Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, differential diagnosis can be difficult. Complementing immune-histological diagnosis with current ancillary diagnostic techniques, including FISH and RT-PCR, can lead to inconclusive results in a significant number of cases. We describe here the design and validation of a novel sequencing tool to improve sarcoma diagnosis. A NGS DNA capture panel containing probes for 87 fusion genes and 7 genes with frequent copy number changes was designed and optimized. A cohort of 113 DNA samples extracted from soft-tissue and bone sarcoma FFPE material with clinical FISH and/or RT-PCR results positive for either a translocation or gene amplification was used for validation of the NGS method. Sarcoma-specific translocations or gene amplifications were confirmed in 110 out of 113 cases using FISH and/or RT-PCR as gold-standard. MDM2/CDK4 amplification and a total of 25 distinct fusion genes were identified in this cohort of patients using the NGS approach. Overall, the sensitivity of the NGS panel is 97% with a specificity of 100 and 0% failure rate. Targeted NGS appears to be a feasible and cost-effective approach to improve sarcoma subtype diagnosis with the ability to screen for a wide range of genetic aberrations in one test.

中文翻译：

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一种改进肉瘤诊断的新型下一代测序方法。

肉瘤是一种同时影响骨骼和结缔组织的罕见疾病，有超过 100 种病理实体，鉴别诊断可能很困难。用当前的辅助诊断技术（包括 FISH 和 RT-PCR）补充免疫组织学诊断，可能会导致大量病例得出不确定的结果。我们在这里描述了一种改进肉瘤诊断的新型测序工具的设计和验证。设计并优化了包含 87 个融合基因和 7 个拷贝数变化频繁的基因的探针的 NGS DNA 捕获面板。从软组织和骨肉瘤 FFPE 材料中提取的一组 113 个 DNA 样本，临床 FISH 和/或 RT-PCR 结果对易位或基因扩增呈阳性，用于验证 NGS 方法。使用 FISH 和/或 RT-PCR 作为金标准，在 113 例病例中的 110 例中确认了肉瘤特异性易位或基因扩增。使用 NGS 方法在这组患者中鉴定出 MDM2/CDK4 扩增和总共 25 个不同的融合基因。总体而言，NGS panel 的灵敏度为 97%，特异性为 100，失败率为 0%。靶向 NGS 似乎是改善肉瘤亚型诊断的一种可行且具有成本效益的方法，能够在一次测试中筛查各种遗传畸变。