Spatially Resolved and Quantitative Analysis of VISTA/PD-1H As a Novel Immunotherapy Target in Human Non–Small Cell Lung Cance

Published OnlineFirst December 4, 2017; DOI: 10.1158/1078-0432.CCR-17-2542

Personalized Medicine and Imaging Clinical Cancer Research Spatially Resolved and Quantitative Analysis of VISTA/PD-1H as a Novel Immunotherapy Target in Human Non–Small Cell Lung Cancer Franz Villarroel-Espindola1, Xiaoqing Yu2, Ila Datar1, Nikita Mani1, Miguel Sanmamed3, Vamsidhar Velcheti4, Konstantinos Syrigos5, Maria Toki1, Hongyu Zhao2, Lieping Chen3, Roy S. Herbst6, and Kurt A. Schalper1,6

Abstract

Purpose: Determine the localized expression pattern and clin- Expression in tumor and stromal cells was seen in 21% and ical significance of VISTA/PD-1H in human non–small cell lung 98% of cases, respectively. The levels of VISTA were positively þ þ cancer (NSCLC). associated with PD-L1, PD-1, CD8 TcellsandCD68 Experimental Design: Using multiplex quantitative immuno- macrophages. VISTA expression was higher in T-lymphocytes fluorescence (QIF), we performed localized measurements of than in macrophages; and in cytotoxic T cells than in T-helper VISTA, PD-1, and PD-L1 protein in 758 stage I–IV NSCLCs from cells. Elevated VISTA was associated with absence of EGFR 3 independent cohorts represented in tissue microarray format. mutations and lower mutational burden in lung adenocarci- þ The targets were selectively measured in cytokeratin tumor nomas. Presence of VISTA in tumor compartment predicted þ þ þ epithelial cells, CD3 T cells, CD4 T-helper cells, CD8 cytotoxic longer 5-year survival. þ þ T cells, CD20 B lymphocytes and CD68 tumor-associated Conclusions: VISTA is frequently expressed in human NSCLC macrophages. We determined the association between the targets, and shows association with increased tumor-infiltrating lympho- clinicopathological/molecular variables and survival. Genomic cytes, PD-1 axis markers, specific genomic alterations and out- analyses of lung cancer cases from TCGA were also performed. come. These results support the immunomodulatory role of Results: VISTA protein was detected in 99% of NSCLCs with VISTA in human NSCLC and suggests its potential as therapeutic a predominant membranous/cytoplasmic staining pattern. target. Clin Cancer Res; 1–12. 2017 AACR.

Introduction the tumor immune microenvironment have used nonhuman model systems and/or methods requiring tissue grinding such as The antitumor immune response is essential to control tumor flow cytometry/CyTOF and mRNA profiling. This has limited the growth and progression. Blockade of immune coregulatory sig- understanding of the spatial context and evaluation of key cell/ nals in the tumor immune microenvironment such as the PD-1/ molecular interactions in human malignancies. For instance, PD-L1 axis has revolutionized the treatment of diverse tumor multiple potentially actionable immune-modulatory proteins, types (1–3). Notably, expression of PD-L1 protein or metrics of such as CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, and other can be tumor immune infiltration in pretreatment samples can be used coexpressed on immune cells and functionally interact with each to predict sensitivity/resistance to treatment (1–6). Therefore, other to modulate the response against tumors (7–9). careful evaluation of the tumor immune landscape could provide Due to its clinical relevance, the PD-L1/PD-1 pathway has been essential information to understand the role of the immune extensively studied in human malignancies. Engagement of PD-1 system during tumor progression and to define the optimal receptor by its ligand PD-L1 (and also PD-L2) mediates inhibitory treatment modalities using both immune and nonimmune ther- signals in T cells and antiapoptotic signals in tumor cells (10–13). apies. To date, most studies characterizing multiple cell/targets in In human neoplasms, PD-L1 is predominantly expressed in malignant tumor cells and macrophages in response to IFNg 1Department of Pathology, Yale School of Medicine, New Haven, Connecticut. stimulation or as a result of constitutive oncogenic signaling. 2Department of Public Health, Yale School of Medicine, New Haven, Connecticut. Elevated levels of PD-L1 are found with variable frequency in 3Immunobiology, Yale School of Medicine, New Haven, Connecticut. 4Solid human malignancies and blockade of this pathway produces Tumor Oncology, Cleveland Clinic Foundation, Cleveland, Ohio. 5Oncology Unit prominent and lasting antitumor responses in a proportion of 6 GPP, Athens School of Medicine, Greece. Medical Oncology and Yale Cancer patients with cancer. This supports a fundamental role of the PD-1 Center, New Haven, Connecticut. pathway in the antitumor immune evasion (2, 3, 10–12). PD-L1 Note: Supplementary data for this article are available at Clinical Cancer (also known as B7-H1 or CD274) belongs to the B7 family of Research Online (http://clincancerres.aacrjournals.org/). immune-regulatory molecules. Since its initial description, addi- Corresponding Author: Kurt A. Schalper, Yale University School of Medicine, PO tional members of this family have been reported containing Box 208023, 310 Cedar Street, New Haven, CT 06520-8023. Phone: 203-988- variable degree of sequence homology including PD-L2/B7-DC, 5773; Fax: 203-737-5089; E-mail: [email protected] B7-H2/ICOS-L, B7-H3, B7-H4, B7-H5, VISTA/PD-1H, and B7-H6 doi: 10.1158/1078-0432.CCR-17-2542 (13). To date, little is known about the expression, biological role, 2017 American Association for Cancer Research. and therapeutic potential of these targets in human neoplasms.

