Curability and transferability of atopy with allogeneic hematopoietic cell transplantation.

Atopy is excessive production of IgE in response to allergens. We evaluated in patients undergoing allogeneic hematopoietic cell transplantation (HCT) the following hypotheses: (1) Atopy is "curable" in atopic patients receiving HCT from a nonatopic donor (D-R+), and (2) Atopy is transferable from atopic donors to nonatopic recipients (D+R-). Atopic patients with atopic donors (D+R+) and non-atopic patients with non-atopic donors (D-R-) served as controls. We measured levels of multiallergen-specific IgE (A-IgE, atopy defined as ≥0.35 kUA/L) in sera from 54 patients and their donors pre HCT and from the patients at ≥2 years post HCT. Only 7/12 (58%) D- R+ patients became nonatopic after HCT. Only 1/11 (9%) D+R- patients became atopic. Eleven of 13 (85%) D-R- patients remained nonatopic. Unexpectedly, 11/18 (61%) D+R+ patients became nonatopic. In conclusion, contrary to our hypothesis and previous reports, the "cure" of atopy may occur in only some D-R+ patients and the transfer of atopy may occur rarely. The "cure" may not be necessarily due to the exchange of atopic for nonatopic immune system, as the "cure" may also occur in D+R+ patients.

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Mutations in the Sterile alpha motif domain containing 9 (SAMD9) gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in SAMD9 (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel SAMD9 mutation was confirmed experimentally. Expression of mutant SAMD9 caused a significant decrease in proliferation and increase in cell death of the transfected cells. Conclusion: We describe a novel SAMD9 mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with SAMD9 mutations and multisystem involvement.

Paediatric Home Parenteral Nutrition in the Czech Republic and Its Development: Multicentre Retrospective Study 1995-2011.

BACKGROUND: Treatment quality and outcomes of paediatric home parenteral nutrition (HPN) program during its development in the Czech Republic. METHODS: A retrospective study of patients receiving HPN from May 1995 till June 2011. RESULTS: Sixty-six patients were treated in 8 centres. In 48 patients, long-term PN began in the first year of life and in 35 of them in the first month. Sixty children had gastrointestinal and 6 had non-gastrointestinal disease. In a majority of the patients, the Broviac catheter was used. Thirty-two (48.5%) patients were weaned from PN after 1-117 months, 21 (32.8%) continued on HPN after 7-183 months, and 13 (19.7%) patients died, all on PN. The mortality in patients with primary gastrointestinal disease was significantly lower than in patients with non-gastrointestinal disease. Thirty-one paediatric patients were receiving HPN for 14,480 catheter days in 2009-2010. Fourteen patients had 23 Catheter Related Blood Stream Infections (CRBSI) episodes. The incidence of CRBSI in 2009-2010 was 1.58/1,000 catheter days. CONCLUSION: Submitted data showed that even in the absence of expert centres, patient care may achieve results comparable to countries with well-developed HPN program. A majority of Czech HPN patients are at present treated in specialized centres, following the most desirable pattern of care.

Appearance of cytomegalovirus-specific T-cells predicts fast resolution of viremia post hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) specific T-cells are known to provide long-term control of CMV reactivation, which is a frequent complication of hematopoietic stem cell transplantation. We have studied 58 pediatric patients after hematopoietic stem cell transplantation who suffered from CMV reactivation to reveal which functional T cell subset is best correlating with successful reactivation resolution and which protects from reactivation episode. METHODS: Detection of 30 combinatorial subsets of four types of response to ex vivo CMV stimulation (IFNγ secretion, IL-2 secretion, CD40L upregulation and degranulation) that were detectable on either CD8+ or CD4+ T cells through flow cytometry intracellular cytokine staining was used. RESULTS: We found that the presence of CD8+ dual positive (IFNγ+ and IL-2+) cells is the most accurate functional parameter that can predict fast resolution of CMV reactivation. Next, we show that the presence of CD8+ dual positive (IFNγ+ and IL-2+) and CD8+ IFNγ+ cells provides a protective effect (a hazard risk of 0.28 (confidence interval 0.18 - 0.43) and 0.45 (CI 0.27 - 0.75), respectively) and the presence of corticotherapy increases the risk of reactivation (HR 2.47 (CI 1.82-3.36)). Thus, a patient without corticotherapy and with both of the critical T cell subsets present has a cumulative 19.6 times lower risk of developing CMV reactivation than a patient on corticotherapy and without CD8+ dual positive (IFNγ+ and IL-2+) or CD8+ IFNγ+ cells. CONCLUSIONS: We have established parameters of CMV specific functional response ex vivo that can be used in assisting clinical management of patients with CMV reactivation. © 2015 International Clinical Cytometry Society.

The TREC/KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome.

