Anna Llort

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.

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.

Secondary malignancies and quality of life after stem cell transplantation.

Summary:Malignant diseases (MD) occurring after stem cell transplantation (SCT) are of particular concern as increasing number of patients survive and remain free of their original disease. The cumulative incidence at 15 years is 10–12%. The B-cell proliferative disorders (BCLP) are the most common MD in the first year after SCT; the incidence probability is 1% in allogeneic transplants but is much higher (until 14%) after HLA-identical, T-cell-depleted SCT in which Campath 1G or ATG are given. BCLP develop because of reactivation of the EBV and a depressed cellular immunity. Prediction of risk of BCLP can be made by frequent monitoring of EBV load in patients with risk factors. The most effective therapies are the early administration of anti-CD20 monoclonal antibody and adoptive immunotherapy with in vitro generated EBV-specific cytotoxic T cells. Myelodysplasia and acute myeloid leukemia with very poor prognosis have been described in 4–18% of patients with non-Hodgkin lymphoma and Hodgkin disease, 12–24 months after autologous SCT. The risk of development of solid tumors increases over time and the cumulative incidence among children who underwent an SCT at less than 10 years of age is 6–11% at 15 years. There are few studies evaluating quality of life (QOL) in children and adolescents who had received an SCT. The findings of these studies can be summarized as follows: (a) The majority of long survivors enjoy good QOL and return successfully to school or work. (b) A minority (10–15%) complain of physical problems or present moderate cognitive or psychological dysfunctions. (c) The importance of family, other social support and psychological adjustments is generally recognized. More extensive, longitudinal and comparative studies with other alternative therapies are required.

Resultados del trasplante de progenitores hematopoyéticos en hemoglobinopatías: talasemia maior y enfermedad drepanocítica

BACKGROUND The prevalence of hemoglobinopathies in Spain is increasing as a result of immigration. Thalassemia major presents with chronic hemolytic anemia that requires regular red blood cell transfusions within the first year of life. Patients with sickle cell disease suffer from chronic anemia, vasculopathy and progressive damage in almost any organ. There is decreased life expectancy in both conditions. Allogeneic hematopoietic stem cell transplantation represents the only potentially curative option. PATIENTS Seventeen patients (fourteen thalassemia major, and three sickle cell disease) underwent allogeneic hematopoietic stem cell transplantations. RESULTS In the thalassemia group, nine donors were HLA-geno-identical siblings, two were partially matched related donors (one HLA allele mismatch), and three unrelated donors. All three patients with sickle cell disease were transplanted from HLA-geno-identical siblings. The source of stem cells was bone marrow in sixteen cases. Median patient age at transplant was six years (range: 1-16) in the thalassemia group, and twelve years (range: 8-15) in the sickle cell disease group. The graft was successful in all patients. Secondary graft rejection was observed in two thalassemia patients rendering them dependent on blood transfusions. Complete chimerism was observed in thirteen patients and, although mixed chimerism occurred in two, with all of them showing normal hemoglobin levels after transplantation and not requiring further transfusion support. Patients affected by sickle cell disease did not present with new vaso-occlusive crises, and stabilization of pulmonary and neurological function was observed. Chronic graft-versus-host disease was detected in three patients affected by thalassemia, and hypogonadotrophic hypogonadism in five patients. CONCLUSIONS We conclude that for thalassemia major and sickle cell disease, allogenic hematopoietic stem cell transplantation from HLA-geno-identical siblings offers a high probability of complication-free survival. Despite good results, morbidity and mortality associated with transplantation from unrelated donors is a risk that might be considered, in contrast to a non-curative medical treatment that offers a long term survival. For thalassemia major groups it could be an option, but not for sickle cell disease, which is still in the investigational phase.

