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Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. Significance: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977

Background: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [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 3562.

BACKGROUND: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) has been identified as a candidate structural variant (SV) driver in a subset of pediatric cancers including CNS embryonal tumors and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation, other non-SV mechanisms of activating aberrant expression, and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. METHODS: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. RESULTS: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and surprisingly found FOXR2 to be aberrantly upregulated in 70% of all cancer types (including diffuse midline gliomas), and 8% of all individual tumors. FOXR2 expression occurred predominantly in the absence of rearrangement/fusions, single nucleotide variants, or copy number aberrations at the DNA level. Transcriptomic and epigenomic analyses show the vast majority of tumors (78%) aberrantly express FOXR2 through a previously undescribed epigenetic mechanism via hypomethylation of a novel promoter. Using both in vitro and in vivo models, we demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, including DMGs. CONCLUSION: Taken together, this study demonstrates that FOXR2 is a novel and potent oncogene across pediatric and adult cancers, and highlights a new epigenetic mechanism by which its expression is activated.

Background: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) expression has been associated with a subset of cancers including CNS and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. Methods: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. Results: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and found FOXR2 to be aberrantly upregulated in 70% of all cancer types, and 8% of all individual tumors. We identified genetic and epigenetic mechanisms that induce its expression, including hypomethylation of a novel promoter in the vast majority (78%) of FOXR2-expressing cases. We demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, using both in vitro and in vivo models. Conclusion: Taken together, this study demonstrates the role of FOXR2 as a potent oncogene across human cancers, and highlights a novel mechanism by which its expression is activated. Citation Format: Jessica W. Tsai, Paloma Cejas, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, J'Ya Hunter, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. FOXR2 is an oncogenic driver across adult and pediatric cancers [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 5730.

Current estimates indicate that approximately 8–10% of cancer incidence in young patients can be attributed to constitutional alterations in genes linked to tumor initiation. The rarity and highly heterogeneous nature of high-grade glioma (HGG) in children and adolescents requires coordinated efforts to capture the diversity of subtypes in a normal-tumor matched sequencing cohort. Combining sequencing data of two registries based at the Hopp Children's Cancer Center (KiTZ) Heidelberg (INFORM and MNP2.0) allowed us to investigate the landscape of constitutional alterations across 350 children with high-grade gliomas. Both cohorts independently showed a surprisingly high proportion of patients (17%) with constitutional pathogenic alteration variants in cancer predisposition genes (n=40/233 and 23/128). Our results confirm a high frequency of alterations affecting genes of the DNA mismatch repair (MMR) pathway in 44% of IDH-mutant tumors (n=12/27). Additionally, we observed an almost exclusive correlation of MSH6 constitutional variation and somatic mutation in the IDH1 gene (n=7/8). A hypermutator phenotype defined by a tumor mutational burden >10 mutations/mb) was linked in 14/17 patients to a constitutional alteration in one of the following MMR genes: MLH1, MSH2, MSH6 or PMS2, with IDH-wildtype cases mostly classified as ‘RTK1 subtype’ by DNA methylation analysis. Constitutional alterations in TP53 were found in 5% of the total cohort. Furthermore, these tumors occurred exclusively in the group of H3-/IDH-wildtype glioblastoma (12%), and 8/18 tumors belong to the pedHGG_MYCN subgroup (conversely, this represents 40% of all ‘MYCN subtype’ patients, a highly significant enrichment over other subtypes (p

INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. Significance: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes. See related commentary by Eggermont et al., p. 2677 . This article is highlighted in the In This Issue feature, p. 2659

