Compound mutations involving T315I and P-loop mutations are the major components of multiple mutations detected in tyrosine kinase inhibitor resistant chronic myeloid leukemia.

To analyze the pattern of multiple mutations detected by Sanger sequencing (SS), we performed subcloning sequencing using 218 samples from 45 patients with tyrosine kinase inhibitor resistant chronic myeloid leukemia. At the first time of multiple mutation detection by SS (baseline), a total of 19 major mutations from 45 samples were detected; these mutations were found in the following order: T315I (68.9%), E255 K (33.3%), Y253H (13.3%), G250E (13.3%), and F317 L (11.1%). Subcloning sequencing of 900 baseline colonies identified 556 different mutant types, and 791 among the 900 were colonies with major mutations (87.9%). The mutations were found in the following order: T315I (36.4%), E255 K (16.2%), Y253H (7.0%), G250E (6.7%), M351 T (6.6%), and E255 V (5.3%). In subcloning sequencing with 4357 colonies of 218 serial samples, 2506 colonies (57.5%) had compound mutations, among which 2238 colonies (89.3%) had at least one major mutation. The median number of mutations in compound mutant colonies was 2 (range, 2-7), and most were double (52.9%) or triple (28.7%) mutations. Additionally, some mutations in allosteric binding sites were detected as low level mutation in 13 patients. With the available retrospective samples before baseline, subcloning sequencing identified low-level mutations of various frequencies (median, 10%) to be major mutations in 20 patients. Thus, compound mutations involving T315I and P-loop mutations were the major components of multiple mutations, and some low-level mutations with potential clinical significance were detected by subcloning sequencing. Hence, more sensitive sequencing assays are needed in patients with multiple mutations.

BCR-ABL1 transcript levels at 4 weeks have prognostic significance for time-specific responses and for predicting survival in chronic-phase chronic myeloid leukemia patients treated with various tyrosine kinase inhibitors.

The present study aimed to assess the clinical impact of BCR-ABL1 transcript levels determined at an earlier time point than the 3-month early molecular response (EMR) in chronic-phase chronic myeloid leukemia (CML-CP) patients. BCR-ABL1 transcript levels of CML-CP patients (n = 258; median age, 43 [range, 18-81] years) treated with various tyrosine kinase inhibitors (TKIs) were determined at 4 weeks (28 ± 3 days) and at every 3 months of treatment initiation. At 4 weeks, receiver operating characteristic curves revealed that cutoff values of BCR-ABL1 transcripts for achieving major molecular responses (MMRs) by 12 and 60 months were 40.89% and 39.16%, respectively (95% CI, 0.658-0.772 and 95% CI, 0.643-0.758; P < 0.0001). With 40% of BCR-ABL1 transcripts at 4 weeks (very early MR; VEMR), patients with VEMR achieved higher 3-month EMR and 4-week VEMR significantly associated with higher cumulative incidences of 5-year MMR (89.1% vs 72.3%; P < 0.001) and 5-year deep molecular response (DMR) (56.5% vs 29.4%; P = 0.001). Furthermore, event-free survival (EFS)-a (93.0% vs 84.8%; P = 0.068) and EFS-b (71.1% vs 57.9%; P = 0.061) by 5 years were also marginally significant. VEMR and 3-month EMR were achieved in 89 patients, with significantly superior outcomes. In multivariate analyses, lower leukocyte count (P = 0.008) and frontline second-generation TKI therapy size (P < 0.001) were significantly associated with VEMR achievement, but not baseline BCR-ABL1 level and CML duration. In conclusion, the 4-week BCR-ABL1 transcript levels including VEMR could be important to predict long-term outcomes and may provide additional information about innate intrinsic sensitivity to CML among individuals.

PMK-S005 Alleviates Age-Related Gastric Acid Secretion, Inflammation, and Oxidative Status in the Rat Stomach.

BACKGROUND/AIMS: The aim of this study was to evaluate the effect of the synthetic S-allyl-L-cysteine (SAC) PMK-S005 on gastric acid secretion, inflammation, and antioxidant enzymes in aging rats. METHODS: The rats were divided into four groups at 31 weeks of age and were continuously fed a diet containing a vehicle control, PMK-S005 (5 or 10 mg/kg), or lansoprazole (5 mg/kg). Gastric acid secretion and connective tissue thickness of the lamina propria were evaluated at 74 weeks and 2 years of age. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and COX-2 levels were measured by using enzyme-linked immunosorbent assays (ELISAs) or Western blot assays. Levels of antioxidant enzymes, including heme oxyganase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1), were also measured. RESULTS: As the rats aged, gastric acid secretion significantly decreased, and the connective tissue of the lamina propria increased. However, 74-week-old rats in the PMK-S005 group exhibited greater levels of gastric acid secretion than those of the control and lansoprazole groups. The increase of TNF-α, IL-1ß, and COX- 2 expression in 74-week and 2-year-old control rats were inhibited by PMK-S005. In addition, the decrease in HO-1 and NQO-1 protein expression that occurred with aging was inhibited by PMK-S005 in the 74-week-old rats. CONCLUSIONS: These results suggest that PMK-S005 has therapeutic potential as an antiaging agent to ameliorate age-related gastric acid secretion, inflammation, and oxidative stress in the stomach.

