Mitsuo Goto

Rap1GAP Promotes Invasion via Induction of Matrix Metalloproteinase 9 Secretion, Which Is Associated with Poor Survival in Low N-Stage Squamous Cell Carcinoma

The objective of the current study was to investigate the effects of Rap1GAP on invasion and progression of head and neck squamous cell carcinoma (SCC) and the role of matrix metalloproteinase (MMP) 9 and MMP2 in this process. Rap1GAP functions by switching off Rap1, the Ras-like protein that has been associated with carcinogenesis. Previous findings suggest that Rap1GAP acts as a tumor suppressor protein in SCC by delaying the G(1)-S transition of the cell cycle. However, cells transfected with Rap1GAP exhibit a more invasive phenotype than corresponding vector-transfected control cells. MMP2 and MMP9 are enzymes that mediate SCC invasion via degradation of the extracellular matrix. Using SCC cells transfected with empty vector or Rap1GAP, cell invasion and MMP secretion were determined by Matrigel assays and gelatin zymography, respectively. Rap1GAP up-regulated transcription and secretion of MMP2 and MMP9, as assayed by quantitative reverse transcription-PCR and zymography. Furthermore, chemical and RNA interference blockade of MMP2/MMP9 inhibited invasion by Rap1GAP-transfected cells. Immunohistochemical staining of a human oropharyngeal SCC tissue microarray showed that Rap1GAP and MMP9 expression and staining intensity are correlated (P < 0.0001) and that, in early N-stage lesions of SCC, high MMP9 is prognostic of poor disease-specific survival (P < 0.05). Furthermore, Rap1GAP staining is correlated with MMP2 (P < 0.03). MMP2 in combination with N stage has a prognostic effect on time to indication of surgery at primary site. MMP2 intensity is also positively correlated with T stage (P < 0.015). In conclusion, Rap1GAP inhibits tumor growth but induces MMP2- and MMP9-mediated SCC invasion and tumor progression, suggesting a role for this protein as a biomarker for early N-stage, aggressive SCCs.

Sentinel lymph node radiolocalization in clinically negative neck oral cancer

The sentinel node concept has become one of the most interesting topics in the treatment of head and neck cancer. The aim of this article is to report the results of our feasibility study and clinical application of sentinel lymph node (SLN) radiolocalization and biopsy in patients with clinically negative neck oral cancer.

Rap1 Stabilizes β-Catenin and Enhances β-Catenin–Dependent Transcription and Invasion in Squamous Cell Carcinoma of the Head and Neck

Purpose: In head and neck squamous cell carcinoma (HNSCC) cells, Rap1 shuttles between the nucleus and cytoplasm. Prior findings suggested that Rap1 may modulate the β-catenin–independent Wnt pathway in some settings, but the role of Rap1 in β-catenin–dependent Wnt signaling remains undefined. Experimental Design and Results: We observed that β-catenin bound to active Rap1 in vitro and Rap1 activated β-catenin/T-cell factor (TCF)–dependent transcription. Immunofluorescence studies showed that ectopic expression of Rap1 increased nuclear translocation of β-catenin. Overexpression of active Rap1 facilitated an increase in β-catenin–mediated transcription that was abrogated by dominant-negative TCF4. Conversely, small interfering RNA–mediated inhibition of endogenous Rap1 expression inhibited β-catenin/TCF–mediated transcription as well as invasion of HNSCC. Furthermore, inhibition of Rap1 expression downregulated the expression of matrix metalloproteinase 7, a transcriptional target of β-catenin/TCF. In HNSCC cells stably transfected with β-catenin or treated with lithium chloride or Wnt3A to stabilize endogenous β-catenin, inhibition of Rap1 expression led to decreases in the free pool of β-catenin. Immunohistochemical studies of tissue from HNSCC patients revealed that increased β-catenin intensity correlated with higher tumor stage. Furthermore, the prognostic effect of active Rap1 on tumor N stage was found to depend on cytosolic β-catenin expression (P < 0.013). When β-catenin is high, higher Rap1GTP intensity is associated with more advanced N stage. Conclusions: The findings suggest that Rap1 enhances β-catenin stability and nuclear localization. In addition to indicating that Rap1 has a significant role in regulating β-catenin and β-catenin–dependent progression to more advanced N-stage lesions, these data highlight Rap1 as a potential therapeutic target in HNSCC. Clin Cancer Res; 16(1); 65–76

