Federico Bonetti

Disseminated Neuroblastoma in Children Older Than One Year at Diagnosis: Comparable Results With Three Consecutive High-Dose Protocols Adopted by the Italian Co-Operative Group for Neuroblastoma

PURPOSE To compare the outcomes associated with modifications in three consecutive protocols employed by the Italian Co-Operative Group for Neuroblastoma (ICGNB) in disseminated neuroblastoma. PATIENTS AND METHODS Between January 1985 and November 1997, a total of 359 children aged 1 to 15 years with newly diagnosed stage 4 neuroblastoma were enrolled in three consecutive protocols. Compared with ICGNB-85, the ICGNB-89 protocol contained two more chemotherapy cycles, and some drugs were given at greater doses, whereas in the ICGNB-92 protocol, the induction phase included a chelating agent, and individual cycles contained four drugs instead of two. RESULTS A total of 330 of 359 evaluable children were included in this analysis; 106 children were treated with ICGNB-85, 65 children were treated with ICGNB-89, and 159 children were treated with ICGNB-92 protocols. Radical resection of primary tumor was carried out in 59.4%, 50.8%, and 57.9% of the patients, respectively. Major tumor response after induction therapy was achieved in 66.7%, 69.2%, and 68.6% of the patients, respectively. A total of 218 of 232 patients received consolidation therapy consisting of conventional chemotherapy in 65 patients and of high-dose chemotherapy in 153 patients. Disease recurrence or progression occurred in 82.1%, 69.2%, and 74.8% of the patients, respectively. Therapy-related deaths occurred in 1.9%, 12.3%, and 6.9% of the patients, respectively. Five-year overall survival (OS) for the three studies was 26%, 23%, and 28%, and event-free survival (EFS) was 19%, 17%, and 17%, respectively. CONCLUSION The therapeutic modifications adopted in the ICGNB-89 and ICGNB-92 protocols were not associated with a significant improvement in response rate or in the 5-year OS and EFS as compared with the ICGNB-85 protocol. Attempts at intensifying chemotherapy were associated with greater toxicity.

Improvement over time in outcome for children with acute lymphoblastic leukemia in second remission given hematopoietic stem cell transplantation from unrelated donors

Aims of this study were to verify whether reduction in transplant-related mortality (TRM) of children with acute lymphoblastic leukemia (ALL) in second complete remission (CR) given allogeneic hematopoietic stem cell transplantation (HSCT) from unrelated volunteers has occurred over time and to investigate the role of other variables on the probabilities of relapse, TRM and event-free survival (EFS). We compared results obtained in 26 children given HSCT before January 1998 with those of 37 patients transplanted beyond that date. In all donor–recipient pairs, histocompatibility was determined by serology for HLA-A and -B antigens and by high-resolution DNA typing for DRB1 antigen. High-resolution molecular typing of HLA class I antigens was employed in 20 of the 37 children transplanted more recently. Probability of both acute and chronic GVHD was comparable in the two groups of patients. In multivariate analysis, children transplanted before January 1998, those with T-lineage ALL and those experiencing grade II–IV acute GVHD had a higher relative risk of TRM at 6 months after transplantation. Relapse rate was unfavorably affected by a time interval between diagnosis and relapse <30 months. The 2-year probability of EFS for children transplanted before and after 1 January 1998 was 27% (10–44) and 58% (42–75), respectively (P = 0.02), this difference remaining significant in multivariate analysis. EFS of unrelated donor HSCT in children with ALL in second CR has improved in the last few years, mainly due to a decreased TRM. This information is of value for counseling of patients with relapsed ALL.

Bloodstream infections in children with cancer: a multicentre surveillance study of the Italian Association of Paediatric Haematology and Oncology

A one-year prospective, multicentre surveillance study on aetiology, main clinical features and outcome of bloodstream infections in children with cancer was conducted in 18 paediatric haematology centres belonging to the Italian Association for Paediatric Haematology and Oncology. A total of 191 bloodstream infections were reported during the study period. Of them, 123 (64%) occurred in neutropenic and 68 (36%) in non-neutropenic patients. Gram-positive cocci caused 45% (85/191) of the episodes, gram-negative rods 41% (78/191), and fungi 9% (18/191). The remaining 5% (10/191) of the episodes were poly-microbial infections. A total of 204 pathogens were isolated (46% gram-positive cocci; 44% gram-negative rods; and 10% fungi). The aetiologic distribution was similar among neutropenic and non-neutropenic patients. A correlation between the infection and the presence of an indwelling central venous catheter was found in 20% (23/114) of the episodes among neutropenic patients and in 55% (23/62) among non-neutropenic patients. Gram-negative micro-organisms were isolated in an unusually high proportion of catheter-related infections (48%). The overall mortality rate from any cause within 30 days from the first positive blood culture was 11%, and was higher among patients who were neutropenic at the onset of the infection than among those who were not neutropenic (15 versus 4%, P = 0.03). In addition, the mortality was significantly higher in recipients of bone marrow transplantation than in patients with acute leukaemia or solid tumour (21, 11 and 6%, respectively) and was also higher in fungaemias and poly-microbial infections (22 and 30%) than in single gram-positive and gram-negative bacteraemias (11 and 6%).

