Kaname Yamashita

Expression and tissue localization of matrix metalloproteinase 7 (matrilysin) in human gastric carcinomas. Implications for vessel invasion and metastasis

We examined the production and tissue localization of matrix metalloproteinase‐7 (MMP‐7 = matrilysin) in human gastric carcinomas and analyzed the data in connection with the clinicopathological factors. Sandwich‐enzyme immunoassay for the zymogen of MMP‐7 (proMMP‐7) showed enhanced production of MMP‐7 in carcinoma tissues compared with control normal gastric mucosa. Immunohistochemical studies demonstrated that MMP‐7 is localized predominantly to the carcinoma cells in 71% of the carcinoma samples (30/42 cases). The percentage of immunoreactive carcinoma cells to total carcinoma cells (positive ratio) was significantly higher in intestinal‐type carcinomas (26%, median) than in diffuse‐type carcinomas (3%, median) (p < 0.05). The positive ratio was markedly higher in carcinoma groups with vascular invasion (28%) or lymphatic permeation (12%) than in those without invasion (6%) or permeation (0%) (p < 0.05). It was also significantly higher in carcinoma groups with liver (49%) or lymph‐node metastases (15%) than in those without metastases (6 and 2% respectively) (p < 0.05). Both proMMP‐7 of 28 kDa and active MMP‐7 of 19 kDa were detected in the carcinoma tissues by immunoblotting. Reverse‐transcription‐PCR showed specific amplification in 50% of the carcinoma cases (6/12 cases) and 8% of the normal control specimens (1/12 cases). In situ hybridization demonstrated that the carcinoma cells almost selectively express MMP‐7 mRNA. These data suggest that enhanced production of proMMP‐7 and its activation are implicated in invasion and metastasis of human gastric carcinomas. Int. J. Cancer (Pred. Oncol.)79:187–194, 1998.© 1998 Wiley‐Liss, Inc.

Expression of Telomerase Activity in Human Endometrium Is Localized to Epithelial Glandular Cells and Regulated in a Menstrual Phase- Dependent Manner Correlated with Cell Proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.

Enhanced production and activation of progelatinase A mediated by membrane-type 1 matrix metalloproteinase in human oral squamous cell carcinomas: Implications for lymph node metastasis

We measured the production levels of seven different matrix metalloproteinases (MMP-1, 2, 3, 7, 8, 9 and 13) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in the homogenates of human oral squamous cell carcinomas and control normal squamous epithelia by the corresponding sandwich enzyme immunoassay systems. The levels of MMP-1, 2, 3, 8, 9, 13 and TIMP-1 were significantly higher in the carcinoma samples than in the control. Among them, only the production level of MMP-2 was significantly higher in the carcinomas with cervical lymph node metastasis than in those without metastasis (P < 0.05). Gelatin zymography demonstrated that activation ratio of the zymogen of MMP-2 (proMMP-2) is significantly higher in the carcinomas with lymph node metastasis than in those without metastasis (P < 0.05) or normal control (P < 0.01). Quantitative RT-PCR for membrane-types 1, 2 and 3 MMPs (MT1, 2 and 3-MMPs), which activate proMMP-2 in vitro, demonstrated that MT1-MMP is predominantly expressed in the carcinoma tissues, and the expression level is significantly higher in the carcinomas with lymph node metastasis than in those without metastasis (P < 0.05) or the control samples (P < 0.05). Although MT2-MMP and MT3-MMP were detected in approximately 30% of the carcinoma cases, their expression levels were extremely lower compared with that of MT1-MMP. There was a direct correlation between the MT1-MMP expression level and proMMP-2 activation ratio (r = 0.62, P < 0.01). In situ hybridization and immunohistochemistry indicated that carcinoma cells and stromal cells adjacent to carcinoma cell nests express MT1-MMP transcripts and protein. MMP-2 and TIMP-2 were also immunolocalized to the carcinoma cells in the carcinoma samples. By in situ zymography, gelatinolytic activity was demonstrated in the carcinoma cell nests and abolished by the treatment with an MMP inhibitor, BB94. These results suggest that among seven different MMPs, the production of proMMP-2 and its activation mediated by MT1-MMP play an important role in the cervical lymph node metastasis of the human oral squamous cell carcinomas.

