CN113804901A - 用于口腔鳞癌早期无创诊断的血清脂质标志物及其应用 - Google Patents
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Abstract
本发明公开了用于口腔鳞癌早期无创诊断的血清脂质标志物及其应用。具体地，本发明公开了可通过CPSI‑MS原位质谱检测的特征脂质分子化合物，其中包括Arginyl‑methionine(精胺酰‑甲硫氨酸)、glycerophosphocholine(甘油磷酰胆碱)、Propionylcholine(丙酰胆碱)，以上三个脂质标志物下调表达时则表明受试者为口腔鳞癌患者或潜在口腔鳞癌患者。本发明的生物标志物可用于口腔鳞癌早期诊断和筛查，且方法简单，易于操作，准确性高，为口腔鳞癌的早期诊断和筛查提供了新的方法和思路。
Description
技术领域
本发明属于生物医药领域，涉及血清脂质标志物及其在口腔鳞癌早期无创诊断中的应用。
背景技术
头颈肿瘤是全球第八大常见恶性肿瘤，全球范围内每年有超过60万例新发病例，而口腔鳞状细胞癌(oral squamous cell carcinoma，OSCC)是头颈部恶性肿瘤中最为常见的类型之一。全球范围内，其在发展中国家的发病率要相对高于发达国家，据报道，口腔鳞癌在美国的发病率约为10例/10万，而在印度的发病率则超过了20例/10万。近几十年来，OSCC的发病率呈逐年递增趋势，在中国大陆，以上海地区为例，女性口腔鳞癌发病率为3.3/10万，男性发病率为3.8/10万。虽然口腔鳞癌的发病率相比其他肿瘤并不算高，但我国人口众多，病员基数大，口腔鳞癌的预防和治疗任重而道远。
临床上，口腔鳞癌目前公认的首选治疗方法是手术治疗，辅以放化疗或生物治疗。由于放射性治疗会导致所照射区域的组织发生纤维化，影响后期的手术操作，因此对于口腔鳞癌，放射性治疗一般建议于术后6周进行，常用的放射剂量是60cGy。对于已经发生局部淋巴结转移的口腔鳞癌病人，对其颈部进行放射性治疗可以预防可能的转移及复发。近年来，化学治疗已经成为局部进展性晚期口腔鳞癌重要的辅助治疗手段，化疗方式包括：术前诱导化疗或同期放化疗(术前或术后放化疗)。术前诱导化疗或术前同期放化疗几乎已成为巨大型晚期口腔鳞癌术前的主要治疗方式，期望通过化疗或同期放化疗达到减瘤的目的，为手术创造条件。目前，最常用的化疗药物是铂类药物(顺铂)，推荐的化疗方案为TPF(多西他赛+顺铂+5-Fu)。生物靶向疗法近年来开始用于口腔鳞癌的辅助治疗。研究表明部分口腔鳞癌病例高表达表皮样生长因子受体(epidermal growth factor receptor,EGFR)，而且研究表明其高表达是口腔鳞癌较差预后的独立预测因素。针对EGFR设计出的靶向药物西妥昔单抗(EGFR的单克隆抗体)是最常见的用于口腔鳞癌的生物治疗药物，有研究报道该药可以提高口腔鳞癌患者预后。但是，一些口腔鳞癌患者可能会对西妥昔单抗产生耐药，从而影响该药的临床治疗效果。
近几十年来，尽管口腔鳞癌的治疗技术和治疗理念不断改进，但其整体治疗效果并没有取得显著提高。口腔鳞癌患者的5年生存率并没有显著改善,仍徘徊在60％左右，晚期患者甚至还不足30％,其中的主要的原因是大多数患者在诊断时已经处于肿瘤晚期阶段。文献报道，口腔鳞癌的术后局部复发率在6.9-22％，而一旦出现术后复发，患者的生存率将明显下降。既往的研究表明，术后复发患者的3年总体生存率(Overall Survival,OS)约52.6％,5年总体生存率仅24-50％。因此，实现对口腔鳞癌的早期诊断、早期治疗对于提高口腔癌的预后，减少患者的经济负担和心理压力具有重要意义。
发明内容
为了实现对口腔鳞癌的早期快速、无创诊断，本发明的目的在于提供一组能准确区分口腔癌病人和健康志愿者的血清脂质标志物，利用该标志物组合，能实现对口腔鳞癌的早期快速、无创诊断。
本发明的目的还在于提供上述血清脂质标志物在制备早期无创诊断口腔鳞癌的试剂盒中的应用。
本发明解决其技术问题所采用的技术方案是：
用于口腔鳞癌早期无创诊断的血清脂质标志物，所述标志物包括精胺酰-甲硫氨酸Arginyl-methionine、甘油磷酰胆碱glycerophosphocholine、丙酰胆碱Propionylcholine三者任意之一或者三者任选其二或者三者共同的组合。
进一步地，所述标志物为精胺酰-甲硫氨酸Arginyl-methionine、甘油磷酰胆碱glycerophosphocholine和丙酰胆碱Propionylcholine三者共同的组合。
一种早期无创诊断口腔鳞癌的试剂盒，所述试剂盒包括上述任意之一所述的血清脂质标志物。
