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WO2014190449A1 - 辅助诊断非小细胞肺癌患者的试剂盒 - Google Patents

辅助诊断非小细胞肺癌患者的试剂盒 Download PDF

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Publication number
WO2014190449A1
WO2014190449A1 PCT/CN2013/000629 CN2013000629W WO2014190449A1 WO 2014190449 A1 WO2014190449 A1 WO 2014190449A1 CN 2013000629 W CN2013000629 W CN 2013000629W WO 2014190449 A1 WO2014190449 A1 WO 2014190449A1
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WIPO (PCT)
Prior art keywords
protein marker
concentration
protein
small cell
lung cancer
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PCT/CN2013/000629
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English (en)
French (fr)
Inventor
赫捷
谭锋维
陈照丽
孙楠
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中国医学科学院肿瘤医院
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Priority to PCT/CN2013/000629 priority Critical patent/WO2014190449A1/zh
Publication of WO2014190449A1 publication Critical patent/WO2014190449A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to a kit for assisting diagnosis of a patient with non-small cell lung cancer.
  • Lung cancer is a major disease that seriously threatens human life and health.
  • WHO World Health Organization
  • IAHO International Agency for Research on Cancer
  • lung cancer is the leading malignant tumor with the highest morbidity and mortality.
  • the number of new cases worldwide is 1,600,000, and the number of deaths is 1,377,000, accounting for 12.7% and 18.2% of all malignant tumors.
  • WHO World Health Organization
  • IAHO International Agency for Research on Cancer
  • lung cancer is the leading cause of death in malignant tumors in the sample area, and the crude death rate is 30.83/100,000, of which 41.34/100,000 are male. Women 19.84/100,000; the first cause of cancer death among men and women. With the acceleration of global aging and the increase of environmental pollution, the incidence of lung cancer will continue to rise. Therefore, the demand for the development of diagnosis and treatment of lung cancer must be more and more intense.
  • lung cancer can be divided into non-small cell lung cancer (NSCLC, about 85%) and small cell lung cancer (SCLC, about 15%).
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • lung cancer screening and diagnosis methods are mainly imaging techniques such as chest X-ray examination and CT scanning. Although these imaging data play an important role in diagnosis, there are also many limitations, such as high false positive rate, inability to detect recessive lesions, subclinical lesions, and micrometastatic lesions.
  • diagnosis of lung cancer also includes invasive procedures such as bronchoscopy and perforating biopsy. These methods take a long time and increase patient suffering. Therefore, the search for non-invasive, non-radiative, low-cost, rapid diagnosis, sensitive and specific screening and diagnosis methods is an urgent need for early diagnosis of cancer.
  • Tumor marker refers to a biologically active substance produced by tumor cells or tissues due to abnormal expression of oncogenes or other tumor-related genes and their products, or is itself detached from cancerous tissues, and has a certain degree of expression or yield in normal tissues or benign diseases. Very little. It reflects the development of cancer The process can be detected in tumor tissues, body fluids and excretions, and is widely used in the diagnosis of tumors, monitoring recurrence, metastasis, prognosis, and predictive efficacy.
  • Several major markers that are widely recognized in clinical practice are: Tumor antigen 125 ( CA125 ) for ovarian cancer, antigen 19-9 (CA19-9) for pancreatic cancer, for colorectal cancer.
  • CA125 Tumor antigen 125
  • CA19-9 antigen 19-9
  • PSA prostate specific antigen
  • Other cancers, such as breast and lung cancer still lack credible tumor markers for screening.
  • the markers that have been used in the clinical diagnosis of lung cancer are only protein markers, which are the following:
  • CEA Gold and Freeman first discovered CEA in colorectal cancer in 1965. It is a glycoprotein with a molecular weight of 180KD and a half-life of 3-4 days. It is an antigen produced during embryonic development and drops significantly after birth. Its specificity is not strong, but it can be used for monitoring tumor development, efficacy judgment and prognosis estimation. It is not sensitive to early diagnosis. Although lung cancer patients have elevated, there are high false negatives and false positives, which are not suitable for lung cancer screening. Used alone in the diagnosis;
  • CA125 Bast first discovered CA125, a macromolecular glycoprotein with a half-life of about 4. 8 days, mainly used in ovarian cancer, and also in lung cancer.