Enhanced production and activation of matrix metalloproteinase-7 (matrilysin) in human endometrial carcinomas.

We examined production and tissue localization of 7 different matrix metalloproteinases (MMP‐1, ‐2, ‐3, ‐7, ‐8, ‐9 and ‐13) and 2 tissue inhibitors of metalloproteinases (TIMP‐1 and ‐2) in human endometrial‐carcinoma tissues. Sandwich enzyme immunoassays showed enhanced production of MMP‐7, MMP‐8 and MMP‐9 as well as TIMP‐1 in the carcinoma tissues compared with non‐carcinoma endometrial tissues. Among these MMPs, only the amount of MMP‐7 correlated with clinicopathological factors of the carcinomas. The level was significantly 6.8‐fold higher in the patient group with lymph‐node metastases than in that without metastases (p < 0.05), and also increased with the progress of the clinical stage. MMP‐7 was immunolocalized predominantly to the carcinoma cells in 73% of the cases, while MMP‐8 and MMP‐9 were immunostained in the inflammatory cells infiltrated in the carcinoma tissues. Immunoblotting revealed a definite band for the zymogen of MMP‐7 (proMMP‐7) of 28 kDa in 82% of the carcinoma samples, while only a faint band for proMMP‐7 was seen in 57% of the non‐carcinoma endometrial samples. Active MMP‐7 species of 19 kDa and its activity were demonstrated in the carcinoma samples with proMMP‐7 production by immunoblotting and zymography, respectively. RT‐PCR using a specific primer pair for MMP‐7 demonstrated expression in 86% of the carcinoma tissue and in 57% of the control tissue samples. In situ hybridization showed carcinoma cells selectively expressing MMP‐7 mRNA. These data suggest that, among the 7 MMPs examined, MMP‐7 may play a key role in invasion and lymph‐node metastasis of human endometrial carcinomas. Int. J. Cancer (Pred. Oncol.) 84:470–477, 1999.

Abnormal expression of E-cadherin, β-catenin, and c-erbB-2 in advanced gastric cancer: its association with liver metastasis

Abstract Background and aims. We investigated expression of E-cadherin, β-catenin, and c-erbB-2 in gastric cancer to identify molecular factor(s) relevant to development of liver metastasis, which is a frequent cause of mortality in gastric cancer patients. Patients and methods. We analyzed by immunohistochemistry and compared expression patterns of E-cadherin, β-catenin, and c-erbB-2 in the tumor between 40 cases of gastric cancer (GC) without (GC-H–) and 16 with concurrent liver metastasis (GC-H+). Results. Loss of E-cadherin expression in the primary tumor was found in 18% of GC-H– and in 19% of GC-H+. Oncogenic β-catenin activation, represented by its nuclear translocation, was detected in 13% of GC-H– and in 31% of GC-H+. There was no statistical difference in incidence of alteration in these molecules between the two groups of patients. c-erbB-2 overexpression was more frequently observed in GC-H+ (10/16, 63%) than in GC-H– (5/40, 13%) while the distribution of histological types of the tumors was similar in the two groups of patients. This overexpression was also detected in metastatic liver tumors and biopsy specimens in the ten of the former group of patients. Conclusion. Our results strongly suggest a role of activated c-erbB-2 in the process of liver metastasis, and an importance of detection of this overexpression in biopsy specimens to identify GC patients who are at high risk of developing liver metastasis.

