Kazuyuki Kawakami

BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status

BackgroundBRAF is a member of RAF family of serine/threonine kinases and mediates cellular responses to growth signals through the RAS-RAF-MAP kinase pathway. Activating mutations in BRAF have recently been found in about 10% of colorectal cancers, with the vast majority being a V600E hotspot mutation. The aim of the present study was to evaluate the clinical, pathological and molecular phenotype of colorectal tumors with BRAF mutations.ResultsMutations in BRAF were identified in 8% (23/275) of colorectal cancers. They were 5–10-fold more frequent in tumors with infiltrating lymphocytes, location in the proximal colon, poor histological grade and mucinous appearance (P < 0.002 for each). Tumors with BRAF mutation were also 10-fold more likely to show microsatellite instability and frequent DNA methylation (P < 0.0001) compared to tumors without this mutation. The characteristic morphological features of tumors with BRAF mutation (infiltrating lymphocytes, poor grade, mucinous) remained after stratification according to microsatellite instability and methylator phenotypes. Mutations in BRAF were mutually exclusive with mutations in KRAS but showed no clear association with the presence of TP53 mutation.ConclusionBRAF mutation identifies a colorectal cancer subgroup with distinctive phenotypic properties independent of microsatellite instability status and thus could be a valuable marker for studies into the clinical properties of these tumors.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Long Interspersed Nuclear Element 1 Hypomethylation Is a Marker of Poor Prognosis in Stage IA Non–Small Cell Lung Cancer

Purpose: Global hypomethylation and the hypermethylation of gene promoter regions are common events in tumor DNA. The aim of this study was to evaluate the prognostic significance of both global hypomethylation and gene promoter hypermethylation in DNA from non–small cell lung cancer (NSCLC). Experimental Design: Genomic DNA was obtained from the tumor tissue of 379 NSCLC patients who underwent surgery. Methylation levels were measured by real-time PCR following bisulfite modification of DNA and were correlated with clinicopathologic parameters and patient prognosis. Methylation of long interspersed nuclear element 1 (LINE-1) was used as a surrogate marker for global methylation. Hypermethylation of the APC, CDH13, and RASSF1 promoter regions was also evaluated. Results: Tumor tissue showed significantly higher CDH13 and RASSF1 methylation levels compared with normal lung tissue, but lower LINE-1 methylation levels. APC, RASSF1, and LINE-1 methylation levels were significant prognostic factors in univariate analysis of an initial cohort of 234 cases. APC and LINE-1 methylation remained significant prognostic factors in multivariate analysis that included age, gender, smoking history, histologic type, and pathologic stage. LINE-1 methylation showed marginally significant prognostic value in stage IA and IB disease. Expansion of the study cohort to 364 cases revealed that LINE-1 methylation had significant prognostic value for stage IA NSCLC patients in multivariate analysis. Conclusions: LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC. Validation of this finding in additional tumor cohorts could have clinical relevance for the management of early-stage NSCLC. Clin Cancer Res; 16(8); 2418–26. ©2010 AACR.

Loss of Heterozygosity at the Thymidylate Synthase (TS) Locus on Chromosome 18 Affects Tumor Response and Survival in Individuals Heterozygous for a 28-bp Polymorphism in the TS Gene

Thymidylate synthase (TS), the target enzyme of the fluoropyrimidine class of drugs, has a 28-bp repeat polymorphism in the promoter region that has been associated with response of tumors to 5-fluorouracil-based therapy. Patients homozygous for the double repeat (2R/2R) in the TS gene have an overall better outcome from treatment than patients homozygous for the triple repeat (3R/3R). However, due to loss of heterozygosity at the TS locus on chromosome 18 in cancer cells, heterozygous 2R/3R individuals can acquire the 2R/loss or the 3R/loss genotype in their tumors. The purpose of this study was to determine whether the response of colorectal cancer to fluoropyrimidine therapy is associated with the resulting tumor TS genotype when loss of heterozygosity occurs in tumor DNA. A total of 30 colorectal cancer patients treated with the fluoropyrimidine-based combination S-1, all of whom had stage IV disease, were studied. The response rate to S-1 in this group of patients was 13 of 30 (43%). The heterozygous 2R/3R genotype was found in 22 of 30 normal tissues, whereas 10 (45%) of the matched cancer tissues showed only the 2R-sequence band (2R/loss), and 7 cancer tissues (32%) showed only the 3R-sequence band (3R/loss). The response rate of the 2R/loss tumor genotype patients was 80% (8 of 10) compared with 14% (1 of 7) in the 3R/loss genotype group (P = 0.029). Patients with tumor 3R/loss genotypes had significantly lower survival than 2R/loss genotypes. Heterozygous patients with a 2R/loss tumor genotype had the same survival as 2R/2R patients, whereas patients with a 2R/3R tumor genotype had a short survival similar to homozygous 3R/3R genotypes. These results show that: (a) response to 5-fluorouracil-based therapy is determined by tumor genotype; and (b) the 3R repeat is a direct negative determinant of outcome.

