Published OnlineFirst May 3, 2019; DOI: 10.1158/1078-0432.CCR-18-4142 Precision Medicine and Imaging Clinical Cancer Research Expression Analysis and Significance of PD-1, LAG-3, and TIM-3 in Human Non–Small Cell Lung Cancer Using Spatially Resolved and Multiparametric Single-Cell Analysis Ila Datar1,2, Miguel F. Sanmamed3,4,5, Jun Wang3, Brian S. Henick1,2, Jungmin Choi6, Ti Badri3, Weilai Dong6, Nikita Mani1, Maria Toki1, Luis D. Mejías4, Maria D. Lozano4, Jose Luis Perez-Gracia4,Vamsidhar Velcheti7, Matthew D. Hellmann8,9,10, Justin F. Gainor11, Kristen McEachern12, David Jenkins12, Konstantinos Syrigos13, Katerina Politi1,2, Scott Gettinger2, David L. Rimm1,2, Roy S. Herbst2, Ignacio Melero4,5, Lieping Chen3, and Kurt A. Schalper1,2 Abstract Purpose: To determine the tumor tissue/cell distribution, ing immunotherapy. Expression of the markers was lower in functional associations, and clinical significance of PD-1, LAG- EGFR-mutated adenocarcinomas and displayed limited asso- 3, and TIM-3 protein expression in human non–small cell lung ciation with tumor mutational burden. In single-cell CyTOF cancer (NSCLC). analysis, PD-1 and LAG-3 were predominantly localized on Experimental Design: Using multiplexed quantitative T-cell subsets/NKT cells, whereas TIM-3 expression was higher immunofluorescence, we performed localized measure- in NK cells and macrophages. Coexpression of PD-1, LAG-3, ments of CD3, PD-1, LAG-3, and TIM-3 protein in >800 and TIM-3 was associated with prominent T-cell activation clinically annotated NSCLCs from three independent (CD69/CD137), effector function (Granzyme-B), and prolif- cohorts represented in tissue microarrays. Associations eration (Ki-67), but also with elevated levels of proapoptotic between the marker's expression and major genomic altera- markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL tions were studied in The Cancer Genome Atlas NSCLC subsets lacking PD-1 expression and showed a distinct func- dataset. Using mass cytometry (CyTOF) analysis of leuko- tional profile. In baseline samples from 90 patients with cytes collected from 20 resected NSCLCs, we determined the advanced NSCLC treated with PD-1 axis blockers, elevated levels, coexpression, and functional profile of PD-1, LAG-3, LAG-3 was significantly associated with shorter progression- and TIM-3 expressing immune cells. Finally, we measured free survival. the markers in baseline samples from 90 patients with Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/ advanced NSCLC treated with PD-1 axis blockers and cell distribution, functional implications, and genomic corre- known response to treatment. lates in human NSCLC. Expression of these immune inhibi- Results: PD-1, LAG-3, and TIM-3 were detected in tumor- tory receptors in TILs is associated with prominent activation, infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of but also with a proapoptotic T-cell phenotype. Elevated LAG-3 NSCLC cases, respectively. These markers showed a prominent expression is associated with insensitivity to PD-1 axis block- association with each other and limited association with major ade, suggesting independence of these immune evasion clinicopathologic variables and survival in patients not receiv- pathways. 1Department of Pathology, Yale University School of Medicine, New Haven, Note: Supplementary data for this article are available at Clinical Cancer Connecticut. 2Department of Medical Oncology, Yale University, Yale Cancer Research Online (http://clincancerres.aacrjournals.org/). Center, New Haven, Connecticut. 3Department of Immunobiology, Yale Univer- sity School of Medicine, New Haven, Connecticut. 4Clinic University of Navarra, I. Datar and M.F. Sanmamed contributed equally in this article. Pamplona, Spain. 5CIBERONC, Madrid, Spain. 6Department of Genetics, Yale Corresponding Author: Kurt A. Schalper, Yale School of Medicine, PO Box University School of Medicine, New Haven, Connecticut. 7Department of Tho- 208023, 310 Cedar Street, New Haven, CT 06520-8023. Phone: 203-988-5773; racic Oncology, New York University, Langone Medical Center, New York, New Fax: 203-737-5089; E-mail: [email protected] York. 8Memorial Sloan Kettering Cancer Center, New York, New York. 9Weill 10 Cornell Medical College, New York, New York. Parker Institute for Cancer Clin Cancer Res 2019;XX:XX–XX Immunotherapy, San Francisco, California. 11Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. 12Tesaro Inc., Boston, doi: 10.1158/1078-0432.CCR-18-4142 Massachusetts. 