Iara S. Rodrigues

Evaluation of the influence of polymorphic variants CYP1A1 2B, CYP1B1 2, CYP3A4 1B, GSTM1 0, and GSTT1 0 in prostate cancer.

BACKGROUND/OBJECTIVE Genetic polymorphisms in cytochrome P-450 (CYPs) and glutathione S-transferase (GSTs) genes can influence the appearance of tumors by the formation of new enzymes with altered activities. In the present study, 5 polymorphic variants were examined in 154 patients with prostate carcinoma and in 154 controls. MATERIALS AND METHODS DNA analysis was carried out through PCR-based methods. The statistical methods used were odds ratio and confidence interval (95% CI), χ(2), Fisher, and Mann-Whitney. RESULTS The study showed absence of association for CYP1A1 2B, CYP1B1 2, GSTM1 0, and GSTT1 0. The statistical analysis implied a positive association of variant CYP3A4 1B for prostate cancer. The combined analysis of CYP1A1 2B, CYP1B1 2, and CYP3A4 1B genotypes showed positive association. The analysis of histopathologic parameters detected statistically significant differences for Gleason score and biochemistry recurrence risk. The presence of the GSTT1 0 genotype in red meat consumers increased the risk for this disease. CONCLUSION Some polymorphic variants analyzed can influence the development and the progression of prostate cancer.

Polymorphisms in CYP17 and CYP3A4 and prostate cancer in men of African descent

A meta and pooled analysis of published and unpublished case–control studies was performed to evaluate the association of CYP17 (rs743572) and CYP3A4 (rs2740574) polymorphisms and prostate cancer (PCa) in men from the USA, Caribbean, and Africa.

EGFR expression in vulvar cancer: clinical implications and tumor heterogeneity ☆,☆☆

Epidermal growth factor receptor (EGFR) protein expression was assessed by immunohistochemistry (IHC) in 150 cases of invasive vulvar squamous cell carcinoma. In addition, gene copy number status by fluorescence in situ hybridization was performed in a smaller set of samples. Results were correlated with patients clinical data and prognostic factors. EGFR overexpression (2+ and 3+) was observed on the membrane in 24.66% and 21.33% of all cases, respectively. Higher EGFR expression was associated with depth of invasion (P = .0409) and disease recurrence (P = .0401). Cytoplasm staining was found in 21.33% of the cases and was associated with absence of nodal metastasis (P = .0061) and better survival (P = .0199). Intratumor heterogeneity of EGFR IHC staining was frequently observed (55.33%) and was associated with the presence of nodal metastasis (P = .0207) and tumor invasion (P = .0161). Worse survival outcomes have been demonstrated in tumors with EGFR heterogeneity (P = .0434). EGFR gene status evaluated by fluorescence in situ hybridization did not correlate with protein expression evaluated by IHC. In conclusion, EGFR cytoplasm staining has no link with poorer outcome; still, this pattern of staining is even more related to better prognosis. EGFR heterogeneity of staining correlated with more aggressive tumors, and presented to be an important marker of poor prognosis in vulvar squamous cell carcinoma. The usage of small biopsies or even tissue microarrays for vulvar cancer evaluation should be carefully reconsidered for the assessment of EGFR as the results may be misleading. Protein overexpression may be independent on gene amplification, showing that other molecular mechanisms than copy number variation may regulate protein expression of EGFR in vulvar cancer.

Polymorphisms in the AR and PSA Genes as Markers of Susceptibility and Aggressiveness in Prostate Cancer

ABSTRACT The study of genes involved in androgen pathway can contribute to a better knowledge of prostate cancer. Our aim was to examine if polymorphisms in prostate-specific antigen (PSA) and androgen receptor (AR) genes were involved in prostate cancer risk and aggressiveness. Genotypes were determined by PCR-RFLP (PSA) or using a 377 ABI DNA Sequencer (AR). PSA(G/G) genotype (OR = 1.78, 95% CI = 1.06–2.99) and AR short CAG repeats (OR = 1.89, 95% CI = 1.21–2.96) increased risk for prostate cancer and were related with tumor aggressiveness. About 38.3% of tumors showed microsatellite instability. In conclusion, polymorphisms in these genes may be indicated as potential biomarkers for prostate cancer.

