Immunohistochemical expression of cyclin-dependent kinase inhibitors p16 and p57 in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly malignant cancer and is the most common soft tissue sarcoma in children and adolescents, but it is rare in adults (<1% of all adult malignancies). Altered expression and molecular abnormalities of cell-cycle-regulatory proteins are one of the most prominent features in RMS. Therefore, we evaluated the expression of cyclin-dependent kinase inhibitors p57 and p16, as well as p16 methylation status, along with clinicopathological characteristics and overall survival (OS) in RMS patients. This analysis was conducted on 23 pediatric and 44 adult patients. There was a male predominance in both groups and extremities were the most frequent tumor site. In adults, alveolar and pleomorphic types were almost equally represented. The majority of pediatric tumors were low grade, whereas, in adults, only one patient had a low-grade tumor. Seven pediatric (30.43%) and eight adult (18.18%) patients had a low p16 expression. The analysis of methylation status of the p16 promoter showed the presence of methylated allele only in one sample with pleomorphic histology. Six (26.1%) pediatric and 15 (34.1%) adult patients had low p57 expression, while in 17 (73.9%) pediatric and 29 (65.9%) adult patients it was assessed as high. Ninetyone percent of the pediatric patients and 32.6% of adults were alive at the end of the observational period. In adults, significant associations were found between OS and age (P = 0.020), gender (P = 0.027), tumor size (P < 0.001), lymph node status (P < 0.001), presence of metastases (P = 0.015), and p57 expression (P = 0.039). Stratification by histological type showed the correlation of low p57 expression (P = 0.030) and worse OS of patients with alveolar RMS. Univariate analysis identified age > 50 yrs. (HR 2.447), tumors > 5 cm (HR 21.31), involvement of regional lymph nodes (HR 3.96), the presence of metastases (HR 2.53), and low p57 expression (HR 2.11) as predictors of lower OS. Tumor size, regional lymph nodes involvement, and metastases were the independent predictors after multivariate analysis, while p57 did not predict OS in an independent way. In summary, although p57 was not confirmed to be an independent predictor of OS, our results indicate that its low expression may be the marker of aggressive phenotype and poor prognosis in adult RMS patients. Also, our findings suggest that epigenetic inactivation of p16 is not important in the pathogenesis of rhabdomyosarcoma.

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.

MicroRNA in breast cancer: The association with BRCA1/2.

Breast cancer (BC) is a heterogeneous disease in an urgent need for developing novel research, classification, and therapy approaches. Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins are well described tumor suppressors with great potential to be the subjects of different therapies. MicroRNAs (miRNAs) are genetic elements that might be used to solve the complex BC puzzle. BRCA1 was described to be the target of up to 100 miRNAs. BRCA1 may directly repress miR-155 activity. In addition, miR-15/107/182-mediated downregulation of BRCA1 interrupt DNA repair and may change the course of BC therapy. miR-146a and miR-146-5p silencing BRCA1 may trigger formation of triple-negative and basal-like sporadic BC cases. miR-182 might effect the therapy outcome. miR-21 targeted therapy might be useful for the treatment of BRCA2 mutation carriers. miR-342 overexpression and the absence of functional BRCA1 gene might cause synthetic lethality, which might be used as a base for future therapies. The present review discusses the latest data from studies that focus on the complex network of miRNAs and BRCA1/2 related BCs, which might be important for improving the therapy within the patients with triple-negative BC (TNBC) and basal-like BC, and for understanding the formation of TNBC.

Recurrence of giant cell tumour of bone: role of p53, cyclin D1, ß-catenin and Ki67.

PURPOSE: To determine various clinical, radiographic, and pathological parameters which may indicate an increased risk of Giant cell tumour of bone (GCTB) recurrence after surgical therapy. METHODS: The study included a total of 164 GCTB samples; 118 (72 %) primary tumours, and 46 (28 %) recurrences; which were analyzed on immunohistochemistry for expression of Ki67, p53, cyclin D1, and ß-catenin. RESULTS: Among 13 analyzed clinical, radiological, and histological variables, which presented possible predictive factors for the incidence of GCTB relapse, univariate logistic regression (ULR) extract three highly statistically significant parameters: 1) lesion localization, 2) nuclear p53 expression in mononuclear cells, and 3) nuclear cyclin D1 expression in giant multinuclear cells. The multivariate logistic regression (MLR), revealing that p53 expression in mononuclear cells was the most significant predictive factor (HR = 6,181 p < 0,001), the positivity of which indicated six times higher probability for recurrence in GCTB. The expression of cyclin D1 in giant cells, containing less than 15 nuclei, was also statistically significant (HR = 8,398, p = 0,038) for predicting the recurrence, and demonstrated eight times more frequent recurrence in positive tumours. CONCLUSIONS: This study confirmed independent predicting factors for GCTB reccurence: p53 expression in mononuclear tumour cells and cyclin D1 expression in giant multinuclear cells. Results are new addition to generally known parameters, such as: localization of lesion, number of surgical interventions, clear destruction of cortex with the presence of extracompartmental lesion, and histological criteria for malignancy and can help in further research and treatment of GCTB.

Changes in miR-221/222 Levels in Invasive and In Situ Carcinomas of the Breast: Differences in Association with Estrogen Receptor and TIMP3 Expression Levels.

