Expression of pannexin1 in lung cancer brain metastasis and immune microenvironment.

Brain metastases are the most common central nervous system malignancy, and the leading cause of cancer-related deaths. Non-small cell lung carcinomas (NSCLC) comprise the most common cell of origin. Immunotherapy, particularly checkpoint inhibitors, has emerged as the standard of care for many patients with advanced lung cancer. Pannexin1 (PANX1) is a transmembrane glycoprotein that forms large-pore channels and has been reported to promote cancer metastasis. However, the roles of PANX1 in lung cancer brain metastases and tumor immune microenvironment have not been characterized. 42 patient-matched formalin-fixed paraffin-embedded tissue samples from lung carcinomas and the subsequent brain metastases were constructed into three tissue microarrays (TMAs). PANX1 and markers of tumor-infiltrating immune cells (CD3, CD4, CD8, CD68, and TMEM119) were assessed using immunohistochemistry and digital image analysis. The expression of PANX1 was significantly higher in brain metastases than in their paired primary lung carcinoma. The high levels of PANX1 in lung carcinoma cells in the brain inversely correlated with infiltration of peripheral blood-derived macrophages. Our findings highlight the role of PANX1 in the progression of metastatic NSCLC, and the potential therapeutic approach of targeting PANX1 enhances the efficacy of immune checkpoint inhibitors in brain metastasis.

Immunohistochemical Characterization of Gingival Fibromas.

PURPOSE: Gingival fibromas (GFs) are fibrous lesions of the gingiva that are not well defined in the literature. They are histologically similar to peripheral ossifying fibromas (POFs), both being characterized as cellular proliferations of dense fibrous tissue, with POFs differing in that they demonstrate foci of calcification. This study aims to expand upon the immunohistochemical characterization of GFs, and to confirm their osteoblastic phenotype. METHODS: Formalin fixed, paraffin embedded GFs, POFs and fibroepithelial polyps (FEPs) of the gingiva were examined. Immunohistochemical staining was performed for special AT-rich sequence binding protein 2 (SATB2), runt-related transcription factor 2 (RUNX2), osteocalcin and alpha-smooth muscle actin (αSMA). Sections were evaluated by light microscopy and the immunohistochemical staining patterns were assigned immunoreactive scores (IRS) based on percentage of stained cells and intensity of staining. RESULTS: GFs, POFs, and FEPs of the gingiva expressed osteoblastic markers SATB2, RUNX2 and osteocalcin. GFs and POFs expressed αSMA while FEPs of the gingiva did not. GFs and POFs had similar staining patterns of SATB2, RUNX2 and αSMA. DISCUSSION: These findings demonstrate that GFs and POFs exhibit a similar immunohistochemical profile, and supports a theory that GFs are osteoblastic lesions possibly related to POFs.

Investigation of the Molecular Profile of Granular Cell Tumours and Schwannomas of the Oral Cavity.

Granular cell tumours (GCTs) are rare submucosal lesions, thought to develop from Schwann cells, characterised by large polygonal cells with abundant lysosomes. The objectives of this study are to investigate whether GCTs have an antigen-presenting cell (APC) phenotype or a neural crest phenotype using immunohistochemistry and to compare expression profiles with Schwannomas. Immunoreactivity to CD68, HLA-DR, CD163, CD40 and CD11c (APC phenotype) and markers of neural crest cell (NCC) origin S100, SOX10, NSE and GAP43 in 23 cases of GCTs and 10 cases of Schwannomas were evaluated. RT-qPCR was used to identify a possible NCC developmental phenotype in 6 cases of GCTs. GAP43 was identified as a new NCC marker for GCTs, and some evidence was found for an APC phenotype from CD68 and HLA-DR immunoreactivity. RT-qPCR failed to identify an NCC developmental phenotype of GCTs, likely due to technical issues.

Human Tissue Kallikreins in Polymorphous Adenocarcinoma: A Polymerase Chain Reaction and Immunohistochemical Study.

