Efficient plasma metabolic fingerprinting as a novel tool for diagnosis and prognosis of gastric cancer: a large-scale, multicentre study.

OBJECTIVE: Metabolic biomarkers are expected to decode the phenotype of gastric cancer (GC) and lead to high-performance blood tests towards GC diagnosis and prognosis. We attempted to develop diagnostic and prognostic models for GC based on plasma metabolic information. DESIGN: We conducted a large-scale, multicentre study comprising 1944 participants from 7 centres in retrospective cohort and 264 participants in prospective cohort. Discovery and verification phases of diagnostic and prognostic models were conducted in retrospective cohort through machine learning and Cox regression of plasma metabolic fingerprints (PMFs) obtained by nanoparticle-enhanced laser desorption/ionisation-mass spectrometry (NPELDI-MS). Furthermore, the developed diagnostic model was validated in prospective cohort by both NPELDI-MS and ultra-performance liquid chromatography-MS (UPLC-MS). RESULTS: We demonstrated the high throughput, desirable reproducibility and limited centre-specific effects of PMFs obtained through NPELDI-MS. In retrospective cohort, we achieved diagnostic performance with areas under curves (AUCs) of 0.862-0.988 in the discovery (n=1157 from 5 centres) and independent external verification dataset (n=787 from another 2 centres), through 5 different machine learning of PMFs, including neural network, ridge regression, lasso regression, support vector machine and random forest. Further, a metabolic panel consisting of 21 metabolites was constructed and identified for GC diagnosis with AUCs of 0.921-0.971 and 0.907-0.940 in the discovery and verification dataset, respectively. In the prospective study (n=264 from lead centre), both NPELDI-MS and UPLC-MS were applied to detect and validate the metabolic panel, and the diagnostic AUCs were 0.855-0.918 and 0.856-0.916, respectively. Moreover, we constructed a prognosis scoring system for GC in retrospective cohort, which can effectively predict the survival of GC patients. CONCLUSION: We developed and validated diagnostic and prognostic models for GC, which also contribute to advanced metabolic analysis towards diseases, including but not limited to GC.

Integrative proteomic characterization of adenocarcinoma of esophagogastric junction.

The incidence of adenocarcinoma of the esophagogastric junction (AEG) has been rapidly increasing in recent decades, but its molecular alterations and subtypes are still obscure. Here, we conduct proteomics and phosphoproteomics profiling of 103 AEG tumors with paired normal adjacent tissues (NATs), whole exome sequencing of 94 tumor-NAT pairs, and RNA sequencing in 83 tumor-NAT pairs. Our analysis reveals an extensively altered proteome and 252 potential druggable proteins in AEG tumors. We identify three proteomic subtypes with significant clinical and molecular differences. The S-II subtype signature protein, FBXO44, is demonstrated to promote tumor progression and metastasis in vitro and in vivo. Our comparative analyses reveal distinct genomic features in AEG subtypes. We find a specific decrease of fibroblasts in the S-III subtype. Further phosphoproteomic comparisons reveal different kinase-phosphosubstrate regulatory networks among AEG subtypes. Our proteogenomics dataset provides valuable resources for understanding molecular mechanisms and developing precision treatment strategies of AEG.

Exosomal miR-590-5p in Serum as a Biomarker for the Diagnosis and Prognosis of Gastric Cancer.

