Gastric Cancer Serum Samples Research Articles

2-Dimensional ultra-high performance liquid chromatography and DMT-MM derivatization paired with tandem mass spectrometry for comprehensive serum N-glycome characterization

Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation. The difficulty in comprehensively characterizing the serum N-glycome arises from the absence of a biosynthetic template resulting in great structural heterogeneity and complexity. To help overcome these challenges we developed a 2-dimensional liquid chromatography platform which utilizes offline weak anion exchange (WAX) chromatography in the first dimension and hydrophilic interaction liquid chromatography (HILIC) in the second dimension to separate N-glycans by charge, corresponding to degree of sialylation, and size, respectively. Performing these separations offline enables subsequent derivatization with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) for sialic acid linkage determination and the identification of sialic acid linkage isomers. Subsequent tandem mass spectrometry analysis revealed the identification of 212 complete and partial N-glycan structures including low abundant N-glycans containing acetyl and sulphate modifications. The identifications obtained through this platform were then applied to N-glycans released from a set of stage 3 gastric cancer serum samples obtained from patients before (pre-op) and after (post-op) tumour resection to investigate how the serum N-glycome can facilitate differentiation between the two pathological states.

Diagnostic value of macrophage inhibitory cytokine 1 as a novel prognostic biomarkers for early gastric cancer screening.

BackgroundEarly diagnosis is very important to improve the survival rate of patients with gastric cancer (GC), especially in asymptomatic participants. However, low sensitivity of common biomarkers has caused difficulties in early screening of GC. In this study, we explored whether MIC‐1 can improve the detection rate of early GC.MethodsWe screened 8257 participants based on risk factors such as age, gender, and family history for physical examination including gastroscopy. Participant blood samples were taken for measure MIC‐1, CA‐199, CA72‐4, and PG1/PG2 levels. The diagnostic performance of MIC‐1 was assessed and compared with CA‐199, CA72‐4, and PG1/PG2, and its role in early GC diagnosis and the assessment of the risk of precancerous lesions have also been studied.ResultsBased on endoscopic and histopathological findings, 55 participants had GC, 566 participants had low‐grade neoplasia, and 2605 participants had chronic gastritis. MIC‐1 levels were significantly elevated in GC serum samples as compared to controls (P < .001). The sensitivity of serum MIC‐1 for GC diagnosis was much higher than that of CA‐199 (49.1% vs 20.0%) with similar specificities. Moreover, receiver operating characteristic (ROC) curve analysis also showed that serum MIC‐1 had a better performance compared with CA‐199, CA72‐4, and PG1/PG2 in distinguishing early‐stage GC (AUC: 72.9% vs 69.5%, 67.5%, 44.0%, respectively).ConclusionsSerum MIC‐1 is significantly elevated in most patients with early GC. MIC‐1 can serve as a novel diagnostic marker of early GC and value the risk of GC.

Integrating endogenous peptides analysis and protease mapping for identification of potential serum biomarkers in gastric adenocarcinoma.

e15564 Background: Gastric cancer (GC) is the third cause of cancer death in the world. In Brazil 21.000 new cases had been diagnosed in 2018 and the mortality rate is associated with late diagnosis that contributed to poor prognosis. The objective was to study endogenous peptides in serum of patients with GC. Methods: Fifteen serum samples of patients with diagnosis of gastric adenocarcinoma (TNM stage I-IV) and 15 controls were included. Endogenous peptides were extracted and pooled of random mode in 5 biological replicates (n = 3 samples) for each group. The mixture peptides was submitted to nLC-MS/MS analysis and peptide sequences identified were used to protease mapping. Results: A total of 191 peptides (≥ 7 amino acids) were identified corresponding to 36 proteins involved mainly on metabolic and immune system processes, signal transduction, platelet and neutrophils degranulation. Peptidome-based protease mapping identified 59 proteases (29 serine-, 19 metallo-, 8 cisteine- and 3 aspartic-), in which Prothrombin, Plasminogen, MMP14, MMP7 and MMP3 proteases yielded most of peptides. Twenty sequences derived from Fibrinogen A, Fibrinogen B, Complement C3 (C3f), Apolipoprotein A-I (N-terminal), Prothrombin (C-terminal of Activation peptide fragment 2; N-terminal of Thrombin light chain) and Coagulation factor XIII A (N-terminal) were significantly different (p ≤ 0.5). Between these peptides, 14 were up regulated and six downregulated in GC patients. The phosphopeptide 20ADSpGEGDFLAEGGGVR35 (Fibrinopeptide A) was significantly increased (p&lt; 0.03) in GC serum samples, while the non-phosphorylated Fibrinopeptide A was decreased (p&lt;0.04), suggesting a higher phosphorylation rate in this protein in GC patients. Further, the peptide 31QGVNDNEEGFFSAR44 (Fibrinopeptide B) was downregulated (p&lt;0.04) in GC serum samples; in counterpart, the Fibrinopeptide B derived peptides NDNEEGFF (p&lt;0.0007) and QGVNDNEEGFFS (p&lt;0.0013) was up regulated, suggesting a higher proteolysis in GC serum samples. Conclusions: The investigation of protease/substrate activity relationship may be described such as a novel panorama for discovery of serum biomarkers.

