1078-0432.CCR-19-3104.Full.Pdf

Published OnlineFirst May 19, 2020; DOI: 10.1158/1078-0432.CCR-19-3104

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

CD8þ T-cell–Mediated Immunoediting Inﬂuences Genomic Evolution and Immune Evasion in Murine Gliomas Joshua R. Kane1,†, Junfei Zhao2,3, Takashi Tsujiuchi4, Brice Lafﬂeur5, Víctor A. Arrieta1,6, Aayushi Mahajan4, Ganesh Rao7, Angeliki Mela8, Crismita Dmello1, Li Chen1, Daniel Y. Zhang1, Edgar Gonzalez-Buendia 1, Catalina Lee-Chang1, Ting Xiao1, Gerson Rothschild5, Uttiya Basu5, Craig Horbinski1, Maciej S. Lesniak1, Amy B. Heimberger7, Raul Rabadan2,3,9, Peter Canoll7, and Adam M. Sonabend1

ABSTRACT ◥ Purpose: Cancer immunoediting shapes tumor progression by depletion. In contrast, gliomas that developed under pressure þ þ the selection of tumor cell variants that can evade immune recog- from CD8 T cells were able to fully engraft in both CD8 nition. Given the immune evasion and intratumor heterogeneity T-cell–depleted mice and immunocompetent mice. Remarkably, þ þ characteristic of gliomas, we hypothesized that CD8 T cells gliomas developed in the absence of CD8 T cells exhibited mediate immunoediting in these tumors. increased aneuploidy, MAPK pathway signaling, gene fusions, Experimental Design: We developed retrovirus-induced and macrophage/microglial infiltration, and showed a pro- þ PDGF Pten / murine gliomas and evaluated glioma progression inflammatory phenotype. MAPK activation correlated with þ and tumor immunogenicity in the absence of CD8 T cells by macrophage/microglia recruitment in this model and in the depleting this immune cell population. Furthermore, we charac- human disease. terized the genomic alterations present in gliomas that developed in Conclusions: Our studies indicate that, in these tumor models, þ þ the presence and absence of CD8 T cells. CD8 T cells influence glioma oncogenic pathways, tumor geno- Results: Upon transplantation, gliomas that developed in the type, and immunogenicity. This suggests immunoediting of þ absence of CD8 T cells engrafted poorly in recipients with immunogenic tumor clones through their negative selection by þ þ intact immunity but engrafted well in those with CD8 T-cell CD8 T cells during glioma formation.

Introduction regulation, apoptosis, and sequestration in the bone marrow (2, 3). As a consequence, the efficacy of immunotherapy is hindered. Glioblastoma (GBM) is the most common and malignant brain Cancer immunoediting has been proposed as a mechanism where cancer in adults, with a median overall survival of approximately the adaptive and innate immunity sculpts the immunogenicity of 21 months (1). This tumor develops multiple mechanisms of immune developing tumors, selecting for tumor cell variants that can evade suppression including induction of T-cell anergy, exhaustion, negative immune surveillance (4–6). This understudied mechanism could help explain why many tumors do not respond to immunotherapies. If antitumoral immunity is capable of recognizing some tumor cell fl 1Department of Neurosurgery, Northwestern University Feinberg School of variants and not others, cancer immunoediting likely in uences the Medicine, Chicago, Illinois. 2Department of Systems Biology, Columbia Univer- evolutionary path of the cancer. Cancer immunoediting relies on sity, New York City, New York. 3Department of Biomedical Informatics, Columbia tumor genomic heterogeneity, a hallmark of GBM (7). We recently University, New York City, New York. 4Department of Neurosurgery, Columbia described the disappearance of immunogenic glioma cell variants 5 University, New York City, New York. Department of Microbiology and Immu- following PD-1 blockade in recurrent patients with GBM who 6 nology, Columbia University, New York City, New York. PECEM, Facultad de responded to treatment (8). This suggests that immunoediting may Medicina, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico. 7Department of Neurological Surgery, The University of Texas MD Anderson occur not only during formation of these gliomas, but also in the Cancer Center, Houston, Texas. 8Department of Pathology and Cell Biology, setting of immunotherapy. In spite of these and other clinical observa- Columbia University, New York City, New York. 9Department of Mathematical tions that suggest immunoediting in gliomas, this phenomenon has Genomics, Columbia University, New York City, New York. not been experimentally investigated in this disease. þ Note: Supplementary data for this article are available at Clinical Cancer We hypothesized that CD8 T cells subject gliomas to immunoe- Research Online (http://clincancerres.aacrjournals.org/). diting. By depleting this immune cell population during development J.R. Kane, J. Zhao, and T. Tsujiuchi contributed equally to this article. of transgenic murine gliomas, we investigated how these immune effectors influence tumor immunogenicity and progression. Upon †Deceased. þ transplantation, gliomas developed in the absence of CD8 T cells Corresponding Author: Adam M. Sonabend, Northwestern University, 676 N St. engrafted poorly in recipients with intact immunity, but readily grafted þ Clair, Suite 2210, Chicago, IL 60611. Phone: 312-695-8143; E-mail: in hosts with CD8 T-cell depletion. On the other hand, gliomas that [email protected] þ developed in the presence of CD8 T cells engrafted well in immu- Clin Cancer Res 2020;XX:XX–XX nocompetent mice. This implies a more immunogenic profile of þ doi: 10.1158/1078-0432.CCR-19-3104 gliomas that develop without the pressure exerted by CD8 T cells. 2020 American Association for Cancer Research. Moreover, these gliomas exhibited increased activation of MAPK

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þ continued twice per week to achieve consistent CD8 T-cell depletion. Translational Relevance Depletion was monitored at three time points starting from intracra- Immune evasion renders gliomas resistant to several types of nial virus injection, 3 weeks apart. immunotherapy. As part of the cancer immunoediting process, many cancer cells are eliminated before they develop into a RNA extraction, sequencing, and gene expression analysis clinically evident malignancy, while other tumor cells prevail After 7 tumor growth/days, gliomas treated either with the anti- evading immune recognition. In this context, therapeutic inter- mouse CD8a (8 mice) or the anti-rat IgG2a isotype control (8 mice) vention often fails for gliomas because tumor cell variants that were dissected from the brain of the animals. RNA extraction was resist immune surveillance get selected by the immune system. The performed from the tumor bulk using the RNeasy Mini Kit (Qiagen). elucidation of how cancer immunoediting leads to immune evasion Paired-end transcriptome reads were processed using STAR (10) in gliomas may allow optimization of immunotherapeutic aligner on the basis of the Ensembl GRCm38 mouse genome assembly approaches to induce sustained clinical responses in these patients. with default parameters. Normalized gene expression values were Our results demonstrate immune evasion in murine gliomas can be calculated by featureCounts (11) as RPKM. Single-sample Gene Set þ attributed to selection pressure by CD8 T cells during the Enrichment Analysis (ssGSEA) was performed using the R package development and progression of these tumors. GSVA (12). Raw sequencing data are available for download at https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA634767.