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three cohorts represented in tissue microarray (TMA) format. The Translational Relevance first two cohorts included NSCLC cases with detailed clinico- VISTA/PD-1H participate in immune regulation and clinical pathological annotation and have been previously reported (23– studies are under way to assess the antitumor effect of VISTA 25). The TMAs were constructed by selecting areas containing blockade. However, the biological impact of VISTA expression viable tumor cells and stromal elements on hematoxylin and in human malignancies remains largely unexplored. Our eosin–stained preparations (as assessed by a pathologist) and results demonstrate that VISTA is frequently expressed in without enriching for specific tumor regions, tissue structures or human non–small cell lung cancer (NSCLC) and shows dif- immune-related features. The first collection is from Greek hos- ferential distribution in tumor and immune cells. Elevated pitals between 1991 and 2001 and included 329 specimens. The VISTA in NSCLC is associated with increased PD-1 axis mar- second cohort was from Yale and included 297 samples collected þ kers, effector T cells and CD68 macrophages, supporting its between 1988 and 2003. The clinicopathological characteristics of modulation by local proinflammatory responses. VISTA pro- these cohorts are described in Supplementary Table S2. A third tein levels are associated with specific genomic alterations in cohort from Yale (n ¼ 139, Supplementary Table S3) including lung adenocarcinomas and its expression in tumor cells pre- advanced lung adenocarcinomas clinically tested for EGFR and dicts longer survival in NSCLC patients. Taken together, our KRAS mutations was also studied. The final number of cases used findings support the immunomodulatory role of VISTA in for each analysis is different from the total number of cases in the human NSCLC and suggests its potential as therapeutic target. cohorts (758 cases included) due to inevitable loss of histospots during the staining procedure or exclusion of cases after quality control. For the QIF measurements, we used two different TMA blocks from the cohorts, each containing two independent 0.6- – Human VISTA is a 311 amino acid long Ig domain-containing mm tumor cores. Serial sections from each TMA were stained in type I transmembrane protein able to suppress T-cell activation twice. Therefore, the results presented included integrated data vitro in vivo. and Notably, this protein shares sequence homology from 2 to 4 independent tumor cores stained at least twice. To with both PD-1 and PD-L1 (14, 15) and can act as a ligand on determine the levels of VISTA in nontumor lung tissue relative to antigen presenting cells and as receptor in T lymphocytes (14–19). þ paired NSCLC from the same cases, we also included a cohort of Previous studies have shown high VISTA expression in CD11b þ þ 46 primary resected NSCLCs (Supplementary Fig. S5). All tissues myeloid cells and lower levels on CD4 and CD8 T cells and were used after approval from the Yale Human Investigation þ þ – – CD4 /Foxp3 T-regulatory lymphocytes (Treg; refs. 14 15, 18 Committee protocols #9505008219 and #1608018220, which 19). To date, there is no clear evidence of VISTA expression in approved the patient consent forms or in some cases waiver of – fl tumor cells (16, 18 20). Chronic in ammation and spontaneous consent. activation of T cells have been reported in PD-1 or VISTA single knockout animals, and this phenotype is enhanced in double Quantitative immunofluorescence (QIF) knockout mice (21). In addition, the magnitude of T-cell Using previously validated/standardized multiplexed QIF responses after a challenge with foreign antigens is synergistically panels and serial TMA sections, we measured the levels of increased in the double knockout mice compared with the single cytokeratin (clone AE1/AE3, eBioscience), VISTA (clone D1L2G, knockout animals, supporting nonredundant immune suppres- CST), PD-L1 (clone 405.9A11, CST), PD-1 (clone EH33, CST), sive effects of VISTA and PD-1 pathway (21). Despite these CD3 (polyclonal, DAKO), CD4 (clone SP35, Spring Biosc.), CD8 functional associations, the expression pattern and possible link (clone C8/144B, DAKO), CD20 (clone L26, DAKO), and CD68 between PD-1, PD-L1, and VISTA expression in human tumors (clone KP1, DAKO). The specific QIF panels used and experi- remains unexplored. Recent studies in melanoma mouse models mental conditions are outlined in Supplementary Table S1 and show that blockade of VISTA using monoclonal antibodies were stained using a previously reported protocol (25–27). fi increases the proportion of circulating tumor-speci c T-cell, pro- Briefly, freshly cut TMA serial sections were deparaffinized and fi motes tumor immune in ltration and reduces tumor growth antigen retrieval was carried on with 1 mmol/L EDTA pH8 (15). Furthermore, an additive antitumor effect with differential (Sigma Aldrich) and boiled for 20 minutes at 97C (PT module, contribution of VISTA, CTLA-4, and PD-1 was reported in a Lab Vision, Thermo Sci.). Inactivation of endogenous peroxidase squamous cell carcinoma model (22). activity was carried on using a solution of 0.3% hydrogen Here, we performed quantitative and spatially resolved mea- peroxide in methanol for 20 minutes and then slides were surements of VISTA protein and additional immune markers in 3 incubated with a blocking solution containing 0.3% BSA in – large cohorts of human non small cell lung cancers (NSCLC). To 0.05% Tween-20 and Tris-buffered solution for 30 minutes. fi further support our ndings, we also performed analyses of The Primary antibody dilution and incubation were carried on as Cancer Genome Atlas (TCGA) lung cancer dataset. Our data described in Supplementary Table S1. Isotype-specific HRP-con- demonstrate frequent expression of VISTA in human lung carci- jugated antibodies and tyramide-based amplification systems nomas and differential expression in tumor and immune cell (Perkin Elmer) were used for signal detection. Residual HRP subsets. We also show association of VISTA with local antitumor activity between sequential detection protocol was eliminated fi immune responses, expression of PD-1 axis markers, speci c incubating the slides twice with a solution containing 100 tumor genomic alterations, and survival in human lung cancer. mmol/L benzoic hydrazide and 50 mmol/L hydrogen peroxide Materials and Methods in phosphate-buffered solution. Patient cohorts and tissue microarrays Fluorescence signal quantification and cases stratification We included retrospectively collected formalin-fixed, paraffin- Quantitative measurement of the fluorescence signal was per- embedded (FFPE) tumor samples from 765 stage I–IV NSCLCs in formed using the AQUA method of QIF, as previously reported

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(25–27). Each slide was visually examined to exclude samples Association was defined based on linear regression factor (R2) with tissue/staining artifacts and those with less than 5% tumor and the statistical significance using the linear correlation by content. Cases were considered to display detectable levels of each GraphPad 5.0 software. target when the QIF score was above the signal to noise threshold determined measuring negative control preparations and by Statistical analysis visual examination of the sample. For VISTA, PD-1 and PD-L1 All statistical and survival studies were conducted using JMP measurement, we considered the signal detected in the tumor 10.0 software for Windows. Graphs and images were prepared compartment (cytokeratin-positive cells), stromal (cytokeratin- using GraphPad 5.0 and Photoshop 9.0 for Windows. A standard negative cells) compartment, and the total tissue area signal two-tailed Student t test was used for all statistical analyses. All detected in the whole tumor sample (tumor and stroma). In samples sizes were appropriate for assumption of normal distri- addition, VISTA was selectively measured in immune cell sub- bution, and variance was similar between compared groups. The fi populations de ned by its colocalization with the immune cell statistical values of P < 0.05 was considered statistically significant. phenotype markers CD3, CD4, CD8, CD20, or CD68. For strat- Adjustment for multiple comparisons was performed using the fi i cation purposes and statistical analysis, VISTA, PD-1 and PD-L1 Bonferroni method and statistical significance was met with P ¼ fi detection were classi ed as high and low using the median score as 0.002. Values of mean determinants are presented as mean cutoff point. SEM.

