Shifting from open surgical cut down to ultrasound-guided percutaneous central venous catheterization in children: learning curve and related complications.

PURPOSE: Tunneled indwelling central venous catheters (CVC) are essential in the management of children with cancer, hematological, nephrological disorders and for parenteral nutrition. The aim of this study is to present the experience of a single center of the transition from traditional open surgical cut down procedure (OSC) to ultrasound (US)-guided percutaneous CVC insertion, focusing on learning curve and related complications. METHODS: All CVCs inserted between April 2008 and November 2009 in children at the Gaslini Children Hospital were revised, and data on methods of cannulation, intraoperative and device-related complications and re-intervention were recorded. RESULTS: 194 CVCs were positioned in 188 patients. 128 out of 194 CVCs were positioned through an OSC technique, whereas the remaining 66 CVCs were inserted percutaneously with US guidance. Of the 27 recorded complications, 15 were mechanical events, 7 cases developed infection, whereas the remaining 5 (2.6%) were classified as intraoperative complications. A second surgical procedure was described in 23 (11.8%) cases. CONCLUSION: Shifting from OSC to US-guided percutaneous CVC insertion inevitably involves a challenging learning curve which is generally associated with high complication rates. Complications progressively decrease once a good experience in US guidance and percutaneous technique has been obtained.

Hepatitis B reactivation in allogeneic hemopoietic stem cell transplantation setting: a pediatric experience.

Reactivation of HBV is a well known complication in patients undergoing HSCT. Lamivudine treatment appears to prevent hepatitis B virus reactivation and to decrease the mortality in at risk HSCT patients. We describe HBV reactivation occurred in three allogeneic HSCT pediatric patients coming from Eastern Europe, one of whom was successfully treated with lamivudine. Our experience confirms that HBV-DNA may persist as intra-hepatic infection or in extra-hepatic sites and that HBV reactivation may appear during immunodepression. Careful and complete screening for HBV markers is mandatory before HSCT, especially in children coming from countries at risk for HBV. Furthermore, a treatment with lamivudine could also represent an efficacious prophylaxis in pediatric patients to avoid HBV reactivation and to decrease the development of severe hepatic disease.

Strategies of the donor search for children with second CR ALL lacking a matched sibling donor.

During the last 10 years, the number of alternative Haematopoietic stem cell transplantations (HSCTs) performed on children in Europe has increased significantly and has reached 61% of the allografts. In this paper, we provide practical guidelines to help define an algorithm for the treatment of children relapsing during or after first-line chemotherapy for ALL and lacking a matched sibling donor. A simultaneous search for an unrelated donor and for a cord blood unit should be started. This study focuses mainly on the effects of some factors on survival in an effort to highlight the influence that these factors have on our choices. Matching the patient for HLA-A, -B, -C and -DRB1 alleles remains the top priority: a single HLA class I or II allele mismatch has no influence on survival, while multiple mismatching for more than one class I allele and simultaneous disparities in class I and II alleles increase mortality. The impact of additional mismatches for HLA-DQ and -DP loci on survival is still controversial. Young donor age is the most important factor that has a significant effect on better survival from among several other factors, including CMV sero-status, gender and ABO. An 18- to 30-year-old, 8/8 allele-matched donor (excluding allele matching at DQB1) or for many teams 10/10 allele-matched donor; or a 4 out of 6 (considering Ag HLA-A, -B and allelic typing of DRB1) CB unit containing more than 3.0 x 10(7) nuclear cells is considered by most institutions. The choice should be made on the basis of urgency. If a donor or a CB unit is not found within an appropriate time frame, generally less than 3 months after obtention of remission, haploidentical HSCT should be offered. Some institutions consider haploidentical HSCT the second therapeutic option when a matched donor is not available.

Bacteremias in children receiving hemopoietic SCT.

The incidence of bacteremia following hemopoietic SCT (HSCT) changes over time from the procedure. The first 30 days have the highest incidence, both in autologous and allogeneic HSCT recipients. In the following periods, bacteremia is a frequent complication in allogeneic HSCT, especially from alternative donors. Gram-positive cocci represent the most frequent cause of single-agent bacteremia. Knowledge of epidemiology (incidence and etiology) of bacteremias following HSCT is pivotal for planning management strategies (prevention, diagnosis and therapy) that must be distinct in the different post-transplant period.

