Abstract 2526: Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients
Authors: Barkhordarzadeh Ainaz Dory , Hsu Robert C. , Wang Rongfu , Martin Sue E. , Nieva Jorge J. , Wallace William Dean 
Published: June 2022

Abstract
Introduction: KK-LC-1 is a cancer testis antigen expressed notably in lung, breast, and gastric cancer making it an attractive target for T-cell receptor therapy. Here, we evaluate cell densities of NSCLC tissue specimens with known KK-LC-1 gene expression to better characterize the tumor immune microenvironment in such patients.
Methods: A seven-plex mIF panel (FoxP3, PDL1, PanCK, PD1, CD8, KK-LC-1, and CD68) was developed based on a previously established mIF Lung-cancer panel and addition of KK-LC-1 following rigorous optimization of KK-LC-1 antibody titer and panel order with qualitative assessment of monoplex immunofluorescent assays. Serial sections from 23 NSCLC cases (14 biopsy samples and 9 excision specimens) with known KK-LC-1 gene expression values were collected. Tissue sections were stained and imaged on an Akoya Biosciences Vectra Polaris multispectral imaging platform. The regions of interest were identified and stamped with the Phenochart mIF image management software. Spectral unmixing, tissue segmentation and cell phenotyping were performed with Akoya Biosciences InForm image analysis software using the multispectral fields workflow. The 4-tier positive staining thresholds for PD-L1 and KK-LC-1 were determined by a pathologist (WDW). Cell densities in the tumor and adjacent stroma as well as the KK-LC-1 H-score were assessed by linear regression analysis. Student’s t-test was used to compare between the top quartile of tumors based on KK-LC-1 gene expression (Q4) and the remaining 3 quartiles (Q1-Q3).
Results: The study consisted of 17/23 adenocarcinoma (73.9%), 4/23 squamous cell carcinoma (SCC) (17.3%), and 2/23 (8.6%) unclear or mixed histology tumors. Adenocarcinoma trended to have higher KK-LC-1 gene expression compared to SCC tumors (mean 17.34 vs. 0.65, p-value = 0.204). 13/23 (56.5%) were pan-wild type, 6/23 (26.0%) were EGFR mutated, 2/23 (8.7%) were KRAS mutated, and 1/23 (4.4%) were MET Exon 14 skip and multiple alterations. We saw significantly higher cell density of FoxP3 in tumor cells in Q4 versus Q1-Q3 (mean 7.17 vs. 0.8 cells/mm2, p-value = 0.004), and there was a trend towards a higher FoxP3/CD8 cell density ratio (mean 3.28 vs. 0.05, p = .110) and higher PD-1 cell density (mean 186.7 vs. 32.8 cells/mm2, p=0.1548) in tumor cells in Q4 versus Q1-Q3. There was significant correlation towards increased CD8+ T cell density ratio between tumor and stroma cells in cases with higher KK-LC-1 gene expression (r2= 0.307, p = 0.011).
Conclusion: KK-LC-1 expression in lung cancer is seen in multiple molecular subsets and is associated with increase in both regulatory T cells in tumor and in CD8+ T-cells in tumor relative to stroma. Strategies to deplete regulatory T cells and to further amplify CD8+ T cells should be pursued in the development of T-cell receptor therapy targeting KK-LC-1.
Citation Format: Ainaz Dory Barkhordarzadeh, Robert C. Hsu, Rongfu Wang, Sue E. Martin, Jorge J. Nieva, William Dean Wallace. Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2526.