Glandular orientation and shape determined by computational pathology could identify aggressive tumor for early colon carcinoma: a triple-center study.

BACKGROUND: Identifying the early-stage colon adenocarcinoma (ECA) patients who have lower risk cancer vs. the higher risk cancer could improve disease prognosis. Our study aimed to explore whether the glandular morphological features determined by computational pathology could identify high risk cancer in ECA via H&E images digitally. METHODS: 532 ECA patients retrospectively from 2 independent data centers, as well as 113 from The Cancer Genome Atlas (TCGA), were enrolled in this study. Four tissue microarrays (TMAs) were constructed across ECA hematoxylin and eosin (H&E) stained slides. 797 quantitative glandular morphometric features were extracted and 5 most prognostic features were identified using minimum redundancy maximum relevance to construct an image classifier. The image classifier was evaluated on D2/D3 = 223, D4 = 46, D5 = 113. The expression of Ki67 and serum CEA levels were scored on D3, aiming to explore the correlations between image classifier and immunohistochemistry data and serum CEA levels. The roles of clinicopathological data and ECAHBC were evaluated by univariate and multivariate analyses for prognostic value. RESULTS: The image classifier could predict ECA recurrence (accuracy of 88.1%). ECA histomorphometric-based image classifier (ECAHBC) was an independent prognostic factor for poorer disease-specific survival [DSS, (HR = 9.65, 95% CI 2.15-43.12, P = 0.003)]. Significant correlations were observed between ECAHBC-positive patients and positivity of Ki67 labeling index (Ki67Li) and serum CEA. CONCLUSION: Glandular orientation and shape could predict the high risk cancer in ECA and contribute to precision oncology. Computational pathology is emerging as a viable and objective means of identifying predictive biomarkers for cancer patients.

Nuclear shape, architecture and orientation features from H&E images are able to predict recurrence in node-negative gastric adenocarcinoma.

BACKGROUND: Identifying intestinal node-negative gastric adenocarcinoma (INGA) patients with high risk of recurrence could help perceive benefit of adjuvant therapy for INGA patients following surgical resection. This study evaluated whether the computer-extracted image features of nuclear shapes, texture, orientation, and tumor architecture on digital images of hematoxylin and eosin stained tissue, could help to predict recurrence in INGA patients. METHODS: A tissue microarrays cohort of 160 retrospectively INGA cases were digitally scanned, and randomly selected as training cohort (D1 = 60), validation cohort (D2 = 100 and D3 = 100, D2 and D3 are different tumor TMA spots from the same patient), accompanied with immunohistochemistry data cohort (D3' = 100, a duplicate cohort of D3) and negative controls data cohort (D5 = 100, normal adjacent tissues). After nuclear segmentation by watershed-based method, 189 local nuclear features were captured on each TMA core and the top 5 features were selected by Wilcoxon rank sum test within D1. A morphometric-based image classifier (NGAHIC) was composed across the discriminative features and predicted the recurrence in INGA on D2. The intra-tumor heterogeneity was assessed on D3. Manual nuclear atypia grading was conducted on D1 and D2 by two pathologists. The expression of HER2 and Ki67 were detected by immunohistochemistry on D3 and D3', respectively. The association between manual grading and INGA outcome was analysis. RESULTS: Independent validation results showed the NGAHIC achieved an AUC of 0.76 for recurrence prediction. NGAHIC-positive patients had poorer overall survival (P = 0.017) by univariate survival analysis. Multivariate survival analysis, controlling for T-stage, histology stage, invasion depth, demonstrated NGAHIC-positive was a reproducible prognostic factor for poorer disease-specific survival (HR = 17.24, 95% CI 3.93-75.60, P < 0.001). In contrast, human grading was only prognostic for one reader on D2. Moreover, significant correlations were observed between NGAHIC-positive patients and positivity of HER2 and Ki67 labeling index. CONCLUSIONS: The NGAHIC could provide precision oncology, personalized cancer management.

Adiponectin polymorphisms and non-alcoholic fatty liver disease risk: a meta-analysis.

