PO.TB06.01 · 肿瘤生物学
Lipocalin 2 作为头颈部鳞状细胞癌（HNSCC）放疗抵抗生物标志物的潜在作用
Potential role of Lipocalin 2 as a biomarker of radio resistance in head and neck squamous cell carcinoma (HNSCC)
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摘要 Abstract
中文摘要
背景：HNSCC 疾病负担沉重，发病率与死亡率均较高。大多数 HNSCC 患者接受放射治疗（RT），而放疗常伴随放射抵抗的发生及疾病复发，这一问题需要深入评估。在我们此前采用 LC/MS 鸟枪法蛋白质组学的研究中，发现 Lipocalin2（LCN2）在 HNSCC 患者唾液中表达失调，同时也被发现与放射抵抗（RR）相关。为确立其作为 RR 预测指标的潜在作用，我们对 HNSCC 患者中的 LCN2 进行了评估。
方法：本研究共纳入 356 例经活检证实、初治的 HNSCC 患者、26 例口腔潜在恶性疾病（OPMDs）患者及 118 名健康受试者。患者接受根治性意图的 66Gy 放疗。在基线及随访期间，采集 5ml 非刺激性唾液和 3ml 全血，随访对象包括治疗完成后无疾病证据（NED）和残留病灶（RD）的患者。为评估 LCN2 作为 RR 标志物的作用，通过将 Cal27 细胞反复亚致死剂量暴露于放射性 Co 60 释放的伽马射线，生成放射抵抗细胞。放射抵抗表型通过 Western Blot（WB）和免疫荧光（IF）检测磷酸化 H2AX（gammaH2AX）表达予以确认。对成功生成的 RR 细胞进行多种 RR 检测，如克隆形成存活实验、凋亡实验及细胞周期分析，随后分析其 LCN2 表达。
结果：与 OPMD（唾液及血清中位数分别为 358.47ng/ml 和 124.05ng/ml）和健康对照（中位数分别为 95.70ng/ml 和 85.53ng/ml）相比，患者唾液和血清中 LCN2 水平显著升高（p≤0.0001，中位数分别为 609.15ng/ml 和 186.18ng/ml）。这些蛋白水平的升高还与患者治疗完成后的疗效相关，RD 组 LCN2 显著高于（p≤0.0001）NED 组（RD 组中位数分别为 1919.61ng/ml 和 133.48ng/ml，NED 组分别为 537.27ng/ml 和 90.68ng/ml），提示治疗完成后测得的 LCN2 水平升高与治疗抵抗相关。在 IHC 分析中，低分化患者的 LCN2 水平（H-score 148.12）显著高于（p≤0.0001）中分化（H-score 34.04）和高分化患者（H-score 17.12）。在所生成的 RR 细胞系中，IF 分析显示 gammaH2AX 灶显著增多，并经 Western Blot 验证。进一步通过克隆形成存活实验、凋亡实验及细胞周期分析实验，证实所生成的细胞系具有 RR 特征。对已证实的 RR 细胞系进一步检测 LCN2 表达，发现其表达较野生型细胞系显著升高（3.6 倍），该结果经 Western Blot 验证。
结论：LCN2 被提出作为一种预测性生物标志物，用于预判 HNSCC 患者对放射的抵抗。
查看英文原文 English abstract
Background: HNSCC has significant burden with high morbidity and mortality. Majority of the HNSCC patients receive Radiotherapy (RT), which is associated with development of radiation resistance and disease-recurrence, which needs in-depth evaluation. Lipocalin2(LCN2), found to be dysregulated in the saliva of HNSCC patients in our previous study using LC/MS shotgun proteomics, is also found associated with radio resistance (RR). To establish the potential role as a predictor of RR, we evaluated LCN2 in HNSCC patients.
Methodology: 356 biopsy-proven treatment-naïve HNSCC, 26 Oral Pre-Malignant Diseases (OPMDs) and 118 healthy subjects were recruited for the study. The patients were treated with curative intent with 66Gy of RT. 5ml of unstimulated saliva and 3ml of whole blood were collected at baseline and during follow-ups in No evidence of disease (NED) and Residual Disease (RD)patients post therapy completion. To evaluate the role LCN2 as a marker of RR, radioresistant cells were generated by repeated sub-lethal exposure of Cal27 cells to gamma-rays from radioactive Co 60 . The RR phenotype was confirmed by phosphorylated H2AX(gammaH2AX) expression using Western Blot (WB) and Immunofluorescence (IF). Various RR assays such as clonogenic survival assay, apoptosis assay and cell cycle analysis, were performed on the successfully generated RR cells, which were later analysed for the expression of LCN2.
Results: Significantly higher( p≤0.0001 ) levels of LCN2 in saliva and serum (median- 609.15ng/ml & 186.18ng/ml) of patients compared to OPMD (median-358.47ng/ml & 124.05ng/ml) and healthy controls (median-95.70ng/ml & 85.53ng/ml) respectively. The increased level of these proteins also correlated with patients' response post therapy completion with significantly higher( p≤0.0001 ) LCN2 in RD (median-1919.61ng/ml & 133.48ng/ml) group compared to NED (median-537.27ng/ml & 90.68ng/ml), indicating that the increased LCN2 level estimated after therapy completion is associated with therapy resistance. Poorly differentiated patients had significantly higher( p≤0.0001 ) LCN2(H-score148.12) as compared to moderately(H-score34.04) and well differentiated(H-score17.12) on IHC analysis. In the generated RR cell line, on IF analysis gammaH2AX foci was significantly higher validated by western blot. Further using clonogenic survival assay, apoptosis and cell cycle analysis assay it was henceforth proven that the cell lines generated had RR characteristics. The proven RR cell lines were further checked for LCN2 expression, which had significantly higher (3.6 fold) expression as compared to wild type cell lines. The results were validated with western blot.
Conclusion: LCN2 is proposed as a predictive biomarker to prognosticate resistance to radiation in patients with HNSCC.
利益披露 Disclosure
S. Suri, None..
S. Ghoshal, None..
J. Bakshi, None..
A. Pal, None.