Overexpression Of P53 Protein Research Articles

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.

Newly synthesised Schiff base metal complexes, characterisation, and contribution as enhancers of colon cancer cell apoptosis by overexpression of P53 protein

Fluoroquinolones emerged as one of potent therapeutic agents, targeting DNA‐signalling pathways. Thus, efforts were spent to create novel quinolones for optimizing their antimicrobial and anticancer properties. Therefore, the new compoundN,N′‐phenylene (bis1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐1,4‐dihydroquinoline‐3‐carboxylic acid) (H2Enro‐o‐phdn) in addition to its related chelates‐contained metals were synthesised and described by spectroscopic, physical methods and thermal analyses. The reaction between H2Enro‐o‐phdn and trivalent La(III), Y(III), and Fe(III) and tetravalent Zr(IV) chloride with molar ratio 1:1 (M:H2Enro‐o‐phdn) yielded the production of chelates. The infrared results elucidate the binding mode of H2Enro‐o‐phdn by means of azomethine nitrogen and carboxylato oxygen atoms as tetradentate. The thermal analyses assured the proposed formula as well as existence of coordinated and latticed H2O molecules. Kinetic parameters for investigated metal complexes were quantified utilisation Horowitz–Metzger in addition to Coats–Redfern methods. Regarding antibacterial efficacy, Y(III) and Fe(III) complexes showed a robust inhibitory activity towards negative‐gram stained (Escherichia coliandSalmonella typhi) and positive‐Gram stained (Staphylococcus aureusand “spore‐forming”Bacillus cereus) strains. Then, we further tested the cytotoxicity of the complexes against the survival of colon‐cancer cells line (CT26). In this regard, in the descending order, Fe(III) &gt; Zr(IV) &gt; La(III) complexes possessed a powerful antitumour activity with IC50values 6.38, 5.36, and 4.03 μM, respectively, compared with the anticancer drug‐reference cisplatin (16.77 μM). To understand the precise mechanism behind the tumour cell death, Western‐blotting analysis was conducted, to monitor the changes in levels of pro‐apoptotic “P53” protein after drug‐treatment. Results indicated that H2Enro‐o‐phdn with higher covalency metals, like La(III) and Zr(IV) complexes, had the highest upregulation of P53 expression in a threefold to fourfold compared cisplatin, paving the way for a novel P53‐based colon‐cancer therapies.

A New Insight Into p53-Inhibiting Genes in Epstein–Barr Virus-Associated Gastric Adenocarcinoma

The p53 mutation is uncommon in Epstein–Barr virus-linked gastric carcinoma, but its suppression occurs through mechanisms such as ubiquitin specific peptidase 7 (USP7) inhibitions via Epstein–Barr virus nuclear antigen-1 (EBNA1) activity. This study aimed to evaluate the effect of EBNA1 on p53-inhibiting gene expression and the impact of USP7 inhibition on p53 suppression. MKN-45 cells were transfected with the EBNA1 plasmid. A stable EBNA1 expression cell line was developed through selection based on hygromycin B resistance. Murine double minute (MDM)4, MDM2, sirtuin (SIRT)3, histone deacetylase (HDAC)1, proteasome 26S subunit, Non-ATPase (PSMD)10, USP7, and p53 expression were checked using real-time PCR. Also, cells containing EBNA1 or control plasmid were treated with GNE-6776, and the expression of the interested genes and cell survival were assessed. MDM4, MDM2, and PSMD10 were significantly upregulated in the MKN-45 cell line following EBNA1 transfection. Morphological changes were observed in the cells harboring EBNA1 after 20 days. In the control cells, USP7 inhibition significantly upregulated the HDAC1, PSMD10, MDM4, and MDM2 genes after 24 h, but downregulated these genes after four days. In the EBNA1-harboring cells, MDM2, MDM4, and PSMD10 genes were significantly upregulated after 24 h, and this effect was sustained for all genes except for MDM4, even after four days. Furthermore, USP7 inhibition induced apoptosis in both cell groups. EBNA1 enhances the expression of p53-inhibiting genes. Two events—p53 protein overexpression and apoptosis activation—followed the suppression of the USP7 protein and provided evidence for its possible function. The significance of the EBNA1-USP7 interaction in p53 suppression warrants additional investigation and possibly reconsideration.

