Analysis Of P53 Expression Research Articles

Molecular cloning, characterization and expression analysis of P53 from high latitude fish Phoxinus lagowskii and its response to hypoxia.

As an intermediate link between multiple cellular stresses and cellular responses, p53, together with its upstream and downstream regulators and related genes, constitutes a complex network that regulates cellular stresses and cellular responses. However, no studies have investigated p53 in Phoxinus lagowskii, particularly the expression of p53 under different hypoxic conditions. In the present study, the cDNA of p53 from the Phoxinus lagowskii was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Pl-p53 was 1878bp, including an open reading frame (ORF) of 1116bp encoding a polypeptide of 371 amino acids with a predicted molecular weight of 41.22kDa and a theoretical isoelectric point of 7.38. Quantitative real-time (qRT) PCR assays revealed that Pl-p53 was commonly expressed in all tissues examined, with highest expression in the heart. In addition, we investigated the expression of Pl-p53 in different tissues under different hypoxic conditions. In the short-term hypoxia group, Pl-p53 expression was down-regulated in both the brain and heart. The Pl-p53 expression was significantly elevated at 6h in the muscle and liver, and was significantly up-regulated at 24h in spleen. These results suggest that Pl-p53 plays different regulatory roles and provide a theoretical basis for the changes of p53 in fish facing hypoxic environments.

Circulating p16-Positive and p16-Negative Tumor Cells Serve as Independent Prognostic Indicators of Survival in Patients with Head and Neck Squamous Cell Carcinomas.

Background: Decisions regarding the staging, prognosis, and treatment of patients with head and neck squamous cell carcinomas (HNSCCs) are made after determining their p16 expression levels and human papillomavirus (HPV) infection status. Methods: We investigated the prognostic roles of p16-positive and p16-negative circulating tumor cells (CTCs) and their cell counts in HNSCC patients. We enrolled patients with locally advanced HNSCCs who received definitive concurrent chemoradiotherapy for final analysis. We performed CTC testing and p16 expression analysis before chemoradiotherapy. We analyzed the correlation between p16-positive and p16-negative CTCs and HPV genotyping, tissue p16 expression status, response to chemoradiotherapy, disease-free survival, and overall survival. Results: Forty-one patients who fulfilled the study criteria were prospectively enrolled for final analysis. The detection rates of p16-positive (>0 cells/mL blood) and p16-negative (≥3 cells/mL blood) CTCs were 51.2% (n = 21/41) and 70.7%, respectively. The best responses of chemoradiotherapy and the p16 positivity of CTCs are independent prognostic factors of disease progression, with hazard ratios of 1.738 (95% confidence interval (CI): 1.031–2.927), 5.497 (95% CI: 1.818–16.615), and 0.176 (95% CI: 0.056–0.554), respectively. The p16 positivity of CTCs was a prognostic factor for cancer death, with a hazard ratio of 0.294 (95% CI: 0.102–0.852). Conclusions: The p16-positive and p16-negative CTCs could predict outcomes in HNSCC patients receiving definitive chemoradiotherapy. This non-invasive CTC test could help stratify the risk and prognosis before chemoradiotherapy in clinical practice and enable us to perform de-intensifying therapies.

A Novel RNA Synthesis Inhibitor, STK160830, Has Negligible DNA-Intercalating Activity for Triggering A p53 Response, and Can Inhibit p53-Dependent Apoptosis.

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.

Role of p53 Expression in the Progression of Gastric Cancer in the Indian Population

