CN116200491A - 隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒 - Google Patents
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Abstract
本发明提供了一种隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒。可全面针对隆突性皮肤纤维肉瘤诊断相关的特征基因、预后相关基因进行准确检测,提高检出率,提供预后相关信息,具有重要的临床价值。
Description
技术领域
本发明属于体外诊断试剂领域,具体涉及隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒。
背景技术
隆突性皮肤纤维肉瘤(dermatofibrosarcoma protuberans,DSFP)是一种局部浸润皮肤和皮下组织的中度恶性纤维母细胞肿瘤,是最常见的皮肤肉瘤之一。肿瘤常表现为结节状或多结节状肿物。DSFP的组织学分型种类较多,包括经典型隆突性皮肤纤维肉瘤、斑块样隆突性皮肤纤维肉瘤、粘液样隆突性皮肤纤维肉瘤、色素型隆突性皮肤纤维肉瘤、纤维肉瘤型隆突性皮肤纤维肉瘤、巨细胞纤维母细胞瘤(Giant cell fibroblastoma,GCF)。该肿瘤治疗方式包括手术切除,靶向药物治疗(靶点为COL1A1::PDGFB融合基因)。手术切除后复发率高,纤维肉瘤型DFSP具有转移潜能。
其中,GCF好发于儿童(平均年龄为6岁),偶可见发生于成年人。瘤细胞主要由温和的梭形细胞和多核巨细胞构成,间质呈粘液样至胶原化,可见多核巨细胞衬复于假脉管性腔隙中。混合型GCF可含有其它DFSP亚型成分。GCF自1982年被首次报道以来,病例较为少见,且与其他软组织肿瘤形态学相似度较高,诊断难度大。
近年来,随着分子检测手段的进步,通过基因检测诊断隆突性皮肤纤维肉瘤的报道逐渐增多,例如,研究发现在90%以上的隆突性皮肤纤维肉瘤中含有特征性融合基因COL1A1::PDGFB,公开号为CN113337588A的专利申请即公开了一种基于捕获测序的COL1A1::PDGFB融合基因检测方法,获得了比临床常用的荧光原位杂交技术更高的准确率,可用于检测临床组织中极微量的肿瘤变异。此外,还存在部分隆突性皮肤纤维肉瘤存在罕见融合或其他罕见分子机制,对该类肿瘤的诊断及治疗造成巨大挑战。随着研究的深入,还发现了可能与隆突性皮肤纤维肉瘤向纤维肉瘤型隆突性皮肤纤维肉瘤转化相关的基因,有利于肿瘤预后的评估。
然而,目前对隆突性皮肤纤维肉瘤的诊断及相关预后基因进行靶向检测的试剂盒还鲜有报道。特别是,对于巨细胞纤维母细胞瘤这一罕见的亚型而言,其基因诊断试剂盒尚未见报道。
发明内容
本发明的目的在于提供隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒,以及一种与巨细胞纤维母细胞瘤这一特定亚型的致病相关的融合基因:COL3A1::PDGFB。
本发明提供了一种检测如下35个基因中的一种或多种的试剂在制备隆突性皮肤纤维肉瘤诊断试剂盒中的用途:
COL1A1、COL1A2、COL6A3、COL3A1、COL5A1、COL5A2、COL5A3、CSPG2、EMILIN2、ELN、FGF6、FGF23、FN1、FBN1、LAMB1、PTK2B、PDGFB、PDGFD、TNXB、TNC、UBAP1L、CCND2、CDKN2A、CDKN2B、CHD4、EZH2、FRS2、FOXM1、KDM5A、MDM2、TP53、RAD52、WNK1、RAC1、MTAP。
进一步地,上述试剂是同时或分别检测35个基因的试剂,优选地,所述试剂是同时检测35个基因的试剂,更优选为基因检测panel。
进一步地,上述试剂是检测COL3A1和PDGFB的试剂,所述试剂盒是检测巨细胞纤维母细胞瘤诊断试剂盒。
更进一步地,上述检测COL3A1和PDGFB的试剂是检测COL3A1-PDGFB融合基因的试剂。
更进一步地,上述检测COL3A1::PDGFB融合基因的试剂是逆转录聚合酶链式反应用试剂、一代测序用试剂或二代测序用试剂。
