Large volume leukapheresis is efficient and safe even in small children up to 15 kg body weight.

BACKGROUND: The collection of peripheral blood stem cells, although now a routine procedure, is still a challenge in low body weight children because of specific technical and clinical issues. For paediatric patients it is crucial to obtain an adequate number of CD34+ cells with the minimum number of procedures: this can be done using large volume leukapheresis (LVL). MATERIALS AND METHODS: We analysed the efficacy and safety of 54 autologous LVL performed in 50 children (33 [66%] males and 17 [34%] females), median age 2 years (range, 1-5) and median body weight 12 kg (range, 6-15). The procedures were performed with a COBE Spectra previously primed with red blood cells; ACD-A solution and heparin were used as anticoagulants. RESULTS: The target CD34+ cell dose (≥5×10/kg body weight) were collected with one LVL in 46 (92%) patients, while four (8%) patients needed another procedure. All our LVL were well tolerated. Side effects were observed in five (9.2%) patients and one procedure had to be discontinued because of catheter-related haemorrhage. The platelet count decreased significantly (p<0.001) after each procedure but without bleeding or need for transfusion support. DISCUSSION: Our experience confirms that LVL is efficient and safe even in small children, if the procedure is adjusted considering the weight and age of child. The most important factors are good venous access, adequate preparation of the child's electrolyte status, and surroundings in which the small child as well as parents feel comfortable, and can tolerate the procedure better. Although a median platelet loss of 50% can be expected, LVL is safe and reduces the overall number of procedures required. It can be recommended for peripheral blood stem cell collection even in small body weight children with malignant diseases, particularly those who mobilise low numbers of CD34+ cells.

Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3'-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

[Treatment of langerhans cell histiocytosis in children]. / Lijecenje histiocitoze langerhansovih stanica u djece.

Langerhans' cell histiocytosis (LCH) is a disease characterised by pathologic accumulation and proliferation of histiocytes, cells from the monocyte-macrophage system, in various tissues and organs. In this retrospective study we analyzed patients charts treated in the Department of pediatric hematology and oncology at the University Hospital Zagreb with the diagnosis of LCH. Twenty-two children were diagnosed between January 1st 1996 and December 31st 2010, and all were treated with chemotherapy. 19 patients survived (86%) and the remaining 3 (14%), all under the age of 2 with multisystem disease, died. At the time of diagnosis 12 children (55%) presented with single-system disease, the most common were bone lesions in 8 children (36%). All children were treated according to protocols LCH-I and LCH -III. Eight children had mild complications of treatment and the disease itself. Diabetes insipidus remains in 4 children.

Subcutaneous panniculitis-like T-cell lymphoma in a 19 month-old boy: a case report.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare type of T-cell lymphoma of CD3+CD8+ phenotype characterized by deep-seated skin nodules or plaques mimicking panniculitis, a result of neoplastic lymphocytes infiltrating the subcutaneous fatty tissue. We present a case of a 19-month year old boy with SPTCL diagnosed and successfully treated in our institution. Disease first presented with symptoms of high fever and painful erythematous nodule located below the umbilicus. Later on the infiltrates appeared on the face, legs, arms and the back of the body. As the most decisive in obtaining the diagnosis, skin biopsy showed atypical, small to medium-sized lymphatic cells infiltrating the deeper dermal layers as well as the subcutaneous adipous tissue surrounding the adipocytes. Immunohystochemical analysis showed neoplastic lymphocytes positive for CD2, CD3, CD5, CD7, CD8, Tia-1, granzyme B and perforine, and negative for CD20, CD34, TDT and CD56. No infiltration of blood vessels or epidermis was evident. Specific T-cell lymphomas protocol (EURO-LB 02) was then initiated which resulted with rapid regression of all general and local symptoms. The treatment was completed according to schedule and the child is now, 24 months after the initiation of the treatment, in complete remission.

CD20 positive childhood B-non Hodgkin lymphoma (B-NHL): morphology, immunophenotype and a novel treatment approach: a single center experience.

Lymphomas represent the third most common group of cancers in childhood and adolescence, mature B non Hodgkin's lymphoma (B-NHL) accounting for up to 60% of newly diagnosed patients. The diagnosis of specific entities of B-NHL is based on well-defined morphologic analysis, immunophenotyping, cytogenetics and molecular genetics, which determine the optimal treatment strategy. In adult population a major turning point in treatment of B-NHL has been achieved since rituximab, in combination with CHOP has improved the survival rate up to 19%. Rituximab is a chimeric monoclonal antibody that targets CD20, a transmembrane calcium channel expressed on normal and malignant B-cells that mediates cytotoxic, apoptotic and anti-proliferative effects. The effect of rituximab in pediatric population is still not well enough investigated. Based on morphology and immunophenotype of malignant cells, seven children with B-NHL in our institution were eligible for treatment with modified B-NHL-Berlin-Frankfurt-Münster (BFM)-95-based protocol with rituximab administered on day -5. The complete remission was achieved in all seven patients. Six patients are still in complete remission at least 12 months after having finished chemotherapy and one patient relapsed two months after the last cycle and subsequently died. Major adverse effects observed during treatment were prolonged B-cell depletion and myelosuppression. Rituximab in combination with B-NHL-BFM-95 protocol was otherwise well tolerated and proved to be effective in children and adolescents with B-NHL. The number of our patients is too small and the follow-up of a larger group of patients will help in defining the role of rituximab in the treatment of childhood B-NHL.

