Identification of genetic variants in pharmacokinetic genes associated with Ewing Sarcoma treatment outcome.

BACKGROUND: Despite the effectiveness of current treatment protocols for Ewing sarcoma (ES), many patients still experience relapse, and survival following recurrence is <15%. We aimed to identify genetic variants that predict treatment outcome in children diagnosed with ES. PATIENTS AND METHODS: We carried out a pharmacogenetic study of 384 single-nucleotide polymorphisms (SNPs) in 24 key transport or metabolism genes relevant to drugs used to treat in pediatric patients (<30 years) with histologically confirmed ES. We studied the association of genotypes with tumor response and overall survival (OS) in a discovery cohort of 106 Spanish children, with replication in a second cohort of 389 pediatric patients from across Europe. RESULTS: We identified associations with OS (P < 0.05) for three SNPs in the Spanish cohort that were replicated in the European cohort. The strongest association observed was with rs7190447, located in the ATP-binding cassette subfamily C member 6 (ABCC6) gene [discovery: hazard ratio (HR) = 14.30, 95% confidence interval (CI) = 1.53-134, P = 0.020; replication: HR = 9.28, 95% CI = 2.20-39.2, P = 0.0024] and its correlated SNP rs7192303, which was predicted to have a plausible regulatory function. We also replicated associations with rs4148737 in the ATP-binding cassette subfamily B member 1 (ABCB1) gene (discovery: HR = 2.96, 95% CI = 1.08-8.10, P = 0.034; replication: HR = 1.60, 95% CI = 1.05-2.44, P = 0.029), which we have previously found to be associated with poorer OS in pediatric osteosarcoma patients, and rs11188147 in cytochrome P450 family 2 subfamily C member 8 gene (CYP2C8) (discovery : HR = 2.49, 95% CI = 1.06-5.87, P = 0.037; replication: HR = 1.77, 95% CI = 1.06-2.96, P = 0.030), an enzyme involved in the oxidative metabolism of the ES chemotherapeutic agents cyclophosphamide and ifosfamide. None of the associations with tumor response were replicated. CONCLUSION: Using an integrated pathway-based approach, we identified polymorphisms in ABCC6, ABCB1 and CYP2C8 associated with OS. These associations were replicated in a large independent cohort, highlighting the importance of pharmacokinetic genes as prognostic markers in ES.

[Liver transplatation for malignant tumors in children]. / Trasplante hepático en tumores malignos en el niño.

OBJECTIVE: To analyse our results on liver transplantation (LTX) in primitive malignant unresectable liver tumours in children and discussing its controversial indications in order to our experience. METHODS/PATIENTS: We report 12 patients with ages ranging from 6 months to 14 years old. They had hepatoblastoma (11) and fibrolamellar hepatocelullar carcinoma (1) without cirrhosis. LTX was considered as primary treatment in 10 patients (PRETEXT IV or any grade if extension to retrohepatic cava vein, 3 hepatic veins or porta vein were assessed) and as rescue therapy after recurrence (1) or persistence of unresectable macroscopic rests (2). One of the patients who underwent a LTX as primary therapy had lung metastases previously resolved with chemotherapy. We used entire liver (5), left lateral segment from cadaveric donor (3), live related donor (3, 2 segments II-III and 1 right liver) and left lateral segment from split (1). All children received chemotherapy prior and post-transplantation following SIOPEL protocol. OUTCOMES ANALYSED: Procedure tolerance, survival, recurrence rate, disease-free period and risk factors for adverse evolution. RESULTS: All patients overcame the LTX and no early loss of the graft was assessed. 2 patients died because of tumoral relapse, 1 after primary LTX and 1 after rescue LTX (survival rate of both groups 90% vs 50%). Graft and patients 1-year, 3-year, 5-year and 14-year survival were 91%, 91%, 82% and 82% respectively. The boy who presented lung metastases developed new ones one year after LTX that were removed and he actually is free of disease. The disease-free period has a probability for 1, 3 and 5 years of 91%, 75% and 75%. Tumoral tissue persistence is the only risk factor for an adverse evolution in our series. CONCLUSIONS: LTX is possible therapeutic approach for unresectable malignant liver tumours. It provides better results as a primary treatment than as a rescue one, being these outcomes comparable to those from resectable tumours. A right staging and referring patients to an expertise centre contribute to optimize results. LTX for patients presenting with lung metastases could be a controversial option. Live-related donor transplantation is an excellent alternative to avoid disease progression during cadaveric waiting list.

