Ryota Oka

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.

Abstract 145: MicroRNA-361-3p functions as an oncogenic microRNA in human oral cancer cells

MicroRNAs (miRNAs) are small non-coding double-stranded RNA with sizes of 20-25 nucleotides, and inhibit protein translation by binding the 39-untranslated region of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes (OncomiR). Most recent study has demonstrated OncomiR addiction in mouse pre-B-cell lymphoma. OncomiR addiction may also provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify an OncomiR in human oral cancer cells through functional screening and considered whether targeting miRNA can be possible for cancer therapy. First, we performed functional screening for OncomiR in human oral cancer cells by the use of miRCURY LNATM microRNA Knockdown Library (Exiqon). We transfected 918 locked nucleic acid (LNA) antisense oligonucleotides for specific human mature miRNAs into human oral squamous cell carcinoma cells (GFP-SAS) and salivary gland cancer cells (GFP-ACCM). After transfection for 80 hours, each cell growth was evaluated. LNA antisense oligonucleotides against microRNA-361-3p (LNA-miR-361-3p) showed a remarkable growth inhibition in both types of cells as compared with non-targeting LNA oligonucleotides. We also observed the change of cell morphology, diminution of colony size, and a number of non-adherent cells after transfection of LNA-miR-361-3p. Subsequently, we examined the knockdown effect of LNA-miR-361-3p in GFP-SAS cells by quantitative RT-PCR. Compared with control oligonucleotides, the expression of miR-361-3p was significantly reduced by 71%. These effects of LNA-miR-361-3p were not observed by transfection of DNA or RNA antisense oligonucleotides for miR-361-3p. Next, we transfected synthetic human mature miR-361-3p into GFP-SAS cells to investigate the effect of miR-361-3p overexpression. Cell growth resulted in a significant 20% increase compared with non-targeting control miRNA. Furthermore, co-transfection of LNA-miR-361-3p and its decoy oligonucleotides abrogated the growth inhibitory effect by LNA-miR-361-3p in GFP-SAS cells. These results suggest that miR-361-3p functions as an OncomiR in human oral cancer cells and LNA antisense oligonucleotides are useful and efficient for silencing miRNA. Targeting miR-361-3p with LNA antisense oligonucleotides may be a useful therapeutic approach for patients with oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2011-145

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.

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.

Abstract 3003: Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent experiments have showed that it is possible to use pharmacological agents that inactivate oncogenes to treat at least some types of human malignancies. However, the development of molecular targeted therapy in oral squamous cell carcinoma (OSCC) is lagging behind compare to other cancers. In this study, we attempted to identify an appropriate target molecule and to determine whether targeting such a molecule is plausible therapeutic approach for the treatment of patients with OSCC. We determined the gene expression profiles of 9 human OSCC cell lines and non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene. AURKA has been shown to be related to the progression, survival, histological differentiation, and metastasis in various tumors. We revealed the overexpression of AURKA mRNA and protein in human OSCC cell lines and tissues. To clarify the function of AURKA in the cell proliferation of OSCC cells, we investigated the effect of small interfering RNA (siRNA) specific for AURKA (siAURKA) and MLN8237, an AURKA selective inhibitor, on the growth of human OSCC cells in vitro and in vivo. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of these cells compared to non-targeting siRNA. MLN8237 also markedly reduced the growth rate of human OSCC cells. Next, we assessed the growth inhibitory effect of siAURKA and MLN8237 using mouse model. Synthetic siAURKA/atelocollagen complexes (40 μM) were administrated into mouse tail vain every 3 days, and MLN8237 (20 mg/kg) was received orally for 14 consecutive days. We found both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors. To confirm the usefulness of targeting AURKA in OSCC, we attempted to culture the resected tumor tissues from 3 patients with OSCC and obtained primary cultured cells. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of OSCC primary cultured cells. Furthermore, the expression of AURKA mRNA in OSCC tissues resected from patients was examined. In 37 of 50 primary OSCC tissues, the expression levels of AURKA mRNA were more than 2-fold increase compared to normal oral mucosa tissues, and notably we found significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. These results suggest that AURKA plays a critical role in the growth of human OSCC cells, and targeting AURKA appears to be a potentially useful therapeutic approach for patients with OSCC. Citation Format: Hiroshi Tanaka, Koh-ichi Nakashiro, Kazuki Iwamoto, Norihiko Tokuzen, Yohei Fujita, Rikimaru Shirakawa, Ryota Oka, Hiroyuki Goda, Hiroyuki Hamakawa. Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2013-3003

