Normal Mucosa Samples Research Articles

Aldehyde Dehydrogenase: An Off-Label Marker of Endothelial Activation in Oral Squamous Cell Carcinoma.

The vascular endothelial (VE) expression of aldehyde dehydrogenase (ALDH) 1/2 family in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cases has not been studied so far. The aim of this study was to illustrate the "off-label" endothelial expression of cancer stem cell (CSC) biomarker, ALDH1/2, adjacent to oral potentially malignant and malignant lesions in order to shed some light on the mechanisms leading to oral carcinogenesis. Materials and methods: The expression of CSC protein-biomarker ALDH1/2 was detected through immunohistochemistry (IHC) in 30 paraffin-embedded samples of OL and 21 samples of OSCC compared to five samples of normal oral mucosa. Statistical analysis was done using SPSS, Pearson Chi-square, and Fischer's exact test. The significance level was set at 0.05 (p≤ 0.05). Results: In oral mucosal vessels, ALDH1/2 was not expressed. It was expressed significantly more in the vessels of OSCCs compared to the OLs (Fisher's exact test, p-value= 0,001). However, when endothelial expression of ALDH1/2 in the vasculature of OLs was compared with that of the normal oral mucosa, no significant change was noticed (Fisher's exact test, p-value=1.000). Discussion: The IHC VE expression of ALDH1/2 in OSCCvasculature but not in OL indicates a possible significantly stronger activation of endothelial cells during carcinogenesis, which could be an indicator of the role of inflammation in the development of field cancerization and of prognostic value for (vascular/lymphatic) metastasis.

Antimicrobial and Defense Proteins in Chronic Rhinosinusitis with Nasal Polyps.

Background and Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) presently remains a difficult disease to manage. Antimicrobial and defense proteins are important factors that could help characterize the role of microorganisms in CRSwNP pathogenesis, as the concept of microbial dysbiosis in CRS is still being considered. Our aim is to investigate the complex appearance, relative distribution and interlinks of human β defensin 2 (HBD-2), human β defensin 3 (HBD-3), human β defensin 4 (HBD-4), and cathelicidin LL 37 (LL 37) in chronic rhinosinusitis with nasal polyps (CRSwNP)-affected human nasal mucosa. Materials and Methods: The study group consisted of 48 samples from patients with CRSwNP. Samples were collected during functional endoscopic sinus surgery. The control group consisted of 17 normal healthy nasal mucosa samples gathered during routine septoplasty. β-defensin-2, β-defensin-3, β-defensin-4 and cathelicidin LL 37 in tissue were detected via immunohistochemical analysis. Results: HBD-2, HBD-3 and LL 37 were significantly decreased in epithelial cells in both primary and recurrent nasal polyp samples (p < 0.001) in comparison to control samples. HBD-2 was decreased in the subepithelial connective tissue of primary nasal polyp samples when compared to both recurrent polyp (p = 0.050) and control (p = 0.033) samples. In subepithelial connective tissue, significantly more HBD-3-positive structures were observed in primary nasal polyp samples (p = 0.049) than in control samples. In primary polyp samples, moderate correlations between connective tissue HBD-3 and connective (R = 0.584, p = 0.001) and epithelial tissue LL 37 (R = 0.556, p = 0.002) were observed. Conclusions: Decreased HBD-2, HBD-3 and LL 37 concentrations in the epithelium suggest a dysfunction of the epithelial barrier in patients with nasal polyps. Decreased subepithelial connective tissue HBD-2 suggests different responses to nasal microbiota in patients with primary nasal polyps compared to recurrent nasal polyps. Increased HBD-3 in subepithelial connective tissue suggests a possible role of this antimicrobial peptide in the pathogenesis of primary nasal polyps.

Sampling Methods and their Prospects in Liquid-Based Cytology of Oral Mucosa: A Comparative Study.

Examining epithelial cells in the mouth cavity is simple and non-invasive with exfoliative cytology. Liquid-based cytology is an additional diagnostic technique that can improve the specificity and sensitivity of conventional cytology. The purpose of our study was to describe the consistency of normal oral mucosa samples obtained with three different tools: the Cytobrush®, dermatological curette, and OralCDx® for liquid-based cytology. Literature review was done, and reporting of the improvements in the field of investigation and diagnosis has been reported. The present communication aims toward comparing three different sampling instruments such as Cytobrush, curette, and OralCDx for liquid-based cytology of the oral mucosa.

