Objective: To screen and perform preliminary clinical validation of biomarkers of activity based on positron emission tomography/computed tomography (PET-CT) and transcriptomics in sputum-negative pulmonary tuberculosis lesion tissue. Methods: Nine patients with sputum-negative pulmonary tuberculosis treated surgically at the Shanghai Public Health Clinical Center for Thoracic Surgery from January 1, 2017 to December 31, 2019 were retrospectively collected as the discovery group, including four males and five females, aged 20-57 years (mean 36 years). All of the patients underwent PET-CT scanning before surgery, and the resected specimens were postoperatively classified according to preoperative PET-CT. The resected specimens were divided into areas with increased fluorodeoxyglucose (FDG) metabolism (SUVmax>3) and areas with normal FDG metabolism (SUVmax ≤ 3) according to the preoperative PET-CT performance. After sample processing, total RNA was extracted from the tissues of different regions, and then whole gene transcriptome sequencing was performed. Bioinformatics analysis of the two sets of data was performed to discover the expression profiles of the differences in whole gene transcriptome data between the two regions and to screen for candidate biomarkers. Eighty patients with sputum-negative pulmonary tuberculosis admitted to Shanghai Public Health Clinical Center from January 1, 2019 to January 1, 2021 were retrospectively collected as the validation group, including 37 males and 43 females, aged 20-62 years, with an average age of 39 years. The validation group was divided into a group with increased SUV (n=40) and a group without lesions on CT imaging (n=40). Enzyme-linked immunosorbent assay (ELISA) was used to determine the protein levels of candidate biomarkers in the peripheral plasma of patients. The effect of biomarkers was assessed using subject operating characteristic (ROC) curves. Student's t-test was used to determine whether the difference in protein levels between the two groups was statistically significant. Results: Bioinformatics analysis revealed that the expression levels of C1QB, CCL19, CCL5 and HLA-DMB correlated with the metabolic activity of sputum-negative tuberculosis lesion tissue. Further screening and validation by the validation group confirmed that the difference in C1QB protein levels in the peripheral plasma of patients was statistically significant between the group with increased SUV and the group without lesions on CT imaging [(3.55±0.34) mg/L vs. (2.75±0.21) mg/L, t=4.12, P<0.001]. And the ROC curve showed that the area under the curve for C1QB protein levels was 0.731, which had potential clinical value. Conclusion: The C1QB protein level can be used to assess the activity of lesions in patients with sputum-negative tuberculosis and is a potential biomarker.
目的： 基于正电子发射断层显像（PET-CT）和转录组学对痰菌阴性肺结核病变组织的活动性生物标志物筛选并进行初步临床验证。 方法： 回顾性收集2017年1月1日至2019年12月31日在上海市公共卫生临床中心胸外科手术治疗的9例痰菌阴性肺结核患者作为发现组，其中男4例，女5例，年龄20~57岁，平均36岁，术前均行PET-CT扫描，术后将切除的标本按照术前PET-CT表现分为氟代脱氧葡萄糖（FDG）代谢增高区域［标准摄取值（SUV）max>3］和FDG代谢基本正常区域（SUVmax≤3）。样本处理后先对不同区域组织进行总RNA的提取，然后行全基因转录组测序。对两组数据进行生物信息学分析，发现两个区域全基因转录组数据差异的表达谱，并筛选出候选生物标志物。回顾性收集2019年1月1日至2021年1月1日上海市公共卫生临床中心收治的80例痰菌阴性肺结核患者作为验证组，其中男37例，女43例，年龄20~62岁，平均39岁，分为SUV增高组（40例）和CT影像无病灶组（40例），采用酶联免疫吸附试验（ELISA）测定患者外周血浆中候选生物标志物的蛋白水平。利用受试者工作特征（ROC）曲线对生物标志物进行效果评估。使用student t检验来判断两组之间蛋白水平差异是否具有统计学意义。 结果： 生物信息学分析发现，C1QB、CCL19、CCL5和HLA-DMB的表达水平与痰菌阴性肺结核病变组织的代谢活性相关。通过验证组进一步筛选验证，证实SUV增高组患者外周血浆中C1QB蛋白水平高于CT影像无病灶组，差异有统计学意义［（3.55±0.34）mg/L比（2.75±0.21）mg/L，t=4.12，P<0.001］。ROC曲线显示，C1QB蛋白水平的曲线下面积为0.731，具有潜在的临床价值。 结论： C1QB蛋白水平可用于评估痰菌阴性肺结核患者病变的活动性，是一种潜在的生物标志物。.