First Transcriptome Analysis of Hepatoblastoma in Brazil: Unraveling the Pivotal Role of Noncoding RNAs and Metabolic Pathways.

Hepatoblastoma stands as the most prevalent liver cancer in the pediatric population. Characterized by a low mutational burden, chromosomal and epigenetic alterations are key drivers of its tumorigenesis. Transcriptome analysis is a powerful tool for unraveling the molecular intricacies of hepatoblastoma, shedding light on the effects of genetic and epigenetic changes on gene expression. In this study conducted in Brazilian patients, an in-depth whole transcriptome analysis was performed on 14 primary hepatoblastomas, compared to control liver tissues. The analysis unveiled 1,492 differentially expressed genes (1,031 upregulated and 461 downregulated), including 920 protein-coding genes (62%). Upregulated biological processes were linked to cell differentiation, signaling, morphogenesis, and development, involving known hepatoblastoma-associated genes (DLK1, MEG3, HDAC2, TET1, HMGA2, DKK1, DKK4), alongside with novel findings (GYNG4, CDH3, and TNFRSF19). Downregulated processes predominantly centered around oxidation and metabolism, affecting amines, nicotinamides, and lipids, featuring novel discoveries like the repression of SYT7, TTC36, THRSP, CCND1, GCK and CAMK2B. Two genes, which displayed a concordant pattern of DNA methylation alteration in their promoter regions and dysregulation in the transcriptome, were further validated by RT-qPCR: the upregulated TNFRSF19, a key gene in the embryonic development, and the repressed THRSP, connected to lipid metabolism. Furthermore, based on protein-protein interaction analysis, we identified genes holding central positions in the network, such as HDAC2, CCND1, GCK, and CAMK2B, among others, that emerged as prime candidates warranting functional validation in future studies. Notably, a significant dysregulation of non-coding RNAs (ncRNAs), predominantly upregulated transcripts, was observed, with 42% of the top 50 highly expressed genes being ncRNAs. An integrative miRNA-mRNA analysis revealed crucial biological processes associated with metabolism, oxidation reactions of lipids and carbohydrates, and methylation-dependent chromatin silencing. In particular, four upregulated miRNAs (miR-186, miR-214, miR-377, and miR-494) played a pivotal role in the network, potentially targeting multiple protein-coding transcripts, including CCND1 and CAMK2B. In summary, our transcriptome analysis highlighted disrupted embryonic development as well as metabolic pathways, particularly those involving lipids, emphasizing the emerging role of ncRNAs as epigenetic regulators in hepatoblastomas. These findings provide insights into the complexity of the hepatoblastoma transcriptome and identify potential targets for future therapeutic interventions.

Plasma immune mediators as laboratorial biomarkers for Sickle Cell Disease patients according to the hydroxyurea therapy and disease severity.

In the present work, the impact of Sickle Cell Disease (SCD) degrees of severity, as well hydroxyurea treatment on the systemic immunological signatures of patients was evaluated. Based on a high-throughput chemokine, cytokine and growth factor multiplex analysis, it was possible to obtain the systemic immunological profile of patients with SCD (n = 40), treated or not with hydroxyurea, as compared to healthy controls (n = 40). Overall, SCD patients with severe disease displayed increased levels of almost all biomarkers analyzed. Our data demonstrated that CXCL8, CCL3 and CXCL10 were pointed out as universal biomarkers of SCD. The results also indicated that HU-untreated patients with indication of HU-therapy display a more prominent increase on plasma immune mediators in a similar way as those with severe SCD disease. Together, these findings provided a comprehensive landscape of evidence that may have implications for further therapeutic strategies and SCD clinical management.

Quantitative expression evaluation of PRAME gene in osteosarcoma.

