Background Imagining ways to prevent or treat glioblastoma (GBM) has been hindered by a lack of understanding of its pathogenesis. Although overexpression of platelet derived growth factor with two A-chains (PDGF-AA) may be an early event, critical details of the core biology of GBM are lacking. For example, existing PDGF-driven models replicate its microscopic appearance, but not its genomic architecture. Here we report a model that overcomes this barrier to authenticity. Methods Using a method developed to establish neural stem cell cultures, we investigated the effects of PDGF-AA on subventricular zone (SVZ) cells, one of the putative cells of origin of GBM. We microdissected SVZ tissue from p53-null and wild-type adult mice, cultured cells in media supplemented with PDGF-AA, and assessed cell viability, proliferation, genome stability, and tumorigenicity. Results Counterintuitive to its canonical role as a growth factor, we observed abrupt and massive cell death in PDGF-AA: wild-type cells did not survive, whereas a small fraction of null cells evaded apoptosis. Surviving null cells displayed attenuated proliferation accompanied by whole chromosome gains and losses. After approximately 100 days in PDGF-AA, cells suddenly proliferated rapidly, acquired growth factor independence, and became tumorigenic in immune-competent mice. Transformed cells had an oligodendrocyte precursor-like lineage marker profile, were resistant to platelet derived growth factor receptor alpha inhibition, and harbored highly abnormal karyotypes similar to human GBM. Conclusion This model associates genome instability in neural progenitor cells with chronic exposure to PDGF-AA and is the first to approximate the genomic landscape of human GBM and the first in which the earliest phases of the disease can be studied directly.

BackgroundTemozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ.MethodsUsing patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888.ResultsCells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis.ConclusionIn laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

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Background: In breast cancer (BC), HER2 expression does not only differ within various primary tumor (PT) areas, but also with respect to the expression on circulating tumor cells (CTCs). Despite clinical guidelines defined by CAP/ASCO, PT HER2 analysis displays limitations due to pre-analytical sampling, methodology and intra-tumor heterogeneity. Fluorescence in situ hybridization (FISH) is used for analyzing HER2/neu (ERBB2) gene amplification and for re-solving inconclusive HER2 immunohistochemistry results. In this study, we aimed to evaluate BC intra-tumor heterogeneity using the DEPArray™ - HER2 FISH workflow in BC patients characterized as i) PT HER2 neg / CTC HER2/neu pos (n=25). Methods: 50 µm FFPE tumor curls (n=25) were cut, deparaffinized and underwent antigen retrieval. After dissociation into a single cell suspension, the cells were stained for cytokeratin (CK-AF488), vimentin (Vim-AF647) and DAPI to distinguish between stroma and tumor cells. These two cell populations were separated using the DEPArray™, an image-based single cell sorting system. Samples containing ~ 200 intact CK+/Vim-/DAPI+ tumor cells were deemed suitable for pure tumor cell recovery. Subsequently, HER2 FISH analysis was performed on the recovered single cell tumor sample for HER2 and chromosome 17 signal evaluations using a dual HER2/CC17 probe. The results obtained with the DEPArray™ - HER2 FISH workflow were then compared with the HER2 status obtained by routine pathology and correlated with the HER2 status on CTCs, enriched from 10 ml blood using positive immunomagnetic selection followed by RNA isolation and subsequent gene expression analysis by reverse transcription and Multiplex-PCR (AdnaTest, Qiagen). Results: A concordant result for HER2 analysis comparing routine pathology and DEPArray™ - HER2 FISH analysis was found for 84% (21/25) of the patients. A discordant result was identified in 4/25 patients using the DEPArray™ - HER2 FISH workflow. 2/25 BC patients were proven to be HER2 positive in our approach, despite being tested HER2 negative in routine pathology. Two other patients showed an equivocal HER2 status in the DEPArray™ - HER2 FISH workflow, but a negative result in routine pathology. Consequently, four BC patients would have been eligible for anti-HER2 treatment. Conclusion: The DEPArray™ system allows the recovery of a pure tumor cell fraction for subsequent HER2 FISH analysis and shows a high concordance with the HER2 status performed by routine pathology. However, the DEPArray™ - HER2 FISH workflow offers a more precise and accurate diagnostic test and could be used for BC patients at high risk, e.g. triple negative BC patients, patients with HER2/HER3 positive CTCs or in HER2 inconclusive BC cases to offer anti-HER2 treatment. Citation Format: Lisa Koenig, Amanda Gerber, Aditi Khurana, Farideh Z. Bischoff, Sabine Kasimir-Bauer. Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2 FISH workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4530.