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Supplementary Data from DIPG Harbors Alterations Targetable by MEK Inhibitors, with Acquired Resistance Mechanisms Overcome by Combinatorial Inhibition

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation.Significance:We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic.This article is highlighted in the In This Issue feature, p. 587

Figure S1 - PDF file 105K, Figure S1. Neuroblastoma cell lines do not express 4-1BB, CD40 or CTLA-4. The expression of 4-1BB, CD40 and CTLA-4 on naive and activated splenocytes (0.5 mg anti-CD3 i.v. 48 hr) and the neuroblastoma cell lines was determined by indirect flow cytometry. Cells were incubated with 10 microg/ml unlabelled mAb, either isotype control (Mc39/16) anti-4-1BB (LOB12.3), anti-CD40 (3/23) or anti-CTLA-4 (UC10 4F10-11) prior to washing and incubation with PE-conjugated anti-mouse Fc. Expression of 4-1BB, CD40 and CTLA-4 could be observed on T-cells from activated splenocytes, but not on naive T-cells as expected. Expression of 4-1BB, CD40 and CTLA-4 was not evident on Neuro2a, AgN2a or NXS2 cell lines. Data represents an example of three experiments

Purpose: Neuroblastoma is one of the commonest extracranial tumors of childhood. The majority of patients present with metastatic disease for which outcome remains poor. Immunotherapy is an attractive therapeutic approach for this disease, and a number of neuroblastoma tumor antigens have been identified. Here, we examine the therapeutic potential of combining immunomodulatory monoclonal antibodies (mAb) with peptide vaccination in murine neuroblastoma models.Experimental Design: Neuroblastoma-bearing mice were treated with mAb targeting 4-1BB, CD40, and CTLA-4 alone, or in combination with a peptide derived from the tumor antigen survivin (GWEDPPNDI). Survivin-specific immune response and therapeutic efficacy were assessed.Results: In the Neuro2a model, treatment of established tumor with anti-4-1BB, anti-CD40, or anti-CTLA-4 mAb results in tumor regression and long-term survival in 40% to 60% of mice. This is dependent on natural killer (NK) and CD8+ T cells and is associated with tumor CD8+ lymphocyte infiltrate. Successful therapy is achieved only if mAb is given to mice once tumors are established, suggesting dependence on sufficient tumor to provide antigen. In the more aggressive AgN2a and NXS2 models, single-agent mAb therapy provides ineffective therapy. However, if mAb (anti-CTLA-4) is given in conjunction with survivin peptide vaccination, then 60% long-term survival is achieved. This is associated with the generation of survivin-specific T-cell immunity, which again is only shown in the presence of tumor antigen.Conclusions: These data suggest that the combination of antigen and costimulatory mAb may provide effective immunotherapy against neuroblastoma and may be of particular use in the minimal residual disease setting. Clin Cancer Res; 19(13); 3545–55. ©2013 AACR.

The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of ‘hematopoietic’ and ‘adipocyte rich’ bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.

Background: National advisory panels (NAPs) have been established for the care of children and young people (CYP) with cancer in the United Kingdom since 2011, with an increase in panel number in recent years. Their practice has not previously been reviewed; therefore, we sought to evaluate the role, practice and impact of six selected NAPs offering expertise in ependymoma, histiocytosis, leukaemia, neuroblastoma, renal tumours and sarcoma. Procedure: This service evaluation used mixed methodology, including review of NAP documentation, semi-structured interviews with the NAP chairs and an analysis of the cases referred for discussion. Results: Total 1110 referrals were analysed. Results demonstrated the significant scope and amount of work undertaken by the NAPs, largely testament to the commitment of the panel members. Specific roles fulfilled have been highlighted, and NAP recommendations have been shown to influence clinical decision-making and be implemented in the majority of cases. Despite widespread good practice, areas to address have been identified; these include clarity regarding NAP membership, consistency in recommendations, the consideration of holistic information to promote personalised management and the exploration of wider multidisciplinary team roles. Conclusions: In the context of increasing demand and the escalating number of NAPs, it is timely to consider how service improvement can be facilitated. Best practice guidelines have been formulated as a product of this study, to promote a sustainable and effective model for NAPs. Review and benchmarking national panel performance against these guidelines will drive high standards of care going forward and they should be embedded as standard practice.

