Zezulinski Daniel, Hoteit Maarouf A, Kaplan David E, Simeone Angela, Zhan Tingting, Doria Cataldo, Ahmed Fowsiyo Y, Roberts Lewis R, Block Timothy M, Sayeed Aejaz
Baruch S. Blumberg Institute, Doylestown, PA, USA.
Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Liver Cancer. 2025 Apr 4:1-32. doi: 10.1159/000545366.
Mutations in circulating nucleic acids can be used as biomarkers for the early detection and management of hepatocellular carcinoma (HCC). However, while circulating tumor DNA and microRNA have been extensively explored, circulating tumor mRNA and circulating mRNA mutants (ctmutRNA), which may provide advantages over other analytes, remain less well described. We previously reported the identification of 288 HCC selective ctmutRNA variants, called "candidates," from a small cohort of HCC patients using total RNAseq. The objective of the current study was to use targeted RNAseq to validate the specificity and sensitivity of these HCC selective variants in an independent cohort of patients with liver cirrhosis (LC).
Several methods to isolate small extracellular vesicles and amplify mRNA from the circulation were compared. RNA was isolated, and the primers and probes selective for the 288 regions of interest were used with RNA from HCC ( = 50) and LC and no HCC ( = 35) patients. HCC tumor tissues ( = 11), a normal liver tissue and 3 cell lines were also studied. cDNA synthesis was followed by library construction using QIAseq RNA Fusion XP panel. QC analysis was carried out with an Agilent Bioanalyzer before sequencing on a NextSeq 550 instrument. A GATK HaplotypeCaller was used for variant calling and annotation carried out using snpEff.
Among the test panel of 288 ctmutRNA candidates in the original cohort, 75 were detected in the new cohort of plasma samples. Moreover, 388 other variants in proximity to the original lesions were also found in multiple HCC but not LC plasma samples. A subset of 36 HCC selective variants was able to identify all HCC patients. The most common tumor specific variants were Indels and SNPs. Novel mRNA fusion variants, corresponding to SENP7, HYI, SAR1A, RASA2, TUBA transcripts, etc., were identified in HCC and LC patients.
Circulating RNA could be a robust analyte for noninvasive early detection of HCC and circulating RNA panels could be powerful tools in the entire spectrum of clinical management.
循环核酸中的突变可作为肝细胞癌（HCC）早期检测和管理的生物标志物。然而，尽管循环肿瘤DNA和微小RNA已得到广泛研究，但循环肿瘤mRNA和循环mRNA突变体（ctmutRNA）可能比其他分析物具有优势，目前对其描述较少。我们之前报道了通过全RNA测序从一小群HCC患者中鉴定出288个HCC选择性ctmutRNA变体，称为“候选物”。本研究的目的是使用靶向RNA测序在一个独立的肝硬化（LC）患者队列中验证这些HCC选择性变体的特异性和敏感性。
比较了几种从小细胞外囊泡中分离并从循环中扩增mRNA的方法。分离RNA，并将针对288个感兴趣区域的引物和探针与来自HCC患者（n = 50）、LC且无HCC患者（n = 35）的RNA一起使用。还研究了HCC肿瘤组织（n = 11）、正常肝组织和3种细胞系。使用QIAseq RNA Fusion XP面板进行cDNA合成，随后构建文库。在NextSeq 550仪器上测序之前，使用安捷伦生物分析仪进行质量控制分析。使用GATK HaplotypeCaller进行变异检测，并使用snpEff进行注释。
在原始队列的288个ctmutRNA候选物测试组中，在新的血浆样本队列中检测到75个。此外，在多个HCC但非LC血浆样本中还发现了靠近原始病变的388个其他变体。36个HCC选择性变体的一个子集能够识别所有HCC患者。最常见的肿瘤特异性变体是插入缺失和单核苷酸多态性。在HCC和LC患者中鉴定出了与SENP7、HYI、SAR1A、RASA2、TUBA转录本等相对应的新型mRNA融合变体。
循环RNA可能是用于HCC无创早期检测的强大分析物，循环RNA检测组可能是整个临床管理领域的有力工具。