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.

Experimental study on heat production by a 23.5-kHz ultrasonically activated device for endoscopic surgery

Abstract. An experimental study was carried out to evaluate heat production by an ultrasonically activated device (USAD) using an animal model. In an anesthetized living pig, the gastroepiploic and mesenteric vessels were coagulated and cut by an USAD at a power level of 70% (n= 8) or 100% (n= 8). During the division, the time-discrete temperature change on the surface of the animal tissue adjacent to the blade was measured by thermography. To compare the USAD with conventional electrocautery (EC), a full-thickness incision of the gastric wall was performed by each device, and the temperature change was measured. With the USAD, the temperature increased gradually and remained below 150°C during the entire activating time at both power levels. By contrast, with EC at 30 W, the temperature increased rapidly and exceeded 350°C within only a few seconds. The area above 60°C reached a final width of 10 mm for the USAD, as compared with 22 mm for EC. Microscopically, thermal alterations such as carbonization and vaporization were much more severe and extensive in the adjacent tissue when using EC rather than the USAD. With the USAD, heat production is much slower and more limited than with conventional EC; thus, the USAD causes fewer thermal alterations in adjacent tissue. USAD should be preferred for tissue coagulation and cutting during endscopic surgery.

Potential Therapeutic Effect of Glycogen Synthase Kinase 3β Inhibition against Human Glioblastoma

Purpose: Glioblastoma represents the malignant brain tumor that is most refractory to treatment and in which the identification of molecular target(s) is urgently required. We investigated the expression, activity, and putative pathologic role of glycogen synthase kinase 3β (GSK3β), an emerging therapeutic target for neurodegenerative diseases, in human glioblastoma. Experimental Design: The active fraction of GSK3β that is phosphorylated at the tyrosine 216 residue (pGSK3βY216) was identified in glioblastoma cell lines. GSK3β activity for phosphorylating its substrate was detected in these cells by nonradioisotopic in vitro kinase assay. Results: Higher expression levels of GSK3β and pGSK3βY216 were frequently detected in glioblastomas compared with nonneoplastic brain tissues. Inhibition of GSK3β activity by escalating doses of a small-molecule inhibitor (AR-A014418) or inhibition of its expression by RNA interference induced the apoptosis and attenuated the survival and proliferation of glioblastoma cells in vitro. Inhibition of GSK3β was associated with increased expression of p53 and p21 in glioblastoma cells with wild-type p53 and with decreased Rb phosphorylation and expression of cyclin-dependent kinase 6 in all glioblastoma cell lines. Administration of AR-A014418 at a low dose significantly sensitized glioblastoma cells to temozolomide and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea, chemotherapeutic agents used in the clinical setting, as well as to ionizing radiation. Conclusion: These results indicate that GSK3β exerts a pathologic role by promoting the survival and proliferation of glioblastoma cells and by protecting them from apoptosis via the inactivation of p53- and/or Rb-mediated pathways. Consequently, we propose that GSK3β provides a potential therapeutic target in glioblastoma.

Small amount of low-residue diet with parenteral nutrition can prevent decreases in intestinal mucosal integrity

OBJECTIVE To investigate the suitable combination ratio of low-residue diet (LRD) and parenteral nutrition (PN) for nutritional support of surgical patients. SUMMARY BACKGROUND DATA Bacterial translocation (BT) is a severe complication of total parenteral nutrition (TPN). However, it is sometimes impossible to supply sufficient amounts of nutrients to surgical patients by the enteral route. The authors reported previously that concomitant use of LRD with PN provided preferable nutritional support for patients undergoing surgery for colorectal cancer. METHODS Ninety male Donryu rats were used for three experiments. In experiment 1, rats were divided into two groups to receive TPN or total enteral nutrition with LRD. In experiment 2, rats were divided into six groups, receiving variable amounts of LRD. In experiment 3, rats were divided into five groups to receive isocaloric nutritional support with variable proportions of PN and LRD. Intestinal permeability was assessed by monitoring urinary excretion of phenolsulfonphthalein. BT was assessed in tissue cultures of mesenteric lymph nodes and spleen. RESULTS In experiment 1, increases in intestinal permeability and BT were observed in rats maintained on 7-day TPN, but not in those maintained on total enteral nutrition for up to 14 days. In experiment 2, the changes in body weight of rats were correlated with the dose of LRD. However, the intestinal permeability was increased only in rats receiving LRD at 15 kcal/kg per day. In experiment 3, additive LRD corresponding to 15% of total caloric intake prevented increases in intestinal permeability and BT. CONCLUSION Combined nutritional therapy consisting of PN and small amounts of LRD can provide better nutritional support than TPN for surgical patients.