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

Expression of heat shock protein 70 in oral epithelial dysplasia and squamous cell carcinoma.

Heat shock proteins (HSPs) are overexpressed in a variety of human malignancies. They are involved in tumor cell proliferation, differentiation, invasion, metastasis, death, and immune system detection. HSP 70 has been shown to resist cytotoxicity in cancer cells and even enhance tumor development through an immune escape mechanism, suggesting that HSP70 may play a role in carcinogenesis. The aim of our study was to evaluate the role of HSP70 as a predictive marker for malignant transformation in oral epithelial dysplasia. Thirty samples of epithelial dysplasia (10 mild dysplasia, 10 moderate dysplasia, and 10 severe dysplasia/carcinoma-in-situ cases), 10 samples of well-differentiated oral squamous cell carcinoma (OSCC), and 10 samples of normal oral mucosa were routinely processed, formalin-fixed, paraffin-embedded, and immunohistochemically examined for HSP70 expressions. To determine the statistical difference between two groups, a one-way analysis of variance (ANOVA) and the Mann-Whitney test were used. HSP70 expression was high but not homogenous in normal mucosa. Dysplasia showed an initial drop, and the expression increased with increasing degrees of dysplasia. There was no statistically significant difference across various types of epithelial dysplasia. From dysplasias to well-differentiated carcinoma, HSP70 exhibited a considerable rise. Overexpression of HSP70 in clinically suspicious and histologically established epithelial dysplasia may suggest a likelihood of transformation to well-differentiated OSCC and may have a prognostic value. However, more studies with a bigger sample size are needed to prove HSP70's role as a predictor.

Abstract 5230: Comprehensive genetic analysis using clinical specimens of gastric adenocarcinoma and proximal polyposis of stomach

Abstract Background and Purpose: Gastric adenocarcinoma and proximal polyposis (GAPPS) is an autosomal dominant genetic disorder that causes gastric carcinoma from fundus gland polyposis. Although germline mutation of APC promoter 1B was reported as a pathogenic variant in 2016, there are still many unknowns about the carcinogenesis. In this study, we performed a comprehensive Next generation sequencing (NGS) analysis of GAPPS clinical specimens to investigate the molecular profile characteristic of carcinogenesis. Patients and Methods: Seven cases from three GAPPS families experienced in our department were included in this study. In each case, tissues were collected from normal mucosa, polyps, and carcinoma, and FFPE and fresh-frozen samples were prepared. After pathological diagnosis was performed on each sample, RNA and DNA were extracted and analyzed based on data obtained by next generation sequence (NGS). Results: In the RNA sequence, gene expression analysis was performed on 45 pathologically diagnosed samples, which were classified into four clusters by K-means clustering. Gene Set Enrichment Analysis (GSEA) of the four clusters showed that cancer-related genes such as p53 and KRAS were significantly enriched in the cluster of cancer, and genes related to immune response were significantly enriched in the cluster of cancer compared to the and clusters of polyp and normal. Gene mutation analysis using whole-exome sequence (read depth/coverage&amp;gt;20) confirmed mutation of APC promoter 1B in all samples. On the other hand, in tumor samples (N=10), the percentage of mutation of genes previously suggested to be associated with GAPPS (TP53, FBXW7, GNAS, and KRAS) were 50%, 0%, 30%, and 60%, respectively. Conclusion: Comprehensive gene expression analysis by RNA sequence and whole-exome sequence was performed using clinical samples of GAPPS. In the future, we plan to identify specific gene-pathways involved in carcinogenesis. (2460/2600 characters) Citation Format: Chihiro Matsumoto, Masaaki Iwatsuki, Koshi Mimori, Hideo Baba. Comprehensive genetic analysis using clinical specimens of gastric adenocarcinoma and proximal polyposis of stomach. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5230.

Expression of Connexins 37, 40 and 45, Pannexin 1 and Vimentin in Laryngeal Squamous Cell Carcinomas.

Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC.

