Carl Koschmann

Mechanisms of Glioma Formation: Iterative Perivascular Glioma Growth and Invasion Leads to Tumor Progression, VEGF-Independent Vascularization, and Resistance to Antiangiogenic Therapy

As glioma cells infiltrate the brain they become associated with various microanatomic brain structures such as blood vessels, white matter tracts, and brain parenchyma. How these distinct invasion patterns coordinate tumor growth and influence clinical outcomes remain poorly understood. We have investigated how perivascular growth affects glioma growth patterning and response to antiangiogenic therapy within the highly vascularized brain. Orthotopically implanted rodent and human glioma cells are shown to commonly invade and proliferate within brain perivascular space. This form of brain tumor growth and invasion is also shown to characterize de novo generated endogenous mouse brain tumors, biopsies of primary human glioblastoma (GBM), and peripheral cancer metastasis to the human brain. Perivascularly invading brain tumors become vascularized by normal brain microvessels as individual glioma cells use perivascular space as a conduit for tumor invasion. Agent-based computational modeling recapitulated biological perivascular glioma growth without the need for neoangiogenesis. We tested the requirement for neoangiogenesis in perivascular glioma by treating animals with angiogenesis inhibitors bevacizumab and DC101. These inhibitors induced the expected vessel normalization, yet failed to reduce tumor growth or improve survival of mice bearing orthotopic or endogenous gliomas while exacerbating brain tumor invasion. Our results provide compelling experimental evidence in support of the recently described failure of clinically used antiangiogenics to extend the overall survival of human GBM patients.

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

The loss of ATRX impairs DNA repair, promoting glioma growth but enhancing sensitivity to DNA-damaging therapies. Aggressive gliomas’ Achilles’ heel ATRX is a protein that is often mutated in glioma, a lethal and relatively common brain tumor. Koschmann et al. developed a mouse model of ATRX-deficient glioma and discovered that these tumors grow more aggressively than their counterparts with wild-type ATRX. The reason this happens is that the loss of ATRX impairs DNA repair, resulting in genetically unstable tumors that can accumulate oncogenic mutations more quickly. However, because of their DNA repair defect, these tumors also proved to be more sensitive to treatments that damage the DNA, such as radiation and some types of chemotherapy. Consistent with these findings, the presence of ATRX mutation correlated with better outcomes in patients, because these tumors were more susceptible to treatment. Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Recent advances and future of immunotherapy for glioblastoma

ABSTRACT Introduction: Outcome for glioma (GBM) remains dismal despite advances in therapeutic interventions including chemotherapy, radiotherapy and surgical resection. The overall survival benefit observed with immunotherapies in cancers such as melanoma and prostate cancer has fuelled research into evaluating immunotherapies for GBM. Areas covered: Preclinical studies have brought a wealth of information for improving the prognosis of GBM and multiple clinical studies are evaluating a wide array of immunotherapies for GBM patients. This review highlights advances in the development of immunotherapeutic approaches. We discuss the strategies and outcomes of active and passive immunotherapies for GBM including vaccination strategies, gene therapy, check point blockade and adoptive T cell therapies. We also focus on immunoediting and tumor neoantigens that can impact the efficacy of immunotherapies. Expert opinion: Encouraging results have been observed with immunotherapeutic strategies; some clinical trials are reaching phase III. Significant progress has been made in unraveling the molecular and genetic heterogeneity of GBM and its implications to disease prognosis. There is now consensus related to the critical need to incorporate tumor heterogeneity into the design of therapeutic approaches. Recent data also indicates that an efficacious treatment strategy will need to be combinatorial and personalized to the tumor genetic signature.