BACKGROUND AND AIM: The adiponectin polymorphism has been implicated in susceptibility to non-alcoholic fatty liver disease (NAFLD), but the results remain inconclusive. The aim of this meta-analysis is to investigate the association between adiponectin polymorphisms and NAFLD risk. METHODS: All eligible case-control studies published up to September 2013 were identified by searching PubMed, Web of Science, and CNKI. Effect sizes of odds ratio (OR) and 95% confidence interval (95% CI) were calculated by using a fixed- or random-effect model. RESULTS: A total of 10 case-control studies were included; of those, there were nine studies (1223 cases and 1580 controls) for +45T>G polymorphism, seven studies (876 cases and 989 controls) for +276G>T polymorphism, and three studies (299 cases and 383 controls) for -11337C>G polymorphism. Overall, a significantly increased risk was found for +45T>G and -11377C>G polymorphism (+45T>G: OR = 1.45, 95% CI: 1.06-2.00 for recessive model, OR = 1.48, 95% CI: 1.07-2.06 for GG vs TT; -11377C>G: OR = 1.52, 95% CI: 1.10-2.09 for dominant model, OR = 3.88, 95% CI: 1.29-11.68 for GG vs CC), while for +276G>T polymorphism, we found a significantly decreased risk between them (OR = 0.65, 95% CI: 0.45-0.94 for recessive model, OR = 0.58, 95% CI: 0.40-0.84 for TT vs GG). In subgroup analysis by ethnicity, significant association was detected among Asians for +276G>T polymorphism, but not for +45T>G polymorphism. Besides, none of the three adiponectin polymorphisms was associated with the serum adiponectin levels. CONCLUSION: This meta-analysis suggests that adiponectin +45T>G and -11377C>G polymorphisms might be a risk factor for NAFLD, while +276G>T polymorphism may be a protective factor for NAFLD among Asians.

Double-nylon purse-string suture in closing postoperative wounds following endoscopic resection of large (≥ 3 cm) gastric submucosal tumors.

BACKGROUND: Endoscopic full-thickness resection (EFTR) of gastric submucosal tumors (SMTs) is safe and effective; however, postoperative wound management is equally important. Literature on suturing following EFTR for large (≥ 3 cm) SMTs is scarce and limited. AIM: To evaluate the efficacy and clinical value of double-nylon purse-string suture in closing postoperative wounds following EFTR of large (≥ 3 cm) SMTs. METHODS: We retrospectively analyzed the data of 85 patients with gastric SMTs in the fundus of the stomach or in the lesser curvature of the gastric body whose wounds were treated with double-nylon purse-string sutures after successful tumor resection at the Endoscopy Center of Renmin Hospital of Wuhan University. The operative, postoperative, and follow-up conditions of the patients were evaluated. RESULTS: All tumors were completely resected using EFTR. 36 (42.35%) patients had tumors located in the fundus of the stomach, and 49 (57.65%) had tumors located in the body of the stomach. All patients underwent suturing with double-nylon sutures after EFTR without laparoscopic assistance or further surgical treatment. Postoperative fever and stomach pain were reported in 13 (15.29%) and 14 (16.47%) patients, respectively. No serious adverse events occurred during the intraoperative or postoperative periods. A postoperative review of all patients revealed no residual or recurrent lesions. CONCLUSION: Double-nylon purse-string sutures can be used to successfully close wounds that cannot be completely closed with a single nylon suture, especially for large (≥ 3 cm) EFTR wounds in SMTs.

Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through ß-catenin/E-cadherin pathway in human hepatocellular cancer.

AIM: To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) in hepatocellular carcinoma (HCC). METHODS: Three human HCC cell lines, i.e., SM-MC7721, HepG2 and Hep3B, were used. We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50. Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of ß-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells. In vitro cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution was assessed with flow cytometry. Invasion and migration ability of before and after the transfection were determined with a transwell assay. Cell apoptosis was demonstrated with Annexin V-FITC. The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. RESULTS: The transfection efficiency was confirmed with Western blotting (1.36 ± 0.07 vs 0.81 ± 0.09, P < 0.01). The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells (P < 0.01). Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50 (61.3% ± 3.1% vs 54.0% ± 2.4%, P < 0.05). The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells (5.8 ± 0.8 vs 21.6 ± 1.3, P < 0.01). Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells (14.8% ± 2.7% vs 3.4% ± 1.3%, P < 0.05). The expression of ß-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells (0.28 ± 0.07 vs 0.56 ± 0.12, P < 0.05; 0.55 ± 0.08 vs 0.39 ± 0.07, P < 0.05). In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells (28.9 ± 7.2 vs 70.1 ± 7.2, P < 0.01). CONCLUSION: The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating ß-catenin, E-cadherin. EBP50 may serve as a potential therapeutic target in HCC.