Cell culture and antibody validation The VISTA antibody validation was carried out using anti- Results body preabsorption with the recombinant protein and by Validation of an anti-VISTA assay and multiplexed quantitative immunodetection in FFPE cell line preparations immunodetection containing parental cells and cells with siRNA-induced VISTA Stringent validation and optimization of a VISTA monoclonal silencing (Supplementary Fig. S1). VISTA monoclonal antibody antibody for use in formalin-fixed paraffin-embedded (FFPE) was preabsorbed with 2.5 and 5.0 mg of human recombinant tissue samples was performed. To determine the target specificity, VISTA protein (R&D Systems). Preabsorption was carried on we measured VISTA protein levels using QIF in Ramos cell overnight at 4 C in a solution of BSA 0.3% and Tris-buffered preparations with known (endogenous) VISTA expression using solution (0.05% Tween-20) containing a ratio 1:2 and 1:1 of the antibody in control conditions or after preabsorption with antigen and antibody. Absorbed antibody was diluted to 94 ng/ recombinant human VISTA protein. As shown in Supplementary mL and used to detect the target by QIF in positive and negative Fig. S1A, overnight incubation of the primary antibody with 2.5 control preparations. HPB-all, Ramos and Karpas cell lines were mg of VISTA protein significantly reduced the target detection and cultured in RPMI-1640 culture medium containing 10% FBS, 1 incubation with 5 mg completely abolished VISTA signal. Pre- mmol/L glutamine and 10 IU streptomycin/ampicillin. VISTA incubation of the antibody did not affect PD-L1 levels in Karpas knockdown in these was achieved using two single siRNAs (ID cells (known to express PD-L1) ruling out a nonspecific effect of 261239 and ID 258598 from Ambion) and a mixed pool (SC- the recombinant peptide and cross reactivity of the primary 90756 from Santa Cruz Biotechnology). A scrambled siRNA antibody with PD-L1 protein (Supplementary Fig. S1B and was used as negative control (#12935-100, Invitrogen). Cells S1E). PD-L1 was not detected in Ramos cells using both control were transfected using Lipofectamin and 10 nmol of each and preabsorbed antibodies. To further assess the assay specificity, siRNA for 24 hours, and then cultured with low FBS OPTI- we performed VISTA measurements in Ramos cells with and MEM medium for additional 24 hours. Control and knocked- without siRNA-induced VISTA silencing. As shown in Supple- down cells were used fresh for protein extraction and immu- mentary Fig. S1C, lower VISTA levels were detected in cells noblotting; or fixed in 10% neutral buffered formalin for 8 to exposed for 48 hours to 2 different VISTA-specific siRNAs but no 12 hours and embedded in paraffinforVISTAquantification by change was seen after transfection with a nonspecific/scrambled QIF. The antibodies against additional targets used in this study siRNA. As expected, the levels of PD-L1 protein were not affected have been previously validated by our group using comparable by incubation with VISTA-specific or scrambled siRNA (Supple- strategies (25–30).Celllinesusedinthisstudywerepurchased mentary Fig. S1D). After the specificity assessment, the VISTA in the ATCC, and authentication was performed every 3 to 6 assay was titrated and optimized for use in multiplexing QIF months using the GenePrint 10 System in the Yale University panels with previously validated antibodies for PD-1, PD-L1, and DNA Analysis Facility. immune cell markers. A detailed description of the antibodies, experimental conditions, fluorescence channels and panels used TCGA data analysis for mRNA expression and genomic is shown in Supplementary Table S1. Human tonsil and placenta alterations samples were used as positive human tissue controls and for We analyzed the NSCLC samples from the TCGA (http:// standardization of the multiplexed QIF experiments. As shown in cancergenome.nih.gov/). Briefly, we downloaded the RNA-seq Supplementary Fig. S2A, VISTA was detected preferentially in the and DNA whole exome sequencing data from 370 NSCLC interfollicular tonsil areas and in trophoblastic cells of the pla- cases, including 250 adenocarcinomas and 120 squamous cell centa. Prominent PD-L1 signal was seen in germinal centers carcinomas. Then, we aligned the transcripts and performed (Supplementary Fig. S2A, top) and trophoblastic cells of the variant calling using defaults TCGA pipelines. Then, we con- chorionic villi, but not in the mesenchymal areas of the placenta ducted single Scatterplot analysis between log2 transformed (Supplementary Fig. S2A, bottom). Representative captions from mRNA FPKM scores of VISTA, PD-L1, PD-1, and CD8A. The the 4 different multiplexing panels described in Supplementary number of nonsynonymous mutations detected in the whole Table S1 stained in the NSCLC cases are shown in Supplementary exome sequencing data was used as the mutational burden. Fig. S2B.

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Figure 1. VISTA is highly expressed in NSCLC. QIF for VISTA protein was performed. The protein expression, immunodetection pattern, and QIF scores were analyzed based on distribution and correlation between different compartments in two different retrospective NSCLC cohorts (cohort #1, N ¼ 324 and cohort #2, N ¼ 295). A–D, Representative immunolocalization of VISTA in NSCLC cases with high and low stromal detection are shown (A and B, respectively). Overall 80% of total NSCLC cases showed a stromal VISTA detection (C) and 20% of cases expressed VISTA on cytokeratin positive cells (D). For both studied cohorts, total number of cases were represented in a distribution curve showing the lowest and highest measured total QIF score using DAPI as mask (E and F). Correlation between stromal and tumor VISTA detection was analyzed and linear regression factor (R2) is indicated; QIF scores were measured using the respective compartment (cytokeratin negative and positive, respectively). Images are representative of 619 cases.

VISTA expression in NSCLC quantitative analysis, VISTA protein levels displayed a continuous To evaluate the expression of VISTA and its biological signif- distribution and the scores were comparable in both studied icance in human NSCLC, 636 cases represented in TMAs from two cohorts (Fig. 1E and F). Overall, VISTA protein was detected in independent cohorts were studied (Supplementary Table S2). As 99% of NSCLC cases with 19.4% and 22.8% of cases showing a shown in Fig. 1A–C, VISTA protein was detected with variable mixed tumor/stromal staining pattern in cohorts #1 and #2, levels across NSCLCs. It was expressed predominantly in stromal respectively. Notably, there was a positive association between cells and showed a cytoplasmic/membranous staining pattern. A the levels of VISTA in tumor and stromal cells [linear regression fraction of cases showed a mixed pattern with simultaneous coefficient (R2) ¼ 0.32 in cohort #1 and 0.33 in cohort #2, P < expression of VISTA in stromal cells and in cytokeratin-positive 0.001, Fig. 1G and H]. As shown in Supplementary Fig. S4, the tumor epithelial cells (Fig. 1D). Some cases also displayed VISTA levels of stromal VISTA were significantly higher in the tumor than expression in immune cells infiltrating or abutting cytokeratin- in morphologically normal lung tissue form the same cases (P ¼ positive tumor regions (Supplementary Fig. S3, bottom). In the 0.035). In the nontumor lung tissue, the majority of VISTA signal

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Role of VISTA/PD-1H in NSCLC

VISTA protein measured in all cells of the sample (e.g., total score) þ or exclusively in CD3 tumor-infiltrating lymphocytes (TIL) was þ þ significantly associated with prominent CD3 /CD8 TILs, þ þ CD20 B lymphocytes and CD68 macrophages, but with low þ levels of CD4 TILs. A similar trend was seen with VISTA in CD4, þ CD8, CD20, and CD68 cells but without reaching statistical significance for all cell type categories, suggesting a weaker association. Altogether, these results indicate an association between higher levels of VISTA expression and increased tumor immune infiltration in human lung cancer.