Serum biomarker panels for the diagnosis of gastric cancer.

Gastric cancer is a leading cause of mortality due to neoplastic disease. Although early detection of gastric cancers can decrease the mortality rate, it remains a diagnostic challenge because of the lack of effective biomarkers. In this study, fifteen gastric cancer patients and ten healthy subjects were recruited to assess novel serum biomarkers for gastric cancer using antibody microarray technology. ELISA was utilized to validate the antibody array results. As a result, compared to the controls, eleven cytokines were found to be significantly increased in gastric cancer, including interferon gamma receptor 1 (IFNGR1), neurogenic locus notch homolog protein 3 (Notch‐3), tumor necrosis factor receptor superfamily member 19L (TNFRSF19L), growth hormone receptor (GHR), signaling lymphocytic activation molecule family 8 (SLAMF8), folate receptor beta (FR‐beta), integrin alpha 5, galectin‐8, erythropoietin‐producing hepatocellular A1 (EphA1), epiregulin, and fibroblast growth factor 12 (FGF‐12) with P < 0.05. ELISA validation supported the results of the antibody array. More importantly, most of these eleven cytokines, including IFNGR1, TNFRSF19L, GHR, SLAMF8, FR‐beta, and integrin alpha 5 were discovered to be elevated in gastric cancer serum samples for the first time in this study, suggesting that these proteins may serve as novel biomarkers for the early diagnosis and prognosis determination of gastric cancer.

High serum Ephrin-B2 levels predict poor prognosis for patients with gastric cancer.

Gastric cancer is an intractable disease with a poor prognosis and limited treatment options. Its treatment remains a major clinical challenge worldwide. Ephrin-B2 is upregulated and involved in tumor growth in various types of cancer. However, the association between ephrin-B2 and prognosis of gastric cancer, and the potential of ephrin-B2 as a therapeutic target remains unknown. The present study investigated ephrin-B2 as a prognostic factor and a therapeutic target for gastric cancer. Reverse transcription-quantitative polymerase chain reaction was performed to detect the protein expression level of ephrin-B2 in gastric cancer serum samples (n=162) and healthy serum samples (n=165). It was revealed that the protein expression level of ephrin-B2 was significantly upregulated in gastric cancer serum samples compared with the healthy samples. Ephrin-B2 protein expression was associated with tumor size (P<0.001), metastasis (P=0.02) and TNM stage (P=0.03), and was indicated to be an independent prognostic factor for gastric cancer. Furthermore, the Kaplan-Meier survival curve demonstrated that patients with high ephrin-B2 protein expression had shorter overall and progression-free survival rates than those with low ephrin-B2 protein expression. Ephrin-B2 protein expression was induced by small interfering RNA (siRNA) transfection of HGC27 and MKN-45 cells, significantly impeding cell viability and inducing apoptosis of HGC27 and MKN-45 cells compared with the respective negative control (NC) group. Thus, to the best of our knowledge, the present study indicates that ephrin-B2 functions as an oncogene in gastric cancer, and that serum ephrin-B2 level may be a promising non-invasive prognostic indicator, as well as a therapeutic target for gastric cancer.