Recombinant activated factor VII controls chemotherapy-related hemorrhage in patients with solid intra-abdominal tumors: a report of three pediatric cases.

Recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) is used predominantly for the treatment of bleeding in patients with hemophilia and inhibitors, and in patients with traumatic injury. There are also literature reports of its use in chemotherapy-related bleeding in leukemia patients and intra- or postoperative bleeding in patients with solid tumors. We describe three pediatric patients where rFVIIa was successfully used to manage bleeding following the failure of conventional hemostatic treatments during chemotherapy for intra-abdominal tumors (hepatoblastoma, rhabdomyosarcoma and non-classified malignant sarcoma). Recombinant FVIIa proved effective and maintained hemostasis in two of three cases, with no evidence for toxic or adverse events in any of the treated patients.

[Hematopoietic stem cell transplantation in children with primary immunodeficiencies]. / Transplantacija krvotvornih maticnih stanica u djece s primarnim imunodeficijencijama.

Hematopoietic stem cell transplantation is the optimal treatment for patients with primary immunodeficiencies. Best results are achieved with stem cells from a HLA-identical donor, but it is only possible in a small number of patients. Until recently, HLA-mismatched/haploidentical hematopoietic stem cell transplantation was reserved exclusively for patients with severe combined immunodeficiency (SCID). However, as there are many haploidentical donors, it has become the treatment of choice for many other severe primary immunodeficiencies. Apart from appropriate choice of the donor, treatment of infections and pre-transplantation patient conditioning have major impact on transplantation outcome in patients with primary immunodeficiencies.

[Biphenotypic and bilineal acute leukemias]. / Bifenotipske i bilinijske akutne leukemije.

Human acute leukemias (AL) are classified as myeloid or lymphoid according to cytomorphology and the expression of leukocyte differentiation antigens/CD-markers. However, in the minority of cases leukemic cells express markers of more than one lineage, which has led to the introduction of a new subgroup of acute leukemias termed mixed or biphenotypic acute leukemias (BAL). In an effort to distinguish between BAL and those AL with aberrant expression of markers of other lineage, the European Group for the Immunological Characterization of Acute Leukemias (EGIL) has proposed a scoring system in which CD-markers are assigned a score of 0.5, 1.0 or 2.0, depending on the specificity of a particular antigen for myeloid, B- and/or T-lymphoid lineage, respectively. The new WHO classification of hematologic tumors has adopted the EGIL criteria for BAL and introduced a new group of AL termed 'AL of ambiguous lineage'. In addition to BAL in which a single cell population expresses both myeloid and lymphoid differentiation markers, this new group of leukemias also comprises cases that present with two separate blast populations (acute bilineal leukemia, aBLL). In general, BAL accounts for less than 5% of all AL cases, whereas aBLL is a rare disease constituting 1%-2% of AL cases that contains B- or T-lymphoid along with myeloid blasts. Chromosome abnormalities are frequent in both entities with a relatively high incidence of Philadelphia chromosome and rearrangements involving 11q23, especially in cases with B- and myeloid involvement. Other biological features include CD34 expression and multi-drug resistance P-glycoprotein overexpression. The prognosis of BAL and aBLL is unfavorable, with poor prognostic factors being age, high WBC and the presence of Philadelphia chromosome. Unfortunately, optimal therapy is not known, although regimens designed for acute lymphoblastic leukemia may result in a better response rate. Collaborative studies are needed for better understanding of the biology of these entities and establishment of standard therapeutic protocols.

[Characteristics of autologous peripheral blood stem cells in pediatric patients]. / Osobitosti skupljanja autolognih krvotvornih maticnih stanica iz periferne krvi u pedijatrijskih bolesnika.

Autologous peripheral blood stem cells are increasingly used for transplantation instead of bone marrow, even in small children and adolescents. We analyzed 73 autologous leukaphereses performed in 25 children (36% males, 64% females). The median age was 15 years (range 3-18) and the median body weight 48 kg (range 16-67). The apheresis procedures were carried out with cell separator COBE Spectra. Each patient underwent a median of 2 collections (range 1-6). The median total nucleated cell yield was 11.86x108/kg (range 1.94-21.21), mononuclear cell yield was 6.01x10(8)/kg (range 0.97-12.73) and CD34+ cell yield was 3.5 x10(6)/kg (range 0.19-28.01). During 6 (8.22%) procedures the patients had experienced apheresis-related side effects. The citrate-induced reactions were most commonly observed. The reactions were mild and cessation of collection was required only in one case, because of catheter related complication. Our results show that leukapheresis in pediatric patients is a safe procedure, well tolerated and with a very low risk of serious adverse events.