GEIS-21: a multicentric phase II study of intensive chemotherapy including gemcitabine and docetaxel for the treatment of Ewing sarcoma of children and adults: a report from the Spanish sarcoma group (GEIS).

BACKGROUND: First Spanish trial of Ewing sarcoma (ES) including adults and children with the aim to test the efficacy of Gemcitabine and Docetaxel (G/D) in newly diagnosed high-risk (HR) patients. METHODS: This was a prospective, multicentric, non-randomised, open study for patients ⩽40 years with newly diagnosed ES. HR patients (metastatic, axial-pelvic primaries or bone marrow micrometastasis) received 2 window cycles of G/D. Patients with an objective response (OR) to G/D received 12 monthly cycles of G/D after completion of mP6. The primary end point was the OR rate to the G/D window phase and the event-free survival (EFS) and overall survival (OS) for all patients. The study is registered at ClinicalTrials.gov (identifier: NCT00006734). RESULTS: Forty-three patients were enroled, median age 17 years (range, 3-40). After a median follow-up of 43.4 months, the 5-year OS rate is 55.0% (95% CI, 41-74%) with an EFS of 50.0% (95% CI, 36-68%). The 5-year OS and EFS rates for standard risk (SR) patients was 76.0% (95% CI, 57-100%) and 71.0% (CI, 54-94%); for HR 36.0% (CI, 20-65%) and 29.0% (CI, 15-56%). Twelve of 17 (70.6%) high-risk (HR) patients showed an OR (7 PR and 5 SD) to G/D window therapy. The 5-year OS rate for patients ⩽18 years of age was 74.0% (CI, 56-97%) and 31.0% for >18 years (95% CI, 15-66%), P<0.001. Grade 4 adverse events during mP6 occurred in 28/39 of patients (72%) and did not correlate with age. Multivariate survival analyses with <18 vs ⩾18 and risk groups significant differences, P<0.00001. Using a Cox model for OS, both age and risk group were statistically significant (P=0.0011 and P=0.0065, respectively). CONCLUSIONS: Age at diagnosis is an independent prognostic factor superior to the presence of metastases with 18 years as the strongest cut-off. The mP6 regimen provided survival curves that plateau at 3 years and G/D produced significant responses in HR-ES that is worth further exploring.

Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7).

PURPOSE: We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. METHODS: From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. RESULTS: After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, (123)I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. CONCLUSIONS: mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival.

Effect of freeze-drying and oven-drying on volatiles and phenolics composition of grape skin.

Grape skins are the part of the fruit with the highest amount of volatile and polyphenolic compounds. Volatile compounds give the fruit and other grape derivatives their flavour. Polyphenolic compounds are responsible for the colour of the fruit, juice and wine, and also act as very important natural antioxidant compounds. Dehydration is a method used to prevent the damage of these compounds over time. Nevertheless, in the case of volatile compounds, removing water can cause compound degradation or the evaporation of such compounds. This work studied two drying methods, freeze-drying and oven-drying, at 60 degrees C, as skin preservation methods. The skins from two grape varieties, Carménère and Cabernet Sauvignon, were dried. Many volatile compounds, which are of interest in the aroma profile, were identified in both varieties as terpenes (linalool, etc.), sesquiterpenes (farnesol), norisoprenoids (vitispirane, etc.), C(6) alcohols (1-hexanol, etc.), etc., and their amount decreased significantly with the oven-drying method, in contrast to the freeze-drying method. Both phenolic compounds, anthocyanins and flavonols, were identified in fresh and dehydrated samples, thus resulting in the freeze-drying method being less aggressive than oven-drying methods.

Comprehensive analysis of the 9p21 region in neuroblastoma suggests a role for genes mapping to 9p21-23 in the biology of favourable stage 4 tumours.