Prediction of Chemosensitivity Using Multigene Analysis in Head and Neck Squamous Cell Carcinoma

Aims: The main purpose of the current study was to find predictive biomarkers that can be routinely used for the response to chemotherapy in head and neck squamous cell carcinoma (HNSCC). Methods: From this standpoint, we selected the histoculture drug response assay (HDRA) to assess in vitro chemosensitivity, and real-time reverse transcription polymerase chain reaction to investigate the gene expression profile of individual tumors as available predictive biomarkers. Using both surgery and biopsy specimens, we analyzed their gene expression profiles using the 18 markers that we thought were likely predictors of the response to anti-cancer agents. Results: Statistically significant associations were found between 5-fluorouracil (5-FU) sensitivity in HDRA and HER-2 mRNA expression level (p = 0.0030). Moreover, HER-2 expression was significantly associated with cisplatin sensitivity (p = 0.0089). Cisplatin sensitivity in HDRA was also demonstrated to have a significant association with epidermal growth factor receptor (EGFR) expression in which the group with cisplatin resistance tended to have a higher expression level than the sensitive group (p = 0.0385). Conclusion: HER-2 and EGFR may be possible reliable predictive biomarkers for anti-cancer therapy, and might help in the decision-making process for individual patients with HNSCC.

A novel approach to biomarker discovery in head and neck cancer using an autoantibody signature

Despite the dismal prognosis for patients with squamous cell carcinoma of the head and neck (SCCHN), there have been no novel treatments in over 40 years. Identification of novel tumor antigens in SCCHN will facilitate the identification of potential novel treatment targets. Tumor antigens are proteins selectively expressed by tumor cells and recognized by the host immune system. Phage-displayed tumor antigens were enriched by biopanning with normal and then SCCHN-specific serum. Ninety-six phage clones were sequenced for identification, and 21 clones were validated using Luminex. One of these proteins, L23, a novel tumor antigen in SCCHN, was validated as an oncogene. L23 is upregulated in SCCHN compared with normal keratinocytes. Knockdown of L23 inhibited proliferation, invasion and cell survival. Overexpression of L23 had the reverse effect. Overexpression of L23 in non malignant cells led to transformation. Injection of SCCHN cells with knockdown of L23 in mice, induced tumors that were significantly smaller than control tumors. In conclusion, the immunomic screen yielded a panel of antigens specific to SCCHN; one of these proteins, L23, is a novel oncogene in SCCHN.

Prognostic factors and outcomes for salvage surgery in patients with recurrent squamous cell carcinoma of the tongue

Recurrence rates of oral cancer following primary treatment have been reported in the range of 25–48%. However, salvage therapy remains a critical challenge to improving outcomes. Here, we investigated prognostic factors and outcomes for salvage surgery in patients with recurrent oral tongue squamous cell carcinoma (OTSCC).

Computed Tomographic Estimation of Particulate Cancellous Bone and Marrow Weight for Successful Transplant in Unilateral Cleft Lip and Palate Patients.

Objective The defect volume measured on computed tomography (CT) for secondary bone graft (SBG) is well correlated to the actual amount of particulate cancellous bone and marrow (PCBM) transplanted in unilateral cleft lip and palate (UCLP) patients. However, the validity of such measurements have not been completely verified due to lack of evaluation of treatment results. The objective of this study was to propose an estimation method by CT based on the data of successfully treated patients. For this purpose, the association was initially verified between the weight of transplanted PCBM and the defect volume measured on CT using the results of successfully treated patients. Methods Treatment results were evaluated 1 year after SBG by intraoral radiography in 50 UCLP patients. For the patients with good results, the correlation was investigated between the defect volume on CT and the transplanted PCBM weight, and a method was proposed based on PCBM density, calculated as PCBM weight divided by defect volume on CT. Results In successfully treated patients showing level 3 or 4 alveolar resorption, a strong correlation (r = .87) was found between the volume on CT and the PCBM weight. Level 4 results were observed in 22 of 23 (95.7%) patients who had calculated PCBM densities of more than 6 g/cm3. Conclusions Volume estimation on preoperative CT was confirmed to have sufficient validity. The weight of PCBM transplanted should be greater than the defect volume on CT multiplied by 6.

