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Fig. S1: MDM2 transcript expression. RNA extracted from flank tumor xenografts from 51 PDX models was assessed by qRT-PCR for MDM2 expression in pooled triplicate samples and compared to expression in normal brain (NB). Fig. S2: MDM2 Amplification in PDX lines. Copy-number profiles were generated from whole exome sequencing of flank xenografts. MDM2 exons are highlighted in red. GBM10, GBM12, and GBM102 show normal MDM2 copy number. GBM108 and GBM143 demonstrate high-level amplification of MDM2. Fig. S3: Apoptosis by TUNEL assay in GBM108. Established flank tumor xenografts were randomized and treated for 5 days with placebo (n=5 mice) versus 50 mg/kg SAR405838 (n=5 mice). Tumor samples were then processed for TUNEL staining. TUNEL-positive cells were quantitated in six high-powered fields (HPF) for each sample. Results are plotted as mean and standard error of the mean. * P

Controversy exists surrounding whether heterogeneous disruption of the blood–brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893–901. ©2018 AACR.

Genetics of recurrent PDX

Purpose:Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization.Experimental Design:PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models.Results:PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection.Conclusions:Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Figures S1-S8, Supplementary Methods, Supplementary Bibliography

Background High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. Methods We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. Results Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. Conclusion The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.

Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.

The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor–associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase–related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.

T cell prolymphocytic leukemia (T-PLL) is a rare disease with aggressive clinical course. Cytogenetic analysis, whole-exome and whole-genome sequencing have identified primary structural alterations in T-PLL, including inversion, translocation and copy number variation. Recurrent somatic mutations were also identified in genes encoding chromatin regulators and those in the JAK-STAT signaling pathway. Epigenetic alterations are the hallmark of many cancers. However, genome-wide epigenomic profiles have not been reported in T-PLL, limiting the mechanistic study of its carcinogenesis. We hypothesize epigenetic mechanisms also play a key role in T-PLL pathogenesis. To systematically test this hypothesis, we generated genome-wide maps of regulatory regions using H3K4me3 and H3K27ac ChIP-seq, as well as RNA-seq data in both T-PLL patients and healthy individuals. We found that genes down-regulated in T-PLL are mainly associated with defense response, immune system or adaptive immune response, while up-regulated genes are enriched in developmental process, as well as WNT signaling pathway with crucial roles in cell fate decision. In particular, our analysis revealed a global alteration of regulatory landscape in T-PLL, with differential peaks highly enriched for binding motifs of immune related transcription factors, supporting the epigenetic regulation of oncogenes and genes involved in DNA damage response and T-cell activation. Together, our work reveals a causal role of epigenetic dysregulation in T-PLL.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a complex heterogeneous neurodegenerative disorder for which mechanisms are poorly understood. To explore transcriptional changes underlying FTLD-TDP, we performed RNA-sequencing on 66 genetically unexplained FTLD-TDP patients, 24 FTLD-TDP patients with GRN mutations and 24 control participants. Using principal component analysis, hierarchical clustering, differential expression and coexpression network analyses, we showed that GRN mutation carriers and FTLD-TDP-A patients without a known mutation shared a common transcriptional signature that is independent of GRN loss-of-function. After combining both groups, differential expression as compared to the control group and coexpression analyses revealed alteration of processes related to immune response, synaptic transmission, RNA metabolism, angiogenesis and vesicle-mediated transport. Deconvolution of the data highlighted strong cellular alterations that were similar in FTLD-TDP-A and GRN mutation carriers with NSF as a potentially important player in both groups. We propose several potentially druggable pathways such as the GABAergic, GDNF and sphingolipid pathways. Our findings underline new disease mechanisms and strongly suggest that affected pathways in GRN mutation carriers extend beyond GRN and contribute to genetically unexplained forms of FTLD-TDP-A.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. It has a strong genetic basis, showing a ~ 8-fold increased risk of CLL in first-degree relatives. Genome-wide association studies (GWAS) have identified 41 risk variants across 41 loci. However, for a majority of the loci, the functional variants and the mechanisms underlying their causal roles remain undefined. Here, we examined the genetic and epigenetic features associated with 12 index variants, along with any correlated (r2 ≥ 0.5) variants, at the CLL risk loci located outside of gene promoters. Based on publicly available ChIP-seq and chromatin accessibility data as well as our own ChIP-seq data from CLL patients, we identified six candidate functional variants at six loci and at least two candidate functional variants at each of the remaining six loci. The functional variants are predominantly located within enhancers or super-enhancers, including bi-directionally transcribed enhancers, which are often restricted to immune cell types. Furthermore, we found that, at 78% of the functional variants, the alternative alleles altered the transcription factor binding motifs or histone modifications, indicating the involvement of these variants in the change of local chromatin state. Finally, the enhancers carrying functional variants physically interacted with genes enriched in the type I interferon signaling pathway, apoptosis, or TP53 network that are known to play key roles in CLL. These results support the regulatory roles for inherited noncoding variants in the pathogenesis of CLL.