Background Pediatric patients with relapsed or refractory disease represent a population with a desperate medical need. The aim of the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) program is to translate next generation molecular diagnostics into a biomarker driven treatment strategy. The program consists of two major foundations: the INFORM registry providing a molecular screening platform and the INFORM2 series of biomarker driven phase I/II trials. The INFORM2 NivEnt trial aims to determine the recommended phase 2 dose (RP2D) of the combination treatment of nivolumab and entinostat (phase I) and to evaluate activity and safety (phase II). Methods This is an exploratory non-randomized, open-label, multinational and multicenter seamless phase I/II trial in children and adolescents with relapsed / refractory or progressive high-risk solid tumors and CNS tumors. The phase I is divided in 2 age cohorts: 12–21 years and 6–11 years and follows a 3 + 3 design with two dose levels for entinostat (2 mg/m2 and 4 mg/m2 once per week) and fixed dose nivolumab (3 mg/kg every 2 weeks). Patients entering the trial on RP2D can seamlessly enter phase II which consists of a biomarker defined four group basket trial: high mutational load (group A), high PD-L1 mRNA expression (group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (group D). A Bayesian adaptive design will be used to early stop cohorts that fail to show evidence of activity. The maximum number of patients is 128. Discussion This trial intends to exploit the immune enhancing effects of entinostat on nivolumab using an innovative biomarker driven approach in order to maximize the chance of detecting signs of activity. It prevents exposure to unnecessary risks by applying the Bayesian adaptive design for early stopping for futility. The adaptive biomarker driven design provides an innovative approach accelerating drug development and reducing exposure to investigational treatments in these vulnerable children at the same time. Trial registration ClinicalTrials.gov, NCT03838042. Registered on 12 February 2019.

Purpose: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive paediatric brain tumour with fatal outcome. The Individualised Therapy For Relapsed Malignancies In Childhood (INFORM) registry study offers comprehensive molecular profiling of high-risk tumours to identify target alterations for potential precision therapy. We analysed molecular characteristics and clinical data after brainstem biopsy of all enrolled newly diagnosed DIPGs. Patients and methods: From -February 2015 to February 2018, 21 subsequent primary DIPG cases were enrolled in the nation-wide multicentre INFORM registry study after brainstem biopsy. Whole-genome, whole-exome sequencing and DNA methylation analysis were performed, and RNA-sequencing was added in case of sufficient material. Clinical data were obtained from standardised questionnaires and the INFORM clinical data bank. Results: Tumour material obtained from brainstem biopsy was sufficient for DNA analysis in all cases and RNA analysis in 16 of 21 cases. In 16 of 21 cases (76%), potential targetable alterations were identified including highly relevant MET and NTRK1 fusions as well as an EZH2 alteration not previously described in DIPG. In 5 of 21 cases, molecular information was used for initiation of targeted treatment. The majority of patients (19/21) presented with neurological deficits at diagnosis. Newly arising or worsening of neurological deficits post-biopsy occurred in nine patients. Symptoms were reversible or improved notably in eight cases. Conclusion: In this multicentre study setting, brainstem biopsy of DIPG was feasible and yielded sufficient material for comprehensive molecular profiling. Relevant molecular targets were identified impacting clinical management in a substantial subset. Death or severe bleeding occurred in none of the cases. One of 20 patients experienced unilateral paraesthesia possibly related to biopsy.

Background After collecting data on diminishing S. pneumoniae susceptibility rates, the Madison VA aimed to optimize azithromycin prescribing practices by enhancing the involvement of outpatient pharmacists. This study aimed to develop effective pharmacy-led stewardship teams in the outpatient setting and assess their collective impact on promoting judicious azithromycin prescribing. Methods Madison VA outpatient pharmacists initiated an azithromycin stewardship protocol in 4/2019 to intervene on prescriptions suspected to be discordant with expert guidelines for COPD, pneumonia, sinusitis, or bronchitis. After pharmacist follow-up with providers to discuss potentially inappropriate prescriptions, recommendations and outcomes were subsequently documented in the electronic health record. Given the longitudinal nature of outpatient pharmacist interventions, a post-hoc survey was provided to assess pharmacists’ perceptions of this protocol, barriers to intervention, and areas for improvement. Results Between 10/2018 and 4/2020, 18 pharmacists intervened on 42 outpatient azithromycin prescriptions to recommend alternative antibiotics with improved streptococcal coverage or supportive care alone. Indications warranting the most intervention included COPD exacerbations, upper respiratory infections, and bronchitis. Factors most often cited by pharmacists as barriers to intervention included negative impact on workload, provider reluctance, and insufficient time for follow-up. All surveyed pharmacists believed that prescribers, most commonly primary care providers, were fairly or very receptive to their recommendations. Data evaluated from 10/2018 to 12/2019 revealed a 45% decrease in azithromycin prescribing. Conclusion Azithromycin prescribing has steadily declined at the Madison VA, reinforced by the implementation of an outpatient pharmacist stewardship team. To more seamlessly integrate recommendation-making into pharmacist workflow, determining solutions to identified barriers is currently underway. It is hoped that continued pharmacist involvement in outpatient antibiotic stewardship can be a sustainable practice and transferrable strategy to other antimicrobial agents in the future. Disclosures All Authors: No reported disclosures

