Hemizygosity of transsulfuration genes confers increased vulnerability against acetaminophen-induced hepatotoxicity in mice.

The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs(+/-) or Cth(+/-)) and homozygous (Cth(-/-)) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth(-/-) mice at 150 mg/kg dose, and also in Cbs(+/-) or Cth(+/-) mice at 250 mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth(-/-) mice but not wild-type mice, although glutamate-cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth(-/-) mice with lower Km values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150 mg acetaminophen/kg into Cth(-/-) mice; the profiles were similar to 1000 mg acetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200-300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities.

Gene expression changes in initiation and progression of oral squamous cell carcinomas revealed by laser microdissection and oligonucleotide microarray analysis.

Oral carcinogenesis is a complex process involving multiple genes. However, the genetic changes involved in this process are not apparent in identical oral squamous cell carcinomas (OSCCs). According to pathological characteristics, samples of normal tissue, oral dysplastic lesions (ODLs), and invasive cancers were obtained from identical OSCCs using laser microdissection (LMD). Large-scale gene expression profiling was carried out on 33 samples derived from 11 OSCCs. We analyzed genes differentially expressed in normal tissues vs. ODLs and in ODLs vs. invasive tumors and identified 15 candidate genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these genes, ISG15, was chosen for further characterization. Real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that ISG15 expression consistently increased during oral tumorigenesis. An ISG15 high-expression level was significantly associated with poor prognosis (p = 0.027). In addition, patients with high-expression tumors had a poorer 5-year survival rate than patients with low expression levels (p = 0.019). In conclusion, we identified 15 genes with continuously increasing or decreasing expression during oral carcinogenesis. One of these, ISG15, is likely to be associated with both dysgenesis and tumorigenesis and may be a potential prognostic marker for oral cancer.

General rules for clinical and pathological studies on oral cancer: a synopsis.

For the doctors and other medical staff treating oral cancers, it is necessary to standardize basic concepts and rules on oral cancers to progress in the treatment, research and diagnosis. Oral cancers are integrated in head and neck cancers and are applied to the general rules on head and neck cancer, but it is considered that more detailed rules based on the characteristics of oral cancers are essential. The objectives of this 'General Rules for Clinical and Pathological Studies on Oral Cancer' are to contribute to the development of the diagnosis, treatment and research of oral cancers based on the correct and useful medical information of clinical, surgical, pathological and image findings accumulated from individual patients at various institutions.

[Clinicopathological study of calcifying cystic odontogenic tumors].

Calcifying cystic odontogenic tumors are benign tumors, characterized by the presence of ghost cells and calcified materials. We evaluated clinical characteristics of calcifying cystic odontogenic tumors in 21 cases at the Maxillofacial Surgery, Tokyo Medical and Dental University Hospital, between January 1979 and December 2006. Of the 21 lesions that were studied, 12 were observed in male patients, and 9 in female patients. The median age was 13.0 years (range, 4-69 years). Of the 21 lesions, 11 were located in the maxilla (intraosseous), 9 in the mandible (intraosseous), and 1 in the lower gingiva (extraosseous). Radiographically, 18 lesions appeared as unilocular radiolucencies, and 2 lesions as multilocular radiolucencies. Impacted teeth were observed in 15 cases. In 20 cases, the lesions were treated by enucleation. The follow-up duration ranged from 2 years, 5 months to 28 years, 8 months, and in 1 case, the lesion recurred and showed a malignant transformation 2 years 10 months after the treatment. Histopathologically, the lining epithelium consisted of cuboidal or columnar odontogenic cells. Ghost cells were frequently calcified, and the tissue was hardened. In 14 cases, the tumor was associated with odontoma.

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.

[A clinico-pathological study of 20 cases of Warthin's tumors of the parotid gland].

PURPOSE: The purpose of this study was to clarify the clinico-pathological findings of Warthin's tumors. SUBJECTS AND METHODS: Twenty cases of Warthin's tumors treated at our clinic during the past 22 years and their medical charts and imaging films were reviewed. RESULTS: Warthin's tumors occurred more frequently in middle-aged or elderly men than in women. Solitary tumors were significantly larger (p < 0.05) than multiple tumors. Warthin's tumors that accumulated 99mTc were significantly larger (p < 0.05) than those that did not. In addition, there was no difference in clinical findings between the two histopathologic types of Warthin's tumors. CONCLUSION: The frequent occurrence of multiple Warthin's tumors indicated the importance of an accurate clinical and radiological examination of parotid glands, in order to detect possible multiple lesions prior to treatment.