Introduction: Gastric carcinogenesis frequently affects tumor suppressor TP53 gene, resulting in increased expression of p53 protein and mutations towards later stages of Gastric Carcinoma. Objectives: To analyze p53 protein expression in different stages of gastric cancer to determine if it could be used as a biomarker to allow targeted screening of high risk groups with intestinal metaplasia. Methods: We retrieved 101 formalin fixed paraffin embedded tissue blocks of histopathology proven Gastric Carcinoma specimens and conducted an immunohistochemical study on a subset of 25 cases. Tumor sections were taken with adjacent normal mucosa. p53 expression was evaluated using immunohistochemical markers. With the observations from the study, analysis of p53 expression in different areas of the mucosa was done. Result: Thirteen carcinoma cases (52%) expressed p53 protein with a majority of eight (66.7%) cases showing intensity 3. Normal and metaplastic mucosa did not express p53 protein. In dysplastic mucosa, seven (28%) cases showed p53 expression with intensity ranging from 1 to 3. On comparing the data after excluding the p53 negative regions of normal and metaplastic mucosa, there was no difference with statistical significance. Conclusion: p53 expression is negative in normal and metaplastic gastric mucosa but shows expression in dysplastic and cancer cells. Thus, TP53 mutation is most likely an event towards later stages of carcinogenesis. We are dubious on the ability of p53 to be used as an authentic screening tool at the metaplastic stage to predict progression to carcinoma.

Regeneration Potential of Bone Marrow-derived Mesenchymal Stromal Cells Lysate for H2O2 (In-Vitro) Injured Cells

Background: Mesenchymal stromal cells (MSCs) and their paracrine factors make them a suitable cell-free-therapeutic candidate. Cellular lysate usage could be an effective treatment strategy that circumvents the possible associated drawbacks of stem cell therapies. Objective: Thus, this research aims to examine the injury regeneration potential of MSCs cellular lysate derived from bone marrow by studying its anti-apoptotic, proliferative, and anti-oxidative effects. Methods: Hydrogen peroxide (H2O2) was used to induce cellular injury. MTT assay, trypan blue, and crystal violet assays were used to assess bone marrow-derived mesenchymal stromal cell (BMSCs) lysate treated cells' viabilities. Next, to investigate the BMSCs lysate anti-oxidative potential anti-oxidants, ascorbate peroxidase (APX), glutathione reductase (GR), and superoxide dismutase (SOD) assays were performed. Simultaneously, the proliferative and anti-apoptotic potential was measured via vascular endothelial growth factor (VEGF A) and p53 expression analysis through immunostaining and ELISA. Results: It was observed that BMSCs lysate enhances the viability of H2O2 injured cells. APX, GR, and SOD's results indicated that after H2O2 injury, the anti-oxidant status decreased significantly and was uplifted by BMSCs lysate treatment. Additionally, the results of p53, BAX, and caspase-3 expression revealed that BMSCs lysate inhibits apoptosis by downregulating their expression in treated cells. The VEGF protein expression findings demonstrated that BMSCs lysate upregulates the downregulated expression of VEGF in H2O2 injured cells. The expression of proliferative markers (TOP2A, PCNA, and Ki-67) was also elevated in BMSCs treated cells. Conclusion: To conclude this study's findings, it was observed that BMSCs lysate could decrease H2O2 injury and possibly regenerate the injured cells by enhancing their viability and proliferation, improving anti-oxidants levels, and alleviating apoptosis.

P53 Expression analysis in Low Grade and High Grade Urothelial Bladder Carcinoma and the Association with Tumor Stages

BackgroundUrothelial carcinoma is the most common bladder malignancy in men. Risk factors include smoking, chemicals exposure, radiation,schistosoma hematobium infection. P53 mutation is the most common mutation in urothelial bladder carcinoma that cause p53 proteinaccumulation which are detected through immunohistochemical examination. The aim of this study is to analyze the differencesbetween p53 expression in low and high grade urothelial and the carcinomas association of p53 expression with tumor stage.MethodsThis was a cross sectional study with 47 cases (22 cases of low grade and 25 cases of high grade) urothelial carcinoma in AnatomicalPathology Department Faculty Medicine of Universitas Indonesia/Cipto Mangunkusumo Hospital (FKUI/RSCM) in 2009-2017. Allcases stained with p53 antibody with immunohistochemical methods with positive cut off ≥20%.ResultsP53 positive expression are found in 33 samples, 20 cases of high grade and 13 cases in low grade; 22 cases of Nonmuscle invasivebladder cancer and 11 cases of Muscle invasive bladder cancer.ConclusionThe expression of p53 in low grade and high grade urotelial carcinoma of the bladder and its association with tumor stage showed nosignificant difference.

