Knockdown RPL29 Gene Can Inhibit the Proliferation, Invasion of Squamous Cell Carcinomas.

OBJECTIVE: Gingival squamous cell carcinoma (GSCC) is one of the most common malignancies. Endogenous ribosomal protein L29 (RPL29) has been previously proven to be up-regulated in cancer tissues. However, RPL29 expression in GSCC has not been described. METHODS: The knockdown of RPL29 gene in GSCC cells was carried out to study the influence of RPL29 silencing on GSCC cells. RESULTS: We investigated the influence on cell proliferation, invasion, and related biomarkers, which led us to confirm that all were repressed by the knockdown of RPL29. CONCLUSION: The role of RPL29 should lead to a better understanding of the development and progression of human GSCC.

Intratumor genetic heterogeneity in squamous cell carcinoma of the oral cavity.

BACKGROUND: We sought to evaluate intratumor heterogeneity in squamous cell carcinoma of the oral cavity (OCC) and specifically determine the effect of physical separation and histologic differentiation within the same tumor. METHODS: We performed whole exome sequencing on five biopsy sites-two from well-differentiated, two from poorly differentiated regions, and one from normal parenchyma-from five primary OCC specimens. RESULTS: We found high levels of intratumor heterogeneity and, in four primary tumors, identified only 0 to 2 identical mutations in all subsites. We found that the heterogeneity inversely correlated with physical separation and that pairs of well-differentiated samples were more similar to each other than analogous poorly differentiated specimens. Only TP53 mutations, but not other purported "driver mutations" in head and neck squamous cell carcinoma, were found in multiple biopsy sites. CONCLUSION: These data highlight the challenges to characterization of the mutational landscape of OCC with single site biopsy and have implications for personalized medicine.

Profiling of genomic alterations of mitochondrial DNA in gingivobuccal oral squamous cell carcinoma: Implications for disease progress.

We have identified 164 somatic mutations in mitochondrial DNA in gingivobuccal oral cancer by deep sequencing the mitochondrial genome from paired tumor and blood DNA samples from 89 patients. We have found evidence of positive selection of somatic nonsynonymous mutations. Non-synonymous mutations in mitochondrial respiratory genes were found to increase the risk of lymph node metastasis (P = 0.0028). We have observed a significant reduction in mitochondrial DNA copy number in tumor DNA of these patients compared to the DNA from adjacent normal tissue samples (P < 1 × 10-6). Analysis of transcriptome data of tumor and adjacent normal tissue revealed patients harboring mutations in mitochondrial protein-coding genes exhibited reduced expression of mitochondrial transcripts.

Comparison of gene expression profiles of gingival carcinoma Ca9-22 cells and colorectal adenocarcinoma HT-29 cells to identify potentially important mediators of SLPI-induced cell migration.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor whose expression level is positively correlated with tumor aggressiveness and metastatic potential. However, the mechanism underlying SLPI-induced enhancement of malignant phenotype is not completely understood. The malignancy of cancer cells is highly dependent on cell migration activity. Our previous study revealed that gingival carcinoma Ca9-22 cells, but not colorectal adenocarcinoma HT-29 cells, expressed SLPI. Therefore, we investigated the migration activity of these two cell types to understand the nature of SLPI-mediated tumor aggressiveness and metastatic potential. In vitro wound healing assay indicated that HT-29 cells and SLPI-deleted Ca9-22 cells showed lower migration activity than wild-type Ca9-22 cells, suggesting that SLPI-induced cell migration plays an important role in tumor aggressiveness and metastatic potential. In addition, our gene expression profiling study based on microarray data for the three cell types identified a number of candidates, including LCP1 and GLI, that could be key molecules in the mechanism of SLPI-induced cell migration.

Spontaneous regression of plasmablastic lymphoma in an elderly human immunodeficiency virus (HIV)-negative patient.

Plasmablastic lymphoma (PBL) is an aggressive lymphoma commonly associated with human immunodeficiency virus (HIV) infection. Herein we describe a rare case of PBL that spontaneously regressed. An 80-year-old man was referred to our hospital owing to an exophytic gingival tumor in the right maxillary second molar region. He had no significant past medical history, and a screening test for HIV was negative. Imaging showed that the tumor measured 26 × 23 × 16 mm and was confined in the alveolar bone. The tumor was histologically comprised of highly proliferative immunoblastic cells positive for CD138 and Epstein-Barr virus (EBV)-encoded RNA. Monoclonal IgH chain gene rearrangement was detected via polymerase chain reaction. After biopsy and diagnosis of PBL, the tumor began to decrease in size and had apparently disappeared at the time of surgery. There was no histological evidence of a residual lesion in the surgical specimen. In conclusion, a minority of immunosenescence-associated PBLs in the elderly should be recognized as a unique clinicopathological entity distinct from common aggressive PBL.

