Yanfang Gong

Combination of plasma microRNAs with serum CA19-9 for early detection of pancreatic cancer

This study was performed to identify plasma microRNAs (miRNAs) as diagnostic biomarkers for pancreatic cancer (PCa) and to assess their supplementary role with serum CA19‐9 in early identification of tumors. Plasma RNAs were extracted from 140 PCa patients, 111 chronic pancreatitis (CP) patients and 68 normal controls, and the relative abundances of seven miRNAs (miR‐16, 21, 155, 181a, 181b, 196a and 210) were measured using real‐time PCR. Their diagnostic utility for PCa and correlation with clinical characteristics were analyzed. All seven miRNAs were significantly aberrantly upregulated in the PCa group compared with both the CP and normal groups, between which only four miRNAs (miR‐155, 181a, 181b and 196a) were significantly different. Logistic modeling proved that only miR‐16 and miR‐196a possessed an independent role in discriminating PCa from normal and CP. Furthermore, after including serum CA19‐9 in the logistic model, the combination of miR‐16, miR‐196a and CA19‐9 was more effective for discriminating PCa from non‐PCa (normal+CP) (AUC‐ROC, 0.979; sensitivity, 92.0%; specificity, 95.6%), and for discriminating PCa from CP (AUC‐ROC, 0.956; sensitivity, 88.4%; specificity, 96.3%) compared with the miRNA panel (miR‐16+miR‐196a) or CA19‐9 alone. Most significantly, the combination was effective at identification of tumors in Stage 1 (85.2%). In conclusion, plasma miRNAs were effective for distinguishing PCa from non‐PCa (normal+CP). The combination of miR‐16, miR‐196a and CA19‐9 was more effective for PCa diagnosis, especially in early tumor screening.

Epidemiology of Peptic Ulcer Disease: Endoscopic Results of the Systematic Investigation of Gastrointestinal Disease in China

OBJECTIVES:Complications of peptic ulcer disease (PUD) are common in China. Population-based estimates of the prevalence of PUD are needed to quantify and characterize the population at risk of these complications.METHODS:As part of a large epidemiological study, 3,600 randomly selected residents of Shanghai (aged 18–80 years) were asked to undergo endoscopy and to provide blood samples for Helicobacter pylori serology. All participants also completed a general information questionnaire and Chinese versions of the reflux disease questionnaire (RDQ) and Rome II questionnaire. Associations between PUD and other factors were analyzed using a multiple logistic regression model.RESULTS:In total, 3,153 individuals (87.6%) completed the survey. All underwent blood tests, and 1,030 patients (32.7%) agreed to undergo endoscopy. Results from 1,022 patients were suitable for analysis. In all, 176 participants (17.2%) had PUD (62 with gastric ulcer; 136 with duodenal ulcer). The prevalence of H. pylori infection was 73.3% in the total population and 92.6% among those with PUD. H. pylori infection was associated with the presence of PUD (odds ratio (OR), 6.77; 95% confidence interval (CI), 2.85–16.10). The majority (72.2%) of individuals with PUD had none of the upper gastrointestinal symptoms assessed by the RDQ. PUD was not significantly associated with symptom-defined gastroesophageal reflux disease (GERD) (OR, 0.80; 95% CI, 0.32–2.03), reflux esophagitis (OR, 1.46; 95% CI, 0.76–2.79) or dyspepsia (OR, 1.69; 95% CI, 0.94–3.04).CONCLUSIONS:The prevalence of endoscopically confirmed PUD in this Shanghai population (17.2%) is substantially higher than in Western populations (4.1%). The majority of individuals with PUD were asymptomatic.

Diagnostic value of mucins (MUC1, MUC2 and MUC5AC) expression profile in endoscopic ultrasound-guided fine-needle aspiration specimens of the pancreas.

