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BackgroundThe prevalence of Lynch syndrome and the use of universal tumor screening to identify Lynch syndrome among unselected patients with upper urinary tract urothelial carcinoma, which is associated with Lynch syndrome, have not been closely investigated yet.MethodsA total of 166 tumors from 164 upper urinary tract urothelial carcinoma patients were tested for microsatellite instability and expression of mismatch repair proteins (MLH1, MHS2, MSH6 and PMS2) by immunohistochemistry. Genetic testing was performed for patients suspected of having Lynch syndrome. Clinicopathological factors, including familial and personal cancer history associated with mismatch repair deficiency, were evaluated.ResultsThe frequency of high-level microsatellite instability and loss of at least one mismatch repair protein was 2.4% (4/164); the microsatellite instability and immunohistochemistry results showed complete concordance. Of these four patients, three were genetically proven to have Lynch syndrome, while the remaining one was highly suggestive for Lynch syndrome based on their personal cancer history. Univariate analysis showed that ageConclusionsThe prevalence of Lynch syndrome among unselected upper urinary tract urothelial carcinoma patients was at least 1.8% in our study population. The screening efficacies of the microsatellite instability test and immunohistochemistry appear equivalent. Universal tumor screening may be a valid approach; however, selective screening methods that consider factors associated with mismatch repair loss/high-level microsatellite instability tumors require further investigation.

Desmoglein (DSG) 3 is overexpressed in oral squamous cell carcinoma (OSCC). Epidermal growth factor receptor (EGFR) inhibitor cetuximab is widely used for OSCC treatment. Several evidences suggest a correlation between DSG3 and EGFR in epidermal keratinocytes. EGFR inhibition has been shown to enhance cell-cell adhesion and induce terminal differentiation in epidermal cells. Thus, here we investigated the DSG3-EGFR interaction in OSCC and its effect on cetuximab treatment. Cell lines established from the primary tumor and metastatic lymph nodes of four OSCC patients and three commercial OSCC cell lines were used for the experiments. Cells from metastatic lymph nodes of each patient expressed increased DSG3 and EGFR than cells from the primary tumor in the same patient. Cetuximab treatment increased DSG3 expression by up to 3.5-fold in seven of the 11 cell lines. A high calcium concentration increased the expression of DSG3 and EGFR in a dose-dependent manner. Strikingly, a high calcium-associated DSG3 induction enhanced cetuximab efficacy by up to 23% increase in cetuximab-low-sensitive cell lines. Our findings also suggest a correlation between DSG3 and EGFR in OSCC, and this affects cetuximab treatment efficacy.

Microsatellite instability (MSI) is an effective biomarker for diagnosing Lynch syndrome (LS) and predicting the responsiveness of cancer therapy. MSI testing is conventionally performed by capillary electrophoresis, and MSI status is judged by visual assessment of allele size change. Here, we attempted to develop a quantitative evaluation model of MSI using next-generation sequencing (NGS). Microsatellite markers were analyzed in tumor and non-tumor tissues of colorectal cancer patients by NGS after a single multiplex polymerase chain reaction amplification. The read counts corresponding to microsatellite loci lengths were calculated independently of mapping against a reference genome, and their distribution was digitized by weighted mean. Weighted mean differences between tumor and non-tumor samples with different MSI status were assessed, and cut-off values for each marker in the discovery cohort were determined. Each microsatellite maker was defined as unstable if the weighted mean difference was greater than the cut-off value. In the discovery cohort, the evaluation model demonstrated sensitivity and specificity of 100% for all markers. In the validation cohort, MSI status determined by the new model was consistent with the outcome of the conventional method in 29/30 cases (97%). The single inconsistent case was classified as low-frequency MSI by the conventional method but considered MSI-high by NGS. Genetic testing for mismatch repair genes revealed a pathogenic variant in MSH6 in the discordant case. We successfully developed a quantitative evaluation method for determining MSI status using NGS. This is a robust and sensitive method and could improve LS diagnosis.