Polymorphous adenocarcinoma (PAC) is the second most common malignant salivary gland tumour of minor salivary glands. Human tissue kallikreins (KLKs) are a family of highly conserved serine proteases expressed by various tissues and organs. The literature demonstrates a link between KLKs and salivary gland neoplasms. The purpose of this study was to determine levels of KLK mRNA in tissue samples of PAC and to determine if KLK expression is limited to tumour cells. Nineteen cases of PAC were reviewed (1987-2013). The diagnosis was confirmed, demographic data was collected, and formalin fixed paraffin-embedded PAC and normal salivary gland tissue samples were obtained. RNA isolation was achieved, followed by conversion to complementary DNA via reverse transcription. Using PCR, the quantitative level of expression of KLKs1-15 was recorded. Samples exhibiting high and low KLK expression were selected for immunohistochemistry staining. Results revealed a statistically significant increase in mean KLK mRNA expression for KLK1, KLK4, KLK10, KLK12 and KLK15 in PAC tissue samples, compared with normal salivary gland tissue (Mann-Whitney U test, p < 0.05). Immunohistochemistry results demonstrated that KLKs were present in tumor cells. Notably, all samples demonstrating relatively higher KLK mRNA expression showed equivalent or increased staining scores relative to the low KLK mRNA expression samples. In conclusion, there appears to be aberrant kallikrein expression in polymorphous adenocarcinoma, suggesting the possibility of a kallikrein cascade influence on tumor development and progression.

Surrogate markers for high-risk human papillomavirus infection in oral epithelial dysplasia: A comparison of p16, Ki-67, and ProExC.

OBJECTIVE: The aim of this study was to determine the utility of surrogate markers of human papillomavirus (HPV) infection in the diagnosis of HPV-associated oral epithelial dysplasia (OED). STUDY DESIGN: Twelve cases of oral dysplasia with histologic features of HPV infection were stained with surrogate markers for HPV (p16, Ki-67, and ProExC) on immunohistochemistry. A second group of 12 cases of oral dysplasia without histologic features of HPV infection was used for comparison. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to confirm the presence of high-risk HPV (HR HPV) in p16-positive cases. RESULTS: All of the surrogate markers showed a statistically significant association with HPV-positive OED (P < .001). The agreement between p16 and HPV positivity was the strongest (κ = 1.00), whereas Ki-67 showed very good association with HPV (κ = 0.83), and ProExC showed good association (κ = 0.75). In each case, the agreement was statistically significant (P < .001). Overall, each of the 3 markers showed good sensitivity; however, ProExC showed the lowest specificity. CONCLUSIONS: The clinicopathologic features of 12 cases of HPV OED are reported. Diffuse p16 positivity is an accurate and reliable method for predicting HR HPV infection in both high and low grade cases of epithelial dysplasia with histopathologic features of HPV OED. The use of Ki-67 and ProExC did not demonstrate any additional diagnostic benefit in the diagnosis HPV OED.

Identifying Candidate Biomarkers for Pleomorphic Adenoma: A Case-Control Study.

Pleomorphic adenoma (PA) is the most common benign salivary gland tumor. Kallikrein-related peptidases have been identified as biomarkers in many human tumors and may influence tumor behavior. We investigated KLK1-15 messenger ribonucleic acid and proteins in PA specimens to determine a KLK expression profile for this tumor. Fresh frozen PA tissue specimens (n = 26) and matched controls were subjected to quantitative real-time reverse transcription polymerase chain reaction to detect KLK1-15 mRNA. Expression of KLK1, KLK12, KLK13, and KLK8 proteins were then evaluated via immunostaining techniques. Statistical analyses were performed with the level of significance set at P < .05. We observed downregulation of KLK1, KLK12, and KLK13 mRNA expression, and immunostaining studies revealed downregulation of the corresponding proteins. Histologic evidence of capsular perforation was associated with increased KLK1 protein expression. Tumor size was not associated with capsular invasion and/or perforation. This study is the first to detail a KLK expression profile for PA at both the transcriptional level and the protein level. Future work is required to develop clinical applications of these findings.

Kallikrein-related peptidase expression in odontogenic cysts and tumors: An immunohistochemical comparative study.

