Vaubel, Rachael A.

Abstract Genetic variation can be considered a driver of somatic evolution in glioblastoma (GBM); however, the tumor microenvironment (TME) is a key contributor to the selection process and evolutionary potential of cancer cells. Although diverse ecosystems exist in GBM which impact tumor cell fitness and characterize an aggressive pathophysiology, little is known about how genetically distinct subpopulations of GBM cells interact with the surrounding TME. Therefore, we sought to investigate how the varying clonal genotypes of GBM cells are influenced by different types of selective pressures within the TME. Our imaging-based strategy is centered around Specific-to-Allele PCR Florescence In Situ Hybridization (STAR-FISH), an in situ PCR based probe hybridization method for detection of point mutations and copy number variations at the single-cell level. Using STAR-FISH to illuminate the spatial distribution of clones with mutations in the TERT promoter, we integrated multicolor immunofluorescent staining, DNA-FISH, reflectance imaging, and computational cellular analysis methods to achieve highly-multiplexed single cell spatial profiling of intact GBM specimens. With this strategy we are able to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, nuclear morphology, mutational status, and several oncogenic amplifications on the same slide and location. We analyzed 20 matched primary and recurrent GBM samples, imaging several distinct regions of the same tumor. Our initial results have revealed interesting and potentially novel distributions of subclones residing within specific microenvironments. These results include TERT mutation positive clones enriched in areas possessing more structured extracellular matrix, as well as unique arrangements of infiltrating immune cells in the angiogenic tumor niche. This research underscores the importance of the TME in selecting for certain phenotypes that characterize somatic evolution, highlighting novel genetic and spatial dependencies which sustain heterogeneity in GBM.

Abstract BACKGROUND Adequate treatment options are limited for patients with meningiomas once they have exhausted surgery and radiation. As our molecular understanding of meningiomas and their progression has improved, CDK, FAK, and RAF/MEK pathways have emerged as potential avenues for therapeutic intervention. However, patient-derived cell and xenograft models of meningiomas are difficult to establish, presenting a challenge in preclinical testing and validation of new therapeutic targets. The objective of this study is to develop informative preclinical models of meningiomas and use those to evaluate the activity of potent and selective inhibitors of CDK4/6, FAK, and RAF/MEK. METHODS We used CellTiter-Glo assay to assess the anti-tumor effects of CDK4/6 inhibitor abemaciclib, FAK inhibitor defactinib (VS-6063) and dual RAF/MEK inhibitor RO5126766 (VS-6766) in BEN-MEN-1 (WHO Grade I, NF2-mutant), CH157-MN (WHO Unknown, NF2-mutant), and IOMM-Lee (WHO Grade III, CDKN2A loss) cell lines. Additionally, we used CyQUANT assays to quantify anti-proliferation activity of these inhibitors in a newly established patient-derived cell line MN14 (WHO Grade II, NF2 mutant). In parallel, we continue our attempts to establish patient-derived cell and xenograft models using freshly isolated surgical specimens of progressive meningioma. RESULTS Defactinib monotherapy significantly decreased cell viability across a 10nM-10μM range for all three established cell lines. RO5126766 showed improved efficacy (by 10-fold) in IOMM-Lee, compared to CH157-MN and Ben-Men-1. Abemaciclib showed potent effects at 10nM in IOMM-Lee and BEN-MEN-1, compared to 100nM for CH157-MN. In the MN14 patient-derived cell line, established from an NF2-mutant atypical meningioma, we observed a significant decrease in cell proliferation with defactinib. A decrease in cell proliferation was also observed with RO5126766 and abemaciclib. CONCLUSION Selective inhibitors of CDK4/6, FAK, and RAF/MEK showed anti-meningioma activity against traditional and patient-derived cell models in vitro. Molecular characterization and establishment of patient-derived models that encompass the diversity of clinical meningiomas are ongoing.

