Development of cDNA array for diagnosis of sarcoma and its application to routine pathological diagnosis

肉瘤可分为许多亚型，反映了其起源的间充质组织的多样性。因此，肉瘤的病理诊断往往是困难的，尽管肉瘤中包含了许多高度恶性的亚型，但每种亚型都很罕见。另一方面，正确的病理诊断对于肉瘤的适当临床管理是必不可少的。最近，在许多肿瘤中发现了对单个肉瘤亚型具有高度特异性的染色体易位和嵌合基因的形成。为了提高肉瘤病理诊断的准确性，我们采用DIN阵列技术开发了一种新的嵌合基因分子检测方法，分别从滑膜肉瘤、透明细胞肉瘤和Ewing肉瘤中提取RNA，转化为cDNA，用多种嵌合基因的引物混合多重PCR扩增每个嵌合基因，同时，将SP6启动子插入到PCR中，利用SP6启动子进行体外转录，得到更多生物素标记的cRNA。然后将其杂交到macroarray载玻片上，macroarray载玻片是一种附着膜的玻璃载玻片，每个嵌合基因的外显子对应的探针在上面被发现。阳性信号通过碱性磷酸酶显示。通过这种多重PCR-cDNA阵列方法，在大多数肿瘤中检测到正确的嵌合基因和断点。这些结果通过对嵌合基因的PCR产物直接测序得到验证。该方法适用于常规病理实验室，各工序简单，除PCR机外不需特殊设备。所有程序约在3天内完成，生成的macroarray切片可与其他组织病理切片一起保存。在诊断方面，该方法可以独立于组织病理诊断进行分子病理诊断，提高肉瘤病理诊断的准确性。少

Sarcoma is classified into numerous subtypes, reflecting the variety of mesenchymal tissue firm which they are originated. For this reason Pathological diagnosis of sarcoma is often difficult, combined with rarity of each subtype, despite many highly malignant subtypes are comprised in sarcoma On the other hand, correct pathological diagnosis is indispensable far appropriate clinical management of sarcoma Recently, reciprocal chromosomal translocation and formation of chimeric gene, which are highly specific to individual sarcoma subtypes, were found in many tumors. In order to improve the accuracy of pathological diagnosis of sarcoma, we developed a new method for molecular detection of chimeric genes using DIN array technology RNA was extracted from synovial sarcoma, clear cell sarcoma and Ewing sarcoma, followed by conversion to cDNA Each chimeric gene was amplified by multiplex PCR with mixture of primers of various chimeric genes, at the same time, SP6 promoter was inserted to PCR … More product Biotin-labeled cRNA was generated them PCR products by in vitro transcription using SP6 promoter; then it was hybridized to macroarray slides, a membrane-attached glass slides on which the probes corresponding to exons of each chimeric gene were spotted. Positive signals were visualized by alkaline phosphatase.By this multiplex PCR-cDNA array method, correct chimeric genes and breakpoints were detected in most tumors. These results were validated by direct sequencing of PCR products of chimeric genes. Moreover, this method was applicable to routine pathological laboratory, because each procedure was simple and no special equipments was required except for a PCR machine. All the procedures are to be finished in about 3 days and resulted macroarray slides are able to store with other histopathological slides.As for diagnostic aspects, this method might improve accuracy of pathological diagnosis of sarcoma, because it enables molecular pathological diagnosis independent from histopathological diagnosis. Less