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Purpose To retrospectively evaluate skeletal stability after Le Fort I maxillary impaction surgery and mandibular autorotation without bilateral sagittal split osteotomy (BSSO) in high-angle class II patients. Materials and methods Seven female high-angle class II patients who underwent maxillary impaction surgery and mandibular autorotation without bilateral sagittal split osteotomy were included in this study. Surgical changes and relapse were measured on lateral cephalograms taken preoperatively and at 1 month, 6 months and 1 year postoperatively. Results The horizontal movement of the maxilla at point A was 5.8 ± 3.3 mm backward, and the upward movement at the posterior nasal spine was 3.3 ± 1.4 mm. The mean horizontal change at point A during the 1-year follow-up period was 0.1 ± 0.2 mm, and the vertical change at posterior nasal spine was 0.2 ± 1.3 mm, which were not statistically significant. The horizontal surgical change at point B was 4.0 ± 1.8 mm forward and the vertical surgical change at point B was 4.7 ± 1.8 mm upward. Postoperative relapse was 10.9% and 13.7% in the horizontal and vertical directions, respectively. Conclusions Le Fort I maxillary impaction surgery with mandibular autorotation may be 1 of the suitable procedures for high-angle class II patients.

To evaluate the relationship between the changes in condylar volume and maxillofacial skeletal morphology according to sex as well as the relationship between condylar volume reduction and skeletal relapse in patients who underwent orthognathic surgery.Ninety-five patients were categorized into skeletal Class III, Class II, and facial asymmetry groups. Computed tomography scans taken preoperatively and at 1 year postoperatively were used for quantitative measurement.Postoperative condylar volume was reduced in both the Class II group and the deviated side of the asymmetry group. Both female and Class II deformity were significant predictors of postoperative reduction in the condylar volume. There was a significant correlation between skeletal relapse and postoperative change in condylar volume in the Class II group.Postoperative condylar resorption may be associated with preoperative maxillofacial skeletal morphology and sex and also with skeletal relapse in the Class II group.

Chronic nonbacterial osteomyelitis is a rare bone disorder that can be found in the jaw. It is often associated with systemic conditions, including autoimmune deficiencies. However, little is known about how the genetic and immunologic background of patients influences the disease. Here, we focus on human leukocyte antigen (HLA), killer cell immunoglobulin-like receptors (KIRs), and their specific combinations that have been difficult to analyze owing to their high diversity. We employed a recently developed technology of simultaneous typing of HLA alleles and KIR haplotype and investigated alleles of the 35 HLA loci and KIR haplotypes composed of centromeric and telomeric motifs in 18 cases and 18 controls for discovery and 472 independent controls for validation. We identified an amino acid substitution of threonine at position 94 of HLA-C in combination with the telomeric KIR genotype of haplotype tA01/tB01 that had significantly higher frequency (>20%) in the case population than in both control populations. Multiple logistic regression analysis based on a dominant model with adjustments for age and sex revealed and validated its statistical significance and high predictive accuracy (C-statistic ≥0.85). Structure-based analysis revealed that the combination of the amino acid change in HLA-C and the telomeric genotype tA01/tB01 could be associated with lower stability of HLA-C. This is the first case-control study of a rare disease that employed the latest sequencing technology enabling simultaneous typing and investigated amino acid polymorphisms at HLA loci in combination with KIR haplotype.

BACKGROUND This study aimed to elucidate the correlation between gene amplification, protein expression of receptor tyrosine kinase, and prognosis of patients with oral squamous cell carcinoma (OSCC) using immunohistochemistry (IHC) and next-generation sequencing data. METHODS We evaluated data pertaining to 208 patients with OSCC using IHC for epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET). RESULTS High expressions of EGFR and MET were detected in 60 and 41 patients, respectively. We evaluated the association of clinicopathological variables with high expressions of EGFR and/or MET. Distant metastasis was found in 9 of 41 patients (22.0%) and 6 of 15 patients (40.0%) with high expression of MET and high co-expressions of EGFR and MET, respectively; statistically significant differences were detected in both high expression of MET (P = .003) and high co-expressions of EGFR and MET (P = 3.41 × 10-5 ). The cumulative 5-year survival rate of patients with high and low expressions of EGFR or MET was approximately 65% and 85%, respectively. Conversely, among cases with high expressions of EGFR or MET, there was no additional decrease in the survival rate of patients harboring TP53 mutations. Moreover, the survival rate of patients with high co-expression of both EGFR and MET was very poor (22.0%) (P

