CN115011673A - A method for detecting abnormal amplification of HER2 gene in circulating tumor DNA and its application - Google Patents
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Abstract
本发明提供了一种循环肿瘤DNA中HER2基因异常扩增的检测方法及其应用。所述检测方法包括：构建定量检测HER2基因的反应体系、优化反应条件、确定HER2基因异常扩增的初定判读阈值和最佳判读阈值，确定HER2基因的扩增情况。本发明利用数字PCR技术开发出一种ctDNA HER2基因拷贝数变异的定量检测方法及检测试剂盒，能够指导胃癌药物的开发和胃癌HER2阳性患者的靶向药物个体化治疗，并为患者肿瘤复发监测、预后判断等提供更为精准、无创的分子分型方法。
The invention provides a method for detecting abnormal amplification of HER2 gene in circulating tumor DNA and its application. The detection method includes: constructing a reaction system for quantitatively detecting the HER2 gene, optimizing the reaction conditions, determining a preliminary judgment threshold and an optimal judgment threshold for abnormal amplification of the HER2 gene, and determining the amplification of the HER2 gene. The invention utilizes digital PCR technology to develop a quantitative detection method and detection kit for ctDNA HER2 gene copy number variation, which can guide the development of gastric cancer drugs and the individualized treatment of targeted drugs for patients with gastric cancer HER2 positive, and monitor the tumor recurrence of patients. , prognosis judgment, etc., to provide more accurate and non-invasive molecular typing methods.
Description
技术领域technical field
本发明属于临床医学诊断技术领域，具体涉及肿瘤的监测和预后判断，尤其涉及一种循环肿瘤DNA中HER2基因异常扩增的检测方法及其应用。The invention belongs to the technical field of clinical medical diagnosis, and specifically relates to tumor monitoring and prognosis judgment, in particular to a method for detecting abnormal amplification of HER2 gene in circulating tumor DNA and its application.
背景技术Background technique
胃癌(Gastric cancer，GC)是消化道恶性肿瘤之一，许多胃癌患者在发现时己处于进展期，5年生存率<10％。目前临床上常用的胃肠肿瘤蛋白标志物如癌坯抗原(CEA)、糖类抗原CA-19-9在GC患者中的阳性率<20％，灵敏度和特异度较低，难以满足“精准医疗”的要求，因此，开展胃癌诊断、监测、评价手术疗效及疾病预后的新型生物标志物检测技术及其应用研究具有重要意义。Gastric cancer (GC) is one of the malignant tumors of the digestive tract. Many patients with gastric cancer are in the advanced stage when they are discovered, and the 5-year survival rate is less than 10%. At present, the commonly used clinical gastrointestinal tumor protein markers such as cancer cell antigen (CEA) and carbohydrate antigen CA-19-9 have a positive rate of less than 20% in GC patients, with low sensitivity and specificity, which are difficult to meet the requirements of "precision medicine". Therefore, it is of great significance to carry out new biomarker detection technology and application research for gastric cancer diagnosis, monitoring, evaluation of surgical efficacy and disease prognosis.
研究表明，在肿瘤进展阶段可释放肿瘤细胞和DNA至外周血，形成循环肿瘤DNA(ctDNA)，其携带有肿瘤特异性遗传学改变的自由基因组片段，与肿瘤发生发展、抵抗性耐药及复发转移等具有相关性，对于肿瘤的诊断、治疗及预后评估具有重要价值，检测肿瘤患者血液中ctDNA可以提供快速、高效、相对无创性的“液体活检”。Studies have shown that tumor cells and DNA can be released to peripheral blood during tumor progression to form circulating tumor DNA (ctDNA), which carries free genome fragments with tumor-specific genetic changes, and is closely related to tumor development, resistance, drug resistance and recurrence. Metastasis is relevant and has important value for tumor diagnosis, treatment and prognosis evaluation. Detection of ctDNA in the blood of tumor patients can provide a rapid, efficient and relatively non-invasive "liquid biopsy".
近年来，与胃癌分子靶向治疗及预后评估有关的人表皮生长因子受体-2(HER2)过表达的作用越来越受到关注，主要参与肿瘤生长、转移、浸润相关基因的调控，目前研究发现HER2在乳腺癌、结肠癌、肺癌、胃癌等多种恶性肿瘤中存在不同程度的基因扩增和/或蛋白过表达，其中，HER2胃癌患者中约20％HER2表达阳性。In recent years, the role of human epidermal growth factor receptor-2 (HER2) overexpression related to molecular targeted therapy and prognosis evaluation of gastric cancer has attracted more and more attention, and it is mainly involved in the regulation of genes related to tumor growth, metastasis and infiltration. It has been found that HER2 has different degrees of gene amplification and/or protein overexpression in various malignant tumors such as breast cancer, colon cancer, lung cancer, and gastric cancer. Among them, about 20% of HER2 gastric cancer patients are positive for HER2 expression.
