Gene expression data from melanoma cell lines and melanocyte controls. Homo sapiens

The two most common melanoma histopathologic subtypes, superficial spreading (SSM) and nodular melanoma (NM), are believed to represent sequential phases of linear progression from radial to vertical growth. Studies suggest, however, that SSM and NM are biologically distinct. We utilized an integrative genomic approach to examine the possibility that SSM and NM are the result of independent pathways characterized by unique molecular alterations. Cell lines including SSM, NM, metastatic melanoma, and melanocyte controls were evaluated for copy number changes and differential mRNA expression using single nucleotide polymorphism array (SNP 6.0, Affymetrix) and gene array (U133A 2.0, Affymetrix). Data sets were integrated to identify copy number alterations that correlated with gene expression, and array results were validated using immunohistochemistry on human tissue microarrays (TMAs) and an external data set. The functional effect of genomic deletion was assessed by lentiviral overexpression. Integrative genomics revealed 8 genes in which NM/SSM-specific copy number alterations were correlated with NM/SSM differential gene expression (P<0.05, Spearman’s rank). Pathways analysis of differentially expressed genes (N=114) showed enrichment for metabolic-related processes. SSM-specific genomic deletions (DIS3, MTAP, G3BP2, SEC23IP, USO1) were verified in an expanded panel of cell lines, and forced overexpression of MTAP in SSM resulted in reduced cell growth. Metabolism-related gene ALDH7A1 was verified as overexpressed in NM using human TMAs.The identification of recurrent genomic deletions in SSM not present in NM challenges the linear model of melanoma progression and supports the unique molecular classification of SSM and NM. Overall design: Gene expression profiling using Affymetrix U133A 2.0 arrays was performed on 18 melanoma cell lines including 2 primary superficial spreading melanoma, 2 primary nodular melanoma, 2 metastatic nodular melanoma, and 12 metastatic cell lines. Four melanocyte control lines were also evaluated including 2 immortalized melanocyte cell lines (Hermes 1 and 2B) and 2 normal melanocyte lines cultured from neonatal foreskin (HEM-N and HEM-LP).

两种最常见的黑色素瘤组织病理亚型——浅表播散型黑色素瘤（superficial spreading melanoma, SSM）与结节性黑色素瘤（nodular melanoma, NM），曾被认为是从放射状生长到垂直生长的线性进展连续阶段。不过诸多研究表明，SSM与NM在生物学层面存在显著差异。本研究采用整合基因组学方法，探究SSM与NM是否由以独特分子改变为特征的独立通路所导致。

研究使用单核苷酸多态性阵列（SNP 6.0，Affymetrix）与基因表达阵列（U133A 2.0，Affymetrix），对涵盖SSM、NM、转移性黑色素瘤细胞系及黑素细胞对照在内的样本开展拷贝数变异与差异mRNA表达分析。通过整合多组数据集，筛选出与基因表达水平相关的拷贝数改变，并利用人类组织微阵列（tissue microarrays, TMAs）及外部数据集通过免疫组化对芯片结果进行验证。通过慢病毒过表达实验，评估基因组缺失的功能效应。

整合基因组学分析显示，共8个基因的NM/SSM特异性拷贝数改变与对应的NM/SSM差异基因表达显著相关（P<0.05，斯皮尔曼秩相关分析）。对114个差异表达基因进行通路富集分析，结果显示其显著富集于代谢相关生物学过程。研究在扩大的细胞系面板中验证了SSM特异性基因组缺失（DIS3、MTAP、G3BP2、SEC23IP、USO1），且在SSM细胞中强制过表达MTAP可显著抑制细胞增殖。通过人类TMAs验证，代谢相关基因ALDH7A1在NM中呈过表达状态。

本研究在SSM中发现的复发性基因组缺失（且NM中未检测到该类缺失），对黑色素瘤进展的线性模型提出了挑战，并支持SSM与NM具有独特分子分类的观点。

整体实验设计：采用Affymetrix U133A 2.0芯片对18株黑色素瘤细胞系进行基因表达谱分析，其中包括2株原发性浅表播散型黑色素瘤细胞系、2株原发性结节性黑色素瘤细胞系、2株转移性结节性黑色素瘤细胞系及12株转移性细胞系；同时还检测了4株黑素细胞对照细胞系，包括2株永生化黑素细胞系（Hermes 1与2B）及2株从新生儿包皮培养得到的正常黑素细胞系（HEM-N与HEM-LP）。