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VAF concordance

Supplementary Data file including supplementary methods and clinical case vignettes.

Reasons for not using recommended targeted therapy.

Patient sequencing data, by patient number and diagnosis.

Introduction Pediatric Graves’ disease (GD) is associated with hyperthyroid symptoms broadly impacting psychosocial functioning. Total thyroidectomy (TT) is a definitive treatment option that replaces long-term antithyroid medication (ATD) for GD. While studies have examined health-related quality of life (hrQOL) in adults, there are no data describing impacts of definitive surgery in pediatrics. In this prospective, longitudinal study, we explored the impact of definitive surgery on hrQOL and psychosocial functioning in adolescent patients with GD. Methods Patients 12-19 years old pursuing definitive surgery for GD and their parents were recruited to complete surveys before and at least six months following TT. Parents pre-operatively completed a demographic questionnaire, parent rating scale assessing perceived symptom severity, and motivations for pursuing TT questionnaire. Patients pre- and post-operatively completed the Pediatric Quality of Life Scales (PedsQL), Quality of Life Questionnaire for Patients with Thyroid Disease (ThyPRO), Perceived Stigmatization Questionnaire (PSQ), Body Esteem Scale for Adolescents and Adults (BESAA), and European Group on Graves’ Orbitopathy GO Quality of Life Questionnaire (EUGOGO-QOL). Patients and parents post-operatively completed the Patient and Observer Scar Assessment Scale (POSAS) and a post-surgical satisfaction scale. Longitudinal paired scores were compared using Wilcoxon rank-sum test with Bonferroni-Holm correction. Survey subscore associations were assessed using Spearman tests with Benjamin-Hochberg correction. P-values < 0.10 for survey association analyses and all other p-values < 0. 05 were considered statistically significant. Results Thirty-six patient/parent dyads completed baseline surveys, including 19 patient/parent dyads completing both pre- and post-TT surveys. Families reported that patients’ GD and ATD significantly interfered with quality of life. Motivations for definitive surgery included ease of monitoring hypothyroidism as opposed to hyperthyroidism, access to a 'high-volume' surgeon, desire to reduce goiter symptoms, and opposition to radioactive medication. At baseline, patients reported high physical and cognitive GD symptomology via ThyPRO, specifically hyperthyroid symptoms, tiredness, anxiety, and emotional susceptibility. Psychosocial functioning via PedsQL, specifically in school settings, was depressed. Disease-specific hrQOL (ThyPRO) significantly improved following TT, with notable improvements in goiter (p=0. 002), hyperthyroid symptoms (p=0. 002), tiredness (p=0. 003), and anxiety (p=0. 003). Physical (p=0. 006) and school-related functioning (p=0. 003) improved post-TT (PedsQL), while variables captured by PSQ, BESAA, and EUGOGO-QOL were not significant pre- and post-TT. Reported GD-associated eye symptomology was minimal at baseline and further improved following surgery (p=0. 007). ThyPRO and PedsQL subscores were significantly associated at baseline and change after surgery, suggesting interconnectedness between disease-specific hrQOL and psychosocial functioning. Patients and parents reported median recovery of two months and high satisfaction with the outcomes of definitive surgery, with minimal scar appearance concerns. Conclusions In the setting of a high-volume surgeon with low complication rates, definitive surgery for GD in pediatric populations has substantial, beneficial effects on disease-specific hrQOL and psychosocial functioning, with minimal adverse scar appearance complaints. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:48 p.m. - 12:53 p.m.