The EGFR Ligands Amphiregulin and Heparin-Binding EGF-like Growth Factor Promote Peritoneal Carcinomatosis in CXCR4-Expressing Gastric Cancer

Purpose: Peritoneal carcinomatosis, often associated with malignant ascites, is the most frequent cause of death in patients with advanced gastric cancer. We previously showed that the CXCR4/CXCL12 axis is involved in the development of peritoneal carcinomatosis from gastric cancer. Here, we investigated whether epidermal growth factor receptor (EGFR) ligands are also involved in the development of peritoneal carcinomatosis from gastric cancer. Experimental Design: The functional involvement of expression of the ErbB family of receptors and/or EGFR ligands was examined in CXCR4-expressing human gastric cancer cells and fibroblasts, clinical samples (primary tumors and ascites), and an animal model. Results: High concentration of the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF), as well as of CXCL12, were present in malignant ascites. Human gastric cancer cell lines and primary gastric tumors, with high potential to generate peritoneal carcinomatosis, expressed high levels of EGFR and CXCR4 mRNA and protein. Both amphiregulin and HB-EGF enhanced the proliferation, migration, and functional CXCR4 expression in highly CXCR4-expressing gastric cancer NUGC4 cells. Amphiregulin strongly enhanced the proliferation of NUGC4 cells, whereas HB-EGF markedly induced the migration of fibroblasts. Moreover, HB-EGF and CXCL12 together enhanced TNFα-converting enzyme (TACE)-dependent amphiregulin shedding from NUGC4 cells. In an experimental peritoneal carcinomatosis model in mice, cetuximab effectively reduced tumor growth and ascites formation. Conclusions: Our results strongly suggest that the EGFR ligands amphiregulin and HB-EGF play an important role, interacting with the CXCL12/CXCR4 axis, in the development of peritoneal carcinomatosis from gastric cancer, indicating that these two axes may be potential therapeutic targets for peritoneal carcinomatosis of gastric carcinoma. Clin Cancer Res; 17(11); 3619–30. ©2011 AACR.

Epidermoid cyst of the intrapancreatic accessory spleen producing CA19-9

We report a rare case of an epidermoid cyst in an accessory spleen at the pancreatic tail with producing CA19‐9. A 55‐year‐old female was admitted to our hospital, Cancer Research Institute, Kanazawa University, for close examination of a cystic lesion at the pancreatic tail and a high serum CA19‐9‐value (176 U/mL). There were almost no abdominal symptoms related to the cystic lesion. A cystic tumor approximately 3 cm in diameter and composed of multilocular cysts without a protruding portion of the inner surface was found at the pancreatic tail by ultrasound sonography, computed tomography, and magnetic resonance imaging. Endoscopic retrograde pancreatography revealed that the main pancreatic duct shifted at the pancreatic tail and there was no communication between the main pancreatic duct and cystic lesion. Based on a preoperative diagnosis of mucinous cystic tumor, distal pancreatectomy with splenectomy was performed. Histological findings suggested an epidermoid cyst (3.5 × 3.0 cm) originating from an intrapancreatic accessory spleen. Immunohistochemical analysis of CA19‐9 in the epidermoid cyst showed clear staining of the inner epithelium of the cyst and amorphous or hyalinous cystic contents. The serum CA19‐9 value was confirmed to decline to normal 2 months after resection. Physicians should not forget this disease during differential diagnosis related to pancreatic cystic lesions with elevated levels of serum tumor markers, such as CA19‐9 or carcinoembryonic antigen, although this disease is extremely rare.

Non-Hodgkin's lymphoma in a patient with probable hereditary nonpolyposis colon cancer: report of a case and review of the literature.

AbstractPURPOSE: Hereditary nonpolyposis colorectal cancer kindreds are frequently associated with cancers in various organs, including endometrium, stomach, and ovary. However, hematologic malignancy has rarely been reported in association with this cancer syndrome. We present here the case of a probable hereditary nonpolyposis colon cancer patient in whom non-Hodgkin’s lymphoma developed after curative resection of colon cancer. Our experience with this rare case encouraged us to review the literature for reports indicating a possible relationship between these diseases. RESULTS: A 52-year-old male whose family history was consistent with the criteria for hereditary nonpolyposis colon cancer underwent right hemicolectomy for ascending colon cancer. Histologically the tumor consisted of adenocarcinoma that was moderately differentiated with mucinous foci and that invaded beyond the muscularis propria. Neither metastasis nor lymphoma was found in paracolonic lymph nodes. Eight months after surgery, the patient developed non-Hodgkin’s lymphoma of T-cell origin involving the ileum and lungs. Both colon cancer and lymphoma frequently showed microsatellite DNA instability, sharing alteration in a locus of chromosome 7 (D7S501). CONCLUSION: A possible association of hematologic malignancy with hereditary nonpolyposis colon cancer reported in the literature, together with a report that MSH2-deficient mice are susceptible to malignant lymphoma, strongly supports the finding that this patient’s lymphoma was related to hereditary nonpolyposis colon cancer. Overall, this case manifested a distinct clinical course similar to that observed in an animal model that is deficient in DNA mismatch repair machinery, thus providing scientific and clinical implications for understanding the molecular basis of these tumors and for critical management of hereditary nonpolyposis colorectal cancer patients, respectively.

