To evaluate prognostic factors in pediatric patients with gonadal germ cell tumors (GCT).

Retinoblastoma and other eye tumors in childhood are rare diseases. Many eye tumors are the first signs of a genetic tumor predisposition syndrome and the affected children carry a higher risk of developing other cancers later in life. Clinical and genetic data of all children with eye tumors diagnosed between 2013-2018 in Germany and Austria were collected in a multicenter prospective observational study. In five years, 300 children were recruited into the study: 287 with retinoblastoma, 7 uveal melanoma, 3 ciliary body medulloepithelioma, 2 retinal astrocytoma, 1 meningioma of the optic nerve extending into the eye. Heritable retinoblastoma was diagnosed in 44% of children with retinoblastoma. One child with meningioma of the optic nerve extending into the eye was diagnosed with neurofibromatosis 2. No pathogenic constitutional variant in DICER1 was detected in a child with medulloepithelioma while two children did not receive genetic analysis. Because of the known association with tumor predisposition syndromes, genetic counseling should be offered to all children with eye tumors. Children with a genetic predisposition to cancer should receive a tailored surveillance including detailed history, physical examinations and, if indicated, imaging to screen for other cancer. Early detection of cancers may reduce mortality.

Risk classification plays a crucial role in clinical management and therapy decisions in children with neuroblastoma. Risk assessment is currently based on patient criteria and molecular factors in single tumor biopsies at diagnosis. Growing evidence of extensive neuroblastoma intratumor heterogeneity drives the need for novel diagnostics to assess molecular profiles more comprehensively in spatial resolution to better predict risk for tumor progression and therapy resistance. We present a pilot study investigating the feasibility and potential of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to identify spatial peptide heterogeneity in neuroblastoma tissues of divergent current risk classification: high versus low/intermediate risk. Univariate (receiver operating characteristic analysis) and multivariate (segmentation, principal component analysis) statistical strategies identified spatially discriminative risk-associated MALDI-based peptide signatures. The AHNAK nucleoprotein and collapsin response mediator protein 1 (CRMP1) were identified as proteins associated with these peptide signatures, and their differential expression in the neuroblastomas of divergent risk was immunohistochemically validated. This proof-of-concept study demonstrates that MALDI-MSI combined with univariate and multivariate analysis strategies can identify spatially discriminative risk-associated peptide signatures in neuroblastoma tissues. These results suggest a promising new analytical strategy improving risk classification and providing new biological insights into neuroblastoma intratumor heterogeneity.

Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Only half of patients with relapsed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) currently survive with standard treatment protocols. Predicting individual patient responses to defined drugs prior to application would help therapy stratification and could improve survival. With the purpose to aid personalized targeted treatment approaches, we developed a human-zebrafish xenograft (ALL-ZeFiX) assay to predict drug response in a patient in 5 days. Leukemia blast cells were pericardially engrafted into transiently immunosuppressed Danio rerio embryos, and engrafted embryos treated for the test case, venetoclax, before single-cell dissolution for quantitative whole blast cell analysis. Bone marrow blasts from patients with newly diagnosed or relapsed BCP-ALL were successfully expanded in 60% of transplants in immunosuppressed zebrafish embryos. The response of BCP-ALL cell lines to venetoclax in ALL-ZeFiX assays mirrored responses in 2D cultures. Venetoclax produced varied responses in patient-derived BCP-ALL grafts, including two results mirroring treatment responses in two refractory BCP-ALL patients treated with venetoclax. Here we demonstrate proof-of-concept for our 5-day ALL-ZeFiX assay with primary patient blasts and the test case, venetoclax, which after expanded testing for further targeted drugs could support personalized treatment decisions within the clinical time window for decision-making.

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.