Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T-Cell Receptors in High-Risk MYCN-Not-Amplified Human Neuroblastoma Jun S
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Published OnlineFirst May 21, 2018; DOI: 10.1158/1078-0432.CCR-18-0599 Biology of Human Tumors Clinical Cancer Research Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T-Cell Receptors in High-Risk MYCN-Not-Amplified Human Neuroblastoma Jun S. Wei1, Igor B. Kuznetsov2, Shile Zhang1, Young K. Song1, Shahab Asgharzadeh3, Sivasish Sindiri1, Xinyu Wen1,RajeshPatidar1, Sushma Najaraj1, Ashley Walton1, Jaime M. Guidry Auvil4, Daniela S. Gerhard4,AysenYuksel5, Daniel Catchpoole5, Stephen M. Hewitt6, Paul M. Sondel7, Robert Seeger3,JohnM.Maris8,andJavedKhan1 Abstract Purpose: High-risk neuroblastoma is an aggressive disease. compared with MYCN-amplified (MYCN-A) tumors. DNA sequencing studies have revealed a paucity of action- A reported MYCN activation signature was significantly able genomic alterations and a low mutation burden, posing associated with poor outcome for high-risk patients with challenges to develop effective novel therapies. We used RNA MYCN-NA tumors; however, a subgroup of these patients þ sequencing (RNA-seq) to investigate the biology of this disease, who had elevated activated natural killer (NK) cells, CD8 T including a focus on tumor-infiltrating lymphocytes (TIL). cells, and cytolytic signatures showed improved outcome Experimental Design: We performed deep RNA-seq on and expansion of infiltrating TCR clones. Furthermore, we pretreatment diagnostic tumors from 129 high-risk and 21 observed upregulation of immune exhaustion marker genes, low- or intermediate-risk patients with neuroblastomas. We indicating an immune-suppressive microenvironment in used single-sample gene set enrichment analysis to detect gene these neuroblastomas. expression signatures of TILs in tumors and examined their Conclusions: This study provides evidence that RNA sig- association with clinical and molecular parameters, including natures of cytotoxic TIL are associated with the presence of patient outcome. The expression profiles of 190 additional activated NK/T cells and improved outcomes in high-risk pretreatment diagnostic neuroblastomas, a neuroblastoma neuroblastoma patients harboring MYCN-NA tumors. Our tissue microarray, and T-cell receptor (TCR) sequencing were findings suggest that these high-risk patients with MYCN-NA used to validate our findings. neuroblastoma may benefit from additional immunothera- Results: We found that MYCN-not-amplified (MYCN-NA) pies incorporated into the current therapeutic strategies. Clin tumors had significantly higher cytotoxic TIL signatures Cancer Res; 1–12. Ó2018 AACR. Introduction 1Oncogenomics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 2Cancer Neuroblastomas are pediatric solid tumors originating from Research Center and Department of Epidemiology and Biostatistics, School of neurons of the sympathetic nervous system. The disease course 3 Public Health, University at Albany, Rensselaer, New York. Division of Hema- can vary drastically, ranging from relentless progression of high- tology/Oncology, the Children's Hospital Los Angeles, Los Angeles, California. 4Office of Cancer Genomics, National Cancer Institute, Bethesda, Maryland. 5The risk tumors despite aggressive treatment to spontaneous regres- Tumour Bank, Children's Cancer Research Unit, Kids Research Institute, the sion of low-risk stage 4S tumors in infants with no cytotoxic Children's Hospital at Westmead, Westmead, New South Wales, Australia. therapy interventions (1, 2). Despite recent FDA approval of anti– 6Experimental Pathology Laboratory, Laboratory of Pathology, Center for GD2 antibody-based immunotherapy for high-risk neuroblasto- Cancer Research, National Cancer Institute, National Institutes of Health, ma patients in first remission, the cure rates remain less than 50% 7 Bethesda, Maryland. Departments of Pediatrics, Human Oncology and Genet- accompanied by significant morbidity in survivors (3). Previous ics, the University of Wisconsin, Madison, Wisconsin. 