Immunohistochemical Expression Research Articles

Evaluation of the diagnostic utility of MCAM-1 (CD146) in a group of common gynecological cancers: A case-control study.

MCAM-1 (CD146) is an endothelial cell adhesion molecule belonging to the immunoglobulin superfamily. Recent studies have identified CD146 expression as a critical marker for tumor progression, migration, and metastasis in various malignancies. This study aimed to evaluate CD146 immunohistochemical expression in various gynecological cancers. This study was conducted in a tertiary medical center in central India. A total of 49 gynecological cancer cases and 16 site-matched controls were included. The cases comprised 27 cervical, 10 endometrial, 10 ovarian, and two miscellaneous cancers. CD146 immunohistochemistry was performed and assessed for immunoreactivity score (IRS), microvascular density (MVD), and microvascular caliber (MVC). An IRS of 5 or more was considered CD146 positive. The p-values for CD146 positivity for cases vs. control were 0.0531, 0.0580, and 0.007 for cervical, endometrial, and ovarian sites, respectively. The mean MVD was found to be significantly higher in cases compared with benign tissues (p-value <0.00001), and the mean MVC of cases was found to be smaller when compared with the controls (p-value <0.0001). MVD by CD146 was found to be higher in gynecological malignancies, highlighting its role in cancer neo-angiogenesis and its potential therapeutic role. CD146 epithelial expression was also significantly higher in ovarian cancers. Further studies with a larger sample size are required to confirm that this protein may be a potential theognostic target in gynecological cancers.

Neutrophils and NADPH Oxidases Are Major Contributors to Mild but Not Severe Ischemic Acute Kidney Injury in Mice.

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1β and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1β and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

278 (PB-094) Poster - A comparison between immunohistochemistry and mRNA expression to detect HER2-low breast cancer

278 (PB-094) Poster - A comparison between immunohistochemistry and mRNA expression to detect HER2-low breast cancer

In Vivo Assessment of Hepatic and Kidney Toxicity Induced by Silicon Quantum Dots in Mice.

In the last decade, silicon-based quantum dots (SiQDs) have attracted the attention of researchers due to their unique properties for which they are used in medical applications and in vivo imaging. Detection of cytotoxic effects in vivo is essential for understanding the mechanisms of toxicity, a mandatory step before their administration to human subjects. In this context, we aimed to evaluate the in vivo hepatic and renal acute toxicity of SiQDs obtained by laser ablation. The nanoparticles were administrated at different doses (0, 1, 10, and 100 mg of QDs/kg of body weight) by intravenous injection into the caudal vein of Swiss mice. After 1, 6, 24, and 72 h, the animals were euthanatized, and liver and kidney tissues were used in further toxicity tests. The time- and dose-dependent effects of SiQDs on the antioxidant defense system of mice liver and kidney were investigated by quantifying the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase) in correlation with the morphological changes and inflammatory status in the liver and kidneys. The results showed a decrease in the activities of antioxidant enzymes and histopathological changes, except for superoxide dismutase, in which no significant changes were registered compared with the control. Furthermore, the immunohistochemical expression of TNF-α was significant at doses over 10 mg of QDs/kg of body weight and were still evident at 72 h after administration. Our results showed that doses under 10 mg of SiQDs/kg of b.w. did not induce hepatic and renal toxicity, providing useful information for further clinical trials.

A deep-learning workflow to predict upper tract urothelial carcinoma protein-based subtypes from H&E slides supporting the prioritization of patients for molecular testing.