BackgroundDespite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc region of anti-PD-1 mAbs and Fcγ receptors (FcγRs) affect therapeutic activity and how these are impacted by the immune environment.MethodsMouse and human anti-PD-1 mAbs with different Fc binding profiles were generated and characterized in vitro. The ability of these mAbs to elicit T-cell responses in vivo was first assessed in a vaccination setting using the model antigen ovalbumin. The antitumor activity of anti-PD-1 mAbs was investigated in the context of immune ‘hot’ MC38 versus ‘cold’ neuroblastoma tumor models, and flow cytometry performed to assess immune infiltration.ResultsEngagement of activating FcγRs by anti-PD-1 mAbs led to depletion of activated CD8 T cells in vitro and in vivo, abrogating therapeutic activity. Importantly, the extent of this Fc-mediated modulation was determined by the surrounding immune environment. Low FcγR-engaging mouse anti-PD-1 isotypes, which are frequently used as surrogates for human mAbs, were unable to expand ovalbumin-reactive CD8 T cells, in contrast to Fc-null mAbs. These results were recapitulated in mice expressing human FcγRs, in which clinically relevant hIgG4 anti-PD-1 led to reduced endogenous expansion of CD8 T cells compared with its engineered Fc-null counterpart. In the context of an immunologically ‘hot’ tumor however, both low-engaging and Fc-null mAbs induced long-term antitumor immunity in MC38-bearing mice. Finally, a similar anti-PD-1 isotype hierarchy was demonstrated in the less responsive ‘cold’ 9464D neuroblastoma model, where the most effective mAbs were able to delay tumor growth but could not induce long-term protection.ConclusionsOur data collectively support a critical role for Fc:FcγR interactions in inhibiting immune responses to both mouse and human anti-PD-1 mAbs, and highlight the context-dependent effect that anti-PD-1 mAb isotypes can have on T-cell responses. We propose that engineering of Fc-null anti-PD-1 mAbs would prevent FcγR-mediated resistance in vivo and allow maximal T-cell stimulation independent of the immunological environment.

Introduction Neuroblastoma is a cancer of the sympathetic nervous system that causes up to 15% of cancer-related deaths among children. Among the ~1,000 newly diagnosed cases per year in Europe, more than half are classified as high-risk, with a 5-year survival rate Areas covered The authors provide a critical review on methodological advances aimed at providing new therapeutic opportunities for neuroblastoma patients, including preclinical models of human disease, generation of omics data to discover new therapeutic targets, and artificial intelligence-based technologies to implement personalized treatments. Expert opinion While survival of childhood cancer has improved over the past decades, progress has been uneven. Still, survival is dismal for some cancers, including high-risk neuroblastoma. Embracing new technologies (e.g. molecular profiling of tumors, 3D in vitro models, etc.), international collaborative efforts and the incorporation of new therapies (e.g. RNA-based therapies, epigenetic therapies, immunotherapy) will ultimately lead to more effective and safer therapies for these subgroups of neuroblastoma patients.

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation. Significance: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587

There is increasing recognition that contralateral metachronous tumor may occur following treatment of unilateral mature ovarian teratoma. We aimed to define this risk to guide appropriate surveillance strategies. We undertook a systematic review of three large medical databases (Ovid Medline, Embase, and Cochrane Controlled Trials Register) to April 2020 using a defined search strategy. From 1831 articles retrieved, 23 were included, reporting 1101 girls with unilateral mature ovarian teratomas. The intensity and duration of follow-up varied between studies, with only five reporting close surveillance. Overall prevalence of metachronous contralateral mature teratoma was 2.1%, with a prevalence per study of 0%–23% (median 0%). Prevalence was higher (7%) among studies with more robust surveillance. These data suggest a small but real risk of metachronous contralateral tumors. Surveillance ultrasonography is proportionate and indicated alongside further prospective data collection to record the natural history and impact of surveillance in greater detail.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

