Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

FGFR4 Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Mouth and Oropharynx

Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. Patients and Methods: DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays. Results: Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. Conclusion: Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

Periocular basal cell carcinoma: cost of topical immunotherapy versus estimated cost of surgical treatment

Purpose: The objective of this study was to compare the estimated cost of clinical and surgical treatment for basl cell carcinoma of the eyelid. Methods: This was a pilot study of 12 patients with basal cell carcinoma receiving treatment with 5% imiquimod cream at the ocular plastic surgery center, medical school University of Sao Paulo (HC-FMUSP, Brazil). The cost of clinical treatment was estimated based on the time of treatment and amount of medication consumed by patients in the home setting. The cost of surgical treatment was estimated by ophthalmologists with experience in reconstructive plastic surgery based on analysis of images of the same patients. Surgeons responded to a questionnaire with four questions about surgical technique, surgical materials required, estimated duration of surgery and type of anesthesia. Results: Immunotherapy lasted from 8 to 12 weeks. All patients reported each cold-stored sachet with 5% imiquimod cream lasted 3 days. According to the institution, a box with 12 sachets costs BRL 480.00. Patients required 1.58-3.11 boxes for complete treatment, corresponding to a total cost of BRL 758.40-1,492.80. Based on image analysis, surgeons evaluated surgery would require 1-3 hours. The estimated cost of surgery room and staff was BRL 263.00, to which the cost of supplies was added. Thus, the total cost of surgical treatment was BRL 272.61-864.82. On the average, immunotherapy was 57,64% more costly than surgical treatment. Conclusions: Malignant eyelid tumors are a common finding in clinical ophthalmology. Surgery is still the treatment of choice at our institution, but immunotherapy with 5% imiquimod cream may be indicated for patients with multiple lesions or high surgical risk and for patients declining surgery for reasons of fear or esthetic concerns. The ability to estimate costs related to the treatment of malignant eyelid tumors is an important aid in the financial planning of health care institutions. Further studies should evaluate the possibility of institutions equating the cost of immunotherapy and surgical treatment by acquiring similar but less expensive medications.

Hyaluronidase splice variants are associated with histology and outcome in adenocarcinoma and squamous cell carcinoma of the lung

Heterogeneity of hyaluronidase (HYAL) expression has been identified in tumors and shows promise as an indicator of disease progression. The expression profile of alternatively spliced forms of HYAL was evaluated in tumors and normal lung tissue from 69 resected tumors of patients with adenocarcinomas and squamous cell carcinomas. HYAL1-wild-type (wt) and variants 1 to 5, HYAL2-wt, and HYAL3-wt, and variants 1 to 3 were identified by polymerase chain reaction and direct sequencing. Different proportions of the 3 HYAL-wt and variants were expressed in tumor and normal lung tissues. HYAL1-wt was associated with a poorer prognosis and HYAL3-vl with a better prognosis. HYAL splice variants are associated with histology and outcome, suggesting that strategies aimed at modulating their levels may be effective for lung cancer treatment. (C) 2012 Elsevier Inc. All rights reserved.

Photodynamic therapy in periocular basal cell carcinoma: Case report

The authors report the use of photodynamic therapy with methyl aminolevulinate (Metvix (R)) in a patient with nodular and infiltrative basal cell carcinoma in the right lower eyelid. Side effects on the eye were evaluated weekly. After 12 weeks of treatment, to confirm cure the patient was submitted to a 2-mm punch biopsy, the anatomopathological findings of which were negative for neoplasia. Photodynamic therapy with methyl aminolevulinate was shown to be an attractive alternative to surgical excision(-)the current gold standard treatment worldwide.