Upper tract urothelial carcinoma (UTUC) is a rare and aggressive, yet understudied, urothelial carcinoma (UC). The more frequent UC of the bladder comprises several molecular subtypes, associated with different targeted therapies and overlapping with protein-based subtypes. However, if and how these findings extend to UTUC remains unclear. Artificial intelligence-based approaches could help elucidate UTUC's biology and extend access to targeted treatments to a wider patient audience. Here, UTUC protein-based subtypes were identified, and a deep-learning (DL) workflow was developed to predict them directly from routine histopathological H&E slides. Protein-based subtypes in a retrospective cohort of 163 invasive tumors were assigned by hierarchical clustering of the immunohistochemical expression of three luminal (FOXA1, GATA3, and CK20) and three basal (CD44, CK5, and CK14) markers. Cluster analysis identified distinctive luminal (N = 80) and basal (N = 42) subtypes. The luminal subtype mostly included pushing, papillary tumors, whereas the basal subtype diffusely infiltrating, non-papillary tumors. DL model building relied on a transfer-learning approach by fine-tuning a pre-trained ResNet50. Classification performance was measured via three-fold repeated cross-validation. A mean area under the receiver operating characteristic curve of 0.83 (95% CI: 0.67-0.99), 0.8 (95% CI: 0.62-0.99), and 0.81 (95% CI: 0.65-0.96) was reached in the three repetitions. High-confidence DL-based predicted subtypes showed significant associations (p < 0.001) with morphological features, i.e. tumor type, histological subtypes, and infiltration type. Furthermore, a significant association was found with programmed cell death ligand 1 (PD-L1) combined positive score (p < 0.001) and FGFR3 mutational status (p = 0.002), with high-confidence basal predictions containing a higher proportion of PD-L1 positive samples and high-confidence luminal predictions a higher proportion of FGFR3-mutated samples. Testing of the DL model on an independent cohort highlighted the importance to accommodate histological subtypes. Taken together, our DL workflow can predict protein-based UTUC subtypes, associated with the presence of targetable alterations, directly from H&E slides.

Comparison of Immunohistochemical Expression of Calretinin, Map2, S-100 and Glut1 in Rectal Biopsies from Suspected Hirschsprung’s Disease

Background: This study aimed to compare the immunohistochemical expression of calretinin, Map2, S-100, and Glut1 in rectal biopsy samples from patients suspected of Hirschsprung’s disease (HD).Methods and Results: Rectal biopsy samples from 40 patients with suspected HD were analyzed using hematoxylin and eosin (H&amp;E) and immunohistochemistry (IHC). Immunohistochemical stains were assessed after previous routine histology interpretation, which was classified as “Positive or in favor for HD” and “Equivocal or negative for HD.” The staining patterns for calretinin, Map2, S-100, and Glut1 were analyzed regarding the following structures: lamina propria small nerve fibrils, submucosal small nerve fibrils,submucosal nerve fibers, and submucosal ganglia. The IHC stains for calretinin and Map2 were score-ranked as 0 – negative and 1 – positive. The IHC stain for S-100 was score-ranked as 0 – normal and 1 – hypertrophic. The IHC stain for Glut1 was ranked as 0 – normal perineural and 1 – conspicuous perineural accentuation Calretinin had 92% accuracy, the highest sensitivity (80%) and specificity (92.00%).Map2 also had the same accuracy as calretinin but lower sensitivity (46.67%). Regarding S-100 and Glut1, these two markers did not support a conclusive diagnosis. The accuracy for S-100 and Glut1 was 66.7% and 60%, respectively. Conclusion: Calretinin remains the currently most valuable single IHC marker in diagnosing difficult cases of HD.

The combined immunohistochemical expression of AMBRA1 and SQSTM1 identifies patients with poorly differentiated cutaneous squamous cell carcinoma at risk of metastasis: A proof of concept study.

