Yorihisa Imanishi

Activation of matrix metalloproteinase-2 in head and neck squamous cell carcinoma: Studies of clinical samples and in vitro cell lines co-cultured with fibroblasts

We undertook this present study to investigate the activation of matrix metalloproteinase-2 (MMP-2) in human head and neck squamous cell carcinomas (HNSCC) tissues and cell lines. Gelatinolytic activities of active MMP-2 were significantly higher in carcinoma samples than in normal portions. Furthermore, the activation ratio of proMMP-2 significantly correlated with cervical lymph node metastasis. In vitro studies revealed an HNSCC cell line, HEp-2, to produce neither the pro form nor the active form of MMP-2, but human fibroblasts were found to produce proMMP-2. However, coculture of HEp-2 cells with fibroblasts resulted in the production of not only proMMP-2 but also activeMMP-2 in the culture medium. Northern blot analysis revealed a stronger expression of membrane-type 1 matrix metalloproteinase (MT1-MMP),which is a specific activator of MMP-2, mRNA in HEp-2 cells than in fibroblasts. These results suggest the activation of proMMP-2 as an important event in the process of HNSCC metastasis. They also suggest MMP-2 is secreted in its pro form by stromal fibroblasts surrounding the cancer cells and activated by MT1-MMP localized on the cancer cells.

Granulocyte-macrophage colony-stimulating factor upregulates matrix metalloproteinase-2 (MMP-2) and membrane type-1 MMP (MT1-MMP) in human head and neck cancer cells.

Matrix metalloproteinase-2 (MMP-2) and membrane type 1-MMP (MT1-MMP) play an important role in the invasion and metastasis of head and neck squamous cell carcinoma (HNSCC), but the mechanism of their regulation is not clearly understood. Recently, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be associated with cancer invasion and metastasis. We hypothesized that GM-CSF may upregulate MMP-2 and/or MT1-MMP expression in HNSCC cells, and may thereby influence their ability to invade and metastasize. We studied the effects of GM-CSF on the production of MMP-2 and MT1-MMP in HNSCC cell lines SAS and HSC-2. Gelatin zymography of conditioned media derived from HNSCC cells revealed a major band of 68 kDa, which was characterized as proMMP-2. GM-CSF stimulated the production of proMMP-2 in both cell lines in a dose-dependent manner. Treatment with 50 ng/ml GM-CSF for 24 h increased the proMMP-2 activity 3.4-fold in SAS cells and 2.3-fold in HSC-2 cells compared with untreated controls. Northern blot analyses demonstrated that GM-CSF led to elevated mRNA levels of MMP-2 and MT1-MMP in both cell lines. The results identify GM-CSF as a regulator of MMP-2 and MT1-MMP expression in certain types of HNSCC, and suggest that GM-CSF may contribute to the invasiveness of HNSCC through the regulation of MMP-2 and MT1-MMP expression.

Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Multicenter Study in Japan

Background: The incidence rates of oropharyngeal squamous cell carcinoma (OPSCC) have risen steadily in the USA and in northern Europe. These increases are thought to be a consequence of persistent infection with high-risk human papillomavirus (HPV) in OPSCC patients. HPV is an emerging etiologic factor in OPSCC. In Japan, the incidence of OPSCC has significantly increased over the last three decades. However, the population of HPV-positive OPSCC patients is currently unknown. We examined the nationwide trends with regard to HPV incidence in OPSCC patients at 21 specific sites, and examined the relationship between the presence of HPV and survival in OPSCC patients in Japan. Methods: Tumor samples were obtained from patients with OPSCC prior to treatment, and HPV infection was investigated by polymerase chain reaction (PCR). Hybrid Capture 2 (HC2) was also adopted for swab examination on the surface of fresh tumors. Results: HPV was detected by PCR in 79 (50.3%) out of 157 OPSCC patients. The clinical features of HPV-positive OPSCC were low differentiation, a tendency to involve the lateral wall, and high nodal staging. The sensitivity and specificity of HC2 were 93.7 and 96.2%, respectively, indicating its utility as a screening test. HPV-positive patients had significantly better overall survival and disease-free survival than HPV-negative patients.