Sprouty 4 expression in human oral squamous cell carcinogenesis

Background/purposeReviewing literature, sprouty 4 (SPRY4) has not been studied in human oral squamous cell carcinomas (OSCCs). The study aimed to examine SPRY4 expression in human oral squamous cell carcinogenesis. Materials and methodsA total of 95 OSCCs, 10 OPMDs with malignant transformation (MT), 17 OPMDs without MT, and six normal oral mucosa (NOM) samples were recruited for immunohistochemical staining; three OSCC tissues with normal tissue counterpart NOM were employed for Western blotting. Three human oral cancer cell lines (OCCLs), an oral precancer cell line (dysplastic oral keratinocyte, DOK), and a primary culture of normal oral keratinocytes (HOK) were used for Western blotting; OCCLs and HOK were employed for real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated in terms of proliferation, migration, and invasion assays. ResultsSPRY4 protein expression was significantly increased in OSCCs compared with NOM. Protein and mRNA SPRY4 expression in OCCLs were significantly elevated compared with HOK. Significant increases in the degrees of proliferation, migration, and invasion were noted in OCCLs with SPRY4 siRNA transfection compared with those without transfection. SPRY4 protein level was increased in OPMD with MT compared to OPMD without MT. SPRY4 protein was significant increase in DOK in comparison with HOK. SPRY4 protein expression was significantly increased from NOM and OPMD without MT to OSCC. SPRY4 protein expression in OCCLs was significantly enhanced compared with DOK and HOK respectively. ConclusionOur results indicate that SPRY4 expression is possibly involved in human oral squamous cell carcinogenesis.

Clinical significance of galectin-3 expression in urinary bladder carcinoma

To uncover the clinical significance of galectin-3 in the evolution of urinary bladder cancer by defining galectin-3 expression and examining the relationship between its expression in a group of urothelial carcinomas versus normal tissues along with clinicopathological factors. This retrospective study included histopathological reports and archival blocks and slides of all patients with urinary bladder cancer treated at King Abdulaziz University Hospital (Jeddah, Saudi Arabia). An anti-galectin-3 monoclonal antibody was used for immunohistochemical staining of tissue microarray slides comprising 128 cases of urothelium carcinoma and 24 specimens of normal bladder mucosa. Galectin-3 was downregulated during transformation, with positive expression found in 50 (39%) urinary bladder neoplasms, of which 33 (66%) showed weak immunostaining. All positively-stained malignant tumor and normal bladder mucosa samples showed cytoplasmic staining; a few samples also showed nuclear staining. No correlation was noted between galectin-3 and histotype, grade, stage, muscularis propria invasion, lymph node invasion, vascular invasion, or metastasis. A Cox proportional hazards model and Kaplan-Meier survival curves did not show differences in survival on the basis of galectin-3 expression. Galectin-3 is down-regulated in bladder cancer but is not a helpful marker for the diagnosis or prognosis of urinary bladder cancer.

Recurrence risk assessment for stage III colorectal cancer based on five methylation biomarkers in plasma cell-free DNA.

For stage III colorectal cancer (CRC) patients with a high risk of recurrence, intensified adjuvant chemotherapy can improve overall survival. We aimed to develop a circulating tumor DNA (ctDNA) methylation marker model for predicting the relapse risk of stage III CRC patients. Differentially methylated markers identified between 53 normal mucosa samples and 165 CRC tissue samples, as well as between plasma samples from 75 stage I/II (early-stage) CRC patients and 55 stage IV (late-stage) CRC patients, were analyzed using Student's t-tests. The overlapping methylation markers shared by plasma and tissue samples were used to establish a methylation marker model to evaluate the tumor burden in the peripheral blood of CRC patients using the random forest method. This model was verified in the validation cohort (n = 44) and then applied to predict recurrence risk in 50 stage III CRC patients and monitor the clinical disease course in serial samples from four CRC patients. We built a five-marker-based ctDNA methylation model that had high sensitivity (84.21%) and specificity (84%) in identifying late-stage CRC in a validation cohort containing 24 stage I/II CRC patients and 20 stage IV CRC patients. The model achieved high sensitivity (87.5%) and specificity (94.12%) in predicting tumor relapse in an independent cohort of 50 stage III CRC patients and could be an independent recurrence risk factor for stage III patients [Hazard ratio (HR), 60.4; 95% confidence interval (CI): 7.68-397; p = 9.73e-5]. Analysis of serial blood samples of CRC showed that the model could monitor disease relapse earlier than imaging examination and serum carcinoembryonic antigen (CEA) and so may provide an opportunity for the early adjustment of therapeutic strategies. Moreover, the model could potentially monitor the clinical course and treatment response dynamically. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Panomics reveals patient individuality as the major driver of colorectal cancer progression

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity.MethodsWe analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results.ResultsAlthough panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes.ConclusionOur research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.

