Doctor of Medicine, Ruprecht Karl Universitat Heidelberg (2010)
Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.
View details for DOI 10.1038/s41591-020-0821-8
View details for PubMedID 32341579
Insufficient reactivity against cells with low antigen density has emerged as an important cause of CAR resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and the CAR construct in axicabtagene-ciloleucel (CD19-CD28z) outperforms that in tisagenlecleucel (CD19-4-1BBz) against antigen low tumors. Enhancing signal strength by including additional ITAMs in the CAR enables recognition of low antigen density cells, while ITAM deletions blunt signal and increase the antigen density threshold. Further, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBz CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunological synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBz-CARs with enhanced capacity to recognize antigen low targets while retaining a superior capacity for persistence.
View details for DOI 10.1158/2159-8290.CD-19-0945
View details for PubMedID 32193224
CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivoare currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used "off label" as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p<0.01) and significantly increased F4/80+CD80+ M1 macrophages on histopathology (p<0.01). CD47 mAb-treated F4/80+ macrophages demonstrated significantly augmented phagocytosis of ferumoxytol nanoparticles (p<0.01). Thus, we conclude that ferumoxytol-MRI can detect TAM response to CD47 mAb in mouse models of osteosarcoma. The ferumoxytol-MRI imaging test could be immediately applied to monitor CD47 mAb therapies in clinical trials.
View details for PubMedID 30674867
The long-term survival of osteosarcoma patients with metastatic or recurrent disease remains dismal and new therapeutic options are urgently needed. The purpose of our study was to compare the efficacy of CD47 mAb plus doxorubicin combination therapy in mouse models of osteosarcoma with CD47 mAb and doxorubicin monotherapy. Forty-eight NOD scid gamma (NSG) mice with intratibial MNNG/HOS tumors received CD47 mAb, doxorubicin, combination therapy or control IgG treatment. Twenty-four mice (n=6 per group) underwent pre- and post-treatment MRI scans with the macrophage marker ferumoxytol, bioluminescence imaging and histological analysis. Tumor ferumoxytol enhancement, tumor flux and tumor TAM density were compared between different groups using a one-way ANOVA. Twenty-four additional NSG mice underwent survival analyses with Kaplan-Meier curves and a log-rank (Mantel-Cox) test. Intratibial osteosarcomas demonstrated significantly stronger ferumoxytol enhancement and significantly increased TAM quantities after CD47 mAb plus doxorubicin combination therapy compared to CD47 mAb (p = 0.02) and doxorubicin monotherapy (p = 0.001). Tumor bearing mice treated with CD47 mAb plus doxorubicin combination therapy demonstrated significantly reduced tumor size and prolonged survival compared to control groups that received CD47 mAb (p = 0.03), doxorubicin monotherapy (p = 0.01) and control IgG (p = 0.001). In conclusion CD47 mAb plus doxorubicin therapy demonstrates an additive therapeutic effect in mouse models of osteosarcomas, which can be monitored with an immediately clinically applicable MRI technique. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/1878-0261.12556
View details for PubMedID 31376208
Purpose To develop a positron emission tomography (PET)/magnetic resonance (MR) imaging protocol for evaluation of the brain, heart, and joints of pediatric cancer survivors for chemotherapy-induced injuries in one session. Materials and Methods Three teams of experts in neuroimaging, cardiac imaging, and bone imaging were tasked to develop a 20-30-minute PET/MR imaging protocol for detection of chemotherapy-induced tissue injuries of the brain, heart, and bone. In an institutional review board-approved, HIPAA-compliant, prospective study from April to July 2016, 10 pediatric cancer survivors who completed chemotherapy underwent imaging of the brain, heart, and bone with a 3-T PET/MR imager. Cumulative chemotherapy doses and clinical symptoms were correlated with the severity of MR imaging abnormalities by using linear regression analyses. MR imaging measures of brain perfusion and metabolism were compared among eight patients who were treated with methotrexate and eight untreated age-matched control subjects by using Wilcoxon rank-sum tests. Results Combined brain, heart, and bone examinations were completed within 90 minutes. Eight of 10 cancer survivors had abnormal findings on brain, heart, and bone images, including six patients with and two patients without clinical symptoms. Cumulative chemotherapy doses correlated significantly with MR imaging measures of left ventricular ejection fraction and end-systolic volume, but not with the severity of brain or bone abnormalities. Methotrexate-treated cancer survivors had significantly lower cerebral blood flow and metabolic activity in key brain areas compared with control subjects. Conclusion The feasibility of a single examination for assessment of chemotherapy-induced injuries of the brain, heart, and joints was shown. Earlier detection of tissue injuries may enable initiation of timely interventions and help to preserve long-term health of pediatric cancer survivors. (©) RSNA, 2017 Online supplemental material is available for this article.
