The Successful Sequential Use of Plerixafor and Eltrombopag For Hematopoietic Cell Transplantation in a Child With High-risk Neuroblastoma.

High-dose chemotherapy with autologous hematopoietic stem cell transplantation improves event-free survival in patients with high-risk neuroblastoma. However, in heavily pretreated patients, poor marrow function can be an obstacle in the successful proceeding of therapy. Priming with plerixafor plus filgrastim is an option for effective mobilization and collection of stem cells. In addition, thrombopoietin agonist eltrombopag can improve the outcome of posttransplantation thrombocytopenia and poor graft function in the posttransplant setting. We describe a case of a child with high-risk neuroblastoma, for whom plerixafor and eltrombopag were used as an effective and safe supportive therapy.

Complex profile of multiple hepatobiliary and gastrointestinal complications after hematopoietic stem cell transplantation in a child with Nijmegen breakage syndrome.

Patients with Nijmegen breakage syndrome (NBS) can develop life-threatening immunodeficiency, which should be treated with hematopoietic stem cell transplantation (HSCT). We report the case of a 14-year-old girl with NBS who due to an increasing number of severe complications was referred for HSCT from a matched unrelated donor. After reduced-intensity conditioning and transplantation of peripheral blood hematopoietic cells, during the early post-transplant period (days 0-30), the girl suffered from severe mucositis, fever episodes, mild acute renal injury and facial vasculitis. All these complications were managed successfully. During the intermediate post-transplant period (days 30-100) a number of hepatic and gastrointestinal complications occurred, including cholecystitis, cholelithiasis with choledocholithiasis, pancreatitis as well as acute bleeding from the lower gastrointestinal tract caused by rectal and recto-sigmoid junction ulcers. All the obstacles were obviously attributable both to the primary congenital disease, its complications, and transplantation itself. We overcame these complications and treated the patient with the best possible and safe methods. The multidisciplinary approach based on combined surgical, endoscopic and conservative management of multiple post-transplant complications was successful for the patient.

Impact of CMV and EBV on Immune Recovery After Allogeneic Hematopoietic Cell Transplantation in Children.

BACKGROUND/AIM: Immune recovery is a key factor in the management of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study analyzed the factors contributing to immune reconstitution after allo-HSCT. PATIENTS AND METHODS: Overall, 65 children with malignant or non-malignant diseases were included in multivariate analyses. RESULTS: The following factors contributed to a faster immune recovery: peripheral blood as a stem cell source and reactivation of CMV infection for CD3+ and CD4+ lymphocyte subpopulations; reactivation of CMV infection for CD8+ subset; donor EBV-IgG+ and no EBV reactivation for CD19 lymphocytes; recipient age below 10 years and peripheral blood as a stem cell source for NK cells. For CD2 and CD4/CD8 ratio no factor was significant in multivariate analysis. CONCLUSION: Patients receiving a graft from an EBV-IgG-positive donor and not having early EBV post-transplant viremia show faster recovery of the B-cells, while patients with early CMV-DNA-emia have a better re-establishment of T-cell subsets.

X-linked TLR7 gene polymorphisms are associated with diverse immunological conditions but not with discoid lupus erythematosus in Polish patients.

INTRODUCTION: Toll-like receptor 7 (TLR7) is an important molecule involved in the development of autoimmunity and the response to different pathogens. Several polymorphisms within the TLR7 gene were previously found to be associated with systemic lupus erythematosus (SLE). However, none of those studies investigated the TLR7 promoter flanking variants rs1634318 and rs1616583. TLR7 gene diversity has not been analyzed with respect to discoid lupus erythematosus (DLE) development, while its role in the human immunological response to fungal infection is not fully known. AIM: To clarify the potential involvement of two novel single-nucleotide polymorphisms (SNPs) located in the TLR7 gene (rs1634318 and rs1616583) in a variety of immune-related conditions, we studied the variability of these loci in patients from a Polish population with SLE and DLE, as well as in immunocompromised patients who were affected by invasive aspergillosis (IA) and those who were not affected. MATERIAL AND METHODS: Real-time polymerase chain reaction was used to genotype SNPs. Statistically significant differences between case and control groups for both allele and genotype frequencies were assessed using the χ2 test with Yates' correction or two-tailed Fisher's exact test. The results were Bonferroni-corrected for multiple comparisons and odds ratios were calculated. RESULTS: Two polymorphisms located in TLR7 might be associated with the development of SLE but not DLE within the Polish population. Moreover, variation of the two investigated SNPs was found to be associated with IA in immunocompromised Polish patients. CONCLUSIONS: In Polish patients, TLR7 promoter flanking gene polymorphisms might be associated with IA and SLE but not DLE.