LBA10503 Background: Several pediatric precision oncology programs have identified molecular actionable variants. However, the clinical benefit is largely unknown. We here report a target prioritization algorithm and associated clinical outcome. Methods: INFORM is a prospective, non-interventional, multi-center, multi-national, and feasibility registry collecting clinical and molecular data. Patients with refractory/relapsed/progressive malignant disease, including primary diagnosis high-risk entities, can be enrolled. Fresh frozen tumor material (incl. germline DNA) was subjected to WES, lcWGS, RNA-Seq, RNA expression array and DNA-methylation. A weekly interdisciplinary molecular board reviewed and prioritized alterations based on a 7- step scale from ‘very high’ to ‘very low’, depending on the type of alteration and its entity specific relevance (described by Worst et al. Eur J Cancer 2016). Results: To date, more than 1300 patients were enrolled. 525 patients finished follow-up and were included in this analysis. They were enrolled in 72 centers in 8 countries. The median age was 12.0 (range 0 - 40) years. Average turnaround time from submission to report was 25.4 days. Median PFS and OS were 116 (95% CI 105 – 135) and 289 (95% CI 250 – 335) days. The distribution of the highest priority target per patient was: very high 8.0%, high 14.8%, moderate 20.3%, intermediate 23.6%, borderline 14.4%, low 2.5%, very low 1.0% and no actionable target 15.4%. 149 patients received targeted treatment on the basis of identified targets, of which 20 had a very high priority target (mostly ALK, BRAF and NRAS mutations and MET and NTRK-fusions) with a median PFS of 204.5 (95% CI 91.0 – 628.0) compared to 114 (95% CI 103 – 133) days in all other 505 patients (p = 0.0095). OS did not show clinically relevant differences. Explorative analysis of the time to progression (TTP) ratio (before compared to after enrollment) showed that patients treated according to a very high priority target had a higher TTP ratio (1.0) compared to all other patients (0.7). Possible predisposition syndromes were identified in 7.8% of patients, half of which were newly diagnosed. Methylation analysis provided a diagnosis refinement in 8% of CNS tumors. Conclusions: Pediatric precision oncology in a real world, multi-national setting is feasible. The prioritization algorithm identifies subgroups benefitting from molecularly matched targeted treatment. Still, for the patients without a very high priority target further layers of molecular and functional data should be incorporated in future programs.