Effective staining method with iodine for leukoplakia and lesions surrounding squamous cell carcinomas of the tongue assessed by colorimetric analysis.

To determine whether staining with iodine solution provides an efficient criterion for determining the area of resection for the lesions surrounding squamous cell carcinoma (SCC) and leukoplakia of the tongue, we determined the optimum density of iodine solution and staining procedure and analyzed the color of lightly stained lesions (LSLs) in relation to the histopathologic findings. Sixty-five patients with SCC or leukoplakia of the tongue were divided into two groups: lesions stained with 3% Lugol solution and restained with either 5% Lugol solution (n=38) or 10% iodine glycerin (n=27). Among the lesions stained with 5% Lugol solution, significant differences were found in all color values. Color difference values (DeltaE*ab) using 3% and 5% Lugol solutions were significantly different between epithelial hyperplasia/mild epithelial dysplasia and moderate to severe dysplasia (P < 0.05). According to the evaluations of five clinicians in 46 LSLs, a distinctive boundary was most often obtained using 5% Lugol solution. These results suggest that the most effective method for obtaining a clear boundary and distinguishing moderate to severe dysplasia from mild or no epithelial dysplasia according to the measured color value was to stain with 3% followed by 5% Lugol solution.

Altered expression of desmocollin 3, desmoglein 3, and beta-catenin in oral squamous cell carcinoma: correlation with lymph node metastasis and cell proliferation.

Desmocollin 3 (Dsc3) and desmoglein 3 (Dsg3) are both transmembrane glycoproteins that belong to the cadherin family of calcium-dependent cell adhesion molecules. beta-Catenin is a member of the cadherin-catenin complex that mediates homotypic cell-cell adhesion and is also an important molecule in the wnt signaling pathway. In this study, we examined the simultaneous expression level of Dsc3, Dsg3, and beta-catenin in oral squamous cell carcinomas (OSCCs) and normal oral epithelia using immunohistochemistry. There was a significant correlation (p < 0.05) among the following variables in OSCCs: reduced or loss of expression of Dsc3, Dsg3, and beta-catenin compared to normal oral epithelium, reduced or loss of expression of Dsc3 and histological grade (moderately or poorly differentiated), and reduced or loss of expression of beta-catenin and lymph node metastasis. Furthermore, a positive correlation was found between reduced or loss of beta-catenin staining and reduced or loss of Dsc3 staining in lymph node metastatic cancer tissue (r = 0.734, p < 0.05). These results suggest an abnormal expression of Dsc3, Dsg3, and beta-catenin induced in the progression of oral carcinomas and that the Dsc3 expression level might be related to the regulation of beta-catenin in lymph node metastasis and cell proliferation in OSCCs.

Activation of ERK1/2 and cyclin D1 expression in oral tongue squamous cell carcinomas: relationship between clinicopathological appearances and cell proliferation.

We report an immunohistochemical investigation of the expression of activated extracellular signal-regulated kinase (ERK1/2) and cyclin D1 protein in both oral tongue squamous cell carcinomas (OTSCCs) and normal tongue epithelium. The expression of Ki-67 labeling index (LI) was also examined in order to evaluate cell proliferation activity. The expression of activated ERK1/2, cyclin D1 protein and Ki-67 LI were significantly stronger in OTSCCs than in normal oral mucosa (P<0.05). Both over-expression of activated ERK1/2 and positive expression of Ki-67 in OTSCCs were significantly associated with a moderately or poorly differentiated grade of carcinoma (P<0.05). Cyclin D1 immunostaining showed statistically significant association with both lymph node metastasis (P<0.05) and a tumor thickness >5mm (P<0.05). Over-expression of activated ERK1/2 was positively correlated with cyclin D1 protein expression (P<0.05, r=0.624) and cell proliferation-related indexes Ki-67 (P<0.05, r=0.723). Our results suggest that over-expression of activated ERK1/2 and cyclin D1 protein are involved in oral tongue carcinogenesis, and that activation of ERK1/2 might be related to cell cycle regulation and cell proliferation in OTSCCs.

Basal cell adenocarcinoma arising from the minor salivary gland in the soft palate: a case report.