Cutaneous squamous cell carcinoma (cSCC) incidence continues to increase globally with, as of yet, an unmet need for reliable prognostic biomarkers to identify patients at increased risk of metastasis. The aim of the present study was to test the prognostic potential of the combined immunohistochemical expression of the autophagy regulatory biomarkers, AMBRA1 and SQSTM1, to identify high-risk patient subsets. A retrospective cohort of 68 formalin-fixed paraffin-embedded primary cSCCs with known 5-year metastatic outcomes were subjected to automated immunohistochemical staining for AMBRA1 and SQSTM1. Digital images of stained slides were annotated to define four regions of interest: the normal and peritumoral epidermis, the tumor mass, and the tumor growth front. H-score analysis was used to semi-quantify AMBRA1 or SQSTM1 expression in each region of interest using Aperio ImageScope software, with receiver operator characteristics and Kaplan-Meier analysis used to assess prognostic potential. The combined loss of expression of AMBRA1 in the tumor growth front and SQSTM1 in the peritumoral epidermis identified patients with poorly differentiated cSCCs at risk of metastasis (*p < 0.05). Collectively, these proof of concept data suggest loss of the combined expression of AMBRA1 in the cSCC growth front and SQSTM1 in the peritumoral epidermis as a putative prognostic biomarker for poorly differentiated cSCC.

Effect of platelet rich plasma versus melatonin on testicular injury induced by Busulfan in adult albino rats: a histological and immunohistochemical study

ABSTRACT This study was done to estimate the testicular histological alterations induced by Busulfan (BUS) and compare the possible protective effects of melatonin (MT) and platelet rich plasma (PRP) in a rat model. Sixty-four male rats were dispersed into: control group, BUS group, melatonin group, and PRP group. Blood samples were processed for biochemical analysis. Tissue specimens were managed for light and electron microscopic studies. Immunohistochemical expression of vimentin and proliferating cell nuclear antigen (PCNA) was performed. Busulfan induced severe testicular damage in all studied methodologies. It showed a statistically significant decrease in serum testosterone and elevation of MDA when compared to the control group. Abnormal testicular cytostructures suggesting defective spermatogenesis were observed: distorted seminiferous tubules, deformed spermatogenic cells, low germinal epithelium height, few mature spermatozoa, and also deformed barrier. Vimentin and PCNA expressions were reduced. Ultrastructurally, Sertoli cells and the blood testis barrier were deformed, spermatogenic cells were affected, and mature spermatozoa were few and showed abnormal structure. Both melatonin and PRP induced improvement in all the previous parameters and restoration of spermatogenesis as confirmed by improvement of Johnsen’s score from 2.6 ± .74 to 7.6 ± .92. In conclusion, melatonin and PRP have equal potential to ameliorate the testicular toxicity of BUS. Melatonin can provide a better noninvasive way to combat BUS induced testicular injury.

Immunohistochemical Investigation of P16 Expression in Curettage Biopsies

Objective: Our study aims to investigate the immunohistochemical expression of the P16 molecule, which is involved in the cell cycle and plays a role in developing endometrial cancer. Materials and Method: A total of 68 patients who underwent endometrial sampling for various reasons at the Department of Obstetrics and Gynecology, Faculty of Medicine, Kafkas University, between 2020 and 2021 were included in the study. The selected cases were categorized into four groups: proliferative endometrium, simple non-atypical endometrium, atypical endometrial hyperplasia, and endometrial polyp. There were no cases with a diagnosis of endometrial tumors in our study. All patients' pathology samples were re-evaluated, and P16 immunohistochemistry was applied to tissue samples. Results: Among patients diagnosed with atypical endometrial hyperplasia, 72.7% exhibited moderate P16 protein expression, 18.2% had low expression, and 9.1% had high protein expression. The number of patients diagnosed with atypical endometrial hyperplasia had a very low frequency in the study population. Among patients diagnosed with endometrial polyps, 50.0% showed moderate P16 protein expression, 20.0% exhibited low protein expression, and 30.0% had high protein expression. Conclusion: High P16 expression has been reported to be significantly associated with endometrial cancer in the literature. P16 expression is significant in precancerous lesions and stages of cancer development. Larger-scale studies with more cases are needed in this regard.

