Table_3_Biological Determinants of Chemo-Radiotherapy Response in HPV-Negative Head and Neck Cancer: A Multicentric External Validation.PDF

Purpose: Tumor markers that are related to hypoxia, proliferation, DNA damage repair and stem cell-ness, have a prognostic value in advanced stage HNSCC patients when assessed individually. Here we aimed to evaluate and validate this in a multifactorial context and assess interrelation and the combined role of these biological factors in determining chemo-radiotherapy response in HPV-negative advanced HNSCC.
Methods: RNA sequencing data of pre-treatment biopsy material from 197 HPV-negative advanced stage HNSCC patients treated with definitive chemoradiotherapy was analyzed. Biological parameter scores were assigned to patient samples using previously generated and described gene expression signatures. Locoregional control rates were used to assess the role of these biological parameters in radiation response and compared to distant metastasis data. Biological factors were ranked according to their clinical impact using bootstrapping methods and multivariate Cox regression analyses that included clinical variables. Multivariate Cox regression analyses comprising all biological variables were used to define their relative role among all factors when combined.
Results: Only few biomarker scores correlate with each other, underscoring their independence. The different biological factors do not correlate or cluster, except for the two stem cell markers CD44 and SLC3A2 (r = 0.4, p < 0.001) and acute hypoxia prediction scores which correlated with T-cell infiltration score, CD8+ T cell abundance and proliferation scores (r = 0.52, 0.56, and 0.6, respectively with p < 0.001). Locoregional control association analyses revealed that chronic (Hazard Ratio (HR) = 3.9) and acute hypoxia (HR = 1.9), followed by stem cell-ness (CD44/SLC3A2; HR = 2.2/2.3), were the strongest and most robust determinants of radiation response. Furthermore, multivariable analysis, considering other biological and clinical factors, reveal a significant role for EGFR expression (HR = 2.9, p < 0.05) and T-cell infiltration (CD8+T-cells: HR = 2.2, p < 0.05; CD8+T-cells/Treg: HR = 2.6, p < 0.01) signatures in locoregional control of chemoradiotherapy-treated HNSCC.
Conclusion: Tumor acute and chronic hypoxia, stem cell-ness, and CD8+ T-cell parameters are relevant and largely independent biological factors that together contribute to locoregional control. The combined analyses illustrate the additive value of multifactorial analyses and support a role for EGFR expression analysis and immune cell markers in addition to previously validated biomarkers. This external validation underscores the relevance of biological factors in determining chemoradiotherapy outcome in HNSCC.

研究目的：与缺氧、增殖、DNA损伤修复及干细胞性相关的肿瘤标志物，单独评估时对晚期头颈部鳞状细胞癌（Head and Neck Squamous Cell Carcinoma, HNSCC）患者具有预后价值。本研究旨在多因素背景下对上述结论进行评估与验证，并探讨这些生物学因素在人乳头瘤病毒（Human Papillomavirus, HPV）阴性晚期头颈部鳞状细胞癌患者放化疗应答中的相互关系及联合作用。
方法：本研究分析了197例接受根治性放化疗的HPV阴性晚期头颈部鳞状细胞癌患者的治疗前活检组织RNA测序（RNA Sequencing）数据。采用已发表并验证的基因表达特征为患者样本赋值生物学参数评分。以区域控制率评估这些生物学参数在放射应答中的作用，并与远处转移数据进行对比。采用自举法（Bootstrap）及纳入临床变量的多因素Cox回归分析，根据生物学因素的临床影响对其进行排序。纳入全部生物学变量的多因素Cox回归分析用于明确各因素联合时的相对贡献。
结果：仅少数标志物评分间存在相关性，证实了各标志物的独立性。不同生物学因素之间无关联或聚类现象，仅两种干细胞标志物CD44与SLC3A2（相关系数r=0.4，p<0.001），以及急性缺氧预测评分与T细胞浸润评分、CD8+T细胞丰度及增殖评分存在相关性（相关系数分别为0.52、0.56和0.6，均p<0.001）。区域控制关联分析显示，慢性缺氧（风险比（Hazard Ratio, HR）=3.9）与急性缺氧（HR=1.9），随后是干细胞性（CD44/SLC3A2；HR=2.2/2.3），是放射应答最强且最稳定的预测因素。此外，纳入其他生物学及临床因素的多变量分析显示，表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）表达评分（HR=2.9，p<0.05）及T细胞浸润相关评分（CD8+T细胞：HR=2.2，p<0.05；CD8+T细胞/调节性T细胞（Regulatory T cell, Treg）：HR=2.6，p<0.01）在接受放化疗的头颈部鳞状细胞癌患者的区域控制中具有显著作用。
结论：肿瘤急性与慢性缺氧、干细胞性及CD8+T细胞相关参数是相关且基本独立的生物学因素，共同影响区域控制效果。多因素联合分析证实了多因素分析的附加价值，并支持在已验证的生物标志物基础上，增加表皮生长因子受体表达分析及免疫细胞标志物检测。本项外部验证进一步明确了生物学因素在决定头颈部鳞状细胞癌患者放化疗结局中的相关性。