更进一步地,上述二代测序用试剂包括COL3A1和PDGFB的捕获探针,优选为DNA探针。
更进一步地,上述一代测序用试剂包括序列如SEQ ID NO.1和SEQ IDNO.2所示的引物。
进一步地,上述试剂是检测人类肿瘤组织中35个基因中的一种或多种的试剂。
本发明还提供了一种隆突性皮肤纤维肉瘤诊断试剂盒,包括检测如下35个基因中的一种或多种的试剂:
COL1A1、COL1A2、COL6A3、COL3A1、COL5A1、COL5A2、COL5A3、CSPG2、EMILIN2、ELN、FGF6、FGF23、FN1、FBN1、LAMB1、PTK2B、PDGFB、PDGFD、TNXB、TNC、UBAP1L、CCND2、CDKN2A、CDKN2B、CHD4、EZH2、FRS2、FOXM1、KDM5A、MDM2、TP53、RAD52、WNK1、RAC1、MTAP。
进一步地,上述试剂是检测COL3A1和PDGFB的试剂,所述试剂盒是检测巨细胞纤维母细胞瘤诊断试剂盒;优选地,检测COL3A1和PDGFB的试剂是检测COL3A1::PDGFB融合基因的试剂。
本发明的有益效果:本发明提供了一种隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒,可全面针对隆突性皮肤纤维肉瘤的特征基因、预后相关基因进行准确检测,提高检出率,为临床提供重要预后信息;特别是,提供了与巨细胞纤维母细胞瘤这一特定亚型致病相关的融合基因:COL3A1::PDGFB,通过检测COL3A1::PDGFB,可以有效进行人类巨细胞纤维母细胞瘤的疾病诊断或辅助诊断,具有重要的临床意义。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为案例1的COL1A1::PDGFB融合基因验证结果。
图2为案例2的COL3A1::PDGFB融合基因验证结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
本发明隆突性皮肤纤维肉瘤案例共2例,来源于四川大学华西医院,均为病理确诊案例。案例1为纤维肉瘤型隆突性皮肤纤维肉瘤;案例2为巨细胞纤维母细胞瘤。
本发明35个基因的检测panel由Twist Bioscience公司进行制备提供。
实施例1、本发明试剂盒进行基因检测
1、检测方法
1.1样本处理和提取
检测样本不限于石蜡包埋肿瘤组织。现以石蜡包埋肿瘤组织为例,将组织蜡块切为4-6μm的卷片,收集6-10张卷片置于1.5ml EP管(RNase-free)中备用。用QIAGEN DNAFFPE Kit和QIAGEN RNeasy FFPE Kit试剂盒按说明书操作流程提取样本DNA和RNA。使用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒,按照说明书操作流程将RNA逆转录为cDNA。
1.2 DNA/cDNA文库构建
通过超声破碎仪将DNA/cDNA片段化;片段纯化后进行末端修复、加A尾及加接头处理;接头连接产物通过AMPureXP磁珠进行纯化;对接头纯化产物进行PCR扩增,PCR产物再次通过AMPureXP磁珠进行纯化。
1.3文库杂交
对35个目的基因进行panel设计(见表1),参考基因组为GRCh37.p13(此为人类基因组标准参考序列,捕获探针的编号对应标准参考序列编号,则可以找出捕获探针序列),设计区域为各基因外显子。通过TWIST提供网站设计捕获探针进行panel合成,捕获探针设计区域为基因外显子区,捕获探针目标区域信息见表2。杂交后对片段进行特异性洗脱,PCR扩增后获得扩增文库。
表1隆突性皮肤纤维肉瘤35基因panel
表2捕获探针目标区域信息
1.4二代测序平台测序
选用Illumina Hiseq4000二代测序平台进行测序,测序深度1000×。
1.5测序数据分析
通过Burrows-Wheeler Aligner将测序数据与人基因组参考序列进行比对。BAM文件由Genome Analysis Toolkit(GATK)、Picard suite进一步对数据进行过滤,插入缺失序列重排等处理。MuTect2进行单核苷酸突变(SNVs)突变分析,Oncotator tool对SNVs进行注释;CNVKit、GISTIC进行拷贝数变异(CNVs)分析;fusionScan进行融合基因分析。