Restoration of E-cadherin expression by selective Cox-2 inhibition and the clinical relevance of the epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma

BackgroundThe epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin has been thought to promote metastasis. Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. The aim of the present study was to investigate whether selective Cox-2 inhibitors restore the expression of E-cadherin in head and neck squamous cell carcinoma (HNSCC) cells, and to examine the possible correlations of the expression levels of EMT-related molecules with clinicopathological factors in HNSCC.MethodsWe used quantitative real-time PCR to examine the effects of three selective Cox-2 inhibitors, i.e., celecoxib, NS-398, and SC-791 on the gene expressions of E-cadherin (CDH-1) and its transcriptional repressors (SIP1, Snail, Twist) in the human HNSCC cell lines HSC-2 and HSC-4. To evaluate the changes in E-cadherin expression on the cell surface, we used a flowcytometer and immunofluorescent staining in addition to Western blotting. We evaluated and statistically analyzed the clinicopathological factors and mRNA expressions of Cox-2, CDH-1 and its repressors in surgical specimens of 40 patients with tongue squamous cell carcinoma (TSCC).ResultsThe selective Cox-2 inhibitors upregulated the E-cadherin expression on the cell surface of the HNSCC cells through the downregulation of its transcriptional repressors. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. A univariate analysis showed that higher Cox-2 mRNA expression (p = 0.037), lower CDH-1 mRNA expression (p = 0.020), and advanced T-classification (p = 0.036) were significantly correlated with lymph node metastasis in TSCC. A multivariate logistic regression revealed that lower CDH-1 mRNA expression was the independent risk factor affecting lymph node metastasis (p = 0.041).ConclusionsThese findings suggest that the appropriately selective administration of certain Cox-2 inhibitors may have an anti-metastatic effect through suppression of the EMT by restoring E-cadherin expression. In addition, the downregulation of CDH-1 resulting from the EMT may be closely involved in lymph node metastasis in TSCC.

An anatomical study of the three-dimensional structure of the nasal septum in patients with alveolar clefts and alveolar-palatal clefts.

Background: The present study was performed to quantitatively analyze the three-dimensional morphology of the nasal septa of patients with alveolar and alveolopalatal clefts. Methods: Twenty-five unilateral complete cleft lip patients with alveolar clefts only (alveolar cleft group) and 35 unilateral complete cleft lip patients with alveolar and palatal clefts (alveolar and palatal cleft group) were included in the study. Although no patient in either group had undergone alveoloplasty, all patients had undergone palatoplasty. The degree of nasal septum deviation was studied for each patient at three different depths along the anteroposterior axis using three-dimensional computed tomographic data; the data were compared between the two groups to elucidate whether the difference in cleft type affects the morphologic patterns of the nasal septum. Results: The nasal septa of the alveolar and palatal cleft group patients presented more uneven morphologic patterns than those of the alveolar cleft group patients. In the alveolar cleft group, the nasal septa did not present significantly different degrees of deviation at their anterior and posterior parts. In the alveolar and palatal cleft group, however, the posterior parts of the nasal septa presented greater deviation than the anterior parts. In the alveolar and palatal cleft group, furthermore, a significant correlation was observed between the severity of the cleft and the degree of the nasal septum deviation. Conclusions: The nasal septa present different three-dimensional morphologic patterns between the patients with alveolar clefts only and those with alveolopalatal clefts. This difference should be considered when performing surgical treatments for these patients.