Abnormal Vasculature Development in Zebrafish Embryos with Reduced Expression of Pantothenate Kinase 2 Gene.

Mutations in pank2 gene encoding pantothenate kinase 2 determine a pantothenate kinase-associated neurodegeneration, a rare disorder characterized by iron deposition in the globus pallidus. To extend our previous work, we performed microinjections of a new pank2-specific morpholino to zebrafish embryos and thoroughly analyzed vasculature development. Vessels development was severely perturbed in the head, trunk, and tail, where blood accumulation was remarkable and associated with dilation of the posterior cardinal vein. This phenotype was specific as confirmed by p53 expression analysis and injection of the same morpholino in pank2-mutant embryos. We can conclude that pank2 gene is involved in vasculature development in zebrafish embryos. The comprehension of the underlining mechanisms could be of relevance for understanding of pantothenate kinase-associated neurodegeneration.

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2020.007, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Molecular cloning, characterization and expression analysis of p53 from turbot Scophthalmus maximus and its response to thermal stress.

The tumor suppressor protein, p53 plays a crucial role in protecting genetic integrity. Once activated by diverse cell stresses, p53 reversibly activates downstream target genes to regulate cell cycle and apoptosis. However, few studies have investigated the effects of thermal stress in turbot, specifically the p53 signaling pathway. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot p53 gene (Sm-p53) and perform bioinformatics analysis. The results showed that the cDNA of the Sm-p53 gene was 2928 bp in length, encoded a 381 amino acid protein, with a theoretical isoelectric point of 6.73. It was composed of a DNA binding and a tetramerization domain. Expression of Sm-p53 in different tissues was detected and quantified by qRT-PCR, and was highest in the liver. We also investigated the expression profiles of Sm-p53 in different tissue and TK cells after thermal stress. These result suggested that Sm-p53 plays a key role, and provides a theoretical basis for Sm-p53 changes in environmental stress responses in the turbot.

The Expression of P53, MDM2, c-myc, and P14ARF Genes in Newly Diagnosed Acute Lymphoblastic Leukemia Patients.

The treatment response of acute lymphoblastic leukemia (ALL) depends on the percentage of lymphoblasts, cytogenetic aberrations, and altered gene expression. The analysis of the gene expression is applicable for determination of risk stratification and prognosis of cancers. c-MYC, P14ARF, MDM2, and P53 play a vital role in cell survival through a functional network. This study aimed to investigate the expression of these genes, also their correlation with immunophenotypic subtypes of ALL and the percentage of blasts. Real-time PCR was performed for the expression analysis of P53, MDM2, c-MYC, and P14ARF in the bone marrow or peripheral blood samples of 52 ALL patients and 13 normal samples as controls. The morphological analysis and flow cytometry were carried out to examine the phenotypes and percentage of lymphoblasts. The decreased expression levels of P53 and MDM2 were seen in ALL patients compared with control group. In T cell subgroup of ALL the expression of P14ARF gene was more decreased among other subgroups. The expression of MDM2 was decreased in ALL patients who were under the age of 16. Based on our study, the interaction between P53 and MDM2 might be more complex and different from reports published in previous studies. Our findings showed that MDM2 is not negatively correlated with P53, at least in our samples. It can be very effective on the current and future studies to use different techniques for analysis of genome, transcriptome, and proteome in definitive risk stratification and prognosis determination.

Analysis of p53 expression in partial hydatidiform mole and hydropic abortion

Gestational trophoblastic disease (GTD) is a heterogeneous group of disorders characterized by abnormal trophoblast tissue. Molar and non-molar hydropic placental changes are the most common forms of GTD. Differential diagnosis of GTD is sometimes problematic. Recently, p53 expression was identified as a good marker for distinguishing GTD types. Comparison of p53 expression in partial hydatidiform mole (PHM) and hydropic abortion. In this prospective cross-sectional study, molar and non-molar hydropic pregnancy specimens were collected. Immunohistochemical staining, based on the Labeled Streptavidin Biotin (LSAB) technique, was carried out on multiple 4 mm paraffin block sections prepared from formalin-fixed trophoblastic tissues. Polymer-based Envision was used to assess p53 tumor suppressor protein immunoreactivity. p53 expression was then compared between both groups. In the study, 40 patients were included: 20 with confirmed PHM and 20 with hydropic pregnancy. p53 protein was positive in 60% of patients with PHM and 25% of patients with hydropic pregnancy. The p53 positive rate was significantly higher in patients with PHM (p = 0.027). Moreover, patients with PHM had a significantly high grade of staining (p<0.001). Our findings indicate that immunohistochemical analysis of p53 protein can be used to distinguish PHM and hydropic pregnancy.

