Off-Label Use of Sirolimus and Everolimus in a Pediatric Center: A Case Series and Review of the Literature.

BACKGROUND: It has been 15 years since sirolimus, an mTOR inhibitor, received Food and Drug Administration approval to prevent acute rejection in kidney transplantation, and 8 years since its analog everolimus acquired the same status. Since then, these drugs have become more and more utilized and their immunosuppressive and antiproliferative properties have been tested in a great variety of clinical conditions, often achieving excellent results. Despite such positive evidence, the on-label indications for these rapalogs are still very restrictive, especially in children. AIMS: The aims of this study were to describe our center's experience with sirolimus and everolimus in managing rare pediatric conditions for which mTOR inhibitors have been reported as a therapeutic option, although without conclusive approval from regulatory agencies, and to evaluate safety and tolerability of the treatment at the prescribed doses. METHODS: All the subjects who received off-label sirolimus or everolimus at the Pediatric Department of the IRCCS Burlo Garofolo in the last 13 years were included. For each disease found in our case series, we reviewed the current scientific literature. RESULTS: Off-label treatment with rapalogs was prescribed in 16 children (11 males, 5 females, median age of 9.5 years, range 1-16 years). Seven had immunologic disorders: four autoimmune lymphoproliferative syndrome (ALPS), one multicentric Castleman disease (mCD), one activated PI3K delta kinase syndrome (APDS), and one immunodysregulation with polyendocrinopathy enteropathy X-linked (IPEX). Eight had proliferative disorders or vascular anomalies: one cystic lymphangioma, two Bannayan-Riley-Ruvalcaba syndrome (BRRS), one blue rubber bleb nevus syndrome (BRBNS), two tuberous sclerosis complex (TSC), and one low-flow mixed arterial and venous malformation. One case had congenital hyperinsulinism (CHI). The average dosage administered was 1 mg/m2 for sirolimus and 7 mg/m2 for everolimus. We experienced a good measurable clinical improvement in 14 patients. Nobody experienced serious adverse events (SAEs). The therapy was interrupted in two cases, for lack of efficacy and poor tolerance in one case and for occurrence of bacterial pneumonia in the other one. A review of the literature identified 101 published reports that met our inclusion criteria. CONCLUSIONS: Although use of mTOR inhibitors has been considered to be complicated, our experience shows that, using low dosages, it is possible to obtain relevant clinical improvements, with a good profile of safety and tolerability.

Pharmacogenetics and induction/consolidation therapy toxicities in acute lymphoblastic leukemia patients treated with AIEOP-BFM ALL 2000 protocol.

Drug-related toxicities represent an important clinical concern in chemotherapy, genetic variants could help tailoring treatment to patient. A pharmacogenetic multicentric study was performed on 508 pediatric acute lymphoblastic leukemia patients treated with AIEOP-BFM 2000 protocol: 28 variants were genotyped by VeraCode and Taqman technologies, deletions of GST-M1 and GST-T1 by multiplex PCR. Toxicities were derived from a central database: 251 patients (49.4%) experienced at least one gastrointestinal (GI) or hepatic (HEP) or neurological (NEU) grade III/IV episode during the remission induction phase: GI occurred in 63 patients (12.4%); HEP in 204 (40.2%) and NEU in 44 (8.7%). Logistic regression model adjusted for sex, risk and treatment phase revealed that ITPA rs1127354 homozygous mutated patients showed an increased risk of severe GI and NEU. ABCC1 rs246240 and ADORA2A rs2236624 homozygous mutated genotypes were associated to NEU and HEP, respectively. These three variants could be putative predictive markers for chemotherapy-related toxicities in AIEOP-BFM protocols.

Pharmacogenomic approaches for tailored anti-leukemic therapy in children.

Several lympholytic and cytotoxic agents are used in acute lymphoblastic leukemia (ALL) polychemotherapy. Genetic variants for cellular components involved in the pharmacokinetics and pharmacodynamics of these drugs can influence the pharmacological response, and molecular characterization of these genetic variants could be helpful for the comprehension of the mechanisms of resistance or increased sensitivity. The purpose of this review is to carry out an update of recent publications on genes that might influence ALL treatment in terms of outcome and/or toxicity and to underlie the role of genetic variants, particularly single nucleotide polymorphisms (SNP), in predicting clinical response, with particular reference to the current protocol for ALL therapy used in Italy, AIEOP-BFM ALL 2009.

Haematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation.