Background O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma patients. Glioblastoma without hypermethylated MGMT promoter is largely resistant to treatment with temozolomide. These patients are in particular need of new treatment approaches, which are offered by biomarker-driven clinical trials with targeted drugs based on molecular characterization of individual tumors. Methods In preparation for an upcoming clinical study, a comprehensive molecular profiling approach was undertaken on tissues from 43 glioblastoma patients harboring an unmethylated MGMT promoter at diagnosis. The diagnostic pipeline covered various levels of molecular characteristics, including whole-exome sequencing, low-coverage whole-genome sequencing, RNA sequencing, as well as microarray-based gene expression profiling and DNA methylation arrays. Results Complex multilayer molecular diagnostics were feasible in this setting with a median turnaround time of 4-5 weeks from surgery to the molecular tumor board. In 35% of cases, potentially relevant therapeutic decisions were derived from the data. Alterations were most frequently found in receptor tyrosine kinases, members of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin and mitogen-activated protein kinase pathway as well as cell cycle control and p53 regulation cascades. Individual tumors harbored clonal alterations such as oncogenic fusions of tyrosine kinases which constitute promising targets for targeted therapies. A prioritization algorithm is proposed to allocate patients with multiple targets to the potentially best treatment option. Conclusion With this feasibility study, a comprehensive molecular profiling approach for patients with newly diagnosed glioblastoma harboring an unmethylated MGMT promoter is presented. Analyses in this pilot cohort serve as a basis for trials based on targetable alterations and on the question of allocation of patients to the best treatment arm.

Background Currently, there is minimal literature detailing the utility or approach to antibiotic stewardship interventions at the transition of care. We sought to evaluate the utility of a stewardship approach where appropriateness of choice and duration of oral antibiotics prescribed at the time of discharge was assessed and, when indicated, recommendations for change provided. Methods Between June 2017 and April 2018, an antimicrobial stewardship team, comprising of a pharmacist and infectious disease provider, reviewed the electronic health record (EHR) of patients discharged on oral antibiotics from the Medical Service at the William S Middleton VA Hospital for appropriateness of antibiotic choice and total duration of therapy. Depending on availability of team members, reviews occurred twice weekly and included patients discharged within the previous 4 days. If an antibiotic was felt to be inappropriate, the case was discussed with the prescribing service and/or pharmacist. Recommendations were documented in the form of a note placed in the EHR with an emphasis on education. These interventions were logged and information regarding prescribing team/provider, antibiotic, indication, and type of intervention was collected. Intervention types included (but were not limited to) antibiotic stop, change of antibiotic, dose, or duration, and laboratory recommendations. Results Stewardship rounds evaluated 463 patients discharged on oral antibiotics from the Medical Service over 177 hospital days. Forty-one interventions were logged in 38 (8.2%) patients, i.e., approximately 1 intervention for every 12 patients discharged on oral antibiotics. The most common intervention type was antibiotic stop (49%), followed by a change in duration (15%). Interventions occurred most commonly in patients treated for COPD (27%), UTI (22%), and pneumonia (15%). Azithromycin (27%), cefpodoxime (12%), and trimethoprim–sulfamethoxazole (12%) were the antibiotics most frequently intervened upon. Conclusion Assessing postdischarge antibiotic therapy with feedback to prescribers is an additional area where Stewardship programs can focus to better optimize usage of antimicrobials. Disclosures All authors: No reported disclosures.

Background: Checkpoint inhibitors for cancer immunotherapy have been shown to unleash a subdued endogenous T-cell response against mutated tumor antigens, so-called neoepitopes. However, tumors with a low burden of non-synonymous mutations - including gliomas - are poorly presented to the immune system, and therefore unlikely to respond to checkpoint inhibition alone. It is therefore important to identify the signature of relevant immunogenic neoepitopes and the T cell receptors (TCRs) recognizing these epitopes to employ a more specific immunotherapy. Limited availability of tumor-infiltrating lymphocytes (TILs) in turn prompts the identification of neoepitope-specific T-cells in peripheral blood. Aim: This study aimed at establishing a platform for the identification of immunogenic neoepitopes and corresponding neoepitope-specific T cells in the peripheral blood of glioma patients. Methods and Results: Putative neoepitopes were predicted from next generation sequencing mutanome data of tumor samples from three glioma patients. Patient-specific neoepitopes were synthesized as codon-optimized tandem mini gene vector (TMG) optimized for neoepitope presentation, with up to 10 neoeptiopes per TMG. Autologous PBMC-derived dendritic cells were transfected with in vitro transcribed TMG RNA or loaded with peptides to test T-cell neoepitope-reactivity in co-culture assays. On probing the reactivity of T-cells from peripheral blood, we found patient-specific reactivity against neoepitopes. Conclusion and scope: We investigated the T-cell reactivity towards patient specific neoepitopes in the peripheral blood of 3 glioma patients and the phenotypic identity of these neoepitope-reactive T-cells. We are currently probing CD8+ T-cell subpopulations to characterize the neoepitope-reactive T-cell phenotype. Ongoing studies aim to further characterize the neoepitope-specific T-cells with respect to their TCR sequences and expand this platform to a larger cohort of glioma patients to provide a proof-of-principle for a minimally invasive approach to identify tumor-reactive T-cells for personalized glioma immunotherapy.