Trombocitemia esencial en pediatría: tratamiento con anagrelide

Introduccion La trombocitemia esencial (TE) es un sindrome mieloproliferativo cronico. La prevalencia es de 0,09 casos por millon de ninos entre 0 y 16 anos. La cefalea es el sintoma clinico mas descrito. Existe una incidencia aumentada de fenomenos tromboticos graves (15 %), hemorragicos moderados-graves (10%) y leves (15 %). Las indicaciones de tratamiento son discutidas, existiendo como opciones la hidroxiurea y el anagrelide. Anagrelide es una imidazoquinazolina que tiene un efecto de inhibicion de la maduracion medular de los megacariocitos y un efecto antiagregante plaquetario. Material y metodos Revision retrospectiva de las historias clinicas de los pacientes diagnosticados de TE en nuestro Centro entre los anos 1997-2006. Se utilizaron los criterios diagnosticos de TE descritos por el Polycythemia Vera Study Group (1997). En todos los casos se practicaron los siguientes estudios: hemograma y formula leucocitaria (paciente y familiares de primer grado), PCR, VSG, metabolismo del hierro, estudio basico de coagulacion, serologias virales, parasitos, dosificacion de vitamina B12, ecografia abdominal, radiografia pulmonar, aspirado de medula osea, citogenetica convencional, reordenamiento bcr/abl, cultivo de progenitores hematopoyeticos, biopsia osea, dosificacion de trombopoyetina y estudio de agregacion plaquetaria. Se administro tratamiento con anagrelide a dosis de 1 mg/dia dosis inicial (modificandola en funcion de la respuesta) y monitorizacion de efectos secundarios atribuibles al farmaco. Resultados Se han diagnosticado 4 pacientes. Todos ellos de sexo femenino. El rango de edad oscilaba entre 4 y 13 anos. El sintoma clinico mas frecuente en nuestras pacientes fue la cefalea (3/4). Una paciente presento epistaxis. En todas, la cifra de plaquetas al diagnostico fue superior a 1.500 × 109/l. Las 4 pacientes recibieron tratamiento con anagrelide, requiriendo dosis entre 1-3 mg, para conseguir un descenso eficaz de la cifra plaquetas (400 × 109/l), y desaparicion de la sintomatologia clinica. Ninguna paciente ha presentado fenomenos hemorragicos/tromboticos durante el tratamiento. No se han objetivado efectos secundarios asociados al farmaco. Disponemos de datos de seguimiento de hasta 6 anos. Conclusiones Anagrelide es una opcion de tratamiento util en la TE, obteniendose una reduccion de la cifra de plaquetas en un periodo de 2 semanas-4 meses, en nuestra serie. No se han descrito efectos secundarios atribuibles al farmaco, en nuestras pacientes. Se precisan estudios cooperativos internacionales, en la poblacion pediatrica, para poder establecer indicaciones claras de tratamiento de estos pacientes.

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence‐activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

Infantile Fibrosarcoma of the Retroperitoneum: A Site of Unfavorable Prognosis?

Infantile fibrosarcoma (IF) is a rare soft tissue sarcoma affecting mainly infants. Presence of the ETV6-NTRK3 fusion gene is characteristic but not specific of this tumor [1]. This fusion is also present in cellular mesoblastic nephroma (CMN). Presence of the same genetic alteration in IF and CMN, together with their similar clinical and phenotypic characteristics, suggests that they belong to the same pathologic entity [2]. A 3-month-old girl was admitted with a tumor occupying the right abdomen, hypotension, and pallor. Laboratory tests confirmed severe anemia (hemoglobin = 5.4 g/dL). Imaging tests showed a large solid cystic mass with hemorrhagic areas extending from the liver to the pelvis. Urgent laparotomy detected a tumor involving the right kidney composed by multiple hemorrhagic cysts. Right nephrectomy and partial tumor resection were performed. Light microscopy showed a densely cellular tumor composed of spindle cells exhibiting a fascicular pattern and frequent mitoses. After surgery, she was treated with vincristine and actinomycin D for 8 months, when she presented local progression. A second surgery was performed with a partial resection. Chemotherapy was changed to carboplatin, etoposide, and ifosfamide (4 cycles) with partial response, followed by a complete resection and treatment was stopped. A year later, she presented with a local recurrence and was reoperated on with microscopic residue; thus, she received right abdominal radiation to 15 Gy. She is currently diseasefree after 17 years of follow-up. The second patient, a 5-month-old boy, was admitted with a tumor occupying the whole abdomen, severe anemia (hemoglobin = 4.9 g/dL), and coagulopathy. Imaging studies demonstrated a large abdominal mass (Figure 1). Emergency laparotomy identified a highly friable tumor composed of cystic areas filled with hematic content adhering to the right kidney, liver, and duodenum, which could only partially be excised. Microscopically, the tumor showed moderately atypical spindle cells, with abundant mitoses. Two cycles of vincristine, actinomycin D, and cyclophosphamide were administered with no response. A month later, he suffered another episode of