Incidence of bacteremias and invasive mycoses in children undergoing allogeneic hematopoietic stem cell transplantation: a single center experience.

We performed a retrospective single center study to define the epidemiology of bacteremias or invasive mycoses in pediatric allogeneic hematopoietic SCT (HSCT) from matched related donors (MRD) or alternative donors (AD). During 119 213 days of follow-up, 156 infections were observed: 130 bacteremias (27 in MRD-HSCT and 103 in AD-HSCT recipients) and 26 invasive mycoses (8 in MRD-HSCT and 18 in AD-HSCT recipients). Overall, the risk of bacteremia was fivefold that of invasive mycosis (P<0.001). AD-HSCT recipients had a higher percentage of infections (89 vs 27%; P<0.001), a higher rate/100 days of immunosuppression (infection rate (IR): 0.21 vs 0.06; P<0.001) and a higher proportion of repeated infections (44 vs 9%; P=0.001). In AD-HSCT, the relative risk of bacteremia was 2.87 in the pre-engraftment period, 5.84 in the early post-engraftment period and 6.46 in the late post-engraftment period (P<0.001) compared to MRD-HSCT. Only after 1 year did the epidemiology become similar. The epidemiology of invasive mycoses did not differ significantly between the two types of transplant.

Rare viral infections in children receiving hemopoietic stem cell transplant.

Viral infections are a rare complication in autologous hemopoietic stem cell transplant (HSCT) recipients but represent a frequent cause of disease after allogeneic HSCT. In the last years, there has been an increase in the number of viral diseases observed in these patients. This fact may be at least partially due to an improvement in diagnostic facilities, but the increasing number of transplant procedures and the more severe immunosuppression may also have played an important role.

Invasive mycoses in children receiving hemopoietic SCT.

Invasive mycoses represent a rare but severe complication following hemopoietic SCT (HSCT) in children. Their incidence is related to the type of donor, being higher after allogeneic transplant, especially from alternative donors. Moreover, the incidence of invasive mycoses varies in the different post transplant phases. Neutropenia, lymphopenia, GvHD, high-dose steroids or other immunosuppressive drugs represent well-known risk factors. The clinical features of invasive mycoses after HSCT in children are similar to those observed in adults, and the diagnostic tools, including Aspergillus galactomannan antigen detection, are feasible also in pediatrics. Mortality due to invasive mycoses after HSCT in children is high.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Nebulized liposomal amphotericin B and combined systemic antifungal therapy for the treatment of severe pulmonary aspergillosis after allogeneic hematopoietic stem cell transplant for a fatal mitochondrial disorder.

Nebulized liposomal amphotericin B (20-15 mg twice daily by nebulizer) was combined with high dose intravenous liposomal amphotericin B (10 mg/kg/day) and high dose caspofungin (100 mg/m(2)) for the treatment of severe, recurrent pulmonary aspergillosis following allogeneic hematopoietic stem cell transplantation from alternative donor in a patient with mitochondrial disease (Pearson's syndrome). This combined treatment was administered for 8 days. Nebulized liposomal amphotericin B was well tolerated. Since severe transplant complications developed, nebulized administration was withdrawn and intravenous doses of liposomal amphotericin B and caspofungin were tapered to usual schedules. Pulmonary aspergillosis responded well to 45 days of combined intravenous antifungal therapies which were maintained for 2 years with secondary prophylaxis, because of persistent immunosuppressive treatment.

A novel leukemia T-cell line (PF-382) with phenotypic and functional features of suppressor lymphocytes.