Massively parallel targeted resequencing reveals novel genetic variants associated with aspergillosis in paediatric patients with haematological malignancies.

This study aimed to find novel genetic variants of susceptibility to aspaergillosis in paediatric patients with haematological malignancies. Complete sequences of fifteen genes of human innate immunity (CCL2, CCR2, CD209, CLEC6A, CLEC7A and ten TLR genes) were studied in 40 patients diagnosed with haematological disorders (20 unaffected and 20 affected by aspergillosis). All samples were sequenced with MiSeq (Illumina) and 454 (Roche Diagnostics) technologies. Statistical significance of the differences between studied groups was determined using the two-tailed Fisher's exact test. Sixty variants of potential importance were identified, the vast majority of which are located in non-coding parts of the targeted genes. At the threshold of p < 0.000005, one intergenic (TLR2 rs4585282) and one intronic variant (CLEC6A rs12099687) were found significant between the case and control groups for genotype and allele frequencies, respectively. Rs12099687 in CLEC6A was predicted to constitute an alternative isoform or cryptic splice site, which potentially changes activity of the Dectin-2 protein. Overall, we assume that the two strongest associations reported in this study are expected to be reproducible even in the absence of other evidence, while another twelve associations may be strong enough to justify additional research in larger cohorts.

RNA isolation from bloodstains collected on FTA cards - application in clinical and forensic genetics.

Aim of the study: In recent years, RNA analysis has been increasingly used in clinical and forensic genetics. Nevertheless, a major limitation of RNA-based applications is very low RNA stability in biological material, due to the RNAse activity. This highlights the need for improving the methods of RNA collection and storage. Technological approaches such as FTA Classic Cards (Whatman) could provide a solution for the problem of RNA degradation. However, different methods of RNA isolation from FTA cards could have diverse effects on RNA quantity and quality. The purpose of this research was to analyze the utility of three different methods of RNA isolation from peripheral blood collected on FTA Classic Cards (Whatman). The study also aimed at assessing RNA stability in bloodstains deposited on FTA cards. Material and methods: The study was performed on peripheral bloodstains collected from 59 individuals on FTA Classic Cards (Whatman). RNA was isolated with High Pure RNA Isolation Kit (Roche Diagnostics), Universal RNA/miRNA Purification (EURx) and TRIzol Reagent (Life Technologies). RNA was subjected to quantitative analysis followed by reverse transcription and Real - Time PCR reaction. Results: The study has shown that FTA Classic Cards (Whatman) are useful tools for storing bloodstains at room temperature for RNA analysis. Moreover, the method of RNA extraction employing TRIzol Reagent (Life Technologies) provides the highest efficiency and reproducibility for samples stored for no more than 2 years. Conclusions: The FTA cards are suitable for collecting and storing bloodstains for RNA analysis in clinical and forensic genetics.

Palifermin in children undergoing autologous stem cell transplantation: a matched-pair analysis.