Decreased EGR3 expression is related to poor prognosis in patients with gastric cancer.

The early growth response (EGR) family has a highly conserved DNA-binding domain and encodes zinc finger proteins, which show suppressive effects on tumour growth. However, the expression and significance of EGR3 in gastric cancer are still unknown. In this study, real-time PCR, immunohistochemistry and western blot assays were performed to detect the expression of EGR3 in gastric cancer tissues and matched non-tumour tissues and to further analyse the EGR3 expression associated with clinical pathological factors, including prognosis. Our results showed that EGR3 expression was significantly lower in gastric cancer tissues compared with matched non-tumour tissues and that patients with lower EGR3 expression had a poorer prognosis compared with patients with higher EGR3 expression. Our results suggest that decreased EGR3 expression might play a critical role in the differentiation, proliferation, metastasis and progression of gastric cancer cells and may also be a potential diagnostic marker for gastric cancer.

Clinical significance of EBP50 overexpression assessed by quantum dot analysis in gastric cancer.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a postsynaptic density-95/disc-large/zonula occludens-1 (PDZ) homologous domain-containing protein that is involved in cell signaling. EBP50 regulates cell apoptosis, proliferation and invasion. In the present study, the prognostic impact factor of EBP50 expression was evaluated using a quantum dot (QD)-based assay and immunohistochemistry (IHC). The EBP50 protein expression in gastric cancer (GC) tissues was evaluated using IHC and QD-IHC. The study included 101 patients with GC (29 females and 72 males, aged 24-81 years), diagnosed and treated at the General Surgery Department of Renmin Hospital of Wuhan University (Wuhan, China) between 2000 and 2005. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. IHC and QD analyses of 101 GC tissue specimens revealed that EBP50-positive tumor cells were frequently present in GC. Increased EBP50 immunostaining was observed in 63 specimens (62.4%). The EBP50 expression levels were correlated with increased tumor size and the male gender. EBP50 was well distributed in the cytoplasm and nuclei of the GC cells. However, EBP50 protein expression exhibited no correlation with age, differentiation, stage or lymph node metastasis. There were no associations between the expression of EBP50 and the mean survival rates (IHC, 50.5 vs. 58.1 months, P>0.05; QD, 55.4 vs. 63.2 months, P>0.05). These findings suggest that EBP50 protein expression is not correlated with the prognosis of patients with GC. QD-IHC and IHC have similar advantages for the detection of EBP50 protein expression.

Hugl-1 induces apoptosis in esophageal carcinoma cells both in vitro and in vivo.