The purpose of this study is to explore the expression of miRNA-590-5p, an exosome of gastric cancer (GC), and to evaluate the suitability of miR-590-5p, an exosome with its own clinical characteristics. Serum samples from 168 gastric cancer patients and 50 matched controls were collected and exosomal RNAs were extracted. After that, miR-590-5p is analyzed by quantitative polymerase chain reaction (qRT-PCR), which is more related to clinical and pathological parameters and patient monitoring data. MGC-803 and HGC-27 cells were treated by miR-590-5p mimics, and then the changes of cell fluidity and invasiveness were monitored. The results showed that the expression level of miR-590-5p in exosomes of healthy observation group, early (I and II) stage group, and late stage (III) group was 30.34 ± 6.35, 6.19 ± 0.81, and 2.9 ± 0.19, respectively (all p < 0.05). ROC (receiver-operating characteristic curve) showed that the AUC (area under the curve) of exosomal miR-590-5p was 0.810 with 63.7% sensitivity and 86% specificity. The expression of exosomal miR-590-5p in serum was related to clinical stage (p = 0.008), infiltration depth, and the expression level of ki-67 (p < 0.001). In addition, Kaplan-Meier analysis showed that the decrease of explicit level of exosomal miR-590-5p was related to the decrease of overall survival rate (p < 0.001). Cox regression analysis showed that miR-590-5p can be used as an independent predictor. Furthermore, upregulation of miR-590-5p inhibited cell migration and invasion in MGC-803 cells and HGC-27 cells. The serum expression level of exosomal miR-590-5p may be a biomarker, which is potentially useful and noninvasive for early detection and prediction of GC. In addition, miR-590-5p can play a role in eliminating carcinogens by actively regulating the malignant potential of gastric cancer.

p-MEK expression predicts prognosis of patients with adenocarcinoma of esophagogastric junction (AEG) and plays a role in anti-AEG efficacy of Huaier.

The incidence rate of adenocarcinoma of the esophagogastric junction (AEG) is increasing worldwide with poor prognosis and unclear pathogenesis. Trametes robiniophila Murr. (Huaier), a traditional Chinese medicine has been used in the clinical treatment of a variety of solid tumors, including AEG. However, its anticancer components and molecular mechanisms are still unclear. In our previous studies, we have found that Huaier n-butanol extract (HBE) shows the most potent anticancer activity among different extracts. In the present study, we aimed to investigate the clinical relevance of p-MEK expression in AEG patients and the role of the MEK/ERK signaling pathway in the anti-AEG efficacy of HBE in vitro and in vivo. We herein demonstrate that p-MEK expression in AEG tissues was significantly higher than that in paracancerous tissues and correlated with a poor prognosis in AEG patients. We further found that HBE inhibited the colony formation, migration, and invasion in AEG cell lines in a concentration-dependent manner in vitro. HBE also suppressed the growth of AEG xenograft tumors without causing any host toxicity in vivo. Mechanistically, HBE caused the inactivation of the MEK/ERK signaling pathway by dephosphorylating MEK1 at S298, ERK1 at T202, and ERK2 at T185 and modulating the expression of EMT-related proteins. In summary, our results demonstrate that the high expression of p-MEK may be an independent factor of poor prognosis in patients with AEG. The clinically used anticancer drug Huaier may exert its anti-AEG efficacy by inhibiting the MEK/ERK signaling pathway.

1H NMR-based metabolomics analyses in children with Helicobacter pylori infection and the alteration of serum metabolites after treatment.

BACKGROUND AND AIMS: Helicobacter pylori (H. pylori) infection can occur in early childhood, without eradication therapies such infection can persist throughout life and cause many different diseases. This study investigated the metabolic characteristics and explored the underlying mechanism of children with H. pylori infection, and identified potential biomarkers for evaluating the efficacy of H. pylori eradication therapies. METHODS: We performed 1H NMR-based metabonomics coupled with multivariate analysis to investigate the metabolic profiling of serum samples between Children with and without H. pylori infection. In the same manner, we compared the alternations of metabolites in H. pylori-infected children before and after H. pylori eradication therapies. RESULTS: 21 metabolites from serum in H. pylori-infected and H. pylori-uninfected children were identified, which were mainly involved in energy, amino acid, lipid and microbial metabolism. We found that the serum levels of trimethylamine N-oxide and alanine were significantly higher in H. pylori-infected children compared to uninfected sera, whereas lactate was significantly lower. We also found that the levels of trimethylamine N-oxide and creatine in H. pylori-infected children was significantly decreased after H. pylori eradication therapies, whereas lactate and low-density lipoprotein/very low-density lipoprotein was significantly increased. CONCLUSIONS: This is the first study using 1H NMR-based metabolomics approach to explore the effects of H. pylori infection in children. Our results demonstrated that the disturbances of metabolism in energy, amino acids, lipids and microbiota could play an important role in the pathogenesis of gastrointestinal and extragastric diseases caused by H. pylori infection. Trimethylamine N-oxide and lactate might serve as potential serum biomarkers for evaluating the efficacy of H. pylori eradication therapies.