在临床治疗中，针对HER2阳性的乳腺癌靶向治疗己达成共识，HER2过表达与淋巴转移、浸润相关，但HER2表达而且与胃癌的临床病理特征之间关系亦无统一的结论，主要原因可能与样本量、免疫组化所用抗体、评分标准、肿瘤异质性等有关。In clinical treatment, a consensus has been reached on targeted therapy for HER2-positive breast cancer. HER2 overexpression is related to lymphatic metastasis and invasion, but there is no unified conclusion on the relationship between HER2 expression and the clinicopathological characteristics of gastric cancer. The main reason may be It is related to the sample size, antibodies used in immunohistochemistry, scoring standards, tumor heterogeneity, etc.
目前，临床应用的HER2分型方法主要有基于抗原抗体反应的免疫组化法(IHC，Immunohistochemistry)和基于核酸杂交的荧光原位杂交(FISH，Fluorescence in situhybridization)。但这两种方法均存在取样量少、结果判断存在主观误差以及检测结果的判断标准不一致等问题。Currently, clinically used HER2 typing methods mainly include immunohistochemistry (IHC, Immunohistochemistry) based on antigen-antibody reaction and fluorescence in situ hybridization (FISH, Fluorescence in situhybridization) based on nucleic acid hybridization. However, these two methods have problems such as small sampling amount, subjective error in the judgment of the results, and inconsistent judgment standards of the test results.
因此，利用数字PCR技术发展一种新的胃癌HER2分型和疗效评价方法，对胃癌进行精准分子分型、对靶向药物治疗的动态监测和评估至关重要，是临床肿瘤治疗的发展方向。Therefore, the use of digital PCR technology to develop a new method for HER2 typing and efficacy evaluation of gastric cancer is crucial for accurate molecular typing of gastric cancer and dynamic monitoring and evaluation of targeted drug therapy, which is the development direction of clinical tumor therapy.
发明内容SUMMARY OF THE INVENTION
针对现有技术存在的不足，本发明的目的在于提供一种循环肿瘤DNA中HER2基因异常扩增的检测方法及其应用。所述检测方法能够监测体内循环肿瘤DNA中HER2基因的扩增情况，通过其扩增情况判断受试对象的健康状态，进而选择合适的治疗方案，达到“精准治疗”的目的。In view of the deficiencies in the prior art, the purpose of the present invention is to provide a detection method for abnormal amplification of HER2 gene in circulating tumor DNA and its application. The detection method can monitor the amplification of the HER2 gene in circulating tumor DNA in the body, judge the health status of the subject based on the amplification, and then select an appropriate treatment plan to achieve the purpose of "precise treatment".
为达此目的，本发明采用以下技术方案：For this purpose, the present invention adopts the following technical solutions:
第一方面，本发明提供一种用于扩增人外周血循环肿瘤DNA(ctDNA)中HER2基因的试剂盒，所述试剂盒包括：扩增HER2基因的特异性引物对和探针、扩增参比基因的特异性引物对和探针和标准品。In a first aspect, the present invention provides a kit for amplifying the HER2 gene in human peripheral blood circulating tumor DNA (ctDNA), the kit comprising: a specific primer pair and a probe for amplifying the HER2 gene, an amplification parameter Gene-specific primer pairs and probes and standards.
优选地，所述内参基因包括CEP17基因。Preferably, the internal reference gene includes the CEP17 gene.
本发明中所述试剂盒用于扩增人外周血循环肿瘤DNA中HER2基因和参比基因，对循环肿瘤DNA中的HER2基因进行精准定量，进而确定HER2基因和参比基因的拷贝数比值。The kit described in the present invention is used to amplify the HER2 gene and the reference gene in the circulating tumor DNA of human peripheral blood, accurately quantify the HER2 gene in the circulating tumor DNA, and then determine the copy number ratio of the HER2 gene and the reference gene.
