Ana Sastre

Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma

Ewings sarcoma (ES) is characterized by specific chromosome translocations, the most common being t(11;22)(q24;q12). Additionally, other type of genetic abnormalities may occur and be relevant for explaining the variable tumour biology and clinical outcome. We have carried out a high-resolution array CGH and expression profiling on 25 ES tumour samples to characterize the DNA copy number aberrations (CNA) occurring in these tumours and determine their association with gene-expression profiles and clinical outcome. CNA were observed in 84% of the cases. We observed a median number of three aberrations per case. Besides numerical chromosome changes, smaller aberrations were found and defined at chromosomes 5p, 7q and 9p. All CNA were compiled to define the smallest overlapping regions of imbalance (SORI). A total of 35 SORI were delimited. Bioinformatics analyses were conducted to identify subgroups according to the pattern of genomic instability. Unsupervised and supervised clustering analysis (using SORI as variables) segregated the tumours in two distinct groups: one genomically stable (⩽3 CNA) and other genomically unstable (>3 CNA). The genomic unstable group showed a statistically significant shorter overall survival and was more refractory to chemotherapy. Expression profile analysis revealed significant differences between both groups. Genes related with chromosome segregation, DNA repair pathways and cell-cycle control were upregulated in the genomically unstable group. This report elucidates, for the first time, data about genomic instability in ES, based on CNA and expression profiling, and shows that a genomically unstable group of Ewings tumours is correlated with a significant poor prognosis.

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.

High Survival Rate in Infant Acute Leukemia Treated With Early High-Dose Chemotherapy and Stem-Cell Support

PURPOSE Infants with acute leukemia have a poor prognosis when treated with conventional chemotherapy. It is still unknown if stem-cell transplantation (SCT) can improve the outcome of these patients. In the present study, we review our experience with SCT in infant acute leukemia to clarify this issue. PATIENTS AND METHODS We report the results of 26 infants who were submitted to a SCT for acute leukemia. There were 15 cases of acute myeloid leukemia and 10 cases of acute lymphoid leukemia. One patient had a bilineal leukemia. Twenty-two patients were in their first complete response (CR1), three were in their second CR, and one was in relapse. Eight patients were submitted to allogeneic SCT, and 18 underwent autologous SCT. RESULTS With a median follow-up of 67 months, the 5-year overall survival and disease-free survival (DFS) are 64% (SE = 9%) and 63% (SE = 10%), respectively. Autologous and allogeneic SCT offered similar outcome. There was not any transplant-related mortality, and all deaths were caused by relapse in the first 6 months after SCT. In multivariate analysis, the single factor associated with better DFS was an interval between CR1 and SCT of less than 4 months (P: <.025). CONCLUSION SCT is a valid option in the treatment of infant acute leukemia, and it may overcome the high risk of relapse with conventional chemotherapy showing very reduced toxicity. This study suggests that SCT should be performed in CR1 in the early phase of the disease.

Autologous stem cell transplantation for advanced Hodgkin's disease in children

This study evaluates the outcome of myeloablative chemo-radiotherapy and autologous stem cell transplantation (ASCT) in children with Hodgkins disease (HD). Twenty children aged 5 to 18 years (median 10.8 years) at diagnosis, with relapsed, refractory or very poor prognosis HD, underwent ASCT in eight hospitals of our country. Status at transplant was: second complete remission (CR2): n = 12; further cr (cr >2): n = 3, partial remission (PR): n = 2, relapse: n = 2 and first CR (CR1): n = 1. Eighteen patients received chemotherapy-based conditioning regimens: cyclophosphamide, carmustine and etoposide (CBV): 11 (55%), carmustine, etoposide, cytarabine and melphalan (BEAM): 5, other: 2; and two patients were conditioned with TBI/Cy. Peripheral blood (PB) was the source of progenitor cells in 12 patients, BM in seven, and BM plus PB, in one. All patients engrafted. One patient died of sepsis and multiorgan failure at day 28 after transplantation. All four patients with measurable disease (PR or relapse) at transplantation attained complete remission. Five patients relapsed 5–34 months after transplant (median: 11 months). Eighteen children remain alive with a median survival time of 40 months. The projected 5-year overall survival and event-free survival (EFS) rates were 0.95 and 0.62. High-dose therapy with stem cell rescue can lead to durable remissions in children with advanced HD. Bone Marrow Transplantation (2000) 25, 31–34.