BACKGROUND: In a previous study, our group observed that 68% of the osteosarcoma (OS) samples presented PRAME (Preferentially Expressed Antigen in Melanoma) gene expression. In this work, we propose to investigate quantitatively gene expression of PRAME in distinct patients groups. METHODS AND RESULTS: 61 osteosarcoma samples, from 3 distinct patients groups were selected for this study: (1) Patients younger than 10 years old at diagnosis, (2) Patients that had poor evolution of disease and (3) Patients that were in remission of disease and had treatment with no intercurrences) PRAME gene expression levels were obtained using quantitative Real-Time Polymerase Chain Reaction method (qRT-PCR). Clinical parameters were collected from patient's medical charts. Results demonstrated an increase in PRAME gene expression in all samples, with high variation in expression levels, when considering all samples and when analyzed in each group. In addition, no statistical difference was found when considering clinical data collected or patients groups. CONCLUSION: PRAME gene expression quantitative investigation did not bring any complementary information beyond of what had already been observed in other qualitative investigations published by our group, there is no relation between PRAME gene expression levels and disease evolution. However, the findings in this work contribute for validation PRAME gene expression as a good biomarker to OS, which, in the future, may allow identification circulating tumor cell or molecules to contribute with early diagnostic of metastasis, a genuine problem in OS that determinate flattening in survival curves.

Pharmacogenetics of the Primary and Metastatic Osteosarcoma: Gene Expression Profile Associated with Outcome.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. In recent decades, OS treatment has reached a plateau and drug resistance is still a major challenge. Therefore, the present study aimed to analyze the expression of the genes related to pharmacogenetics in OS. The expression of 32 target genes in 80 paired specimens (pre-chemotherapeutic primary tumor, post-chemotherapeutic primary tumor and pulmonary metastasis) obtained from 33 patients diagnosed with OS were analyzed by the real-time PCR methodology. As the calibrators (control), five normal bone specimens were used. The present study identified associations between the OS outcome and the expression of the genes TOP2A, DHFR, MTHFR, BCL2L1, CASP3, FASLG, GSTM3, SOD1, ABCC1, ABCC2, ABCC3, ABCC5, ABCC6, ABCC10, ABCC11, ABCG2, RALBP1, SLC19A1, SLC22A1, ERCC1 and MSH2. In addition, the expression of the ABCC10, GGH, GSTM3 and SLC22A1 genes were associated with the disease event, and the metastasis specimens showed a high expression profile of ABCC1, ABCC3 and ABCC4 genes and a low expression of SLC22A1 and ABCC10 genes, which is possibly an important factor for resistance in OS metastasis. Therefore, our findings may, in the future, contribute to clinical management as prognostic factors as well as possible therapeutic targets.

Endothelial dysfunction biomarkers in sickle cell disease: is there a role for ADMA and PAI-1?

Within the spectrum of sickle cell disease (SCD) are sickle cell anemia (SCA), presence of hemoglobin SS (HbSS), hemoglobin SC disease (HbSC), and sickle cell ß-thalassemia (Sß-thal). Asymmetric dimethylarginine (ADMA) competitively inhibits the binding of arginine to NOS, reducing NO production. In patients with HbSS, increased levels of ADMA have been reported, as well as changes in many hemostatic biomarkers, including the plasminogen activator inhibitor type 1 (PAI-1). We hypothesized that high levels of ADMA and PAI-1 may be associated with more severe SCD. Thus, ADMA and PAI-1 levels were determined in 78 individuals including 38 adult patients with SCD and 40 control subjects. Higher levels of ADMA were shown in HbSS and Sß-thal patients compared to controls. Concerning PAI-1, all patients showed high levels of PAI-1 compared to controls. As a role of NO in the pathogenesis of SCD has already been established, we concluded that high levels of ADMA should compromise, at least in part, NO synthesis, resulting in endothelial dysfunction. Elevated plasma levels of PAI-1 in all patients may indicate not only endothelial dysfunction but also a hypofibrinolytic state favoring thrombotic complications. Finally, high levels of ADMA and PAI-1 may be associated with more severe SCD.

Molecular profiling of pediatric and adolescent ependymomas: identification of genetic variants using a next-generation sequencing panel.

