Abstract B20: Premalignant lung lesions demonstrate enhanced PD-L1 upregulation in response to interferon-gamma exposure
Authors: Yanagawa Jane , Fung Eileen , Lee Mi-Heon , Wallace W. Dean , Fishbein Michael C. , Paul Manash , Krysan Kostyantyn , Minna John D. , Guo Rong , Elashoff David , Lee Jay M. , Dubinett Steven M. 
Published: February 2019

Background: Interferon-gamma (IFN-g) is known to play a pivotal role in PD-L1 expression and immune evasion in cancer cells, but there is little known regarding its interactions with premalignant lung lesions.

Methods: Immortalized human bronchial epithelial cells (HBEC-vector control), KRAS-mutated (KRASv12) HBEC cells (HBEC-KRAS), p53 knockdown HBEC cells (HBEC-p53), and p53 knockdown/KRAS mutated cells (HBEC-p53/KRAS) were used to assess mRNA expression as well as surface and total protein expression levels of PD-L1 by RT-PCR, flow cytometry, and Western blot before and after treatment with IFN-g. For STAT-1 knockdown, cells were transiently transfected using Lipofectamine RNAiMAX (Thermo Scientific). After 48 hours of transfection, cells were incubated with IFN-g (50 ng/mL) or PBS with 0.1% BSA for 48 hours, then harvested and analyzed by RT-PCR, flow cytometry, and Western blot. An FFPE tissue block from a patient with known premalignant lesions and lung adenocarcinoma was obtained from the UCLA Lung Cancer Tissue Repository and sectioned to create slides for H&E staining and to identify areas of atypical adenomatous hyperplasia. Serial sectioning was performed to create slides for multiplex staining, targeting anti-PD-L1, anti-p-STAT1-Ser747, and anti-CD8. Quantitative image analysis was performed with Visiopharm.

Results: Using RT-PCR, flow cytometry, and Western blot, exposure to IFN-g led to upregulation of PD-L1 in HBECs with various gene mutations (HBEC-vector compared to HBEC-Kras, HBEC-p53, HBEC-KRAS/p53, HBEC-EGFR-L858R) (p&lt;0.05). These experiments demonstrated that PD-L1 is elevated at the mRNA and protein levels with IFN-g exposure, with the greatest upregulation in HBECs with KRAS mutation (p&lt;0.013). To investigate potential mechanisms of PD-L1 upregulation in premalignant cell lines after IFN-g exposure, we evaluated levels of STAT1, pSTAT1, ERK, pERK, AKT, and pAKT in the paired HBEC3 vector and KRAS cell lines. On Western blot, pSTAT1 was the only signaling protein that was upregulated after IFN-g exposure. We then performed STAT-1 knockdown of HBEC3-vector and HBEC3-KRAS cell lines. With IFN-g exposure, there was significantly diminished PD-L1 upregulation in the HBEC3-KRAS with STAT-1 knockdown as compared to the HBEC3-KRAS control (p&lt;0.002). In our human staining, there was increased CD8 T cells (11% versus 2.5%) and pSTAT1 (80% versus 32%) in the vicinity of premalignant lesions as compared to normal tissues. In addition, although we identified sporadic premalignant epithelial cells with membranous PD-L1 staining, no PD-L1 staining was identified in any normal epithelial cells.

Conclusions: Premalignant lung lesions may contribute to immune evasion by responding to IFN-gamma exposure with enhanced PD-L1 upregulation via pSTAT1 signaling. Further studies are required to establish the role of IFN-gamma in the immune microenvironment of premalignant lung lesions.

Citation Format: Jane Yanagawa, Eileen Fung, Mi-Heon Lee, W. Dean Wallace, Michael C. Fishbein, Manash Paul, Kostyantyn Krysan, John D. Minna, Rong Guo, David Elashoff, Jay M. Lee, Steven M. Dubinett. Premalignant lung lesions demonstrate enhanced PD-L1 upregulation in response to interferon-gamma exposure [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B20.