Julián Sevilla

High-dose chemotherapy with autologous stem cell rescue for children with high risk and recurrent medulloblastoma and supratentorial primitive neuroectodermal tumors

Current treatment for high risk and recurrent medulloblastoma (MB) and supratentorial primitive neuroectodermal tumors (stPNET) has a very poor prognosis in children. High dose chemotherapy (HDCT) and autologous stem cell rescue have improved survival rates. We present 19 patients (thirteen classified in the high risk group and six patients with recurrent disease) that received HDCT and autologous stem cell rescue.In the high risk group [Med Pediatr Oncol 38 (2002) 83], all patients underwent neurosurgical debulking. Standard chemotherapy was prescribed in 10 patients. Radiotherapy was given to 4 patients (all older than 4 years old). In the recurrence disease group [Childs Nerv Syst 15 (1999) 498], five patients underwent surgery. Radiotherapy was given to those who were not previously irradiated. The HDCT in twelve patients consisted of busulfan 4 mg/kg/day, orally over 4 days in 6-hourly divided doses and melphalan at a dose of 140 mg/m2/day by intravenous infusion over 5 min on day −1. Three patients additionally received thiotepa 250 mg/m2/day intravenously over 2 days and four patients additionally received topotecan 2 mg/m2/day over 5 days by intravenous infusion over 30 min. The other seven patients received busulfan and thiotepa at the same doses.Patient’s stem cells were mobilized with granulocyte colony-stimulating factor at a dose of 12 μg/kg twice daily subcutaneously for four consecutive days. Cryopreserved peripheral blood progenitor cells were re-infused 48 h after completion of chemotherapy. With a median follow-up of 34 months (range 5–93) eight complete responses and one partial response were observed. Three patients died of treatment-related toxicities (15%). The 2 year event-free survival was 37.67 ± 14% in all patients and 57 ± 15% for the high risk group.Therefore we conclude that HDCT may improve survival rates in patients with high risk/recurrent MB and stPNET despite treatment toxicity.

KIR-HLA receptor-ligand mismatch associated with a graft-versus-tumor effect in haploidentical stem cell transplantation for pediatric metastatic solid tumors.

Killer immunoglobulin‐like receptors (KIRs) on natural killer cells (NKs) recognize groups of human leukocyte antigen (HLA) class I alleles. Cells without an inhibitory HLA ligand may trigger NK activation. Reduced risk of relapse has been reported in malignant hematologic diseases after haploidentical transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient. We performed haploidentical transplant in three children with refractory solid tumors. Our results showed that beneficial antitumor effects could be observed in the presence of inhibitory KIR–HLA mismatch. These preliminary results suggest a possible association between disease control and NK cell alloreactivity. Pediatr Blood Cancer 2009;53:120–124.

Increasing incidence of invasive aspergillosis in pediatric hematology oncology patients over the last decade: a retrospective single centre study.

There is scanty information about invasive aspergillosis (IA) in the pediatric population. A review of IA at Hospital Infantil Universitario Niño Jesús between 1996 and 2006 was undertaken to analyze incidence, risk factors, and treatment response. Twenty patients were diagnosed with probable or proven IA during the study period, with a cumulative incidence of 1.96%. Incidence was higher in hematopoietic stem cell transplantation (HSCT) recipients: 2.26% (3.5% in allogeneic HSCT and 1.2% in autologous HSCT). A significative increase in IA incidence was observed along the study period (P=0.013), although this increase did not reach signification if only proven cases were compared (P=0.058). Most patients presented multiple risk factors for IA (87% more than 1, and 47% more than 3). The most frequently described risk factor was chemotherapy (90%), after by long-term neutropenia (90%), and corticotherapy (75%). Main locations of the infection were pulmonary (8 patients), cutaneous (3 patients) and intestinal (3 patients). Six patients presented disseminated IA. Initial response to treatment was 55%, although 3 of these cases had a subsequent episode. Global antifungal response, at the end of the follow-up, was 45%. IA-related mortality was 55%. Global mortality was 90%. Only 2 patients (isolated cutaneous IA cases) survived. Seven patients died due to their underlying malignant disease without active fungal disease. Incidence of IA in oncology children is increasing, and in adults. In our experience, IA is a marker of poor outcome even for patients who initially respond to antifungal treatment.

Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

Previous clinical trials based on the genetic correction of purified CD34(+) cells with gamma-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34(+) cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34(+) cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34(-) mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).Previous clinical trials based on the genetic correction of purified CD34+ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34+ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34+ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34- mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).

Gene therapy for Fanconi anemia: one step closer to the clinic.

Game-changing trends in biomedical science usually start as daring, visionary ideas that struggle through the confusion of trials and experiments, experience a crisis at the collision of an expectant public and disbelieving colleagues, and—if successful—finally emerge as widely accepted new standards (Evans, 2011). The gene therapy field has followed this sequence and, in the teens of the twenty-first century, seems to be emerging as a realistic new therapy for genetic disorders of hematopoiesis, such as congenital immunodeficiencies and bone marrow failure syndromes (Naldini, 2011; Sheridan, 2011). Fanconi anemia (FA) is a prototypical inherited bone marrow failure disorder characterized by aplastic anemia and a dramatically increased risk of hematological and solid malignancies. FA is due to defects in one of at least 15 genes that encode the proteins involved in the cellular response to DNA damage and the maintenance of genomic integrity. More than half of the reported cases of FA are due to FANCA gene mutations. FANCA protein is a member of a ‘‘functional core complex’’ (formed with seven other proteins) that is essential in the DNA damage response. A lack of core complex results in a phenotype characterized by bone marrow failure, myelodysplasia, leukemia, and other cancers (D’Andrea, 2010). With support from the FA Research Fund and Fanconi Hope Charitable Trust, two dozen scientists and clinicians convened in Barcelona on November 22, 2011 for the second meeting of the FA Gene Therapy International Work Group. This International Work Group, chaired by Jakub Tolar (University of Minnesota, Minneapolis, MN), has been established with the goal of coordinating the best available knowledge in gene therapy with the best format of clinical trial for FA. The first meeting, a year ago in London, brought together researchers from fields that rarely interact. The objective of that meeting was to establish an open platform whereby teams initiating gene therapy trials in FA, institutions that already have strong track records in gene therapy, and groups that are developing novel strategies in genome modification could be brought together. We found common ground in our efforts to accelerate the transition of gene therapy research into clinical trials for patients with FA. The initial FA gene therapy platform was outlined as follows: FANCA gene delivered by third-generation lentiviral vector pseudotyped with vesicular stomatitis virus (VSV-G); short transduction without prolonged prestimulation with growth factors; and exclusion of individuals who have a human leukocyte antigen-matched sibling donor, an abnormal karyotype, or a serious infection (Tolar et al., 2011). The particular focus of this year’s event was to synthesize the data to inform the impending clinical trials and make the future iterations of FA gene therapy trials possible. Hematopoietic stem cell gene therapy has the potential to transform conventional therapy for FA, which for decades has involved transfusion support, anabolic steroids, and hematopoietic cell transplantation (HCT). Androgens can

Engraftment syndrome in children undergoing autologous peripheral blood progenitor cell transplantation.

There is limited experience on engraftment syndrome (ES) in children. The present study analyzes the characteristics of ES in pediatric patients undergoing autologous peripheral blood progenitor cells transplantation (PBPCT). From 1993 to 2001, 30 of 156 patients (19.2%) who underwent PBPCT developed ES (skin rash which involved more than 27% of the body surface and temperature >38.3°C with no compatible infectious disease etiology, during neutrophil recovery). Of the 30 patients who developed ES, 20 (66%) developed hypoxia and/or pulmonary infiltrates, seven (23%) had hepatic dysfunction, six (20%) developed renal insufficiency, 16 (53%) showed weight gain and three (10%) experienced transient encephalopathy. Multivariate analysis showed that the only positive predictive factor for developing ES was mobilization with high-dose G-CSF (12 μg/kg twice daily) (RR 3.88, CI 95% 1.73–8.67; P < 0.0005). The overall transplant-related mortality (TRM) was 8.33% and this was significantly higher in the patients who developed ES than in those who did not (23% vs 4.76%; P < 0.0001). We also found a higher morbidity in patients who developed ES, expressed as a statistically significant increase in supportive care (transfusion requirement, parenteral nutrition) and increase in the length of hospital stay. In summary, we have found ES to be the most important cause of morbidity and mortality in children undergoing autologous PBPCT.

