Citations

Abstract High-grade gliomas (HGGs) are aggressive tumors with poor outcomes and limited treatment options. Here, we combined genomic and transcriptomic tumor profiling with drug testing in a patient-derived 3-dimensional culture model to identify individualized treatments and predictive biomarkers. Activity of single agents targeting frequently dysregulated glioma pathways was relatively poor ex vivo and generally reflected historical patient data. However, compounds targeting PI3K, epigenetic, and survival/senescence signaling were effective in some cases. Drug sensitivity correlated with transcriptional rather than genomic features and suggested heterogeneity as a resistance mechanism. Bromodomain and extraterminal domain inhibition was particularly effective in tumors enriched in the mesenchymal transcriptional subtype, promoted proneural transition, and was overcome by upregulated PI3K signaling. Notably, combinations were largely effective, with 6 strategies exhibiting stronger efficacy than corresponding single agents in most cases (58-77%). This study identifies HGG vulnerabilities and associated biomarkers, resistance mechanisms, and effective combination strategies that warrant further clinical validation. Graphical Abstract

Abstract Purpose: Germline pathogenic variants (PV) in ATM increase the risk of pancreatic ductal adenocarcinoma (PDAC), but the underlying tumor biology of PDAC associated with germline PV in ATM has not been adequately explored. Experimental Design: Whole-genome, whole-exome, and RNA sequencing were performed on PDAC tumors from 25 germline ATM PV carriers diagnosed at Mayo Clinic between 2007 and 2017. Somatic and copy-number alterations, mutational signatures, transcriptomic subtypes, and the immune landscape were evaluated. Results: High-quality whole-exome and whole-genome sequencing were obtained from 21 and 15 tumors, respectively. Biallelic inactivation of ATM was observed in 87%, KRAS PV in 90%, CDKN2A homozygous loss in 60%, and TP53 alterations in <10% of these tumors. A predominant clock-like mutational signature was present in all samples. Whole-transcriptome analysis identified that the aberrantly differentiated endocrine exocrine subtype accounted for 18% of PDAC and was consistently associated with >5-year overall survival. In addition, a 28-gene expression–based signature associated with overall survival was identified and further validated in The Cancer Genome Atlas cohort. Immune landscape analysis through CODEX identified enriched CD4 T-helper cell/tumor interactions and reduced B7H3-high cell/tumor interactions in ATM PV carriers compared with noncarriers. Conclusions: The observed absence of TP53 PV and enrichment for CDKN2A alterations in ATM tumors, along with differences in the mutational signatures, transcriptomic subtypes and immune landscape, improve our understanding of the mechanistic pathways involved in PDAC development in germline ATM PV carriers and help identify potential targeted therapeutic strategies.

Abstract Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications. Experimental Design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase–mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples. Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression. Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

Abstract Accurate response assessment in patients with high-grade gliomas (HGGs) is complicated by post-treatment radiographic changes that are equivocal for treatment effect versus progression. Plasma-based liquid biopsy assays for circulating tumor DNA (ctDNA) have had limited sensitivity to date, due in part to the presence of the blood-brain barrier that limits the diffusion of tumor-derived analytes out of the central nervous system. To address this challenge, we developed a ctDNA plasma-based assay to detect individualized tumor-derived amplified junctions in patients with HGGs. These junctions occur due to large-scale chromosomal rearrangements, such as those associated with epidermal growth factor receptor (EGFR) or cyclin-dependent kinase 4 (CDK4) amplification. To identify these amplified junctions, whole genome sequencing was performed on resected tissue. Primers were designed to flank junction breakpoints and were then utilized on each patient’s plasma samples to detect the amplified junction via qPCR. Tumor-specific amplified junctions were robustly detected in plasma from 14/14 (100%) patients with IDH-mutant or wild-type grade 4 astrocytomas whose tumor genomes demonstrated amplification-related junctions, located at chromosomes 7, 11, or 12. In comparison, ctDNA could not be detected in three control cases that did not have an amplification or did not have high copy number junctions. Importantly, serial plasma samples obtained prior to and at disease recurrence in 4 patients demonstrated an increasing abundance of amplified junctions that accurately correlated with disease progression in 4/4 patients (100%). In two of these patients, increasing abundance of amplified junctions preceded radiographic changes compatible with disease progression by approximately 2 or 4 months, respectively. In conclusion, tumor-specific amplified junctions could reliably be detected in plasma from patients with IDH-mutant or wild-type grade 4 astrocytomas and correlated with changes in disease burden. Further work is currently ongoing to evaluate these promising results in a larger cohort of patients with serial plasma samples.

