Annexin A8 is a novel molecular marker for detecting lymph node metastasis in oral squamous cell carcinoma.

Cervical lymph node metastasis is an important prognostic factor in oral squamous cell carcinoma (OSCC), but its accurate assessment after sentinel node biopsy or neck dissection is often limited to the histopathological examination of only one or two sections. Previous our study showed the usefulness of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting keratin 19 (KRT19) mRNA for the genetic detection of lymph node metastasis, but the sensitivity was insufficient. Here, we have attempted to identify novel molecular markers for OSCC cells in lymph nodes. We performed microarray analysis to identify genes overexpressed in 7 metastatic lymph nodes from OSCC patients, compared to 1 normal lymph node and 5 salivary glands from non-cancer patients. We then used real-time quantitative RT-PCR (qRT-PCR) and RT-LAMP to compare the expression of these genes in newly resected metastatic and normal lymph nodes. Of 4 genes identified by microarray analysis, annexin A8 (ANXA8) and desmoglein 3 mRNA were detected by qRT-PCR in metastatic lymph nodes but not in normal lymph nodes. Furthermore, ANXA8 mRNA expression was detected in all KRT19-negative metastatic lymph nodes. Both KRT19 and ANXA8 mRNA may be useful markers for detecting lymph node metastases in OSCC patients.

Targeting Aurora kinase A suppresses the growth of human oral squamous cell carcinoma cells in vitro and in vivo.

OBJECTIVES: Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not yet available. In this study, we attempted to identify an appropriate target molecule for treatment of patients with OSCC. MATERIALS AND METHODS: Microarray analysis was performed to determine the gene expression profiles in nine human OSCC cell lines and a non-neoplastic keratinocyte cell line. The expression levels of Aurora kinase A (AURKA) mRNA and protein in human OSCC cells and tissues were examined. We investigated the effect of small interfering RNAs specific for AURKA (siAURKAs) and MLN8237, an AURKA selective inhibitor on the growth of OSCC cells in vitro and in vivo. We also analyzed clinical significance in AURKA mRNA expression levels in OSCC. RESULTS: AURKA was overexpressed in human OSCC cell lines and tissues. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of OSCC cells by 31-89%. MLN8237 also reduced the cellular growth rate by 38-74%. Both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors by 66% and 40%. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of primary cultured cells established from patients' OSCC tumors. Furthermore, we found a significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. CONCLUSIONS: AURKA plays a critical role in the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC.

One-step nucleic acid amplification for detecting lymph node metastasis of head and neck squamous cell carcinoma.

OBJECTIVES: Lymph node stage is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC). We previously reported the clinical usefulness of sentinel lymph node biopsy diagnosed by genetic analysis using quantitative RT-PCR. However, this method takes about 3h. In this study, we attempted to develop a more efficient method for the intraoperative genetic detection of lymph node metastasis in HNSCC. MATERIALS AND METHODS: A total of 312 lymph nodes (65 patients) were diagnosed by the one-step nucleic acid amplification (OSNA) method using GD-100. OSNA consists of a short homogenization step followed by amplification of cytokeratin 19 (CK19) mRNA directly from the lysate. Each lymph node was divided into two to diagnose metastasis. One half was used for the OSNA assay, and the other was subjected to semi-serial sectioning, sliced at 200-µm intervals and examined by H&E and cytokeratin AE1/AE3 immunohistochemical staining. The accuracy of OSNA assay was evaluated based on histopathological diagnosis. RESULTS: Sixty-one of 312 lymph nodes were pathologically metastasis-positive. The overall concordance rate between the OSNA assay using breast cancer criteria and histopathology was 94.2%. The optimal cut-off for the copy number of CK19 mRNA in assessing lymph node metastasis of HNSCC was 300 copies/µl, which had the highest diagnostic accuracy (95.2%). The OSNA assay can be completed within 30 min. CONCLUSION: The OSNA assay, which shows high sensitivity and specificity, suggests the possibility to be used as a novel tool for the genetic detection of lymph node metastasis in HNSCC patients.

Human FAT1 cadherin controls cell migration and invasion of oral squamous cell carcinoma through the localization of ß-catenin.

FAT1 [Homo sapiens FAT tumor suppressor homolog 1 (Drosophila)] is an intrinsic membrane protein classified as a member of the cadherin superfamily. The FAT1 gene is a tumor suppressor in humans as well as being the pivotal gene for cell morphogenesis and migration. Deletion of this gene could play a role in the characteristics of oral squamous cell carcinomas (OSCCs), involving cell adhesion, migration and/or invasion. This study investigated the mechanisms by which FAT1 is involved in the biological behavior of OSCCs. First, a rat monoclonal antibody was developed against a FAT1 intra-cellular domain epitope, and used for an immunohistochemical study of FAT1 in clinically obtained OSCC samples. FAT1 was localized at lamellipodial edges or cell-cell boundaries in normal cells and well differentiated OSCCs, but showed a diffuse cytoplasmic and nuclear distribution in moderately-poorly differentiated OSCCs. FAT1-siRNA was transfected into OSCCs resulting in a drastic inhibition of cell migration and invasion based on the suppression of FAT1 expression and disorganized localization of ß-catenin which is associated with cell polarity and migration. These results suggested that FAT1 may be involved in the migration and invasion mechanisms of OSCCs and, therefore, it could be an important target for the development of new therapeutic strategies.

Massive subcutaneous emphysema developing before surgery for mandibular fracture: a case report.

Preoperative massive subcutaneous emphysema before intubation is extremely rare. However, this complication may be potentially lethal, depend on the condition of air spreading. Subcutaneous emphysema which occurs intra- or postoperative period is sometimes iatrogenic because the air is introduced into the tissue space through the hole injured by the operation. But the emphysema in this case occurred preoperatively by the pressure of the bag valve mask, because the patient had an intra-oral wound, which reaches the submental space. In this report, we describe an extremely rare case of preoperative massive emphysema of the patient with the mandibular fracture.