Beate Winkler

Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

Immune function in children under chemotherapy for standard risk acute lymphoblastic leukaemia – a prospective study of 20 paediatric patients

Multidrug chemotherapy is a highly effective treatment for paediatric acute lymphoblastic leukaemia (ALL), but at the same time compromises immunity of patients. Immune function in a homogenous cohort of 20 children with standard‐ and intermediate‐risk ALL was analysed by immunophenotyping, intracellular cytokine staining, assessment of serum cytokine concentrations, T‐cell receptor (TCR) repertoire diversity and thymic function. B‐cells were most severely affected by chemotherapy, rapidly declined under induction and did not recover until the cessation of maintenance therapy. This recovery was paralleled by a relative increase in naive IgM+IgD+CD27− B‐cells, indicating de novo B‐cell generation as the major pathway for B‐cell reconstitution. T‐ and Natural Killer‐cells were less severely affected. Although numerically diminished by chemotherapy, they had partially recovered at the end of induction. Interestingly, CD4:CD8 ratio, distribution of naive versus memory T‐cells, cytokine production, TCR‐repertoire complexity and thymic function were all only marginally affected by chemotherapy. Patients receiving dexamethasone had significantly less IFNγ+ T‐cells than those receiving prednisone. Our data show that during chemotherapy in standard‐ and intermediate‐risk paediatric ALL patients the T‐cell system remains relatively well preserved. Future studies will show if this effect can be exploited for inclusion of immunotherapy in standard ALL treatment protocols.

Diffusion-weighted MRI for detection and differentiation of musculoskeletal tumorous and tumor-like lesions in pediatric patients.

BackgroundMRI is the diagnostic mainstay for detection and differentiation of musculoskeletal tumors. However, a projection regarding the biological dignity of lesions based on standard MRI sequences remains difficult and uncertain. This study was undertaken to analyse whether diffusion-weighted MRI (DWI) can distinguish between benign and malignant musculoskeletal tumorous and tumor-like lesions in pediatric patients.MethodsMR examinations of 44 consecutive pediatric patients (26 girls, mean age 11±6 years) including standard sequences and DWI (b=50/800 s/mm2) at 1.5 or 3 Tesla were retrospectively evaluated. The study group contained 10 patients with non-treated malignant tumors and 34 patients with benign lesions. Size, relative signal intensity and apparent diffusion coefficient (ADC, unit ×10−3 mm2/s) were determined in one lesion per patient.ResultsMean ADC was 0.78±0.45×10−3 mm2/s in patients with malignant tumors and 1.71±0.75 ×10−3 mm2/s in patients with benign lesions (P<0.001). Relative operating characteristics (ROC) analysis showed a sensitivity of 90% and a specificity of 91% for malignancy, based on an ADC cut-off value of ≤1.03. On logistic regression, mean ADC and lesion size accounted for 62% of variability in benign vs. malignant tumors. For malignant tumors, the signal intensity ratio was higher on DWI than on T1w post-contrast images (P<0.002). Two cases of local tumor recurrence were diagnosed by DWI only.ConclusionsDWI shows promising results for determination of biological dignity in musculoskeletal tumors. Mean ADC ≤1.03×10−3 mm2/s is a strong indicator of malignancy at the first diagnosis. The use of DWI for early diagnosis of tumor recurrence in comparison with standard MRI sequences should be evaluated in prospective studies.

Age-related changes in intracellular cytokine expression in healthy children

Cytokine production by human lymphocytes from healthy children (ages 0-18 years) was assessed using a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. To establish valid cytokine values for intracellular cytokine expression in healthy children in the different age groups, we measured 117 samples after 24 h in vitro stimulation with PMA, ionomycin and brefeldin followed by staining with intracellular anti-cytokine and surface antibodies. We found decreasing IL-2 expression, increasing IFN-gamma and TNF-alpha production and stable IL-4, Ki67 and TGFb levels with advancing age. The cytokines were mainly produced by memory T-cells. Apart from age, there was a differential expression in boys and girls: boys (< 6 years) produce significantly more IL-2 (p < 0,04), while girls > 12 years produce more IFNg than boys of the same age (p < 0.05). This systematic analysis of cytokine profiles during childhood allows a better understanding of immune maturation and will contribute significantly to the interpretation of cytokine data from children with pathological conditions.

Immunotherapy with the trifunctional anti‐CD20 x anti‐CD3 antibody FBTA05 (Lymphomun) in paediatric high‐risk patients with recurrent CD20‐positive B cell malignancies

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow‐up of ten patients diagnosed with relapsed or refractory mature B‐cell Non Hodgkin Lymphoma (B‐NHL), Burkitt leukaemia (B‐AL) or pre B‐acute lymphoblastic leukaemia (pre B‐ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti‐CD3 x anti‐CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo‐SCT, n = 6) to induce sustained long‐term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo‐SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow‐up. The other patients still maintain CR with a current overall survival of 874–1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.