Curcumin combined with cis-platinum promote the apoptosis of human colorectal cancer HT29 cells and mechanism.

Colorectal carcinoma is one of the common malignant carcinoma that severely harms the public health. Curcumin (Cur) exhibits anti-tumor, antioxidant, anti-inflammatory and antiviral effects, which result in the promotion of the apoptosis of cancer cells. The present study aimed to investigate the effect of cis-platinum (DDP) combined with Cur on the apoptosis of colorectal carcinoma cells, and to elucidate its underlying mechanism of action. The cell viability of human colorectal cancer HT29 cells was detected by MTT after DDP treatment. The effect of DDP/Cur co-treatment on cell proliferation and apoptosis were investigated using CCK-8 assay and flow cytometry assay. HT29 cells growth was significantly inhibited after DDP treatment and DDP combined with Cur treatment for 24 h. In our results, DDP in combination with Cur significantly promoted the apoptosis of HT29 colorectal carcinoma cell in a dose dependent manner. Bcl-2-associated X protein (Bax) expression was promoted and B-cell lymphoma-2 (Bcl-2) expression was suppressed, In addition, Notch1, Notch1 intracellular domain (NICD1) and hairy and enhancer of split 1 (Hes-1) were decreased dramatically in HT29 cells treated with Cur combined with DDP. The effect of Cur combined with DDP treatment was better than the effect of DDP treatment. It showed that DDP combined with Cur promote the apoptosis of HT29 cells via regulating the expression of related apoptotic genes and inhibiting the activation of Notch1 signaling pathway.

A Nomogram to Predict Prognostic Value of Red Cell Distribution Width in Patients with Esophageal Cancer.

OBJECTIVES: The prognostic value of inflammatory index in esophageal cancer (EC) was not established. In the present study, we initially used a nomogram to predict prognostic value of red cell distribution width (RDW) in patients with esophageal squamous cell carcinoma (ESCC). METHODS: A total of 277 ESCC patients were included in this retrospective study. Kaplan-Meier method was used to calculate the cancer-specific survival (CSS). A nomogram was established to predict the prognosis for CSS. RESULTS: The mean value of RDW was 14.5 ± 2.3%. The patients were then divided into two groups: RDW ≥ 14.5% and RDW < 14.5%. Patients with RDW < 14.5% had a significantly better 5-year CSS than patients with RDW ≥ 14.5% (43.9% versus 23.3%, P < 0.001). RDW was an independent prognostic factor in patients with ESCC (P = 0.036). A nomogram could be more accurate for CSS. Harrell's c-index for CSS prediction was 0.68. CONCLUSION: RDW was a potential prognostic biomarker in patients with ESCC. The nomogram based on CSS could be used as an accurately prognostic prediction for patients with ESCC.

[The clinical patterns and RET proto-oncogene identification of pheochromocytoma in 13 multiple endocrine neoplasia type 2A pedigrees].

