北山 丈二

Background: The stromal cell-derived factor-1/CXC chemokine receptor-4 (SDF-1/CXCR4) signal has been shown to be important in various immunological reactions. Recent studies have suggested that CXCR4 is expressed in certain cancer cells and that they use this chemokine receptor efficiently for metastasis formation. Method: The expression of CXCR4 was evaluated by immunohistochemical study in 79 surgically resected invasive ductal carcinomas, and the relation between the staining pattern and clinicopathological features was examined. Results: CXCR4 was diffusely and homogeneously expressed in 59 cancers, which were further divided into 28 high-expression and 31 low-expression cancers by their staining intensity. The other 20 cancers showed heterogeneous immunoreactivity in tumor tissue, which was defined as focal type. In comparison with the diffuse type, focal type tumors showed significantly more extensive lymph node metastasis, because the number and extent of metastatic nodes were larger in the focal than the diffuse type. In the diffuse type, the rate of node-positive cases did not show a difference in staining intensity. However, high-CXCR4 tumors showed more extensive nodal metastasis in comparison with low-expression tumors. In contrast, the expression pattern of CXCR4 did not have a significant correlation with hematogeneous metastasis. The overall survival of these patients tended to be better in the diffuse type than in the focal type, although the difference was not statistically significant. Conclusion: The expression pattern of CXCR4 was significantly correlated with the degree of lymph node metastasis in breast cancers. Our data suggest that CXCR4 might be particularly important in facilitating metastasis through the lymphatic system.

Background: Vascular endothelial growth factor C (VEGF-C) and D (VEGF-D) are considered to be potentially lymphangiogenic and can selectively induce hyperplasia of the lymphatic vasculature. In this study, we aimed to clarify the relation between expression of VEGF-C and -D and lymphatic metastasis in early gastric cancers. Methods: Using the specific antibodies, we classified 105 cases which were treated as gastrectomy with standard lymphadenectomy at the First Department of Surgery, Tokyo University Hospital, between 1994 and 2001, into three groups (diffuse type, focal type and negative type) for VEGF-C and two groups (positive and negative) for VEGF-D. Results: There was a significant correlation between the expression of VEGF-C and -D and lymphatic invasion but not with venous invasion. All of the 22 cases that were negative for VEGF-C and -D were histologically classified as adenocarcinoma of undifferentiated type and showed negative lymph node metastasis and also negative lymphatic invasion. VEGF-C was positive in all tumors of differentiated type, while its expression varied in tumors of undifferentiated type. The VEGF-D positive rate is much lower than that of VEGF-C. In undifferentiated tumors in particular, VEGF-D was positive only in 4/64 (6%) and three of these four had nodal metastasis. Therefore, in tumors of differentiated type, expression of VEGF-C and -D had no clinical relevance. In tumors of undifferentiated type, the negative expression of VEGF-C suggests lack of nodal metastasis, while the positive expression of VEGF-D suggests nodal metastasis. The lymph node metastasis was significantly related to the expression of VEGF-C and -D in adenocarcinomas of undifferentiated type but not in those of differentiated tumors. Conclusions: In early gastric cancers of histologically undifferentiated type with negative expression of VEGF-C and -D, limited surgery might be safely applied because the possibility of nodal metastasis is very low. These observations are based only on retrospective analysis of a small case series and further evaluation with a larger number of cases is necessary.

Peritoneal dissemination is the most frequent type of recurrence in patients with gastric cancer with serosal exposure, irrespective of whether they have undergone curative gastrectomy. The purpose of this study was to establish a method to detect micrometastatic cells in the abdominal cavity and predict peritoneal recurrence in patients with such gastric carcinomas. A total of 86 patients with gastric carcinoma, undergoing gastrectomy, were examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect carcinoembryonic antigen (CEA) mRNA in abdominal lavage fluid. Twenty-four cases without serosal exposure were negative, while all 13 cases with macroscopic peritoneal dissemination were positive for CEA mRNA. Among the 49 cases with macroscopic serosal invasion and without peritoneal metastasis, cancer cells were detected in 27 cases with RT-PCR while in only 6 cases with conventional cytology. All cytologically-positive cases were also positive for CEA mRNA. Among the 27 CEA-positive cases, 15 patients (56%) relapsed with peritoneal metastasis within 12 months after gastrectomy. In contrast, none of the 22 CEA-negative cases had peritoneal recurrence within 16-60 months of observation, whereas in 43 cytologically-negative cases, 10 patients relapsed with peritoneal recurrence. As compared with conventional cytological examination, this method would be clinically more beneficial for detecting free cancer cells in the peritoneal cavity and for predicting peritoneal recurrence in gastric carcinoma with serosal invasion.

BACKGROUND. Preoperative radiotherapy reduces the rate of local recurrence and improves the chance of survival in patients with resectable, advanced rectal carcinoma. However, because not all tumors respond similarly to radiation, sorting out suitable patients is required to irradiate tumors rationally. The authors examined the possible role of Ku protein in determining tumor radiosensitivity and disease free survival in patients with rectal carcinoma. METHODS. The authors studied 96 patients with advanced rectal carcinoma. In preradiation biopsy specimens of tumor samples, the number of cells that were stained positive for Ku protein was evaluated by immunohistochemistry. The expression pattern of Ku protein was examined for an association between tumor radiosensitivity (which was determined according to T classification downstaging, complete pathologic response, or Response Evaluation Criteria in Solid Tumors) and disease free survival. RESULTS. There was a high degree of correlation between the percentage of cells that expressed the 70-kDa Ku protein (Ku70) and the 86-kDa Ku protein (Ku86) in the tumor sections (correlation coefficient = 0.85; P < 0.001). The expression pattern of Ku protein was correlated not only with tumor radiosensitivity but also with disease free survival. Pathologic TMN classification, histopathologic grade, and Ku70 expression were significant prognostic variables for disease free survival in a multivariate analysis (P = 0.0031, P = 0.030, and P = 0.023, respectively). CONCLUSIONS. Ku70 and Ku86 raise the predictive possibility of tumor radiosensitivity. Ku may be a useful parameter for selecting patients with rectal carcinoma for preoperative radiotherapy. (C) 2002 American Cancer Society.