The third multistakeholder Paediatric Strategy Forum organised by ACCELERATE and the European Medicines Agency focused on immune checkpoint inhibitors for use in combination therapy in children and adolescents. As immune checkpoint inhibitors, both as monotherapy and in combinations have shown impressive success in some adult malignancies and early phase trials in children of single agent checkpoint inhibitors have now been completed, it seemed an appropriate time to consider opportunities for paediatric studies of checkpoint inhibitors used in combination. Among paediatric patients, early clinical studies of checkpoint inhibitors used as monotherapy have demonstrated a high rate of activity, including complete responses, in Hodgkin lymphoma and hypermutant paediatric tumours. Activity has been very limited, however, in more common malignancies of childhood and adolescence. Furthermore, apart from tumour mutational burden, no other predictive biomarker for monotherapy activity in paediatric tumours has been identified. Based on these observations, there is collective agreement that there is no scientific rationale for children to be enrolled in new monotherapy trials of additional checkpoint inhibitors with the same mechanism of action of agents already studied (e.g. anti-PD1, anti-PDL1 anti-CTLA-4) unless additional scientific knowledge supporting a different approach becomes available. This shared perspective, based on scientific evidence and supported by paediatric oncology cooperative groups, should inform companies on whether a paediatric development plan is justified. This could then be proposed to regulators through the available regulatory tools. Generally, an academic-industry consensus on the scientific merits of a proposal before submission of a paediatric investigational plan would be of great benefit to determine which studies have the highest probability of generating new insights. There is already a rationale for the evaluation of combinations of checkpoint inhibitors with other agents in paediatric Hodgkin lymphoma and hypermutated tumours in view of the activity shown as single agents. In paediatric tumours where no single agent activity has been observed in multiple clinical trials of anti-PD1, anti-PDL1 and anti-CTLA-4 agents as monotherapy, combinations of checkpoint inhibitors with other treatment modalities should be explored when a scientific rationale indicates that they could be efficacious in paediatric cancers and not because these combinations are being evaluated in adults. Immunotherapy in the form of engineered proteins (e.g. monoclonal antibodies and T cell engaging agents) and cellular products (e.g. CAR T cells) has great therapeutic potential for benefit in paediatric cancer. The major challenge for developing checkpoint inhibitors for paediatric cancers is the lack of neoantigens (based on mutations) and corresponding antigen-specific T cells. Progress critically depends on understanding the immune macroenvironment and microenvironment and the ability of the adaptive immune system to recognise paediatric cancers in the absence of high neoantigen burden. Future clinical studies of checkpoint inhibitors in children need to build upon strong biological hypotheses that take into account the distinctive immunobiology of childhood cancers in comparison to that of checkpoint inhibitor responsive adult cancers. (C) 2020 The Authors. Published by Elsevier Ltd.

Background Antimicrobial resistance (AMR) is being recognized as a priority by healthcare organizations across the world. However, many children are managed on IV antimicrobials in hospital with very little consideration of antimicrobial stewardship issues. Objectives A nurse-led paediatric ambulatory outpatient parenteral antimicrobial therapy (OPAT) service, managing children with common infections being ambulated on short courses of IV antimicrobials, was introduced within Southampton Children’s Hospital in January 2018. We evaluated the impact of this service in terms of the quality of antimicrobial prescribing and timing of ambulation in children presenting with common infections. Methods All cases managed within the service were reviewed in two separate 2 month time periods: prior to introduction of the service (September–October 2016) and then prospectively after its introduction (September–October 2018). Results A total of 96% of IV antibiotic management decisions at 48 h were deemed appropriate in 2018, compared with 75% in 2016. A total of 64% of patients were ambulated on IV antibiotics at some point during their treatment course in 2018, compared with 19% in 2016. However, a significant proportion of antimicrobial decisions made at the point of presentation to hospital remained suboptimal in 2018. Conclusions Children are commonly managed with IV antibiotics in hospital. We demonstrate marked improvements in appropriate antimicrobial use through the introduction of a nurse-led ambulatory OPAT service. In addition, such a service can promote a greater proportion of children being ambulated from hospital, freeing up valuable inpatient beds and potentially delivering cost savings that can be used to fund such services.

Checkpoint blockade (CPB) immunotherapy has shown unprecedented success in a wide range of adult malignancies, and is increasingly being employed in the treatment of advanced cancers. However, the experience in the paediatric population remains limited and the small number of single agent studies reported have shown disappointing response rates. Paediatric cancers offer unique challenges that can hinder the translation of CPB into the paediatric clinic, and combinational therapies are likely to be needed to achieve therapeutic success. As the number of paediatric trials using CPB rapidly increases, understanding the challenges that these agents may encounter in this population is of special significance to allow the design of optimal combinatorial strategies for each tumour type. Here, we offer an overview of the unique biological and immunological features of paediatric cancers as compared to adult malignancies, and how these might impact the overall success of CPB in the paediatric population. We review the growing body of pre-clinical and clinical experiences to date, and discuss future strategies involving the combination of CPB with traditionally used therapies (chemotherapy and radiotherapy) or with other newly developed immunotherapies.