Introduction: Risk of malignancy for pediatric thyroid nodules classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is not well defined. Correlations between risk of malignancy and ancillary clinical data remain inconclusive. We report a single institutional experience of fine-needle aspiration (FNA) to improve upon current management paradigm of thyroid nodules. Methods: A retrospective chart review of 575 thyroid nodules was performed of 324 patients who underwent 340 FNAs between 2008 and 2018 at the Children’s Hospital of Philadelphia. Demographics, ultrasound (US) characteristics, FNA cytology, surgical pathology, and ancillary data were reviewed. Results: The rate of malignancy according to TBSRTC was 0.0% for category I, 0.8% for category II, 15.6% for category III, 54.5% for category IV, 100.0% for category V, and 100.0% for category VI. The cumulative Thyroid Imaging Reporting and Data System (TI-RADS) score was significantly correlated with benign and malignant nodules on pathology (p < 2.2e−16). Distribution of TI-RADS for cytologically indeterminate nodules with benign or malignant pathology revealed significant differences for composition (p = 3.20e−8) and echogenic foci (p = 0.005) but not for echogenicity (p = 0.445), shape (p = 0.160), margins (p = 0.220), and size (p = 0.105). Distributions of thyroid-stimulating hormone levels between benign and malignant patients was significant (p = 1.58e−3). Conclusions: Nodules with TI-RADS scores >3 should undergo FNA, irrespective of size; surgical resection is recommended for nodules classified as TBSRTC category IV and V due to high risk of malignancy. US surveillance instead of FNA can be performed for nodules with TI-RADS scores ≤3.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Introduction: Thyroid lobectomy reduces risks of surgical complications and need for levothyroxine (LT4). We aimed at identifying the clinical course and risk factors for postlobectomy hypothyroidism to optimize surgical counseling and management in pediatric patients undergoing lobectomy. Methods: Clinical and biochemical presentations pre- and postlobectomy were retrospectively reviewed for 110 patients who underwent thyroid lobectomy between 2008 and 2020 at the Children's Hospital of Philadelphia. Results: Approximately 28.2% of patients (31/110) developed postlobectomy hypothyroidism defined by an elevated thyrotropin (TSH) level, including 24.5% (27/110) with subclinical hypothyroidism (TSH >4.5 and 10.0 mIU/L). LT4 was initiated in 12.7% (14/110) of cases. Most patients (81.6%; 84/103) recovered euthyroidism within 12 months postlobectomy. When excluding patients with autonomous nodule(s), median preoperative TSH was 1.09 (interquartile range [IQR] = 0.70–1.77) mIU/L and 1.80 (IQR = 1.02–2.68) mIU/L in euthyroid and hypothyroid patients, respectively, with multivariate logistic regression confirming the association between an increased preoperative TSH and postlobectomy hypothyroidism (odds ratio = 1.8 [confidence interval 1.08–3.13], p = 0.024). Of the patients who underwent thyroid lobectomy and developed postoperative hypothyroidism (n = 31), 38.7% (12/31) had a preoperative diagnosis of an autonomously functioning thyroid nodule. Conclusions: Thyroid function should be evaluated postlobectomy to assess the need for LT4. LT4 should be considered if the TSH remains elevated, especially if an upward trend is observed or TSH is >10.0 mIU/L. Suppressed preoperative TSH associated with autonomous nodules is an independent risk factor for postlobectomy hypothyroidism.

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. Significance: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations. See related commentary by Bornhauser and Bourquin, p. 1322. This article is highlighted in the In This Issue feature, p. 1307

Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [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 6051.

BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [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 699.