13Oncology Unit GPP, Athens School of Medicine, Athens, Greece. Ó2019 American Association for Cancer Research. www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 3, 2019; DOI: 10.1158/1078-0432.CCR-18-4142 Datar et al. variable N-terminal Ig domain and a mucin stalk domain (24). Translational Relevance An immunosuppressive effect of TIM-3 signaling on T cells was Our results reveal that PD-1, LAG-3, and TIM-3 show reported to be related with the binding of Galectin-9 and CEA- differential tissue/cell distribution, functional impact, and CAM1 (25, 26). Additional candidate ligands have also been clinical significance in NSCLCs. In addition, single-cell studies shown to be able to modulate TIM-3 functions including phos- show that simultaneous coexpression of these immune-inhib- phatidyl serine (PtdSer) and high-mobility group protein B1 itory receptors is associated with prominent tumor-infiltrating (HMGB1; ref. 14).Clinical studies assessing the safety and anti- lymphocyte activation, but also with acquisition of a proa- tumor effect of TIM-3 blockers alone or in combination with other poptotic phenotype. Finally, we determine the prognostic therapies are currently ongoing: NCT02817633, NCT03099109, value of the markers and identify a negative association NCT03066648. The expression, tissue distribution, and associa- between elevated baseline LAG-3 and sensitivity to PD-1 axis tion of TIM-3 with other immune-inhibitory receptors in human blockade. Taken together, our results support independence of lung cancer are not well understood. these immune-inhibitory pathways and expand the current Here, we used multiplexed tissue imaging of large tumor understanding of their interplay in cancer. This information collections and single-cell phenotypic analysis of primary cancers could be used to guide and interpret results from ongoing and to evaluate the distribution and significance of PD-1, LAG-3, and future clinical trials. TIM-3 expression in NSCLC. Our results reveal complex func- tional associations and support independent functions of these receptors to suppress T-cell function. Introduction Materials and Methods Immunostimulatory therapies blocking the PD-1 axis pathway Patients, cohorts, and tissue microarrays have become major antitumor treatment options in diverse Formalin-fixed, paraffin-embedded (FFPE) samples from pre- malignancies including non–small cell lung cancer (NSCLC; viously reported retrospective collections of NSCLC not treated refs. 1–5). To date, single-agent treatment using mAbs targeting with immune checkpoint blockers and represented in tissue PD-1 receptor or its ligand PD-L1 induce lasting clinical responses microarrays (TMA) were analyzed (27, 28). The first collection in approximately 18% of patients with advanced NSCLC. How- includes samples from 426 patients with NSCLC seen at Yale ever, primary resistance occurs in the majority of patients and Pathology between 1988 and 2012 (cohort #1). The second acquired adaptation of tumors to immune pressure in patients cohort includes samples from 304 patients with NSCLC initially responding to therapy has also become a clinical chal- collected at Sotiria General Hospital and Patras University lenge (6–9). Therefore, identification of biomarkers for patient General Hospital (Greece) between 1991 and 2001 (cohort selection and characterization of additional nonredundant #2). All cases in the cohorts were reviewed by a local pathologist actionable immunostimulatory targets are needed. using hematoxylin and eosin–stained preparations and tumor Various immune and tumor genomic metrics are associated histology variant was confirmed by morphology analysis. Tumor with sensitivity to PD-1 axis blockers including tumor PD-L1 cores for TMA construction were obtained from case areas expression, measurement of tumor-infiltrating lymphocytes (TIL) selected by a pathologist to represent the disease. Tumor core or inflammation-related mRNA expression profiles, tumor muta- selection was not based on specific tumor segments or location. tional burden, and microsatellite instability (3, 4, 10–13). To Clinicopathologic information from patients in both cohorts date, however, only detection of PD-L1 protein using IHC and was collected from clinical records and pathology reports. mismatch repair deficiency are approved by the FDA as compan- Analysis of mRNA expression and nonsynonymous mutations ion biomarkers for PD-1–blocking antibodies. was performed using the lung cancer dataset from TCGA (cohort Additional immune coinhibitory receptors beyond PD-1, such #3, n ¼ 370). Another TMA-based cohort from Yale (cohort #4) as LAG-3 and TIM-3, are induced after T-cell receptor (TCR) including retrospective samples from 108 lung adenocarcinomas stimulation and mediate T-cell suppression/dysfunction (14–17). clinically tested for EGFR and KRAS mutations was also The potential role of these receptors in