IHC signaling, mitoses, aneuploidy, gene fusions, and macrophage/micro- Formalin-fixed, paraffin-embedded material from animals was þ glia infiltration. Our results reveal that CD8 T cells mediate immu- deparaffinized with xylene and antigen retrieval was performed with noediting in gliomas, influencing the selection of genomic alterations 10 mmol/L sodium citrate buffer (pH ¼ 6). IHC was performed with a and modulating the tumor microenvironment during gliomagenesis, 1:150 diluted rat mAb against MHC-I (AB15680; Abcam), 1:150 leading to a nonimmunogenic phenotype observed at the end of tumor diluted rabbit polyclonal antibody against MHC-II (AB180779; development. Abcam), 1:2,000 diluted rabbit mAb against CD11b (AB133357; Abcam), 1:3,000 diluted rabbit mAb against Iba1 (AB178846; Abcam), 1:250 diluted rabbit mAb against p38 (4511; Cell Signaling Technol- Materials and Methods ogy), and 1:250 diluted rabbit mAb against pERK (4370; Cell Signaling þ Generation and transplantation of PDGF Pten / gliomas Technology). Counterstaining was performed on the same material þ PDGF Pten / gliomas were generated by intracranial injection of with hematoxylin (Abcam). IHC staining was performed on a Leica a PDGF–IRES–Cre retrovirus into the subcortical white matter of Bond-Max automatic immunostainer. Images were acquired using a adult mice with the Ptenlox/lox genotype as described previously standard light microscope (Leica DM2000 LED, Leica Microsystems) (C57BL/6 background, Jackson Lab Stock No.: 006440; ref. 9). Gliomas mounted with a digital microscope camera (Leica DFC450 C, Leica generated in hosts with intact immunity can grow in syngeneic Microsystems). immune-competent recipients. Transplantation was done by extract- Machine learning of IHC quantification was used by setting para- þ ing the gliomas that developed in the absence or presence of CD8 T meters to identify positivity and subtracting counterstained/negative cells that were dissociated with 2.5% TrypLE in PBS upon which they background using ImageJ software (available from the NIH). The were cultured with DMEM (Gibco) þ 0.5% FBS (Gibco), 1% N2 substrate optical density and intensity was determined by quantifying (Gibco), 1% penicillin/streptomycin (Gibco), 10 ng/mL PDGF-AA the positive nuclei or cytoplasm (dependent on particular antibody (PeproTech), and 10 ng/mL FGF (Basal FGF PDGF; PeproTech) for up assessed) paired with the intensity of the staining in neoplastic tumor to a week prior to transplant. Animals were anesthetized with ketamine cell regions of the brain at 4 light microscopic magnification to infer (115 mg/kg) and xylazine (17 mg/kg), a skin incision was made in the immunoreactivity index. middle of the head, and a burr hole was made with a drill. þ PDGF Pten / glioma cells were then intracranially implanted Bioanalysis of aneuploidy through a stereotactic injection using a 10-mL Hamilton syringe Mutational burden was called by analysis of germline and (Hamilton) with a 30-gauge needle while animals were mounted on somatic variation by Strelka2 Small Variant Caller (Illumina). The a stereotactic apparatus (Harvard Apparatus). For survival analysis, germline identification employed a tiered haplotype model adap- animals losing ≥30% of their body weight or having trouble ambu- tively selected between assembly and alignment-based haplotyping lating, feeding, or grooming were euthanized by CO2 administration at each variant locus. The aneuploidy score from somatic copy- followed by cervical dislocation. After conclusion of survival analysis, number alterations was calculated by calling the presence or animals were sacrificed. Brains were harvested, sectioned, and stained absence of amplifications or deletions as described previously (13). by IHC and hematoxylin and eosin (H&E). All the animal procedures Aneuploidy score as a comparison between two groups was called were performed and approved in compliance with institutional guide- by copy-number detection, implemented in the software package lines under the Institutional Animal Care and Use Committee of CNVkit, using both targeted reads and nonspecifically captured off- Columbia University (New York). target reads to infer copy number evenly across the genome (14). STAR-Fusion was used to identify candidate fusion events (10). The þ CD8 T-cell depletion impact of the fusion event on coding regions was explored by Animals were treated with either anti-mouse CD8a (BE0004-1, invoking the “examine_coding_effect” parameter. Only the candi- BioXCell) or anti-rat IgG2a isotype control, anti-trinitrophenol dates that affected the coding regions were retained for fusion load (BE0089, BioXCell). The antibody concentration given to each mouse analysis. Copy-number variance events were counted and the was 2 mg/mL diluted in PBS at a final volume of 100 mL administered percentage of the genome affected by such events was calculated via intraperitoneal injection one week prior to glioma induction and as described previously (13).

