Chinese Consensus Report on Family-Based Helicobacter pylori Infection Control and Management (2021 Edition).

OBJECTIVE: Helicobacter pylori infection is mostly a family-based infectious disease. To facilitate its prevention and management, a national consensus meeting was held to review current evidence and propose strategies for population-wide and family-based H. pylori infection control and management to reduce the related disease burden. METHODS: Fifty-seven experts from 41 major universities and institutions in 20 provinces/regions of mainland China were invited to review evidence and modify statements using Delphi process and grading of recommendations assessment, development and evaluation system. The consensus level was defined as ≥80% for agreement on the proposed statements. RESULTS: Experts discussed and modified the original 23 statements on family-based H. pylori infection transmission, control and management, and reached consensus on 16 statements. The final report consists of three parts: (1) H. pylori infection and transmission among family members, (2) prevention and management of H. pylori infection in children and elderly people within households, and (3) strategies for prevention and management of H. pylori infection for family members. In addition to the 'test-and-treat' and 'screen-and-treat' strategies, this consensus also introduced a novel third 'family-based H. pylori infection control and management' strategy to prevent its intrafamilial transmission and development of related diseases. CONCLUSION: H. pylori is transmissible from person to person, and among family members. A family-based H. pylori prevention and eradication strategy would be a suitable approach to prevent its intra-familial transmission and related diseases. The notion and practice would be beneficial not only for Chinese residents but also valuable as a reference for other highly infected areas.

STAT3 signaling drives EZH2 transcriptional activation and mediates poor prognosis in gastric cancer.

BACKGROUND: STAT3 signaling plays the pivotal role in tumorigenesis through EZH2 epigenetic modification, which enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. Here, another possible feedback mechanism and clinical significance of EZH2 and STAT3 were investigated in gastric cancer (GC). METHODS: STAT3, p-STAT3 (Tyr 705) and EZH2 expression were examined in 63 GC specimens with matched normal tissues by IHC staining. EZH2 and STAT3 were also identified in five GC cell lines using RT-PCR and western blot analyses. p-STAT3 protein was detected by western blotting. In order to investigate whether EZH2 expression was directly regulated by STAT3, EZH2 expression was further detected using siRNA for STAT3 or IL-6 stimulation, with dual luciferase reporter analyses, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The clinical significance of STAT3, p-STAT3 and EZH2 expression was evaluated by multi-factor COX regression and Kaplan-Meier analyses. RESULTS: Hyper-activation of STAT3, p-STAT3 and EZH2 expression were observed in GC cells and tissues. STAT3 signaling was correlated with EZH2 expression in GC (R = 0.373, P = 0.003), which was consistent with our data showing that STAT3 as the transcriptional factor enhanced EZH2 transcriptional activity by binding the relative promoter region (-214 ~ -206). STAT3 was an independent signature for poor survival (P = 0.002). Patients with STAT3+/EZH2+ or p-STAT3+/EZH2+ had a worse outcome than others (P < 0.001); Besides, high levels of STAT3 and EZH2 was associated with advanced TNM staging (P = 0.017). Moreover, treatment with a combination of siSTAT3 and EZH2-specific inhibitor, 3-deazaneplanocin A (DZNEP), increased the apoptotic ratio of cells. It is benefit for targeting STAT3-EZH2 interplay in GC treatment. CONCLUSIONS: Our results indicate that STAT3 status mediated EZH2 upregulation, associated with advanced TNM stage and poor prognosis, suggesting that combination with knockdown of STAT3 and EZH2 inhibitor might be a novel therapy in GC treatment. Collectively, STAT3, p-STAT3 and EZH2 expression were provided for the precision medicine in GC patients.

Role of C9orf140 in the promotion of colorectal cancer progression and mechanisms of its upregulation via activation of STAT5, ß-catenin and EZH2.