BackgroundThe prevalence and molecular characteristics of defective DNA mismatch repair endometrial cancers in the Japanese population have been underexplored. Data supporting clinical management of patients with Lynch-like syndrome and germline variant of uncertain significance of mismatch repair genes are still lacking.MethodsImmunohistochemistry of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed on formalin-fixed paraffin-embedded sections prepared from resected primary endometrial cancers in 395 women with a median age of 59 years. Genetic and/or epigenetic alterations of the mismatch repair genes were also investigated.ResultsLoss of expression of one or more mismatch repair proteins was observed in 68 patients (17.2%). A total of 17 out of 68 patients (25%, 4.3% of all cases) were identified as candidates for genetic testing for Lynch syndrome after excluding 51 patients with MLH1 hypermethylated cancer. Fourteen of these 17 patients subjected to genetic testing were found to have Lynch syndrome (n = 5), germline variant of uncertain significance (n = 2) or Lynch-like syndrome (n = 7). Compared with patients with Lynch syndrome, those with germline variant of uncertain significance and Lynch-like syndrome tended to demonstrate an older age at the time of endometrial cancer diagnosis (P = 0.07), less fulfillment of the revised Bethesda guidelines (P = 0.09) and lower prevalence of Lynch syndrome-associated tumors in their first-degree relatives (P = 0.01).ConclusionsThis study provides useful information for management in patients with DNA mismatch repair endometrial cancer. Specifically, cancer surveillance as recommended in patients with Lynch syndrome might not be necessary in patients with germline variant of uncertain significance and Lynch-like syndrome and their relatives.

The relationship between thymidine phosphorylase (TP) and angiogenesis at the early stage of esophageal squamous cell carcinoma has been unclear. Using 14 samples of normal squamous epithelium, 11 samples of low-grade intraepithelial neoplasia, and 64 samples of superficial esophageal cancer, microvessel density (MVD) was estimated using immunostaining for CD34 and CD105. TP expression was also evaluated in both cancer cells and stromal monocytic cells (SMCs). We then investigated the correlation between MVD and TP expression in both cancer cells and SMCs. On the basis of the above parameters, MVD was significantly higher in cancerous lesions than in normal squamous epithelium. In terms of CD34 and CD105 expression, MVD showed a gradual increase from normal squamous epithelium, to low-grade intraepithelial neoplasia, and then to M1 and M2 cancer, and M3 or deeper cancer. M1 and M2 cancer showed overexpression of TP in both cancer cells and SMCs. There was no significant correlation between TP expression in cancer cells and MVD estimated from CD34 (rS = 0.16, P = 0.21) or CD105 (rS = 0.05, P = 0.68) expression. Significant correlations were found between TP expression in SMCs and CD34-related (rS = 0.46, P

To clarify the prevalence and clinicopathologic/molecular characteristics of mismatch repair (MMR)-deficient colorectal cancer in the young Japanese population. Immunohistochemical analyses for MMR proteins (MLH1, MSH2, MSH6, and PMS2) were performed in formalin-fixed paraffin-embedded sections prepared from the resected CRC specimens of 119 consecutive patients aged

Objective Oral squamous cell carcinoma is characterized by diverse histopathological features, tumorigenesis, and invasion. Gingiva squamous cell carcinoma (GSCC) is characterized by easily invading the jawbone. Epithelial–mesenchymal transition (EMT) is a crucial event for invasion of cancer cells and progression of carcinogenesis, inducing stem-like properties in epithelial cells. Here, expression of BMI1, which controls stem cell self-renewal, and ZEB1, a transcription factor that regulates EMT, was evaluated for its role in EMT and invasion of GSCC. Materials and methods We analyzed 36 gingival specimens (24 carcinomas and 12 dysplasias) by measuring protein and mRNA expression levels of BMI1, ZEB1, vimentin, and E-cadherin, and examining associations between their expression and clinicopathological parameters. Results Compared with adjacent normal squamous epithelium, E-cadherin expression was decreased significantly ( P P P = 0.027), and vimentin and BMI1 expression was significantly increased in grade 4 mode of invasion ( P P = 0.014) and between BMI1 and ZEB1 mRNA expression ( P = 0.034). Conclusion The present study shows that the expression levels of several biomarkers of gingiva carcinogenesis have unique characteristics that differ from those in tongue carcinogenesis. BMI1 and ZEB1 may be important factors associated with EMT in the invasion and progression of GSCC.

A 42‑year‑old woman presented with ~30 adenomatous polyps of the left sided‑colon with early rectosigmoid cancer. The patient had no previous medical history and no familial history of inherited colorectal disease. No germline gene mutations associated with colorectal adenomatous polyposis, including APC regulator of WNT signaling pathway, mutY DNA glycosylase, DNA polymerase‑e, catalytic subunit, DNA polymerase δ1, catalytic subunit, and mismatch repair genes, were detected via germline genetic testing. A heterozygous germline mutation in methyl‑CpG binding domain 4, DNA glycosylase (MBD4), c.217C>T/p.Gln73*, which resulted in the generation of a stop codon, was identified by genetic analyses including whole‑exome sequencing. Immunohistochemical staining analysis revealed that the expression of MBD4 protein was absent in the cancer tissue, while it was expressed in the normal epithelium. Sequencing and copy‑number analyses demonstrated the loss of the remaining allele of MBD4 in the cancer tissue. Furthermore, somatic mutation signature analysis showed preferential transition of cytosine to thymine residues at CpG dinucleotides in cancer tissues. Although it has been previously reported that germline missense mutations and somatic mutations of MBD4 are associated with the development of colorectal cancer, this is the first report, to the best of our knowledge, in which a germline nonsense mutation of the MBD4 gene has been identified in an early‑onset colorectal cancer patient with oligopolyposis.

