TGF-ß3 from fibroblasts promotes necrotising sialometaplasia by suppressing salivary gland cell proliferation and inducing squamous metaplasia.

Necrotising sialometaplasia (NSM) is a non-neoplastic lesion mainly arising in the minor salivary glands of the oral cavity. In the clinical features, NSM shows swelling with or without ulceration, and can mimic a malignant disease such as squamous cell carcinoma. Histopathologically, NSM usually shows the lobular architecture that is observed in the salivary glands. Additionally, acinar infarction and squamous metaplasia of salivary ducts and acini are observable. The aetiology of this lesion remains unknown, although it has a characteristic feature that sometimes requires clinical and histopathological differentiation from malignancy. In this study, we investigated upregulated genes in NSM compared with normal salivary glands, and focused on the TGF-ß3 (TGFB3) gene. The results of the histopathological studies clarified that fibroblasts surrounding the lesion express TGF-ß3. Moreover, in vitro studies using mouse salivary gland organoids revealed that TGF-ß3 suppressed salivary gland cell proliferation and induced squamous metaplasia. We demonstrated a possible aetiology of NSM by concluding that increased TGF-ß3 expression during wound healing or tissue regeneration played a critical role in cell proliferation and metaplasia. © 2024 The Pathological Society of Great Britain and Ireland.

Salivary gland polymorphous adenocarcinoma: Clinicopathological features and gene alterations in 36 Japanese patients.

BACKGROUND: Polymorphous adenocarcinoma is a common intraoral minor salivary gland carcinoma in Western countries but is extremely rare in Japan. The current study aimed to characterize the clinicopathological features and status of molecular alterations of polymorphous adenocarcinoma-associated genes, such as PRKD1/2/3, ARID1A, and DDX3X, in a large cohort of Japanese patients with polymorphous adenocarcinoma. METHODS: We examined the cases of 36 Japanese patients with salivary gland polymorphous adenocarcinoma and 26 cases involving histopathological mimics. To detect gene splits, fluorescence in situ hybridization was carried out for polymorphous adenocarcinoma-associated genes. Additionally, we applied a SNaPshot multiplex assay to identify PRKD1 hotspot mutations. RESULTS: This study revealed the indolent clinical course of polymorphous adenocarcinoma with a high 10-year overall survival rate (92.9%), accompanied by occasional local recurrences and cervical lymph node metastasis (23.3%). Twenty cases (55.6%) of polymorphous adenocarcinoma (but none of the mimics) exhibited alterations in at least one polymorphous adenocarcinoma-associated gene. Rearrangement of polymorphous adenocarcinoma-associated genes and PRKD1 E710D were identified in 17 (47.2%) and 4 (11.1%) cases, respectively; one case showed coexisting PRKD3 split and PRKD1 E710D. In the multivariate analysis, high clinical stage (p = 0.0005), the presence of prominent nucleoli (p = 0.0003), and ARID1A split positivity (p = 0.004) were independent risk factors for disease-free survival. CONCLUSION: Japanese patients with polymorphous adenocarcinoma showed clinicopathological features similar to those reported in Western countries. This study disclosed that polymorphous adenocarcinoma-associated genetic alterations were common and specific findings in polymorphous adenocarcinomas. The diagnostic role and possible prognostic significance of polymorphous adenocarcinoma-associated genetic alterations in polymorphous adenocarcinomas were suggested.

White opaque substance represents an intracytoplasmic accumulation of lipid droplets: immunohistochemical and immunoelectron microscopic investigation of 26 cases.