Comparative study of the cytotoxicity, apoptotic, and epigenetic effects of Boswellic acid derivatives on breast cancer

This study aimed to compare the effect of Boswellic acid derivatives on the viability, apoptosis, and epigenomic profiling of breast cancer. According to the viability assays, 3-O-acetyl-11-keto-β-Boswellic acid (AKBA) showed more toxicity against MDA-MB-231 cells when compared with the 3-O-acetyl-β-Boswellic acid (ABA). In contrast, ABA revealed less toxicity against MCF-10A. Cell cycle and apoptosis assays determined the maximum apoptotic effect of AKBA on MCF-7, and MDA-MB-231 cells. Interestingly, β-Boswellic acid (BA) and ABA did not promote the apoptosis in MCF-10A cells. Transwell migration assay indicated the greatest normalized inhibition (around 160%) in the migration of MDA-MB-231 cells induced by AKBA. The expression of P53, BAX, and BCL2 genes in cancerous cell lines has affirmed that both AKBA and ABA could induce the maximal apoptosis. Western-blot investigation demonstrated that the maximum over-expression of P53 protein (1.96 times) was caused by AKBA in MDA-MB-231 cells, followed by ABA in MCF-7 cells. The BCL2 protein expression was in agreement with the previously reported results. The global DNA methylation in both cancerous cells was reduced by ABA. These results suggest that ABA represented more epigenetic modulatory effect while AKBA shows more cytotoxic and apoptotic effect against breast cancer cell lines.

EXPRESSION OF P53 IN COLORECTAL CARCINOMAS AND ITS CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS

Colorectal cancer is one of the most life-threatening gastrointestinal diseases encountered in clinical practice.1 The rectum is one of the most frequently involved sites and accounts for 25% of primary colorectal cancers.2 Accumulation of molecular alterations, including K-ras, P53, Bcl-2 and adenomatous polyposis coli, contribute to colorectal carcinogenesis.3 According to the literature, the deletion of p53 with the overexpression of p53 protein is correlated with a low rate of survival, thus being an independent prognosis factor.17 Summary: Overexpression of p53 protein is associated with high grade of colorectal carcinoma.

Comparison of biomarker expression in oral lichen planus and oral lichenoid lesions.

Oral lichen planus (OLP) and oral lichenoid lesions (OLL) comprise a group of oral mucosal disorders that have similar clinical and histological features. To compare the levels of investigated biomarkers in biopsied OLP and OLL, and to determine the pattern of biomarkers, which could be useful for the biological characterization of these 2 disorders. A total of 56 biopsy specimens in 2 groups were analyzed in this study. One group consisted of 25 idiopathic OLP lesions, and the other included 31 OLL from patients treated with antihypertensive and cardiac medications. The expression of protein p53, topoisomerase I (topo I), heat shock protein 90 (HSP90), and E-cadherin was analyzed using immunohistochemistry. The p53 protein expression showed a trend to a positive correlation with topo I expression in the total sample (p = 0.067, R = 0.25). The p53 protein and HSP90 expression was higher in the OLL group compared to the OLP group, but the difference was not statistically significant. No association was found between topo I and E-cadherin expression for either the OLP or OLL group. The findings of this study suggest that the slightly higher protein p53 and HSP90 expression in the OLL group might be caused by the medications used. The slight association between p53 and topo I expression indicates that the cooperation between these proteins might be essential for the growth of OLP/OLL in general. We conclude that the overexpression of p53 protein and high expression of topo I found in both types of lesions might induce their biologically aggressive behavior.

Cytotoxicity, genotoxicity, and immunohistochemical expression of p53 in the oral mucosal epithelium of adults following cone-beam computed tomography.