The proband was a 62-year-old man with ureter cancer. He had a history of metachronous colorectal and gastric cancer. Immunohistochemical staining showed the absence of both MSH2 and MSH6 proteins in the ureter cancer and other available cancer tissue specimens. Genetic testing was conducted to identify the causative genes of hereditary gastrointestinal cancer syndromes including mismatch repair genes. We detected a germline variant, c.2635-3delC, within the splice acceptor site of exon 16, in the MSH2 gene. To investigate whether this variant affected splicing of the gene, RNA sequencing was performed using blood samples. We observed a substantial amount of the transcripts that lacked proper splicing of intron 15 in the indexed case, whereas, a very low amount of such aberrant transcripts was detected in the controls, strongly indicating an association between the variant and splicing defect. These results indicate that MSH2 c.2635-3delC affects normal splicing and might be a cause of Lynch syndrome.

Epithelial-mesenchymal transition (EMT) is a crucial event required for the invasion and progression of carcinogenesis, inducing stem-like properties in epithelial cells. In the present study, the expression of BMI1, which controls self-renewal in stem cells, as well as that of ZEB1, a transcription factor that regulates EMT, was evaluated for its role in EMT and the carcinogenic processes of tongue squamous cell carcinoma (TSCC). Collagen invasion assays using two TSCC cells and 64 tongue specimens (32 carcinomas and 32 dysplasias) were employed and analyzed in the present study. We assessed the protein and mRNA expression levels of BMI1, ZEB1, vimentin and E-cadherin in the two cell lines and tumor tissues. The protein and mRNA expression of BMI1 and ZEB1 occurred at the invasion of TSCC. The elevated levels of BMI1 and ZEB1 were accompanied by the downregulation of E-cadherin and upregulation of vimentin at the invasive front, indicative of EMT in vitro and in vivo. The results showed that BMI1 and ZEB1 are important factors in association with the promotion of EMT and invasion of TSCC.

Background The prevalence and molecular characteristics of defective mismatch repair epithelial ovarian cancers in the Japanese population have scarcely been investigated. Methods Immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed in formalin-fixed paraffin-embedded sections prepared from resected primary epithelial ovarian cancers in patients who underwent oophorectomy at our institution between April 2005 and September 2014. Genetic and/or epigenetic alterations of the mismatch repair genes were investigated in patients with loss of any mismatch repair proteins in the tumor. Results There were 305 patients with a median age of 54 years (range, 18-83 years). Loss of expression in the ovarian tumor of one or more mismatch repair proteins was observed in 3 of the 305 patients (0.98%): 2 patients MLH1/PMS2 loss and 1 patient showed MSH2/MSH6 loss. Genetic testing of these three patients failed to reveal any pathogenic germline mutations of MLH1 or MSH2. One patient with MLH1/PMS2 loss showed hypermethylation of the promoter region of MLH1. Somatic mutations were found in each of the alleles of MLH1 (c.545dupG and deletion of exons 2-19) in the other patient with MLH1/PMS2 loss. In the patient with MSH2/MSH6 loss, two somatic mutations were detected in MSH2 (c.229_230delAG and c.1861C>T), although we could not determine whether these mutations were biallelic or not. Conclusions The prevalence of defective mismatch repair epithelial ovarian cancer in the Japanese hospital-based population was extremely low. Molecular mechanism involved in such defective mismatch repair ovarian cancers seems to be epigenetic events through MLH1 promotor hypermethylation or somatically mutated mismatch repair genes without germline mismatch repair mutation.

Background Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes. Materials and methods Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted. Results HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation. Conclusion HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP) cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment.

BRAF V600E mutation plays an important role in the serrated neoplasia pathway of colorectal tumorigenesis and is a negative predictive factor for chemotherapy response as well as a prognostic factor in patients with colorectal cancer. To evaluate BRAF V600E mutations, a conventional polymerase chain reaction(PCR)is performed but recently immunohistochemistry (IHC)with a BRAF antibody has been used. Although similarities between the PCR and IHC methods have been reported, some investigators have doubts about the usefulness of IHC for BRAF mutation analysis. The subjects were 38 colorectal cancer patients with tumors demonstrating loss of both MLH1 and PMS2, and high-level microsatellite instability. Of the original 39 patients, 1 was excluded due to Lynch syndrome, which was identified using germline mutation testing. The mutation rate of BRAF V600E was 57.9% using both methods, but the concordance rate was 68.4%, with a kappa-value of 0.33. We should consider the usefulness of the IHC method in the evaluation of BRAF mutations in colorectal cancer patients.

