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Hemifacial spasm (HFS) is caused by pulsative vascular compression of the root exit zone (REZ) of the facial nerve. However, the mechanism that causes the offending vessels to compress the REZ has not been clarified. Elongation of intracranial arteries due to arteriosclerosis is one possibility, but such arteriosclerotic changes are not observed very frequently among patients with HFS. The aim of the present study was to investigate whether arteriosclerotic changes would contribute to the pathogenesis of HFS. This study included 111 HFS patients, all of whom were Japanese. The prevalence rates of hypertension, hyperlipidemia, and diabetes mellitus were examined as risk factors of atherosclerosis, and the cardio-ankle vascular index (CAVI) was measured as an indicator of arteriosclerotic change. The severity of white matter lesions (WMLs) in HFS patients was measured by magnetic resonance imaging. These data were compared with data from healthy Japanese controls. The prevalence rates of the risk factors for atherosclerosis in the HFS patients were not higher than those in the general Japanese population. The CAVI scores for the HFS patients were similar to, or lower than those in the healthy controls for all age groups except 60 to 69-year-old men. The severity of WMLs in the HFS patients was not significantly worse than that in the controls. It is suggested that arteriosclerotic changes are not involved in the pathogenesis of HFS, and that vascular compression syndromes are attributable to anatomical features of the intracranial arteries and facial nerves formed during the prenatal stage.

RAS signaling is frequently deregulated in human neoplasms. However, RAS mutations have been found in only a small proportion of human gastric cancers, implicating other mechanisms in the activation of RAS signaling in gastric tumorigenesis. We have previously reported that decreased expression of RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, contributes to colon tumor progression. In our study, we explored the involvement of decreased RASAL1 expression in gastric tumorigenesis. RASAL1 expression was reduced in 6 of 10 gastric cancer cell lines examined by immunoblotting. Knockdown of RASAL1 increased mitogen-activated protein kinase signaling in response to growth factor stimulation, and the forced expression of RASAL1 reduced proliferation of gastric cancer cells. Immunohistochemical analyses in primary gastric tumors showed that RASAL1 expression was reduced in 23 of 48 (48%) of the gastric cancers but in none of the adenomas (0/10). Methylation of the RASAL1 promoter region and loss of heterozygosity (LOH) at the RASAL1 locus were examined to investigate the causes of RASAL1 silencing. All cell lines with reduced RASAL1 had RASAL1 methylation, and two had LOH. In primary gastric cancers, methylation or LOH was detected in 50% (6/12) of those with reduced RASAL1. Furthermore, RASAL1 expression was restored in some cell lines by histone deacetylase inhibitor treatment. Our findings demonstrate that reduced RASAL1 expression, partly due to genetic and epigenetic changes, contributes to gastric carcinogenesis, and also re-emphasize the importance of RAS signaling in gastric cancer development.

Cytochrome P450 2C19 (CYP2C19) is clinically important for the metabolism of many therapeutic drugs. CYP2C19 has two main point mutation sites leading to low metabolic capacity. Several CYP enzymes are also important for the metabolism of chemical carcinogens, and several studies have reported associations between CYP polymorphism and cancer susceptibility. Speculating on a potential association between CYP2C19 polymorphism and cancer susceptibility, we conducted this study in two phases. Cell lines of various gastroenterological cancers were screened in the first phase. A clinical investigation was then conducted to confirm the association with the candidate cancer in the second phase. Genetic polymorphism of CYP2C19 was investigated in a total of 114 cell lines of five gastroenterological cancers. Based on this screening investigation suggesting an association with biliary tract cancer, we conducted a related study by recruiting 65 patients with biliary tract cancer and 566 patients with benign diseases as controls. Among the 114 cell lines investigated, biliary tract cancer was suggested to be most strongly associated with poor metabolizers of CYP2C19. Among 65 patients with biliary tract cancer, 18 (28%) were poor metabolizers of CYP2C19, whereas 87 (15%) of 566 control patients were poor metabolizers. The age- and gender-adjusted odds ratios for intermediate and poor metabolizers regarding the risk of biliary tract cancer were 1.5 (95% CI: 0.8–3.0, P = 0.17) and 2.7 (1.3–5.9, P = 0.006) compared to extensive metabolizers. A genetic polymorphism of CYP2C19 is associated with susceptibility to biliary tract cancer.