Anti-inflammatory and Anti-tumorigenic Effects of Açai Berry in Helicobacter felis-infected mice.

BACKGROUND: The aim of this study was to evaluate the anti-inflammatory and anti-tumorigenic effect of açai berry after chronic Helicobacter felis colonization in the stomachs of C57BL/6 mice. METHODS: A total of 57 four-week-old female C57BL/6 mice (18 control mice and 39 experimental mice) were used. The mice were administered orogastrically with vehicle only or vehicle containing H. felis, 5 times every other day. After inoculation of H. felis, mice were fed either a standard or an açai-containing diet and then sacrificed at 4, 24, and 52 weeks. The infection status and degree of inflammation were determined by culture and histopathology. The level of gastric mucosal myeloperoxidase (MPO), TNF-α, and interleukin-1ß (IL-1ß) were measured by ELISA. RESULTS: At 24 weeks after inoculation, mucosal atrophy and mucous metaplasia appeared in all infected mice. At 52 weeks after inoculation, dysplastic change was noted in 10%, 25%, and 50% of mice in the H. felis-control, H. felis-açai 5%, and H. felis-açai 10% groups, respectively. The neutrophil, monocyte, atrophy, and metaplasia grades of infected mice showed no significant difference among the H. felis-infected groups. H. felis-infected mice fed with açai berry showed no significant difference compared with H. felis-infected control mice in gastric mucosal MPO, TNF-α, and IL-1ß levels. CONCLUSIONS: H. felis that colonized the stomachs of C57BL/6 mice provoked inflammation, and induced mucosal atrophy, metaplasia, and dysplasia. However, açai berry did not effectively prohibit the gastric carcinogenesis which was induced by chronic H. felis infection.

Comparison of the efficacy of culture-based tailored therapy for Helicobacter pylori eradication with that of the traditional second-line rescue therapy in Korean patients: a prospective single tertiary center study.

OBJECTIVE: The effectiveness of Helicobacter pylori therapies has declined with an increase in antibiotic resistance. To overcome this problem, the efficacy of tailored H. pylori eradication therapy based on antimicrobial susceptibility testing was compared with that of empirical second-line rescue regimens. MATERIAL AND METHODS: Patients who had persistent H. pylori infection after the first eradication were recommended to undergo culture for determining the minimal inhibitory concentration (MIC) via gastroscopy, which increased the cost by 300%. Fourteen-day esomeprazole, tripotassium dicitrate bismuthate, metronidazole and tetracycline (EBMT) therapy or esomeprazole, moxifloxacin and amoxicillin (MEA) therapy was performed according to the results of antibiotic susceptibility testing. In case of refusal to undergo culture, the participants were treated with either 14-day empirical EBMT or MEA regimen for second eradication after explaining the complexity, side effects and costs associated with each regimen. This trial was registered at ClinicalTrials.Gov (NCT 02349685). RESULTS: In the 219 patients included, the intention to treat (ITT) and per protocol (PP) eradication rates was 75.3% and 79.8% in the 14-day EBMT group (n = 89), 70.8% and 72.4% in the 14-day MEA group (n = 89) and 87.8% and 100.0% in the 14-day tailored therapy group (n = 41), respectively. Based on the PP analysis, the 14-day tailored therapy group showed a significantly higher eradication rate than the 14-day EBMT or MEA group (both p ≤ 0.001). CONCLUSIONS: Tailored therapy based on H. pylori culture and MIC test could be an option as a second-line eradication regimen in the presence of high level of antimicrobial resistance.

Adequate Dextran Sodium Sulfate-induced Colitis Model in Mice and Effective Outcome Measurement Method.