Molecular cloning, characterization, and expression analysis of p53 from the oriental river prawn, Macrobrachium nipponense, in response to hypoxia.

The tumor suppressor gene p53 plays a critical role in safeguarding the integrity of the genome in mammalian cells. It acts as a sequence-specific transcription factor. Once p53 is activated by a variety of cellular stresses, it transactivates downstream target genes and regulates the cell cycle and apoptosis. However, little is known about the functions of the p53 pathway in prawns in response to hypoxia. In this study, the cDNA of p53 from the oriental river prawn, Macrobrachium nipponense, (Mnp53) was cloned using a combination of homology cloning and rapid amplification of cDNA ends. The full-length cDNA of Mnp53 has 2130bp, including an open reading frame of 1125bp that encodes a polypeptide of 374 amino acids with a predicted molecular weight of 41.9kDa and a theoretical isoelectric point of 6.9. Quantitative real-time (qRT)-PCR assays revealed that Mnp53 was ubiquitously expressed in all examined tissues, but at high levels in the hepatopancreas. In addition, we studied respiratory bursts and reactive oxygen species (ROS) production in the hepatopancreas of M. nipponense. Our results suggest that oxidative stress occurred in prawns in response to hypoxia and that apoptosis was associated with an increase in caspase-3 mRNA expression. qRT-PCR and western blot results confirmed that hypoxic stress induced the upregulation of Mnp53at mRNA and protein levels. Furthermore, immunohistochemistry showed remarkable changes in immunopositive staining after the same hypoxic treatment. These results suggest that hypoxia-induced oxidative stress may cause apoptosis and cooperatively stimulate the expression of Mnp53.

Cyclooxygenase-2 (COX2) and p53 protein expression are interdependent in breast cancer but not associated with clinico-pathological surrogate subtypes, tumor aggressiveness and patient survival.

In the last decade, different molecular subtypes of breast cancer have been proposed. Although displaying appreciable association with disease prognosis and the prognostic value of cytotoxic and endocrine therapeutic modalities, the subtypes seem to fail at completely explaining disease behavior and response to treatment. Molecules such as those of the cyclocooxigenase (COX) family, currently composed of three entities (COX 1, 2 and 3) have been shown to be associated with breast carcinogenesis, and the analysis of p53 expression in breast tumors may also offer some additional prognostic clues. Our study is aimed at assessing COX2 and p53 expression in these clinico-pathological surrogate subtypes, and to evaluate whether the expression of these molecules can help further explain the variability in prognosis still found within the clinico-pathological subtypes groups of breast cancer. MethodsA total of 183 breast cancer samples were obtained from women treated at the Womeńs Hospital of Campinas State University, Campinas, Brazil, between June 2008 and January 2011. Immunohistochemistry was performed to detect the expression of ER, PR, ki67, COX2, and p53 and the HER2 status of the 183 specimens was assessed using FISH. Two COX2 staining thresholds were used to define COX2 positivity: low threshold (LT): moderate and intense staining were considered positive; high-threshold (HT): only intense staining was considered positive. ResultsThere was no trend in COX2 overexpression from Luminal A-like to Triple-negative subtypes. By contrast, p53 was expressed in roughly 67% of the Luminal A-like tumors, 50% of the Luminal B-like HER2 positive tumors, 60.9% of the Luminal B-like HER2 negative, approximately 82% of the HER2 positive (non-luminal) and 87% of the Triple-negative tumors (p for trends=0.06). There was a significantly higher proportion of COX2 positive (LT) tumors (66.9%) when p53 was also positive compared to when the tumor was negative for p53 (in which case only18.0% of the tumors were positive for COX2; p<0.001). Neither marker was found to be associated with patients‘ survival. Conclusions: There seems to be a positive association between the expressions of COX2 and p53. Otherwise, neither the expression of COX nor that of p53 was associated with clinico-pathological subtypes, tumor features and prognosis. It seems to be too early to elect the detection of COX2 using IHC as prognostic or predictive tool, but incipient evidence points toward a possible role for the marker.