Recent advances in colonoscopic techniques have resulted in more frequent detection of superficial-type colorectal tumors, that is, laterally spreading tumors (LSTs), although little is known about the characteristic clinical features and genetic alterations of LSTs. To elucidate the molecular characteristics of LSTs, genetic alterations in the KRAS, BRAF and PIK3CA genes and abnormal expression of the p53, beta-catenin and MYC proteins were analyzed using direct DNA sequencing and immunohistochemistry for 50 protruded-type tumors (Protruded), 35 granular-type LSTs (LST-G) and 19 nongranular-type LSTs (LST-NG). In addition, loss of heterozygosity (LOH) close to the adenomatous polyposis coli (APC) gene (5q21) was examined in these tumors. In univariate analyses, significant differences were noted in the percentages with KRAS mutations (Protruded, LST-G, LST-NG = 30.0%, 54.3%, 21.1%, respectively, p = 0.0156), nuclear accumulation of beta-catenin (Protruded, LST-G, LST-NG = 50.0%, 37.1%, 68.4%, respectively, p = 0.0267), expression of MYC (Protruded, LST-G, LST-NG = 26.0%, 17.1%, 42.1%, respectively, p = 0.0456) and LOH at the APC gene locus (Protruded, LST-G, LST-NG = 22.0%, 20.0%, 47.4%, respectively, p = 0.0302). Multivariate analysis demonstrated that the macroscopic subtype of LST was significantly associated with KRAS mutation (for LST-NG: odds ratio [OR] 0.23, 95% CI 0.06-0.90) and nuclear accumulation of beta-catenin (for LST-NG: OR 4.05, 95% CI 1.11-14.8). Our data revealed that the 2 subtypes of LST have different molecular characteristics, suggesting that 2 or more different molecular mechanisms result in colorectal tumors with a similar growth pattern.

The hedgehog and mitogen-activated protein kinase (MAPK) signaling pathways regulate growth in many tumors, suggesting cooperation between these two pathways in the regulation of cell proliferation. However, interactions between these pathways have not been extensively studied. We assessed cross-talk between hedgehog and MAPK signaling in the regulation of cell proliferation in gastric cancer. We showed that PTCH expression was significantly correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P = 0.016) as well as SHH expression (P = 0.034) in the 35 gastric cancers assessed by immunohistochemistry. Indeed, MAPK signaling increased the GLI transcriptional activity and induced the expression of hedgehog target genes in gastric cancer cells. The inductive effect of activated KRAS and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1 was blocked by the suppressor of fused (SUFU), indicating that MAPK signaling regulates GLI activity via a SUFU-independent process. Moreover, the deletion of the NH2-terminal domain of GLI1 gene resulted in reduced response to MEK1 stimulation. Our results suggest that the KRAS-MEK-ERK cascade has a positive regulatory role in GLI transcriptional activity in gastric cancer.

Background & Aims Although colorectal cancer (CRC) progression has been associated with alterations in KRAS and RAS signaling, not all CRC cells have KRAS gene mutations. RAS activity is modulated by RAS-GTPase-activating proteins (RASGAPs), so we investigated the role of RASGAPs in CRC progression. Methods The level of RASGAP expression in CRC cells was analyzed using quantitative real-time polymerase chain reaction. The expression of the RAS protein activator like-1 (RASAL1) was examined in clinical colorectal neoplasms using immunohistochemistry. The clinicopathologic (age, sex, and tumor site and grade) and molecular ( KRAS gene mutation, as well as CTNNB1 and TP53 expression patterns) factors that could affect RASAL1 expression were examined. Results Of 12 RASGAPs examined, expression levels of only RASAL1 decreased in CRC cells; RASAL1 expression decreased in most CRC cells with wild-type KRAS gene but rarely in those with mutant KRAS gene. A transfection assay showed that RASAL1 repressed RAS/mitogen-activated protein kinase signaling in response to growth factor stimulation and reduced proliferation of CRC cells that contained wild-type KRAS gene. RASAL1 expression was detected in 46.9% (30/64) of adenocarcinoma, 17.4% (8/46) of large adenoma, and no (0/42) small adenoma samples. RASAL1 expression levels were correlated with the presence of wild-type KRAS gene in CRC tumor samples ( P = .0010), distal location ( P = .0066), and abnormal expression of TP53 ( P = .0208). Conclusions RASAL1 expression is reduced in CRC cells that contain wild-type KRAS gene. Reductions in RASAL1 expression were detected more frequently in advanced lesions than in small adenomas, suggesting that RASAL1 functions in the progression of benign colonic neoplasms.

