[Febrile neutropenia in pediatric and adolescent cancer patients]. / Fieber während der Granulozytopenie bei krebskranken Kindern und Jugendlichen.

Febrile neutropenia is the most common potential emergency situation in children and adolescents with cancer. The host response of these patients is severely compromised by treatment-induced immunosuppression resulting in a lack of important defence mechanisms, so that bacterial infections and in certain risk groups also fungal infections can be life threatening. As the clinical course of these infectious complications may be rapid and fatal, early antibiotic treatment can save lives. This article aims to raise awareness to this emergency situation and gives an overview of the management of pediatric cancer patients with febrile neutropenia.

Percentiles of Lymphocyte Subsets in Preterm Infants According to Gestational Age Compared to Children and Adolescents.

Preterm newborns show an increased susceptibility to infections, conceivably related to their immature immune system. To gain further knowledge about the immune development in early preterm infants, we aimed to establish references for lymphocyte subsets and compare the maturation process during hospitalization to healthy term-born children and adolescents. For this purpose, peripheral blood samples (n = 153) were collected from 40 preterm infants, gestational age (GA) 26-30 week between 2nd and 6th day of life, and were monitored in intervals of every 2 or rather 4 weeks until the end of hospitalization. Furthermore, we analysed single sample controls of 10 term neonates. We compared these data with results of a study in healthy children and adolescent (n = 176). Flow cytometry of immune cell subsets was performed as single-platform analysis using 10-colour flow cytometry. Based on preterm's age, our percentile model allows readout of absolute cell count for lymphocytes, B cells, T cells, NK cells, T8 and T4 cells. The median (minimum) value of T-, B- and NK cells after birth was 2800 (600), 790 (120) and 140 (20) cells/µl, respectively. Major differences were found in absolute cell numbers of B cells, and in the frequency of regulatory T cells, most pronounced in the earliest preterm infants (GA 26). Compared to healthy children and adolescents, preterm infants reached lymphocyte counts in between the 5th and 50th percentile when discharging the hospital. This prospective observational study provides reference percentiles for lymphocytes subsets of preterm infants. These data are conducive to interpret immunological capability of preterm infants with possible immune disorders appropriate.

Infectious complications in children with acute myeloid leukemia: decreased mortality in multicenter trial AML-BFM 2004.

Infections are an important cause for morbidity and mortality in pediatric acute myeloid leukemia (AML). We therefore characterized infectious complications in children treated according to the trial AML-BFM 2004. Patients with Down syndrome were excluded from the analysis. Data were gathered from the medical records in the hospital where the patients were treated. A total of 405 patients (203 girls; median age 8.4 years) experienced 1326 infections. Fever without identifiable source occurred in 56.1% of the patients and clinically and microbiologically documented infections in 17.5% and 32.4% of the patients, respectively. In all, 240 Gram-positive (112 viridans group streptococci) and 90 Gram-negative isolates were recovered from the bloodstream. Invasive fungal infection was diagnosed in 3% of the patients. Three children each died of Gram-negative bacteremia and invasive aspergillosis, respectively. As compared with the results of AML-BFM 93 with lower dose intensity, infection-related morbidity was slightly higher in AML-BFM 2004 (3.3. versus 2.8 infections per patient), whereas infection-related mortality significantly decreased (1.5% versus 5.4%; P=0.003). Specific anti-infective recommendations included in the treatment protocol, regular training courses for pediatric hematologists and increasing experience may be the reason for reduced infection-related mortality in children with AML. Further studies are needed to decrease infection-related morbidity.

Micafungin twice weekly as antifungal prophylaxis in paediatric patients at high risk for invasive fungal disease.