Angioimmunoblastic T-cell lymphoma of the oral cavity presenting as gingival mass: report of the histopathologic and molecular characteristics of an unusual case featuring clonal T-cell receptor γ gene rearrangement by polymerase chain reaction.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare neoplastic process constituting 15% to 20% of peripheral T-cell lymphomas. We report the clinicopathologic and molecular characteristics of an unusual intraoral manifestation of AITL. A 35-year-old white man with a history of AITL presented with a 2.5-cm, poorly circumscribed, erythematous, exophytic lesion occupying the free and attached buccal gingiva of the right maxillary lateral incisor and canine. Histopathologically, the tumor showed diffuse and intense polymorphic infiltration by small to medium-sized lymphocytes admixed with numerous eosinophils. The neoplastic cells showed strong and diffuse reactivity for CD2, CD3, CD4, CD10, and PD-1 (programmed cell death 1 [PDCD1]). Rare immunopositivity was seen with BCL6 (B-cell CLL/lymphoma 6) and CXCL13 (chemokine [C-X-C motif] ligand 13). Neoplastic cells were negative for CD7 and EBER ISH (Epstein-Barr virus-encoded small RNA in situ hybridization). CD21 did not show any increased follicular dendritic cell component. Polymerase chain reaction-based assay found monoclonal T-cell receptor γ (TRG) gene rearrangements. Diagnosis of recurrent/residual AITL was rendered. Chemotherapy was administered, with the intraoral tumor resolving completely 3 months later.

Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva.

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.

Identification of prognosis-related proteins in gingival squamous cell carcinoma by twodimensional gel electrophoresis and mass spectrometry-based proteomics.

OBJECTIVES: The aim of this study was to identify differentially expressed proteins in oral squamous carcinoma cells that could be potential prognosis-related cancer biomarkers. MATERIALS AND METHODS: We compared protein expression patterns from gingival squamous cellc carcinoma (GSCC) tissues and adjacent non-cancerous matched tissues by proteomic analysis using two-dimensional gel electrophoresis coupled to mass spectrometry (2D-PAGE/MS). RESULTS: Seventeen protein spots were found to be over-expressed and eight were under-expressed in cancerous tissue compared to the normal counterpart. Of these, annexin A2 and ezrin were validated by Western blot. We also demonstrated by immunohistochemistry that POSTN is highly expressed in the neoplastic tissues examined. Among the differentially expressed proteins, we focused our attention on Chloride intracellular channel 1 (CLIC1). CONCLUSION: The 2D-PAGE/MS-based proteomics appears an efficient approach in detecting and identifying differentially expressed proteins that might function as potential biomarkers and/or molecular targets for early cancer diagnosis and prognosis and that might contribute to a innovative therapeutic strategies in GSCC. However, further validation and functional studies are needed to confirm and to support these promising, still preliminary data. KEY WORDS: Cancer biomarkers, Oral squamous cell carcinoma, Proteomics.

Randomized, controlled phase II study of post-surgery radiotherapy combined with recombinant adenoviral human p53 gene therapy in treatment of oral cancer.

The aim of this study is to evaluate clinical benefits of recombinant adenoviral human p53 (rAd-p53) gene therapy combined with radiotherapy in prevention of oral cancer recurrence after a radical resection. A total of 51 patients with tongue cancer (TCa) and 56 patients with gingival carcinoma (GCa) satisfying the inclusion criteria were randomly assigned to two groups: the experiment group (EG) and the control group (CG). The EG group received multipoint injections of rAd-p53 into the surgical wound surface at a dose of 1 × 10¹² viral particles after a radical resection. Patients in both EG and CG were given radiotherapy at a total dose of 60 Gy at 3 weeks after surgery. All these patients were followed up for at least 3 years. Two cases (2/27) of TCa and 2 (2/30) in GCa patients had a local recurrence in EG, but 8 (8/24) TCa and 8 (8/26) GCa patients in CG had a local recurrence. Both recurrent rates of TCa (33.3%) and GCa (30.8%) in CG are statistically significantly higher than those of TCa (7.4%) and GCa (6.7%) in EG, respectively. The overall recurrent rate in EG is 7%, which is also statistically significantly lower than that (32%) in CG. The 3-year overall survival (OS) rate and 3-year disease-free survival (DFS) rate of EG is 100% and 93%, respectively. The 3-year OS and DFS rates of CG are 94 and 68%, respectively. Mild or medium fever and flu-like symptoms were more frequently observed in EG and were considered to be associated with application of rAd-p53. Post-tumorectomy wound surface injection of rAd-p53 combining with radiotherapy is a safe and effective regimen for the patients with TGa or GCa.