A human leukemia T-cell line (PF-382) spontaneously derived from the pleural effusion of a child with T-cell acute lymphoblastic leukemia is described. The cell line, which has been maintained in culture for over 10 months, has a modal number of 46 chromosomes and is characterized by a chromosomal abnormality, present in most of the cells, consisting of a translocation between chromosome X and chromosome 15 (46X,Xq-,15p+). The cells are not recognized by the OKT3 and OKT11 monoclonal antibodies (MoAb), nor do they form rosettes with sheep erythrocytes. By contrast, they react with the OKT6, Leu-1, and Leu-9 MoAb, which detect early T-lymphocytes, and express the more mature OKT8 antigen. The presence of the OKT8 marker is associated with suppressor activity on the pokeweed mitogen-induced proliferation and differentiation of normal B-cells, both by the PF-382 cells and by their supernatant. However, no cytotoxic activity against natural killer (NK)-sensitive target cells (K562) was found, indicating that the proliferating cells do not correspond to the subset of NK cells expressing the OKT8 antigen. Furthermore, the cells are incapable of both spontaneous and mitogen-induced interleukin-2 and interferon production. The ability of the PF-382 cell line to release a soluble factor(s) capable of modulating the differentiation of the B-cell compartment suggests that this new cell line represents a valuable model for the investigation of the interrelationships between T-cell subsets and other hematopoietic cell lineages.

Myeloablative therapy and unpurged autologous bone marrow transplantation for poor-prognosis neuroblastoma: report of 34 cases.

From October 1984 to November 1987, 34 patients aged from 1 year 1 month to 7 years 7 months with resistant or relapsed neuroblastoma (NB) (group 1, 10 patients), unselected disseminated NB (group 2, 14 patients), or selected disseminated NB (group 3, 10 patients) received myeloablative therapy (MAT) followed by unpurged autologous bone marrow transplantation (ABMT) at the end of an intensive protocol, which included high-dose chemotherapy and surgery to the primary tumor. Median time from diagnosis to MAT and ABMT was 6 months (5 months from last relapse to MAT and ABMT in the relapsed patients). The MAT regimen included vincristine, fractionated total body irradiation (TBI), and melphalan. Seventeen patients were grafted in complete remission (CR), five in very good partial remission (VGPR), 10 in partial remission (PR), and two in progressive disease (PD). The acute toxic death rate was 2.9%. The overall progression-free survival was 29%. The median progression-free survival was 20 months for the 17 patients grafted in CR, 6 months for the five patients grafted in VGPR, and 12 months for the 10 patients grafted in PR.

Total-body irradiation and melphalan is a safe and effective conditioning regimen for autologous bone marrow transplantation in children with acute myeloid leukemia in first remission. The Italian Association for Pediatric Hematology and Oncology-Bone Marrow Transplantation Group.

PURPOSE: To evaluate the safety and efficacy of a preparative regimen consisting of fractionated total-body radiation (9.9 to 12 Gy) and melphalan (140 mg/m(2) in a single dose) in children with acute myeloid leukemia in first complete remission (CR) given autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Fifty-three children (30 males and 23 females; age range, 1.5 to 18 years) were enrolled onto the study. The median time from first CR to ABMT was 3.5 months (range, 1.4 to 13 months), with 45 patients (85%) undergoing transplantation within 6 months from the diagnosis. Forty-five patients received in vitro marrow purging with standard-dose mafosfamide (100 microg/mL), seven patients were treated with interleukin-2 before marrow collection, and in the remaining child, the marrow was unmanipulated. The median infused cell dose was 1.8 x 10(8)/kg (range, 0.4 to 5.8 x 10(8)/kg). RESULTS: All patients but one achieved hematopoietic engraftment, with a median time to neutrophil recovery of 24 days (range,11 to 66 days). Treatment-related toxicity was moderate and consisted mainly of mucositis. One patient died from cytomegalovirus interstitial pneumonia, and one died from pulmonary hemorrhage. Fourteen patients (26%) relapsed at a median time of 6 months after ABMT (range, 2 to 17 months), with a cumulative relapse probability of 29% (95% confidence interval, 16% to 42%). The 5-year Kaplan-Meier estimate of survival for all 53 patients was 78% (range, 65% to 90%), whereas the overall 5-year disease-free survival was 68% (range, 55% to 81%), with a median follow-up duration of 40 months (range, 7 to 130 months). CONCLUSIONS: These data suggest that, in our cohort of patients, the combination of total-body irradiation and melphalan is safe and associated with good antileukemia activity, making ABMT an appealing alternative for postremission therapy in children with acute myeloid leukemia in first CR.

Update on extracorporeal photochemotherapy for graft-versus-host disease treatment.