  • CYFRA211 is a water-soluble fragment of cytokeratin 19 with a half-life of about 4 days;
  • IDH1 is a NADP + -dependent isocitrate dehydrogenase located in the cytoplasm and peroxisomes that catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate.
  • the inventors of the present invention have found in previous studies that the levels of IDH1 in patients with lung squamous cell carcinoma and lung adenocarcinoma are significantly higher than those in the normal population and benign lung tumors, and can be used as a new diagnostic marker for lung cancer.
  • kits for assisting diagnosis of a patient with non-small cell lung cancer comprising a product for detecting protein marker IDH1, a product for detecting protein marker CA125, a product for detecting protein marker CYFRA21-1, and having recorded
  • the following functional formula: P -
  • the protein marker IDH1 may specifically be a protein represented by GENBANK ACCESSION NO. CAG46496. 1 (sequence 1 of the sequence listing; GENBANK ACCESSION NO. CAG46496. 1; 01: 49456351; linear PRI 16-OCT-2008).
  • the protein marker CA125 may specifically be a protein represented by GENBANK ACCESSION NO. AAL65133. 2 (GENBANK ACCESSION NO.
  • the protein marker CYFRA21-1 may specifically be a protein represented by GENBANK ACCESSION NO. NP-002267. 2 (Sequence Listing 2; 01: 24234699; linear PRI 06- MAY-2012).
  • the unit of the concentration of the protein marker IDH1 is specifically U/L
  • the unit of the concentration of the protein marker CA125 is specifically U/mL
  • the unit of the concentration of the protein marker CYFRA21-1 is specifically ng/mL.
  • the following diagnostic criteria can also be recorded on the carrier: When P 0. 846, the patient to be tested is a candidate for non-small cell lung cancer, when? ⁇ 0. At 846, the patient to be tested was a candidate for non-small cell lung cancer.
  • the present invention also protects a product for detecting the protein marker IDH1, a product for detecting the protein marker CA125, a product for detecting the protein marker CYFRA21-1, and a carrier having a functional formula and a diagnostic standard for preparing a non-diagnostic diagnosis.
  • a product for detecting the protein marker IDH1 a product for detecting the protein marker CA125
  • a product for detecting the protein marker CYFRA21-1 a carrier having a functional formula and a diagnostic standard for preparing a non-diagnostic diagnosis.
  • Xl represents the concentration of the protein marker IDH1, the unit is U/L, x2 represents the concentration of the protein marker CA125, the unit is U/mL, and x3 represents the concentration of the protein marker CYFRA21-1, and the unit is ng/mL;
  • the patient to be tested is a candidate for non-small cell lung cancer, when? ⁇ 0.
  • the patient to be tested was a candidate for non-small cell lung cancer.
  • the protein marker IDH1 may specifically be a protein represented by GENBANK ACCESSION NO. CAG46496. 1 (sequence 1 of the sequence listing; GENBANK ACCESSION NO. CAG46496. 1; 01 : 49456351; Linear PRI 16- OCT- 2008).
  • the protein marker CA125 may specifically be a protein represented by GENBANK ACCESSION NO. AAL65133. 2 (GENBANK ACCESSION NO.
  • the protein marker CYFRA21-1 may specifically be a protein represented by GENBANK ACCESSION NO. NP-002267. 2 (Sequence Listing 2; 01: 24234699; linear PRI 06- MAY-2012).
  • the product for detecting the protein marker IDH1 may specifically be an IDH1 enzyme-linked immunosorbent assay kit (ELISA Kit; including IDH1 standard) purchased from Wuhan Yousheng Technology Co., Ltd.
  • IDH1 enzyme-linked immunosorbent assay kit ELISA Kit; including IDH1 standard
  • the product for detecting the protein marker CYFRA21-1 as described above may specifically be a CYFRA21-1 quantitative assay kit purchased from Roche Diagnostics (Shanghai) Co., Ltd.
  • the product for detecting the protein marker CA125 may specifically be a CA125 quantitative assay kit of Roche Diagnostics (Shanghai) Co., Ltd.
  • the invention also protects a method for assisting diagnosis of a patient with non-small cell lung cancer, comprising the steps of:
  • Xl represents the concentration of the protein marker IDH1, the unit is U/L, x2 represents the concentration of the protein marker CA125, the unit is U/mL, x3 represents the concentration of the protein marker CYFRA21-1, and the unit is ng/mL;
  • the patient to be tested at 846 is a candidate for non-small cell lung cancer, when? ⁇ 0. At 846, the patient to be tested was a candidate for non-small cell lung cancer.