Objective We investigated the prevalence of Lynch syndrome and Lynch-like syndrome among Japanese colorectal cancer patients, as there have been no credible data from Japan. Methods Immunohistochemical analyses for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were carried out in surgically resected, formalin-fixed paraffin-embedded specimens obtained from 1,234 newly diagnosed colorectal cancer patients between March 2005 and April 2014. The presence/absence of the BRAF V600E mutation and hypermethylation of the MLH1 promoter was analyzed where necessary. Genetic testing was finally undertaken in patients suspected as having Lynch syndrome. Results By the universal screening approach with immunohistochemical analysis for mismatch repair proteins followed by analyses for the BRAF V600E mutation and MLH1 promoter methylation status, 11 (0.9%) of the 1,234 patients were identified as candidates for genetic testing. Out of the 11 patients, 9 (0.7%) were finally diagnosed as having Lynch syndrome; the responsible genes included MLH1 (n = 1), MSH2 (n = 4), EPCAM (n = 1) and MSH6 (n = 3). The remaining two patients (0.2%) were regarded as having Lynch-like syndrome, since biallelic somatic deletion of the relevant mismatch repair genes was detected in the absence of germline mismatch repair alterations. None of the cases was identified as having germline MLH1 epimutation. Conclusions The prevalence of Lynch syndrome among all newly diagnosed cases of colorectal cancer in Japan is in the same range as that recently reported by studies in Western population. The prevalence of Lynch-like syndrome seems to be extremely low.

In the pathological diagnosis of oral squamous cell carcinoma, we often confront the difficulty of determining whether it is invasive carcinoma or epithelial dysplasia. Recently, myelin and lymphocyte protein (MAL; T-cell differentiation-related gene) has been reported to be a candidate gene suppressed in esophageal carcinoma. When we performed cDNA microarray analysis, we found that gene expression of MAL was significantly downregulated in oral squamous cell carcinoma (OSCC). We evaluated the expression of the MAL gene by laser microdissection and real-time PCR methods and protein localization by immunohistochemistry. The gene expression of MAL was significantly decreased in OSCC compared with normal epithelium (P < 0.05). Furthermore, protein expression of MAL disappeared gradually in proportion to malignancy. The results suggest that MAL plays an important role during oral carcinogenesis and that the gene may have potential as a biomarker target for OSCC.

Tsukamoto Y, Usui M, Yamamoto G, Takagi Y, Tachikawa T, Yamamoto M, Nakamura M. Role of the junctional epithelium in periodontal innate defense and homeostasis. J Periodont Res 2012; 47: 750–757. © 2012 John Wiley & Sons A/S Background and Objective: The junctional epithelium provides the front-line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ-free mice. Material and Methods: Germ-free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr-1, macrophage inflammatory protein-2 (MIP-2/CXCL2) and proliferating cell nuclear antigen-positive cells in the two groups of mice. Laser capture microdissection and RT-PCR analysis were used to evaluate the expression of keratinocyte-derived chemokine (KC/CXCL1), MIP-2, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) mRNAs in the two groups of mice. Results: Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up-regulation of KC and MIP-2 were detected in the junctional epithelium of conventional mice than in germ-free mice, whereas the expression of Il-1β and Tnfα mRNAs was not affected. Conclusion: Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy. We performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from patients with PTC and compared the results with those from normal thyroid tissue validated by real-time (RT) PCR and immunohistochemistry (IHC). We detected 524 types of protein in PTC and 432 in normal thyroid gland. Among these proteins, 145 were specific to PTC and 53 were specific to normal thyroid gland. We have also identified two important new markers, nephronectin (NPNT) and malectin (MLEC). Reproducibility was confirmed with several known markers, but the one of two new candidate markers such as MLEC did not show large variations in expression levels. Furthermore, IHC confirmed the overexpression of both those markers in PTCs compared with normal surrounding tissues. Our protein data suggest that NPNT and MLEC could be a characteristic marker for PTC.