Combining high-coverage whole-genome sequencing with functional analyses, Giessler et al. demonstrate that tumor initiation and long-term tumor formation in human colorectal cancer are driven by multiple genomic subclones and that the functional heterogeneity of colorectal cancer tumor clone–initiating cells is not based on genomic architecture., A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

TPS10065 Background: Immune checkpoint inhibition in children has shown limited success rates until now. This is most likely due to the fact that the vast majority of pediatric cancers are so-called immunologic cold tumors, and that patients have been enrolled in an unselected manner in single agent trials. Recently, it has been shown that the class I selective HDAC inhibitor entinostat has significant immune enhancing activity in vitro and in vivo. This is mediated through multiple mechanisms including depletion of myeloid-derived suppressor cells, activation of neoantigen transcription and increase of MHC expression. Methods: INFORM2 NivEnt is an exploratory nonrandomized, open-label, multinational and multicenter seamless phase I/II basket trial of nivolumab and entinostat in children and adolescents with relapsed, refractory or progressive high-risk solid and CNS tumors. Patients aged 6-21 will be allocated to the following biomarker-defined groups: high mutational load ( > 100 somatic SNVs/exome; group A), high PD-L1 mRNA expression (RPKM > 3; group B), focal MYC(N) amplification (group C), low mutational load and low PD-L1 mRNA expression and no MYC(N) amplification (Biomarker low group D). Phase I determines the recommended phase 2 dose for the combination for the age groups 6-11 and 12-21 years. Patients will receive nivolumab 3mg/kg every 2 weeks. Entinostat has 2 dose levels: 2mg/m2 and 4mg/m2 once per week. Patients can seamlessly enter the phase II which investigates activity (defined as best response during the first 6 cycles) in the 4 biomarker groups A-D. The duration of treatment is 12 cycles, preceded by 1 entinostat priming week. Interim analyses for futility will be performed after every 10 patients in each group. The study will enroll a maximum of 128 patients in Germany, The Netherlands, Sweden, France, Australia and additional countries under discussion. A comprehensive accompanying biomarker program will investigate a series of immune and epigenetic pharmacodynamic biomarkers. Clinical trial information: NCT03838042.

Background Approximately 2 million Americans develop a diabetic foot ulcer (DFU) annually; >50% die and 5% lose a limb within 5 years. IDSA guidelines recommend multidisciplinary team care for these patients (moderate evidence). Little is known about who should compose the team or how the team should function (low evidence). We conducted a systematic review following PRISMA guidelines to evaluate the effect of multidisciplinary team care on major amputation in patients with DFU’s and describe team composition and function. Methods A medical reference librarian searched databases without date limits through May 26, 2017. Two independent reviewers screened abstracts and then full text using the following inclusion criteria: original article; reported the effect of multidisciplinary teams (≥2 specialties) on major amputation; included a control group; >50% of study patients had diabetes; in English. Abstracted data included study design, patient characteristics, team composition and function, and major amputation rates. Results We included 33 studies (Figure 1). Five (15%) were in the United States, and 27 (82%) were historically controlled trials. Thirty-two (97%) documented lower major amputation rates among patients cared for by a multidisciplinary team (Figure 2). Relative reductions ranged from 11 to 90%. A 12% relative increase was observed in the single study documenting increased rates of major amputation following multidisciplinary care. Thirty-six different specialties were represented in the 26 studies reporting team composition, including: endocrinology (85%), vascular surgery (73%), orthopedic surgery (65%), podiatry (54%), and infectious disease (50%). Teams functioned in the following settings: inpatient (30%), outpatient (15%), or both (55%). Among 12 studies reporting team function, the following topics were addressed: surgical debridement/offloading (66%), vascular disease (63%), infection (59%), and glycemic control (41%). Conclusion Care by multidisciplinary teams may help prevent major amputation for patients with DFUs. Team composition and function, and reductions in major amputation rates, varied considerably. Research directly comparing different models of multidisciplinary care is needed. Disclosures All authors: No reported disclosures.

