Molecular analyses of cell origin and detection of circulating tumor cells in the peripheral blood in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a distinct, rare soft tissue tumor with an unknown histogenesis and a tendency for late widespread metastases to lung, bone, and brain. It is now clear that they are caused by a specific unbalanced translocation, der(17)t(X;17)(p11;q25), which results in the formation of an ASPSCR1-TFE3 (alias ASPL-TFE3) fusion gene. The rearrangement results in the expression of chimeric transcripts, which can be identified by means of reverse transcriptase-polymerase chain reaction (RT-PCR). We investigated the histogenesis of ASPS and attempted to detect circulating ASPS tumor cells in peripheral blood. The immunohistochemical and genetic details of four cases and one cell line of ASPS were examined. An immunohistochemical analysis and RT-PCR did not detect myogenic differentiation gene MYOD1. The sensitivity of nested RT-PCR for detection of circulating ASPS cells was assessed by demonstrating that the tumor cell-associated gene translocation could be detected in 50 tumor cells/2 mL of blood. Clinically, it was detectable in a peripheral blood sample (2 mL) of ASPS patient with distant metastases. The findings suggest that ASPS is not of skeletal muscle origin. ASPS tumor cells in the peripheral blood could be monitored by RT-PCR.

Antitumor activity of a combination of trastuzumab (Herceptin) and oral fluoropyrimidine S-1 on human epidermal growth factor receptor 2-overexpressing pancreatic cancer.

The cytotoxic effect of trastuzumab in combination with oral fluoropyrimidine S-1 on human epidermal growth factor receptor 2 (HER2)-overexpressing human pancreatic cancer cell line TRG in vitro and in vivo was investigated. HER2 expression in TRG was analyzed by RT-PCR and flow cytometry. For in vitro experiments, 5-fluorouracil (5-FU) was used instead of S-1. In vivo studies were conducted with TRG xenografts in athymic mice. Trastuzumab (10 mg/kg) was administered intraperitoneally once a week for 4 weeks. S-1 (10 mg/kg) was administered orally 5 days a week for 4 weeks. The results showed that TRG cells were positive for HER2 mRNA and overexpressed HER2 protein. Either trastuzumab or 5-FU concentration-dependently inhibited the growth of TRG cells. The combination of trastuzumab and 5-FU resulted in a significant inhibition of growth of TRG cells compared to either agent alone (P<0.001). Incubation of TRG cells with peripheral blood mononuclear cells after treatment with trastuzumab enhanced the antiproliferative effect of trastuzumab, which could be the result of antibody-dependent cellular cytotoxicity. The combination of trastuzumab and S-1 resulted in a significant reduction in xenograft volume compared to each agent alone (P<0.0001). In conclusion, this study showed that combination therapy with trastuzumab and S-1 may be effective for HER2-overexpressing pancreatic cancer patients.

Expression of MMP-7 and MT1-MMP in peritoneal dissemination of gastric cancer.

BACKGROUND/AIMS: In this study, we examined the expression of MMP-2, MMP-7, and MT1-MMP in peritoneal dissemination of gastric cancer, so as to clarify a possible role of these MMPs in developing peritoneal dissemination, using culture cells and an animal model with peritoneal dissemination. METHODOLOGY: Total RNA was extracted from tumor tissues of disseminated foci from 7 patients with gastric cancer and human gastric cancer cell lines of STSA, STKM-1, MKN-28, MKN-45, and KATOIII. Expressions of mRNA for MMP-2, MMP-7, and MT1-MMP were analyzed by reverse transcriptase-polymerase chain reaction. To examine relationships between the expression of these mRNAs and the ability to establish peritoneal dissemination, nude mice were injected into the intraperitoneal cavity with 106 cultured cells of those 5 gastric cancer cell lines. RESULTS: MMP-7 was expressed in 6 of 7 tissues (85.7%) and MT1-MMP in 2 of 7 tissues (28.6%), while MMP-2 was not detected in any of 7 tumor tissues. All 7 tumors had either MMP-7 or MT1-MMP. MMP-7 was recognized in 4 of 5 cells (80%) and MT1-MMP in 2 of 5 cells (40%), while MMP-2 was not found at all. All 5 cancer cells expressed at least one MMP mRNA. In the animal experiments, nude mice inoculated with STKM-1 or MKN-45 cells developed peritoneal dissemination, while those with other cell lines did not. MMP-7 was found both in STSA and MKN-28 (dissemination negative), and in STKM-1 and MKN-45 (dissemination positive). MT1-MMP mRNA was detected in one of two dissemination positive cell lines and in one of three dissemination negative ones. CONCLUSIONS: Our results suggested the importance of MMP-7 and MT1-MMP in the peritoneal metastases of gastric cancer, however, it has to be further dissected what role these MMPs might play in detaching of cancer cells from the gastric wall and establishing peritoneal disseminating foci.