BACKGROUND AND AIM: White opaque substance (WOS) in gastric neoplasias is a unique finding visualized in magnifying endoscopy (ME) with narrow band imaging (NBI) and it represents intramucosal accumulation of lipid droplets using oil red O staining. METHODS: Subjects were 26 WOS-positive (13 adenomas and 13 well-differentiated adenocarcinomas) and 27 WOS-negative gastric epithelial neoplasias. We carried out immunohistochemical staining using a monoclonal antibody specific for adipophilin as a marker of lipids. Immunoelectron microscopy was used to evaluate morphology of the lipid droplets. RESULTS: Adipophilin was detected in 24 of 25 (96.0%) WOS-positive neoplasias, but it was detected in only two of 27 (7.4%) WOS-negative neoplasias. Lipid droplets were only seen in the surface epithelium in 10 of 11 (91.1%) adenomas, whereas the lipid droplets also existed in the cryptal epithelium in seven of 13 (53.8%) adenocarcinomas. Immunoelectron microscopy revealed numerous lipid droplets mainly existing in the subnuclear cytoplasm of the epithelium. The shape of the lipid droplets in adenomas was round and uniform, whereas that in adenocarcinomas was irregular. CONCLUSIONS: The present study confirmed that the presence of WOS in gastric neoplasias was dependent upon intramucosal accumulation of lipid droplets using anti-adipophilin staining. Intraepithelial distribution and morphology of the lipid droplets differed between adenoma and adenocarcinoma.

Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

Cyclic anthraquinone derivatives, unique G-quadruplex binders, selectively induce cancer cell apoptosis and inhibit tumor growth.

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

Human telomerase reverse transcriptase protein expression is associated with survival in patients with oral squamous cell carcinoma.

Despite improvements in diagnosis and treatment, the 5-year survival of oral squamous cell carcinoma (OSCC), no matter the location, remains low, averaging 50%. Telomerase is expressed in 85% of malignancies and may play an important role in human carcinogenesis. Its catalytic component is human telomerase reverse transcriptase (hTERT), which has been thought, but not proven, to be involved in survival with OSCC. We investigated whether hTERT protein was a prognostic factor in OSCC by evaluating its association with clinicopathologic findings and OSCC survival. We found that in comparison to patients with high hTERT expression, patients with low hTERT expression survived significantly longer, including a longer 5-year overall survival. In addition, overall survival was significantly correlated to hTERT expression and the histologic grade and N status of the tumor. Disease-free survival was significantly related to hTERT expression, the histologic grade and N status of the tumor, and mode of invasion. These results suggest that hTERT protein is involved in cervical lymph node metastasis, that its levels may be increased during carcinogenesis, and that it may influence tumor invasion. We believe that this study is the first to demonstrate that OSCC with high hTERT expression carries a worse prognosis than cases with low hTERT expression.

Assessing the Expression of Aquaporin 3 Antigen-Recognition Sites in Oral Squamous Cell Carcinoma.

Aquaporin 3 (AQP3) serves as a water and glycerol transporter facilitating epithelial cell hydration. Recently, the involvement of AQP3 in cancers has been reported. However, the immunohistochemical expression of AQP3 in carcinomas remains controversial. We hypothesized that differences in aquaporin 3 antigen recognition (AQP3 AR) may influence their expressions. Thus, our study aimed to assess the immunostaining patterns of 3 AQP3 AR sites in oral squamous cell carcinoma (OSCC) and to compare the adjacent areas of high-grade epithelial dysplasia (HG-ED) and normal oral mucosa (NOM). The study group included formalin-fixed OSCC samples (n=51) with adjacent regions of HG-ED (n=12) and NOM (n=51). The tissues were stained with anti-AQP3 antibodies (AR sites at amino acid (AA) 250-C terminus, AA180-228, and N terminus AA1-80) by immunohistochemistry. Our results showed that strong membranous immunostaining was observed for AQP3 AR sites at the AA250-C terminus and AA180-228 in all the samples for NOM and weak AQP3 immunostaining for both the AR sites in all the 12 samples for HG-ED. The invasive front of OSCC samples showed that AQP3 AR at the AA250-C terminus decreased in 42/51 samples (82.4%) and AA180-228 in 47/51 samples (92.2%). Conversely, in the AQP3 AR site at N terminus AA1-80, all samples of the NOM showed negative or slightly positive staining in the cytoplasm of the lower layers. AQP3 expression was increased in 12/12 cases (100%) and 46/51 cases (90.2%) in the HG-ED and invasive front of OSCC, respectively. AQP3 may be used as a biomarker for detecting malignant transformations. AQP3 AR site differences influence their immunohistochemical expression in OSCC.

Merkel cell carcinoma co-existent with sebaceous carcinoma of the eyelid.

