Analysis of the p53 gene mutation status and methylation status of the promoters of p14 and p16 genes in liposarcoma

Липосаркоми, тумори мезенхималног порекла, представљају најучесталији хистолошки тип саркома меких ткива. Групу карактерише велика разноликост како у погледу патохистолошке организације тумора тако и биологије понашања. Уобичајена подела липосаркома обухвата следећа три подтипа: а.) добро диферентовани / дедиферентовани, б.) миксоидни / округлоћелијски и в.) плеоморфни. С обзиром на значајне клиничко-патолошке разлике између три подтипа липосаркома, вероватно је да су различити механизми укључени у њихов настанак и развој. На основу студија које су се фокусирале на истраживање генетике саркома меких ткива и костију, може се закључити да сигнални путеви p53-p14 и Rb-p16 играју одређену улогу у свим типовима саркома. Разјашњавање улоге ових сигналних путева у патогенези липосаркома, може да буде допринос како дијагностици и прогностици липосаркома, тако и дефинисању потенцијалних терапеутских циљева. Студија је обухватила испитивање мутационог статуса гена р53 и метилационог статуса гена р14 и р16, на 33 узорка липосаркома, сва три подтипа. У циљу свеобухватније анализе испитивана је и експресија протеина р16, циклина D1 и фактора пролиферације Ki-67, применом имунохистохемијског бојења. Резултати експеримента су показали да промене у сигналним путевима р53-р14 и р16-Rb могу имати значаја у патогенези сва три подтипа липосаркома. Разлике се огледају у квалитету и квантитету промена.Liposarcoma, tumors of mesenchymal origin, represent the most frequent type of soft tissue sarcomas. The group is characterized by a great diversity regarding pathohistology and biological behavior. The group can be divided into three different subtypes: a.) well differentiated / dedifferentiated, b.) myxoid / round cell and c.) pleomorphic. Given the significant clinical and pathological differences between the three subtypes of liposarcoma, it is likely that different mechanisms are involved in their formation and development. Based on the studies that have been focused on genetic research of sarcoma of soft tissues and bones, conclusion can be drawn that the p53-p14 and Rb-p16 signaling pathways play a certain role in sarcomagenesis. Clarification of the role of these signaling pathways in the pathogenesis of liposarcoma, could be a contribution towards better diagnostic and prognostic criteria and the identification of potential therapeutic targets. The study included examination of the p53 gene mutation status and assessment of the p14/p16 gene methylation status in 33 liposarcoma samples of all three subtypes. For the purpose of more comprehensive analysis, protein expression of the p16, cyclin D1 and Ki-67 has been evaluated by immunohistochemical staining. The results of the study have showed that the alterations of the targeted signal pathways are important in the pathogenesis of all three subtypes of liposarcoma. The differences are reflected in the quality and quantity of the detected alterations

Significance of Beta-Catenin Expression for the Incidence of Pathological Fractures in Giant Cell Tumors of Bone

Aim of the study is to determine the possible roles of p53, cyclin D1, B-catenin and Ki-67 in the increase in risk of fractures in patients with giant cell tumor of bone. The study included a total of 164 patients with giant cell tumor of bone (GCTB), 21 (12.8%) with and 143 (87.2%) without fracture. The samples were analyzed immunohistochemically for expression of Ki-67, p53, cyclin D1 and beta-catenin. According to the immunohistochemical expression of p53 and Ki-67 in mononuclear stromal cells, as well as of cyclin D1 in multinuclear giant cells, there was no significant association with immunopositivity and risk of fractures. However, our research revealed that patients with cytoplasmic expression of beta-catenin in stromal cells had three times more frequent occurrence of pathological fractures, which was highly statistically significant (chi(2) = 7.065; p = 0.008). Moreover, a highly statistically significant correlation between the nuclear expression of beta-catenin in giant cells and the incidence of pathological fractures was also found (chi(2) = 8.824; p = 0.003). The study showed that beta-catenin expression highly correlates with the incidence of pathological fractures in patients with GCTB. Taking into account that beta-catenin is closely linked to activation of the Wnt signaling pathway in GCTB pathogenesis, one could postulate that activation of the Wnt pathway is one of the contributing factors to locally destructive behavior of this tumor, as well as to the incidence of pathological fractures