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þ þ Calculation of Shannon index in the fraction of CD4 as well as CD8 T cells in the glioma at 35 d.p.i. þ For each tumor, the Shannon diversity was estimated using the compared with that exhibited at 21 d.p.i. [P ¼ 0.0189 (CD4 ), P ¼ þ command “entropy.empirical” from the “entropy” R package. This was 0.0026 (CD8 ), t test; Supplementary Fig. S1D]. þ calculated on the basis of the estimated prevalence of different immune To investigate the effects of CD8 T cells on glioma development cell compartments found in the tumor. The Shannon diversity score, and immunogenicity, we depleted this immune cell population by þ H, followed the formula H ¼Spi log(pi). administration of an anti-CD8 antibody starting one week prior to retroviral injection, and throughout glioma development. In addition, Prediction of neoantigen binders we injected an isotype control antibody to another cohort of mice Novel 9–11merpeptidesthatcouldarisefromidentified non- (intact immunity). Then, we induced gliomas in these hosts treated þ silentmutationsorgenefusionspresentinthetumorsampleswere with and without the anti-CD8 T-cell antibody. As a second exper- determined. We used the pVAC-Seq54 pipeline with the NetMHC- iment, we isolated and generated primary cultures of these tumors, cons55 binding strength predictor to identify neoantigens (15, 16). and subsequently transplanted these glioma cells into recipients with fi þ The predicted IC50 inhibitory concentration binding af nities and or without CD8 T-cell depletion (Fig. 1A shows a schematic of þ rank percentage scores were calculated for all peptides that bound to both experiments). CD8 T-cell depletion was confirmed by flow þ each of the tumor's human leukocyte antigen alleles. Using estab- cytometry analysis of splenocytes (Fig. 1B). The absence of CD8 lished thresholds, predicted binders were considered to be those T cells during glioma development had no effect on glioma growth, as þ peptides that had a predicted binding affinity <500 nmol/L. mice treated with the anti-CD8 antibody had comparable survival to mice treated with the isotype control antibody (P ¼ 0.7563, log-rank Statistical analysis test; Fig. 1C). This is consistent with the results of the transgenic þ þ All statistical analysis was conducted using GraphPad Prism 8 PDGFB RCAS Stat3 / glioma model, in which CD8 T cells had no (GraphPad Software) and R version 3.1.2 (R Core Team). Numer- effect on overall survival in tumor-bearing mice (Rao and colleagues, ical data were reported as mean SEM. Mann–Whitney U test or unpublished data). þ unpaired Student t test was used for two-group comparisons. Implantation of glioma cells that were originated in mice with CD8 ANOVA was used for more than two groups. Kaplan–Meier T-cell depletion into recipients treated with the anti-CD8 antibody þ survival curves were generated, and a log-rank test was utilized to (which have CD8 T-cell depletion) led to growth of gliomas in all compare survival distribution. Mutational and gene fusion counts cases. Implantation of the same glioma cells developed in mice with þ were analyzed using a binomial model for count data with over- CD8 T-cell depletion into recipients treated with the isotype control dispersion. All reported q values were calculated by statistical antibody (intact immunity) led to engraftment in only 25% of the mice, adjustment of P values using the Benjamini–Hochberg constant. with the remaining animals surviving past 220 days without any The Pearson coefficient of correlation (r) was calculated by the evidence of glioma or neurologic symptoms (P ¼ 0.018, log-rank linear regression of the data points according to the goodness of fit test; Fig. 1C bottom left, and D). In contrast, transplantation of a þ where significance was determined at which point the slope was tumor generated in a mouse host with intact CD8 T cells (treated with significantly nonzero. In silico analysis was performed with 161 isotype antibody), led to growth and similar survival for recipients that þ cataloged, human GBM samples provided by The Cancer Genome had intact CD8 T cells (isotype antibody treatment) or hosts with þ Atlas (TCGA) with mRNA expression data available (RNAseq CD8 T-cell depletion (anti-CD8 antibody treatment; Fig. 1C bottom V2 RSEM). right). þ Interestingly, gliomas that developed in the absence of CD8 T cells exhibited a distinct histology relative to those that devel- þ Results oped in the presence of CD8 T cells (Fig. 1E). In the former þ Gliomas that develop in the absence of CD8 T cells are more group, H&E staining revealed increased lymphohistiocytic infil- immunogenic and show more malignant histologic features trates in addition to a higher extent of necrosis. Vasculature in þ We investigated the effects of CD8 T cells on glioma development these gliomas was coopted by neoplastic cells that were dysmor- þ using a transgenic PDGF Pten / murine glioma model. In this phic and multinucleated. We observed more mitotic activity in þ model, gliomas are induced de novo by retroviral vector-based expres- gliomas that developed in the absence of CD8 T cells [mean sion of PDGF and Cre recombinase, which leads to loss of the Pten mitoses per high power field (HPF): 22.5] compared with gliomas þ tumor suppressor gene (in Ptenlox/lox mice) and overexpression of that developed in the presence of CD8 T cells (mean mitoses per PDGF. Resultant gliomas accumulate genetic alterations that resemble HPF: 12.4; P ¼ 0.0079, t test; Fig. 1F). Furthermore, mitotic þ those of the human disease (17). CD8 T-cell–mediated immunoedit- activity showed an inverse correlation with survival [r ¼ ing would require glioma infiltration by these lymphocytes. To inves- 0.8931, P ¼ 0.0005 (anti-CD8); r ¼0.9238, P ¼ 0.0001 (isotype þ þ tigate this hypothesis, we evaluated the presence of CD4 and CD8 T control), Pearson r; Fig. 1G]. cells at different stages of glioma progression. At 21 days postvirus In accordance to the cancer immunoediting paradigm, these results þ injection (d.p.i) for glioma induction, which is an early phase of glioma show that gliomas developed without CD8 T cells display increased þ development that precedes the accumulation of the genetic alterations immunogenicity compared with tumors developed under the CD8 and histologic features of high-grade gliomas (17), there was both T-cell pressure. Along with these findings, these immunogenic tumors þ þ CD4 and CD8 T-cell infiltration in the glioma (Supplementary exhibited more malignant histologic characteristics than the less Fig. S1A). We then evaluated T-cell infiltration at 35 d.p.i., a period in immunogenic tumors. which this model exhibits the genetic alterations and histology of high- þ grade gliomas (17). Given that these gliomas exhibit considerable Gliomas that develop in the absence of CD8 T cells have more growth within weeks, we normalized T-cell infiltration by the average chromosomal deletions and gene fusions þ glioma size (estimated by bioluminescence) for each time point We investigated whether the absence of CD8 T cells during (Supplementary Fig. S1B and S1C). This analysis revealed a decline glioma development has an effect on the tumor genome. We

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Figure 1. Gliomas that develop in the absence of CD8þ T cells are more immunogenic yet display increased oncogenic features. A, Scheme of the experimental design to investigate glioma immunoediting. Mice were treated with either isotype control antibody or anti-CD8þ T-cell antibody prior to the injection of the PDGFþ–IRES–Cre retrovirus, and throughout tumor development. Tumors were harvested and used to create primary cultures. These cell lines were intracranially implanted into recipients treated with either anti-CD8þ antibody or isotype control antibody prior tumor implantation, and until death. B, Representative ﬂow cytometry dot plots of CD4þ and CD8þ T cells in the spleen after administration of anti-CD8þ antibody and the isotype control antibody. C, Kaplan–Meier plots of animals treated with anti-CD8þ and isotype control antibodies prior to and during de novo tumor formation (n ¼ 28; top). Kaplan–Meier plot of animals that received the transplant of glioma originated in a host treated with anti-CD8þ antibody (depleted of CD8þ T cells; n ¼ 8; bottom left). (Continued on the following page.)

