Régia Caroline Peixoto Lira

Low Expression of Human Histocompatibility Soluble Leukocyte Antigen-G (HLA-G5) in Invasive Cervical Cancer With and Without Metastasis, Associated With Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC w )] and absence [ICC without metastasis (ICC wt )] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC wt (n=52 patients) and ICC w (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

The larynx is the most common site of malignancy in the upper aerodigestive tract. In Brazil, malignant laryngeal lesions represent 2% of all cancers, with ∼3000 annual deaths. The association between human papillomavirus (HPV) and laryngeal cancer is still controversial. The aim of the present retrospective study was to determine the expression of galectin-3 immunoperoxidase in laryngeal carcinoma by examining paraffin-embedded larynx biopsies from 65 patients, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, and 28 with metastases. Twenty-eight cervical lymph nodes from patients with metastatic lesions were also evaluated. Nested PCR was performed to detect and type HPV DNA. Galectin-3 expression was assessed by immunohistochemistry using a computer-assisted system. Among 65 patients, 55 (84.6%) were positive to beta-globin (internal control); 10 (15.4%) patients were beta-globin negative and were excluded from the HPV evaluation. Thus, 7 (12.7%) out of 55 patients were HPV positive and 48 (87.3%) out of 55 patients were HPV negative. High expression of galectin-3 was observed in invasive laryngeal tumors, suggesting that galectin-3 could be associated with the invasiveness and aggressiveness of laryngeal carcinomas. (J Histochem Cytochem 57:665–673, 2009)

Inhibition of nuclear factor-κB by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-&kgr;B (NF-&kgr;B) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-&kgr;B blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases.

Mucoepidermoid Carcinoma of the Lung Arising at the Primary Site of A Bronchogenic Cyst: Clinical, Cytogenetic, and Molecular Findings

Primary lung tumors are rare in children, and mucoepidermoid carcinoma (MEC) represents less than 10% of them. Additionally, MEC arising from bronchogenic cysts (BC) is particularly unusual. We describe the clinical and genetic findings on a MEC occurring within a previous location of a BC in an adolescent. This particular association has not been previously reported. The lesion revealed normal karyotype without the typical t(11;19)(q21;p13) translocation. Cyclin D1 overexpression (165‐fold increase) was demonstrated by real‐time PCR although FISH assessment showed normal hybridization at 11q13. Information on these unusual clinical presentations may present relevant insight on tumorigenesis of infrequent pediatric pulmonary tumors. Pediatr Blood Cancer 2011;56:311–313.

Low expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 is associated with poor prognosis in pediatric adrenocortical tumors (ACT).

Low expression of HLA class II antigens has been associated with more aggressive disease in several human malignancies including adult adrenocortical tumors (ACT), but their clinical relevance in pediatric ACT needs to be investigated.

Chromosomal heterogeneity and instability characterize pediatric medulloblastoma cell lines and affect neoplastic phenotype

Chromosomal heterogeneity is a hallmark of most tumors and it can drive critical events as growth advantages, survival advantages, progression and karyotypic evolution. Medulloblastoma (MB) is the most common malignant central nervous system tumor in children. This work attempted to investigate chromosomal heterogeneity and instability profiles of two MB pediatric cell lines and their relationship with cell phenotype. We performed GTG-banding and cytokinesis-block micronucleus cytome assays, as well as morphological characterization, cell population doubling time, colony-forming efficiency, and chemo-sensitivity assays in two pediatric MB cell lines (UW402 and UW473). Both MB cells showed a high chromosomal heterogeneity. UW473 cells showed ~2 fold higher both clonal- and non-clonal chromosomal alterations than UW402 cells. Besides, UW473 showed two clonal-groups well-differentiated by ploidy level (<2n> and <4n>) and also presented a significantly higher number of chromosomal instability biomarkers. These results were associated with high morphological heterogeneity and survival advantages for UW473 and proliferation advantages for UW402 cells. Moreover, UW473 was significantly more sensitive to methotrexate, temozolomide and cisplatin while UW402 cells were more sensitive to doxorubicin. These data suggest that distinct different degrees of karyotypic heterogeneity and instability may affect neoplasic phenotype of MB cells. These findings bring new insights into cell and tumor biology.

HIF1A is overexpressed in medulloblastoma and its inhibition reduces proliferation and increases EPAS1 and ATG16L1 methylation.

BACKGROUND Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. OBJECTIVE Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. METHOD qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. RESULTS A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. CONCLUSION HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patients clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1μM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.

Perioperative Conjunctival Inflammation and Trabeculectomy Outcome

Abstract Purpose: To correlate subclinical conjunctival inflammation and trabeculectomy results. Methods: Prospective case series of 28 patients with primary open-angle glaucoma (28 eyes) under topical anti-glaucoma medication who underwent trabeculectomy. During surgery, a sample from the inferior bulbar conjunctiva was collected and the expression of HLA-DR together with the presence of inflammatory cells was correlated with trabeculectomy outcomes after 24 months. Surgical success was defined as intraocular pressure between 6 and 20 mmHg irrespective of the use of anti-glaucoma medication. Results: Five patients missed follow-up visits and were removed from the study. Ten eyes (43.5%) were HLA-DR+, but no significant differences were observed between eyes with successful and failed surgeries (p = 0.214). There was no significant association between the number of neutrophils and surgical outcomes (p = 0.353). Conclusions: The presence of inflammatory cells and expression of the inflammation marker HLA-DR in the conjunctiva did not correlate with the prognosis of trabeculectomy in this study.

Prognostic value and functional role of ROCK2 in pediatric Ewing sarcoma

Ewings sarcoma (EWS) is a highly aggressive bone cancer that affects children and adolescents. Despite advances in multimodal management, 5-year event-free survival rates for patients presenting with metastases at diagnosis remain at 25%. As key regulators of actin organization, the Rho-associated coiled-coil containing protein kinases, ROCK1 and ROCK2, have been associated with cancer dissemination and poorer prognosis. Recently, in vitro data indicating ROCK2 as a molecular target for the treatment of EWS has been presented. Nonetheless, a deeper exploration of the contribution of this kinase dysregulation in EWS is still necessary. In this regard, the present study aimed to evaluate the expression of ROCK1 and ROCK2 in 23 pediatric tumor samples and to verify the prospect of using their pharmacological inhibition through functional assays. Our results showed positive immunostaining for ROCK1 and ROCK2 in the majority samples (75 and 65%, respectively). A significantly increased risk of incomplete remission in patients with positive immunostaining for ROCK2 was found (P=0.026), though no correlations with other prognostic features (huvos classification, FLI1/EWS status, relapse, metastasis or death) were observed. Associations with survival were merely suggestive. Apparent protein expression of both kinases was also found in EWS cell lines (SK-ES-1 and RD-ES). Treatments with selective ROCK inhibitors did not alter cell viability or migration in vitro. However, a significant increase in invasion was observed after treatment with SR3677 (ROCK2 inhibitor) and hydroxyfasudil (pan-inhibitor). Consequently, even though the majority of EWS samples included in our study showed positivity for ROCK1 and ROCK2, the lack of significant associations with prognosis and absence of appropriate responses to their inhibition in vitro does not support their prospective use as therapeutic targets for the treatment of this metastatic tumor. Larger cohort studies might provide more evidence on whether there is a specific role of ROCK kinases in EWS physiopathology.