基于单细胞RNA-seq技术寻找t(8;21)白血病预后相关基因及其临床应用研究

Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy. The most common chromosomal translocation of AML is t(8;21)(q22;q22), generating the AML1-ETO fusion protein. It is reported that 60% t(8;21) AML patients have a better prognosis, yet 40% patients have a poor prognosis. The study of prognostic genes of t(8;21) AML remains unclear. As single-cell RNA-seq technique has an advantage in studying heterogeneity, we chose three untreated or relapse t(8;21) AML patients, and established single-cell RNA-Seq profiles of 193 single-cells from the peripheral blood or bone marrow of these patients. We aim to screen subclones belonged to different prognosis group by unsupervised clustering analysis and obtain approximately 20 genes that are most significantly related to prognosis by principal component analysis. By combining the treatment and prognosis information of these patients as well as the GO (gene ontology) database information, we can obtain the unfavorable prognostic genes that are significantly upregulated in malignant subclone. After verifying in 150 clinical samples by real-time quantitative PCR, we aim to determine 3 to 5 most significantly unfavorable prognostic genes. By comparing the expression profile of subclones that belonged to different stages in disease, we can also study the molecular mechanism of relapsed t(8;21) AML. Eventually, we can provide the prognostic stratification indicators and potential therapeutic targets for AML.

AML是高度异质性的恶性肿瘤，其中t(8;21)(q22;q22)为最常见的染色体易位，形成 AML1-ETO融合蛋白。临床发现，约60% t(8;21) AML患者预后好，但40%患者预后很差。目前该疾病的预后相关基因研究尚不清楚，而单细胞RNA-seq技术在研究疾病异质性方面有很大优势。因此本项目选取3位 t(8;21)AML初治和复发患者，应用单细胞RNA-seq技术对其外周血或骨髓中共193例单细胞进行测序。我们拟应用无监督聚类分析得到预后不同的亚克隆；应用主成分分析等获得预后相关的约20个差异最大基因；结合患者和GO数据库信息选出恶性亚克隆中显著上调的预后不良基因，在150例临床样本中进定量PCR验证，最终确定3-5个t(8,21)AML预后不良显著相关基因，并通过比较不同疾病阶段的亚克隆基因表达谱来揭示t(8,21)AML复发的分子机制，为其预后分层和潜在基因治疗靶点提供依据。

AML是高度异质性的恶性肿瘤，其中t(8;21)(q22;q22)为最常见的染色体易位，形成 AML1-ETO融合蛋白。临床发现，约60% t(8;21) AML患者预后好，但40%患者预后很差。目前该疾病的预后相关基因研究尚不清楚，而单细胞RNA-seq技术在研究疾病异质性方面有很大优势。因此本项目选取3位 t(8;21)AML初治和复发患者，应用单细胞RNA-seq技术对其外周血或骨髓中共193例单细胞进行测序。我们拟应用无监督聚类分析得到预后不同的亚克隆；应用主成分分析等获得预后相关的约50个差异最大基因，基于原始读段数量的SC3聚类结果也显示6个细胞亚群的区分度较高，且鉴定出了2138个差异表达基因。图1C列出了表达差异最显著的50个基因，其中有免疫相关基因（HLA-DRA, HLA-DRB1, HLA-E, 和NCAM1）和DNA甲基化相关基因（IDH2）；结合患者和GO数据库信息选出恶性亚克隆中显著上调的预后不良基因，在生物学过程部分，ZGG的差异表达基因主要参与细胞周期或RNA催化过程。在ZGG_3 vs ZGG_2比较组中表达上调的基因参与RNA转运、剪切体和细胞周期等信号通路。在150例临床样本中进定量PCR验证，最终确定3-5个t(8,21)AML预后不良显著相关基因，并通过比较不同疾病阶段的亚克隆基因表达谱来揭示t(8,21)AML复发的分子机制，为其预后分层和潜在基因治疗靶点提供依据。