ObjectiveTo determine the likely rate of patient randomisation and to facilitate sample size calculation for a full-scale phase III trial of varicella zoster immunoglobulin (VZIG) and aciclovir as postexposure prophylaxis against chickenpox in children with cancer.DesignMulticentre pilot randomised controlled trial of VZIG and oral aciclovir.SettingEngland, UK.PatientsChildren under 16 years of age with a diagnosis of cancer: currently or within 6 months of receiving cancer treatment and with negative varicella zoster virus (VZV) serostatus at diagnosis or within the last 3 months.InterventionsStudy participants who have a significant VZV exposure were randomised to receive PEP in the form of VZIG or aciclovir after the exposure.Main outcome measuresNumber of patients registered and randomised within 12 months of the trial opening to recruitment and incidence of breakthrough varicella.ResultsThe study opened in six sites over a 13-month period. 482 patients were screened for eligibility, 32 patients were registered and 3 patients were randomised following VZV exposure. All three were randomised to receive aciclovir and there were no cases of breakthrough varicella.ConclusionsGiven the limited recruitment to the PEPtalk2 pilot, it is unlikely that the necessary sample size would be achievable using this strategy in a full-scale trial. The study identified factors that could be used to modify the design of a definitive trial but other options for defining the best means to protect such children against VZV should be explored.Trial registration numberISRCTN48257441, EudraCT number: 2013-001332-22, sponsor: University of Birmingham.

10536 Background: We previously developed a risk factor model in HR-NBL1/SIOPEN patients treated without DB including age, LDH and metastatic site index (MSI). We tested if this score (Morgenstern, PBC 2018) would provide meaningful information in DB treated HR-NBL1/SIOPEN patients. Methods: High-risk patients (stage 4 ≥1yr.; stage 4 < 1yr. with MYCN amplification (MNA); stage 2, 3, 0-21y with MNA) received intensive induction, surgery, high dose therapy (HDT) and local radiotherapy (21Gy). Patients with a ≤9 months intervall till HDT/SCT, ≥ partial response prior and no progression received up to 5 cycles of 100mg/m2 DB: as short-term (STI: 5 days) or long-term infusion (LTI: 10 days ) ± 6 (STI) or 3 (LTI) x 106 IU/m2 subcutaneous interleukin 2 (scIL2 over 2 x 5 days) followed by 2 weeks of 160mg/m2 oral isotretinoin. Results: DB was used in 1018 patients [512 males; 89% Stage, 1% Stage 4s, 10% loc. MNA; 2.8 yrs median age at diagnosis] in 18 countries. STI was used in 61% patients, LTI in 39% and DB without scIL2 in 62% patients. 2-yrs. event-free (EFS)/overall survival (OS) was 0.65±0.02/0.78±0.01 and 5-yrs. EFS/OS of 0.56±0.02/0.63±0.02. 891/1018 patients were evaluable for risk factor analysis. EFS multivariate analysis included age, MSI, LDH ≥1250U/L, HDT typ, response prior DB, DB schedule and use of scIL2: only age > 1.5yrs.(p = 0.002) and MSI > 1 (p = 0.002) had independent prognostic prediction. If restricted to stage 4 only, LDH ≥1250U/L was also an independent factor (p = 0.049). Points were allocated in proportion to the log-cumulative hazard ratio (cHR): cHR for LDH > 1250U/L was 1.33 (score of 1), cHR for age > 5 years and metastatic site involvement (MSI) > 1 were 1.79 and 1.86 (scores of 2). Using the score (0 to 5) relevant risk groups can be identified: 5-yrs. EFS/OS for scores 0&1 are 0.80±0.06&0.77±0.04, score 2/3 are 0.58±0.03&0.57±0.04 and for scores 4/5 are 0.50±0.06&0.43±0.08. Conclusions: The score adds value in DB treatment groups as it differentiates prognostic subgroups facilitating decision making for future DB add on treatments. Clinical trial information: NCT01704716.