Loss of genomic imprinting is an epigenetic alteration of some cancers involving the absence of parental origin-specific expression of imprinted genes. Loss of genomic imprinting of insulin-like growth factor II is often detected in colorectal cancer. However, the genetic alterations accompanied by colorectal cancer with insulin-like growth factor II loss of genomic imprinting have not been fully determined. Genomic DNA samples were collected from 52 colorectal cancer tissues and analyzed. The loss of insulin-like growth factor II genomic imprinting status was determined by assessing the demethylation of the insulin-like growth factor II differentially methylated region using bisulfite sequencing. The molecular signatures were also examined: genetic mutations of KRAS, BRAF, and PIK3CA; the expression of CTNNB1 and TP53; and microsatellite instability status. Several cases of colorectal cancer with normal insulin-like growth factor II imprinting were located in the distal colon; in contrast, colorectal cancer with loss of genomic imprinting tended to be found in the proximal colon (22.7 versus 56.6%). The PIK3CA gene mutation was highly detected in normal imprinting tumors compared to colorectal cancers with insulin-like growth factor II loss of genomic imprinting (27.3% versus 6.7%). In multivariate analysis of these clinicopathologic and molecular factors of tumors, statistically significant relationships were observed among the proximal location of the tumor (odds ratio, 12.9; 95% confidence interval, 1.52-110.13), PIK3CA genetic mutation (odds ratio, 0.082; 95% confidence interval, 0.01-0.73), and insulin-like growth factor II genomic imprinting status. Our findings indicate that colorectal cancers with demethylation of the insulin-like growth factor II gene are distinct from normal imprinting tumors, both in clinical and genetic features.

Objective: To chart molecular genetic events in pancreatic cancer. Methods: We analyzed genome-wide copy number alterations and loss of heterozygosity (LOH) in 25 established pancreatic cancer cell lines using a high-density single nucleotide polymorphism (SNP) array. We verified the data using genomic PCR and applied them to clinical samples. Results: Twenty-six homozygous deletion regions were detected in at least 1 cell line and LOH was found at 9p, 18q, 17p, 8p, 13q, 6q, 3p, 6p, 22q, 9q and 12q with high frequency (>50%), consistent with a previous study. Moreover, we found 23 amplified regions in at least 2 cell lines, including 8 unreported loci. We then examined representative genes at the 8 amplified loci in matched pairs of pancreatic cancer and normal tissues. The amplification was detected in 1 (7.1%) to 5 (35.7%) of 14 microdissected tissue specimens. Conclusion: Using high-resolution SNP arrays, we studied genome-wide copy number alterations and LOH simultaneously. We identified several novel and minute genomic amplifications, which contained candidate oncogenes in human pancreatic cancers.

BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.

