Tsunekazu Hishima

Higher frequency of smad4 gene mutation in human colorectal cancer with distant metastasis

We have previously detected an increased frequency of loss of heterozygosity (LOH) on chromosome 18q during progression of colorectal carcinomas. To clarify the target of 18qLOH, mutation of Smad4 and Smad2 genes was analysed in 176 colorectal tumors with different stages, including liver metastasis, from 111 sporadic, 52 familial adenomatous polyposis (FAP) and nine hereditary nonpolyposis colorectal cancer (HNPCC) patients. Mutation of other Smad gene families in the TGF-β signaling pathway was also examined. Twenty-one Smad4 mutations and one Smad2 mutation were detected, whereas mutation of Smad3, 6 and 7 genes was not detected. Smad4 mutations included seven frameshift, one inframe deletion, four nonsense and nine missense mutations, 95% of which resulted in alteration of Smad4 protein regions included in homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44 (7%) in primary invasive carcinoma without distant metastasis, 6/17 (35%) in primary invasive carcinoma with distant metastasis, and 11/36 (31%) in distant metastasis (metastatic/non-metastatic: P=0.006 ∼0.01). Loss of the other allele was observed in 19 of 20 (95%) invasive and metastasized carcinomas with Smad4 mutations. In four cases both primary and metastasized carcinomas in the same patients showed the same mutations. The present results suggest that Smad4 gene is one of true targets of 18qLOH, and that its inactivation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis.

Pathogenesis of Hepatitis C Virus Infection in Tupaia belangeri

ABSTRACT The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.

Tumor cell-derived angiopoietin-like protein ANGPTL2 is a critical driver of metastasis

Strategies to inhibit metastasis have been mainly unsuccessful in part due to insufficient mechanistic understanding. Here, we report evidence of critical role for the angiopoietin-like protein 2 (ANGPTL2) in metastatic progression. In mice, Angptl2 has been implicated in inflammatory carcinogenesis but it has not been studied in human tumors. In patients with lung cancer, elevated levels of ANGPTL2 expression in tumor cells within the primary tumor were associated with a reduction in the period of disease-free survival after surgical resection. Transcription factors NFATc, ATF2, and c-Jun upregulated in aggressive tumor cells promoted increased Angptl2 expression. Most notably, tumor cell-derived ANGPTL2 increased in vitro motility and invasion in an autocrine/paracrine manner, conferring an aggressive metastatic tumor phenotype. In xenograft mouse models, tumor cell-derived ANGPTL2 accelerated metastasis and shortened survival whereas attenuating ANGPTL2 expression in tumor cells-blunted metastasis and extended survival. Overall, our findings showed that tumor cell-derived ANGPTL2 drives metastasis and provided an initial proof of concept for blockade of its action as a strategy to antagonize the metastatic process.

CD70 expression in thymic carcinoma.

CD70, a type II transmembrane glycoprotein, is a member of the tumor necrosis factor (TNF) family that mediates the interaction between B- and T-lymphocytes. CD70 has been shown to be expressed by malignant lymphoma, especially Hodgkins disease, and by nasopharyngeal carcinoma, both of which are frequently associated with Epstein-Barr virus (EBV). In this study, we investigated the expression of CD70 in epithelial cells of various types of thymic epithelial tumors and its association with EBV. Immunohistochemical expression of CD70 was studied on frozen tissue. In a series of 27 thymic epithelial tumors, including thymic carcinomas (n = 8), atypical thymomas (n = 5), thymomas (n = 13), and thymic carcinoid (n = 1), 7 (88%) thymic carcinomas and 1 (20%) atypical thymoma showed positive immunoreactivity for CD70, whereas CD70 was not detected in other tumors. Twenty-four intrathoracic malignant epithelial tumors of nonthymic origin, including lung (n = 17), esophagus (n = 5), and mesothelium (n = 2), showed no immunoreactivity for CD70. Northern blot analysis also revealed that CD70 messenger RNA was expressed in 2 of 2 thymic carcinomas, 0 of 2 atypical thymomas. and 0 of 2 thymomas. All of the 27 thymic epithelial tumors were EBV-negative as assessed by EBV-encoded small RNA in situ hybridization. The expression of CD70 is closely related to the pathogenesis of thymic carcinoma but unrelated to EBV infection in the thymus.

