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Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets., Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies but their origin are still unclear. Here, the authors define the genomic, epigenetic and transcriptional landscape of 32 RIGs cases.

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Despite extensive research, the molecular alterations leading to PCNSL have not been fully elucidated. In order to provide a comprehensive description of the genomic and transcriptional landscape of PCNSL, we here performed whole-genome and transcriptome sequencing and integrative analysis of 51 lymphomas presenting in the CNS, including 42 EBV-negative PCNSL, 6 secondary CNS lymphomas (SCNSL) and 3 EBV+ CNSL and matched controls. The results were compared to an independent validation cohort of 31 FFPE CNSL specimens (PCNSL, n = 19; SCNSL, n = 9; EBV+ CNSL, n = 3) as well as 39 FL and 36 systemic DLBCL cases outside the CNS. Somatic genomic alterations in PCNSL mainly affect the JAK-STAT, NFkB, and B-cell receptor signaling pathways, with hallmark recurrent mutations including MYD88 L265P (67%) and CD79B (63%), CDKN2A deletions (83%) and also non-coding RNA genes such as MALAT1 (70%), NEAT (60%), and MIR142 (80%). Kataegis events, which affected 15 of 50 identified driver genes and 21 of the top 50 mutated ncRNAs, played a decisive role in shaping the mutational repertoire of PCNSL. Compared to systemic DLBCL, PCNSLs exhibited significantly more focal deletions in 6p21 targeting the HLA-D locus that encodes for MHC class II molecules as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis (SBS1, ID1 and ID2) were significantly enriched in PCNSL (SBS1: p = 0.0027, ID1/ID2: p < 1×10−4). Furthermore, TERT gene expression was significantly higher in PCNSL compared to ABC-DLBCL (p = 0.027). Although PCNSL share many genetic alterations with systemic ABC-DLBCL in the same signaling pathways, transcriptome analysis clearly distinguished both into distinct molecular subtypes. EBV+ CNSL cases may be distinguished by lack of recurrent mutational hotspots apart from IG and HLA-DRB loci.

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

Breast cancer is the most common type of cancer affecting women. The formation of lumps in the breast is one of the first signs of the presence of this disease. These tumors can either be cancerous or benign and hence a breast tissue biopsy is conducted to determine their nature. Advancements in the field of vision-based Deep Learning have facilitated the wide adoption of automated diagnostic systems in hospitals, for tasks such as cancer and COVID detection from lung X-ray scans, diabetic retinopathy detection from retinal fundus images, brain MRI segmentation, etc. Moving forward, reduction in training, validation and development times, and efficient usage of training resources for these models will be more in focus. The EfficientNet architecture proposed by Google has recently outperformed prior state-of-the-art architectures such as DenseNet and ResNet on the ImageNet classification task while using fewer parameters and epochs to converge faster. In this paper, we compare the performance of the EfficientNetB3 architecture with the above-mentioned architectures for the tasks of binary and multinomial tumor classification on the benchmark BreakHis dataset, which consists of around 8000 breast histopathology images of varying magnification. Our results show that under similar training conditions, the EfficientNetB3 can converge faster and outperform the previous benchmark models by a significant margin. Our best models achieved 100% sensitivity and accuracy on certain binary classification tasks and a sensitivity of 95.45% and precision of 95.15% on 8-ary classification tasks.

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.

Our stewardship team evaluated 19 months of discharge antibiotic prescriptions to determine prescribing appropriateness and to characterize the interventions made. Intervention occurred in 9.7% of patients, with a 58% acceptance rate. Most interventions were educational (antibiotic course was complete at time of intervention). Discharge antibiotic review is a potential stewardship tool.

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children’s Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.

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.

Incomplete understanding of the metastatic process hinders personalized therapy. Here we report the most comprehensive whole-genome study of colorectal metastases vs. matched primary tumors. 65% of somatic mutations originate from a common progenitor, with 15% being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. Tumor- and metastasis-specific mutations harbor elevated levels of BRCAness. We confirm multistage progression with new components ARHGEF7/ARHGEF33. Recurrently mutated non-coding elements include ncRNAs RP11-594N15.3, AC010091, SNHG14, 3’ UTRs of FOXP2, DACH2, TRPM3, XKR4, ANO5, CBL, CBLB, the latter four potentially dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Actionable metastasis-specific lesions include FAT1, FGF1, BRCA2, KDR, and AKT2-, AKT3-, and PDGFRA-3’ UTRs. Metastasis specific mutations are enriched in PI3K-Akt signaling, cell adhesion, ECM and hepatic stellate activation genes, suggesting genetic programs for site-specific colonization. Our results put forward hypotheses on tumor and metastasis evolution, and evidence for metastasis-specific events relevant for personalized therapy., The evolution and genetic nature of metastatic lesions is not completely characterized. Here the authors perform a comprehensive whole-genome study of colorectal metastases in comparison to matched primary tumors and define a multistage progression model and metastasis-specific changes that, in part, are therapeutically actionable.

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.

In this Article, author Benedikt Brors was erroneously associated with affiliation number '8' (Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee, USA); the author's two other affiliations (affiliations '3' and '7', both at the German Cancer Research Center (DKFZ)) were correct. This has been corrected online.

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.

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

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.

Introduction: Vitamin D deficiency has reached epidemic proportions in India with prevalence rates of 70-100% in the general population. Low vitamin D levels are associated with worse outcomes in ischemic strokes. The outcome of stroke after vitamin D replenishment has not been much studied. Aim: To compare the stroke outcome between patients receiving vitamin D supplementation and patients without vitamin D supplementation by Scandinavian Stroke Scale (SSS) and thus, to assess the long term outcome of ischemic stroke patients with low vitamin D level by supplementation of vitamin D. Materials and Methods: This is a non blinded randomized controlled trial conducted in ischemic stroke patients. The patients were divided into two groups (A and B) where Group A received a single dose of 6 lac IU of Cholecalciferol Intramascular (IM) injection and Group B were not given vitamin D. Finally 30 patients in each group were analyzed. SSS was applied to evaluate the stroke severity at the onset and at the follow up after three months in both groups. The difference in SSS was analyzed using SPSS software. Independent t-test was applied and p-value < 0.05 was taken as significant. Results: Mean±SD of vitamin D levels in Group A and Group B were 17.98±3.81ng/ml and 18.44±4.69 ng/ml respectively. Mean±SD of SSS at time of admission and after three months for Group A was 32.50±11.61 and 38.89±8.34; and for Group B 35.82±8.56 and 38.32±7.10. The difference in SSS from time of admission and after three months, in Group A (6.39±4.56) and Group B (2.50±2.20) were analyzed statistically and found to be highly significant (p

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

Acute intraperitoneal administration of ethanol to rats causes a dose-dependent transient hypothermia. On repeated exposure, however, rats develop tolerance to hypothermic effects of ethanol. Cyclo(His-Pro), an endogenous brain peptide, modifies both acute and chronic themomodulatory effects of alcohol. For example, a) acute pretreatment of rats with increasing amounts of cyclo(His-Pro) produces a progressive decrease in ethanol hypothermia, and b) chronic cyclo(His-Pro) administration augments the development of tolerance to hypothermic effects of alcohol. While the mechanism of cyclo(His-Pro) action is not clear, these data are interpreted to suggest that this peptide may play important roles in ethanol intoxication, preference, tolerance, and/or addiction.