血清脂质标志物在制备早期无创诊断口腔鳞癌的试剂盒中的应用。
进一步地，所述血清脂质标志物下调表达时则表明受试者为口腔鳞癌患者或潜在口腔鳞癌患者。
进一步地，所述精胺酰-甲硫氨酸、甘油磷酰胆碱和丙酰胆碱均下调表达时则表明受试者为口腔鳞癌患者或潜在口腔鳞癌患者。
本发明具有的有益效果如下：
(1)本发明选择Arginyl-methionine(精胺酰-甲硫氨酸)、glycerophosphocholine(甘油磷酰胆碱)、Propionylcholine(丙酰胆碱)作为血清分子标志物组合，可以实现对口腔鳞癌的早期诊断，早期治疗，从而提高口腔鳞癌患者的治疗效果。
(2)本发明基于Arginyl-methionine(精胺酰-甲硫氨酸)、glycerophosphocholine(甘油磷酰胆碱)、Propionylcholine(丙酰胆碱)开发成口腔鳞癌的血清检测试剂盒，能实现对口腔鳞癌的早期快速、无创诊断，可以满足绝大多数医疗机构的检测需求，具有广阔的临床应用前景。
综上，本发明的生物标志物可用于口腔鳞癌早期诊断和筛查，且方法简单，易于操作，准确性高，为口腔鳞癌的早期诊断和筛查提供了新的方法和思路。
附图说明
图1为Arginyl-methionine对实验开发组口腔鳞癌和健康志愿者的区分能力示意图；(a)：为ROC曲线分析口腔鳞癌组和健康组(AUC＝0.83)；(b):为Arginyl-methionine在口腔鳞癌组和健康组相对含量柱状图分析(P＜0.05)；
图2为Propionylcholine对实验开发组口腔鳞癌和健康志愿者的区分能力示意图；(a)：为ROC曲线分析口腔鳞癌组和健康组(AUC＝0.79)；(b)：为Propionylcholine在口腔鳞癌组和健康组相对含量柱状图分析(P＜0.05)；
图3为glycerophosphocholine对实验开发组口腔鳞癌和健康志愿者的区分能力示意图；(a)：为ROC曲线分析口腔鳞癌组和健康组(AUC＝0.79)；(b)：为glycerophosphocholine在口腔鳞癌组和健康组相对含量柱状图分析(P＜0.05)；
图4为Arginyl-methionine、Propionylcholine、glycerophosphocholine三个代谢物作为一个组合对口腔鳞癌组(C)和健康组(H)的区分能力(logistic回归分析)，其中，实验开发组作为训练集，验证组作为测试集。
图5为基于Arginyl-methionine、Propionylcholine、glycerophosphocholine三个差异代谢物进行PLS-DA分析示意图，其能显著分开口腔鳞癌组和健康志愿者，其中，C:口腔鳞癌组(Cancer)，H:健康组(Health)。
具体实施方式
下面结合附图和实施例对本发明做进一步的说明。
1.实施例一：实验开发组初步确定口腔鳞癌早期诊断血清脂质代谢标志物。
本实施例中，总共纳入556例血清样本进行研究，其中早期口腔鳞癌病例315例和健康志愿者241例(表1)，所有入组病例均签有知情同意书。
表1纳入研究的早期口腔癌和健康志愿者临床病例资料(n＝556)
将所有病例分为实验开发组和验证组进行研究，其中，实验开发组包括早期口腔鳞癌病例84例，健康志愿者100例；验证组包括早期口腔鳞癌病例231例，健康志愿者141例。
注：提取血清样本的方法：术前收集原发早期口腔鳞癌病人或健康志愿者的全血，放入促凝管，3000转离心5min，离心后用移液枪吸上层的血清10ul进行CPSI-MS分析，若暂时不用，可存放于-80度冰箱长期保存。
注：导电聚合物喷雾离子化质谱技术(CPSI-MS)是一种先进的原位质谱分析技术，可以在常压、开放的环境中，仅需少量样本，即可高通量分析样本中的脂质分子。相比于传统的纸喷雾离子化，该技术具有以下优点：(1)样本消耗量少(≥100nL)；(2)极性、水溶性代谢物及药物分子；(3)操作快速简便(4)定性、半定量；(5)灵敏度较高。上述特点使该技术满足了临床样本的及时检测(Point of Care Test,POCT)要求。
本实验首先纳入184例病例的血清样本(口腔鳞癌病例84例，健康志愿者100例)作为实验开发组。利用CPSI-MS技术对184例口腔鳞癌和健康组的血清样本进行高通量脂质代谢谱分析，筛选差异表达的代谢物。利用MetaboAnalyst数据库总共得到511个m/z离子。使用student’s t检验口腔鳞癌组和健康组进行比较分析，初步筛选出161个差异代谢物(p＜0.05)；紧接着进一步利用ROC曲线从上述161差异代谢物中确定了58个能显著区分口腔鳞癌组和健康组的生物标志物(p＜0.05,且AUC＞0.70具体见表2)。