Methylation-specific PCR: four steps in primer design

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design

Alignment-free method for functional annotation of amino acid substitutions: Application on epigenetic factors involved in hematologic malignancies

For the last couple of decades, there has been a significant growth in sequencing data, leading to an extraordinary increase in the number of gene variants. This places a challenge on the bioinformatics research community to develop and improve computational tools for functional annotation of new variants. Genes coding for epigenetic regulators have important roles in cancer pathogenesis and mutations in these genes show great potential as clinical biomarkers, especially in hematologic malignancies. Therefore, we developed a model that specifically focuses on these genes, with an assumption that it would outperform general models in predicting the functional effects of amino acid substitutions. EpiMut is a standalone software that implements a sequence based alignment-free method. We applied a two-step approach for generating sequence based features, relying on the biophysical and biochemical indices of amino acids and the Fourier Transform as a sequence transformation method. For each gene in the dataset, the machine learning algorithm–Naïve Bayes was used for building a model for prediction of the neutral or disease-related status of variants. EpiMut outperformed state-of-the-art tools used for comparison, PolyPhen-2, SIFT and SNAP2. Additionally, EpiMut showed the highest performance on the subset of variants positioned outside conserved functional domains of analysed proteins, which represents an important group of cancer-related variants. These results imply that EpiMut can be applied as a first choice tool in research of the impact of gene variants in epigenetic regulators, especially in the light of the biomarker role in hematologic malignancies. EpiMut is freely available at https://www.vin.bg.ac.rs/180/tools/epimut.php.EpiMut is freely available at [https://www.vin.bg.ac.rs/180/tools/epimut.php]

Critical assessment of protein intrinsic disorder prediction

Intrinsically disordered proteins, defying the traditional protein structure–function paradigm, are a challenge to study experimentally. Because a large part of our knowledge rests on computational predictions, it is crucial that their accuracy is high. The Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiment was established as a community-based blind test to determine the state of the art in prediction of intrinsically disordered regions and the subset of residues involved in binding. A total of 43 methods were evaluated on a dataset of 646 proteins from DisProt. The best methods use deep learning techniques and notably outperform physicochemical methods. The top disorder predictor has F max = 0.483 on the full dataset and F max = 0.792 following filtering out of bona fide structured regions. Disordered binding regions remain hard to predict, with F max = 0.231. Interestingly, computing times among methods can vary by up to four orders of magnitude

Immunohistochemical expression of nestin in rhabdomyosarcoma: implications for clinicopathology and patient outcome

Rhabdomyosarcoma (RMS) is a highly malignant cancer. Over the last two decades, prognosis for RMS patients has significantly improved, with the exception of those in the high-risk group. In order to identify new prognostic factors, we investigated the expression of nestin in RMS cells and its correlation with clinicopathological features and patient outcome. The analysis of overall survival for all patients (N = 30) revealed 1-, 2-, 3-, 4-, and 5-year survival rates of 93.3, 83.3, 66.7, 63.3, and 63.3%, respectively. Nestin overexpression significantly correlated with survival (P = 0.044). Survival of patients with = 50% nestin-positive cells was 90, 70, and 40% after 1, 2, and 3 years, respectively, and remained unchanged until the end of the investigation period. The study group composed of patients exhibiting nestin expression in GT 50% of cells showed 1-, 2-, 3-, and 4-year survival rates of 95, 90, 80, and 75%, respectively, remaining stable at 75% for the fifth year of observation. A nestin-positive expression rate lower than 50% was observed more frequently in older patients (43.60 +/- 27.58 years; P = 0.028). In addition, higher rates of nestin expression were observed in most embryonal RMS specimens and low-grade tumors, in early stages of the disease, and among younger patients. Our results lead us to propose nestin as possible positive prognostic factor in RMS

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure. © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research

Immunohistochemical expression of nestin in rhabdomyosarcoma: implications for clinicopathology and patient outcome

