P53-positive Cells Research Articles

Analysis of Adenoviral p53 Gene Therapy Clinical Trials in Recurrent Head and Neck Squamous Cell Carcinoma.

BackgroundWe conducted an analysis of previous adenoviral p53 (Ad-p53) treatment data in recurrent head and neck squamous cell carcinoma (HNSCC) patients to identify optimal Ad-p53 treatment methods for future clinical trials.MethodsThe analysis involved recurrent HNSCC patients treated with Ad-p53 for whom p53 genotyping and immunohistochemistry tumor biomarker studies had been performed (n = 70). Ad-p53 tumor treatment responses defined by RECIST 1.1 criteria were correlated with Ad-p53 dose and tumor p53 biomarkers. Gene expression profiles induced by Ad-p53 treatment were evaluated using the Nanostring IO 360 panel.ResultsAd-p53 dose based upon the injected tumor volume had a critical effect on tumor responses. All responders had received Ad-p53 doses greater than 7 × 1010 viral particles/cm3 of tumor volume. There was a statistically significant difference in tumor responses between patients treated with greater than 7 × 1010 viral particles/cm3 compared to patients treated at lower Ad-p53 doses (Tumor Response 31% (9/29) for Ad-p53 > 7 × 1010 viral particles/cm3 versus 0% (0/25) for Ad-p53 < 7 × 1010 viral particles/cm3; p = 0.0023). All responders were found to have favorable p53 biomarker profiles defined by less than 20% p53 positive tumor cells by immunohistochemistry (IHC), wild type p53 gene sequence or p53 deletions, truncations, or frame-shift mutations without functional p53 tetramerization domains. Preliminary gene expression profiling results revealed that Ad-p53 treatment increased interferon signaling, decreased TGF-beta and beta-catenin signaling resulting in an increased CD8+ T cell signature which are associated with increased responses to immune checkpoint blockade.ConclusionsOur findings have important implications for future p53 targeted cancer treatments and identify fundamental principles to guide Ad-p53 gene therapy. We discovered that previous Ad-p53 clinical trials were negatively impacted by the inclusion of patients with unfavorable p53 biomarker profiles and by under dosing of Ad-p53 treatment. Future Ad-p53 clinical trials should have favorable p53 biomarker profiles inclusion criteria and Ad-p53 dosing above 7 × 1010 viral particles/cm3 of injected tumor volume. Preliminary gene expression profiling identified p53 mechanisms of action associated with responses to immune checkpoint blockade supporting evaluation of Ad-p53 in combination with immune checkpoint inhibitors.

Melatonin Protects against the Side-Effects of 5-Fluorouracil on Hippocampal Neurogenesis and Ameliorates Antioxidant Activity in an Adult Rat Hippocampus and Prefrontal Cortex.

Melatonin is an endogenous hormone that exhibits antioxidant functions and neuroprotective effects. The hippocampus and the prefrontal cortex (PFC) play an important role linked to working memory. 5-fluorouracil (5-FU) can induce oxidative stress and reduce neurogenesis in the subgranular zone (SGZ) of the dentate gyrus in a rat hippocampus and these alterations are related to working memory deficits. This study aimed to determine the effect of melatonin on 5-FU-induced oxidative stress that interferes with the antioxidant enzymes and protein expression levels in a rat hippocampus and PFC. A total of 68 male Sprague Dawley rats were divided into four groups: vehicle, 5-FU, melatonin and melatonin+5-FU groups. Rats were administered 5-FU (25 mg/kg, i.v.) on days 9, 12, 15, 18 and 21 and received melatonin (8 mg/kg, i.p.) at 19:00 from day 1 to day 21 of the experiment. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels. Antioxidant enzyme levels including glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) were determined. p21 immunofluorescence staining and Western blotting were used to detect the cell cycle arrest and protein expression of the nuclear factor erythroid 2-related factor 2 (Nrf2), doublecortin (DCX) and brain derived neurotrophic factor (BDNF), respectively. The results showed that melatonin reduced the number of p21-positive cells in the SGZ of the dentate gyrus and increased Nrf2, DCX and BDNF protein expression in rats treated with 5-FU. Moreover, melatonin restored antioxidant enzyme levels and reduced oxidative stress in the hippocampus and PFC caused by 5-FU. These findings reveal a mechanism of the neuroprotective properties of melatonin against 5-FU in a rat hippocampus and PFC.

