cancer

Tumor angiogenesis plays an important role in cancer cell proliferation and metastasis. In gastric cancer, among the numerous clinical trials investigating various anti‐angiogenic therapies, such as antivascular endothelial growth factor (VEGF) or anti‐VEGF receptor (VEGFR)‐2 monoclonal antibodies, VEGF‐Trap and VEGFR tyrosine kinase inhibitors, the anti‐VEGFR‐2 antibody ramucirumab was shown to prolong overall survival not only as a single agent but also in combination with paclitaxel as a second‐line chemotherapy. Additionally, apatinib, a selective VEGFR‐2 tyrosine kinase inhibitor, prolonged survival as a third‐line or later treatment option in patients with advanced gastric cancer. Preliminary results of studies investigating ramucirumab plus immune checkpoint inhibitors in gastric cancer were encouraging, and further investigations are ongoing. In China, apatinib in combination with cytotoxic agents is being investigated for systemic chemotherapy or maintenance therapy as an earlier treatment option. The clinical activity in gastric cancer of the multikinase inhibitor regorafenib was suggested in a randomized phase II study. A global phase III trial comparing regorafenib with placebo is currently ongoing. Further studies of anti‐angiogenic therapy combined with not only chemotherapy but also immune checkpoint inhibitors are also being pursued, providing hope for improved survival in patients with gastric cancer.     

A prospective evaluation of the utility of 2‐deoxy‐2‐[18F]fluoro‐D‐glucose positron emission tomography and computed tomography in staging locally advanced gastric cancer

BACKGROUND:

The aim of this study was to examine prospectively the utility of adding preoperative [¹⁸F]fluorodeoxyglucose positron emission tomography (FDG‐PET)/computed tomography (CT) to routine CT, endoscopic ultrasound (EUS), and laparoscopic staging of localized gastric cancer.

METHODS:

Patients with locally advanced gastric/gastroesophageal cancer were screened for 2 institutional review board–approved Memorial Sloan‐Kettering Cancer Center neoadjuvant chemotherapy protocols. Locally advanced disease was defined as T3 or T4, or lymph node–positive, based on EUS and high‐resolution CT scan. All patients underwent both standard FDG‐PET/CT and laparoscopy with cytological examination of washings. The sensitivity and specificity of FDG‐PET/CT for the identification of metastatic disease not seen on CT was determined. An economic model using Medicare/Medicaid reimbursement charges was developed to assess the cost‐effectiveness of these interventions.

RESULTS:

A total of 113 patients were enrolled from 2003 to 2010. All patients were assessed as having locally advanced disease by CT/EUS. FDG uptake in the primary tumor was associated with male sex, proximal tumors, and nondiffuse Lauren's subtype. 31 (27%) patients had occult metastatic disease detected by PET/CT (n = 11, 10%) and/or laparoscopy (n = 21, 19%), with a single overlap. Economic modeling suggests that the addition of FDG‐PET/CT to the standard staging evaluation of patients with locally advanced gastric cancer resulted in an estimated cost savings of ～US $13,000 per patient.

CONCLUSIONS:

FDG‐PET/CT identifies occult metastatic lesions in approximately 10% of patients with locally advanced gastric cancer. Because of reduced morbidity from fewer futile surgeries and lower patient care costs, PET/CT should be considered as a component of the standard staging algorithm for localized gastric cancer. Cancer 2012. © 2012 American Cancer Society.