Abstract 5395: MicroRNA-196b-3p identified from serum microRNA expression profiling is a potential diagnostic biomarker for gastritis and early gastric cancer

Abstract Background: Gastric cancer is one of the most common forms of cancer worldwide. The progression of chronic atrophic gastritis (CAG) is involved in gastric carcinogenesis. With the progression of chronic atrophic gastritis, the risk of gastric cancer increased in a stepwise fashion. Non-invasive tools for early gastric cancer are required to improve the prognosis. MicroRNAs (miRNAs), a class of small non-coding RNAs of 19-22 nucleotides in length, function as post-transcriptional regulators by directly cleaving target messenger RNA or translational repression. MiRNAs in serum have been shown to be potential biomarkers in various type of cancers and several miRNAs have been reported in gastric cancer. The purpose of present study was to identify a serum miRNA expression profile that can serve as a novel diagnostic biomarker for early gastric cancer. Materials and Method: We defined a normal gastric mucosa as grade0, CAG classified C-1, 2 in Kimura-Takemoto classification as grade1, CAG classified C-3, O-1 in Kimura-Takemoto classification as grade2, and CAG classified O-2, 3 in Kimura-Takemoto classification as grade3. We obtained 344 serum samples, which gathered from 19 health people (grade0), 125 patients with chronic atrophic gastritis (grade1: 54, grade2: 36, grade3: 35), and 200 patients with early gastric cancer. The miRNAs were extracted from serums and miRNA microarray analysis was performed. In the discovery phase, we analyzed 71 cases (grade0: 11, grade1: 20, grade2: 20, grade3: 20). Next, in the training phase, we analyzed 73 cases (grade0: 8, grade1: 34, grade2: 16, grade3: 15). Then, we analyzed 200 early gastric cancer serum samples. Result: The microarray analysis identified a profile of miRNA (miRNA-196b-3p) as a biomarker for early gastric cancer. The analysis results showed that the expression level of the serum miRNA was correlated to degree of CAG and carcinogenesis. When compared with grade0,1,2 and grade3, the area under the receiver operating characteristic (ROC) curve of this serum miRNA signature was 0.730 and 0.763 for the two sets of serum samples. There was significant difference in serum value of miRNA-196b-3p between grade1-3 and early gastric cancer (p=0.003). Conclusion: Serum miRNA-196b-3p is good biomarker for early gastric cancer. Citation Format: Atsushi Fujii, Sho Nambara, Yosuke Kuroda, Shuhei Ito, Hidetoshi Eguchi, Takaaki Masuda, Takao Otsuka, Masafumi Nakamura, Koshi Mimori. MicroRNA-196b-3p identified from serum microRNA expression profiling is a potential diagnostic biomarker for gastritis and early gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5395.

A serum exosomal microRNA panel as a potential biomarker test for gastric cancer

The findings from several studies have suggested that circulating miRNAs are imbalanced with the genesis of gastric cancer (GC). Both normal and cancer cells can generate and secrete exosomes, which are nanosized membrane vesicles that can transport microRNAs and proteins. Emerging evidence indicates that the exosomes secreted by cancer cells can be released into the circulatory system. In this study, we investigated whether circulating exosomal miRNAs can be used to discriminate individuals with GC from healthy controls (NCs). Based on the quantitative reverse transcription polymerase chain reaction (qRT-PCR), four miRNAs (miR-19b-3p, miR-17-5p, miR-30a-5p, and miR-106a-5p) related to GC pathogenesis were identified in serum-circulating exosomes from a cohort of 20 healthy controls and 20 individuals with GC in the initial screening phase. The distinguished miRNAs were further validated in the training (90 GC vs. 90 NCs) and blinded phases 20 GC vs. 20 NCs), and the area under receiver operating characteristic (ROC) curves of these miRNAs were analyzed. We found that miR-19b and miR-106a were markedly overexpressed in individuals with GC compared to NCs (P < 0.0001). Besides, the ROC analyses yielded the AUC values of 0.786 for miR-106a-5p, 0.769 for miR-19b-3p and combined ROC analysis revealed the highest AUC value of 0.814 in discriminating GC patients from NCs. Furthermore, based on the model developed from the data, a signature composed of the 2 miRNAs (miR-19b-3p and miR-106a-5p) correctly discriminated 19 out of 20 GC serum samples (95% sensitivity) and 18 out of 20 normal samples (90% specificity) in the blinded phase. Moreover, the validated miRNAs were related to GC lymphatic metastasis (P < 0.01) and expressed at higher levels in stages III and IV compared to I and II stages (P < 0.05). These results suggest that serum exosomal miR-19b-3p and miR-106a-5p are novel potential biomarkers for detecting GC.

High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression.