Abstract Atypical meningiomas (CNS WHO grade 2) show an intermediate risk of recurrence/progression, and molecular parameters informing management following gross total resection (GTR) remain to be established. We performed comprehensive genomic analysis in 63 patients who underwent GTR of an atypical meningioma. Median age was 59.8 years, 41 patients (65.1%) were female, and 8/62 patients (12.9%) underwent radiation therapy following resection. Follow-up was available for all patients (median 10 years), with recurrence/progression in 17 (5-year PFS: 84.2%; 10-year PFS: 71.0%). Tumors were evaluated by next generation sequencing (n=61), chromosomal microarray (n=63), DNA methylation profiling (n=62), and H3K27me3 immunohistochemistry (n=62). Next generation sequencing using a CLIA-certified 50-gene Neuro-Oncology specific panel identified pathogenic alterations in NF2 (n=31; 50.8%), SMARCB1 (n=5; 8.2%), AKT1 (n=2; 3.3%), TERT (n=1, 1.6%), SMO (n=1), SUFU (n=1), TP53 (n=1), and PTEN (n=1). There was trend toward decreased PFS in patients with NF2 alterations (p=0.078) which was not statistically significant. Copy number variants (CNVs) were identified using OncoScan (ThermoFisher). The most frequent CNVs were loss -22 (n=45; 71.4%), -1p (n=30, 47.6%), -14 (n=21, 33.3%), -6q (n=18, 28.6%), -10q (n=15, 23.8%), -X (n=13, 20.6%), and -18 (n=11, 17.4%). CNVs associated with decreased PFS included -1p (p=0.015), -7p (p=0.007), -10q (p=0.0007), and -18 (p=0.0376). DNA methylation profiling was performed using the Illumina MethylationEPIC array (n=62) and evaluated using the DKFZ classifier (v12.3). Based on the highest score, tumors matched to benign (n=32; 51.6%) or intermediate (n=29; 46.7%) meningioma subclasses or showed non-contributory profiles (n=1; 1.6%). Methylation class (benign vs. intermediate) showed no association with PFS (p=0.96). H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 (6.5%) tumors, which was insufficient for PFS analysis. Overall, our study supports CNV profiling of atypical meningiomas following GTR, which can be implemented using existing, clinically validated technologies. Molecular predictors could guide use of radiotherapy in these patients.

Abstract Oligodendroglioma is a subtype of diffuse glioma defined by a mutation in the isocitrate dehydrogenase (IDH) genes and a co-deletion of chromosome arms 1p and 19q. These tumors primarily occur in adult patients in their third and fourth decade of life and are universally fatal due to an inevitable recurrence that follows a treatment regimen of surgical resection and an optional combination of alkylating chemotherapy and/or radiation therapy. While initially slow growing, recurrent tumors exhibit increasingly aggressive phenotypes that become progressively more difficult to treat with conventional therapy. Currently, the molecular mechanisms and cellular phenotypes that drive this recurrence remain unknown. To understand these factors, we assembled a cohort of matched initial and recurrent oligodendroglioma samples from over 100 patients and performed whole-genome sequencing and whole-exome sequencing on each of them. To link these molecular profiles to cell state changes, we additionally performed bulk and single-nucleus RNA-sequencing on a subset of these tumor pairs. In nearly 40% of alkylating chemotherapy-treated patients, recurrent tumors presented with hypermutation that corresponded with an increase in neoplastic cell proliferation. Additionally, while individual somatic alterations specific to recurrence were relatively rare, we observed a subset of tumors that acquired deletions in the cell cycle regulator CDKN2A following treatment with radiotherapy. Acquisition of either of these features associated with shorter patient survival and higher grade at recurrence, implicating cell cycle dysregulation as a mechanism of treatment resistance and increased tumor severity. Together, these results indicate that oligodendrogliomas evolve in a treatment-specific manner following chemo- and radiation therapy and highlight key pathways that can be targeted to delay the onset of recurrence.

