[Joint analysis of invasive margins and tumor center to evaluate the prognostic value of bystander CD8

Objective: The impact of bystander CD8+ T cells (CD8+ Tbys) within the tumor microenvironment on the prognosis of early-stage non-small cell lung cancer (NSCLC) remains unclear, particularly concerning their infiltration differences at the invasive margin (IM) and tumor center (TC). Methods: We retrospectively collected postoperative specimens from 173 patients with primary early-stage NSCLC who underwent radical surgery at the Affiliated Tumor Hospital of Shandong First Medical University from 2014 to 2018. Tissue microarrays encompassing IM and TC regions were prepared and subjected to multicolor immunofluorescence staining (CK/CD8/CD103/DAPI). Image processing and phenotype recognition (CD8+ T cells, CK-CD8+; CD8+ Tbys, CK-CD8+CD103-) were performed using inFrom software, and automatic quantitative cell density analysis was conducted using R language. Differences in CD8+ T and CD8+ Tbys densities at IM and TC were analyzed using the Mann-Whitney U test. The relationship between CD8+ T, CD8+ Tbys and clinicopathological features was examined using the Kruskal-Wallis H test. The impact of CD8+ T and CD8+ Tbys on recurrence-free survival (RFS) was evaluated using Kaplan-Meier, log-rank, and Cox proportional hazards models. Results: A total of 173 patients with stage ⅠA-ⅡA NSCLC were included, with a recurrence rate of 26.6% (46/173) and a median RFS of 62.3 months (range: 44.7-71.9 months). CD8+ T and CD8+ Tbys densities (1/1 000) were significantly higher in the IM region than in the TC region [70(32, 155) vs. 37(18, 88), P＜0.001; 25(11, 46) vs. 18(7, 34), P=0.002]. No significant association was found between CD8+ T, CD8+ Tbys and age, gender, smoking history, histological type, or pathological stage (all P＞0.05). Patients with low-density IM CD8+ T cells had lower RFS compared to those with high-density IM CD8+ T cells (P=0.021; median RFS not reached), Further analysis revealed that patients with low-density IM CD8+ Tbys cell had lower RFS compared to those with high-density IM CD8+ Tbys (P=0.047; median RFS not reached), and low-density IM CD8+ T cell was an independent risk factor for postoperative recurrence (HR=1.836, 95% CI:1.007-3.347, P=0.048). Joint analysis of IM and TC revealed that patients with low IM CD8+ Tbys and high TC CD8+ Tbys had significantly lower RFS compared to the other three groups (P=0.006), and this combination was an independent risk factor for postoperative recurrence in early-stage NSCLC (HR=2.090, 95% CI:1.162-3.760, P=0.014). Conclusions: The spatial distribution of bystander CD8+ T cells within the primary tumor influences the prognosis of patients with early-stage NSCLC. Patients with low-density IM CD8+ Tbys and high-density TC CD8+ Tbys are more prone to recurrence after radical surgery.