The treatment of relapses from high-risk entities remains a major clinical challenge, thus the desperate need for precision medicine approaches. To serve this need, we have developed the INFORM registry study (INdividualized therapy FOr Relapsed Malignancies in Childhood), which attempts to rapidly generate personalized tumor profiles and identify therapeutic targets in a clinical diagnostic environment for relapse patients. The INFORM study assesses the feasibility of integrating rapid molecular profiling in the clinical management of pediatric cancer patients with progressive or relapsed high-risk malignancies. Whole-exome and low-coverage whole-genome sequencing are being performed on tumor and normal DNA, complemented with matched tumor RNA sequencing (Illumina HiSeq4000), DNA methylation profiling, and gene expression profiling (for outlier gene expression). To date, more than 400 patients were enrolled from >50 centers in seven different countries (Germany, The Netherlands, Switzerland, Austria, Sweden, Finland, and Australia). The average turnaround time from tissue arrival to molecular results is 3 weeks. Actionable targets with at least “borderline” evidence (according to a prioritization score harmonized with the other major pediatric precision oncology programs across Europe) are being identified in ~50% of patients. Based on these findings, several patients were recruited onto ongoing clinical trials, or targeted therapeutics and/or patient-specific peptide vaccines were incorporated into individualized treatment regimes, with first reports of marked responses. Furthermore, we have established a systematic workflow for the reporting of hereditary predisposition, which is detected in ~7% of cases. In 2018, we will start recruiting patients onto several target-defined (entity independent) subtrials of the INFORM2 interventional trial series as well as the complementary counterpart conducted in France, the eSMART trial, which will collectively provide a portfolio of ~10 mechanism-of-action defined, investigator-initiated early phase clinical (combination) trials for pediatric patients at relapse within the European Innovative Therapies for Children with Cancer (ITCC) Consortium. Citation Format: David T. W. Jones, Barbara C.Worst, Elke Pfaff, Cornelis M. Van Tilburg, Gnana Prakash Balasubramanian, Petra Fiesel, Kristian W. Pajtler, Angelika Freitag, Ruth Witt, Andreas E. Kulozik, Felix Sahm, Andreas von Deimling, Angelika Eggert, Uta Dirksen, Peter Lichter, David Capper, Olaf Witt, Stefan M. Pfister. Pediatric precision oncology programs in Germany and Europe [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA11.