AIM: To determine whether the human giant larvae homolog 1 gene (Hugl-1/Llg1/Lgl1) exerts tumor suppressor effects in esophageal cancer. METHODS: We constructed a Hugl-1 expression plasmid, pEZ-M29-Hugl1, for gene transfection. We transfected the pEZ-M29-Hugl1 plasmid into Eca109 esophageal cancer cell lines with Lipofectamine 2000 to overexpress Hugl-1. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the effects of the plasmid on Hugl-1 expression. In vitro cell proliferation and apoptosis were examined separately by cell counting Kit-8 (CCK-8) assay, flow cytometry, and Western blotting before and after the transfection of the plasmid into Eca109 cells. Cell cycle distribution was assessed with flow cytometry. The effect of Hugl-1 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. Expression of Hugl-1 in xenograft tumor was analyzed by immunohistochemistry. The transferase-mediated dUTP nick end-labeling (TUNEL) technique was performed to detect and quantitate apoptotic cell. RESULTS: The transfection efficiency was confirmed with real-time RT-PCR and Western blotting. Our results show that compared with control groups the mRNA levels and protein levels of Hugl-1 in pEZ-M29-Hugl1-treated group were remarkably increased (P < 0.05). The CCK-8 assay demonstrated that the growth of cells overexpressing Hugl-1 was significantly lower than control cells. Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing Hugl-1 (64.09% ± 3.14% vs 50.32% ± 4.60%, 64.09% ± 3.14% vs 49.13% ± 2.24%). Annexin V-fluorescein isothiocyanate revealed that apoptosis was significantly increased in cells overexpressing Hugl-1 compared with control group (17.33% ± 4.76% vs 6.90% ± 1.61%, 17.33% ± 4.76% vs 6.27% ± 0.38%). Moreover, we found that Hugl-1 changes the level of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax and the activation of both caspase-3 and caspase-9. With a TUNEL assay, we found that Hugl-1 markedly increased the apoptosis rate of Eca109 cells in vivo (60.50% ± 9.11% vs 25.00% ± 12.25%). It was shown that Hugl-1 represents a significantly more effective tumor suppressor gene alone in a xenograft tumor mouse model. This data suggest that Hugl-1 inhibited tumor growth and induced cell apoptosis in vivo. CONCLUSION: These results suggest that Hugl-1 induces growth suppression and apoptosis in a human esophageal squamous cell carcinoma cell line both in vitro and in vivo.

TNF-α-308 polymorphism and risk of digestive system cancers: a meta-analysis.

AIM: To evaluate the association between the tumour necrosis factor alpha-308 (TNF-α-308) gene polymorphism and the risk of digestive system cancers. METHODS: All eligible case-control studies published up to December 2012 were identified by searching PubMed, Web of Science, Embase and China National Knowledge Internet without language restrictions. The risk of digestive system cancers associated with the TNF-α-308 polymorphism was estimated for each study using odds ratio (OR) together with its 95%CI, respectively. Cochrane Collaboration RevMan 5.1 was used to perform the analysis. A χ²-test-based Q statistic test and an I² test were performed to assess the between-study heterogeneity. When the Q test was significant (P < 0.05) or I² > 50%, the random effects model was used, otherwise the fixed effects model was used. RESULTS: Fifty-eight studies from fifty-five publications with a total of 9986 cancer patients and 15511 healthy controls were included. Overall, a significant association was found between the TNF-α-308 polymorphism and the risk of digestive system cancers [dominant model: OR = 1.23, 95%CI: 1.09-1.39, (G/A) vs (G/G): OR = 1.15, 95%CI: 1.02-1.28, (A/A) vs (G/G): OR = 1.44, 95%CI: 1.19-1.73, recessive model: OR = 1.38, 95%CI: 1.15-1.66]. Furthermore, when the analysis was stratified by ethnicity, similar results were observed in both the Asian and Caucasian populations, except for the dominant model and heterozygote comparisons in the Asian population [dominant model: OR = 1.24, 95%CI: 0.99-1.56, (G/A) vs (G/G): OR = 1.09, 95%CI: 0.96-1.24]. When the cancer type subgroups were examined, similar results were detected in gastric and hepatocellular carcinomas; however, no significant association was observed among other digestive system cancers. CONCLUSION: The TNF-α-308 gene polymorphism may be significantly associated with the risk of gastric and hepatocellular carcinomas, but not colorectal, pancreatic, or oesophageal cancer, in the Asian population.

Meta-analysis of the ADH1B and ALDH2 polymorphisms and the risk of colorectal cancer in East Asians.