Background: Biomarkers predicting therapeutic response to immunotherapy have been widely explored via monitoring the liquid and tissue-derived components. Increasing treatment options for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) mandates prediction of the therapeutic response of anti-PD-1 antibody alone as well as optimization of the treatment sequence. In view of improving biomarkers predicting the efficacy of immunotherapy for R/M HNSCC, we hypothesized that biomarkers can be personalized depending on clinicopathological backgrounds and treatment sequence. Methods: In this study, we retrospectively included formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood cell counts at treatment, clinicopathological information, and outcome data for patients with R/M HNSCC receiving nivolumab across 22 institutions in Japan (N = 100). FFPE samples were subjected to 14-marker multiplex immunohistochemistry (IHC) and image cytometry analysis (Tsujikawa T et al. Cell Reports, 2017) to quantitatively evaluate CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, CD66b+ granulocytes, mast cells, programmed death ligand 1 (PD-L1) and PD-1 expression in a single slide. Intratumoral and circulating immune cell frequencies were comparatively analyzed between responders (CR, n = 14; PR, n = 39) and non-responders (SD, n = 2; PD, n = 45). Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-L1 expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. Next, focusing on the history of prior therapy, stratified analysis revealed that the frequency of NK cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Furthermore, stratified analysis by patient age revealed that nivolumab response was significantly associated with high CPS and lymphoid-inflamed profiles based on cell densities of nine immune cell lineages in the group aged 65 years or older, but not in the group under 65 years of age. On the contrary, the neutrophil/lymphocyte ratios (NLR) in peripheral blood counts at nivolumab treatment were significantly lower in responders (mean 4.96) than those in non-responders (mean 10.46) in the group under 65 years of age, but not in those over 65 years of age (7.41 versus 8.47). Conclusions: Using peripheral blood data and tumor tissue profiling stratified by patient age and prior treatment might provide better predictive biomarkers in nivolumab response to HNSCC. Further preclinical and clinical studies elucidating immune mechanisms in different patient backgrounds will be warranted. Citation Format: Takahiro Tsujikawa, Kazuchika Ohno, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Alisa Kimura, Hiroki Morimoto, Gaku Ohmura, Akihito Arai, Hiroshi Ogi, Saya Shibata, Yosuke Ariizumi, Akihisa Tasaki, Ryosuke Takahashi, Yumiko Tateishi, Hiroaki Kawabe, Sadakatsu Ikeda, Kei-ichi Morita, Tatsuhiko Tsunoda, Takumi Akashi, Morito Kurata, Issei Imoto, Yasushi Shimizu, Akihito Watanabe, Yukinori Asada, Ryuichi Hayashi, Yuki Saito, Hiroyuki Ozawa, Kiyoaki Tsukahara, Nobuhiko Oridate, Arata Horii, Takashi Maruo, Nobuhiro Hanai, Hidenori Inohara, Hiroshi Iwai, Takashi Fujii, Ken-ichi Nibu, Shigemichi Iwae, Tsutomu Ueda, Ryuji Yasumatsu, Hirohito Umeno, Muneyuki Masuda, Kyoko Itoh, Shigeru Hirano, Takahiro Asakage. Tumor immune characterization identifies age-stratified biomarkers for nivolumab in patients with head and neck squamous cell carcinoma: A nationwide collaborative study in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5210.

Adenoid cystic carcinoma (AdCC) is a rare, indolent salivary gland tumor that is reported to be driven by fusion genes. However, MYB/MYBL1-NFIB fusions have been detected in60% of all AdCC cases and the oncogenic driver mutations in approximately 40% of AdCC remain unknown. Our aim was to identify novel gene fusions in AdCC.We investigated 20 AdCC cases using a targeted RNA sequencing panel to identify gene fusions and performed quantitative real-time reverse transcription polymerase chain reaction to assess MYB, MYBL1, and NFIB expression levels.A total of 36 fusion transcripts in 15 cases were detected and validated by Sanger sequencing. The MYB-NFIB and MYBL1-NFIB fusion genes were detected in 9 and 3 cases, respectively, in a mutually exclusive manner. Furthermore, novel gene fusions, namely, NFIB-EPB41L2, MAP7-NFIB, NFIB-MCMDC2, MYBL1-C8orf34, C8orf34-NFIB, and NFIB-CASC20, were identified. Among them, NFIB-EPB41L2 and NFIB-MCMDC2 are thought to activate MYB and MYBL1 expression, respectively, through the insertion of a genomic segment in proximity to MYB and MYBL1 genes, respectively.Six novel gene fusions other than MYB/MYBL1-NFIB were identified. The detection of novel fusion genes and investigation of the molecular mechanism will contribute to the development of novel molecular targeted therapies for this disease.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

Autoimmune regulator (AIRE) is a transcriptional regulator that is primarily expressed in medullary epithelial cells, where it induces tissue-specific antigen expression. Under pathological conditions, AIRE expression is induced in epidermal cells and promotes skin tumor development. This study aimed to clarify the role of AIRE in the pathogenesis of oral squamous cell carcinoma (OSCC). AIRE expression was evaluated in six OSCC cell lines and in OSCC tissue specimens. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was elevated in 293A cells stably expressing AIRE, and conversely, was decreased in AIRE-knockout HSC3 OSCC cells when compared to the respective controls. Upregulation of STAT1, and ICAM in OSCC cells was confirmed in tissue specimens by immunohistochemistry. We provide evidence that AIRE exerts transcriptional control in cooperation with ETS1. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was increased in 293A cells upon Ets1 transfection, and coexpression of AIRE further increased the expression of these proteins. AIRE coprecipitated with ETS1 in a modified immunoprecipitation assay using formaldehyde crosslinking. Chromatin immunoprecipitation and quantitative PCR analysis revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitates. These results indicate that AIRE is induced in OSCC and supports cancer-related gene expression in cooperation with ETS1. This is a novel function of AIRE in extrathymic tissues under the pathological condition.