VISTA levels and PD-1 axis in NSCLC To evaluate the association between VISTA and major PD-1 axis components, we simultaneously measured the levels of cytokeratin, VISTA, PD-1 and PD-L1 protein in the NSCLC cohorts. As expected, PD-1 was located exclusively in immune cells of the stroma and PD-L1 was predominantly recognized in tumor epithelial cells and tumor associated macrophages (Fig. 3A and B). No cases showed simultaneous coexpression of all three markers in the same cell population. The most frequent phenotype seen was the coexpression of two of the targets in specific cell types. Notably, we found a proportion of cases containing stromal cells with simultaneous expression of VISTA/PD-1 and morphological features consistent with lymphocytes (Fig. 3A); and cells coexpressing VISTA/PD-L1 and features consistent with tumor- associated macrophages (Fig. 3B). Overall and measuring the markers in the whole tumor tissue sample, the levels of VISTA protein were significantly correlated with the levels of both PD-1 and PD-L1 (R2 ¼ 0.53–0.61, P < 0.0001, Fig. 3C–F). In additional analysis using the median marker levels as stratification cutoff point, elevated expression of VISTA was significantly associated þ with high PD-1, PD-L1, and CD8 TILs; and this association was Figure 2. VISTA is selectively expressed in immune cells subset. Simultaneous QIF for consistent in both studied cohorts (Table 2). VISTA and most relevant immune cell subtype markers were performed in a To further support our findings, we studied the association single unrelated NSCLC cohort (N ¼ 340). QIF scores for VISTA expression in a between the levels of VISTA, PD-1, PD-L1, and CD8A mRNA cellular subset were measured using the specific immune marker as mask. transcripts in the NSCLC datasets from TCGA. As shown in Fig. 4A þ In A, VISTA is detected predominantly in T lymphocytes (CD3 cells) and and B, we found a positive association between the levels macrophages (CD68þ cells) than B lymphocytes (CD20þ cells) population. þ þ of VISTA, PD-1, and CD8 mRNA in both lung adenocarcinomas In B, CD8 cytotoxic cells expressed more VISTA than CD4 T regulatory N ¼ N ¼ lymphocytes. C, Representative images of VISTA, immune marker, and ( 250, Fig. 4A) and squamous cell carcinomas ( 120, tumor cells (cytokeratin, CKþ cells) codetection are shown. , P ¼ 0.001; Fig. 4B). However, a significant association between VISTA and , P < 0.0001. PD-L1 transcript was seen only in lung adenocarcinomas from TCGA, but not in squamous tumors.

Genotype/phenotype associations of VISTA expression in was located in nonepithelial cells underlying cytokeratin-positive NSCLC pneumocytes with morphological features consistent with To explore the association of VISTA levels with major molecular macrophages. subtypes of lung cancer, we studied a third cohort including 139 To explore the possible role of VISTA in the immune micro- primary lung adenocarcinomas clinically tested for activating þ environment of NSCLC, we measured its levels in CD3 tumor mutations in KRAS and EGFR (Supplementary Table S3). As þ þ infiltrating T cells, CD4 T-helper cells, CD8 cytotoxic T lym- shown in Fig. 5A, total or tumor-specific VISTA levels were þ þ phocytes, CD20 B cells and CD68 tumor-associated macro- comparable across cases with and without KRAS/EGFR muta- phages. As shown in Fig. 2A–C and in Supplementary Fig. S5, tions. However, elevated VISTA protein in stromal cells was detectable levels of VISTA were recognized in all studied immune significantly lower in tumors harboring oncogenic mutations in þ cell subsets but were significantly higher in CD3 T cells than in EGFR than in neoplasms lacking KRAS and EGFR mutations (Fig. þ CD68-positive macrophages or CD20 B lymphocytes (Fig. 2A). 5B). þ In addition, higher levels of VISTA were found in CD8 cytotoxic To explore the association between VISTA levels and the þ cells than in CD4 helper T lymphocytes (Fig. 2B). somatic genomic landscape in NSCLC, we studied the relation- To understand the relationship between VISTA expression and ship between VISTA mRNA expression and the nonsynonymous the immune composition of NSCLCs, we studied the association mutational burden in lung cancer cases from TCGA. As shown between cell-specific VISTA levels and tumor infiltration by dif- in Fig. 5C and D, we found an inverse relationship between VISTA ferent immune cell populations. As shown in Table 1, elevated transcript levels and the number of nonsynonymous DNA

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clincancerres.aacrjournals.org Table 1. VISTA is expressed in immune cells and correlated with TILs CD8þ cells CD4þ cells CD3þ cells CD20þ cells CD68þ cells infiltration infiltration infiltration infiltration infiltration 462 Low High 462 Low High 675 Low High 503 Low High 297 Low High N 231 231 PN232 230 PN326 349 PN252 251 PN149 148 P total VISTA Low 241 142/31.7% 99/21.4% <0.0001 241 109/23.6% 132/28.6% 0.025 339 229/33.9% 97/14.4% <0.0001 242 138/27.4% 104/20.7% 0.0027 111 75 25.2% 36 12.1% <0.0001 High 221 89/19.3% 132/28.6% 221 123/26.6% 98/21.2% 336 110/16.3% 239/35.4% 261 114/22.7% 147/29.2% 186 74/24.9% 112/37.7% 462 Low High 462 Low High 414 Low High 411 Low High 95 Low High N 231 231 P N 232 230 P N 212 202 P N 207 204 P N 52 43 P VISTA in Low 231 130/28.1% 101/21.9% 0.0069 231 115/24.9% 116/25.1% 0.8524 208 122/29.5% 86/20.8% 0.002 205 110/26.8% 95/23.1% 0.1826 46 24/25.3% 22/23.2% 0.6267 CD8þ cells High 231 101/21.9% 130/28.1% 231 117/25.3% 114/24.7% 206 90/21.7% 116/28.0% 206 97/23.6% 109/26.5% 49 28/29.5% 21/22.1% 462 Low High 462 Low High 414 Low High 411 Low High 95 Low High N 231 231 P N 232 230 P N 212 202 P N 207 204 P N 52 43 P