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performed paired somatic and germline exome sequencing, as well ERK and p38 signaling is activated in gliomas that develop in the þ as paired-end bulk RNA sequencing (RNA-seq), of gliomas absence of CD8 T cells induced de novo that developed in the presence or absence of Serine/threonine kinase BRAF, as well as phosphatase PTPN11, are þ CD8 T cells. Overall, there were only a few nonsynonymous upstream activators of the MAPK signaling pathway that have onco- mutations detected, mostly in TP53, consistent with our pre- genic properties (20). Both genes are involved in the activation of ERK. vious characterization of this glioma model (17). There was no We recently described the overrepresentation of activating mutations change in the number of point mutations between these gliomas in these genes in recurrent patients with GBM who responded to PD-1 (P ¼ 0.7047, t test; Fig. 2A). Yet gene fusions occurred more blockade (8). Given that these mutations were associated with sus- þ frequently in the gliomas that developed in the absence of CD8 ceptibility to immunotherapy, we hypothesized that murine gliomas þ T cells. Specifically, the mean fusion count of gliomas that devel- that developed in the absence of CD8 T cells may exhibit an over- þ oped in the absence of CD8 Tcellswas2.5versus0.5inthe expression and activation of MAPK/ERK. Murine gliomas did not þ presence of CD8 T cells (P ¼ 0.0309, Mann–Whitney U test; have mutations in these genes, yet RNA-seq analysis revealed that both Fig. 2B). In the group of gliomas that developed in the absence of Braf (P ¼ 0.0232, t test) and Ptpn11 (P ¼ 0.0256, t test) were over- þ þ CD8 T cells, one glioma showed two neoantigens derived from expressed in gliomas that developed in the absence of CD8 T cells two separate gene fusion events with predicted high binding (Fig. 3A). The oncogenic MAPK signaling pathway has distinct affinity to MHC-1 (H2-Kb and H2-Db). branches that include the ERK, p38, and JNK effectors. We next We assigned an aneuploidy score to each tumor as determined by investigated the expression of other known activators of these signaling Davoli and colleagues (13). Gliomas that developed in the absence cascades. Nras is an activator of the ERK cascade and was over- þ of CD8 T cells exhibited increased aneuploidy relative to those that expressed in these gliomas (P ¼ 0.0483, t test). Traf6, an activator þ developed in the presence of CD8 T cells. The mean aneuploidy of the p38 cascade, was also overexpressed (P ¼ 0.0483, t test; Fig. 3A). þ score of gliomas that developed in the absence of CD8 T cells There were no differences in expression of known activators of the JNK þ was 3.7, while it was 0.8 in the presence of CD8 T cells (P ¼ 0.0485, cascade (data not shown). Given the overexpression of these activators þ t test; Fig. 2C). We next used a complementary aneuploidy score of MAPK signaling in gliomas that developed in the absence of CD8 T calculation to infer somatic copy-number alterations evenly across cells, we then investigated the functional activity of the ERK and p38 þ the genome as described previously (14). This analysis revealed that cascades. Gliomas that developed in the absence of CD8 T cells had þ gliomas that developed in the absence of CD8 T cells had elevated activation of these cascades, as demonstrated by immunore- aneuploidy in 8.587% of the genome while that of the isotype activity for phosphorylation of ERK (pERK) and p38 (p-p38). pERK in þ control was only in 1.281% (P ¼ 0.0404, Mann–Whitney U test). gliomas that developed in the absence of CD8 T cells revealed intense Copy-number variation events were mapped in the genome. The diffuse positivity with a mean immunoreactivity score of 25.91, as þ increase in aneuploidy exhibited by gliomas that developed in the compared with gliomas that developed in the presence of CD8 T cells þ absence of CD8 T cells primarily consisted of chromosomal losses that had a mean immunoreactivity score of 13.41 (P ¼ 0.0229, (Fig. 2D). As hyperploidy has been reported to drive innate t test; Fig. 3B). Phospho-p38 expression in gliomas that developed þ immune activation via cGAS–STING signaling (18), we evaluated in the absence of CD8 T cells demonstrated scattered focal positivity the gene expression associated with cGAS–STING (Tbk1) activation with a mean immunoreactivity score of 14.50, as compared with þ by RNA-seq (19). This analysis revealed increased expression of gliomas that developed in the presence of CD8 T cells that had a Tbk1, as well as evidence-based associated genes in gliomas that mean immunoreactivity score of 9.41 (P ¼ 0.0285, t test). To determine þ þ developed in the absence of CD8 T cells (P ¼ 0.0138, ANOVA; whether this signaling was unique to the PDGF Pten / murine Fig. 2E). glioma model, we evaluated the phosphorylation of the ERK and þ In line with the malignant histologic features revealed in gliomas p38 cascades in a PDGFB RCAS Stat3 / transgenic glioma model, þ þ developed in the absence of CD8 T cells, oncogenes and tumor in which CD8 T cells were depleted by germline homozygous suppressor genes were examined by their relative expression between knockout of the Cd8a gene (21). In this model, pERK had a mean þ gliomas that developed in the presence versus absence of CD8 T cells. immunoreactivity score of 95.39 and 73.76 in the gliomas from either þ þ/þ We found that the gliomas that developed in the absence of CD8 the CD8 / or the CD8 background, respectively (P ¼ 0.4848, þ T cells exhibited differential expression of both oncogenes and tumor t test; Fig. 3C). Similar to the PDGF Pten / murine glioma model, þ þ þ suppressors relative to gliomas that developed in the setting of intact PDGFB RCAS Stat3 / gliomas demonstrated similar findings in / immunity (Fig. 2F). p-p38. More specifically, gliomas in the CD8 background revealed þ Jointly, our results indicate that CD8 T cells influence expres- scattered to moderate p-p38 positivity with a mean immunoreactivity sion of certain oncogenes and tumor suppressor genes, in addition score of 71.56, as compared with a mean immunoreactivity score of þ/þ to reducing aneuploidy and gene fusions. In contrast, the absence of 9.18 in the CD8 gliomas (P ¼ 0.0022, t test). We next investigated þ CD8 T cells during glioma formation is associated with these whether the expression of upstream regulators of the MAPK pathway genomic alterations and the increased gene expression of the correlated with phosphorylation of effectors of this pathway in the þ STING pathway. PDGF Pten / murine glioma model. Braf (r ¼ 0.7276; P ¼ 0.0408,