EWS-FLI1-mediated suppression of the RAS-antagonist Sprouty 1 (SPRY1) confers aggressiveness to Ewing sarcoma.

Ewing sarcoma is characterized by chromosomal translocations fusing the EWS gene with various members of the ETS family of transcription factors, most commonly FLI1. EWS-FLI1 is an aberrant transcription factor driving Ewing sarcoma tumorigenesis by either transcriptionally inducing or repressing specific target genes. Herein, we showed that Sprouty 1 (SPRY1), which is a physiological negative feedback inhibitor downstream of fibroblast growth factor (FGF) receptors (FGFRs) and other RAS-activating receptors, is an EWS-FLI1 repressed gene. EWS-FLI1 knockdown specifically increased the expression of SPRY1, while other Sprouty family members remained unaffected. Analysis of SPRY1 expression in a panel of Ewing sarcoma cells showed that SPRY1 was not expressed in Ewing sarcoma cell lines, suggesting that it could act as a tumor suppressor gene in these cells. In agreement, induction of SPRY1 in three different Ewing sarcoma cell lines functionally impaired proliferation, clonogenic growth and migration. In addition, SPRY1 expression inhibited extracellular signal-related kinase/mitogen-activated protein kinase (MAPK) signaling induced by serum and basic FGF (bFGF). Moreover, treatment of Ewing sarcoma cells with the potent FGFR inhibitor PD-173074 reduced bFGF-induced proliferation, colony formation and in vivo tumor growth in a dose-dependent manner, thus mimicking SPRY1 activity in Ewing sarcoma cells. Although the expression of SPRY1 was low when compared with other tumors, SPRY1 was variably expressed in primary Ewing sarcoma tumors and higher expression levels were significantly associated with improved outcome in a large patient cohort. Taken together, our data indicate that EWS-FLI1-mediated repression of SPRY1 leads to unrestrained bFGF-induced cell proliferation, suggesting that targeting the FGFR/MAPK pathway can constitute a promising therapeutic approach for this devastating disease.

Liver transplantation for malignant tumours in children.

OBJECTIVE: The object of this study was to analyse our results with liver transplantation (LTX) for primitive malignant unresectable liver tumours in children and to discuss the controversial indications, based on our experience. METHODS/PATIENTS: We report on 12 patients, aged 6 months to 14 years, with hepatic malignant tumours: 11 with hepatoblastoma and 1 with fibrolamellar hepatocelullar carcinoma without cirrhosis. LTX was the primary treatment in 10 patients (PRETEXT IV or any grade, if there was extension to the retrohepatic vena cava, 3 hepatic veins or portal vein) and a rescue therapy after recurrence for 1 and for persistence of unresectable macroscopic residuals in 2 patients. One of the patients who underwent a LTX as primary therapy had lung metastases previously resolved with chemotherapy. We used entire liver (n = 5), left lateral segment from cadaveric donor (n = 3), living related donor (n = 3; 2 segments II-III and 1 right lobe) and left lateral segment from split liver (n = 1). All children received chemotherapy prior and post transplantation following the SIOPEL protocol. We analysed procedure tolerance, survival, recurrence rate, disease-free period and risk factors for adverse evolution. RESULTS: All patients overcame LTX and no early graft loss was recorded. Two cases died because of tumour relapse, 1 after primary LTX and 1 after rescue LTX (survival rate of both groups, 90% and 50%). Graft and patient survival rates at 1 year, 3 years, 5 years and 14 years were 91%, 91%, 82% and 82% respectively. The boy who presented with lung metastases developed new ones one year after LTX that were removed and he is currently free of disease. The disease-free period has a probability at 1, 3 and 5 years of 91%, 75% and 75%, respectively. Tumour tissue persistence was the only risk factor for an adverse clinical course in our series. CONCLUSIONS: LTX is a reasonable therapeutic approach for unresectable malignant liver tumours, providing outcomes comparable to those for resectable tumours. Results obtained with LTX are better when it is used as a primary treatment than when used as a rescue procedure. Proper staging and early referral to centres with enough expertise optimise the results. LTX for patients with lung metastases could be a controversial option. Living related-donor transplantation is an excellent alternative to avoid disease progression while on the waiting list for cadaveric donors.