Marked and sustained improvement two years after autologous stem cell transplantation in a girl with systemic sclerosis

Autologous transplantation of hematopoietic stem cells has recently been proposed as a possible treatment for autoimmune diseases that are associated with a very severe prognosis. A 12-year-old girl who, since 4 years of age, had systemic sclerosis with progressive pulmonary involvement underwent autologous peripheral blood-derived stem cell transplantation (aPBSCT) using CD34+ selection, cyclophosphamide, and the infusion of the monoclonal antibody CAMPATH-1G. Following transplantation, in the absence of any treatment other than symptomatic therapy, the patients exertional dyspnea and alveolitis disappeared and she experienced a marked improvement in skin score, height velocity, and general well-being that has persisted 2 years after the transplantation procedure. Autologous PBSCT associated with the infusion of the monoclonal antibody CAMPATH-1G appears to be a useful therapy for otherwise intractable forms of progressive systemic sclerosis.

Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation

Fifty-three pediatric patients given an allogeneic or an autologous bone marrow transplantation (BMT) were immunized with a polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax II). Vaccine was administered six months or more after BMT and the pneumococcal IgM, total IgG, and IgG subclasses levels were evaluated before and three weeks after immunization. Immunization promoted a significant rise in antibody serum levels (P<0.000001), and all children vaccinated more than two years after transplantation responded to pneumococcal polysaccharides, whereas only 20–30% and 50% of patients given BMT between six months and one year and one and two years, respectively, mounted an eifective antibody production (P<0.0001). In univariate analysis, lapse of time from BMT to vaccination, chronic graft-versus-host disease occurrence, and female sex influenced the response rate. However, in multivariate analysis, only time between marrow transplant and immunization was a powerful predictor of response. Interestingly, four of 11 patients with IgG2 deficiency before immunization normalized serum levels of this IgG subclass after the pneumococcal antigenic challenge. Our study suggests that time after transplant is the major factor influencing the recovery of immune reactivity to polysaccharide antigens. This seems to confirm the hypothesis that ontogeny of the B-cell repertoire follows a predetermined sequential program in which polysaccharide antigens are some of the last to evoke an antibody response.

Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease

BACKGROUND AIMS Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohns disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application. METHODS MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD). RESULTS MSC were successfully expanded from all patients. Colony-forming unit-fibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [12-84% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens. CONCLUSIONS CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease.

Disposition of high-dose busulfan in pediatric patients undergoing bone marrow transplantation

We studied the pharmacokinetics of busulfan (16 mg/kg) in 16 pediatric patients affected by malignant and nonmalignant disorders between 6 months and 19 years of age (mean ± SD, 5.7 ± 6.5 years) who were undergoing allogeneic (15 patients) and autologous (one patient) bone marrow transplantation. In all children, the conditioning regimen consisted of busulfan given orally at a dose of 1 mg/kg every 6 hours for 16 doses (total dose, 16 mg/kg), associated with other drugs. The pharmacokinetics of busulfan was studied during the 6‐hour dosing interval on the third day of therapy by use of a high‐performance liquid chromatographie assay. The value for the time to reach maximum concentration, expressed as mean ± SD, was 1.1 ± 0.5 hour; maximum concentration was 609.6 ± 225.3 ng/ml; steady‐state concentration was 358.9 ± 135.5 ng/ml; area under the plasma concentration–time curve was 2153.6 ± 813.1 ng · hr/ml; oral clearance was 0.535 ± 0.226 L/hr/kg; and half‐life was 2.4 ± 0.8 hours. Age‐related differences in busulfan disposition were observed. The mean busulfan oral clearance in a group of 10 patients with an age range from 6 months to 3 years was 0.619 L/hr/kg, whereas six patients whose ages ranged from 7 to 19 years had a oral clearance of 0.396 L/hr/kg. The half‐lives for busulfan during infancy decrease continuously until early childhood but were prolonged in older children. No significant relationship between systemic exposure to busulfan and drug effect was observed.

Prophylactic co-trimoxazole versus norfloxacin in neutropenic children - perspective randomized study