Merkel cell carcinoma is occasionally associated with other types of cutaneous malignancies including squamous cell carcinoma, basal cell carcinoma and lentigo maligna. We report a case of Merkel cell carcinoma co-existent with sebaceous carcinoma in the right upper eyelid of a 61-year-old Japanese man. Histopathologically, the resected tumor consisted of three nodules located in the tarsal plate, showing two distinct histopathological types. Two nodules were Merkel cell carcinoma and located in the proximal part of the palpebral conjunctiva. The third was sebaceous carcinoma located in the distal transitional zone between the epidermis and the conjunctiva. No features of transition between these two components were noted. Metastatic deposits were identified in the regional lymph nodes, which solely consisted of Merkel cell carcinoma without sebaceous carcinoma. This is the first report of such co-existent lesions.

Macrophage-mediated repair of intraepithelial damage in rat oral mucosa.

OBJECTIVE: It is important to protect the patient's oral mucosa from injury caused by inappropriate oral care. We established anin vivo model of intraepithelial injury of the oral mucosa and assessed the effects of inappropriate dental apparatus use on inflammatory response of macrophages in rats. DESIGN: Using this model, swabbing was performed to the labial mucosa of rats with the interdental brush, and the tissue samples were processed for histological evaluation. RESULTS: CD 68-positive macrophage-like cells appeared only in the subepithelial region after intraepithelial injury in the presence and absence of antibiotics. After intraepithelial injury, interleukin-1ß levels did not increase beyond those observed in intact rats. The number of bacteria in the lip increased slightly and decreased promptly. In the ear, intraepithelial injury caused an inflammatory response with macrophages infiltrating into the intraepithelial region as well as the subepithelial region, increased interleukin-1ß production, and high bacterial levels around tissues. CONCLUSION: Our results highlight the importance of protecting against the intraepithelial injury during oral care procedures, and these findings would contribute to oral care risk management in the future.

In vitro and in vivo effects of a novel bioactive glass-based cement used as a direct pulp capping agent.

Direct pulp capping is an important procedure for preserving pulp viability. The pulp capping agent must possess several properties, including usability, biocompatibility, and the ability to induce reparative dentin formation. In this study, a novel bioactive glass-based cement was examined to determine whether the cement has the necessary properties to act as a direct pulp capping agent. Physicochemical properties of the bioactive glass-based cement and in vitro effects of the cement on odontoblast-like cells, as well as in vivo effects on the exposed dental pulp, were analyzed. The cement immersed in water stabilized at pH10, and hydroxyapatite-like precipitation was induced on the surface of the cement in simulate body fluid. There were no cytotoxic effects on the viability, alkaline phosphatase activity, or calcium deposition ability of odontoblast-like cells. In the in vivo rat study of an exposed dental pulp model, the cement induced a sufficient level of reparative dentin formation by odontoblast-like cells expressing odontoblastic markers at the exposed area of the dental pulp. These results suggest that the newly developed bioactive glass-based cement provides favorable biocompatibility with the dental pulp and may be useful as a direct pulp capping agent. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 161-168, 2019.

Characteristics of diffusion-weighted images and apparent diffusion coefficients of ranulas and other masses in and around the floor of the mouth.

OBJECTIVE: The aim of this study was to evaluate the characteristics of diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) values of ranulas. In addition, to elucidate DWI findings and ADC values of other representative masses in and around the floor of the mouth. STUDY DESIGN: DWI findings and ADC values in 35 patients with ranulas and 33 patients with other masses were retrospectively reviewed with a central focus on cystic masses or lesions that may have cyst-like components in and around the floor of the mouth based on the diagnosis of each respective disease. RESULTS: Ranulas were all well-defined, homogeneous masses with high signal intensity on DWI. The mean ± standard deviation ADC value of the 35 ranulas was 2.59 ± 0.31 × 10-3 mm2/s. There was a significant difference in ADC values between simple and plunging ranulas. On DWI, most other masses were heterogeneous, and most ADC values, except those for thyroglossal duct cysts, hemangiomas, and pleomorphic adenomas, were significantly lower than those for ranulas. CONCLUSIONS: The characteristic DWI and ADC findings of ranulas can be determined accurately, and these data can be significantly useful in the differential diagnosis of many kinds of diseases in and around the oral floor.

Aberrant methylation in promoter regions of cyclin-dependent kinase inhibitor genes in adenoid cystic carcinoma of the salivary gland.

