Abstract Background: Efforts are needed to discover novel immunotherapeutic targets in non-responders of current immunotherapies. VTCN1 (B7-H4/B7x/B7S1) and HHLA2 (B7-H7/B7y) are B7 family co-inhibitory molecules reported to be expressed in PD-L1-negative or EGFR-mutant lung adenocarcinoma (LUAD). To elucidate the clinicopathological implications of these two immune checkpoints, we checked their detailed expression patterns including intratumoral heterogeneity. Methods: VTCN1 and HHLA2 expression were evaluated in public single-cell RNA sequencing (scRNAseq) and The Cancer Genome Atlas (TCGA) dataset. Their immunohistochemistry (IHC) was performed on tissue microarrays of 413 LUAD and 382 lung squamous cell carcinoma (LUSC) specimens. Intratumoral heterogeneity was evaluated by IHC on representative sections of 59 surgically resected LUAD and 49 LUSC specimens. Results: In LUAD scRNAseq, VTCN1 and HHLA2 were predominantly expressed on epithelial cells and were not co-expressed with PD-L1. However, IHC showed their expression in both tumor and immune cells. In TCGA analysis, there was a negative correlation between VTCN1 and PD-L1 (LUAD, r = -0.094, p = 0.033; LUSC, r = -0.227, p <0.001). VTCN1 and HHLA2 showed positive correlations in both TCGA (LUAD, r = 0.211, p <0.001; LUSC, r = 0.182, p <0.001) and IHC analysis (LUAD, p <0.001; LUSC, p = 0.016). They were more frequently expressed in EGFR-mutant LUAD (52.9% vs. 40.3%, p = 0.015 for VTCN1 in EGFR-mutant vs. wildtype; 59.8% vs. 40.5%, p <0.001 for HHLA2). CD8+ T-cell infiltration was significantly higher in VTCN1- or HHLA2-positive EGFR-mutant LUAD, but there was no significant relationship in EGFR-wildtype. In 60% of cases expressing both VTCN1 and HHLA2, the two showed spatially concordant expression (15 out of 24 LUAD; 3 out of 6 LUSC). However, VTCN1 and PD-L1 showed concordant expression in 37.2% of cases expressing both (6 out of 16 LUAD; 10 out of 27 LUSC). HHLA2 expression was associated with better overall survival in LUSC, but there was no significant difference in survival according to VTCN1 expression. Conclusions: VTCN1 and HHLA2 are frequently co-expressed in PD-L1-negative NSCLC, especially EGFR-mutant LUAD rich in CD8+ T-cells and can serve as potential novel immunotherapeutic targets. Citation Format: Sojung Lim, Jaemoon Koh, Seung Geun Song, Jeemin Yim, Bogyeong Han, Young A Kim, Doo Hyun Chung, Yoon Kyung Jeon. Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6656.