Whole Genome Characterization of Circulating Tumor Cells Identifies Novel Prognostic Genomic Alterations in Melanoma Metastasis. Homo sapiens

Circulating tumor cells (CTCs) are critical in the development of distant organ tumor metastasis, and are associated with advanced cancer stage and poor patient outcome. Here, we present the first genome-wide nucleotide-level characterization of CTCs. Our single-nucleotide polymorphism (SNP) analysis in patients with melanoma involved: 1) global comparative genomic analysis of CTCs and matched regional metastases, 2) identification of key genomic aberrations in CTCs, 3) verification of these target genes in aggressive distant tumor metastases, and 4) evidence of selective expression and functional consequence of CTC-associated genes in melanomas. We report 131 aberrant loci in CTCs that are potentially pro-metastatic, and show that such expression of a 5-marker gene panel (CSMD2, CNTNAP5, FLJ14051, ADAM6, TRPM2) in melanomas confers prognostic utility. Successful treatment of melanoma requires understanding of the metastatic process and identification of patients with tumors most likely to develop aggressive metastatic disease. Melanomas are heterogeneous, and CTCs have long been recognized as vehicles for cancer spread, representing particularly aggressive tumor clones that can evolve into successful clinical metastases. Elucidation of genomic aberrations in CTCs will aid in the development of prognostic biomarkers and therapeutic strategies to target CTCs to prevent or control distant cancer spread. This study provides the first detailed genomic confirmation of the close relation between CTCs and tumor metastases, and illustrates how CTCs can be utilized as a novel approach and rational source for identification of pro-metastatic genes in cancer research. Overall design: Three individual patient cohorts were utilized in the study. CNV and LOH loci were evaluated initially in metastatic melanoma patients (n=13) in a discovery cohort. SNP loci that harbored CNV/LOH in CTCs were then separately verified for: (a) presence in distant organ metastasis (AJCC Stage IV melanoma) (n=27), and (b) relevance to prognosis in regional melanoma metastasis (AJCC Stage III melanoma) (n=35). The first discovery patient cohort group was utilized for capture of CTCs, and consisted of peripheral blood mononuclear cell (PBMC) and tumor specimens from metastatic melanoma patients (n=13). CTC-related loci were verified in a second cohort of patients with Stage IV distant organ metastases (n=27), including 15 brain, 4 lung, and 8 gastrointestinal (bowel, liver) metastases. The third patient cohort consisted of early passage (<12) established melanoma cell lines derived from 35 regional melanoma metastases (Stage III) for evaluation of the prognostic utility of the CTC-associated aberrant loci.

循环肿瘤细胞（Circulating Tumor Cells, CTCs）在远处器官肿瘤转移的发生过程中发挥关键作用，且与癌症晚期阶段及患者不良预后密切相关。本研究首次完成了循环肿瘤细胞全基因组核苷酸级别的特征分析。针对黑色素瘤患者开展的单核苷酸多态性（Single-Nucleotide Polymorphism, SNP）分析包含以下内容：1）循环肿瘤细胞与配对区域转移灶的全基因组比较分析；2）循环肿瘤细胞中关键基因组畸变的鉴定；3）在侵袭性远处肿瘤转移灶中对上述靶基因进行验证；4）证实黑色素瘤中循环肿瘤细胞相关基因的选择性表达及其功能影响。本研究鉴定出循环肿瘤细胞中131个潜在促转移的畸变基因座，并证实黑色素瘤中5个标志物基因组合（CSMD2、CNTNAP5、FLJ14051、ADAM6、TRPM2）的表达具有预后价值。黑色素瘤的有效治疗需要深入理解转移过程，并甄别出最可能发生侵袭性转移的患者群体。黑色素瘤具有异质性，而循环肿瘤细胞长期以来被视为癌症扩散的载体，其代表了具有高度侵袭性的肿瘤克隆，可进一步发展为临床可见的转移灶。解析循环肿瘤细胞中的基因组畸变，将有助于开发预后生物标志物及靶向循环肿瘤细胞的治疗策略，从而预防或控制癌症的远处扩散。本研究首次通过基因组学手段详细证实了循环肿瘤细胞与肿瘤转移之间的紧密关联，并阐明了循环肿瘤细胞可作为癌症研究中鉴定促转移基因的全新方法与可靠来源。整体实验设计：本研究共纳入3个独立患者队列。首先在发现队列（n=13）的转移性黑色素瘤患者中评估拷贝数变异（Copy Number Variation, CNV）与杂合性缺失（Loss of Heterozygosity, LOH）基因座。随后，针对循环肿瘤细胞中携带拷贝数变异/杂合性缺失的单核苷酸多态性基因座，分别从两方面进行验证：(a) 在远处器官转移灶（AJCC IV期黑色素瘤，n=27）中确认其存在；(b) 在区域黑色素瘤转移（AJCC III期黑色素瘤，n=35）中验证其与预后的相关性。首个发现队列用于循环肿瘤细胞的捕获，其样本来自13例转移性黑色素瘤患者的外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）与肿瘤组织标本。在第二个队列（n=27，IV期远处器官转移患者）中验证循环肿瘤细胞相关基因座，该队列包含15例脑转移、4例肺转移及8例胃肠道（肠道、肝脏）转移患者。第三个患者队列包含35株来自III期区域黑色素瘤转移灶的早期传代（<12代）黑色素瘤细胞系，用于评估循环肿瘤细胞相关畸变基因座的预后价值。