Evaluation of soluble adhesion molecules CD44 (CD44st, CD44v5, CD44v6), ICAM-1, and VCAM-1 as tumor markers in head and neck cancer.

PURPOSE: Standard CD44 (CD44st), CD44 variant 5 (CD44v5), and CD44 variant 6 (CD44v6), intercellular adhesion molecule 1(ICAM-1), and vascular cell adhesion molecule 1(VCAM-1) are expressed in human malignant cells and tissues. Their mechanism remains unclear but has been reported to be associated with the progression and metastasis of malignancies. MATERIALS AND METHODS: In this study, we investigated any correlations between the soluble adhesion molecule CD44 (st, v5, and v6), ICAM-1, and VCAM-1 and the clinicopathologic variables (eg, age, sex, histological grading, tumor size, lymph node status, distant metastasis, and TNM staging) and evaluated the difference between the pretreatment level in the patients with head and neck cancer and that in the control group. Furthermore, we examined the difference between the pretreatment serum levels and the after-treatment serum levels in the group with head and neck cancer. The pretreatment and after-treatment serum levels of soluble CD44st, CD44v5, CD44v6, ICAM-1, and VCAM-1 were measured in 81 patients with head and neck cancer and in 20 healthy volunteers as controls. RESULTS: There were no significant differences between the serum levels of sCD44st, sCD44v5, sCD44v6, sICAM-1, and sVCAM-1 and the clinicopathologic variables in cancer patients. However, the higher serum level of sCD44v6 was significantly associated with distant metastasis (P = .02). Especially, we found that the pretreatment serum levels of sCD44st, sCD44v5, and sCD44v6 were markedly associated with TNM staging (CD44st P = .0017, CD44v5 P = .0005, CD44v6 P = .0046). Furthermore, the median serum levels of sCD44st, sCD44v5, sCD44v6, sICAM-1, and sVCAM-1 before treatment of head and neck cancer were significantly higher than those of the control group (CD44st P = .0067, CD44v5 P = .0048, CD44v6 P = .0007, ICAM-1 P = .0089, VCAM-1 P = .0178). The median serum level of sCD44st after treatment in the group of patients was significantly lower than that of pretreatment (CD44st P = .0001). And, both the median serum levels of sCD44v5 and sCD44v6 after treatment were also lower than those of pretreatment (CD44v5 P = .0004, CD44v6 P = .0025). CONCLUSIONS: The possible roles of soluble adhesion molecules in the prognosis of head and neck carcinoma deserve further elucidation and evaluation with long-term patient follow-up.

Expression of SPARC in tongue carcinoma of stage II is associated with poor prognosis: an immunohistochemical study of 86 cases.