Basal cell adenocarcinomas (BCACs) of the oral minor salivary gland are very rare neoplasms. We report on an 86-year-old woman with BCAC arising from the minor salivary gland in the soft palate. Histologically, the tumor was located in the submucosa and showed microinvasion into the adjacent soft tissue without encapsulation. It contained tiny tumor islands with solid and tubular patterns, as well as myxoid stroma. The neoplastic cells were basaloid cells and were composed of large pale cells and small dark cells. They were positive for alpha-smooth muscle actin, cytokeratin 14, and vimentin in the periphery of the tumor island, showing a myoepithelial differentiation. The myxoid stroma was positive for alcian blue and colloidal iron. Apical membranes of the neoplastic cells were positive for MUC1 and CEA. The present case is the 14th documented case of oral BCAC (the fifth case of palatal BCAC).

Abnormal expression of Rb pathway-related proteins in salivary gland acinic cell carcinoma.

Salivary gland acinic cell carcinoma (ACC) is a relatively rare neoplasm, and limited information is available regarding its molecular pathogenesis. Because the deregulation of Rb pathway is common to most human tumors, we immunohistochemically investigated the expression of Rb pathway-related proteins, including Rb, Rb proteins phosphorylated at serine 780 and 795 (pRb-S780 and pRb-S795, respectively), cyclin D1, and p16INK4a in 18 cases of ACC. The expression of topoisomerase II-alpha and Ki-67 was also examined to evaluate cell proliferation. All the ACCs exhibited substantial numbers of positive cells against Rb antibody that recognizes both unphosphorylated and phosphorylated Rb proteins. The numbers of positive cells for pRb-S795 and cyclin D1 significantly increased in ACCs as compared with normal salivary glands. Double immunofluorescent staining demonstrated that pRb-S795 was colocalized with cyclin D1 in most tumor cells. However, neither significant change of the expression of Rb protein phosphorylated at serine 780 nor its colocalization with cyclin D1 was observed. The loss of p16INK4a is infrequent, but its expression was correlated with phosphorylated Rb proteins. Our results suggest that serine 795 but not serine 780 is the preferred phosphorylation site induced by cyclin D1. This phosphorylation appeared to be critical for inactivation of Rb-mediated growth suppression and may play an important role in the pathogenesis of ACC.

Neck node metastasis after successful brachytherapy for early stage tongue carcinoma.

BACKGROUND AND PURPOSE: The accuracy of factors for predicting lymph node metastasis in patients with early-stage (stage I and II) mobile tongue carcinoma and prognostic factors associated with the clinical and pathological findings of lymph node metastasis were examined. MATERIAL AND METHODS: Between 1971 and 1998, 616 patients with early stage mobile tongue carcinoma were treated by brachytherapy with or without external irradiation. Neck lymph node metastasis occurred in a total of 237 cases, and 191 of them were not associated with primary failure. Neck dissection was performed in 169 of these 191 cases, and 16 cases were treated by radiotherapy. A pathological analysis was possible in 159 of the 169 neck dissection cases. RESULTS: There were 88 tongue cancer recurrences, and the incidence of neck metastasis was 38% (191/528) in the cases of primary controlled early tongue carcinoma, and 25% (38/151) and 41% (153/377), in stage-I and -II carcinoma, respectively. Neck metastasis was diagnosed within 12 months in 80% of cases, and within 24 months in 95%. Macroscopic appearance, tumor thickness and tumor length were identified as significant risk factors by a univariate analysis, but macroscopic appearance was the only significant risk factor identified by a multivariate analysis (P<0.001). The incidence of cervical lymph node metastasis was 62% among the invasive/ulcerative type tongue carcinomas, and was lower among the superficial type and exophytic/nodular type (20 and 35%, respectively). Regional and/or distant failure occurred in 75 of the 169 neck dissection cases (44%). The incidence of regional/distant failure was extremely high (49/68=72%) in the extra-nodal invasion group, and extra-nodal invasion was found even in small metastatic node less than 1 cm in length (20%). CONCLUSIONS: The macroscopic appearance of the primary tongue carcinoma has a major impact on the incidence of lymph node metastasis in patients with early tongue cancer, and extra-nodal invasion was the dominant risk factor for regional and distant failure. Treatment policy for clinically negative neck metastasis in early tongue cancer patients should be determined after considering the possibility of neck metastases and the morbidity associated with elective neck dissection.