Adenoid cystic carcinoma (ACC) is a common malignant neoplasm of the salivary gland. The mechanism underlying ACC carcinogenesis is not fully elucidated, although data on associated genetic alterations are accumulating. Cyclin-dependent kinase inhibitors (CKIs) act as tumor suppressors in various cancers, and aberrant methylation in the CKI gene promoter region has been linked to gene silencing and downregulation of expression. The present study investigated methylation of CKI genes, p15, p18, p19, p21, and p27, in 34 cases of ACC. We found frequent and plural methylations of these genes in most cases (68.8% in p15, 90.3% in p18, 78.8% in p19, 92.3% in p21, and 26.5% in p27). Cell cycle disruption induced by these epigenetic aberrations might be important in the tumorigenesis of ACC.

Mutational analysis of Wnt signaling molecules in ameloblastoma with aberrant nuclear expression of beta-catenin.

BACKGROUND: To clarify the genetic background of ameloblastoma, expression of beta-catenin, and mutational status of genes involved in Wnt signaling pathway were investigated. METHODS: We analyzed beta-catenin and cyclin D1 in 18 cases of ameloblastoma by immunohistochemical staining, and searched for mutations in CTNNB1 (gene for beta-catenin), APC, AXIN1, and AXIN2 by polymerase chain reaction (PCR) and direct sequencing method. RESULT: We detected membranous and occasionally cytoplasmic expression of beta-catenin in 16 of 18 cases (89%), and nuclear expression of beta-catenin principally in the peripheral columnar cells in 11 of 18 cases (61%). In nine of the 18 cases (50%), we detected the expression of cyclin D1 principally in the peripheral columnar cells. However, there was no correlation between nuclear expressions of beta-catenin and cyclin D1. No missense mutations were found in CTNNB1, APC, AXIN1, and AXIN2 in all cases except for silent mutation and already-known single nucleotide polymorphism. CONCLUSION: Mutations in CTNNB1, APC, AXIN1, and AXIN2 are not implicated in nuclear accumulation of beta-catenin, and that the expression of cyclin D1 is accelerated independently of beta-catenin in ameloblastomas. Other Wnt signaling members or alternative pathways involved in the degradation of beta-catenin should be subject of further investigation.

A case of sebaceoma with extensive apocrine differentiation.

Apocrine differentiation is a rare event in sebaceoma, and only 3 cases have been reported. We report a case of sebaceoma with extensive apocrine differentiation on the scalp in a 73-year-old Japanese woman. The resected tumor was located entirely within the dermis and subcutis as a well-circumscribed, lobulated, solid, and partially cystic mass, measuring 35 mm at the largest diameter. Histopathologically, it was composed of uniform basaloid cells with clusters of sebocytes, squamous islands of ductal structures, and apocrine cells with apparent decapitation secretion. Nuclear atypia of all types of cells was inconspicuous, and mitotic figures were infrequent. We considered the lesion to be a sebaceoma with apocrine differentiation.

Significance and usefulness of imaging characteristics of gubernaculum tracts for the diagnosis of odontogenic tumors or cysts.

We aimed to determine the significance and usefulness of imaging characteristics of gubernaculum tracts (GT) for the diagnosis of odontogenic tumors or cysts. This was a retrospective analysis of relationships between odontogenic or non-odontogenic tumors or cysts and the GT that were visualized using multi-detector computed tomography (MDCT). The relationship between the size of a mass and expansion of the GT in all odontogenic tumors or cysts to which GTs were contiguous on MDCT, was statistically analyzed. Intact or expanded GTs were detected in MDCT images on the top of almost all odontogenic tumors or cysts, but not on non-odontogenic tumors or cysts. Characteristic image findings regarding the relationship between the GT and the odontogenic mass were detected for the respective odontogenic tumors or cysts in which the GTs were contiguous to the mass on MDCT. In ameloblastomas, expansion of the GTs significantly and very strongly correlated with tumor size (r = 0.741, p = 0.0001), but this correlation was very weak in dentigerous cysts (r = 0.167, p = 0.028) and there was no correlation between these parameters in odontogenic keratocysts (r = -0.089, p = 0.557). The imaging characteristics of GTs at the top of masses should be very useful for both the differential diagnosis of the pathological diagnosis of odontogenic masses and for differentiation between odontogenic and non-odontogenic masses.