表2实验开发组确定的口腔鳞癌组和健康组生物标志物
2.实施例二：验证组对口腔鳞癌早期诊断候选标志物进一步验证确定。
使用验证组372例血清样本(早期口腔鳞癌病例231例，健康志愿者141例)对实验开发组确定的58个候选标志物进行验证，其中有6个代谢物被再次检测到有统计学差异且ROC曲线分析能显著区分癌症组和健康组(t test,p＜0.05且AUC＞0.70)。
其中3个代谢物即精胺酰-甲硫氨酸(Arginyl-methionine)、甘油磷酰胆碱(glycerophosphocholine)、丙酰胆碱(Propionylcholine)通过二级质谱鉴定出代谢物结构和名称，并且有统计学差异(p＜0.05且AUC＞0.70具体见表3)。
使用PLS-DA对上述代谢物进行组别聚类分析，发现同一类型组别的病例能很好的聚类在一起(如图5所示)，即上述代谢物能显著区分口腔鳞癌组和健康志愿者。
表3实验组和验证组共同确定的代谢标志物(t test,p＜0.05且AUC＞0.70)
Table:Differential metabolites detected in Development group andvalidation group by CPSI-MS untargeted analysis
经上述统计分析得到的目标差异代谢物的结构鉴定，主要通过以下方法及流程进行鉴定：
1)通过在线数据库检索，进一步匹配与推断其可能的分子结构；
2)根据分子离子峰的精确质量数与同位素丰度比，推测代谢物可能的分子式，主要针对一级高分辨质谱获得代谢物的[M+H]+或[M-H]-离子峰，；
3)对于可以购买到标准品的差异代谢物离子，进一步对目标代谢物的标准品进行LC-MS/MS分析，并与二级质谱的裂解结果进行比对从而确认代谢物的结构名称。
3.实施例三：血清脂质代谢标志物组合对口腔鳞癌病例和健康志愿者的鉴别能力。
对上述3个标志物精胺酰-甲硫氨酸(Arginyl-methionine)、甘油磷酰胆碱(glycerophosphocholine)、丙酰胆碱(Propionylcholine)分别进行ROC曲线分析，结果表明虽然单个标志物也能区分实验开发组中早期口腔鳞癌病例和健康志愿者(如图1a、图2a和图3a所示)，但区分能力并不是最佳的状态(AUC:0.79-0.83)。进一步分析发现，此3个代谢物在口腔鳞癌组中的含量低于健康志愿者(如图1b、图2b和图3b所示)，表明从健康人到癌症演变过程需要大量消耗上述3个代谢物，为肿瘤的生长提供能量和原材料。
更进一步地，当将此3个标志物作为一个组合进行Logistic Regression回归建模,采用glmnet的Binomial Models对第一批样本进行建模(184例)，并用第二批样本进行验证(372例)，能准确区分早期口腔鳞癌病例和健康志愿者(图4)，对早期口腔鳞癌诊断效果达到最佳，从而达到对早期口腔鳞癌的精准诊断。
以上所述仅为本发明的优选例实施方式，并不构成对本发明保护范围的限定。任何在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的权利要求保护范围之内。
Claims (6)
1.用于口腔鳞癌早期无创诊断的血清脂质标志物，其特征在于，所述标志物包括精胺酰-甲硫氨酸Arginyl-methionine、甘油磷酰胆碱glycerophosphocholine、丙酰胆碱Propionylcholine三者任意之一或者三者任选其二或者三者共同的组合。
2.如权利要求1所述的用于口腔鳞癌早期无创诊断的血清脂质标志物，其特征在于，所述标志物为精胺酰-甲硫氨酸Arginyl-methionine、甘油磷酰胆碱glycerophosphocholine和丙酰胆碱Propionylcholine三者共同的组合。
3.一种早期无创诊断口腔鳞癌的试剂盒，其特征在于，所述试剂盒包括权利要求1-2任意之一所述的血清脂质标志物。
4.血清脂质标志物在制备早期无创诊断口腔鳞癌的试剂盒中的应用。
5.如权利要求4所述的应用，其特征在于，所述血清脂质标志物下调表达时则表明受试者为口腔鳞癌患者或潜在口腔鳞癌患者。
6.如权利要求4或5所述的应用，其特征在于，所述精胺酰-甲硫氨酸Arginyl-methionine、甘油磷酰胆碱glycerophosphocholine和丙酰胆碱Propionylcholine均下调表达时则表明受试者为口腔鳞癌患者或潜在口腔鳞癌患者。
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