Cancer Research. VISTA in Low 231 137/29.6% 94/20.4% <0.0001 231 99/21.4% 132/28.6% 0.0015 205 109/26.3% 96/23.2% 0.4287 203 109/26.5% 94/22.9% 0.1821 45 25/26.3% 20/21.0% 0.8791 CD4þ cells High 231 94/20.4% 137/29.6% 231 133/28.8% 98/21.2% 209 103/24.9% 106/25.6% 208 98/23.8% 110/26.8% 50 27/28.4% 23/24.2% on September 29, 2021. © 2017American Association for 414 Low High 414 Low High 675 Low High 503 Low High 297 Low High N 204 210 P N 216 198 P N 339 336 P N 252 251 P N 149 148 P VISTA in Low 206 117/28.3% 89/21.5% 0.002 206 95/22.9% 111/26.8% 0.014 339 226/33.5% 113/16.7% <0.0001 255 147/29.2% 108/21.5% 0.0006 114 71/23.9% 43/14.5% 0.0009 CD3þ cells High 208 87/21.0% 121/29.2% 208 121/29.2% 87/21.0% 336 113/16.7% 223/33.0% 248 105/20.9% 143/28.4% 183 78/26.3% 105/35.3% 411 Low High 411 Low High 503 Low High 503 Low High 125 Low High N 203 208 P N 215 196 P N 255 248 P N 252 251 P N 63 62 P VISTA in Low 205 117/28.5% 88/21.4% 0.0018 205 101/24.6% 104/25.3% 0.2177 252 136/27.0% 116/23.1% 0.1412 252 143/28.4% 109/21.7% 0.002 65 37/29.6% 28/22.4% 0.1284 CD20þ cells High 206 86/20.9% 120/29.2% 206 114/27.7% 92/22.4% 251 119/23.7% 132/26.2% 251 109/21.7% 142/28.2% 60 26/20.8% 34/27.2% 95 Low High 95 Low High 297 Low High 125 Low High 297 Low High N 50 45 P N 46 49 P N 110 187 P N 70 55 P N 149 148 P VISTA in Low 46 30/31.6% 16/16.8% 0.0167 55 28/29.5% 18/18.9% 0.5692 149 62/20.9% 87/29.3% 0.1011 62 32/25.6% 30/24% 0.3267 149 83/27.9% 66/22.2% 0.0553 CD68þ cells High 49 20/21.1% 29/30.5% 40 27/28.4% 22/23.2% 148 48/16.2% 100/33.7% 63 38/30.4% 25/20% 148 66/22.2% 82/27.6% NOTE: Simultaneous QIF was performed in a single unrelated NSCLC cohort. QIF scores for VISTA expression in a cellular subset were measured in the respective mask. Studied cohort was separated in cases with high and low expression using the median QIF score as cut point. Contingency analysis between immune cell infiltration and VISTA expression was performed. The number of cases per marker/mask (N) and P values for each category are indicated. Adjustment for multiple comparisons was performed using the Bonferroni method and statistical significance was met with P ¼ 0.002. Bold P values are statistically significant after Bonferroni test. lnclCne Research Cancer Clinical Published OnlineFirst December 4, 2017; DOI: 10.1158/1078-0432.CCR-17-2542

Role of VISTA/PD-1H in NSCLC

Figure 3. PD-1 axis markers are coexpressed and correlated with VISTA expression in NSCLC. Simultaneous QIF for VISTA, PD-L1 and PD-1 was performed in two different unrelated retrospective NSCLC cohorts (cohort #1, N ¼ 324; cohort #2, N ¼ 295). For both studied cohorts, the respective target coexpression, distribution and total QIF scores were measured. A–D, Representative coimmunolocalization of VISTA, PD-L1 and PD-1 proteins in NSCLC cases with high and low stromal detection are shown (A and B, respectively). Selected area shows an ampliﬁed view of the multiplex immunodetection. QIF scores for each studied target were analyzed by a scatterplot matrix, representing the correlation between PD-L1 and VISTA coexpression (C and D), and PD-1 and VISTA coexpression (E and F) in both NSCLC cohorts. Linear regression factor (R2)andP values are indicated. Images are representative of 619 cases.

variants in lung adenocarcinomas (Fig. 5C). However, no signif- Biological implications of VISTA expression in NSCLC icant association was seen in squamous lung malignancies To assess the biological relevance of VISTA expression in lung (Fig. 5D). carcinomas, we determined the association between VISTA levels,

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Table 2. VISTA and PD-1 axis molecules are associated and correlated with tumor-inﬁltrating CD8þ cytotoxic lymphocytes Cohort #1 Cohort #2 VISTA expression VISTA expression NHighLow P N High Low P PD-1 324 162 162 292 146 146 High 162 41.4% 8.6% <0.0001 147 43.0% 6.8% <0.0001 Low 162 8.6% 41.4% 145 6.8% 43.4% PD-L1 324 162 162 292 146 146 High 162 41.0% 9.0% <0.0001 147 41.4% 8.5% <0.0001 Low 162 8.9% 41.1% 145 8.5% 41.6% CD8 277 145 132 288 147 141 High 140 32.5% 18.1% <0.0001 145 33.6% 15.5% 0.001 Low 137 19.9% 29.5% 143 20.0% 30.9% NOTE: Simultaneous QIF was performed and QIF scores for the respective target were measured in two different unrelated retrospective NSCLC cohorts (cohort #1, N ¼ 324 and cohort #2, N ¼ 295). Studied cohorts were separated in cases with high and low expression using the median QIF score as cut point. Contingency analysis for each cohort was independently performed and the P values for each category are indicated.

major clinicopathological variables and survival in the NSCLC consistent association between stromal VISTA levels or VISTA cohorts. As shown in Table 3 and using the median score as measured in the total tissue area and survival were detected stratification cutoff point, we found no consistent association using the median score as stratification cutoff point (Supple- between the level of total VISTA in the samples and major mentary Fig. S6). clinicopathological variables in the cohorts. However, elevated expression of VISTA measured exclusively in the tumor area was significantly associated with longer 5-year overall survival, and Discussion this was consistent in both the training cohort #1 and in the Despite the prominent interest and possible role of VISTA/PD- validation set/cohort #2 (log-rank P ¼ 0.027–0.038, Fig. 6). No 1H as a novel anticancer immunotherapy target in human

Figure 4. VISTA mRNA is correlated with PD-L1 and PD-1 molecules and CD8þ cytotoxic lymphocytes in The TCGA data set. Scatterplot analysis of 250 cases of lung adenocarcinomas and 120 cases of lung squamous cell carcinoma from the TCGA data set were analyzed. Linear regression factor (R2)andP values for the association between VISTA, PD-L1, PD-1, and CD8a are indicated.

