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

肉瘤分为许多亚型，反映了它们起源的间充质组织的多样性。由于这个原因，尽管肉瘤中包含许多高度恶性的亚型，但肉瘤的病理诊断往往很困难，并且每个亚型都很罕见。另一方面，正确的病理诊断对于肉瘤的临床治疗是必不可少的。最近，在许多肿瘤中发现了染色体相互易位和嵌合基因的形成，这些基因对各个肉瘤亚型具有高度特异性。为了提高肉瘤病理诊断的准确性，我们建立了一种利用DIN芯片技术检测嵌合基因的新方法。从滑膜肉瘤、透明细胞肉瘤和尤文肉瘤中提取RNA，转化为cDNA，用多种嵌合基因的混合引物进行多重PCR扩增，同时将SP 6启动子插入到PCR中。 关于我们 PCR产物经SP 6启动子体外转录产生生物素标记的cRNA，然后将其与macroarray载玻片杂交，macroarray载玻片是一种膜附着的载玻片，其上点有对应于每个嵌合基因外显子的探针。用碱性磷酸酶检测阳性信号，多重PCR-cDNA芯片法在大多数肿瘤中检测到正确的嵌合基因和断裂点。这些结果通过嵌合基因的PCR产物的直接测序得到验证。该方法操作简单，除PCR仪外，不需要特殊设备，适用于常规病理实验室。在诊断方面，该方法使肉瘤的分子病理学诊断独立于组织病理学诊断，提高了肉瘤病理学诊断的准确性。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