In 1997, the first consensus guidelines for haematopoietic SCT (HSCT) in autoimmune diseases (ADs) were published, while an international coordinated clinical programme was launched. These guidelines provided broad principles for the field over the following decade and were accompanied by comprehensive data collection in the European Group for Blood and Marrow Transplantation (EBMT) AD Registry. Subsequently, retrospective analyses and prospective phase I/II studies generated evidence to support the feasibility, safety and efficacy of HSCT in several types of severe, treatment-resistant ADs, which became the basis for larger-scale phase II and III studies. In parallel, there has also been an era of immense progress in biological therapy in ADs. The aim of this document is to provide revised and updated guidelines for both the current application and future development of HSCT in ADs in relation to the benefits, risks and health economic considerations of other modern treatments. Patient safety considerations are central to guidance on patient selection and HSCT procedural aspects within appropriately experienced and Joint Accreditation Committee of International Society for Cellular Therapy and EBMT accredited centres. A need for prospective interventional and non-interventional studies, where feasible, along with systematic data reporting, in accordance with EBMT policies and procedures, is emphasized.

Haematopoietic SCT in autoimmune diseases in children: rationale and new perspectives.

The possible role of haematopoietic SCT (HSCT) for the treatment of severe autoimmune diseases was originally supported by animal experiments and remission of concomitant autoimmune diseases in patients undergoing transplantation for haematological disorders. Since 1996, over 100 procedures were performed in children with different severe autoimmune diseases such as juvenile idiopathic arthritis, systemic lupus erythematosus, systemic sclerosis, immune cytopaenias and Crohn's disease. This review tries to summarize the published data on efficacy and toxicity of HSCT in this group of patients.

Haematopoetic stem cell transplantation for refractory autoimmune cytopenia.

This study describes the outcome of patients receiving haematopoietic stem cell transplantation (HSCT) to treat severe refractory autoimmune cytopenia. The registry of the European Group of Blood and Marrow Transplantation holds data on 36 patients receiving 38 transplants, the first transplant was autologous for 27 and allogeneic for nine patients. Patients had autoimmune haemolytic anaemia (autologous: 5; allogeneic: 2), Evans's syndrome (autologous: 2; allogeneic: 5); immune thrombocytopenia (autologous: 12), pure red cell aplasia (autologous: 4; allogeneic: 1), pure white cell aplasia (autologous: 1; allogeneic 1), or thrombotic thrombocytopenic purpura (autologous: 3). Patients had longstanding disease having failed multiple prior treatments. Among 26 evaluable patients mobilized for autologous HSCT, three died of treatment-related causes, one died of disease progression, seven were non-responders, six patients had transient responses and nine had continuous partial or complete remission. Of the seven evaluable patients receiving allogeneic HSCT, one died of treatment-related complications, one with transient response died of progressive disease and five had a continuous response. Autologous and allogeneic HSCT may induce a response in a subset of patients with autoimmune cytopenia of long duration albeit at the price of considerable toxicity.

Impact of marrow unrelated donor search duration on outcome of children with acute lymphoblastic leukemia in second remission.

We analyzed the outcome of 167 consecutive children with second CR acute lymphoblastic leukemia (ALL), for whom an unrelated donor (UD) search was activated between 1989 and 1998 at a median time of 2 months after relapse. A suitable donor was identified for 70 patients at 1 year and 6.5 months before and after 1995 from search activation, respectively; a further leukemia relapse occurred during the search in 94 children at a median of 4 months after search activation, 36 of whom underwent UD (14) or other types of transplant (22), beyond second CR, while 58 died of progressive disease. Of 73 patients not experiencing a second relapse, 64 underwent UD (46) or other types of transplant (18), while nine proceeded with chemotherapy, and only four of them survived. The 3-year disease-free survival (DFS) from second CR for the 167 patients is 15.1%, whereas 3-year DFS after transplant for the 60 UD and 40 alternative donor transplanted children is 31.6 and 25.4%, respectively. In conclusion, a further relapse is the main factor adversely affecting outcome of children with second CR ALL. Thus, for these patients, the search should be activated early after relapse and either a UD or an alternative transplant should be performed as early as possible.

Hematopoietic stem cell transplantation for multiple sclerosis. A retrospective multicenter study.

