Akihiro Katayama

Expressions of Matrix Metalloproteinases in Early-Stage Oral Squamous Cell Carcinoma as Predictive Indicators for Tumor Metastases and Prognosis

Purpose: Matrix metalloproteinase (MMP)-2 and MMP-9 are considered to play an important role in the metastasis of malignant tumors. Membrane type 1-MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) are essential factors for the activation of pro-MMP-2. There are some reports about expressions of MMP family in relationship to clinical features of head and neck squamous cell carcinoma (SCC), but the results were not uniform and the prognostic value of their expressions remains unclear. Experimental Design: The study group consisted of 53 Japanese patients with oral SCC of early stage (T1-2N0M0). Expressions of MMP-2, MMP-9, MT1-MMP, and TIMP-2 were examined using immunohistological methods on the sections of tumor biopsy samples. The intensity of MMP expression was categorized into four grades (score 0–3) by semiquantitative analysis using a computer with NIH image, and correlation between this grade and clinical aspects such as tumor recurrence, metastasis, and prognosis were examined. Results: The expression score of MMP-2 correlated with that of MMP-9 (r = 0.291; P = 0.036), MT1-MMP (r = 0.286; P = 0.039), and TIMP-2 (r = 0.257; P = 0.050). Patients who developed regional lymph node and/or distant metastasis showed significantly higher scores in the expressions of MMP-9 and TIMP-2 than patients without any tumor metastases (P = 0.036 and P = 0.043, respectively). Kaplan-Meier analyses as well as univariate analyses using the Cox proportional hazards model showed that expression of MMP-9 (P = 0.0143 and P = 0.0418, respectively) and marked expression of TIMP-2 (P < 0.0001 and P = 0.0004, respectively) correlated with worse-cause-specific survival. Multivariate analysis confirmed that marked expression of TIMP-2 was the only independent factor for cause-specific death (hazard ratio, 7.543; confidence interval, 1.693–33.610; P = 0.0080). Conclusions: Expressions of MMP-9 and TIMP-2 have predictive value for tumor metastases and cause-specific survival. High expression of TIMP-2 is the most independent factor for worse prognosis in early-stage oral SCC.

CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer

BACKGROUND The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb. METHODS CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided. RESULTS CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis. CONCLUSIONS This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma

Background:On the basis of the microRNA (miRNA) expression signature of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-874 was significantly reduced in cancer cells. We focused on the functional significance of miR-874 in cancer cells and identification of miR-874-regulated novel cancer networks in MSSCC.Methods:We used PCR-based methods to investigate the downregulated miRNAs in clinical specimens of MSSCC. Our signature analyses identified 23 miRNAs that were significantly reduced in cancer cells, such as miR-874, miR-133a, miR-375, miR-204, and miR-1. We focused on miR-874 as the most downregulated novel miRNA in our analysis.Results:We found potential tumour suppressive functions such as inhibition of cancer cell proliferation and invasion. A molecular target search of miR-874 revealed that PPP1CA was directly regulated by miR-874. Overexpression of PPP1CA was observed in MSSCC clinical specimens. Silencing of the PPP1CA gene significantly inhibited cancer cell proliferation and invasion.Conclusion:The downregulation of miR-874 was a frequent event in MSSCC, which suggests that miR-874 functions as a tumour suppressive miRNA, directly regulating PPP1CA that has a potential role of an oncogene. The identification of novel miR-874-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Expression of CXCR4 and Its Down-Regulation by IFN-γ in Head and Neck Squamous Cell Carcinoma

