Soledad Gallego

Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells.

Tumors of the Ewings sarcoma family (ESFT), such as Ewings sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

Cholecystokinin Down-Regulation by RNA Interference Impairs Ewing Tumor Growth

Purpose: Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1 oncoprotein and assess its functional role in Ewing tumor pathogenesis. Experimental Design: Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1 expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycycline-inducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth. Results: Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK. Conclusions: Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.

First-night effect in the chronic fatigue syndrome

Since the magnitude of the first-night effect has been shown to be a function of medical conditions and of settings in which polysomnographies are performed, it is essential to evaluate the habituation phenomenon in each case in order to determine the optimal recording methodology. A first-night effect was evidenced in certain cases of chronic fatigue syndrome, but not in others. To clarify this issue, a large group of patients with chronic fatigue syndrome who had no primary sleep disorders were selected and recorded for two consecutive nights in a hospital sleep unit. Several parameters, frequently associated with the first-night effect, were found to be influenced by the recording methodology: Total Sleep Time, Sleep Efficiency, Sleep Efficiency minus Sleep Onset, Sleep Onset Latency, Wake Time, Slow Wave Sleep, Rapid Eye Movement Sleep, Rapid Eye Movement Sleep Latency and Number of Sleep Cycles. Bland and Altman plots determined that the difference scores between the nights included a systematic bias linked to the order of recordings (first-night effect). Factorial analysis grouped the difference scores into three factors. No significant difference was observed between patients with generalized anxiety comorbidity and those with no psychiatric comorbidity, or between those with and without psychiatric comorbidity. Chronic fatigue syndrome must thus be added on the list of conditions where a clinically significant habituation effect takes place.

Notch Pathway Inhibition Significantly Reduces Rhabdomyosarcoma Invasiveness and Mobility In Vitro

Purpose: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal and alveolar RMS. Patients with metastatic disease continue to have very poor prognosis although aggressive therapies and recurrences are common in advanced localized disease. The oncogenic potential of the Notch pathway has been established in some cancers of the adult and in some pediatric malignancies. Experimental Design: A real-time PCR assay was used to ascertain the expression of several Notch pathway components in a wide panel of RMS and cell lines. Four γ-secretase inhibitors (GSIs) were tested for pathway inhibition and the degree of inhibition was assessed by analysis of Hes1 and Hey1 expression. The putative effects of Notch pathway inhibition were evaluated by wound-healing, matrigel/transwell invasion, cell-cycle, and apoptosis assays. Results: The Notch pathway was widely expressed and activated in RMS and underwent substantial inhibition when treated with GSIs or transfected with a dominant negative form of MAML1. RMS cells showed a significant decrease in its mobility and invasiveness when the Notch pathway was properly inhibited; conversely, its inhibition had no noticeable effect on cell cycle or apoptosis. Conclusion: Pharmacological or genetic blockage of the pathway significantly reduced invasiveness of RMS cell lines, thereby suggesting a possible role of the Notch pathway in the regulation of the metastatic process in RMS. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

A seroepidemiologic study of hepatitis a in Spanish children. Relationship of prevalence to age and socio-environmental factors

Three child population groups from the Madrid area were studied for anti-HAV antibodies. Analysis was carried out with respect to age and socio-environmental factors. The population under study was composed of 156 children, with ages ranging from 1 to 14 years; they were stratified in three socio-environmental groups (white-family unit, gypsy-family unit and orphanage), and also divided into subgroups according to age. As a whole, an age-related increase in prevalence was found. The overall seroprevalence by socio-environmental groups was: gypsy-family unit 63%, orphanage 46%, and white-family unit 23%. Significant differences between groups appeared from seven years on, being more marked among the eldest subgroups. Among the factors evaluated, hygienic-sanitary conditions and overcrowding influenced the high prevalence rate found in the gypsy-family unit subjects, whereas overcrowding appeared to be responsible for the higher prevalence in orphanage residents, as compared to white-family unit children. Kinder aus der Region von Madrid, die drei Bevölkerungsgruppen angehören, wurden auf anti-HAV-Antikörper untersucht und Beziehungen zwischen Prävalenz, Alter und Milieufaktoren analysiert. Die Gruppe bestand aus 156 Kindern im Alter von 1 bis 14 Jahren. Sie gehörten drei Gruppen mit unterschiedlichen sozialen Gegebenheiten an: in der Familie lebende weiße Kinder, in der Familie lebende Zigeunerkinder und Waisenkinder. Die Analyse nach Altersgruppen zeigte einen deutlichen altersabhängigen Anstieg der Seroprävalenz mit signifikant höheren Infektionsraten in den Altersgruppen 7–14 als bei 1–6jährigen. Die Durchseuchung mit Hepatitis A war in den Zigeunerfamilien am höchsten (63%), gefolgt von Waisenkindern (46%) und weissen Kindern, die in Familien leben (23%). Für die Zigeunerkinder wurden unzureichende sanitäre Verhältnisse und Übervölkerung der Wohnräume als wichtige Einflußfaktoren identifiziert. Bei Waisenkindern wird angenommen, daß enge räumliche Verhältnisse für die im Vergleich zu den in Familien lebenden Kindern höhere Seroprävalenz verantwortlich sind.SummaryThree child population groups from the Madrid area were studied for anti-HAV antibodies. Analysis was carried out with respect to age and socio-environmental factors. The population under study was composed of 156 children, with ages ranging from 1 to 14 years; they were stratified in three socio-environmental groups (white-family unit, gypsy-family unit and orphanage), and also divided into subgroups according to age. As a whole, an age-related increase in prevalence was found. The overall seroprevalence by socio-environmental groups was: gypsy-family unit 63%, orphanage 46%, and white-family unit 23%. Significant differences between groups appeared from seven years on, being more marked among the eldest subgroups. Among the factors evaluated, hygienic-sanitary conditions and overcrowding influenced the high prevalence rate found in the gypsy-family unit subjects, whereas overcrowding appeared to be responsible for the higher prevalence in orphanage residents, as compared to white-family unit children.ZusammenfassungKinder aus der Region von Madrid, die drei Bevölkerungsgruppen angehören, wurden auf anti-HAV-Antikörper untersucht und Beziehungen zwischen Prävalenz, Alter und Milieufaktoren analysiert. Die Gruppe bestand aus 156 Kindern im Alter von 1 bis 14 Jahren. Sie gehörten drei Gruppen mit unterschiedlichen sozialen Gegebenheiten an: in der Familie lebende weiße Kinder, in der Familie lebende Zigeunerkinder und Waisenkinder. Die Analyse nach Altersgruppen zeigte einen deutlichen altersabhängigen Anstieg der Seroprävalenz mit signifikant höheren Infektionsraten in den Altersgruppen 7–14 als bei 1–6jährigen. Die Durchseuchung mit Hepatitis A war in den Zigeunerfamilien am höchsten (63%), gefolgt von Waisenkindern (46%) und weissen Kindern, die in Familien leben (23%). Für die Zigeunerkinder wurden unzureichende sanitäre Verhältnisse und Übervölkerung der Wohnräume als wichtige Einflußfaktoren identifiziert. Bei Waisenkindern wird angenommen, daß enge räumliche Verhältnisse für die im Vergleich zu den in Familien lebenden Kindern höhere Seroprävalenz verantwortlich sind.