  • the protein marker IDH1 may specifically be a protein represented by GENBANK ACCESSION NO. CAG46496. 1 (sequence 1 of the sequence listing; GENBANK ACCESSION NO. CAG46496. 1; 01: 49456351; linear PRI 16-OCT-2008).
  • the protein marker CA125 may specifically be a protein represented by GENBANK ACCESSION NO. AAL65133. 2 (GENBANK ACCESSION NO.
  • the protein marker CYFRA21-1 may specifically be a protein represented by GENBANK ACCESSION NO. NP-002267. 2 (Sequence Listing 2; 01: 24234699; linear PRI 06- MAY-2012).
  • the product for detecting the concentration of the protein marker IDH1 may specifically be an IDH1 enzyme-linked immunosorbent assay kit (ELISA Kit; purchased from Wuhan Youersheng Technology Co., Ltd.; IDH1 standard).
  • IDH1 enzyme-linked immunosorbent assay kit ELISA Kit; purchased from Wuhan Youersheng Technology Co., Ltd.; IDH1 standard.
  • the product for detecting the concentration of the protein marker CYFRA21-1 may specifically be a CYFRA21-1 quantitative assay kit purchased from Roche Diagnostics (Shanghai) Co., Ltd.
  • the product for detecting the concentration of the protein marker CA125 may specifically be a CA125 quantitative assay kit of Roche Diagnostics (Shanghai) Co., Ltd.
  • Figure 1 shows the R0C curve of the NSCLC diagnostic model.
  • the ordinate is sensitivity and the abscissa is specific.
  • Plasma samples There were 943 plasma samples from NSCLC patients, and healthy people (normal control). A total of 479 plasma samples were taken from patients with lung cancer who were diagnosed by pathology in the Department of Thoracic Surgery, Cancer Hospital of Chinese Academy of Medical Sciences and the normal population who participated in the cancer screening. The experiment was approved by the ethics committee, and the subjects were informed of the informed consent to sign the informed consent form. Plasma samples were obtained before surgery, and patients had not received chemotherapy, radiotherapy, intervention, etc. before.
  • R0C curve (receiver operat ing characteri ic curve) with sensitivity as the ordinate (Y-axis) and specificity as the abscissa (X-axis).
  • the corresponding coordinate points can be generated, and the curve generated by the line is generated. That is the ROC curve.
  • the ROC curve can compare the diagnostic efficiency of different tumor markers, and the larger the area under the curve (AUC), the higher the diagnostic efficiency.
  • the data was analyzed by Logistic binomial regression of SPSS software, and the model was tested using SAS, and a model was finally determined for the diagnosis of NSCLC.
  • the calculated results of the model were plotted on the R0C curve (see Figure 1).
  • the area under the curve was 0.896 (95% confidence interval: 0.871-0.918, p ⁇ 0.0001), and the marker with the highest diagnostic efficiency for NSCLC - IDH1 (AUC Compared with 0.817), there is a significant difference (/ 0.0001).
  • step 1 by performing a large amount of data simulation and model establishment on a large number of clinical samples for detection of various protein markers, the functional formula for assisting diagnosis of non-small cell lung cancer is as follows:
  • Xl represents the concentration of the protein marker IDH1 (unit: U/L)
  • ⁇ 2 represents the concentration of the protein marker CA125 (unit: U/mL)
  • x3 represents the concentration of the protein marker CYFRA21-1 (unit: ng/mL).
  • the protein marker IDH1 concentration, the protein marker CA125 concentration, and the protein marker CYFRA211 concentration in the obtained sample are calculated, and then the p value is calculated by the above functional formula, and the patient to be tested is candidate when P 0.846 Non-small cell lung cancer patients, when? ⁇ 0.846 hours
  • the patient to be tested was a candidate for non-small cell lung cancer.
  • Example 2 verification of the method of the invention
  • the patients used in this example were 150 patients with clinically diagnosed non-small cell lung cancer (peripheral venous blood was obtained before surgery, patients had not received chemotherapy, radiotherapy, intervention, etc.) and 50 healthy people (not non-small) Patients with cell lung cancer) are volunteers with informed consent.
