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.

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.

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.

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.

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.

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.

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.

Lower TGFß serum levels and higher frequency of IFNγ-producing T cells during early immune reconstitution in surviving children after allogeneic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (SCT) is increasingly used as a salvage therapy for patients with high-risk malignancies as well as life-threatening non-malignant diseases. However, only limited data about the association between outcome and functional parameters of recovering lymphocytes are available so far. PROCEDURES: In this prospective study of 19 pediatric SCT recipients, we serially evaluated immune parameters quantitatively and qualitatively before and throughout allogeneic SCT. These data were analyzed with respect to survival. RESULTS: Age, gender, GvHD, and type of graft were not different between surviving and non-surviving patients. Notably, in our cohort there was no case of transplant-related or infectious mortality. However, with the exception of two patients with advanced MDS, all patients not in complete remission (CR) relapsed in addition to three patients in higher CR (n = 7). All seven patients relapsing after allogeneic SCT later succumbed to their disease recurrence. Uni- and multivariate analysis showed that relapsing patients had higher TGFß serum levels as well as lower percentages of IFNγ-producing T cells before and early after transplantation. Furthermore, relapsing patients had a further decline in their thymic function between day 60 and 120 whereas non-relapsing patients already showed increasing TREC values during this time interval. CONCLUSIONS: Collectively, patients who later relapse show a different pattern of immune reconstitution before and at early time points post-transplantation.

Immune function in newly diagnosed children with malignancy.

BACKGROUND: Tumor genesis of many pediatric malignancies remains unclear. Data of immune function are lacking at diagnosis. We prospectively analyzed 109 pediatric patients with malignancy at diagnosis. METHODS: Lymphocyte subpopulations were characterized by FACS, TREC-assay, and Immunoscope, cytokines by FACS and ELISA. OBSERVATIONS: We detected higher values of CD4(+) T cells and consecutively shifted CD4(+)/CD8(+) ratio in all patients compared with the control group. In patients with lymphoma, interleukin-2 was upregulated in all subpopulations. CONCLUSIONS: On the basis of these findings an altered immune function could be found in children with different malignancies at diagnosis. Further investigations are necessary to identify tumor-related immune deficiency for novel therapeutic approaches.

TH1 predominance is associated with improved survival in pediatric medulloblastoma patients.

Medulloblastoma, a primitive neuro-ectodermal tumor that arises in the posterior fossa, is the most common malignant brain tumor occurring in childhood. Even though 60-70% of children with medulloblastoma will be cured with intensive multimodal therapy, including surgery, radiotherapy, and chemotherapy, a significant proportion of surviving patients may suffer from long-term treatment-related sequelae. Therapeutic success is limited especially in younger children by radiotherapy-induced neurocognitive longterm deficits. In order to avoid or delay craniospinal radiotherapy, high-dose chemotherapy followed by autologous stem cell transplantation (HSCT) has become an established treatment modality. Data on the host immunologic environment in medulloblastoma patients are rare, notably data on cytokine expression and immune reconstitution in patients with medulloblastoma undergoing HSCT are lacking. In this present study, we therefore decided to prospectively assess immune function following 24 consecutive autologous HSCT in 17 children with medulloblastoma treated according to the German-Austrian-Swiss HIT-2000-protocol. TH1 predominance was found to be the most important factor for probability of survival. Already before HSCT, survivors showed higher IFNγ levels in sera as well as higher numbers of IFNγ-positive T-cells. After transplantation, this effect was even more pronounced. Patients with higher numbers of IFNγ- and TNFα-positive T-cells had a more favorable outcome at all analyzed time points. In addition, patients in complete remission (CR) before transplantation, known to have a better prognosis a priori, showed higher expression of IFNγ in T-cells. Taken together, this is the first report to demonstrate that high expression of IFNγ and TNFα in T-cells of medulloblastoma patients in the early post-transplant period correlates with a better prognosis. Our data point toward a potentially important influence of TH1-cytokine expression before and after transplantation on the survival of pediatric medulloblastoma patients.

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 IFNgamma(+) 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.