(Continued.)Kaplan–Meier plot of animals that received the transplant of glioma originated in a host treated with the isotype control antibody (immunocompetent host for de novo tumor formation; n ¼ 8; bottom right). D, Representative MRI of mice implanted with gliomas generated in the absence of CD8þ T cells. E, Histopathologic micrographs with H&E staining in tumors from mice treated with isotype control and anti-CD8þ antibodies. F, Barplotshowingmitoticfigures identified per 10 HPFs compared between tumors treated with the anti-CD8þ and isotype control antibody (n ¼ 20). G, Scatter plot showing the correlation between mitotic activity and survival from mice treated with isotype control and anti-CD8þ antibodies. P ¼ 0.7563 and P ¼ 0.0180 in (C), log-rank test. Data in F are shown as mean SEM, unpaired Student t test with points representing individual mice. Data in G [r ¼0.8931, P ¼ 0.0005 (anti-CD8þ); r ¼0.9238, P ¼ 0.0001 (isotype control)] obtained by Pearson correlation coefficient.

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Figure 2. Loss of CD8þ T cells during glioma formation is associated with increase in tumor genomic instability. Bee swarm plots showing the number of point mutations (n ¼ 33, A) and gene fusions (n ¼ 16, B) compared between tumors from animals treated with anti-CD8þ and isotype control antibodies. Predicted neoantigens derived from gene fusions with high binding affinity to H2-Kb and H2-Db are shown via schematic representation of the neoantigen prediction pipeline in the right. C, Bee swarm plots showing the aneuploidy score (n ¼ 21; left) and percentage of aneuploidy across the genome (n ¼ 21; right) compared between tumors from animals treated with anti-CD8þ and isotype control antibodies. D, Circos plot showing the chromosomal losses and amplifications compared between tumors from animals treated with anti-CD8þ and isotype control antibodies (n ¼ 21; P ¼ 0.034). Each wheel represents a track of a tumor for the copy-number variation analysis. Blue represents deletion while red represents amplification. The left schematic of each panel depicts only deep copy-number variations while the right also depicts shallow variations. E, Heatmap and box plot of the gene expression of the cGAS–STING signaling. The RPKM z-score of genes involved in cGAS–STING signaling is compared between tumors developed in the absence of CD8þ T cells and the isotype control (n ¼ 15). F, Volcano

plots show the relative expression of all oncogenes and tumor suppressors as plotted by their RPKM log2 fold change versus adjusted P value ¼ 0.05 by differential expression analysis in each group. Data in A–C, E are shown as mean SEM, P values in (A and C)byStudentt test, P value in B by Mann–Whitney U test. Points in A–C represent individual mice. P ¼ 0.0138 in E, two-way ANOVA.

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Figure 3. MAPK pathway (ERK and p38) activation is increased in gliomas that develop in the absence of CD8þ T cells and is correlated with increased macrophage/microglial inﬁltration. A, Box plots showing the Braf, Ptpn11, Nras,andTraf6 (n ¼ 16) expression compared between tumors developed from mice treated with anti-CD8 and isotype control antibodies by analysis of RNA-seq data in a Pten/ transgenic murine glioma model. B, Representative micrographs showing the pERK (left) and p-p38 (right) immunoreactivity index comparing anti-CD8þ and isotype control groups. C, Representative micrographs showing pERK (left) and p-p38 (right) immunoreactivity comparing tumors from CD8/ and CD8þ/þ mice in a Stat3/ transgenic murine glioma model. Bee swarm plot showing the quantiﬁcation of pERK and p-p38 (n ¼ 16; middle). D, Box plot showing the Shannon diversity index of immune cell populations in tumors developed in the absence of CD8þ T cells compared with the isotype control (n ¼ 20). E, Violin plots showing the macrophage, NK cell, and eosinophil (n ¼ 20) gene expression signature between groups by CIBERSORT of RNA-seq data. (Continued on the following page.)