Serotonin and Melatonin in Human Lower Gastrointestinal Tract.

Melatonin is a ubiquitous hormone produced not only by the pineal gland but also by other organs and tissues. It is involved in the regulation of several gastrointestinal functions. The main cells responsible for the production and release of extrapineal melatonin are the enterochromaffin (EC) cells that produce serotonin. They are involved in the pathogenesis of neuromotor disorders that characterize functional gastrointestinal disorders and in the pathophysiology of inflammatory intestinal diseases. Our aim was the immunohistochemical highlighting on biopsy samples of normal gastrointestinal mucosa and in ulcerative colitis (UC) of immunoreactive cells for melatonin and serotonin in order to identify any differences in their distribution. Our prospective case-control study involves the highlighting on human mucosal biopsies of immunoreactive cells for melatonin and serotonin. All patients undergoing colonoscopy + ileoscopy were considered eligible for the study, divided into two groups: 1. patients with active ulcerative colitis (UC); 2. control group consisting of patients undergoing endoscopic examination for colorectal cancer screening. Twenty-one patients were enrolled. The controls had a higher concentration of EC cells containing 5HT particularly in the rectum (p value ≤ 0.05). In patients with active colitis the expression of 5-HT-iR was greater in all tracts of the colon. The correlation analysis in UC patients shows that a higher expression of 5-HT-iR+ cells corresponds to a lower extension of the disease and a greater severity of the same. 5HT+ cells decreased in the case of UC compared to healthy controls. In the severe disease, there was an increase in the expression of melatonin-secreting cells, probably as a compensatory response to the inflammation and oxidative stress. This increase is negatively correlated with the extent of the disease and positively with the severity of the same.

Genomically Stable Gastric Cancer Characterized by Hypomethylation in Wnt Signal Cascade

Introduction: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. Methods: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. Results: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3–2.9]; EBV: 2.1 [1.2–3.1]; CIN: 2.4 [2.2–2.7]; GS: 1.3 [0.8–0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3–12.4]; EBV: 5.7 [1.7–9.7]; CIN: 4.4 [3.6–5.1]; GS: 3.4 [2.2–4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. Conclusions: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Изменение экспрессии мРНК панели генов в слизистой оболочке толстой кишки в процессе естественного старения

Background: during natural aging, cellular and molecular changes in the colonic mucosa contribute to chronic damage and the development of various neoplasia, including colorectal cancer (CRC). Aim: to assess the age peculiarities of mRNA expression in samples of normal mucosa from various parts of the colon. Patients and Methods: the study included 95 patients. Normal colonic mucosa biopsies were derived during videocolonoscopy from different parts of the colon, i.e., the right colon (n=91), left colon (n=95), and rectum (n=87). In total, 273 samples were examined. The relative level of mRNA expression of 63 genes potentially involved in various pathways of CRC carcino-genesis was evaluated by reverse transcription polymerase chain reaction. Results: a linear regression model for predicting passport age, including data on gene ex-pression, was characterized by moderate reliability (R2 0.45, DW test 1.54). Age estimation er-rors in the model arise proportionally to its increase, which is associated with the accumulation of epigenetic changes. Maximum accuracy in age prediction is typical for rectal mucosa samples. The first statistically significant division (primary classification node) of patients by age in indi-viduals over or under 60 years provides the level of TERT gene expression. Telomerase activity in association with HLA-G5 gene overexpression is typical in older patients, whereas HLA-G5 gene hypoexpression and simultaneous granulysin (GNLY) overexpression are typical in patients younger than 60 years. A molecular cascade involving a decrease in telomerase activity due to TERT hypoexpression with simultaneous overexpression of the cell cycle regulator and tumor suppressor P14ARF is also typical for normal colonic mucosa in patients aged 60 years and over. Conclusion: our findings support molecular genetic changes in the colonic mucosa that occur with age. The specifics of gene expression involved in CRC carcinogenesis are the basis for assessing CRC risk. KEYWORDS: gene expression, mRNA, RT-PCR, normal colon mucosa, age, epigenetic changes, colorectal cancer. FOR CITATION: Goncharov S.V., Bozhenko V.K., Zakharenko M.V., Chaptykov A.A., Kulinich T.M., Solodkiy V.A. Changes in mRNA expression of colon mucosa genes during natural aging. Russian Medical Inquiry. 2023;7(6):347–352 (in Russ.). DOI: 10.32364/2587-6821-2023-7-6-2.