The 'Individualized Therapy for Relapsed Malignancies in Childhood' (INFORM) precision medicine study is a nationwide German program for children with high-risk relapsed/refractory malignancies, which aims to identify therapeutic targets on an individualised basis. In a pilot phase, reported here, we developed the logistical and analytical pipelines necessary for rapid and comprehensive molecular profiling in a clinical setting. Fifty-seven patients from 20 centers were prospectively recruited. Malignancies investigated included sarcomas (n = 25), brain tumours (n = 23), and others (n = 9). Whole-exome, low-coverage whole-genome, and RNA sequencing were complemented with methylation and expression microarray analyses. Alterations were assessed for potential targetability according to a customised prioritisation algorithm and subsequently discussed in an interdisciplinary molecular tumour board. Next-generation sequencing data were generated for 52 patients, with the full analysis possible in 46 of 52. Turnaround time from sample receipt until first report averaged 28 d. Twenty-six patients (50%) harbored a potentially druggable alteration with a prioritisation score of 'intermediate' or higher (level 4 of 7). Common targets included receptor tyrosine kinases, phosphoinositide 3-kinase-mammalian target of rapamycin pathway, mitogen-activated protein kinase pathway, and cell cycle control. Ten patients received a targeted therapy based on these findings, with responses observed in some previously treatment-refractory tumours. Comparative primary relapse analysis revealed substantial tumour evolution as well as one case of unsuspected secondary malignancy, highlighting the importance of re-biopsy at relapse. This study demonstrates the feasibility of comprehensive, real-time molecular profiling for high-risk paediatric cancer patients. This extended proof-of-concept, with examples of treatment consequences, expands upon previous personalised oncology endeavors, and presents a model with considerable interest and practical relevance in the burgeoning era of personalised medicine.

12090Background: Gliomas regularly evade current therapies through primary and acquired resistance and the effect of temozolomide is mainly restricted to the subgroup of methylguanin-O6-methyltrans...

10509 Background: Relapses from high-risk tumors pose a major clinical challenge in pediatric oncology. The German INFORM registry (INdividualized therapy FOr Relapsed Malignancies in children) addresses this problem using integrated next-generation sequencing to rapidly identify patient-specific therapeutic targets. Methods: Whole-exome, low-coverage whole-genome and RNA sequencing is complemented with microarray-based DNA methylation profiling. Identified alterations are discussed and prioritized according to biological significance and potential druggability in a weekly molecular tumor board. Results: To date, 214 tumor samples of high-risk pediatric cancer patients have been profiled from 47 German centers, with 39% being sarcomas, 30% brain tumors, 13% neuroblastoma and 18% hematological or other malignancies. Turnaround time from tissue arrival to molecular results was 21 calendar days on average. In 14/214 patients (7%) we identified an underlying germline predisposition syndrome. In several cases there were discrepancies between the original histological diagnosis and our molecular findings, especially in brain tumors. We detected one or more potentially druggable alterations in 147/214 (69%) cases. Tyrosine kinases, the PI3K/mTOR pathway, MAPK pathway, and cell-cycle as well as transcriptional regulators were commonly affected. Based on these findings, targeted therapeutics were incorporated into the therapy regime in one-third of patients, with anecdotal reports of marked responses, including a patient with a pleomorphic sarcoma, where we detected a previously undescribed RAF-fusion, showing a partial remission upon RAF-inhibition. Conclusions: In summary, real-time comprehensive profiling of pediatric tumors provides valuable diagnostic information and identifies potential therapeutic targets. In parallel, the implementation of a systematic program for reverse-translational evaluation is ongoing. Recently, this nationwide effort has expanded to include patients from other countries. We will also recruit patients to the complementary eSMART and INFORM2 biomarker-driven, phase I/II combination trial series, to provide unprecedented access to targeted therapies in Europe.

A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hübschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 910.

