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The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Supplementary Figures and Tables.

The central nervous system (CNS) antigen-presenting cell (APC) that primes antitumor CD8+ T-cell responses remains undefined. Elsewhere in the body, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain parenchyma cDC1 are extremely rare; cDCs localize to the choroid plexus and dura. Thus, whether the cDC1 play a function in presenting antigen derived from parenchymal sources in the tumor setting remains unknown. Using preclinical glioblastoma (GBM) models and cDC1-deficient mice, we explored the presently unknown role of cDC1 in CNS antitumor immunity. We determined that, in addition to infiltrating the brain tumor parenchyma itself, cDC1 prime neoantigen-specific CD8+ T cells against brain tumors and mediate checkpoint blockade-induced survival benefit. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1 equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same APC subsets, we identified a tumor-specific fluorescent metabolite of 5-aminolevulinic acid, which fluorescently labeled tumor cells during fluorescence-guided GBM resection. Together, these data elucidate the specialized behavior of cDC1 and suggest that cDC1 play a significant role in CNS antitumor immunity.

Clonal clustering assignments generated from sciClone.

Supplemental Table 2. List of genes contained in genomic region 12q15 (chr12: 69692322-71120515) that are recurrently amplified in high-grade serous ovarian cancers.

SMA-560 Composite

Supplemental Table 1. List of 50 genes that are specifically essential and amplified in ovarian cancer.

Supplementary Figure S1. Mutation Spectra of each subclonal population. Supplementary Figure S2. Somatic Copy Number calls. Top: Primary, Middle: C7-T2 Metastasis, Bottom: T7-8 Metastasis. Supplementary Figure S3. Mutation signatures derived for each subclone independently. Supplementary Figure S4: Expression of immune-related genes.

Supplementary Figure from Characterization of the Genomic and Immunologic Diversity of Malignant Brain Tumors through Multisector Analysis

Supplemental Figure 1. Correlation between FRS2 expression and copy number in primary high-grade ovarian serous adenocarcinoma.

Supplemental Figure 2. FRS2 suppression induces apoptosis in FRS2 amplified cell lines, shown by propidium iodide staining and flow cytometry.

GL261 Composite

Legends for supplemental Figure 1 and 2

Supplementary Table from Characterization of the Genomic and Immunologic Diversity of Malignant Brain Tumors through Multisector Analysis

The “cancer immunogenomics” paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific “neoantigens” in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db–restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb–restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell–activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007–15. ©2016 AACR.

Despite some success in secondary brain metastases, targeted or immune-based therapies have shown limited efficacy against primary brain malignancies such as glioblastoma (GBM). Although the intratumoral heterogeneity of GBM is implicated in treatment resistance, it remains unclear whether this diversity is observed within brain metastases and to what extent cancer cell–intrinsic heterogeneity sculpts the local immune microenvironment. Here, we profiled the immunogenomic state of 93 spatially distinct regions from 30 malignant brain tumors through whole-exome, RNA, and T-cell receptor sequencing. Our analyses identified differences between primary and secondary malignancies, with gliomas displaying more spatial heterogeneity at the genomic and neoantigen levels. In addition, this spatial diversity was recapitulated in the distribution of T-cell clones in which some gliomas harbored highly expanded but spatially restricted clonotypes. This study defines the immunogenomic landscape across a cohort of malignant brain tumors and contains implications for the design of targeted and immune-based therapies against intracranial malignancies.Significance:This study describes the impact of spatial heterogeneity on genomic and immunologic characteristics of gliomas and brain metastases. The results suggest that gliomas harbor significantly greater intratumoral heterogeneity of genomic alterations, neoantigens, and T-cell clones than brain metastases, indicating the importance of multisector analysis for clinical or translational studies.This article is highlighted in the In This Issue feature, p. 1

High-grade serous ovarian cancers (HGSOC) are characterized by widespread recurrent regions of copy-number gain and loss. Here, we interrogated 50 genes that are recurrently amplified in HGSOC and essential for cancer proliferation and survival in ovarian cancer cell lines. FRS2 is one of the 50 genes located on chromosomal region 12q15 that is focally amplified in 12.5% of HGSOC. We found that FRS2-amplified cancer cell lines are dependent on FRS2 expression, and that FRS2 overexpression in immortalized human cell lines conferred the ability to grow in an anchorage-independent manner and as tumors in immunodeficient mice. FRS2, an adaptor protein in the FGFR pathway, induces downstream activation of the Ras–MAPK pathway. These observations identify FRS2 as an oncogene in a subset of HGSOC that harbor FRS2 amplifications.Implications: These studies identify FRS2 as an amplified oncogene in a subset of HGSOC. FRS2 expression is essential to ovarian cancer cells that harbor 12q15 amplification. Mol Cancer Res; 13(3); 502–9. ©2014 AACR.

