Molecular Signatures of Multiple Myeloma Progression through Single Cell RNA-Seq

Multiple myeloma (MM) is a malignant plasma cell disorder with well-defined clonal genetic/cytogenetic abnormalities. However, cellular heterogeneity is a key factor in MM's progression, therapeutic decision, and response to treatment. Single cell whole transcriptome profiling (scRNA-Seq) offers an opportunity to dissect this molecular heterogeneity during MM progression to better understand the disease and guide rational therapy. Here, we examined 597 CD138 positive cells from 15 patients at different stages of MM progression using scRNA-Seq. We selected 790 genes based on a Coefficient of Variation (CV) approach which organized cells into four clusters (L1-L4) based on unsupervised clustering. Plasma cells from each patient contained a mixed population of plasma cells at different state of aggressiveness based on gene expression signature reflecting the inter-cellular heterogeneous nature of MM. Cells in the L1 group is characterized by low level expression of genes involved in the oxidative phosphorylation, Myc targets, and mTORC1 signaling pathway having most cells from MGUS patients (p < 1.2x10-14). In contrast, low level of these genes in L1 group increased progressively and were the highest in the L4 group containing only cells from high-risk MM patients with t(4;14) translocations. Furthermore, 44 genes consistently overexpressed by pair-wised comparisons of the four groups strongly associated with a reduced overall survival in MM patients (APEX trial, p < 0.0001; Hazard Ratio (HR), 1.83; 95% CI, 1.33 to 2.52), particularly those in the bortezomib treated group (p < 0.0001; HR, 2.00; 95% CI, 1.39 to 2.89). No survival significance was observed for the dexamethasone treated group. Our study at the resolution of single cells showed that there is a mixed population of cells in each patient at different stages of MM progression and these cells can be organized into four different subgroups (L1 to L4). Consistent overexpression of the 44 genes from L1 to L4 groups is associated with patient outcome and treatment response. Our results show that oxidative phosphorylation, Myc target, and mTORC1 signaling genes are significant pathways for MM progression and affect MM prognosis and treatment stratification. Overall design: 597 single cell libraries passed QC and were included in the downstream analysis

多发性骨髓瘤（Multiple myeloma, MM）是一种具有明确克隆性遗传/细胞遗传学异常的恶性浆细胞疾病。然而，细胞异质性是影响MM进展、治疗决策及治疗响应的关键因素。单细胞全转录组测序（Single cell whole transcriptome profiling, scRNA-Seq）为解析MM进展过程中的分子异质性提供了契机，有助于更深入地理解该疾病并指导合理治疗。本研究通过单细胞全转录组测序，对15例不同进展阶段MM患者的597个CD138阳性细胞进行了分析。基于变异系数（Coefficient of Variation, CV）方法筛选出790个基因，并通过无监督聚类将细胞划分为4个亚群（L1-L4）。每名患者的浆细胞均呈现出不同侵袭状态的混合群体，这一特征通过基因表达特征得以体现，反映了MM的细胞间异质性本质。L1组细胞的特征为氧化磷酸化、Myc靶基因及mTORC1信号通路相关基因的低表达，该亚群绝大多数细胞来自意义未明的单克隆丙种球蛋白血症（MGUS）患者（p < 1.2×10^-14）。与之相反，L1组中这些基因的低表达水平呈渐进式升高，在仅包含伴t(4;14)易位的高危MM患者细胞的L4组中达到峰值。此外，通过对4个亚群进行两两比较，筛选出的44个持续高表达基因与MM患者较差的总生存期显著相关（APEX临床试验，p < 0.0001；风险比（Hazard Ratio, HR）=1.83；95%置信区间（95% CI）：1.33~2.52），在接受硼替佐米治疗的患者亚组中这一关联尤为显著（p < 0.0001；HR=2.00；95% CI：1.39~2.89）。而在地塞米松治疗组中未观察到此类生存相关显著性。本研究以单细胞分辨率揭示了每名MM患者体内均存在不同进展阶段的混合细胞群体，且这些细胞可被划分为L1至L4共4个不同亚群。L1至L4亚群中44个基因的持续高表达与患者预后及治疗响应密切相关。研究结果表明，氧化磷酸化、Myc靶基因及mTORC1信号通路相关基因是MM进展的关键通路，同时可影响MM的预后及治疗分层。总体实验设计：本研究共纳入597个通过质量控制的单细胞文库用于后续下游分析。