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.

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.

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.

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.

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.

[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.

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.

Comprehensive keratin profiling reveals different histopathogenesis of keratocystic odontogenic tumor and orthokeratinized odontogenic cyst.

Keratocystic odontogenic tumor is a cystic lesion that behaves more aggressively than other jaw cysts. One of its characteristic histologic features is a parakeratinized uniform layer of lining epithelium. A jaw cyst lined with orthokeratinized epithelium is called an orthokeratinized odontogenic cyst. These keratinized jaw cysts are thought to be separate entities, although their histopathogenesis has not been fully assessed. To better understand these lesions, we performed comprehensive immunohistochemical profiling of the keratin expression of each. Orthokeratinized odontogenic cysts expressed keratin 1, keratin 2, keratin 10, and loricrin, suggesting differentiation toward normal epidermis. Keratocystic odontogenic tumors expressed keratin 4, keratin 13, keratin 17, and keratin 19, which is a unique expression pattern reminiscent of a mucosal squamous epithelium and an epithelial appendage. In neonatal rat tooth germ, cells strongly positive for keratin 17 and keratin 19 were observed, specifically in the dental lamina, implying the origin of keratocystic odontogenic tumor. GLI2, a downstream effector of hedgehog signaling, was significantly expressed in keratocystic odontogenic tumor and basal cell carcinoma, accompanied with robust expression of keratin 17, mammalian target of rapamycin, and BCL2. The expression of these GLI2- or keratin 17-related factors was not significantly observed in orthokeratinized odontogenic cysts. These findings provide evidence to support the viewpoint that keratocystic odontogenic tumor and orthokeratinized odontogenic cyst are separate entities, and furthermore suggest their characteristic histology, pathogenesis, and biological behaviors.

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.

Nerve sheath myxoma (neurothekeoma) arising in the oral cavity: histological and immunohistochemical features of 3 cases.

This report describes the histological and immunohistochemical characteristics of 3 cases of nerve sheath myxoma (NSM)/neurothekeoma arising in the oral cavity. Histopathologically, 3 distinct variants were observed based on the amount of myxoid matrix: classic/hypocellular, cellular, and mixed types. Immunohistochemically, all types expressed strong immunoreactivity to S-100 protein (3/3), NSE (neuron-specific enolase) (3/3), and NGFR (nerve growth factor receptor) (3/3), whereas all cases were negative for SMA (smooth muscle actin) and FVIII. The number of Ki-67 positive cells was less than 5% in all lesions confirming the slow growing characteristics of NSM. The results suggest that NSMs of the oral cavity are true peripheral nerve sheath tumors and show close relationship to schwannoma.

A case of basal cell adenocarcinoma of the upper gingiva.

Basal cell adenocarcinoma arising from the minor salivary gland is extremely rare. We report a 76-year-old Japanese man with basal cell adenocarcinoma originating in the upper gingiva. He underwent subtotal maxillectomy combined with resection of the coronoid process, and reconstruction was performed using a rectus abdominis microvascular flap. The patient has been followed for 40 months after operation without any evidence of disease.

Diagnostic accuracy of cone-beam CT in the assessment of mandibular invasion of lower gingival carcinoma: comparison with conventional panoramic radiography.

PURPOSE: To evaluate the diagnostic accuracy of cone-beam CT in assessing mandibular invasion by lower gingival carcinoma and compare it with that of panoramic radiography. PATIENTS AND METHODS: Fifty patients with squamous cell carcinoma of the lower gingiva who were examined by both panoramic radiography and cone-beam CT before surgery were included in this study. Five radiologists used a 6-point rating scale to independently evaluate cone-beam CT and panoramic images for the presence or absence of alveolar bone and mandibular canal involvement by tumor. Using the histopathogical findings as the gold standard, we calculated and compared the area under the receiver operating characteristic curve (Az value) and the sensitivity and specificity of the two imaging modalities. RESULTS: In evaluations of both alveolar bone and mandibular canal involvement, the mean Az value for cone-beam CT (0.918 and 0.977, respectively) was significantly higher than that for panoramic radiography (0.793 and 0.872, respectively). The mean sensitivity for cone-beam CT (89% and 99%, respectively) was significantly higher than that for panoramic radiography (73% and 56%, respectively). There was no significant difference in the mean specificity. While cone-beam CT could provide high-resolution three-dimensional images, the image quality around the alveolar crest was often hampered by severe dental artifacts and image noise, resulting in difficulties in detecting subtle alveolar invasion. CONCLUSION: Cone-beam CT was significantly superior to panoramic radiography in evaluating mandibular invasion by lower gingival carcinoma. Its diagnostic value in detecting subtle alveolar invasion, however, may be limited by severe dental artifacts and image noise.

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.

A male infant case of lipofibromatosis in the submental region exhibited the expression of the connective tissue growth factor.

PURPOSE: We encountered a case of lipofibromatosis in a 21-month-old male patient and examined its histopathologic properties. METHODS: We examined morphological aspects of the tumor and immunohistochemical patterns. RESULTS: Tumor proliferation was infiltrative, which did not show apparent encapsulation. Positive immunoreactivity was found for CD-34, CD-99, Ki-67, and connective tissue growth factor/CCN2 in the fibrous region, S-100 in the adipose region, and Notch1 stain was observed in the eccrine sweat gland cells juxtaposed to the tumor adipose tissue, but no reactivity for Bcl-2, alphaSMA, Notch 2-4, CCN1, and CCN3. CONCLUSIONS: The clinical process and immunohistochemical pattern of this case was consistent with the criteria of reported lipofibromatosis. Specific expression of CCN2 might be significant for the development of the tumor.