Abstract Diffuse hemispheric glioma, H3 G34-mutant (DHG_H3G34) is a novel tumor type in the 2021 WHO CNS classification. We describe a comprehensive sequencing and high-resolution genome-wide copy number analysis of a series of cases clinically tested by a single laboratory (2018-2022). Cases included tumors from 47 unique patients (1 reportedly recurrent) that had an H3-3A G34 mutation detected using an 187-gene mutation and fusion targeted neuro-oncology NGS panel (n=47). A subset (n=18) of cases was also tested Oncoscan chromosomal microarray. Median age at testing was 20 years (range, 12-50). H3-3A G34 mutations included G34R (n=44; 94%), G34V (n=2) and a novel G34E variant considered to be likely clinically relevant (n=1). All DHG_H3G34 were hemispheric tumors, and one tumor was multifocal with midline involvement. Concurrent mutations recurrently involved ATRX (n=38), TERT promoter (n=3; mutually exclusive with ATRX mutations), TP53 (n=43), PDGFRA (n=26), PTEN (n=5), NF1 (n=4, all patients over 20 years), PIK3CA (n=4) and CDKN2A (n=3). The single recurrent tumor also had an MSH6 mutation, and in addition to 3 other cases, had increased number of sequence variants suggestive of a high tumor mutational burden. A single case also showed an FGFR3::FAM184B (exon 17::exon 2) fusion. All cases with available chromosomal microarray data had unbalanced genomes with multiple chromosomal gains/losses, and regions of copy-neutral loss of heterozygosity. The most frequent recurrent (at least 50% of cases) abnormalities were losses involving 3q, 4q, 10q, 13q and 18q, and 17p copy-neutral loss of heterozygosity encompassing TP53 (all but one case had concomitant TP53 mutation), a pattern reminiscent of IDH-mutant astrocytomas. Oncogene amplifications (PDGFRA and PIK3CA) were detected in 3 (of 18) cases. CDKN2A/B deletion was observed in 8 cases (2 homozygous). In conclusion, we describe additional sequence and copy-number abnormalities in DHG_H3G34, expanding the spectrum of genetic changes associated with this novel tumor type.

Abstract Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Abstract The Mayo Clinic collection of patient-derived xenograft (PDXs) has been developed from the direct implantation of glioblastoma (GBM) samples into immunodeficient mice. Over 105 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated infiltration of tumor cells into the brain parenchymal in 90% of models with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed the PDX collection captures the genetic heterogeneity of GBM, with the majority of typical molecular alterations seen at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations (86%) and homozygous deletion of CDKN2A (70%) were the most frequent. Additional alterations in the p53 pathway included TP53 mutations (36%) and MDM2/4 amplification (10%/2%). IDH mutations were found in three models while MGMT promoter methylation was observed in 45% of PDXs. The available molecular sequencing results are viewable on cBioPortal. These genetically diverse models are extremely useful for biomarker discovery and efficacy evaluations. Using an innovative 1x1x1 PDX preclinical trial design, we are establishing a workflow to evaluate control and drug treatment in an individual animal across PDX lines in the flank. As our first use of this strategy, a small molecule MDM2 inhibitor was evaluated in 17 PDX lines with variable MDM2 and TP53 statuses. Tumors harboring MDM2 amplification without TP53 mutation (n=5) showed profound response, with 4 PDXs showing a complete response that lasted >150 days. Response in TP53 wild-type tumors lacking MDM2 amplification was variable, with a 1.9 to 4.8 fold longer time to endpoint (mean 3; median 2.7). A more traditionally powered intracranial efficacy study performed with one MDM2 amplified (GBM108) and two non-amplified (GBM14, GBM10) PDX lines confirmed these findings of reduced and more variable response to therapy in MDM2 non-amplified TP53 wild-type tumors. Future studies will aim to examine novel biomarkers indicative of therapeutic response to better understand molecular features associated with MDM2 inhibitor efficacy. With over 20 years of experience, our group has extensive expertise in development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Our diverse collection of clinically relevant GBM PDX models provides a highly sought-after resource which has been utilized by the pharmaceutical community with 58 working relationships to date (26 contracts, 32 collaborations). Through these collaborations, we aim to expand the use of our highly characterized GBM PDX panel for biomarker discovery using similar strategies. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Rachael Vaubel, Jann N. Sarkaria. Evaluation of novel therapeutics using the Mayo Clinic GBM Patient-Derived Xenograft (PDX) collection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4001.