Expression of p53 as Potential Biomarker in Oral Submucous Fibrosis: An Immunohistochemical Study.

Oral submucous fibrosis (OSMF) is potentially malignant disorder known to transform into oral cancer. The malignant transformation is often associated with changes at the genetic level that in turn is reflected by the altered expression of proteins related to cell cycle, proliferation, and apoptosis. Expression of p53 tumor suppressor gene is one of the common findings in human cancers including the oral cancer. Therefore, the early detection of potentially malignant OSMF has been crucial in the inhibition of oral cancer. To determine the main pathological logical factors and expression of aberrant p53 in OSMF, oral squamous cell carcinomas (OSCC) and in normal patients, to study correlation between p53 expression with clinical staging and histological grading of OSMF. An immunohistochemical (IHC) study was performed for p53 expression on 35 cases of OSMF, 10 cases of OSCC with history of habits and 10 normal patients without any habits. The expression of p53 showed a significant difference between normal oral mucosa, OSMF and OSCC samples. The study demonstrated a high incidence of p53 over expression in OSMF and OSCC. The results indicate that p53 over expression may play a role in pathogenesis of OSMF and in the development of Oral squamous cell carcinoma. With early detection of the high-risk patients with OSMF, we can expect to develop more intensive treatment modalities, leading to the reduction in cancer transformation rate from OSMF.

Evaluation of Her-2 protein in histologically tumor-free margins of oral squamous cell carcinoma: An immunohistochemical study.

The status of the 5-year survival rate and loco-regional recurrence in oral squamous cell carcinoma (OSCC) has remained unchanged over the decades. Recent advances in oral cancer research have revealed that the presence of molecular alterations in histologically tumor-free margins of OSCC has a prognostic significance and can aid in designing therapeutic strategies. However, the literature on molecular studies on histologically tumor-free margins is scant, especially in the Indian population. Considering the prognostic implications of Her-2 in malignancies of the breast, ovary, and OSCC, we aimed to assess the expression of Her-2 protein in histologically tumor-free margins of OSCC and to establish correlation with clinico-pathological parameters. 4 μ m thick sections from formalin-fixed paraffin-embedded tissue blocks of 40 histologically tumor-free margins of OSCC affecting the buccal mucosa and/or lower gingiva-buccal sulcus and 40 normal oral mucosa samples were subjected to immunohistochemical analysis for the Her-2 antibody. The obtained data were analyzed statistically. The mean ages in study and control groups were 49.83 years (SD ± 10.43) and 37.28 years (SD ± 8.61), respectively, with male predominance. Local recurrence was seen in 52.5% of patients. Follow-up data revealed that a total of 71.4% of patients succumbed to mortality and all of them had reported local recurrence. Overall, the presence of local recurrence and the status of survival showed a statistically significant association (p = 0.0001). All the samples in the study and control groups were negative for Her-2 immuno-expression. The study indicated the lack of Her-2 immuno-expression in histologically tumor-free margins of OSCC with several speculated explanations. As it is a preliminary study, further studies employing both immunohistochemistry (IHC) and gene amplification in histologically tumor-free margins of OSCC affecting different anatomical sites are warranted. This will aid in identifying the subset of patients that may benefit from targeted therapy.

Overexpression of transient receptor potential melastatin 6 during human oral squamous cell carcinogenesis

Background/purposeTransient receptor potential melastatin (TRPM) channel is involved in cell proliferation and cell survival. Eight members (TRPM1–8) are within the TRPM subfamily. The current study is aimed to investigate TRPM6 expression in human oral carcinogenesis. Materials and methodsSixty-six oral squamous cell carcinomas (OSCCs), 47 oral potentially malignant disorders (OPMD) with moderate-severe epithelial dysplasia (ED), 28 OPMD with mild ED, and 33 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot analysis. OCCLs were evaluated for proliferation, migration, invasion assays, and intracellular calcium concentration. ResultsTRPM6 protein expression in OSCC was significantly increased as compared with normal samples. Protein expression of TRPM6 in OCCLs was significantly higher as compared with HOK. Significant decreases in degrees of proliferation, migration, invasion, and intracellular calcium concentration were noted in OCCLs with TRPM6 siRNA transfection as compared with those without transfection. Significantly increased TRPM6 protein level was noted in OPMD with moderate-severe ED as compared with those with mild ED. ConclusionOur results implicate that TRPM6 overexpression is potentially related to human oral carcinogenesis.