Sleep bruxism is a sleep-related movement disorder that can be responsible for various pains and dysfunctions in the orofacial region. The aim of the current case-control association study was to investigate the association of genetic, psychological and behavioral factors with sleep bruxism in a Japanese population. Non-related participants were recruited and divided into either a sleep bruxism group (n = 66) or control group (n = 48) by clinical diagnoses and 3-night masseter electromyographic recordings by means of a portable miniature device. The Epworth Sleepiness Scale, Temperament and Character Inventory, NEO-Five Factor Inventory and custom-made questionnaires that asked about familial aggregation, alcohol intake, caffeine intake, cigarette smoking, past stressful life events, daytime tooth-contacting habit, temporomandibular disorder, daily headache, snoring, apnea/hypopnea symptoms, leg-restlessness symptoms and nocturnal-myoclonus symptoms were administered. In addition, 13 polymorphisms in four genes related to serotonergic neurotransmission (SLC6A4, HTR1A, HTR2A and HTR2C) were genotyped. These factors were compared between case (sleep bruxism) and control groups in order to select potential predictors of sleep-bruxism status. The statistical procedure selected five predictors: Epworth Sleepiness Scale, leg-restlessness symptoms, rs6313 genotypes, rs2770304 genotypes and rs4941573 genotypes. A multivariate stepwise logistic regression analysis between the selected predictors and sleep-bruxism status was then conducted. This analysis revealed that only the C allele carrier of HTR2A single nucleotide polymorphism rs6313 (102C>T) was associated significantly with an increased risk of sleep bruxism (odds ratio = 4.250, 95% confidence interval: 1.599-11.297, P = 0.004).This finding suggests a possible genetic contribution to the etiology of sleep bruxism.

Objective We have searched for the factor to diagnose malignant potential of epithelial dysplasia. Recent studies have suggested that determination of the expression of cytokeratin 13 and 17 would be useful for the early diagnosis of oral squamous cell carcinoma. In this study, we focused on the diagnosis based on the malignant potential of epithelial dysplasia and evaluated the feasibility of combined assessment of cytokeratin 13 and 17. Patients and methods The expression of cytokeratin 13 and 17 observed in 8 patients with clinically diagnosed T1 or T2 tongue squamous cell carcinoma was analyzed using real-time PCR and immunohistochemical staining of resected specimens. Areas in each specimen were identified as Normal, Dysplasia and Cancer. Results Real-time PCR showed that the expression of cytokeratin 13 decreased beginning with Dysplasia, whereas the expression of cytokeratin 17 increased beginning with Dysplasia. Immunohistochemical staining revealed that cytokeratin 13-positive cells decreased beginning with Dysplasia, while there were almost no cytokeratin 13-positive cells in Cancer. Conversely, cytokeratin 17-positive cells increased beginning with Dysplasia, and almost all cells were cytokeratin 17-positive in Cancer. These results thus indicate that the expression of cytokeratin 13 and 17 is associated with carcinogenesis in epithelial dysplasia. Conclusions The findings of this study indicate that assessment of the expression of cytokeratin 13 and 17 might be useful in the diagnosis of malignant potential in epithelial dysplasia. Moreover, evaluating changes in the expression of cytokeratin 13 and 17 could be effective in evaluating surgical margins.

Hayashi Y, Matsunaga T, Yamamoto G, Nishii K, Usui M, Yamamoto M, Tachikawa T. Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis. J Periodont Res 2010; 45: 618–625. © 2010 John Wiley & Sons A/S Background and Objective: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. Material and Methods: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. Results: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. Conclusion: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

The cell adhesion extracellular matrix (ECM), and the cell adhesive system mediate various adhesion molecules. Cell adhesion regulates proliferation and differentiation. However, the cell adhesive systems between titanium and epithelial cells are still not clearly understood. In this study, we cultured human epithelial cells on type IV collagen-coated titanium (TiCol-4) and non-coated titanium (Ti). We used titanium disks of 30 mm diameter and 1.0 mm thickness. The titanium disks were first abraded using #800, #1200, #2400, and #4000 diamond pads and 0.3 μm alumina, and then washed with acetone, ethanol, and ultra-pure water in an ultrasonic cleaner for 10 min each. Human oral keratinocytes (hOMK) were seeded with 5.0 × 104 cells on TiCol-4 and Ti and placed in 6-well culture dishes. The cell adhesion examination was conducted with a Cell Coulter Counter after 1, 3, and 5 h. hOMK were observed using scanning electron microscopy (SEM). Real-time PCR was performed with four primers: gene laminin β3, integrin β4, integrin α1, and integrin α3. The results suggest that TiCol-4 could be used as a means for obtaining better hOMK than Ti. Type IV collagen could provide an excellent substratum for hOMK attachment on titanium surfaces. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

A case of sclerosing odontogenic carcinoma (SOC) admixed with a benign fibro-osseous lesion (BFOL) is reported herein. A 67-year-old male had paresthesia in the mental region. Computed tomography detected an intragnathic mass that was focally expansile with disappearance of cortical bone, and contained admixed radiolucency and radio-opacity. Under the pathological diagnosis as benign fibro-osseous lesion, it was surgically removed by curettage. Microscopic analysis showed that a few parts of the resected materials contained dispersed thin cords and small nests of epithelial cells accompanied by fibrous stroma. Cellular atypia and mitotic figures were not evident. The diagnosis of BFOL with hyperplastic and metaplastic odontogenic epithelia was ultimately made. Eight months after the operation, the lesion recurred and segmental mandibulectomy was carried out. Histologically, the lesion was predominantly occupied by the fibro-osseous component with irregular-shaped foci of epithelial component. The epithelial component exhibited mostly thin cord or small nest patterns and showed definite perineural infiltration. Immunohistochemically, the epithelial cells were positive for p63, cytokeratin (CK) 6 and CK19, and focally positive for CK7 but negative for vimentin. MIB-1 positive nuclei were inconspicuous. To the best of our knowledge, this report is the first case of SOC with BFOL.