BACKGROUND: Breast cancer (BC) is a heterogeneous group of diseases that still represents a major cause of death in the female population. MicroRNAs (miRNAs, miRs), such as miR-221 and miR-222, have been shown to be involved in BC pathology by acting via its target genes such as tissue inhibitor of metalloproteinase 3 (TIMP3). OBJECTIVES: The main goals of this study were to find differences in miR-221/222 levels of expression in BC groups based on invasiveness, and to investigate the association with estrogen receptor (ER), TIMP3 messenger RNA (mRNA) levels, and clinicopathological characteristics of patients and tumors. METHODS: In this study, we measured levels of miR-221/222 in 63 breast tissue samples by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using TaqMan® technology and immunohistochemistry. RESULTS: miR-221/222 levels varied significantly across groups based on invasiveness (P < 0.001). In in situ tumors, miR-221 and miR-222 were negatively associated with ER (P = 0.001, r = -0.714, and P = 0.013, r = -0.585, respectively). In invasive breast carcinomas associated with non-invasive tumors, miR-222 was inversely associated with ER (P = 0.039, r = -0.620). Pure invasive BCs showed a positive correlation of miR-221 and miR-222 with TIMP3 mRNA levels (P = 0.008, r = 0.508, and P = 0.010, r = 0.497, respectively). CONCLUSION: An increase in miR-221/222 might be an important event for in situ carcinoma formation, and miR-221/222 may be important molecules that highlight potential differences between invasive breast carcinomas associated with non-invasive and pure invasive BCs.

Significance of ß-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, ß-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 ß-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 b-catenin in stromal cells had three times more frequent occurrence of pathological fractures, which was highly statistically significant (χ2 = 7.065; p = 0.008). Moreover, a highly statistically significant correlation between the nuclear expression of ß-catenin in giant cells and the incidence of pathological fractures was also found (χ2 = 8.824; p = 0.003). The study showed that ß-catenin expression highly correlates with the incidence of pathological fractures in patients with GCTB. Taking into account that ß-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.

p14(ARF) methylation is a common event in the pathogenesis and progression of myxoid and pleomorphic liposarcoma.

Liposarcoma represents the most frequent group of soft tissue sarcomas. The group can be divided into three different classes: (1) differentiated/undifferentiated (WDLPS/DDLPS), (2) myxoid/round cell (MLPS/RCLPS) and (3) pleomorphic liposarcoma (PLS). It has become apparent that p53-p14 and Rb-p16 pathways play important roles in the pathogenesis of various sarcoma types. Molecular studies of the genes involved in these two pathways showed wide variations between the liposarcoma subtypes or even within the same subtype. We sought to examine mutational status of p53 and methylation status of p16 (INK4a) /p14 (ARF) genes in primary and recurrent liposarcoma tumors. There were twelve myxoid (12/18, 66.7 %) and six pleomorphic liposarcoma (6/18, 33.3 %) samples. Immunohistochemical analysis revealed that p53 protein was overexpressed in 3/12 MLPS (25 %) and 6/6 PLS (100 %). Mutational analysis showed that 2/11 MLPS (18.2 %) and 2/6 PLS (33.3 %) contained mutated p53 gene. On the other hand, 3/18 samples (16.7 %) had methylated p16 promoter. However, the frequencies of the p14 (ARF) gene methylation were 83.3 % (10/12) and 50 % (3/6) in myxoid and pleomorphic group, respectively. Overall, 15 out of 18 (83.3 %) samples had either p53 gene mutation or methylated p14 (ARF) promoter. The results from the current study suggest significant impact of the p14 (ARF) gene methylation on the pathogenesis and progression of myxoid and to a lesser extent pleomorphic liposarcoma. Despite the limited number of samples, our study points to necessity of further investigation of p53-p14 and Rb-p16 pathways in liposarcoma.

Prognostic significance of epigenetic inactivation of p16, p15, MGMT and DAPK genes in follicular lymphoma.

In this study, methylation-specific polymerase chain reaction was used to investigate the role and potential prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes in 32 specimens of follicular lymphoma (FL). Hypermethylation of p15 gene was associated with lower hemoglobin level (P = 0.020) and MGMT/DAPK comethylation with relapsed disease (P = 0.018). Among all patients with FL, there was no significant difference in the overall survival between those with hypermethylated and unmethylated of any examined genes. Therefore, we analyzed methylation in the different groups according to FL International Prognostic Index (FLIPI) and tumor grade. In the high-risk group, patients with hypermethylated p16 gene had significant lower overall survival than those with unmethylated p16 (P = 0.006) and trend toward shorter failure-free survival (P = 0.068). In the same risk group, there was a trend toward longer overall survival for patients with hypermethylated MGMT gene, compared to those with unmethylated MGMT gene (P = 0.066). p15 methylation had impact on shorter overall survival in grade I group of patients (P = 0.013), and DAPK methylation tended to have impact on shorter failure-free survival in the whole examined group (P = 0.079). Our results suggest that promoter methylation of p16 and MGMT genes could have prognostic value when used in combination with the FLIPI and p15 methylation in combination with tumor grade. Concurrent methylation of MGMT and DAPK genes could be the marker of tumor chemoresistance and disease recurrence.

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.