GD2-directed immunotherapies improve survival of high-risk neuroblastoma (NB) patients (pts). Treatment with chimeric anti-GD2 antibodies (Ab), such as ch14.18, can induce development of human anti-chimeric Ab (HACA). Here, we report HACA effects on ch14.18/CHO pharmacokinetics, pharmacodynamics and pain intensity in pts treated by long-term infusion (LTI) of ch14.18/CHO combined with IL-2. 124 pts received up to 5 cycles of ch14.18/CHO 10 days (d) infusion (10 mg/m2/d, d8&ndash, 18) combined with s.c. IL-2 (6 &times, 106 IU/m2/d, d1&ndash, 5, d8&ndash, 12). HACA, treatment toxicity, ch14.18/CHO levels, Ab-dependent cellular- (ADCC) and complement-dependent cytotoxicity (CDC) were assessed using respective validated assays. HACA-negative pts showed a steadily decreased pain in cycle 1 (74% pts without morphine by d5 of LTI) with further decrease in subsequent cycles. Ch14.18/CHO peak concentrations of 11.26 &plusmn, 0.50 &micro, g/mL found in cycle 1 were further elevated in subsequent cycles and resulted in robust GD2-specific CDC and ADCC. Development of HACA (21% of pts) resulted in strong reduction of ch14.18/CHO levels, abrogated CDC and ADCC. Surprisingly, no difference in pain toxicity between HACA-positive and -negative pts was found. In conclusion, ch14.18/CHO LTI combined with IL-2 results in strong activation of Ab effector functions. Importantly, HACA response abrogated CDC but did not affect pain intensity indicating CDC-independent pain induction.

Background Langerhans Cell Histiocytosis (LCH) describes a clinical spectrum of disease caused by accumulation of pathologicallangerhan’scells infiltrating tissues. Further understanding regarding the patho-physiology and natural history of langerhans cell histiocytosis has emerged with the understanding of its status as a clonal BRAF stimulated malignancy. Objectives To characterise the disease presentations of Langerhans Cell Histiocytosis in a UK Principal treatment centre (PTC) over the past 10 years. Methods Our PTC serves a population of 3.6 million sharing care with 11 regional shared care centres. A retrospective case review was performed for all patients treated for LCH over the last 10 years. Data were collected on demographics, disease location, treatment modality, intervals of remission free outcome and latest evaluation at follow up. Results 1041 children have been treated for cancerover the past 10 years (1/1/2006–31/12/2016). We identified 45 cases of LCH from children aged 2 mths to 16 years (median age of 2 years 6 mths) in our database accounting for 4% of our patients. There were 44% females and 58% males. 64% of cases presented with single-system disease at diagnosis and 36% with multi-system disease. 4 children were identified as having diabetes insipidus at diagnosis 2.5%. 33/45 (73%) had a biopsy performed consistent with LCH, 10 had no biopsy performed and 2 had a biopsy which was negative but were treated as per LCH due to typical clinic-radiological correlation. 2 cases of neurodegenerative LCH were identified in this case series. Multi-system patients have a more challenging course and in our series 10/18 (56%) went on to have a recurrence and of these 2 patients have had 4 relapses and are still undergoing further therapy. Conclusions Our centres experience over the past 10 years which shows the full spectrum of presentation from the benign self-limiting single system bony disease to that of extensive multi-system disease leading to secondary HLH. Novel strategies to evaluate and monitor BRAF in blood and urine such as those being employed in the latest international LCH IV trial offer the opportunity to better understand the risk factors for disease severity.

Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor.

Monoclonal antibodies (mAbs) have inaugurated the concepts of tumor-targeted therapy and personalized medicine. A new family of mAbs is currently emerging in the clinic, which target immune cells rather than cancer cells. These immune-targeted therapies have recently demonstrated long-term tumor responses in adults with refractory/relapsing metastatic solid tumors. Pediatric cancers are different from their adult counterparts in terms of histological features and immune infiltrates. However, the same immune checkpoint targets can be expressed within the microenvironment of pediatric tumors. The benefits of immune checkpoint blockade in pediatric cancers are currently under evaluation in early phase clinical trials.

Purpose To determine the prevalence of osteonecrosis (ON) in children following treatment of acute lymphoblastic leukaemia (ALL), characterise these cases and review treatment methods. Methods All children diagnosed and treated for ALL between 01 January 2003 and 31 December 2013 at our centre were retrospectively reviewed. Logistic regression was used to investigate risk factors for ON occurrence. Results Of 235 children treated for ALL, 48/235 (20.4%) children suffered musculoskeletal symptoms necessitating radiological investigation. A total of 13 (5.5%) had MRI-diagnosed ON, with a median diagnosis time of 12 months (interquartile range 10 to 14) following initiation of chemotherapy. ON affected 40 joints in 13 children. The most commonly involved joints were hips (14 joints in eight patients) and knees (12 joints in seven patients). Older age at ALL diagnosis was associated with significantly increased risk of development of ON per year (odds ratio 1.35, 95% confidence interval 1.17 to 1.57, p < 0.001). Eight children underwent at least one surgical intervention. Joint arthroplasty was undertaken in nine joints of four children at a mean age of 18.3 years. All patients who underwent hip arthroplasty had previously received core decompression, with a mean time of 27.8 months (18 to 33) between treatments. Conclusions ON is a significant complication of ALL treatment. Our results suggest risk stratification for development of ON by age, and targeted monitoring of high-risk joints is possible. ON treatment is varied with little evidence base.