Neonatal maternal separation increases the number of p27-positive Sertoli cells in prepuberty

Neonatal maternal separation (NMS) is an experimental model for early life stress, which affects the growth and development of various organs, resulting in adverse health effects in humans and animals. In our previous study, we demonstrated that NMS [(0.5-, 1-, 2-h/day NMS, from postnatal day (PND) 1–10] induced morphological changes to the male reproductive system, including decreased Sertoli cell numbers in mouse testes at PND 70. To clarify the mechanism by which NMS decreases Sertoli cell numbers, we evaluated the effects of NMS on mouse testes at PNDs 10 and 16. At PND 10, the Sertoli cell number was not significantly different among experimental groups; however, it decreased in 0.5- and 2-h/day NMS mice at PND 16. The termination of Sertoli cell proliferation in prepuberty can be induced by p27, a cyclin-dependent kinase inhibitor. At PND 10, we observed an increase in the number of p27-positive Sertoli cells in 2-h/day NMS mice. The seminiferous tubule diameters decreased significantly in 1- and 2-h/day NMS mice, and the relative interstitial area increased in 2-h/day NMS mice. Serum corticosterone level significantly increased, and serum testosterone level significantly decreased in the 2-h/day NMS mice. At PND 16, the tubule diameters and height of seminiferous epithelium were significantly higher in 0.5- and 2-h/day NMS mice. Our results suggest that NMS disturbs serum corticosterone and testosterone levels and increases the number of p27-positive Sertoli cells at PND 10, resulting in a decrease in the number of Sertoli cells at PND 16.

Decreased number of p16-positive senescent cells in human endometrium as a marker of miscarriage

The aim of this study was to evaluate whether the number of p16-positive cells in the functional layer of the endometrium could be a useful biomarker to identify women with recurrent implantation failure (RIF) undergoing in vitro fertilization (IVF) at risk of miscarriage. Immunohistochemical staining was performed in 311 endometrial biopsies taken during mid-luteal phase using antibody against p16INK4A. The percentage of p16-positive cells was determined in luminal, glandular and stromal endometrial cells. After embryo transfer, women were divided into the following groups: unsuccessful embryo implantation (n = 151), miscarriage (n = 66) and live birth (n = 94). The percentage of p16-positive cells in all endometrial compartments was compared among these groups. We found that the percentages of p16-positive glandular and luminal epithelial endometrial cells were significantly higher in patients with live births compared to women with miscarriage (9.3% vs. 2.9%, P < 0.001; and 35.2% vs. 11.7%, P = 0.001, respectively). This tendency was not confirmed in thе stroma. The cut-off values with p16-positive luminal cells lower than 12.5% and p16-positive glandular cells lower than 3.2% could be predictive factors for miscarriage (AUC 0.80 and 0.79; sensitivity 71.3% and 74.5%; specificity 74.2% and 71.2%, respectively). A decreased number of senescent p16-positive cells could be involved in the implantation failures and aetiology of recurrent miscarriage. Women with history of RIF with reduced populations of p16-positive cells in the endometrial glandular and luminal epithelium may be at greater risk for unsuccessful implantation and miscarriage. The percentage of p16-positive luminal epithelial cells may be clinically useful as a biomarker of miscarriage.

LAMELLAR HISTOLOGICAL AND IMMUNOHISTOCHEMICAL EVALUATION OF THE HOOF WALL IN HORSES WITH CHRONIC LAMINITIS TREATED WITH MESENQUIMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE BY REGIONAL PERFUSION