Purpose: The functional expression of CXCR4, which plays roles in cell migration and proliferation in response to its unique ligand stromal cell–derived factor-1 (SDF-1), has been reported in variety of carcinomas. However, CXCR4 expression and its functional role in head and neck squamous cell carcinomas (HNSCC) remain unclear. In this study, we investigated CXCR4 expression and analyzed its functions in HNSCC cell lines. We also attempted to regulate CXCR4 expression using cytokines, such as interleukin-1β, tumor necrosis factor-α, and IFN-γ. Finally, we investigated correlation between CXCR4 expression and clinical features in patients with HNSCC. Experimental Design: Six HNSCC cell lines were used in this study. Reverse transcription-PCR and flow cytometry analysis were shown for CXCR4 expressions with or without stimulations of cytokines. SDF-1-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. The SDF-1-mediated cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The SDF-1-mediated signaling pathways were analyzed by Western blot analysis. Biopsy specimens from 56 patients with HNSCC were used for immunohistologic analysis. Results: The significant CXCR4 expression was found in HSQ-89, IMC-3, and Nakamura cells. The SDF-1-mediated cell migration and proliferation were observed in CXCR4-positive cells. SDF-1 also promoted rapid phosphorylation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways in CXCR4-positive cells. The SDF-1-mediated cell migration and proliferation of CXCR4-positive cells were inhibited by neutralization of CXCR4. Among three cytokines tested, IFN-γ significantly reduced CXCR4 expression and SDF-1-induced cell migration and proliferation of CXCR4-positive cells. Immunohistologic analysis revealed that patients with advanced neck status and patients who developed distant metastases showed significantly higher CXCR4 expression, and the cause-specific survival of patients with CXCR4-expression was significantly shorter. Furthermore, multivariate analysis confirmed that CXCR4 positive was the independent factor for cause-specific death. Conclusion: Our results may provide an insight into future therapeutic agent that inhibits tumor metastasis and progression via down-regulating CXCR4 expression in patients with HNSCC.

Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

Production of Interferon-γ–Inducible Protein-10 and Its Role as an Autocrine Invasion Factor in Nasal Natural Killer/T-Cell Lymphoma Cells

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with Epstein-Barr virus and has poor prognosis because of local invasion and/or multiple dissemination. Recently, the role of chemokines/chemokine receptors in tumor proliferation and invasion has been shown. In this study, we examined whether the specific chemokines were related to the tumor behaviors in nasal NK/T-cell lymphoma. Experimental Design: A chemokine protein array was used to examine specific chemokines produced by SNK-6 and SNT-8 (Epstein-Barr viruspositive nasal NK/T-cell lymphoma lines). The expression of interferon inducible protein 10 (IP-10) and the IP-10 receptor CXCR3 was investigated by ELISA and flow cytometry. Cell growth and invasion were assessed by the MTT and Matrigel invasion assays, respectively. Immunohistologic staining and ELISA were used to examine IP-10 expression in biopsies and sera from patients, respectively. Results: IP-10 was specifically produced by SNK-6 and SNT-8. Moreover, CXCR3 was expressed on the NK cell lines. Functionally, IP-10 did not affect cell proliferation but enhanced cell invasion. In biopsy samples, IP-10 and CXCR3 expressions were detected in the lymphoma cells. Serum IP-10 levels in the patients were much higher than those of healthy controls and the levels were decreased during the complete remission phase after treatments. Conclusions: These results suggest that IP-10 may play an important role in cell invasion in nasal NK/T-cell lymphoma through an autocrine mechanism. (Clin Cancer Res 2009;15(22):67719)

Expression of B7-H3 in hypopharyngeal squamous cell carcinoma as a predictive indicator for tumor metastasis and prognosis

B7-H3 is a member of the B7 family thought to be a co-regulatory factor of antigen-specific T-cell immune response via co-stimulatory and co-inhibitory receptors. We evaluated its potential expression in head and squamous cell carcinoma (SCC) cell lines, and in clinical tissue samples obtained from 37 patients with human hypopharyngeal SCC. All head and neck SCC cell lines tested expressed both the B7-H3 gene and cell surface protein. The staining intensity of immunoreactivity by tumor cells was blindly evaluated by two head and neck surgeons and the results were categorized into 4 grades according to staining intensity. Eighty-seven percent of patients expressed B7-H3. B7-H3 expression was inversely correlated with the number of tumor infiltrating CD8+ T-cells (r=-0.4339, p=0.023). Patients who developed distant metastasis after tumor-free periods showed significantly higher B7-H3 expression scores compared to patients who did not develop distant metastasis during follow-up periods (p=0.048). Distant metastasis control ratio in patients with strong B7-H3 expression was significantly lower compared to that in patients with no to intermediate B7-H3 expression (p=0.040). Cause-specific survival ratio in patients with strong B7-H3 expression was significantly lower compared to that in patients with no to intermediate B7-H3 expression (p=0.028). Moreover, multivariate analysis revealed that strong B7-H3 expression was an independent prognostic factor in tumor-specific death in hypopharyngeal SCC (hazard ratio: 9.803, confidence interval: 0.018-0.539, p=0.0110).