Purpose: It is important to identify the definitive molecular switches involved in the malignant transformation of premalignant tissues. Cellular senescence is a specific characteristic of precancerous tissues, but not of cancers, which might reflect tumorigenesis-protecting mechanisms in premalignant lesions. Polycomb protein Bmi1, which is a potent negative regulator of the p16INK4 gene, suppresses senescence in primary cells and is overexpressed in various cancers. We hypothesized that Bmi1 expression would also be dysregulated in precancerous lesions in human digestive precancerous tissues. Experimental Design: Bmi1 expression was investigated in cancerous and precancerous tissues of the digestive tract. The expression of p16, β-catenin, and Gli1 and the in vivo methylation status of the p16 gene were also analyzed in serial sections of colonic precancerous lesions. Results: Bmi1 was clearly overexpressed across a broad spectrum of gastrointestinal cancers, and the expression of Bmi1 increased in a manner that reflected the pathologic malignant features of precancerous colonic tissues (low-grade dysplasia, 12.9 ± 2.0%; high-grade dysplasia, 82.9 ± 1.6%; cancer, 87.5 ± 2.4%). p16 was also strongly expressed in high-grade dysplasia, but not in cancers. p16 promoter methylation was detected only in some Bmi1-positive neoplastic cells. Conclusions: Bmi1 overexpression was correlated with the malignant grades of human digestive precancerous tissues, which suggests that advanced Bmi1 dysregulation might predict malignant progression. The abnormal Bmi1 expression might link to malignant transformation via the disturbance of orderly histone modification.

Hemorrhage is among the most serious complications of colorectal polypectomy and may occur after a longer postprocedure interval.We aimed to elucidate the risk factors for delayed postpolypectomy hemorrhage, including both polyp characteristics and the general condition of the patients.Retrospective cohort study.A total of 6617 cases of colorectal polypectomy was performed in 3138 consecutive patients in Japan.The risk factors for delayed postpolypectomy hemorrhage were assessed among polyp characteristics (form, size, histologic features) and the method of resection by unconditional logistic regression. Patient conditions (smoking, alcohol, hypertension, diabetes mellitus, hyperlipidemia) were compared between case-control pairs matched on polyp-related characteristics by conditional logistic regression.Hemorrhage occurred in 38 lesions (0.57%) of 37 patients (1.2%): 22 required endoscopic hemostasis and 1 required blood transfusion. Although polyp size was associated with the occurrence of delayed hemorrhage (10.0 +/- 6.9 mm in hemorrhage cases vs 5.6 +/- 3.8 mm in others, P.0001), other polyp-related factors were not significant. Hypertension was a complication in 25 of 37 (68%) cases and in 21 of 74 (28%) matched controls, showing an adjusted odds ratio of 5.6 (95% CI 1.8-17.2, P = .001). Other patient characteristics were not significant. The interval between polypectomy and hemorrhage was significantly longer in patients with hypertension (median 6 days, range 2-14 days) than in those without hypertension (2.5 days, 1-9 days; P = .019).This study does not provide information regarding prevention of hemorrhage.Hypertension is a significant risk factor for delayed colorectal postpolypectomy hemorrhage. The interval between polypectomy and hemorrhage can be as long as 14 days in the presence of hypertension.

The activation of Hedgehog (Hh) signaling has been implicated in the growth of various tumor types, including gastric carcinoma. However, the precise mechanisms of Hh activation and suppression of tumor growth by the blockade of Hh signaling in gastric carcinoma cells remain unknown. The aim of this study was to elucidate the mechanism of abnormal Hh signaling and the key molecules contributing to dysregulated growth of gastric carcinoma. The Sonic hedgehog (Shh) ligand and its receptor Patched were expressed in all five gastric carcinoma cell lines examined (MKN1, MKN7, MKN45, MKN74, and AGS cells). The blockade of Hh signaling with anti-Shh antibody inhibited the growth of all five gastric carcinoma cell lines. Shh was overexpressed (mean, 12.8-fold) in 8 of 14 (57.0%) cancerous tissue samples from patients with gastric carcinoma as compared with expression in the surrounding noncancerous tissues. The disruption of glioma-associated oncogene 1 (Gli1) by small interfering RNA induced an increase in p21/cyclin-dependent kinase–interacting protein 1 (CIP1), interfered with the G1-S transition, and suppressed cell proliferation. The stimulation or inhibition of Hh signaling did not affect p53 activity and the induction of p21/CIP1 expression and the G1 arrest by inhibition of Hh signaling were not affected by the p53 status. These findings suggest that the overexpression of Shh contributes to constitutive Hh activation and that this signaling pathway negatively regulates p21/CIP1 through a Gli1-dependent and p53-independent mechanism in gastric carcinoma cells.