The majority of the clinico-pathological variability observed in patients harboring a repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) remains unexplained. This expansion, which represents the most common genetic cause of frontotemporal lobar degeneration (FTLD) and motor neuron disease (MND), results in a loss of C9orf72 expression and the generation of RNA foci and dipeptide repeat (DPR) proteins. The C9orf72 protein itself plays a role in vesicular transport, serving as a guanine nucleotide exchange factor that regulates GTPases. To further elucidate the mechanisms underlying C9orf72-related diseases and to identify potential disease modifiers, we performed an extensive RNA sequencing study. We included individuals for whom frontal cortex tissue was available: FTLD and FTLD/MND patients with (n = 34) or without (n = 44) an expanded C9orf72 repeat as well as control subjects (n = 24). In total, 6706 genes were differentially expressed between these groups (false discovery rate [FDR] C9orf72 (FDR = 1.41E-14), which was roughly two-fold lower in C9orf72 expansion carriers than in (disease) controls. Co-expression analysis revealed groups of correlated genes (modules) that were enriched for processes such as protein folding, RNA splicing, synaptic signaling, metabolism, and Golgi vesicle transport. Within our cohort of C9orf72 expansion carriers, machine learning uncovered interesting candidates associated with clinico-pathological features, including age at onset (vascular endothelial growth factor A [VEGFA]), C9orf72 expansion size (cyclin dependent kinase like 1 [CDKL1]), DPR protein levels (eukaryotic elongation factor 2 kinase [EEF2K]), and survival after onset (small G protein signaling modulator 3 [SGSM3]). Given the fact that we detected a module involved in vesicular transport in addition to a GTPase activator (SGSM3) as a potential modifier, our findings seem to suggest that the presence of a C9orf72 repeat expansion might hamper vesicular transport and that genes affecting this process may modify the phenotype of C9orf72-linked diseases.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p < 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

Background Currently, there is no biologically based rationale for drug selection in migraine prophylactic treatment. Methods To investigate the genetic variation underlying treatment response to verapamil prophylaxis, we selected 225 patients from a longitudinally established, deeply phenotyped migraine database (N = 5983), and collected uninterrupted quantitated verapamil treatment response data and DNA for these 225 cases. We recorded the number of headache days in the four weeks preceding treatment with verapamil and for four weeks, following completion of a treatment period with verapamil lasting at least five weeks. Whole‐exome sequencing (WES) was applied to a discovery cohort consisting of 21 definitive responders and 14 definitive non‐responders, and the identified single nucleotide polymorphisms (SNPs) showing significant association were genotyped in a separate confirmation cohort (185 verapamil treated patients). Statistical analysis of the WES data from the discovery cohort identified 524 SNPs associated with verapamil responsiveness (p, Whole exome sequencing in 21 definitive verapamil responders and 14 definitive non‐responders identified 524 SNPs showing significant (p

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.

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.

Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin’s lymphoma cell lines and in 113 primary MM samples. Ex vivo drug sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This “direct to drug” screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.