Neuroendocrine differentiation in thymic epithelial tumors with special reference to thymic carcinoma and atypical thymoma

To determine the neuroendocrine (NE) features of thymic epithelial tumor, immunohistochemistry and electron microscopy studies were performed on eight NE tumors (thymic carcinoids) and 26 non-NE tumors (nine thymic carcinomas, five atypical thymomas, and 12 thymomas other than lymphocytic thymoma). Immunohistochemical studies were performed with antibodies against general markers for NE cells (synaptophysin, alpha subunit of a guanine nucleotide-binding protein, Go, and small-cell lung carcinoma cluster 1 antigen), and a broad panel of antibodies for hormonal substances. Thymic carcinoid showed synchronous diffuse immunoreactivity for the three NE markers and contained cells that were positive for a variety of hormonal products: human chorionic gonadotropin (hCG) alpha-subunit (eight of eight), hCG beta-subunit (three of eight), adrenocorticotropic hormone (ACTH) (three of eight), calcitonin (two of eight), calcitonin gene-related peptide (two of eight), and serotonin (one of eight). Conversely, although positivity for NE markers was neither synchronous nor diffuse in non-NE tumors, seven of nine thymic carcinomas, three of five atypical thymomas (focal or dispersed distribution), and none of the five thymomas were positive for at least two of these NE markers. A small number of neoplastic cells were positive for hCGalpha-subunit or ACTH in three thymic carcinomas and one atypical thymoma. Ultrastructurally, dense core granules (DCG) were much more frequent in thymic carcinoid, but several DCG-like granules were identified in 12 of 13 non-NE tumors with or without immunoexpression of NE markers. The presence of focal or dispersed NE cells in thymic carcinoma and atypical thymoma may reflect multidirectional differentiation within the tumor, which, like cytological atypia, epithelial CD5 expression, and lack of immature T cell infiltration, may be another feature of this group at thymic tumors.

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes

BACKGROUND & AIMS We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. METHODS We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. RESULTS Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. CONCLUSIONS DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target.

MEN1 gene mutations in sporadic neuroendocrine tumors of foregut derivation

Foregut‐derived neuroendocrine (NE) tumors occur sporadically or in association with multiple endocrine neoplasia type 1 (MEN1) syndrome. Thirty‐nine sporadic NE tumors of foregut derivation (six thymic, 21 bronchial, three gastric, and nine pancreatic tumors) as well as two hindgut‐derived rectal carcinoids for somatic MEN1 gene mutation were analyzed by direct sequencing analysis. Five tumors showed mutations: nonsense mutations (Q393X and R98X) in thymic and pancreatic NE tumors, respectively, a 4 b.p. deletion (357del4) in a gastric NE carcinoma, and missense mutations (D172Y and S178Y) in pancreatic NE tumors. No mutation was identified in pulmonary or rectal NE tumors. In a patient with a pancreatic NE tumor (D172Y), the corresponding germline DNA showed the same mutation, suggesting that sporadic MEN1 syndrome was masked in this case. Somatic MEN1 gene mutations and deletions may play a crucial role in the tumorigenesis of a subset of foregut‐derived NE tumors. Sporadic MEN1 syndrome may occur as a sporadic NE tumor of the pancreas.

Accumulation profile of frameshift mutations during development and progression of colorectal cancer from patients with hereditary nonpolyposis colorectal cancer

