IL-22 produced by cancer-associated fibroblasts promotes gastric cancer cell invasion via STAT3 and ERK signaling.

BACKGROUND: Interleukin-22 (IL-22) has been recently highlighted owing to its biological significance in the modulation of tissue responses during inflammation. However, the role of IL-22 in carcinogenesis has remained unclear. Here, we investigated the pathophysiological significance of IL-22 expression in gastric cancer tissues and examined the mechanism by which IL-22 promotes gastric cancer cell invasion. METHODS: Human gastric cancer specimens were analysed by immunohistochemistry for expression of IL-22 and IL-22 receptor 1 (IL-22R1). The effects of IL-22-induced STAT3 and ERK signalling on invasive ability of gastric cancer cells were examined using a small-interfering RNA system and specific inhibitors. AGS cells were co-cultured with cancer-associated fibroblasts (CAFs) from human gastric cancer tissues and assessed by invasion assay. RESULTS: Interleukin-22 and its receptor were expressed in α-smooth muscle actin-positive stromal cells and tumour cells at the invasive front of gastric cancer tissues, respectively. The expression of IL-22 and IL-22R1 was significantly related to lymphatic invasion. Interleukin-22 treatment promoted the invasive ability of gastric cancer cells through STAT3 and ERK activation. The invasive ability of gastric cancer cells was significantly enhanced by co-culture with IL-22-expressing CAFs. CONCLUSIONS: Interleukin-22 produced by CAFs promotes gastric cancer cell invasion via STAT3 and ERK signalling.

Involvement of REG Ialpha protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome.

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi-gene family in humans. REG Ialpha protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Ialpha expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Ialpha protein, Ki67 and single-strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Ialpha protein, Ki67 and ssDNA in the MSG were then analysed. REG Ialpha protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Ialpha-positive than in negative SS patients. REG Ialpha protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.

Expression of SDF-1 alpha and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer.

Although stromal cell-derived factor (SDF)-1 alpha and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1 alpha and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1 alpha and CXCR4. The relationships between clinicopathological features and SDF-1 alpha or CXCR4 expression were then analysed. Stromal cell-derived factor-1 alpha and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1 alpha and nuclear CXCR4 predicts LN metastasis in CRCs.

Methylation of the oestrogen receptor gene in non-neoplastic epithelium as a marker of colorectal neoplasia risk in longstanding and extensive ulcerative colitis.

BACKGROUND: Surveillance colonoscopy is widely recommended in patients with longstanding and extensive ulcerative colitis (UC) in order to detect colorectal neoplasia at an early stage. However, it still remains questionable whether surveillance colonoscopy effectively enables early detection of UC associated neoplasia. There is a great need for sensitive markers to identify individuals at increased risk of neoplasia. The oestrogen receptor (OR) gene shows age related methylation in the colorectal epithelium and is methylated frequently in sporadic colorectal neoplasia, suggesting that OR methylation may predispose to colorectal neoplasia. AIM: To clarify whether analysis of methylation of the OR gene in non-neoplastic epithelium can contribute to prediction of increased neoplasia risk in UC patients. MATERIALS AND METHODS: A total of 165 non-neoplastic colorectal epithelia from 30 patients with longstanding and extensive UC, including 13 UC patients with neoplasia and 17 patients without, were evaluated. Methylation specific polymerase chain reaction was performed to determine the methylation status of the OR gene. RESULTS: Methylation of the OR gene was detected in 54 of 70 (77.1%) non-neoplastic colorectal epithelia in UC with neoplasia but in only 23 of 95 (24.2%) without neoplasia. Methylation of the OR gene was significantly more frequent in non-neoplastic epithelium from UC with neoplasia than in chronic colitic epithelium from UC without neoplasia. Furthermore, in UC with neoplasia, the OR gene was extensively methylated in non-neoplastic epithelia throughout the colorectum compared with those in UC without neoplasia. CONCLUSION: These results suggest that analysis of OR gene methylation may have potential as a useful marker for identifying individuals at increased risk of neoplasia among those with longstanding and extensive UC.