Increased p53 Expression in Patients with Therapy-Related MDS/AML Is Common and May be Associated with a Poor Prognosis

Therapy-related myelodysplastic syndromes and acute myeloid leukaemia (t-MDS/AML) occur as a late complication of cytotoxic chemotherapy and/or radiation for neoplastic and non-neoplastic disorders and are a major cause of non-relapse mortality. The prognosis of t-MDS/AML is poor because of an increased incidence of adverse cytogenetic features and relative resistance/intolerance to conventional therapies. TP53 is a tumour suppressor and cell cycle regulatory protein. Mutations in the TP53 gene are known to be associated with adverse outcomes in a variety of cancers including haematological malignancies.We examined p53 expression using immunohistochemical staining, as a surrogate marker for TP53 mutation, in marrow biopsies of 39 patients with t-MDS/AML from a single institution and correlated p53 expression with survival. Formalin fixed, paraffin embedded marrow trephine samples at diagnosis of t-MDS/AML were stained with DO-7 mouse p53 monoclonal antibody. Positive expression was defined as per Modified Quick Score. Of 39 patients, 35 had t-MDS and 4 had t-AML. 51% of patients had marrows at diagnosis that showed p53 positivity and 49% were negative. In a control group of 47 MDS patients in our centre, 22% were p53 positive and 78% were p53 negative. Of the t-MDS/AML group, age at diagnosis and gender distribution was similar in p53 positive and negative patients. Median latency (time from diagnosis of primary malignancy to diagnosis of t-MDS/AML) was similar between p53 positive and negative patients (66 vs. 52 months, p=0.51). A similar distribution was noted for the type of primary tumours (haematological vs. solid tumour) among the two groups, but all patients with more than one prior malignancy were noted to be p53 positive (N=4) whereas all patients who developed t-MDS after cytotoxic therapy for non-neoplastic disorders were p53 negative(N=3). There was no difference in p53 expression based on the type of primary therapy received (chemotherapy and/or radiotherapy) but a greater proportion (71%) of patients who received combined chemotherapy and radiation was p53 positive. A higher proportion of p53 negative patients had lower risk cytogenetics and normal karyotype whereas in p53 positive group there was lower incidence of good risk and a higher proportion to intermediate risk cytogenetics. However, equal numbers of patients with higher risk cytogenetics were found between p53 positive and negative groups. These findings were again reflected by IPSS-R risk groups (Table 1). Median overall survival was 10.5 months in p53 positive group compared to 22.5 months in p53 negative patients, p= 0.208. (Figure 1)In conclusion, our study demonstrated that a higher proportion of patients with t-MDS/AML were positive for p53 than in de novo MDS which may be related to poor outcomes in this group. p53 positive t-MDS/AML patients tend to have lower incidence of favourable karyotypes and showed poor survival compared to their counterparts who were p53 negative. Analysis of p53 expression by immunohistochemistry is a readily accessible, cost-effective method of assessment without the need for expensive gene sequencing and is a clinically useful prognostic tool. It may be particularly useful in patients with t-MDS/AML.Table 1p53 expression in Therapy-related Myeloid Neoplasmsp53 Positive (N=20, 51%)p53 Negative (N=19, 49%)Primary TumourHaematological (22) Solid Tumour (10) Autoimmune disease (3) More than one disorder (4)45% 60% 0 100%55% 40% 100% 0Primary TherapyChemotherapy alone (27) Radiotherapy alone (5) Combined therapy (7)44% 60% 71%56% 40% 29%Cytogenetic RisksLow (very good, good) (14) Intermediate (8) High (poor, very poor) (12)29% 75% 50%71% 25% 50%IPSS-RLower (very low, low)(12) Intermediate (4) Higher (high, very high) (16)33% 75% 50%67% 25% 50% [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in p53 Expression By Immunohistochemistry during Azacitidine Therapy in Patients with MDS Can Predict Overall Prognosis