<h3>Background</h3> The objective was to evaluate the histological and immunohistochemical effects of regional perfusion of characterized mesenchymal stem cells derived from adipose tissue (Ad-MSC) in biopsy hoof samples from horses with laminitis. <h3>Methods</h3> Six mares with stable chronic laminitis in standard treatment for distal phalangeal realignment were subjected to regional perfusion of the digital vein with Ad-MSC (20 million cells, 3 infusions every 15 days) in one forelimb compared to placebo infusion in the contralateral one. Evaluations occurred in two 30-day periods, before and after treatment (from the second infusion). Dorsal hoof lamellar biopsies were taken 30 days before and 15 days after the therapeutic protocols. Histological 5-μm cuts stained with H&E and PAS were classified based on a crescent 1-5 scale lesion score system. The sum of each lesion grade represented the global lesion score. Immunohistochemistry for p63 positive cells were also performed in formalin-fixed samples. <h3>Results</h3> In the placebo limbs, there were increases over time in following histological variables: global lesion (P=0.01), keratinized axis loss (P=0.0001), stratum medium hyperplasia (P=0.001), primary epidermal lamellae homogeneity loss (P=0.003), and hyperkeratosis (P=0.05). Only the stratum medium hyperplasia score was increased in the Ad-MSC-treated hooves. P63-positive cells could be detected in all formalin-fixed paraffin-embedded samples, but background unspecific stain made counts imprecise to be made. <h3>Conclusion</h3> In conclusion Ad-MSC regional vascular perfusion prevented histological lesion progression in the lamellar tissue of horses with chronic laminitis. Our results confirm the therapeutic potential of Ad-MSC in the treatment of horses with stable chronic laminitis.

A novel p53 regulator, C16ORF72/TAPR1, buffers against telomerase inhibition.

Telomere erosion in cells with insufficient levels of the telomerase reverse transcriptase (TERT), contributes to age‐associated tissue dysfunction and senescence, and p53 plays a crucial role in this response. We undertook a genome‐wide CRISPR screen to identify gene deletions that sensitized p53‐positive human cells to telomerase inhibition. We uncovered a previously unannotated gene, C16ORF72, which we term Telomere Attrition and p53 Response 1 (TAPR1), that exhibited a synthetic‐sick relationship with TERT loss. A subsequent genome‐wide CRISPR screen in TAPR1‐disrupted cells reciprocally identified TERT as a sensitizing gene deletion. Cells lacking TAPR1 or TERT possessed elevated p53 levels and transcriptional signatures consistent with p53 upregulation. The elevated p53 response in TERT‐ or TAPR1‐deficient cells was exacerbated by treatment with the MDM2 inhibitor and p53 stabilizer nutlin‐3a and coincided with a further reduction in cell fitness. Importantly, the sensitivity to treatment with nutlin‐3a in TERT‐ or TAPR1‐deficient cells was rescued by loss of p53. These data suggest that TAPR1 buffers against the deleterious consequences of telomere erosion or DNA damage by constraining p53. These findings identify C16ORF72/TAPR1 as new regulator at the nexus of telomere integrity and p53 regulation.

Micronodular thymic carcinoma with lymphoid hyperplasia: relevance of immunohistochemistry with a small panel of antibodies for diagnosis-a RYTHMIC study.

Micronodular thymic carcinoma with lymphoid hyperplasia (MNTCLH) is a rare form of thymic carcinoma. We present the experience of RYTHMIC, the French national network devoted to the treatment of thymic epithelial tumors through multidisciplinary tumor boards with a review of all tumors by pathologists for classification and staging. Six cases of MNTCLH were diagnosed during a review of 1007 thymic epithelial tumors. Histologically, epithelial cells with atypia and mitoses formed micronodules that were surrounded by an abundant lymphoid background with follicles. There was neither obvious fibro-inflammatory stroma nor necrosis. Spindle cells areas were common. Initial diagnosis was micronodular thymoma in two cases, cellular atypia being overlooked, eclipsed by the micronodular pattern. Immunohistochemistry with a panel of five antibodies showed that cytokeratins (AE1-AE3) and p63-positive epithelial cells also expressed CD5 and that there was no TdT-positive cells within the tumors. CD20 highlighted the lymphoid hyperplasia. Additionally epithelial cells also expressed CD117 and diffusely Glut 1. Twenty-seven micronodular thymomas with lymphoid stroma diagnosed during the same period did not show the CD5 and CD117 positivities seen in MNTCLH and contained TdT-positive lymphocytes. Three of the 6 patients with MNTCLH had adjuvant radiotherapy. Three patients with follow-up information were alive without recurrence at 38, 51, and 95 months. Our study shows that immunohistochemistry, such as that used in the RYTHMIC network with a small panel of antibodies, may easily help to confirm the correct diagnosis of MNTCLH, a rare and low-aggressive form of thymic carcinoma, and avoid the misdiagnosis of micronodular thymoma.