OBJECTIVE: The aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) genes have been implicated in the development of colorectal cancer (CRC). However, the results are inconsistent. In this study, a meta-analysis was performed to assess the associations between the ALDH2 and ADH1B polymorphisms and the risk of CRC. METHODS: Relevant studies were identified using PubMed, Web of Science and CNKI up to February, 2013. The pooled odds ratio (OR) with a 95% confidence interval (CI) was calculated using the fixed- or random-effects model. RESULTS: A total of 11 case-controlled studies were selected. Of these, 11 studies included 2,893 cases and 3,817 controls concerning the ALDH2 Glu487Lys polymorphism and six studies included 1,864 cases and 3,502 controls concerning the ADH1B polymorphism. The results indicated that there was a statistically significant link between the ALDH2 polymorphism and the risk of CRC (Glu/Lys+Lys/Lys vs. Glu/Glu: OR=0.87, 95%CI: 0.78-0.96, p=0.10; Glu/Lys vs. Glu/Glu: OR=0.87, 95%CI: 0.77-0.97, p=0.38); however, no significant associations were observed between the ADH1B polymorphism and the risk of CRC win any of the genetic models. CONCLUSION: This meta-analysis demonstrated that the ALDH2 polymorphism, but not the ADH1B polymorphism, significantly increases the risk of CRC in East Asians.

Proton pump inhibitor for non-erosive reflux disease: a meta-analysis.

AIM: To evaluate the efficacy, safety and influential factors of proton pump inhibitor (PPI) treatment for non-erosive reflux disease (NERD). METHODS: PubMed, MEDLINE, EMBASE and the Cochrane Library were searched up to April 2013 to identify eligible randomized controlled trials (RCTs) that probed into the efficacy, safety and influential factors of PPI treatment for NERD. The rates of symptomatic relief and adverse events were measured as the outcomes. After RCT selection, assessment and data collection, the pooled RRs and 95%CI were calculated. This meta-analysis was performed using the Stata 12.0 software (Stata Corporation, College Station, Texas, United States). The level of evidence was estimated by the Grading of Recommendations Assessment, Development and Evaluation system. RESULTS: Seventeen RCTs including 6072 patients met the inclusion criteria. The results of the meta-analysis showed that PPI treatment was significantly superior to H2 receptor antagonists (H2RA) treatment (RR = 1.629, 95%CI: 1.422-1.867, P = 0.000) and placebo (RR = 1.903, 95%CI: 1.573-2.302, P = 0.000) for the symptomatic relief of NERD. However, there were no obvious differences between PPI and H2RA (RR = 0.928, 95%CI: 0.776-1.110, P = 0.414) or PPI and the placebo (RR = 1.000, 95%CI: 0.896-1.116, P = 0.997) regarding the rate of adverse events. The overall rate of symptomatic relief of PPI against NERD was 51.4% (95%CI: 0.433-0.595, P = 0.000), and relief was influenced by hiatal hernia (P = 0.030). The adverse rate of PPI against NERD was 21.0% (95%CI: 0.152-0.208, P = 0.000), and was affected by hiatal hernia (P = 0.081) and drinking (P = 0.053). CONCLUSION: PPI overmatched H2RA on symptomatic relief rate but not on adverse rate for NERD. Its relief rate and adverse rate were influenced by hiatal hernia and drinking.

EBP50 gene transfection promotes 5-fluorouracil-induced apoptosis in gastric cancer cells through Bax- and Bcl-2-triggered mitochondrial pathways.

5-Fluorouracil (5-FU) plays an important role in the chemotherapy of advanced gastric cancer. However, genetic factors that affect therapeutic efficacy of 5-FU warrant further investigation. In the present study, using stable transfection of the ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) gene, we explored the genetic influences on 5-FU-induced apoptosis of human gastric cancer cells. Stable overexpression of the EBP50 gene was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and western blot analysis. After treatment with 5-FU, cell growth activities in vitro were investigated by MTT assay. Cell apoptosis was evaluated by Hoechst 33258 staining and flow cytometry of Annexin V-FITC/PI staining. Compared with the BGC823 or BGC823/neo cells, EBP50 mRNA and protein levels in the BGC823/EBP50 cells (EBP50-transfected BGC823 cells) were markedly higher. Chemosensitivity and apoptosis rates of the BGC823/EBP50 cells were higher compared to the BGC823 and BGC823/neo cells following treatment with 5-FU. Stable overexpression of extrinsic EBP50 distinctly increases the 5-FU-induced apoptosis of gastric cancer cells, and is a novel strategy by which to improve the chemosensitivity of gastric cancer to 5-FU.

The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated ß-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.