  • step 2 Dissolve the sample from step 1 for 3 h at room temperature, then centrifuge at 3000 r/min for 5 min.
  • the protein marker IDH1 may specifically be a protein represented by GENBANK ACCESSION NO. CAG46496. 1 (sequence 1 of the sequence listing; GENBANK ACCESSION NO. CAG46496. 1; 01: 49456351; linear PRI 16-OCT-2008).
  • the protein marker CA125 may specifically be a protein represented by GENBANK ACCESSION NO. AAL65133. 2 (GENBANK ACCESSION NO.
  • the protein marker CYFRA21-1 may specifically be a protein represented by GENBANK ACCESSION NO. NP-002267. 2 (sequence 2 of the list; 01: 24234699; linear PRI 06- MAY-2012).
  • the protein marker CEA can specifically be a protein represented by GENBANK ACCESSION NO. CAE75559. 1 (sequence 3 of the sequence listing; GENBANK ACCESSION NO. CAE75559. 1; 01 : 38522495; linear PAT 25-NOV- 2003).
  • the IDH1 enzyme-linked immunosorbent assay kit (ELISA Kit; including IDH1 standard; catalog number E97839Hu 96T) purchased from Wuhan Youersheng Technology Co., Ltd. was used and the concentration of the protein marker IDH1 was determined according to the instructions of the kit.
  • the CYFRA21-1 Quantitative Assay Kit (catalog number 11820966122) purchased from Roche Diagnostics (Shanghai) Co., Ltd. was used and the concentration of the protein marker CYFRA21-1 was measured according to the kit instructions.
  • the concentration of the protein marker CEA was determined using the CEA Quantitative Assay Kit from Roche Diagnostics (Shanghai) Co., Ltd. and following the instructions in the kit (Cat. No. 11731629322).
  • the CA125 Quantitative Assay Kit (Cat. No. 11776223822) from Roche Diagnostics (Shanghai) Co., Ltd. was used and the concentration of the protein marker CA125 was measured according to the kit instructions.
  • the results of steps 3 and 4 are shown in Tables 1 and 2 (in gender, 1 represents male and 2 represents female).
  • Tables 1 and 2 the diagnosis results of the respective individual protein markers were used as a control for the results of the diagnosis using the functional formula of Example 1.
  • the protein marker IDH1 was used alone, according to the conventional threshold, when the concentration was 2.19, the patient to be tested was a candidate non-small cell lung cancer patient, and when the concentration was ⁇ 2.19, the patient to be tested was a candidate for non-small cell lung cancer.
  • Protein marker alone when the concentration is 35, the patient to be tested is a candidate non-small cell lung cancer patient, and when the concentration is ⁇ 35, the patient to be tested is a candidate for non-small cell lung cancer.
  • the protein marker CYFRA21-1 when used alone, according to the conventional threshold, when the concentration was 3.3, the patient to be tested was a candidate non-small cell lung cancer patient, and when the concentration was ⁇ 3.3, the patient to be tested was a candidate for non-small cell lung cancer.
  • the protein marker CEA when used alone, according to the conventional threshold, when the concentration is 5, the patient to be tested is a candidate non-small cell lung cancer patient, and when the concentration is ⁇ 5, the patient to be tested is a candidate for non-small cell lung cancer.
  • the protein marker IDH1 and its threshold were used alone for the determination.
  • the sensitivity was 43.33% and the specificity was 92%.
  • the protein marker CA125 and its threshold were used alone to determine the sensitivity, with a sensitivity of 13.33% and a specificity of 98%.
  • the protein marker CYFRA21-1 and its threshold were used alone to determine the sensitivity, with a sensitivity of 55.33% and a specificity of 82%.
  • the protein marker CEA and its threshold were used alone to determine the sensitivity, with a sensitivity of 25.33% and a specificity of 72%. Taking into account the sensitivity and specificity, the functional formula and diagnostic method of Example 1 is significantly superior to the use of several separate protein markers for diagnosis.
  • Lung cancer ranks first in the world for cancer deaths and is the number one cancer killer that harms the health of our people. Therefore, looking for early diagnosis of lung cancer, individual treatment, prognosis and monitoring of recurrence Tumor markers have become a hot spot in recent years.
