Dentithecamides A-H, Diacylated Zoanthoxanthin Derivatives with PAX3-FOXO1 Inhibitory Activity from the Hydroid Dentitheca habereri.

Chemical investigation of the marine hydroid Dentitheca habereri led to the identification of eight new diacylated zoanthoxanthin alkaloids, named dentithecamides A-H (1-8), along with three previously reported analogues, zoamides B-D (9-11). The structures of compounds 1-11 were elucidated by spectroscopic and spectrometric analyses, including IR, HRESIMS, and NMR experiments, and by comparison with literature data. Compounds 1-11 are the first zoanthoxanthin alkaloids to be reported from a hydroid. Dentithecamides A (1) and B (2) along with zoamides B-D (9-11), which all share a conformationally mobile cycloheptadiene core, inhibited PAX3-FOXO1 regulated transcriptional activity and thus provided a structural framework for the potential development of more potent PAX3-FOXO1 inhibitors.

Clonality and evolutionary history of rhabdomyosarcoma.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Frequent inactivating germline mutations in DNA repair genes in patients with Ewing sarcoma.

PURPOSE: Ewing sarcoma is a small round blue cell tumor that is highly malignant and predominantly affects the adolescent and young adult population. It has long been suspected that a genetic predisposition exists for this cancer, but the germ-line genetic underpinnings of this disease have not been well established. METHODS: We performed germline variant analysis of whole-genome or whole-exome sequencing of samples from 175 patients affected by Ewing sarcoma. RESULTS: We discovered pathogenic or likely pathogenic germline mutations in 13.1% of our cohort. Pathogenic mutations were highly enriched for genes involved with DNA damage repair and for genes associated with cancer predisposition syndromes. CONCLUSION: Our findings reported here have important clinical implications for patients and families affected by Ewing sarcoma. Genetic counseling should be considered for patients and families affected by this disease to take advantage of existing risk management strategies. Our study also highlights the importance of germline sequencing for patients enrolled in precision-medicine protocols.Genet Med advance online publication 26 January 2017.

Absence of STAT1 in donor-derived plasmacytoid dendritic cells results in increased STAT3 and attenuates murine GVHD.

Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. Transplantation of STAT1(-/-) bone marrow (BM) prevented GVHD induced by STAT1(+/+) T cells, leading to expansion of B220(+) cells and regulatory T cells. STAT1(-/-) BM also preserved GVT activity and enhanced overall survival of tumor-challenged mice in the setting of GVHD. Furthermore, recipients of allogeneic STAT1(-/-) BM demonstrated increased CD9(-)Siglec H(hi) plasmacytoid dendritic cells (pDCs), and depletion of pDCs after STAT1(-/-) BM transplantation prevented GVHD resistance. STAT1(-/-) pDCs were found to produce decreased free radicals, IFNα, and interleukin (IL)-12, and increased IL-10. Additionally, STAT1(-/-) pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9(-/-) BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1(-/-) pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity.

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.

A stem cell epigenome is associated with primary nonresponse to CD19 CAR T cells in pediatric acute lymphoblastic leukemia.

CD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin-sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma.

Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.

Synthetic lethal screen identifies NF-κB as a target for combination therapy with topotecan for patients with neuroblastoma.

BACKGROUND: Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. METHODS: We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. RESULTS: We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. CONCLUSIONS: Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma.

Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.

Systematic proteome analysis identifies transcription factor YY1 as a direct target of miR-34a.

MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by two or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3' UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53, and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities.

Murine rhabdomyosarcoma is immunogenic and responsive to T-cell-based immunotherapy.

BACKGROUND: Immunotherapies targeting cellular immunity are currently approved for treatment of melanoma, renal cell carcinoma, and prostate cancer. Studies on the immunogenicity and immune responsiveness of pediatric tumors are limited, therefore, it remains unclear to what extent T-cell-based immunotherapy holds promise for pediatric solid tumors. PROCEDURE: A new rhabdomyosarcoma cell line (M3-9-M) was derived from an embryonal rhabdomyosarcoma (ERMS) occurring in a C57BL/6 mouse transgenic for hepatocyte growth factor and heterozygous for mutated p53. Primary tumors and metastases derived from M3-9-M were studied for similarities to human ERMS, and for immunogenicity and immune responsiveness. RESULTS: Primary and metastatic tumors develop after orthotopic injection of M3-9-M into immunocompetent C57BL/6 mice, which mirror human ERMS with regard to histology, gene expression, and metastatic behavior. Whole cell vaccination using irradiated M3-9-M cells or M3-9-M-pulsed dendritic cells (DC)-induced tumor-specific T-cell responses that prevent tumor growth following low-dose tumor injection, and slow tumor growth following higher doses. Administration of anti-CD25 moAbs to deplete CD4(+)CD25(+)FOXP3(+) regulatory T cells prior to tumor vaccination enhanced the potency of the ERMS tumor vaccine. Adoptive immunotherapy with M3-9-M primed T cells plus DC-based vaccination resulted in complete eradication of day 10 M3-9-M derived tumors. CONCLUSIONS: M3-9-M derived murine ERMS is immunogenic and immunoresponsive; regulatory T cells contribute to immune evasion by murine rhabdomyosarcoma. Adoptive immunotherapy with DC vaccination can eradicate low tumor burdens. Future work will seek to identify the tumor-associated antigens that mediate protective and therapeutic immunity in this model.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

Tumor Mutation Burden, Expressed Neoantigens and the Immune Microenvironment in Diffuse Gliomas.