The transcription factor tumour protein 63, encoded by the TP63 gene, is a regulator of epidermal development. Heterozygous mutations in TP63 cause a variety of human ectodermal dysplasia disorders, including ankyloblepharon-ectodermal defects-cleft lip/palate syndrome, ectrodactyly, ectodermal dysplasia, cleft lip/palate syndrome, split hand/foot malformation 4, Rapp-Hodgkin syndrome, limb mammary syndrome, and acro-dermato-ungual-lacrimal-tooth syndrome. There are genotype-phenotype correlations in some of these syndromes, and the number of cases with overlapping symptoms has been increasing. The phenotypic spectrum and expressivity of TP63 mutation-associated syndromes complicate its clinical diagnosis and classification. Here, we present an updated review of TP63 mutation mapping information, together with a comprehensive overview of TP63 mutation-associated syndromes. We show that several cases with the same mutation have been previously diagnosed with different syndromes. This study will be useful for the diagnosis and classification of TP63 mutation-associated syndromes.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

This study aimed to clarify the genomic factors associated with the diagnosis and prognosis of oral squamous cell carcinoma via next-generation sequencing. We evaluated data from 220 cases of oral squamous cell carcinoma. Genomic DNA was eluted using formalin-fixed, paraffin-embedded samples, and targeted resequencing of 50 cancer-related genes was performed. In total, 311 somatic mutations were detected in 220 patients, consisting of 68 synonymous mutations and 243 non-synonymous mutations. Genes carrying mutations included TP53, CDKN2A, and PIK3CA in 79 (35.9%), 35 (15.9%), and 19 patients (8.6%), respectively. Copy number analysis detected amplification of PIK3CA and AKT1 in 38 (17.3%) and 11 patients (5.0%), respectively. Amplification of receptor tyrosine kinases was found in 37 patients (16.8%). Distant metastasis was noted in nine of 37 patients (24%) with receptor tyrosine kinase amplification, accounting for 43% of the 21 cases of distant metastasis. The cumulative 5-year survival rate was 64.6% in the receptor tyrosine kinase amplification group vs 85.2% in the no receptor tyrosine kinase amplification group. Moreover, we identified significantly poorer prognosis in the TP53 mutation/receptor tyrosine kinase amplification group, for which the cumulative 5-year survival rate was 41.6%. In conclusion, the results of this study demonstrated that receptor tyrosine kinase amplification is a prognostic factor for distant metastasis of oral squamous cell carcinoma, indicating the necessity of using next-generation sequencing in clinical sequencing.

Autoimmune regulator (AIRE) is a transcriptional regulator that is primarily expressed in medullary epithelial cells, where it induces tissue-specific antigen expression. Under pathological conditions, AIRE expression is induced in epidermal cells and promotes skin tumor development in association with stress-responsive keratin KRT17. This study aimed to clarify the role of AIRE in the pathogenesis of oral squamous cell carcinoma (OSCC). AIRE expression was evaluated in seven OSCC cell lines and in OSCC tissue specimens. Transient or constitutive expression of AIRE in 293A cells induced KRT17 expression. cDNA microarray analysis of 293A cells stably expressing AIRE revealed that STAT1 and ICAM1 were significantly upregulated by AIRE. Expression of KRT17, STAT1, ICAM1, MMP9, CXCL10, and CXCL11 was elevated in 293A cells stably expressing AIRE, and conversely, was decreased in AIRE-knockout HSC3 OSCC cells when compared to the respective controls. Upregulation of KRT17, STAT1, and ICAM in OSCC cells was confirmed in tissue specimens by immunohistochemistry. We provide evidence that AIRE exerts transcriptional control in cooperation with ETS1. Expression of STAT1, ICAM1, CXCL10, and MMP9 was increased in 293A cells upon Ets1 transfection, and coexpression of AIRE resulted in enhanced expression of STAT1. AIRE coprecipitated with ETS1 in a modified immunoprecipitation assay using formaldehyde crosslinking. Chromatin immunoprecipitation and quantitative PCR analysis revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitates. In oral cancer cells, ETS1 was diffusely located in the nucleus and partially overlapped with dot-like AIRE accumulation sites. Nuclear translocation of AIRE was promoted by cotransfection with Ets1. These results indicate that AIRE is induced in OSCC and supports cancer-related gene expression in cooperation with ETS1. This is a novel function of AIRE in extrathymic tissues under the pathological condition.