Sinonasal low-grade non-intestinal-type adenocarcinoma: A retrospective analysis and literature review

Sinonasal low-grade non-intestinal-type adenocarcinomas (LG non-ITACs) are uncommon tumors with unclear histogenesis, although they are presumed to arise from seromucous glands or respiratory epithelium. We investigated the clinicopathological and immunohistochemical features of the tumors, with particular attention to the transition area from the normal epithelium to neoplastic cells and concurrent lesions; these features were compared with those of 10 patients with chronic sinusitis, who served as a control group. Seventeen patients with LG non-ITACs (17 tumors) were enrolled in this retrospective study (9 male patients and 8 female patients; mean age, 48 years [range, 16–74 years]). Tumor cells continuous with respiratory epithelium were detected in 10 tumors composed of a single layer of cells with papillary, tubular, or cystic growth pattern. The tumor cells were uniformly cuboidal to columnar and polar. In seven tumors without transition areas discerned, three tumors consisted of polygonal and flat cells with a solid, acinar, micropapillary and cribriform pattern. The others had the same morphology as those with transition areas. The tumor cells were positive for SOX10 (15/17), S100 protein (8/17), and CK7 (17/17). The normal epithelium connected to the respiratory epithelium was the terminal duct in the control group. Except for the lack of p63-positive cells, the immunophenotype and histomorphology of transition areas with LG non-ITACs were similar to those of the continuous areas between the terminal duct and the respiratory epithelium in the control group. LG non-ITACs are seromucinous tumors, some of which may originate from the terminal ducts of seromucinous glands.

Cellular senescence evaluated by P16INK4a immunohistochemistry is a prevalent phenomenon in advanced calcific aortic valve disease

BackgroundFibrosis, calcification, and ossification are histopathologic hallmarks of calcific aortic valve disease (CAVD), a leading cause of morbidity and mortality in the aging population. Cellular senescence contributes to a functional decay in chronic diseases by intensifying tissue remodeling and impairing tissue regeneration. We evaluated the expression of P16INK4A and P53 as surrogate markers of senescence in CAVD. MethodsAortic valves from 27 individuals with severe CAVD requiring aortic valve replacement were selected for routine histologic processing. Immunohistochemical expression of P16INK4A and P53 was quantified using computerized image analysis on fields matching compartments with varying degrees of tissue remodeling. ResultsAll aortic valves demonstrated P16INK4A and P53-positive cells. The percentage of P16INK4A -positive cells, but not of P53, was higher in areas of calcification and/or ossification (57.21%±26.31, n=40) and severe fibrosis (54.79%±27.19, n=25) than in areas with minimal to mild tissue remodeling (13.69% ± 11.88, n=16, P<.0001). P16INK4A expression was observed in interstitial valve cells within all compartments proportional to the degree of fibrosis and did not correlate with age, severity of aortic stenosis, or P53 expression. Multiple linear regression analysis by backward elimination revealed P16INK4A expression was lower among statin users (P<.01). ConclusionsP16INK4A- expression is ubiquitous in calcified aortic valves and correlates with severity of tissue remodeling, suggesting a role of cellular senescence in the progression of CAVD. Further research is needed to identify possible treatment modalities as disease modifying agents for CAVD.

Proliferating Pilar Tumors: Can Immunohistochemistry Differentiate Benign and Malignant Forms?

Proliferating pilar tumor (PPT) is an adnexal tumor of purported differentiation toward the follicular outer root sheath. Immunohistochemistry has been suggested to differentiate between benign and malignant forms. Eleven benign (PPT) and 9 malignant PPT lesions were reviewed; Ki67, p27, and p53 were applied. The staining intensity (strong, moderate, weak, and negative), positive cell numbers, and marker indexes (%) were scored using image-analysis software (ViraSoft). Overall, there was no significant correlation between Ki67 and p53 and histopathological features. However, malignant PPTs had significantly lower numbers of p27-positive cells (P = 0.030). Our study includes the largest group of patients in whom image analysis of p53, Ki67, and p27 has been used to try to separate benign from malignant lesions. Although there were no significant differences regarding Ki67 and p53, malignant lesions have a statistically lower expression of p27. Further studies may be needed to determine the clinical usefulness of image analysis in this differential diagnosis.

