Naoki Katase

NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation.

Diagnostic value of MRI for odontogenic tumours

OBJECTIVES To evaluate the diagnostic value of MRI for odontogenic tumours. MATERIALS AND METHODS 51 patients with odontogenic tumours were subjected to pre-operative MRI examinations. For tumours with liquid components, i.e. ameloblastomas and keratocystic odontogenic tumours (KCOTs), the signal intensity (SI) uniformity of their cystic components (UΣ) was calculated and then their UΣ values were compared. For tumours with solid components that had been examined using dynamic contrast-enhanced MRI (DCE-MRI), their CImax (maximum contrast index), Tmax (the time when CImax occurred), CIpeak (CImax × 0.90), Tpeak (the time when CIpeak occurred) and CI300 (i.e. the CI observed at 300 s after contrast medium injection) values were determined from CI curves. We then classified the odontogenic tumours according to their DCE-MRI parameters. RESULTS Significant differences between the UΣ values of the ameloblastomas and KCOT were observed on T1 weighted images, T2 weighted images and short TI inversion recovery images. Depending on their DCE-MRI parameters, we classified the odontogenic tumours into the following five types: Type A, CIpeak > 2.0 and Tpeak < 200 s; Type B, CIpeak < 2.0 and Tpeak < 200 s; Type C, CI300 > 2.0 and Tmax < 600 s; Type D, CI300 > 2.0 and Tmax > 600 s; Type E, CI300 < 2.0 and Tmax > 600 s. CONCLUSION Cystic component SI uniformity was found to be useful for differentiating between ameloblastomas and KCOT. However, the DCE-MRI parameters of odontogenic tumours, except for odontogenic fibromas and odontogenic myxomas, contributed little to their differential diagnosis.

Usefulness of MRI and dynamic contrast-enhanced MRI for differential diagnosis of simple bone cysts from true cysts in the jaw

INTRODUCTION It can be difficult to differentiate simple bone cysts (SBCs) from true cysts in the jaw when these lesions appear unilocular. The present study reports the MR imaging of subjects with SBCs and describes the diagnostic value of the MRI findings. MATERIALS AND METHODS Ten subjects with SBCs in the jaw were examined using MRI. T1- and T2-weighted images (T1WI, T2WI) were obtained, and contrast-enhanced images and dynamic contrast-enhanced MRI (DCE-MRI) were acquired. RESULTS In all cases, the contrast-enhanced T1WI acquired approximately 6 minutes after the administration of Gd-DTPA showed marked enhancement of the margin and slight enhancement of the inner part of the cyst cavity. In all cases, the time-signal intensity (SI) curves show a gradual increase in the SI until approximately 15 minutes after the administration of Gd-DTPA. These findings might not be observed on the DCE-MRIs of the other true cysts with epithelial lining that show no enhancement in a cavity. CONCLUSION MRI, especially DCE-MRI, can provide useful information for distinguishing SBCs from other cysts.

Loss of Heterozygosity at the 9p21-24 Region and Identification of BRM as a Candidate Tumor Suppressor Gene in Head and Neck Squamous Cell Carcinoma

We have analyzed allelic loss of the short arm of chromosome 9 in 39 head and neck cancers using 13 polymorphic markers and found two deletions of hot spots at 9p21 and 9p24. Loss of heterozygosity was detected at least at one locus in 28 of 39 cases (71.8%). P16, a well-known tumor suppressor gene, is considered to be a target for deletion of the 9p21 region. However, novel frequent chromosomal deletion and a candidate tumor suppressor gene, BRM at the 9p24 region, were detected. Moreover, comparison of clinicopathological variables demonstrated that loss of heterozygosity at the BRM locus was associated with a worse prognosis.

