超出了三级淋巴结结构，在非小细胞肺癌中观察到了大量不具有生发中心样结构的免疫活跃区。在此，我们整合转录组数据和数字病理图像来研究935名肺癌患者中免疫活跃区（免疫热点）的预后影响、空间位置和组成。高肿瘤内免疫热点评分，它衡量免疫热点与肿瘤海岛相接触的比例，与肺鳞癌的总生存率呈负相关，但与肺腺癌无关。高肿瘤内免疫热点评分的肺鳞癌特征是B细胞标志物的持续上调。串行多重免疫组织化学切片的空间统计分析进一步揭示出围肿瘤区域中只有4.87%的免疫热点和肿瘤内区域中只有0.26%的免疫热点是三级淋巴结结构。肿瘤内免疫热点中CD20 + CXCR5 + 和CD79b + B细胞密度较低，免疫细胞相互作用较少，这与围肿瘤免疫热点相比有很大不同。此外，在肿瘤内免疫热点中，CD8 + T细胞和T调节细胞的百分比呈负相关，而在围肿瘤免疫热点中不呈负相关（除了三级淋巴结结构）。这些发现表明，肿瘤内免疫热点反映出免疫抑制的环境，而不考虑三级淋巴结结构的分布，相比之下围肿瘤免疫热点则有更好的免疫活性。肺鳞癌中高肿瘤内免疫热点评分的平衡表明，抗肿瘤免疫反应受损，并且预后不良。

Beyond tertiary lymphoid structures, a significant number of immune rich areas without germinal center-like structures are observed in non-small cell lung cancer. Here, we integrated transcriptomic data and digital pathology images to study the prognostic implications, spatial locations, and constitution of immune rich areas (immune hotspots) in a cohort of 935 lung cancer patients from the TCGA. A high intratumoral immune hotspot score, which measures the proportion of immune hotspots interfacing with tumor islands, was correlated with poor overall survival in lung squamous cell carcinoma but not in lung adenocarcinoma. Lung squamous cell carcinomas with high intratumoral immune hotspot scores were characterized by consistent upregulation of B cell signatures. Spatial statistical analyses conducted on serial multiplex immunohistochemistry slides further revealed that only 4.87% of peritumoral immune hotspots and 0.26% of intratumoral immune hotspots were tertiary lymphoid structures. Significantly lower densities of CD20+CXCR5+ and CD79b+ B cells and less diverse immune cell interactions were found in intratumoral immune hotspots compared to peritumoral immune hotspots. Furthermore, there was a negative correlation between the percentages of CD8+ T cells and T regulatory cells in intratumoral but not in peritumoral immune hotspots, with tertiary lymphoid structures excluded. These findings suggest that the intratumoral immune hotspots reflect an immunosuppressive niche compared to peritumoral immune hotspots, independent of the distribution of tertiary lymphoid structures. A balance towards increased intratumoral immune hotspots is indicative of a compromised anti-tumor immune response and poor outcome in lung squamous cell carcinoma.