Abstract 5600: Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC)
Authors: Datar Ila J. , Wang Jun , Mani Nikita , Villarroel-Espindola Franz , Ryan Patrick , Sanmamed Miguel F. , McEachern Kristen , Jenkins David , Rimm David L. , Chen Leiping , Herbst Roy , Schalper Kurt 
Published: July 2017

Introduction: The ineffective anti-tumor immune response is characterized by increased immune suppressive signals in the tumor microenvironment. In particular, T-cells recognizing tumor antigens can express diverse immune inhibitory receptors mediating lymphocyte inactivation and limiting tumor rejection. Blockade of these receptors such as PD-1 induces prominent clinical benefit in patients with NSCLC. However, the expression and significance of additional potentially actionable immune inhibitory receptors in lung cancer is poorly understood.

Methods: After careful validation of assays and using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (clone EH33, CST), LAG-3 (Clone 17B4, Abcam) and TIM-3 (clone D5D5R, CST) in 698 stages I-IV formalin-fixed paraffin embedded (FFPE) lung carcinomas represented in three tissue microarrays (cohort #1 [Yale n=186], cohort #2 [Yale n=192, and cohort #3 [Greece n=320]). We also included a collection of lung adenocarcinomas with molecular annotation (cohort #4 [Yale n=106]). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers and with major clinico-pathological variables, driver mutations and survival were studied.

Results: All the targets were detected predominantly in CD3+ T-cells with membranous staining. Expression of PD-1, LAG-3 and TIM-3 in T-cells across all NSCLC cohorts was 68.7%, 39.7% and 55.8%, respectively. Elevated levels of PD-1, LAG-3 or TIM-3 were significantly associated with increased tumor infiltrating lymphocytes and with the co-expression of one or more of the other inhibitory receptors (P&lt;0.001). Simultaneous co-expression of all 3 markers was identified in 32.6% of cases. No consistent association was seen between the targets and patient age, gender, smoking status, clinical stage, tumor histology and overall survival. PD-1, TIM-3 and CD3 expression was significantly lower in EGFR and KRAS mutant lung adenocarcinomas than in tumors lacking mutations in these oncogenic drivers (P&lt;0.05).

Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in NSCLC, show frequent co-expression and association with elevated CD3+ T-cells. Our results support the biological role of PD-1, LAG-3 and TIM-3 in NSCLC and suggest co-activation of these immune inhibitory pathways in a proportion of cases. Modulation of these receptors could enhance the anti-tumor immune response in lung cancer.

Citation Format: Ila J. Datar, Jun Wang, Nikita Mani, Franz Villarroel-Espindola, Patrick Ryan, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, David L. Rimm, Leiping Chen, Roy Herbst, Kurt Schalper. Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5600. doi:10.1158/1538-7445.AM2017-5600