2、检测结果
如表3所示,为2例案例的检测结果。案例1使用荧光原位杂交技术未检出染色体易位;测序中DNA和RNA水平均检测出COL1A1::PDGFB融合基因,并检测出其他多种异常基因,融合基因验证引物为COL1A1-F:5’-GTCCCCAAGGCTTCCAAGGTC-3’(SEQ ID NO.3);PDGFB-R:5’-ATCTTCCTCTCCGGGGTCTCC-3’(SEQ ID NO.4),用一代测序进行验证,确认了COL1A1::PDGFB融合基因异常,判断为隆突性皮肤纤维肉瘤相关基因,病理形态学考虑为隆突性皮肤纤维肉瘤,与基因检测结果一致。其COL1A1::PDGFB融合基因验证结果如图1所示。
案例2使用荧光原位杂交技术未检出染色体易位;测序中DNA水平检测出COL3A1::PDGFB罕见融合基因,COL3A1::PDGFB融合基因验证引物为COL3A1-F:5’-CTTCAGGGTGAGACAGCCAA-3’(SEQ ID NO.1);PDGFB-R:5’-CATAAGCCCCCGGATTTGGT-3’(SEQID NO.2),确认了COL3A1::PDGFB融合基因异常,判断为巨细胞纤维母细胞瘤相关基因。病理形态学考虑为巨细胞纤维母细胞瘤,与基因检测结果一致。其COL3A1::PDGFB融合基因验证结果如图2所示。
表3临床隆突性皮肤纤维肉瘤样本使用该试剂盒的检测结果
图2所示,DNA水平检测出COL3A1::PDGFB罕见融合基因。
巨细胞纤维母细胞瘤与隆突性皮肤纤维肉瘤为同一家族肿瘤,病理组织学形态有各自的特点,但具有相似的染色体和基因异常。该家族肿瘤主要致病机制为17号和22号染色体重排,形成COL1A1(17q21.33)和PDGFB(22q13.1)融合基因,导致PDGFB基因异常转录上调,功能性PDGFB蛋白产生增加,通过PDGFB介导的自分泌或旁分泌途经活化PDGFRB及下游信号通路,导致肿瘤发生。目前研究发现在90%以上的隆突性皮肤纤维肉瘤中含有特征性融合基因COL1A1::PDGFB。除了COL1A1::PDGFB,在隆突性皮肤纤维肉瘤中还存在罕见融合或其他罕见分子机制,如COL1A2::PDGFB、COL6A3::PDGFD、EMILIN2::PDGFD、TNC::PDGFD、涉及CSPG2和PTK2B基因的复杂性染色体异位。此外,出现罕见融合基因的DFSP在临床病理特征上与出COL1A1::PDGFB融合基因DFSP并无差异,因此提示PDGFB和PDGFD相关融合基因均是隆突性皮肤纤维肉瘤发生发展重要的遗传学特征。巨细胞纤维母细胞瘤遗传学研究中进行了基因确诊的病例主要存在COL1A1::PDGFB融合基因。案例2病理形态学符合巨细胞纤维母细胞瘤特征,测序结果只存在COL3A1::PDGFB这一融合基因,为PDGFB重排基因,高度提示COL3A1::PDGFB为该例巨细胞纤维母细胞瘤的相关致病基因,本病例为GCF的遗传学贡献了新的特征,并为GCF的遗传学检测提供了新的线索。
结论:通过检测COL3A1::PDGFB融合基因,可以实现对巨细胞纤维母细胞瘤的诊断或辅助诊断。
综上,本发明提供了一种隆突性皮肤纤维肉瘤诊断及预后相关基因靶向检测试剂盒,可全面针对隆突性皮肤纤维肉瘤的特征基因、预后相关基因进行准确检测,提高检出率,为临床提供重要预后信息;特别是,提供了与巨细胞纤维母细胞瘤这一特定亚型致病相关的融合基因:COL3A1::PDGFB,通过检测COL3A1::PDGFB,可以有效进行人类巨细胞纤维母细胞瘤的疾病诊断或辅助诊断,具有重要的临床意义。
Claims (10)
1.检测如下35个基因中的一种或多种的试剂在制备隆突性皮肤纤维肉瘤诊断试剂盒中的用途:
COL1A1、COL1A2、COL6A3、COL3A1、COL5A1、COL5A2、COL5A3、CSPG2、EMILIN2、ELN、FGF6、FGF23、FN1、FBN1、LAMB1、PTK2B、PDGFB、PDGFD、TNXB、TNC、UBAP1L、CCND2、CDKN2A、CDKN2B、CHD4、EZH2、FRS2、FOXM1、KDM5A、MDM2、TP53、RAD52、WNK1、RAC1、MTAP。