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma.

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained (P < 0.05). Correlations were obtained between the CENP-F LI and tumor size (P < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G2 to M cells, compared to Ki-67.

Decreased thyroid uptake of Tc-99m pertechnetate in patients with advanced-stage Sjögren syndrome: evaluation using salivary gland scintigraphy.

PURPOSE: The authors assessed the uptake of Tc-99m pertechnetate in the thyroid using salivary gland scintigraphy in patients with Sjögren syndrome and in healthy controls. MATERIALS AND METHODS: Salivary gland scintigraphy and a labial biopsy were performed in 73 patients with Sjögren syndrome. Based on the labial biopsy findings, 32 patients with a histopathologic grade of 1 or 2 were regarded as having early-stage Sjögren syndrome and 41 patients with a grade of 3 or 4 were regarded as having an advanced stage. After the administration of 370 MBq (10 mCi) Tc-99m pertechnetate, dynamic salivary gland scintigraphy was performed for 50 minutes. Lemon juice was used to stimulate the salivary glands, and the thyroid gland was included in the imaging area. Scintigraphy was also performed in an age- and sex-matched control group of 25 healthy persons. The thyroid uptake ratio was calculated for the scintigraphic images and compared among the three groups: healthy controls, patients with early-stage Sjögren syndrome, and those with advanced-stage Sjögren syndrome. RESULTS: When compared with the control group, the thyroid uptake ratio of the early-stage Sjögren syndrome group was not significantly different, whereas that of the advanced-stage group was significantly lower. CONCLUSIONS: Thyroid uptake of Tc-99m pertechnetate was less in patients with advanced-stage Sjögren syndrome than in patients with early-stage Sjögren syndrome or in healthy controls. Measuring the thyroid uptake of Tc-99m pertechnetate using salivary gland scintigraphy is an easy and useful method for assessing thyroid disorders in Sjögren syndrome and thus should be performed routinely.

Clinical significance of lymphatic and blood vessel invasion in oral tongue squamous cell carcinomas.

Although vascular invasion (VI) is recognized as an important predictor of lymph node metastasis and a significant prognostic factor in head and neck squamous cell carcinoma (HNSCC), there is currently no common definition for the pathological evaluation of VI status. We reviewed the medical records of 63 consecutive resected primary oral tongue SCCs (OTSCCs) without preoperative treatment between June 1999 and April 2008, and evaluated VI status by investigating lymphatic vessel invasion (LVI) and blood vessel invasion (BVI) by using immunohistochemistry (IHC) with monoclonal antibody D2-40 (D2-40) and Elastica van Gieson (EVG) staining, respectively. Subsequently, we analyzed their correlations with cervical lymph node metastasis and prognosis. LVI was found in 16 of the 63 tumors (25.4%) and BVI was in 32 tumors (50.8%). Univariate analysis revealed that the presence of LVI is statistically correlated with lymph node metastasis. Moreover, multivariate logistic regression analysis revealed that LVI is an independent risk factor of nodal metastasis (odds ratio=4.262, 95% confidence interval=1.262-14.397, p=0.020). In contrast, Kaplan-Meier survival analysis revealed that patients with BVI had a significantly shorter disease-free survival (DFS) and overall survival (OS) rates than those without BVI (68.6% versus 90.3%, p=0.028 and 68.6% versus 93.5%, p=0.013, respectively). The present study clearly demonstrated that LVI at primary OTSCC had significant correlation with lymph node metastasis, and that BVI was significantly associated with recurrence and poor prognosis. Evaluation of VI status, as LVI and BVI status separately, using IHC with D2-40 and EVG staining may be useful in predicting lymph node metastasis and poor prognosis in OTSCCs.