Abstract Introduction: EGFR targeted antibody-drug conjugates (ADCs) show promise as a novel treatment in a subset of glioblastoma (GBM). Two EGFR targeting ADCs include first generation Depatux-M, with an antimitotic toxin monomethyl auristatin F (MMAF), and ABBV-321, with a DNA crosslinking agent pyrrolobenzodiazepine dimer (PBD) toxin. Due to the large molecular weight, poor drug distribution across the blood-brain barrier significantly limits the efficacy in EGFR-amplified GBM. We studied whether convection enhanced delivery (CED) can be used to safely infuse these two EGFR-targeted ADCs in patient-derived xenograft (PDX) models of EGFR-amplified GBM. Methods: The efficacy of Depatux-M and ABBV-321 was evaluated in vitro and in vivo in two EGFRviii-amplified PDXs (GBM6 and GBM108). Immunofluorescence staining was used to evaluate drug distribution along with pharmacodynamics of the ADCs. CED was performed by stereotactic placement of an infusion catheter in the same location as the original tumor implantation. Immunohistochemistry was used to explore mechanisms of normal cell toxicity. Results: Despite potent activity in vitro (high ng/ml IC50), systemic administration of either ADC conferred minimal extension in survival for either GBM6 or GBM108. In contrast, CED significantly enhanced ADC delivery to tumor and peri-tumoral regions and extended survival. In a pilot study in GBM6 with n=3 mice per group, a single CED infusion of 0.002mg ABBV-321 resulted in extended survival beyond 365 days for two mice, while other doses and placebo were associated with shorter median survival (0 mg - 39 days; 0.03 mg - 105 days; 0.3 mg - 49 days). In a subsequent trial evaluating serial infusions performed every 21 days, four infusions at 0.002 mg ABBV-321 resulted in lethal toxicity. In contrast, limiting ABBV-321 to only two serial CED infusions in GBM108 was associated with extended survival of more than 300 days vs. 53 days for AB095 antibody control infusion. In contrast, serial infusion with Depatux-M was much better tolerated. In a single infusion in GBM6, a 0.06 mg dose was well tolerated and associated with a 49-day extension in median survival. Further, four serial infusions at 0.06 mg given every 21 days in GBM6 and GBM108 was associated with 100 day and more than 250-day extension in survival as compared to AB095 antibody control infusion. To investigate the mechanism of toxicity, infusion of non-tumor bearing C57Bl6 mice with ABBV-321 resulted in a marked loss in NeuN staining and elevated CD68 and GFAP staining 7 days later; Depatux-M infusion was only associated with modest elevation in GFAP without loss of NeuN staining or elevated CD68-positive cells. Conclusion: Depatux-M is well tolerated when infused into normal brain and results in extended survival in orthotopic GBM PDXs. In contrast, ABBV-321, with a distinct PBD toxin, had a much narrower therapeutic window when delivered by CED. Citation Format: Kendra A. Porath, Ann Mladek, Rachael A. Vaubel, Michael S. Regan, Sonia Jain, Danielle Burgenske, Katrina Bakken, Brett Carlson, Margaret Connors, Zeng Hu, Lihong He, Paul A. Decker, Gaspar Kitange, Shiv Gupta, Nathalie Y. Agar, Thomas M. Feldsien, Didier R. Lefebvre, Jann N. Sarkaria. Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 334.