Trisomy 7 as sole aberration in peripheral ameloblastoma of the mandible.

Peripheral ameloblastoma (PA) is a rare, extraosseous odontogenic tumor with histologic features similar to those of the more common intraosseous ameloblastoma. The exact nature and tumorigenesis of PA are unclear. Although there are some reports on the cytogenetics of intraosseous ameloblastoma, to the authors' knowledge, there are no studies on the cytogenetic analysis of PA. The cytogenetic analysis of a PA occurring in the gingiva of a 56-year-old man is presented. Trisomy 7 was the only cytogenetic aberration.

Expression level of pre-B-cell leukemia transcription factor 2 (PBX2) as a prognostic marker for gingival squamous cell carcinoma.

OBJECTIVE: In this study, we investigated the interrelationship between clinicopathologic findings and pre-B-cell leukemia transcription factor 2 (PBX2) expression in gingival squamous cell carcinoma (GSCC). METHODS: Expression level of PBX2 was immunohistochemically examined in 66 GSCC subjects (30 men and 36 women) with ages ranging from 42 to 85 (median 64.5) years, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells. RESULTS: PBX2 expression is correlated with valosin-containing protein (VCP) expression. Univariate and multivariate analyses revealed a high level of PBX2 expression to be a poor prognosticator for disease-free survival (DFS) and overall survival (OS), and PBX2 expression was an independent prognostic factor for both DFS and OS in GSCC. CONCLUSIONS: PBX2 expression level in GSCC is prognostic. PBX2 may be a useful marker to identify the potential for progression in GSCC.

Genome-wide expression and copy number analysis identifies driver genes in gingivobuccal cancers.

The molecular mechanisms contributing to the development and progression of gingivobuccal complex (GBC) cancers-a sub-site of oral cancer, comprising the buccal mucosa, the gingivobuccal sulcus, the lower gingival region, and the retromolar trigone-remain poorly understood. Identifying the GBC cancer-related gene expression signature and the driver genes residing on the altered chromosomal regions is critical for understanding the molecular basis of its pathogenesis. Genome-wide expression profiling of 27 GBC cancers with known chromosomal alterations was performed to reveal differentially expressed genes. Putative driver genes were identified by integrating copy number and gene expression data. A total of 315 genes were found differentially expressed (P ≤ 0.05, logFC > 2.0) of which 11 genes were validated by real-time quantitative reverse transcriptase-PCR (qRT-PCR) in tumors (n = 57) and normal GBC tissues (n = 18). Overexpression of LY6K, in chromosome band 8q24.3, was validated by immunohistochemical (IHC) analysis. We found that 78.5% (2,417/3,079) of the genes located in regions of recurrent chromosomal alterations show copy number dependent expression indicating that copy number alteration has a direct effect on global gene expression. The integrative analysis revealed BIRC3 in 11q22.2 as a candidate driver gene associated with poor clinical outcome. Our study identified previously unreported differentially expressed genes in a homogeneous subtype of oral cancer and the candidate driver genes that may contribute to the development and progression of the disease. © 2011 Wiley Periodicals, Inc.

Expression of cytokeratins in the epithelium of canine odontogenic tumours.

Odontogenic tumours are considered to be relatively rare; however, several histologically distinct types have been identified in dogs. The more common canine odontogenic tumours are peripheral odontogenic fibroma and canine acanthomatous ameloblastoma. The expression of cytokeratins (CKs) has been established for the human dental germ and odontogenic tumours. The aim of the present study was to describe the immunohistochemical expression of a panel of CKs in the epithelium of the canine dental germ, normal gingiva and odontogenic tumours arising in this species. Samples from 20 odontogenic tumours, 12 tooth germs and three normal gingival tissues were obtained. Each sample was stained with haematoxylin and eosin and subjected to immunohistochemistry for CK expression. The typical expression pattern of CKs in the odontogenic epithelium and gingiva of dogs was CK14 and CK5/6. CKs 7, 8, 18 and 20 were generally absent from the canine dental germ, gingiva and odontogenic tumours. Dogs and man therefore exhibit similar CK expression in the odontogenic epithelium.