Time-course changes in 5-fluorouracil-induced neural progenitor cell damages in the developing rat brain.

5-Fluorouracil (5-Fu) is a DNA-damaging agent and teratogenic in rodents. This study aimed to investigate its influence on neural progenitor cells (NPCs) in the developing fetal rat brain. Dams were intraperitoneally injected with 5-Fu (50 mg/kg b.w.) on gestation day 13 and its effects on fetal NPCs were observed from 3 to 72 hours after treatment (HAT), via periodic examination at six intervals. In NPCs of the fetal brain, the p53-labeling index (LI%) was markedly elevated at 3 HAT. Pyknosis and cleaved caspase-3-LI% also increased at 3 HAT, reaching peak values at 9 and 12 HAT. These parallel changes suggested the induction of apoptosis through a p53-mediated pathway. Pyknotic NPCs were distributed across the ventricular zone (VZ) of the telencephalic wall until 12 HAT, and became localized in the medial and dorsal layers at 12 and 48 HAT. Significant decreases in the numbers of mitotic NPCs and BrdU-LI% were noted from 3 HAT and 24 HAT, respectively. BrdU-positive NPCs were located in the ventral and middle layer at 24 and 48 HAT. p21-positive cells were detected at 12 and 24 HAT. The present results demonstrated that p53-mediated apoptosis was induced in all phases of the cell cycle of the NPCs in the early stage after 5-FU treatment. Furthermore, apoptosis of NPCs and suppression of cell proliferative activity are the events that take place in parallel leading to prominent reduction in the width of the telencephalic wall.

Melatonin Ameliorates Valproic Acid-Induced Neurogenesis Impairment: The Role of Oxidative Stress in Adult Rats.

Background Valproic acid (anticonvulsant medication) has been found to inhibit histone deacetylase activity and suppress hippocampal neurogenesis, which causes memory impairment in both humans and rodents. The neurohormone melatonin, which regulates mammalian seasonal and circadian physiology, has recently been shown to have neuroprotective properties, counteracting memory impairment associated with VPA-caused hippocampal neurogenesis reduction. This study is aimed at investigating the molecular mechanisms of melatonin associated with VPA-induced hippocampal neurogenesis and memory impairment. Methods Male Spraque-Dawley rats received VPA (300 mg/kg) twice daily or melatonin (8 mg/kg/day) or some rats were given melatonin for 14 days during VPA administration. Results The VPA-treated rats showed a significant increase in malondialdehyde (MDA) levels in the hippocampus and p21-positive cells in the subgranular zone (SGZ) of the dentate gyrus (DG) but decreased superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities. Moreover, VPA significantly decreased levels of nestin, Notchl, nuclear factor erythroid 2-related factor 2 (Nrf2), doublecortin (DCX), sex determining region Y-box 2 (SOX2), and brain-derived neurotrophic factor (BDNF). Conclusions We found that melatonin was able to counteract these neurotoxic effects, acting as a neuroprotectant in VPA-induced memory hippocampal neurogenesis impairment by preventing intracellular oxidative stress and increasing antioxidant activity.

The antitumor activity of cannabidiol on lung cancer cell lines A549 and H1299: the role of apoptosis

In the recent years, the application of new antitumor drugs has focused on the replacement of conventional chemotherapeutics with compounds derived from natural products. Cannabidiol (CBD) is one of the 113 cannabinoids derived from the plant Cannabis sativa and is characterized with complex and not entirely understood biological function. Unlike the other most abundant cannabinoid in Cannabis sativa – tetrahydrocannabinol, cannabidiol has low affinity to the endocannabinoid receptors and the manifestation of its activity does not appear to rely on the endocannabinoid system. Cannabidiol is used in the treatment of many diseases including some types of cancer. The aim of our study was to evaluate the cytotoxic activity of cannabidiol and its effect on the process of programmed cell death. This process is directly involved in the antitumor effect of many drugs. Two lung cancer cell lines, A549 expressing р53 and H1299-p53 negative, were used. Apoptosis was monitored by Anexin V assay and the activation of Caspase 3/7. We found that CBD treatment led to a dose-dependant apoptosis increase in p53 positive A549 cells. The level of apoptosis in p53 negative H1299 cells was much lower and did not seem to be dose dependent. However, the total cell viability was similar for the two cell lines, which was due to the increased necrosis observed in H1299 cells.