Systems for "protein transduction," intracellular delivery of functional proteins, are needed to address deliverability challenges of protein therapeutics. However, in vivo protein transduction remains challenging because of instability in serum, extracellular protease digestion and rapid excretion from the bloodstream. Here, a magnetically guided in vivo protein transduction using magnetic nanogel chaperone (MC) composed of iron oxide nanoparticles and a polysaccharide nanogel, a protein carrier inspired by "catch and release" mechanisms of molecular chaperones is demonstrated. The MC system enables efficient delivery of anti-cancer proteins, saporin and RNaseA, into cultured tumor lines and inhibits cell proliferation, mainly via apoptosis. Magnetic in vivo protein transduction via intravenous whole body administration is demonstrated in a fibrosarcoma model. By in vivo optical imaging, MC accumulated in tumor tissues under magnetic field three times more than without irradiation. With subcutaneous injection, saporin is delivered by MC to the cytoplasm in magnetically targeted tissues. In an oral cancer model, MC-delivered magnetically targeted saporin decreased tumor volume without significant body weight changes and no regrowth of tumor at 3 months after complete regression. Protein transduction with MC shows promise for cancer therapeutics and, potentially, for regenerative medicine and other biomedical applications.

Background LAMC2 plays an important role in cancer invasion. The aim of this study was to (i) compare the immunoexpression of LAMC2 in different stages of oral squamous cell carcinoma (OSCC), early and advanced, and (ii) to evaluate LAMC2 as a marker of malignant transformation in leukoplakia. Materials and Methods The expression of LAMC2 was examined by immunohistochemistry in 50 surgical specimens of advanced OSCC assembled as tissue microarrays, and by cDNA microarray in 43 surgical specimens of advanced OSCC. LAMC2 expression was further examined in 39 surgical specimens of early OSCC and in 93 incisional biopsy specimens of leukoplakia of the tongue, which exhibited epithelial dysplasia. The relationship of LAMC2 expression score with clinico-pathological characteristics was analyzed. Results LAMC2 was remarkably upregulated in OSCC at the cancer–stroma interface. The grade of LAMC2 expression was significantly associated with the pattern and depth of invasion of OSCC. Foci of LAMC2-positive cells were observed in some cases of leukoplakia. The number and size of LAMC2-positive foci were significantly associated with the grade of dysplasia. The presence of LAMC2-positive foci was a significant predictive factor for the malignant progression of leukoplakia. LAMC2-positive leukoplakia had an approximately 11-fold increased risk of malignancy compared with LAMC2-negative leukoplakia. Conclusions The results of this study highlight the value of LAMC2 as a marker of cancer invasion. LAMC2-positive foci in leukoplakia suggest an imminent risk of cancer. LAMC2 immunostaining is expected to contribute to a more precise assessment of the malignancy of leukoplakia.

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

Background THBS1 (thrombospondin-1) is the extracellular matrix (ECM) protein that affects diverse cellular activities. It constitutes the tumor stroma, but the role of THBS1 in oral squamous cell carcinoma (OSCC) development is unclear. The aim of this study was to clarify the relevance of THBS1 in the pathogenesis of OSCC. Materials and methods The expression of THBS1 was examined in 44 OSCC by immunohistochemical analysis and in 43 OSCC by cDNA microarray analysis. Cell culture experiments were conducted using human OSCC cell lines HSC3 and HO1N1 and mouse fibroblast ST2 cells to examine the effect of TGFB1 on THBS1 expression, and the effect of THBS1 on cellular behaviors. Results THBS1 was specifically induced in the tumor microenvironment of OSCC. THBS1 appeared to be produced mainly by the stromal cells, but also by OSCC cells. TGFB1 stimulated THBS1 expression in ST2, primary fibroblasts, and the OSCC cells. THBS1 promoted migration and invasion of HSC3 and HO1N1 in transwell migration assays. THBS1 stimulated the expression of MMP3 (matrix metalloprotease 3), MMP9, MMP11, and MMP13 in ST2 cells and MMP3, MMP11, and MMP13 in HO1N1 cells. The RGD peptide suppressed the THBS1-stimulated migration and upregulation of MMP11 and MMP13. Conclusions THBS1 is a tumor-specific ECM protein that is induced by TGFB1 and promotes migration of cancer cells and stimulates the expression of MMPs partly through the integrin signaling, thereby favoring OSCC invasion.

Notch signaling functions in diverse developmental and homeostatic processes, including stem cell self-renewal and cell fate determination. Notch1-inactivating mutations are frequently detected in skin and oro-esophageal cancers, suggesting a role for Notch1 as a tumor suppressor. Here, we clarify the contribution of Notch1 deficiency to oro-esophageal tumorigenesis using a physiological experimental model. Tongue and esophageal tumors induced in mice by 4-nitroquinoline-1-oxide (4-NQO) showed pathophysiological similarities to human tumors, including decreased Notch1 expression in the basal cells. We created mutant mice (N1cKO), in which the Notch1 gene was disrupted specifically in the squamous epithelium. The epithelium formed normally in N1cKO mice, and although multiple skin tumors were detected at 65 weeks, no tumors developed in the tongue and esophagus. However, 4-NQO-induced tumorigenesis assays revealed that tumor onset occurred earlier in N1cKO mice than in wild-type littermates, and the tumors arose preferentially from the Notch1-negative epithelium, indicating the tumor susceptibility of Notch1-deficient epithelium. Notch1 regulates the expression of TERT, and age-related telomere erosion was more rapid in Notch1-deficient basal cells. Our results indicated that although Notch1 deficiency had little effect on squamous epithelium formation, it predisposed the affected epithelium to tumor development, at least in part through accelerated telomere erosion.