RATIONALE: Phase I/II studies of autologous hematopoietic stem cell transplantation (HSCT) for multiple sclerosis ( MS) were initiated, based on results of experimental transplantation in animal models of multiple sclerosis and clinical observations in patients treated concomitantly for malignant disease. PATIENTS: Eighty-five patients with progressive MS were treated with autologous HSCT in 20 centers and reported to the autoimmune disease working party of the European Group for Blood and Marrow Transplantation (EBMT). 52 (61 %) were female, median age was 39 [20-58] years. The median interval from diagnosis to transplant was 7 [1-26] years. Patients suffered from severe disease with a median EDSS score of 6.5 [4.5-8.5]. Active disease prior to transplant was documented in 79 of 82 evaluable cases. RESULTS: The stem cell source was bone marrow in 6 and peripheral blood in 79, and stem cells were mobilized into peripheral blood using either cyclophosphamide combined with growth factors or growth factors alone. Three patients experienced transient neurological complications during the mobilization phase. The high dose regimen included combination chemotherapy, with or without anti-lymphocyte antibodies or, with or without, total body irradiation. The stem cell transplants were purged of lymphocytes in 52 patients. Median follow-up was 16 [3-59] months. There were 7 deaths, 5 due to toxicity and infectious complications, 2 with neurological deterioration. The risk of death of any cause at 3 years was 10 (+/-7)% (95 % confidence interval). Neurological deterioration during transplant was observed in 22 patients; this was transient in most but was associated with MS progression in 6 patients. Neurological improvement by > or = 1 point in the EDSS score was seen in 18 (21 %) patients. Confirmed progression-free survival was 74 (+/-12)% at 3 years being 66 (+/-23)% in patients with primary progressive MS but higher in patients with secondary progressive or relapsing-remitting MS, 78 (+/-13)%; p = 0.59. The probability of confirmed disease progression was 20 (+/-11)%. MRI data were available in 78 patients before transplant showing disease activity (gadolinium enhancing, new or enlarging lesions) in 33 %. Posttransplant MRI showed activity at any time in 5/61 (8 %) evaluable cases. CONCLUSION: Autologous HSCT suggest positive early results in the management of progressive MS and is feasible. These multicentre data suggest an association with significant mortality risks especially in some patient groups and are being utilised in the planning of future trials to reduce transplant related mortality.

Mycophenolate mofetil (MMF) as therapy for refractory chronic GVHD (cGVHD) in children receiving bone marrow transplantation.

Mycophenolate mofetil (MMF) is an alternative immunosuppressant which inhibits the proliferation of T and B lymphocytes. The purpose of the present study was to evaluate the safety and efficacy of MMF as salvage therapy for chronic GVHD (cGVHD) in children receiving allogeneic bone marrow transplantation. Fifteen children, 3-16 years of age, who had received grafts from HLA-compatible siblings (n = 8), partially matched related donors (n= 2) or matched unrelated donors (n = 5), developed extensive cGVHD which had proved unresponsive to standard immunosuppressive therapy. Patients were treated with MMF at the dose of 15-40 mg/kg/day in combination with other immunosuppressive therapy for a median of 4 months (range 1-15 months). The overall response rate (complete or partial response) was 60%. Thirteen percent had only minor responses, whereas 27% of patients had progressive disease. Best responses were seen in patients with GI tract (60% of complete responses) or mouth (33% of complete responses) cGVHD and skin involvement (43% of complete responses) that did not include sclerodermatous manifestations. Once MMF was started, improvements in the clinical manifestations of cGVHD allowed a significant reduction of steroids in 45% of patients and discontinuation in 27% of cases. Six patients (40%) experienced adverse events, with gastrointestinal symptoms predominating. Five patients experienced opportunistic infections. MMF was discontinued after 35-180 days in six patients for the following reasons: parents choice (n = 2), liver toxicity (n = 1), poor compliance (n = 2), and no response (n = 1). In conclusion, these preliminary results suggest that MMF in combination with other immunosuppressive agents may have a role to play in patients with cGVHD. Prospective clinical trials are needed to establish exact indications for therapy and dosage scheduling. Bone Marrow Transplantation (2000).

Hematopoietic stem cell transplantation in childhood: report from the bone marrow transplantation group of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP).