A comparative study of morphological and Immunohistochemical expression of P40 and P63 immunomarkers in squamous cell carcinoma and adenocarcinoma lung

Background: Lung cancer is the leading cause of cancer related death worldwide and accounts for 28% of all cancer mortality and around 1.8 million new cases were diagnosed in 2012. The morphological distinction between pulmonary adenocarcinoma (ADC) and squamous cell carcinomas (SCC) is sometimes difficult, mainly in cases of poorly differentiated tumors or when degenerative changes, necrosis and crushing may obscure the cell characteristics. p63 is a homologue of the p53 tumour suppressor gene that is responsible for proliferation and differentiation of epithelial progenitor cells. p40 is consistently the predominant isoform expressed in squamous cell carcinoma; thus, it offers improved specificity for diagnosing squamous cell carcinoma. Material and Methods: This is a prospective and observational study conducted in the Department of Pathology, Tertiary care Teaching Hospital over a period of 1 year. Primary lung carcinoma cases included with unequivocal morphological diagnosis irrespective of age, gender and nature of biopsy material (endoscopic biopsy/ needle core biopsy / resected specimen). Cases diagnosed as Small cell carcinoma of lung, as metastatic lung cancers, poorly differentiated NSCLC-NOS and with inadequate material for IHC study were excluded from present study. Results: In the present study, a total of 150 patients were included out of which 112 (74.6%) were males and 38 (25.4%) were females. In our study, most of the patients were &gt;61 years i.e., 63 out of 150 (42%), followed by 51-60 years, i.e., 40 out of 150 (26.7%). All 8 cases of well differentiated Adenocarcinoma were positive for P40 and 5 cases showed P63 expression. Out of 13 cases of moderately differentiated Adenocarcinoma, 3 cases were positive for P40 and 10 cases were positive for p63 marker. Out of 30 cases of well differentiated Squamous cell carcinoma 15 cases were P40 positive and 15 cases were P63 positive. All 53 cases of moderately differentiated Squamous cell carcinoma showed positive P40 and P63 expression.

Impact of L-Arginine on diabetes-induced neuropathy and myopathy: Roles of PAI-1, Irisin, oxidative stress, NF-κβ, autophagy and microRNA-29a

BackgroundT2DM is a chronic disorder with progressive neuromuscular alterations. L-arginine (ARG) is the most common semi-essential amino acid having several metabolic functions. Aimto investigate the impact of L-arginine in combating diabetic-induced neuromyopathy and its possible mechanisms. Materials & Methods24 rats were divided into CON, CON+ARG, DC, DC+ARG. Behavioral tests, Body weight (BW), fasting blood glucose (FBG), insulin, total antioxidant capacity (TAC), malondialdehyde (MDA), plasminogen activator inhibitor-1 (PAI-1), and irisin were done. Creatine kinase-MM (CK-MM), interleukin 4 (IL-4), interleukin 6 (IL-6), TAC, MDA, expression of microRNA-29a mRNA & light chain 3 protein were determined in muscle. Histological and NF-κβ immunohistochemical expression in muscle and nerve were assessed. ResultsARG supplementation to diabetic rats improved altered behavior, significantly increased BW, insulin, TAC, irisin and Il-4, decreased levels of glucose, microRNA-29a, NF-κβ and LC3 expression, PAI-1, CK-MM and restored the normal histological appearance. ConclusionsARG supplementation potently alleviated diabetic-induced neuromuscular alterations.

Exploring the Relationship between E-Cadherin and β-Catenin Cell Adhesion Proteins and Periacinar Retraction Clefting in Prostatic Adenocarcinoma.