[An immunological approach to acute myeloid leukaemia]. / Aspectos inmunológicos de la leucemia mieloblástica aguda.

INTRODUCTION: Acute myeloid leukaemia (AML) is the second haematological malignancy in the paediatric population, and one of the leading causes of childhood cancer mortality. Survival is currently around 60%, with no improvement in last decades, suggesting that new therapeutic approaches are needed. The anti-leukaemia effect mediated by the lymphocytes and natural killer (NK) cells of the immune system has been established in haematopoietic stem cell transplantation, and also as adoptive immunotherapy after consolidation chemotherapy schemes. PATIENTS AND METHODS: A retrospective study was conducted on the clinical characteristics of patients diagnosed and treated for AML in our centre during 1996-2014. The mean fluorescence intensities of HLA-I, MICA/B and ULBP1-4, ligands for NK cell receptors, were also analysed in ten new diagnosed leukaemia cases, five myeloid and five lymphoid. RESULTS: A total of 67 patients were used in this analysis. With a median follow up of 25 months, the event-free survival was 62% (95% CI: 55-67). Secondary AML, non-M3 phenotype, and the absence of favourable cytogenetic markers had a lower survival. The probability of relapse was 38% (95% CI: 31-45). The expression of HLA-I and ULBP-4 was significantly lower in myeloid than in lymphoid blast cells. CONCLUSIONS: Our clinical results are similar to those described in the literature. Survival did not significantly change in recent decades, and the likelihood of relapse remains high. Myeloid blasts might be more susceptible to the cytotoxicity of NK cells through their lower expression of HLA-I. NK therapy strategies in minimal disease situation could be effective, as reported by other groups.

Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients: Phenotypic and molecular epidemiological implications.

Mutation analysis of retinoblastoma is considered important for genetic counseling purposes, as well as for understanding the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of an analysis of 43 hereditary retinoblastoma Spanish patients and kindred, using direct PCR sequencing, we have observed 29 mutations; most of them (62%) have not been reported previously. Of the mutations, 69% correspond to nonsense mutations (mainly CpG transitions) and frameshifts, with the expected outcome of a truncated Rb protein that lacks the functional pocket domains and tail. The remainder corresponds to splicing mutations, most of them (62%) targeted to invariant nucleotides, with the predicted consequence of out of frame exon skipping. Two of the splicing mutations in our study were found associated to families with a low-penetrance phenotype. Additionally, most of the mutations affecting splice junctions corresponded to retinoblastoma cases of either sporadic or hereditary nature with delayed onset (32 months on average). In contrast, most of the nonsense and frameshift mutations are associated with an early age at diagnosis (8.7 months on average). These differences are discussed in the context of the relationships between genotype and low expressivity phenotype. The differences in the spectrum of RB1 mutations found in this and other European surveys are also discussed in the context of alternate DNA methylation and mismatch repair phenotypes.

