Katia Candido Carvalho

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p=0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p=0.010 and p=0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC.

Glial fibrillary acidic protein in tumor types with cartilaginous differentiation

Glial fibrillary acidic protein (GFAP) is a member of the intermediary filament protein family. It is an important component of astrocytes and a known diagnostic marker of glial differentiation. GFAP is expressed in other neural tumors and pleomorphic adenoma and, less frequently, in cartilage tumors, chordomas, and soft tissue myoepitheliomas. The aim of this study was to evaluate the role of GFAP and its reliability in nonglial tumors as an immunohistochemical marker. We evaluated GFAP gene and protein expression using Q-PCR and immunohistochemistry, respectively, in 81 and 387 cases of soft tissue, bone tumors, and salivary pleomorphic adenomas. Immunohistochemistry staining for GFAP was observed in all osteosarcomas (8 cases), all pleomorphic adenomas (7 cases), in 5 of 6 soft tissue myoepitheliomas, and in 21 of 76 chondrosarcomas. By Q-PCR, GFAP was highly expressed in pleomorphic adenomas and, to a lesser extent, chondrosarcomas, soft tissue myoepitheliomas, and chondroblastic osteosarcomas. The results that we obtained by immunohistochemistry and Q-PCR were well correlated. GFAP is a potential marker for tumors with cartilaginous differentiation, supported by evidence that GFAP is expressed in certain cases of myoepithelial tumors by immunohistochemistry, including soft tissue myoepitheliomas, which are related to cartilaginous differentiation. These findings contribute significantly to the diagnosis of soft tissue myoepitheliomas with cartilaginous differentiation and chondroblastic osteosarcoma in mesenchymal tumors.

May Sonic Hedgehog proteins be markers for malignancy in uterine smooth muscle tumors

Several studies have demonstrated that the Sonic Hedgehog signaling pathway (SHH) plays an important role in tumorigenesis and cellular differentiation. We analyzed the protein expression of SHH pathway components and evaluated whether their profile could be useful for the diagnosis, prognosis, or prediction of the risk of malignancy for uterine smooth muscle tumors (USMTs). A total of 176 samples (20 myometrium, 119 variants of leiomyoma, and 37 leiomyosarcoma) were evaluated for the protein expression of the SHH signaling components, HHIP1 (SHH inhibitor), and BMP4 (SHH target) by immunohistochemistry. Western blot analysis was performed to verify the specificity of the antibodies. We grouped leiomyoma samples into conventional leiomyomas and unusual leiomyomas that comprise atypical, cellular, mitotically active leiomyomas and uterine smooth muscle tumors of uncertain malignant potential. Immunohistochemical analysis showed that SMO, SUFU, GLI1, GLI3, and BMP4 expression gradually increased depending on to the histologic tissue type. The protein expression of SMO, SUFU, and GLI1 was increased in unusual leiomyoma and leiomyosarcoma samples compared to normal myometrium. The inhibitor HHIP1 showed higher expression in myometrium, whereas only negative or basal expression of SMO, SUFU, GLI1, and GLI3 was detected in these samples. Strong expression of SHH was associated with poorer overall survival. Our data suggest that the expression of SHH proteins can be useful for evaluating the potential risk of malignancy for USMTs. Moreover, GLI1 and SMO may serve as future therapeutic targets for women with USMTs.

Oncomirs Expression Profiling in Uterine Leiomyosarcoma Cells

MicroRNAs (miRNAs) are small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. They play a key role in several biological processes. Their abnormal expression may lead to malignant cell transformation. This study aimed to evaluate the expression profile of 84 miRNAs involved in tumorigenesis in immortalized cells of myometrium (MM), uterine leiomyoma (ULM), and uterine leiomyosarcoma (ULMS). Specific cell lines were cultured and qRT-PCR was performed. Thirteen miRNAs presented different expression profiles in ULM and the same thirteen in ULMS compared to MM. Eight miRNAs were overexpressed, and five were underexpressed in ULM. In ULMS cells, five miRNAs exhibited an overexpression and eight were down-regulated. Six miRNAs (miR-1-3p, miR-130b-3p, miR-140-5p, miR-202-3p, miR-205-5p, and miR-7-5p) presented a similar expression pattern in cell lines compared to patient samples. Of these, only three miRNAs showed significant expression in ULM (miR-1-3p, miR-140-5p, and miR-7-5p) and ULMS (miR-1-3p, miR-202-3p, and miR-7-5p). Our preliminary approach identified 24 oncomirs with an altered expression profile in ULM and ULMS cells. We identified four differentially expressed miRNAs with the same profile when compared with patients’ samples, which strongly interacted with relevant genes, including apoptosis regulator (BCL2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), insulin like growth factor 1 receptor (IGF1R),serine/threonine kinase (RAF1), receptor tyrosine kinase (MET), and bHLH transcription factor (MYCN). This led to alterations in their mRNA-target.