Aims: The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is involved in the morphogenesis of normal salivary gland as well as in the mechanisms of tumour invasion and metastasis. The role of MMPs and TIMPs in pleomorphic adenoma has not been elucidated sufficiently. Our aim was to analyse the mRNA and protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the epithelium and stroma of pleomorphic adenoma and to evaluate their roles. Methods and results: In each sample from six patients, cells from the epithelium and stroma were obtained by laser microdissection. The mRNA expression of MMPs and TIMPs was determined by real-time quantitative reverse transcriptase-polymerase chain reaction and protein expression was confirmed by immunohistochemistry. Results showed that mRNA expression of MMPs and TIMPs was significantly higher in stroma than in epithelium in most patients. MMPs and TIMPs were immunoreactive mainly in epithelium rather than in stroma. Conclusions: Our results provide preliminary evidence that stromal myoepithelium may be the primary source of MMPs and that the stroma has the potential to play a more important role than ductal epithelium in biological behaviour of pleomorphic adenomas. These findings seem worthy of further investigation.

summary It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.

Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous-cell carcinoma (OSCC) cell lines for genome-wide copy-number aberrations by array-CGH using our in-house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A (MTNR1A) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2′-deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor-suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis. (Cancer Sci 2008; 99: 1390–1400)

The patient was a 65-year-old man without any noteworthy medical history. A colonoscopy conducted after a positive fecal occult blood test revealed approximately 100 polyps in the large intestine. A biopsy of some these polyps revealed serrated and hyperplastic polyps, which were histologically determined to be well-differentiated adenocarcinoma. Based on these findings, a diagnosis of serrated polyposis syndrome (SPS) was made, and the patient underwent laparoscopic pancolectomy/ileoproctostomy. Histopathological analysis revealed a total of 91 lesions, out of which 15 were ≥10 mm. A 30 mm lesion in the ascending colon was a well-differentiated adenocarcinoma, stage Ⅰ colon cancer (T1a [sm], ly0, v0, N0, and M0). No germline mutations were found on genetic testing of the adenomatous polyposis coli (APC), mutY homolog (MUTYH), mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6), and postmeiotic segregation increased 2 (PMS2) genes. No loss of MLH1 protein expression or expression of mutated B-Raf (BRAF) V600E protein was observed in the cancer regions after immunostaining. This case is important because not only is the condition rare but also because it showed that the serrated pathway may not necessarily be the mechanism by which serrated lesions become cancerous in patients with SPS.

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses.

Although RAD21 is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth, its role in cancer has been unclear. In this study, the relevance of RAD21 gene expression to the invasion and metastasis of oral squamous cell carcinoma was clarified using laser microdissection and real-time polymerase chain reaction (PCR). Using two different metastatic potential oral squamous cells [high-metastatic-potential squamous cell carcinoma cells (SAS-Ly) and low-metastatic-potential squamous cell carcinoma cells (SAS)], the relation of RAD21 gene expression to apoptosis, invasion, and metastasis was examined. The results showed that RAD21 gene expression was significantly decreased in oral squamous cell carcinoma when it expressed the INFbeta and INFgamma invasion patterns in comparison with the INFalpha invasion pattern (p0.01). In addition, in comparison with SAS cells, SAS-Ly cells indicated tolerance to cell death induced by an apoptosis induction reagent, while the expression level of the RAD21 gene in SAS cells was increased by the apoptosis induction reagent. However, in SAS-Ly cells, the reagent induced no significant difference. Our findings indicate that the RAD21 gene was closely related to the invasion and metastasis of cancer cells.

