In situ assessment of Mindin as a biomarker of podocyte lesions in diabetic nephropathy.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and end-stage renal failure worldwide. Several mechanisms are involved in the pathogenesis of this disease, which culminate in morphological changes such as podocyte injury. Despite the complex diagnosis and pathogenesis, limited attempts have been made to establish new biomarkers for DN. The higher concentration of Mindin protein in the urine of patients with type 2 diabetes mellitus suggests that it plays a role in DN. Therefore, this study investigated whether in situ protein expression of Mindin can be considered a potential DN biomarker. Fifty renal biopsies from patients diagnosed with DN, 57 with nondiabetic glomerular diseases, including 17 with focal segmental glomerulosclerosis (FSGS), 14 with minimal lesion disease (MLD) and 27 with immunoglobulin A nephropathy (IgAN), and 23 adult kidney samples from autopsies (control group) were evaluated for Mindin expression by immunohistochemistry. Podocyte density was inferred by Wilms' tumor 1 (WT1) immunostaining, while foot process effacement was assessed by transmission electron microscopy. Receiver operative characteristic (ROC) analysis was performed to determine the biomarker sensitivity/specificity. Low podocyte density and increased Mindin expression were observed in all cases of DN, regardless of their class. In the DN group, Mindin expression was significantly higher than that in the FSGS, MCD, IgAN and control groups. Higher Mindin expression was significantly positively correlated with foot process effacement only in class III DN cases. Furthermore, Mindin protein presented high specificity in the biopsies of patients with DN (p < 0.0001). Our data suggest that Mindin may play a role in DN pathogenesis and is a promising biomarker of podocyte lesions.

MicroRNA-149-3p expression correlates with outcomes of adrenocortical tumor patients and affects proliferation and cell cycle progression of H295A adrenocortical cancer cell line.

Pediatric adrenocortical tumor (ACT) is a rare and aggressive neoplasm, with incidence in southern and southeastern Brazil 10-15 times higher than worldwide. Although microRNAs (miRNAs) have been reported to act as tumor suppressors or oncogenes in several cancers, the role of miR-149-3p in ACT remains unknown. In this study, we evaluated the expression of miR-149-3p in 67 pediatric ACT samples and 19 non-neoplastic adrenal tissues. The overexpression of miR-149-3p was induced in H295A cell line, and cell viability, proliferation, colony formation, and cell cycle were assessed by in miR-149-3p mimic or mimic control. In silico analysis were used to predict miR-149-3p putative target genes. CDKN1A expression at the mRNA and protein levels was evaluated by qRT-PCR and western blot, respectively. Higher miR-149-3p expression was associated with unfavorable ACT outcomes. Compared to the mimic control, miR-149-3p overexpression increased cell viability and colony formation, and affected cell cycle progression. Also, we identified CDKN1A as a potential miR-149-3p target gene, with decreased expression at both the gene and protein levels in miR-149-3p mimic cells. Collectively, these findings suggest that miR-149-3p promotes H295A cell viability by downregulating CDKN1A and provide evidence that miR-149-3p may be useful as a novel therapeutic target for pediatric ACT.

MicroRNA expression profile predicts prognosis of pediatric adrenocortical tumors.

Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. Despite extensive efforts, identifying reliable prognostic factors for pediatric patients with ACT remains a challenge. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and prognosis of several types of cancer. However, the role of miRNAs has been poorly explored in pediatric ACT. In this study, we performed miRNA microarray profiling on a cohort of 37 pediatric ACT and nine nonneoplastic adrenal (NNA) samples and evaluated the prognostic significance of abnormally expressed miRNAs using Kaplan-Meier plots, log-rank test, and Cox regression analysis. We identified a total of 98 abnormally expressed miRNAs; their expression profile discriminated ACT from NNAs. Among the 98 deregulated miRNAs, 17 presented significant associations with patients' survival. In addition, higher expression levels of hsa-miR-630, -139-3p, -125a-3p, -574-5p, -596, -564, -1321, and -423-5p and lower expression levels of hsa-miR-377-3p, -126-3p, -410, -136-3p, -29b-3p, -29a-3p, -337-5p, -143-3p, and 140-5p were significantly associated with poor prognosis, tumor relapse, and/or death. Importantly, the expression profile of these 17 miRNAs stratified patients into two groups of ACTs with different clinical outcomes. Although some individual miRNAs exhibit potential prognostic values in ACTs, only the 17 miRNA-based expression clustering was considered an independent prognostic factor for 5-year event-free survival (EFS) compared to other clinicopathological features. In conclusion, our study reports for the first time associations between miRNA profiles and childhood ACT prognosis, providing evidence that miRNAs could be useful biomarkers to discriminate patients with favorable and unfavorable clinical outcomes.

A coordinated approach for the assessment of molecular subgroups in pediatric ependymomas using low-cost methods.

Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (C11orf95-RELA, YAP1-MAMLD1, and YAP1-FAM118B) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of CXorf67, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with LAMA2/NELL2 markers. RELA cases and YAP1-MAMLD1 fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note, LAMA2 and NELL2 gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN. KEY MESSAGES: Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups. RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs. Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs. Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups. A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.

High-throughput microRNA profile in adult and pediatric primary glioblastomas: the role of miR-10b-5p and miR-630 in the tumor aggressiveness.

Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients' prognoses remain dismal. The miRNA expression profiles have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.

Arsenic Trioxide exerts cytotoxic and radiosensitizing effects in pediatric Medulloblastoma cell lines of SHH Subgroup.