Cancer cell adhesion to lymphatic endothelial cells (LEC) was examined under shear stress mimicking lymph flow. An established rat gastric adenocarcinoma cell line, BV9, was perfused over a primary cultured monolayer of LEC, which were explanted from the rat thoracic duct, and the adhesion pattern was observed. BV9 preferably adhered to LEC, at a level 8-fold greater than that to vascular endothelium in the unstimulated condition. When shear stress was increased after adhesion, a considerable number of BV9 on LEC withstood shear up to 50 dyn/cm(2), while BV9 attached on vascular endothelium did not remain adherent under 5 dyn/cm2. Adhesion was significantly augmented by prestimulation of LEC with 10 ng/ml IL1-beta or 500 ng/ml TNF-alpha. Our study indicates high affinity between cancer cells and LEC, and suggests the possibility that lymph node metastasis arises from cancer cells adherent to LEC, which can be augmented by an inflammatory stimulus.

The function of dendritic cells (DCs). antigen-presenting cells that can initiate and regulate cellular and humoral responses, is highly influenced by their level of maturation. Immature DCs may be harmful in anti-tumor immunotherapy, because they can induce immunotolerance rather than immunostimulation. In this study, the authors sought to determine the optimal culture conditions for obtaining fully mature DCs. When DCs were cultured in agonistic anti-CD40 monoclonal antibody-immobilized plates, they showed a higher expression of the maturation marker CD83 than DCs cultured without CD40 ligation or those cultured in medium supplemented with anti-CD40 monoclonal antibody. In addition, when interferon-gamma (IFN-gamma) was added to the medium, additive up-regulation of CD83 expression was observed. These DCs treated with both maturation signals showed a higher secretion of interleukin-12. To evaluate the capacity of antigen presentation. specific cytotoxic T lymphocytes were generated using autologous DC pulsed with a human lymphocyte antigen-A24-restricted peptide epitope derived from carcinoembryonic antigen. Interferon-gamma-secreting CD8+ T cells were analyzed by now cytometry using the cellular affinity matrix technology. Dendritic cells, matured with CD40 ligation and IFN-gamma, were more efficient at eliciting an antigen-specific T-cell response in vitro than DCs stimulated with anti-CD40 monoclonal antibody or IFN-gamma alone. A cytotoxicity assay using carcinoembryonic antigen-expressing tumor cell lines also showed that DCs matured with both signals were more efficient at inducing cytotoxic T lymphocytes. These results demonstrate that DC culture in an anti-CD40 monoclonal antibody-immobilized plate in medium supplemented with IFN-gamma has a positive impact oil DC maturation and may be optimal for eliciting an antigen-specific T-cell response without the need for CD4+ T-helper epitopes.

Caspase-8 is a member of the cysteine protease family that plays a critical role in death receptor-mediated apoptosis. We previously demonstrated that adenovirally transduced caspase-8 efficiently induced apoptosis in tumor cells (Shinoura et al. (2000) Hun. Gene Ther. 11, 1123-1137). However, to ensure safety in clinical applications some devise for minimization of the dose of adenoviral vector required for sufficient antitumor effect is needed. In this study, we evaluated the proapoptotic effect in DLD-1 colon cancer cells of a combination of low-dose infection with an adenoviral vector expressing caspase-8 and X-ray irradiation. Under these conditions, X-ray irradiation strongly induced apoptosis whereas irradiation without transduction only had a trace proapoptotic effect. Overexpression of bcl-xL strongly blocked the activation of caspase-8 and induction of apoptosis, suggesting that adenovirally transduced caspase-8 was activated at a point downstream of mitochondria. This combination strategy may be a useful modality for gene therapy of colorectal cancer. (C) 2002 Elsevier Science (USA).

Purpose. Local resection of the stomach for early gastric cancer is being performed more frequently, despite which no report focusing on the Postoperative complications has been published. The purpose of this study was to investigate the incidence and factors affecting postoperative complications after local resection of the stomach. Methods. Local resection of the stomach was performed in 37 patients with gastric cancers, submucosal tumors (SMT), or bleeding gastric ulcers, 24 of whom underwent gastroscopy at least once after their operation. We retrospectively examined the complications and background relating to the operations performed in those 24 patients. Results. Postoperative hemorrhage occurred in 2 patients, an open ulcer developed on the suture line in 2 and leakage developed in 1. The hemorrhage and open ulcer were observed only when wide resection with regional lymph node dissection was performed for early gastric cancers in the middle third of the stomach. Conclusion. These findings show that the possibility of postoperative hemorrhage and open ulcers on the suture line should be borne in mind, especially when wide local resection with lymph node dissection is performed for cancers in the middle part of the stomach.