PURPOSE: Ependymoma (EPN) accounts for approximately 10% of all primary central nervous system (CNS) tumors in children and in most cases, chemotherapy is ineffective and treatment remains challenging. We investigated molecular alterations, with a potential prognostic marker and therapeutic target in EPNs of childhood and adolescence, using a next-generation sequencing (NGS) panel specific for pediatric neoplasms. METHODS: We selected 61 samples with initial diagnosis of EPN from patients treated at Pediatric Oncology Institute-GRAACC/UNIFESP. All samples were divided according to the anatomical compartment of the CNS - 42 posterior fossa (PF), 14 supratentorial (ST), and five spinal (SP). NGS was performed to identify somatic genetic variants in tumor samples using the Oncomine Childhood Cancer Research Assay® (OCCRA®) panel, from Thermo Fisher Scientific®. RESULTS: Genetic variants were identified in 24 of 61 (39.3%) tumors and over 90% of all variants were pathogenic or likely pathogenic. The most commonly variants detected were in CIC, ASXL1, and JAK2 genes and have not been reported in EPN yet. MN1-BEND2 fusion, alteration recently described in a new CNS tumor type, was identified in one ST sample that was reclassified as astroblastoma. Additionally, YAP1-MAMLD1 fusion, a rare event associated with good outcome in ST-EPN, was observed in two patients diagnosed under 2 years old. CONCLUSIONS: Molecular profiling by the OCCRA® panel showed novel alterations in pediatric and adolescent EPNs, which highlights the clinical importance in identifying genetic variants for patients' prognosis and therapeutic orientation.

Abnormal spindle-like microcephaly-associated (ASPM) gene expression in posterior fossa brain tumors of childhood and adolescence.

PURPOSE: In neurogenesis, ASPM (abnormal spindle-like microcephaly-associated) gene is expressed mainly in the ventricular zone of posterior fossa and is the major determinant in the cerebral cortex. Besides its role in embryonic development, ASPM overexpression promotes tumor growth, including central nervous system (CNS) tumors. This study aims to investigate ASPM expression levels in most frequent posterior fossa brain tumors of childhood and adolescence: medulloblastoma (MB), ependymoma (EPN), and astrocytoma (AS), correlating them with clinicopathological characteristics and tumor solid portion size. METHODS: Quantitative reverse transcription (qRT-PCR) is used to quantify ASPM mRNA levels in 80 pre-treatment tumor samples: 28 MB, 22 EPN, and 30 AS. The tumor solid portion size was determined by IOP-GRAACC Diagnostic Imaging Center. We correlated these findings with clinicopathological characteristics and tumor solid portion size. RESULTS: Our results demonstrated that ASPM gene was overexpressed in MB (p = 0.007) and EPN (p = 0.0260) samples. ASPM high expression was significantly associated to MB samples from patients with worse overall survival (p = 0.0123) and death due to disease progression (p = 0.0039). Interestingly, two patients with AS progressed toward higher grade showed ASPM overexpression (p = 0.0046). No correlation was found between the tumor solid portion size and ASPM expression levels in MB (p = 0.1154 and r = - 0.4825) and EPN (p = 0.1108 and r = - 0.3495) samples. CONCLUSION: Taking in account that ASPM gene has several functions to support cell proliferation, as mitotic defects and premature differentiation, we suggest that its overexpression, presumably, plays a critical role in disease progression of posterior fossa brain tumors of childhood and adolescence.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

MAPK7 variants related to prognosis and chemotherapy response in osteosarcoma.

Osteosarcoma (OS) is a class of cancer originating from the bone, affecting mainly children and young adults. Our previous study showed that MAPK7 gene overexpression was significantly associated with tumor progression, poor treatment response, and worse overall survival, suggesting that MAPK7 could play an important role in OS tumorigenesis. We have investigated if MAPK7 overexpression was a result of any genomic changes in OS tumor specimens. We identified five SNPs (Single Nucleotide Polymorphism) previously described in databases, dbSNP and COSMIC, and identified two single nucleotide substitution not yet described. We found, in prechemotherapy specimens, a significant association of MAPK7 rs2233072G allele variant with metastasis at diagnosis and relapse (0.0909 and 0.0455, respectively). In post-chemotherapy, rs1054206GG specimen's genotype was associated with osteoblastic histological type (P= 0.0249) and presented decreased MAPK7 gene expression when compared with pre-chemotherapy specimens of same patients (P = 0.0095). Interestingly, it was observed some SNPs genotype exchange after chemotherapy. Our data indicated that MAPK7 gene expression associated with genotype exchange after chemotherapy, and these SNPs associated with important clinical parameters might be a valuable indicator for predicting in OS.

Genome-wide association study identifies the GLDC/IL33 locus associated with survival of osteosarcoma patients.

Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. We conducted a multi-institutional genome-wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma, including 523 patients of European ancestry and 109 from Brazil. We conducted a time-to-event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. The results were combined across the European and Brazilian case sets using a random-effects meta-analysis. The strongest association after meta-analysis was for rs3765555 at 9p24.1, which was inversely associated with overall survival (HR = 1.76; 95% CI 1.41-2.18, p = 4.84 × 10-7 ). After imputation across this region, the combined analysis identified two SNPs that reached genome-wide significance. The strongest single association was with rs55933544 (HR = 1.9; 95% CI 1.5-2.4; p = 1.3 × 10-8 ), which localizes to the GLDC gene, adjacent to the IL33 gene and was consistent across both the European and Brazilian case sets. Using publicly available data, the risk allele was associated with lower expression of IL33 and low expression of IL33 was associated with poor survival in an independent set of patients with osteosarcoma. In conclusion, we have identified the GLDC/IL33 locus on chromosome 9p24.1 as associated with overall survival in patients with osteosarcoma. Further studies are needed to confirm this association and shed light on the biological underpinnings of this susceptibility locus.

Valproic acid treatment response in vitro is determined by TP53 status in medulloblastoma.

PURPOSE: Histone deacetylate inhibitors (HDACi), as valproic acid (VA), have been reported to enhance efficacy and to prevent drug resistance in some tumors, including medulloblastoma (MB). In the present study, we investigated VA role, combined to cisplatin (CDDP) in cell viability and gene expression of MB cell lines. METHODS: Dose-response curve determined IC50 values for each treatment: (1) VA single, (2) CDDP single, and (3) VA and CDDP combined. Cytotoxicity and flow cytometry evaluated cell viability after exposure to treatments. Quantitative PCR evaluated gene expression levels of AKT, CTNNB1, GLI1, KDM6A, KDM6B, NOTCH2, PTCH1, and TERT, before and after treatment. Besides, we performed next-generation sequencing (NGS) for PTCH1, TERT, and TP53 genes. RESULTS: The most effective treatment to reduce viability was combined for D283MED and ONS-76; and CDDP single for DAOY cells (p < 0.0001). TERT, GLI1, and AKT genes were overexpressed after treatments with VA. D283MED and ONS-76 cells presented variants in TERT and PTCH1, respectively and DAOY cell line presented a TP53 mutation. CONCLUSIONS: MB tumors belonging to SHH molecular subgroup, with TP53MUT, would be the ones that present high risk in relation to VA use during the treatment, while TP53WT MBs can benefit from VA therapy, both SHH and groups 3 and 4. Our study shows a new perspective about VA action in medulloblastoma cells, raising the possibility that VA may act in different patterns. According to the genetic background of MB cell, VA can stimulate cell cycle arrest and apoptosis or induce resistance to treatment via signaling pathways activation.

MAPK pathways regulation by DUSP1 in the development of osteosarcoma: Potential markers and therapeutic targets.

Osteosarcoma (OS) is the most frequent primary bone tumor that affect children and adolescents. This tumor is highly aggressive with high risk of metastasis and the implementation of new drugs has not been successful. The search for biomarkers or new therapeutic targets is urgently needed and can help in advances of OS treatment. MAPKs are major signaling transduction molecules that play an important role in regulating a variety of cellular responses. DUSP1 is a phosphatase that dephosphorylates the MAPKs. Both MAPKs and DUSPs have been implicated as major modulators of critical signaling pathways that are dysregulated in various diseases. In a previous study, we found an increase in MAPK7 gene expression contributed for worst overall survival and treatment response. We analyzed gene expression of MAPK pathways that participate in MAPK7 regulation, and DUSP1 gene using paired 28 pre/post-chemotherapy and 12 metastasis OS samples. To understand the DUSP1 role in the pathogenesis of OS, we assessed the function of DUSP1 in four OS cell lines through a series of cellular assays combined with gene silencing technique. Our findings showed increased MAP2K6, MAP4K3, and DUSP1 gene expression in post-chemotherapy OS samples presenting poor prognosis. We also found that the suppression of DUSP1 gene expression resulted in decreased proliferation, migration, and invasion in OS cells. These results suggest that members of MAPK family may be possible prognostic markers in OS and DUSP1 has a relevant role in the OS pathogenesis and can be an attractive therapeutic target in new strategies of OS treatment.

MAPK7 gene controls proliferation, migration and cell invasion in osteosarcoma.