10049 Background: Childhood cancer survivors (CCS) are at risk for radiotherapy (RT) late effects, including second malignancies. Optimal screening for thyroid cancer (TC) in CCS post-RT remains controversial. We assessed the clinical benefit of thyroid ultrasound (US) surveillance in CCS exposed to RT. Methods: 316 CCS (175 males) were prospectively surveilled with thyroid US between 2002 and 2021 at the Children’s Hospital of Philadelphia. Patients were screened upon referral to the Survivorship Program. Thyroid US, clinicopathologic features, and endocrine-related outcomes were ascertained. Outcomes were compared using primary CCS diagnosis age cohorts of ≤ 3, > 3 to ≤ 10, and > 10 years. Risk factors for thyroid nodule(s) and TC were evaluated using Kruskal Wallis and ANOVA [OR (95% CI)]. Results: The most common CCS diagnoses were leukemia (32%), CNS tumor (26%), and neuroblastoma (18%). Patients received TBI (43%) and/or RT to craniospinal (43%), chest (13%), and neck regions (7%). About 48% (n = 152) of patients presented thyroid nodule(s) (Table). Forty-six patients underwent surgery, and 28 had TC, including 19 with ATA low-risk, 2 with ATA intermediate-risk, and 7 with ATA high-risk disease. Of the 9 patients with intermediate- or high-risk disease, 5 were ≤ 3 years, 3 were > 3 to ≤ 10 years, and 1 was > 10 years at the time of RT exposure. Eight patients with TC demonstrated pathogenic variant(s). RT exposure at ≤ 3 years old conferred 2-fold increased risk for nodule(s) compared to RT at > 10 years [OR = 2.14 (1.44-2.84) p = 0.03]. Female sex [OR = 1.73 (1.25-2.21) p = 0.02] and greater interval between RT and first US [OR = 1.10 (1.04-1.15) p = 0.001] were additional independent risk factors. Conclusions: Younger age at RT exposure is associated with increased risk of and shorter latency for developing TC. Thyroid US surveillance appears most beneficial in CCS exposed to RT ≤ 3 years old in an effort to diagnose TC at an earlier stage prior to metastasis. [Table: see text]

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

This study dissected the complexity of the immune architecture of acute myeloid leukemia (AML) at high resolution and assessed its influence on therapeutic response. Using 387 primary bone marrow samples from three discovery cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease subtypes and unraveled critical differences in immune gene expression across age groups and disease stages. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles sheds novel insights into the immuno-biology of AML and will inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Chemotherapy resistance is common, and relapse is the major cause of treatment failure. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. Methods Immune gene expression profiling for the high-dimensional analysis of the immunological landscape of bone marrow (BM) samples from patients with newly-diagnosed (de novo) non-promyelocytic AML (n=387) was employed to analyze the tumor microenvironment (TME). We derived immune scores from mRNA expression levels and devised an RNA-based, quantitative metric of immune infiltration, as previously published (Danaher P, et al. JITC 2017 and 2018). The PanCancer IO360™ gene expression assay was used to profile BM samples collected prior to and during flotetuzumab (FLZ) treatment from 30 AML patients treated at the RP2D (500 ng/kg/day) in the CP-MGD006-01 clinical trial (NCT#02152956), primary refractory, n=23 or relapsed, n=7. IO360 score comparisons are presented as mean ± SD and significance was assessed by the Mann Whitney U test. Results Analysis of pre-treatment BM samples from de novo AML revealed distinct immune signature modules, reflecting the co-expression of genes associated with 1) an IFNγ-dominant TME, 2) adaptive immune responses, and 3) myeloid cell abundance. When considered in aggregate, the relative intensity of gene expression in the immune modules stratified BM samples into two subgroups, which will be herein termed immune-infiltrated and immune-depleted. When AML patients were dichotomized based on median immune scores, high versus low, a higher percentage of primary refractory patients was observed within the IFNγ high module (65.4% versus 34.6%; p=0.0022). In multivariate logistic regression, an IFNγ high profile (derived from the IFNγ-dominant module) was predictive of therapeutic resistance to induction chemotherapy, even more than ELN risk categories (AUROC = 0.815 versus 0.702 with ELN risk only). In a validation cohort, Beat AML series, the IFNγ high profile when compared to other clinically established prognostic indicators in AML, i.e. disease type (primary versus secondary), WBC count, patient age at diagnosis, and ELN risk categories was significantly more predictive of therapeutic resistance to induction chemotherapy (AUROC=0.921 versus 0.709 with ELN cytogenetic risk alone; two-tailed p value=0.002673). Similarly, a higher percentage of patients with an IFNγ high profile AML in the HOVON series failed to achieve CR in response to induction chemotherapy when compared to AML cases with an IFNγ low profile (27.2% versus 15.2%; p=0.0004). We hypothesized that higher expression of IFNγ inducible genes, while underpinning chemotherapy resistance, might identify AML patients who derive benefit from immunotherapy with FLZ. BM samples from 92% of patients with evidence of FLZ anti-leukemic activity (ALA), which was defined as either CR, CRh, CRi, PR or overall benefit (>30% reduction in BM blasts), had an immune infiltrated TME relative to non-responders (SD or PD). Interestingly, the IFNγ-signaling score was significantly higher in patients with chemotherapy-refractory AML compared with relapsed AML at time of FLZ treatment, and in individuals with evidence of ALA compared to non-responders (p Conclusions Our findings to date suggest that microenvironmental immune gene profiles could be used to inform the delivery of personalized immunotherapies to patients with IFNγ-dominant AML subtypes, and identify patients less likely to respond to cytotoxic chemotherapy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Hood:NanoString Technologies, Inc.: Employment. Church:NanoString Technologies, Inc.: Employment, Equity Ownership. Sullivan:NanoString Technologies, Inc.: Employment. Viboch:NanoString Technologies, Inc.: Employment. Warren:NanoString Technologies, Inc.: Employment. Liang:NanoString Technologies, Inc.: Employment. Cesano:NanoString Technologies, Inc.: Employment. Löwenberg:Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. DiPersio:Celgene: Consultancy; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Bioline Rx: Research Funding, Speakers Bureau. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding.

Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells. Acute myeloid leukemia (AML) is the second most common leukemia of childhood. Although intensive multi-agent chemotherapy in conjunction with improved supportive care has increased survival rates to 70%, 30-40% of children with AML relapse and only one-third of them will survive to adulthood. Investigation of new therapeutic strategies for high-risk AML, including immunotherapy, remains a priority and is being actively pursued. Importantly, AML-induced immune suppression poses a fundamental barrier to successful immune-based interventions. We previously showed that in vitro treatment of primary AML cells with interferon (IFN)-γ induces functional indoleamine 2,3-dioxygenase-1 (IDO1), a molecule with potent immunosuppressive properties, in 50% of unselected cases (IFN-γ responders). With a median follow-up of 8 years, IFN-γ responders experienced a significantly shorter event-free survival and overall survival (OS). Herein, we aimed to get insights into the immune landscape of childhood AML using multi-scale immune profiling strategies with the aim to identify molecular circuits that can be targeted to revert leukemia-induced immune dysfunction and improve clinical outcome. We employed a novel high-throughput digital platform, the nCounter system (nanoString Technologies, Seattle, USA), and Optimized Multi-color Immuno-phenotyping Panels (OMIPs) to comprehensively characterize the bone marrow immune infiltrate in 34 children diagnosed with non-promyelocytic AML (17 males, 17 females; median age at diagnosis=10 years; 28 de novo AML, 4 infant leukemia, 1 secondary AML, 1 congenital leukemia; 7 patients with favorable-risk cytogenetics, 21 patients with intermediate-risk cytogenetics and 6 patients with unfavorable-risk cytogenetics). The RNA Pan-Cancer Immune Profiling Panel, which includes 109 cell surface markers for 24 immune cell types, 30 cancer testis antigens, and >500 immune response genes, allowed us to measure immune gene expression levels on the clinic-ready nCounter® FLEX platform (nanoString Technologies). Transcriptomic data were normalized and analyzed using the nSolver software package. Relapse-free survival (RFS) and OS were selected as covariates. For immune phenotyping studies, viable bone marrow mononuclear cells were labeled with fluorochrome-conjugated antibodies and then analyzed on a 10-color Gallios flow cytometer (Beckman Coulter Life Sciences, Buckinghamshire, UK) with standard settings. Hierarchical clustering of immune gene expression data highlighted patient subgroups with heightened expression of T-cell and natural killer (NK)-cell function genes, as well as Toll-like receptor (TLR) genes, interleukin genes and tumor necrosis factor (TNF) family genes. Patients with high levels of CD8A mRNA also expressed IFNG, CXCL9, CXCL10, FOXP3 and negative checkpoints, including LAG3, CTLA4, IDO1 and CD279 (PD-L1), consistent with T-cell inflamed phenotypes that might underpin the establishment of adaptive immune resistance mechanisms of immune escape. In-depth phenotyping studies showed higher percentages of bone marrow-resident naïve B cells, plasmablasts, intermediate and non-conventional monocytes, plasmacytoid dendritic cells (DCs) and CXCR3-expressing Th1 cells at diagnosis compared with complete remission (CR). By contrast, both type 1 and type 2 myeloid DCs were more represented in the TME of patients with AML in CR. The frequencies of innate lymphoid cells (ILCs) and Vγ9+Vδ2+ T cells were not significantly different when comparing diagnosis and CR samples. Relapses occurred in 71.4% of children with “inflamed” AML compared with 28.6% of patients with “non-inflamed” AML (p=0.012; two-sided Fisher’s exact test). Importantly, RFS was remarkably shorter in patients with inflamed compared with non-inflamed AML (median RFS=394 days vs. undefined; hazard ratio = 0.266; 95% CI=0.11-0.64; p=0.0039, log-rank test). A non-significant association with OS was also observed. In conclusion, inflamed or “hot” AMLs are associated with shorter RFS and might be amenable to immunotherapy approaches tailored to the bone marrow TME, including PD-1/PD-L1 blockade and/or small-molecule IDO1 inhibitors. Grant support: Roger Counter Foundation, Dorset, UK and Qatar National Research Fund (grant #NPRP8-2297-3-494). Citation Format: Jayakumar Vadakekolathu, Gemma A. Foulds, Tasleema Patel, Stephen Reeder, Alan Graham Pockley, Sarah K. Tasian, Sergio Rutella. Immune gene expression profiling identifies predictors of relapse in childhood acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B62.