Purpose. In the present study, we investigated the effect of troglitazone, a selective ligand and agonist of PPAR-gamma on the metastatic potential of human colon cancer cells. Methods. High- and low-PPAR-gamma expression clones of the colon cancer cell line, HT29, namely clones 21 and 3 respectively, were used. We investigated the effect of troglitazone on the proliferation, on the adhesion to extracellular matrix proteins and on the synthesis of matrix metalloproteinases (MMPs) of colon cancer cells. Results. Troglitazone inhibited the proliferation of both subclones, in a dose-dependent manner, and the inhibitory effect correlated with the level of PPAR-gamma expression. Troglitazone strongly inhibited the production of MMP-7, an enzyme associated with invasiveness of cancer cells, by both subclones. In addition, troglitazone caused a strong decrease in the adhesion of clone 21 to extracellular matrix (ECM) proteins, laminin and type IV collagen. This effect was independent of beta1-integrins expression. Conclusion. In addition to inhibition of cancer cell growth, troglitazone had an inhibitory effect on two important events associated with the metastatic potential of cancer cells, production of MMPs and adhesion to ECM proteins. Consequently, troglitazone is a promising agent for the treatment and prevention of colon cancer metastasis.

Cholesterol granuloma of the breast is a rare benign condition which is often clinically and radiologically indistinguishable from breast carcinoma. We herein report the case of a 62-year-old asymptomatic woman who was found on a routine breast examination to have an elastic hard mass, measuring 0.9 cm in diameter, in the upper outer quadrant of the left breast. Physical examination and ultrasonography strongly suggested a carcinomatous lesion. A cytological examination of a fine-needle aspiration biopsy specimen was inconclusive because of the paucity of epithelial cells. A histological examination of excisional biopsy materials showed scattered cholesterol crystals arranged in irregular, parallel arrays, surrounded by histiocytes and giant cells, which were consistent with a diagnosis of cholesterol granuloma. This case report indicates the importance of performing a histological examination to establish the final diagnosis of cholesterol granuloma. We believe that a better awareness of this breast disease might help to prevent both a misdiagnosis and unnecessary surgery.

Dendritic cells (DCs) are essential antigen-presenting cells with a wide variety of functions relating to both adaptive and innate immunity. Recently, interactions of DCs with natural killer (NK) cells and NK1.1-positive T cells have been reported in mice. However, in humans, this interaction is not well understood. Here we report the use of a coculture method to analyze the modulation of NK cell function in antitumor immunity by DCs. We found that peripheral blood DCs (PDCs) enhanced NK cell activity in cytotoxicity assay, even without direct contact between DC and NK cells. In contrast, neither monocyte-derived DCs (MoDCs), nor TNF-alpha-treated MoDCs, stimulated NK lytic activity. Secretion of IL-12 and TNF-alpha into the PDC-NK coculture supernatant was increased. However, blocking antibodies against these cytokines could not completely abolish the upregulation of NK activity, suggesting the presence of other soluble factor(s) that affect DC-NK cell interaction. To summarize, this study demonstrates for the first time the direct activation of human NK cells by DC-NK cell interaction in vitro, suggesting that DCs may have a central role linking the innate and adaptive immune responses. Moreover, in stimulating NK cell function, PDCs appear to have a different potential from MoDCs. (C) 2001 Elsevier Science.

It remains a question whether hematogeneous metastasis arises from a single cancer cell attached to the local endothelium or from a cluster of cancer cells trapped in the vascular bed in the target organ. Adhesive interaction of the single cell form and the clustered form of cancer cells was examined under flow conditions, using two subclones of mouse colon adenocarcinoma Colon 26. A subclone NL17, but not NL14, formed many clusters composed of tumor cells and platelets just after the addition of platelet rich plasma (PRP). Under the shear of 1.0 dyn/cm(3), the clustered form of NL17 tethered on laminin or mouse endothelial cell line in hepatic sinusoids (HSE) more frequently than the single cell form of NL17 and NL14. However, all of the clusters showed only transient attachment and never underwent stable arrest on coated laminin, while the single cell form of NL14 and NL17 underwent immediate arrest under shear conditions. On HSE stimulated with TNF-alpha, a small number of NL17 clusters made stable adhesion, although all the clusters detached if the shear stress was increased above 4.0 dyn/cm(2). In contrast, the single form of arrested NL17 as well as NL14 remained adherent even at shear of 8.0 dyn/cm(2). Compared with single cell, binding of cancer cell clusters to laminin and HSE showed lower resistance to shear stress, although they had adhesive interactions more frequently inflow condition. Since NL17 cells form significantly more metastases by intravenous injection in vivo, our data suggest that "stable adhesion" observed in our flow assay system is not always a prerequisite for clustered cancer cells to develop into metastatic lesions.

Background/Aims: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas (HCCs), GS expression may be related to tumor recurrence, since GS-expressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply, However, there are no studies concerning the prognostic value of GS expression in patients with HCC. Methods: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier product-limit method and multivariate analysis based on the Cox regression model was performed. Results: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group), Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=0.042). Multivariate analysis revealed that GS expression (p=0.021), as well as Child's classification (p=0.005) and portal invasion (p=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. Conclusion: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.

Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), known to inhibit cyclooxygenase (COX), reduce the risk of colorectal cancer. COX is a key enzyme in prostaglandin biosynthesis, and two isoforms of COX, COX-1 and COX-2, have been identified. Recently COX-2 has been reported to frequently overexpress in colorectal neoplasms and to play a role in colorectal tumorigenesis and tumour progression. In this study, using immunohistochemistry, we examined COX-2 expression in advanced human colorectal cancer and its correlation with clinicopathological features. COX-2 expression was observed mainly in the cytoplasm of cancer cells in all the specimens examined, but some stromal cells and endothetial cells were also stained. According to the grade of COX-2 expression of the cancer cells, patients were divided into high- and low-COX-2 expression groups. High-COX-2 expression significantly correlated with tumour recurrence, especially haematogenous metastasis. These results suggest that a selective COX-2 inhibitor can be a novel class of therapeutic agents not only for tumorigenesis but also for haematogenous metastasis of cololectal cancer. To our knowledge, this is the first report on the correlation between COX-2 overexpression and recurrence of colorectal cancer. (C) 2000 Cancer Research Campaign.

alpha-Glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR(+)NK1.1(+) T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR(+)NK1.1(+)cells, are rarely seen among freshly isolated human hepatic lymphocytes, Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro, The direct effector cells of the elicited cytotoxicity were CD3(-)CD56(+) NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.

Polymorphonuclear cell (PMN) transmigration across the TNF-alpha-stimulated endothelial cell (HUVEC) monolayer in the presence of shear flow was monitored with time-lapse videotapes. More than half of the PMN that arrested on HUVEC transmigrated through endothelial cell junctions within the following 15 min. The kinetics of transmigration was significantly faster than that of PMN placed under static conditions. Once PMN crept into the subendothelial space, they showed random migration beneath the HUVEC monolayer. PMN that did not transmigrate moved on the apical surface of HUVEC in the direction of flow down stream. Anti-beta 1 integrin mAb (4B4) and RGD peptide inhibited the transmigration more effectively than anti-beta 2 integrin mAb (TS1/18) and almost totally abrogated transmigration. When HUVEC were cultured on fibronectin or laminin, the transmigration was significantly inhibited by anti-alpha 5 or alpha 6 integrin mAbs, respectively. Our data clearly indicate that shear stress affects the migration behavior of PMN arrested on endothelium and suggest that binding to subendothelial extracellular matrix via beta 1 integrins is another essential step in leukocyte extravasation. (C) 2000 Academic Press.

Background: Analysis of serum cytokine levels has shown that cancer-bearing hosts have lower levels of IL-2 and IFN-gamma, suggesting that Th1-type immunity is impaired by cancer. However, the mechanisms of the Th1 dysfunction are not clearly understood. Method: The frequencies of Th1 cells in CD4(+) helper T cells were evaluated with an intracytoplasmic cytokine staining method in peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) of patients with gastrointestinal cancer. Results: Activation of lymphocytes with PMA + lonomycin induced the expression of IL-2 and IFN-gamma in each lymphocyte population. Compared with PBL of non-malignant donors, PBL in cancer patients contained significantly lower frequencies of CD4(+) T cells that produced IL-2 and IFN-gamma. LNL in cancer patients also contained lower levels of IL-2- and IFN-gamma-producing CD4(+) T cells, although the percentages did not show significant differences from those of PBL in the same patients. Conclusion: Our data suggest that suppression of Th1 cytokine in cancer patients is,at least in part, due to the decreased frequency of Th1 cells with CD4(+) phenotype.

Background: Thymidylate synthase (TS) is regarded as a parameter of 5-fluorouracil (5-FU) chemosensitivity for colorectal carcinoma. Recent researchers indicate that the chemosensitivity of 5-FU for colorectal carcinoma with low expression of TS is better than tumors with high expression of TS. But the relation between TS expression and overall survival of curatively resected colorectal cancer patients has been less studied. Methods: Specimens of curatively resected colon carcinoma from 148 patients were included in this study. TS expression in the tumor was assessed by immunohistochemical staining technique, and the patients were categorized into TS-(+) and TS-(-) groups. First, the relation between TS expression and survival of patients was examined. Next, for each group, we compared survival between the chemotherapy-(+) and the chemotherapy-(-) subgroup. Results: Overall survival was significantly better in the TS-(-) group (n = 107) than in the TS-(+) group (n = 41) (P = .0003). In the TS-(-) group, there was Little difference between the chemotherapy-(+) and the chemotherapy-(-) subgroup. In the TS-(+) group, the survival of the chemotherapy-(+) subgroup was significantly better than the chemotherapy-(-) subgroup (P = .0439). Conclusions: TS, itself, may be a prognostic factor for colon carcinoma; and 5-FU adjuvant chemotherapy may be appropriate for colon carcinoma with high expression of TS.

Background: Dihydropyrimidine dehydrogenase (DPD) is the first enzyme that metabolizes 5-fluorouracil (5-FU). Until now, enzymatic activity or mRNA expression of DPD has been investigated. However, there are no papers on immunohistochemical evaluation of DPD. We investigated DPD staining on immunohistochemistry, and examined the relationship among immunohistochemical score, protein level and mRNA expression of DPD. Materials and methods: Forty-seven resected colon cancer specimens, four colon cancer cell lines, two xenografts by colon cancer cell lines, and human mononuclear cells were used. Immunohistochemistry was performed using DPD monoclonal antibody. Protein levels were determined by Western blot analysis. And mRNA levels were calculated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: DPD was strongly expressed in the cytoplasm of cancer cells, and in the cytoplasm of macrophage and plasma cells. The immunohistochemical score was more correlated with protein levels (P = 0.0054) than mRNA expression (P = 0.9028). Conclusions: We investigated the characterization of DPD immunohistochemically, and showed that immunohistochemical expression of DPD can be used to predict the sensitivity of colorectal carcinomas to 5-FU.