Osteosarcomas (OS) are the most common malignant bone tumors, and the identification of useful tumor biomarkers and target proteins is required to predict the clinical outcome of patients and therapeutic response as well as to develop novel therapeutic strategies. In our previous study, MAPK7 has been identified as a candidate oncogene, and a promising prognostic marker for OS. Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. In this study, we investigated the behavior of MAPK7 gene in OS cell lines. Technical viability, proliferation, migration, invasion, and apoptosis were used to evaluate the function of the MAPK7 gene. We evaluated the behavior of the OS cells with MAPK7 gene silenced, not silenced, and exposed to the main chemotherapy drugs used in OS treatment. We found that silenced MAPK7 gene is effective at suppressing cell proliferation, inhibiting cell migration, and invasion. Furthermore, MAPK7 is an important activator of transcription factors and is the main expression modulator of other key genes in the MAPK pathway. In summary, our study suggests that MAPK7 might be a promising therapeutic target for OS. © 2015 Wiley Periodicals, Inc.

GNAS mutations are not detected in parosteal and low-grade central osteosarcomas.

Parosteal osteosarcoma, low-grade central osteosarcoma, and fibrous dysplasia share similar histological features that may pose a diagnostic challenge. The detection of GNAS mutations in primary bone tumors has been useful in clinical practice for diagnosing fibrous dysplasia. However, the recent report of GNAS mutations being detected in a significant proportion of parosteal osteosarcoma challenges the specificity of this mutation. As the number of cases reported in this study was small we set out to determine if these results could be reproduced. We studied 97 formalin-fixed paraffin-embedded low-grade osteosarcomas from 90 patients including 62 parosteal osteosarcomas, of which MDM2 amplification was detected in 79%, 11 periosteal osteosarcomas and 24 low-grade central osteosarcoma samples. The mutational status of GNAS was analyzed in codons p.R201, p.Q227, and other less common GNAS alterations by bidirectional Sanger sequencing and/or next generation sequencing using the Life Technologies Ion Torrent platform. GNAS mutations were not detected in any of the low-grade osteosarcomas from which informative DNA was extracted. Our findings therefore support prior observations that GNAS mutations are highly specific for fibrous dysplasia and occur rarely, if ever, in parosteal and other low-grade osteosarcomas.

Analysis of KIAA1549-BRAF fusion gene expression and IDH1/IDH2 mutations in low grade pediatric astrocytomas.

Low-grade astrocytomas comprise about 30 % of the central nervous system tumors in children. Several investigations have searched a correlation between the BRAF gene fusions alterations and mutations at IDH1 and IDH2 genes in low grade pediatric astrocytomas. This study identified the expression of KIAA1549-BRAF fusion gene and BRAF V600E mutation, mutations at exon 4 of the IDH1 and IDH2 genes in samples of pilocytic astrocytomas (PA) and grade-II astrocytomas (A-II) pediatric patients. The correlation between these alterations and the clinical profile of the patients was also evaluated. Eighty-two samples of low-grade astrocytomas (65 PA and 17 A-II) were analyzed by PCR and sequencing for each of the targets identified. We identified the KIAA1549-BRAF fusion transcript in 45 % of the samples. BRAF V600E and BRAFins598T mutations were detected in 7 and 1 % of the samples, respectively. Mutations in the R132/R172 residues of the IDH1/IDH2 genes were detected in only two samples, and the G105G polymorphism (rs11554137:C>T) was identified in ten patients. Additionally, we observed two mutations out of the usual hotspots at IDH1 and IDH2 genes. We observed a smaller frequency of mutations in IDHs genes than previously described, but since the prior studies were composed of adult or mixed (adults and children) samples, we believe that our results represent a relevant contribution to the growing knowledge in low grade childhood astrocytomas.

SHH, WNT, and NOTCH pathways in medulloblastoma: when cancer stem cells maintain self-renewal and differentiation properties.