Tumors acquire numerous mutations during development and progression. When translated into proteins, these mutations give rise to neoantigens that can be recognized by T cells and generate antibodies, representing an exciting direction of cancer immunotherapy. While neoantigens have been reported in many cancer types, the profiling of neoantigens often focused on the class-I subtype that are presented to CD8 + T cells, and the relationship between neoantigen load and clinical outcomes was often inconsistent among cancer types. In this study, we described an informatics workflow, REAL-neo, for identification, quality control (QC), and prioritization of both class-I and class-II human leukocyte antigen (HLA) bound neoantigens that arise from somatic single nucleotide mutations (SNM), small insertions and deletions (INDEL), and gene fusions. We applied REAL-neo to 835 primary breast tumors in the Cancer Genome Atlas (TCGA) and performed comprehensive profiling and characterization of the detected neoantigens. We found recurrent HLA class-I and class-II restricted neoantigens across breast cancer cases, and uncovered associations between neoantigen load and clinical traits. Both class-I and class-II neoantigen loads from SNM and INDEL were found to predict overall survival independent of tumor mutational burden (TMB), breast cancer subtypes, tumor-infiltrating lymphocyte (TIL) levels, tumor stage, and age at diagnosis. Our study highlighted the importance of accurate and comprehensive neoantigen profiling and QC, and is the first to report the predictive value of neoantigen load for overall survival in breast cancer.

Next-generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL-IPI) in newly diagnosed CLL and high-count monoclonal B-cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi-stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time-to-first treatment (TTT), adjusted for CLL-IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17-1.39, P = 2.6 × 10-8 ; c-statistic = 0.76). When stratified by CLL-IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37-1.72, P = 7.0 × 10-14 ). Overall, 80% of low/intermediate CLL-IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12-2.07, P = .007; c-statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL-IPI, especially among low/intermediate CLL-IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.

Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

T-cell prolymphocytic leukemia (T-PLL) is a rare disease showing a rapid clinical course, with a median survival of =2.5 kb away from transcription start sites) that are lost and gained in T-PLL, respectively, compared to normal. The differential H3K27ac peaks are preferentially associated with the nearest genes differentially expressed between T-PLL and normal (chi-square test p Citation Format: Shulan Tian, Henan Zhang, Eric Klee, Huihuang Yan, Wei Ding. Genome-wide epigenetic alterations in T-cell prolymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2119.

Chronic lymphocytic leukemia (CLL) is a neoplastic disease of mature B-cells with a highly heterogeneous clinical course. While European ancestry (EA) populations present an increased incidence of CLL, African ancestry (AA) populations have a younger median age of onset, higher frequency of adverse prognostic factors, and inferior clinical outcomes. Despite the considerable effort to characterize the genetic landscape of CLL, AA are overwhelmingly underrepresented. Our hypothesis is that the clinical differences observed between AA and EA populations are, in part, explained by underlying genetic features. To address this imbalance, we identified 90 AA patients diagnosed with CLL, 64% of which were untreated at sample collection. RNA and DNA were extracted from CD5+/CD19+ clonal B-cells. We performed mRNA-seq and targeted sequencing in 59 recurrently mutated somatic CLL driver genes. Differentially expressed genes were identified using edgeR. Data was compared to our previously analyzed EA CLL cohort (N=445). The median age at diagnosis was 59 years for AA and 66 for EA and 74% of AA and 50% of EA had unmutated IGHV (u-IGHV) status. When evaluating the entire AA and EA cohorts, there was a significant increased frequency of mutations in TP53, SF3B1, and NFKBIE, identified in 29%, 24%, and 20% of AA CLLs, compared to 5%, 9%, and 9%, respectively, in EA CLLs (p Citation Format: Cecilia Bonolo de Campos, Daniel R. O'Brien, Chantal E. McCabe, Huihuang Yan, Geffen Kleinstern, Zhiquan Wang, Laura A. Bruins, Cristine Allmer, Nicholas J. Boddicker, Charla R. Secreto, Aaron D. Norman, Shulan Tian, Kari G. Rabe, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Richard Furman, J Brice Weinberg, James R. Cerhan, Celine M. Vachon, Neil E. Kay, Susan L. Slager, Esteban Braggio. Characterization of underlying genomic features among African ancestry populations diagnosed with chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2209.

Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies >0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.