Transient loss of consciousness in pediatric recipients of dimethylsulfoxide (DMSO)-cryopreserved peripheral blood stem cells independent of morphine co-medication.

Toxicity related to the infusion of dimethylsulfoxide-cryopreserved peripheral blood stem cells (DMSO-PBSC) manifests mostly as cardiovascular side effects. Neurotoxicity1 including transient global amnesia,2,3 seizures,4,5 and stroke6,7 has been reported as a rare complication primarily in adults. In children, data8 are sparse. In light of a recent report implicating morphine co-medication as a major contributing factor,8 we evaluated retrospectively our own data base, including all infusions of DMSO-PBSC applied in our pediatric center between January 1st 2002 and December 31st 2008. We report on 2 incidences of transient loss of consciousness following 131 infusions of DMSO-PBSC.

Impaired B-cell reconstitution in children after chemotherapy for standard or medium risk acute precursor B-lymphoblastic leukemia

Abstract Chemotherapy for childhood acute lymphoblastic leukemia (ALL) is a highly effective treatment, but at the same time causes significant suppression of the patients immunity. Immune reconstitution was studied in a homogeneous cohort of 48 children with standard or medium risk ALL treated according to the ALL-Berlin–Frankfurt–Münster (BFM) protocol. Whereas the T-cell compartment was only moderately affected and recovered to normal levels quickly after treatment cessation, B-cells were significantly reduced during and after therapy. In particular, the naive B-cell compartment declined. Even 5 years after the end of therapy, B-cell distribution was disturbed and patients showed an ongoing reconstitution. Thus, even standard regimens for chemotherapy cause severe B-cell depletion that resolves only gradually.

Fast MR Imaging of the Paediatric Abdomen with CAIPIRINHA-Accelerated T1w 3D FLASH and with High-Resolution T2w HASTE: A Study on Image Quality

The aim of this study was to explore the applicability of fast MR techniques to routine paediatric abdominopelvic MRI at 1.5 Tesla. “Controlled Aliasing in Parallel Imaging Results in Higher Acceleration-” (CAIPIRINHA-) accelerated contrast-enhanced-T1w 3D FLASH imaging was compared to standard T1w 2D FLASH imaging with breath-holding in 40 paediatric patients and to respiratory-triggered T1w TSE imaging in 10 sedated young children. In 20 nonsedated patients, we compared T2w TIRM to fat-saturated T2w HASTE imaging. Two observers performed an independent and blinded assessment of overall image quality. Acquisition time was reduced by the factor of 15 with CAIPIRINHA-accelerated T1w FLASH and by 7 with T2w HASTE. With CAIPIRINHA and with HASTE, there were significantly less motion artefacts in nonsedated patients. In sedated patients, respiratory-triggered T1w imaging in general showed better image quality. However, satisfactory image quality was achieved with CAIPIRINHA in two sedated patients where respiratory triggering failed. In summary, fast scanning with CAIPIRINHA and HASTE presents a reliable high quality alternative to standard sequences in paediatric abdominal MRI. Paediatric patients, in particular, benefit greatly from fast image acquisition with less breath-hold cycles or shorter sedation.

Hit the mark with diffusion-weighted imaging: metastases of rhabdomyosarcoma to the extraocular eye muscles

BackgroundRhabdomyosarcoma is the most frequent malignant intraorbital tumour in paediatric patients. Differentiation of tumour recurrence or metastases from post-therapeutic signal alteration can be challenging, using standard MR imaging techniques. Diffusion-weighted MRI (DWI) is increasingly considered a helpful supplementary imaging tool for differentiation of orbital masses.Case presentationWe report on a 15-year-old female adolescent of Caucasian ethnicity who developed isolated bilateral thickening of extraocular eye muscles about two years after successful multimodal treatment of orbital alveolar rhabdomyosarcoma. Intramuscular restricted diffusion was the first diagnostic indicator suggestive of metastatic disease to the eye muscles. DWI subsequently showed signal changes consistent with tumour progression, complete remission under chemoradiotherapy and tumour recurrence.ConclusionsRestricted diffusivity is a strong early indicator of malignancy in orbital tumours. DWI can be the key to correct diagnosis in unusual tumour manifestations and can provide additional diagnostic information beyond standard MRI and PET/CT. Diffusion-weighted MRI is useful for monitoring therapy response and for detecting tumour recurrence.

TGFβ and IL10 have an impact on risk group and prognosis in childhood ALL

Cytokines and their genes have been described to have an influence on incidence and prognosis in malignant, infectious and autoimmune disease. We previously described the impact of cytokine production on prognosis in paediatric standard‐risk acute lymphoblastic leukaemia (ALL).