Clinicopathological Significance of Expression of CD44 Variants in Head and Neck Squamous Cell Carcinoma

Splice variants of the cell surface glycoprotein CD44 have been reported to be associated with the progression of various human tumors. The aim of this study is to determine the correlation between the expression of CD44 isoforms, especially CD44 variant 2 (CD44v2), and the clinicopathological features of head and neck squamous cell carcinomas (HNSCCs). The expression of CD44 isoforms was evaluated immunohistochemically in paraffin‐embedded tissues from 89 primary lesions, using monoclonal antibodies against CD44 standard (CD44st), CD44 variant 6 (CD44v6) and CD44v2. Cancer tissues from 89 (100%), 85 (95.5%) and 59 (66.3%) patients showed positive immunoreactivity for CD44st, CD44v6 and CD44v2, respectively. A significant correlation was observed between the down‐regulation of CD44v2 and poorer differentiation of the tumor cells (P=0.02). We could not find any significant correlation between the expression of CD44v2 and T stage or N stage (lymph node status). However, the rate of positive cervical lymph node metastasis tended to increase with reduced expression of CD44v2 (P=0.08). Down‐regulation of CD44v2 expression was correlated with shorter overall survival (P=0.01). Furthermore, Coxs multivariate analysis revealed that only CD44v2 expression and lymph node status were independent prognostic factors. These findings suggest that down‐regulation of CD44v2 expression may be one of the biological markers for the degree of malignancy in HNSCCs.

Intramucosal PCO2 measurement as a new monitoring method of free jejunal transfer following pharyngo-laryngo-esophagectomy.

The choices for practical monitoring of free jejunal transfer have been quite limited because of its own characteristics, such as buried form, lack of skin surface, and the structure of a hollow viscous tract. Physiologically, it is known that tissue hypoxia caused by compromised perfusion leads to an increase of partial pressure of carbon dioxide (PCO2). Because of its physiological properties, the diffusion of carbon dioxide is always equilibrated between the mucosa of a hollow viscous organ and its lumen. The intramucosal PCO2 (PiCO2) of the gastrointestinal tract can therefore be determined indirectly from the intraluminal PCO2, which is measured with the aid of the tonometer catheter. To develop an optimal monitoring method for free jejunal transfer, the authors proposed the application of PiCO2 measurement by a modified use of a tonometer catheter. Since May of 1999, the authors performed postoperative PiCO2 monitoring on 20 cases of reconstructed pharyngoesophageal tracts in 18 patients who underwent radical tumor resection and one-stage reconstruction at the Shizuoka Red Cross Hospital. All 20 cases were safely monitored by PiCO2 measurement without any complications associated with the use of the tonometer catheter. In the 17 cases that succeeded uneventfully, the mean values of PiCO2 were kept lower than 40 mmHg throughout the monitoring period. On the other hand, the other three cases (15 percent) needed reexploration due to development of vascular complications, which was alerted by an abrupt increase of PiCO2 in each case (229, 130, and 99.6 mmHg). Two of the patients were fortunately successfully treated by immediate reexploration, leading to a 95 percent overall success rate. No false-negative or false-positive cases were observed. The authors’ experience suggests that PiCO2 measurement using a tonometer catheter can provide the surgeon with reliable information for evaluating the perfusion and viability of a free jejunal transfer. Simplified manipulation and the objectivity of the numerical data allow stable measurement of PiCO2 and prompt judgment of the adequacy of the perfusion, which could minimize the burden and anxiety of the surgeon, particularly in the early postoperative period.

Biomarker immunoprofile in salivary duct carcinomas: clinicopathological and prognostic implications with evaluation of the revised classification