2.如权利要求1所述的用途,其特征在于,所述试剂是同时或分别检测35个基因的试剂,优选地,所述试剂是同时检测35个基因的试剂,更优选为基因检测panel。
3.如权利要求1所述的用途,其特征在于,所述试剂是检测COL3A1和PDGFB的试剂,所述试剂盒是检测巨细胞纤维母细胞瘤诊断试剂盒。
4.如权利要求3所述的用途,其特征在于,所述检测COL3A1和PDGFB的试剂是检测COL3A1::PDGFB融合基因的试剂。
5.如权利要求4所述的用途,其特征在于,所述检测COL3A1-PDGFB融合基因的试剂是逆转录聚合酶链式反应用试剂、一代测序用试剂或二代测序用试剂。
6.如权利要求5所述的用途,其特征在于,所述二代测序用试剂包括COL3A1和PDGFB的捕获探针,优选为DNA探针。
7.如权利要求5所述的用途,其特征在于,所述一代测序用试剂包括序列如SEQ IDNO.1和SEQ ID NO.2所示的引物。
8.如权利要求1所述的用途,其特征在于,所述试剂是检测人类肿瘤组织中35个基因中的一种或多种的试剂。
9.一种隆突性皮肤纤维肉瘤诊断试剂盒,其特征在于,包括检测如下35个基因中的一种或多种的试剂:
COL1A1、COL1A2、COL6A3、COL3A1、COL5A1、COL5A2、COL5A3、CSPG2、EMILIN2、ELN、FGF6、FGF23、FN1、FBN1、LAMB1、PTK2B、PDGFB、PDGFD、TNXB、TNC、UBAP1L、CCND2、CDKN2A、CDKN2B、CHD4、EZH2、FRS2、FOXM1、KDM5A、MDM2、TP53、RAD52、WNK1、RAC1、MTAP。
10.如权利要求9所述的试剂盒,其特征在于,所述试剂是检测COL3A1和PDGFB的试剂,所述试剂盒是检测巨细胞纤维母细胞瘤诊断试剂盒;优选地,检测COL3A1和PDGFB的试剂是检测COL3A1::PDGFB融合基因的试剂。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109811055A (zh) * | 2019-01-08 | 2019-05-28 | 广州金域医学检验中心有限公司 | 肉瘤融合基因检测试剂盒及系统 |
CN113462763A (zh) * | 2021-04-21 | 2021-10-01 | 四川大学华西医院 | 靶向检测软组织肿瘤小圆细胞肿瘤融合基因panel设计试剂盒 |
US20220290242A1 (en) * | 2018-11-05 | 2022-09-15 | Centre Henri Becquerel | Method for diagnosing a cancer and associated kit |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20220290242A1 (en) * | 2018-11-05 | 2022-09-15 | Centre Henri Becquerel | Method for diagnosing a cancer and associated kit |
CN109811055A (zh) * | 2019-01-08 | 2019-05-28 | 广州金域医学检验中心有限公司 | 肉瘤融合基因检测试剂盒及系统 |
CN113462763A (zh) * | 2021-04-21 | 2021-10-01 | 四川大学华西医院 | 靶向检测软组织肿瘤小圆细胞肿瘤融合基因panel设计试剂盒 |
Non-Patent Citations (1)
Title |
---|
RONAN ULVÉ等: "Discovery of Human-Similar Gene Fusions in Canine Cancers", PRIORITY REPORT, vol. 77, no. 21, pages 5721 * |
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