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Immune checkpoint therapy has resulted in minimal clinical response in many pediatric cancers. We sought to understand the influence of immune checkpoint inhibition using anti-PD-1 and anti-CTLA-4 antibodies individually, in combination, and after chemotherapy on immune responses in minimal and established murine neuroblastoma models. We also sought to understand the role of the tumor microenvironment (TME) and PD-L1 expression and their alteration post-chemotherapy in our models and human tissues. PD-L1 expression was enriched in human tumor-associated macrophages and up-regulated after chemotherapy. In a murine minimal disease model, single and dual immune checkpoint blockade promoted tumor rejection, improved survival, and established immune memory with long-term anti-tumor immunity against re-challenge. In an established tumor model, only dual immune checkpoint blockade showed efficacy. Interestingly, dual immune checkpoint therapy distinctly influenced adaptive and innate immune responses, with significant increase in CD8+CD28+PD-1+ T cells and inflammatory macrophages (CD11bhiCD11c−F4/80+Ly6Chi) in tumor-draining lymph nodes. Adding chemotherapy before immunotherapy provided significant survival benefit for mice with established tumors receiving anti-PD-1 or dual immune checkpoint blockade. Our findings demonstrate anti-PD-1 and anti-CTLA-4 therapy induces a novel subset of effector T cells, and support administration of induction chemotherapy immediately prior to immune checkpoint blockade in children with high-risk neuroblastoma.

// Michael D. Hadjidaniel 1, * , Sakunthala Muthugounder 1, * , Long T. Hung 1, * , Michael A. Sheard 1 , Soheila Shirinbak 1 , Randall Y. Chan 1, 3 , Rie Nakata 1 , Lucia Borriello 1 , Jemily Malvar 1 , Rebekah J. Kennedy 1 , Hiroshi Iwakura 2 , Takashi Akamizu 2 , Richard Sposto 1, 3 , Hiroyuki Shimada 1, 3 , Yves A. DeClerck 1, 3 and Shahab Asgharzadeh 1, 3 1 Children's Hospital Los Angeles, Children's Center for Cancer and Blood Diseases, Division of Hematology, Oncology and Blood & Marrow Transplantation, and The Saban Research Institute, Los Angeles, CA, USA 2 The First Department of Medicine, Wakayama Medical University, Wakayama, Japan 3 Keck School of Medicine, University of Southern California, Los Angeles, CA, USA * These authors have contributed equally to this work Correspondence to: Shahab Asgharzadeh, email: sasgharzadeh@chla.usc.edu Keywords: neuroblastoma, tumor-associated macrophages, tumor microenvironment, STAT3, MYC Received: March 17, 2017 Accepted: July 19, 2017 Published: September 16, 2017 ABSTRACT Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6 ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.

DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p

SummaryDNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

13-cis-Retinoic acid (13-cis-RA) is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immunoblotting. Pretreatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC99) increased from clinically achievable levels to nonachievable levels, greater than 5-fold (cisplatin) to greater than 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxicity for neuroblastoma in vitro, mediated at least in part via the antiapoptotic Bcl-2 family of proteins. Mol Cancer Ther; 9(12); 3164–74. ©2010 AACR.

Background: How exosomic microRNAs (miRNAs) contribute to the development of drug resistance in the context of the tumor microenvironment has not been previously described in neuroblastoma (NBL). Methods: Coculture experiments were performed to assess exosomic transfer of miR-21 from NBL cells to human monocytes and miR-155 from human monocytes to NBL cells. Luciferase reporter assays were performed to assess miR155 targeting of TERF1 in NBL cells. Tumor growth was measured in NBL xenografts treated with Cisplatin and peritumoral exosomic miR-155 (n = 6 mice per group) CD163, miR-155, and TERF1 levels were assessed in 20 NBL primary tissues by Human Exon Arrays and quantitative real-time polymerase chain reaction. Student’s t test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. Results: miR-21 mean fold change (f.c.) was 12.08 ± 0.30 (P < .001) in human monocytes treated with NBL derived exosomes for 48 hours, and miR-155 mean f.c. was 4.51 ± 0.25 (P < .001) in NBL cells cocultured with human monocytes for 48 hours. TERF1 mean luciferase activity in miR-155 transfected NBL cells normalized to scrambled was 0.36 ± 0.05 (P

Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

Epigenetic modifications have proven to play a significant role in cancer development, as well as fetal development. Taking advantage of the knowledge acquired during the last decade, great interest has been shown worldwide in deciphering the fetal epigenome towards the development of methylation-based non-invasive prenatal tests (NIPT). In this review, we highlight the different approaches implemented, such as sodium bisulfite conversion, restriction enzyme digestion and methylated DNA immunoprecipitation, for the identification of differentially methylated regions (DMRs) between free fetal DNA found in maternal blood and DNA from maternal blood cells. Furthermore, we evaluate the use of selected DMRs identified towards the development of NIPT for fetal chromosomal aneuploidies. In addition, we perform a comparison analysis, evaluate the performance of each assay and provide a comprehensive discussion on the potential use of different methylation-based technologies in retrieving the fetal methylome, with the aim of further expanding the development of NIPT assays.

Purpose Children diagnosed at age ≥ 18 months with metastatic MYCN-nonamplified neuroblastoma (NBL-NA) are at high risk for disease relapse, whereas those diagnosed at age < 18 months are nearly always cured. In this study, we investigated the hypothesis that expression of genes related to tumor-associated inflammatory cells correlates with the observed differences in survival by age at diagnosis and contributes to a prognostic signature. Methods Tumor-associated macrophages (TAMs) in localized and metastatic neuroblastomas (n = 71) were assessed by immunohistochemistry. Expression of 44 genes representing tumor and inflammatory cells was quantified in 133 metastatic NBL-NAs to assess age-dependent expression and to develop a logistic regression model to provide low- and high-risk scores for predicting progression-free survival (PFS). Tumors from high-risk patients enrolled onto two additional studies (n = 91) served as independent validation cohorts. Results Metastatic neuroblastomas had higher infiltration of TAMs than locoregional tumors, and metastatic tumors diagnosed in patients at age ≥ 18 months had higher expression of inflammation-related genes than those in patients diagnosed at age < 18 months. Expression of genes representing TAMs (CD33/CD16/IL6R/IL10/FCGR3) contributed to 25% of the accuracy of a novel 14-gene tumor classification score. PFS at 5 years for children diagnosed at age ≥ 18 months with NBL-NA with a low- versus high-risk score was 47% versus 12%, 57% versus 8%, and 50% versus 20% in three independent clinical trials, respectively. Conclusion These data suggest that interactions between tumor and inflammatory cells may contribute to the clinical metastatic neuroblastoma phenotype, improve prognostication, and reveal novel therapeutic targets.

Background: The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Our group has demonstrated the significance of inflammation-related genes in poor outcome of children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). We investigated the effect of TAMs on neuroblastoma growth using a novel murine model. Methods: We have established a novel 100% penetrant NBL-NA murine model driven by SV40's Large T-antigen (NBL-Tag) and characterized expression of immune-related genes in tumors at various developmental stages. Single cell suspensions of murine tumors were analyzed using flow cytometry for determination of immune cells frequency and IL6 levels. NBL-Tag mice were crossed with IL6 knockout mice (NBL-Tag/IL6-/-) to investigate the role of IL6 in tumor pathogenesis. Results: NBL-Tag tumor growth coincided with IL6 levels becoming detectable and increasing in blood. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 week old) showed age-dependent increase in expression of IL6 and CCL2 (MCP1) compared to wild-type adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. FACS of tumors showed high expression of IL6, and infiltration by IL6-producing TAMs. In vitro co-culture of peritoneal macrophages from wild type mice increased proliferation of a NBL-Tag mice driven cell line (NBT2) by average of 50% over its basal rate, as measured by Brdu incorporation. This effect coincided with two-fold increase in IL6 protein level in the co-cultured media. However, the proliferative changes were only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. IL6 genetic ablation studies were conducted to generate NBL-Tag/IL6-/- mice, which convincingly demonstrated IL6 was not required for tumor growth and development. Macrophages from IL6-/- mice increased in vitro proliferation of tumor to similar levels as those from wild-type mice, while IHC staining of NBL-Tag/IL6-/- tumors revealed similar level of TAM infiltration and positivity for phospo-STAT3, indicating an IL6-independent pathway for STAT3 activation. Lastly, gene expression analyses revealed upregulation of MYC in tumor cells during co-culture and was confirmed by western analysis. Conclusions: This study demonstrates the recruitment and growth-promoting effects of TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The macrophage and neuroblastoma cell cross-talk lead to tumor cell proliferation with phosphorylation of STAT3 and MYC upregulation in tumor cells. These effects were found to be independent of IL6 activity. Citation Format: Michael D. Hadjidaniel, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Michael Sheard, Janahan Gnanachandran, Jin Kim, Richard Sposto, Hiroyuki Shimada, Shahab Asgharzadeh. Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3668. doi:10.1158/1538-7445.AM2014-3668