Abstract 113: Effect of targeting Aurora kinase A in human oral squamous cell carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not available. In this study, we identified an appropriate molecule for treatment of patients with OSCC. We determined the gene expression profiles in 10 human OSCC cell lines and a non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene which was commonly overexpressed in OSCC cells. We also confirmed the overexpression of AURKA mRNA in OSCC tissues and compared to normal oral mucosa tissues by quantitative RT-PCR. AURKA plays an essential role in centrosome duplication, mitoic entry, and spindle assembly. Overexpression of AURKA in human malignancies is associated with gene amplification, genetic instability, poor histological differentiation, and poor prognosis. To determine if the AURKA is a plausible therapeutic target, we investigated the effect of small interfering RNAs specific for AURKA(siAURKA) and an AURKA selective inhibitor MLN8237 on the growth of human OSCC cells in vitro and in vivo. Knockdown of AURKA by RNA interference (RNAi) markedly inhibited the growth of OSCC cells and observed the change of colony formation and cell size looked like cell cycle arrest. We examined the RNAi effect of siAURKA in OSCC cells by Western blotting. Compared with non-target siRNA, the protein expression level of AURKA was almost completely suppressed. MLN8237 also reduced the cell growth, but showed relatively resistant to cells expressing AURKA at high levels. Transfection of siAURKA inhibited the growth of OSCC cells more potent than treatment with MLN8237 in vitro. Furthermore, we confirmed the effect of siAURKA and MLN8237 on the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice. Atelocollagen-siAURKA (8 nmol) complexes were injected into the tail vein every 3 days and MLN8237 (30 mg/kg) were received orally for 14 consecutive days. We found that siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSSC tumors. Interestingly, the combination of siAURKA and MLN8237 induced remarkable anti-tumor activity compared with single agent groups. These results suggest that AURKA functions as a critical gene for supporting the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 113. doi:1538-7445.AM2012-113

Abstract 720: Identification of novel molecular markers for detecting lymph node metastasis of oral squamous cell carcinoma

Cervical lymph node metastasis is a potent prognostic factor in oral squamous cell carcinoma (OSCC). Management of occult metastasis is an important issue for the surgeons. Sentinel lymph node biopsy (SLNB) is highlighted for its role in deciding making of neck dissection. We have performed the SLNB for cN0 cases routinely and reported the clinical usefulness. Accurate diagnosis of the metastasis in sentinel lymph nodes is important but depends on a heavy workload of the pathologist. We carried out a clinical study to evaluate a novel automated assay system for cytokeratin 19 (CK19) mRNA, the one-step nucleic acid amplification (OSNA) method, to detect the lymph node metastasis of OSCC. The accuracy of OSNA assay was nearly 95%, but it could not detect occult metastasis in case of no or low expression of CK19 mRNA in the primary tumor. In this study, we attempted to identify novel molecular markers to find the harboring cancer cells in the lymph node. First, we determined the gene expression profiles in 7 metastatic lymph nodes from patients with OSCC, 1 benign lymph node, and 5 salivary gland tissues from non-cancerous patients by microarray analysis. We found the overexpression of 36 genes in all metastatic lymph nodes but not in benign lymph node and salivary glands. Subsequently, we examined the expression of these genes in newly 15 metastatic lymph nodes and 9 benign lymph nodes by real-time quantitative RT-PCR method. Among the 36 genes, the expression of annexin A8-like 2 (ANXA8L2) and desmoglein 3 (DSG3) was commonly detected in metastatic lymph nodes at much higher level but not in benign lymph nodes at all. Furthermore, we retrospectively evaluated the usefulness of these markers with the use of 330 lymph nodes that were histopathologically metastasis-positive in 62 and metastasis-negative in 268. The accuracy of each marker CK19, ANXA8L2 and DSG3 is all near 90%. However, the combination of these markers improved the sensitivity up to 96-100%. Expression of ANXA8L2 and DSG3 was also detected in approximately 3% of the histopathologically metastasis-negative lymph nodes. These results suggested that ANXA8L2 and DSG3 could be useful molecular markers and improve detection rate of occult metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 720. doi:1538-7445.AM2012-720