This study sought to assess the cytotoxicity, genotoxicity, and immunohistochemical (IHC) expression of p53 in the oral mucosal epithelium of adults following cone-beam computed tomography (CBCT). This before-and-after observational study evaluated 30 patients (15 males and 15 females); mean age 35 years, who required CBCT. Exfoliating epithelial cells of the buccal mucosa were collected with a cotton swab immediately before and 10 days after CBCT. Following Papanicolaou staining, genotoxicity was evaluated by the micronucleus assay. The frequencies of pyknosis, karyorrhexis, karyolysis, budding and binucleation as cytotoxicity factors were also recorded. Expression of p53 was evaluated by IHC staining. Data were analyzed by paired samples t-test. Micronucleated cells, pyknosis, karyorrhexis, karyolysis, budding, cytotoxicity, and expression of p53 increased significantly after CBCT. CBCT exerts genotoxic and cytotoxic effects and leads to overexpression of p53 protein in the oral buccal mucosal cells.

LMP1 Induces p53 Protein Expression via the H19/miR-675-5p Axis.

ABSTRACTEpstein-Barr virus (EBV), a ubiquitous oncogenic herpesvirus, infects more than 90% of the adult population worldwide. The long noncoding RNA H19 is downregulated in EBV-positive gastric cancer (EBVaGC) and nasopharyngeal cancer (NPC). In this study, we found that loss of H19 is caused by hypermethylation status of the H19 promoter in EBV-positive GC and NPC cell lines. Furthermore, latent membrane protein 1 (LMP1), encoded by EBV, induced H19 promoter hypermethylation and deregulated the expression of H19 by upregulating DNMT1 expression. Transwell assays showed that H19 promoted cell migration. Furthermore, H19 promoted cell proliferation and inhibited apoptosis in CCK-8 and flow cytometry assays, respectively. p53, a well-known tumor suppressor, was upregulated in EBVaGC and NPC cell lines. miR-675-5p derived from H19 inhibited p53 protein expression by targeting the 3′ untranslated region of the gene. Overall, we found that LMP1 induced p53 protein expression via the H19/miR-675-5p axis in EBVaGC and NPC. LMP1 induced H19 promoter hypermethylation, which repressed the expression of H19 and miR-675-5p and caused p53 protein overexpression in EBVaGC and NPC cells.IMPORTANCE Epstein-Barr virus (EBV) is the first virus to be known to have direct association with human cancer and to be considered as an important DNA tumor virus. The EBV life cycle consists of both latent and lytic modes of infection in B lymphocytes and epithelial cells. The persistence of EBV genomes in malignant cells promoted cell growth. p53, acting as a critical gatekeeper tumor suppressor, is involved in multiple virus-mediated tumorigeneses. Overexpression of p53 inhibits the ability of BZLF1 (EBV-encoded immediate early gene) to disrupt viral latency. In our study, we found LMP1 induces H19 promoter hypermethylation, which represses the expression of H19 and miR-675-5p and results in p53 protein overexpression in EBVaGC and NPC cells. These observations suggest a new mechanism of aberrant expression of p53 by LMP1, which facilitates EBV latency.

The Proportion of p16 Expression in Penile Squamous Cell Carcinoma Based on Immunohistochemistry Examination in the Balinese Population

Background: : Penile cancer is a rare type of cancer in men and was the highest in Bali compared to other regions in Indonesia. Around 95% of malignancies in the penis are squamous cell carcinoma (SCC) type. One of the risk factors associated with penile squamous cell carcinoma is an infection caused by Human Papillomavirus (HPV). Human Papillomavirus E6 and E7 oncoproteins play a significant role in penile squamous cell carcinoma carcinogenesis. Overexpression of p16 protein can be used as a marker of HPV infection. This study aims to determine the expression of p16 protein in penile squamous cell carcinoma.Methods: This study was conducted to determine the prevalence of p16 protein expression was assessed by immunohistochemical examination from paraffin block tumors of penile cancer patients. Expression of p16 was observed to be positive when stained with a strong brown color in the nucleus and cytoplasm of tumor cells. The analysis method used in this study is a descriptive statistic to provide a general description of the characteristics of each research variable. The data were processed using SPSS to determine the frequency and median of the age of patients with penile squamous cell carcinoma and the prevalence of p16 protein expression in cell penile squamous cell carcinoma. The data shown in this study are the age range and prevalence of p16 protein expression in penile squamous cell carcinoma using the prevalence table.Results: This study used 142 penile squamous cell carcinoma samples derived from biopsy, partial, and total penectomy. The results of the IHC examination showed that the expression of p16 protein was positive in penile cancer tumor cells in 68 cases (47.9%), with the highest age distribution in the 50–59-year age group (28.2%).Conclusions: Further research is needed to confirm the correlation between HPV infection and p16 protein overexpression in penile squamous cell carcinoma, including HPV genotyping