Chromosome 9p21 is frequently deleted in many cancers. Previous reports have indicated that 9p21 LOH is an uncommon finding in neuroblastoma (NB), a tumour of childhood. We have performed an extensive analysis of 9p21 and genes located in this region (cyclin-dependent kinase inhibitor 2A - CDKN2A/p16(INK4a), CDKN2A/p14(ARF), CDKN2B/p15(INK4b), MTAP, interferon alpha and beta cluster). LOH was detected in 16.4% of 177 NB. The SRO was identified between markers D9S1751 and D9S254, at 9p21-23, a region telomeric to the CDKN2A and MTAP genes. A significantly better overall and progression-free survival was detected in stage 4 patients displaying 9p21-23 LOH. Hemizygous deletion of the region harbouring the CDKN2A and CDKN2B loci was identified in two tumours by means of fluorescent in situ hybridisation and MTAP was present by immunostaining in all but one tumour analysed. The transcriptional profile of tumours with 9p21-23 LOH was compared to that of NB displaying normal 9p21-23 status by means of oligonucleotide microarrays. Four of the 363 probe sets downregulated in tumours with 9p21-23 LOH were encoded by genes mapping to 9p22-24. The only well-characterised transcript among them was nuclear factor I-B3. Our results suggest a role for genes located telomeric of 9p21 in good risk NB.

[The pattern of DNA fragmentation in pediatric neuromuscular disorders]. / Análisis del patrón de fragmentación del ADN en enfermedades neuromusculares pediátricas.

INTRODUCTION: An apoptotic mode of cell death has been identified in the developing skeletal muscle of invertebrates. It has been also involved in the pathogenesis of muscle degeneration in the dystrophin-deficient mdx mouse. This has raised an interest on the possible role of apoptosis in human neuromuscular disorders, but the existing studies have offered conflicting results. OBJECTIVE: To analyze the presence of apoptosis in several pediatric neuromuscular disorders. PATIENTS AND METHODS: Identification of cells with apoptotic morphology, detection of DNA fragmentation in situ by means of the TUNEL assay and study of the pattern of DNA fragmentation by agarose gel electrophoresis and Southern blot hybridization in muscle biopsies of 29 patients and three normal controls. RESULTS: No morphological features of apoptosis were found in any of the samples examined. The TUNEL method did not label the myonuclei from any of the biopsies, though some mastocytes stained positively. Agarose gel electrophoresis of extracted DNA and Southern blot hybridization with a genomic DNA probe did not show oligonucleosomal fragmentation. However, smearing of DNA was observed in samples from three patients affected with Duchenne's muscular dystrophy, one with Becker's muscular dystrophy and one with infantile spinal muscular atrophy. CONCLUSIONS: The present results do not favor the hypothesis of persistent apoptosis in some of the pediatric neuromuscular disorders, although they do not exclude that some sort of programmed cell death occurs either excessively or anachronically at other stages of the pathogenesis of these diseases.

Cell death and associated c-jun induction in perinatal hypoxia-ischemia. Effect of the neuroprotective drug dexamethasone.

Previous studies in a model of unilateral hypoxia-ischemia in the developing rat brain have shown induction of the mRNAs of c-fos and c-jun and presence of apoptotic DNA fragmentation. In this same model, dexamethasone confers neuroprotection if given before the insult. Since c-fos and c-jun have been involved in several models of cell death, we investigated whether the neuroprotective effect of dexamethasone could be associated with changes in expression of these genes. Rat pups, pre-treated with either 0.5 mg/kg dexamethasone or vehicle 48 h, 24 h and immediately before the injury, were subjected to ligation of the left common carotid artery followed by 3 h hypoxia. Analysis of c-fos and c-jun expression at 2 h, by means of in situ hybridization, revealed diminished induction in dexamethasone-treated animals. Jun immunoreactivity, but not Fos, and DNA fragmentation, assessed by in situ end-labeling of fragmented DNA, were present at 24 h only in vehicle-injected animals. Electrophoresis of brain extracted DNA revealed a ladder pattern in all the animals. Our results show a relationship between Jun overexpression and cell-death in the hypoxic-ischemic developing brain and suggest that dexamethasone exerts its protective effect anteceding immediate early gene induction, at some early point in post-ischemic signal transduction.

Identification of necrotic cell death by the TUNEL assay in the hypoxic-ischemic neonatal rat brain.

The time course and localization of DNA fragmentation in a neonatal rat model of unilateral hypoxia-ischemia were assessed by means of the terminal transferase-mediated biotin dUTP nick end labeling (TUNEL) assay. TUNEL-positive cells were detected in the hemisphere ipsilateral to the ligation immediately after the injury and increased to reach a maximum 1-3 days later, then decreasing until day 10, in parallel with cell death identified by standard histological methods. Cells showing any of the different morphologies of chromatin condensation and fragmentation were labeled, particularly within the core of the ischemic lesion. These results, obtained in a paradigm of necrosis in the immature brain, add to previous evidence suggesting that some forms of non-apoptotic DNA fragmentation are labeled by the TUNEL assay.