Adjuvant Immunochemotherapy with Protein-Bound Polysaccharide K for Colon Cancer in Relation to Oncogenic β-Catenin Activation

PurposeProtein-bound polysaccharide K is an immunotherapeutic agent that promotes apoptosis by inhibiting nuclear factor-kappaB activation in cancer cells. We previously showed that oncogenic β-catenin activates nuclear factor-kappaB and inhibits apoptosis by up-regulating β-transducin repeat-containing protein. We investigated whether the activation state of β-catenin in the primary tumor is associated with differences in survival rates of patients with colon cancer undergoing immunochemotherapy with 5-fluorouracil plus polysaccharide K vs. chemotherapy with 5-fluorouracil alone.MethodsWe assessed the activation states of β-catenin and nuclear factor-kappaB in the primary tumors of 202 colon cancer patients, and analyzed the data in terms of the clinicopathologic characteristics and survival of patients undergoing the two forms of adjuvant therapy.ResultsWe found two distinct patterns of nuclear accumulation of activated β-catenin in the tumor cells: diffuse nuclear accumulation in 89 cases (44 percent) and selective nuclear accumulation at the tumor invasion front in 18 cases (9 percent). Nuclear factor-kappaB activation was found in 64 cases (32 percent). In patients with diffuse nuclear accumulation-type β-catenin activation, immunochemotherapy significantly improved recurrence-free survival, cancer death survival, and overall survival rates compared with patients receiving chemotherapy alone. No survival benefit was found in cases with nuclear accumulation at the tumor invasion front-type β-catenin activation or no activation. Similarly, immunochemotherapy favored the survival of patients with nuclear factor-kappaB activation. Multivariate analysis established the TNM stage and administration of polysaccharide K as independent prognostic factors in the patients with diffuse nuclear accumulation-type β-catenin activation.ConclusionsThe presence of diffuse nuclear accumulation-type β-catenin activation identifies patients with colon cancer who respond better to immunotherapy with polysaccharide K.

Pilot Study of Low-Dose, Divided Maximum Tolerated Dose of CPT-11 in 21 Consecutive Patients with Metastatic Colorectal or Gastric Cancer

PurposeWe devised a new treatment regimen, delivering a frequent low dose of CPT-11, calculated by dividing the maximum tolerated dose (MTD) to reduce its toxicity without impairing its efficacy.MethodsCPI-11, 25 mg/m2, determined by dividing the MTD dose per month by 12, was given on days 1, 2, and 3 of every week, to 21 consecutive patients; 12 with metastatic colon cancer and 9 with metastatic gastric cancers.ResultsThe total delivered dose of CPI-11 per patient was more than 1 000 mg in 17 (80.1%) of the 21 patients. Grade 3 marrow depression developed in 3 (14.3%) patients, and although nausea, vomiting, alopecia, and diarrhea developed in some patients, these side effects were all categorized as grade 2 or milder. The antitumor effect was evaluated in 18 patients with measurable lesions, who had received CPI-11 according to our regimen for at least 3 weeks. Of these 18 patients, 10, 7, and 1, respectively, had a found to have partial response, no change, or progression of disease, demonstrating a 55.6% efficacy rate [colon 6/10 (60.0%) and stomach 4/8 (50.0%)]. Moreover, time to progression (TTP) was greater than 90 days in 12 (75.0%) of these 18 patients.ConclusionThese results show that our low-dose, divided MTD of CPI-11 regimen is a promising method of reducing toxicity and strengthening the antitumor effect, justifying further large-scale comparative clinical studies to verify this potential.