Development of a gastric cancer risk calculator for questionnaire-based surveillance of Iranian dyspeptic patients.

BACKGROUND: Gastric cancer (GC) is considered a silent killer, taking more than three quarters of a million lives annually. Therefore, prior to further costly and invasive diagnostic approaches, an initial GC risk screening is desperately in demand. METHODS: In order to develop a simple risk scoring system, the demographic and lifestyle indices from 858 GC and 1132 non-ulcer dyspeptic (NUD) patients were analysed. We applied a multivariate logistic regression approach to identify the association between our target predictors and GC versus NUD. The model performance in classification was assessed by receiver operating characteristic (ROC) analysis. Our questionnaire covering 64 predictors, included known risk factors, such as demographic features, dietary habits, self-reported medical status, narcotics use, and SES indicators. RESULTS: Our model segregated GC from NUD patients with the sensitivity, specificity, and accuracy rates of 85.89, 63.9, and 73.03%, respectively, which was confirmed in the development dataset (AUC equal to 86.37%, P < 0.0001). Predictors which contributed most to our GC risk calculator, based on risk scores (RS) and shared percentages (SP), included: 1) older age group [> 70 (RS:+ 241, SP:7.23), 60-70 (RS:+ 221, SP:6.60), 50-60 (RS:+ 134, SP:4.02), 2) history of gastrointestinal cancers (RS:+ 173, SP:5.19), 3) male gender (RS:+ 119, SP:3.55), 4) non-Fars ethnicity (RS:+ 89, SP:2.66), 5) illiteracy of both parents (RS:+ 78, SP:2.38), 6) rural residence (RS:+ 77, SP:2.3), and modifiable dietary behaviors (RS:+ 32 to + 53, SP:0.96 to 1.58). CONCLUSION: Our developed risk calculator provides a primary screening step, prior to the subsequent costly and invasive measures. Furthermore, public awareness regarding modifiable risk predictors may encourage and promote lifestyle adjustments and healthy behaviours.

Alteration in Serum Levels of Tumor Necrosis Factor Alpha is associated with Histopathologic Progression of Gastric Cancer

Background: The role of inflammatory cytokines, such as tumor necrosis-α (TNF-α) and IL-8, in gastric carcinogenesis has been investigated, but their impact remains to be further elucidated. Methods: In this study, we measured the serum concentrations of these cytokines and H. pylori serostatus in dyspeptic patients, presenting with normal mucosa (NM = 53), chronic gastritis (CG = 94), and gastric cancer (GC = 82), by ELISA. Results: Moderate levels of TNF-α were detected in the NM group (19.9 ± 19.5 pg/ml), which were nearly doubled in patients with CG (35.7 ± 28.0 pg/ml) and drastically declined in GC patients (1.8 ± 5.9 pg/ml). The serum levels of IL-8, however, were not statistically different amongst these three groups. Conclusion: TNF-α serum concentration seemed to undergo up- and downregulation, when moving from NM to CG and from CG to GC, respectively. If confirmed in a prospective study, this cytokine can behave as a serum indicator of gastric inflammation and malignant transformation.

Infection with a hypervirulent strain of Helicobacter pylori primes gastric cells toward intestinal transdifferentiation.

BACKGROUND: Intestinal metaplasia, gastric-to-intestinal transdifferentiation, occurs as a result of the misexpression of certain regulatory factors, leading to genetic reprogramming. Here, we have evaluated the H. pylori-induced expression patterns of these candidate genes. METHODS: The expression levels of 1) tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), 2) stemness factors (TNFRSF19, LGR5, VIL1) and 3) tissue-specific mucins (MUC5AC, MUC2) were evaluated by quantitative real-time PCR in gastric primary cells (GPCs), in parallel with two gastric cancer (MKN45 and AGS) cell lines, up to 96h following H. pylori infection. RESULTS: Following H. pylori infection of GPCs, RUNX3 declined at 24h post infection (-6.2 ± 0.3) and remained downregulated for up to 96h. Subsequently, overexpression of self-renewal and pluripotency transcription factors, KLF5 (3.6 ± 0.2), SOX2 (7.6 ± 0.5) and SALL4 (4.3 ± 0.2) occurred. The expression of TNFRSF19 and LGR5, demonstrated opposing trends, with an early rise of the former (4.5 ± 0.3) at 8h, and a simultaneous fall of the latter (-1.8 ± 0.5). This trend was reversed at 96h, with the decline in TNFRSF19 (-5.5 ± 0.2), and escalation of LGR5 (2.6 ± 0.2) and VIL1 (1.8 ± 0.3). Ultimately, CDX1 and CDX2 were upregulated by 1.9 and 4.7-fold, respectively. The above scenario was, variably observed in MKN45 and AGS cells. CONCLUSION: Our data suggests an interdependent gene regulatory network, induced by H. pylori infection. This interaction begins with the downregulation of RUNX3, upregulation of self-renewal and pluripotency transcription factors, KLF5, SOX2 and SALL4, leading to the downregulation of TNFRSF19, upregulation of LGR5 and aberrant expression of intestine-specific transcription factors, potentially facilitating the process of gastric-to-intestinal transdifferentiation.