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Role of VISTA/PD-1H in NSCLC

Figure 5. VISTA shows a minimal association with lung cancer mutational status. An unrelated and retrospective cohort with major molecular adenocarcinoma variants was analyzed (cohort #3, N ¼ 139). A and B, Levels of VISTA protein in the whole tumor tissue (A)orinthe stromal area (B) in primary lung adenocarcinomas lacking mutations in KRAS and EGFR (nonmutated) and in tumors with oncogenic KRAS or EGFR variants (mutants). Scatterplot analysis of 250 cases of lung adenocarcinomas (C) and 120 cases of lung squamous cell carcinoma (D) from the TCGA data set were analyzed. Linear regression factor (R2)andP values for the association between VISTA mRNA expression and global mutational load are indicated. , P < 0.05.

malignancies, little is known about its expression in cancer tissues. upregulated by TLR3-, TLR5-ligand, IL10 and IFNg (19). The Here, we have carefully validated a protein detection assay and latter indicates the inducible nature of VISTA under inflamma- interrogated the expression, tissue distribution, and role of VISTA tory conditions and supports the need for context-specific in a sizable number of human lung carcinomas from 4 indepen- measurements. dent populations. Our data demonstrate that VISTA is expressed To our knowledge, no studies reliably evaluating the expression in the vast majority of human NSCLCs with specific geographical/ and location of VISTA protein in human tumors have been spatial patterns and positively associated with tumor immune reported. For instance, previous reports failed to demonstrate infiltration and PD-1/PD-L1 expression. The levels of VISTA are VISTA expression in colon and lung cancer samples using a not associated with specific NSCLC histological subtypes or noncommercial monoclonal antibody (clone GG8) after seeing patient clinical/demographical variables. However, compartment limited sensitivity and affinity of the assay for use in immuno- specific expression of VISTA in stromal or tumor epithelial cells is histochemistry studies (16, 20). In our work and using a validated associated with oncogenic EGFR mutations and with the non- commercially available assay that is suitable for FFPE tissue synonymous mutational burden in lung adenocarcinomas; and samples, we found expression of VISTA in >99% of lung cancer with overall survival in two independent NSCLC cohorts. Taken samples from over 700 cases studied. Notably, we found distinct together, these findings suggest an immune-modulatory effect of expression patterns with most cases showing stromal-only stain- VISTA in human lung cancer. ing and nearly 20% showing staining in stromal and epithelial Most studies addressing VISTA expression in tissues have used tumor cells. The expression in tumor cells was predominantly mRNA expression or flow cytometry of mechanically/enzymati- focal and spatially connected with immune infiltration and PD-1/ cally disaggregated samples, thus potentially altering the native PD-L1 expression, suggesting that locally secreted factors such as cell conditions and precluding assessment of the spatial context. interleukins or interferons could mediate VISTA upregulation. In In mice, high expression of VISTA has been detected in hemato- support of this notion, VISTA expression has been shown to be poietic cells, lung and small intestine, and very low levels of directly and synergistically induced by IL10 and IFNg (19). mRNA have been found in the heart, brain, muscle, kidney, testis, Additional studies will be required to determine the effect of and placenta (14, 15). In humans, VISTA has also been reported to multiple cytokines/chemokines (alone or in combination) in be mainly expressed in hematopoietic cells (14–19). In immune VISTA expression on tumor and immune cells. To date, only few cells, the highest level of VISTA expression is detected in mono- studies have directly assessed the role of VISTA protein in tumor cytes and dendritic cells, intermediate levels on neutrophils, and cells. One study described that VISTA overexpression in a murine very low levels on natural killer (NK) cells (16). VISTA expression fibro-sarcoma model promotes tumor growth and the effect was was also detected with variable levels in both parenchymal and T-cell dependent (15). However, the biological (e.g., immune) stromal/immune cells from morphologically normal human determinants of this finding and the ligand/receptor function of brain, thyroid, stomach, spleen, liver and in human peripheral VISTA in tumor cells remain unclear. blood mononuclear cells (PBMC) (19). Notably, the levels of In human lung cancer, VISTA protein was found in different TIL VISTA in PBMCs or isolated human monocytes were prominently populations and in tumor-associated macrophages. Moreover,

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Table 3. Clinical relevance of VISTA expression in NSCLC Cohort #1 Cohort #2 VISTA expression VISTA expression N High Low PNHigh Low P Total patients 324 167 157 292 150 142 Age (years) <70 247 39.9% 36.5% 0.589 125 29.7% 27.7% 0.814 70 77 11.5% 12.1% 167 21.5% 21.2% Sex Male 287 45.7% 42.9% 0.980 139 25.0% 22.6% 0.708 Female 37 5.9% 5.6% 153 26.4% 26.0% Smoking status Never-smoker 26 4.5% 4.5% 0.911 39 7.6% 5.9% 0.529 Smoker 262 46.5% 44.4% 249 44.1% 42.4% Unknown 36 4 Histology Adenocarcinoma 122 23.2% 20.4% 0.516 162 37.9% 31.9% 0.315 Squamous cell carcinoma 158 27.9% 28.6% 70 14.2% 16.0% Others 44 57 Unknown 0 3 Stage I–II 191 29.2% 30.7% 0.293 229 41.7% 37.2% 0.462 III–IV 128 21.9% 18.2% 61 10.0% 11.0% Unknown 5 2 NOTE: Contingency analysis between major clinicopathological variables and total QIF scores for VISTA expression from two unrelated retrospectiveNSCLC cohorts (cohort #1, N ¼ 324 and cohort #2, N ¼ 295) was performed. Studied cohorts were separated in cases with high and low expression using the median QIF score as cutoff point. P values for each category are indicated.