OBJECTIVE: To explore the clinical patterns and clinical significance for RET screening in adrenal pheochromocytoma (PHEO) associated with multiple endocrine neoplasia type 2A (MEN2A). METHODS: The clinical data of 32 PHEO patients with MEN2A from 13 unrelated MEN2A pedigrees from August 1989 to January 2013 were analyzed. The comprehensive medical data included systemic examinations and germline RET gene screening. RESULTS: Among 68 patients belonging to 13 MEN2A families, 32 (47.1%) presented with PHEO. There were 19 males and 13 females with a mean age of (41 ± 12) years. And the mean maximum diameter of PHEO was (4.6 ± 2.2) cm. The diagnosis of PHEO was made after medullary thyroid carcinoma (n = 12, 37.5%), simultaneously (n = 12, 37.5%), initially (n = 7, 21.9%) and death during appendectomy for PHEO-induced hypertensive crisis (n = 1, 3.1%). The diagnosis of PHEO was made before (n = 22) or after (n = 10) clinical screening. The former had 12 symptomatic cases while the latter only 1 case (12/22 vs 1/10, P = 0.024).Except for 5 asymtomatic fatal cases during non-PHEO operations, bilateral PHEO was found in 17 cases including 3 unilaterally treated cases developing another PHEO in contralateral adrenal with a lag period of 5, 10 and 17 years. There were 7 symptomatic patients in bilateral cases versus 6 in unilateral cases (7/17 vs 6/10, P = 0.440). Twenty-five patients underwent PHEO surgery: laparascopic approach in 14 cases (8 with bilateral simultaneous adrenalectomy) and open approach in 11 (2 with bilateral simultaneous adrenalectomy). And 10 patients undergoing bilateral adrenal-sparing operations or adrenalectomy required hormonal replacement therapy. During a mean observation period of 72 (1-282) months, no local recurrence, distant metastasis or Addisonian crisis were noted in 25 cases (contralateral relapse in 3 cases). Among them, 2 cases developed adrenocortical insufficiency unresponsive to an adjustment of hormonal doses.RET screening showed 4 recurrent missense substitutions in 32 MEN2A-PHEO patients: p. C634Y exon 11 (n = 27, 84.4%), p. C634R exon 11 (n = 3, 9.4%), p. C634F exon 11 (n = 1, 3.1%) and p. C618R exon 10 (n = 1, 3.1%). CONCLUSIONS: The mutations of RET proto-oncognene of PHEO in MEN2A are frequently located at codon 634. A combination of pedigree examination and RET gene screening may facilitate an early diagnosis and early treatment of asymptomatic PHEO patients in MEN2A.Laparoscopic cortical-sparing adrenalectomy for preserving adrenocortical function is a preferred surgical approach.

Consumption of hydrogen-rich water protects against ferric nitrilotriacetate-induced nephrotoxicity and early tumor promotional events in rats.

The aim of this work was to test whether consumption with hydrogen-rich water (HW) alleviated renal injury and inhibited early tumor promotional events in Ferric nitrilotriacetate (Fe-NTA)-treated rats. Rats were injected with Fe-NTA solution (7.5mg Fe/kg body weight) intraperitoneally to induce renal injury and simultaneously treated with HW (1.3 ± 0.2mg/l). We found that consumption with HW ameliorated Fe-NTA-induced renal injuries including suppressing elevation of serum creatinine and blood urea nitrogen and inhibited early tumor promotional events including decreasing ornithine decarboxylase activity and incorporation of [3H]thymidine into renal DNA. Consumption with HW suppressed Fe-NTA-induced oxidative stress through decreasing formation of lipid peroxidation and peroxynitrite and activities of NADPH oxidase and xanthine oxidase, increasing activity of catalase, and restoring mitochondrial function in kidneys. Consumption with HW suppressed Fe-NTA-induced inflammation marked by reduced NF-κB, IL-6, and MCP-1 expression and macrophage accumulating in kidneys. In addition, consumption with HW suppressed VEGF expression, STAT3 phosphorylation and PCNA expression in kidneys of Fe-NTA-treated rats. Consumption with HW decreased the incidence of renal cell carcinoma and suppressed tumor growth in Fe-NTA-treated in rats. In conclusion, drinking with HW attenuated Fe-NTA-induced renal injury and inhibited early tumor promotional events in rats.

[Double-mutated oncolytic adenovirus combined with gemcitabine for treating an orthotopic nude mouse model of bladder cancer].