Mutations in the B-raf gene have been reported in a number of human cancers, including melanoma and lung cancer. More than 80% of the reported B-raf mutations were V599E; however, non-V599E mutations have been frequently found in non-small cell lung cancers as compared with melanoma. Some non-V599E mutations have been found surrounding Thr439, which is thought likely to be one of the three Akt phosphorylation sites in the B-raf protein. However, as a previous report indicated that Thr439 was not phosphorylated by Akt, the functional consequences of these mutations have been unclear. Here, we examined the effects of cancer-related B-raf mutations surrounding Thr439 on the activation of the mitogen-activated protein/ extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (Erk) pathway and the transformation of NIH 3T3 fibroblasts. Among the three reported mutations (K438Q, K438T, and T439P) found in non-small cell lung carcinoma and melanoma, none elevated the activity of the MEK/Erk cascade as determined by in vitro kinase assays, immunoblots using antibody specific for phosphorylated Erk, or Elk1-dependent reporter assays. The inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling by LY294002 increased the Erk activation induced by the mutant B-raf proteins, as well as by wild-type B-raf. Furthermore, the B-raf mutants did not have increased NIH 3T3-transforming activities, as determined by colony-formation assays. These results suggest that the B-raf mutations surrounding Thr439 found in human cancers are unlikely to contribute to increased oncogenic properties of B-raf.

The BRAF gene is mutated in 66% of melanomas and less frequently in various human cancers. More than 80% of these mutations are T to A transversions at nucleotide 1796 (T1796A), leading to a substitution of glutamic acid for valine at amino acid 599 (V599E). We established a new method for rapidly detecting V599E mutations using real-time polymerase chain reaction and melting curve analysis. Furthermore, we examined mutations in gastrointestinal cancer cell lines using this method. We found a mutation in 1 of 12 (8%) colorectal cancer cell lines, but no mutation was detected in 9 gastric cancer cell lines. These results suggest that the BRAF mutation is unlikely to be involved in gastric carcinogenesis.

Tyrosine kinases, which are important regulators of intracellular signal-transduction pathways, have mutated forms that are often associated with oncogenesis and are attractive targets for therapeutic intervention. Recently, systematic mutational analyses of tyrosine kinases revealed that a minimum of 30% of colorectal cancer contain at least one mutation in the tyrosine kinases. To further explore these mutations, we examined all reported mutations of NTRK3, FES, KDR, EPHA3, NTRK2, JAK1, PDGFRA, EPHA7, EPHA8, ERBB4, FGFR1, MLK4 and GUCY2F genes in the 24 colorectal cancer cell lines. Unexpectedly, among 24 colorectal cancer cell lines, only two cell lines (LoVo and CaR1) harbored mutation C1408T (R470C) in MLK4 gene. The mutation rate was extremely low compared to that previously reported. Therefore, we analyzed mutations in 46 colorectal cancer samples resected from the same number of Japanese patients. Surprisingly, none of the 46 samples contained any of the mutations reported. Based on our study, we advise that a more comprehensive tyrosine kinase gene mutation assay is necessary in the future.

A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.

Microvascular decompression (MVD) has become a well-established surgical procedure for hemifacial spasm (HFS). Before surgery, it is essential to evaluate any possible deformity of the brainstem and establish the precise location of the offending vessels. In the present study of HFS patients we examined coronal sections taken by heavily T2-weighted MR cisternography in addition to routine axial sections, and assessed the usefulness of these images through comparison with intraoperative findings. Eighty patients with HFS underwent preoperative coronal heavily T2-weighted MR cisternography before microvascular decompression surgery. Three neurosurgeons examined the preoperative axial and coronal MR images and evaluated vessel invagination into the brainstem. The usefulness of coronal sections was assessed statistically by the Mann-Whitney U test. Invagination of the offending vessel into the brainstem was observed in 24 cases (30.0%). In 19 patients, it was predicted preoperatively that compression of the flocculus and brainstem would be required in order to approach the offending vessels. Coronal MR cisternography was significantly more useful in cases with vessel invagination into the brainstem than in cases without invagination. Coronal sections obtained by MR cisternography are able to demonstrate the severity of vessel invagination into the brainstem as well as revealing the presence of the offending vessel. This information is helpful for planning a suitable approach to the root exit zone.