þ higher levels of VISTA were seen in effector CD8 T cells than in such as alternatively polarized macrophages (e.g., M2-type macro- CD4-helper TILs and expression of VISTA in the whole sample or phages) and MDSCs. Studies are under way to determine the in immune cells was in general associated with increased tumor optimal strategy to accurately interrogate such populations. þ þ þ immune infiltration by CD8 , CD20 , and CD68 cells, but not After major clinical success of blocking the PD-1 axis using þ by CD4 helper T lymphocytes. The latter suggest that VISTA monoclonal antibodies, the current landscape of anticancer expression in the tumor immune microenvironment could be immunotherapies includes numerous combinations of immune related with antitumor immune pressure. We also observed and nonimmune agents. Therefore, identification of indepen- limited association between the tumor mutational burden and dent or redundant immune evasion pathways could be used to VISTA levels in lung carcinomas. The determinants for this are support optimal treatment modalities and rational design of currently under investigation. clinical trials. Previous work using VISTA and PD-1 knockout Several preclinical mouse models have reported that VISTA mice demonstrated that the immune-regulatory pathways for upregulation in stromal cells can act as an immune evasion PD-1 and VISTA are functionally nonredundant in antigen- mechanism. For instance, VISTA expression is higher on immature specific responses and during autoimmune inflammatory con- dendritic cells, myeloid-derived suppressor cells (MDSC) and ditions (21). However, little is known about the association Tregs than in peripheral tissues (18). Due to prominent limita- between VISTA and PD-1 pathways in human malignancies. In tions in the current understanding and validation of markers to our study, we found a consistent and prominent association characterize specific myeloid cell subsets in human specimens, between the levels of VISTA protein, PD-1 and PD-L1 in NSCLC our study did not include major immune suppressive cell types cohorts represented in TMA format and also measuring mRNA

Figure 6. Association between VISTA levels and overall survival in NSCLC. Kaplan–Meier graphical analysis of the 5-year overall survival in NSCLC cases from cohort #1 (A) and cohort #2 (B) based on the levels of VISTA protein expression in the tumor area. Patients from two independent cohorts were stratiﬁed by VISTA QIF score (low vs. high expression) using the median as a cutoff point. P values comparing risk groups were calculated with the log-rank test.

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Role of VISTA/PD-1H in NSCLC

transcripts in whole tissue lung cancer specimens from TCGA. Disclosure of Potential Conflicts of Interest The latter indicates the robustness of the association and V. Velcheti is a consultant/advisory board member for Amgen, AstraZeneca, excludes a possible impact/bias of measuring the targets in Bristol-Myers Squibb, Celgene, Fulgent Health, Genentech, Merck, and Navigate relatively small TMA histospots. Although we noted distinct Biopharma. L. Chen reports receiving commercial research grants from Boeh- ringer Ingelheim, NextCure, and Pfizer, and is a consultant/advisory board cellular coexpression patterns of VISTA, PD-1 and PD-L1 in the member for GenomiCare, Pfizer, and VcanBio. K.A. Schalper reports receiving human tumors (Fig. 3A and B), our results demonstrate frequent commercial research grants from Navigate, Onkaido, Surface Oncology, Takeda, regional colocalization of the markers in lung cancer and suggest Tesaro, and Vasculox, speakers bureau honoraria from Merck and Takeda, and is a synergetic/cooperative immune evasion effect. The previously a consultant/advisory board member for Celgene and Shattuck Labs. No reported induction of VISTA and PD-L1 by proinflammatory potential conflicts of interest were disclosed by the other authors. cytokines such as IFNg could mechanistically support the markers coexpression and the positive association with TILs. There- Authors' Contributions fore, simultaneous blockade of both VISTA and PD-1 pathway Conception and design: F. Villarroel-Espindola, K. Syrigos, L. Chen, R.S. Herbst, could represent an effective antitumor strategy in human K.A. Schalper NSCLC. The results from ongoing and future human clinical Development of methodology: F. Villarroel-Espindola, I. Datar, V. Velcheti, trials using anti-VISTA monotherapy or combination therapies L. Chen, R.S. Herbst, K.A. Schalper Acquisition of data (provided animals, acquired and managed patients, will be required to support this observation. provided facilities, etc.): F. Villarroel-Espindola, M. Sanmamed, V. Velcheti, ConflictingresultsabouttheantitumoreffectofdualVISTA/ K. Syrigos, R.S. Herbst PD-1 axis blockade have been reported using two different Analysis and interpretation of data (e.g., statistical analysis, biostatistics, monoclonal VISTA antibodies in preclinical models (21, 22). A computational analysis): F. Villarroel-Espindola, X. Yu, V. Velcheti, K. Syrigos, synergisticeffectofthecombinationofamonoclonaltherapy H. Zhao, L. Chen, R.S. Herbst, K.A. Schalper against VISTA and PD-L1 was described in a colon cancer Writing, review, and/or revision of the manuscript: F. Villarroel-Espindola, I. Datar, M. Sanmamed, V. Velcheti, K. Syrigos, M. Toki, H. Zhao, L. Chen, mouse model, showing a reduction in the tumor growth and R.S. Herbst, K.A. Schalper an increased overall survival in comparison to monotherapy Administrative, technical, or material support (i.e., reporting or organizing regimens (21). However, and despite seeing increased CD8 T- data, constructing databases): F. Villarroel-Espindola, N. Mani, M. Sanmamed, cell function, no effect on tumor growth was detected after M. Toki treatment with the combination of anti-VISTA/PD-1 treatment Study supervision: F. Villarroel-Espindola, L. Chen, R.S. Herbst, K.A. Schalper in an oral squamous cell carcinoma model (22). In support of Acknowledgments fi our ndings in human tumors, a recent study showed a positive Thanks to Lori Charette and Yale Pathology Tissue Services for the technical association between VISTA and PD-L1 protein levels; and a support. þ cooperative effect of VISTA and CD8 cells to predict survival in This study was supported by the Lung Cancer Research Foundation (LCRF), a retrospective cohort of human oral squamous carcinomas Yale SPORE in Lung Cancer (P50CA196530), Department of Defense-Lung (31). Taken together, these data suggest that the VISTA/PD-1H Cancer Research Program Career Development Award (LC150383), and Stand – pathway may contribute to the adaptive resistance mechanisms Up to Cancer American Cancer Society Lung Cancer Dream Team Transla- tional Research Grant (grant number: SU2C-AACR-DT17-15). Stand Up To operating in the tumor microenvironment. Cancer is a program of the Entertainment Industry Foundation. Research grants Increased VISTA expression could also participate in acquired are administered by the American Association for Cancer Research, the scientific resistance to immune checkpoint blockade. In this regard, partner of SU2C, sponsored research support by Navigate Biopharma and Yale VISTA upregulation after CTLA-4 blockade with ipilimumab Cancer Center Support Grant (P30CA016359). was recently reported in prostate carcinomas (32). In this study, VISTA was upregulated in tumor-infiltrating immune cells, The costs of publication of this article were defrayed in part by the payment of þ particularly in CD68 tumor-associated macrophages. The page charges. This article must therefore be hereby marked advertisement in possible role of VISTA in mediating resistance to immune accordance with 18 U.S.C. Section 1734 solely to indicate this fact. checkpoint blockers in human lung carcinomas is currently Received August 31, 2017; revised October 18, 2017; accepted November 28, under investigation. 2017; published OnlineFirst December 4, 2017.