Development of colonic neoplasia in p53 deficient mice with experimental colitis induced by dextran sulphate sodium.

BACKGROUND: Several animal models for human ulcerative colitis (UC) associated neoplasia have been reported. However, most neoplasias developed in these models have morphological and genetic characteristics different from UC associated neoplasia. AIMS: To establish a new colitis associated neoplasia model in p53 deficient mice by treatment with dextran sulphate sodium (DSS). METHODS: DSS colitis was induced in homozygous p53 deficient mice (p53(-/-)-DSS), heterozygous p53 deficient mice (p53(+/-)-DSS) and wild-type mice (p53+/+-DSS) by treatment with 4% DSS. Numbers of developed neoplasias were compared among the experimental groups, and macroscopic and microscopic features of the neoplasias were analysed. Furthermore, K-ras mutation and beta-catenin expression were assessed. RESULTS: p53(-/-)-DSS mice showed 100% incidence of neoplasias whereas the incidences in p53(+/-)-DSS and p53+/+-DSS mice were 46.2% and 13.3%, respectively. No neoplasias were observed in the control groups. The mean numbers of total neoplasias per mouse were 5.0 (p53(-/-)-DSS), 0.62 (p53(+/-)-DSS), and 0.2 (p53+/+-DSS). The number of neoplasias per mouse in the p53(-/-)-DSS group was significantly higher than that in the other DSS groups. The incidences of superficial type neoplasias were 91.7% in p53(-/-)-DSS mice, 75.0% in p53(+/-)-DSS mice, and 33.3% in p53+/+-DSS mice. The K-ras mutation was not detected in any of the neoplasias tested. Translocation of beta-catenin from the cell membrane to the cytoplasm or nucleus was observed in 19 of 23 (82.6%) neoplasias. CONCLUSIONS: The p53(-/-)-DSS mice is an excellent animal model of UC associated neoplasia because the morphological features and molecular genetics are similar to those of UC associated neoplasia. Therefore, this model will contribute to the analysis of tumorigenesis related to human UC associated neoplasia and the development of chemopreventive agents.

Dedifferentiation and decreased expression of adhesion molecules, E-cadherin and ZO-1, in colorectal cancer are closely related to liver metastasis.

Carcinoma cells with high metastatic potential often show a dedifferentiated phenotype at the primary site. In this study, a total of 48 cases (24 primary tumors of colorectal cancer (Pr-CRC) with liver metastasis, 24 without) were examined for E-cadherin and ZO-1 expression by immunohistochemical staining, and for their dedifferentiated phenotype. The expression levels of E-cadherin and ZO-1 were markedly decreased in the cancer cells of tumors with liver metastasis. Moreover, dedifferentiation of cancer cells, which was evaluated by the modified Gleason score, was also related to liver metastasis. However, none of the conventional clinicopathologic parameters of invasion, except lymph node metastasis, showed any relationship with liver metastasis. These results indicate that dedifferentiation and a decreased expression of E-cadherin and ZO-1 are closely related to liver metastasis.

Down-regulated p16 expression predicts poor prognosis in patients with extrahepatic biliary tract carcinomas.

The prognosis of extrahepatic biliary tract cancer (EBT) patients is generally accepted to be poor. We immunohistochemically evaluated expression of p16, a cyclin-depend kinase inhibitor, in tumor specimens surgically removed from 99 EBT patients. We also examined whether there was any relationship between expression of p16 and biological malignancy of the tumor by comparing its clinicopathological factors. Consequently, we found that there were three types of p16 expression in the tumor cells; diffuse, heterogeneous and negative types, the percentages of which were 19, 41 and 39%, respectively. Heterogeneous and negative types, whose expression of p16 was considered to be down-regulated, showed scirrhous (p=0.022) and infiltrating growth (p=0.002). In addition, we found that the proportion of down-regulated expression of p16 was different, depending on the location of the tumor. We also observed that the down-regulated p16 expression was the highest in a proportion of patients with the extrahepatic bile duct carcinoma. In contrast, the proportion of down-regulated p16 expression was the least among the patients in the region of the ampulla of Vater with better prognosis, and we showed that the prognosis of patients with down-regulated expression of p16 was the poorest in terms of the cancer location where it is limited to the region of ampulla of Vater. These findings suggest that down-regulated p16 expression is evaluated as a factor of poorer prognosis and also that immunohistochemical pattern of p16 expression becomes a marker reflecting the biological malignancy of EBT patients.