Periacinar retraction clefts represent a histopathological criterion supporting the diagnosis of prostatic adenocarcinoma. The origin of these clefts in prostatic adenocarcinoma remains unclear. Exploring the established functions of E-cadherin and β-catenin as intercellular adhesion proteins, and aiming to elucidate the origin of periacinar retraction clefting, we conducted a correlation study between the immunohistochemical expression of E-cadherin and β-catenin and the presence of periacinar retraction clefts in prostatic adenocarcinoma. We examined 53 cases of morphologically diagnosed prostatic adenocarcinoma, assessing both the neoplastic and adjacent nonneoplastic prostatic tissues for the existence and degree of periacinar retraction clefts. Additionally, we analyzed the immunohistochemical expression of E-cadherin and β-catenin proteins in prostatic tissue and explored their correlation with periacinar retraction clefts, and Gleason score, Grade Group, preoperative serum prostate specific-antigen (sPSA) levels, surgical margin status, and Tumor, Node, Metastasis (TNM) stage in prostatic adenocarcinoma. Our study confirms that periacinar retraction clefting is significantly more extensive in prostatic adenocarcinoma than in nonneoplastic prostatic tissue (p < 0.001). We report a decreased expression of E-cadherin and β-catenin immunostaining in prostatic adenocarcinoma and a negative correlation with Gleason score and Grade Group. Periacinar retraction clefting positively correlated with E-cadherin and β-catenin ((rho = 0.350; p = 0.010) and (rho = 0.340; p = 0.012)) immunostaining in prostatic adenocarcinoma. Periacinar retraction clefts stand out as a dependable criterion in the diagnosis of prostatic adenocarcinoma. E-cadherin and β-catenin proteins are potential markers indicative of tumor progression and invasiveness in prostatic adenocarcinoma. Our discovery of a positive correlation between immunostaining of E-cadherin and β-catenin proteins and periacinar retraction clefts in prostatic adenocarcinoma aligns with the notion that periacinar retraction clefting is more characteristic of Gleason Grade3 pattern in prostatic adenocarcinomas, whereas the immunohistochemical expression of E-cadherin and β-catenin shows a decrease with increasing histopathological tumor grade.

Prevention of DMBA-induced Mammary Tumors from Pulmonary Metastases by Saussurea costus (Falc.) Lipsc Root Ethanolic Extract in Sprague Dawley Rats

Background: Breast cancer is an important problem in human females and therapies used for treatment often found to produce side effects. The present study was conducted to evaluate the effect of Saussurea costus root extract (SC) against metastatic breast cancer to the lungs. Methods: The anti-neoplastic activity of SC extract was assessed by calculating the % cytotoxicity on Leydig cell testicular tumour cells-540 (LC-540). The anti-metastatic effect of SC extract at 3 different doses (100, 250 and 500 mg/kg BW) for a period of 18 weeks against 12-dimethylbenz (a) anthracene (DMBA) induced mammary tumours in Sprague Dawley (SD) female rats was studied through radiography, gross lesion scoring, histopathology and immunohistochemistry (MMP-9). Result: SC extract exhibited the highest anti-neoplastic activity in a time and concentration-dependent manner. After 72 h of duration SC extract exhibited highest anti-neoplastic activity at 200 μg of concentration. On radiological and gross pathological examination, the inhibition of metastases was maximal in the rats treated with highest dose of the extract as compared with only DMBA treated group. The histopathological changes and immunohistochemical expression of metastases marker MMP-9 in lung tissue were more severe in the rats treated with DMBA only and were reduced in the groups treated with SC extract in a dose-dependent manner. SC extract has potential anti-neoplastic activity and prevents the rate of metastases from mammary tumours to lungs. Further research in exploring the possible therapeutic usage of SC extract against various neoplastic conditions is warranted.

Novel drug therapy of acute hepatic failure induced in rats by a combination of tadalafil and Lepidium sativum