C9orf140 is a newly identified and characterized gene which is associated with cell proliferation and tumorigenicity. Expression of C9orf140 is upregulated in human gastric cancer and colorectal cancer (CRC); however, little is known about its role in CRC progression. We have investigated the clinical significance, biological effects and mechanisms of C9orf140 signaling. We found that the expression of C9orf140 is dramatically increased in a subset of CRC and correlates significantly with vascular invasion and lymph node metastasis. Our finding showed that knockdown of C9orf140 significantly reduced cell proliferation and invasion in vitro and dramatically increased overall survival and decreased lung metastasis in vivo. Conversely, overexpression of C9orf140 significantly increased lung metastasis and shortened overall survival when compared with control tumors. C9orf140-induced CRC cell invasion may depend on promoting the epithelial-mesenchymal transition progression. STAT5 may directly interact with the enhancer of zeste homolog 2 (EZH2) and ß-catenin to enhance C9orf140 gene transactivation. Furthermore, C9orf140 may participate in cell invasion which is induced by STAT5, EZH2 or ß-catenin activation. We describe the role of C9orf140 in CRC progression and find that C9orf140 overexpression may be regulated by STAT5, EZH2 and ß-catenin interaction.

Patient-Derived Tumor Organoids Combined with Function-Associated ScRNA-Seq for Dissecting the Local Immune Response of Lung Cancer.

In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient-derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function-associated single-cell RNA sequencing (FascRNA-seq) platform allowing both phenotypic evaluation and scRNA-seq at single-organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA-seq data of individual pLCOs with their responses to anti-PD-1 (αPD-1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti-tumor immunity, to identify potential tumor-reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA-seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.

Let-7c functions as a metastasis suppressor by targeting MMP11 and PBX3 in colorectal cancer.

Accumulating evidence shows that microRNAs, functioning as either oncogenes or tumour suppressors by negatively regulating downstream target genes that are actively involved in tumour initiation and progression, may be promising biomarkers and therapy targets. Data mining through a microRNA chip database indicated that let-7c may be associated with tumour metastasis. Here, we confirmed that down-regulation of let-7c in primary cancer tissues was significantly associated with metastases, advanced TNM stages and poor survival of colorectal cancer patients. Moreover, ectopic expression of let-7c in a highly metastatic Lovo cell line remarkably suppressed cell migration and invasion in vitro by the down-regulation of K-RAS, MMP11 and PBX3, as well as tumour growth and metastases in vivo, whereas inhibition of let-7c in low-metastatic HT29 cells increased cell motility and invasion by the enhanced gene expression of K-RAS, MMP11 and PBX3. Interestingly, the luciferase reporters' activities with the 3'-UTRs of K-RAS, MMP11 and PBX3 were inhibited significantly by let-7c. Importantly, rescue experiments involving the over-expression of these genes without their 3'-UTRs completely reversed the effects of let-7c on tumour metastasis, both in vitro and in vivo. Finally, the levels of let-7c were inversely correlated with those of MMP11 and PBX3, but not with those of K-RAS. Taken together, these results demonstrate that let-7c, apart from its tumour growth suppression role, also functions as a tumour metastasis suppressor in colorectal cancer by directly destabilizing the mRNAs of MMP11 and PBX3 at least.