UNLABELLED: DiGeorge syndrome (DGS) presents with a wide spectrum of thymic pathologies. Nationwide neonatal screening programs of lymphocyte production using T-cell recombination excision circles (TREC) have repeatedly identified patients with DGS. We tested what proportion of DGS patients could be identified at birth by combined TREC and kappa-deleting element recombination circle (KREC) screening. Furthermore, we followed TREC/KREC levels in peripheral blood (PB) to monitor postnatal changes in lymphocyte production. METHODS: TREC/KREC copies were assessed by quantitative PCR (qPCR) and were related to the albumin control gene in dry blood spots (DBSs) from control (n = 56), severe immunodeficiency syndrome (SCID, n = 10) and DGS (n = 13) newborns. PB was evaluated in DGS children (n = 32), in diagnostic samples from SCID babies (n = 5) and in 91 controls. RESULTS: All but one DGS patient had TREC levels in the normal range at birth, albeit quantitative TREC values were significantly lower in the DGS cohort. One patient had slightly reduced KREC at birth. Postnatal DGS samples revealed reduced TREC numbers in 5 of 32 (16%) patients, whereas KREC copy numbers were similar to controls. Both TREC and KREC levels showed a more pronounced decrease with age in DGS patients than in controls (p < 0.0001 for both in a linear model). DGS patients had higher percentages of NK cells at the expense of T cells (p < 0.0001). The patients with reduced TREC levels had repeated infections in infancy and developed allergy and/or autoimmunity, but they were not strikingly different from other patients. In 12 DGS patients with paired DBS and blood samples, the TREC/KREC levels were mostly stable or increased and showed similar kinetics in respective patients. CONCLUSIONS: The combined TREC/KREC approach with correction via control gene identified 1 of 13 (8%) of DiGeorge syndrome patients at birth in our cohort. The majority of patients had TREC/KREC levels in the normal range.

The importance of therapeutic drug monitoring (TDM) for parenteral busulfan dosing in conditioning regimen for hematopoietic stem cell transplantation (HSCT) in children.

BACKGROUND: Series of observations indicate PK/PD variability challenging the accuracy of the body-weight based busulfan (Bu) dosing schedule for (HSCT) conditioning therapy. The purpose of this communication is to describe the frequency of dose changes in initially body-weight-based fixed IV Bu dose and to emphasize the importance of TDM. MATERIAL AND METHODS: Sixty-two children (ages 2 months-18 years) were treated with IV busulfan doses based on body weight for myeloablation. TDM utilizing a limited sample strategy (trough concentration immediately before the 5th dose, followed by samples immediately after the end of the 2-h infusion peak, 4 h, and 6 h from initiation of the infusion) was performed in 46 of 62 subjects. Busulfan concentrations were determined by high-performance liquid chromatography (HPLC). AUC was calculated according to the trapezoidal rule. RESULTS: We observed trough levels of 25-1244 µg/L, peak levels of 849-4586 µg/L, and AUC of 2225-12818 µg/L·h following body weight-based high-dose busulfan. The doses were changed in 54% of cases. AUC in 5 of 9 patients with VOD were within target, in 3 patients AUS was higher, and in 1 patient AUC was lower. One of the 2 patients with neurotoxicity had higher AUC. Engraftment was 100%, but relapse occurred in 25% of cases. CONCLUSIONS: Our results demonstrate that even with IV busulfan, intra-individual PK/PD variability is challenging. Although AUC does not necessarily correspond with outcomes (due to the role of other factors the fact that doses were changed in 54% of cases underlines the importance of TDM.

[Guidelines for antifungal prophylaxis, diagnosis and therapy in pediatric hematology and oncology - a review of literature and expert recommendations].

OBJECTIVES: Problems with importing non-registered medicines for treating rare life-threatening infectious diseases led to establishment of the Emergency Anti-Infective Drug Reserve (EAIDR) for the Czech Republic. METHODS: Thirteen anti-infective drugs are included in the project: antisera against rabies virus, varicella-zoster virus, and botulinum toxin; antituberculosis drugs (intravenous rifampicin and isoniazid; capreomycin, cycloserine, and clofazimine); antiparasitics (intravenous quinine, primaquine, meglumine antimoniate, and praziquantel); and pentamidine. These drugs are imported according to the Czech drug legislation (specific drug availability programs). Realization: The project, approved by the Czech Ministry of Health in September 2013, was started in January 2014. The anti-infective drugs sufficient for 2-4 patients are permanently available in the Toxicological Information Center (TIC) in Prague. The medicines can be applied in any hospital throughout the Czech Republic within several hours. CONCLUSIONS: All but three drugs are available at present; the remaining ones will be imported after new batches of these drugs are released.

[Invasive mucormycosis in pediatric hematology patients--single-center experience from 2005-2010]. / Invazivní mukormykóza u detských hematoonkologických pacientu: zhusenosti 2005-2010.

