Gene expression profiles of multiple myeloma plasma cell fractions from bone marrow II. Homo sapiens

Today’s diagnostic tests for multiple myeloma (MM) reflect the criteria of the updated WHO classification based on biomarkers and clinicopathologic heterogeneity. To that end, we propose a new subtyping of myeloma plasma cells (PC) by B-cell subset associated gene signatures (BAGS), from the normal B-cell hierarchy in the bone marrow (BM). To do this, we combined FACS and GEP data from normal BM samples to generate classifiers by BAGS for the PreBI, PreBII, immature (Im), naïve (N), memory (M) and PC subsets. The resultant tumor assignments in available clinical datasets exhibited similar BAGS subtype frequencies in four cohorts across 1302 individual cases. The prognostic impact of BAGS was analyzed in patients treated with high dose melphalan as first line therapy in three prospective trials: UAMS, HOVON65/GMMG-HD4 and MRC Myeloma IX with Affymetrix U133 plus 2.0 microarray data available from diagnostic myeloma PC samples. The BAGS subtypes were significantly associated with progression free (PFS) and overall survival (OS) (PFS, P=3.05e−06 and OS, P=1.06e−11) in a meta-analysis of 926 pts. The major impact was observed within the PreBII and M subtypes conferred with significant inferior prognosis compared to the Im, N and PC subtypes. Cox proportional hazard meta-analysis documented that the BAGS subtypes added significant and independent prognostic information to the TC classification system and ISS staging. BAGS subtype analysis identified transcriptome differences and a number of novel differentially spliced genes. We have identified hierarchal subtype differences in the myeloma plasma cells, with prognostic impact. This observation support an acquired reversible B-cell trait and phenotypic plasticity as a hallmark, also in MM. Overall design: Gene expression profiles of multiple myeloma plasma cell fractions from bone marrow were normalized together with B-cell subsets of PreBI, PreBII, Immature, Naive, Memory and Plasma cells obtained using FACS from mononuclear cells isolated from bone marrow of healthy persons.

当前多发性骨髓瘤（multiple myeloma, MM）的诊断检测，均以兼顾生物标志物与临床病理异质性的更新版世界卫生组织（World Health Organization, WHO）分类标准为依据。为此，我们以骨髓（bone marrow, BM）中的正常B细胞谱系为参照，提出了一套基于B细胞亚群相关基因特征（B-cell subset associated gene signatures, BAGS）的骨髓瘤浆细胞（plasma cell, PC）新分型方法。

为构建该分型体系，我们整合了正常骨髓样本的荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）与基因表达谱（gene expression profiling, GEP）数据，针对前BI（PreBI）、前BII（PreBII）、未成熟（Im）、初始（N）、记忆（M）及浆细胞亚群生成了基于BAGS的分类器。

对覆盖1302例个体的4个队列的可用临床数据集分析后发现，各队列的肿瘤BAGS亚型分布频率较为相似。

我们在三项前瞻性临床试验（UAMS、HOVON65/GMMG-HD4及MRC Myeloma IX）中，对接受大剂量美法仑一线治疗的患者开展了BAGS预后影响分析，上述试验的诊断性骨髓瘤浆细胞样本均带有Affymetrix U133 Plus 2.0基因芯片检测数据。

对926例患者的荟萃分析表明，BAGS亚型与无进展生存期（progression-free survival, PFS）及总生存期（overall survival, OS）显著相关（PFS：P=3.05e−06；OS：P=1.06e−11）。其中，前BII与记忆亚型患者的预后显著差于未成熟、初始及浆细胞亚型患者，这是本次分析的核心发现。

Cox比例风险荟萃分析证实，BAGS亚型可为TC分类系统与国际分期系统（International Staging System, ISS）分期提供显著且独立的预后信息。

BAGS亚型分析还揭示了骨髓瘤浆细胞的转录组差异，以及一批全新的差异剪接基因。我们明确了骨髓瘤浆细胞的谱系分型差异及其预后价值，该发现支持“获得性可逆B细胞特性与表型可塑性是多发性骨髓瘤的标志性特征”这一论点。

实验整体设计：对从骨髓分离得到的多发性骨髓瘤浆细胞组分，与从健康人骨髓单核细胞经FACS分选获得的前BI、前BII、未成熟、初始、记忆及浆细胞亚群的基因表达谱进行联合归一化处理。