第二方面，本发明提供一种循环肿瘤DNA中HER2基因异常扩增的检测方法，所述检测方法包括：In a second aspect, the present invention provides a detection method for abnormal amplification of HER2 gene in circulating tumor DNA, the detection method comprising:
(1)合成HER2基因和参比基因的特异性扩增引物和检测探针，并分别构建含有所述HER2基因和参比基因的质粒作为标准品；(1) synthesizing specific amplification primers and detection probes of the HER2 gene and the reference gene, and constructing a plasmid containing the HER2 gene and the reference gene as a standard respectively;
(2)采用数字PCR的方法对所述标准品进行定量检测，得到检测循环肿瘤DNA中HER2基因的反应条件；(2) quantitatively detecting the standard substance by means of digital PCR to obtain the reaction conditions for detecting the HER2 gene in circulating tumor DNA;
(3)依据步骤(2)所得的反应条件，检测正常对照样品和胃癌样品中HER2基因和参比基因的含量，得到定量检测结果，并将所述HER2基因和参比基因的拷贝数比值作为异常扩增的初定判读阈值；(3) according to the reaction conditions obtained in step (2), detect the content of the HER2 gene and the reference gene in the normal control sample and the gastric cancer sample, obtain a quantitative detection result, and use the copy number ratio of the HER2 gene and the reference gene as The initial judgment threshold of abnormal amplification;
(4)比较步骤(3)所得的定量检测结果与临床病理分型结果，并进行敏感性评价和特异性评价，得到异常扩增的最佳判读阈值；(4) comparing the quantitative detection results obtained in step (3) with the clinical pathological typing results, and performing sensitivity evaluation and specificity evaluation to obtain the best interpretation threshold for abnormal amplification;
(5)检测待测样品循环肿瘤DNA中HER2基因和参比基因的含量，并根据步骤(4)所述的最佳判读阈值判断所述待测样品循环肿瘤DNA中HER2基因的扩增情况。(5) Detecting the contents of the HER2 gene and the reference gene in the circulating tumor DNA of the sample to be tested, and judging the amplification of the HER2 gene in the circulating tumor DNA of the sample to be tested according to the optimal interpretation threshold described in step (4).
本发明中，所述检测方法在检测循环肿瘤DNA中HER2基因的扩增情况中具有重要的应用价值，所得HER2基因的扩增情况能够反映待测对象(包括人和动物)中循环肿瘤DNA的情况，进而对待测对象的生理状况进行判断；因此，所述检测方法不仅可以判断胃癌患者的预后情况，在胃癌药物的研发阶段(该检测方法可以检测动物的HER2基因的扩增情况)也具有重要的使用价值。In the present invention, the detection method has important application value in detecting the amplification of HER2 gene in circulating tumor DNA, and the obtained amplification of HER2 gene can reflect the amplification of circulating tumor DNA in the subject (including humans and animals) to be tested. Therefore, the detection method can not only judge the prognosis of gastric cancer patients, but also has the ability to detect the HER2 gene amplification in animals in the development stage of gastric cancer drugs (the detection method can detect the amplification of the HER2 gene in animals). important use value.
作为本发明优选的技术方案，步骤(3)中所述胃癌样品为经免疫组化、FISH确诊HER2表达情况的胃癌组织的外周血循环肿瘤DNA。As a preferred technical solution of the present invention, the gastric cancer sample in step (3) is the peripheral blood circulating tumor DNA of gastric cancer tissue confirmed by immunohistochemistry and FISH for HER2 expression.
优选地，步骤(4)中所述临床病理分型结果包括IHC分型结果和/或FISH分型结果。Preferably, the clinicopathological typing results in step (4) include IHC typing results and/or FISH typing results.
优选地，步骤(5)所述待测样品循环肿瘤DNA的提取方法包括：Preferably, the method for extracting circulating tumor DNA of the sample to be tested in step (5) includes:
取外周静脉血并加入抗凝剂，采用QIAamp通过裂解、结合、洗涤和洗脱，获得纯化和浓缩游离的循环肿瘤DNA。Peripheral venous blood was taken and anticoagulant was added, and purified and concentrated free circulating tumor DNA was obtained by lysis, binding, washing and elution using QIAamp.
优选地，步骤(5)中所述判断的标准为：Preferably, the criterion for judgment described in step (5) is:
所述HER2基因和参比基因的拷贝数比值小于等于最佳判读阈值，则所述HER2基因未出现异常扩增；If the copy number ratio between the HER2 gene and the reference gene is less than or equal to the optimal interpretation threshold, the HER2 gene does not have abnormal amplification;
所述HER2基因和参比基因的拷贝数比值大于最佳判读阈值，则所述HER2基因出现异常扩增。If the copy number ratio between the HER2 gene and the reference gene is greater than the optimal reading threshold, the HER2 gene is abnormally amplified.
本发明中，以正常对照和胃癌患者HER2基因和参比基因的拷贝数比值为初步的检测标准，并分别在健康人血中定量添加NCI-N87或MKN-45等癌细胞，排除不同细胞的特异性因素，提高检测方法的准确度和灵敏度，得到最终的最佳判读阈值；将检测结果与最佳判读阈值相比较，进而判断受试样品中HER2基因的扩增情况。In the present invention, the ratio of the copy number of the HER2 gene and the reference gene in the normal control and gastric cancer patients is used as the preliminary detection standard, and cancer cells such as NCI-N87 or MKN-45 are quantitatively added to the blood of healthy people respectively to exclude the different cells. The specificity factor improves the accuracy and sensitivity of the detection method, and the final optimal interpretation threshold is obtained; the detection result is compared with the optimal interpretation threshold, and then the amplification of the HER2 gene in the test sample is judged.