BACKGROUND: A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. METHODS: Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). RESULTS: Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5% of IDH-wildtype gliomas (p = 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R = -0.46, p = 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. The Antigen Processing and Presentation (APP) score correlated with immune score and was surprisingly higher in NHMP versus HMP samples in IDH-wildtype gliomas, but higher in HMP versus NHMP in IDH-mutant gliomas. CONCLUSION: TMB was inversely correlated with immune score in IDH-wildtype gliomas and showed no correlation in IDH-mutant tumors. APP was correlated with immune score and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a larger glioma cohort are warranted.

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

PURPOSE: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome. PATIENTS AND METHODS: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. RESULTS: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data. CONCLUSION: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.

Global genomic and proteomic analysis identifies biological pathways related to high-risk neuroblastoma.

Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1-) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1- tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p < 0.0001), as approximately half of the up-regulated proteins in 4+ tumor have elevated mRNA level (n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma.

Outcome-Related Signatures Identified by Whole Transcriptome Sequencing of Resectable Stage III/IV Melanoma Evaluated after Starting Hu14.18-IL2.

PURPOSE: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence. EXPERIMENTAL DESIGN: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing. RESULTS: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors. CONCLUSIONS: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy.

Case Report: Single-Cell Transcriptomic Analysis of an Anaplastic Oligodendroglioma Post Immunotherapy.

Glioma is the most common primary malignant brain tumor with a poor prognosis. Immune checkpoint inhibitors have been of great interest in investigation of glioma treatments. Here, we report single-cell transcriptomic analyses of two tumor areas from an oligodendroglioma taken from a patient who had multiple tumor recurrences, following several chemotherapies and radiation treatments. The patient subsequently received nivolumab and was considered have disease progression based on conventional diagnostic imaging after two cycles of treatment. He underwent a debulking surgical resection and pathological diagnosis was recurrent disease. During the surgery, tumor tissues were also collected from the enhancing and non-enhancing areas for a scRNAseq analysis to investigate the tumor microenvironment of these radiographically divergent areas. The scRNAseq analysis reveals a plethora of immune cells, suggesting that the increased mass observed on MRI may be partially a result of immune cell infiltration. The patient continued to receive immunotherapy after a short course of palliative radiation and remained free of disease progression for at least 12 months after the last surgery, suggesting a sustained response to immunotherapy. The scRNAseq analysis indicated that the radiological progression was in large part due to immune cell infiltrate and continued immunotherapy led to a positive clinical outcome in a patient who would have otherwise been admitted to hospice care with halting of immunotherapy. Our study demonstrates the potential of scRNAseq analyses in understanding the tumor microenvironment, which may assist the clinical decision-making process for challenging glioma cases following immunotherapy.

An integrated cross-platform prognosis study on neuroblastoma patients.

There have been several reports about the potential for predicting prognosis of neuroblastoma patients using microarray gene expression profiling of the tumors. However these studies have revealed an apparent diversity in the identity of the genes in their predictive signatures. To test the contribution of the platform to this discrepancy we applied the z-scoring method to minimize the impact of platform and combine gene expression profiles of neuroblastoma (NB) tumors from two different platforms, cDNA and Affymetrix. A total of 12442 genes were common to both cDNA and Affymetrix arrays in our data set. Two-way ANOVA analysis was applied to the combined data set for assessing the relative effect of prognosis and platform on gene expression. We found that 26.6% (3307) of the genes had significant impact on survival. There was no significant impact of microarray platform on expression after application of z-scoring standardization procedure. Artificial neural network (ANN) analysis of the combined data set in a leave-one-out prediction strategy correctly predicted the outcome for 90% of the samples. Hierarchical clustering analysis using the top-ranked 160 genes showed the great separation of two clusters, and the majority of matched samples from the different platforms were clustered next to each other. The ANN classifier trained with our combined cross-platform data for these 160 genes could predict the prognosis of 102 independent test samples with 71% accuracy. Furthermore it correctly predicted the outcome for 85/102 (83%) NB patients through the leave-one-out cross-validation approach. Our study showed that gene expression studies performed in different platforms could be integrated for prognosis analysis after removing variation resulting from different platforms.