Characterizing and targeting PDGFRA alterations in pediatric high-grade glioma

Pediatric high-grade glioma (HGG, WHO Grade III and IV) is a devastating brain tumor with a median survival of less than two years. PDGFRA is frequently mutated/amplified in pediatric HGG, but the significance of this finding has not been fully characterized. We hypothesize that alterations of PDGFRA will promote distinct prognostic and treatment implications in pediatric HGG. In order to characterize the impact of PDGFR pathway alterations, we integrated genomic data from pediatric HGG patients (n=290) from multiple pediatric datasets and sequencing platforms. Integration of multiple human datasets showed that PDGFRA mutation, but not amplification, was associated with older age in pediatric HGG (P= <0.0001). In multivariate analysis, PDGFRA mutation was correlated with worse prognosis (P = 0.026), while PDGFRA amplification was not (P = 0.11). By Kaplan-Meier analysis, non-brainstem HGG with PDGFRA amplification carried a worse prognosis than non-brainstem HGG without PDGFRA amplification (P = 0.021). There were no pediatric patients with PDGFRA-amplified HGG that survived longer than two years. Additionally, we performed paired molecular profiling (germline / tumor / primary cell culture) and targeting of an infant thalamic HGG with amplification and outlier increased expression of PDGFRA. Dasatinib inhibited proliferation most effectively. In summary, integration of the largest genomic dataset of pediatric HGG to date, allowed us to highlight that PDGFRA mutation is found in older pediatric patients and that PDGFRA amplification is prognostic in non-brainstem HGG. Future precision-medicine based clinical trials for pediatric patients with PDGFRA-altered HGG should consider the optimized delivery of dasatinib.

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227-33. ©2017 AACR.

ATRX mutations and glioblastoma: Impaired DNA damage repair, alternative lengthening of telomeres, and genetic instability

ABSTRACT Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is mutated in nearly a third of pediatric glioblastoma (GBM) patients. We developed an animal model of ATRX-deficient GBM. Using this model combined with analysis of multiple human glioma genome-wide datasets, we determined that ATRX mutation leads to genetic instability, impaired non-homologous end joining, and alternate lengthening of telomeres (ALT).

Survival after relapse of medulloblastoma

Survival after recurrence of medulloblastoma has not been reported in an unselected cohort of patients in the contemporary era. We reviewed 55 patients diagnosed with medulloblastoma between 2000 and 2010, and treated at Seattle Children’s Hospital to evaluate patterns of relapse treatment and survival. Fourteen of 47 patients (30%) over the age of 3 experienced recurrent or progressive medulloblastoma after standard therapy. The median time from diagnosis to recurrence was 18.0 months (range, 3.6 to 62.6 mo), and site of recurrence was metastatic in 86%. The median survival after relapse was 10.3 months (range, 1.3 to 80.5 mo); 3-year survival after relapse was 18%. There were trend associations between longer survival and having received additional chemotherapy (median survival 12.8 vs. 1.3 mo, P=0.16) and radiation therapy (15.4 vs. 5.9 mo, P=0.20). Isolated local relapse was significantly associated with shorter survival (1.3 vs. 12.8 mo, P=0.009). Recurrence of medulloblastoma is more likely to be metastatic than reported in previous eras. Within the limits of our small sample, our data suggest a potential survival benefit from retreatment with cytotoxic chemotherapy and radiation even in heavily pretreated patients. This report serves as a baseline against which to evaluate novel therapy combinations.

Single vs. combination immunotherapeutic strategies for glioma

ABSTRACT Introduction: Malignant gliomas are highly invasive tumors, associated with a dismal survival rate despite standard of care, which includes surgical resection, radiotherapy and chemotherapy with temozolomide (TMZ). Precision immunotherapies or combinations of immunotherapies that target unique tumor-specific features may substantially improve upon existing treatments. Areas covered: Clinical trials of single immunotherapies have shown therapeutic potential in high-grade glioma patients, and emerging preclinical studies indicate that combinations of immunotherapies may be more effective than monotherapies. In this review, the authors discuss emerging combinations of immunotherapies and compare efficacy of single vs. combined therapies tested in preclinical brain tumor models. Expert opinion: Malignant gliomas are characterized by a number of factors which may limit the success of single immunotherapies including inter-tumor and intra-tumor heterogeneity, intrinsic resistance to traditional therapies, immunosuppression, and immune selection for tumor cells with low antigenicity. Combination of therapies which target multiple aspects of tumor physiology are likely to be more effective than single therapies. While a limited number of combination immunotherapies are described which are currently being tested in preclinical and clinical studies, the field is expanding at an astounding rate, and endless combinations remain open for exploration.