Background: Despite substantial progress in treating primary childhood malignancies, relapses from high-risk entities remain a major clinical challenge. The German INFORM study (INdividualized therapy FOr Relapsed Malignancies) is attempting to address this problem using an integrated next-generation sequencing analysis to rapidly generate personalized tumor profiles and identify therapeutic targets. Methods: The INFORM pilot phase assessed the feasibility of integrating rapid molecular profiling into the clinical management of progressive or relapsed high-risk pediatric cancer patients. Whole-exome and low-coverage whole-genome sequencing of tumor and normal DNA was complemented with tumor RNA sequencing (Illumina HiSeq2500, ‘rapid’ mode). This allowed reliable detection of copy-number changes, point mutations, InDels, fusion genes and deregulated gene expression. Identified alterations were prioritized according to tumor biological relevance and potential as an actionable drug target, with results discussed in a weekly molecular tumor board. Results: From Oct 2013 to Jan 2015, 57 patients (average age 13 years) were enrolled from 20 centers throughout Germany. Entities included: high-grade glioma (n = 12), Ewing's sarcoma (n = 11), rhabdomyosarcoma/DSRCT (n = 7), medulloblastoma (n = 5), ependymoma (n = 5), osteosarcoma (n = 4), neuroblastoma (n = 4), and others (n = 9). Tumor tissue was sufficient for analysis of 52 cases. Average turnaround time from tissue arrival to molecular results was 25 days. Actionable targets with at least ‘borderline’ evidence (according to a prioritization score harmonized with other pediatric precision oncology programs across Europe) were identified in 28 patients (49%). The most commonly affected targets included: tyrosine kinases (ALK, n = 5; FGFR, n = 4; MET, n = 4; others, n = 4), the PI3K/mTOR-pathway (PIK3CA, n = 4; PIK3R1, n = 1; TSC2, n = 1), the MAPK pathway (BRAF, n = 1; KRAS, n = 1), the SHH-pathway (PTCH1, n = 3) and cell-cycle control (CCND1/2, n = 4; CDK4, n = 4; CDKN2A/B, n = 3). Based on these findings, targeted therapeutics were incorporated into the therapy regime of several patients (as single experimental treatments or in clinical trials), with anecdotal reports of marked responses. For example, one patient with a previously inoperable myofibroblastic sarcoma is now in complete remission more than 12 months after entering an ALK-inhibitor trial on the basis of our identification of an ALK fusion. Conclusion: A nationwide individualized diagnostic and treatment approach for pediatric cancer patients based on rapid next-generation sequencing is feasible. Our pilot phase results show that actionable targets can be identified in roughly half of the patients. The INFORM registry study has now opened (www.dkfz.de/en/inform/), to collect molecular and clinical data and establish the infrastructure for prospective clinical trials on personalized pediatric oncology. Citation Format: Barbara C. Worst, Cornelis M. van Tilburg, Gnana P. Balasubramanian, Petra Fiesel, David Capper, Miream Boudalil, Stephan Wolf, Sabine Schmidt, Melanie Bewerunge-Hudler, Matthias Schick, Angelika Freitag, Ruth Witt, Lenka Taylor, Andreas von Deimling, Matthias Schlesner, Angelika Eggert, Peter Lichter, David TW Jones, Olaf Witt, Stefan M. Pfister. The INFORM personalized medicine study for high-risk pediatric cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2015-LB-114

Background: The Breast Cancer Association Consortium (BCAC) is an international collaboration that was established to provide large sample sizes for examining genetic associations. We conducted combined analyses on all single-nucleotide polymorphisms (SNPs) whose associations with breast cancer have been investigated by at least three participating groups. Methods: Data from up to 12 studies were pooled for each SNP ( ADH1C I350V, AURKA F31I, BRCA2 N372H, CASP8 D302H, ERCC2 D312N, IGFBP3 −202 c > a , LIG4 D501D, PGR V660L, SOD2 V16A, TGFB1 L10P, TP53 R72P, XRCC1 R399Q, XRCC2 R188H, XRCC3 T241M, XRCC3 5′ UTR, and XRCC3 IVS7-14). Genotype frequencies in case and control subjects were compared, and genotype-specific odds ratios for the risk of breast cancer in heterozygotes and homozygotes for the rare allele compared with homozygotes for the common allele were estimated with logistic regression. Statistical tests were two-sided. Results: The total number of subjects for analysis of each SNP ranged from 12 013 to 31 595. For five SNPs— CASP8 D302H, IGFBP3 −202 c > a , PGR V660L, SOD2 V16A, and TGFB1 L10P—the associations with breast cancer were of borderline statistical significance ( P = .016, .060, .047, .056, and .0088 respectively). The remaining 11 SNPs were not associated with breast cancer risk; genotype-specific odds ratios were close to unity. There was some evidence for between-study heterogeneity ( P