BACKGROUND: Current guidelines recommend antifungal prophylaxis for children at high risk for invasive fungal disease (IFD), but the use of polyenes and triazoles may not be feasible in some patients due to toxicities and drug-drug interactions. Micafungin is well tolerated, with intravenous daily dosing being the current standard. Recent reports indicate safety and efficacy of intermittent dosing of micafungin. METHODS: We analysed safety, efficacy and micafungin serum concentrations of children at high risk for IFD receiving prophylactic micafungin between 3 and 4 mg/kg twice weekly. All children were intolerant or had contraindications to polyenes and triazoles. RESULTS: A total of 21 children (median age = 9 years) at high risk for IFD were included in the analysis. No significant clinical adverse event occurred, and end of treatment values of parameters of renal and hepatic function in serum were not different from baseline. Proven or probable breakthrough IFD did not occur in any of the patients. In 9 out of 11 patients in whom plasma micafungin concentrations were assessed, the first trough concentration exceeded 150 ng/mL, a concentration proposed to be effective for prophylaxis. CONCLUSIONS: Our data indicate that micafungin administered twice weekly at a dosage of 3-4 mg/kg of bodyweight could be a convenient, safe and efficient alternative for antifungal prophylaxis in children at high risk for IFD.

Response of children with stage IV soft tissue sarcoma to topotecan and carboplatin: a phase II window trial of the cooperative soft tissue sarcoma group.

To investigate antitumor activity and toxicity associated with combined topotecan and carboplatin treatment in children and adolescents with metastasized, untreated soft tissue sarcoma (STS).Patients (n=34) less than 21 years old and untreated, stage IV STS. Patients were treated with topotecan (1 mg/m²/d for 4 days) and carboplatin (150 mg/m²/d for 4 days) (TC course) during week 1 and 4 of a chemotherapy window trial, which was followed by chemotherapy and local therapy from week 6 on. We evaluated the side effects, toxicity and tumor response (using RECIST criteria) 6 weeks after starting the 2 TC chemotherapy courses.The objective response rate (ORR) was 38% (n=13 patients with a partial response (PR)), and a stable disease (SD) was reached in 11 cases. No patient showed a complete response (CR) of all metastatic lesions, although 1 patient showed a CR of the target lesion. 2 patients died of progress of disease (PD). Toxicity was mainly hematological (grade III/IV toxicity 79%), and nonhematological toxicities mainly included infection, fever, nausea,and vomiting. Regarding adverse events, 4 probable and 8 possible events related to study medication occurred among the 66 courses of TC.In conclusion, TC was potent against high-risk STS, but results and toxicity data were not superior to former published monotherapeutic topotecan therapies.

Impact of minimal residual disease detection prior to autologous stem cell transplantation for post-transplant outcome in high risk neuroblastoma.

Autologous stem cell transplantation (SCT) has become standard therapy in high risk stage IV neuroblastoma (NB) patients. Residual NB cells in the bone marrow (BM) shortly before SCT may shape the overall survival.Thus, we sought to thoroughly investigate minimal residual disease (MRD) in BM prior to SCT using conventional and real time RT-PCR for tyrosine hydroxylase (TH) as well as morphology. To avoid influence of residual NB cells in the stem cell harvest, 17 patients transplanted with MRD negative grafts (n=11 CD34-selected and n=6 unmanipulated) are included in the final analysis, only.35% of these patients are alive with a median follow up of 8.6 years. In the BM of 9/17 patients residual NB cells could be detected < 40 d before SCT. These patients had a significant lower overall survival compared to patients without BM involvement based on combined RT-PCR and morphology results (11% vs. 62%, p=0.026) or using RT-PCR, only (p=0.01). In contrast morphology on its own did not lead to a significant discrimination between both groups.Our results obtained in a small cohort of stage IV NB patients suggest that MRD diagnostic in the BM shortly before SCT might be a valuable predictive tool for these patients but requires conformation in a multicenter study.

Detection of neuroblastoma cells during clinical follow up: advanced flow cytometry and rt-PCR for tyrosine hydroxylase using both conventional and real-time PCR.

PURPOSE: Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. METHODS: We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). RESULTS: Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. CONCLUSION: TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.

Liposomal amphotericin B twice weekly as antifungal prophylaxis in paediatric haematological malignancy patients.