BACKGROUND AND OBJECTIVE: Transplantation of hematopoietic stem cells from different sources is being increasingly used to treat a variety of diseases in children. Transplant procedures and indications have changed considerably during recent years. Monitoring of information about these changes is useful for interpretation of nationwide collected data. DESIGN AND METHODS: Since 1985, Centers belonging to the AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica), performing hematopoietic stem cell transplants (HSCT) in children, and members of the AIEOP-Bone Marrow Transplant (BMT) Group annually report data on their transplant activity to the AIEOP-BMT Registry employing specially prepared patient-oriented forms. RESULTS: From January 1985 to December 1998, a total of 2,474 bone marrow (BM), peripheral blood (PB) or umbilical cord blood (CB) transplants were reported: 1,296 (52%) were allogeneic (Allo) and 1,178 (48%) autologous (Auto) transplants. These transplants were performed in 19 Italian Centers on 2,249 patients aged less than 17 years. Among Allo-transplants, 1,198 (92%) were performed using BM progenitor cells, whereas 49 (4%) CB, 42 (3%) were PB, 4 BM plus PB, and 3 BM plus CB allografts; they were performed using HLA-identical sibling donors in 867 cases (67%) and alternative donors (i.e. partially-matched relatives or unrelated donors) in the remaining 429 (33%) cases. Allogeneic transplants were performed on 786 (67%) patients with malignancy and on 395 (33%) patients with non-malignant disorders. In the last 6 years, the number of Allo-transplants per year exceeded that of Auto-transplants. Of the Auto-transplants, 775 (66%) were performed using BM, and 403 (34%) using PB alone or combined with BM hematopoietic stem cells. Indications for Auto-BMT were myelo-lymphoproliferative disorders in 524 (49%) cases, solid tumor in 533 (50%) cases and non-malignant disease in 11 (1%) cases. In the last 5 years, the use of PB for autografts has increased from 7% to 70%. INTERPRETATION AND CONCLUSIONS: These data reflect the development and present status of HSCT in Italy and provide a basis for patient counseling and health care planning.

Haploidentical bone marrow transplantation in leukemia and genetic diseases.

From 1986 to June 2000, sixty children suffering from acute and chronic leukemia (n = 42, 33 of which in resistant relapse), genetic diseases (n = 11), aplastic anemia (n = 2, one of which with platelet refractoriness and bleeding), myelodysplasia (n = 5) received an haploidentical bone marrow, mismatched for 2-3 HLA loci. The donor's marrow was treated in vitro with vincristine and methylprednisolone to obtain a functional T depletion (MLC and CTL inhibition, functional blockade of Th1 and Th2). The prevalence of infectious complications and GVHD was similar to that recorded in matched unrelated donor (MUD) transplants. In situations of high risk of rejection (chronic leukemia, genetic diseases) we infused immediately one half of the harvest and then frozen aliquots from the second week. Of the 25 ALL and 8 AML in resistant relapse, 3 survived, disease-free at 14, 8 and 1 years respectively. Of the 3 ALL, transplanted during remission, 1 is surviving at 18 months. Of the 6 CML, 1 had fractionated bone marrow and is surviving at 3 years, and 5 had standard single dose infusion and died of progression of their disease after rejection of the graft (4) or blast crisis after complete engraftment (1). The 2 patients with aplastic anemia, those with myelodysplasia, and 6 of the 10 with genetic disorders died of transplant-related complications or disease progression. 4 patients with osteopetrosis (n = 2), MLD (n = 1), Wiskott Aldrich dis. (n = 1) survive at 8, 2, 5 and 1.5 years respectively. In patients transplanted with fractionated marrow GVHD > 2nd grade occurred in 15%. Only one patient rejected the graft. Compared with MUD transplantation, mismatched BMT whenever performed in patients in good conditions provides similar outcome and widens the donor availability.

In vitro incubation of bone marrow and peripheral stem cells with vincristine and methylprednisolone: functional T-cell depletion for haploidentical and autologous transplants.

A mismatched bone marrow transplantation is feasible only if the donor's marrow lymphocytes are eliminated from the graft. This can be achieved by several methods, but all have the disadvantage of inducing a long-lasting immune deficiency while the risk of graft rejection and leukemic relapse increase. We use a sort of functional T-cell depletion by treating the cells with vincristine and methylprednisolone. This method is surely the cheapest and has allowed us to perform 60 transplants with a tolerable risk of GVHD. The treatment of the donor's lymphocytes has already been demonstrated to be able to block the mixed lymphocyte culture reaction in vitro. In this experiment Th1 and Th2 activities were almost completely blocked without reduction of lymphocyte viability and apoptosis induction.