Summary The standard treatments for oral leukoplakia range from careful observation to complete resection. No surgical intervention is chosen for several supposable reasons. Surgical treatment and no surgical treatment for oral leukoplakia have no defined basis for comparisons, and few studies have reported on the long-term outcomes of oral leukoplakia without surgery. Objectives This study aimed to identify the important factors using a long-term wait-and-see policy in patients with oral leukoplakia. Materials and methods In total, 237 lesions from 218 patients selected for non-surgical therapy between 2001 and 2010 were analyzed. On the basis of long-term follow-up data, lesions were classified as unchanged, reduced, disappeared, expanded, and malignantly transformed. Results In total, 135 (57.0%) lesions remained unchanged, 30 (12.7%) lesions were characterized by a reduction in size or clinical severity, and 44 (18.6%) lesions had disappeared. Another 17 (7.2%) lesions resulted in spread or clinical deterioration, and 11 (4.6%) lesions developed oral squamous cell carcinoma. Conclusions We demonstrated a cumulative malignant transformation rate of 11.6% in 10 years without resection. The lesions that were nonhomogeneous, and higher degree of epithelial dysplasia, located on the tongue were likely to progress into cancer. In addition, 32.5% of lesions without surgical treatment were reduced or disappeared. There is a possibility that removal of considerable irritation for a long time contributes to the treatment of this disease. The development of appropriate treatments for oral leukoplakia is required, which will enable successful differentiation between surgical and observation cases.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic tumors that form abnormally keratinized ghost cells. Mutations in CTNNB1, which encodes beta-catenin, have been implicated in the development of these tumors, but a causal relationship has not been definitively established. Thus, mutational hot spots in 50 cancer genes were examined by targeted next-generation sequencing in 11 samples of CCOT. Mutations in CTNNB1, but not in other genes, were observed in 10 of 11 cases. These mutations constitutively activate beta-catenin signaling by abolishing the phosphorylation sites Asp32, Ser33, or Ser37, and are similar to those reported in pilomatrixoma and adamantinomatous craniopharyngioma. In contrast, BRAF or NRAS mutations were observed in 12 and two control samples of ameloblastoma, respectively. In HEK293 cells, overexpression of mutated CTNNB1 also upregulated hair keratin, a marker of ghost cells. Furthermore, ghost cells were present in two cases of ameloblastoma with BRAF and CTNNB1 mutations, indicating that ghost cells form due to mutations in CTNNB1. The data suggest that mutations in CTNNB1 are the major driver mutations of CCOT, and that CCOT is the genetic analog of pilomatrixoma and adamantinomatous craniopharyngioma in odontogenic tissue.

Objective Peripheral odontogenic keratocyst (POKC) is a rare gingival cyst showing histologic features identical to those of keratocystic odontogenic tumor. A rare case of POKC associated with nevoid basal cell carcinoma syndrome (NBCCS) is presented. Study Design A 24-year-old woman with NBCCS presented with a pigmented papule, 3 mm in size, involving the lingual gingiva of the right canine area of the mandible. Based on a clinical diagnosis of benign pigmentation, an excisional biopsy was performed, and a histopathologic diagnosis of POKC was rendered. Results The lining cells were positive for the proteins GLI2, BCL2, keratin 8, keratin 17, and mTOR. TP53 and Ber-EP4 were also weakly positive. Gene mutational analysis on a buccal swab sample revealed 2 missense mutations in the PTCH1 gene. Conclusions This case is a distinctive example of a genuine soft tissue counterpart of keratocystic odontogenic tumor, in which an aberrant PTCH1-GLI pathway played a considerable role in the pathogenesis.

Treatment of advanced oral squamous cell carcinoma (OSCC) requires the integration of multimodal approaches. The aim of this study was to identify predictors of tumor sensitivity to preoperative radiotherapy/chemotherapy for OSCC in order to allow oncologists to determine optimum therapeutic strategies without the associated adverse effects. Here, the protein expression profiles of formalin-fixed paraffin-embedded (FFPE) tissue samples from 18 OSCC patients, termed learning cases, who received preoperative chemotherapy and/or radiotherapy followed by surgery were analyzed by quantitative proteomics and validated by immunohistochemistry in 68 test cases as well as in the 18 learning cases. We identified galectin-7 as a potential predictive marker of chemotherapy and/or radiotherapy resistance, and the sensitivity and specificity of the galectin-7 prediction score (G7PS) in predicting this resistance was of 96.0% and 39.5%, respectively, in the 68 test cases. The cumulative 5-year disease-specific survival rate was 75.2% in patients with resistant prediction using G7PS and 100% in patients with sensitive prediction. In vitro overexpression of galectin-7 significantly decreased cell viability in OSCC cell line. Therefore, our findings suggest that galectin-7 is a potential predictive marker of chemotherapy and/or radiotherapy resistance in patients with OSCC. Identification of proteins differentially expressed in OSSC samples from patients sensitive or resistant. The samples were processed by LC-MS and analyzed with 2DICAL.