Deletion at Dickkopf (dkk)-3 locus (11p15.2) is related with lower lymph node metastasis and better prognosis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a frequently occurring cancer, and despite improvement of its treatment methods, including chemotherapy, radiotherapy, and surgery, the improvement of survival remains poor. Recent advances in molecular biology of human cancer indicated various molecular abnormalities in HNSCC, including activation of oncogenes and inactivation of tumor suppressor genes (TSGs). Dickkopf (Dkk)-3 gene is known as a negative regulator of Wnt signaling and is suggested to function as TSG in several kinds of malignancies. We hypothesized that Dkk-3 might play an important role in HNSCC, too. Thus, in the current study, we analyzed allelic alteration of Dkk-3 locus (chromosome 11p15.2) by means of loss of heterozygosity (LOH) analysis. The study population consisted of 50 patients with HNSCC (mean age of 65 years old). Furthermore, we also examined the correlation between LOH findings of Dkk-3 locus with clinicopathological parameters to investigate its use as a biomarker in HNSCC. A remarkable LOH ratio (57%) was detected in the cases studied, implying that Dkk-3 is likely to be involved in HNSCC carcinogenesis. However, interestingly and in contrast to the expectations, we found that the group with LOH of Dkk-3 locus had less lymph node metastasis, and showed a favorable overall survival compared to the patients with retention of Dkk-3 area in survival analysis. These results indicate that Dkk-3 can play a role in HNSCC carcinogenesis with unknown mechanism. Moreover, allelic loss at Dkk-3 locus may also be used as a novel prognostic biomarker in HNSCC.

Clinical Evaluation of an Unsintered Hydroxyapatite/Poly-L-Lactide Osteoconductive Composite Device for the Internal Fixation of Maxillofacial Fractures

Introduction:OSTEOTRANS MX (Takiron Co, Ltd, Osaka, Japan) is a resorbable osteosynthetic material composed of an unsintered hydroxyapatite/poly-L-lactide composite, and its osteoconductive capacity has been documented. The authors here report their clinical experience using OSTEOTRANS MX. Methods:The authors treated 35 patients (19 men, 16 women; age, 14–88 years; mean ± standard deviation, 38.4 ± 19.9 years) with maxillofacial fractures. The authors used standard surgery to stabilize fractures in all patients, fitting resorbable plates (thickness, 1.0 or 1.4 mm) and screws (diameter, 2 mm) according to Arbeitsgemeinschaft für Osteosynthesefragen/Association (AO) for the Study of Internal Fixation guidelines. Results:All patients eventually achieved satisfactory healing with favorable restoration of form and function without foreign body reaction. Complications occurred in 3 patients—plate exposure in 2 and discomfort in 1. However, fracture sites healed in all patients. Scanning electron microscopy revealed that the devices bonded directly to the bone without interposition of nonmineralized tissue. Conclusion:OSTEOTRANS MX is a useful material with few complications. Its osteoconductive bioactivity is advantageous for the early functional improvement of maxillofacial fractures.

Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

Background. Wnt signaling is involved in muscle formation through β-catenin-dependent or -independent pathways, but interactions with other signaling pathways including transforming growth factor β/Smad have not been precisely elucidated. Results. As Wnt4 stimulates myogenic differentiation by antagonizing myostatin (GDF8) activity, we examined the role of Wnt4 signaling during muscle differentiation in the C2C12 myoblast cell line. Among several extrinsic signaling molecules examined in a microarray analysis of C2C12 cells during the transition from cell proliferation to differentiation after mitogen deprivation, bone morphogenetic protein 4 (BMP4) expression was prominently increased. Wnt4 overexpression had similar effects on BMP4 expression. BMP4 was able to inhibit muscle differentiation when added to the culture medium. BMP4 and noggin had no effects on the cellular localization of β-catenin induced by Wnt3a; however, the BMP4-induced phosphorylation of Smad1/5/8 was enhanced by Wnt4, but not by Wnt3a. The BMP antagonist noggin effectively stimulated muscle differentiation through binding to endogenous BMPs, and the effect of noggin was enhanced by the presence of Wnt3a and Wnt4. Conclusion. These results suggest that BMP/Smad pathways are modified through Wnt signaling during the transition from progenitor cell proliferation to myogenic differentiation, although Wnt/β-catenin signaling is not modified with BMP/Smad signaling.