Mucins are aberrantly expressed in various malignancies. We immunohistochemistrically tested mucins expression (MUC1, MUC2 and MUC5AC) in EUS‐FNA samples from pancreatic occupying lesions for the diagnostic utility. The prevalence of MUC1, MUC2 and MUC5AC expression in pancreatic cancers were 77.5% (31/40), 10.0% (4/40) and 80.0% (32/40), respectively, and in the benign pancreatic diseases 25% (4/16), 31.3% (5/16) and 43.8% (7/16). MUC1 and MUC5AC significantly overexpressed in pancreatic cancer, and MUC1 negatively related with tumor differentiation degree (p < 0.05). The prevalence of MUC1, MUC2 and MUC5AC expression in pancreatic mucinous neoplasms were 66.7% (12/18), 38.9% (7/18) and 88.9% (16/18), respectively, and in the pancreatic nonmucinous neoplasms 60.5% (23/38), 5.3% (2/38) and 57.9% (22/38). MUC2 and MUC5AC significantly overexpressed in pancreatic mucinous neoplasms, especially MUC2 in benign mucinous neoplasms (p < 0.05). Compared with cytology alone, the combination test of MUC1+cytology, and MUC5AC+cytology could achieve higher sensitivity (85 vs. 65%, 100 vs. 65%) and accuracy (89.3% vs. 73.2%, 91.1% vs. 73.2%) for pancreatic cancer diagnosis; the combination test of MUC2 + cytology, and MUC5AC + cytology could achieve higher sensitivity (77.8% vs. 38.9%, 100% vs. 38.9%), and specificity (97.4% vs. 60.5%, 71.1% vs. 60.5%) accuracy (100% vs. 51.8%, 80.4% vs. 51.8%) for mucinous neoplasm diagnosis. The panel MUC1+/MUC2−/MUC5AC+/ was higher specific in pancreatic cancer diagnosis, as well as MUC1−/MUC2+/MUC5AC+/ in pancreatic mucinous neoplasms. Our observations suggest the mucins expression profile in EUS‐FNA specimens has higher value for the diagnosis of pancreatic cancer and mucinous neoplasms.

Epidemiology of symptom-defined gastroesophageal reflux disease and reflux esophagitis: the systematic investigation of gastrointestinal diseases in China (SILC).

Abstract Objective. Gastroesophageal reflux disease (GERD) is thought to be less prevalent in China than in Western countries. However, essential population-based endoscopy data are lacking for this country. Material and methods. As part of a wider study, 3600 individuals selected randomly from the Shanghai region were asked to undergo endoscopy. Participants completed a general information questionnaire and a Chinese version of the Reflux Disease Questionnaire. When sufficient numbers were available, associations were assessed using multiple logistic regression or the Wilcoxon rank-sum test. Results. Of 3153 (87.6%) individuals who completed the survey, 1030 (32.7%) agreed to endoscopy and 1029 endoscopies were suitable for analysis. Symptom-defined GERD was more prevalent in the endoscopy group (4.7%) than in the non-endoscopy group (1.7%). Prevalence estimates were 6.4% for reflux esophagitis, 1.8% for endoscopically suspected esophageal metaplasia and 0.7% for hiatus hernia. Reflux esophagitis was more prevalent in patients with symptom-defined GERD than in those without (12.5% [6/48] vs. 6.1% [60/981]), and was significantly associated with reflux symptoms of any frequency or severity (OR = 2.10, 95% CI 1.13–3.89) and with negative Helicobacter pylori infection (OR = 0.44, 95% CI 0.25–0.80). Only 28.8% of participants with reflux esophagitis had heartburn and/or regurgitation symptoms. Epigastric burning was significantly more severe and frequent in participants with reflux esophagitis than in those without (p = 0.05). Conclusions. Reflux esophagitis is less prevalent in China than reported in Western countries. Further work is needed to establish why reflux esophagitis appears less symptomatic in China than in Western countries.

Adjuvant probiotics improve the eradication effect of triple therapy for Helicobacter pylori infection

AIM To investigate whether the addition of probiotics can improve the eradication effect of triple therapy for Helicobacter pylori (H. pylori) infection. METHODS This open randomized trial recruited 234 H. pylori positive gastritis patients from seven local centers. The patients were randomized to one-week standard triple therapy (omeprazole 20 mg bid, clarithromycin 500 mg bid, and amoxicillin 1000 mg bid; OCA group, n = 79); two weeks of pre-treatment with probiotics, containing 3 × 10(7)Lactobacillus acidophilus per day, prior to one week of triple therapy (POCA group, n = 78); or one week of triple therapy followed by two weeks of the same probiotics (OCAP group, n = 77). Successful eradication was defined as a negative C13 or C14 urease breath test four weeks after triple therapy. Patients were asked to report associated symptoms at baseline and during follow-up, and side effects related to therapy were recorded. Data were analyzed by both intention-to-treat (ITT) and per-protocol (PP) methods. RESULTS PP analysis involved 228 patients, 78 in the OCA, 76 in the POCA and 74 in the OCAP group. Successful eradication was observed in 171 patients; by PP analysis, the eradication rates were significantly higher (P = 0.007 each) in the POCA (62/76; 81.6%, 95% CI 72.8%-90.4%) and OCAP (61/74; 82.4%, 95% CI 73.6%-91.2%) groups than in the OCA group (48/78; 61.5%, 95% CI 50.6%-72.4%). ITT analysis also showed that eradication rates were significantly higher in the POCA (62/78; 79.5%, 95% CI 70.4%-88.6%) and OCAP (61/77; 79.2%, 95% CI 70%-88.4%) groups than in the OCA group (48/79; 60.8%, 95% CI 49.9%-71.7%), (P = 0.014 and P = 0.015). The symptom relieving rates in the POCA, OCAP and OCA groups were 85.5%, 89.2% and 87.2%, respectively. Only one of the 228 patients experienced an adverse reaction. CONCLUSION Administration of probiotics before or after standard triple therapy may improve H. pylori eradication rates.