CD25(+) T cell depletion impairs murine squamous cell carcinoma development via modulation of antitumor immune responses

Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1 beta, IL-10, IL-12, IL-13, interferon-gamma, transforming growth factor-beta and tumor necrosis factor-alpha, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

The density of metastatic lymph node as prognostic factor in squamous cell carcinoma of the tongue and floor of the mouth

The presence of metastatic lymph nodes is a relevant prognostic factor in oral cancer. Objective: This paper aims to assess metastatic lymph node density (pN+) in patients with tongue and floor-of-mouth squamous cell carcinoma (SCC) and the association of this parameter with disease-free survival (DFS). Materials and Methods: A group of 182 patients seen between 1985 and 2007 was included, 169 of which were males. Five were on stage I, 35 on stage II, 56 on stage III, and 85 on stage IV. Median values were considered in lymph node density assessment, and the Kaplan-Meier curve was used to evaluate DFS; survival differences within the group were elicited through the log-rank test. Results: An average 3.2 metastatic lymph nodes were excised from the patients in the group. Density ranged from 0.009 to 0.4, with a mean value of 0.09. Five-year DFS rates were of 44% and 28% for the groups with lymph node densities below and above the median respectively (p = 0.006). Two-year local/regional control was achieved for 71% and 49% for the patients below and above the median density respectively (p = 0.01). In terms of pN staging, local/regional control was achieved in 70% and 54% of pN1 and pN2 patients respectively, albeit without statistical significance (0.20%). Conclusion: Lymph node density may be used as a prognostic indicator for tongue and floor-ofmouth SCC.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

TWIST and p-Akt immunoexpression in normal oral epithelium, oral dysplasia and in oral squamous cell carcinoma

Objectives: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. Study design: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. Results: Kruskal-Wallis and Dunn's post tests revealed a significant difference in TWIST and p-Akt immunoexpression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearson's correlation test. Conclusions: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multi-step process of oral carcinogenesis since its early stages.

Topoisomerase expression in oral squamous cell carcinoma: relationship with cancer stem cells profiles and lymph node metastasis

BACKGROUND: The relationship between predictive proteins and tumors presenting cancer stem cells (CSCs) profiles in oral tumors is still poorly understood. This study aims to identify the relationship between topoisomerases I, II alpha, and III alpha and putative CSCs immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. METHODS: The following data were retrieved from 127 patients: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, and survival. An immunohistochemical study for topoisomerases I, II alpha, and III alpha was performed in a tissue microarray containing 127 paraffin blocks of OSCCs. RESULTS: In univariate analysis, topoisomerases expression showed significant differences according to CSCs profiles and p53 immunoexpression, but not with survival. Topoisomerases II alpha and III alpha also showed significant relationship with lymph node metastasis. The multivariate test confirmed these associations. CONCLUSIONS: The results that all topoisomerases correlates with OSCC CSCs may indicate a role for topoisomerases in head and neck carcinogenesis. Notwithstanding, it is plausible that other members of topoisomerases family could represent novel therapeutical targets in oral squamous cell carcinoma. J Oral Pathol Med (2012) 41: 762-768

Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma

Background. Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. Material and methods. We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. Results. Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN+) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN+ with an accuracy of 80.0% (p = 0.012). Conclusions. The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma

Background Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. Methods This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. Results The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. Conclusion The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. (c) 2011 Wiley Periodicals, Inc. Head Neck, 2012

Oral candidiasis mimicking an oral squamous cell carcinoma: report of a case

Oral candidiasis is a significant problem in immune-compromised patients. The most common forms of mucosal candidiasis are oropharyngeal, oesophageal and vaginal, and more than 90% of HIV positive persons will manifest at least one episode of oropharyngeal candidiasis. Local and systemic factors such as uninterrupted daily use of a prosthesis by patients, smoking habit, as well as high glucose intake may contribute to the development of the lesion. The aim of this article is to report an uncommon case of oral candidiasis presenting an aggressive clinical behaviour in a 64-year-old male patient, with a significant smoking habit and a medical history of non-controlled diabetes. The lesion affected the hard and soft palate of the right side, revealing erythematous and ulcerated areas, elevated borders and central portions resembling necrosis, mimicking the clinical features of oral squamous cell carcinoma. However, the correct diagnosis of oral candidiasis was obtained after histopathological and cytological examinations and the patient was easily treated with traditional antifungal drugs and correction of his glucose levels.