The expression of thymidine phosphorylase (TP) in carcinoma of the papilla of Vater was studied to clarify its significance in tumor progression and in determining prognosis. Fifty-nine cases of surgically resected carcinoma of the papilla of Vater were studied. Immunohistochemical staining was performed to evaluate the expression of TP, microvessel count and p53 overexpression. TP expression was demonstrated in tumor cells in 62.7% (37/59) of the cases. A higher frequency of regional lymph node metastasis was found in TP-positive tumors than in TP-negative tumors (P=0.006). TP-positive tumors were more advanced than TP-negative tumors with regard to clinical stage (P=0.035). TP-positive tumors had significantly higher microvessel density (27.6+/-10.1) than TP-negative tumors (20.4+/-10.0, P=0.01). Moreover, TP expression was significantly correlated with a poor prognosis (P=0.02). These suggest that in carcinoma of the papilla of Vater, TP production by tumor cells is correlated with tumor progression through its regulatory effect on neovascularization.

Polymorphonuclear leukocytes (PMN) were perfused over extracellular matrix protein substrates under laminar shear flow. Under shear below 1.5 dyn/cm(2), many PMN tethered to immobilized laminin but not to fibronectin or vitronectin, Almost all the tethered PMN immediately arrested on laminin. The number of tethered PMN was mostly abrogated by mAbs to integrin alpha 6 Or beta 1 chains at concentrations of more than 5 mu g/ml. Addition of the two mAbs together produced no further inhibition compared with each mAb alone. In contrast, none of the mAbs to alpha 2, alpha 3, and beta 4 chains showed significant inhibition, indicating that PMN tethering to laminin is mostly dependent on alpha 6 beta 1 integrin. The addition of 10-100 ng/ml IL-8 in the assay medium before perfusion partially reduced PMN tethering to laminin. Stimulation with IL-8 also induced detachment of some tethered PMN within 30 s. Thus, IL-8 partially weakens the adhesiveness of alpha 6 beta 1 integrin on PMN in how conditions. (C) 2000 Academic Press.

Background. Degradation of basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be an essential step in the complicated process of hematogenous metastasis. MMP-1 is a member of collagenases, a family of MMPs that degrades collagens type I, II, and III, main components of the interstitial stroma. The purpose of this study was to investigate the expression of MMP-1 in colorectal cancer and its correlation with hematogenous metastasis. Patients and Methods. We examined 133 cases of colorectal cancer (Dukes A: 72 Dukes B: 26 Dukes C: 23 Dukes D: 12). Sections were cut from formalin-fixed, paraffin-embedded samples containing the deepest site of cancer invasion and stained immunohistochemically with a monoclonal antibody to MMP-1. According to the area of the tumor that was stained, patients were divided into high- and low-MMP-1 expression groups. Results. MMP-1 expression was observed in the cytoplasm of cancer cells, some stromal cells, and a few normal epithelial cells of colonic mucosa. High MMP- 1 expression was found in 47 (35.3%) cases and low in 86 (64.7%). Hematogenous metastasis was identified in 14 (29.8%) of high-MMP-1 groups and 12 (13.9%) of low-MMP-1 groups. MMP-1 expression significantly correlated with hematogenous metastasis of colorectal cancer, but no correlation was found between MMP-1 expression and the other clinicopathological features investigated. Conclusions. MMP-1 expression may be a novel marker for hematogenous metastasis of colorectal cancer, and its inhibition may be a strategy for prevention of metastasis.

BACKGROUND. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that has potent chemotactic activity for endothelial cells. Although it is expressed in the majority of colorectal tumors, and some reports suggest that its high expression is related to poor prognosis, to the authors' knowledge there is yet no consensus regarding whether PD-ECGF expression is a prognostic factor. To investigate the prognostic value of PD-ECGF and its role in tumor angiogenesis, an immunohistochemical study of PD-ECGF expression and tumor vasculature was performed and their relation with the clinicopathologic factors in patients with advanced colorectal carcinoma was evaluated. METHODS. Formalin fixed, paraffin embedded specimens from 86 colorectal carcinoma patients (40 cases in the muscularis propria and 46 cases in the subserosa) were immunostained for PD-ECGF and CD31 as a marker for vascular endothelial cells and expression of PD-ECGF was evaluated using an image analysis system. Patients were divided into high expression and low expression groups based on PD-ECGF expression, and were divided into high vascular grade and low vascular grade groups based on the microvessel density. Correlations between PD-ECGF expression and vascular grade and between PD-ECGF: expression,vascular grade, and the clinicopathologic features of the patients were evaluated statistically. RESULTS. PD-ECGF expression was observed predominantly in the turner stroma and not in tumor cells. The cells that stained strongly for PD-ECGF were confirmed to be macrophages infiltrating the interstitial tissue of the tumor. High PD-ECGF expression was found in 56 cases (65.1%) and low expression was detected in 30 cases (34.9%). Thirty-one of 86 tumors (36.0%) showed high vascular grade and 55 (64.0%) showed lo iv vascular grade. No correlation between PD-ECGF expression and vascular grade was found, but there was an inverse correlation between PD-ECGF expression and the rate of incidence of lymph node and hematogenous metastasis. These correlations were statistically significant. Vascular grade was not found to correlate with the clinicopathologic features, CONCLUSIONS. Patients with high PD-ECGF expression had a lower rate of incidence of lymphatic and hematogenous metastasis, with a consequently better prognosis than patients with low PD-ECGF expression. PD-ECGF expression did not correlate with vascular grade, suggesting that PD-ECGF plays little role in tumor angiogenesis of colorectal carcinoma. Based on these data, the authors conclude that macrophages infiltrating the tumor stroma produce PD-ECGF and play important roles in the immune reaction against the tumor rather than in tumor angiogenesis. Cancer 2000;88:42-9, (C) 2000 American Cancer Society.

