Array comparative genomic hybridization (aCGH) study of formalin-fixed paraffin-embedded (FFPE) small gastrointestinal stromal tumors (GISTs) less than 1 cm in greatest diameter (microGISTs)

To investigate the cytogenetic and large-scale chromosomal changes in involuted or non-involuted microGISTs using post-whole genome amplification (WGA) FFPE DNA materials Sixteen patients, total 19 FFPE tumor samples (block storage time 4 months to 9 years), including 16 microGISTs and 3 GISTs larger than 1 cm from the same patients harboring microGISTs. All FFPE tumor samples underwent DNA extraction and WGA (modified degenerate oligonucleotide PCR (DOP) method, provided by Sigma). For each tumor sample, a post-WGA DNA extract from the normal tissue in the same block (or block from the same patient with a block storage time differences less than 2 years) was obtained for tumor sample DNA co-hybridization. Tumor and normal areas of interest were marked and collected from 5- to 10-micron unstained or hematoxylin-stained sections by manual or laser (PixCell IITM, Arcturus Bioscience, CA, USA) microdissection. DNAs were then extracted. WGA was performed using GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA; http://www.sigmaaldrich.com/) in parallel in accordance with the manufacturer's protocols. At least four independent experiments were concurrently performed per template amplification. Four separate WGA reaction products were pooled for each sample.

本研究旨在借助全基因组扩增（Whole Genome Amplification, WGA）后的福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）DNA样本，探究退缩性与非退缩性微型胃肠道间质瘤（microGISTs）的细胞遗传学特征及大规模染色体变异情况。本研究纳入16名患者，共计19份FFPE肿瘤样本，样本包埋块的存储时长为4个月至9年，其中包含16份微型胃肠道间质瘤样本，以及来自同一名携带微型胃肠道间质瘤患者的3份直径大于1cm的胃肠道间质瘤（GIST）样本。所有FFPE肿瘤样本均进行DNA提取与全基因组扩增，扩增采用Sigma公司提供的改良简并寡核苷酸引物PCR（Degenerate Oligonucleotide Primed PCR, DOP）方法。针对每份肿瘤样本，获取同一块包埋物中正常组织的全基因组扩增后DNA提取物，或来自同一患者、包埋块存储时长差异不超过2年的其他包埋块的正常组织提取物，用于肿瘤样本DNA的共杂交实验。研究人员通过手工或激光显微切割技术（PixCell II™，美国加利福尼亚州Arcturus Bioscience公司），在5至10微米厚的未染色或苏木精染色的切片上标记并收集目标肿瘤与正常组织区域，随后提取上述区域的DNA。本研究同时采用GenomePlex®组织全基因组扩增WGA5试剂盒（Sigma公司，美国密苏里州圣路易斯市；官网：http://www.sigmaaldrich.com/）进行全基因组扩增，操作严格遵循试剂盒制造商的实验方案。每份模板的扩增实验至少并行开展四次独立重复，随后将同一样本的四份独立全基因组扩增反应产物进行混合。