Predicting clinical outcome of 5-fluorouracil-based chemotherapy for colon cancer patients: is the CpG island methylator phenotype the 5-fluorouracilresponsive subgroup?

The CpG island methylator phenotype (CIMP+) of colorectal cancer (CRC) occurs predominantly in the proximal colon and is characterized by frequent hypermethylation of gene promoter regions. In this review, we present evidence suggesting CIMP+ represents the subgroup of colon cancers that are responsive to 5-fluorouracil (5-FU)-based treatments. CIMP+ has been associated with survival benefit from 5-FU in a clinical study of CRC, with additional evidence coming from studies on gastric cancer and tumor cell lines. Elevated concentrations of 5-10-methylene tetrahydrofolate (CH2FH4) occur in CIMP+ tumors and are probably due to low expression levels for γ-glutamyl hydrolase (GGH). Clinical and in vitro work has previously shown that high CH2FH4 and low GGH expression levels correlate with good response to 5-FU. Methylation-induced silencing of dihydropyrimidine dehydrogenase, the rate-limiting enzyme in 5-FU degradation, may also provide a link between CIMP+ and good response to 5-FU. The CIMP+-related phenotype referred to as microsatellite instability (MSI+) has been widely investigated as a predictive marker of response to 5-FU, with contradictory results. The interpretation of these studies is likely to be confounded by the fact that some MSI+ tumors occur in the background of CIMP+, but a significant proportion of others do not. Further studies on tumors from randomized clinical trials are required to confirm the value of CIMP+ and associated molecular features for the prediction of clinical outcome to 5-FU-based chemotherapy.

Prognostic Role of Thymidylate Synthase Polymorphisms in Gastric Cancer Patients Treated with Surgery and Adjuvant Chemotherapy

Purpose: To investigate the prognostic role of thymidylate synthase (TS) polymorphisms in gastric cancer patients treated with radical surgery and fluorouracil-based adjuvant chemotherapy. Experimental Design: Ninety gastric cancer cases were identified among 187 patients previously enrolled in prospective case-control studies for disease susceptibility. Patients were genotyped for a G/C nucleotide change within a triple 28 bp variable number of tandem repeat sequence in the TS 5′-untranslated region (5′-UTR) and a 6 bp deletion in the TS 3′-untranslated region (3′-UTR). According to available functional data, patients with 5′-UTR 2R/2R, 2R/3C, 3C/3C genotypes were classified as low TS producers (5′-UTRlow) and patients with 5′-UTR 3G/3G, 3G/3C, 2R/3G genotypes as high TS producers (5′UTRhigh). Patients with 3′-UTR del6/del6 and del6/ins6 genotypes were classified as low TS producers (3′-UTRlow) and patients with 3′-UTR ins6/ins6 genotype as high TS producers (3′-UTRhigh). The prognostic analysis was based on 5′-UTR/3′-UTR combined genotypes. Results: Ten patients (11%) were 5′-UTRhigh/3′-UTRhigh, 36 patients were 5′-UTRhigh/3′-UTRlow, 19 patients were 5′-UTRlow/3′-UTRhigh, and 25 patients were 5′-UTRlow/3′-UTRlow. 5′-UTRlow/3′-UTRlow patients showed the best outcome and the threshold of statistical significance was achieved in the comparison of disease-free survival and overall survival with 5′-UTRhigh/3′-UTRlow patients and 5′-UTRhigh/3′-UTRhigh patients. The presence of at least one high TS expression genotype showed independent adverse prognostic role in multivariate analysis. Conclusions: The prognostic role of TS polymorphisms in gastric cancer deserves further investigation because the adverse effect of high TS expression genotypes may be a relevant information to improve adjuvant chemotherapeutic strategies.