The aim of this study was to determine whether colon cancer cells flowing in blood exhibit the same adhesion pattern to the vascular bed as leucocytes using a flow adhesion system. In shear flow conditions, five colon cancer cell lines showed less tethering to E-selectin substrates than polymorphonuclear cells (PMN). However, some of the Colo201 cells formed complete arrest on E-selectin in continuous shear flow which was never observed in PMN cells. Colo201 cells expressed both sialyl Le-x and sialyl Le-a at similar levels in flow cytometry. However, the staining pattern showed marked contrast under the fluorescein microscope. The cell membrane of Colo201 cells was uniformly stained with anti-salyl Le-a MAb, whereas anti-sialyl Le-x MAb only stained in the patchy areas. Pretreatment of Colo201 cells with anti-ale-a decreased tethering, while anti-sle-x significantly inhibited the arrest formation. Our data suggest that E-selectin alone can mediate colon cancer cell lodgement and subsequent metastasis without the contribution of integrin molecules and that the different distribution of E-selectin ligands may affect the adhesion behaviour of colon cancer cells in flow conditions. (C) 2000 Elsevier Science Ltd. All rights reserved.

Under the physiological shear condition, cultured colon cancer cells bound to laminin (LM), but not to fibronectin or vitronectin. Most of the tethered cells did not roll, but arrested immediately and spread within 10-30 min on LM under the continuous presence of shear flow. The tethering of Colo201 was partially inhibited by monoclonal antibodies (mAbs) to alpha 6 integrin and a combination of mAbs to beta 1 and beta 4 integrins, but not by mAb to 67KD laminin receptor. Some Colo201 cells still tethered at 4 degrees C. This suggests that alpha 6 beta 1 and alpha 6 beta 4 integrins participate in Colo201 tethering on LM, although other non-integrin molecules play roles. In contrast, the spread of Colo201 was effectively inhibited by the mAbs to integrin alpha 2, alpha 6 and beta 1 chains. The effect of anti-alpha 2 plus anti-alpha 6 mAbs was almost equal to anti-beta 1, suggesting that Colo201 cells mainly use alpha 2 beta 1 and alpha 6 beta 1 integrins for spreading on LM. When the cells were perfused on subconfluent endothelial cells (HUVEC) cultured on LM, they did not tether on HUVEC but did on coated LM exposed at intercellular gap area. Immunohistochemistry revealed that LM abundantly existed in the cytosol of human portal and hepatic vein endothelial cells. These data suggest that LM can mediate from tethering to spreading of colon cancer cells under the blood flow and plays an essential role in haematogeneous metastasis.

A unique subset of T cells that co-express NKR-P1, which is a lectin type of NK receptor and is thought to have a major role in triggering NK activity, has been identified. In mice, NK1.1 (mouse NKR-P1C)(+) T cells, called NKT cells, preferentially accumulate in the liver and bone marrow. They predominantly use invariant V alpha 14 chain VCR and phenotypically are CD4(+)CD8(-) or CD4(-)CD8(-) T cells. In this study, we analyzed, phenotypically and functionally, the NKR-P1A (analogue of murine NKR-P1C)(+) T cells resident: in the human liver. Here, we show that in complete contrast to the NKT cells in the mouse liver, the majority of NKR-P1A(+) T cells in the human liver are CD8(+) and their TCR repertoire is not skewed to V alpha 24 TCR, the homologue of murine V alpha 14 TCR. Almost all of the NKR-P1A(+) T cells in the human liver expressed CD69, suggesting that they were activated. Furthermore, the NKR-P1A(+) T cells in the human liver exhibited strong cytotoxicity against a variety of tumor cell lines including K562, Molt4 and some colonic adenocarcinoma cell lines.

Integrins play an important role in various lymphocyte functions. In this study, we isolated lamina propria lymphocytes (LPL) and tumor-infiltrating lymphocytes (Tn) from normal and malignant tissues in patients with colorectal cancer, and examined the expression of beta 1 and beta 2 integrins on these lymphocytes quantitatively with two-color flow cytometry, Both LPL and Tn expressed a lower level of common beta 1 chain (CD29) in CD4 and CD8 subpopulations than did peripheral blood lymphocytes (PBL). Among the associated alpha chains, the expression levels of alpha 1 (CD49a) and alpha 2 (CD49b) were slightly higher, whereas those of alpha 4 (CD49d) and alpha 6 (CD49f) were markedly reduced in LPL and Tn. No significant differences were observed in expressions of any beta 1 integrin chains between these two lymphocytes populations. Similarly, both alpha L (CD11a) and beta 2 (CD18) were down-regulated in TIL and LPL with CD8(+) cytotoxic phenotype, but not in these with CD4 phenotype. CD8(+) TIL expressed a slightly but significantly higher level of alpha L beta 2 than did CD8 LPL. CD8(+) LPL and CD8(+) TIL consistently showed significantly decreased binding to purified ICAM-1, VCAM-1 and HT29 colon cancer cells as compared with CD8(+) PBL. Although CD8(+) TIL showed a slightly higher level of adhesion to these substrates than did CD8(+) LPL, the level was much lower than that in PBL. The expression pattern and functional down-regulation of these integrins may be one of the reasons why TIL cannot eradicate the cancer cells in colorectal cancer.