Mir-pharmacogenetics of methotrexate in childhood B-cell acute lymphoblastic leukemia

Objectives Methotrexate (MTX), the key drug in childhood B-cell acute lymphoblastic leukemia (B-ALL) therapy, often causes toxicity. An association between genetic variants in MTX transport genes and toxicity has been found. It is known that these transporters are regulated by microRNAs (miRNAs), and miRNA single nucleotide polymorphisms (SNPs) interfere with miRNA levels or function. With regard to B-cell ALL, we have previously found rs56103835 in miR-323b that targets ABCC4 associated with MTX plasma levels. Despite these evidences and that nowadays a large amount of new miRNAs have been annotated, studies of miRNA polymorphisms and MTX toxicity are almost absent. Therefore, the aim of this study was to determine whether there are other variants in miRNAs associated with MTX levels. Patients and methods Blood samples of 167 Spanish patients with pediatric B-cell ALL treated with the LAL-SHOP protocol were analyzed. We selected all the SNPs described in pre-miRNAs with a minor allele frequency more than 1% (213 SNPs in 206 miRNAs) that could regulate MTX transporters because the miRNAs that target MTX transporter genes are not completely defined. Genotyping was performed with VeraCode GoldenGate platform. Results Among the most significant results, we found rs56292801 in miR-5189, rs4909237 in miR-595, and rs78790512 in miR-6083 to be associated with MTX plasma levels. These miRNAs were predicted, in silico, to regulate genes involved in MTX uptake: SLC46A1, SLC19A1, and SLCO1A2. Conclusion In this study, we detected three SNPs in miR-5189, miR-595, and miR-6083 that might affect SLC46A1, SLC19A1, and SLCO1A2 MTX transport gene regulation and could affect MTX levels in patients with pediatric B-cell ALL.

Long-term outcome of allogeneic or autologous haemopoietic cell transplantation for acute lymphoblastic leukaemia in second remission in children. GETMON experience 1983–1998

Summary:We present a retrospective study of long-term outcome and predictive factors of survival and relapse in 219 paediatric patients with acute lymphoblastic leukaemia (ALL) in second remission. They received allogeneic (allo) or autologous (auto) haemopoietic cell transplantation (HCT) depending on the availability of a matched sibling donor. The probability of event-free survival (EFS) for the total patient group was 0.35+0.03 at 14 years. No significant differences were observed for EFS between allo- and auto-HCT: 0.39+0.05 vs 0.32+0.04 (P=0.43). A better EFS was seen in patients with a late relapse (LR) (P=0.06 and 0.02, for allogeneic and autologous respectively). Significantly better EFS was observed in allo-HCT patients under 10 years of age and in auto-HCT patients with leukocytes at diagnosis below 25 × 109/l and late relapse. Predictive factors of failure in both groups were early relapse (ER), medullary relapse and age over 10 years. The probability of relapse (RP) for the total group of patients was 0.57+0.03, and it was significantly higher in auto-HCT patients: 0.65+0.04 vs 0.42+0.06 (P=0.002). Factors predictive for relapse were medullary and early relapse, auto-HCT and WBC >25 × 109/l at diagnosis.

ALLOGENEIC HEMOPOIETIC STEM CELL TRANSPLANTATION (HSCT) FOR WISKOTT-ALDRICH SYNDROME: A Report of the Spanish Working Party for Blood and Marrow Transplantation in Children (GETMON)

Allogeneic stem cell transplantation is the only curative treatment for Wiskott-Aldrich syndrome. The authors retrospectively analyzed the outcome with this procedure in 13 patients with severe Wiskott-Aldrich syndrome transplanted in 5 Spanish centers from 1989 to 2006. A patient was transplanted twice from the same donor due to a late engraftment failure. Age at transplant ranged from 7 to 192 months (median 30 months). There were 10 matched donors (3 related and 7 unrelated), 2 mismatched unrelated, and 1 haploidentical. Conditioning regimen consisted of busulfan and cyclophosphamide (BuCy) in 11 cases and fludarabine and melfalan (1) or BuCy (1). ATG was added in transplants from non-genetically matched donors. GvHD prophylaxis consisted of cyclosporine and methotrexate in most patients plus T-cell depletion in the haploidentical HSCT. Nine of the 13 transplanted patients are alive with complete clinical, immunologic, and hematologic recovery 8–204 months (median 101 months) after HSCT. Eight surviving patients had been transplanted from matched donors (3 related and 5 unrelated) and 1 from a haploidentical donor. Four patients died, 2 transplanted from matched donors (1 from acute GvHD and organ failure, 1 from a lymphoproliferative disorder after a second transplant), and 2 transplanted from mismatched unrelated donors (1 from acute GvHD and organ failure, 1 from graft failure and infection). Allogeneic hemopoietic stem cell transplantation must be utilized in all patients with severe Wisckott-Aldrich syndrome, using the most suitable graft variant for each patient.

Efficacy and safety of liposomal cytarabine in children with primary CNS tumours with leptomeningeal involvement

PurposeTo assess the efficacy and safety of liposomal cytarabine in the treatment of de novo and relapsed leptomeningeal involvement in children with primary CNS tumours.MethodsData from clinical charts were entered into a database for consecutive unselected patients (n=20) from nine Spanish centres. Diagnosis of leptomeningeal involvement was confirmed by cytology, MRI and/or CT scan. The dose of liposomal cytarabine used varied from 20 to 50 mg, by age.ResultsThere were 8 females and 12 males, mean age 7.3 years (range 8 months to 18 years). The tumours were: 10 medulloblastomas, 4 ependymomas, 3 primitive neuroectodermal tumours and 3 other tumours. Fourteen had undergone previous chemotherapy and 12 radiotherapy. Nine received concurrent chemotherapy and 2 concurrent radiotherapy. Median follow-up was 244.5 days (range 12–869). Patients received a median of 5 doses (range 1–9) of liposomal cytarabine. A neurological response (complete or partial) was seen in 11/19 (58%) and a cytological response in 7/10 (64%). Median time to neurological progression exceeded 180 days (range 12–869). Adverse effects were reported in 11/20 patients, but none was grade IV.DiscussionLiposomal cytarabine was well tolerated and efficacious in this patient group, but prospective randomised trials are needed.