View details for PubMedID 28777701
Considering the improved outcome, a better understanding of the late effects in Wilms tumor survivors (WT-S) is needed. This study was aimed at evaluating renal function and determining the prevalence of clinical and subclinical renal dysfunction in a cohort of WT-S using a multimodal diagnostic approach.Thirty-seven WT-S were included in this prospective cross-sectional single center study. To evaluate renal function, glomerular filtration rate (GFR) and urinary protein excretion were assessed. Additionally, kidney sonomorphology and blood pressure were analyzed.All examined WT-S (mean age 28.7 years, mean follow-up 24.8 years) had been treated with a combination of surgery and chemotherapy; 59.5% had received adjuvant radiotherapy. Impaired glomerular renal function was detected in a considerable proportion of WT-S, with age-adjusted cystatin-based GFR estimation below age norm in 55.9%. A lower cystatin-based estimated GFR (eGFR) correlated with longer follow-up time and higher irradiation dose. In 5 patients (13.5%) albuminuria was identified. Analysis of sonomorphology detected compensatory contralateral renal hypertrophy in 83.3% of WT-S. Chronic kidney disease (CKD) ? stage II was present in 55.9% of WT-S. Blood pressure measurements revealed arterial hypertension in 15 (40.5%) WT-S (newly diagnosed n=10). In 24.3% both CKD ? stage II and arterial hypertension were determined.Even though WT-S are believed to carry a low risk for end-stage renal disease, in this study, a remarkable number of WT-S presented with previously unidentified subclinical signs of renal function impairment and secondary morbidity. Therefore, it is important to continue regular follow-up, especially after transition into adulthood.
View details for DOI 10.1007/s00467-017-3673-9
View details for PubMedID 28451896
Although neuro- and nephroblastoma are common solid tumors in children, the simultaneous occurrence is very rare and is often associated with syndromes. Here, we present a unique case of synchronous occurrence of neuro- and nephroblastoma in an infant with no signs of congenital anomalies or a syndrome. We performed genetic testing for possible candidate genes as underlying mutation using the next-generation sequencing (NGS) approach to target 94 genes and 284 single-nucleotide polymorphisms (SNPs) involved in cancer. We uncovered a novel heterozygous germline missense mutation p.F58L (c.172T?C) in the anaplastic lymphoma kinase (ALK) gene and one novel heterozygous rearrangement Q418Hfs(*)11 (c.1254_1264delins TTACTTAGTACAAGAACTG) in the Fanconi anemia gene FANCD2 leading to a truncated protein. Besides, several SNPs associated with the occurrence of neuroblastoma and/or nephroblastoma or multiple primary tumors were identified. The next-generation sequencing approach might in the future be useful not only in understanding tumor etiology but also in recognizing new genetic markers and targets for future personalized therapy.
View details for DOI 10.1080/08880018.2016.1184362
View details for PubMedID 27285993
High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches.We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO, PTCH1 and SUFU, three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC50 of 1.3 ?M.In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.
View details for DOI 10.18632/oncotarget.13092
View details for PubMedID 27825128
View details for PubMedCentralID PMC5347776
Liver regeneration after partial hepatectomy (PH) occurs in conditions of reduced oxygen supply. HIF prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) are oxygen sensors involved in adaptive response to hypoxia. Specific functions of these PHD enzymes in liver regeneration have, however, remained enigmatic. Here, we investigated the significance of PHD1 in liver regeneration following hepatectomy.Liver regeneration was studied in PHD1-deficient (PHD1(-/-)) and wild type (WT) mice subjected to 80% hepatectomy. For in vitro analyses, hepatocytes were isolated from PHD1(-/-) and WT livers. Cell cycle progression was studied via FACS-based analysis of nuclear DNA profile. Transcription factor binding assays, qRT-PCR, and immunoblotting were applied to study the relevance of PHD1 downstream effectors during liver regeneration.Liver regeneration was significantly enhanced in PHD1(-/-) mice compared to WT littermates. This effect was due to enhanced proliferation rather than to hypertrophy of liver cells. Cell cycle progression was significantly enhanced, and transcriptional activity of the cell cycle regulator c-Myc was increased in PHD1-deficient hepatocytes. These changes coincided with increased expression of cyclin D2, a cell cycle-promoting c-Myc target, and decreased expression of the cell cycle-delaying c-Myc target p21.Loss of PHD1 enhances liver regeneration by boosting hepatocyte proliferation in a c-Myc-dependent fashion. PHD1 might, therefore, represent a potential target to facilitate liver regeneration after surgical resection.
View details for DOI 10.1007/s00423-012-0998-5
View details for PubMedID 22961008
Hypoxia and HIFs (HIF-1? and HIF-2?) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3(-/-)) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1(-/-)) or PHD2 (PHD2(+/-)). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1? protein stabilization and increased NF-?B activity, and interference with the expression of HIF-1? or the canonical NF-?B pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1?- and NF-?B-mediated enhancement of the innate immune response.
View details for DOI 10.4049/jimmunol.1103471
View details for PubMedID 22786772