BackgroundHepatocyte death and a systemic inflammatory response are the outcome of a complex chain of events mediated by numerous inflammatory cells and chemical mediators. The point of this study was to find out if tadalafil and/or Lepidium sativum (L. sativum) could help people who have been exposed to carbon tetrachloride (CCL4) and are experiencing acute moderate liver failure. This was especially true when the two were used together.Method and materialsTo cause mild liver failure 24 h before sacrifice, a single oral dosage of CCL4 (2.5 mL/kg b.w.) (50% in olive oil) was utilized. Furthermore, immunohistochemical expression of nuclear factor kappa B (NF-κB) as well as histological abnormalities were performed on liver tissue.ResultsThe results showed that tadalafil and/or L. sativum, especially in combination, performed well to cure acute mild liver failure caused by CCL4. This was demonstrated by a decrease in NF-κB expression in the liver tissue and an improvement in organ damage markers observed in the blood and liver tissues. Furthermore, such therapy reduced interleukin1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels in the liver tissue. It’s worth noting that the tested combination resulted in greater liver improvement.ConclusionsAccording to the findings, tadalafil and L. sativum, particularly in combination, have the ability to protect the liver from the negative effects of CCL4 exposure. Because of its capacity to improve liver function, restore redox equilibrium, and decrease inflammatory mediators, it is a prospective option for mitigating the negative effects of common environmental pollutants such as CCL4.

Identification and Evaluation of Cancer Stem Cells in Oral Squamous Cell Carcinoma and Oral Epithelial Dysplasia Using NANOG: An Immunohistochemical Study.

Squamous cell carcinoma of the oral cavity may show precursor lesions, termed as potentially malignant disorders, of which leukoplakia is the most frequent one. Oral leukoplakia is a clinical diagnosis for which the histological diagnosis may be either hyperplasia or oral epithelial dysplasia (OED) and sometimes even oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs), identified in various tumors, are a specific group of cells that exhibit the properties of self-renewal and differentiation. Among the various biomarkers that identify CSCs, the transcription factor NANOG is considered to be a significant one. In this study, we intend to identify and compare the immunohistochemical expression of NANOG in OSCC, OED, and normal oral mucosa. Tissue blocks of OSCC (n=28), OED (n=28), and normal oral mucosa (n=28) were used in this study. Specimens were immunohistochemically analyzed for NANOG expression. The results were statistically analyzed using one-way ANOVA, Games-Howell post hoc, and Student t-test. Statistical Product and Service Solutions (SPSS, version 21; IBM SPSS Statistics for Windows, Armonk, NY) software was used for performing the statistical analysis, and the level of significance was set as 0.05. NANOG expression was higher in OSCC when compared to oral dysplasias and normal oral mucosa, in decreasing order. A significantly higher histo-score and labeling index score were observed in OSCC and oral dysplasias compared to normal oral mucosa (p=<0.001). The expression levels of NANOG were positively correlated with disease progression in OSCC, implicating that NANOG can be used as a surrogate marker of oral oncogenesis and prognosis. Therefore, decoding the molecular mechanisms ofNANOG regulation in the progression of cancer helps in developing new therapeutic strategies for oral cancer.

Differential Upregulation of Th1/Th17-Associated Proteins and PD-L1 in Granulomatous Mycosis Fungoides.

Granulomatous Mycosis Fungoides (GMF) is a rare form of mycosis fungoides (MF) characterized by a granulomatous infiltrate associated with the neoplastic lymphoid population and is considered to have a worse prognosis compared with regular MF. The upregulation of the T helper (Th) axis, especially Th17, plays an important role in the pathogenesis of several inflammatory/infectious granulomatous cutaneous diseases, but its role in GMF is still not elucidated to date. In this study, we evaluated the immunohistochemical expression of Th1 (Tbet), Th2 (GATA-3), Th17 (RORγT), T regulatory (Foxp3), and immune checkpoint (IC) (PD-1 and PD-L1) markers in a cohort of patients with GMF and MF with large cell transformation (MFLCT). Skin biopsies from 49 patients (28 GMF and 21 MFLCT) were studied. Patients with GMF were associated with early clinical stage (p = 0.036) and lower levels of lactate dehydrogenase (p = 0.042). An increased percentage of cells positive for Tbet (p = 0.017), RORγT (p = 0.001), and PD-L1 (p = 0.011) was also observed among the GMF specimens, while a stronger PD-1 intensity was detected in cases of MFLCT. In this cohort, LCT, RORγT < 10%, Foxp3 < 10%, age, and advanced stage were associated with worse overall survival (OS) in univariate analysis. GMF demonstrated Th1 (cellular response) and Th17 (autoimmunity) phenotype, seen in early MF and granulomatous processes, respectively, which may be related to the histopathological appearance and biological behavior of GMF. Further studies involving larger series of cases and more sensitive techniques are warranted.