SummaryCo-trimoxazole or norfloxacin were randomly administered to 44 granulocytopenic children with malignancies in order to prevent bacterial infections. Although more patients in the co-trimoxazole group had febrile episodes (p<0.01), the mean of febrile days and the mean of days with systemic antibiotics did not differ significantly in the two groups. Five patients in the co-trimoxazole group had a microbiologically documented infection (four with septicemia) due toEscherichia coli (n=2),Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus sp. There were four septicemic episodes in the norfloxacin group due toP. aeruginosa, Streptococcus pneumoniae, Streptococcus mitis andStreptococcus faecalis. Compliance was good during administration of both drugs. No signs or symptoms of arthropathy were seen in the norfloxacin group. The number of gram-negative bacilli resistant to co-trimoxazole isolated from stools significantly increased during prophylaxis with co-trimoxazole (p<0.001). Norfloxacin did not select resistant strains and was very active in eradicating gram-negative bacilli from stools (27.5% of positive cultures).Zusammenfassung44 Kinder, die an malignen Erkrankungen litten, erhielten zur Prävention bakterieller Infektionen während der granulozytopenischen Phase nach Randomverfahren entweder Co-trimoxazol oder Norfloxacin. Fieberzustände traten bei der mit Co-trimoxazol behandelten Gruppe häufiger auf (p<0,01), aber die Anzahl der Tage mit Fieber und die Anzahl der Tage, an denen eine systemische Antibiotikatherapie durchgeführt werden mußte, wiesen im Mittel keine signifikanten Unterschiede auf. Unter Co-trimoxazol-Prophylaxe traten fünf mikrobiologisch dokumentierte Infektionen auf (vier mit Septikämie); die Erreger warenEscherichia coli (n=2)Klebsiella pneumoniae, Pseudomonas aeruginosa undStreptococcus sp. In der Norfloxacin-Gruppe traten vier Septikämien auf, die durchP. aeruginosa, Streptococcus pneumoniae, Streptococcus mitis undStreptococcus faecalis verursacht wurden. Bei beiden Prophylaxeregimen war eine gute Compliance festzustellen. In der Norfloxacin-Gruppe waren keine Hinweise für eine Arthropathie zu finden. Unter Cotrimoxazol-Prophylaxe war eine signifikante Zunahme von gramnegativen Stuhlisolaten mit Resistenz gegen Co-trimoxazol festzustellen (p<0,001). Bei Norfloxacin war dagegen keine Selektion resistenter Stämme festzustellen, gramnegative Stäbchenbakterien wurden sehr effizient aus dem Stuhl eliminiert (27,5% der positiven Kulturen).

Total Body Irradiation, Thiotepa, and Cyclophosphamide as a Conditioning Regimen for Children With Acute Lymphoblastic Leukemia in First or Second Remission Undergoing Bone Marrow Transplantation With HLA-Identical Siblings

PURPOSE Allogeneic hematopoietic stem-cell transplantation (HSCT) from HLA-identical siblings can be used to treat children with acute lymphoblastic leukemia (ALL). However, a significant proportion of patients with ALL who undergo HSCT relapse. For this reason, we prospectively evaluated a preparative regimen that included total body irradiation (TBI), thiotepa (TT), and cyclophosphamide (CY) in patients with high-risk ALL in first complete remission (CR) and in children with ALL in second CR. PATIENTS AND METHODS Forty children (median age, 9 years; range, 1 to 18 years) with ALL in first or second CR who underwent allogeneic HSCT from HLA-identical siblings were conditioned with a combination of fractionated TBI, TT (10 mg/kg), and CY (120 mg/kg over 2 days). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine administered intravenously at a dose of 1 to 3 mg/kg/d for the first 21 days and subsequently orally at a dose of 6 mg/kg/d. RESULTS All assessable patients were engrafted, with a median time of 11 and 24 days for neutrophil and platelet recovery, respectively. The preparative regimen was well tolerated. Only one patient died as a result of regimen-related causes. Eight patients relapsed at a median time of 8 months after transplantation (range, 3 to 9 months), and this determined a cumulative probability of relapse of 23%. Twenty-six of 40 patients (65%) are alive and in complete hematologic remission, with a median observation time of 36 months (range, 14 to 57 months), which results in a disease-free survival (DFS) at 3 years of 65%. The 13 patients who underwent transplantation in first CR had a DFS of 85%, whereas the 27 patients who underwent HSCT in second CR had a DFS of 56%. CONCLUSION These data suggest that TT is an effective cytotoxic drug that can be safely added to the classical TBI-CY regimen. Because of its cell cycle-independent action, good CNS diffusion, and limited extramedullary toxicity, TT may contribute to increasing the percentage of children with ALL who are successfully cured with allogeneic BMT.

Does the emergence and persistence of donor-derived leukaemia-reactive cytotoxic T lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study

The frequency of CTL precursors (CTLp) directed towards recipient-derived pre-transplant leukaemic blasts (LB) was measured in the peripheral blood of nine children with acute leukaemia and given BMT from either an HLA-identical sibling or a matched unrelated donor (MUD). Patients were evaluated at various time points between 1 month and more than 2 years after transplantation. A high frequency of donor-derived LB-reactive CTLp was detectable 2–6 months after BMT in all children and persisted for at least up to 18 months in the eight patients in haematological remission, while it rapidly declined in the only patient who relapsed. Generation of LB-reactive T cell clones obtained from some of these patients demonstrates that various T lymphocyte subsets, either HLA class I-restricted/TCR-dependent or HLA-unrestricted, contribute to this phenomenon. The in vitro GVL effect here described seems to be at least partially separated from GVHR, since no correlation was observed between the emergence of LB-reactive CTLp and the development and/or severity of GVHD. Development of LB-reactive CTL in the patients was independent of the frequency of these cells in the donor. These data suggest that donor-derived CTL activity specifically directed towards leukaemic blasts may develop in patients given allogeneic BMT and contribute to the maintenance of a state of haematological remission.