SAFU Unit, IRCCS - Regina Elena National Cancer Institute, via Elio Chianesi 53, 00144, Rome, Italy.
Clinical Trial Center - Biostatistics & Bioinformatics, IRCCS - Regina Elena National Cancer Institute, Rome, Italy.
J Exp Clin Cancer Res. 2022 Apr 21;41(1):151. doi: 10.1186/s13046-022-02359-5.
Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC).
The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades.
We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen.
Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.
癌症死亡率主要是由器官衰竭和血栓事件引起的。已经证明，中性粒细胞实施的染色质释放机制 NETosis 可能导致这些致命的全身效应。我们的目的是研究子宫内膜癌 (EC) 中的 NETosis 生物标志物。
该实验在 21 名没有妇科疾病的健康受试者 (HS) 和 63 名 EC 患者中进行。为了评估 NETosis 特征的存在，使用针对瓜氨酸化组蛋白 H3（citH3）、中性粒细胞弹性蛋白酶 (NE) 和组蛋白 2B 的抗体进行 IHC 和 IF。使用来自失活血清的一微升通过 qPCR 测量细胞游离 DNA (cfDNA)、细胞游离线粒体 DNA (cfmtDNA) 和 citH3 的血清水平，并分别通过 ELISA 测定。使用 Agilent 2100 Bioanalyzer 和 High Sensitivity DNA Chips 分析血清 cfDNA 的片段化模式。使用接收器操作特性 (ROC) 分析确定能够区分 EC 和 HS 的 cfDNA 和 cfmtDNA 值的截止值。使用相关分析和多元对应分析 (MCA) 分析 cfDNA、mtcfDNA、citH3 和血液参数之间的关系，以确定 EC 分级中血清参数之间的潜在关联。
我们证明了所有 EC 分级组织中都存在 NETosis 特征。血清 cfDNA 和 cfmtDNA 水平可区分 EC 和 HS，并且在 EC 血清中观察到 citH3 与 cfDNA 含量之间的直接相关性和 cfmtDNA 与 citH3 之间的负相关性，而在 HS 中则没有检测到。MCA 表示 cfDNA、cfmtDNA 和 citH3 是与 G1 和 G2 分级相关的特征。发现 cfDNA、citH3 和炎症特征的水平升高之间存在相关性。最后，EC 血清中的核小体 cfDNA 片段化模式发生变化，并且与 cfDNA、citH3、淋巴细胞和纤维蛋白原水平升高相关。
我们的数据强调了 NETosis 在 EC 中的发生，并表明血清 cfDNA 和 citH3 是子宫内膜癌中肿瘤诱导的全身效应的非侵入性生物标志物。