Co-expression of immune checkpoints in murine glioma

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Purpose:Pembrolizumab improved survival in patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)–unrelated HNSCC.Patients and Methods:Neoadjuvant pembrolizumab (200 mg) was administered and followed 2 to 3 weeks later by surgical tumor ablation. Postoperative (chemo)radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (Results:Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3–4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among 18 patients with high-risk pathology was 16.7% (95% confidence interval, 3.6%–41.4%). pTR ≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFNγ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0 and confirmed clonal loss in some patients.Conclusions:Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The 1-year relapse rate in patients with high-risk pathology was lower than historical.

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM.Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers.Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations.Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR.See related commentary by Jackson and Lim, p. 4059

PDF file - 486KB, Figure S1 (A) Average statistics for depth of coverage at 20X, 30X and 40X of all MOC lines. Robust reads were obtained in all samples with a range of 97-98 percent for 20X, 93-95 percent for 30X and 88-92 percent for 40X. (B) Putative SNVs, nsSNVs (Table S1 and S2) and Indels (Table S4) of all MOC lines. Figure S2 Oncoprints from cBio of AKAP9, MED12L, THSD7A, MUC5B, MYH6, LAMA1, LRP2, and 3 RAS (Table S6) genes representing other candidate tumor promoters as compared to the 3 RAS genes. Note that for AKAPs and MED components, we found 9 AKAP family member mutations in 20.4 percent of tumors, with AKAP9 changes in 7 percent (Fig. 1D). Six components of the mediator complex were mutated in 14.7% of cases, with MED12L changes in 5% (Fig. 1E). Figure S3 Number of nsSNV's per node negative (N0) and node positive (N+) tumor in (A) all TCGA OSCC (note that patient TCGA-D6-6516 (N0) with 1463 mutations is not included in this graph) and (B) TCGA OSCC patients who had smoking history reported. There was no significant difference in the average number of mutations between N0 (3272 nsSNVs) and N+ (3097 nsSNVs) patients regardless of smoking status (Tables S8, S9). This analysis showed 17 genes commonly mutated in mouse indolent and human N0 tumors and 55 common genes mutated in mouse aggressive and human N+ tumors (Supplementary Table S10). However, none of these common genes were mutated at high frequency in the human N0 or N+ datasets (Supplementary Table S11 and 12). Finally, comparing N0 and N+ tumors from human TCGA data also showed that specific mutations occur infrequently in both the metastatic and non-metastatic tumors (data not shown). Figure S4 SAM plotsheet of MOC line microarray data with estimated miss rates for delta=4.68 Figure S5 (A) GSEA data of OCAMP-A on UW/FHCRC data with (B) first condensation of enriched genes. (C) GSEA data of OCAMP-A on MDA data with (D) first condensation of enriched genes. (E) GSEA data of OCAMP-A on TCGA data with (F) first condensation of enriched genes. Figure S6 (A) Disease specific survival (DSS) after weighted voting classification of OCAMP-B signature on the MD Anderson dataset shows worse outcome for those with aggressive classification (p=0.028). Figure S7 (A) Disease specific survival (DSS) and (B) overall survival (OS) after weighted voting classification of OCAMP-B signature on the UW/FHCRC dataset shows worse outcome for those with aggressive classification (p

Cytokine production across immunologic compartments

Gating strategy for tumor infiltrating lymphocytes

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Immune checkpoint and cytokine expression on human GBM CD4+ TIL