Patients with hepatocellular carcinoma (HCC) sometimes suffer from obscure gastrointestinal bleeding. Portal hypertension (PH), common in cirrhosis, induces esophagogastric varices. Because of the location, PH also may influence mucosal abnormalities in the small intestine. The objective of this study is to estimate the prevalence of small intestinal mucosal abnormalities in HCC patients using capsule endoscopy (CE).We prospectively conducted CE in HCC patients, and analyzed the findings in relation to hepatic function, the number and size of HCC tumor and findings obtained by conventional endoscopy.Thirty-six patients (aged 66.7 ± 7.5 years, 29 men) underwent CE. Abnormal findings in the small bowel were found in 16 patients (44%), angioectasias in eight patients (22%), erosions in five (14%), varices in four (11%), polyps in four (11%), and submucosal tumor in one (3%). The patients with angioectasia had a larger spleen index than the no abnormal lesions group (85.4 ± 15.8 vs 59.0 ± 24.4, P = 0.02). The former group had been more frequently treated for esophageal varices endoscopically (62% vs 15%, P = 0.02). Large HCC nodules seemed more common in the patients with angioectasia than subjects without abnormal lesions (38% vs 5%, P = 0.06). Small intestinal varices also seemed to have a positive association with large HCC. During the follow up after CE, one patient with small intestinal polyps suffered from obscure gastrointestinal bleeding.CE revealed that HCC patients frequently have small intestinal mucosal lesions. In particular, small intestinal angioectasia, which may cause obscure gastrointestinal bleeding, seems to be associated with portal hypertension.

A 44-year-old man presented with a very rare case of skull base osteomyelitis manifesting as persistent diplopia. He initially had the symptom with fever after dental extraction. Biopsy from the cervix and upper pharynx performed in a previous hospital had showed negative findings by histological and bacterial examinations. Magnetic resonance (MR) imaging disclosed enhanced lesions in the right cavernous sinus, clivus, and right cervical regions. Computed tomography revealed osteolysis, and fluorodeoxyglucose positron emission tomography (FDG-PET) showed areas of increased uptake. Bacteriological examination of the isolated clival lesion disclosed Actinomyces israelii, and he was treated with intravenous penicillin, 18 million units a day for 6 weeks. MR imaging revealed reduction of intensity in the enhanced areas, and FDG-PET showed disappearance of the increased uptake. After 6 months of oral antibiotics administration, MR imaging disclosed disappearance of the enhanced lesions, and the patient had no sign of neurological deficits. Skull base osteomyelitis resembles neoplasm or inflammatory disease of this region in neurological and radiographic findings. The biopsy specimen should be ideally obtained from an isolated region, and prepared to identify a wide range of organisms and to differentiate other diseases. The serum level of C-reactive protein and FDG-PET are useful to follow up the efficacy of antibiotic therapy.

We sought to identify genomic changes that could be useful for clinical application, focusing on chromosomal instability and using a high-density single nucleotide polymorphism (SNP) array. We analyzed 34 gastric cancer cell lines for areas of DNA that exhibited copy number changes using the Affymetrix GeneChip Human Mapping 50 K Arrays. The results obtained with the cell lines were verified in 42 gastric cancer tissues using genomic PCR, quantitative real-time PCR, and loss of heterozygosity (LOH) analyses. Twenty-six local homozygous deletion regions, including 13 novel loci, and 31 recurrent high-grade gain regions, encompassing 14 novel loci, were found in the gastric cancer cell lines. Among the genes detected for high-grade gain in the cell lines, MYC, PAK1, and ITGB4BP showed copy number gain in more than 40% of gastric cancer tissues. LOH of AB051467, PTPRD, A2BP1, and C20orf133 was detected in more than 35% of gastric cancer tissues. The number of LOH was significantly greater in tumors with lymph node metastasis. In the early stage, the prognosis of patients with LOH of less than two genes was significantly better than that of those with LOH of two genes or more. Using high-density SNP arrays, we identified several novel and minute genomic alterations. LOH of four genes could be useful for prediction of lymph node metastasis and prognosis in early stage gastric cancers.