Methylation of the SPARC gene promoter and its clinical implication in pancreatic cancer

BackgroundThe secreted protein acidic and rich in cysteine (SPARC) plays a pivotal role in regulating cell-matrix interactions and tumor angiogenesis, proliferation, and migration. Detection of SPARC gene methylation may be useful as a tumorigenesis marker for early detection of pancreatic cancer.MethodsMethylation of the SPARC gene transcriptional regulation region (TRR) was detected using bisulfite-specific (BSP) PCR-based sequencing analysis in 40 cases of pancreatic cancer and the adjacent normal tissues, 6 chronic pancreatitis tissues, and 6 normal pancreatic tissues. BSP cloning-based sequencing analysis was also performed in selected cases. Clinicopathological data from the cancer patients were collected and analyzed.ResultsAnalysis of SPARC gene TRR methylation showed two hypermethylation wave peak regions: CpG Region 1 (CpG site 1-7) and CpG Region 2 (CpG site 8-12). Pancreatic tissues have shown methylation in both regions with gradual increases from normal, chronic pancreatitis, and adjacent normal tissues to cancerous tissues. However, Methylation of CpG Region 2 was more sensitive than CpG Region 1 in pancreatic tumorigenesis. Furthermore, the methylation level of CpG Region 2 was associated with increased tumor size and exposure to the risk factors (tobacco smoke and alcohol consumption) for developing pancreatic cancer.ConclusionMethylation of the SPARC gene, specifically CpG Region 2, may be an early event during pancreatic tumorigenesis and should be further evaluated as a tumorigenesis marker for early detection of pancreatic cancer.

Aberrant expression miR-196a is associated with abnormal apoptosis, invasion, and proliferation of pancreatic cancer cells.

Objectives MiR-196a levels inversely correlated with survival in pancreatic adenocarcinoma patients. However, the functional contributions of miR-196a to pancreatic cancer remain unclear. Methods Three lentiviral vectors encoding microRNA miR-196a precursor, inhibitor, and scrambled microRNA oligomer were transfected into Panc-1 cells, respectively. Then we explored the regulation of inhibitor of growth 5 (ING5) expression by miR-196a and its impact on apoptosis, invasion, and growth of pancreatic cancer cells. The lentiviral transfected Panc-1 cells were surgically implanted into the pancreas of mice. In vivo tumor growth and ING5 expression were measured. Results Down-regulation of ING5 expression was detected in cells transfected with miR-196a precursor (P < 0.01), accompanied by less apoptosis, increased invasion, and proliferation compared with control cells (P < 0.05). Cells transfected with miR-196a inhibitor revealed an opposite trend. Smaller detectable tumors were found in only 60% of mice after implantation of Lenti.miR-196a inhibitor–transfected Panc-1 cells compared with controls (360.7 ± 303.6 mm3 vs 511.58 ± 365.9 mm3 in controls; P < 0.01). Conclusion Our results provide experimental evidence to support aberrant expression of miR-196a is associated with abnormal apoptosis, invasion, and proliferation of pancreatic cancer cells.

Differential signature of fecal microRNAs in patients with pancreatic cancer.

The potential value of microRNAs as new biomarkers for pancreatic cancer (PCa) screening was explored in this study. Fecal microRNAs from stool samples obtained from 29 PCa patients, 22 chronic pancreatitis (CP) patients and 13 normal individuals were extracted, and 7 microRNAs (miR-16, miR-21, miR-155, miR-181a, miR-181b, miR-196a and miR-210) were detected. miR-181b and miR-210 discriminated PCa from normal individuals with receiver operating characteristic (ROC) curves and area under curve (AUC-ROC) of 0.745 and 0.772, respectively. There was a significant correlation between miR‑196a and the maximum tumor diameter (Spearman r = 0.516, P = 0.041). These findings suggest that fecal microRNAs such as miR-181b and miR-210 may have potential to be used as new biomarkers for PCa screening.