MiR-223-5p works as an oncomiR in vulvar carcinoma by TP63 suppression

MiR-223-5p has been previously mentioned to be associated with tumor metastasis in HPV negative vulvar carcinomas, such as in several other tumor types. In the present study, we hypothesized that this microRNA would be important in vulvar cancer carcinogenesis and progression. To investigate this, we artificially mimicked miR-223-5p expression in a cell line derived from lymph node metastasis of vulvar carcinoma (SW962) and performed in vitro assays. As results, lower cell proliferation (p < 0.01) and migration (p < 0.001) were observed when miR-223-5p was overexpressed. In contrast, increased invasive potential of these cells was verified (p < 0.004). In silico search indicated that miR-223-5p targets TP63, member of the TP53 family of proteins, largely described with importance in vulvar cancer. We experimentally demonstrated that this microRNA is capable to decrease levels of p63 at both mRNA and protein levels (p < 0.001, and p < 0.0001; respectively). Also, a significant inverse correlation was observed between miR-223-5p and p63 expressions in tumors from patients (p = 0.0365). Furthermore, low p63 protein expression was correlated with deeper tumor invasion (p = 0.0491) and lower patient overall survival (p = 0.0494). Our study points out miR-223-5p overexpression as a putative pathological mechanism of tumor invasion and a promising therapeutic target and highlights the importance of both miR-223-5p and p63 as prognostic factors in vulvar cancer. Also, it is plausible that the evaluation of p63 expression in vulvar cancer at the biopsy level may bring important contribution on prognostic establishment and in elaborating better surgical approaches for vulvar cancer patients.

Prognostic value of GRIM-19, NF-κB and IKK2 in patients with high-grade serous ovarian cancer

AIMS High grade serous carcinoma (HGSC) is an aggressive tumour, and most patients relapse after treatment, acquiring resistance to platinum-based chemotherapy. One of the resistance mechanisms proposed is apoptosis evasion triggered by drug-related cytotoxic effect in the cell. In this context, this study aims to evaluate the protein expression of GRIM-19, NF-κB and IKK2, their association with chemotherapy response and to determine their prognostic values in HGSC. METHODS GRIM-19, NF-κB and IKK2 expression was evaluated by immunohistochemistry (IHC) in 71 patients with HGSC selected between 2003 and 2013, whose underwent primary debulking surgery with complete cytoreduction. Protein expression was analyzed in relation to platinum response groups, tumour progression, clinicopathological data and survival. RESULTS Positive IKK2 expression was related to resistance (p = 0.011), shorter disease-free survival (p = 0.001) and overall survival (p = 0.026) and was also a risk factor for relapse (p = 0.002) and death (p = 0.032). The association between IKK2 and NF-κB positivity predicted a subgroup with shorter overall survival (p = 0.004), disease-free survival (p = 0.003) and resistance to platinum-based chemotherapy (p = 0.036). NF-κB positivity was associated with worse overall survival (p = 0.005) and disease-free survival (p = 0.027) and was a positive predictor for relapse (p = 0.032) and death (p = 0.008). Higher expression of GRIM-19 was associated with higher disease-free survival (p = 0.039) and was a negative predictor for relapse (p = 0.046). CONCLUSIONS GRIM-19 is a potential predictor of prognosis and disease recurrence in HGSC. IKK2 and NF-κB are related to poor prognosis and are potential predictors of response to platinum-based chemotherapy in HGSC. IHC analyses of GRIM19, IKK2 and NF-κB may be important in the attempt to provide prognostic values for relapse and response to treatment in patients with HGSC.

Abstract 1106: Mir-223-5p works as an oncomir in vulvar carcinoma by TP63 regulation

Recently, miR-223-5p was associated with metastasis in HPV negative vulvar samples and in several other tumor types. In the present study, we hypothesized that this microRNA would have a potential impact on in vitro vulvar cancer behavior. We artificially mimicked microRNA expression in vulvar cancer cell line, SW962, derived from lymph node metastasis of vulvar carcinoma, and performed in vitro assays as follows in the result section. Impaired cell proliferation (p Citation Format: Rafael Rocha, Beatriz De Melo Maia, Iara S. Rodrigues, Nayra S. Amaral, Hui Ling, George A. Calin, Fernando A. Soares. Mir-223-5p works as an oncomir in vulvar carcinoma by TP63 regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1106.