SPARC (secretory protein acidic and rich in cysteine), also known as osteonectin or BM-40, associates with progression in various kinds of tumors. We have examined whether SPARC expression can be a prognostic marker for patients with head and neck squamous cell carcinomas (HN-SCC). We examined immunolocalization of SPARC in 86 clinical specimens of tongue carcinoma. Although there was no correlation between SPARC positivity in the tumor cells and tumor stages, the 5-year overall survival rate was significantly lower in the SPARC positive cases (28.6%) than in the SPARC negative cases (91.7%), confined to stage II patients (p < 0.001, Wilcoxon test). Additionally, in stage II cases (n = 3), frequency of the postoperative metastasis was significantly higher in SPARC positive cases (5/8, 62.5%) than in the negative cases (1/15, 6.7%) (p < 0.01, chi2 test). Together with these results, SPARC can be a beneficial prognostic marker for the stage II tongue carcinoma, of which clinical outcomes are sometimes difficult to predict.

The F(ab)'2 fragment of an Abeta-specific monoclonal antibody reduces Abeta deposits in the brain.

This work examines whether administering the F(ab' )2 fragment of an IgG1 monoclonal antibody (mAb) targeting the N-terminal 1-13 amino acids of the beta-amyloid peptide (Abeta mAb) reduces amyloid deposition in Alzheimer's disease (AD). The F(ab')2 fragment was injected intraperitoneally or intracranially into Tg2576 mice, a murine model of human AD. Both routes of administration significantly reduced Abeta plaque formation in the brain, as determined immunohistochemically and by monitoring levels of Abeta1-40 and Abeta1-42 peptide. Use of the F(ab')2 fragment significantly reduced phagocytic infiltration in the CNS when compared to intact mAb. Since IgG1 Abs do not fix complement, these findings suggest that effective in vivo clearance of amyloid deposits can be achieved without stimulation of FcR-reactive phagocytes or activation of the complement cascade.

Soluble CD44 standard, CD44 variant 5 and CD44 variant 6 and their relation to staging in head and neck cancer.

CONCLUSION: The possible roles of CD44st, CD44v5 and CD44v6 in the prognosis of head and neck cancer deserve further elucidation and evaluation with long-term patient follow-up. OBJECTIVE: Standard CD44 (CD44st), CD44 variant 5 (CD44v5) and CD44 variant 6 (CD44v6) are expressed in human malignant cells and tissues. The mechanism of their expression remains unclear, but has been reported to be associated with the progression and metastasis of malignancies. Recently, it has frequently been reported that the prognosis of head and neck cancer is associated with expression of the cell adhesion molecule CD44. MATERIAL AND METHODS: We investigated correlations between the soluble adhesion molecule CD44 and clinicopathologic variables, for example, age, sex, histologic grade, tumor size, lymph node status, distant metastasis and TNM stage. The pre- and post-treatment serum levels of CD44st, CD44v5 and CD44v6 were determined by means of ELISAs in 81 patients with head and neck cancer and 20 healthy volunteers (controls). RESULTS: In the cancer patients, the pre-treatment median serum levels of CD44st, CD44v5 and CD44v6 were 327 +/- 134, 312 +/- 118 and 211 +/ 110 ng/ml, respectively. The corresponding post-treatment levels were 185 +/- 103, 177 +/- 90 and 110 +/- 65 ng/ml. In the healthy volunteers, the median serum levels of CD44st, CD44v5 and CD44v6 were 133 +/- 40, 142 +/- 39 and 86 +/- 22 ng/ml, respectively. In the cancer patients, there was no significant correlation between the serum levels of CD44st, CD44v5 and CD44v6 and the clinicopathological variables. The pre-treatment serum levels of CD44st, CD44v5 and CD44v6 were closely associated with TNM stage (p = 0.0017, 0.0005 and 0.0046, respectively). The median pre-treatment serum levels of CD44st, CD44v5 and CD44v6 were significantly higher than those in the control group (p = 0.0002, 0.0065 and 0.0038, respectively). The median post- treatment serum levels of CD44st, CD44v5 and CD44v6 were significantly lower than the pre-treatment levels (p = 0.0003, 0.0027 and 0.0034, respectively).

Induction of lymphokine-activated cytotoxic T lymphocytes stimulated by dendritic cells and autologous tumor from a patient with gastric cancer and their effects in vitro.