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Pearson r) and Ptpn11 (r ¼ 0.8499; P ¼ 0.0075, Pearson r) expression proteins known to interact with AIF1 (Iba1; ref. 25) were no by RNA-seq correlated with pERK in gliomas that developed in the different between the groups of mice (P ¼ 0.5421, ANOVA; þ absence of CD8 T cells (Supplementary Fig. S2A and S2B). Nras Fig. 3H). expression did not correlate with pERK in the absence or presence of These results suggest increased infiltration of cells from innate CD8 T cells (Supplementary Fig. S2C). Traf6 showed a statistical trend immunity, including macrophages/microglia, with strong expression þ in expression with p-p38 in gliomas that developed in the absence of of CD11b in gliomas that developed in the absence of CD8 T cells. þ CD8 T cells (r ¼ 0.6496; P ¼ 0.0813, Pearson r; Supplementary Fig. S2D). MAPK signaling correlates with macrophage infiltration in þ Together, these results indicate that the overexpression and acti- gliomas that develop in the absence of CD8 T cells vation of the p38 and ERK cascade of the MAPK signaling pathway, are Because we encountered robust yet variable macrophage infiltration þ oncogenic features that are suppressed by CD8 T cells during the and MAPK pathway activation in the gliomas that developed in the þ progression of gliomas. Our results also confirm the integrity of the absence of CD8 T cells, we investigated whether this signaling MAPK signaling cascade in these gliomas, as expression of upstream correlated with the degree of immune infiltration. CD11b expression members of the pathway are associated with phosphorylation of the highly correlated with pERK in gliomas that developed in the absence þ downstream effectors, which is indicative of activation. of CD8 T cells (r ¼ 0.6512; P ¼ 0.0414, Pearson r) but not in those þ gliomas that developed in the presence of CD8 T cells (r ¼ 0.3252; Macrophage/microglial infiltration increases in gliomas that P ¼ 0.3591, Pearson r; Fig. 3I). CD11b also correlated with p-p38 in þ þ develop in the absence of CD8 T cells gliomas that developed in the absence of CD8 T cells (r ¼ 0.8950; P ¼ þ We hypothesized that the immunogenicity of gliomas that devel- 0.0005, Pearson r) but not in gliomas developed with CD8 T cells þ oped in the absence of CD8 T cells relates to a distinct immune present (r ¼ 0.4044; P ¼ 0.2464, Pearson r). Similarly, Iba1 expression microenvironment. We determined the Shannon diversity index correlated with pERK in gliomas that developed in the absence of þ between the immune cell populations within glioma groups. Gliomas CD8 T cells (r ¼ 0.8591; P ¼ 0.0014, Pearson r) but not in gliomas þ þ that developed in the absence of CD8 T cells had a collective index that developed in the presence of CD8 T cells (r ¼ 0.3087; P ¼ 0.3855, ranging from 1.228 to 1.652 while those that developed with intact Pearson r). Iba1 also correlated with p-p38 in gliomas that developed in þ immunity had an index ranging from 1.223 to 2.081 (P ¼ 0.1578, t the absence of CD8 T cells (r ¼ 0.8934; P ¼ 0.0005, Pearson r) but not test; Fig. 3D), indicating that the diversity of glioma-infiltrating in gliomas with intact immunity (r ¼ 0.1560; P ¼ 0.6670, Pearson r). immune cell populations between both groups of mice is similar. We We then investigated the relationship between MAPK signaling and then used CIBERSORT to estimate the immune cell populations in the macrophage/microglial infiltration in human GBM. mRNA expres- tumors by RNA-seq, as done previously (22). This analysis showed an sion analysis of 161 patients with GBM in TCGA revealed a correlation increase in the expression signature of natural killer (NK) cells (q ¼ between ITGAM (CD11b) and MAPK13 (p38delta), an activator of the 0.04), macrophages (q ¼ 0.04), and eosinophils (q ¼ 0.021) in gliomas p38 cascade (r ¼ 0.6194; P < 0.0001, Pearson r; Supplementary þ that developed while undergoing treatment with anti-CD8 antibody Fig. S3A). mRNA expression analysis of patients with GBM in TCGA (Fig. 3E). revealed a correlation between AIF1 (Iba1) and RRAS, an activator of IHC staining confirmed that macrophage/microglia infiltration the ERK cascade (r ¼ 0.6141; P < 0.0001, Pearson r; Supplementary þ in gliomas was enhanced in the absence of CD8 T cells. Iba1, a Fig. S3B). AIF1 gene expression also correlated with MAPK13 (r ¼ macrophage/microglia marker, showed higher expression with a 0.6008; P < 0.0001, Pearson r; Supplementary Fig. S3C), and MAP3K8, mean immunoreactivity score of 22.12 in gliomas that developed in an activator of the ERK cascade (r ¼ 0.8159; P < 0.0001, Pearson r; þ the absence of CD8 T cells, relative to a mean immunoreactivity Supplementary Fig. S3D). These results suggest that MAPK activation þ score of 11.22 in gliomas that developed in the presence of CD8 and macrophage/microglial recruitment are highly interrelated in both T cells (P ¼ 0.0003, t test; Fig. 3F). In addition, we evaluated the murine gliomas and human patients with GBM. Furthermore, this þ integrin subunit CD11b highly expressed on monocytes, granulo- association appears to be strengthened by the absence of CD8 T cells cytes, and/or tissue resident macrophages, which has been recently during glioma development. reported to be implicated with proinflammatory polarization of þ tumor-associated macrophages (23, 24). This marker revealed Gliomas that develop in the absence of CD8 T cells have a diffuse positivity with a mean immunoreactivity score of 27.87 in proinflammatory macrophage gene signature þ gliomas that developed in the absence of CD8 T cells, as compared Given that macrophage/microglial infiltration was robust in glio- þ with a mean immunoreactivity score of 20.79 in gliomas that mas that developed in the absence of CD8 T cells, we explored þ developed in the presence of CD8 T cells (P ¼ 0.0355, t test; whether this immune cell infiltration was associated with an inflam- Fig. 3F). To strengthen this point, genes coding for proteins known matory milieu. Inflammatory chemokines and cytokines released to interact with ITGAM (CD11b; ref. 25) showed a trend for during the elimination phase of cancer immunoediting (4) were þ elevated mRNA expression in gliomas that developed in the absence increased in gliomas that developed in the presence of CD8 T cells þ þ of CD8 T cells (P ¼ 0.0693, ANOVA; Fig. 3G). Genes coding for relative to the CD8 T-cell–depleted background (P ¼ 0.0106,

(Continued.) F, Representative micrographs showing the Iba1 (left) and CD11b (middle) immunoreactivity index compared between groups by IHC. Quantiﬁcation of CD11b and Iba1 (n ¼ 20) IHC expression (right). G, RNA-seq–based expression analysis of genes of proteins known to interact with ITGAM (CD11b; H) and of genes of proteins known to interact with AIF1 (Iba1) in tumors developed in the absence of CD8þ T cells versus the isotype control (n ¼ 15). I, Correlation between CD11b expression and pERK (n ¼ 10). Correlation between CD11b expression and p-p38 (n ¼ 10). Correlation between Iba1 expression and pERK (n ¼ 10). Correlation between Iba1 expression and p-p38 (n ¼ 10). Data are shown as mean SEM, P values in A, B, C, D, F, G,andH by unpaired Student t test. For B, C, F,andG, points represent individual mice. q values by statistical adjustment of P values using the Benjamini–Hochberg constant. r and P values in I by Pearson correlation coefﬁcient. Points represent individual mice.

