Spatial Biomarkers Associated with PNI and LVI in Stage II Colon Cancer

Multiple retrospective studies and meta-analyses have shown that presence of lymphovascular invasion (LVI) and/or perineural invasion (PNI) is associated with significantly worse disease-free and overall survival in stage II colorectal cancer (CRC), and these features are commonly considered when deciding whether to recommend adjuvant chemotherapy for stage II disease. Current study reviewed 32 stage II CRC patient archived FFPE samples to investigate immune cell features among LVI, PNI or no-invasion groups. IHC control, multiplex immunofluorescence (mIF) panel control and the followed mIF staining were conducted using PN 7-Plex Detection Kit (PhenoVision Bio Co., Itd) targeted CD68, CD3, CD20, panCK, PD-1, PD-L1 and CD68, panCK, CD3, CD8, CD163, αSMA, respectively. The QC procedures made sure each marker from mIF results exhibited identical location and density of the same marker from IHC staining. The 64 staining slides were scanned, tumor areas were lined out by pathologist based on H&E staining from the serial section of each sample. Non-tumor region, tumor invasive margin (IM) , which was defined as 360μm within and outside of tumor boundary i.e. total 720μm, tumor parenchyma, tumor stromal, 30μm region inside and outside of tumor parenchyma boundary were analyzed using PhenoVision mIF AI analysis system trained from Oncotopix Discovery system (Visiopharm). Positive cell percentage were analyzed for further analysis. Statistical analysis exhibited correlation of CD20 and CD20+/PD-1- cell percentage to PNI condition (p=0.046 and 0.046, respectively) in tumor invasive region and CD68+/CD163- cell percentage to PNI (p=0.048) of 30μm outside tumor parenchyma based on binary logistic regression. There were higher CD20+ positive cell percentage and CD20+/PD-1- cell percentage in PNI group vs. non PNI group (p=0.013 and 0.013, respectively) at tumor invasive region. Whereas lower CD68+CD163- cell percentage was observed in PNI group vs. non PNI group of 30μm outside of tumor parenchyma region (p=0.01). There was significant more panCK cell percentage in LVI positive group at tumor parenchyma region compared to non LVI group (p=0.000). Current study indicates that spatial biomarkers are correlated to PNI and LVI in tumor invasive margin and tumor parenchyma region of stage II colon cancers which may be related to stage II CRC prognosis. Increased sample number of the spatial marker distribution feature to disease prognosis analysis are scheduled. Citation Format: Hanqing Hu, Hongzhe Sun, Lin Zhu, Shuo Han, Zhifu Zhang, Enkai Zhang, Na Li, Guiyu Wang. Spatial biomarker related to PNI and LVI in stage II colon cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6225.