同时，本发明中，为了进一步验证所述检测方法的准确性，将所得检测结果与IHC联合FISH检测相比较，所得结果的一致性检验用Kappa值表示，得到Kappa＞0.75，说明两法检验一致性好。At the same time, in the present invention, in order to further verify the accuracy of the detection method, the obtained detection results were compared with the IHC combined FISH detection. good sex.
第三方面，本发明提供一种如第一方面所述的试剂盒或如第二方面所述的检测方法在制备用于诊断、治疗或预后检测胃癌的药物或装置中的应用。In a third aspect, the present invention provides an application of the kit according to the first aspect or the detection method according to the second aspect in the preparation of a medicament or a device for diagnosing, treating or prognosing gastric cancer.
第四方面，本发明还提供一种检测循环肿瘤DNA中HER2基因异常扩增的装置，其特征在于，所述装置包括：扩增单元、检测单元和分析单元；In a fourth aspect, the present invention also provides a device for detecting abnormal amplification of HER2 gene in circulating tumor DNA, characterized in that the device includes: an amplification unit, a detection unit, and an analysis unit;
其中，所述扩增单元包括如第一方面所述的试剂盒。Wherein, the amplification unit includes the kit according to the first aspect.
优选地，所述检测单元使用数字PCR方法对HER2基因的拷贝数进行定量检测。Preferably, the detection unit uses a digital PCR method to quantitatively detect the copy number of the HER2 gene.
优选地，所述分析单元用于分析所述HER2基因与参比基因的拷贝数比值，并将所述拷贝数比值与最佳判读阈值比较，得到待测样品循环肿瘤DNA中HER2基因的扩增情况。Preferably, the analysis unit is used to analyze the copy number ratio between the HER2 gene and the reference gene, and compare the copy number ratio with the optimal interpretation threshold to obtain the amplification of the HER2 gene in the circulating tumor DNA of the sample to be tested. Happening.
本发明所提供的检测方法以及构建该检测方法的技术路线，为胃癌的诊断、治疗以及预后等提供了更为精准的分型方法。The detection method and the technical route for constructing the detection method provided by the present invention provide a more accurate typing method for the diagnosis, treatment and prognosis of gastric cancer.
本发明所述的数值范围不仅包括上述列举的点值，还包括没有列举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本发明不再穷尽列举所述范围包括的具体点值。The numerical range described in the present invention not only includes the above-mentioned point values, but also includes any point value between the above-mentioned numerical ranges that are not listed. Due to space limitations and for the sake of brevity, the present invention will not exhaustively list the above-mentioned ranges. The specific point value to include.
与现有技术相比，本发明的有益效果为：Compared with the prior art, the beneficial effects of the present invention are:
(1)本发明中，利用数字PCR技术开发出一种ctDNA HER2基因拷贝数变异的定量检测方法，并将其应用于胃癌的精准诊治和胃癌药物的研发中，能够指导胃癌HER2阳性患者的靶向药物个体化治疗，并为肿瘤复发监测、预后判断等提供更为精准、无创的分子分型新方法，对于肿瘤精准医学临床应用而言具有重要的意义；(1) In the present invention, a quantitative detection method for ctDNA HER2 gene copy number variation is developed by using digital PCR technology, and it is applied to the precise diagnosis and treatment of gastric cancer and the research and development of gastric cancer drugs, which can guide the target of gastric cancer HER2 positive patients. It is of great significance for the clinical application of tumor precision medicine to provide individualized treatment of drugs and provide more accurate and non-invasive new molecular typing methods for tumor recurrence monitoring and prognosis judgment;
(2)本发明结合分子生物学、肿瘤学、检验医学等相关理论和技术，采用分子生物学的最新技术，利用双荧光digital PCR技术同时定量HER2基因和参比基因，解决荧光定量PCR定量CNV时定量不准的问题，构建HER2基因异常扩增检测新方法，有利于形成一套完整的基于Digital PCR整套实验方案和标准；(2) The present invention combines the relevant theories and technologies of molecular biology, oncology, laboratory medicine, etc., adopts the latest technology of molecular biology, and uses the dual-fluorescence digital PCR technology to simultaneously quantify the HER2 gene and the reference gene, so as to solve the quantitative PCR quantitative CNV In order to solve the problem of inaccurate quantification, the construction of a new method for the detection of abnormal HER2 gene amplification is conducive to the formation of a complete set of experimental protocols and standards based on Digital PCR;
(3)本发明利用数字PCR技术，确立胃癌患者外周血ctDNA的HER2基因异常扩增阈值，分析胃癌患者术前术后ctDNA的HER2基因异常扩增变化与肿瘤者复发时长、治疗反应性的关系，以期为指导胃癌患者靶向用药、肿瘤复发早期诊断以及预后判断等提供更为简便、无损伤和便于动态监测的新方法。(3) The present invention uses digital PCR technology to establish a threshold for abnormal amplification of HER2 gene in peripheral blood ctDNA of gastric cancer patients, and analyzes the relationship between abnormal amplification changes of HER2 gene in ctDNA of gastric cancer patients before and after surgery and the recurrence time and treatment responsiveness of tumor patients It is expected to provide a new method that is more simple, non-invasive and convenient for dynamic monitoring for guiding targeted drug use, early diagnosis of tumor recurrence and prognosis judgment of gastric cancer patients.