所属リンパ節転移は, 口腔癌の悪性度に最も相関する因子の一つであり, リンパ節転移の制御は臨床において重要な治療戦略である。従って, 原発巣における微小リンパ管動態について高い関心が持たれてきた。しかし, 微小血管との鑑別が容易でないため, なお不明な点が多い。今回われわれは, 口腔扁平上皮癌における血管新生およびリンパ管新生と腫瘍の浸潤や増殖の関係を明らかにする目的で, 5'-nucleotidase (5'-Nase) リンパ管染色-alkaline phosphatase (ALP) 血管染色の二重染色法を用いて微少血管, リンパ管密度と臨床的, 病理組織学的因子および癌浸潤様式との関係について検討した。結果: 1) 正常粘膜の血管, リンパ管密度はそれぞれ27.4±4.4, 12.8±4.0で, また癌組織の血管, リンパ管密度はそれぞれ9.5±6.3, 6.4±1.7であった。2) 腫瘍の大きさ, 転移の有無および分化度ではいずれも有意差は認められなかったが, 血管密度は非転移症例や高分化型に高い傾向にあった。一方, 癌浸潤様式との関連性においては, 浸潤傾向が高度のものほど血管密度が低くなる傾向がみられたが, リンパ管密度はほぼ一.定で推移し, 高度浸潤癌においてやや増加する傾向が認められた。

目的: 骨組織再生のための新たな生体材料の開発や成長因子の応用が期待されている. そこで, 本研究は各種成長因子の担体として高分子複合化材料の作製を行い, この材料の骨髄由来未分化間葉系細胞への影響を検討した.方法: 脱アセチル化キチン50とType I-Aコラーゲンをクーロン反応させることで, DAC50-TYPEIコラーゲン複合化ゲルを作製した. そして, この材料内にて骨髄由来未分化間葉系細胞の三次元培養を試み, 細胞増殖そして分化という観点から細胞増殖性試験, H-E染色による形態学的観察, ALP染色によるALP活性の観察およびRT-PCR法によるBSP, ALP, Osteopontin, Osteocalcin, およびGAPDH遺伝子の発現を検索した.結果: 細胞増殖性試験では細胞増殖は認めたが, 著しい増殖傾向を示すものはなかった. H-E染色では三次元的な細胞伸展所見を呈した. ALP染色では明らかなALP陽性所見が観察された. また, RTPCR法ではOsteocalcin以外の標的遺伝子の発現を確認した.結論: 今回作製した材料は骨髄由来未分化間葉系細胞に対して為害作用がなく, 三次元培養を可能とし, 骨組織再生への新たな生体材料として期待できる物質であることが示唆された.

Peritoneal metastasis is an important prognostic factor in cases of gastric cancer. Although studies on comparative genomic hybridization (CGH) in gastric cancer have been reported, there are few reports on the peritoneal metastasis (P) and peritoneal cytology (CY) factors in this cancer. In this study, we analyzed the chromosomal changes in the primary tumor with a combination of laser microdissection analysis and CGH in an attempt to detect the unknown abnormal chromosomal regions. We analyzed 34 primary tumors, including 13 primary tumors with peritoneal metastasis (P1) and/or positive peritoneal cytology (CY1) using a combination of laser microdissection and CGH. The minimal overlapping regions in gains were assigned to 5p14 (46.2%), 7q21.3 (61.5%), 7q31 (46.2%), 7q36 (46.2%), 8q23 (53.8%), 15q26 (46.2%), 20q12 (61.5%), 20q13.1 (53.8%), and 20q13.2 (53.8%) in primary tumors with P1 and/or CY1. The minimal regions of losses that occurred most frequently were 4q34-q35 (23.1%) and 22q11.2 (23.1%). There were significant differences in the minimal regions of 5p14 ( P =0.033), 7q21.3 ( P P =0.013), 7q36 ( P =0.033), and 22q11.2 ( P =0.048) between primary tumors with and without P1 and/or CY1. In this study, gain/amplification of 5p14, 7q21.3, 7q31, and 7q36, and loss of 22q11.2 were significant in gastric cancer cases with peritoneal dissemination and/or positive peritoneal cytology.

With islet transplantation having grown in popularity since the introduction of the Edmonton protocol, how to secure an unlimited source of islets has become an urgent problem. To resolve this problem, techniques to induce or proliferate islets are urgently required. To achieve this goal, gene expression analysis using a cDNA microarray in islets of partially pancreatectomized mice, in which the remaining islets regenerate and proliferate with insulin secretion and glucose responsiveness, provides us with valuable information. However, those experiments have two critical problems: first, how to selectively collect the regenerating or proliferating islets, and second, the shortage of total RNA extracted from one islet for a microarray analysis. A useful system was thus designed which combined laser microdissection, cDNA amplification by SMART PCR, which can maintain the relative expression profile of transcripts throughout reactions, and a cDNA microarray. Furthermore, this system is expected to contribute to future studies regarding not only islet regeneration but also the function of the islet itself, and this system may also be applicable to many other types of endocrine tissue. In this review, the details of this system are presented and discussed.

Angiolipoma usually develops in the trunk and extremities and rarely arises in the head and neck region, especially in the oral cavity. A case of angiolipoma of the cheek is reported. A 12-year-old girl was referred to our hospital because of swelling of the left cheek. Clinical examination revealed a rather ill-defined soft mass in the left cheek. Magnetic resonance (MR) imaging and MR angiography (MRA) suggested the mass to be a hemangioma. The mass was extirpated transorally with the patient under general anesthesia. Microscopically, the mass consisted of mature fat tissue intermixed with abundant vascular elements. A pathologic diagnosis of angiolipoma was made.