Biological Heterogeneity

Studies of aging in invertebrates, mammalian animal models and humans have demonstrated increasing heterogeneity with aging in terms of varied facets of biological aging. In addition to growing heterogeneity, aging is also associated with qualitative and quantitative changes involving DNA methylation captured in epigenetic clocks of aging which seek to predict chronological and biological aging. Increased heterogeneity with aging is also evident in terms of posttranslational histone modification, gene expression, somatic clonal expansion, and increased degree of tissue mosaicism. Senescent cells accumulating with aging demonstrate significant heterogeneity. For example, while most studies targeting senescent cells have focused on cells expressing p16 (CDKN2A), not all p16-positive cells are senescent and not all senescent cells express p16. Further studies are needed to better define heterogeneity involving other hallmarks of aging and to also explore associations between heterogeneity involving these biological measures with clinical manifestations or outcomes.

DNA damage in lens epithelial cells exposed to occupationally-relevant X-ray doses and role in cataract formation

The current framework of radiological protection of occupational exposed medical workers reduced the eye-lens equivalent dose limit from 150 to 20 mSv per year requiring an accurate dosimetric evaluation and an increase understanding of radiation induced effects on Lens cells considering the typical scenario of occupational exposed medical operators. Indeed, it is widely accepted that genomic damage of Lens epithelial cells (LEC) is a key mechanism of cataractogenesis. However, the relationship between apoptosis and cataractogenesis is still controversial. In this study biological and physical data are combined to improve the understanding of radiation induced effects on LEC. To characterize the occupational exposure of medical workers during angiographic procedures an INNOVA 4100 (General Electric Healthcare) equipment was used (scenario A). Additional experiments were conducted using a research tube (scenario B). For both scenarios, the frequencies of binucleated cells, micronuclei, p21-positive cells were assessed with different doses and dose rates. A Monte-Carlo study was conducted using a model for the photon generation with the X-ray tubes and with the Petri dishes considering the two different scenarios (A and B) to reproduce the experimental conditions and validate the irradiation setups to the cells. The simulation results have been tallied using the Monte Carlo code MCNP6. The spectral characteristics of the different X-ray beams have been estimated. All irradiated samples showed frequencies of micronuclei and p21-positive cells higher than the unirradiated controls. Differences in frequencies increased with the delivered dose measured with Gafchromic films XR-RV3. The spectrum incident on eye lens and Petri, as estimated with MCNP6, was in good agreement in the scenario A (confirming the experimental setup), while the mean energy spectrum was higher in the scenario B. Nevertheless, the response of LEC seemed mainly related to the measured absorbed dose. No effects on viability were detected. Our results support the hypothesis that apoptosis is not responsible for cataract induced by low doses of X-ray (i.e. 25 mGy) while the induction of transient p21 may interfere with the disassembly of the nuclear envelop in differentiating LEC, leading to cataract formation. Further studies are needed to better clarify the relationship we suggested between DNA damage, transient p21 induction and the inability of LEC enucleation.

TAp63 and ΔNp63 (p40) in prostate adenocarcinomas: ΔNp63 associates with a basal-like cancer stem cell population but not with metastasis.

Like other malignancies, prostate tumors are thought to contain cancer stem-like cells (CSCs) that are responsible for growth, metastasis, and therapy resistance. ΔNp63 (also called p40) is a regulator of normal prostate stem/progenitor cell activities and a marker of normal basal epithelial cells. The levels of ΔNp63 are reduced in prostate adenocarcinomas, although there is also evidence that ΔNp63 is involved in CSC regulation and drives metastasis to the bone. We studied metastatic deposits of prostate cancers with isoform-specific ΔNp63 and TAp63 antibodies. We identified p63-positive cells in only 3 of 42 metastatic prostate tumors (7%), including 2/38 (5.3%) "usual-type" adenocarcinomas. ΔNp63 and TAp63 isoforms were present in the nuclei of a small subpopulation (< 1%) of tumor cells in these metastases. ΔNp63-positive cells showed a basal-like cell phenotype (cytokeratin 8- and androgen receptor-negative, high molecular weight cytokeratin- and cytokeratin 19-positive), distinct from the tumor bulk. TAp63-positive cells were similar but were sometimes cytokeratin 8-positive. A subset of ΔNp63-positive tumor cells were CD44-positive, a marker of "basal" CSCs but were not positive for the "epithelial" CSC marker ALDH1. TAp63 was not associated with either of these CSC markers. None of the tumors containing p63-positive cells showed evidence of bone metastasis, compared with 28% of the p63-negative tumors. These data show that both ΔNp63 and TAp63 are present in only a small proportion of prostate adenocarcinomas and do not associate with metastasis. The data suggest heterogeneity of CSCs in prostate cancer, similar to other cancer types.