Abstract 516: S100P: Cause or effect of vulvar carcinoma prognostic status

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Vulvar squamous cell carcinoma (VSCC) is a rare disease accounting for 3 to 5% of female genital-tract malignancy. Conventional surgical treatment of VSCC can be very aggressive and mutilating, resulting in relevant psychosexual implications for patients. S100 family comprises genes involved in several molecular pathways including proliferation, differentiation and migration. Among them, S100P has a significant role during the development and progression of different cancers being able to promote migration. Aims: To evaluate the role of S100P in VSCC and associate it with clinical outcomes. Methods: 5 samples of frozen VSCC tissue were microdissected after carefully revision of the HE slides. Two areas of the same tumor - invasion front (IF) and center of the tumor (CT)- were selected for different dissections. Each of the samples had their total RNA extracted, amplified and analyzed using Whole Human Genome 8×60K array (Agilent Technologies). Results of RNA expression were compared between both groups (IF and CT) from the same tumor. The differential expressed genes were selected by RankProduct methodology, which pointed out S100P as one of the main ones. Then, 66 VSCC formalin-fixed paraffin-embbebed samples were used for immunohistochemical (IHC) analysis of S100P. The stained slides were digitalized and evaluated using Pannoramic250 Flash II and Pannoramic Viewer, respectively (3DHistechTM). HScore was calculated based on the percentage and the intensity of stained cells. The statistical analysis was performed on SPSS® Statistics software, version 21.0.0.0, with a confidence interval of 95%. Results: The gene wide expression analysis presented a ranked list with 69 genes, one of them, S100P. The IHC results showed that IF had significant (p = 0,002) higher HScore values (mean = 132,79) compared to CT (mean = 112,34). Analysis of IF staining hot spots showed that higher expression of S100P is related with worse prognosis (p = 0,041). In addition, the greater is the difference in S100P expression between IF compared to CT of the same tumor, the worse is the patient prognosis in cancer specific survival analysis (p = 0,035). Conclusion: S100P is a marker of worse prognosis in VSCC. Also, its heterogeneity of expression between IF and CT seems to be related to a more aggressive component of the disease, maybe due to its relation with cell motility and migration. A comprehensive IHC assessment of S100P can bring important contribution for stablishing prognosis of women with vulvar cancer. Note: This abstract was not presented at the meeting. Citation Format: Mayara C. Botelho, Andre M. Lavorato-Rocha, Iara S. Rodrigues, Beatriz M. Maia, Katia C. Carvalho, Renato Puga, Fernando A. Soares, Rafael M. Rocha. S100P: Cause or effect of vulvar carcinoma prognostic status. [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 516. doi:10.1158/1538-7445.AM2015-516

Abstract 3427: Uncovering vulvar cancer: Integrated analysis of genomic and transcriptomic data