Involvement of ferroptosis‐defensive xCT/GPX4 axis in radioresistance and its impacts on prognosis in oral squamous cell carcinoma

AbstractAimRadiotherapy, the mainstay treatment for patients with advanced oral squamous cell carcinoma (OSCC), has low clinical efficacy, resulting in poor prognosis. Radiation induces ferroptosis, a form of cell death driven by iron‐dependent lipid peroxidation. Since the xCT/glutathione peroxidase 4 (GPX4) axis is predominantly involved in the ferroptosis defense system, targeting this axis may potentiate cancer cell vulnerability to ferroptosis.MethodsWe examined the expression of xCT and GPX4 in the tumor cells of biopsy specimens using immunohistochemistry and evaluated the radiation‐mediated antitumor effects of the ferroptosis inducers, erastin and RSL3, that act by inhibiting xCT and GPX4, respectively.ResultsUnivariate analysis revealed that high immunohistochemical expression of xCT was correlated with shorter survival, and high GPX4 expression was an independent poor prognostic factor, indicating that the xCT/GPX4 axis influences the clinical outcome of OSCC. Analyses using two types of OSCC cell lines revealed that the clonogenic survival of irradiated cells was increased by the ferroptosis inhibitor ferrostatin‐1 and decreased by erastin and RSL3. The enhanced antitumor effects of erastin and RSL3 were accompanied by increased lipid peroxidation, which was suppressed by the iron chelator deferoxamine. Erastin and RSL3 enhance the effects of radiation on OSCC cells in a mouse xenograft model; these effects were associated with increased expression of the lipid peroxidation marker 4‐hydroxynonenal in tumor cells.ConclusionsxCT/GPX4 axis is involved in the tumor resistance against radiation through the inhibition of ferroptosis in OSCC. Furthermore, radiotherapy combined with ferroptosis induction by targeting the xCT/GPX4 axis may improve patient prognosis in advanced OSCC.

Evaluation of Cellular Changes and Immunohistochemistry Expression of p53 and p16 in the Oral Mucosa Among Saudi Smokers.

Smoking is a well-known risk factor for various health problems, including oral cancer. P16 and P53 proteins are involved in cell cycle regulation and proliferation, and their expression levels can provide insights into cellular health. This study aims to evaluate the cellular changes and immunohistochemistry expression of p53 and p16 in the oral mucosa among Saudi smokers. In a cross-sectional study obtained by scraping the buccal mucosa, 1000 samples were collected from 2022 to 2023. All of the study's participants were Saudi citizens of both genders. Seven hundred cigarette smokers and 300 nonsmokers made up the controls, using two sampling techniques: initially purposive and then snowball sampling. The materials were subjected to immunohistochemical analysis for P16 and P53 protein overexpression. The samples were scored based on the percentage of positively stained cells and staining intensity. The data were analyzed using SPSS, and categorical variables were identified as frequencies and percentages using the chi-squared test; a value of (P<0.05) was considered significant. Cigarette smokers demonstrate significantly higher rates of cytological inflammation, reverse cytological infection, atypia, and binucleated/multinucleated cells compared to nonsmokers, with an overall abnormal result rate of 46% versus 18.7%, respectively (P=0.024). The study found higher P53 and P16 expression among smokers (7.14% and 2.14%, respectively) compared to nonsmokers (0.1% and 0.33%) (P=0.038). No significant differences were observed in P53/P16 expression across age groups (P=0.72) or between male and female participants (P=0.25). These findings highlight the detrimental effects of smoking on cellular health and reinforce the importance of smoking cessation in reducing the risk of developing cytological abnormalities and associated diseases. These results highlight the association of smoking with increased biomarker expression, emphasizing its relevance in understanding oral health risks.