Kan Kishibe

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.

Prognostic value of p53 mutations, bax, and spontaneous apoptosis in maxillary sinus squamous cell carcinoma

Many researchers have attempted to correlate p53 mutation and spontaneous apoptosis with the effectiveness of radiochemotherapy and with prognosis in several malignancies.

Nasal natural killer (NK)/T-cell lymphoma: clinical, histological, virological, and genetic features

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is a clinical illness characterized by progressive unrelenting ulceration and necrosis of the nasal cavity and midline facial tissues. Histological features of the lymphoma include angiocentric and polymorphous lymphoreticular infiltrates, called polymorphic reticulosis. Surface antigens and the NK-cell marker, CD56, as well as pan-T antigen CD2, cytoplasmic CD3 (CD3ɛ), and CD45 are expressed in the lymphoma cells. The origin of the lymphoma is thought to be either NK-cell linkage without T-cell receptor (TCR) rearrangement or γδT-cell linkage with γδTCR rearrangement. Since the authors of this study first demonstrated the presence of Epstein Barr virus (EBV)-DNA and EBV oncogenic proteins in NNKTL, the lymphoma has been classified as one of the EBV-associated malignancies. The NNKTL cells produce interleukin (IL)-9, IL-10, and interferon-γ-inducible protein-10 (IP-10), possibly due to EBV-oncogenic proteins in the lymphoma cells, and such cytokines take an important part in the cell proliferation and invasion, acting in an autocrine manner. Clinically, the serum EBV-DNA copy number is useful as a specific tumor marker and a predictive prognostic factor. Even in early clinical stages, the lymphoma shows poor prognosis caused by the rapid progression of the lesion into distinct organs. Our newly designed arterial infusion chemotherapy, from the superficial temporal artery, in combination with radiotherapy, has shown a favorable outcome in patients with NNKTL. In this article, the clinical, pathological, and virological characteristics of the lymphoma are reviewed, along with a report of our investigations.

Expression of Interleukin-9 in Nasal Natural Killer/T-Cell Lymphoma Cell Lines and Patients

Purpose: Nasal natural killer (NK)/T-cell lymphoma is associated with EBV and has distinct clinical and histologic features. However, little is known about its genetic features. In this study, we examined the genes expressed by SNK-6 and SNT-8 cells, which were established from nasal NK/T-cell lymphomas, and found that interleukin (IL)-9 was specifically expressed in these two cell lines. Experimental Design: cDNA array was used to examine the genes expressed by SNK-6 and SNT-8 cells. Expression of IL-9 and IL-9 receptor was investigated by reverse transcription-PCR, ELISA, and flow cytometry. Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunohistologic staining and ELISA were used to examine IL-9 expression in biopsies and sera from patients, respectively. Results: In cDNA array, expression of IL-9 mRNA was much higher in SNK-6 and SNT-8 cells than in NK-92 cells from non-nasal NK-cell lymphoma and peripheral blood mononuclear cells from healthy volunteers. Furthermore, IL-9 was specifically expressed by SNK-6 and SNT-8 cells but not by other NK-cell, NK-like T-cell, and T-cell lymphoma/leukemia cell lines. IL-9 receptor was also expressed on the surfaces of SNK-6 and SNT-8 cells. An IL-9-neutralizing antibody inhibited the growth of these two cell lines, whereas recombinant human IL-9 enhanced their growth. Most significantly, IL-9 was present in biopsies and sera from patients with this lymphoma. Conclusions: These results suggest that IL-9 plays an important role in nasal NK/T-cell lymphoma possibly via an autocrine mechanism.

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)

Monocytes enhance cell proliferation and LMP1 expression of nasal natural killer/T‐cell lymphoma cells by cell contact‐dependent interaction through membrane‐bound IL‐15

Nasal natural killer (NK)/T‐cell lymphoma (NNKTL) is an Epstein‐Barr virus (EBV)‐related malignancy with poor prognosis and has distinct histological features characterized by angiocentric and polymorphous lymphoreticular infiltrates including inflammatory cells such as granulocytes, monocytes, macrophages and lymphocytes. Here, we show that the monocytes enhance proliferation as well as LMP1 expression of NNKTL cells by cell contact‐dependent interaction through membrane‐bound interleukin (IL)‐15. We used two EBV‐positive NK‐cell lines, SNK6 and KAI3, which originated from two patients—SNK6 from a patient with NNKTL and KAI3 from a patient with a severe mosquito allergy. We cocultured the cell lines with granulocytes or monocytes and examined whether proliferation, survival and LMP1 expression of the cells changed. Although cocultured granulocytes did not affect proliferation, survival or LMP1 expression of the cells, cocultured monocytes enhanced both proliferation and LMP1 expression in a dose‐dependent manner. These phenomena were not seen when monocytes were placed in a separate chamber. Moreover, the monocyte‐inducible proliferation and LMP1 expression were inhibited by treatment with an antibody against IL‐15. Furthermore, production of interferon‐gamma‐inducible protein (IP)‐10 were enhanced by coculture with monocytes and were inhibited by the antibody. Immunohistological studies confirmed that a number of infiltrating CD14‐positive monocytes contacted CD56‐positive lymphoma cells in all of 20 NNKTL tissues tested. These results suggest that monocytes enhance cell growth as well as LMP1 expression of NNKTL cells by cell contact‐dependent interaction through membrane‐bound IL‐15. In the microenvironment of NNKTL tissue, a positive feedback loop of interaction between lymphoma cells and monocytes may be present and contribute to lymphoma progression.