BACKGROUND/AIMS: The purpose of the study was to generate lymphokine-activated cytotoxic T lymphocytes stimulated by dendritic cells (DC) and autologous tumor from a patient with gastric cancer and to clarify their cytotoxic effects in vitro. METHODOLOGY: DC was induced by interleukin-4 (IL-4) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) from the peripheral blood mononuclear cells (PBMC). Then, PBMC was incubated with mitomycin C-treated tumor cells and DC, and following that was activated with IL-2 and anti-CD3. Induction of DC and cytotoxic T cells (CTL) were confirmed by the analyses of the cell surface antigens, killing activities, and blocking tests. RESULTS: Induction of DC and cytotoxic T cells (CTL) was confirmed by the analyses of the cell surface antigens, killing activities, and blocking tests. In vitro study demonstrated that lymphokine-activated lymphocytes pulsed by DCs and autologous tumor contained the largest population of CTLs, the greatest production of IFN-gamma, and the greatest ATK activity. CONCLUSIONS: Those results indicated that CTLs could be generated in vitro from a patient with gastric cancer more successfully by this method than by conventional methods, suggesting the possibility of a new immunotherapy for the treatment of gastric cancer.

Reovirus oncolysis in human head and neck squamous carcinoma cells.

OBJECTIVE: To determine whether reovirus, a double-standed RNA virus is effective on the growth of a human head and neck squamous cell carcinoma cell line. DESIGNS: In vitro cell proliferation assay, KB cells, a human oral floor squamous cell carcinoma cell line, were treated with reovirus and the number of cells was quantitated by an assay, using trypan blue staining. In vivo tumor growth assay, KB cells were injected subcutaneously into athymic nude mice, which were given an intratumoral injection of reovirus to a maximum four times in every week. The tumor size was measured once a week. Simultaneously, apoptosis and necrosis of KB cells were investigated, using technique of immunohistochemistry. RESULTS: In vitro, the multiplication of the KB cell was inhibited depending on the concentration of reovirus. In vivo, athymic nude mice bearing KB tumors were injected with the virus intratumorally, and the tumor growth was suppressed proportionally depending on the injection time of reovirus. Necrosis was recognized extensively in the pathological specimen. On the other hand, apoptosis-inducing effect was not obvious in these specimen. CONCLUSIONS: Reovirus suppressed tumor growth of KB cells in vivo as well as in vitro. The possibility that reovirus could become the means of treatment for head and neck carcinoma, was suggested with further work.

Effects of TS-1 on peritoneal dissemination of gastric cancer in nude mice.

BACKGROUND/AIMS: We investigated the effects of TS-1 on the survival of nude mice developing peritoneal dissemination of gastric cancer. METHODOLOGY: MKN-45 cells were injected into the peritoneal cavity of nude mice and a model of peritoneal dissemination was developed. TS-1 was administered orally every day from day 1 to day 10 or day 10 to day 19. RESULTS: Survival time of these treatment groups was significantly longer than untreated controls. In a pharmacokinetic study, TS-1 was administered on day 10 and the 5-fluorouracil levels were retained and maintained for a longer time, in the ascites and tumor than in plasma. The area under the concentration curve for 5-FU in the tumor was higher, than in plasma or ascites. CONCLUSIONS: TS-1 could be effective in treating peritoneal dissemination of gastric cancer, due to the supply of 5-fluorouracil in the tumor by systemic and intraperitoneal circulation.

Prognostic impact of tissue inhibitor of matrix metalloproteinase-1 in plasma of patients with colorectal cancer.

