Keita Kai

Expression of hypoxic marker CA IX is regulated by site-specific DNA methylation and is associated with the histology of gastric cancer.

The hypoxic marker carbonic anhydrase (CA) IX has been recognized as a tumor-associated protein and is essential for cancer development. However, because CA IX expression does not always correlate with hypoxia, its regulatory mechanism remains unclear. The objective of the present study was to clarify the role and regulation of CA IX expression in gastric cancer. The immunohistochemical expression of CA IX and hypoxia-inducible factor-1α was assessed in 77 patients with gastric cancer. A methylation-sensitive restriction enzyme method was used to quantify site-specific methylation at -74 bp in the CA9 promoter in tissue from patients with gastric cancer and in corresponding normal tissue. CA9 expression in cell lines was strongly dependent on methylation status but not hypoxic stimuli. In tissue from patients with gastric cancer, the quantity of methylation was significantly correlated with the protein expression (P = 0.003). Moreover, the methylation value was significantly lower in intestinal-type compared with diffuse-type cancer (P = 0.003). Compared with normal mucosa, intestinal-type cancer demonstrated significant hypomethylation, whereas diffuse-type cancer exhibited hypermethylation. In conclusion, expression of CA IX in gastric cancer is predominantly regulated by methylation of a single CpG rather than by hypoxia. Furthermore, epigenetic alterations in CA9 differ between the intestinal and diffuse types of gastric cancer.

HIF‐1α is an unfavorable determinant of relapse in gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy

Among several chemotherapeutic agents, 5‐fluorouracil (5‐FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5‐FU in an adjuvant setting, has been identified. Hypoxia‐induced drug resistance, via upregulation of HIF‐1α, is a major obstacle in the development of effective cancer therapy. However, few clinical studies have so far assessed the relationship between the HIF‐1α expression and the chemo‐resistance of gastric cancer patients in an adjuvant setting. We established 2 HIF‐1α knockdown gastric cancer cell lines in order to clarify the role of HIF‐1α in chemo‐resistance against 5‐FU. Furthermore, expression of HIF‐1α was immunohistochemically assessed in 91 resected specimens. Sixty‐four of 91 patients received 5‐FU adjuvant chemotherapy after surgery. HIF‐1α expression was associated with the significantly shorter relapse‐free survival and disease‐specific survival in the 64 patients of adjuvant group (p = 0.026, 0.014, respectively), but not in the 27 of surgery group. Multivariate analysis showed that HIF‐1α was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). In conclusion, the current study confirmed, for the first time that HIF‐1α expression is an independent risk factor for relapse in high‐risk gastric cancer patients who underwent curative surgery followed by adjuvant 5‐FU chemotherapy. A favorable effect of 5‐FU might therefore be expected in patients that do not express HIF‐1α, whereas, other types of chemotherapy or additional treatments, such as HIF‐1α inhibitors, should be considered in patients that do express HIF‐1α.

HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer

The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1α in the development of peritoneal dissemination of gastric cancer. HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1α exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1α was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1α played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1α-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1α activates tumor angiogenesis.

Hepatoid carcinoma of the pancreas penetrating into the gastric cavity: A case report and literature review

A 79‐year‐old Japanese woman was admitted to our hospital for treatment of a pancreatic tumor measuring approximately 7 × 5 cm. The tumor had invaded the left adrenal gland and gastric wall and had penetrated into the gastric cavity. Surgical resection was performed. The tumor was composed of a brown to whitish solid area and a zone of hemorrhage, necrosis, and cystic degeneration resembling the gross features of solid pseudopapillary tumor (SPT). Histologically, the tumor showed a heterogeneous growth pattern with a combination of seat‐like, trabecular, papillary and hemorrhagic‐necrotic areas in various proportions. The differential diagnoses first considered were acinar cell carcinoma, neuroendocrine carcinoma and SPT with malignant transformation. Immunohistochemistry showed tumor cells were negative for pancreatic exocrine enzymes and endocrine markers. Tumor cells diffusely expressed cytokeratin 19, alpha‐fetoprotein, carcinoembryonic antigen and glypican‐3, but lacked vimentin or β‐catenin expression. Small proportions of tumor cells expressed hepatocyte paraffin‐1. Although typical morphological features of well‐differentiated hepatocellular carcinoma (HCC) were not distinctly apparent, the tumor morphology partly resembled poorly differentiated HCC. Given these findings and considerations, the tumor was finally diagnosed as poorly differentiated hepatoid carcinoma of the pancreas.