Purpose: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. Experimental Design: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription–PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. Results: HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. Conclusions: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants.TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method.The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results.We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.

Background/Aims The aim of this study is to identify genomic changes that might be implicated in hepatocellular carcinoma (HCC) progression, and evaluate the associations with clinico-pathological features. Methods The genomic DNA of 17 hepatoma cell lines was analyzed using Affymetrix GeneChip Human Mapping 50K high-density oligonucleotide arrays. We selected representative genes from recurrent amplified regions and measured the copy number of these genes in 70 HCC clinical samples. Results We found 10 recurrent high-grade gain regions spanning less than 3Mb in at least two hepatoma cell lines, and selected 10 representative genes. The copy number was almost normal in non-cancerous tissue and frequently amplified in Edmondson grade II or III HCC compared to Edmondson grade I HCC. Gain of TAX1BP1 in 7p15.2-1 was associated with larger tumor size and positivity of HCV antibody, and gain of CCND1 in 11q13.2-3 was associated with larger tumor size by multivariate analysis. Furthermore, a gain of GRHL2 in 8q22.3 was associated with early recurrence of HCC, controlling for clinical parameters. Decreased GRHL2 expression by RNA interference inhibits the growth of hepatoma cells, suggesting its association with cell proliferation. Conclusions A gain of GRHL2 might be a predictive marker for HCC recurrence.

Considering the increasing demand for colonoscopy, auxiliary devices that could facilitate the examination would be useful. A hood attached to the tip of the colonoscope has been reported to be helpful in detecting and removing colorectal polyps. However, its usefulness in aiding scope intubation has not been fully evaluated.Patients for colonoscopy between July 2004 and May 2005 in Tokyo University Hospital were enrolled to this randomized controlled trial, and assigned to colonoscopy with a transparent hood, a short hood, or no hood. Colonoscopies were conducted by trainees without sedation. The evaluated outcomes were cecal intubation rate, trainee intubation rate (cecal intubation within 15 min), cecal intubation time, and polyp detection rate.Enrolled 684 patients were randomly assigned to transparent hood (N = 221), short hood (N = 228), and no hood (N = 235) groups. The overall cecal intubation rate was 95.3% (652/684) and did not differ among the groups. The overall trainee intubation rate was 55.1% (377/684) and significantly higher in the transparent hood group than in the no hood group for female patients (60.7%vs 37.4%, P = 0.003). Cecal intubation time was 11.5, 13.5, and 14.0 min in the transparent, short, and no hood groups, respectively, and significantly shorter in the transparent hood group than in the no hood group among overall (P = 0.008), female (P = 0.001), and old (P = 0.04) patients. Polyp detection rate was higher in the transparent hood group than in the no hood group (49.3%vs 39.1%, P = 0.04).The transparent hood was useful in shortening the cecal intubation time especially in difficult cases.

Colonic diverticulosis, although usually asymptomatic, sometimes causes diverticular hemorrhage. Studies about risk factors, other than nonsteroidal anti-inflammatory drugs, for colonic diverticular hemorrhage are limited. We conducted the present study to elucidate their significance as a risk factor.Colonic diverticulosis was found in 1,753 patients and diverticular hemorrhage in 44 patients among 9,499 total colonoscopy examinees at the authors' institutions between September 1995 and December 2005. After reviewing their clinical features, we chose two controls for each case with diverticular hemorrhage matched for age, gender, and the location of diverticulosis. We evaluated the effects of comorbidities (hypertension, hyperlipidemia, diabetes mellitus, cerebrovascular disease, ischemic heart disease), habits (alcohol, smoking), and medications, including nonsteroidal anti-inflammatory drugs, by using conditional logistic regression analysis.There were no significant differences between patients with diverticular hemorrhage and those with nonbleeding diverticulosis regarding age (67 +/- 13 vs. 64 +/- 11 years) or gender ratio (male/female ratio: 36/8 vs. 1,237/472). As for location, the proportion of bilateral diverticulosis was larger among patients with hemorrhage (43 vs. 22 percent). In the case-control study, nonsteroidal anti-inflammatory drugs (odds ratio, 15.6; 95 percent confidence interval, 1.1-214; P = 0.04), hypertension (odds ratio, 6.6; 95 percent confidence interval, 2.1-20.5; P = 0.0011), and aspirin and/or other anticoagulant (odds ratio, 3; 95 percent confidence interval, 1.04-8.6; P = 0.042) were shown to be significant risk factors by multivariate analysis.Hypertension, nonsteroidal anti-inflammatory drugs, and anticoagulants, including aspirin, are independent risk factors for colonic diverticular hemorrhage.