The prognostic role of p53 and its correlation with CDK9 in urothelial carcinoma

PurposeThe mutation of p53 is considered a pivotal step in bladder cancer pathogenesis. Recently, distinct interactions between p53 and CDK9, a transcription regulator, have been described. In this work, we explored the prognostic role of p53 expression and evaluated its associations with CDK9 in urothelial carcinoma.Materials and methodsThe research group consisted of 67 bladder cancer samples and 32 normal urothelial mucosa samples. All specimens were analyzed using ImageJ and the IHC profiler plugin. To validate the results, 406 cases from The Cancer Genome Atlas database were analyzed.ResultsP53 and CDK9 are overexpressed in urothelial cancer tissues when compared to normal urothelial tissues (p < 0.05). High p53 expression was observed in metastatic tumors and tumors with high CDK9 expression (p < 0,05). High p53 expression was predictive for shorter survival in patients with non-muscle-invasive bladder cancer (HR = 0.107 [0.012–0.96]; p = 0.046) but did not correlate with prognosis in the muscle-invasive group. In high CDK9 cancers, high p53 expression correlated with the occurrence of high-grade and muscle-invasive tumors (p < 0.05).ConclusionHigh expression of p53 correlates with unfavorable clinical features of bladder cancer. CDK9 is associated with the expression of p53, possibly through interactions with p53 inhibitors. Since the blockade of CDK9 in other malignancies reactivates wild-p53 activity, confirming the crosstalk between p53 and CDK9 in bladder cancer may be another step to explain the mechanism of tumor progression in its early stages.

The Effect of Bevacizumab and Propranolol on Nasal Polyposis.

Objective This study aims to evaluate the effects of bevacizumab and propranolol from the point of view of a possible antiangiogenic effect in a model of primary nasal polyp (NP) tissue culture. Methods NP samples of 21 patients and normal healthy nasal mucosa samples of 7 patients were cultured. Samples were divided into four groups as follows (healthy nasal mucosa, NP without any treatment, NP treated with propranolol, NP treated with bevacizumab). Cultured tissues were formalin fixed and paraffin embedded. Tissue sections and immunohistochemical VEGF-A, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) expressions were evaluated. ELISA was also performed for each one of them. Results Both propranolol and bevacizumab significantly decreased the expressions of VEGF-A and Ang-1, and they significantly increased the expression of Ang-2 in comparison to the control NP group. In the healthy nasal mucosa group, no significant expression of VEGF-A was seen, a slight (+) Ang-1 expression, and a high (+++) Ang-2 expression were observed. Conclusion Bevacizumab and propranolol exert an antiangiogenic effect on NP tissues, mainly by decreasing VEGF-A and Ang-1 expression, increasing Ang-2 expression.

Immunohistochemical expression of putative cancer stem cell markers aldehyde dehydrogenase 1, SOX2, CD44 and OCT4 in different grades of oral epithelial dysplasia.

The hypothesized existence of cancer stem cells (CSC) and its markers aldehyde dehydrogenase 1 (ALDH1), CD44, SOX2 and OCT4 in oral dysplastic tissues provides the potential for a more reliable assessment of malignant transformation of oral epithelial dysplasia (OED). Thus, the present study is intended to evaluate the immunohistochemical expression of four different CSC markers ALDH1, CD44, SOX2 and OCT4 in different grades of OED and to investigate the co-expression of these putative stem cell markers in OED. A total of 35 samples of varying grades of OED which included 7 mild, 11 moderate and 17 severe dysplasia samples and 10 samples of normal oral mucosa without dysplasia were used. Four sections each from all 45 samples were stained with ALDH1, CD44, SOX2 and OCT4, respectively, by immunohistochemistry. The acquired data were analyzed and evaluated using the Chi-square test and unpaired t-test and the P < 0.05 was taken significant. ALDH1 and SOX2 expression percentages showed statistically significant differences among study groups (P < 0.05). Statistical comparison of percentage expression of CD44 and OCT4 between OED and normal was nonsignificant (P > 0.05). Co-expression of all four markers was found in 15 cases of OED with none of the normal cases showing co-expression. The expression of CSC markers in OED and normal mucosa differs significantly with co-expression of all four markers located only in dysplastic tissues. Until now, no single protein marker has been able to unequivocally identify the CSCs. Thus, a panel of putative CSC markers will help in identifying the patients with high risk for malignant transformation in OED.