BACKGROUND: Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in plasma has been reported to be related to disease progression in patients with colorectal cancer. However, the prognostic significance of plasma TIMP-1 has not been clarified. PATIENTS AND METHODS: Concentrations of TIMP-1 protein were measured by enzyme-linked immunosorbent assay in plasma samples of 87 preoperative patients who subsequently underwent resection, and prognosis was compared. The cut-off value of plasma TIMP-1 was defined as 170 ng/ml. RESULTS: When clinicopathological factors between patients with positive and those with negative plasma TIMP-1 were analyzed, significant differences were observed in lymph node metastasis, serosal invasion, curability and Dukes' classification. Univariate analysis of these factors demonstrated that depth of invasion, metastases to lymph nodes, peritoneum, liver and distant organ, lymphatic and vessel invasions, curability. Dukes' classification and plasma TIMP-1 concentration were significant. By multivariate analysis excluding patients with distant or peritoneal metastases, histological type, lymphatic invasions, lymph node metastasis and plasma TIMP-1 were retained in the final model. CONCLUSION: These results suggested that plasma TIMP-1 may be a useful prognostic marker for survival in patients with colorectal carcinoma.

Expression of E-cadherin, and CD44s and CD44v6 and its association with prognosis in head and neck cancer.

OBJECTIVES: In the current study, the expression of E-cadherin, CD44s, and CD44v6 has been noted as markers for tumor metastasis and prognosis in several tumors, so we examined whether or not E-cadherin, CD44s, and CD44v6 are useful markers for evaluating the prognosis of mesopharyngeal cancer patients. METHODS: The expression of E-cadherin, CD44s, and CD44v6, was evaluated immunohistochemically using monoclonal antibodies against epitopes of standard and variant proteins, in paraffin-embedded mesopharyngeal cancer tissues from 57 patients who had received curative therapy. RESULTS: Tumor tissues from 47 (82.5%) patients showed positive immunoreactivity with monoclonal antibody against E-cadherin, 43 (75.4%) patients showed positive expression with CD44, and 45 (78.9%) patients showed positive expression with CD44v6. The expression of CD44v6 was slightly correlated with tumor volume, and lymph node metastasis, and stage classification (P > 0.05). However, there was no significant correlation between the expression of E-cadherin, CD44s and CD44v6 and clinicopathological characteristics. Concerning the prognosis, the survival period of patients with CD44s positive tumors was shorter than that of patients with CD44s negative tumors (18.2% versus 52.1%, 5-year survival, P > 0.05). The survival period of patients with CD44v6 positive tumors was also shorter than that of patients with CD44v6 negative tumors (12.8% versus 55.6%, 5-year survival, P > 0.05). CONCLUSION: These results suggest that CD44v6 may be related to tumor invasion and metastasis, and both CD44s and CD44v6 may be useful markers for poor prognosis in head and neck cancer.

Reduction of in vivo tumor growth by MMI-166, a selective matrix metalloproteinase inhibitor, through inhibition of tumor angiogenesis in squamous cell carcinoma cell lines of head and neck.

Matrix metalloproteinases (MMPs) have been implicated in tumor invasion, metastasis, and angiogenesis. We have recently shown that MMI-166, a new orally active MMP inhibitor specific for MMP-2 and -9, suppressed experimental metastasis of Lewis lung cancer, C-H1 human colon cancer, and pancreatic cancer without affecting tumor growth in vitro. In the present study, we determined whether oral administration of MMI-166 reduces tumor growth not only in such tumors but also in squamous cell carcinoma of head and neck (SCCHN). MMI-166 inhibited both activity of MMP-2 and -9 without affecting steady state levels of their mRNAs in SCCHN. Interestingly, protein levels of MMP-2 and -9 from the cultures were drastically diminished by culturing with MMI-166. This was also observed in xenografts of MMI-166-administered mice. In addition, daily oral administration of MMI-166 (100mg/kg) inhibited local tumor growth accompanied by the reduction of blood vessel density and Ki-67-positivity and increase in terminal deoxynucleotidyl transferase-mediated cUDP nick-end labeling (TUNEL)-positivity. These results suggested that orally administered MMI-166 reduced in vivo tumor growth of SCCHN through inhibition of angiogenesis and induction of apoptosis accompanied by the reduction of MMP productions and activities. Therefore, MMI-166 seems to be useful for tumor dormancy therapy of SCCHN.