Chinese expert consensus on the application of next-generation sequencing for bone and soft tissue tumors (2021 version)
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摘要: 骨与软组织肿瘤临床罕见，亚型众多，且肉瘤性病变恶性程度高、预后差，给临床诊治带来巨大挑战。近年来，随着分子检测技术的发展，二代基因测序（next-generation sequencing，NGS）已广泛应用于肿瘤的分子诊断、靶向基因筛选以及表观遗传学分析等领域，使得骨与软组织肿瘤的诊治获得改善。目前，NGS技术在骨与软组织肿瘤中的应用尚存疑问。基于相关循证医学证据和专家共识，中国抗癌协会肉瘤专业委员会从NGS检测、临床应用及实验室质控角度制定了《骨与软组织肿瘤二代测序中国专家共识（2021年版）》，旨在规范NGS检测在骨与软组织肿瘤领域内的应用，更好地服务于临床诊治，使患者受益。Abstract: Bone and soft tissue tumors are a group of clinically rare malignancies with abundant subtypes. These tumors, particularly sarcomatous lesions, are challenging to diagnose and treatment because of their aggressiveness and poor prognosis. In recent years, next-generation sequencing (NGS) has been widely used for molecular diagnosis, targeted gene screening, and epigenetic analysis of tumors with the development of molecular detection technology, which improves the diagnosis and treatment of bone and soft tissue tumors to some extent. Currently, there are still many doubts about the application of NGS for bone and soft tissue tumors. Based on available medical evidence and expert opinions, the sarcoma committee of the China Anti-Cancer Association Committee of Sarcoma issued a consensus regarding the application of NGS for bone and soft tissues with respect to detection using NGS, clinical application, and laboratory quality control. The consensus aimed to standardize the utility of NGS in detecting bone and soft tissue tumors and facilitate clinical diagnosis and treatment of these tumors for the benefit of patients.
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表 1 常规病理学手段难以明确诊断需要进行NGS检测的骨与软组织肿瘤
肿瘤类型 涉及的分子遗传学改变 EWSR1-SMAD3阳性纤维母细胞性肿瘤 EWSR1-SMAD3* GAB1-ABL1阳性纤维母细胞性肿瘤 GAB1-ABL1 ALK阴性炎性肌纤维母细胞瘤 TFG/YWHAE-ROS1**，ETV6-NTRK3 上皮样炎性肌纤维母细胞肉瘤 RANBP2/RRBP-ALK 少数隆突性皮纤维肉瘤 COL6A3/EMILIN2-PDGFD MUC4阴性硬化性上皮样纤维肉瘤 YAP1-KMT2A 上皮样纤维组织细胞瘤 SQSTM1/VCL-ALK 上皮样血管瘤 MBNL1/VIM/lincRNA-FOS，ZFP36/WWTR1/ACTB-FOSB TFE3重排上皮样血管内皮瘤 YAP1-TFE3 假肌源性血管内皮瘤 SERPINE1/ACTB-FOSB 网状和复合性血管内皮瘤 YAP1-MAML2 伴有神经内分泌分化复合性血管内皮瘤 PTBP1-MAML2 先天性梭形细胞横纹肌肉瘤 SRF/TEAD1/VGLL2-NCOA2，VGLL2-CITED2 梭形细胞/硬化性横纹肌肉瘤 MYOD1（p.L122R）突变 骨上皮样和梭形细胞横纹肌肉瘤 EWSR1/FUS-TFCP2，MEIS1-NCOA2 NTRK重排梭形细胞肿瘤 LMNA/TPR/TPM3-NTRK1，SPECC1L/STRN-NTRK2，ETV6/EMAL4-NTRK3 其他双表达CD34和S-100的梭形细胞肿瘤 SEPT7/CUX1/CDC42SE2-BRAF，PDZRN3/SLMAP/TMF1/MTAP-RAF1，TFG/MYH10/NCOA4/VCL/CLIP2/KIAA121/KHDRBS1/SPECC1L/CCDC6-RET，PPP1CB-ALK EWSR1-非ETS融合肉瘤 EWSR1/FUS-NFATC2，EWSR1-PATZ1/POU5F1/SP3/SMARCA5 BCOR重排肉瘤 BCOR-CCNB3/MAML3，ZC3H7B-BCOR，YWHAE-NUTM2B GLI1重排/扩增恶性上皮样肿瘤 ACTB/MALAT1/PTCH1-GLI1，GLI1扩增 胃母细胞瘤/胃丛状纤维黏液瘤 MALAT1-GLI1 野生型胃肠道间质瘤 SDHx/BRAF/NF1/KRAS突变，FGFR1-HOOK3/TACC1，ETV6-NTRK3 富于细胞性肌样肿瘤 SRF-ICA1L 肌上皮瘤样玻璃样变肿瘤 OGT-FOXO3 部分不能分类的圆细胞肉瘤 EWSR1-CREB 肾脏原始梭形细胞肉瘤 MEIS1-NCOA2 *：EWSR1基因与SMAD3基因发生融合；**：TFG/YWHAE两种不同的伴侣基因亚型分别与ROS1基因发生融合 