AIM: The aim of the present study was to profile the expression of human kallikrein (KLK)-related peptidases (KLK) in odontogenic lesions. METHODS: Paraffin-embedded, formalin-fixed, non-odontogenic (control) and odontogenic lesions were stained for KLK using a standard immunohistochemical technique. The intensity and proportion of epithelial cells stained was scored. Reverse transcription-polymerase chain reaction was utilized to evaluate KLK 1-15 mRNA expression in ameloblastomas. RESULTS: KLK 3, 4, 9, 11, and 14 were present in all lesions. KLK 3 staining was increased in ameloblastomas and keratocystic odontogenic tumors. KLK 5 was present only in Keratocystic odontogenic tumor. KLK 6 was significantly higher in ameloblastomas than in other lesions. For KLK 7, keratocystic odontogenic tumors and nasopalatine duct cysts were significantly different. KLK 6, 8, 10, 11, and 13 were significantly higher in ameloblastomas than in other lesions. KLK 9 was increased in keratocystic odontogenic tumors and dentigerous cysts. The expression of KLK 1, 4, 7, 8, 10, and 12 mRNA was found in ameloblastomas. CONCLUSION: The results suggested that KLK 6, 8, 10, and 13 could be involved in the progression of ameloblastomas. KLK 10 could have a greater role in odontogenic lesions, rather than non-odontogenic lesions. Future studies aim to define the specific roles of KLK cascades in odontogenic lesions.

Ameloblastoma: a multicentric study.

OBJECTIVE: The objective of this study was to supplement the current ameloblastoma database by reporting the clinicopathologic features of ameloblastoma from Asia and North America. MATERIALS AND METHODS: Biopsy records of the participating institutes were reviewed for lesions diagnosed as ameloblastoma during the years 1993 to 2009. Slides were reclassified according to the World Health Organization Classification of Odontogenic Tumors in 2005. Clinical information and radiographic features were collected and analyzed. RESULTS: The mean age of the patients ± SD was 38.27 ± 17.78 years; 662 patients (51.36%) were men. Mandible (84.26%) outnumbered maxilla and other locations combined in all countries. The number of multilocular radiolucencies (43.40%) was comparable with that of unilocular radiolucencies (42.04%). Follicular pattern was the most common histopathologic pattern (27.70%), followed by plexiform (21.10%) and unicystic pattern (20.71%), respectively. CONCLUSIONS: The clinicopathologic features of ameloblastomas in the present study show some similarities with previous studies; however, minor differences exist.

Markers of potential malignancy in chronic hyperplastic candidiasis.

AIM: To examine the presence of markers associated with malignancy, including p53, p21 cyclin-dependent kinase inhibitor 1A, murine double minutes-2, and others, in chronic hyperplastic candidiasis. METHODS: Immunohistochemical methods were used to examine the expression of p53, murine double minutes-2, p21 cyclin-dependent kinase inhibitor 1A, metallothionein, and proliferating cell nuclear antigen in 42 chronic hyperplastic candidiasis lesions and 11 non-infected control tissues. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was used to examine apoptosis, which was correlated with p53 expression. These markers were measured in lesions of chronic hyperplastic candidiasis that did not show any epithelial dysplasia or histological signs of malignancy. RESULTS: p53 scores were higher in chronic hyperplastic candidiasis than in controls (P = 0.0046). Murine double-minutes 2 levels were not elevated. p21 cyclin-dependent kinase inhibitor 1A was increased in parabasal (P < 0.0001) and basal epithelial cells. Chronic hyperplastic candidiasis lesions showed a similar basal/parabasal metallothionein staining pattern to that seen in normal squamous epithelium. Proliferating cell nuclear antigen was increased (P = 0.0007), as was apoptosis (P = 0.0033). CONCLUSION: Increased p53 in oral chronic hyperplastic candidiasis suggests an increased potential for malignant change in the epithelium, above that of normal tissues. Further functional investigation is required, as well as clinical follow-up studies.

Oral elastofibromatous lesions: a review and case series.

Elastofibromas of the oral cavity are rare, with only 5 cases reported. In this paper, we present a series of five new cases of oral elastofibromatous lesions, occurring in 4 males and 1 female, with ages ranging from 33 to 76 years. The clinical differential diagnosis includes fibroepithelial polyp or fibroma, among other connective tissue tumours. Elastofibromas probably develop as reactive lesions, for which surgical treatment is definitive.

Periostin localizes to cells in normal skin, but is associated with the extracellular matrix during wound repair.