References 1. Sznol M, Chen L. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the 6. Larkin J, Lao CD, Urba WJ, McDermott DF, Horak C, Jiang J, Wolchok JD. treatment of advanced human cancer–response. Clin Cancer Res Efﬁcacy and safety of nivolumab in patients with BRAF V600 mutant and 2013;19:5542. BRAF wild-type advanced melanoma: a pooled analysis of 4 clinical trials. 2. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. JAMA Oncol 2015;1:433–40. PD-1 blockade induces responses by inhibiting adaptive immune resis- 7. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. tance. Nature 2014;515:568–71. Combined nivolumab and ipilimumab or monotherapy in untreated 3. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Programmed melanoma. N Engl J Med 2015;373:23–34. death-1 ligand 1 interacts speciﬁcally with the B7-1 costimulatory molecule 8. Lavin Y, Kobayashi S, Leader A, Amir ED, Elefant N, Bigenwald C, et al. to inhibit T cell responses. Immunity 2007;27:111–22. Innate immune landscape in early lung adenocarcinoma by paired single- 4. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. cell analyses. Cell 2017;169:750–65.e17. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J 9. Hirsh V. New developments in the treatment of advanced squamous Med 2015;372:2018–28. cell lung cancer: focus on afatinib. Onco Targets Ther 2017;10: 5. Herbst RS, Baas P, Kim DW, Felip E, Perez-Gracia JL, Han JY, et al. 2513–26. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, 10. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 advanced non-small-cell lung cancer (KEYNOTE-010): a randomised con- family, costimulates T cell proliferation and interleukin-10 secretion. Nat trolled trial. Lancet 2016;387:1540–50. Med 1999;5:1365–9.

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11. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. ways to antitumor responses against squamous cell carcinoma. Oral Oncol Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family 2016;57:54–60. member leads to negative regulation of lymphocyte activation. J Exp Med 23. Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, Syrigos KN, Sznol M, 2000;192:1027–34. et al. Programmed death ligand-1 expression in non-small cell lung cancer. 12. Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen L. B7-H1 is a ubiquitous Lab Invest 2014;94:107–16. antiapoptotic receptor on cancer cells. Blood 2008;111:3635–43. 24. Anagnostou VK, Syrigos KN, Bepler G, Homer RJ, Rimm DL. Thyroid 13. Zou W, Chen L. Inhibitory B7-family molecules in the tumour microen- transcription factor 1 is an independent prognostic factor for patients with vironment. Nat Rev Immunol 2008;8:467–77. stage I lung adenocarcinoma. J Clin Oncol 2009;27:271–8. 14. Flies DB, Wang S, Xu H, Chen L. Cutting edge: a monoclonal antibody 25. Schalper KA, Brown J, Carvajal-Hausdorf D, McLaughlin J, Velcheti V, specific for the programmed death-1 homolog prevents graft-versus-host Syrigos KN, et al. Objective measurement and clinical significance of disease in mouse models. J Immunol 2011;187:1537–41. TILs in non-small cell lung cancer. J Natl Cancer Inst 2015;107. pii: 15. Wang L, Rubinstein R, Lines JL, Wasiuk A, Ahonen C, Guo Y, et al. VISTA, a dju435. novel mouse Ig superfamily ligand that negatively regulates T cell 26. Carvajal-Hausdorf DE, Schalper KA, Neumeister VM, Rimm DL. Quanti- responses. J Exp Med 2011;208:577–92. tative measurement of cancer tissue biomarkers in the lab and in the clinic. 16. Lines JL, Pantazi E, Mak J, Sempere LF, Wang L, O'Connell S, et al. VISTA is Lab Invest 2015;95:385–96. an immune checkpoint molecule for human T cells. Cancer Res 2014;74: 27. Schalper KA, Carvajal-Hausdorf D, McLaughlin J, Altan M, Velcheti V, 1924–32. Gaule P, et al. Differential expression and significance of PD-L1, IDO-1, 17. Flies DB, Han X, Higuchi T, Zheng L, Sun J, Ye JJ, et al. Coinhibitory receptor and B7-H4 in human lung cancer. Clin Cancer Res 2017;23:370–8. PD-1H preferentially suppresses CD4⁺ T cell-mediated immunity. J Clin 28. Mani NL, Schalper KA, Hatzis C, Saglam O, Tavassoli F, Butler M, Invest 2014;124:1966–75. et al. Quantitative assessment of the spatial heterogeneity of tumor- 18. Le Mercier I, Chen W, Lines JL, Day M, Li J, Sergent P, et al. VISTA regulates infiltrating lymphocytes in breast cancer. Breast Cancer Res 2016; the development of protective antitumor immunity. Cancer Res 2014; 18:78–84. 74:1933–44. 29. Gaule P, Smithy JW, Toki M, Rehman J, Patell-Socha F, Cougot D, et al. A 19. Bharaj P, Chahar HS, Alozie OK, Rodarte L, Bansal A, Goepfert PA, et al. quantitative comparison of antibodies to programmed cell death 1 ligand Characterization of programmed death-1 homologue-1 (PD-1H) expres- 1. JAMA Oncol 2017;3:256–9. sion and function in normal and HIV infected individuals. PLoS One 30. Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, et al. 2014;9:e109103. Antibody validation. Biotechniques 2010;48:197–209. 20. Lines JL, Sempere LF, Broughton T, Wang L, Noelle R. VISTA is a novel 31. Wu L, Deng WW, Huang CF, Bu LL, Yu GT, Mao L, et al. Expression of VISTA broad-spectrum negative checkpoint regulator for cancer immunotherapy. correlated with immunosuppression and synergized with CD8 to predict Cancer Immunol Res 2014;2:510–7. survival in human oral squamous cell carcinoma. Cancer Immunol 21. Liu J, Yuan Y, Chen W, Putra J, Suriawinata AA, Schenk AD, et al. Immune- Immunother 2017;66:627–36. checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T- 32. Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, et al. cell responses. Proc Natl Acad Sci U S A 2015;112:6682–7. VISTA is an inhibitory immune checkpoint that is increased after 22. Kondo Y, Ohno T, Nishii N, Harada K, Yagita H, Azuma M. Differential ipilimumab therapy in patients with prostate cancer. Nat Med 2017; contribution of three immune checkpoint (VISTA, CTLA-4, PD-1) path- 23:551–5.

OF12 Clin Cancer Res; 2018 Clinical Cancer Research

Spatially Resolved and Quantitative Analysis of VISTA/PD-1H as a Novel Immunotherapy Target in Human Non −Small Cell Lung Cancer

Franz Villarroel-Espindola, Xiaoqing Yu, Ila Datar, et al.

Clin Cancer Res Published OnlineFirst December 4, 2017.

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