Background: p21Waf1/Cip1 (p21), p27Kip1 (p27), p53, and Rb play critical roles in cell cycle regulation and may influence the clinical behavior of tumors. We examined whether their expression is useful to predict survival of patients with esophageal squamous cell carcinoma (ESC). Methods: Expression of p21, p27, p53, and Rb was studied by the immunohistochemical method in specimens from 62 patients with curatively resected ESC tumors and scored by a computerized image analysis system. Results: The median expression scores of p21, p27, p53, and Rb (14, 12, 27, and 50, respectively) were used as cut-off points to define low and high expression groups for each protein. The 5-year survival rate for the high p21 expression group was 68%; that for the low expression group was 31% (P = .0062). p27, p53, and Rb were not correlated with overall survival. When patients were categorized into four groups based on p21 expression level and lymph node involvement (pN), the survival curves were significantly different (P = .0017). Thus, patients without lymph node involvement but with low p21 expression had survival similar to that of patients with lymph node involvement and high p21 expression. Multivariate analysis showed that age (P = .0102), lymph node involvement (P = .0076), and p21 (P = .0276) were independent prognostic factors, Conclusions: Expression of p21 is an independent prognostic factor in curatively resected ESC. Definition of new subgroups of patients based on p21 expression may help to enhance the stratification of stage.

Integrins play an important role in various lymphocyte functions. In this study, tumor-infiltrating lymphocytes (TIL) were isolated from colorectal cancer tissues and the expression of beta 1 and beta 2 integrins on the TIL was quantitatively examined with two-color flow cytometry. In comparison with peripheral blood lymphocytes (PBL), TIL expressed a lower level of common beta 1 chain (CD29) in both CD4 and CD8 subpopulations. Among the associated a chains, the expressions of alpha 1 (CD49a) and alpha 2 (CD49b) were slightly higher in TIL than in PBL, whereas alpha 4 (CD-49d) and alpha 6 (CD49f) were markedly downregulated in TIL. Both alpha L (CD11a) and beta 2 (CD18) were reduced in CD8(+) TIL but not in CD4(+) TIL, TIL with the CD8(+) cytotoxic phenotype showed significantly decreased binding to purified intracellular adhesion molecules (ICAM)-1, and vascular adhesion cell molecule (VCAM)-1, and HT29 colon cancer cells, compared with the in counterparts in PBL, The peculiar expression pattern and functional down regulation of these integrins may explain why TIL in colorectal cancer cannot eradicate the malignant cells.

Background: The frequency of lymph node metastasis in mucosal gastric cancers 2-4 cm in diameter was low (three (1.3 per cent) of 234) in patients treated in this unit between 1966 and 1995. This study was a prospective report on local resection with lymphadenectomy for early gastric cancer. Methods: Eight patients with a single early gastric cancer underwent local resection with lymphadenectomy. The tumour was excised with a non-cancerous rim of approximately 2 cm. The extent of lymphadenectomy depended on tumour location. Intraoperative endoscopic examination and frozen-section analysis of the dissected nodes were used to determine the resection line and evaluate nodal status. Results: Mean operating time, blood loss and number of dissected nodes were 171 min, 87 ml and 8 respectively. There were no operative complications. Cancer invasion was confined to the mucosa in six tumours but two patients had minute submucosal invasion. The maximum diameter of the resected specimens was 10 cm and no nodal involvement was detected. No patient developed postgastrectomy syndrome. Conclusion: For selected patients with early gastric cancer, local resection with lymphadenectomy can provide a good quality of life without compromising cure rate.

It has been established that lymphocytes obtained from tumor-draining lymph nodes (DLN) are sensitized to the tumor antigen in vivo. Moreover, after being activated in vitro, these cells can be utilized for adoptive immunotherapy. In the present study, DLN cells, obtained from C57BL/6 mice with fibrosarcoma (MC-1), were activated and expanded with anti-CD3 monoclonal antibody followed by culture with recombinant interleukin-2 (rIL-2), These CD4(-) CD8(+) CD25(+) CD44(+) T-cells shelved specific antitumor efficacy to the pulmonary micrometastases of an autologous tumor, against which lymphokine-activated killer cells were ineffective; however, they did not show cytolytic activity in vitro. The supernatant, obtained by coculturing the activated DLN cells with MC-1 cells, exhibited the specific production of interferon-gamma (IFN-gamma) which was enhanced by rIL-2, The therapeutic effect of the activated DLN cells correlated with the specific IFN-gamma production better than with the cytolytic activity.

A technique, by which the root of the right gastric artery can be clearly exposed and the area of the right gastric artery can thus be completely dissected, is presented herein, In conventional procedures, the approach to the right gastric artery is performed from the lessor omentum, and a lymphadenectomy along the common hepatic artery is carried out after the division of the right gastric artery. Using the present technique, the common hepatic artery is exposed before the exposure of the right gastric artery and lymph node dissection is thus continued peripherally to the proper hepatic artery, During these procedures, the root of the right gastric artery arising from the hepatic artery can be easily identified. The lymphatic flow from the area of the right gastric artery to that of the common hepatic artery is transected using the conventional procedure, but not with the present technique. In addition, an en bloc dissection of the regional lymph nodes is also possible when using this procedure.