Epidermal tissue repair represents a complex series of temporal and dynamic events resulting in wound closure. Matricellular proteins, not normally expressed in quiescent adult tissues, play a pivotal role in wound repair and associated extracellular matrix remodeling by modulating the adhesion, migration, intracellular signaling, and gene expression of inflammatory cells, pericytes, fibroblasts and keratinocytes. Several matricellular proteins show temporal expression during dermal wound repair, but the expression pattern of the recently identified matricellular protein, periostin, has not yet been characterized. The primary aim of this study was to assess whether periostin protein is present in healthy human skin or in pathological remodeling (Nevus). The second aim was to determine if periostin is expressed during dermal wound repair. Using immunohistochemistry, periostin reactivity was detected in the keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin. In pathological nevus samples, periostin was present in the extracellular matrix. In excisional wounds in mice, periostin protein was first detected in the granulation tissue at day 3, with levels peaking at day 7. Periostin protein co-localized with alpha-smooth muscle actin-positive cells and keratinocytes, but not CD68 positive inflammatory cells. We conclude that periostin is normally expressed at the cellular level in human and murine skin, but additionally becomes extracellular during tissue remodeling. Periostin may represent a new therapeutic target for modulating the wound repair process.

Human kallikrein 8 expression in salivary gland tumors.

The human kallikrein 8 protein (KLK8) is expressed in many normal tissues including esophagus, skin, testis, tonsil, kidney, breast, and salivary gland, and is found in biological fluids including breast milk, amniotic fluid, seminal fluid and serum. It has also been shown to be a biomarker and prognostic factor for breast cancer. The aim of this study was to determine whether KLK8 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas, adenoid cystic carcinomas, polymorphous low grade adenocarcinomas, acinic cell carcinomas, mucoepidermoid carcinomas, and adenocarcinomas NOS of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of KLK8.

The adhesion molecules NCAM, HCAM, PECAM-1 and ICAM-1 in normal salivary gland tissues and salivary gland malignancies.

BACKGROUND: Some malignant salivary gland tumors are known for their propensity to exhibit perineural invasion and vascular metastases. It was hypothesized that alterations in the expression of cell adhesion molecules are involved in these processes. METHODS: The expression and distribution of neural cell adhesion molecule (NCAM), HCAM (CD44), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and intercellular cell adhesion molecule-1 (ICAM-1) in normal salivary gland tissues and selected salivary gland malignancies, especially adenoid cystic carcinoma (AdCyCa) and polymorphous low-grade adenocarcinoma (PMLG), were determined immunohistochemically, and their influence on histologically demonstrated perineural invasion, vascular invasion, and tumor recurrence/patient death were investigated. RESULTS: NCAM, HCAM, and ICAM-1 were often found to be expressed by neoplastic cells, but no correlation to perineural invasion, tumor behavior, or patient prognosis was found. PECAM-1 was rarely and only focally expressed in three tumors, all of which were related to tumor metastases and patient death. CONCLUSIONS: Immunohistochemical demonstration of NCAM, HCAM, and ICAM-1 is not related to perineural invasion or tumor behavior. PECAM-1 expression was related to vascular invasion and poor patient prognosis in three cases.

Immunohistochemical localization of growth factors fibroblast growth factor-1 and fibroblast growth factor-2 and receptors fibroblast growth factor receptor-2 and fibroblast growth factor receptor-3 in normal oral epithelium, epithelial dysplasias, and squamous cell carcinoma.

OBJECTIVES: Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been identified in a variety of carcinomas, but there are few studies concerning their presence in oral cancers. The objective of this study was to determine whether FGF-1, FGF-2, and high affinity receptors FGFR2 and FGFR3 are present in the pathogenesis of oral epithelial dysplasias and oral squamous cell carcinoma. STUDY DESIGN: Sections from formalin-fixed, paraffin-embedded samples of oral normal mucosa (n = 14), epithelial dysplasia (n = 20), carcinoma in situ (n = 10), and squamous cell carcinoma (n = 12) were tested for cytoplasmic staining by standard in situ immunohistochemistry with antibodies for FGF-1, FGF-2, FGFR2, and FGFR3. RESULTS: Staining for FGF-1 is decreased or lost in the development of epithelial dysplasia and carcinoma. Staining for FGF-2 showed increased intensity (although not statistically significant) in oral epithelial dysplasias and squamous cell carcinomas and showed a significant increased expression in the upper layers of dysplasias and stratum spinosum-like cells in squamous cell carcinomas. Staining for FGFR2 showed a statistically significant increase in intensity in all layers of epithelial dysplasias and squamous cell carcinomas. Staining for FGFR3 was found in the upper stratum spinosum cells of normal and dysplastic epithelium and well-differentiated squamous cells in squamous cell carcinomas, with a statistically significant increase in staining intensity in dysplastic and carcinomatous tissues. CONCLUSIONS: The loss of FGF-1 is consistent with loss of differentiation in dysplasias and some squamous cell carcinomas. Changes in the localization of FGF-2 and FGFR2 into upper epithelial layers with increasing dysplasia suggest increased mitotic potential of high level cells. The co-localization of FGF-2 and its high affinity receptors in neoplastic tissues suggests an autocrine mechanism of influence on carcinogenesis.