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表 2 临床检测技术的全面对比
检测技术 IHC FISH RT-PCR NGS（DNA+RNA） 检测层面 蛋白检测 DNA RNA DNA RNA 融合基因 主基因 主基因 主基因+伴侣基因 主基因+伴侣基因 主基因+伴侣基因 优势 直接展现表达结果 灵敏度高、特异度高；空间定位准确，可同时分析多个细胞，并进行定量 单位点精准度非常高；结果判读简单 大规模，高通量；可检测点突变、小片段插入/缺失；大片段CNV等变异 大规模，高通量；可检测已知、未知融合基因；可以发现新融合基因 局限性 只能检测已知的融合蛋白，不能检测未知的融合蛋白；无法区分融合伴侣；假阴性率高 可检基因有限；需要多次检测；无法区分融合伴侣；距离<2兆的融合检出性能差；假阴性率较高 仅限于已知融合，不能发现新融合；无法识别未知和罕见的融合基因；存在假阴性 panel需要特殊设计；部分融合基因无法检测出，存在假阴性；对数据注释和报告解读要求高 样本质量要求高；对数据注释和报告解读要求高 应用场景 主要用于确定细胞分化来源；少量指标用于检测遗传学改变 对已知基因判断是否融合 融合验证 肿瘤分子全面检测 肿瘤分子全面检测
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表 3 靶向药物临床试验结果分析
靶向药物 临床试验 主要结局 NTRK抑制剂 拉罗替尼（larotrectinib） 实体瘤1/2期临床试验[23] 肉瘤RR 87%，其中软组织肉瘤88%和骨肉瘤50% 恩曲替尼（entrectinib） 实体瘤1/2期临床试验[24] 肉瘤RR 46% EZH2抑制剂 他泽司他（tazemetostat） 上皮样肉瘤2期试验[25] ORR 15%，其中CR率1.6%，PR率13%；中位响应时间3.9个月，mPFS 5.5个月，mOS 19个月 CDK4/6抑制剂 帕博西尼（palbociclib） 高分化或去分化脂肪肉瘤2期试验[26] 12周时PFS率57.2%，mPFS 17.9周，CR率1.8% 阿贝西利（abemaciclib） 去分化脂肪肉瘤2期试验[27] 12周时PFS率76%，mPFS 6.3个月，PR率3.4% ALK抑制剂 克唑替尼（crizotinib） ALK阳性炎性肌纤维母细胞瘤临床试验[28] ORR 86%，CR率36%，PR率50% PDGFR抑制剂 伊马替尼（imatinib） 中危或高危原发性胃肠道间质瘤临床试验[29] 5年RFS 90%，OS率95% 帕唑帕尼（pazopanib） 转移性软组织肉瘤3期试验[30] 帕唑帕尼组：mPFS 4.6个月，OS 12.5个月；慰剂组：mPFS 1.6个月，OS 10.7个月 瑞普替尼（ripretinib） 晚期胃肠道间质瘤3期试验[31] 瑞普替尼组：mPFS 4.6个月，ORR 0%，中位TTP 1.0个月，mOS 15.1个月；安慰剂组：mPFS 1.0个月，ORR 9.4%，中位TTP 6.4个月，mOS 6.6个月 RR：response rate，总有效率；ORR：overall responses rate，总客观缓解率；CR：complete response，完全缓解；PR：partial response，部分缓解；mPFS：median progression-free survival，中位无疾病进展时间；mOS：median overall survival，中位生存期；DOR：duration of response，响应时间；DCR：disease control rate，疾病控制率；RFS：relapse-free rate，无疾病复发率；TTP：time to progression，疾病进展时间
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表 4 骨与软组织肿瘤二代测序中国专家共识要点
序号 推荐要点 诊断 共识1 推荐常规病理学检查不能明确诊断的骨与软组织肿瘤患者进行NGS检测 共识2 推荐常规分子学检测结果为阴性的骨与软组织肿瘤患者使用（DNA+RNA）NGS技术或平台进行复检 共识3 推荐常规分子学检测与NGS检测有差异的骨与软组织肿瘤患者，进行第3种检测进行验证 治疗 共识4 推荐考虑接受特异性靶向治疗的骨与软组织肿瘤患者，通过NGS技术或平台验证靶向药物相关的基因或潜在基因 共识5 推荐进展期骨与软组织肿瘤患者，分别采用IHC和NGS检测PD-L1、MSI、TMB等免疫治疗相关的分子标志物，根据结果辅助免疫治疗 共识6 推荐既往治疗失败且无有效替代方案的骨与软组织肿瘤患者通过NGS检测，以寻找匹配的临床试验机会 NGS检测样本类型和流程规范 共识7 骨与软组织肿瘤的NGS样本采集应符合规范要求 共识8 骨与软组织肿瘤的NGS生物信息学分析应符合规范要求，配备完善的标准分析及质量控制流程 共识9 推荐有CAP/CLIA/CNAS认证或认可的实验室进行NGS检测 NGS检测报告的临床解读 共识10 倡导各单位组建分子肿瘤专家委员会（molecular tumor boards，MTB），依据国内外专家共识及解读流程，正确解读NGS检测结果，制定精准诊疗方案
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