Could miRNA Signatures be Useful for Predicting Uterine Sarcoma and Carcinosarcoma Prognosis and Treatment

Changes in microRNA (miRNA) expression may lead to cancer development and/or contribute to its progression; however, their role in uterine sarcomas is poorly understood. Uterine sarcomas (US) belong to a rare class of heterogeneous tumors, representing about 1% of all gynecologic neoplasms. This study aimed to assess the expression profile of 84 cancer-related miRNAs and to evaluate their correlation with clinical pathological features. Eighty-two formalin-fixed paraffin-embedded (FFPE) samples were selected. In leiomyosarcoma (LMS), there was an association of lower cancer-specific survival (CSS) with the downregulation of miR-125a-5p and miR-10a-5p, and the upregulation of miR-196a-5p and miR-34c-5p. In carcinosarcoma (CS), lower CSS was associated with the upregulation of miR-184, and the downregulation of let-7b-5p and miR-124. In endometrial stromal sarcomas (ESS), the upregulation of miR-373-3p, miR-372-3p, and let-7b-5p, and the down-expression of let-7f-5p, miR-23-3p, and let-7b-5p were associated with lower CSS. Only miR-138-5p upregulation was associated with higher survival rates. miR-335-5p, miR-301a-3p, and miR-210-3p were more highly expressed in patients with tumor metastasis and relapse. miR-138-5p, miR-146b-5p, and miR-218-5p expression were associated with higher disease-free survival (DFS) in treated patients. These miRNAs represent potential prediction markers for prognosis and treatment response in these tumors.

Abstract 541: Involvement of miRNA expression may contribute for FoxO3a oncogenic role in uterine leiomyosarcoma

Introduction: Leiomyoma (LM) and leiomyosarcoma (LMS) are uterine mesenchymal tumors that present variable clinical behavior. LM are extremely common estrogen and progesterone dependent benign tumors. On the other hand, LMS are rare and aggressive malignant tumors with limited available treatment options. FoxO3a seems to have a tumor suppressor role and its loss of function may determine deregulation in the cell proliferation, with DNA damage accumulation. Several studies demonstrated FoxO3a with an important role in several tumors development; however, its regulation or function is unknown in uterine mesenchymal tumors. Objective: Here, our goals were to assess the FoxO3a expression profile in uterine LM and LMS, to evaluate the mechanisms involved on FoxO3a regulation, and to assess its use as a marker for diagnosis, prognosis and malignancy risk prediction. Methods: Formalin-Fixed Paraffin-Embedded tissues of 56 leiomyosarcomas (LMS), 80 leiomyomas (LM) and 20 myometrium (MM) from patients, were used for FOXO3a protein evaluation by immunohistochemistry (IHQ). Epigenetic regulation of FoxO3a expression was assessed by 84 miRNAs gene analysis, using Real Time PCR method (MIHS 109Z, Qiagen). MM, LM and LMS cells were evaluated for FoxO3a gene expression by Real Time PCR. Results: FOXO3a protein showed an increased expression profile in MM, LM and LMS. Higher protein levels were observed in the comparison between LMS and MM (p Citation Format: Natalia Garcia, Anamaria Ritti Ricci, Bruna Cristine de Almeida, Thais Gomes Almeida, Isabela Werneck Cunha, Edmund Chada Baracat, Katia Candido Carvalho. Involvement of miRNA expression may contribute for FoxO3a oncogenic role in uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 541.

Abstract 4882: Differential BMP4 and GREM1 protein expression in uterine leiomyosarcoma and leiomyoma.

Background: Uterine leiomyosarcoma (ULMS) and leiomyoma (ULM) are smooth muscle tumors with distinct clinical behavior. Little is known about the factors that influence these tumors biology. Differential diagnosis among low grade ULMS and the several clinical forms of ULM is a challenge both in laboratory and pathological routine. Thus, the identification of new molecular markers can help in the differential diagnosis between ULM from ULMS and clinical management. Our aim was to evaluate expression of bone morphogenetic protein - 4 (BMP-4) and Gremlin-1 (GREM1) in uterine leiomyosarcoma and leiomyoma (including leiomyoma with atypia) using immunohistochemistry. Method: 57 patients with ULMS and 30 with ULM, among them 10 ULM with atypia (ULMA) were select. The tissues were used in tissue microarray construction. Immunohistochemistry reaction was performed with monoclonal antibody against BMP-4 and GREM-1 and evaluated using semi-quantitative method. Results: BMP-4 was positive in 43% of the ULMS, 5% ULM and 80% ULMA, respectively. GREM-1 was positive in 67% of the ULMS, 20% ULM and 10% ULMA. BMP-4 is a potent growth factor that is involved in many important biological processes. Gremlin-1 can specifically bind to BMP-4 precursor protein inside cells, which prevents the production and secretion of mature BMP-4. This regulation determines BMP-4 biological effects on cells in the local microenvironment. However, the molecular mechanism of GREM-1/BMP-4 interaction remains unexplored. Conclusion: Ours preliminary results showed that expression of BMP-4 and GREM-1 play an important role in uterine smooth tumors, in while atypical leiomyomas presented higher levels of BMP-4 protein expression and lower levels of GREM-1. However, the role of these proteins interactions must be clarified. Citation Format: Natalia Garcia, Faila Catarina Souza, Isabela Werneck Cunha, Fernando Augusto Soares, Gustavo Arantes Rosa Maciel, Edmund Chada Baracat, Katia Candido Carvalho. Differential BMP4 and GREM1 protein expression in uterine leiomyosarcoma and leiomyoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2013-4882