Czyż Zbigniew T, Hoffmann Martin, Schlimok Günter, Polzer Bernhard, Klein Christoph A
Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
PLoS One. 2014 Jan 21;9(1):e85907. doi: 10.1371/journal.pone.0085907. eCollection 2014.
Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape.
METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points.
CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.
播散癌细胞（DCCs）和循环肿瘤细胞（CTCs）极其罕见，但却是远处转移或治疗抗性细胞的前体细胞。对这些细胞进行详细的分子分析可能有助于识别癌细胞播散、转移灶形成及全身治疗逃逸的关键事件。
方法/主要发现：我们使用Ampli1™全基因组扩增（WGA）技术和高分辨率寡核苷酸aCGH微阵列优化了结构拷贝数变化分析的条件。本文介绍的方案能够可靠地检测单个细胞中低至0.1 Mb的数字基因组改变。对特征明确的细胞系中的单个细胞和单个正常细胞进行分析，证实了扩增和杂交方案严格的定量性质。重要的是，用于检测DCCs的固定和染色程序对分析结果没有显著影响，证明了我们方法的临床实用性。在一项原理验证研究中，我们通过在四个不同时间点分离和分析一名乳腺癌患者的DCCs，追踪了单个DCCs在高剂量化疗全过程中的染色体变化。
结论/意义：该方案能够对DCCs进行详细的基因组分析，从而评估疾病自然进程中和选择压力下的克隆进化。一名典型患者的结果表明，在选择性治疗条件下存活的DCCs可能来自基因组较不先进的细胞群体，这些细胞表现出特定基因组改变的稳定子集。