Clinicopathologic features of advanced gallbladder cancer associated with adenomyomatosis

Adenomyomatosis of the gallbladder has not been considered to have malignant potential, but gross features of adenomyomatosis are sometimes encountered in gallbladders resected under a diagnosis of gallbladder carcinoma. The purpose of this study was to determine the clinicopathologic features and survival rates in cases of gallbladder cancer with gross features of adenomyomatosis. The study subjects were 97 surgically treated gallbladder carcinoma patients. Only gallbladder showing typical gross features of adenomyomatosis was judged as adenomyomatosis-positive gallbladder cancer. In terms of gross findings, 25 cases (25.8%) were classified as adenomyomatosis-positive. The status of adenomyomatosis was significantly associated with the T stage (P = 0.0004), lymph node (LN) metastasis (P < 0.0001), distant metastasis (P = 0.008), and stage (P = 0.0005). In the adenomyomatosis-positive group, 16 of the 25 cases (64.0%) were classified as segmental type and 9 cases (36.0%) were classified as fundal type. No diffuse-type cases were present in this series. The status of adenomyomatosis correlated significantly with survival (P = 0.0007). However, the multivariate analysis of significant variables identified from the univariate analysis identified only T stage (P = 0.0178) and LN metastasis (P = 0.0048) as independent prognostic factors. Subset analysis with T stage according to the status of adenomyomatosis showed no significant impact on survival. These results indicate that adenomyomatosis-positive gallbladder cancer is more often diagnosed clinically in the advanced stages. Since preceding adenomyomatosis may prevent the early detection of gallbladder cancer, the usefulness of preventive cholecystectomy in cases of asymptomatic adenomyomatosis should be considered.

Loss of trefoil factor 1 is regulated by DNA methylation and is an independent predictive factor for poor survival in advanced gastric cancer

Trefoil factor 1 (TFF1) is considered to be a tumor suppressor gene in gastric cancer. However, the role of TFF1 expression and its regulation in gastric cancer patients remain unclear. The aims of this study were to clarify the clinical significance of TFF1 and to determine its regulatory mechanisms. We assessed the immunohistochemical expression of TFF1 in 182 gastric cancer patients and examined whether or not TFF1 is associated with the clinicopathological factors and patient survival. In vitro study using TFF1 knockdown gastric cancer cells evaluated the role of TFF1 in cancer invasion. Bisulfite sequencing was performed to assess DNA methylation of TFF1 in cells and resected tissues. Patients with low expression of TFF1 showed a significantly deeper invasion of the tumor than those with high expression (p=0.037). Low expression of TFF1 was also associated with a poor survival (p=0.029) in 108 patients who were treated by surgery alone. Both TFF1 expression and lymph node metastasis are independent predictive factors for disease-specific survival in a multivariate analysis. In an in vitro study, invasive power of the cells was significantly increased in the TFF1‑deficient cells compared with the control cells. Bisulfate sequencing showed that TFF1 expression is strongly dependent on DNA methylation in both gastric cancer cells and tissues. Interestingly, methylation status of two specific CpG sites, which are located close to a TATA box and hypoxia response element (HRE), determined the TFF1 expression in the resected tissues. TFF1 expression is silenced by DNA methylation and is associated with tumor invasion and a poor survival in gastric cancer patients. The expression and̸or methylation status of TFF1 may, therefore, serve as a useful biomarker for predicting survival in patients with advanced gastric cancer.

Fumagillin inhibits colorectal cancer growth and metastasis in mice: in vivo and in vitro study of anti-angiogenesis.