A new in vivo model for studying invasion and metastasis of esophageal squamous cell carcinoma.

The aim of our current study was to establish an orthotopic inoculation model for studying invasion and metastasis of esophageal squamous cell carcinoma (SCC). Male BALB/c nude mice were used for the experiment. A midline incision was made from the upper to middle abdomen. The abdominal esophagus was carefully exposed. Human esophageal T.Tn SCC cells or human cervical HeLa SCC cells, were injected into the submucosa of the lower esophagus. One of the mice injected with T.Tn cells was sacrificed at 5 weeks, and the remaining five sacrificed at 13 weeks after inoculation. The mice injected with HeLa cells were sacrificed at 3-4 weeks after inoculation. T.Tn cells and HeLa cells formed tumors at the esophagus, but did not metastasize to lymph nodes or lungs. HeLa cells produced peritoneal implants, and directly invaded the stomach and the liver. In the present study, we established a novel orthotopic inoculation model of esophageal SCC. This system is an appropriate and a useful model for studying invasion and metastasis of esophageal SCC, and can also be used as a model for developing therapeutic strategies for esophageal cancer in vivo.

Beta-catenin expression as a prognostic indicator in cervical adenocarcinoma.

The purpose of this study was to assess the prognostic influence of beta-catenin expression by immunohistochemistry in patients with cervical adenocarcinomas. The study group comprised of 51 patients who underwent total hysterectomy for cervical cancer. The median follow-up was 39 months (range 1-138 months). beta-catenin was expressed strongly on the membranes of normal cervical epithelial and glandular cells. Uniform membranous beta-catenin staining localized to intercellular borders was observed in 35% of tumors, whereas 65% of tumors demonstrated an abnormal pattern of reduced or aberrant beta-catenin expression (i.e., cytoplasmic and/or nuclear staining patterns). Abnormal beta-catenin immunoreactivity was associated statistically with advanced pathologic stage (p=0.018). The 10-year disease-free survival was 51.0% in patients with preserved expression of beta-catenin. On the other hand, a poorer prognosis was noted in the group with abnormal expression of beta-catenin with a 10-year disease-free survival of 43.4%. By multivariate analysis, low pathologic stage (stages I and II, p=0.001) and preservation of beta-catenin expression (p=0.012) were independently favorable prognostic factors. Our results indicate that changes in beta-catenin expression occur during the progression of cervical adenocarcinoma to an invasive phenotype. These results suggest that beta-catenin is an important intercellular adhesion molecule. Assessment of beta-catenin immunoreactivity may be a useful prognostic tool in cervical adenocarcinoma complementary to established prognostic factors. Furthermore, we developed a strategy for choosing biomarkers representing the steps in malignant progression in an effort to identify patients with occult metastases who will need adjuvant therapy and spare women from unnecessary interventions.

Concurrent occurrence of gastric adenocarcinoma and duodenal neuroendocrine cell carcinoma: a composite tumour or collision tumours ?