The treatment of advanced neuroblastoma. Results of the Spanish Neuroblastoma Study Group (SNSG) studies.

The Spanish Neuroblastoma Study Group has conducted a study on advanced neuroblastoma (N-I-87), which included 33 stage III and 60 stage IV neuroblastoma children more than 1 year of age, enrolled between October 1987 and April 1992. They were staged according to Evans and treated with induction chemotherapy (IC) consisting of 3 courses of cyclophosphamide-doxorubicin alternating with 3 of high-dose cisplatin-teniposide. Evaluation after IC and surgery demonstrated an overall response rate of 88% for stage III and 69% for stage IV. In the latter, complete responses and good partial responses were 33 and 14%, respectively. After surgery, children received maintenance chemotherapy (all stage III except 2 and 30 stage IV) or autologous bone marrow transplantation (ABMT) (11 stage IV), the distribution was not randomised. Probability of survival at 5 years was 0.60 +/- 0.12 for stage III and 0.24 +/- 0.07 for stage IV. A significant difference in survival at 5 years was found between "good responders" and "non-responders" to initial chemotherapy.

Prospective evaluation of the International Neuroblastoma Staging System (INSS) and the International Neuroblastoma Response Criteria (INRC) in a multicentre setting.

The aim of this study was to classify prospectively a series of neuroblastoma tumours according to the International Neuroblastoma Staging System (INSS) and the International Neuroblastoma Response Criteria (INRC) and to evaluate the difficulties and pitfalls involved in a multicentre setting. Each hospital provided their data for central review. The surgical procedures and their complications were reported. Kaplan-Meier estimates of survival and event-free survival were calculated according to stage and response to therapy. From June 1992 to December 1996, 194 patients were included in the study, with a mean age of 2 years. Initial studies were performed according to INSS recommendations without major problems. INSS stage was correctly applied to all patients except for 9 (95%). Post-operative complications were observed in 15 patients (8.3%). Response to therapy (INRC) was studied in 63 stage 4 patients, 11 of whom were not classified correctly (17%). Differences in survival according to stage (INSS) and group of response to therapy (INRC) were statistically significant (P < 0.001). In conclusion the INSS was easy to use and separated different prognostic groups. Surgical complications and mortality did not increase in this series because of using the INSS. The feasibility of INRC was evaluated in a small series of stage 4 patients and the designation of response was problematic in a relatively high proportion of cases. The prognostic value of the different responses was highly significant, but less informative than had been hoped for.

Hematopoietic cell transplantation using plasma and DMSO without HES, with non-programmed freezing by immersion in a methanol bath: results in 213 cases.

A simplified cryopreservation method for bone marrow (BM) and peripheral blood progenitor cells (PBPC) was utilized in hematopoietic cell transplantation of 213 patients with hematological or solid neoplasms after ablative chemotherapy (187 with peripheral blood progenitor cells and 26 with bone marrow). Cells were cryopreserved, after addition of autologous fresh plasma with DMSO, without HES, by freezing to -80 degrees C in a methanol bath and non-programmed freezer. For the patients autotransplanted with PBPC, the median period necessary for recovery of more than 0.5 x 10(9)/l granulocytes was 11 days (range 6-44), and 15 (8-204) days were required to obtain more than 20 x 10(9)/l platelets. For the patients autotransplanted with BM, the median period necessary to recover >0.5 x 10(9)/l granulocytes was 12 days (range 9-33), and 24 (12-57) days to obtain more than 20 x 10(9)/l platelets. These results support this method as being very effective in achieving high-quality cryopreservation. The procedure, which uses a non-programmed freezer, simplifies and reduces enormously the cost of the technical measures currently in use, enabling its adoption in almost any clinical oncological institution.

A microsatellite fluorescent method for linkage analysis in familial retinoblastoma and deletion detection at the RB1 locus in retinoblastoma and osteosarcoma.