OBJECTIVE: To investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model. METHODS: The susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-ß-galactoside (X-Gal). Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopic bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed. RESULTS: Gene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 cells with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal cells infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was (39 ± 3) % and (49 ± 5) % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle (P < 0.05). AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAlacZ, gemcitabine, AxdAdB-3, and AxdAdB-3 + gemcitabine were 400.6, 126.4, 82. 0, 40.4 mg, respectively. Either AxdAdB-3 (P < 0.0001) and gemcitabine (P < 0.0001) suppressed the tumor growth in nude mice, and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P < 0.0001) or gemcitabine (P < 0.0001) alone. CONCLUSIONS: Intravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse, and further relevant clinical studies are guaranteed.

[Identification of Nanfeng mandarin from different origins by using near infrared spectroscopy coupled with principal components analysis].

The identification of Nanfeng mandarins from different origins was developed by using near infrared spectroscopy (NIR) and principal components analysis (PCA). Mandarins from different places in one orchard, and from different orchards in three towns of Nanfeng county were studied. Also differences among Shaowu, Liucheng and Nanfeng were investigated. Furthermore, the effect of storage time of mandarins on the PCA model was considered. The results demonstrate that there was no clear diversity of the mandarins in one origin but great differences existed among different ones. And the storage time of mandarins played little role in the discrimination model. The method of multiple scattering correction (MSC) coupled with second derivative was selected to build PCA discrimination models compared with other data pretreatment methods. The proposed model would be a reference method for origin identification of Nanfeng mandarin.

E1A, E1B double-restricted adenovirus enhances the cytotoxicity and antitumor activity of gemcitabine to renal cell carcinoma.

BACKGROUND: Our previous studies have demonstrated potent oncolysis efficacy of the E1A, E1B double-restricted replication-competent oncolytic adenovirus AxdAdB-3 for treatment of bladder cancer. Here, we reported the feasibility and efficacy of AxdAdB-3 alone, or in combination with gemcitabine for treating renal cell carcinoma. METHODS: Cytopathic effects of AxdAdB-3 were evaluated in human renal cell carcinoma cell lines TOS-1, TOS-2, TOS-3, TOS-3LN, SMKT-R3, SMKT-R4 and ACHN, and in normal human renal proximal tubule epithelial cells (RPTEC). AxdAdB-3 induced down-regulation of the cell cycle was determined by flow cytometry. Combination therapies of AxdAdB-3 with gemcitabine were evaluated in vitro and in vivo on subcutaneous TOS-3LN tumors in a severe combined immunodeficiency disease (SCID) mouse model. RESULTS: AxdAdB-3 was potently cytopathic against the tested most renal cell carcinoma cell lines including TOS-2, TOS-3, TOS-3LN, SMKT-R3 and SMKT-R4, while normal human RPTEC were not destroyed. AxdAdB-3 effectively induced cell cycle S-phase entry. Combined therapy of AxdAdB-3 with gemcitabine demonstrated stronger antitumor effects in vitro and in vivo compared with either AxdAdB-3 or gemcitabine alone. CONCLUSION: AxdAdB-3 alone, or in combination with gemcitabine may be a promising strategy against renal cell carcinoma.

[Prognostic significance of metastatic lymph node ratio in colorectal cancer].