VARIATION IN THE PERCENTAGE OF ENDOMETRIAL p16-POSITIVE SENESCENT CELLS IN CONSECUTIVE MENSTRUAL CYCLES

Recently, it was found that p16ink4a-positive senescent cells are involved in endometrial receptivity and participate in the acute cellular remodelling at the time of embryo implantation. Our previous research showed that the percentage of glandular p16-positive cells and luminal epithelial p16-positive cells is strongly associated with successful implantation and live births. The aim of the present study was to compare the percentage of p16-positive cells in the glandular and luminal epithelial compartments of the human endometrium during the mid-luteal phase in two consecutive menstrual cycles. We measured the percentage of p16ink4a-positive cells by immunohistochemistry in endometrial biopsy samples of 94 women in two consecutive menstrual cycles. This is a prospective observational study of 94 fertile women who had two endometrial biopsies during the mid-lutheal phase (7 days after LH surge) in two consecutive natural cycles. Patients older than 40 years, with BMI<18 kg/m2 or BMI≥30 kg/m2, endometriosis, polycystic ovary syndrome (PCOS), endometrial polyps, abnormal uterine development and hydrosalpinx were excluded from the study. Endometrial biopsies were obtained by pipelle suction and they were immediately fixed in 10% formalin. The endometrial tissue was submitted to paraffin embedding for histological determination and subsequent analysis. Immunohistochemistry (IHC) was performed on the paraffin-embedded sections by Novolink Polymer Detection System (Leica Biosystems, Wetzlar, Hesse, Germany). We used monoclonal antibody against p16ink4a (Master Diagnostica, Granada, Spain) to identify senescent endometrial epithelial cells. The percentage of p16+ cells in each tissue compartment was calculated after enumeration by two independent investigators in multiple endometrial sections. Values were expressed as mean ± SD. Paired t-test was used to compare the percentage of p16-positive cells between the two time points. P<0.05 was considered statistically significant. The percentage of p16-positive cells in the endometrial glands during the mid-luteal phase of the cycle ranged between 0.12% and 53.22%, while it varied between 1.39% and 85.29% in the luminal epithelium. The temporal change in the percentage of p16-positive cells varied at both time points between 0.55% and 15.56% in the endometrial glands, and between 1.58% and 30.74% in the endometrial luminal epithelium. In addition, for both endometrial glands and luminal epithelium, the proportion of p16+ senescent cells was not significantly different between the two time measurements (p=0.56 and p=0.93, respectively). In 80% of the patients the difference in the percentage of p16-positive cells at both time points was lower than 20% in the endometrial luminal epithelium and lower than 9% in the luminal epithelium. The percentage of endometrial epithelial p16-positive senescent cells is relatively constant in human endometrium during the mid-luteal phase in consecutive menstrual cycles.