PURPOSE: Infant medulloblastoma (MB) is a malignant neuroepithelial embryonal tumor of the cerebellum, believed to derive from precursor granule cells with stem or progenitor cells appearance, and caused by a change in expression profile of genes related to the development. This work aims to study the expression profile of these genes in MB tumors, correlating with clinicopathological characteristics. METHODS: We quantified, by qPCR in 40 MB tumor samples, the expression of genes in HH (PTCH1, PTCH2, and GLI1), WNT (APC, CTNNB1, WIF1, and DKK2), and NOTCH pathways (NOTCH2 and HES1), which have a crucial role in development, and genes as MYCC, MYCN, and TERT, correlating this findings to patient's clinicopathological characteristics. RESULTS: Considering the universal RNA as our control sample, and considering the median of gene expression in the control samples as our cutoff, we observed that HES1 gene showed decreased expression compared to control (p = 0.0059), but patients with HES1 overexpression were directly related to a shorter survival (p = 0.0165). Individuals with higher GLI1 gene expression had significant shorter survival (p = 0.0469), and high expression was prevalent in patients up to 5 years old (p = 0.0479). Patients showing high PTCH2 expression were related to worse survival (p = 0.0426), and it was correlated with GLI1 high expression (p = 0.0094). We also observed a concomitant overexpression of WIF1 and DKK2 genes in a subgroup of MB samples (n = 11, p = 0.0118). CONCLUSIONS: Our results suggest the presence of activated developmental signaling pathways in MB, which are important for cell proliferation and maintenance, and that may be targeted for novel therapeutic options.

Expression profiling using a cDNA array and immunohistochemistry for the extracellular matrix genes FN-1, ITGA-3, ITGB-5, MMP-2, and MMP-9 in colorectal carcinoma progression and dissemination.

Colorectal cancer dissemination depends on extracellular matrix genes related to remodeling and degradation of the matrix structure. This investigation intended to evaluate the association between FN-1, ITGA-3, ITGB-5, MMP-2, and MMP-9 gene and protein expression levels in tumor tissue with clinical and histopathological neoplastic parameters of cancer dissemination. The expression associations between ECM molecules and selected epithelial markers EGFR, VEGF, Bcl2, P53, and KI-67 have also been examined in 114 patients with colorectal cancer who underwent primary tumor resection. Quantitative real-time PCR and immunohistochemistry tissue microarray methods were performed in samples from the primary tumors. The gene expression results showed that the ITGA-3 and ITGB-5 genes were overexpressed in tumors with lymph node and distant metastasis (III/IV-stage tumors compared with I/II tumors). The MMP-2 gene showed significant overexpression in mucinous type tumors, and MMP-9 was overexpressed in villous adenocarcinoma histologic type tumors. The ECM genes MMP9 and ITGA-3 have shown a significant expression correlation with EGFR epithelial marker. The overexpression of the matrix extracellular genes ITGA-3 and ITGB-5 is associated with advanced stage tumors, and the genes MMP-2 and MMP-9 are overexpressed in mucinous and villous adenocarcinoma type tumors, respectively. The epithelial marker EGFR overactivity has been shown to be associated with the ECM genes MMP-9 and ITGA-3 expression.

Expression of integrin genes and proteins in progression and dissemination of colorectal adenocarcinoma.

BACKGROUND: This study aimed to evaluate the relationship between the expression levels of selected integrin genes and proteins and cell differentiation, TNM stage, histological type and other variables potentially associated with the progression and dissemination of colorectal carcinoma (CRC). METHODS: A total of 114 patients (63 men and 51 women) were treated for CRC between 2006 and 2009, including 25 (21.9%) TNM I, 39 (34.2%) TNM II, 34 (29.8%) TNM III, and 16 (14.1%) TNM IV. Regarding grade, 91 (79.8%) were grade II, 14 (12.2%) were grade III and nine (7.8%) were grade I. Reverse-transcription polymerase chain reaction (RT-PCR) and tissue microarray (TMA) methods were used to examine the expression levels of the genes ITGAV, ITGA3, ITGA5, ITGB5, and ITGA6, and their proteins, respectively. RESULTS: In relation to TNM staging, ITGB5 and ITGA3 were over-expressed in stages III versus I. These results were confirmed by TMA analysis. In terms of age, ITGA5 was under-expressed according to RT-PCR, but over-expressed by TMA in patients over 60 years, while ITGA5 gene and protein levels were increased in mucinous carcinomas. In addition ITGAV gene and protein levels were elevated in tumors with neural invasion, and ITGA6 gene and protein were over-expressed in cases with venous invasion. All these results were significant at P < 0.05. CONCLUSION: The results of this study suggest that over-expression of some integrins is associated with TNM III stage, increased risk of vascular and neural invasion, and mucinous histology in patients with CRC.

Expression analysis of stem cell-related genes reveal OCT4 as a predictor of poor clinical outcome in medulloblastoma.

Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P ≤ 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.