OBJECTIVE: To investigate the prognostic significance of metastatic lymph node ratio in patients with colorectal cancer. METHODS: The clinicopathological data of 303 surgically treated patients with colorectal cancer were retrospectively analyzed. Spearman correlation analysis was used to determine the correlation coefficient. The survival was analyzed using Kaplan-Meier method, and the survival difference was assessed by Log-rank test. Multivariate analysis was performed using Cox proportional hazard regression model in forward stepwise regression. Receiver working characteristic curve was used to compare the accuracy of the metastatic lymph nodes ratio in predicting the death of patients at 5 years postoperatively with that of the number of metastatic lymph nodes. RESULTS: The MLR was not correlated with the total number of dissected lymph nodes (Spearman correlation coefficient: -0.099, P > 0.05), but the positive rate of metastatic lymph nodes did (correlation coefficient: 0.107, P < 0.05). Kaplan-Meier survival analysis revealed that the MLR significantly influenced the postoperative survival time (Log-rank chi(2) = 42.878, P < 0.01), even in the patients with less than 12 resected lymph nodes. The 5-year survival rates for rN0, rN1, rN2 and rN3 were 90.9%, 68.9%, 54.7% and 39.4%, respectively. There was a significant difference between the different stages (P < 0.01). Cox proportional hazard regression model analysis showed that the metastatic lymph node ratio was an independent prognostic factor. (EXP(B) = 7.809, P < 0.01). There was no significant difference between metastatic lymph node ratio and the number of metastatic lymph nodes in predicting the death of patients at 5 years postoperatively based on the area under the receiver working characteristic curve. CONCLUSION: The metastatic lymph node ratio in colorectal cancer patients is not correlated with the total number of dissected lymph nodes. The metastatic lymph node ratio is a major independent prognostic factor for patients with colorectal cancer. The ability of metastatic lymph node ratio in predicting the death of colorectal cancer patients at 5 years postoperatively is the same as that of the number of metastatic lymph nodes.

[Initial RAPD analysis of Isatis indigotica somatic clones' resistance to root rot disease].

OBJECTIVE: To make initial identification on Isatis Indigotica Somatic Clones' resistant mutant. METHODS: The resistant plants were selected under the treatment of root-rot disease crude toxin. The DNA of the same clones and their parents were analysed by RAPD technology. RESULTS: We got initial resistance of the plants and selected 12 primers of which most were identical but the less were different. The genetic background of the somatic clones was identical but the individuals were mutated on the DNA level. CONCLUSION: It provides experimental foundation for further study on separating resistant mutant and resistant gene.

[Efficacy assessment of preoperative radiochemotherapy and analysis of associated factors in rectal cancer].

OBJECTIVE: To evaluate the efficacy of preoperative radiochemotherapy and investigate the influencing factors in rectal cancer. METHODS: Fifty-three patients with locally advanced rectal cancer were treated with radiochemotherapy before surgery. Three-field technique of radiation therapy was administered with 46 Gy, 2 Gy per fraction, five times a week. Two cycles of chemotherapy were carried out at day 1, 2 and day 21, 22 during the radiation course. Surgery was performed 4-6 weeks after the radiochemotherapy. Response of preoperative radiochemotherapy was evaluated in all the patients by endoscopic ultrasonography (EUS), spiral computed tomography (SCT) and pathology. Influencing factors of the efficacy of radiochemotherapy were evaluated by univariate and Logistic analysis. RESULTS: Univariate analysis revealed that tumor size and histological grading were associated with the efficacy of preoperative radiochemotherapy. Logistic regression analysis showed that only tumor size was the significant predictive factor for response to preoperative radiochemotherapy. All patients underwent surgical resection after preoperative radiochemotherapy. The tumor was reduced by an average of 32.1%. T-level down-staging was 64.2%. Nodal negativity was 58.1%. Complete pathologic remission occurred in 11 patients. CONCLUSIONS: Preoperative radiochemotherapy can shrink the primary tumor and decrease lymph node metastasis rate. Patient with small tumor may have better response to preoperative radiochemotherapy.

[Isolation and characterization of SIR73 gene fragment in salt resistant mutant of wheat involved in salt stress].

cDNA-AFLP (amplified fragment length polymorphism) is used to isolate genes differentially expressed in two wheat lines with the different resistance to NaCl derived from a single seed. A lot of cDNA fragments related to salt tolerance are obtained. Of with the number 73 cDNA fragment encodes for a transcription factors with an 32% similarity to human transcription factors in the relative amino acid which is named SIR73. Northern analysis confirms that SIR73 is strongly induced by NaCl stress and the expression in SR is more strongly induced than in SS.SIR73 may be involved in the regulation of gene expression in salt stress in wheat.