BACKGROUND: Neuroendocrine cell (NEC) carcinoma is occasionally accompanied by adenocarcinoma but the relationship between these two morphologically distinct tumours is unclear. Two hypotheses have arisen regarding the mechanism for the association of adenocarcinoma and NEC carcinoma. One is that both are derived from a common multipotential epithelial stem cell. The second hypothesis is that adenocarcinoma and NEC carcinoma arise from a multipotential epithelial stem cell and a primitive NEC, respectively. AIMS: To elucidate the relationship between the two histologically distinct tumours, adenocarcinoma of the stomach and NEC carcinoma of the duodenum. PATIENT/METHODS: We present a case in which the tumour extended across the pyloric ring, the gastric portion of which revealed adenocarcinoma while the duodenal portion showed argyrophil NEC carcinoma. The two histologically distinct lesions of the tumour were examined by immunohistochemistry and genetic analysis of p53. RESULTS: The gastric region was negative for chromogranin A staining but positive for carcinoembryonic antigen (CEA) staining. In contrast, the duodenal region was positive for chromogranin A but negative for CEA. All tumour regions showed a point mutation in p53 gene at exon 7 (GGC (glycine)-->GTC (valine) at codon 245). The distal portion of the duodenal tumour showed an additional point mutation in p53 gene at exon 5 (GCC (alanine)-->GTC (valine) at codon 129). CONCLUSIONS: The two histologically distinct tumours, adenocarcinoma of the stomach and NEC carcinoma of the duodenum, appear to be derived from a common epithelial cell.

Decreased expression of E-cadherin and Yamamoto-Kohama's mode of invasion highly correlates with lymph node metastasis in esophageal squamous cell carcinoma.

OBJECTIVES: A reduction in cell-cell adhesion in cancer cells is an essential step in the progression from localized malignancy to metastatic disease. E-Cadherin is an important component of cell-cell adhesion molecules and may be a crucial determinant of tumor invasion and metastasis. E-Cadherin expression is reported to be correlated with lymph node metastasis in esophageal squamous cell carcinoma (SCC). The objective of this experiment is to examine the factors that are associated with invasion and metastasis of esophageal SCC. METHODS: Forty-six cases of esophageal SCC were examined by immunohistochemical staining for E-cadherin. The relationship between E-cadherin-staining patterns, conventional clinicopathological parameters and Yamamoto-Kohama's (Y-K's) mode of invasion were examined. RESULTS: The expression of E-cadherin on the cell membrane was reduced or lost in some of the esophageal SCC. Lymph node metastasis was highly correlated with the expression pattern of E-cadherin (p = 0.0002) and also highly correlated with Y-K's mode of invasion (p = 0.0078). However, lymph node metastasis was not correlated with any conventional clinicopathological parameters for invasion. CONCLUSIONS: These results indicate that E-cadherin plays a crucial role in invasion and metastasis in esophageal SCC, and that Y-K's mode of invasion highly reflects the invasiveness and metastatic potentials of esophageal SCC cells. Therefore, examination of the expression of E-cadherin and Y-K's mode of invasion would be helpful in predicting lymph node metastasis in esophageal SCC.

Intracytoplasmic localization of cathepsin D reflects the invasive potential of gastric carcinoma.

BACKGROUND: The present study was undertaken to investigate the role of cathepsin D in the invasiveness of human gastric cancer. METHODS: Immunohistochemical cathepsin D staining was performed in samples from 29 early gastric adenocarcinomas (papillary or tubular adenocarcinoma) and 15 gastric adenomas, and their adjacent nonneoplastic gastric mucosa. We classified the patterns of cathepsin D immunostaining into four types; type A, fine granular staining in the apical portion: type B, intense coarse granular staining in the apical portion; type C, fine granular staining in the basal portion; and type D, diffuse granular staining throughout the cytoplasm. RESULTS: All of the nonneoplastic mucosa showed an apical cytoplasmic distribution pattern (type A or type B). However, 20% (2/10) of low-grade gastric adenomas and 60% (3/5) of high-grade gastric adenomas showed an abnormal staining pattern. i.e., types C and D. Moreover, 82% (9/11) definite intramucosal gastric adenocarcinomas, and the majority of gastric adenocarcinomas with submucosal invasion [83% (15/18) of those in the mucosal part and 100% (14/ 14) of those in the invasive submucosal part] showed an abnormal staining pattern (types C and D). Interestingly, most of the carcinoma cells invading the stroma and lymphatics showed the type D staining pattern. CONCLUSIONS: These results indicate that an abnormal cytoplasmic staining pattern of cathepsin D may reflect the invasive potential of gastric carcinoma cells.