Immunoablation followed by autologous hematopoietic stem cell infusion for the treatment of severe autoimmune disease.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the tolerability and effectiveness of a non-myeloablative conditioning regimen followed by autologous hematopoietic stem cell infusion for the treatment of severe autoimmune diseases. DESIGN AND METHODS: From 1996 patients with severe autoimmune disease not responsive to conventional immunosuppressive treatment were selected. The patients' blood or marrow cells were harvested after incubation with vincristine and methylprednisolone. Two different immunoablative conditioning regimens were employed. The first used cyclophosphamide (2500 mg/m2 in one day) and antilymphocyte globulin (ALG) (15 vials/m2 in three days) and the second used fludarabine (300 mg/m2 in two courses of 5 days) plus ALG (25 vials/m2 in 5 days). RESULTS: Nineteen patients (14 female, 5 male) with severe autoimmune diseases were treated. Nine had a rheumatologic disorder (5 juvenile chronic arthritis, 1 rheumatoid arthritis, 1 systemic vasculitis, 1 Sjögren's syndrome, 1 Behçt's disease), 4 a neurologic disorder (3 multiple sclerosis, 1 myasthenia), 3 a haematologic disease (2 pure red cell aplasia, 1 autoimmune thrombocytopenia), 2 had a gastrointestinal disease (1 Crohn's disease, 1 autoimmune enteropathy) and 1 had a multiple autoimmune disorder. There was no regimen-related toxicity and no opportunistic infections occurred. Ninety percent of the patients improved and/or had a complete remission after the procedure. Fifty percent of the subjects went into complete or partial remission after a median follow-up of 15 months (range 3-25) while 50% relapsed after a median follow-up of 11 months, (range 6-16). The incidence of relapse in the group treated with fludarabine was lower (30%). INTERPRETATION AND CONCLUSIONS: A non-myeloablative conditioning regimen was able to induce persistent remission in some patients with severe autoimmune diseases. There was no mortality or morbidity related to the procedure. The extent of remission does, however, remain to be established.

N-acetylcysteine in the treatment of steroid-resistant acute graft-versus-host-disease: preliminary results. Gruppo Italiano Trapianto di Midollo Osseo (GITMO).

BACKGROUND: Acute graft-versus-host disease (GVHD) results from reactivity of donor immunocompetent cells versus host tissues. Its pathogenesis involves co-stimulatory molecules, cytokines, free radicals, and oxidative stress products. N-Acetylcysteine (NAC) is an antioxidant that inhibits the B7-1/CD28 expression in vitro, and it may contrabalance the effects of free radicals and oxidative stress; it has been tested in eight patients with steroid-resistant acute GVHD. METHODS: NAC was given at the dose of 150 mg/kg bolus intravenously, followed by 50 mg/kg intravenous continuous infusion over 3 weeks or less up, to clinical GVHD resolution. In four patients, flow cytometric analysis of co-stimulatory molecules was performed on peripheral mononuclear cells before and after NAC therapy. RESULTS: We achieved prompt response in six patients: four had complete response, two partial response. Two patients died of acute GVHD, and four of intercurrent disease. We noticed significant decrease in CD80, CD25, and CD8+ cells after NAC therapy. CONCLUSION: NAC therapy is feasible; it may give response in steroid-resistant acute GVHD. More extensive studies are needed to confirm these data.

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G-->A in position -1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the delta254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G-->A (-1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G-->A substitution observed in WD at position + 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive delta254-277 protein. In conclusion, LAL genotypes determine the level of residual enzymatic activity, thus explaining the severity of the phenotype.

Autologous bone marrow transplantation for childhood acute lymphoblastic leukaemia in Italy. AIEOP/FONOP-TMO Group. Italian Association of Paediatric Haemato-Oncology.

From January 1984 to December 1994, ABMT was performed on 154 children (101 males, 53 females; median age 10, range 3-21 years) with ALL and registered for BMT by the AIEOP (Italian Association of Paediatric Haemato-Oncology). All patients were in CR: 98 were in 2nd CR and 56 were in >2nd CR. Fifteen children (9.7%) died of transplant-related mortality. Ninety-five patients (61.6%) relapsed at a median of 5 (range 1-42) months after ABMT. The 8-year EFS according to pre-BMT status was 34.6% (s.e. 4.9) for 2nd CR patients and 10.6% (s.e. 5.6) for patients in >2nd CR. By univariate analysis, site of relapse (isolated extramedullary (IE) vs BM: EFS = 68.5% vs 18.2%; P < 0.0001) and TBI containing regimen (TBI vs no TBI: EFS = 48.1 vs 15.4%; P = 0.0023) were significant factors for 2nd CR patients. When the 2nd CR subset with BM involvement was analysed, TBI became insignificant (EFS = 25.4 vs 11.8%). No factors influenced EFS in patients in >2nd CR. By multivariate analysis, site of relapse was the only significant factor in 2nd CR patients (P < 0.0001). In conclusion, ABMT is an effective treatment after one early IE relapse. Few patients can be rescued after BM relapse.

Bone marrow transplantation in chronic GvHD and autoimmune diseases.

The same ex vivo marrow's treatment that we used in mismatched BMT (incubation with vincristine and methylprednisolone) was used in two patients with chronic GVHD and in one patient with PRCA. The conditioning regimen with cyclophosphamide and ALG was aimed to kill only the patient's lymphocytes, and did not cause a deep myeloid aplasia. The patients achieved a clear improvement of their symptoms.