【摘要】 背景与目的非小细胞肺癌（non-small cell lung cancer,NSCLC）术后复发率较高,传统肿瘤原发灶-淋巴结-转移（tumor-node-metastasis,TNM）分期难以充分反映其生物学异质性,乏氧诱导因子-1α （hypoxia-inducible factor-1alpha,HIF-1α）在肿瘤进展及免疫微环境重塑中发挥重要作用,但其在不同淋巴结转移状态下的空间分布特征及对术后复发的预后意义尚不明确。本研究旨在探讨NSCLC患者原发灶中的表达HIF-1α的肿瘤细胞、CD4⁺T细胞及CD8⁺T细胞的密度差异,以及对淋巴结转移及患者术后复发的影响。方法回顾性收集2014年1月1日至2018年12月31日在山东第一医科大学附属肿瘤医院行根治性手术的256例NSCLC患者的术后石蜡包埋原发灶组织标本,构建包含肿瘤中心（tumor center,TC）和侵袭边缘（invasive margin,IM）区域的组织微阵列,并应用多重免疫荧光染色技术[HIF-1α/CD4/CD8/细胞角蛋白（cytokeratin,CK）/4’,6-二脒基-2-苯基吲哚（4’,6-diamidino-2-phenylindole,DAPI）]定量表达HIF-1α的肿瘤细胞（HIF-1α⁺CK⁺）、表达HIF-1α的CD4⁺T细胞（HIF-1a⁺CD4⁺）及表达HIF-1α的CD8⁺T细胞（HIF-1α⁺CD8⁺）的密度。采用Mann-Whitney U检验分析N₀与N_1-2组、N₁与N₂组中的细胞密度差异,Cox比例风险回归模型分析关键复发因子。结果研究最终纳入256例ⅠA-ⅢB期NSCLC患者,中位随访时间为37.05个月,其中87例（34.0%）患者在随访期间复发。在NSCLC患者的TC及IM区域,与N。组相比,N_1-2组HIF-1α⁺CK⁺细胞密度均更高（P分别为0.039、<0.001）,HIF-1α⁺CD8⁺细胞密度均更低（P分别为<0.001、<0.001）;与N₁组相比,N₂组原发灶内TC区域和IM区域HIF-1α⁺CK⁺细胞、HIF-1α⁺CD4⁺细胞及HIF-1α⁺CD8⁺细胞的密度差异均无统计学意义（均P>0.05）。多因素Cox分析显示,在N₀组患者中,TC区域低HIF-1α⁺CD8⁺密度是NSCLC患者复发的独立危险因素[风险比（hazard ratio,HR）=1.998,95%CI:1.077-3.705,P=0.028]。在N₁、N₂组患者中,TC区域和IM区域HIF-1α⁺CK⁺密度、HIF-1α⁺CD4⁺、HIF-1α⁺CD8⁺密度与NSCLC患者复发无统计学关联。结论在NSCLC患者中淋巴结转移与原发灶HIF-1α相关细胞亚群密度变化密切关联。原发灶TC区域中HIF-1α⁺CD8⁺细胞密度降低与N₀期NSCLC患者术后复发显著关联,可作为术后风险分层的潜在免疫学指标。更多还原

【Abstract】 Background and objective Non-small cell lung cancer（NSCLC） is associated with a high rate of postoperative recurrence,and conventional tumor-node-metastasis（TNM） staging does not fully reflect its biological heterogeneity.Hypoxia-inducible factor-1alpha（HIF-1α） plays a critical role in tumor progression and remodeling of the tumor immune micro environment.However,the spatial distribution of HIF-1α and its prognostic significance in the context of different lymph node metastatic states remain unclear.This study aimed to investigate the densities of HIF-1α-expressing tumor cells,CD4⁺T cells,and CD8+T cells in the primary tumors of NSCLC patients,and to assess their associations with lymph node metastasis and postoperative recurrence.Methods 256 formalin-fixed paraffin-embedded primary tumor specimens from NSCLC patients who underwent radical resection at Shandong First Medical University Affliated Cancer Hospital between January 1,2014 and December 31,2018 were retrospectively collected.Tissue microarrays containing both tumor center（TC） and invasive margin（IM） regions were constructed and multiplex immunofluorescence staining [HIF-1α/CD4/CD8/cytokeratin（CK）/4’,6-diamidino-2-phenylindole（DAPI）] were performed to quantitatively analyze the densities of HIF-1α-expressing tumor cells（HIF-1α⁺CK⁺）,HIF-1α⁺CD4⁺T cells and HIF-1α⁺CD8⁺T cells.Mann-Whitney U tests were used to compare cellular density differences between No versus N_1-2 groups and N₁ versus N₂ subgroups,while Cox proportional hazards regression models were employed to identify critical recurrence-associated factors.Results The study ultimately included 256 patients with stage IA-IIIB NSCLC,with a median follow-up duration of 37.05 months,during which 87 cases（34.0%） experienced recurrence.Comparative analysis revealed that in both TC and IM regions,the N_1-2 group exhibited significantly higher densities of HIF-1α⁺CK⁺cells（P values:0.039 and <0.001,respectively） and lower densities of HIF-1α⁺CD8⁺cells（both P values:<0.001） compared to the No group,while no statistically significant differences were observed in the densities of HIF-1α⁺ CK⁺ cells,HIF-1α⁺ CD4⁺ cells,or HIF-1α⁺CD8⁺cells between N₁ and N₂ subgroups within either TC or IM regions（all P>0.05）.Multivariate Cox regression analysis showed that,among No patients,a low density of HIF-1α⁺CD8⁺ cells in the TC was an independent risk factor for recurrence in NSCLC patients[hazard ratio（HR）=1.998,95%CI:1.077-3.70 5,P=0.028].In contrast,among N₁ and N₂ patients,the densities of HIF-1α⁺CK⁺cells,HIF-1α⁺ CD4⁺T cells,and HIF-1α⁺ CD8⁺ cells in both the TC and IM regions were not significantly associated with NSCLC recurrence.Conclusion In patients with NSCLC,lymph node metastasis is closely associated with alterations in the densities of HIF-1α-related cellular subpopulations in the primary tumor.A reduced density of HIF-1α⁺ CD8⁺ cells in the TC of the primary lesion is significantly associated with postoperative recurrence in N₀-stage NSCLC patients and may serve as a potential immunological marker for postoperative risk stratification.更多还原