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.

Preoperative radiotherapy for cancer of the extrahepatic bile duct.

From January 1988 through June 1996, 12 patients who had extrahepatic bile-duct cancer received preoperative radiotherapy at doses of 40.6 Gy to 58.4 Gy. At restaging, 1 patient was found to have liver metastases and the remaining 11 patients were taken to surgery. Nine patients underwent resection, and 8 of the 9 received intraoperative radiotherapy. Complications occurred in 4 patients, 3 of whom died postoperatively. The 2 patients who died of intraabdominal complications received both preoperative radiation doses of more than 55 Gy and intraoperative radiotherapy doses of 14 Gy or more. Histologic evidence of irradiation effects was present in all specimens. Irradiation effects on perineural invasion were observed in varying degrees. Two of the four patients who had marked irradiation effects on perineural invasion developed local recurrence, which was found at autopsy to have infiltrated the hepatic hilum without obstructing the hepatic ducts. One patient who had minimal irradiation effects on perineural invasion developed local recurrence with obstructing the hepatic ducts. Of the 2 patients who had positive margins, the patient with marked irradiation effects on perineural invasion survived 18 months, but the patient with slight irradiation effects on perineural invasion survived only 5 months. The high complication rate requires modification of this strategy. The propriety of combining preoperative radiotherapy with intraoperative radiotherapy as well as the radiation dose should be reinvestigated.

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen.

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.

An evaluation of serum microelement concentrations in lung cancer and matched non-cancer patients to determine the risk of developing lung cancer: a preliminary study.

In a case-control study to determine the risk of developing lung cancer, the serum levels of vitamins A and E, carotene and selenium were determined in 31 patients, newly diagnosed as having lung cancer, and in matched controls, the said controls being selected from outpatients with no cancer. A significant, inverse association was found between serum vitamins A and E and lung cancer. The relative risk for the low vs high tertiles were, respectively, 5.94 for serum vitamin A and 8.44 for serum vitamin E. Taking histological cancer subtype into account, no relation was revealed between the microelements and squamous cell carcinoma of the lung. The relative risk for lung cancer was 6.50, however, when three, or all four, microelement levels were in the lowest tertile, compared with there being fewer than three in the lowest tertile. Even when three microelements, excluding vitamin E which had the most significant inverse association with lung cancer, were considered, the relative risk was 7.50 when any two or all three were in the lowest tertile, compared with there being just one microelement or none at all in the lowest tertile. A combined effect of vitamins A and E, carotene and selenium on the development of lung cancer has, therefore, been suggested. Further studies will thus be necessary to elucidate the cumulative effect of the serum micronutrients and trace elements, as well as the effect of single elements, on the development of lung cancer.

Studies on the vascular permeability factor of bovine and human platelets.

The vascular permeability factors (VPFs) of bovine and human platelets were studied on the vascular permeability activity in the skin of unanesthetized rabbits. By continuous Urografin or sucrose density gradient method, subcellular organelles were separated from both bovine and human platelets as four fractions. Acid extract prepared from each fraction was used as a provocative for the production of inflammation. After intradermal injection, the alpha-granule extract alone provoked an obvious vascular permeability activity. In skin sites responding to the injection, high concentration of the bovine VPF induced a monophasic response with time, though both human and low concentration of bovine VPFs brought about nearly the same biphasic response. On quantitative analysis of the vascular permeability activity, chromatoscanner method was applied for the first time to the estimation of exuded dye. It was confirmed that the method attains to satisfactory results corresponding to those by the common dye-extraction method. We assume that the bovine and human platelets may play a role in inflammation by releasing the VPF during the aggregation and adhesion of the cells to each other as well as to the endothelium in association with migration.