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Abstract：
BACKGROUND: HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis.
METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors.
RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC.
CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.
METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors.
RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC.
CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.
摘要：
背景：HHLA2（人类内源性逆转录病毒-H长末端重复序列相关蛋白2）代表了最近确定的B7免疫检查点家族成员，其特征是在正常组织中表达有限，但在各种癌症类型中明显过表达。然而，精确的功能和与免疫细胞的相互作用仍然知之甚少，尤其是喉鳞状细胞癌（LSCC）。这项研究试图阐明HHLA2在人类LSCC组织的肿瘤微环境中的生物学意义，并描述HHLA2在LSCC发病机理中的临床相关性和功能作用。
方法：通过对来自LSCC患者（n=72）的组织微阵列进行的多重免疫组织化学分析，进行分析以评估HHLA2的表达水平，CD68HLA-DRCD163-（M1巨噬细胞）的密度和空间模式，CTLA-4+CD4+FoxP3+（CTLA-4+Treg细胞），CTLA-4+CD4+FoxP3-（CTLA-4+Tcon细胞），耗尽的CD8+T细胞，和LSCC组织中终末耗尽的CD8+T细胞。进行生存分析以评估HHLA2和这些免疫检查点或免疫细胞群的预后意义。采用COX回归分析确定独立的预后因素。
结果：Kaplan-Meier(K-M)存活曲线揭示了LSCC中HHLA2表达与总生存期(OS)之间的显著关联。HHLA2水平升高与患者生存率降低有关。表明其作为预后标志物的潜力（HR：3.230，95CI0.9205-11.34，P=0.0067）。值得注意的是,CD68+细胞（总巨噬细胞）的浸润增加，STING+CD68+HLA-DR+CD163-（STING+M1巨噬细胞），CTLA-4+CD4+FoxP3+,CTLA-4+CD4+FoxP3-,PD-1+LAG-3+CD8+T细胞，PD-1+LAG-3+TIM-3+CD8+T细胞与较差的生存结局密切相关(P<0.05)。在这些免疫细胞群的水平之间观察到明显的趋势，STING+CD68+（STING+总巨噬细胞），CD68+HLA-DR+CD163-,STING+CD68+CD163+HLA-DR-（STING+M2巨噬细胞），PD-1+LAG-3-CD8+T细胞，PD-1+TIM-3+CD8+T细胞，PD-1+LAG-3+TIM-3-CD8+T细胞与预后的关系。重要的是,多因素COX分析确定HHLA2是LSCC患者OS的独立预测因子(HR=3.86,95%CI1.08-13.80,P=0.038)。这强调了HHLA2作为预测LSCC患者预后的关键标志物的潜力。
结论：HHLA2作为评估LSCC患者OS的一个有害的预后生物标志物出现。相对于其他免疫检查点，HHLA2对LSCC患者的预后表现出更高的预测功效。
方法：通过对来自LSCC患者（n=72）的组织微阵列进行的多重免疫组织化学分析，进行分析以评估HHLA2的表达水平，CD68HLA-DRCD163-（M1巨噬细胞）的密度和空间模式，CTLA-4+CD4+FoxP3+（CTLA-4+Treg细胞），CTLA-4+CD4+FoxP3-（CTLA-4+Tcon细胞），耗尽的CD8+T细胞，和LSCC组织中终末耗尽的CD8+T细胞。进行生存分析以评估HHLA2和这些免疫检查点或免疫细胞群的预后意义。采用COX回归分析确定独立的预后因素。
结果：Kaplan-Meier(K-M)存活曲线揭示了LSCC中HHLA2表达与总生存期(OS)之间的显著关联。HHLA2水平升高与患者生存率降低有关。表明其作为预后标志物的潜力（HR：3.230，95CI0.9205-11.34，P=0.0067）。值得注意的是,CD68+细胞（总巨噬细胞）的浸润增加，STING+CD68+HLA-DR+CD163-（STING+M1巨噬细胞），CTLA-4+CD4+FoxP3+,CTLA-4+CD4+FoxP3-,PD-1+LAG-3+CD8+T细胞，PD-1+LAG-3+TIM-3+CD8+T细胞与较差的生存结局密切相关(P<0.05)。在这些免疫细胞群的水平之间观察到明显的趋势，STING+CD68+（STING+总巨噬细胞），CD68+HLA-DR+CD163-,STING+CD68+CD163+HLA-DR-（STING+M2巨噬细胞），PD-1+LAG-3-CD8+T细胞，PD-1+TIM-3+CD8+T细胞，PD-1+LAG-3+TIM-3-CD8+T细胞与预后的关系。重要的是,多因素COX分析确定HHLA2是LSCC患者OS的独立预测因子(HR=3.86,95%CI1.08-13.80,P=0.038)。这强调了HHLA2作为预测LSCC患者预后的关键标志物的潜力。
结论：HHLA2作为评估LSCC患者OS的一个有害的预后生物标志物出现。相对于其他免疫检查点，HHLA2对LSCC患者的预后表现出更高的预测功效。