Mitochondrial DNA Copy Number Variations and Serum Pepsinogen Levels for Risk Assessment in Gastric Cancer

Background: Variations in mitochondrial DNA copy number (mtDNA-CN) of peripheral blood leukocytes (PBLs), as a potential biomarker for gastric cancer (GC) screening has currently been subject to controversy. Herein, we have assessed its efficiency in GC screening, in parallel and in combination with serum pepsinogen (sPG) I/II ratio, as an established indicator of gastric atrophy. Methods: The study population included GC (n = 53) and non-GC (n = 207) dyspeptic patients. The non-GC group was histologically categorized into CG (n = 104) and NM (n = 103) subgroups. The MtDNA-CN of PBLs was measured by quantitative real-time PCR. The sPG I and II levels and anti-H. pylori serum IgG were measured by ELISA. Results: The mtDNA-CN was found significantly higher in GC vs. non-GC (OR = 3.0; 95% CI = 1.4, 6.4) subjects. Conversely, GC patients had significantly lower sPG I/II ratio than the non-GC (OR = 3.2; CI = 1.4, 7.2) subjects. The combination of these two biomarkers yielded a dramatic amplification of the odds of GC risk in double-positive (high mtDNA-CN-low sPGI/II) subjects, in reference to double-negatives (low mtDNA-CN-high sPGI/II), when assessed against non-GC (OR = 27.1; CI = 5.0, 147.3), CG (OR = 13.1; CI = 2.4, 72.6), or NM (OR = 49.5; CI = 7.9, 311.6) groups. Conclusion: The combination of these two biomarkers, namely mtDNA-CN in PBLs and serum PG I/II ratio, drastically enhanced the efficiency of GC risk assessment, which calls for further validations.

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes.

BACKGROUND: Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. RESULTS: The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. CONCLUSIONS: We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

The outward shift of clarithromycin binding to the ribosome in mutant Helicobacter pylori strains.

OBJECTIVES: Disruption of protein synthesis, by drug-mediated restriction of the ribosomal nascent peptide exit tunnel (NPET), may inhibit bacterial growth. Here, we have studied the secondary and tertiary structures of domain V of the 23S rRNA in the wild-type and mutant (resistant) H. pylori strains and their mechanisms of interaction with clarithromycin (CLA). METHODS: H pylori strains, isolated from cultured gastric biopsies, underwent CLA susceptibility testing by E test, followed by PCR amplification and sequencing of domain V of 23S rRNA. The homology model of this domain in H pylori, in complex with L4 and L22 accessory proteins, was determined based on the E. coli ribosome 3D structure. The interactions between CLA and 23S rRNA complex were determined by molecular docking studies. RESULTS: Of the 70 H pylori strains, isolated from 200 dyspeptic patients, 11 (16%) were CLA-resistant. DNA sequencing identified categories with no (A), A2142G (B), and A2143G (C) mutations. Docking studies of our homology model of 23S rRNA complex with CLA showed deviated positions for categories B and C, in reference to category A, with 12.19 Å and 7.92 Å RMSD values, respectively. In both mutant categories, CLA lost its interactions at positions 2142 and 2587 and gained two new bonds with the L4 accessory protein. CONCLUSION: Our data suggest that, in mutant H pylori strains, once the nucleotides at positions 2142 and 2587 are detached from the drug, CLA interacts with and is peeled back by the L4 accessory protein, removing the drug-imposed spatial restriction of the NPET.