8Department of Pediatrics, University of Pennsylvania and Center for Childhood Cancer Research, the large-scale sequencing by us and others has revealed a low somatic Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. mutation burden in this disease with only a few recurrently mutated genes (4–6), imposing a substantial challenge for devel- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). oping new targeted therapies for a large proportion of patients with high-risk neuroblastoma. Therefore, there remains a critical J.S. Wei, I.B. Kuznetsov, and S. Zhang contributed equally to this article. need to identify effective novel therapies for these aggressive Corresponding Author: Javed Khan, National Institutes of Health, Room 2016B enigmatic tumors. Building 37, 37 Convent Drive, Bethesda, MD 20892. Phone: 240-760-6135; Fax: Immune checkpoint inhibitor therapies have recently shown 301-480-0314; E-mail:[email protected] significant antitumor activity in several adult human cancers doi: 10.1158/1078-0432.CCR-18-0599 (7–9). Their activity is thought to be correlated with higher tumor Ó2018 American Association for Cancer Research. mutation burden presumably because of the greater probability of www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 21, 2018; DOI: 10.1158/1078-0432.CCR-18-0599 Wei et al. integral part of the Therapeutically Applicable Research to Gen- Translational Relevance erate Effective Treatment (TARGET) initiative to genomically We used RNA sequencing (RNA-seq) and single-sample profile high-risk neuroblastoma (5, 17), we sequenced the mRNA gene set enrichment analysis to characterize tumor-infiltrating of 150 primary neuroblastoma tumors obtained prior to any lymphocyte (TIL) signatures in pretreatment diagnostic neu- therapy (Table 1 and Supplementary Table S1). Of these, 139 roblastomas. We showed, for the first time, that the presence of tumors have matched DNA sequencing data from whole-exome cytolytic TIL signatures in a subgroup of high-risk MYCN-not- and/or whole-genome sequencing (Supplementary Fig. S1). In amplified (MYCN-NA) neuroblastoma patients with addition, we used previously published RNA-seq data for 190 improved survival was not associated with high mutation patients with neuroblastoma (obtained prior to therapy) as an burden. Instead, they were significantly correlated with natural independent cohort to validate our findings (ref.16; validation killer– and T-cell infiltration accompanied by T-cell receptor cohort; Table 1 and Supplementary Table S1). (TCR) clonal expansion. In addition, upregulation of the expression of immune exhaustion marker genes suggested an RNA-seq immune-suppressive microenvironment in these tumors. Fur- Only RNAs with RNA Integrity Number greater than 6.0 were thermore, these cytotoxic TIL signatures were independently used in this study. RNA-seq libraries were prepared using Illumina validated using the data from a previously published neuro- TruSeqRNA Sample Preparation V2 kits according to the manu- blastoma RNA-seq study. Therefore, our study suggests the facturer's protocol (Illumina), and 100 bp paired-end sequencing – potential of immune checkpoint inhibitor based immu- was performed using Illumina HiSeq 2000 sequencers. Each notherapies for treating high-risk neuroblastoma patients with sample was processed through a RNA-seq data analysis pipeline MYCN -NA tumors or TCR-based adoptive cell therapy for where reads were mapped to the ENSEMBL human genome MYCN fi those with -A tumors. Our ndings may also have GRCh37 build 71 using TopHat2 (18). Cufflinks was then used implications for other pediatric cancers with low mutation to quantify gene expression as Fragments Per Kilobase of tran- burden. script per Million mapped reads (FPKM; ref. 19). The final measure of gene expression level is log2(FPKMþ1). FASTQ files for RNA-seq data are available at the NCI's Genomic Data Com- mons (https://portal.gdc.cancer.gov) and the high-level data at infiltrating tumor-reactive T cells recognizing neoantigens pre- https://ocg.cancer.gov/programs/target/data-matrix. sented in these tumors (8). Because neuroblastomas typically The independent validation RNA-seq data used in this study have a low somatic mutation burden at the time of diagnosis, it is were derived from a published RNA-seq dataset generated using postulated that there will be fewer T cells in the tumor microen- the Illumina HiSeq 2000 platform (16). Gene expression levels in vironment (6). However, recent studies by us and other groups log2(FPKMþ1) and the associated clinical information for these reported that intensive chemotherapy and radiotherapy dramat- 190 patients (Table 1) were downloaded from the Gene Expres- ically increase mutation burden in the relapsed setting (10, 11). sion Omnibus database entry GSE49711. Furthermore, DNA-damaging agents in conjunction of dinutux- imab have been reported to show a survival advantage for patients Consensus clustering with relapsed/refractory neuroblastoma, suggesting that immu- We used consensus clustering for genes with nonzero expres- notherapy may be further exploited for future therapies (12). sion variability in the dataset using the R package Consensu- – Remarkably, in synovial sarcoma, a fusion gene driven cancer sClusterPlus for class discovery based on the comparison of gene fi with a low