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CD8þ T-cell–Mediated Immunoediting in Murine Glioma

Figure 4. Macrophages/microglia in tumors that develop in the absence of CD8þ T cells have an enriched proinflammatory phenotype. A, Box plot showing RPKM gene expression standardized score. B, GSEA employing the tumor inflammation signature between animals treated with anti-CD8þ and isotype control antibodies. C, GSEA (left) and heatmap (right) evaluating the expression signature of proinflammatory phenotype in macrophages/microglia in tumors developed in the absence þ of CD8 T cells and the isotype control (left, NES ¼ 1.785; FDR < 0.001; n ¼ 15). Data in A are shown as mean SEM, P ¼ 0.0106 by two-way ANOVA test. P ¼ 0.033 in B and P < 0.001 in C, two-sided Kolmogorov–Smirnov test.

ANOVA; Fig. 4A). However, gene set enrichment analysis (GSEA) environment, including proinflammatory macrophage/microglial employing the tumor inflammation signature, a gene expression polarization. profile indicative of an inflamed tumor microenvironment (TME) developed by Ayers and colleagues (26), revealed that gliomas treated þ with the anti-CD8 antibody had an active inflammatory TME Discussion þ compared with gliomas developed under the presence of CD8 T Cancer immunoediting shapes tumor evolution by eliminating cells (P ¼ 0.033, Kolmogorov–Smirnov test; Fig. 4B). immunogenic tumor cell variants that can evade immune surveillance, As macrophages can exist either in a nonpolarized state or along a while others are selected for resistance to immune recognition (6). þ continuum between proinflammatory and antiinflammatory (27), we Particularly, CD8 T-cell recognition of tumor antigens drives the evaluated such phenotypic changes in these gliomas. GSEA revealed an immunologic sculpting of a developing cancer (4). Whereas this increased proinflammatory macrophage phenotype by a gene signa- concept has previously been suggested in GBM (30–32), our study þ ture reported to contribute to such polarization (28) in gliomas that directly investigated the effects of CD8 T-cell–dependent immunoe- þ developed in the absence of CD8 T cells, with a normalized enrich- diting in the development of gliomas. We generated experimental þ ment score (NES) of 1.785 compared with those that developed in the evidence of immunoediting by CD8 T cells in a transgenic murine þ presence of CD8 T cells (P < 0.001, Kolmogorov–Smirnov glioma model, as depletion of this immune cell population had a test; Fig. 4C). A gene signature associated with an anti- profound effect on tumor genomic evolution, attributable to selection þ inflammatory macrophage phenotype (28, 29) did not reveal signif- of immunogenic tumor clones by CD8 T cells. While we found strong þ icant differences in gliomas developed in a CD8 T-cell–depleted differences between gliomas that developed with or without this background (NES ¼ 1.2, P ¼ 0.232, data not shown). immune cell population, we also found important variation of these Together, these results indicate that gliomas that develop in features within the group of gliomas that developed in the absence of þ þ the absence of CD8 T cells have an inflamed tumor micro- CD8 T cells. Recent evidence has shown that glioma genomic