BACKGROUND: Keratinocyte growth factor (palifermin) is used for prevention of mucositis in adults following autologous and allogeneic hematopoietic stem cell transplantation (HSCT). It is known that palifermin decreases length of initial hospital stay, mean number of days of total parenteral nutrition (TPN) and the use of opioids for pain control in oral mucositis in adults. There are limited data evaluating palifermin use in children following autologous HSCT. AIM: The objective of the present study was the analysis of efficacy and safety of palifermin in children undergoing auto-HSCT. PATIENTS AND METHODS: This matched-pair analysis study included 62 pediatric patients undergoing first auto-HSCT receiving palifermin on a compassionate-use basis (study group, n=31) or not (control group, n=31). RESULTS: Palifermin decreased the incidence of severe (grade 3-4 WHO) oral mucositis (p=0.041), length of hospitalization (p=0.047) and contributed to the shorter duration of oral mucositis (p=0.035) and lower incidence of clinically or microbiologically documented infections (p=0.038). There were no differences between groups in opioid use, neutrophil and platelet recovery, TPN use and gastrointestinal hemorrhage. CONCLUSION: Palifermin decreases the incidence and severity of oral mucositis in children undergoing autologous HSCT.

Intrathecal therapy with rituximab in central nervous system involvement of post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder (PTLD) caused by Epstein-Barr virus (EBV) is a severe complication in high-risk allogeneic hematopoietic stem cell transplant (HSCT) recipients. Central nervous system (CNS) involvement of PTLD is a very rare event in patients with HSCT. As no established standard therapy in CNS-EBV-PTLD is available, the aim of this study was analysis of the safety and efficacy of intrathecal rituximab therapy in a group of eight children and adolescents with CNS-EBV-PTLD. Seven patients responded to therapy: all clinical symptoms and EBV-DNA viral load resolved after a median 2 (range: 1-7) doses of rituximab. However, some magnetic resonance imaging (MRI) changes in brain scan persisted in two patients. In all patients, except one, no adverse events of the therapy were observed. In conclusion, intrathecal rituximab administration seems to be an effective and safe method of treatment of CNS-EBV-PTLD in pediatric stem cell recipients. We recommend this treatment modality for further investigation.

Pneumatosis cystoides interstitialis: A complication of graft-versus-host disease. A report of two cases.

BACKGROUND: Pneumatosis cystoides intestinalis (PCI) is a rare disorder characterized by the presence of multiple gas collections in the subserosal or submucosal intestinal wall of the large or small intestine. We report two cases of PCI in the course of chronic graft-versus-host disease. CASE REPORT: A 5-year-old girl was treated for acute lymphoblastic leukemia. Twenty-four months after the hematopoietic stem cell transplantation, in the course of graft-versus-host disease, she developed subcutaneous emphysema of the right inguinal and pudendal region. PCI was diagnosed based on a CT examination. A 3-year-old boy was treated for juvenile myelomonocytic leukemia. Fourteen months after the hematopoietic stem cell transplantation he presented with an increased severity of intestinal symptoms, including intermittent bleeding from large intestine. PCI was diagnosed based on a CT exam and was confirmed by a colonoscopy. CONCLUSIONS: Pneumatosis cystoides interstitialis in the course of chronic graft-versus-host disease has a heterogeneous clinical presentation that does not correlate with results of imaging. CT is a method of choice to diagnose PCI. In patients with PCI, the presence of free air in the peritoneal cavity does not confirm an intestinal perforation.

[G-CSF - primed bone marrow as a stem-cell source for allogeneic bone marrow transplantation in children]. / Stymulowany szpik kostny jako zródlo allogenicznych komórek hematopoetycznych u dzieci.