Notch, Wnt, and Hedgehog Pathways in Rhabdomyosarcoma: From Single Pathways to an Integrated Network

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Regarding histopathological criteria, RMS can be divided into 2 main subtypes: embryonal and alveolar. These subtypes differ considerably in their clinical phenotype and molecular features. Abnormal regulation or mutation of signalling pathways that regulate normal embryonic development such as Notch, Hedgehog, and Wnt is a recurrent feature in tumorigenesis. Herein, the general features of each of the three pathways, their implication in cancer and particularly in RMS are reviewed. Finally, the cross-talking among these three pathways and the possibility of better understanding of the horizontal communication among them, leading to the development of more potent therapeutic approaches, are discussed.

Anesthetic Efficacy of Eutectic Prilocalne-Lidocaine Cream in Pediatric Oncology Patients Undergoing Lumbar Puncture

OBJECTIVE: To evaluate the efficacy of eutectic mixture of local anesthetics 5% (Emla) in reducing pain associated with lumbar punctures in children. DESIGN: Prospective, double-blind, randomized, placebo-controlled trial. SETTING: University pediatric hospital. PATIENTS: Eleven pediatric oncology patients (mean age 6.6 y, range 4–16) who underwent 31 lumbar punctures. MAIN OUTCOME MEASURES: The analgesic effect was measured by using two methods. The first was a 10-point visual analog scale reported by the patient and the second was an 8-point behavioral pain scale assessed by the nurse who applied the cream. RESULTS: Emla cream was associated with significantly lower pain scores than those with placebo as measured by the patient when the puncture was successful on the first attempt (2.0 +1.6 Emla group, 3.8 +1.9 placebo group; p < 0.05). CONCLUSIONS: The use of Emla cream may reduce pain substantially only in patients who undergo a successful lumbar puncture on the first attempt.

BRG1/SMARCA4 is essential for neuroblastoma cell viability through modulation of cell death and survival pathways

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30–40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.

Post-transplant lymphoproliferative disorders in children: The role of chemotherapy in the era of rituximab

Gallego S, Llort A, Gros L, Sanchez de Toledo Jr J, Bueno J, Moreno A, Nieto J, Sanchez de Toledo J. Post‐transplant lymphoproliferative disorders in children: The role of chemotherapy in the era of rituximab.
Pediatr Transplantation 2010: 14: 61–66.

Detection of bone marrow micrometastasis and microcirculating disease in rhabdomyosarcoma by a real-time RT-PCR assay

Purpose: To assess if molecular detection of minimal disseminated disease by real-time reverse transcription and polymerase chain reaction (RT-PCR) could contribute to a better treatment stratification in patients with rhabdomyosarcoma (RMS). Methods: Relative quantification of the tumor-mRNA present in serial samples of bone marrow (BM) and peripheral blood (PB) from 16 patients with RMS (7 alveolar and 9 embryonal) was performed by a real-time RT-PCR assay. Expression of MyoD1 and acetylcholine receptor (AChR) was analyzed in all samples, along with PAX3/7-FKHR in samples from alveolar tumors. Results: A good correlation was found between the expression of PAX3/7-FKHR and AChR, while MyoD1 was more sensitive but less specific. In this study, patients with positive PB at the end of treatment showed a poorer prognosis than patients with negative PB. Moreover, in this patient cohort, metastatic relapses were preceded by the detection of microcirculating disease in all cases. Conclusion: The detection of minimal circulating and micrometastatic disease by real-time RT-PCR, based on the expression of multiple genes, yields highly reproducible results. Patients with positive PB after treatment show poorer survival than patients without microcirculating disease.