The product of the adenomatous polyposis coli (APC) tumor suppressor gene has been observed to regulate the Wnt signaling pathway through beta-catenin. In the present study, we attempted to clarify the relation between APC and the canceration of oral squamous epithelium. Each target tissue of normal squamous epithelium, epithelial dysplasia, and squamous cell carcinoma (SCC) was recovered the oral SCC case by laser microdissection. In recovered cells, we examined the change in expression of APC and beta-catenin gene transcription products, as well as the existence of mutations in APC gene. We analyzed the localization of each protein of APC and beta-catenin by immunohistochemical study. We found a clear change in the expression level of the gene transcription product of APC in canceration of oral squamous epithelium and the differentiation of oral SCC. In addition, there was some change in the localization of the APC protein in canceration. It was not clear, however, whether the APC was mutated. A change was also observed in the expression level of the beta-catenin gene transcription product during the differentiation of oral SCC. Our results suggest that the changes in the expression level and the intracellular localization of APC are related to the canceration of oral squamous epithelium, and in malignant characterization of oral SCC. Mutations of the APC gene might not be indispensable, however, in canceration of oral squamous epithelium.

Little is known about the mechanisms of mandibular condylar growth. In this study, gene expression in the mandibular condylar cartilage of young post-natal mice was monitored by means of a cDNA microarray, real-time PCR, and laser microdissection before and after the initiation of mastication (newborn, 7 days, 21 days, initiation of mastication, and 35 days). Insulin-like growth factor-1 (IGF-I), transforming-growth-factor-beta-2 (TGFbeta2), and aggrecan mRNAs were clearly expressed at 21 days, while the expression of osteopontin mRNAs was most clear at 35 days. Parathyroid-hormone-related protein (PTHrP), Indian-hedgehog (Ihh), and insulin-like growth factor-2 (IGF-2) mRNAs were clearly expressed during lactation (newborn and 7 days). Heat-shock-protein 84 (HSP-84) and heat-shock-protein 86 (HSP-86) were clearly expressed at 35 days. These results revealed that gene expression changed during mandibular condylar cartilage growth, and that, interestingly, these changes coincided with the initiation of mastication.

Laser microdissection is a method for the procurement of targeted cells from a tissue section, and its use enables one to analyze both nucleic acid and protein from procured cells. However, contamination by cells other than the target cells and damage to mRNA are problems that have been encountered. We have developed a laser pressure cell transfer method that is similar to the laser pressure catapulting method, but it has some advantages with regard to ease of the handling of an original thin film, the procedure for the preparation before laser microdissection, and the use of toluidine blue stain. We demonstrated that the quality of total RNA extracted from procured cells using the laser pressure cell transfer method was excellent for subsequent analysis, and this method could obtain clear morphological images of targeted cells before and after laser microdissection. Furthermore, as trial research for this method, we examined the expression profile of various transcriptional products related to invasive and metastatic potential using surgical materials of oral squamous cell carcinoma (OSCC). Lymph node metastasis tended to be often seen in cases that express VEGF, VEGFC and CD44s (2 of 3 cases) and in cases that express vimentin, keratin 19 and osteopontin (2 of 3 cases). These findings indicate that there may be a close relationship between those molecules in OSCC. This technique seems to be significantly better than the laser microdissection method currently available for obtaining clear morphological images, preserving the form of procured targeted cells, procuring mRNAs efficiently, and preserving them.

Endovascular coil emobolization has emerged as an alternative treatment of ruptured intracranial aneurysms, and it has reduced the therapeutic assault on vessels. However, it is not clear what happens between such therapeutic assault and vascular response on a molecular level. We investigated endothelial cell disorder after subarachnoid hemorrhage (SAH) and the impact of open and endovascular surgery. In rat SAH models, we confirmed the endothelial perturbation such as the expression of interleukin 1-β, plasminogen actibator inhibitor-1 (PAI-1), von Willebrand factor (vWF), E-selectin, and apoptosis-related products that caused the morphological changes of endothelial cells. In a real-time RT-PCR study, direct brain damage by a cold injury after experimental SAH emphasized the expression of p38 mitogen-activated protein kinase (p38 MAPK) in endothelial cells. In 36 SAH patients, the serum markers of endothelial cell disorder such as PAI-1, vWF and E-selectin that exist in the lower stream of p38 MAPK were compared between endovascular coil embolization (18 patients) and open surgery (18 patients). The levels of serum markers of endothelial cell disorder in open surgery were statistically higher than endovascular surgery (p