AbstractAxenfeld‐Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Abstract Sequencing efforts have identified key oncogenic drivers and transcriptomic subtypes of high-grade gliomas (HGGs), but this knowledge has not resulted in individualized therapy or improved patient outcomes. Here, we used a functional genomics approach combining integrated genomic/transcriptomic analyses with patient-derived 3D micro-cancers (mCancers) to rapidly identify individualized therapeutic vulnerabilities and determine predictive response biomarkers. 30 primary/recurrent HGGs, including 20 glioblastomas, 5 astrocytomas, and 5 oligodendrogliomas, were included for drug testing. mCancers recapitulated the structural and molecular alterations, cellularity, and heterogeneity, of parental patient tumors. A total of 119 single-agent drugs and 183 drug combinations were tested, while a pre-selected drug panel comprising 25 glioma-relevant treatment strategies was tested across all cases. In general, mCancer efficacy corresponded well to historical responses to previously tested agents (EGFR, HDAC inhibitors, etc.). Combined, strong efficacy (i.e. >70% growth inhibition at Cmax) of single treatments targeting deregulated receptor tyrosine kinase (RTK), p53/apoptosis, cell cycle, DNA damage repair, IDH, or telomere maintainance pathways, was observed in 14 out of 30 cases. Surprisingly, transcriptional signatures, but not specific genetic alterations, were found to stratify drug responses. For example, mCancers enriched for the most aggressive mesenchymal (MES) and glycolytic/plurimetabolic (GPM) transcriptional subtypes exhibited stronger response to the BET inhibitor JQ1 (and other clinical BET inhibitors) and the DNA-PKcs/mTOR dual inhibitor CC-115. This therapeutic vulnerability was associated with enrichment of YAP/TAZ signaling, and the glioblastoma stem cell G-STEM signature. Notably, individual combination strategies were effective across tumors, with strong responses ranging from 38% to 77% of all tumors tested. Overall, this study underscores the value of functional genomics in identifying novel vulnerabilities of HGGs and associated response biomarkers. It also highlights the need for further clinical validation of several novel therapies and especially drug combination strategies with strong efficacy in the ex vivo setting.