Inducible nitric oxide synthase expression in various laryngeal lesions in relation to carcinogenesis, angiogenesis, and patients' prognosis.

Conclusions: The inducible nitric oxide synthase (iNOS) expression leading to vascular endothelial growth factor (VEGF) overexpression may be useful as a factor for predicting recurrence after initial treatment and prognosis in laryngeal squamous cell carcinoma (SCC). Objective: We analyzed expression of iNOS, p53, and VEGF in various laryngeal lesions. Materials and methods: The study samples consisted of 63 SCC, 20 dysplasia, 7 polyp, and 5 normal epithelium of the larynx. The expression of iNOS, p53, and VEGF was identified by immunohistological methods. Results: No positive immunostaining for iNOS, p53, and VEGF was observed in normal epithelium and polyps. In contrast, with the progression from mild/moderate dysplasia to severe dysplasia to carcinoma, their expression levels increased. In dysplasia, there was a significant positive correlation among expression of iNOS, p53, and VEGF. In SCC, iNOS expression correlated with VEGF overexpression and microvessel density, but not with p53 overexpression. In SCC, the expression of iNOS and VEGF significantly increased in patients who developed local recurrence and/or metastases after initial treatments. Kaplan–Meier analysis showed that disease-free survival was significantly shorter in patients with iNOS or VEGF expression. Multivariate analysis showed expression of iNOS and VEGF as independent indicators for poor disease-free survival.

Laryngeal plexiform schwannoma as first symptom in a patient with neurofibromatosis type 2

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by bilateral vestibular schwannomas. The initial symptoms of NF2 are usually hearing loss and tinnitus caused by vestibular schwannomas. Although other intracranial, spinal, or skin tumors also occur in NF2, laryngeal lesions are very rare. We report a rare case of NF2 with laryngeal plexiform schwannoma as first symptom. A 38-year-old man presented with a smooth-surfaced submucosal supraglottic mass. Two round masses in the left chest wall and left supraclavicular fossa were noted incidentally during investigation of the laryngeal mass. Magnetic resonance imaging (MRI) findings for these masses were identical to those of the laryngeal mass. No typical symptoms related to NF2 were identified. Histologically, the laryngeal tumor represented plexiform schwannoma. We thus considered that the two round masses in the left chest wall and left supraclavicular fossa might also represent plexiform schwannomas. NF2 was suspected, as a high incidence of multiple plexiform schwannomas has been suggested for patients with NF2. MRI of brain lesions demonstrated bilateral vestibular schwannomas and multiple meningiomas. Finally, NF2 with laryngeal plexiform schwannoma was diagnosed. Recognizing that multiple plexiform schwannomas could be associated with NF2 is important.

Loss of p21 expression is associated with p53 mutations and increased cell proliferation and p27 expression is associated with apoptosis in maxillary sinus squamous cell carcinoma

Conclusions. Loss of p21 expression dependent on the p53 mutation may be associated with higher tumor cell proliferation, and low p27 expression may be associated with decreased spontaneous apoptosis, resulting in poorer prognosis in patients with maxillary sinus squamous cell carcinoma (SCC). Objective. We have previously reported that p53 mutations and decreased spontaneous apoptosis were associated with poor prognosis in maxillary sinus SCC. However, whether p21 and p27 expression and cell proliferation correlate with either p53 status, spontaneous apoptosis or prognosis in maxillary sinus SCC has not been evaluated. Material and methods. Seventy patients with maxillary sinus SCC were analyzed. Tumor biopsy specimens were examined for p21 and p27 expression using an immunohistological method. The percentage of proliferating cells labeled by anti-Ki-67 mAb was expressed as the Ki-67 index (KI). Results. Loss of p21 expression correlated with p53 mutations (p=0.0072). The KIs in patients without p21 expression and with p53 mutations were significantly higher than those in patients with p21 expression (p=0.0119) and those without p53 mutations (p=0.0048). Patients with p27 expression showed a significantly higher apoptotic index than those without (p=0.0012). Kaplan–Meier analysis showed that p21 expression was closely associated with prolonged disease-free survival in the group with a normal p53 status (p=0.0472). Multivariate analysis identified high KI as an independent prognostic marker (p=0.047).