Data on antifungal prophylaxis in paediatric cancer patients at high risk for invasive fungal disease (IFD) are scant. Intermittent administration of liposomal amphotericin B (LAMB) has been shown to be safe and effective in adult patients with haematological malignancies. We prospectively evaluated the safety and efficacy of prophylactic LAMB at a dosage of 2.5 mg/kg twice weekly in children at high risk for IFD. Efficacy was compared with that in a historical control group of patients with similar demographic characteristics not receiving LAMB prophylaxis. A total of 46 high-risk patients (24 boys; mean age, 7.7 years) with 187 episodes of antifungal prophylaxis were analysed. The median duration of neutropenia (<500/µL) was 10 days. LAMB was discontinued in four patients because of acute allergic reactions. Median values for creatinine and liver enzymes at end of treatment did not differ significantly from those at baseline. Hypokalaemia (<3.0 mmol/L) occurred with 13.5% of the prophylactic episodes, but was usually mild and always reversible. No proven/probable IFD occurred in patients receiving LAMB prophylaxis. In comparison, five proven and two probable IFDs were observed in 45 historical controls not receiving LAMB prophylaxis (p 0.01). LAMB prophylaxis had no impact on the use of empirical antifungal therapy. Systemic antifungal prophylaxis with LAMB 2.5 mg/kg twice weekly is feasible and safe, and seems to be an effective approach for antifungal prophylaxis in high-risk paediatric cancer patients.

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3- NK cells.

Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient's body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 +/- 0.5 CD3(+) cells/microl with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3(+) T cell number of 221 x 10(3) (0.09%, CD34 selected grafts, n = 187), 900 x 10(3) (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 x 10(3) (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations.

Immune recovery in children undergoing allogeneic stem cell transplantation: absolute CD8+ CD3+ count reconstitution is associated with survival.

To evaluate the correlation between kinetics of immune reconstitution and survival, we prospectively evaluated lymphocyte subsets in 32 paediatric patients undergoing allogeneic stem cell transplantation (SCT) for haematological malignancies. Four-colour flow cytometric analysis was performed at short intervals with a median follow-up of 4 years post SCT. A total of 50% of patients reached age-matched 5th percentile of natural killer, cytotoxic T, B and helper T cells 4, 9, 20 and 28 weeks after SCT, respectively, which increased to more than 80% within 1 year after SCT. Transplantation of peripheral blood stem cells (PBSC) seemed to elicit the fastest reconstitution of CD3+, CD4+ CD3+, CD8+ CD3+ and naïve T cells compared to bone marrow (BM) or CD34-selected PBSC, which did not differ. Most importantly, we observed a significantly higher number of survivors among patients whose CD8+ CD3+ absolute counts rose above the 5th percentile of age-matched normal levels during the first year post SCT compared to patients who never reached these levels (19/25 vs 0/7, P<0.001). This was still present in both subgroups, BM- and CD34-selected grafts (P=0.03, 0.02). These results from a small patient sample underline the importance of particular lymphocyte subsets for the outcome of children undergoing SCT. A larger study with detailed subset analysis is underway.

Diagnostic approaches for immunocompromised paediatric patients with pulmonary infiltrates.

Pulmonary infiltrates in immunocompromised children often pose problems in terms of deciding on further diagnostic and therapeutic procedures, but few studies have evaluated the value of non-invasive and invasive diagnostic methods in paediatric populations. Both galactomannan ELISA and PCR protocols appear to be less useful in children than in adults. Invasive procedures, such as bronchoalveolar lavage or lung biopsy, can yield a pathohistological diagnosis and/or the isolation of a pathogen. Prospective studies in paediatric patients are needed urgently to assess the value of different diagnostic procedures and to define an effective and safe diagnostic strategy for the individual child.

Hourly monitoring of circulating CD34+ cells to optimize timing of autologous apheresis in pediatric patients.

In order to increase the CD34+ cell yield in children undergoing autologous stem cell transplantation, the optimum time of apheresis after G-CSF administration has still to be found. We prospectively studied the mobilization of CD34+ cells and white blood cells in the peripheral blood (PB) of 20 pediatric patients before leukapheresis. The monitoring schedule covered 12 h, with blood samples taken before and at 2, 4, 5, 6, 7, 8, 10 and 12 h after G-CSF administration when 10 CD34+ cells/mul were reached. CD34+ cells were measured by flow cytometric analysis both in the single- and dual-platform setting. Two different patterns of mobilization (POM) emerged: 12 patients showed an increase in CD34+ cells in PB during the first 4 h after G-CSF (POM I), while eight patients had an initial decrease of CD34+ cells. However, all patients together showed a significant increase of CD34+ cells about 10 h after G-CSF administration. Further studies with more patients, using an enhanced monitoring schedule will be required to refine the results.