Background Nasoalveolar cysts are rare non-odontogenic cysts that occur beneath the nasal alar region. Few cases of bilateral nasoalveolar cysts have been described. Case presentation We report a rare case of a 67-year-old Japanese woman with bilateral nasoalveolar cysts who presented to our department with the chief complaint of a swollen left nasal alar base. Panoramic radiography revealed no abnormalities. Computed tomography and magnetic resonance imaging revealed a well-circumscribed oval lesion at both alar bases. Therefore, bilateral nasoalveolar cysts were clinically diagnosed. Furthermore, these cysts were extirpated under general anesthesia; the aforementioned diagnosis was histopathologically confirmed. No recurrence has been observed 1 year after surgery. Conclusions Nasoalveolar cysts are rare. It is necessary to be careful because nasoalveolar cysts can show bilateral occurrence.

Summary Objectives The early detection of oral cancer improves patient outcomes. However, despite our growing knowledge of oral cancers, patients often present with advanced disease. The development of simple screening methods is desirable to provide an alternative to screening examinations by specialists. Thus, we developed a method of oral cancer detection among exfoliated oral mucosal cells, and we evaluated the feasibility of implementing an oral cancer screening test that is examiner independent. Material and methods The study population consisted of 185 subjects: 89 with oral cancer, 18 with oral leukoplakia, and 78 controls. We used real-time polymerase chain reaction (PCR) to detect the biomarkers serpin peptidase inhibitor B3 (SCCA1), interleukin 15 (IL-15), and thrombomodulin (THBD). Results The sensitivities for the detection of oral cancer and oral leukoplakia were 72.0% (77/107) with SCCA1, 75.7% (81/107) with IL-15, and 56.1% (60/107) with THBD, and the specificities were 73.1% (57/78) with SCCA1, 64.1% (50/78) with IL-15, and 78.2% (61/78) with THBD. Analysis of the sensitivity according to tumor size revealed that sensitivity was lower for large tumors. When analyzing the sensitivity according to the clinical growth pattern, the sensitivity was observed to be low for endophytic tumors. Conclusion We developed an oral cancer screening test based on real-time PCR analysis of SCCA1 that is examiner independent, and the sensitivity and specificity were approximately 70%; therefore, we concluded that the performance of this method using a single biomarker was suboptimal.

Background Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder and is characterized by tumorigenesis and physical deformity. Keratocystic odontogenic tumors (KCOTs) of the jaws are a common manifestation of this syndrome. This study involved a pooled analysis of Japanese individuals with NBCCS and was performed with the aim of analyzing the clinical features of NBCCS and the patterns of occurrence and recurrence of KCOTs in Japanese individuals. Methods This study included 25 patients. The relative frequencies of the major symptoms in these patients were compared with those reported in the literature. We also investigated 11 patients with KCOTs (40 lesions) initially treated at Tokyo Medical and Dental University. Results KCOTs (100%) and palmar and/or plantar pits (n = 19; 76.0%) were the most frequently observed manifestations. Eleven patients (44.0%) had a radiologically confirmed rib anomaly. Nineteen patients (76.0%) had a family history of the syndrome within first-degree relatives. Japanese patients had a relatively low frequency of basal cell carcinoma (n = 7; 28.0%) and falx calcification (n = 7; 28.0%) compared with that reported in other populations. Twelve of the total 40 KCOTs (30.0%) that were followed up for 6 months or more recurred. All recurrent cases had undergone conservative treatment, whereas no recurrences occurred in cases that had undergone radical treatment. Conclusions Recurrence of KCOTs associated with NBCCS is frequently encountered, and further investigations are required to confirm the optimal treatment that will ensure a complete cure improving the patient's quality of life.

J Oral Pathol Med (2012) 41: 682–688 Background: Oral leukoplakia can be treated using a variety of treatment procedures; however, the lesions recur in many cases irrespective of the treatment procedure used. The rate of recurrence was from 7.7% to 38.1%. This study aims to identify the important factors that can lower the risk of recurrence of oral leukoplakia treated by curative surgical resection. Methods: The clinical records of 52 patients with oral leukoplakia (53 lesions) who underwent curative surgical resection between 2004 and 2009 were retrospectively analyzed for the rate of recurrence, clinical outcome, epithelial dysplasia, lesion location, and resection margins. Results: The recurrence rate following curative surgical resection was 15.1%, with the most common site being the gingiva. Malignant transformation occurred in a single patient (1.9%). Minimal resection margins (

Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

角化嚢胞性歯原性腫瘍は，歯原性上皮に由来する単房性もしくは多房性の顎骨内腫瘍で，錯角化を呈する重層扁平上皮により裏装されている。多発性の本腫瘍は母斑基底細胞癌症候群の徴候の一つとして知られている。本腫瘍の臨床的な特徴としては，周囲組織に対して侵襲的な態度を示すことや，再発傾向があることなどがある。今回われわれは，2001年から2010年までの10年間に東京医科歯科大学歯学部附属病院顎口腔外科で加療した角化嚢胞性歯原性腫瘍68例（男性39例，女性29例），90腫瘍について臨床的に検討した。このうち8例（男性3例，女性5例），30腫瘍は母斑基底細胞癌症候群例であった。全90腫瘍のうち24腫瘍で再発を認め，再発率は26.7%であった。開窓・摘出術を行った腫瘍の再発率（16.1%）は，開窓療法を行わず摘出術を行った腫瘍の再発率（34.5%）より低かった。腫瘍に接する歯を根治的に処置した腫瘍の再発率（3.4%）は，保存的に処置した腫瘍の再発率（37.7%）より低かった。再発は，その多くが保存した歯の歯根付近から生じていた。これは，この部位は腫瘍の摘出が困難なため，不完全になってしまうことによると考えられた。腫瘍の摘出から再発までの期間は6か月から5年に分布し，ほとんどが3年以内であった。よって，腫瘍摘出後は最低3年間は定期的に経過観察を行う必要があると考えられた。

Sakamoto K, Aragaki T, Morita K-i, Kawachi H, Kayamori K, Nakanishi S, Omura K, Miki Y, Okada N, Katsube K-i, Takizawa T & Yamaguchi A (2011) Histopathology58, 531–542 Down-regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis Aims: This study aimed to identify relevant keratin subtypes that may associate with the pathogenesis of oral epithelial neoplasms. Methods and results: Expression of all the keratin subtypes was examined by cDNA microarray analysis of 43 oral squamous cell carcinoma (OSCC) cases. Immunohistochemical expression of the major keratins was examined in 100 OSCC and oral epithelial dysplasia (OED) cases. Many changes in keratin expression were observed and, significantly, consistent down-regulation of keratin 4 (K4) and K13 expression was observed. Aberrant expression of K4 and K13 was associated with morphological changes in the affected oral epithelium. Experiments with cell cultures transfected with various keratin subtypes suggested that alterations in keratin subtype expression can cause changes in cell shape and movement. Conclusions: Aberrant expression of K4 and K13, which are the dominant pair of differentiation-related keratins in oral keratinocytes, indicates dysregulation of epithelial differentiation in OSCC and OED. These keratins, especially K4, may be useful for pathological diagnosis. We propose that the aberrant expression of K4 and K13 and concomitant up-regulation of the other keratins may be one of the causative factors for morphological alterations in the affected epithelium.

Photodynamic diagnosis (PDD) is a form of cancer detection based on the administration of an exogenous photo-activated compound that accumulates in malignant cells, followed by appropriate photo irradiation. The authors describe a spectroscopic study of 5-aminolevulinic acid (5-ALA)-generated photosensitizer protoporphyrin IX (PpIX) fluorescence in human oral squamous cell carcinoma (OSCC) cell lines to validate its clinical use. 5-ALA-induced PpIX fluorescence intensity was measured in the presence and absence of deferoxamine mesylate (DFO). Two, one and two cell lines produced poorly, moderately and well differentiated carcinomas, respectively, on transplantation in scid mice. The fluorescence intensity was high in the poorly differentiated cell lines, followed by the moderately differentiated cell line; the intensity of the well differentiated cell lines was low and not significantly different from that of normal keratinocytes in the absence of DFO. It was elevated to the level of poorly differentiated cell lines following DFO treatment. This elevation was not observed in normal keratinocytes. The results indicate that DFO enhances the photodynamic sensitivity of 5-ALA in non-responsive carcinoma cells as a result of increased cellular accumulation by inhibiting haeme biosynthesis. This PDD system can be applied clinically to the detection of OSCC irrespective of the degree of differentiation.

We investigated the roles of interleukin-6 (IL-6) and parathyroid hormone-related peptide (PTHrP) in oral squamous cell carcinoma (OSCC)-induced osteoclast formation. Microarray analyses performed on 43 human OSCC specimens revealed that many of the specimens overexpressed PTHrP mRNA, but a few overexpressed IL-6 mRNA. Immunohistochemical analysis revealed that IL-6 was expressed not only in cancer cells but also in fibroblasts and osteoclasts at the tumor-bone interface. Many of the IL-6-positive cells coexpressed vimentin. Conditioned medium (CM) derived from the culture of oral cancer cell lines (BHY, Ca9-22, HSC3, and HO1-u-1) stimulated Rankl expression in stromal cells and osteoclast formation. Antibodies against both human PTHrP and mouse IL-6 receptor suppressed Rankl in ST2 cells and osteoclast formation induced by CM from BHY and Ca9-22, although the inhibitory effects of IL6 antibody were greater than those of PTHrP antibody. CM derived from all of the OSCC cell lines effectively induced IL-6 expression in stromal cells, and the induction was partially blocked by anti-PTHrP antibody. Xenografts of HSC3 cells onto the periosteal region of the parietal bone in athymic mice presented histology and expression profiles of RANKL and IL-6 similar to those observed in bone-invasive human OSCC specimens. These results indicate that OSCC provides a suitable microenvironment for osteoclast formation not only by producing IL-6 and PTHrP but also by stimulating stromal cells to synthesize IL-6.