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Circumscribed/non-diffuse histology confers a better prognosis in H3K27M-mutant gliomas

Diffuse midline gliomas harboring a recurrent H3 lysine 27-to-methionine (p.Lys27Met, H3K27M) constitute a recently defined pathologic entity with a particularly poor prognosis. They mainly occur in midline structures, such as the pons and thalamus of children and young adults, and are highly infiltrative [2]. More recently, case reports have described the H3K27M mutation in circumscribed (non-diffuse) gliomas, many of which are low-grade (e.g., pilocytic astrocytoma, ganglioglioma) [1, 3–5] (additional references are provided in Online Resource 1). Because of the rarity of these tumors, it is unknown whether they carry the poor clinical outcome ascribed to H3K27M-mutant infiltrating gliomas of the midline. Here, we address this gap in our knowledge by performing an integrated meta-analysis on collated clinical and pathologic data from published studies, data repositories, and collaborative efforts. A systematic search of the literature was performed from 2012 to November, 2017. Tumors were categorized based on diagnosis, location, histologic grade, growth pattern, and H3K27M mutation status. IDH 1/2-mutant diffuse gliomas were excluded to avoid bias arising from the good prognosis attributed to this mutation. Circumscribed gliomas included pilocytic astrocytoma, ganglioglioma/glioneuronal tumor/ ganglion cell tumor, pleomorphic xanthoastrocytoma, and ependymoma. Data from The Cancer Genome Atlas (TCGA) (http ://canc erge nome .nih.gov/) were obtained through the cBioPortal and PedcBioPortal for Cancer Genomics. Factors extracted included age, sex, overall survival, tumor location, histopathologic diagnosis, and WHO grade. Patient samples were then cross-referenced across studies to filter out duplicate data. Cases were also acquired from our inhouse sequencing efforts and through collaboration with the Mayo Clinic in Rochester, Minnesota. Co-occurring mutations were also documented. The endpoint extracted from all data sources was overall survival (OS). Survival functions were estimated using the Kaplan–Meier method and differences analyzed with the log-rank (Mantel–Cox) test using GraphPad Prism software (version 7). The Coxproportional hazards model was used to calculate hazard ratios (HR) using SPSS (version 24, IBM). All studies were approved by ethics committees at the respective institutions (see Online Resource 1 for further details of the methods). Among published studies and through institutional collaboration, we identified 28 cases of H3K27M-mutant circumscribed gliomas (grade I: n = 19; grade III, n = 9). Histopathology included pilocytic astrocytoma (n = 7), ganglioglioma (n = 10), anaplastic ganglioglioma (n = 3), glioneuronal tumor (n = 1), anaplastic glioneuronal tumor (n = 1), ganglion cell tumor (n = 1; Fig. 1a–d), anaplastic ependymoma (n = 3), and circumscribed glioma, not further specified (n = 2) (see Online Resource 2 for references and case details). Strikingly, more than 96% (n = 26/27 cases with location provided) of H3K27M-mutant circumscribed gliomas occurred within the midline, including the brainstem (n = 7), thalamus (n = 5), cerebellum (n = 2), spinal cord (n = 8), and other midline regions (peduncle, posterior fossa, and midline-not further specified; n = 4) (Fig. 1g). Of the remaining two cases, one was reported in the cerebrum, while the location was not specified for the other. H3K27M mutations occurred in H3F3A (n = 21), HIST1H3B (n = 1), Electronic supplementary material The online version of this article (http s://doi.org/10.1007 /s004 01-018-1805 -3) contains supplementary material, which is available to authorized users.