A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green–Labeled Podoplanin Antibody

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)–labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma–xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression–dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

Predictive biomarkers for combined chemotherapy with 5‑fluorouracil and cisplatin in oro‑ and hypopharyngeal cancers

The present study aimed to identify significant correlations between gene expression and chemotherapy response to 5-fluorouracil (5-FU)/cisplatin in head and neck squamous cell carcinoma (HNSCC), and to identify patients who would benefit from induction chemotherapy for both organ preservation and survival. A total of 64 patients who underwent radical treatment for HNSCC were enrolled. All patients received induction chemotherapy with 5-FU/cisplatin and tumor responses were evaluated. Pretreatment biopsy specimens from all patients were assayed for mRNA expression of thymidylate synthase, dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, tymidine phosphorylase, glutathione S-transferase-pi, p53, RB Transcriptional Corepressor 1, B-cell lymphoma 2 (Bcl-2), Bcl-xL, E2F Transcription Factor 1, epidermal growth factor receptor, human epidermal growth factor receptor 2, phosphoinositide 3-kinase, phosphatase and tensin homolog, vascular endothelial growth factor (VEGF), cyclooxygenase-2, XPA, DNA Damage Recognition And Repair Factor, excision repair cross-complementing 1 (ERCC1), multidrug resistance gene 1 (MDR1), multidrug resistance-associated protein 1, equilibrative nucleoside transporter 1 and β-tubulin by reverse transcription-quantitative polymerase chain reaction, and the association between the expression levels of these genes and patient response to chemotherapy was determined. The complete response (CR) group and non-CR group for induction chemotherapy comprised 32.8 and 67.2% of patients, respectively. The 5-year overall survival rate was significantly higher for the CR group (95%) compared with the non-CR group (57%). According to univariate analysis, chemotherapy response was associated with T-class and mRNA expressions of DPD, ERCC1, XPA, p53, Bcl-2, VEGF and MDR1. Multivariate analysis identified ERCC1 expression and T-class as significant predictors of response to chemotherapy, indicating that a DNA-repair pathway and apoptosis pathway are pivotal mechanisms governing response to chemotherapy. The findings suggest that ERCC1 expression could be a predictive biomarker for chemotherapy response to 5-FU/cisplatin in HNSCC. Assessing mRNA expression is a standard method for these studies, however further investigations examining polymorphisms and mutations in addition to apoptotic responses are required to determine target gene activation in HNSCC.

A Case of Bisphosphonate-Related Osteonecrosis of the Jaw in a Patient with Subpontic Osseous Hyperplasia

Subpontic osseous hyperplasia (SOH) is a growth of bone occurring on the edentulous ridge beneath the pontics of fixed partial dentures (FPDs). This report describes a case of bisphosphonate- (BP-) related osteonecrosis of the jaw (BRONJ) in a SOH patient followed by deciduation of the bony lesion. A 73-year-old woman visited a dental clinic after experiencing pain and swelling beneath the pontics of a FPD that had been inserted 15 years ago. The pontics were removed, but the symptoms persisted and she was referred to our hospital. There was an osseous bulge and gum swelling around the edentulous ridge of teeth 18 and 19, as well as bone exposure. As she had been taking an oral BP for 6 years, we diagnosed this case as stage 2 BRONJ. Following BP withdrawal, the bony lesion detached from the mandible. The tissue was diagnosed as sequestrum based on the histopathological findings. Two months after deciduation, epithelialization over the area of exposed bone was achieved and no recurrence has been observed.