附图说明Description of drawings
图1为本发明中所述检测方法的构建路径分析图。FIG. 1 is an analysis diagram of the construction path of the detection method described in the present invention.
具体实施方式Detailed ways
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案，但下述的实例仅仅是本发明的简易例子，并不代表或限制本发明的权利保护范围，本发明的保护范围以权利要求书为准。The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and through specific embodiments, but the following examples are only simple examples of the present invention, and do not represent or limit the protection scope of the present invention. The request shall prevail.
以下实施例中，若无特殊说明，所以的试剂及耗材均购自本领域常规试剂厂商；若无特殊说明，所用的实验方法和技术手段均为本领域常规的方法和手段。In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.
本发明所述的检测方法依据图1所示的技术路线构建得到，具体包括步骤如下：The detection method of the present invention is constructed according to the technical route shown in FIG. 1, and specifically includes the following steps:
S1、依据Digital PCR建立定量检测HER2的体系S1. Establish a quantitative detection system for HER2 based on Digital PCR
基于Digital PCR技术的ctDNA HER2基因异常扩增定量检测体系建立，首先通过pubmed查询HER2和CEP17参比基因的序列信息，设计引物，从人基因组中调取相关序列，构建质粒，制成标准品；A quantitative detection system for abnormal amplification of ctDNA HER2 gene based on Digital PCR technology was established. First, the sequence information of HER2 and CEP17 reference genes was queried through pubmed, primers were designed, relevant sequences were extracted from the human genome, and plasmids were constructed to make standard products;
再用Primer3软件针对这HER2基因和CEP17参比基因设计引物和探针，并用荧光定量PCR进行验证；Then use Primer3 software to design primers and probes for the HER2 gene and CEP17 reference gene, and use fluorescence quantitative PCR to verify;
S2、优化HER2的检测体系S2. Optimize the detection system of HER2
利用Digital PCR仪将反应体系分成若干个独立的反应体系，使每个液滴中含有少于1个拷贝的目的基因，在设定好的反应条件下完成扩增反应；Divide the reaction system into several independent reaction systems by using a digital PCR instrument, so that each droplet contains less than 1 copy of the target gene, and complete the amplification reaction under the set reaction conditions;
用验证后的引物在dPCR体系对1.2倍倍比稀释标准品进定量，优化反应条件，构建dPCR定量检测HER2基因异常扩增方法；The 1.2-fold dilution standard was quantified in the dPCR system with the verified primers, the reaction conditions were optimized, and a dPCR quantitative detection method for abnormal amplification of HER2 gene was constructed;
S3、建立从外周血标本中提取ctDNA的流程和方法S3. Establish a process and method for extracting ctDNA from peripheral blood samples
抽取外周静脉EDTA抗凝血5mL，采用QIAamp通过裂解、结合、洗涤和洗脱4步，获得纯化和浓缩游离的DNA。通过比较不同方法的提取效率和提取产物的纯度，找出最佳核酸提取方法。5 mL of EDTA anticoagulant was withdrawn from the peripheral vein, and purified and concentrated free DNA was obtained by using QIAamp through 4 steps of lysis, binding, washing and elution. Find out the best nucleic acid extraction method by comparing the extraction efficiency and the purity of the extracted products of different methods.