Loss of NKX3-1 as a potential marker for an increased risk of occult lymph node metastasis and poor prognosis in oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) is significantly dependent on the existence of cervical lymph node metastasis (LNM), with the overall survival rate being much lower in patients with LNM. Primary causes and molecular mechanisms of LNM are still largely unclear. We hypothesized that factors related with cancer progress and/or prognosis in OSCC are revealed by genome-wide investigation of DNA copy number aberrations (CNAs). In order to find biomarkers for occult LNM of OSCC, we comprehensively investigated genomic DNAs from 60 OSCC patients using Affymetrix mapping arrays and statistically analyzed correlations between CNAs of genes and the presence of occult LNM in the patients. The genome-wide CNA study indicated significant correlations between the presence of occult LNM and CNAs of certain genes. Through a literature survey, we narrowed down the candidates and focused on loss of NKX3-1, which is a homeodomain-containing transcription factor. NKX3-1 is known as a tumor suppressor gene in prostate cancer but has never been reported in OSCC. Quantitative RT-PCR and immunohistochemistry (IHC) analyses also showed significantly lower expression of NKX3-1 in the cases with occult LNM, which was further validated by IHC analysis in independent cases. The survival analyses indicated that NKX3-1 loss is a significant risk factor to decrease the disease-free survival (DFS) and the overall survival (OS) rates. This is the first time that the significant association of NKX3-1 loss and occult LNM was indicated in OSCC. The present results suggest that loss of NKX3-1 may be a potential biomarker for occult LNM of OSCC.

Cementoblastoma arising in the maxilla of an 8-year-old boy: a case report.

Cementoblastoma is an uncommon disease, representing only 1-8% of all odontogenic tumours. Furthermore, this tumour is especially uncommon in children, as only five cases have been reported in this age group. Here, we describe a case of cementoblastoma arising in the maxilla of an 8-year-old boy, that was treated with a partial maxillectomy. The patient's facial appearance has remained satisfactory, and the tumour has not recurred in the 9 years after the operation.

EGFR gene copy number alteration is a better prognostic indicator than protein overexpression in oral tongue squamous cell carcinomas.

Although epidermal growth factor receptor (EGFR) is particularly important in the pathogenesis of head and neck squamous cell carcinomas (HNSCCs), conflicting data have been reported on the correlation between EGFR copy number and survival and the association between EGFR copy number and protein expression. Anatomical site of the tumour in HNSCCs may likely contribute to the discordance of the above points as EGFR expression may differ between the sub-sites of HNSCCs. Thus, in this study, we focused on oral tongue squamous cell carcinomas (OTSCCs). To investigate the association between EGFR copy number alteration and overexpression and to determine which is the more reliable prognostic indicator, Fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were performed at a single institution on samples from 89 patients with OTSCCs undergoing surgery as the primary treatment modality. Thirty-two (36%) of 89 cases demonstrated an EGFR copy number alteration. EGFR protein expression was found in all 89 cases, of which 82.0% showed overexpression. No significant correlation was found between gene copy number and protein overexpression. Gene copy number alteration was significantly associated with reduced disease-free survival (P=0.048) and overall survival (P=0.001). Multivariate Cox proportional hazards analysis demonstrated that EGFR copy number increase was significantly correlated with overall survival (P=0.001). EGFR copy number status is a more reliable indicator than protein overexpression of the survival rate in OTSCCs. FISH analysis of the EGFR status is useful in predicting poor prognosis in OTSCCs.

Immunohistochemical study of ß-catenin and functionally related molecular markers in tongue squamous cell carcinoma and its correlation with cellular proliferation.

ß-catenin plays an important role in the maintenance of cell adhesion and is a key component of the Wnt signaling pathway. However, little is known about its prognostic significance or its role in tumor progression in tongue squamous cell carcinoma (SCC). This study conducted an immunohistochemical analysis of the expression of ß-catenin. Moreover, its possible correlation with clinical parameters and with the expression of the functionally related molecular markers cyclin D1 and p53 was evaluated in 50 cases of tongue SCC and 10 cases of normal tongue epithelium. The ki-67 labeling index (LI) was also examined to evaluate cellular proliferation. Our results showed a higher frequency of abnormal ß-catenin expression, positive cyclin D1 and p53 expression, and a significantly higher ki-67 LI in the tongue SCC samples compared with normal tongue epithelium (P<0.05). Abnormal ß-catenin and a higher ki-67 expression was significantly associated with moderately or poorly differentiated carcinoma (P<0.05). Cyclin D1-positive immunostaining showed a statistically significant association with lymph node metastasis (P<0.05). Furthermore, the abnormal expression of ß-catenin significantly correlated with a higher ki-67 LI and p53 expression (P<0.05); however, there was no correlation with cyclin D1 expression (P>0.05). Taken together, our results suggest that abnormal ß-catenin expression is related to the impaired cellular differentiation and proliferation involved in tumor progression in tongue SCC.