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high‐mobility group box‐1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC‐associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34‐positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1‐treated or HMGB1‐TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti‐HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin‐8 (IL‐8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1‐TF were significantly reversed by IL‐8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL‐8 mediation, and combined targeting of HMGB1 and IL‐8 can control tumor angiogenesis in GC.

The microRNA-423-3p-Bim Axis Promotes Cancer Progression and Activates Oncogenic Autophagy in Gastric Cancer

Human serum microRNAs (miRNAs) have been shown toserve as disease fingerprints for predicting survival ofcancer patients. However, the roles of specific miRNAs involved in gastric cancer (GC) are largely unknown. In this study, miRNA profiling was performed on sera obtained from six patients in good- and poor-survival groups. Expression of miR-423-3p was validated by quantitative RT-PCRinanother 67 GC serum samples and paired normal and cancerous gastric tissues. Luciferase reporter assays were used to identify the target gene Bcl-2-interacting mediator of cell death (Bim). As a result, between the good-survival and poor-survival groups, the expression of nine serum miRNAs was altered more than two-fold. Amongthese, miR-423-3p was significantly increased in the poor-survival group, and its overexpression in GC tissuespredicted poor survival in 119 patients with GC. miR-423-3p was found topromote cell proliferation, migration, and invasion in cell lines and animal models. Mechanistically, knockdown of the autophagy-related gene (Atg) 7 rescued the GC-promoting effect of miR-423-3p. In conclusion, miR-423-3p activates oncogenic and Beclin-1-dependent autophagy and promotes GC progression by reducing theexpression of Bim. The newly identified miR-423-3p-Bim axis might be a potential therapeutic target inGC.

Six Serum-Based miRNAs as Potential Diagnostic Biomarkers for Gastric Cancer.

Circulating miRNAs in serum may serve as promising diagnostic biomarkers for patients with gastric cancer. Using qRT-PCR-based Exiqon panel, we identified 58 differentially expressed miRNAs from three gastric cancer pool samples and one normal control (NC) pool in the initial screening phase. Identified miRNAs were further validated in the training (49 gastric cancer vs. 47 NCs) and validation phases (154 gastric cancer vs. 120 NCs) using qRT-PCR. The expression levels of the miRNAs were also determined in tissues, arterial serum, and exosomes. Consequently, six serum miRNAs (miR10b-5p, miR132-3p, miR185-5p, miR195-5p, miR-20a3p, and miR296-5p) were significantly overexpressed in gastric cancer compared with NCs. The areas under the receiver operating characteristic curve of the six-miRNA panel were 0.764 and 0.702 for the training and validation phases, respectively. miR10b-5p and miR296-5p were significantly upregulated in gastric cancer tissues (n = 188). In addition, patients who did not receive adjuvant chemotherapy with high expression of miR10b-5p or miR296-5p in tissues tended to suffer worse overall survival. Furthermore, the expression levels of miR10b-5p, miR195-5p, miR20a-3p, and miR296-5p were significantly elevated in exosomes from gastric cancer serum samples (n = 30). We identified a six-miRNA panel in serum for the detection of gastric cancer. Our findings provide a novel serum miRNA signature for gastric cancer diagnosis, and will serve as the basis of the application of circulating miRNAs in clinical for the detection of gastric cancer in the future. Cancer Epidemiol Biomarkers Prev; 26(2); 188-96. ©2016 AACR.

CpG island methylator phenotype andHelicobacter pyloriinfection associated with gastric cancer