Cochlear delivery of fibroblast growth factor 1 and its effects on apoptosis and cell cycling in noise-exposed guinea pig ears.

OBJECTIVES: Acidic fibroblast growth factor 1 (FGF-1) is a mitogen and antiapoptotic factor synthesized by cochlear neurons and transported to the organ of Corti. The objectives of this investigation were threefold: (1) to develop an animal model to study the cochlear effects of intratympanic delivery of FGF-1; (2) to determine the distribution, in the mature mammalian cochlea, of FGF-1 and the receptor, FGFR3, to which it binds with high affinity; and (3) to examine the effect of exogenous FGF-1 on cochlear apoptotic and cell-cycling markers in noise and non-noise-exposed guinea pigs ears. METHODS: Fifteen adult Hartley guinea pigs were divided into three groups. Group 1 animals (n = 5) underwent direct placement of FGF-1 in phosphate buffered saline (PBS) (20 pg/mL) soaked Gelfoam pledgets to the right round window membrane. Phosphate buffered saline-soaked Gelfoam pledgets were placed on the left round window membrane as a control. In group 2 animals (n = 5), surgical placement of either FGF-1 or PBS was followed by exposure to 120 dB of white noise for 2 hours. Group 3 animals (n = 5) were subjected to identical noise conditions prior to undergoing round window application of either FGF-1 or PBS. All groups were allowed to recover in a noise-controlled environment for 12 hours following surgery. Anti-FGF-1-stained Western blots and optical densitometry analyses were used to quantitate passage of FGF-1 into cochlear perilymph. Standard in situ immunohistochemical techniques were used to stain each cochlea for FGF-1 and FGFR3, apoptotic markers p53 and p21, Bcl-2, and the cell-cycling marker proliferating cell nuclear antigen (PCNA). Tissue sections were subjected to the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling technique (TUNEL) for apoptosis. RESULTS: Western blot and optical densitometry analyses of cochlear perilymph showed increased concentrations of FGF-1 in 10 of 14 experimental cochleas. Cochlear perilymph FGF-1 was consistently bound to heparan sulphate proteoglycan (HSPG). Immunoreactivity of both FGF-1 and FGFR3 was observed in spiral ganglion neurons, inner and outer hair cells, pillar cells, and Dieter and Hensen's cells. Specific FGF-1 immunostaining to the distal portion of the pars pectinata of the basilar membrane was noted in noise-exposed animals only. Bcl-2 and PCNA immunostaining was not detected in any group. There was no significant nuclear immunoreactivity to proapoptotic markers, p53 and p21, in any group. Semiquantitative analysis of TUNEL staining in block sections of all cochleas demonstrated a 340% increase in nuclear immunoreactivity of noise-exposed outer hair cells and organ of Corti cells. There was no difference between FGF-1 treated and control ears subjected to TUNEL staining. CONCLUSIONS: Exogenous FGF-1 crosses the round window membrane and is bound to HSPG in cochlear perilymph. The specific immunoreactivity of the pars pectinata to FGF-1 may represent a unique reservoir for cochlear FGF-1 in noise-exposed ears of the guinea pig. Noise induces apoptosis of organ of Corti cells as demonstrated with the TUNEL technique. PCNA, Bcl-2, p53, and p21 in noise-exposed and non-noise-exposed guinea pig cochleas are not affected by exogenous FGF-1. Noise-induced hair cell apoptosis appears to be independent of the p53 pathway. Lack of immunoreactivity to Bcl-2 supports the concept that the apoptotic mechanism is likely to involve C-Jun-N-terminal kinase- or caspase-dependent pathways. Exogenous FGF-1 does not alter apoptosis or cell cycling in the mature guinea pig cochlea within 12 hours of acute acoustic trauma.