PurposeRole and timing of frameshift mutations during carcinogenesis in hereditary nonpolyposis colorectal cancer have not been examined. This study was designed to clarify the relationship between frameshift mutations and clinicopathologic features in colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.MethodsThirty-one colorectal cancers from patients with hereditary nonpolyposis colorectal cancer at different clinicopathologic stages were analyzed for frameshift mutation in 18 genes.ResultsThe frameshift mutations of the ACVR2 and PTHLH genes were found to have an extremely high frequency (94–100 percent) in all pathologic stages, and mutation of the MARCKS gene also was high (94 percent) in Dukes B and C cancers. These frequencies were higher than the frequency of TGFβRII gene inactivation (64–88 percent). Mutations of the hMSH3, TCF4, CASP5, RIZ, RAD50, and MBD4 genes were comparatively frequent (>35 percent) in all stages. Frequencies of inactivation of the MARCKS, BAX, IGFIIR, and PTEN genes were significantly higher in Dukes B and C cancers than in Dukes A cancer (P < 0.05). The number of accumulated frameshift mutations was larger in Dukes B and C cancers (9.4) than in Dukes A cancer (6.8) (P = 0.003).ConclusionsThe present data suggest that the disruption of the transforming growth factor-β super-family signaling pathway by the alteration of the ACVR2 and/or TGFβRII genes and the disruption of antiproliferative function by the PTHLH gene alteration contribute to the development of early colorectal cancer. Moreover, the further accumulation of alterations in the MARCKS, BAX, IGFIIR, and PTEN genes seem to be associated with progression from early to advanced colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.

Superficial basaloid squamous carcinoma of the esophagus. A clinicopathological and immunohistochemical study of 12 cases.

Basaloid squamous carcinoma (BSC) is a rare variant of squamous cell carcinoma (SCC). In this study, clinicopathological and immunohistochemical characteristics of 12 superficial esophageal BSCs were examined and compared with those of typical superficial SCCs. Eight cases were classified into an elevated type, and the other four into a depressed type. High-grade intraepithelial neoplasia was not observed around the invasive lesions in five cases, and only BSC components were apparent. High-grade intraepithelial neoplasia was demonstrated in seven cases, five of which had both BSC and SCC components in the invasive lesion. A cribriform growth pattern, comedo-type necrosis, and hyaline deposits were conspicuous histological findings. CK14 was positively stained in 90% of the series, but the proportion of positive cells was small in most cases. Type IV collagen was increased or well preserved in the basement membrane in 70% of cases, but heparan sulfate was decreased in the majority. In comparison with SCCs, lymphatic permeation was observed less frequently. However, regarding the frequencies of venous permeation, nodal metastasis, p53 protein expression, and Ki-67 labeling index, no significant differences were noted. Thus, esophageal BSCs demonstrate the pathological features characteristic of an early stage, but pathological parameters related to biological behavior do not significantly vary from those typical of SCCs.

Clinical outcomes with chemotherapy for advanced thymic carcinoma

BACKGROUND The clinical characteristics and prognostic factors of thymic carcinoma have not been investigated in detail because of its rarity. The aim of this study was to elucidate the disease profile, outcomes, and prognostic factors for survival among patients with advanced thymic carcinoma treated with palliative-intent chemotherapy. PATIENTS AND METHODS A retrospective review was conducted of the medical records of 40 patients treated with palliative-intent chemotherapy for advanced thymic carcinoma between 1991 and 2011 in our institution. Clinical demographics, histology, overall survival, and factors expected to predict survival were analyzed. Differences in survival were assessed using Kaplan-Meier analysis and univariate and multivariate Cox proportional hazards regression analyses. RESULTS The study included 22 males (55.0%) and 18 females (45.0%). The median age at diagnosis was 58.5 years. The most common metastatic sites at diagnosis were lung (45.0%), lymph nodes (20.0%), liver (15.0%), bone (15.0%), and brain (5.0%). The most common histological subtypes were squamous cell carcinoma (70.0%), followed by neuroendocrine carcinoma (17.5%), and mucoepidermoid carcinoma (7.5%). The response rate for first-line chemotherapy was 47.5%. The median survival time was 24.5 months (95% confidence interval 20.9-43.5 months). Overall survival rates at 1-, 2-, and 5-years were 72.5%, 52.5%, and 17.5%, respectively. In uni- and multivariate analyses, the only favorable prognostic factor for overall survival was response to first-line chemotherapy (p=0.01). CONCLUSION Response to first-line chemotherapy may be implicated as a potential surrogate for survival in advanced thymic carcinoma.