S4、确定外周血ctDNA中HER2基因异常扩增的阈值S4. Determine the threshold for abnormal amplification of HER2 gene in peripheral blood ctDNA
提取健康对照、经免疫组化、FISH确诊HER2表达情况胃癌患者的外周血ctDNA，用建立的dPCR定量方法对提取的ctDNA中的HER2基因和CEP17参比基因进行定量检测分析，用两者的比值来确定是否为HER2阳性，即两者比值为1为阴性，两者比值大于1为阳性，以此初定判读阈值；Peripheral blood ctDNA was extracted from healthy controls, patients with gastric cancer confirmed by immunohistochemistry and FISH with HER2 expression. The established dPCR quantitative method was used to quantitatively detect and analyze the HER2 gene and CEP17 reference gene in the extracted ctDNA, and the ratio of the two was used. To determine whether it is HER2 positive, that is, if the ratio of the two is 1, it is negative, and if the ratio of the two is greater than 1, it is positive, and the judgment threshold is initially determined;
针对己有IHC和FISH的HER2分型结果的胃癌病例，将dPCR检测ctDNA的HER2基因定量检测结果与临床病理IHC，FISH的HER2分型结果比较，进行敏感性、特异性评价，调整确定最佳判读阈值；For gastric cancer cases with HER2 typing results of IHC and FISH, the HER2 gene quantitative detection results of ctDNA detected by dPCR were compared with the HER2 typing results of clinicopathological IHC and FISH, and the sensitivity and specificity were evaluated and adjusted to determine the best Interpretation threshold;
S5、监测胃癌患者ctDNA的HER2基因异常扩增动态S5. Monitoring abnormal amplification of HER2 gene in ctDNA of gastric cancer patients
开展中晚期胃癌患者术前术后ctDNA的HER2基因异常扩增动态监测，选取进展期胃癌患者，分别于术前、术后一个月及每隔3个月检测外周血ctDNA的HER2；To carry out dynamic monitoring of abnormal amplification of HER2 gene in ctDNA before and after surgery in patients with advanced gastric cancer, and select patients with advanced gastric cancer to detect HER2 in peripheral blood ctDNA before surgery, one month after surgery, and every 3 months;
S6、确定定量检测HER2拷贝数变异在胃癌进展监测中的应用可行性S6. Determine the feasibility of quantitative detection of HER2 copy number variation in the monitoring of gastric cancer progression
探讨定量检测HER2拷贝数变异在胃癌进展监测中的应用可行性，分析外周血ctDNA的HER2基因异常扩增动态变化与胃癌患者复发时长、治疗反应性的关系；同时，与患者同期影像结果比对，探讨定量检测HER2拷贝数变异在胃癌精准治疗中的临床应用前景。To explore the feasibility of quantitative detection of HER2 copy number variation in the monitoring of gastric cancer progression, and to analyze the relationship between the dynamic changes of abnormal amplification of HER2 gene in peripheral blood ctDNA and the recurrence time and treatment response of gastric cancer patients. To explore the clinical application prospect of quantitative detection of HER2 copy number variation in the precision treatment of gastric cancer.
实施例1Example 1
本实施例用于构建HER2基因的定量检测体系。具体步骤如下：This example is used to construct a quantitative detection system for HER2 gene. Specific steps are as follows:
(1)利用软件针对HER2基因和CEP17基因设计引物和Taqman探针，测定不同引物和探针的特异性和灵敏度；(1) Using software to design primers and Taqman probes for HER2 gene and CEP17 gene, and measure the specificity and sensitivity of different primers and probes;
设计并合成引物，对HER2基因和CEP17基因进行扩增，同时构建质粒作为标准品。Primers were designed and synthesized to amplify the HER2 gene and CEP17 gene, and plasmids were constructed as standards.
(2)利用Digital PCR仪将20μL反应体系分成20,000个独立的反应体系，使每个液滴中含有少于1个拷贝的目的基因，完成扩增反应后，用液滴读取仪读取每个液滴的荧光值利用泊松分布计算出初始样本中目的基因的拷贝数；(2) Divide the 20 μL reaction system into 20,000 independent reaction systems using a Digital PCR instrument, so that each droplet contains less than 1 copy of the target gene. After the amplification reaction is completed, read each droplet reader with a droplet reader. The fluorescence value of each droplet uses the Poisson distribution to calculate the copy number of the target gene in the initial sample;
通过HER2基因与CEP17基因拷贝数的比值来确定HER2基因发生异常扩增定量检测；Quantitative detection of abnormal amplification of HER2 gene by the ratio of HER2 gene to CEP17 gene copy number;
(3)用验证后的引物在dPCR体系对1.2倍倍比稀释标准品进定量，对Digital PCR反应条件进行优化包括液滴生成条件、反应温度、以及反应液中各组分浓度；(3) Use the verified primers to quantify the 1.2-fold dilution standard in the dPCR system, and optimize the Digital PCR reaction conditions, including droplet generation conditions, reaction temperature, and the concentration of each component in the reaction solution;
(4)通过分别在健康人血定量添加NCI-N87和MKN-45癌细胞，考察所述检测方法对不同细胞的检测灵敏度和特异性，最终得到HER2基因异常扩增的最佳判读阈值。(4) By quantitatively adding NCI-N87 and MKN-45 cancer cells to healthy human blood, the detection sensitivity and specificity of the detection method for different cells were investigated, and the optimal interpretation threshold for abnormal HER2 gene amplification was finally obtained.
实施例2Example 2
本实施例中用于计算构建所述检测方法所需的样本量。本发明为基于DigitalPCR技术的ctDNA HER2基因异常扩增检测在胃癌精准诊治中的应用研究，属于单个诊断试验。This example is used to calculate the sample size required to construct the detection method. The invention relates to the application research of abnormal amplification detection of ctDNA HER2 gene based on DigitalPCR technology in precise diagnosis and treatment of gastric cancer, and belongs to a single diagnostic test.