The advent of monoclonal antibody technology in the 1970s brought with it the possibility of generating virtually unlimited amounts of pure antibody targeting almost any antigen of choice, opening the door to widespread application. Since the first monoclonal antibody was licensed for clinical use 30 years ago, there has been an exponential growth in our knowledge of how they may be used therapeutically, particularly in the treatment to cancer. The attraction of antibodies as therapeutics lies in part in their exquisite specificity, with little off target binding, but also in their relative ease of production and storage (as compared to cellular immunotherapies) and their long in vivo half-life (as compared to small molecules). Although the development of monoclonal antibodies for paediatric cancers has lagged behind their use for adult malignancies, there are increasing numbers of antibodies in paediatric clinical trials, and in 2015, dinutuximab, the first monoclonal antibody specifically for a paediatric malignancy (neuroblastoma), was approved.

10014 Background: We determined the role of scIL-2 combined with long term infusion (LTI) of DB in patients (pts) with high-risk relapsed/refractory neuroblastoma. Methods: 160 pts were enrolled into an open label SIOPEN Phase II clinical trial (EudraCT 2009-018077-31). Pts were randomly assigned to receive up to 5 cycles of 100 mg/m2 DB-LTI (d8-17) and 160 mg/m2 oral isotretinoin (d19-32) (81 pts) with and without 6x106IU/m2 scIL-2 (d1-5; 8-12) (79 pts). Endpoints were toxicity, response rates and 2yrs-event free and -overall survival. Results: Between 07/2014 and 07/2017, 160 pts from 11 countries were randomised. Median follow-up is 2.6 years. Pts were well balanced between arms according to stage, age, MYCN amplification, patients with relapse and remission status. The 2yrs-EFS and -OS for DB (81 pts) vs. DB combined with scIL-2 (79 pts) was 59%±6% vs 65%±6% (p = 0.721) and 79%±5% vs 84%±4% (p = 0.904). In 97 pts with evaluable disease, a response rate of 49% (9% CR, 40% PR) vs 52% (26% CR, 26% PR) after treatment with DB vs DB and scIL-2 was observed. Grade 3&4 fever (16% vs 46%, P = 0.000), allergic reaction (1% vs 14%, P = 0.004), hematological toxicity (46% vs 66%, P = 0.013) and neurotoxicity (0% vs 8%, p = 0.003) were significantly worse in the combination arm, but no difference was seen for capillary leak, gastrointestinal, liver enzyme elevation and pain. Paraplegia possibly related to the treatment was observed in 2 pts in the combination arm, none in the arm without scIL-2, and one resolved to baseline. A subgroup of 34 pts who had a relapse and measurable disease at treatment start, showed a 2yrs-EFS and -OS in DB (17 pts) vs DB combined with scIL-2 (17 pts) of 35%±12% vs 69%±12% (p = 0.116) and 59%±12% vs 81%±10% (p = 0.167). However, this trend was statistically not significant. Pharmacokinetic and HACA response between both arms was not different with overlapping antibody concentration-time curves and a HACA response of 15/81 (19%) (DB) vs 16/79 (20%) (DB and scIL-2). Conclusions: No significant difference in efficacy of DB combined with scIL-2 and increased toxicity in this arm suggests that this schedule of scIL-2 is of no additional benefit. Clinical trial information: 2009-018077-31.

We report the case of a 14-year-old male with metastatic alveolar rhabdomyosarcoma, presenting with hypercalcaemia (3.89 mmol/l) and elevated parathyroid hormone (PTH) level (10.2 pmol/l). Imaging demonstrated extensive bony lytic damage, with "floating teeth" in the mandible. Normalisation of calcium levels and bony reformation of the mandible occurred following chemotherapy; PTH levels decreased initially but remained above normal levels. Imaging did not demonstrate any evidence of parathyroid abnormality. Tumour ectopic PTH secretion is a very rare cause of hypercalcaemia of malignancy in children. Hypercalcaemia with an elevated PTH, in the absence of parathyroid-related cause, should prompt investigation for underlying malignancy.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic. Buchan et al. reveal dual anti-tumor activities for antibodies to the co-stimulatory receptor 4-1BB, which depend on antibody isotype and FcγR availability. Sequential scheduling of anti-4-1BB and checkpoint blockade mAbs, and antibodies engineered to harness both Treg cell depleting and effector cell agonism properties show potent anti-tumor activity in preclinical models, laying the groundwork for translation into the clinic.