The residual wide alveolar cleft often causes potential risk in bone grafting and prosthodontic treatment for cleft lip and palate patients. While performing cleft reduction, maxillary advancement is required because closure of the cleft space itself with orthodontic/orthognathic treatment will result in a crossbite. In this paper, we report the case of a 21-year 4-month old male with left cleft lip and palate. The patient had undergone orthodontic treatment once at another institute, and he visited our Orthodontic Clinic at the University Hospital of Dentistry, Tokyo Medical and Dental University with a chief complaint of residual cleft space. Overjet and overbite were both 2.0 mm and the intercuspation of the buccal segments was optimal. However, the residual cleft space was 14.5 mm with no history of bone grafting. The right maxillary lateral incisor lacked congenitally, and the left lateral incisor was a lingually malpositioned conical tooth. The midline of the maxillary had right deviation of 6.5 mm. A multibracket appliance was used for the presurgical orthodontic treatment and a Le Fort I osteotomy was performed when the patient was 22-years and 4-months old. During the operation, the left maxillary lateral incisor in the cleft area was extracted. A Twin-Track device was placed along with the maxillary dental arch to guide the surgical movement of the alveolar segments into place. After complete cleft closure was achieved along this splint, maxillary segments were fixed using mini-plates and bone grafting was simultaneously performed. After 2 years of post-surgical orthodontic treatment, acceptable occlusion was achieved and no further prosthetic treatment was required.

Background: Oral leukoplakias (LP) are the most frequent types of oral pre-cancerous lesions, but there is no accurate assessment of this malignant transformation or even genetic diagnosis of the oral epithelial dysplasia. We need to identify the new genetic diagnosis system of the epithelial dysplasia. Methods: Oligonucleotide microarray was used to analyze expression patterns of 29 952 genes in 10 LP patients. We compared the different gene expressions between mild dysplasia cases and severe dysplasia cases. Results: Ninety-six genes expressed differentially were selected as candidates for up-regulated in severe dysplasia. Subsequently, we further selected 16 genes with highest differentially expression. By hierarchical clustering analysis, the 10 cases were divided mild dysplasia from severe dysplasia. Conclusions: The 16 genes are suggested as biomarker gene sets of efficacy and quickly recognized in the development of oral epithelial dysplasia.

A case of surgical ciliated cyst after Le Fort I osteotomy is described. A 32-year-old man who had undergone Le Fort I osteotomy in 1992 was referred to our hospital because of swelling in the left cheek. Radiographic and computed tomographic examinations showed a unilocular cystic lesion in the left side of the maxilla. The lesion was diagnosed as a maxillary cyst. We treated the lesion surgically. The lesion and thin bony wall between the cystic cavity and maxillary sinus were removed, so that the bony cavity commuted with the inferior nasal meatus. The postoperative course was uneventful, and there was no evidence of recurrence 12 months after surgery. On the basis of the clinical course and histopathological findings of the surgical specimen (i.e., the cyst had a thick fibrous wall lined by stratified ciliated columnar or cuboidal epithelium), the lesion was diagnosed as a surgical ciliated cyst.

Keratin subtypes are selectively expressed depending on the cell type. They not only provide structural support, but regulate the metabolic processes and signaling pathways that control the growth of the epithelium. KRT17 (keratin 17) is induced in the regenerative epithelium and acts on diverse signaling pathways. Here, we demonstrate that KRT17 is invariably and permanently induced in oral squamous cell carcinoma (OSCC), as revealed by immunohistochemistry and cDNA microarray analysis. Two representative OSCC cell lines; KRT17-weakly expressing Ca9-22 and KRT17-highly expressing HSC3 were used to establish KRT17-overexpressing Ca9-22 and KRT17-knockdown HSC3 cells. Analysis of these cells revealed that KRT17 promoted cell proliferation and migration by stimulating the Akt/mTOR pathway. KRT17 also upregulated the expression of SLC2A1 (solute carrier family 2 member 1/Glut1) and glucose uptake. To further investigate the effect of KRT17 on tumorigenesis, KRT17-knockout HSC3 cells were established and were transplanted to the cephalic skin of nude mice. The tumors that developed from KRT17-knockout HSC3 cells had a lower Ki-67 labeling index and were significantly smaller compared to the controls. These results indicate that KRT17 stimulates the Akt/mTOR pathway and glucose uptake, thereby facilitating tumor growth. We could not confirm the relationship between KRT17 and SFN (stratifin) in the cells examined in this study. However, our study reinforces the concept that the cellular properties of cancer are regulated by a series of molecules similar to those found in wound healing. In OSCC, KRT17 acts as a pathogenic keratin that facilitates tumor growth through the stimulation of multiple signaling pathways, highlighting the importance of KRT17 as a multifunctional promoter of tumorigenesis.

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.