Fumagillin is an inhibitor of type 2 methionine aminopeptidase that can block blood vessel formation, but its molecular mechanism and therapeutic value in colon cancer still remain to be elucidated. In this study, male severe combined immunodeficiency (SCID) mice were injected with colon cancer cells in the subcutis and then treated with Fumagillin and Cyclo (Arg‐Gly‐Asp‐D‐Phe‐Val), an integrin αvβ3 antagonist. The tumor weight, microvessel density (MVD), and number of pulmonary metastatic foci were examined. Gene expression profiles were examined by microarray analysis of human umbilical endothelial cells (HUVEC). The Fumagillin‐treated mice had smaller tumor mass, fewer pulmonary metastases, and lower MVD‐CD105 levels than control animals. In vitro proliferation and tube formation of HUVEC was also significantly decreased by Fumagillin. Microarray analysis of Fumagillin‐treated HUVEC showed upregulation of 71 genes and downregulation of 143 genes. Expression changes were involved in cell proliferation, migration, adhesion, and gene transcription. Quantitative real‐time–polymerase chain reaction and western blotting showed decreased expression of cyclin E2, activated leukocyte cell adhesion molecule (ALCAM), and intercellular adhesion molecule‐1 (ICAM‐1) genes in the presence of Fumagillin. This downregulation by Fumagillin may be involved in the anti‐angiogenesis by Fumagillin. In conclusion, Fumagillin was found to suppress colorectal cancer growth and metastasis by suppressing angiogenesis.

Intrahepatic multicystic biliary hamartoma: report of a case.

Multicystic biliary hamartoma is a very rare hamartomatous nodule in the liver, which has recently been described as a new category of hepatic nodular lesion. We herein report the case of a 55‐year‐old man histopathologically diagnosed with this entity following surgery. A solitary multilocular lesion in the liver was pointed out by ultrasonography during a systemic examination for a positive HBs antigen. This nodule could not be definitively diagnosed by radiologic modalities, including computed tomography, magnetic resonance imaging and arteriography. The patient underwent a partial resection of the posterior segment of the liver. The nodule was a localized lesion which measured 5 × 3 cm at the widest point and displayed a honeycomb appearance. Histologically, it consisted of ductal structures, periductal glands, fibrous connective tissues containing blood vessels, and bile‐like materials and xanthogranulomatous inflammation within some ducts. Liver parenchyma was not present in the nodule and the bile ducts were not dilated in the background liver. The ductal epithelium expressed biliary type cytokeratins (CK7 and 19) in immunohistochemical studies. These histopathological features were consistent with multicystic biliary hamartoma, and we discuss this rare case in detail in this report.

Endoglin (CD105) expression and angiogenesis status in small cell lung cancer

It is well established that angiogenesis is crucial for tumor development and progression. Among the angiogenesis immunomarkers defined to date, endoglin (CD105) has been shown to be a useful marker of angiogenesis. To investigate the degree of angiogenesis status in small cell lung cancer (SCLC) tissue, we assessed 35 cases of SCLC at autopsy using immunohistochemical staining of CD31 and CD105. The intratumoral area, peritumoral area, and background pulmonary alveoli were then observed under low magnification, and the microvessel density (MVD) for each area was determined. The MVD-CD31 was the highest in the background alveoli, followed by the intratumoral and peritumoral areas. The MVD-CD105 was highest in the intratumoral area, followed by the peritumoral area and the background lung. The ratio of CD105/CD31 revealed that almost 78% of the intratumoral area, 63% of the peritumoral area, and 4.6% of the background lung alveoli were newly formed and expressed CD105. This result indicated that SCLC is predominantly supported by newly formed vessels that are generated by CD105-mediated angiogenesis. These findings suggest that anti-angiogenic therapy, especially CD105-targeting, may prove an effective form of SCLC treatment.

Gallbladder cancer: Clinical and pathological approach.

Gallbladder cancer (GBC) shows a marked geographical variation in its incidence. Middle-aged and elderly women are more commonly affected. Risk factors for its development include the presence of gallstones, chronic infection and pancreaticobiliary maljunction. Controversy remains in regard to the theory of carcinogenesis from adenomyomatosis, porcelain gallbladder and adenoma of the gallbladder. The surgical strategy and prognosis after surgery for GBC differ strikingly according to T-stage. Discrimination of favorable cases, particularly T2 or T3 lesions, is useful for the selection of surgical strategies for individual patients. Although many candidate factors predicting disease progression, such as depth of subserosal invasion, horizontal tumor spread, tumor budding, dedifferentiation, Ki-67 labeling index, p53 nuclear expression, CD8+ tumor-infiltrating lymphocytes, mitotic counts, Laminin-5-gamma-2 chain, hypoxia-inducible factor-1a, cyclooxygenase-2 and the Hedgehog signaling pathway have been investigated, useful prognostic makers or factors have not been established. As GBC is often discovered incidentally after routine cholecystectomy and accurate preoperative diagnosis is difficult, close mutual cooperation between surgeons and pathologists is essential for developing a rational surgical strategy for GBC.