Resultados del tratamiento con 2-clorodesoxiadenosina en la histiocitosis de células de Langerhans resistente o en recaída. Estudio de 9 pacientes

Fundamento Analizar los resultados del tratamiento con 2-clorodesoxiadenosina (2CdA) en pacientes diagnosticados de histiocitosis de celulas de Langerhans (HCL) multisistemica, refractaria o en recaida,atendidos en 8 hospitales espanoles entre 1993 y 1999 Pacientes y metodo Se recogieron los siguientes datos de los 9 pacientes: edad, sexo, afeccion organica por la HCL, tratamiento inicial y respuesta al mismo, dosis, numero de ciclos y forma de administracion de la 2CdA, respuesta al tratamiento con 2CdA, toxicidad, supervivencia libre de enfermedad y supervivencia global Resultados La edad mediana era de 25 anos (limites, 6-63). Todos ellos presentaban afeccion multiorganica por la HCL, con disfuncion grave de organos en 4 casos. La 2CdA se administrocomo tratamientode segunda linea en 7 casos y de tercera linea en dos. La dosis de 2CdA fue de 0,1 mg/kg y dia durante 5 dias en la mayoria de los pacientes, con un intervalo de 4 semanas entre los ciclos. Hubo respuesta completa (RC) en dos casos y parcial (RP) en 4 (respuesta global del 66%percnt;). La principal toxicidad fue la hematologica, con neutropenia de grado superior a 2 en 5 casos y trombocitopenia mayor de 2 en 5. Cuatro pacientes presentaron infecciones, con evolucion fatal en uno de ellos. Tras una mediana de seguimiento de 8 meses (intervalo, 2-17), dos enfermos se hallan en RC (12 mesesen ambos), 4 en RP (intervalo, 2 12meses) y uno con enfermedad activa (17 meses). Los dos restantes fallecieron por progression de la enfermedad y sepsis por Aspergillus spp., respectivamente. Las probabilidades actuariales de supervivencia libre de enfermedad y supervivencia global al ano fueron del 58%percnt; (intervalo de confianza [IC] del 95%percnt;, 38-78%percnt;) y del 71%percnt; (IC del 95%percnt;, 54-88%percnt;), respectivamente Conclusiones La 2CdA es un farmaco eficaz en pacientes con HCL refractaria o en recaida, y su efecto toxico principal es la mielodepresion. La utilidad de la 2CdA, aislada o en combinacion con otros farmacos, en pacientes con HCL multisistemica, refractaria o en recaida, debe demostrarse en estudios controlados

Diagnóstico molecular del retinoblastoma: epidemiología molecular y consejo genético

Fundamento y objetivo: El retinoblastoma, prototipo de cancer hereditario, puede causar ceguera, por enucleacion terapeutica, segundos tumores en pacientes con mutacion germinal e incluso muerte si no se trata. El diagnostico molecular de 213 pacientes a lo largo de 5 anos ha conducido a la deteccion de 106 mutaciones que se analizan desde la perspectiva de la epidemiologia molecular y consejo genetico. Pacientes y metodo: Estudio mutacional (reaccion en cadena de la polimerasa, secuenciacion y analisis de microsatelites) en pacientes con retinoblastoma procedentes de Espana, Cuba, Colombia y Serbia. Resultados: Un 45% de las mutaciones analizadas son nuevas y corresponden a mutaciones de tipo de corrimiento de pauta de lectura, cambio de aminoacido o procesado del acido ribonucleico. Todas las mutaciones sin sentido corresponden a sitios de alta mutabilidad. La tasa de deteccion de mutaciones en pacientes unilaterales esporadicos es alta (22%). En este grupo de pacientes se detecta una mayor incidencia (p = 0,018) de mutaciones de cambio de aminoacido y procesamiento. Espana y Francia muestran una incidencia mayor de mutaciones del procesado (p = 0,0003) que Alemania y el Reino Unido, paises en los que predominan las mutaciones sin sentido (p = 0,0006). Las mutaciones del procesado se asocian al fenotipo de baja penetracion y retraso en la aparicion de tumores (p = 0,018). Conclusiones: La incidencia de mutaciones germinales en pacientes unilaterales y las relaciones fenotipo/genotipo analizadas indican la necesidad del consejo genetico basado en el diagnostico molecular temprano. La mejora de las tecnicas diagnosticas, la caracterizacion funcional de mutaciones asociadas a baja penetrancia o expresividad y el estudio del transcriptoma de los tumores son objetivos necesarios para definir mejor la patogenia del retinoblastoma.