Linkage analysis at the retinoblastoma locus (RB1) is essential for identifying individuals at risk and to offer adequate genetic counseling in familial retinoblastoma. It can also be used to detect large deletions involving RB1, which accounts for 15% of the genetic alterations in hereditary retinoblastoma. These studies are usually carried out with lengthy Southern blot analyses of relatively uninformative restriction fragment length polymorphisms. The authors report an alternative, reliable protocol for genotyping the RB1 locus using two pairs of highly informative intragenic and flanking microsatellites linked closely to the RB1 gene, and analysis of the fluorescent-labeled polymerase chain reaction products with automatic sizing technology. This methodology has successfully identified high risk carriers in five of the five pedigrees of familial retinoblastoma studied. In addition, gross deletions affecting the RB1 gene were identified in two of 12 sporadic bilateral retinoblastomas, and loss of heterozygosity at the RB1 locus has been detected in one of three osteosarcomas using the same experimental protocol. The described protocol is simpler and faster than conventional Southern blot methodologies and can identify a larger number of informative cases.

Allelic status of chromosome 1 in neoplasms of the nervous system.

By using five highly polymorphic markers, the allelic status of chromosome 1 was established in a series of 236 tumors of the nervous system, including all major histologic subtypes: gliomas, meningiomas, neurinomas, neuroblastomas, medulloblastomas, etc. Loss of alleles at 1p was observed at significant frequencies in neuroblastomas (26% of cases), meningiomas (32%), and malignant gliomas (37%) (primarily oligodendrogliomas [94%]). This anomaly was also detected in two of 23 neurinomas, two of three neurofibrosarcomas, one primary lymphoma, and two metastatic tumors of the brain. The analysis of tumors displaying partial 1p deletions suggests the existence of two distinct regions, 1p36 and 1p35-p32, in which loci nonrandomly involved in the development of neurogenic neoplasms might be located.

Lymphoproliferative disorders in paediatric rheumatic diseases. A report of two cases.

Lymphoproliferative disorders (LPD) are reported with a much lower frequency in children with rheumatic diseases than in their adult counterparts. We describe 2 patients who developed a lymphoma during the course of the disease. The first is a 16-year-old girl diagnosed with systemic juvenile idiopathic arthritis 6 years before who developed a mucosa-associated lymphoid tissue (MALT) lymphoma. The second report involves a boy diagnosed with systemic lupus erythematosus at 9 years of age who developed a Hodgkin's lymphoma 9 years after the disease onset. In spite of the low frequency of LPD in children with rheumatic diseases, these processes do occur.

Ewing's sarcoma of the mandible: radiologic features with emphasis on magnetic resonance appearance.

Ewing's sarcoma is an uncommon malignancy that usually occurs in children. A case of Ewing's sarcoma of the mandible is presented, and the radiologic appearance is described, with special consideration given to the magnetic resonance imaging features.

[Sensibility and specificity of N-myc oncogene with respect to other prognostic factors in 15 neuroblastomas]. / Sensibilidad y especificidad del oncogén N-myc con respecto a otros factores de pronóstico en 15 neuroblastomas.

Several biologic features of tumor cells correlate closely with a favorable or unfavorable outcome. To aid in assessing correlation in the various number of prognostic factors including the age, stage, VMA/HVA ratios, and the serum levels of NSE and ferritin, the histopathological features, ploidy, partial monosomy for the short arm of chromosome 1, and the tumor N-myc gene copy number, are examined. We determined the sensitivity and specificity of classical markers above the amplification of the N-myc oncogene. A striking new observation is the positive correlation between genomic amplification and some prognostic factors (stage, ferritin, NSE, pathologic anatomy and 1p deletion.

[Infant mortality in a third level paediatric hospital. therapeutic effort limitation, clinical-pathological agreement and diagnostic accuracy]. / Mortalidad infantil en un hospital de nivel terciario. Limitación de esfuerzo terapéutico, correspondencia clínico-patológica y precisión diagnóstica.