Diagnostic and Prognostic Performance of MicroRNA-25, Carbohydrate Antigen 19-9, Carcinoembryonic Antigen, and Carbohydrate Antigen 125 in Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic cancer is a malignancy with high mortality due to the difficulties in early detection. We investigated and compared the diagnostic and prognostic performance of several blood biomarkers, including microRNA-25 (miR-25), carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), and carbohydrate antigen 125 (CA125). Methods: A retrospective study was conducted at the Chinese People's Liberation Army General Hospital from May 2014 to September 2018. Serum specimens were collected, and miR-25 expression levels were measured using real-time quantitative polymerase chain reaction. Serum CA19-9, CEA, and CA125 levels were measured using enzyme-linked immunosorbent assay (ELISA). Statistical analyses including nonparametric test, receiver operator characteristic (ROC) curves, Kaplan-Meier analysis, and subsequent log-rank test were performed with PRISM 5.0 software. Univariate and multivariate analyses were performed with the R software. P<0.05 was considered statistically significant. Results: A total of 250 individuals were recruited, including 75 with pancreatic ductal adenocarcinoma (PDAC), 75 with benign lesions, and 100 healthy controls. miR-25, CA19-9, CEA, and CA125 exhibited an area under the curve (AUC) of 0.88, 0.91, 0.81, and 0.76 with a sensitivity of 78.7%, 74.7%, 37.3%, and 35.7% and specificity of 91.5%, 97.0%, 98.2%, and 98.3%, respectively. The combination of miR-25 and CA19-9 further increased the sensitivity to 93.3% with a specificity of 88.5%. Stage-dependent sensitivity was observed with CA19-9, CEA, and CA125. miR-25 levels significantly stratified the prognosis by median level (4,989.97 copies/mL). CA19-9, CEA, and CA125 levels significantly stratified the prognosis by median levels. Univariate and subsequent multivariate analyses identified tumor (T) stage, CA19-9, and CA125 as independent risk factors for PDAC prognosis. Conclusion: The combination of miR-25 and CA19-9 significantly enhanced the detection sensitivity of PDAC. T stage, CA19-9, and CA125 levels were independent risk factors for PDAC prognosis.

CTHRC1 is upregulated by promoter demethylation and transforming growth factor-ß1 and may be associated with metastasis in human gastric cancer.

The gene, collagen triple helix repeat containing 1 (CTHRC1), has been reported to increase in several kinds of human solid cancers and is associated with tumor invasion and metastasis. To date, the expression and function of CTHRC1 in gastric cancer (GC) have not been reported. The aim of this study was to investigate the expression levels and regulatory transcription mechanisms of CTHRC1 in GC. Immunohistochemical analysis revealed that CTHRC1 expression was markedly increased in carcinoma compared with normal gastric mucosa, chronic atrophic gastritis, and intestinal metaplasia (P < 0.05 for all), and this overexpression in tumor was related to depth of tumor invasion. Moreover, RNA interference-mediated knockdown and ectopic expression of CTHRC1 showed that CTHRC1 promoted tumor cell invasion in vitro. We then investigated the mechanisms underlying the aberrant expression of CTHRC1 in GC and found that CTHRC1 expression was restored after GC cell lines were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Transforming growth factor-ß1 led to an increase in levels of CTHRC1 mRNA and protein. Overall, our data revealed that the upregulated expression of CTHRC1 in gastric carcinogenesis contributes to tumor cell invasion and metastasis, and promoter demethylation and transforming growth factor-ß1 may co-regulate the expression of CTHRC1.

Transfection of PDCD5 effect on the biological behavior of tumor cells and sensitized gastric cancer cells to cisplatin-induced apoptosis.

BACKGROUND: Programmed cell death 5 (PDCD5) expression is reduced in various human tumor cells, and the protein concentration and nuclear translocation of PDCD5 is also observed during tumor cell apoptosis. AIMS: The purpose of this study was to investigate the differential expression of PDCD5 in six gastric cell lines, and to explore the changes of biological behavior mechanism underlying enhanced apoptosis-inducing effects of cisplatin by PDCD5 over-expression on gastric cancer BGC823 cells. METHODS: RT-PCR and real-time PCR were used to determine PDCD5 expression. BGC823/PDCD5 cells were assessed the cellular proliferating ability by MTT assay, soft agar cloning experiments and tumorigenicity in nude mice experiments in vivo. The effects of cisplatin in combination with PDCD5 on the proliferation and apoptosis were measured by MTT, Annexin-V-FITC/PI dual labeling and cell cycle analysis, respectively. Immunofluorescence was used to detect co-localization of p53 and PDCD5 protein to explore the mechanism underlying the synergistic therapeutic effect of PDCD5 with cisplatin (5 µg/ml for 24 h). RESULTS: PDCD5 had the highest expression level in the GES1 cell among other cell lines. The growths of BGC823 cells transfected with PDCD5 for six (6th) or 17 (17th) days were both slower than that of BGC823 and BGC823/Neo (P < 0.01). The stable transfection of PDCD5 demonstrated G2/M cell cycle arrest, increased apoptosis and nuclear translocation of PDCD5 and p53 after cisplatin treatment. CONCLUSIONS: Stable transfection of the PDCD5 gene can inhibit the growth of the BGC823 cell line and notably improve apoptosis-inducing effects of cisplatin, indicating a novel strategy for better chemotherapeutic effects on gastric cancer.