Introduction Neuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB. Material and methods The effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma. Results and discussions Chemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice. Conclusion This work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.

10539Background: Randomization of immunotherapy versus standard was not possible in the HR-NBL1/SIOPEN trial. In order to explore an impact of immunotherapy on outcome, we used trial patients prior...

111 Background: Receptors expressed on natural killer (NK) cells and their ligands on target cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC) may influence outcome in patients (pts) with high-risk relapsed/refractory neuroblastoma (NB) treated by long-term infusion (LTI) of anti-GD2 antibody ch14.18/CHO Methods: 53 pts received 6x106 IU/m2 sc IL-2 (d1-5; 8-12), LTI of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in a closed single center program (53 pts) (APN311-303). Polymorphisms in Fcγ-receptor genes 2A (H131R), -3A (V158F) and -3B (NA1/NA2), and KIR- as well as KIR ligand expression (HLA-C1; HLA-C2; HLA-Bw4+) were determined by real-time PCR. Results: The survival update of the APN311-303 cohort revealed a 5-y OS of 56.4±7.1% (mean OS 4.35y [0.3-6.2y]) and a 5-y PFS of 29.1±6.3% (mean PFS 2.4y [0.1 - 5.9y]). Median TTP/PFS was 1.35 y (95% CI: 0.21-2.48 y). This result is clearly superior to historical controls not treated with ch14.18/CHO with a 5-y OS of 14.8±6.8% (p < 0.005), indicating clinical efficacy of the treatment. In this cohort, we identified 26/53 pts with the NK- stimulatory KIR haplotype (2DS2+) who had superior OS- and PFS-rates compared to 18/53 pts with an inhibitory haplotype and KIR/KIR-ligand match (2DS2-, 3DL1+, Bw4+) (p < 0.02). Similarly, we found 33/53 pts with low affinity FCGR alleles (FCGR2A-H131R/R and/or FCGRA3A-V158 F/F). These patients showed lower PFS rates compared to 20/53 patients with high affinity FCGR polymorphisms (p < 0.01). ADCC analysis on day 15 of cycle 1 showed higher levels in the pt group with NK- stimulatory versus NK- inhibitory KIR/KIR ligand haplotypes (23±6 % vs. 6±2%, p < 0.01). A similar effect was observed in pts with high affinity FCGR polymorphisms with an ADCC increase of 20±6% compared to 11±2% in the low affinity FCGR control. Conclusions: KIR-haplotypes and FCGR- polymorphisms correlated with the functional immune parameter ADCC and clinical outcome, and thus support the relevance of ADCC for clinical efficacy of ch14.18/CHO in neuroblastoma.

10500Background: Dinutuximab (ch14.18/SP2/0) in combination with cytokines (IL2 and GM-CSF) resulted in a survival benefit in high risk neuroblastoma (NB) patients (2y EFS 66%±5%, A.Yu, NEJM, 2010)...

10562Background: A new delivery method of anti-GD2 antibody ch14.18/CHO by long term infusion (LTI) may improve the toxicity profile but maintain effective immune modulation and clinical activity i...

10548Background: Receptors expressed on natural killer (NK) cells and their ligands on target cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC) may predict outcome in patients ...

Nat. Genet. 47, 1471–1474 (2015); published online 9 November 2015; corrected after print 8 February 2016 In the version of this article initially published, the authors failed to acknowledge funding from the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS FoundationTrust and University College London to Neil Sebire.