Sequence variations of Epstein–Barr virus LMP1 gene in nasal NK/T-cell lymphoma

Nasal natural killer (NK)/T-cell lymphoma is a peculiar lymphoma with an unique immunophenotype. Etiologically, the authors previously first demonstrated the presence of Epstein–Barr virus (EBV) genomes and their products in this lymphoma (Lancet 1990; 335). It is suggested that some of sequence variations such as a 30-bp deletion and multiple base substitutions and as amino acid changes at HLA-A2 restricted CTL epitopes were associated with an increase in tumorigenicity and with a decrease in immune recognition. In this study, we determined full-length of LMP1 sequence isolated from 7 patients with nasal NK/T-cell lymphoma using polymerase chain reaction (PCR) method and compared the sequences with those referred to previous reports. In the carboxyl-terminal site, all 7 patients showed 4 copies of the 11 amino acids repeat (codon 254–302) and 30-bp deletion corresponding to codon 343–352 of the B95-8 strain. Within the NF-kB-activating domains, all 7 patients showed amino acid changes at codon 189 (Gln to Pro), 192 (Ser to Thr) and 212 (Gly to Ser) on either site of the PXQXT (codon 204–208) motif. In the major HLA-A2 restricted T-cell epitope sequence YLLEMLWRL (codon 125–133), all 7 patients showed amino acid changes at codon 126 (Leu to Phe) and 129 (Met to Ile). In the epitopes ALLVLYSFA (codon 51–59), VLFIFGCLL (codon 110–118) and WLLLFLAIL (codon 173–181), several patients showed novel amino acid changes at codon 59 (Ala to Gly), 110 (Val to Leu) and 174 (Leu to Ile), respectively. Although it is still not clear what the most specific and biologic variation of LMP1 gene in nasal NK/T-cell lymphoma is, the sequence data may be valuable on the study for pathogenesis of nasal NK/T-cell lymphoma and EBV molecular epidemiology.

Downregulation of miR-15a due to LMP1 promotes cell proliferation and predicts poor prognosis in nasal NK/T-cell lymphoma

Nasal NK/T‐cell lymphoma (NNKTL) is an Epstein‐Barr virus (EBV)‐associated malignancy and has distinct clinical and histological features. However, its genetic features are hitherto unclear. MicroRNAs (miRNAs) play a crucial role in the pathogenesis of several malignancies via regulating gene expression. In this study, we investigated whether the specific microRNAs were related to the tumor behaviors in NNKTL. MiRNA array and Quantitative RT‐PCR analyses revealed that miR‐15a was expressed at a much lower level in NNKTL cells (SNK‐1, SNK‐6, and SNT‐8) than in normal peripheral NK cells and EBV‐negative NK cell line KHYG‐1. Quantitative PCR and western blot analyses showed that the expression of MYB and cyclin D1, which are validated targets of miR‐15a, was higher in NNKTL cells. Transfection of NNKTL cells (SNK‐6 and SNT‐8) with a miR‐15a precursor decreased MYB and cyclin D1 levels, thereby blocking G1/S transition and cell proliferation. Knockdown of EBV‐encoded latent membrane protein 1 (LMP1) significantly increased miR‐15a expression in SNK‐6 cells. In NNKTL tissues, we found that reduced miR‐15a expression, which correlated with MYB and cyclin D1 expression, was associated with poor prognosis of NNKTL patients. These data suggest that downregulation of miR‐15a, possibly due to LMP1, implicates in the pathogenesis of NNKTL by inducing cell proliferation via MYB and cyclin D1. Thus, miR‐15a could be a potential target for antitumor therapy and a prognostic predictor for NNKTL. Am. J. Hematol. 89:25–33, 2014.

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).

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

Pustulosis palmaris et plantaris (PPP) is a tonsil‐related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up‐regulated more intensively in PPP patients than in non‐PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α‐streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non‐PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non‐PPP patients (P < 0·05). The percentage of CCR6‐positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6‐positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non‐pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α‐streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6‐positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil‐related disease PPP.