[Expression of peroxiredoxin 6 in gastric cancer and its clinical significance].

OBJECTIVE: To elucidate the significance of anti-oxidant protein peroxiredoxin 6 (Prx6) expression in gastric cancer (GC) tissue. METHODS: Eighty-six GC tissues and 69 para-cancer tissues from surgically resected specimens were included. And the clinico-pathological data were collected by reviewing medical records. Tissue microarray and immunohistochemistry (IHC) were used to detect Prx6 protein expression in tissues. RESULTS: Prx6 protein was predominantly expressed in cytoplasm. Its expression rate in GC tissues (32.6%, n = 28) was significantly lower than that in para-cancer normal tissues (94.2%, n = 65, P < 0.05). And its protein overexpression rate in well and moderately-differentiated GC tissues was significantly higher than that in lowly-differentiated ones (39.6% (21/53) vs 6.1% (2/33), P < 0.05). Prx6 expression in gastric cancer tissues was not significantly related to age and sex of patients, lesion site, depth of invasion, clinical staging, vascular invasion and liver metastasis. CONCLUSIONS: The level of Prx6 protein is lower in GC tissues than that in normal para-cancer ones. And it is significantly correlated with the differentiation degree of GC.

Effect of Hydrogen Inhalation Therapy on Hearing Loss of Patients With Nasopharyngeal Carcinoma After Radiotherapy.

Objective: To evaluate the clinical efficacy and safety of hydrogen inhalation in improving hearing loss in patients with long-term survival of nasopharyngeal carcinoma after radiotherapy. Methods: The eustachian tube dysfunction score, pure tone air conduction threshold, bone conduction threshold, the score of tympanogram and otoscope were prospectively observed in patients with deafness after radiotherapy only or combined radiotherapy and chemotherapy for nasopharyngeal carcinoma. Paired t test and one-way analysis of variance were used to analyze the data before and after treatment. Results: A total of 17 patients were observed. The median time from radiotherapy to now was 228 months, and the median time from the diagnose of deafness to now was 92 months. After 4 weeks of hydrogen inhalation, the score of eustachian tube dysfunction, air conduction and bone conduction hearing thresholds were significantly reduced, P values were 0.0293, 0.0027, 0.0404, respectively. The mean air-bone gap, the score of otoendoscopy and tympanogram were also decreased, but the differences were not significant (P = 0.2079, P = 0.0536, P = 0.1056). Patients with radiotherapy alone and concurrent chemo-radiotherapy had significantly lower air conduction hearing threshold after hydrogen absorption (P = 0.0142, P = 0.0495). The results of air and bone hearing thresholds before, 4 and 12 weeks after hydrogen inhalation showed a descending trend. The air and bone hearing thresholds before hydrogen inhalation were 74.69 ± 27.03 dB and 45.70 ± 21.58 dB, respectively. At the 12th week, the mean values of air and bone hearing thresholds were the lowest, which were 66.88 ± 20.88 dB and 40.94 ± 18.93 dB, respectively, but there was no significant difference in air and bone hearing thresholds among all groups (P = 0.6755, P = 0.7712). After hydrogen inhalation treatment, no adverse reactions such as nosebleed, chest pain, dyspnea, nausea, vomiting, dizziness, earache and allergic reaction were observed. Conclusion: This is the first prospective study on the effect of hydrogen inhalation on hearing improvement in patients with deafness after radiotherapy/chemotherapy for nasopharyngeal carcinoma, suggesting that continuous hydrogen inhalation may be an alternative rehabilitation therapy for these patients.

A narrative review of hydrogen oncology: from real world survey to real world evidence.