BACKGROUND. To evaluate the immunologic activity of regional lymph nodes, the phenotype of lymphocytes and the functional expression of cell adhesion molecules (CAMs) on lymph node lymphocytes (LNL-: uninvolved, LNL+: involved) were investigated in patients with gastrointestinal carcinoma. METHODS. The lymphocyte subpopulation and the expression of CD11a, CD44, and CD29 on CD4+ and CD8+ cells in peripheral blood lymphocytes (PBL), LNL-, and LNL+ derived from 37 patients with gastrointestinal carcinoma were studied. In addition, the adherence of CD8+ cells to ICAM-1 which reflects the adhesive function of CD11a, was examined, and changes in this adherence were studied by experimental coculture with cancer cells (DLD-1). RESULTS. Although there were no differences in the overall proportion of T cells between the groups, CD8+ cells and CD16+ cells were considerably diminished in LNL+. The expression of CD11a and CD29 on CD4+ and CD8+ cells was significantly lower in LNL than in PBL, whereas the expression of CD44 showed no significant differences. The expression levels of these CAMs were almost the same in LNL- and LNL+. Only CD11a expression on CD8+ cells in LNL+ was significantly lower than that in LNL- (P < 0.005). The adherence of CD8+ cells in LNL+ to ICAM-1 was lower than that in PBL and LNL-, and was extremely enhanced by experimental coculture with cancer cells (DLD-1). CONCLUSIONS. These data indicate that the functional expression of CD11a (LFA-1) on CD8+ T cells is suppressed in cancer-involved regional lymph nodes in patients with gastrointestinal carcinoma. (C) 1996 American Cancer Society.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate in duced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1, Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors, Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization, Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1, Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant stimulated adhesion of eosinophils to intercellular adhesion molecule 1, Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1, To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

The effect of basic fibroblast growth factor (b-FGF), one of the commonest angiogenic factors in various cancer types, on lymphocyte adhesion and transmigration across the endothelial cell monolayer was investigated using human umbilical vein-derived endothelial cells (HUVEC) and type I collagen gel. Forty-eight h exposure of HUVEC with 2 ng/ml b-FGF significantly decreased the basal adhesion of lymphocytes to endothelial cells. The decrease ratio is further enhanced by the addition of shear stress in this assay system. When HUVEC was stimulated for the last 24 h with optimal conditions of recombinant interleukin 1 beta, the percentages of transmigration as well as adhesion were also decreased significantly by the presence of b-FGF. The expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 was downregulated by b-FGF exposure in both resting and activated conditions by recombinant interleukin 1 beta, supposedly the main reason for this phenomenon. The migrating cells across b-FGF-stimulated HUVEC contained a markedly lower percentage of CD4(+) T-cells than those across nontreated HUVEC, although the 4B4(+)/2H4(+) ratio in CD4(+) T-cell populations did not differ significantly. These facts suggest that the presence of b-FGF in the angiogenic area suppresses lymphocyte emigration, especially that of CD4(c) T-cells, and thus causes insufficient helper function in local immune response. This effect of b-FGF was possibly one of the critical mechanisms by which cancer cells escape from the host immune reactions in the angiogenic stage of tumor development.

We describe a 56-year-old woman with Stewart-Treves syndrome who had severe dyspnea from a pleural effusion caused by metastatic angiosarcoma in the right lung. Tumorinfiltrating lymphocytes (TIL) in the pleural effusion were cultured and expanded in vitro in the continuous presence of recombinant interleukin 2 with periodic stimulation by CD3 antibody. The expanded TIL were administered intrapleurally seven times at 1- to 4-week intervals in combination with intravenous infusion of recombinant interleukin 2. A panel of T-cell clones was also obtained from TIL. Immunotherapy dramatically improved the patient's dyspnea and pleural effusion. A CD4(+) T-cell clone and a CD8(+) T-cell clone established from TIL had specific cytotoxicity to the tumor cells.

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16(+) natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon gamma (rIFN gamma) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1 beta. rTNF alpha or acidic or basic fibroblast growth factor did not alter the lyric sensitivity of HUVEC. The resistance of rIFN gamma-treated HUVEC was specific to lysis by CD16(+) NK LAK cells, and their lysis by CD3(+) T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1 beta-treated HUVEC, rlFN beta-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN gamma treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.

In six patients with advanced pancreatic carcinoma, TIL and tumour-draining lymphocytes (TDL) were isolated from primary pancreatic tumour and regional lymph nodes. In comparison with TDL and peripheral blood lymphocytes (PBL), TIL contained a comparatively higher percentage of TCR gammadelta+ cells, although they were still a small fraction. By 2 weeks culture with rIL-2 and immobilized OKT-3 antibody, the TCR gammadelta + cells in TIL were preferentially expanded at the early culture periods, although it was temporary. In four cases, the TCR gammadelta+ and CD8+ TCR alphabeta+ TIL were separated by negative sorting using flowcytometry. All the TCR gammadelta+ TIL were CD4-, CD8- (double negative), and one of the TIL lines was mostly composed of deltaTCS1+ cells, while the others were deltaTCS1-. In comparison with CD8+ TCR alphabeta+ TIL, all the TCR gammadelta+ TIL exhibited much stronger lytic activity against freshly isolated autologous pancreatic cancer cells. However, all the gammadelta+ TIL also exhibited a strong non-MHC-restricted cytotoxicity, and there was no correlation between the lytic pattern and the percentage of deltaTCS1+ cells. These data suggest that the TCR gammadelta+ T cells can proliferate vigorously in a certain condition, and if successfully expanded in vitro they might be helpful material for effective adoptive immunotherapy.