Detection of KRAS gene mutations in endoscopic ultrasound-guided fine-needle aspiration biopsy for improving pancreatic cancer diagnosis.

OBJECTIVES:Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) is a useful tool in the diagnosis of pancreatic masses. Genetic analysis of these samples could increase the sensitivity and specificity of diagnosis. This study aimed to evaluate the usefulness of a novel method for the detection of mutations in the KRAS (Kirsten rat sarcoma-2 virus) gene for the diagnosis of pancreatic cancer.METHODS:EUS-FNABs were performed on 82 patients with pancreatic masses, including 54 cases of pancreatic ductal adenocarcinoma and 28 of non-malignant pancreatic masses. The biopsies were histopathologically and cytopathologically evaluated, and the detection of KRAS gene mutations (codons 12 and 13) was performed through peptide nucleic acid-directed polymerase chain reaction clamping and DNA sequencing.RESULTS:In the pancreatic cancer cases, 88.9% (48/54; 95% confidence interval (CI): 80.5–97.2%) had KRAS mutations, while 61.1% (33/54; 95% CI: 48.1–74.1%) were unequivocally diagnosed by histo/cytopathology. In the indeterminate patients (n=49; diagnosed by EUS-FNA as either insufficient material to make a diagnosis, no malignancy, or suspicion of malignancy), there were 10 cases of pancreatic cancer with low serum carbohydrate antigen 19-9 (CA19-9) (<37 U/l) and 6 of these were KRAS mutations. The sensitivity of detection by KRAS mutations (76.2%) and the combination of KRAS mutations and serum CA19-9 (81%) were significantly higher than for serum CA19-9 alone (52.4%). A logistic regression model showed that the KRAS mutation was significant (odds ratio=5.830; CI: 1.531–22.199, P=0.01), but not serum CA19-9. In the non-malignant pancreatic masses (n=28), KRAS mutations were detected in nine precancerous lesions.CONCLUSIONS:Our method for the detection of KRAS gene mutations may be useful to supplement histo/cytopathologic evaluations for pancreatic cancer, and is superior to serum CA19-9 in EUS-FNAB histo/cytopathology-indeterminate patients. Results warrant further verification in other patient populations.

Adenovirus vector-mediated Gli1 siRNA induces growth inhibition and apoptosis in human pancreatic cancer with Smo-dependent or Smo-independent Hh pathway activation in vitro and in vivo.

Activation of Hedgehog (Hh) signaling pathway is a core molecular mechanism in pancreatic carcinogenesis. However, the inhibition of upstream Hh signals does not inhibit the growth of a subset of pancreatic cancer (PC). This study was to examine the effect of siRNA targeting Gli1, the downstream component of Hh pathway, on PC cells and to provide some insight into the underlying mechanisms. A Gli1siRNA-expressing adenovirus (Ad-U6-Gli1siRNA) was constructed, and its effect on PC cells was investigated in vitro and in vivo. Gli1 was expressed in 83.3% (20/24) PC tissues, whereas no expression was found in normal pancreatic ductal epithelium. Gli1 was expressed in SW1990 and CFPAC cells in which Smo was completely absent, as well as in PaTu8988, Panc-1 and BxPC-3 cells in which Smo was concomitantly present. Ad-U6-Gli1siRNA induced cell growth inhibition, strong G0/G1 cell cycle arrest and apoptosis in all five human PC cell lines. Meanwhile, Ad-U6-Gli1siRNA significantly suppressed the expression of Gli1, Ptch1 and two target genes, Cyclin D2 and Bcl-2, in all five lines. Furthermore, two tumor xenograft nude mice models were established by subcutaneously injecting Smo-positive Panc-1 cells or Smo-negative SW1990 cells. The in vivo experimental results demonstrated that Ad-U6-Gli1siRNA inhibited the growth of both Panc1-derived and SW1990-derived tumors and induced cell apoptosis. Our study indicates that Gli1-targeting siRNA could induce growth inhibition and apoptosis in PC through knockdown of Gli1 and its target genes; and this method may represent a more effective therapeutic strategy for PC with Smo-dependent or Smo-independent Hh pathway activation.