BACKGROUND: Mucormycosis is an invasive fungal disease severely complicating treatment of patients with hematologic diseases. Effective therapy is represented by the combination of surgery and amphotericin B administration and early initiation of the therapy is necessary for favorable outcome. The first clinical symptoms are usually non-specific and this can lead to late therapy onset. The objective of this retrospective work was to determine the frequency, risk factors and outcome of invasive mucormycosis in pediatric hematology patients. MATERIAL AND METHODS: The study cohort comprised 399 patients diagnosed with hematologic diseases in the Department of Pediatric Hematology and Oncology (DPHO), University Hospital Motol, Prague between 2005 and 2010. Risk factors for the development of mucormycosis, clinical symptoms and radiology and laboratory results were retrospectively evaluated. So were the therapy used and outcomes. The findings were analyzed using Fisher's exact test. RESULTS: During the selected period, mucormycosis was detected in 8 patients diagnosed with hematologic disease. The incidence of mucormycosis was 1.75 %. These conditions accounted for 20.6 % of all mycoses. In five patients, it was found as isolated infection; three cases were associated with other mycoses (one with candidiasis, two with aspergillosis). The most frequent underlying disease was acute leukemia; the most common risk factor was severe prolonged neutropenia (median duration 21.5 days). Three of eight patients survived mucormycosis, a mortality rate of 62.5 %. The effective therapy was amphotericin B administration in three patients (p = 0.02); in two of them, it was combined with radical surgery. CONCLUSION: In the cohort, the proportion of mucormycosis cases was surprisingly high when compared with other fungal diseases. Continuous surveillance of mucormycosis in the DPHO is needed. There was no significant influence of the combination of radical surgery and amphotericin B administration as compared to administration of amphotericin B alone. Nevertheless, according to the published data, we consider this approach as an optimal strategy for the management of mucormycosis at the present time.

HHV-6 DNA throughout the tissues of two stem cell transplant patients with chromosomally integrated HHV-6 and fatal CMV pneumonitis.

Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.

Cytomegalovirus encephalitis/retinitis in allogeneic haematopoietic stem cell transplant recipient treated successfully with combination of cidofovir and foscarnet.

We report an 18-yr-old female patient with repeated CMV reactivations after HSCT treated by several pre-emptive courses of virostatic therapy. Seven months after HSCT, she developed CMV encephalitis/retinitis. Initial therapy with GCV and hyperimmune globulin failed, and later on GCV-resistant strain was detected. Continual increase of CMV DNA in peripheral blood led us to combined therapy with CDV and FCV, which was successful and free of severe renal toxicity. To our best knowledge, this is the first reported case of successful CMV treatment with a combination of CDV and FCV.

Allogeneic stem cell transplantation in children with leukemia using human leukocyte antigen-mismatched unrelated donors.

Allogeneic HSCT is a curative treatment, when chemotherapy fails, for certain malignant diseases. In Europe, only 15% of the indicated children have an HLA-matched sibling available; in 65-70% of others, HLA allele-matched (9-10/10) UDs can be identified. For the rest, it is necessary to identify other alternative donors (HLA-mismatched family or unrelated cord blood). We present our data of HSCT using HLA partially allele-mismatched (7-8/10) UDs in 24 children with leukemia. Uniform GvHD prophylaxis was used (rATG, CsA and MTX). Acute GvHD grade II was diagnosed in 70.8% of the patients and grade III-IV in 12.5%. Overall incidence of chronic GvHD was 38.7% (extensive in 30%). The probability of EFS was 60.3% (95% CI 35.5-78.1) and OS was 74.9 (95% CI 49.1-88.9). No difference in survival between PBSC and BM recipients was observed. TRM at day + 100 was 4%, and overall was 12.5%. We conclude that used combination of drugs for GvHD prophylaxis is efficient even for patients transplanted with grafts from a HLA-mismatched UDs. It enables stable engraftment, good control of GvHD, full reconstitution of immunity, and is not connected with unacceptable transplant-related mortality.

Unrelated partially matched lymphocyte infusions in a patient with complete DiGeorge/CHARGE syndrome.

We present an infant with cDGS overlapping with CHARGE syndrome, who suffered from T-cell deficiency treated with screened healthy DLI from an unrelated donor (8/10 match). The first dose of DLI (1.1 x 10(6) CD3+/kg) was administered at the age of six months, the second one (0.9 x 10(6) CD3+/kg) 36 days later. No conditioning was employed, GvHD prophylaxis consisting of CsA was used only during the second infusion. Since day+10 after the first DLI, split chimerism showing T-cell engraftment has been documented. Proliferative response to PHA was detected on day+145. The treatment was complicated by severe acute GvHD (grade II-III) after the first DLI and prolonged chronic liver cholestatic GvHD developing after the second DLI. Vigorous EBV proliferation four wk after the second DLI was accompanied by peripheral expansion of CD8+ donor cells. The patient, 26-months old, is clinically well and has slowly started to gain his developmental milestones. We believe that infusions of small doses of DLI from an unrelated donor represent a potentially helpful therapeutic option in patients with cDGS/CHARGE phenotype.