Abstract P5-04-24: Differential expression of novel immune checkpoint receptors expressed on tumor-infiltrating lymphocytes (TIL) in patients with early breast cancer
Authors: Mollavelioglu Baran , Cetin Esin Aktas , Cabioglu Neslihan , Abbasov Aykhan , Onder Semen , Emiroglu Selman , Tukenmez Mustafa , Muslumanoglu Mahmut , Igci Abdullah , Deniz Gunnur , Ozmen Vahit 
Published: March 2020

Abstract
Introduction: High expression of immune checkpoint receptors in tumor microenvironment reduces antitumor immunity and cause immune evasion of tumor cells. In recent years, immunotherapy trials using PD-1 or PDL-1 inhibitors in advanced triple negative (TN) breast cancer evolved very rapidly. The differential expression of novel immune checkpoint receptors such as TIM-3, LAG-3 and TIGIT in addition to PD-1, and CTLA-4 on tumor infiltrating lymphocytes (TIL) in patients with early breast cancer was investigated.
Material and methods: TIL were isolated by using a Tumor Dissociation Kit from fresh tumoral tissue. Flow-cytometric analyses were performed by using CD8, CD16, CD56, PD-1, CTLA-4, TIM-3, LAG-3 and TIGIT specific monoclonal antibodies on isolated TIL. Correlations were estimated between biological and clinical characteristics of tumors and demographic features of patients and flow cytometric findings.
Results: Median age was 47 (range 28-68). There were 7 patients (35%) with HER2+ or triple negative tumors, whereas 13 patients (65%) had HER2 (-) luminal cancers. Our findings showed that patients younger than 45 years were more likely to express high levels of CTLA-4 (p=0.013) and TIGIT (p=0.007) on CD56+ natural killer (NK) cells and TIM-3 (p=0.043) on CD16+ lymphocytes (Table 1), whereas the other high expressions including LAG-3 (p=0,08) and TIM-3 (p=0.06) on CD56+ NK cells did not reach the statistical significance. Furthermore, patients with high Ki-67 proliferation index &gt;35% were found to express higher CTLA-4 (p=0.011) on CD16+ lymphocytes. Patients with Stage II disease expressed higher levels of PD-1 (p=0.018) and LAG-3 (p=0.04) on CD8+ cytotoxic T lymphocytes than patients with Stage I disease. Similarly, patients with lymph node metastasis had higher TIGIT (p=0.04) and PD-1 (p=0.05) levels on CD16+ and CD56+ lymphocytes, respectively. No other significant associations could be found between immune check receptors and other parameters.
Conclusion: Our results suggest TIL in patients with more advanced stages and younger than 45 years old are more likely to express higher levels of immune checkpoint receptors such as LAG-3, TIM-3, CTLA-4, TIGIT and PD-1. Interestingly, no difference could be found in immune checkpoint receptor expressions in TIL between patients with luminal and TN or HER2+, that would justify immunotherapeutical approaches in selected luminal breast cancers in future trials.
Table 1. Significant correlations between immune check point receptor expression and demographic and tumor features CD8PD1CD8LAG3CD16CTLA4CD16TIGITCD16TIM3CD56CTLA4CD56TIGITCD56PD1meanpmeanpmeanpmeanpmeanpmeanpmeanpmeanpAge&lt;45 (n:7)11.930.427120.40513.210.13212.290.3214.140.043150.01315.360.00710.290.905&gt;45 (n:13)9.739.699.049.548.548.087.8810.62Ki-67 (cut off %35)&lt;%35 (n:12)10.710.84711.710.26313.250.01110.920.69810.540.96911.920.18911.880.20312.040.153≥%35 (n:8)10.198.696.389.8810.448,388.448.19N stageN0 (n:13)10.120.69210.770.7819.810.4758.580.04710.270.8129,580.3419.960.5798.620.05N1 (n:7)11.211011.7914.0710.9312,2111.514StageStage 1 (n:5)5.10.0185.90.0447.40.17611.40.69311.20.7690.51312.50.3826.40.073Stage 2 (n:15)12.312.0311.5310.210.27119.8311.87
All statistical analyses were evaluated using the Mann Whitney U test.
Citation Format: Baran Mollavelioglu, Esin Aktas Cetin, Neslihan Cabioglu, Aykhan Abbasov, Semen Onder, Selman Emiroglu, Mustafa Tukenmez, Mahmut Muslumanoglu, Abdullah Igci, Gunnur Deniz, Vahit Ozmen. Differential expression of novel immune checkpoint receptors expressed on tumor-infiltrating lymphocytes (TIL) in patients with early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-24.