IMMUNOHISTOCHEMICAL EXPRESSION OF METABOLIC MARKERS IN PLEOMORPHIC ADENOMA AND CARCINOMA EX PLEOMORPHIC ADENOMA

Objective: To perform a comparative study focusing on the morphologic and immunohistochemical features of pleomorphic adenomas (PAs) and carcinoma ex pleomorphic adenomas (CXPAs) of the minor salivary glands. Study design: Forty PAs and 10 CXPAs were retrieved from the archives of 3 pathology laboratories, and the diagnoses were reviewed and confirmed. Immunohistochemical reactions were done for p53, Ki67, mammaglobin, and the metabolic markers of glucose transporter 1 (GLUT-1) and adipophilin. Results: The most common subtypes of CXPA were ductal carcinoma (n = 4), clear cell carcinoma (n = 3), and myoepithelial carcinoma (n = 2). The morphologic features suggestive of malignancy were the presence of mitosis, cellular pleomorphism, and extracapsular component. Compared to PA, CXPA demonstrated a higher number of Ki67- and p53-positive cells, in addition to higher expression of GLUT-1 and adipophilin. Mammaglobin expression was observed in the malignant areas, mainly in the ductal CXPA, being negative in the benign reminiscent PA. Conclusion: The diagnosis of CXPA based only on the morphologic aspects can be difficult; thus, an association of the morphologic and immunohistochemical features, particularly high number of Ki67- and p53-positive cells, is essential for a proper diagnosis. Other metabolic markers such as GLUT-1 and adipophilin may be also helpful, although their exact utility remains to be determined.

ASSOCIATION BETWEEN THE PERCENTAGE OF p16-POSITIVE SENESCENT CELLS AND THE EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN HUMAN ENDOMETRIUM DURING THE MID-LUTEAL PHASE

Progesterone and estrogen secretion and the expression of their receptors are strongly connected with the cyclic changes in endometrium and its receptivity. We previously reported that the frequency of occurrence of p16-positive senescent cells in human endometrium appear to be also associated with endometrial receptivity and successful embryo implantation. The objective of this study was to analyze the relationship between the percentage of senescent cells and estrogen and progesterone receptors expression in human endometrium during the mid-luteal phase of the menstrual cycle. Prospective observational study. This study includes 383 women who had an endometrial biopsy during the mid-lutheal phase (7 days after LH surge) of the natural cycles. Tissue sections were stained immunohistochemically for ER, PR and p16 using the following monoclonal antibodies: progesterone receptor (PR) (NCL-PGR-312, Leica Microsystems), 1:100 dilution; estrogen receptor (ER) (NCL-L-ER-6F11, Leica Microsystems), 1:100 dilution; p16 ink4a antibody (MAD-000690QD–7, Master Diagnostica), ready-to-use. The percentage of positively stained p16 cells and the levels of ER/PR expression were evaluated by Image-J software (NIH, Maryland, USA) in multiple endometrial sections. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). No correlation was found between the frequencies of occurrence of senescent p16-positive stromal, luminal and glandular epithelial cells and PR (p>0.05). In contrast, the glandular expression of ER showed low, but a significant correlation with the percentage of p16+ glandular epithelial cells (R=0.34; p=0.03). In conclusion, we found that the increased cell senescence in endometrial glands during the mid-luteal phase is associated with increased expression of glandular ER in the mid-luteal phase of the cycle. These results suggest an estrogen related change of endometrial senescent cells during the period of embryo implantation.

Synergistic Effect of Resveratrol with 5-Fluorouracil in Chemotherapy of Induced Oral Squamous Cell Carcinoma

Aim: To evaluate the anticancer effect of resveratrol alone or combined with 5-Fu on experimentally- induced tongue cancer in rats. Methodology: Forty male albino rats were divided into five groups: Group I: untreated (negative control), group II: positive-control, receiving 50 ppm 4-NQO in drinking water, group III: received i.p injection of (5-FU), group IV: oral-administration of resveratrol (RV), and group V: receiving both RV and 5-FU. Before euthanasia, blood samples were withdrawn to evaluate serum level of ALT, AST and creatinine. After euthanasia, the tongues were surgically excised, and processed for H&E, and mutant p53 IHC. The mean of positive cells/group were statistically analyzed. Results: Group I showed normal tongue mucosa with negative immune expression of mutant p53 cells. Group II revealed malignant ulcers in a form of well/moderately differentiated SCC, and the highest mean of mutant p53 immuno-expression. Group III showed mild dysplasia of the tongue epithelium with a significant regression of the mean number of mutant p53 positive-cells. Group IV expressed moderate dysplasia, with a significantly high mean of mutant p53. Group V showed mild dysplasia with comparable mean of mutated p53 positive cells. Regarding toxicity, group II showed the maximum elevation of ALT, AST, and serum creatinine. These levels were decreased significantly in the treated groups, but high compared to Group I. Conclusion: Resveratrol has a promising therapeutic effect against 4NQO- induced tongue cancer as 5-FU alone. Also, it diminished the cytotoxic effect of 5-FU and enhanced normal histologic-appearance of tongue mucosa when used in combination.