For practical reasons, in many studies PD-L1 expression is measured by combined positive score (CPS) from a single tumor sample. This does not reflect the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC). We investigated the extent and relevance of PD-L1 expression heterogeneity in HNSCC analyzing primary tumors and recurrences (LRs), as well as metastases. Tumor tissue from 200 HNSCC patients was immunohistochemically stained for PD-L1 and analyzed using image-analysis software QuPath v3.4 with multiple specimens per patient. CPS was ≥20 in 25.6% of primary tumors. Intra-tumoral heterogeneity led to a therapeutically relevant underestimation of PD-L1 expression in 28.7% of patients, when only one specimen per patient was analyzed. Inter-tumoral differences in PD-L1 expression between primary tumors and lymph node metastasis (LNM) or LR occurred in 44.4% and 61.5% (CPS) and in 40.6% and 50% of cases (TPS). Overall survival was increased in patients with CPS ≥ 1 vs. CPS < 1 in primary tumors and LNM (hazard ratio: 0.46 and 0.35;p< 0.005); CPS in LR was not prognostic. Our analysis shows clinically relevant intra- and inter-sample heterogeneity of PD-L1 expression in HNSCC. To account for heterogeneity and improve patient selection for immunotherapy, multiple sample analyses should be performed, particularly in patients with CPS/TPS < 1.
出于实际操作考虑，许多研究采用单次肿瘤样本的综合阳性评分（CPS）来评估PD-L1表达水平，这种方法无法准确反映头颈部鳞状细胞癌（HNSCC）中PD-L1表达的异质性特征。本研究通过分析原发肿瘤、局部复发灶及转移灶，系统探讨了HNSCC中PD-L1表达异质性的程度及其临床意义。我们对200例HNSCC患者的肿瘤组织进行PD-L1免疫组化染色，采用QuPath v3.4图像分析软件对每位患者的多个样本进行分析。结果显示：25.6%的原发肿瘤CPS≥20；当仅分析单一样本时，28.7%的患者因肿瘤内异质性导致PD-L1表达被低估至影响治疗决策的程度。原发肿瘤与淋巴结转移灶（LNM）或局部复发灶（LR）之间的PD-L1表达差异分别出现在44.4%和61.5%的病例（CPS评估），以及40.6%和50%的病例（TPS评估）。生存分析表明，原发灶和LNM中CPS≥1的患者总生存期显著优于CPS<1者（风险比：0.46和0.35；p<0.005），而LR的CPS评分无预后价值。本研究证实HNSCC中PD-L1表达存在具有临床意义的样本内及样本间异质性。为克服异质性影响并优化免疫治疗患者筛选策略，建议实施多样本分析，尤其对于CPS/TPS<1的患者群体。
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