以病理FISH结果为金标准，选择胃癌患者组和健康对照组进行研究，评价检测试验对胃癌HER异常扩增表达分型的诊断价值，包括灵敏度和特异度。Using pathological FISH results as the gold standard, gastric cancer patient groups and healthy control groups were selected for the study, and the diagnostic value of the detection test for the genotyping of abnormal HER amplification expression in gastric cancer, including sensitivity and specificity, was evaluated.
样本量的计算依据如下：The sample size is calculated based on the following:
(1)首先，选定置信度1-α；一般情况下，默认选取检验水准：α＝0.05(双侧)，即1-α＝0.95。(1) First, select the confidence level 1-α; in general, select the inspection level by default: α=0.05 (two-sided), that is, 1-α=0.95.
(2)根据既往文献或预实验的结果，设定四个参数，包括：(2) According to the results of previous literature or preliminary experiments, set four parameters, including:
1)预计该方法诊断患者的灵敏度：73％；1) Estimated sensitivity of the method for diagnosing patients: 73%;
2)灵敏度的容许误差：5％；2) Allowable error of sensitivity: 5%;
3)预计该方法诊断非患者的特异度：93％；3) The specificity of the method for diagnosing non-patients is expected to be 93%;
4)特异度的容许误差：10％。4) Tolerance of specificity: 10%.
(3)分别估算灵敏度所需要的样本量(N实验，即患者组的样本量)和特异度所需要的样本量(N对照，即非患者组的样本量)，由于患者和非患者采用相等样本量，因此两组研究对象均取上述最大值。(3) Separately estimate the sample size required for sensitivity (N experiment, that is, the sample size of the patient group) and the sample size required for specificity (N control, that is, the sample size of the non-patient group), since patients and non-patients use equal The sample size, so the two groups of research subjects took the above maximum value.
(4)诊断试验的样本例数计算公式为：(4) The formula for calculating the number of samples for the diagnostic test is:
n＝(μα/δ)2×(1-P灵敏度)×P特异度；n=(μα/δ)2×(1-P sensitivity)×P specificity;
其中，参数μα，因α＝0.05，μα＝Zα/2＝1.960；Among them, the parameter μα, because α=0.05, μα=Zα/2=1.960;
δ：判断界值，根据预试验及相关文献估计，综合取灵敏度或特异度的1/5～1/10，本发明中取δ＝0.l。δ: Judgment threshold, estimated according to pre-tests and related literatures, comprehensively taking 1/5 to 1/10 of the sensitivity or specificity, in the present invention, taking δ=0.1.
P：根据预试验及相关文献估计，P灵敏度＝0.73，P特异度＝0.93。P: Estimated based on pre-tests and related literature, P sensitivity = 0.73, P specificity = 0.93.
代入公式计算求出样本量：N试验组＝75，N对照组＝25例，患者组和非患者对照组采用2:1的样本量，故需要纳入75例患者和37例健康对照，共112例研究对象。Substitute into the formula to calculate the sample size: N test group = 75, N control group = 25 cases, the patient group and non-patient control group use a 2:1 sample size, so 75 patients and 37 healthy controls need to be included, a total of 112 case study object.
实施例3Example 3
本实施例中选择TNM分期诊断为III、IV期的进展期胃癌患者纳入动态监测研究。In this example, patients with advanced gastric cancer diagnosed as stage III and IV by TNM staging were selected to be included in the dynamic monitoring study.
本实施例中所有胃癌患者组织病理均进行IHC和FISH检测。所有患者均行术前、术后血浆标本采集。对照组为健康成人志愿者，并通过标准的肘静脉穿刺采集血浆。All gastric cancer patients in this example were histopathologically detected by IHC and FISH. All patients underwent preoperative and postoperative plasma specimen collection. The control group consisted of healthy adult volunteers and plasma was collected by standard cubital venipuncture.
患者的基本临床信息数据、肿瘤复发监测及后续管理均详细记录。根据实体瘤疗效评价标准(RECIST)，采用CT等影像学检查记录治疗反应；根据UICC分类标准确定肿瘤的病理类型。The patients' basic clinical information data, tumor recurrence monitoring and follow-up management were all recorded in detail. According to the Response Evaluation Criteria in Solid Tumors (RECIST), CT and other imaging examinations were used to record the treatment response; the pathological type of the tumor was determined according to the UICC classification criteria.