Abstract Background: Mutations in RAS is reported in embryonal rhabdomyosarcoma (ERMS). Upregulation of IGF2/IGF1R axis and IGF2 overexpression is a common finding. Targeting RAS downstream is associated with high toxicity. Combination therapies targeting parallel pathways is necessary to overcome treatment resistance. Dual inhibition of RAS downstream and IGF2/IGF1R axis can be a potential mechanism of targeted treatment. The efficacy of this mechanism was studied using a microcancer 3D exvivo tumor cell viability assay in a KRAS mutant ERMS. Design: Tumor was collected for a high-risk pediatric ERMS patient. Comprehensive genomic analysis, for identification of targetable genomic alterations, was performed by integrating WES, MPseq and RNAseq. Tumor spheroids grown in 3D environment were tested against multiple single drugs and combinations. The tumor viability was assed employing the Cell Titer Glow Luminescent Cell Viability Assay. Results were expressed as nMATP and were shown as drug-response curve exhibiting the ATP concentration changes at Cmax. Results: Genomic findings included an oncogenic KRAS (G12D) mutation. BAY-293 has a partial cell killing response as a single agent. Given to the presence of IGF2 over-expression, IGF2/IGF1R pathway was targeted by linsitinib. Addition of BAY-293 to linsitinib led to enhanced effectiveness of this agent (Table 1). Conclusion: Combination of comprehensive genomic profiling and a laboratory-based 3D micro-cancer model using the patient’s own tumor is an effective way to determine drug efficacy. Targeting RAS pathway by single agents, increases the possibility of resistance. Dual targeting of downstream pathways by MEK and PI3K pathway inhibition is highly toxic, and the clinical usage of this combination is limited. Our results have indicated inhibition of IGF-1R/IGF2 axis in combination with other agents targeting RAS pathway, can be a potential method to address these limitations. ABEL 1. Summary of molecular alterations and targeting methods Molecular alteration Drug Target Kill% at Cmax KRAS (G12D) Mutation BAY-293 RAS-SOS1 interaction 55.3 Trametinib MEK1/2 88.3 Selumetinib MEK1/2 76 Paxalisib PI3K/mTOR 89.7 Samotolisib PI3K/mTOR 92.9 Copanlisib PI3K 95.2 Perifosine AKT 89.2 IGF2 Over-expression Linsitinib IGF-1R/InsR 84.1 BMS-754807 IGF-1R/InsR 67.4 NTRK2 mutation Larotrectinib NTRK 41.9 KMT2D mutation Panobinostat HDAC 85.3 Combinations Kill% at Cmax BAY-293+Selumetinib 100 BAY-293+Samotolisib 100 BAY-293+Paxalisib 99.7 BAY-293+Linsitinib 97.5 Selumetinib+Samotolisib 93.1 Citation Format: Dorsay Sadeghian, Wan-Hsin Lin, James Smadbeck, Alan Panheiter, Michael Barrett, Alexa F. Mccune, Giannoula Karagouga, Sarah Johnson, Ryan W. Feathers, Faye R. Harris, Lindsey Kinsella, Taylor Berry, John Cheville, Panos Anastasiadis, Candace Granberg, Patricio Gargollo, Vasmatzis George. Dual inhibition of RAS and the IGF2/IGF1R pathways: a potential targeting strategy for RAS-mutated embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3391.

Background Immune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden and no robust predictors of survival with ICI. Since ICIs enable adaptive antitumor immune responses, we investigated T-cell receptor (TCR) associations with survival in participants from two clinical trials treated with ICI. Methods We included patients with pleural mesothelioma who were treated with nivolumab (NivoMes, NCT02497508) or nivolumab and ipilimumab (INITIATE, NCT03048474) after first-line therapy. TCR sequencing was performed with the ImmunoSEQ assay in 49 and 39 pretreatment and post-treatment patient peripheral blood mononuclear cell (PBMC) samples. These data were integrated with TCR sequences found in bulk RNAseq data by TRUST4 program in 45 and 35 pretreatment and post-treatment tumor biopsy samples and TCR sequences from over 600 healthy controls. The TCR sequences were clustered into groups of shared antigen specificity using GIANA. Associations of TCR clusters with overall survival were determined by cox proportional hazard analysis. Results We identified 4.2 million and 12 thousand complementarity-determining region 3 (CDR3) sequences from PBMCs and tumors, respectively, in patients treated with ICI. These CDR3 sequences were integrated with 2.1 million publically available CDR3 sequences from healthy controls and clustered. ICI-enhanced T-cell infiltration and expanded T cell diversity in tumors. Cases with TCR clones in the top tertile in the pretreatment tissue or in circulation had significantly better survival than the bottom two tertiles (p<0.04). Furthermore, a high number of shared TCR clones between pretreatment tissue and in circulation was associated with improved survival (p=0.01). To potentially select antitumor clusters, we filtered for clusters that were (1) not found in healthy controls, (2) recurrent in multiple patients with mesothelioma, and (3) more prevalent in post-treatment than pretreatment samples. The detection of two-specific TCR clusters provided significant survival benefit compared with detection of 1 cluster (HR<0.001, p=0.026) or the detection of no TCR clusters (HR=0.10, p=0.002). These two clusters were not found in bulk tissue RNA-seq data and have not been reported in public CDR3 databases. Conclusions We identified two unique TCR clusters that were associated with survival on treatment with ICI in patients with pleural mesothelioma. These clusters may enable approaches for antigen discovery and inform future targets for design of adoptive T cell therapies.