IntroductionThe TP53 gene encodes the tumour suppressor and cell cycle regulatory protein and is found to be mutated in a variety of carcinomas. Mutation in TP53 gene is associated with resistance to conventional therapy, disease progression and overall poor prognosis in solid tumours and haematological malignancies including Myelodysplastic Syndromes (MDS). TP53 mutated sub-clones in MDS have been demonstrated by deep sequencing technology in prior studies. Strong nuclear staining of p53 protein by immunohistochemistry has been used as a surrogate marker for TP53 gene mutation in haematological and other malignancies.MethodsWe analysed sequential marrow samples for p53 expression on 35 patients with MDS from a single institution pre and post Azacitidine therapy. Formalin fixed, paraffin embedded marrow biopsies were stained with DO-7 mouse p53 monoclonal antibody. 1000 haematopoietic cells were examined under the high power and p53 expression was determined as per Modified Quick Score.ResultsMedian age of the patients was 70 and WHO subgroups were identified as follows: 7 RCMD, 1 5q-syndrome, 1 MDS/MPN, 8 CMML, 6 RAEB-1, 6 RAEB-2 and 6 t-MDS. Cytogenetic risk as per IPSS-R/CPSS showed 17 (50%) lower risk, 4(12%) intermediate risk and 13(38%) higher risk groups. Patients received a median 13 cycles of Azacitidine. Marrows were assessed prior to treatment and after 3-6 cycles. Median overall survival of the study group was 20 months and transformation to AML occurred in 13 patients (37%). At diagnosis, 27 patients (77%) were p53 negative and 8 patients (23%) were p53 positive. At reassessment, 24 patients (69%) remained p53 negative while 6 patients (17%) remained p53 positive. Two patients (6%) became p53 negative and 3 (8%) became p53 positive following Azacitidine treatment. Median overall survival of patients who remained p53 negative during Azacitidine treatment was 28 months compared to 11 months in patients who remained p53 positive, p=0.005. Similarly, median overall survival of patients who remained or became p53 negative was 28 months compared to 18 months for those who remained or became p53 positive during Azacitidine therapy, p=0.012. p53 expression at diagnosis or changes in p53 expression during Azacitidine treatment did not correlate with transformation to Acute Myeloid Leukaemia (AML) or time to progression to AML. Among the p53 positive group, patients who had more than 10% p53 expression had lower overall survival compared to those who were <10% positive (14 vs. 27 months) In addition, changes in p53 positivity pre and post treatment did not seem to influence overall survival or transformation to AML in this small cohort.ConclusionsIn conclusion, our study showed that p53 expression changes in some patients during treatment with Azacitidine. Persistently positive p53 expression correlates with poor survival but does not correlate with transformation to AML. Analysis of p53 expression by immunohistochemistry is a clinically useful tool without employing expensive gene sequencing techniques and is readily available for routine practice. This method may be particularly useful in predicting outcomes for a subgroup of MDS patients on Azacitidine.Table 1Comparison of Median Overall Survival between p53 Positive and Negative groups during Azacitidine TherapyGroupN, %Median OSP53 Negative at DiagnosisP53 Positive at Diagnosis27, 77% 8, 23%23 months 14 monthsp= 0.049 (95% CI)Positive p53 >10% at DiagnosisPositive p53 <10% at Diagnosis10, 29% 23, 66%14 months 27 monthsp=0.039 (95% CI)Remained p53 NegRemained p53 Pos24, 69% 6, 17%28 months 11 monthsp= 0.005 (95% CI)Remained or became p53 Neg Remained or became p53 Pos26, 74% 9, 26%28 months 18 monthsp= 0.012 (95% CI)Decrease in p53 post-Azacitine(≤1%)Increase in p53 post-Azacitidine(≥1%)20, 57% 13, 37%19 months 20 monthsp=0.294 (95% CI) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3520: Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation

Abstract Emerging evidence implicates the role of Ring1B and Bmi1 in the maintenance of embryonic stem cells and regulating adult stem cells self-renewal. Both Ring1B and Bmi1 are part of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (UbH2A K119). In addition, Bmi1 is also required for the self-renewal capacity of leukemic stem cells, suggesting that Poly-comb Group proteins may contribute to oncogenic transformation by regulating stem cell self-renewal. Targeting cancer stem cell self-renewal has been proposed as a therapeutic goal and could be an effective approach to control tumor growth. In this study, we report the development and characterization of small molecule inhibitors against Ring1B-Bmi1 complex which interfere with its E3 ligase function. We developed ligands binding to Ring1B-Bmi1 employing NMR fragment-based screening. We further performed extensive medicinal chemistry optimization and synthesized over 80 analogs of the initial fragment hit and improved its binding affinity to Ring1B-Bmi1 complex by more than 1000-fold. The activity of developed small molecule inhibitors was analyzed by cellular and biochemical studies including in vitro ubiquitination assay and quantitative analysis of p16 expression. Mechanistically, we found that our most potent compounds inhibit E3 ligase activity of Ring1B-Bmi1 at low micromolar concentrations. As expected, treatment of HeLa and leukemic cell lines with these compounds demonstrated reduced UbH2A K119 levels and increased expression of p16. In addition, presence of the lead compounds also reduces leukemic cell proliferation as determined by MTT assays. Furthermore, the comparison between knockdown effects of Ring1B/Bmi1 with small molecule inhibitors revealed on target effect of the lead compounds. To our knowledge, these compounds represent the first small molecule inhibitors that directly bind to Ring1B-Bmi1 and inhibit its E3 ubiquitin ligase activity and have a strong potential for further development into potent anticancer agents. Citation Format: Shirish Shukla, Qingjie Zhao, Weijang Ying, Felicia Gray, Kelly Vandenberg, George Lund, Bohdan Boytsov, Shihan He, Jolanta Grembecka, Tomasz Cierpicki. Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2015-3520

Expression Analysis of p53, Ki-67 and bcl-2 in Pre-Malignant Lesions of the Cervix

Objectives: The present study has tried to analyze the individual and combined expression of the three markers—p53, Ki-67 and bel-2-in HPV associated pre-malignant lesions of the cervix. Methods: Thirty-four cases of high-grade lesions, 21 low-grade cases and 18 normal cases were histologically assessed. HPV-DNA presence and the viral types were determined through PCR and RFLP, respectively. Marker expression was performed by immuno-histochemistry.Results: With regards to viral types, HPV with high oncogenic potential represented the majority, with just one case reporting the low oncogenic potential for viral infection (HPV 6). HPV was the most frequent and could be associated with the high-grade lesion group, however, no significant associations were found between the viral ones and the marker expression. When the expression location, beyond the basal layer, was taken into account, the three markers proved to be good indicators of the lesion grade. The markers’ combined expression: p53(-), Ki-67(+), bcl-2(-) and p53(-), Ki-67(+), bcl-2(-) was statistically significant when associated with the lesion grade.Conclusions: The results suggest that the p53, Ki-67 and bel-2 markers can contribute to differentiate the lesion grades, whether in its isolate form or in combined form.

P16 expression as a surrogate marker for HPV‐related oropharyngeal carcinoma: A guide for interpretative relevance and consistency

Recent studies of oropharyngeal carcinoma have reported remarkable correlation between integrated human papillomavirus (HPV) viral detection and p16 protein overexpression in tumor cells. These findings led to calls for the substitution of p16 expression for the more demanding HPV testing in clinical practice. The rationale for such practice is largely driven by the simplicity, low cost, and the feasibility of the immunohistochemical (IHC) analysis. There are, however, several caveats that need to be fully considered. These include the subjective nature of IHC evaluation, the variable mechanisms of p16 expression in head and neck squamous cell carcinoma, and the lack of scoring and interpretive criteria. This perspective addresses the conceptual and practical issues associated with the p16 expression analysis and provides a broad outline for its application and evaluation in patients with oropharyngeal carcinoma.