根据《胃癌HER2检测指南(2016版)》，组织标本先进行IHC检测，IHC评分为3+，则判读为HER2阳性；同时，对于HER2阳性病例将给予曲妥珠单抗治疗；According to the "Guidelines for HER2 Detection of Gastric Cancer (2016 Edition)", the tissue specimens are first tested by IHC, and the IHC score is 3+, which is interpreted as HER2 positive; at the same time, trastuzumab treatment will be given to HER2 positive cases;
IHC评分为0和1+则判读为阴性；IHC scores of 0 and 1+ are interpreted as negative;
IHC评分为2+时则需采用FISH进行检测，进一步明确HER2表达分型情况。When the IHC score is 2+, FISH should be used to further clarify the HER2 expression typing.
使用本发明所述的检测方法对进展期胃癌患者的ctDNA的HER2基因的扩增动态进行检测和分析，具体步骤如下：The detection method of the present invention is used to detect and analyze the amplification dynamics of the HER2 gene in the ctDNA of patients with advanced gastric cancer, and the specific steps are as follows:
(1)根据外周血标本中提取循环肿瘤DNA的流程和方法，抽取外周静脉血，加入EDTA抗凝血剂，采用QIAamp通过裂解、结合、洗涤和洗脱4步，获得纯化和浓缩游离的DNA；(1) According to the process and method of extracting circulating tumor DNA from peripheral blood samples, peripheral venous blood was extracted, EDTA anticoagulant was added, and purified and concentrated free DNA was obtained by using QIAamp through four steps of lysis, binding, washing and elution ;
(2)分别于术前、术后一个月及每隔3个月检测外周血ctDNA的HER2基因异常扩增动态变化，并详细记录同期CT等影像学及治疗反应性等临床特征信息；(2) Detect the dynamic changes of abnormal amplification of HER2 gene in peripheral blood ctDNA before surgery, one month after surgery, and every 3 months, and record in detail the clinical characteristics such as CT imaging and treatment responsiveness during the same period;
其中，所得的数据使用的数据管理与统计方法包括：Among them, the data management and statistical methods used for the obtained data include:
1、应用SPSS统计软件，IHC联合FISH检测与ddPCR检测HER2结果的一致性检验用Kappa值表示：1. Using SPSS statistical software, the consistency test of the results of IHC combined with FISH detection and ddPCR detection of HER2 is expressed by Kappa value:
当Kappa＞0.75时，认为两法检验一致性好。When Kappa>0.75, the consistency of the two methods is considered to be good.
2、HER2基因扩增与病理学指标的相关性用Spearman相关分析，并计算两者相关系数r和P值；2. The correlation between HER2 gene amplification and pathological indicators was analyzed by Spearman correlation, and the correlation coefficient r and P value of the two were calculated;
3、血浆ctDNA的Her2基因扩增阳性比值与临床病理因素的关系分析采用X2或Fisher精确检验。3. The relationship between the positive ratio of Her2 gene amplification in plasma ctDNA and clinicopathological factors was analyzed by X2 or Fisher's exact test.
本发明中所有进行的统计学检验，除了配对试验，均进行双侧检验，P值<0.05表示差异有统计学意义。All statistical tests carried out in the present invention, except paired tests, were carried out two-sided tests, and a P value <0.05 indicated that the difference was statistically significant.
(3)分析外周血ctDNA的HER2基因异常扩增动态变化与胃癌患者复发时长、治疗反应性的关系；并与患者同期影像结果比对，探讨定量检测HER2拷贝数变异在胃癌精准治疗中的临床应用前景。(3) To analyze the relationship between the dynamic changes of abnormal amplification of HER2 gene in peripheral blood ctDNA and the recurrence time and treatment response of gastric cancer patients; and to compare with the imaging results of patients during the same period to explore the clinical value of quantitative detection of HER2 copy number variation in the precision treatment of gastric cancer. application prospects.
综上所述，本发明所述的检测方法利用数字PCR技术，定量检测ctDNA HER2基因拷贝数，并以正常状态下所得到的拷贝数与参比基因的比值作为判断阈值，同时对判断阈值进行特异性和灵敏度优化得到最佳判读阈值，根据定量检测结果与最佳判读阈值判断HER2基因的扩增情况，将其应用于胃癌的精准诊治中，能够指导胃癌HER2阳性患者的靶向药物个体化治疗，对于肿瘤精准医学临床应用而言具有重要的意义。To sum up, the detection method of the present invention utilizes digital PCR technology to quantitatively detect the copy number of the ctDNA HER2 gene, and uses the ratio of the copy number obtained in the normal state to the reference gene as the judgment threshold, and at the same time, the judgment threshold is carried out. The specificity and sensitivity are optimized to obtain the best interpretation threshold. According to the quantitative detection results and the best interpretation threshold, the amplification of the HER2 gene can be judged, and it can be applied to the precise diagnosis and treatment of gastric cancer, which can guide the individualization of targeted drugs for HER2-positive patients with gastric cancer. Treatment is of great significance for the clinical application of tumor precision medicine.
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。The applicant declares that the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should Changes or substitutions that can be easily conceived within the technical scope all fall within the protection scope and disclosure scope of the present invention.
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