Evaluation of MMP-1, MMP-10, TIMP-1, a-SMA and TGF-b1 in angiofibromas of tuberous sclerosis.

AIM: Tuberous sclerosis is a neurocutaneous syndrome characterized by affect multiple organs such as brain, kidneys, heart, eyes, lungs and skin. The aim of this study was to analyze the pattern of immunolocalization of markers MMP-1, MMP-10, TIMP-1, α-SMA and TGF-ß1 in oral and facial angiofibromas in individuals affected by tuberous sclerosis. METHODS: Microscopical analyses on hematoxilin-eosin and immunohistochemistry reactions were performed to analyze the previously cited biological markers pattern in orofacial angiofibromas. RESULTS: Reactivity was observed for MMP-1, MMP-10 and TGF-ß1, in addition to negative for TIMP-1 and α-SMA, except perivascular and epithelial staining for this. Concerning the intensity, a strong marking for MMP-1 in the basal layer of the epithelium, and a slight positivity in the suprabasal layers predominated. MMP-10 was slightly expressed in all epithelial layers. The connective tissue showed slight to moderate reactivity for MMP-1 and MMP-10. TIMP-1 demonstrated slight to moderate marking in the various layers of a single lesion and to TGF-ß1 expression showed varied in intensity staining both between lesions and between tissue layers. CONCLUSION: MMP-1, MMP-10 and TGF-ß1 exhibited reactivity in oral and cutaneous angiofibromas with heterogeneous distribution patterns among both tissue elements analyzed in the intensity of marking the same among the specimens. TIMP-1 showed reactivity predominantly negative in the specimens analyzed and α-SMA presented restricted to epithelial and perivascular regions of these lesions.

Plasmablastic lymphoma of the oral cavity in an HIV-negative patient.

Plasmablastic lymphoma (PBL) is a rare, highly aggressive lymphoma typified by immunoblast-like cells with abundant basophilic cytoplasm and paranuclear hof. It shows absent expression of CD45 and CD20. In contrast, it displays a constant reaction with CD138 and VS38c. It may be easily misinterpreted as some other lymphoma. An exhaustive integration of clinical, morphologic, phenotypic, and molecular features is important to exclude misdiagnosis and inappropriate treatment. We report a case of HIV-negative PBL arising on the left areas of posterior teeth mucosa of a 58-year-old man. Immunohistochemically, the tumor cell was immunoreactive for CD138, VS38c, VEGF, and vimentin; Ki-67 showed a high proliferation rate. Epstein-Barr virus (in situ hybridization) was nonreactive, and IgH gene rearrangement was identified by polymerase chain reaction amplification products. A diagnosis of PBL was rendered.

High α-defensin and S100A7 expression and missing DOC-1 down-regulation characterize irritation fibromas of the oral cavity and may counteract malignant transformation.

PURPOSE: The purposes of this study were to analyze the gene expression pattern of antimicrobial peptides, tumor suppressors, growth factors, matrix metalloproteases, and inflammatory cytokines and chemokines in oral irritation fibromas and to identify genes with protective effects against malignant transformation in benign proliferating tumors of the oral mucosa. MATERIALS AND METHODS: Biopsies of irritation fibromas (n = 15) and healthy gingiva (n = 15) were obtained during routine surgical procedures. RNA was extracted according to standard protocols, and transcription levels of CCL20, DEFA 1/3, DEFA 4, S100A7, DOC-1, interleukin (IL) 1ß, IL-6, IL-8, IL-10, tumor necrosis factor α, Cox-2, matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, transforming growth factor ß1, transforming growth factor α, and keratinocyte growth factor were analyzed by real-time polymerase chain reaction. In addition, immunostaining was performed to visualize the transcription products of the genes of interest in fibroma tissue as well as in healthy gingiva. RESULTS: The gene expression of S100A7 was 11.3-fold and that of DEFA 1/3 was 14-fold higher in irritation fibromas than in healthy gingiva, whereas the expression of MMP-3 and of inflammation markers IL-1ß, IL-6, IL-8, tumor necrosis factor α, and Cox-2 was reduced. Profound down-regulation of DOC-1 gene expression, characteristic for proliferating malignant tumors of the oral cavity, was in irritation fibromas not verifiable. CONCLUSIONS: Changes in the expression pattern of S100A7, DEFA 1/3, and MMP-3 seem to be involved in the development of irritation fibromas, whereas chronic inflammation might be of less importance. Overexpression of S100A7, but missing down-regulation of the tumor-suppressor gene DOC-1, might exert protective effects and counteract malignant transformation of benign, proliferating lesions of the oral cavity.