Chrysin and Capsaicin induces premature senescence and apoptosis via mitochondrial dysfunction and p53 elevation in Cervical cancer cells

Current studies are focusing on the anti-cancerous properties of natural bioactive compounds, primarily those included in the human diet. These compounds have the potential to alter the redox balance that can hinder cancer cell's growth. In cancer cells, an abnormal rate of ROS production is balanced with higher antioxidant activities, which if not maintained, results in cancer cells being prone to cell death due to oxidative stress. Here, we have analyzed the effects of Chrysin and Capsaicin on the HeLa cells viability and cellular redox signaling. Both these compounds stimulate cellular and mitochondrial ROS overproduction that perturbs the cellular redox state and results in mitochondrial membrane potential loss. Apart from this, these compounds induce cell cycle arrest and induce premature senescence, along with the overexpression of p21, p53, and p16 protein at lower concentration treatment of Chrysin or Capsaicin. Moreover, at higher concentration treatment with these compounds, pro-apoptotic activity was observed with the high level of Bax and cleaved caspase-3 along with suppression of the Bcl-2 protein levels. In-Silico analysis with STITCH v5 also confirms the direct interaction of Chrysin and Capsaicin with target protein p53. This suggests that Chrysin and Capsaicin trigger an increase in mitochondrial ROS, and p53 interaction leading to premature senescence and apoptosis in concentration dependent manner and have therapeutic potential for cancer treatment.

Sarcomatoid Yolk Sac Tumor Harbors Somatic Mutations That Are Otherwise Rare in Testicular Germ Cell Tumors.

In testicular germ cell tumors (TGCTs), components with nonspecific sarcomatous features that express keratins and glypican 3 are classified as sarcomatoid yolk sac tumor (SYST). SYST is most frequently seen in metastatic sites after chemotherapy. Like so-called "somatic-type" malignancies arising in TGCTs, SYST is markedly resistant to systemic therapy and has a more aggressive clinical course than conventional types of TGCT. However, the clinicopathologic and molecular features of SYST remain incompletely described. This study evaluated a multi-institutional series of 20 SYSTs using massively parallel sequencing and p53 immunohistochemistry. The histologic and clinical characteristics of the cases were also assessed, including analyses of disease-specific outcomes. DNA sequencing identified somatic mutations in 12/20 cases (60%), including recurrent TP53 and RIF1 mutations (present in 4/20 cases, 20% each). In 3 of the 4 SYST with TP53 mutations, there was molecular evidence of loss of heterozygosity. Immunohistochemistry demonstrated diffuse overexpression of p53 protein in 3/4 (75%) cases with TP53 mutations. The remaining TP53-mutant case demonstrated multifocal overexpression of p53, suggestive of subclonal inactivation of the gene. Overexpression of p53 protein was not seen in any of 15 TP53 wild-type cases evaluated by immunohistochemistry. A subset of 4 cases underwent RNA sequencing (fusion panel), which demonstrated the absence of oncogenic gene fusions. A 2-tiered grading system based on 3 histologic parameters (cellularity, number of mitoses, and necrosis) demonstrated that high-grade SYSTs have a higher risk of disease-specific death compared to low-grade tumors. The risk of disease-specific mortality was also higher in SYSTs with somatic mutations. In conclusion, this study demonstrated that 60% of SYSTs harbor somatic oncogenic mutations that are otherwise rare in TGCTs, and the presence of these mutations is associated with an aggressive clinical course. In addition, the results presented herein suggest that grading SYSTs may be clinically relevant.