BACKGROUND: Cell dose is one of the most important factors contributing to transplant success. Collection of G-CSF-stimulated haematopoietic stem cells from peripheral blood in adults belongs to standard procedures in transplantology. The data on collection of G-CSF-stimulated bone marrow, both in adults and children, are lacking. OBJECTIVE: Analysis of the possibility and safety of use of G-CSF-stimulated haematopoietic stem cells from bone marrow in paediatric allogeneic sibling donor setting. PATIENTS AND METHODS: The prospective study included 10 children aged 3-17 years (median 13 years). Donors (aged 5-30 years, median 13 years; including 7/10 aged <18 years) were given subcutaneous G-CSF as 5 03BC g/kg/24 h for 2-4 days before bone marrow collection. Follow-up lasted 0.3-2.1 years. RESULTS: In none of the donors any serious adverse effects related to G-CSF or stem cell collection were observed. Median number of transplanted mononuclear cells (MNC) and CD3 4 cells was 10 x 10;8/ /kg (range, 5-12.7 x 10;8/kg) and 1.7 x 10;6/kg (range, 0.5-4.7 x 10;6/kg), respectively. All patients had granulocyte and platelet recovery with median time 16 days (range, 11-23 days) i 16 days (range, 12- -39 days), respectively. Overall, 4/10 patients had acute graft-versus-host disease (GVHD) > or =2 degrees and 2/10 patients had chronic GVHD (1 localized and 1 systemic). Higher cell-dose was not related to higher risk of acute or chronic GVHD. One-year probability of event-free-survival was 51.4%. CONCLUSIONS: Collecting G-CSF primed bone marrow from paediatric donors is a safe and promising concept, and may help to obtain higher MNC and CD 34 cell dose from the donor. Benefits to recipient may occur related to faster engraftment after myeloablative transplantation without increasing the risk of GVHD. This concept requires further studies to evaluate this approach.

[Efficacy and toxicity of intravenous busulfan-based conditioning treatment before hAematopoietic stem cell transplantation in children: preliminary report]. / Ocena skutecznosci i bezpieczenstwa stosowania dozylnego busulfanu u dzieci przed przeszczepieniem komórek hematopoetycznych: doniesienie wstepne.

BACKGROUND: An intravenous form of busulfan has been developed to replace oral busulfan in conditioning treatment prior to haematopoietic stem cell transplantation (HSCT). AIM: Retrospective evaluation of the efficacy and safety of iv busulfan in paediatric patients. METHODS: A total number of 33 patients entered the study: 11 patients received intravenous Bu-based conditioning before HSCT (group A; 3 allo, 8 auto); 22 patients received oral Bu-based conditioning (group B; 5 allo, 17 auto). The median age was 5.3 (1.5-20) years. An intravenous dosing of busulfan based on body weight was used. Patients were conditioned with BuCy+/-Vp+/-ATG or BuMel protocols. Cyclosporin+/-methotrexate were used for GVHD prophylaxis during allo-HSCT. RESULTS: Patients in group A were younger (p=0.005), received comparable amount of MNC and CD34 cells. The median time of neutrophil engraftment was similar in both groups: 12 (10-19) vs 12.5 (10-25) days, while platelet recovery was shorter in group A: 11.5 (7-30) vs 14 (9-187) days (p=0.019). Severe stomatitis occurred in 5/11 patients in group A and in 19/22 patients in group B (p=0.033). Additional analgesics were required, respectively, by 5/11 and 17/22 patients (p=0.117). There were no significant differences in a/cGVHD, gastrointestinal reaction, hepatic toxicity, occurrence of infections and haemorrhagic cystitis between the two groups. With a median follow-up of 9 months in group A and 12 months in group B, 8 (72.7%) patients in group A and 15 (68,2%) patients in group B were alive. 9/10 deaths were related to disease relapse. With this short follow up, the probability of relapse and overall survival were comparable in both groups. CONCLUSIONS: The intravenous Bu-based conditioning treatment before HSCT for paediatric patients has a favorable toxicity profile. It has the potential to accelerate haematological reconstitution and to improve survival and engraftment in children undergoing HSCT. The i.v. administration is convenient, favouring its more widespread application. Further studies are needed.

[Skin biopsy in diagnosis of chronic graft-versus-host disease in patients after allogeneic haematopoietic stem cell transplantation: pathologist's point of view on quantitative scoring system]. / Biopsje skóry w diagnostyce choroby przeszczep-przeciwko-gospodarzowi u pacjentow po transplantacji allogenicznych komórek hematopoetycznych: spojrzenie patologa na klasyfikacje ilosciowa.