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þ evolution is to some extent stochastic (33), that is, is a neutral is affected by the presence of CD8 T cells during glioma develop- þ evolutionary process, with genomic events that can lead to immune ment. Whereas these genes were not mutated in the absence of CD8 recognition or evasion (31). Surprisingly, whereas depletion of T cells, we did find them overexpressed in this setting, along with þ CD8 T cells during glioma development had profound effects on several other known activators of ERK and p38 signaling (8). In þ tumor genotype, phenotype, immunogenicity, and microenviron- PDGFB RCAS Stat3 / gliomas, a separate transgenic murine gli- ment, depletion of these immune effector cells did not lead to shorter oma model, the p38 cascade was also more phosphorylatyed/activated þ þ survival. CD8 T-cell depletion did not alter survival of other trans- when tumors developed in the absence of CD8 T cells. We also þ genic glioma models in which tumors develop de novo (Rao and observed that in the setting of CD8 T-cell depletion, MAPK activa- colleagues, submitted). Previous studies also showed that, whereas tion in gliomas was associated with robust tumor infiltration with þ þ immunosuppression can increase the incidence of cancer, it usually CD11b and Iba1 macrophages/microglia. Moreover, in lung cancer, does not affect survival upon cancer diagnosis (34). These observations activating mutations of KRAS (which leads to MAPK signaling) are in the face of tumor progression are termed the “Hellstrom para- associated with robust tumor infiltration by macrophages (43). MAPK dox” (35, 36). How this paradoxical situation manifests remains activation has been extensively studied as a driver of innate immunity unresolved. De novo tumor formation and the influence of host with effector proinflammatory polarization (44), yet these results immunity have been explored previously (37, 38). For instance, Urban suggest that activation of MAPK signaling in tumors can influence and colleagues described that UV light–related immunosuppression in cancer microenvironment and immunogenicity. These observations þ mice influences grafting of skin tumors upon transplantation, suggest- of active MAPK signaling in gliomas developed in the absence of CD8 ing immune selection (37). Yet our study is different, as we show that T cells add to our recent discovery of MAPK-activating mutations as þ depletion of CD8 T cells during gliomagenesis leads to tumors that molecular features associated with glioblastoma response to immu- exhibit more immunogenicity. notherapy (8). Collectively, this evidence implicates MAPK in immu- We sought to explore genomic differences as a result of tumor- noediting during glioma development and in susceptibility to immu- þ igenesis in the absence of CD8 T cells. Low mutational burden is a notherapy, yet the causal relationship and mechanism that drives this common explanation for the relative lack of immunogenicity in association remains unknown. GBM (39). We found no change in the number of point mutations Our mouse glioma model recapitulates loss of Pten function, a þ as a result of the absence of T cells. Our results imply that CD8 genetic alteration that has been associated with an immunosuppressive T cells do not protect against point mutations or influence this facet microenvironment, and lack of response to immunotherapy, including of immunogenicity to an appreciable extent. Yet, it is important to poor response to PD-1 blockade in recurrent patients with consider that in this particular glioma model, mice may not survive GBM (8, 45, 46). Indeed, PTEN deficiency was recently shown to long enough for the development and selection of mutations other promote macrophage infiltration through lysil oxidase secretion pro- than those with a strong advantage for cell growth (i.e., Trp53). On moting tumor progression (47). Whereas our transgenic model is the other hand, we observed an increase in the frequency of glioma generated through Pten deletion, we observed increased infiltration of þ gene fusions in the absence of CD8 T cells. Gene fusions are macrophages/microglia and a proinflammatory phenotype in gliomas þ known to be strong drivers of neoplasia as well as the formation of that developed in the absence of CD8 T cells. In addition, we neoantigens from junctional epitopes (40, 41). Bioinformatic encountered a strong relationship between MAPK signaling and modeling predicted these gene fusions to have high binding affinity macrophage/microglial recruitment in these gliomas. mRNA expres- to MHC-I (H2-Kb and H2-Db). Whereas the antigenic properties sion analysis of human patients with GBM in silico revealed a strong predicted warrant experimental validation, this finding is interest- correlation between levels of known activators of the MAPK signaling ing as junctional epitopes from gene fusions are a potential source pathway and macrophage markers, asserting the biological signifi- for development of neoantigen-based vaccination. Among other cance of these findings in the human disease. Furthermore, MAPK is genomic alterations, increased levels of aneuploidy were also known to promote a proinflammatory innate immune response (44). þ observed in gliomas developed in the absence of CD8 T cells. Consistent with this, we established an association between MAPK Previous research associated aneuploidy with immune evasion in pathway activation and a proinflammatory macrophage recruitment þ cancer (13). Meanwhile, we show that gliomas developed in the in gliomas. Whereas we observed that CD8 T cells dampen MAPK þ absence of CD8 T cells have both high aneuploidy and a more signaling in gliomas, the effect of MAPK signaling on proinflammatory immunogenic phenotype, reinforcing the biological importance of microenvironment in the context of glioma immunoediting remains to negative immunoselection of cancer cells harboring somatic copy- be elucidated. number alterations during cancer development (18). Chromosomal In sum, our study suggests that glioma immunoediting involves þ instability drives innate immune activation via cGAS–STING in CD8 T-cell–dependent selection of tumor clones, leading to immune cancer (19), and in support of this, we showed upregulation of genes evasion by the time tumors reach a clinical stage. Cancer immunoedit- þ related to this pathway in gliomas that developed in the absence of ing mediated by CD8 T cells simultaneously protects against certain þ CD8 T cells (37). Considering that the aneuploidy content in evolutionary hallmarks of cancer and selects for tumor cell variants human gliomas is relatively low compared with other cancers (42), that are capable of immune evasion. Understanding the interplay þ our results imply that CD8 T cells might be negatively selecting between glioma progression and antitumoral immunity is essential for against high aneuploidy glioma cells during tumor development the development of effective immunotherapy for this disease. Manip- that manifest as low aneuploidy tumors at late tumoral stages ulation of tumor evolution towards a more immunogenic path may be þ þ as observed in the murine gliomas that developed under the CD8 explored by surpassing the CD8 T-cell–mediated immune evasion in T-cell pressure. this disease. We recently reported the presence of MAPK-activating mutations in BRAF and PTPN11 in recurrent patients with GBM who responded Disclosure of Potential Conflicts of Interest to PD-1 blockade (8). Given the implication of these genes and A.M. Sonabend has ownership interest in a patent. No potential conflicts of MAPK in response to immunotherapy, we explored how this pathway interest were disclosed by the other authors.

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CD8þ T-cell–Mediated Immunoediting in Murine Glioma

Authors’ Contributions Acknowledgments Conception and design: T. Tsujiuchi, V.A. Arrieta, P. Canoll, A.M. Sonabend This work was funded by 1R01NS110703-01A1 (A.M. Sonabend), Development of methodology: J.R. Kane, J. Zhao, T. Tsujiuchi, B. Laffleur, P. Canoll, 5DP5OD021356-05 (to A.M. Sonabend), P50CA221747 SPORE for Translational A.M. Sonabend Approaches to Brain Cancer (to A.M. Sonabend), developmental funds from the Acquisition of data (provided animals, acquired and managed patients, provided Robert H. Lurie Cancer Center Support Grant #P30CA060553 (to A.M. Sonabend), facilities, etc.): T. Tsujiuchi, B. Laffleur, A. Mahajan, G. Rao, L. Chen, D.Y. Zhang, Institutional grant from the Lou and Jean Malnati Brain Tumor Institute (to A.M. C. Lee-Chang, G. Rothschild, U. Basu, C. Horbinski, A.B. Heimberger, P. Canoll, Sonabend), and the Matthew Larson Foundation Iron Matt Research Award (to A.M. A.M. Sonabend Sonabend). Histology services were provided by the Northwestern University Mouse Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Histology and Phenotyping Laboratory, which is supported by NCI P30-CA060553 computational analysis): J.R. Kane, J. Zhao, T. Tsujiuchi, V.A. Arrieta, awarded to the Robert H. Lurie Comprehensive Cancer Center. C. Dmello, D.Y. Zhang, E. Gonzalez-Buendia, C. Lee-Chang, T. Xiao, C. Horbinski, M.S. Lesniak, A.B. Heimberger, R. Rabadan, A.M. Sonabend The costs of publication of this article were defrayed in part by the payment of page Writing, review, and/or revision of the manuscript: J.R. Kane, V.A. Arrieta, G. Rao, charges. This article must therefore be hereby marked advertisement in accordance C. Horbinski, M.S. Lesniak, A.B. Heimberger, P. Canoll, A.M. Sonabend with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Mela, L. Chen, A.M. Sonabend Received September 21, 2019; revised March 27, 2020; accepted May 14, 2020; Study supervision: A.M. Sonabend published first May 19, 2020.

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OF12 Clin Cancer Res; 2020 CLINICAL CANCER RESEARCH

CD8+ T-cell −Mediated Immunoediting Influences Genomic Evolution and Immune Evasion in Murine Gliomas

Joshua R. Kane, Junfei Zhao, Takashi Tsujiuchi, et al.

Clin Cancer Res Published OnlineFirst May 19, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-3104

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