Granulocyte colony-stimulating factor alone at 12 μg/kg twice a day for 4 days for peripheral blood progenitor cell priming in pediatric patients

In children, the optimal mobilization schedule for harvesting peripheral blood progenitor cells (PBPC) is an issue of continuous research. We have studied a schedule based on high and daily divided doses of G-CSF (12 μg/kg body weight twice daily) for 4 days for PBPC priming. Toxicity related to G-CSF was observed in 13 patients (23%), mainly mild bone pain and myalgia. The median CD34+cell number collected was 4.4 (0.4–35 × 106/kg body weight), with 46 patients achieving 2 × 106/kg body weight (83.6%) after a single large volume leukapheresis. In conclusion, this mobilization schedule allows safe and efficient collection of the minimum target CD34+ cell dose in most pediatric patients by only one procedure.

Stem cell gene therapy for fanconi anemia: report from the 1st international Fanconi anemia gene therapy working group meeting.

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patients own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Chromosome fragility in patients with Fanconi anaemia: diagnostic implications and clinical impact

Background Fanconi anaemia (FA) is a rare syndrome characterized by bone marrow failure, malformations and cancer predisposition. Chromosome fragility induced by DNA interstrand crosslink (ICL)-inducing agents such as diepoxybutane (DEB) or mitomycin C (MMC) is the ‘gold standard’ test for the diagnosis of FA. Objective To study the variability, the diagnostic implications and the clinical impact of chromosome fragility in FA. Methods Data are presented from 198 DEB-induced chromosome fragility tests in patients with and without FA where information on genetic subtype, cell sensitivity to MMC and clinical data were available. Results This large series allowed quantification of the variability and the level of overlap in ICL sensitivity among patients with FA and the normal population. A new chromosome fragility index is proposed that provides a cut-off diagnostic level to unambiguously distinguish patients with FA, including mosaics, from non-FA individuals. Spontaneous chromosome fragility and its correlation with DEB-induced fragility was also analysed, indicating that although both variables are correlated, 54% of patients with FA do not have spontaneous fragility. The data reveal a correlation between malformations and sensitivity to ICL-inducing agents. This correlation was also statistically significant when the analysis was restricted to patients from the FA-A complementation group. Finally, chromosome fragility does not correlate with the age of onset of haematological disease. Conclusions This study proposes a new chromosome fragility index and suggests that genome instability during embryo development may be related to malformations in FA, while DEB-induced chromosome breaks in T cells have no prognostic value for the haematological disease.

Transient donor cell-derived myelodysplastic syndrome with monosomy 7 after unrelated cord blood transplantation

Abstract:  Donor cell leukaemia or myelodysplastic syndromes are extremely rare complications that have been observed not only after haematopoietic transplantation with progenitor cells harvested from bone marrow and peripheral blood, but also after cord blood transplantation. We describe the early onset of monosomy 7 in donor cells after cord blood transplantation in a patient diagnosed with myelodysplastic syndrome 3 months after transplantation. Fluorescent in situ hybridisation analysis performed in a cryopreserved aliquot of the cord blood showed 2.5% of nuclei with monosomy 7. The cord blood donor was studied and he showed neither peripheral blood cytopenias nor cytological or cytogenetic features of myelodysplasia. The cell blood counts (CBC) of the girl have improved over 2 yr while decreasing the percentage of monosomic cells. The monosomic clone has finally disappeared and the CBC are finally normal. This case of transient monosomy 7 started very early after engraftment emphasises the relevance of clonal instability of specific progenitor cells in the early engraftment, and host immune status, in cytogenetic abnormalities founded in donor cell‐derived MDS and acute leukaemia. Moreover, the clinical follow‐up of this patient, recommends a more conservative treatment for this clonal disease early developed after transplantation.