Pediatric experience with extracorporeal photochemotherapy (ECP) for graft-versus-host disease (GvHD) has mainly been reported by Italian and French groups. Data concerning 41 children with acute GvHD and 63 children affected by chronic GvHD are available. In 73 and 63% of them, respectively, improvement was observed, with addition of ECP to their immunosuppressive regimen. Treatment with ECP was associated with minimal side effects, even in the smallest of patients. In all responded pediatric patients, both with acute and chronic GvHD, ECP allowed progressive reduction or discontinuation of the concomitant pharmacological immunosuppressive therapy without an increase in GvHD activity. These data show that ECP is a useful therapy for children affected by GvHD resistant to conventional treatment and can be safely used.

Fludarabine, cyclophosphamide and anti-thymocyte globulin for alternative donor transplants in acquired severe aplastic anemia: a report from the EBMT-SAA Working Party.

We have developed a reduced-intensity conditioning regimen for patients with severe aplastic anemia (SAA) undergoing alternative donor transplants, which includes fludarabine (120 mg/m(2)), cyclophosphamide (1,200 mg/m(2)) and antithymocyte globulin (7.5 mg/kg). Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine and methotrexate. We have enrolled 38 SAA patients in this trial: median age of 14 (3-37) years, transplanted from unrelated (n=33) or family mismatched (n=5) donors, with unmanipulated marrow (n=36) or peripheral blood (n=2). Seven patients (18%) had evidence of graft failure, 11% developed grade II-III acute GvHD and 27% developed chronic GvHD. The actuarial 2-year survival is 73%, with a median follow-up of 621 days. Younger patients (

Meiotic origin of trisomy in neoplasms: evidence in a case of erythroleukaemia.

Trisomic cells in neoplasms may represent abnormal clones originated from a tissue-confined mosaicism, and arise therefore by a meiotic error. We report on a 16-month-old child with erythroleukaemia (AML-M6), whose marrow karyotype at onset was 48,XX,del(13)(q12q14),del(14)(q22q32),+21,+21. The parental origin of the supernumerary chromosomes 21 was investigated by comparing 10 polymorphic loci scattered along the whole chromosome on the patient's marrow and her parents' leukocytes. Three loci were informative for the presence of three alleles, two of which were of maternal origin; two further loci showed a maternal allele of higher intensity. Lymphocytes and skin fibroblasts showed a normal karyotype, and molecular analysis on leukocytes at remission, buccal smear and urinary sediment cells consistently showed only one maternal allele, whereas neonatal blood from Guthrie spot showed two maternal alleles as in the marrow. An accurate clinical re-evaluation confirmed a normal phenotype. Our results indicate that tetrasomy 21 arose from a marrow clone with trisomy 21 of meiotic origin. To the best of our knowledge, this is the first evidence that supernumerary chromosomes in neoplastic clones may in fact be present due to a meiotic error. This demonstrates that a tissue-confined constitutional mosaicism for a trisomy may indeed represent the first event in multistep carcinogenesis.

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.

Outcome of children with high-risk acute myeloid leukemia given autologous or allogeneic hematopoietic cell transplantation in the aieop AML-2002/01 study.

We analyzed the outcome of 243 children with high-risk (HR) AML in first CR1 enrolled in the AIEOP-2002/01 protocol, who were given either allogeneic (ALLO; n=141) or autologous (AUTO; n=102) hematopoietic SCT (HSCT), depending on the availability of a HLA-compatible sibling. Infants, patients with AML-M7, or complex karyotype or those with FLT3-ITD, were eligible to be transplanted also from alternative donors. All patients received a myeloablative regimen combining busulfan, cyclophosphamide and melphalan; [corrected] AUTO-HSCT patients received BM cells in most cases, while in children given ALLO-HSCT stem cell source was BM in 96, peripheral blood in 19 and cord blood in 26. With a median follow-up of 57 months (range 12-130), the probability of disease-free survival (DFS) was 73% and 63% in patients given either ALLO- or AUTO-HSCT, respectively (P=NS). Although the cumulative incidence (CI) of relapse was lower in ALLO- than in AUTO-HSCT recipients (17% vs 28%, respectively; P=0.043), the CI of TRM was 7% in both groups. Patients transplanted with unrelated donor cord blood had a remarkable 92.3% 8-year DFS probability. Altogether, these data confirm that HSCT is a suitable option for preventing leukemia recurrence in HR children with CR1 AML.