The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Recent studies have demonstrated the prognostic significance of tumor-associated macrophages (TAM) in several cancers. Our group has also demonstrated the significance of TAMs and interleukin-6 receptor in predicting poor outcome in children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). Previous in vitro work has shown that co-culture of a human neuroblastoma MYCN non-amplified cell line with monocytes enhanced proliferation, an effect mediated in part by interleukin-6 (IL6). To address the mechanism by which inflammation contributes to tumorigenesis in neuroblastoma, we have established a novel, 100% penetrant, murine model of metastatic NBL-NA. The tumors of these transgenic mice (NBL-Tag) are detected by MRI only after 12 weeks of age, which coincide with IL6 levels becoming detectable and increasing in blood. DNA profiling of tumors reveal no amplification of the MYCN locus, while RNA profiling and FACS analyses of tumors show high expression of IL6, and infiltration by IL6-producing TAMs. Thus, a pro-inflammatory microenvironment mimicking that observed in human metastatic NBL-NA characterizes the growth of NBL-Tag murine tumors. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 weeks old) show age-dependent increase in expression of CCL2 (aka monocyte chemotactic protein 1) and fibronectin (FN1) compared to wildtype adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. In vitro co-culture of spleen or peritoneal macrophages from wildtype mice increases proliferation of a NBL-Tag cell line (NBT2) by 30 - 50% over its basal rate, as measured by Brdu incorporation. This effect coincides with two-fold increase in IL6 protein level in the co-cultured media. However, these proliferative changes do not require cell-cell contact and are only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. Co-cultured NBT2 cells also increase their expression of MYC by two-fold, providing insight into the tumor proliferative mechanism facilitated by TAMs. In summary, this study demonstrates the recruitment and growth-promoting effects of IL6 producing TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The NBL-Tag mice provide a robust transgenic model to study neuroblastoma pathogenesis and provide a platform to assess therapies targeting tumor cells and tumor-associated inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 383. doi:1538-7445.AM2012-383

Introduction: High-risk neuroblastoma (H-NB) is an aggressive tumor of the sympathetic nervous system for which there are few predictors of therapeutic response. We sought to determine the association of global alternative splicing (AS) gene expression patterns with NB aggressiveness and MYCN gene status. Methods: As part of the NCI-sponsored Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, we profiled 190 primary human NBs using Affymetrix Human Exon arrays. Tumors were obtained from children who participated in Children's Oncology Group trials with informed consent. The high-risk cohort included tumors with (H-NBA n=48) and without MYCN amplification (H-NBN n=111), a key molecular determinant of clinical aggressiveness. Twenty-four samples from low-risk NB lacking MYCN amplification (L-NB) were also profiled as a comparison group. Robust multichip analysis (RMA) was used for summarization of probe selection regions (PSR) and transcript regions. Finding isoforms using RMA (FIRMA) was used to generate PSR isoform scores. After an unsupervised pre-filtering step based on coefficient of variation, ANOVA analyses were conducted comparing H-NBA or H-NBN against the L-NB group for differential transcript and isoform expression. Last, gene set enrichment analyses (GSEA) comparing high-risk to low-risk NBs were conducted using unfiltered transcript data and the Molecular Signature Database's (MsigDB) canonical gene sets. Results: 1914 out of the 22,011 core transcripts passed our strict prefiltering criteria that required a high coefficient of variation. There was a larger number of genes with significant differential expression between H-NBA vs. L-NB than H-NBN vs. L-NB (23.5% versus 4.6%, P Conclusion: Significant differences in gene expression and pathway expression are identified among H-NBA compared to H-NBN tumors. In addition, we show for the first time a high degree of alternative splicing events in the H-NBA tumors. These findings could aid in understanding the regulatory networks in the two subgroups of high-risk neuroblastomas and suggest possible therapeutic targets for further validation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5249.