Determining the probability of malignant transformation of tobacco-induced oral leukoplakia using tissue p53 as a prognostic marker – A cross-sectional study

Context: The concept of field cancerization necessitates substantiation of clinical assessment and histopathological examination by molecular markers. Molecular tumor biomarkers such as p53 protein overexpression aid in assessing the malignant transformation of such potentially malignant oral lesions. Aim: To identify the molecular changes in oral leukoplakia by studying the immunoexpression of tissue p53 and correlating it with the clinical and histological findings to establish an appropriate treatment plan. Methods and Material: Biopsy samples taken from 25 oral leukoplakia cases were subjected to histopathological and immunohistochemical analysis to determine the percentage positivity of p53 expression. Histopathological grading was based on dysplastic features and level of epithelial involvement. Dysplasia grading in IHC sections was estimated by p53% positivity in the cells. The obtained data were subjected to statistical analysis. Results: 24 samples were considered for statistical analysis due to the loss of epithelium in the IHC section of one sample. The results showed a statistically significant association between histopathological and IHC grading of dysplasia with a P value less than 0.05 in Fisher Exact test. Conclusion: Increased expressions of p53 in potentially malignant oral lesions are proportional to the risk of malignancy. The gold standard histopathological result does not reveal the molecular abnormality associated with a potentially malignant oral lesion. Therefore molecular analysis of such lesions will aid in the effective prevention of oral cancer.

Loss of p21ras, p53, and Mdm2 Protein Expression Is Indicative of Worse Prognosis in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis and refractory cytopenias that can progress to acute leukemia. In order to identify molecular events implicated in the pathogenesis of MDS, we analyzed the bone marrow expression and mutational status of ras, p53 and mdm2 genes in a cohort of 25 patients with MDS. The expression of p21ras, p53, and Mdm2 proteins was studied in marrow cytopins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10 to detect p21ras, p53 and mdm2 proteins, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of gene mutations. The quantitative wild-type expression of p21ras, p53 and mdm2 proteins in patient samples (combination of number of cells and staining intensity) was compared to the values of expression of these proteins in 7 normal bone marrow samples. We found overexpression of p21ras and p53 proteins in 12/22 (55%) and 7/23 (30%) of the patients. Five of 7 patients who overexpressed wt-p53 also co-overexpressed wt-p21ras protein. We observed absence of expression of p21ras, p53 and mdm2 in 4/22, 4/23 and 9/19 cases, respectively. Overall survival of patients with absence of p21ras and p53 expression was considerably worse than that of patients with normal expression of these proteins (p=0.0006 to p21ras and p=0.004 to p53 by Log-Rank test; p=0.02 to p21ras and p=0.01 to p53 by Cox proportional hazards model after age, neutrophil and platelet count, and hemoglobin adjustment). We also observed that the patients who exhibited hypoexpression (p=0.055) and absence of mdm2 expression (p=0.07) demonstrated a tendency to have worse prognosis (Log-Rank). There was no correlation between variables p21ras, p53 and mdm2 versus hemoglobin, platelet and neutrophil counts and no association between FAB SMD subcategories and absence of p53 and mdm2 expression by Fisher test (two-tailed), but there was a strong over-representation between absence of p21ras expression and MDS with excess of blasts (p=0.0006). There was a significant correlation between p53 and mdm2 gene expression (p=0.03, Pearson). Only one patient harbored one transversion mutation in codon 12 of K-ras gene and did not correlate with p53 overexpression, but with worse prognosis. No p53 mutation was found. Overall, these findings suggest that the via of signal transduction p21ras/p53 and the auto-regulatory genes p53/mdm2 may be active and play a pivotal role in the pathogenesis of MDS. Hence, loss of bone marrow expression of p21ras, p53 and/or mdm2 proteins is an indicator of worse prognosis in MDS and may serve as early markers of leukemic transformation.

Effect of pharmacodynamical interaction between nutlin-3a and aspirin in the activation of p53

Background and objectivep53, an anti-tumour protein, is significantly inactivated in most tumours. A small molecule of nutlin-3a is used to activate its function by repressing (Mouse double minute 2 homolog) Mdm2 protein which inhibits its activity. In cancer patients, a high risk of drug-drug interactions (DDIs) is observed owing to their multi-dosing prescriptions, which may lead them to harmful effects. In the presented work, we have aimed to investigate the effect of pharmacodynamical interaction between two anti-cancer drugs, nutlin-3a and aspirin in the activation of p53 protein. MethodsWe have adapted control system techniques and designed a Proportional-Integral-Derivative (PID) controller. This controller is used to activate p53 protein. A drug interaction parameter is used to incorporate the effect of both drugs. Extensive simulation is performed using two different doses of aspirin, i.e. a low and a high dose of aspirin. ResultsThe result shows no harmful effects of pharmacodynamical interaction when a low dose is administered along with nutlin-3a. When a high dose of aspirin is administered it acts as input disturbance and leads to undesirable over-expression of p53 protein. This can further harm other growth cells, thus inducing harmful effects. A comparative analysis is also tabulated with different dosing regimens which shows that a combination of nutlin-3a and a low dose of aspirin provides better results than a high dose of aspirin. ConclusionOverall, the work provides an insight to the activation of p53 protein in cancer patients under the presence of pharmacodynamical interaction and might contribute to the effective management of cancer patients.

