Decruyenaere Philippe, Giuili Edoardo, Verniers Kimberly, Anckaert Jasper, De Grove Katrien, Van der Linden Malaïka, Deeren Dries, Van Dorpe Jo, Offner Fritz, Vandesompele Jo
Department of Hematology, Ghent University Hospital, Ghent, Belgium.
OncoRNALab, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium.
Front Oncol. 2023 Oct 25;13:1221471. doi: 10.3389/fonc.2023.1221471. eCollection 2023.
Diffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) are aggressive histological subtypes of non-Hodgkin's lymphoma. Improved understanding of the underlying molecular pathogenesis has led to new classification and risk stratification tools, including the development of cell-free biomarkers through liquid biopsies. The goal of this study was to investigate cell-free RNA (cfRNA) biomarkers in DLBCL and PMBCL patients.
Blood plasma samples (n=168) and matched diagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples (n=69) of DLBCL patients, PMBCL patients and healthy controls were collected between 2016-2021. Plasma samples were collected at diagnosis, at interim evaluation, after treatment, and in case of refractory or relapsed disease. RNA was extracted from 200 µl plasma using the miRNeasy serum/plasma kit and from FFPE tissue using the miRNeasy FFPE kit. RNA was subsequently sequenced on a NovaSeq 6000 instrument using the SMARTer Stranded Total RNA-seq pico v3 library preparation kit.
Higher cfRNA concentrations were demonstrated in lymphoma patients compared to healthy controls. A large number of differentially abundant genes were identified between the cell-free transcriptomes of DLBCL patients, PMBCL patients, and healthy controls. Overlap analyses with matched FFPE samples showed that blood plasma has a unique transcriptomic profile that significantly differs from that of the tumor tissue. As a good concordance between tissue-derived gene expression and the immunohistochemistry Hans algorithm for cell-of-origin (COO) classification was demonstrated in the FFPE samples, but not in the plasma samples, a 64-gene cfRNA classifier was developed that can accurately determine COO in plasma. High plasma levels of a 9-gene signature (, , , , , , , , pseudogene) and a 5-gene signature (, , , , ) were significantly associated with inferior progression-free and overall survival in DLBCL patients, respectively, independent of the NCCN-IPI score.
Total RNA sequencing of blood plasma samples allows the analysis of the cell-free transcriptome in DLBCL and PMBCL patients and demonstrates its unexplored potential in identifying diagnostic, cell-of-origin, and prognostic cfRNA biomarkers.
弥漫性大B细胞淋巴瘤（DLBCL）和原发性纵隔B细胞淋巴瘤（PMBCL）是非霍奇金淋巴瘤的侵袭性组织学亚型。对潜在分子发病机制的深入了解催生了新的分类和风险分层工具，包括通过液体活检开发无细胞生物标志物。本研究的目的是调查DLBCL和PMBCL患者的无细胞RNA（cfRNA）生物标志物。
在2016年至2021年期间收集了DLBCL患者、PMBCL患者和健康对照者的血浆样本（n = 168）以及匹配的诊断性福尔马林固定石蜡包埋（FFPE）组织样本（n = 69）。血浆样本在诊断时、中期评估时、治疗后以及难治性或复发性疾病时采集。使用miRNeasy血清/血浆试剂盒从200μl血浆中提取RNA，使用miRNeasy FFPE试剂盒从FFPE组织中提取RNA。随后使用SMARTer Stranded Total RNA-seq pico v3文库制备试剂盒在NovaSeq 6000仪器上对RNA进行测序。
与健康对照相比，淋巴瘤患者的cfRNA浓度更高。在DLBCL患者、PMBCL患者和健康对照者的无细胞转录组之间鉴定出大量差异丰富的基因。与匹配的FFPE样本进行的重叠分析表明，血浆具有独特的转录组特征，与肿瘤组织的转录组特征有显著差异。由于在FFPE样本中证明了组织来源的基因表达与免疫组化汉斯细胞起源（COO）分类算法之间具有良好的一致性，但在血浆样本中未得到证明，因此开发了一种64基因的cfRNA分类器，该分类器可以准确确定血浆中的COO。DLBCL患者血浆中9基因特征（，，，，，，，，假基因）和5基因特征（，，，，）的高表达水平分别与较差的无进展生存期和总生存期显著相关，独立于NCCN-IPI评分。
血浆样本的全RNA测序能够分析DLBCL和PMBCL患者的无细胞转录组，并证明其在识别诊断、细胞起源和预后cfRNA生物标志物方面具有尚未探索的潜力。
