Classification of Brain Tumors by Nanopore Sequencing of Cell-Free DNA from Cerebrospinal Fluid.

BACKGROUND: Molecular brain tumor diagnosis is usually dependent on tissue biopsies or resections. This can pose several risks associated with anesthesia or neurosurgery, especially for lesions in the brain stem or other difficult-to-reach anatomical sites. Apart from initial diagnosis, tumor progression, recurrence, or the acquisition of novel genetic alterations can only be proven by re-biopsies. METHODS: We employed Nanopore sequencing on cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) and analyzed copy number variations (CNV) and global DNA methylation using a random forest classifier. We sequenced 129 samples with sufficient DNA. These samples came from 99 patients and encompassed 22 entities. Results were compared to clinical diagnosis and molecular analysis of tumor tissue, if available. RESULTS: 110/129 samples were technically successful, and 50 of these contained detectable circulating tumor DNA (ctDNA) by CNV or methylation profiling. ctDNA was detected in samples from patients with progressive disease but also from patients without known residual disease. CNV plots showed diagnostic and prognostic alterations, such as C19MC amplifications in embryonal tumors with multilayered rosettes or Chr.1q gains and Chr.6q losses in posterior fossa group A ependymoma, respectively. Most CNV profiles mirrored the profiles of the respective tumor tissue. DNA methylation allowed exact classification of the tumor in 22/110 cases and led to incorrect classification in 2/110 cases. Only 5/50 samples with detected ctDNA contained tumor cells detectable through microscopy. CONCLUSIONS: Our results suggest that Nanopore sequencing data of cfDNA from CSF samples may be a promising approach for initial brain tumor diagnostics and an important tool for disease monitoring.

Impact of COVID-19 Related Restrictions on Infections in Children with Cancer or after Hematopoietic SCT.

BACKGROUND: Infections are a major concern for immunocompromised children. We investigated whether non-pharmaceutical interventions (NPIs) implemented in the general population during the coronavirus disease 2019 (COVID-19) pandemic in Germany had an impact on frequency, type and severity of infections in these patients. PATIENTS AND METHODS: We analyzed all admissions to the clinic of pediatric hematology, oncology and stem cell transplantation (SCT) with (suspected) infection or fever of unknown origin (FUO) from 2018 to 2021. RESULTS: We compared a 27-month period before NPIs (Pre-COVID: 01/2018-03/2020; 1041 cases) with a 12-month period with underlying NPIs (COVID: 04/2020-03/2021; 420 cases). During the COVID period the number of in-patient stays with FUO or infections decreased (38,6 cases/month vs. 35,0 cases/month), the median duration of hospital stays was longer (8 d (CI95: 7-8 d) vs. 9 d (CI95: 8-10 d) P=0,02)), the mean number of antibiotics per case increased (2,1 (CI95: 2,0-2,2) vs. 2,5 (CI95: 2,3-2,7); P=0,003)) and a substantial reduction of viral respiratory and gastrointestinal infections per case was seen (0,24 vs. 0,13; P<0,001). Notably, there was no detection of respiratory syncytial virus, influenza and norovirus, between May 2020 and March 2021. Based on need of intensive care measures and further parameters we conclude that severe (bacterial) infections were not significantly reduced by NPIs. CONCLUSIONS: Introduction of NPIs in the general population during the COVID-pandemic substantially reduced viral respiratory and gastrointestinal infections in immunocompromised patients, while severe (bacterial) infections were not prevented.

Long-term survival of an adolescent glioblastoma patient under treatment with vinblastine and valproic acid illustrates importance of methylation profiling.

Glioblastoma (GBM) is an exceptionally aggressive brain tumor with a dismal prognosis, demanding fast and precise classification as a base for patient-specific treatment strategies. Here, we report on an adolescent patient with a histologically bona fide GBM that shows a molecular methylation profile suggesting a low-grade glioma-like subgroup. Despite an early relapse, intolerance of temozolomide, and change of treatment strategy to vinblastine and valproic acid (VPA), the patient is now in good clinical condition after more than 5 years since initial diagnosis. This case stresses the merit of methylation array data for clinical prognosis and treatment planning.

Successful Stem Cell Apheresis Using Spectra Optia in a 6 kg Child With Atypical Teratoid/Rhabdoid Tumor.

Peripheral blood stem cell apheresis has become a routine procedure for the collection of peripheral blood stem cells to enable high-dose chemotherapy followed by autologous stem cell transplantation in high-risk pediatric malignancies. However, the procedure remains challenging in very low-weight infants due to high extracorporeal blood volume and citrate toxicity. Our case report demonstrates in detail a successful and complication-free large-volume leukapheresis in a very small infant weighing 6 kg using a Spectra Optia apheresis system after placing a femoral double-lumen Shaldon catheter. Anticoagulation was achieved by citrate dextrose solution without the use of heparin. The total amount of blood being processed during the procedure equaled almost 4 times the total blood volume of the patient. The final apheresis product contained 14.0×10 CD34 cells/kg body weight. The infant was diagnosed with an atypical teratoid/rhabdoid tumor of the thalamus and third ventricle at the age of 3 months and had a history of epileptic seizures.

