Block Timothy, Zezulinski Daniel, Kaplan David E, Lu Jingqiao, Zanine Samantha, Zhan Tingting, Doria Cataldo, Sayeed Aejaz
Department of Translational Medicine, Baruch S. Blumberg Institute, Doylestown, PA, United States.
Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman School of Medicine and The Corporal Michael J. Crescenz Veterans Administration Hospital, Philadelphia, PA, United States.
Front Oncol. 2022 Dec 13;12:963641. doi: 10.3389/fonc.2022.963641. eCollection 2022.
Liver derived messenger ribonucleic acid (mRNA) transcripts were reported to be elevated in the circulation of hepatocellular carcinoma (HCC) patients. We now report the detection of high-risk mRNA variants exclusively in the circulation of HCC patients. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Our findings potentially offer a novel non-invasive platform for HCC surveillance and early detection.
RNAseq analysis was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. Specimens from two cholangiocarcinoma (CCA) patients were also included in our study. HCC specific SNPs and Indels referred as "variants" were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants.
The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. RNAseq analyses in tumors, normal liver tissue, plasma, and plasma derived EVs led to the detection of 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none of these were detected in normal liver, plasma of LC patients or normal healthy individuals. Our results demonstrate selective detection of concordant high-risk HCC-specific mRNA variants in free plasma, plasma derived EVs and tumors of HCC patients. The variants comprise of splicing, frameshift, fusion and single nucleotide alterations and correspond to cancer and tumor metabolism pathways. Detection of these high-risk variants in matching specimens from same subjects with an enrichment in circulating EVs is remarkable. Validation of these HCC selective ctmRNA variants in larger patient cohorts is likely to identify a predictive set of ctmRNA with high diagnostic performance and thus offer a novel non-invasive serology-based biomarker for HCC.
据报道，肝细胞癌（HCC）患者循环中的肝脏来源信使核糖核酸（mRNA）转录物水平升高。我们现在报告在HCC患者循环中仅检测到高风险mRNA变异体。许多基因组等位基因，如单核苷酸多态性（SNP）、核苷酸插入和缺失（称为Indel）以及许多基因中的剪接变异体，都与癌症风险升高有关。我们的发现可能为HCC监测和早期检测提供一个新的非侵入性平台。
对14例诊断为HCC的个体、8例患有肝硬化（LC）但无HCC的个体以及6例未诊断出肝病的个体的血浆进行RNA测序分析。还分析了来自14例HCC患者的6个匹配肿瘤和5个循环细胞外囊泡（EV）样本的RNA。我们的研究还纳入了两名胆管癌（CCA）患者的样本。使用GATK HaplotypeCaller识别被称为“变异体”的HCC特异性SNP和Indel，并通过SnpEff进行注释以筛选出高风险变异体。
对所有RNA样本进行变异体检测，共检测到520万个SNP、91万个插入和81万个缺失。对肿瘤、正常肝组织、血浆和血浆来源的EV进行RNA测序分析，在HCC患者循环中检测到5480个高风险肿瘤特异性mRNA变异体。这些变异体在HCC患者的肿瘤和血浆样本或肿瘤和EV中同时被检测到，但在正常肝脏、LC患者血浆或正常健康个体中均未检测到。我们的结果表明，在HCC患者的游离血浆、血浆来源的EV和肿瘤中可选择性检测到一致的高风险HCC特异性mRNA变异体。这些变异体包括剪接、移码、融合和单核苷酸改变，并且与癌症和肿瘤代谢途径相对应。在来自同一受试者的匹配样本中检测到这些高风险变异体，且在循环EV中有所富集，这很值得关注。在更大的患者队列中验证这些HCC选择性循环肿瘤mRNA（ctmRNA）变异体，可能会识别出一组具有高诊断性能的预测性ctmRNA，从而为HCC提供一种基于血清学的新型非侵入性生物标志物。