Although cholesterol embolism syndrome was recognized as a clinicopathologic entity more than 50 years ago, it is attracting growing attention recently. It is a multisystemic disorder in which cholesterol crystals released from atherosclerotic plaques obstruct small arterioles, resulting in local ischemia and end-organ damage. There are no established treatments, and with the limited treatment options available, it is important to make the diagnosis as early as possible. We present the case of a 68-year-old man with cholesterol embolism who had a few fluttering atheromas in the aorta, as demonstrated by transesophageal ultrasonography. The diagnosis was confirmed by skin biopsy, and treatment with statins and steroids proved effective, as renal failure progressively improved. This case emphasizes the importance of early diagnosis and shows the possible therapeutic effects of statins and steroids for patients with this syndrome.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

Mutations in the PIK3CA gene, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been reported in human cancers, including colorectal cancer. Most of the mutations cluster at hotspots within the helical and kinase domains. Whereas H1047R, one of the hotspot mutants, is reported to have elevated lipid kinase activity, the functional consequences of other mutations have not been examined. In this study, we examined the effects of colon cancer–associated PIK3CA mutations on the lipid kinase activity in vitro, activation of the downstream targets Akt and p70S6K in vivo and NIH 3T3-transforming ability. Of eight mutations examined, all showed increased lipid kinase activity compared with wild-type p110α. All the mutants strongly activated Akt and p70S6K compared with wild-type p110α as determined by immunoblotting using phospho-specific antibodies. These mutants also induced morphologic changes, loss of contact inhibition, and anchorage-independent growth of NIH 3T3 cells. The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities. These results show that almost all the colon cancer–associated PIK3CA mutations are functionally active so that they are likely to be involved in carcinogenesis.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Recently, mutations in the B-Raf gene have been identified in a variety of human cancers, such as melanoma and colorectal carcinoma, and more than 80% of the B-Raf mutations have been V599E. Although other mutations have been reported, their functional consequences are poorly understood. In our earlier study, we demonstrated that colon tumor-associated B-Raf mutations within the kinase activation segment are not necessarily associated with an increase in mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/Erk) or nuclear factor kappaB (NFkappaB) signaling activity or in NIH3T3-transforming ability. In this study, we examined the effect of colon tumor-associated mutations within the B-Raf glycine-rich loop (G loop) on MEK/Erk and NFkappaB signaling and on the transformation of NIH3T3 fibroblasts or IEC-6 intestinal epithelial cells. Of the six G loop mutations examined, only the B-Raf G468A significantly increased MEK/Erk and NFkappaB signaling and NIH3T3 transformation. Only this mutation induced transformed phenotypes of IEC-6 cells. In contrast, the B-Raf G468E mutation significantly decreased MEK/Erk signaling and NIH3T3 transformation and had no effect on NFkappaB signaling. The B-Raf F467C mutation moderately elevated MEK/Erk signaling and NIH3T3 transformation. The other three B-Raf mutations, R461I, I462S, and G463E, did not increase MEK/Erk or NFkappaB signaling or NIH3T3 transformation. Except for F467C, none of the tumors with B-Raf mutations examined in this study had K-Ras mutations. These results suggest that some of the B-Raf G loop mutations reported in colorectal tumors do not increase kinase or transforming activities but might contribute to carcinogenesis via other mechanisms or be irrelevant to carcinogenesis.

Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor kappaB (NFkappaB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFkappaB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFkappaB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild-type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFkappaB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and transforming activities.

Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.