Supplementary Figure S1 (related to Figure 1): PoleP286R mouse model design, validation, and tumor findings. Supplementary Figure S2 (related to Figure 1): PoleS459F mouse model design, validation, and tumor findings. Supplementary Figure S3 (related to Figure 2): WES analysis reveals a stochastic accumulation of mutations in Pole mutant tumors. Supplementary Figure S4 (related to Figure 2): Tumors from Pole mutant mice exhibit mutational signatures found in POLE driven human cancers. Supplementary Figure S5 (related to Figure 2): Exome data from tumor fractions from a single mouse were compared for 3 additional mice. Supplementary Figure S6. Determination of cell of origin for PoleS459F/S459F and PoleS459F/+ mice lymphomas (Related to Figure 3). Supplementary Figure S7. Characterization of T cell malignancies in PoleS459F/S459F and PoleS459F/+ mice (Related to Figure 3). Supplemental Figure S8. Extrinsic effect of 'Group B' malignant T cells on B cell population; (Related to Figure 3).

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Clinical trial protocol

T cell counts per gram of tumor across tumor models

Memory phenotype of human GBM TIL

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype–phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients.Significance:Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459

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Purpose: Improved understanding of the molecular basis underlying oral squamous cell carcinoma (OSCC) aggressive growth has significant clinical implications. Herein, cross-species genomic comparison of carcinogen-induced murine and human OSCCs with indolent or metastatic growth yielded results with surprising translational relevance.Experimental Design: Murine OSCC cell lines were subjected to next-generation sequencing (NGS) to define their mutational landscape, to define novel candidate cancer genes, and to assess for parallels with known drivers in human OSCC. Expression arrays identified a mouse metastasis signature, and we assessed its representation in four independent human datasets comprising 324 patients using weighted voting and gene set enrichment analysis. Kaplan–Meier analysis and multivariate Cox proportional hazards modeling were used to stratify outcomes. A quantitative real-time PCR assay based on the mouse signature coupled to a machine-learning algorithm was developed and used to stratify an independent set of 31 patients with respect to metastatic lymphadenopathy.Results: NGS revealed conservation of human driver pathway mutations in mouse OSCC, including in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Moreover, comparative analysis between The Cancer Genome Atlas and mouse samples defined AKAP9, MED12L, and MYH6 as novel putative cancer genes. Expression analysis identified a transcriptional signature predicting aggressiveness and clinical outcomes, which were validated in four independent human OSCC datasets. Finally, we harnessed the translational potential of this signature by creating a clinically feasible assay that stratified patients with OSCC with a 93.5% accuracy.Conclusions: These data demonstrate surprising cross-species genomic conservation that has translational relevance for human oral squamous cell cancer. Clin Cancer Res; 20(11); 2873–84. ©2014 AACR.

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Purpose:In a head and neck squamous cell carcinoma (HNSCC) “window of opportunity” clinical trial, we reported that trametinib reduced MEK-Erk1/2 activation and resulted in tumor responses in a subset of patients. Here, we investigated resistance to trametinib and molecular correlates in HNSCC cell lines and patient samples.Experimental Design:HNSCC cell lines were treated with trametinib to generate resistant lines. Candidate bypass pathways were assessed using immunoblotting, CRISPR knockout, and survival assays. Effectiveness of combined trametinib and verteporfin targeting was evaluated. Patient-derived xenografts (PDXs) from responder patients were treated with trametinib and resistant tumors were analyzed. Window trial clinical samples were subjected to whole-exome and RNA sequencing.Results:HNSCC cell lines developed resistance (CAL27-TR and HSC3-TR) after prolonged trametinib exposure. Downstream effectors of the Hippo pathway were activated in CAL27-TR and HSC3-TR, and combined trametinib and verteporfin treatment resulted in synergistic treatment response. We defined the Hippo pathway effector Yap1 as an induced survival pathway promoting resistance to trametinib in HSC3-TR. Yap1 was necessary for HSC3-TR trametinib resistance, and constitutively active Yap1 was sufficient to confer resistance in parental HSC3. Analysis of trametinib neoadjuvant trial patient tumors indicated canonical MEK-Erk1/2 pathway activating mutations were infrequent, and Yap1 activity increased following trametinib treatment. Trametinib treatment of a PDX from a responder patient resulted in evolution of resistance with increased Yap1 expression and activity.Conclusions:These studies identify a Yap1-dependent resistance to trametinib therapy in HNSCCs. Combined Yap1 and MEK targeting may represent a strategy to enhance HNSCC response.

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CyTOF panel antibodies