The Role of Immunohistochemical Markers for the Diagnosis and Prognosis of Adrenocortical Neoplasms.

Adrenal cortical carcinoma (ACC) is a rare cancer with poor prognosis that needs to be distinguished from adrenocortical adenomas (ACAs). Although, the recently developed transcriptome analysis seems to be a reliable tool for the differential diagnosis of adrenocortical neoplasms, it is not widely available in clinical practice. We aim to evaluate histological and immunohistochemical markers for the distinction of ACCs from ACAs along with assessing their prognostic role. Clinical data were retrospectively analyzed from 37 patients; 24 archived, formalin-fixed, and paraffin-embedded ACC samples underwent histochemical analysis of reticulin and immunohistochemical analysis of p27, p53, Ki-67 markers and were compared with 13 ACA samples. Weiss and Helsinki scores were also considered. Kaplan−Meier and univariate Cox regression methods were implemented to identify prognostic effects. Altered reticulin pattern, Ki-67% labelling index and overexpression of p53 protein were found to be useful histopathological markers for distinguishing ACAs from ACCs. Among the studied markers, only pathological p53 nuclear protein expression was found to reach statistically significant association with poor survival and development of metastases, although in a small series of patients. In conclusion, altered reticulin pattern and p53/Ki-67 expression are useful markers for distinguishing ACCs from ACAs. Immunohistopathology alone cannot discriminate ACCs with different prognosis and it should be combined with morphological criteria and transcriptome analysis.

Prognostic role of immunohistochemical overexpression of the p16 protein in women under the age of 35 and diagnosed with HSIL (CIN2) subjected to "cervix sparing" excision.

To evaluate the role of immunohistochemical staining overexpression of p16 protein (p16 IHC) as a prognostic factor of persistence or recurrence of intraepithelial disease after excision procedure in young women diagnosed with HSIL (CIN2). 62 women with a histological diagnosis of HSIL (CIN2) subjected to "cervix sparing" excisional procedure were included in this retrospective study. All had age less than or equal to 35 years, negative history of immunosuppression, available follow-up, and assessment of the resection margins state. Immunohistochemical staining for the p16 protein was evaluated on reviewed and confirmed HSIL (CIN2) histological specimens with negative resection margins. The post-treatment follow-up, including cytology, colposcopy, and histology, ranged from a minimum of 6 months to a maximum of 60 months. The persistence or recurrence of SIL during the follow-up period was based on histologic referral and defined as "the presence of SIL", "the presence of HSIL" and "progression to HSIL (CIN3)". 31/62 patients were positive for immunostaining (p16 IHC+), and 31/62 were negative (p16 IHC-). Persistence or recurrence after excision occurred more frequently within the p16 IHC+ than in p16 IHC- group, both as SIL (29% p16 IHC- vs. 32.3% p16 IHC+, p = 0.783) and HSIL (6.5% p16 IHC- vs. 12.9% p16 IHC+, p = 0.671). None of the patients in the p16 IHC- group showed progression to CIN3 for the entire observation period, whereas 9.7% of p16 IHC+ women progressed to CIN3 lesion (p = 0.042). The p16 IHC positivity showed a significant association with progression to CIN3 in 5 years of follow-up (p = 0.029) and with the presence of SIL after two years of follow-up (p = 0.031). The differences between the two groups increased after two years post-treatment: the p16 IHC- patients still had SIL only in 3.2% of cases and no longer had HSIL, while the p16 IHC+ women still showed SIL in 19.4% and HSIL in 6.5% of cases. The negative predictive value (NPV) of p16 IHC in predicting SIL's presence after treatment increased with the severity of the lesion (NPV for SIL 70.97%, for HSIL 93.55%, for CIN3 100%). The study suggests that young patients with p16 IHC- HSIL (CIN2) have a better post-excisional course of the cervical intraepithelial disease compared to p16 IHC+ women and that p16 IHC could have prognostic utility during the long-term follow-up, especially in forecasting progression to CIN3 in consideration of the high NPV (up to 100%). The efficacy of the adjuvant HPV vaccination in the management of HSIL (CIN2) p16+ young women is to be evaluated as part of the fertility-sparing treatment.