Comparative analysis of basal lamina type IV collagen α chains, matrix metalloproteinases-2 and -9 expressions in oral dysplasia and invasive carcinoma

The aim of this study was to compare the expressions of basal lamina (BL) collagen IV α chains and matrix metalloproteinases (MMP)-2 and MMP-9 in oral dysplasia (OED) and invasive carcinoma. Ten cases each of OEDs, carcinomas-in situ and oral squamous cell carcinomas (OSCCs) were examined by immunohistochemistry. Another 5 cases, each of normal and hyperplastic oral mucosa, served as controls. Results showed that α1(IV)/α2(IV) and α5(IV)/α6(IV) chains were intact in BLs of control and OEDs. In BLs of carcinoma-in situ, α1(IV)/α2(IV) chains preceded α5(IV)/α6(IV) chains in showing incipient signs of disruption. OSCCs exhibited varying degrees of collagen α(IV) chain degradation. MMP-2 and MMP-9 were absent in controls and OED, but weakly detectable in carcinoma-in situ. In OSCC, these proteolytic enzymes were expressed in areas corresponding to collagen α(IV) chain loss. Enzymatic activity was enhanced in higher grade OSCC, and along the tumor advancing front. Overall the present findings suggest that loss of BL collagen α(IV) chains coincided with gain of expression for MMP-2 and MMP-9, and that these protein alterations are crucial events during progression from OED to OSCC.

Dickkopf (Dkk)-3 and β-catenin expressions increased in the transition from normal oral mucosal to oral squamous cell carcinoma

Dickkopf (Dkk)-3, an inhibitor of the Wnt/β-catenin pathway, is reported as a potential tumor suppressor gene in many cancers. To gain a better comprehension of the mechanisms involved in the carcinogenesis of oral squamous epithelium, protein expression and localization of Dkk-3 and β-catenin was investigated in normal epithelium, dysplasias and squamous cell carcinoma (SCC). An increase in β-catenin and Ki-67 expressions was observed from dysplasias to poorly differentiated SCC. Interestingly, an increase in Dkk-3 positive cells was also noted, which was correlated to the cancer progression step. A change in Dkk-3 localization during the transformation of normal oral epithelium to SCC was clearly observed. Dkk-3 was localized in the cell membrane in normal oral epithelium and in dysplasias, whereas that was localized in both cell membrane and cytoplasm in SCC. These results suggest that Dkk-3 is involved in the carcinogenesis of SCC with a distinct function from those in other cancers.

Allelic loss of the ING gene family loci is a frequent event in ameloblastoma

Ameloblastoma is the most frequently encountered odontogenic tumor, characterized by a locally invasive behavior, frequent recurrences, and, although rare, metastatic capacity. Loss or inactivation of tumor suppressor genes (TSGs) allows cells to acquire neoplastic growth. The ING family proteins are tumor suppressors that physically and functionally interact with p53 to perform important roles in apoptosis, DNA repair, cell cycle regulation, and senescence. TP53 genetic alterations were reported to infrequently occur in ameloblastoma. Considering that other TSGs related to TP53 could be altered in this tumor, we focused our study on the ING family genes. We analyzed the loss of heterozygosity (LOH) status of the ING family (ING1-ING5) chromosomal loci in a group of ameloblastomas by microsatellite analysis, and correlated the ING LOH status with clinicopathological characteristics. By using specific microsatellite markers, high frequency of LOH was found at the loci of each ING gene family member (33.3-72.2%). A significant relationship was shown between LOH of D2S 140 (ING5 locus) and solid tumor type (p = 0.02). LOH of ING3MS (ING3 locus) was also high in solid type tumors, showing a near significant association. In addition, a notable tendency toward higher LOH for half of the markers was observed in recurrent cases. LOH of ING family genes appears as a common genetic alteration in solid ameloblastoma. The current study provides interesting novel information regarding the potential prognostic significance of the allelic loss of the ING gene family loci in ameloblastoma tumorigenesis.