We evaluated the potential effects of ATO in different pediatric SHH-MB cell lines (ONS-76: TP53-wild type; DAOY and UW402: TP53-mutated). MB cell lines molecular subgroup was confirmed and TP53 mutations were validated. Cell viability, clonogenicity and apoptosis were evaluated after ATO treatment at different concentrations (1-16 µM) alone or combined with irradiation doses (0.5, 1, 2 and 4 Gy). Rad51 and Ku86 proteins were evaluated by WB. ATO treatment reduced cell viability for all SHH-MB cell lines. Significant decrease of clonogenic capacity and higher apoptosis rates were also observed after ATO exposure, being cell death more pronounced (>70%) for the SHH-MB TP53-mutated. Combined treatment of ATO with irradiation also reduced colonies formation in UW402 tumor cells, which was independent of DNA damage repair proteins Rad51 and Ku86. In silico analyses suggested that a set of genes from cell cycle and p53 pathways are differentially expressed in SHH tumor subtypes, suggesting that cell lines may respond to therapies according to the gene expression profiles. Herein, we showed ATO cytotoxicity in pediatric SHH cell lines, with marked radiosensitizing effect for the MB-SHH TP53-mutated cells. These results highlight the potential of ATO, alone or in combination with radiotherapy, supporting further clinical investigations.

Notch pathway in ependymoma RELA-fused subgroup: upregulation and association with cancer stem cells markers expression.

RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

microRNA-138-5p as a Worse Prognosis Biomarker in Pediatric, Adolescent, and Young Adult Osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumor with two peaks of incidence, in early adolescence and the elderly. Patients affected with this malignancy often present metastatic disease at diagnosis, and despite multimodality therapy, survival has not improved substantially over the past 3 decades. Recently, miR-138-5p, proposed as a crucial intracellular mediator of invasion, has been recognized to target the Rho-associated coiled-coil containing protein kinase 2 (ROCK2). Dysregulation of ROCK1 and ROCK2 was also described in OS, being associated to higher metastasis incidence and worse prognosis. Nonetheless, the specific roles of miR-138-5p in pediatric and young adult OS and its ability to modulate these kinases remain to be established. Thus, in the present study, the expression levels miR-138-5p were evaluated in a consecutive cohort of exclusively pediatric and young adult primary OS samples. In contrast to previous reports that included adult tissues, our results showed upregulation of miR-138-5p associated with reduced event-free survival and relapsed cases. In parallel, ROCK1 mRNA levels were significantly reduced in tumor samples and negatively correlated with miR-138-5p. Similar correlations were observed after studying the profiles of ROCK1 and ROCK2 by immunohistochemistry. Our data present miR-138-5p as a consistent prognostic factor in pediatric and young adult OS, reinforcing its participation in the post-transcriptional regulation of ROCK kinases.

CTGF expression is indicative of better survival rates in patients with medulloblastoma.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children and it is subgrouped into 4 entities (SHH, WNT, Group 3, and Group 4). Molecular pathways involved in these different subgroups still are evolving and can be of clinical relevance to therapy. The YAP1-CTGF axis is known to regulate cell proliferation, differentiation, and cell death; however, its role in MB is poorly explored. We aimed to investigate the role of YAP1 gene in the MB SHH cell line DAOY and evaluate cell proliferation, doubling time and 3D spheroids invasion and its consequence on CTGF regulation. We assessed CTGF expression from 22 children with MB. Lastly, we validated our findings through in silico analysis in large cohorts dataset of patients. We observed an increased invasion rate of DAOY cells and CTGF downregulation under YAP1 knockdown (p < 0.0001). Additionally CTGF is overexpressed in MB with extensive nodularity subtype and an indicative of higher survival rates in pediatric MB (p < 0.05). Interestingly, no difference of CTGF expression was observed between molecular subgroups. These results provide new evidence ofCTGF as a potential prognostic marker for MB, corroborating to the role of YAP1 in restricting MB cell.

A simplified approach using Taqman low-density array for medulloblastoma subgrouping.

Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making.

MicroRNA profile of pediatric pilocytic astrocytomas identifies two tumor-specific signatures when compared to non-neoplastic white matter.

PURPOSES: Pilocytic astrocytoma (PA) is a low-grade neoplasm frequently found in childhood. PA is characterized by slow growth and a relatively good prognosis. Genetic mechanisms such as activation of MAPK, BRAF gene deregulation and neurofibromatosis type 1 (NF1) syndrome have been associated with PA development. Epigenetic signature and miRNA expression profile are providing new insights about different types of tumor, including PAs. METHODS: In the present study we evaluated global miRNA expression in 16 microdissected pediatric PA specimens, three NF1-associated PAs and 11 cerebral white matter (WM) samples by the microarray method. An additional cohort of 20 PAs was used to validate by qRT-PCR the expression of six miRNAs differentially expressed in the microarray data. RESULTS: Unsupervised hierarchical clustering analysis distinguished one cluster with nine PAs, including all NF1 cases and a second group consisting of the WM samples and seven PAs. Among 88 differentially expressed miRNAs between PAs and WM samples, the most underexpressed ones regulate classical pathways of tumorigenesis, while the most overexpressed miRNAs are related to pathways such as focal adhesion, P53 signaling pathway and gliomagenesis. The PAs/NF1 presented a subset of underexpressed miRNAs, which was also associated with known deregulated pathways in cancer such as cell cycle and hippo pathway. CONCLUSIONS: In summary, our data demonstrate that PA harbors at least two distinct miRNA signatures, including a subgroup of patients with NF1/PA lesions.

Prognostic value and functional role of ROCK2 in pediatric Ewing sarcoma.

Ewing's 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.

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 patient's 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.

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.

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.

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-κB (NF-κB) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-κB 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.