Rhabdomyosarcoma (RMS) is a highly malignant cancer. Over the last two decades, prognosis for RMS patients has significantly improved, with the exception of those in the high-risk group. In order to identify new prognostic factors, we investigated the expression of nestin in RMS cells and its correlation with clinicopathological features and patient outcome. The analysis of overall survival for all patients (N = 30) revealed 1-, 2-, 3-, 4-, and 5-year survival rates of 93.3, 83.3, 66.7, 63.3, and 63.3%, respectively. Nestin overexpression significantly correlated with survival (P = 0.044). Survival of patients with = 50% nestin-positive cells was 90, 70, and 40% after 1, 2, and 3 years, respectively, and remained unchanged until the end of the investigation period. The study group composed of patients exhibiting nestin expression in GT 50% of cells showed 1-, 2-, 3-, and 4-year survival rates of 95, 90, 80, and 75%, respectively, remaining stable at 75% for the fifth year of observation. A nestin-positive expression rate lower than 50% was observed more frequently in older patients (43.60 +/- 27.58 years; P = 0.028). In addition, higher rates of nestin expression were observed in most embryonal RMS specimens and low-grade tumors, in early stages of the disease, and among younger patients. Our results lead us to propose nestin as possible positive prognostic factor in RMS

Methylation status of p16 and p14 genes in locally advanced rectal cancer: Potential clinical implication

Methylation of p16 and p14 genes is a common event in colorectal cancers; however, their exact role in the prediction of patients' outcome is unclear. We conducted this retrospective study to evaluate their potential predictive and/or prognostic roles. Methylation-specific PCR was used to examine the methylation status of p16 and p14 in pretherapeutic and preoperative biopsy specimens of 60 patients with locally advanced rectal cancer. The methylation status of the examined genes did not affect the response to preoperative chemoradiotherapy (CRT), recurrence rate and overall survival. However, patients with a simultaneous presence of either p16 or p14 methylation and high vascular endothelial growth factor (VEGF) expression showed a significantly worse response to CRT (p=0.005 and p=0.038, respectively). Moreover, patients with both p16 methylation and high VEGF expression had significantly shorter overall survival (p=0.010), while no such association was found in patients with p14 methylation and high VEGF expression. On the other hand, a subgroup of patients with p16 methylation and low VEGF and high epidermal growth factor receptor (EGFR) expression showed a significantly better response to CRT (p=0.024). The obtained results point to the importance of p16 and p14 methylation analyses in combination with VEGF and EGFR expression, aimed at better predicting treatment response and patient outcome. © 2018 by the Serbian Biological Society

The impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen treatment in breast cancer patients

Tamoxifen is a standard therapeutical treatment in patients with estrogen receptor positive breast carcinoma. However, less than 50% of estrogen receptor positive breast cancers do not respond to tamoxifen treatment whereas 40% of tumors that initially respond to treatment develop resistance over time. The underlying mechanisms for tamoxifen resistance are probably multifactorial but remain largely unknown. The primary aim of this study was to investigate the impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen by analyzing loss of heterozygosity (LOH) and immunohystochemical expression of PTEN in 49 primary breast carcinomas of patients treated with tamoxifen as the only adjuvant therapy. The effect of PTEN inactivation on breast cancer progression and disease outcome was also analyzed. Reduced or completely lost PTEN expression was observed in 55.1% of samples, while 63.3% of samples displayed LOH of PTEN gene. Inactivation of PTEN immunoexpression significantly correlated with the PTEN loss of heterozygosity, suggesting LOH as the most important genetic mechanism for the reduction or complete loss of PTEN expression in primary breast carcinoma. Most importantly, LOH of PTEN and consequential reduction of its immunoexpression showed significant correlation with the recurrence of the disease. Besides, our study revealed that LOH of PTEN tumor suppressor was significantly associated with shorter disease free survival, breast cancer specific survival and overall survival. In summary, our results imply that LOH of PTEN could be used as a good prognostic characteristic for the outcome of breast cancer patients treated with tamoxifen