50 Background: Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells. We characterized the immune ecosystem of the bone marrow (BM) TME in patients with acute myeloid leukemia (AML) to correlate transcriptomic and proteomic profiles with patient outcome. Methods: We used the nCounter system (NanoString Technologies) to characterize BM specimens from 42 children and 28 adults with AML. Ninety BM samples (63 from de novo AML, 18 from AML in complete remission [CR] and 9 from relapsed AML) were analyzed using the RNA Pan-Cancer Immune Profiling Panel. In situ leukemia-immune system interactions were visualized using Digital Spatial Profiling (DSP). Results: Hierarchical clustering identified subgroups with heightened expression of lymphocyte-associated genes, including NK cell and cytotoxicity genes (“immune-enriched” samples). They also expressed CD8A, IFNG, FOXP3, the cell chemoattractants CXCL9, CXCL10, and inhibitory molecules including IDO1 and the immune checkpoints LAG3, CTLA4 and PD-L1. Conversely, ‘immune-depleted’ samples over-expressed genes associated with mast cell functions and CD8 T-cell exhaustion and showed low expression of T-cell and B-cell genes. Importantly, relapse-free survival (RFS) and overall survival (OS) were significantly shorter in patients with “immune-enriched” compared with “immune-depleted” AMLs (2.2 vs. 18.3 months, p = 0.0064, and 6.3 vs. 22.4 months, p = 0.017, respectively). DSP of the BM TME showed the co-localization of CD8 T cells with FoxP3 Treg cells and PD-L1- and VISTA-expressing cell types, an immune landscape which is consistent with the establishment of adaptive immune resistance mechanisms of immune escape. Conclusions: Our study has identified heterogeneous immune profiles in children and adults with AML. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment, including blockade of co-inhibitory molecules and/or small-molecule IDO1 inhibitors. Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494).