Severe neurologic complications after hematopoietic stem cell transplantation in children.

OBJECTIVE: To describe and evaluate the incidence and risk factors of severe neurologic events (SNE) in pediatric recipients of allogeneic or autologous hematopoietic stem cell transplantation (HSCT) for hematologic or nonhematologic diseases. METHODS: Retrospective analysis of 272 consecutive children admitted to the G. Gaslini Children's Research Institute and given HSCT (70 from unrelated donors, 115 from related donors, and 87 autologous) between June 1985 and January 2001. RESULTS: Thirty-seven children (13.6%) developed SNE after a median of 90 days (range, 5 days to 8.8 years) after HSCT. Cyclosporine A (CSA) neurotoxicity was the most frequent SNE (n = 21), followed by irradiation or chemotherapy injury (n = 7), CNS infections (n = 7), cerebrovascular events (n = 3), and immune-mediated etiology SNE (n = 2). Eleven patients (30%) died because of the neurologic complications. Type of HSCT, treatment with total body irradiation (TBI), acute graft-vs-host disease (GvHD), GvHD >grade 2, and treatment with CSA were associated with a significant increased risk of SNE. CONCLUSIONS: Severe neurologic complications are frequent (14%) among children receiving HSCT, causing 8.5% of deaths after transplant. Transplant from allogeneic donor, especially if unrelated, the development of severe acute GvHD grade >2, and the use of TBI in the preparative regimen are the main risk factors for such complications.

High-dose chemotherapy with autologous bone marrow rescue in advanced stage IV neuroblastoma.

In order to better evaluate the role of bone marrow purging procedures in the treatment of stage IV neuroblastoma, two similar groups of patients, prospectively treated during the same period at Léon Bérard Center, Lyon, France, and at Giannina Gaslini Institute, Genova, Italy, were reviewed. 18 children were treated in Lyon with a protocol including induction chemotherapy, surgery and a single course of high-dose chemotherapy followed by purged autologous bone marrow rescue. 21 patients were treated in Genoa with a very similar protocol which did not include purging procedures. Progression-free survival at 6 years was 12% (95% confidence interval 0-24%), without any difference between the two series of patients. The only prognostic factor for long-term survival was the persistence (or not) of bone lesions and the presence of metastatic disease (bone or bone marrow) at graft. The small numbers in the two groups and the very poor outcome make it difficult to conclude on the efficacy of purging.

Immunohistochemical study of skin lesions in acute and chronic graft versus host disease following bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) is the therapy of choice for a variety of malignant and nonmalignant disorders; however, a major constraint to successful BMT is graft versus host disease (GVHD). Skin lesions are the earliest presentation of GVHD. Donor-derived cytotoxic T lymphocytes are the effector cells responsible for lesions in the skin and other tissues. Here we show that most skin-infiltrating lymphocytes, in all forms of GVHD, are memory T cells with a predominance of CD4+ cells in the dermis and CD8+ cells in the epidermis. Relatively little attention has been focused on the adhesive phenotype of keratinocytes in GVHD. In this study, immunohistochemical analyses of skin biopsies from BMT patients with acute or chronic GVHD were conducted, with particular emphasis on antigen-presenting cells (APCs) and on keratinocytes. The distribution of APCs in the epidermis (Langerhans' cells) was investigated. Keratinocytes were analyzed for the expression of human leukocyte antigen DR locus (HLA-DR) and of a novel integrin, alpha10.1.2 beta1, which is detected in the basal layer of normal epidermis. Langerhans' cells were decreased in all grades of acute GVHD, but the epidermal APC network was reconstituted in chronic GVHD. HLA-DR was expressed by keratinocytes in grade 2 and 3 acute GVHD lesions, but not in two of three chronic GVHD cases, and in the regression phase of acute GVHD. Integrin chains alpha10.1.2 and beta1 were detected in the epidermal basal cell layer of most GVHD cases but they were also expressed in suprabasal keratinocytes of both acute and chronic GVHD. This latter finding indicates that a proliferative response uncoupled from differentiation occurs in keratinocytes in the course of GVHD.