Vulvar squamous cell carcinoma (VSCC) is a rare gynecological cancer, representing approximately 3-5% of genital tumors. However, its incidence has risen considerably and no consistent profile of this disease was established so far. Also, little is known about the genomic abnormalities of VSCC and how they correlate with gene expression. In order to verify which are the major genomic alterations that lead to gene expression abnormalities we used integrative analysis in VSCC samples. Seventeen frozen samples of VSCC were carried out in genome-wide expression (GWE) profiling using the Agilent Whole Human Genome Microarray 60K (Agilent Technologies, Santa Clara, CA, USA) and in Array-CGH (aCGH) using the Agilent Human Genome CGH Microarray 60K (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer9s protocol. Data were extracted and flagged with Feature Extraction version 10.5 (Agilent Technologies, Santa Clara, CA) and the aCGH data was processed using NEXUS 6.0 (BioDiscovery, Hawthorne, CA, USA). JISTIC was used to classify genes mapped at gains and losses in genomic regions. Differentially expressed genes were identified by SAM statistical test. The CONEXIC algorithm was applied to integrate the data and in silico functional analysis were performed using Ingenuity Pathways Analysis (IPA) (Ingenuity® Systems). The aCGH analysis revealed 216 significant genomic alterations including 196 gains and 20 losses. GWE profile identified 3799 differentially expressed genes in comparison with normal tissue including 1352 up-regulated and 2447 down-regulated. The integrated analysis showed that genomic and transcriptomic results were concordant in 47 genes, in which 46 were up regulated and involved in gain of DNA copy number and only 1 gene (PLXDC2) was down-regulated and associated to genomic loss. According to IPA analysis of the top biological functions associated with the 47 genes, 3 of them were related with “tumor morphology”. Also, “cell death and survival” and “cellular function and maintenance” were included in the list of pathways disrupted by the genes selected as possible drivers of VSCC. Three significant networks could be defined comprising these genes. The first network comprised 18 of the concordant genes and was associated to functions as carbohydrate metabolism, small molecule biochemistry, cellular function and maintenance. The second network (18 genes) was related to RNA post-transcriptional modification, developmental disorder and hereditary disorder. The third network (15 genes) was associated to RNA post-transcriptional modification, gene expression and organ development. Thereby, important signaling networks were found disrupted by these genes which could influence tumor development or progression in VSCC. Therefore, these genes might be useful as a first step to identify molecular markers to improve diagnosis and therapeutic approaches for this tumor. Citation Format: Andre M. Lavorato-Rocha, Beatriz de Melo Maia, Iara S. Rodrigues, Fabio A. Marchi, Gabriel R. Fernandes, Glauco Baiocchi, Fernando A. Soares, Silvia R. Rogatto, Yukie Sato-Kuwabara, Rafael M. Rocha. Uncovering vulvar cancer: Integrated analysis of genomic and transcriptomic data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3427. doi:10.1158/1538-7445.AM2014-3427

Abstract 4051: Prion protein and its ligand STI1/HOP modulate migration and invasion of cell lines derived from colorectal tumors

The colorectal cancer is the fourth most common type of cancer in the world. The search for potential molecular targets for therapy has led to the Cellular Prion Protein (PrPC) as a possible candidate. Its activity depends on the interaction with the secreted form of the stress inducible protein 1 or heat shock organizing protein (STI1/HOP). Previous results indicated the importance of the PrPC-STI1/HOP complex in proliferation of glioblastomas and invasion of melanomas. Therefore, this work aims to understand how PrPC-STI1/HOP complex can influence the migration and invasion of WiDr and HCT8 colorectal tumor cells. PrPC is equally expressed at the cell surface of both cell lines. Cell migration assays were performed using boyden chambers and recombinant STI1/HOP as chemical attractant. For invasion experiments cells were seeded on boyden chambers covered with matrigel and treated with STI1/HOP. The results showed that when stimulated with recombinant STI1/HOP, both tumor cell lines showed higher migration and invasion potentials than non-treated cells or those treated with a STI1/HOP deletion mutant for the PrPC binding domain (STI1Δ230-245). Moreover, migration and invasion mediated by PrPC-STI1/HOP complex were blocked by a STI-1/HOP (230-245) peptide, which mimics STI1 binding site to PrPC or also by a neutralizing anti-PrPC antibody. STI1/HOP is secreted in the conditioned medium (CM) within extracellular microvesicles (MVs) and WiDr cells secreted more MVs and STI1/HOP than HCT8 cells. Remarkable, HCT8 cells treated with CM or isolated MVs from WiDr cells show a higher migration than that induced when the former cells were treated with their own CM or when WiDr cells were treated with CM or MVs derived from HCT8 cells. Together, these results indicate that the PrPC-STI1/HOP complex mediate migration and invasion of colorectal tumor cells and suggest that cellular migration is dependent on the concentration of secreted MVs. The role of STI1/HOP present in MVs on cell migration is still under investigation. Citation Format: Tonielli S. Lacerda, Marcos Vinicios S. Dias, Antuani Rafael Baptistella, Fernanda S. Giudice, Bruna R. Roz, Iara S. Rodrigues, Vilma Regina Martins. Prion protein and its ligand STI1/HOP modulate migration and invasion of cell lines derived from colorectal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4051. doi:10.1158/1538-7445.AM2014-4051