To investigate the association between the CpG island methylator phenotype (CIMP) and serum Helicobacter pylori (H. pylori) levels for clinical prediction of gastric cancer (GC) progression. We analyzed the serum CIMP status of 75 patients with GC using a methylation marker panel and a methylation-specific polymerase chain reaction. Serum samples from 40 healthy persons were examined at the same time. The genes examined were APC, WIF-1, RUNX-3, DLC-1, SFRP-1, DKK and E-cad. H. pylori infection in serum was assayed with an anti-H. pylori immunoglobulin G antibody test and a rapid urease test. The frequencies of high-level methylation in GC tissues for the seven genes were: 48% for APC, 57.33% for WIF-1, 56% for RUNX-3, 50.67% for DLC-1, 52% for SFRP-1, 54.67% for DKK, and 48% for E-cad. The frequencies in GC serum were 30.67% for APC, 34.67% for WIF-1, 37.33% for RUNX-3, 29.33% for DLC-1, 33.33% for SFRP-1, 32% for DKK, and 26.67% for E-cad. CIMP+ (defined as ≥ 3 methylated genes) was associated with 47 (62.67%) GC tissue samples and 44 (58.67%) GC serum samples. CIMP+ was not associated with non-neoplastic mucosal tissues or the serum of healthy persons. Of the 75 GC cases, 51 (68%) were H. pylori+, and 24 (32%) were H. pylori-. Of the 51 H. pylori+ cases, 36 were CIMP+ and 15 were CIMP-. In contrast, for the 24 H. pylori- cases, 11 were CIMP+, and 13 were CIMP-. The difference was significant between the H. pylori+ and H. pylori- groups (χ(2) = 4.27, P < 0.05). Of the 51 H. pylori+ GC patients, 34 were CIMP+ and 17 were CIMP-, while among the 24 H. pylori- GC cases, 10 were CIMP+ and 14 were CIMP-. The difference was significant between the H. pylori+ and H. pylori- groups (χ(2) = 4.21, P < 0.05). A 2-year follow-up showed significant difference in the rates of metastasis and recurrence between H. pylori+/CIMP+ cases and the H. pylori+/CIMP- cases or CIMP- cases associated with H. pylori assayed in serum (P < 0.05). However, there were no significant differences in survival rates between the two groups. H. pylori+/CIMP+ cases are associated with higher rates of metastasis and recurrence than H. pylori+/CIMP- cases. Serum may be useful for examining CIMP status.

Five Serum Proteins Identified Using SELDI-TOF-MS as Potential Biomarkers of Gastric Cancer

The aims of this study were to detect serum proteomic patterns in gastric cancer serum samples using Surface-enhanced Laser Desorption/ionization-Time-of-flight-Mass Spectrometry ProteinChip array technology, to screen biomarker candidates, to build diagnostic models and to evaluate their clinical significance. Serum samples from patients with gastric cancer and normal healthy control subjects (n = 125) were analysed using surface-enhanced laser desorption/ionization technology. The spectra were generated on weak cation exchange (WCX2) chips, and protein peak clustering and classification analyses were established using Ciphergen Biomarker Wizard and Biomarker Pattern software, respectively. The diagnostic models were developed and validated by discriminant analysis. In addition, the results of the surface-enhanced laser desorption/ionization model were compared with the biomarkers carcinoembryonic antigen and carbohydrate antigen 199 in a subset of samples using a microparticle enzyme immunoassay. Five protein peaks at 2046, 3179, 1817, 1725 and 1929 m/z were automatically chosen as components of the best biomarker pattern for diagnosis of gastric cancer. In addition, we identified a single protein peak at 4665 m/z, which could distinguish between stage I/II and stage III/IV gastric cancer with a specificity and sensitivity of 91.6% (11/12) and 95.4% (21/22), respectively. When this biomarker was validated in the second set of samples, the specificity and sensitivity were 91.7% (11/12) and 86.3% (19/22), respectively. The present results suggest that serum surface-enhanced laser desorption/ionization protein profiling can distinguish patients with gastric cancer, and in particular stage I/II patients, from normal subjects with a relatively high sensitivity and specificity. Surface-enhanced Laser Desorption/ionization-Time-of-flight-Mass Spectrometry is a potential new diagnostic tool for the screening of gastric cancer.

Identification of novel serum biomarkers for gastric cancer by magnetic bead

Early diagnosis and early treatment is known to improve prognosis for gastric cancer. Magnetic affinity beads can be used to extract peptides from un-fractionated serum samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can detect the presence and the molecular mass of peptides. MALDI-TOF-MS mass spectra of peptides and proteins were generated after WCX CLINPROT bead fractionation of 62 gastric cancer serum samples. The discovery set consisted of 44 samples while the validation set was 18 serum samples. The spectra were analyzed statistically using flexAnalysisTM and Clin-ProtTM bioinformatic software. The six most significant peaks were selected out by ClinProTool software and utilized to train a Supervised Neural Network to identify gastric cancer sera from control sera. The sensitivity and specificity of the model when tested on the validation set were 100% and 75%, respectively. A set of 6 peptides that can be used to distinguish serum from gastric cancer patients with good sensitivity and specificity were identified, and these peptides may be useful biomarkers to distinguish cancer individuals who may benefit from radiologic or endoscopic examination.