Relapse of a group 4 medulloblastoma after 18 years as proven by histology and DNA methylation profiling.

BACKGROUND: Recent studies on medulloblastomas (MB) suggest that a large fraction of tumors appearing as late recurrence turn out to be secondary malignancies, e.g., malignant gliomas, after thorough molecular investigation. RESULTS: Here, we report of a patient with a group 4 MB that developed a distant recurrence after more than 18 years. The recurrent tumor was confirmed by histology and genome-wide DNA methylation profiling. CONCLUSION: Our case not only illustrates the potential of very late recurrences after seemingly cured group 4 MB, but also illustrates that detailed molecular analyses are indispensable in patients with a history of a previous malignancy.

Comparison of Immune Reconstitution After Allogeneic Versus Autologous Stem Cell Transplantation in 182 Pediatric Recipients.

Hematopoietic stem cell transplantation (HSCT) is a life-saving procedure for children with a variety of malignant and nonmalignant conditions. However, even if immune reconstitution after HSCT has been studied extensively, until now, data on the comparison of immune reconstitution after autologous versus allogeneic HSCT are scarce, but might provide important clinical implications. We examined immune reconstitution (T cells, B cells, and NK cells) at defined timepoints in 147 children who received 182 HSCTs. Differences in the time course of immune reconstitution were analyzed in autologous versus allogeneic HSCT. We identified a quicker immune reconstitution in the T-cell compartment, especially in the CD4 and naive subset after autologous HSCT, whereas recipients of allogeneic transplants showed a higher TCRgd proportion. B-cell reconstitution showed a delayed immune reconstitution after allogeneic HSCT in the first 2 years after HSCT. However, a reconstitution of all lymphocyte subsets after HSCT could be achieved in all patients. Children undergoing an HSCT show a different pattern of immune reconstitution in the allogeneic and autologous setting. This might influence the outcome and should affect the clinical handling of infectious prophylaxis and revaccinations.

Subgroup-specific immune and stromal microenvironment in medulloblastoma.

Knowledge on immune and stromal cells in medulloblastoma microenvironment is still limited as previous work was frequently restricted by low sample size and the lack of molecular subgroup information. We characterized 10 microenvironment cell populations as well as PD-L1 from gene expression in 1422 brain tumors and 763 medulloblastomas. All in all, medulloblastomas showed low expression of immune markers. Still, there were substantial differences with a clustering of medulloblastoma subgroups according to their microenvironment profile. Specifically, SHH medulloblastomas displayed strong signatures of fibroblasts, T cells and macrophages, while markers of cytotoxic lymphocytes were enriched in Group 4 tumors. PD-L1 gene expression appeared to be relatively high in single SHH and WNT cases but was undetectable by immunohistochemistry. In addition, two diverse immuno-stromal patterns were identified, indicating distinct types of local tumor immunosuppression, which were primarily controlled by either macrophage and regulatory T cell-mediated mechanisms or immunosuppressive cytokines and checkpoints, respectively. None of the immune cell signatures had an independent prognostic value in the present dataset after multiple testing correction. These results suggest a mild, but subgroup-specific infiltration of immune cells in medulloblastoma.

Lineage switch under blinatumomab treatment of relapsed common acute lymphoblastic leukemia without MLL rearrangement.

Blinatumomab is a bispecific T-cell engaging αCD19 antibody used in refractory or relapsed B-cell precursor acute lymphoblastic leukemia (ALL). Recently, lineage switch to a myeloid phenotype has been described following CD19 targeting treatment in three pediatric patients with mixed lineage leukemia (MLL) rearranged ALL. We report the case of a female who received blinatumomab for a first relapse of ALL without MLL alterations. She suffered from a second relapse early after hematopoietic stem cell transplantation and was treated with blinatumomab again. During this treatment, the leukemia lost CD19 expression as well as nearly all other B-cell markers, while still harboring the initial minimal residual disease marker, and switched to a myeloid phenotype.

The minimum required level of donor chimerism in hereditary hemophagocytic lymphohistiocytosis.