Salivary duct carcinoma (SDC) is an uncommon, aggressive malignant neoplasm histologically resembling high-grade mammary ductal carcinoma. SDC can arise de novo or ex pleomorphic adenoma. To clarify the correlation of biomarker immunoprofile with clinicopathological findings and clinical outcome of SDC, we conducted immunohistochemistry for EGFR, HER2, HER3, AR, CK5/6, p53, and Ki-67, along with HER2 fluorescence in situ hybridization in 151 SDCs. SDCs ex pleomorphic adenoma more commonly overexpressed EGFR, HER2, HER3, and Ki-67 than de novo SDCs (P = 0.015, < 0.001, 0.045, and 0.02, respectively). In multivariate analysis, AR− and CK5/6+ were associated with shorter progression-free survival (P = 0.027 and 0.004, respectively). Moreover, patients with p53-extreme negative/positive demonstrated poorer overall survival (P = 0.007). On assessing the revised classification by the combination of biomarker expression, the percentages of each subtype were as follows: ‘apocrine A’ (AR+/HER2−/Ki-67-low) (24%), ‘apocrine B’ (AR+/HER2−/Ki-67-high) (18%), ‘apocrine HER2’ (AR+/HER2+) (35%), ‘HER2-enriched’ (AR−/HER2+) (12%), and ‘double negative’ (AR−/HER2−) (11%). ‘Double negative’ was further subclassified into ‘basal-like’ (EGFR and/or CK5/6+) (7%) and ‘unclassified’ (3%). Consequently, patients with ‘apocrine A’ showed a better progression-free survival than those with any other subtypes. Our revised immunoprofiling classification was valuable for predicting the survival and might be useful in personalized therapy for patients with SDC.

Adjuvant Chemotherapy with Oral Tegaful and Uracil for Maxillary Sinus Carcinoma

We have applied tegaful and uracil (UFT) treatment as adjuvant chemotherapy to patients after the completion of primary therapy. UFT was given per os at a dose of 300 or 400 mg/day for more than 1 year. A retrospective study was conducted on 15 patients, assessed as the UFT-treated group, and 24 patients assessed as the UFT-nontreated group. The 5-year survival rate in patients treated or not treated with UFT was 76.6 and 22.6%, respectively. Among those who underwent surgery in combination with other therapy, the 5-year survival rate was 74.1% with UFT and 27.3% without UFT. In patients receiving chemotherapy plus radiotherapy alone, the 5-year survival rate was 80.0% with UFT and 19.2% without UFT. In 23 patients with proven effects of neoadjuvant chemotherapy comprising 18 PR cases and 5 CR cases, a comparison was made between 10 patients treated with UFT and 13 patients not treated with UFT. As a result, the 5-year survival was 76.2 and 17.9%, respectively. In the patients with T3 disease, who occupied the majority, the 5-year survival rate was 71.4 and 23.8%, respectively. Adjuvant chemotherapy with UFT tends to show a substantial significant difference particularly in patients who were successfully treated with radiotherapy plus chemotherapy as the primary treatment. UFT was clearly shown to have a statistically significant effect, despite a small population. The findings of the present investigation point to the value of conducting further study to ascertain the effect of UFT by a randomized trial in a larger population.

Impact of hematological inflammatory markers on clinical outcome in patients with salivary duct carcinoma: a multi-institutional study in Japan

The prognostic role of modified Glasgow Prognostic Score (mGPS), neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in patients with salivary duct carcinoma (SDC) remains unclear. We conducted a multi-institutional retrospective cohort study of 140 SDC patients. The survival impact of these hematological markers was evaluated using multivariate proportional hazard models.High mGPS (≥1) was significantly associated with worse survival (3-year overall survival (OS): 16.7% vs 66.1%, p-value=0.003; 3-year progression-free survival (PFS): 0.0% vs 27.9%, p-value<0.001). Additionally, high C-reactive protein (CRP) (≥0.39 mg/dl) was significantly associated with worse survival (3-year OS: 32.1% vs 68.2%, p-value=0.001; 3-year PFS: 7.1% vs 31.1%, p-value<0.001). These associations were consistent with multivariate analysis adjusted for established prognostic factors. Although we also found significant association of high NLR (≥2.5) with OS (HR 1.80; 95% confidence interval, 1.05-3.08) in multivariate analysis, this association were inconsistent with the results of PFS. In addition, we found no significant associations of PLR with survival. In conclusion, we found that mGPS, CRP and NLR were identified as prognostic factors associated with survival in SDC patients.