The use of hydrogen for cancer control has made great progress in cytology and animal experiments. With the increasing number of hydrogen products on the market, larger numbers of advanced cancer patients have participated in clinical trials or received treatment at home after purchase. Our study reported a real-world survey from 82 patients with good cancer control using hydrogen products, including real world evidence from patients who received ineffective traditional treatment, patients who received traditional treatment that failed, or patients who refused traditional treatment. Two typical cases were reported herein. Subsequently, we included studies on the mechanism of hydrogen oncology. The mechanism of cancer control using hydrogen includes the inhibition of tumor cells and the activation of exhausted lymphocytes. Large-scale real world evidence has shown clinical value, and yet remains to be further developed and researched.

Two weeks of hydrogen inhalation can significantly reverse adaptive and innate immune system senescence patients with advanced non-small cell lung cancer: a self-controlled study.

Following standard treatments, the traditional model for enhancing anti-tumor immunity involves performing immune reconstitution (e.g., adoptive immune cell therapies or immunoenhancing drugs) to prevent recurrence. For patients with advanced non-small cell lung cancer, we report here on two objectives, the immunosenescence for advanced non-small cell lung cancer and hydrogen gas inhalation for immune reconstitution. From July 1st to September 25th, 2019, 20 non-small cell lung cancer patients were enrolled to evaluate the immunosenescence of peripheral blood lymphocyte subsets, including T cell, natural killer/natural killer T cell and gamma delta T cell. Two weeks of hydrogen inhalation was performed during the waiting period for treatment-related examination. All patients inhaled a mixture of hydrogen (66.7%) and oxygen (33.3%) with a gas flow rate of 3 L/min for 4 hours each day. None of the patients received any standard treatment during the hydrogen inhalation period. After pretreatment testing, major indexes of immunosenescence were observed. The abnormally higher indexes included exhausted cytotoxic T cells, senescent cytotoxic T cells, and killer Vδ1 cells. After 2 weeks of hydrogen therapy, the number of exhausted and senescent cytotoxic T cells decreased to within the normal range, and there was an increase in killer Vδ1 cells. The abnormally lower indexes included functional helper and cytotoxic T cells, Th1, total natural killer T cells, natural killer, and Vδ2 cells. After 2 weeks of hydrogen therapy, all six cell subsets increased to within the normal range. The current data indicate that the immunosenescence of advanced non-small cell lung cancer involves nearly all lymphocyte subsets, and 2 weeks of hydrogen treatment can significantly improve most of these indexes. The study was approved by the Ethics Committee of Fuda Cancer Hospital, Jinan University in China (approval No. Fuda20181207) on December 7th, 2018, and was registered on ClinicalTrials.gov (ID: NCT03818347) on January 24th, 2019.

Hydrogen therapy can be used to control tumor progression and alleviate the adverse events of medications in patients with advanced non-small cell lung cancer.

Chemotherapy, targeted therapy, and immunotherapy are used against advanced non-small cell lung cancer. A clinically efficacious method for relieving the adverse events associated of such therapies is lacking. Fifty-eight adult patients were enrolled in our trial to relieve pulmonary symptoms or the adverse events of drugs. Twenty patients who refused drug treatment were assigned equally and randomly to a hydrogen (H2)-only group and a control group. According to the results of tumor-gene mutations and drug-sensitivity tests, 10, 18, and 10 patients were enrolled into chemotherapy, targeted therapy, and immunotherapy groups in which these therapies were combined with H2-therapy, respectively. Patients underwent H2 inhalation for 4-5 hours per day for 5 months or stopped when cancer recurrence. Before study initiation, the demographics (except for tumor-mutation genes) and pulmonary symptoms (except for moderate cough) of the five groups showed no significant difference. During the first 5 months of treatment, the prevalence of symptoms of the control group increased gradually, whereas that of the four treatment groups decreased gradually. After 16 months of follow-up, progression-free survival of the control group was lower than that of the H2-only group, and significantly lower than that of H2 + chemotherapy, H2 + targeted therapy, and H2 + immunotherapy groups. In the combined-therapy groups, most drug-associated adverse events decreased gradually or even disappeared. H2 inhalation was first discovered in the clinic that can be used to control tumor progression and alleviate the adverse events of medications for patients with advanced non-small cell lung cancer. This study was approved by the Ethics Committee of Fuda Cancer Hospital of Jinan University on December 7, 2018 (approval No. Fuda20181207), and was registered at ClinicalTrials.gov (Identifier: NCT03818347) on January 28, 2019.