Reduced-intensity conditioning has improved survival after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) at the cost of more frequent mixed chimerism. The minimum level of donor chimerism (DC) required to prevent HLH reactivation in humans remains to be determined. In a multicenter retrospective study, 103 patients transplanted for hereditary HLH (2000-2013) and DC permanently or transiently <75% (overall, CD3(+), CD56(+)) were analyzed regarding DC, specific immunologic function, occurrence of systemic reactivations (≥5/8 HLH criteria), partial systemic flares (<5 criteria and HLH-directed treatment), isolated central nervous system reactivations, and management. Recurrence was reported in 18 patients (systemic reactivation n = 11, partial flare n = 3, isolated central nervous system reactivation n = 4). Ten events occurred during profound immune suppression before day 180 (median DC, 10%; range, 1-100%; CD3(+) if available, otherwise overall DC), which renders a differentiation between secondary post-HSCT HLH and HLH related to the genetic defect difficult. Eight events occurred between 0.5 and 6.7 years post-HSCT (median DC, 13%; range, 0-30%). In 5 patients, overall and lineage-specific DC were ≤10% for >6 months (median, 5.1; range, 1.1-10 years) without reactivation. A second HSCT was performed in 18 patients (median, DC 4%; range, 0-19%). Death from reactivation occurred in 4 patients (22% of recurrences). Six patients died of transplant complications following a second HSCT (33% of second HSCT). We conclude that a DC >20%-30% is protective against late reactivation. Lower levels do not, however, inescapably result in recurrences. The decision for or against second HSCT must be based on a thorough risk assessment.

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.

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.

Formula-feeding is associated with shift towards Th1 cytokines.

PURPOSE: Breast-feeding (BF) versus formula-feeding (FF) may be a factor for the development and differentiation of T-cell subsets and cytokine production in infancy and childhood. We therefore investigated T-cell subpopulations and their cytokine production by flow cytometry as well as cytokine levels in serum samples in breast-fed versus formula-fed infants and children. METHODS: Heparinised blood was taken from 191 healthy infants and children. Peripheral blood mononuclear cells were stimulated with phorbol-mystriate-acetate and ionomycin in the presence of brefeldin. T-cell subsets and cytokines were determined by flow cytometry. Furthermore, serum concentrations of IFNγ and IL4 were measured using ELISA. An IFNγ/IL4 ratio was calculated to estimate the Th1/Th2 balance. RESULTS: Children who were formula-fed show higher numbers of memory T and T helper cells. After stimulation, the number of IFNγ-positive memory T-cells was increased up to the age of 6 years. Breast-fed infants show higher percentages of IL4-positive T helper cells. At ELISA determination, formula-fed children showed higher IFNγ levels than breast-fed children, while IL4 levels did not differ. The IFNγ/IL4 ratio (FACS and ELISA) was elevated in formula-fed infants and children. CONCLUSION: This systematic analysis of cytokine profiles during childhood in dependency of BF allows a better understanding of immune maturation and demonstrates the influence of early feeding on immune function throughout childhood, even after cessation of BF. FF induces a shift towards Th1 cytokines in children. This may have an influence on the development of autoimmune disease in later life.

Thrombosis as a complication of central venous access in pediatric patients with malignancies: a 5-year single-center experience.

BACKGROUND: Reliable central venous access (CVC) is essential for hematology-oncology patients since frequent puncture of peripheral veins-e.g., for chemotherapy, antibiotic administration, repeated blood sampling, and monitoring-can cause unacceptable pain and psychological trauma, as well as severe side effects in cases of extravasation of chemotherapy drugs. However, CVC lines still carry major risk factors, including thrombosis, infection (e.g., entry site, tunnel, and luminal infections), and catheter dislocation, leakage, or breakage. METHODS: Here we performed a retrospective database analysis to determine the incidence of CVC-associated thrombosis in a single-center cohort of 448 pediatric oncologic patients, and to analyze whether any subgroup of patients was at increased risk and thus might benefit from prophylactic anticoagulation. RESULTS: Of the 448 patients, 269 consecutive patients received a CVC, and 55 of these 269 patients (20%) also had a thrombosis. Of these 55 patients, 43 had at least one CVC-associated thrombosis (total number of CVC-associated thrombosis: n = 52). Among all patients, the median duration of CVC exposure was 464 days. Regarding exposure time, no significant difference was found between patients with and without CVC-associated thrombosis. Subclavia catheters and advanced tumor stages seem to be the main risk factors for the development of CVC-associated thrombosis, whereas pharmacologic prophylaxis did not seem to have a relevant impact on the rate of thrombosis. CONCLUSIONS: We conclude that pediatric surgeons and oncologists should pay close attention to ensuring optimal and accurate CVC placement, as this appears the most effective tool to minimize CVC-associated complications.

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

BACKGROUND: Rhabdomyosarcoma 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 PRESENTATION: We 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. CONCLUSIONS: Restricted 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.

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

Chemotherapy for childhood acute lymphoblastic leukemia (ALL) is a highly effective treatment, but at the same time causes significant suppression of the patient's 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.