Targeting of epigenetic regulators in neuroblastoma

Approximately 15,000 new cases of pediatric cancer are diagnosed yearly in Europe, with 8–10% corresponding to neuroblastoma, a rare disease with an incidence of 8–9 cases per million children <15 years of age. Although the survival rate for low-risk and intermediate-risk patients is excellent, half of children with high-risk, refractory, or relapsed tumors will be cured, and two-thirds of the other half will suffer major side effects and life-long disabilities. Epigenetic therapies aimed at reversing the oncogenic alterations in chromatin structure and function are an emerging alternative against aggressive tumors that are or will become resistant to conventional treatments. This approach proposes targeting epigenetic regulators, which are proteins that are involved in the creation, detection, and interpretation of epigenetic signals, such as methylation or histone post-translational modifications. In this review, we focused on the most promising epigenetic regulators for targeting and current drugs that have already reached clinical trials.Cancer therapy: Exploring an epigenetic approachTreatments that target chromatin, the combination of DNA and proteins, are emerging as alternative ways to treat aggressive neuroblastomas, cancers of neural tissue. Altering the structure and function of chromatin is a form of “epigenetic therapy”, treatment that affects inheritable molecular signals controlling the activity of genes, rather than targeting the genes directly. Researchers in Spain led by Miguel Segura at the Vall d’Hebron Research Institute in Barcelona review progress in developing epigenetic therapies for neuroblastomas. A growing body of fundamental research and evidence from clinical trials suggest this approach could open promising new avenues to treating aggressive and drug-resistant cancers. The authors recommend an increased effort to identify and explore the activities of small molecules that could form the basis of effective epigenetic therapies for various cancers.

Ensayos clínicos precoces en oncología pediátrica en España: una perspectiva nacional

INTRODUCTION Cancer is the leading cause of death between the first year of life and adolescence, and some types of diseases are still a major challenge in terms of cure. There is, therefore, a major need for new drugs. Recent findings in cancer biology open the door to the development of targeted therapies against individual molecular changes, as well as immunotherapy. Promising results in adult anti-cancer drug development have not yet been translated into paediatric clinical practice. A report is presented on the activity in early paediatric oncology trials (phase I-II) in Spain. MATERIAL AND METHODS All members of the Spanish Society of Paediatric Haematology Oncology (SEHOP) were contacted in order to identify early clinical trials in paediatric cancer opened between 2005 and 2015. RESULTS A total of 30 trials had been opened in this period: 21 (70%) in solid tumours, and 9 (30%) in malignant haemopathies. A total of 212 patients have been enrolled. The majority was industry sponsored (53%). Since 2010, four centres have joined the international consortium of Innovative Therapies for Children with Cancer (ITCC), which has as its aim to develop novel therapies for paediatric tumours. A significant number of new studies have opened since 2010, improving the treatment opportunities for our children. Results of recently closed trials show the contribution of Spanish investigators, the introduction of molecularly targeted agents, and their benefits. CONCLUSIONS The activity in clinical trials has increased in the years analysed. The SEHOP is committed to develop and participate in collaborative academic trials, in order to help in the advancement and optimisation of existing therapies in paediatric cancer.