Identification of matrix metalloproteinase 11 as a predictive tumor marker in serum based on gene expression profiling.

PURPOSE: Prognostic markers discovery is a strategy for early diagnosis and individualization therapy for human cancer. In this study, we focus to integrate different methods to identify specific biomarker and elucidate its clinical significance. EXPERIMENTAL DESIGN: A powerful tool named Digital Gene Expression Display online was applied to isolate differentially expressed genes correlated with gastric cancer. Matrix metalloproteinase 11 (MMP11) was selected and confirmed at both mRNA and protein level in 10 cell lines, 123 cases of tumor tissues, and 305 cases of gastric cancer serum specimen by semiquantitative PCR, immunohistochemistry staining, and ELISA techniques, respectively. RESULTS: Our data showed that overexpression of MMP11 at mRNA and protein level was consistently detected in cell lines and primary tumors compared with matched normal tissues. Importantly, serum MMP11 levels were also significantly elevated in gastric cancer patients compared with those of the control subjects (P < 0.001), and the positive expression was well correlated with metastasis in gastric cancer patients (P = 0.009). Furthermore, we have shown that overexpression of MMP11 was associated with the malignant proliferation of AGS cells. CONCLUSIONS: Combination of gene expression profiling and specific clinical resource is a promising approach to validate gene expression patterns associated with malignant phenotype. As a secreted protein, MMP11 may play an important role in carcinogenesis and has potential implication as a biomarker for the diagnosis and prognosis of human cancers including gastric cancer.

[Identification of biomarkers for early detection in gastric cancer and its clinical biological significance.].

OBJECTIVE: To systematically understand the cellular and molecular mechanism of gastric cancer (GC) development and to discover early diagnosis and predictive biomarkers, which will be used for early diagnosis and novel treatment targets. METHODS: 70 mer 22 K-oligonucleotide microarrays and bioinformatic analysis were conducted to recognize gene expression profiles in GC and normal appearing tissue (NAT). The control group was collected from non-tumor patients including 20 specimen mixture as a common reference (CR) and 5 individuals as additional control. Our results showed that 837 different expression genes (DEGs) were identified in GC while 570 DEGs were in NATs by Bayesian analysis (P<0.001, Fold change>2.0) as compared respectively with CR. An interesting finding is that we identified 67 over-expressed genes in both GC and NAT tissues, and these gene expression alterations could not be detected by comparison of GC with NATs, which were normally used in routine experiment design. Most of these genes were involved in the control of cell proliferation, metabolism and differentiation. RESULTS: These differential expressed genes were confirmed at mRNA and protein levels in primary tumors using RT-PCR and immunohistochemistry (IHC). The results showed that three genes, EGR1, CYR61 and ADAMTS1 were over expressed in both GC and NATs at mRNA level. These results were consistent with oligo microarray data. Another interesting finding is that these three genes were also over-expressed in intestinal metaplasia (IM) and dysplasia (DYS), which indicated that these three genes might be potential biomakers for early detection of GC. CONCLUSION: Through the systematic analysis of gene expression profiles in GC tissues, NAT and CR normal tissues, we identified a group of genes over-expressed both in GC and precancerous lesions, which might be potential biomarkers for early GC diagnosis.

Expression status of ataxia-telangiectasia-mutated gene correlated with prognosis in advanced gastric cancer.