Analysis of p16 expression by AQUA in non-small cell lung cancer (NSCLC) among non-smokers.

e21173 Background: The tumor suppressor p16 is a biomarker for HPV-related head and neck squamous cell cancer, especially in patients (pts) without prior tobacco use. Retrospective analyses of NSCLC from unselected pts provide limited data regarding the role of p16 and incidence of HPV, but suggest that p16 expression is associated with a favorable prognosis. Our goal is to estimate the proportion of never/light smoking (< 10 pack years) pts with NSCLC with p16+ tumors and correlate these data with outcomes. We hypothesize that p16+ tumors will be HPV related. Methods: A tissue microarray was constructed with NSCLC surgical specimens from never/light smokers (1995-2009) available in the Fox Chase Cancer Center Biosample Repository. Slides were incubated with anti-p16 antibody (E6H4, MTM Laboratories) and wide-spectrum screening rabbit cytokeratin antibody (Dako Z0622). DAPI was used to define tissue nuclei. p16 levels were measured with fluorescent immunohistochemistry (IHC) on the HistoRx PM-2000 platform (AQUA). After image validation, scores from two nonoverlapping images were obtained per case. p16 AQUA score was analyzed as a dichotomous variable (positive/negative) based on a naturally occurring cutpoint. We used Wilcoxon rank-sum tests, Fisher's exact tests, and Kaplan-Meier estimators with log-rank tests for analyses. Results: 33 pts were included (25 female, ages 47-84). Histology: adenocarcinoma (28), adenosquamous (3), squamous (2). TNM Stage: T1 (4), T2 (26), T3 (1), N0 (15), N1 (8), N2 (8), N3 (1), M1 (2). Perioperative treatment: chemotherapy (9), radiation (5). AQUA score range: 91-7733. A natural cutpoint occurred at 3000 with 25 low (p16-) and 8 high (p16+) expressors. AQUA score correlated with standard IHC H score (p=0.0005). There was no significant relationship between p16 level and gender, T or N stage. When controlling for N stage and p16 level, p16+ was associated with inferior survival (HR 2.68, p=0.03). Conclusions: In NSCLC among never/light smokers, p16 expression can be quantified with a natural cutpoint (high vs. low). p16+ is associated with inferior survival. Analysis of HPV status and EGFR/KRAS mutations in these tumors is ongoing and will be presented at the meeting. Funded by ACS-IRG grant.

Biomarkers in cervical screening: quantitative reverse transcriptase PCR analysis of P16INK4a expression

Molecular insights into the human papillomavirus (HPV)-induced cervical carcinogenesis led to the discovery of biomarkers for cervical disease. The detection of cellular proteins that are overexpressed by HPV-infected cells, such as tumor suppressor protein p16(INK4a), might play an important role in future cervical cancer screening strategies. P16(INK4a) immunostaining correlates with the severity of cytological and histological abnormalities, but shows some methodological shortcomings such as the lack of standardized methodology and interobserver variability. This study evaluated quantitative reverse transcriptase PCR (RT-PCR) as an alternative tool to analyze p16(INK4a) overexpression as a biomarker for transforming HPV-infections in a liquid-based cervical cytology (LBC) setting. Sixty LBC samples, divided in three groups based on their cytological diagnosis, were subjected to HPV typing and analysis of p16 expression by immunocytochemistry and RT-PCR. The analytical sensitivity of the RT-PCR was determined by spiking HeLa and HaCaT cells. P16(INK4a) expression measured by RT-PCR did not correlate with the cytological diagnosis or HPV status (HPV-positivity, infection type and HPV16-positivity). The spiking experiment proved that, to detect increased biomarker expression by RT-PCR, about 1.0% dysplastic cells is required within a pool of normal keratinocytes. In conclusion, RT-PCR analysis of biomarker expression is not appropriate for cervical screening purposes. In typical LBC samples, the biomarker transcripts of the dysplastic cells are diluted by the RNA of the normal cells in such a manner that their overexpression cannot be detected by RT-PCR.