Oral and neurocutaneous phenotypes of familial tuberous sclerosis.

OBJECTIVE: The objective of this study was to describe the pattern of inheritance and the clinical features in a large family with tuberous sclerosis (TS), and to focus on the general diagnosis after the initial oral examination. STUDY DESIGN: To characterize the pattern of inheritance and the clinical features, 61 familial members were systematically evaluated, including dermatologic, ophthalmologic, and orofacial examination. Imaging exams, such as abdomen ultrasonography, echocardiogram, fundoscopy, cranial cone-beam computerized tomography, and brain magnetic resonance, were performed. Hematoxylin and eosin stain and scanning electronic microscopy were performed to characterize TS-associated alterations in the teeth, nails, and hair. RESULTS: The pedigree of the family was constructed including the 4 last generations and revealed nonconsanguineous marriages and an autosomal dominant mode of TS transmission. We identified 13 family members affected by TS, with 6 of them completely fulfilling the diagnostic criteria of this disorder. Hypomelanotic macules in the skin, facial angiofibromas, and dental enamel pits were the most common features of affected patients. Central nervous system alterations were identified in 5 family members, whereas cardiac and renal alterations were found in 1 member each. CONCLUSION: We emphasize, in this study, the importance of oral findings such as dental enamel pits and gingival angiofibromas in the early diagnosis of familial TS which led to complete familial profile and pattern of inheritance establishment.

Mutational analyses of the BRAF, KRAS, and PIK3CA genes in oral squamous cell carcinoma.

OBJECTIVES: The development of oral squamous cell carcinoma (OSCC) is a complex, multistep process. To date, numerous oncogenes and tumor-suppressor genes have been implicated in oral carcinogenesis. Of particular interest in this regard are genes involved in cell cycling and apoptosis, such BRAF, KRAS, and PIK3CA genes. STUDY DESIGN: Mutations of BRAF, KRAS, and PIK3CA were evaluated by direct genomic sequencing of exons 1 of KRAS, 11 and 15 of BRAF, and 9 and 20 of PIK3CA in OSCC specimens. RESULTS: Both BRAF and KRAS mutations were detected with a mutation frequency of 2% (1/42). PIK3CA mutations were detected at 3% (1/35). CONCLUSIONS: This is the first report implicating BRAF mutation in OSCC. Our study supports that mutations in the BRAF, KRAS, and PIK3CA genes make at least a minor contribution to OSCC tumorigenesis, and pathway-specific therapies targeting these 2 pathways should be considered for OSCC in a subset of patients with these mutations.

Bone destruction by invading oral squamous carcinoma cells mediated by the transforming growth factor-beta signalling pathway.

BACKGROUND: Gingival squamous cell carcinoma (SCC) cells frequently invade mandibular bone, and this destruction is associated with a worse prognosis. However, the relationship between bone destruction and associated factors is unclear. In this study, the role and diagnostic utility of transforming growth factor-beta (TGF-beta) type I receptor (TbetaRI) in bone destruction of the mandible was investigated. PATIENTS AND METHODS: The expression of TbetaRI was explored by using an immunohistochemical method on paraffin-embedded tissues from 21 cases of mandibular SCC. An inhibitor of the kinase activity of the TbetaRI (TbetaRI-I) was used to assess the role of TbetaRI in bone destruction by a human oral SCC cell line (HSC-2) that highly expresses TbetaRI. RESULTS: TbetaRI-positive signals were closely associated with destructive invasion of the mandible by oral SCC cells. Consistent with these results, TbetaRI-I greatly reduced HSC-2 cell-induced bone destruction and osteoclast formation in vivo and in vitro. TbetaRI-I treatment reduced the expression of TNF-alpha, RANKL and connective tissue growth factor (CTGF/CCN2), all of which were up-regulated by TGF-beta in HSC-2 cells. CONCLUSION: These data demonstrated an important role for TGF-beta signalling in bone invasion by oral SCC cells, and suggest that the bone destruction is mediated by RANKL, TNF-alpha and CCN2.