Many studies have revealed the ATM alterations involved in cancer development and progression. In order to elucidate ATM deficiency in advanced GC and its clinical significance, a total of 20 exons of ATM gene, including frequently reported variations, were screened in 40 advanced primary GC and matched normal tissues using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing analysis. Furthermore, ATM mRNA level was analyzed using Real-time RT-PCR and in situ hybridization, and protein expression and phosphorylation at Ser1981 were measured by immunohistochemical assessment in tissue microarray of GC. Five variants were identified in 6 of 40 cases (15%), but no hot spot of variation was detected. However, decreased expression and phosphorylation of ATM were consistently presented in tumors. In a cohort of 70 GC samples, low level of phosphorylated ATM was significantly correlated with poor differentiation, lymph node metastasis and poor 5-year survival (P<0.05). These results indicated that ATM phosphorylation status might be a prognostic marker for individual therapy in advanced GC patients.

Mutations in components of the Wnt signaling pathway in gastric cancer.

AIM: To explore the contribution of AXIN1, AXIN2 and beta-catenin, components of Wnt signaling pathway, to the carcinogenesis of gastric cancer (GC), we examined AXIN1, AXIN2 exon7 and CTNNB1 (encoding beta-catenin) exon3 mutations in 70 GCs. METHODS: The presence of mutations was identified by polymerase chain reaction (PCR)-based denaturing high-performance liquid chromatography and direct DNA sequencing. Beta-catenin expression was detected by immunohistochemical analysis. RESULTS: Among the 70 GCs, 5 (7.1%) had mutations in one or two of these three components. A frameshift mutation (1 bp deletion) in exon7 of AXIN2 was found in one case. Four cases, including the case with a mutation in AXIN2, had frameshift mutations and missense mutations in AXIN1. Five single nucleotide polymorphisms (SNPs), 334 C>T, 874 C>T, 1396 G>A, 1690 C>T and 1942 T>G, were identified in AXIN1. A frameshift mutation (27 bp deletion) spanning exon3 of CTNNB1 was observed in one case. All four cases with mutations in AXIN1 and AXIN2 showed nuclear beta-catenin expression. CONCLUSION: These data indicate that the mutations in AXIN1 and AXIN2 may contribute to gastric carcino-genesis.

[Alteration of early growth response 1 expression in gastroenterological cancers and its biological significance].

OBJECTIVE: To investigate the expression of early growth response 1 (EGR1) in gastroenterological cancers and its significance in the pathogenesis. METHODS: RT-PCR was used to determine the expression of EGR1 in normal gastric mucosa tissues from 20 non-tumor patients, gastroenterological tumor tissues and matched para-cancer tissues normal morphologically. RT-PCR and Western blotting were used to analyze the mRNA and protein expression of EGR1 in 20 cancer cell lines. Immunohistochemistry (IHC) was preformed to measure the expression level of EGR1 protein on tissue microarray including 179 tumors and 159 normal tissues. RESULTS: EGR1 was overexpressed in gastric cancer (GC) and its matched adjacent normal tissue (ANT), but not expressed or expressed at a low level in the normal gastric mucosa from the non-tumor patients, which was consistent with the GC gene expression profiling data. Overexpression of EGR1 was seen in the 20 cancer cell lines at both mRNA and protein levels. IHC showed strong positive staining of EGR1 protein in the cytoplasm of both tumor tissues and matched normal tissues and showed negative or weaker nuclear staining in the normal gastric mucosa tissues from non-tumor patients. Overexpression of EGR1 was detected in 87% (49/56) of the GC tissues and 79% (43/54) of their ANTs; 83% (43/52) of the hepatocellular carcinoma tissues and 79% (32/42) of their ANTs; 78% (41/52) of colorectal cancer tissues and 63% (22/35) of their ANTs; and 79% (15/19) of the squamous cell carcinoma tissues and 78% (14/18) of their ANTs. CONCLUSION: EGR1 may be correlated with the abnormal proliferation of cells at the early stage of malignant transformation.

Soluble expression of active human beta-defensin-3 in Escherichia coli and its effects on the growth of host cells.