BACKGROUND: Pathology diagnosis of chronic graft-versus-host-disease (GVHD) after allogeneic haematopoietic stem cell transplantation (allo-HSCT) is an important issue in clinical follow-up, in spite of frequent difficulties in interpretation., related to dynamic changes occurring in the skin during the disease, as well as to sequelae of basic disease and immunosuppressive therapy. Recently presented Consensus NIH (National Health Institute, Bethesda, USA) of histopathologic (HP) analysis is still complex and intrinsically divergent, thus clinically difficult to implement. OBJECTIVE: Analysis of clinical value of histological evaluation results of skin biopsy in children after allo-HSCT and its correlation with clinical status. PATIENTS AND METHODS: Ten skin biopsies were taken from 7 patients (4 boys, 3 girls, age 3-15 years) after allo-HSCT (6 MFD, 1 MMUD) and analyzed after hematoxylin/eosine and immunohistochemical (CD3, CD45T, CD20) staining. Pathology analysis was based on commonly accepted criteria enabling simple and unambiguous interpretation. Results were compared with clinical data and indications for immunosuppressive therapy. RESULTS: It was found that reliable and coherent interpretation can be made when following parameters were taken into account: 1. in epithelium: the presence of apoptosis, archetypical changes and vacuolar degeneration in the basilar layer, presence of CD3/CD45 in the epidermis; 2. in the dermis: the extent of collagenization, presence of melanophages and lymphocyte infiltrations; 3. in the eccrine glands epithelium: eccrine glands atrophy and presence of lymphocytes. CONCLUSIONS: A new scoring system of skin biopsy analysis in patients with chronic GVHD based on the modified NIH Consensus was proposed. The preliminary clinical value of histological results was assessed. Skin biopsy evaluation based on limited qualitative and quantitative analysis of lymphocyte infiltrates together with studies on intensity of apoptosis, collagenization and archetypical changes is a valuable diagnostic method complementary to clinical records, enabling easier undertaking of therapeutic decisions.

[Cancer among adolescents 15-19 years of age. Own experience]. / Nowotwory u mlodziezy w wieku 15-19 lat. Badania wlasne.

BACKGROUND: Adolescents aged 15-19 years are variably included in analyses of childhood cancer. They should be considered separately because tumours that occur in adolescents differ from those in younger children. AIM: To describe the distribution of tumour types and treatment results in this group of patients treated in the Department of Pediatric Oncology at The Children's Memorial Health Institute. METHODS: Retrospective analysis of medical records of patients aged 15-19 years treated in our institution was performed. It included demographic data, tumour types and treatment results. RESULTS: Between 1998-2004, 207 pts, 110 boys and 97 girls aged 15-19 yrs (median--16.5 yrs) were treated. Distribution of tumours was as follows: CNS tumours--74 pts (35.7%), HD -18 pts (8.7%), NHL--13 pts (6.3%), bone tumours--31 pts (15%), STS--23 pts (11.1%), gonadal tumours--12 pts (5.8%), carcinomas--16 pts (7.7%), hepatomas--5 pts (2.4%), neuroblastoma--3pts (1.5%) and other 12 pts (5.8%). Out of 207 pts 130 are alive (62.8%). Seventy seven (37.2%) pts died--64 (83.1%) from disease, 9 (11.6%) from chemotherapy complications, 4 due to other reasons. 111 pts completed treatment and are disease free for 11 months to 7 yrs (median 3 yrs 11 mos) from diagnosis. Nineteen patients are still treated. Treatment results are as follows: CNS tumours--58.1%, HD - 88.8%, NHL--69.2%, bone tumours--51.6%, STS--65.2%, gonadal tumours--83.3%, carcinomas-- 56.25%. CONCLUSIONS: Spectrum of malignancies that occur in adolescents 15-19 years of age differs from younger children. Unlike younger patients epithelial carcinomas of adults are observed in this age group. Outcome of treatment is inferior to younger patients. Adolescents should be offered optimal treatment. This specific group should be studied in many aspects.