OBJECTIVE: To study infant and child mortality in a third level children's hospital treating highly complex patients. PATIENTS AND METHODS: All children dying in the period 2007- 2009 at La Paz Children's Hospital were evaluated. Epidemiological data, autopsy rate, clinical and autopsy diagnoses and their correspondence and the number of, patients with precise final diagnoses were analysed. Therapeutic effort limitation and palliative care were also evaluated as well as if the final result was expected according to the initial disease or clinical condition of the patients. All the variables were prospectively defined at the start of the study period. RESULTS: A total of 253 cases (6.08‰ admissions) were analysed. The two leading causes of death were disorders related to prematurity and low birth weight, and haematology oncology malignant diseases. Most patients (87%) died in an intensive care unit (neonatal or paediatric). During the study period 134 autopsies (53%) were performed, and new clinically significant findings were observed in 12 of these (7.8%) but in only one case the treatment could have possibly modified the prognosis (class I discrepancy). Therapeutic effort limitation and palliative care were implemented in 41.9%. Death was initially expected in 83.9% of cases. An accurate final diagnosis was defined in 92%, and the aetiology of the disease was considered to be identified in 86.4% of all deaths. CONCLUSIONS: Hospital mortality analysis is useful to evaluate the quality of the paediatric care and to detect adverse results that could be corrected. Paediatric autopsy continues to provide clinically significant data for paediatricians and families. Therapeutic effort limitation and palliative care is increasingly applied in paediatric end of life care. The number of infants and children dying without a final aetiological diagnosis is still considerably high.

High-risk hepatoblastoma: results in a pediatric liver transplantation center.

AIM: Aim of the study was assess the results of the treatment of High-Risk Hepatoblastoma (HRH) in a tertiary center where all liver surgery facilities, including pediatric transplantation (LT), are available. METHODS: 91 primary liver tumors treated between 1991 and 2009 were retrospectively reviewed. HRHs as defined by the SIOP criteria (PRETEXT IV or any stage with venous involvement, extrahepatic disease, tumor rupture and <100 ng/ml serum AFP) were identified and imaging and biopsies were reviewed. The treatment consisted of total removal of the tumor, involving extended hepatectomies and LT if necessary, together with SIOPEL-guided chemotherapy. RESULTS: 23/57 hepatoblastomas were HRH (11F/12M). 17 were considered unresectable by standard techniques, 3 had extrahepatic disease, and 3 fulfilled both criteria. Mean age at diagnosis was 2.3 ± 2.4 years. 3 children (referred after chemotherapy) died without surgery. 4 had resections (2 left and 2 right trisegmentectomies). Primary LT was required in 15 children (7 cadaveric donors and 8 living related donor transplantations (LRDT), 2 of them with retrohepatic vena cava replacement), and 1 patient had rescue LT after recurrence. Mean follow-up was 4.8 ± 2.9 years. 2 children who had undergone liver resection developed pulmonary metastases at 1.7 and 1.6 years postoperatively and survived after surgical treatment. 2 children with LT developed EBV-related lymphoma and leukemia respectively but survived. Event-free survival (EFS) at 1, 5, and 10 years was 78.3 ± 8.6%, 63.1 ± 10.5%, and 63.1 ± 10.5%, respectively. 6 children died (3 without surgery, 1 after liver resection, 1 after primary LT and 1 after rescue LT). Overall survival at 1, 5 and 10 years was 78.3 ± 21.7%, 73.2 ± 26.8% and 73.2 ± 26.8%. Of those with primary LT, survival at 1, 5 and 10 years was 93.3 ± 6.4%, 93.3 ± 6.4% and 93.3 ± 6.4%. CONCLUSIONS: Outstanding results in the treatment of HRH are possible in tertiary centers when referral is early (preferably at diagnosis) and specialized liver surgery and transplantation facilities are available.