Abstract PR006: Correlation of immunohistochemistry with TP53 sequence and clinical outcomes in GOG-086P

Abstract Introduction: GOG-086P was one of the first attempts to combine molecular inhibitors such as bevacizumab or temsirolimus with chemotherapy in patients with advanced endometrial cancer. An exploratory analysis of this study indicates that bevacizumab combined with chemotherapy is superior to temsirolimus plus chemotherapy in patients with TP53 mutated tumors as determined by next generation sequencing. The functional status of p53 in a tumor can be inferred by sequence analysis and/or by immunohistochemistry (IHC). In this next study of the GOG-086P cohort, we sought to determine whether p53 IHC alone or integrated with TP53 sequencing was similarly predictive of outcome and whether IHC was predictive of the sequence analysis. Methods: A total of 349 patients were enrolled on GOG-086P. Two hundred and forty-three patients have TP53 sequence data, and 213 have p53 protein expression data measured by IHC. Tumor histology and sequence data were correlated with p53 IHC categorizing each case as p53 negative or null, wild type (WT) or over-expressed (OE), and these variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy + bevacizumab arms versus the chemotherapy + temsirolimus arm. Results: The majority of patients (118) had tumors with WT p53 protein expression by IHC and non-mutated or WT TP53 sequence, with expected concordance between IHC and sequence analysis. However, there were five discordant cases with OE p53 protein in the absence of a TP53 mutation. Fourteen patients had null IHC protein expression and a concordant TP53 sequence mutation expected to result in the total loss of protein. Four cases that were p53 null by IHC had a discordant WT TP53 sequence. Seventeen patients had tumors with a missense TP53 mutation but had WT p53 protein expression by IHC, reflecting a potential discordance between sequencing and IHC versus a missense mutation that did not result in loss of protein function. Six of these 17 discordant cases may be explained by co-mutations in TP53 and POLE or microsatellite instability where p53 proteins, though mutated, function more like WT. Reassuringly and as expected, high concordance was found in the 55 cases that had OE protein by IHC and a missense mutation in TP53. On integrated analysis, a TP53 mutation with p53 protein OE was associated with significantly lower hazard ratio for PFS (HR=0.41; 95% CI 0.22-0.83) and OS (HR=0.28; 95% CI 0.14-0.59), i.e., longer PFS and OS, for patients receiving bevacizumab + chemotherapy relative to temsirolimus + chemotherapy. Conclusions: Mutations in TP53, which are common in patients with advanced endometrial cancer, represent a developing platform upon which to design personalized treatment regimens. We demonstrate that IHC is often but not always predictive of the TP53 sequence. However, most cases with over-expressed p53 protein by IHC had a mutation in TP53, and bevacizumab + chemotherapy prolonged PFS and OS in such patients. Citation Format: Kristina W. Thiel, Eric J. Devor, Virginia Filiaci, Douglas A. Levine, Fanny Dao, Narciso Olvera, David Mutch, Carol Aghajanian, Kimberly K. Leslie. Correlation of immunohistochemistry with TP53 sequence and clinical outcomes in GOG-086P [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR006.

Expression of p53 and Ki-67 proteins in patients with increasing severity and duration of pterygium.