Carcinosarcoma ex recurrent pleomorphic adenoma of the submandibular gland.

We report a case of carcinosarcoma ex recurrent pleomorphic adenoma in the submandibular region of a 56-year-old Japanese man. He presented with a 2-year history of a rapidly growing mass in the submandibular region. He reported undergoing excision of a nodule in the same region 10 years earlier. Incisional biopsy confirmed the diagnosis of pleomorphic adenoma. The lesion was excised surgically. The resected tumor measured 40 x 20 mm and was composed of two large nodules and multiple small satellite nodules in the subcutaneous tissue. Histopathologically, one large nodule was carcinosarcoma while the other large nodules and small satellite nodules were pleomorphic adenoma. The former large nodule showed a variegated pattern with carcinomatous components (poorly differentiated adenocarcinoma, salivary duct carcinoma, squamous cell carcinoma and undifferentiated carcinoma) and sarcomatous components (spindle cell sarcoma, chondrosarcoma, liposarcoma and rhabdomyosarcoma). Based on the clinical history and histopathology, we consider the lesion to have originated from recurrent pleomorphic adenoma.

The methylation status and expression of human telomerase reverse transcriptase is significantly high in oral carcinogenesis.

Telomerase activity is present in most cancers and is tightly regulated by the expression of human telomerase reverse transcriptase (hTERT). Hypermethylation in the promoter region of hTERT contributes to the regulation of hTERT expression. In this study, we investigated the methylation and expression of hTERT in oral squamous cell carcinoma (OSCC), oral leukoplakia, and normal oral mucosa. Furthermore, we investigated the significance of hTERT to the clinicopathological findings of OSCC. 35 OSCC, 50 oral leukoplakia (epithelial dysplasia n = 25, squamous cell hyperplasia n = 25), and 10 normal oral mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analyzed in 35 OSCC, 50 oral leukoplakia, and 4 normal oral mucosa samples. The methylation and expression of hTERT increased from normal oral mucosa to oral leukoplakia to OSCC. In OSCC, all samples were methylated. However, partial methylation (20%) or unmethylation (80%), but never complete methylation, was observed in normal oral mucosa. Additionally, hTERT expression correlated with cervical lymph node metastasis. These results suggested that the methylation and expression of hTERT is high in oral carcinogenesis and may play an important role in oral cancer. hTERT expression may also be predictive of cervical lymph node metastasis.

A spatial association between odontomas and the gubernaculum tracts.

OBJECTIVES: The purpose of this study was to identify the spatial relationship and/or association between odontomas and the gubernaculum tract or the dental sac and the characteristic findings for radiographic diagnosis of odontomas. STUDY DESIGN: The visualizations of the odontomas and the gubernaculum tract were retrospectively analyzed using cone beam computed tomography or multidetector computed tomography. RESULTS: Most of odontomas were within the gubernaculum tract or dental sac of unerupted permanent teeth on computed tomography. In some odontomas, the gubernaculum tract existed as a well-defined low density tract extending from the top of odontomas on computed tomography. CONCLUSIONS: A close spatial relationship and/or association between odontomas and the gubernaculum tract or dental sac on computed tomography may be used as one of the criteria for radiographic diagnosis of odontomas. Development of odontomas may be associated with the gubernaculum tract or dental sac of unerupted permanent teeth.

Detection and imaging characteristics of the gubernacular tract in children on cone beam and multidetector computed tomography.

PURPOSE: To elucidate the appearance and imaging characteristics of the gubernacular tract (GT) during the growth stage of children. Furthermore, this study evaluated the significance of the appearance of the GT. STUDY DESIGN: The visualizations of the GT were retrospectively analyzed by using panoramic radiographs and computed tomography (CT) in children. RESULTS: In patients with normal eruption who had unerupted permanent teeth, except maxillary central supernumerary teeth, the GT was clearly visualized as a well-defined low-density tract on CT but not on panoramic radiographs. In patients with obstructive eruption, including impaction, the GT was deformed and not visible on CT. CONCLUSIONS: This paper describes the frequency of detection and appearance of the GT in unerupted teeth. Preliminary data suggest that any alteration to the GT may be used to predict abnormal eruption of permanent teeth.