Background: Ganglioside GD2 is a glycolipid highly expressed on neuro-ectodermal tumors including melanoma and neuroblastoma (NB). It is ranked on position 12 of the list of tumor associated antigens by the NCI. Anti-GD2 antibody (Ab) ch14.18/CHO targets this antigen and effectively orchestrates innate immune effector mechanisms against GD2 expressing malignancies. However, one on target effect of anti-GD2 Abs is the induction of neuropathic pain when applied as short term infusion (STI) requiring co-medication with intravenous morphine. Here we investigated whether long-term infusion (LTI) of anti-GD2 Ab ch14.18/CHO may have an improved pain toxicity profile and at the same time mediate an effective immune response in patients (pts) with high risk relapsed/refractory NB which translates to objective clinical responses and an improved survival. Methods: 97 pts received 6x106 IU/m2 sc IL2 (d1-5; 8-12), LTI of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in an ongoing SIOPEN Phase II study (APN311-202) (NCT01701479) (44 pts) and a closed single center program (53 pts) (APN311-303). Response assessments followed INRG criteria. Serum ch14.18/CHO concentration-time curves were determined by validated ELISA methods and pharmacokinetics parameter were analyzed using standard non-compartmental methods. Fcγ;-receptor polymorphisms FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) and patient-specific antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and whole blood cytotoxicity (WBT) responses were determined. Results: LTI was associated with low pain scores and a decreasing morphine usage compared to STI. Clinical overall responses were 30% (APN311-303) and 31% (APN311-202). The survival update of the APN311-303 cohort revealed a 1-y & 4-y OS of 94.2±3.2% & 60.9±9.0% (median FU 2.9y [0.7-5.2y]) and a 1-y & 4-y PFS of 54.4±6.9% & 32.3%±6.9% (median FU 2.8y [0.7 - 4.9y]). Median TTP was 571d (95% CI: 232.7d). The comparator is the reported historical gold standard for relapsed refractory NB patients with 1-y & 4-y PFS of 19±2% & 8±3% and OS of 56±3% & 14±4% and a median TTP of 63 d (95% CI: 56.8d). NB pts with high affinity FCGR alleles and an increase in ADCC (cut off 15%) are associated with longer PFS and OS rates (p Parameters of immune modulation (CDC, and WBT) and PK of ch14.18/CHO were comparable between APN311-202 and -303 cohorts. PK of ch14.18/CHO was analyzed in cycle 1: Cmax=12.2±0.4μg/ml, t½=8.4±1.1 d, AUC=145.3± 5.8 μg*d/ml, Vdss=9.3±0.5L/m2. A pro-inflammatory cytokine response (IL-2, IL-6, IL-8, IFNγ) translated into the expansion of effector NK- (3x) and T-cells (2x). We observed HACA in 17/97 pts (17.5%) of which only 9/97 (9.3%) were neutralizing with respect to the inhibition of CDC and WBT activity. In HACA negative patients, levels of ch14.18/CHO and functional parameters (CDC and WBT) analyzed before subsequent treatment cycles indicate persistent anti-NB activity for the entire treatment period. Conclusion: LTI of ch14.18/CHO has an improved pain toxicity profile and at the same time is active and effective in high-risk relapsed/refractory NB. Citation Format: Holger N. Lode, Dominique Valteau-Couanet, Alberto Garaventa, Juliet Gray, Victoria Castel, Isaac Yaniv, Nikolai Siebert, Christian Jensen, Stefanie Endres, Lena Pill, Christin Eger, Diana Seidel, Madlen Jüttner, Silke Kietz, Karoline Ehlert, Evelyne Janzek, Carla Manzitti, Ina Müller, Hans Loibner, Ruth Ladenstein. Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A032.

Recent monoclonal antibody trials targeting the PD1/PD-L1 immune-checkpoint pathway have shown remarkable success in treating adult malignancies, with PD-L1-expressing tumors showing the most objective response. However, little is known as to whether pediatric cancers have also adopted this immune evasion mechanism. We evaluated 115 pediatric tumors (taken at diagnosis) for PD-L1 expression and the presence of CD8+ tumor-infiltrating lymphocytes (TILs). Tumors with >5% PD-L1 membrane staining were scored positive. The presence of CD8+ TILs expressing PD-1 was assessed using dual-labeling immunohistochemistry. Data were evaluated against clinical demographics. The proportion of PD-L1+ tumors was 86% for alveolar rhabdomyosarcoma (12/14), 72% for high-risk neuroblastoma (31/43), 57% for Ewing's sarcoma (8/14), 50% for embryonal rhabdomyosarcoma (8/16) and 47% for osteosarcoma (7/15). Increased proportions of CD8+ TILs significantly correlated with PD-1 expression. When grouped by cancer type, those with the...

TPS10080 Background: Long-term infusion (LTI) of anti-GD2 antibody ch14.18/CHO may improve outcome in patients (pts) with high risk relapsed/refractory neuroblastoma (NB). Methods: 97 pts received ...