BACKGROUND: Human beta-defensin-3 (HBD(3)) is an epithelial peptide that has been demonstrated to have a salt-insensitive broad spectrum of potent antimicrobial activity. Expressing antimicrobial peptides in Escherichia coli (E. coli) is very difficult for it can result in death of the bacterial host cells. Our aim was to establish a prokaryotic system expressing soluble HBD(3) protein and demonstrate the antimicrobial activity of the expressed protein. We then studied whether the host cells would activate the suicide pathways. METHODS: We first cloned the complementary DNA coding for the mature chain of HBD(3), inserted it into the vector PGEX-KG then transformed E. coli BL21 (DE3) with the appropriate recombinant plasmid. After induction with 0.5 mmol/L isopropyl-1-thio-beta-D-galactopyranoside (IPTG) the transformed E. coli produced a recombinant glutathione S-transferase and HBD(3) (GST-HBD(3)) fusion protein. The fusion protein was treated with thrombin to produce pure HBD(3) protein then the antimicrobial activity of HBD(3) was evaluated in a liquid microdilution assay. RESULTS: The fusion protein GST-HBD(3) was efficiently cleaved by thrombin and yielded HBD(3) that had anti-staphylococcus aureus activity with a minimal inhibitory concentration level of 12.5 microg/ml. The E. coli strain expressing the recombinant protein did not grow slower than the empty vector strain. CONCLUSION: Active HBD(3) in E. coli by expressing the recombinant protein GST-HBD(3) could be produced, and suicide did not occur in the E. coli strain expressing the recombinant protein.

[Prognostic significance of clinicopathologic parameters in gastrointestinal stromal tumor: a study of 156 cases].

OBJECTIVE: To evaluate the prognostic significance of various clinicopathologic parameters in gastrointestinal stromal tumor (GIST), and to study the frequency of c-kit exon 11 mutations in this tumor. METHODS: One hundred and fifty-six cases of gastric or small intestinal GIST were retrieved from the archival files of the Department of Pathology, Chinese PLA General Hospital. The clinical features, site of occurrence, tumor diameter, mitotic index, coagulative tumor necrosis, and risk grade were studied and analyzed statistically. Tumor DNA was extracted and c-kit exon 11 was amplified. Upon detection by denaturing high-performance liquid chromatography, the amplified exon 11 was sequenced. RESULTS: For the 83 cases of gastric GIST studied, the mean age of patients was 55.4 years. Follow-up information was available in 62 cases, with 17 cases having local recurrence or distant metastasis. The 5-year survival rate was 66.5% +/- 17.1%. For the 73 cases of small intestinal GIST studied, the mean age of patients was 50.6 years. Follow-up information was available in 43 cases, with 22 cases having local recurrence or distant metastasis. The 5-year survival rate was 61.8% +/- 18.3%. In general, for gastric GIST, age younger than 50 years (P = 0.046), advanced clinical stage (P = 0.0001), large tumor size (P = 0.0001), high mitotic index (P = 0.0001), presence of coagulative tumor necrosis (P = 0.0001), and high risk grade (P = 0.004) were associated with lower survival rate. COX hazard proportional model revealed that advanced clinical stage (P = 0.001), large tumor size (P = 0.001), high mitotic index (P = 0.002) and high risk grade (P = 0.018) indicated worse prognosi. For small intestinal GIST, advanced clinical stage (P = 0.010) and presence of coagulative tumor necrosis (P = 0.036) were associated with lower survival rate. Advanced clinical stage was an independent prognostic factor. A total of 25 cases harbored c-kit mutations. The frequency of c-kit mutations was 32% and 22.5% for gastric and small intestinal GIST respectively. For gastric GIST, c-kit mutations occurred mainly in patients older than 50 years. In contrast, c-kit mutations in small intestinal GIST occurred in the age group of 40 to 49 years. CONCLUSIONS: For gastric GIST, advanced clinical stage, tumor diameter, mitotic index and risk grade are the main prognostic indicators. For small intestinal GIST, advanced clinical stage and presence of coagulative tumor necrosis indicate poor prognosis. In general, small intestinal GIST is more frequently associated with metastasis and tumor relapse than gastric GIST. The occurrence of c-kit mutations also correlates with age of patients.