BACKGROUND: Dextran sodium sulfate (DSS)-induced colitis mouse model is used for research of inflammatory bowel disease. The aim of this study was to establish the adequate conditions for DSS mice model, and to find useful tool to measure inflammation. METHODS: The 2.5% DSS was administered to six male C57BL/6 mice and 4% DSS to eight mice at 5 or 9 weeks of age. Each group was consisted of 6 mice with control group in which vehicle was administered instead of DSS. The mice were sacrificed on the 7th day after DSS or vehicle administration. Body weight, diarrhea, and hematochezia were recorded daily. Disease activity index (DAI) score which was composed of body weight change, diarrhea, and hematochezia was measured every day. Colon length was measured after sacrifice and colon mucosal level of interleukin 1 beta (IL-1ß) was measured by ELISA assay. Histological score was compared between ascending and descending colon in the DSS group. RESULTS: Colon length of five- and nine-week DSS group was significantly shorter than each control group but there was no statistical significance depending on DSS concentration or age. DAI score of 4% DSS group in nine-week was significantly higher than that five-week (P = 0.012) but there was no difference between 2.5% and 4% DSS group. The level of IL-1ß in DSS mice was much higher than control group (P < 0.01), but there was no difference among several DSS groups. The histological score was higher in the descending colon than in the ascending colon but there was no statistical difference between each pair of DSS groups. CONCLUSIONS: The 4% DSS mice in nine-week was adequate for DSS-induced colitis model. DAI score was useful tool and descending colon was more appropriate site for histological evaluation of colitis than ascending colon.

Histologic Findings and Inflammatory Reactions After Long-term Colonization of Helicobacter felis in C57BL/6 Mice.

BACKGROUND: The Helicobacter felis (H. felis) mouse model has been developed for the research regarding pathogenesis of chronic gastritis and gastric cancer. The aim of this study was to investigate long-term H. felis colonization in the stomachs of C57BL/6 mice and subsequent histologic findings and inflammatory reactions including pro-inflammatory cytokines. METHODS: Twenty-three female C57BL/6 mice at 4 weeks of age were gavaged with H. felis, and 13 control mice served as vehicle only. The mice were sacrificed at 4, 24, and 52 weeks after inoculation. The infection status and degree of inflammation were determined by culture and histopathology. The level of gastric mucosal myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-α), and interleukin-1beta (IL-1ß) were measured by ELISA. RESULTS: The overall infection rate was 100%, as determined by the culture and histology. At 4, 24, and 52 weeks, the neutrophil and monocyte scores were significantly higher in infected mice than in control mice. At 24 weeks after inoculation, most of the infected mice showed mucosal atrophy with or without metaplasia, and a few showed focal dysplasia. Adenocarcinoma was observed in one mouse at 52 week post-infection. Gastric mucosal MPO and IL-1ß levels were significantly higher in infected mice than those in control mice at 24 and 52 weeks. However, the expression of gastric mucosal TNF-α was not significantly different between the infected and control mice at any time-point. CONCLUSIONS: Long-term H. felis-infection in C57BL/6 mice provoked a severe inflammatory reaction and it progressed into atrophy, metaplasia, dysplasia and cancer. IL-1ß might play an important role in the inflammatory response of mice to Helicobacter species.

Association of polymorphisms in virulence factor of Helicobacter pylori and gastroduodenal diseases in South Korea.

BACKGROUND AND AIM: Clinical outcomes of Helicobacter pylori (HP) infection have been shown to be dependent on the variability of virulence factors. The aim of this study was to evaluate the prevalence of each virulence factor and the association between polymorphisms of the virulence factors of HP, and the clinical outcome of gastroduodenal diseases in South Korea. METHODS: Four hundred one HP colonies were analyzed (75 colonies from 45 controls; 71 colonies from 39 benign gastric ulcer [BGU] patients; 102 colonies from 54 duodenal ulcer [DU] patients; 121 colonies from 77 stomach cancer patients; and 32 colonies from 25 dysplasia patients). Polymerase chain reaction amplifications for vacA, cagA, iceA, oipA, and dupA were performed using DNA extract from HP isolates cultured from mucosal biopsy specimens. dupA was regarded as positive when all of jph0718, jph0719, and dupA were positive. RESULTS: Most colonies were composed of vacA s1 (100.0%), i1 (100.0%) and m1 (92.9%), cagA-positive (87.2%), iceA1 (95.8%), oipA-positive (91.2%), and dupA-negative (52.0%) genotypes. dupA was more frequently expressed in BGU (81.3%), DU (74.7%), and dysplasia (41.7%) than control (16.7%) (P < 0.001). Infection by dupA-positive HP showed an increased risk of BGU (odds ratio 33.06, 95% confidence interval 11.91-91.79) and DU (odds ratio 15.60, 95% confidence interval 6.49-37.49). CONCLUSION: HP infection in South Koreans appears to be closely related to highly virulent strains (vacA s1/i1/m1, cagA(+), iceA1(+), and oipA(+)), except dupA. dupA has an intimate association with the development of peptic ulcer diseases.