Purpose:Pterygium is a triangular fibrovascular subepithelial ingrowth of degenerative bulbar conjunctival tissue over the cornea. It is now considered to be a result of uncontrolled cellular proliferation as overexpression of p53 protein and Ki-67 nuclear protein was found in the epithelium. This study was done to find the expression of p53 and Ki-67 with the severity and duration of the pterygium to explain the etiopathogenesis.Methods:Data were analyzed from 43 Indian participants of all age groups. All patients were divided according to the severity of pterygium (mild, moderate, and severe groups) and according to the duration of pterygium (<4 years and >4 years). The samples were studied by immunohistochemistry by using antibodies against p53 and Ki-67 proteins considering >5% expression as significant.Results:Of 43 cases, p53 and Ki-67 expression were positive in 33 cases. In mild, moderate, and severe cases p53 positivity was 33.3%, 78.4%, 100%, respectively. P53 expression increased with duration, 79.3% positive in <4 years, and 92.9% positive in >4 years. With increasing severity of pterygium, mild, moderate, and severe cases, Ki-67 positivity was 66.7%, 78.37%, 66.7%, respectively. Ki-67 expression with duration, 79.3% positive in <4 years, and 85.7% positive in >4 years of the duration of pterygium with no statistical significance.Conclusion:Our study revealed that with increasing duration and severity of pterygium, p53 expression was observed to be increasing. Ki-67 expression increased with the duration of pterygium but not with the severity.

Characterization of acquired nutlin-3 resistant non-small cell lung cancer cells.

Aim: The purpose of this manuscript is to study the potential characteristics of acquired nutlin-3 resistant non-small cell lung cancer cells (NSCLC). Nutlin-3 is an inhibitor of the murine-double minute 2 protein, the main negative regulator of wild type p53, of which several derivatives are currently in clinical development.Methods: A549 NSCLC cells were exposed to increasing concentrations of nutlin-3 for a period of 18 weeks. Monoclonal derivates were cultured, and the most resistance subclone was selected for whole transcriptome analysis. Gene set enrichment analysis was performed on differentially expressed genes between A549 nutlin-3 resistant cancer cells and the parental A549 p53 wild type cancer cells. Relevant findings were validated at the gene, protein and/or functional level.Results: All nutlin-3 resistant subclones acquired mutations in the TP53 gene, resulting in overexpression of the mutant p53 protein. The most resistant subclone was enriched for genes related to epithelial to mesenchymal transition (EMT), resulting in increased migratory and invasive potential. Furthermore, these cells were enriched in genes related to inflammation, tissue remodelling, and angiogenesis. Importantly, expression of several immune checkpoints, including PD-L1 and PD-L2, was significantly upregulated, and cisplatin-induced cell death was reduced.Conclusion: Transcriptome analysis of a highly nutlin-3 resistant A549 subclone shows the relevance of studying (1) resistance to standard of care chemotherapy; (2) secretion of immunomodulating chemo- and cytokines; (3) immune checkpoint expression; and (4) EMT and invasion in nutlin-3 resistant cancer cells in addition to acquired mutations in the TP53 gene.

In situ self-assembly of near-infrared-emitting gold nanoparticles into body-clearable 1D nanostructures with rapid lysosome escape and fast cellular excretion

The integration of strong near-infrared (NIR) emission, rapid lysosome escape, fast cellular excretion, and efficient total body clearance is highly desired for nanoparticles (NPs) to achieve synergistic functions in both molecular imaging and delivery. Herein, using a well-designed cyclopeptide (CP) that can spontaneously assemble into controllable nanofibers as template, a facile strategy is reported for in situ self-assembly of NIR-emitting gold NPs (AuNPs) into ordered and well-controlled one-dimensional (1D) nanostructures (AuNPs@CP) with greatly enhanced NIR emission (∼ 6 fold). Comparing with the unassembled AuNPs, the AuNPs@CP are observed to enter living cells through endocytosis, escape from lysosome rapidly, and excrete the cell fast, which shows high gene transfection efficiencies in construction of cell line with ∼ 7.5-fold overexpression of p53 protein. Furthermore, the AuNPs@CP exhibit high in vivo diffusibility and total body clearance efficiency with minimized healthy organ retention, which are also demonstrated to be good nanovectors for plasmid complementary deoxyribonucleic acid 3.1 (pcDNA3.1)(+)-internal ribosome entry site (IRES)-green fluorescent protein (GFP)-p53 plasmid with efficient p53 gene over-expression in tumor site. This facile in situ strategy in fabricating highly luminescent 1D nanostructures provides a promising approach toward future translatable multifunctional nanostructures for delivering, tracking, and therapy.