  • the kit provided by the present invention and assisting diagnosis of non-small cell lung cancer patients according to the corresponding diagnostic criteria, the sensitivity and the specificity are strong, and the reliability of the diagnosis is much higher than that of using each individual protein marker for diagnosis. It has great value and application prospects for the diagnosis and treatment of lung cancer.

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Abstract

本发明公开了一种辅助诊断非小细胞肺癌患者的试剂盒。本发明提供的试剂盒包括用于检测蛋白标志物IDH1的产品、用于检测蛋白标志物CA125的产品、用于检测蛋白标志物CYFRA21-1的产品和记载有如函数式的载体：式I。x1代表IDH1的浓度；x2代表CA125的浓度；x3代表CYFRA21-1的浓度。采用本发明提供的试剂盒并按照相应的诊断标准辅助诊断非小细胞肺癌患者，具有灵敏性高、特异性强的特点，诊断结果可信度远远高于采用各个单独的蛋白质标记物进行诊断，对于非小细胞肺癌的诊断和治疗具有重大价值和应用前景。

Description

辅助诊断非小细胞肺癌患者的试剂盒 技术领域
本发明涉及一种辅助诊断非小细胞肺癌患者的试剂盒。
背景技术
肺癌是严重威胁人类生命和健康的重大疾病。 2011年世界卫生组织 (WHO)国际癌症研究机构 IARC公布的 2008全球统计资料结果显示： 肺癌是 发病率和死亡率都高居榜首的恶性肿瘤。 每年全球新发病例数 160. 8万， 死亡病例数 137. 7万， 占全部恶性肿瘤的 12. 7%和 18. 2%。 美国癌症协会公 布的统计结果显示： 预计在 2012年， 美国新发肺癌病例数将达到 22. 6万， 因肺癌死亡病例数将达到 16万。 在我国， 由卫生部主持的全国第三次死因 回顾抽样调查结果显示： 肺癌为样本地区恶性肿瘤死亡原因之首， 粗死亡 率为 30. 83/10万， 其中男性 41. 34/10万， 女性 19. 84/10万； 在男性、 女性 中均为首位癌症死亡原因。 随着全球老龄化的加速和环境污染加剧等因素， 肺癌发病必将继续呈上升趋势， 因此，对肺癌的诊断治疗研究的进展需求， 必定越来越强烈。
从病理上， 肺癌可分为非小细胞肺癌 （NSCLC, 约占 85%) 和小细胞肺 癌 ( SCLC, 约占 15%) 。
肺癌的发病较为隐匿， 超过 70%的患者在就诊时已发展至中晚期， 失 去了手术机会。 这也是肺癌疗效差， 死亡率高的主要原因之一。 因此， 早 期筛查、 诊断成为防治肺癌的重中之重。 目前， 临床所使用肺癌筛查诊断 方法主要是胸部 X射线检查、 CT扫描等影像学技术。 虽然这些影像资料对 诊断起着重要作用， 但也存在很多局限性， 例如假阳性率高， 无法检出隐 性病灶、 亚临床病灶和微小转移病灶。 除此之外， 对肺癌的诊断方法还包 括支气管镜检查、穿剌活检等有创操作，这类方法耗时长，增加患者痛苦。 因此， 寻找无创、 无辐射、 费用低廉、 诊断快速且敏感性、 特异性均高的 筛查、 诊断方法， 是目前癌症早期诊断的迫切需求。
肿瘤标记物是指肿瘤细胞或者组织由于癌基因或其他肿瘤相关基因 及其产物异常表达所产生的生物活性物质或其本身由癌组织脱落， 而在正 常组织或良性疾病时有一定程度表达或产量甚微。 它反映了癌的发生发展 过程，可在肿瘤患者组织、体液及排泄物中检出，广泛应用于肿瘤的诊断， 监测复发、 转移， 预后， 预测疗效等。 临床上现有的广泛受到认可的几种 主要标志物为： 应用于卵巢癌的肿瘤抗原 125 ( CA125 ) ， 应用于胰腺癌的 类抗原 19-9 (CA19-9) , 应用于结直肠癌的癌胚抗原 （CEA) 和应用于前列 腺癌的前列腺特异性抗原 （PSA) 。 而其他癌症例如乳腺癌和肺癌， 目前 仍然缺少用于筛查的可信的肿瘤标记物。
目前已经应用于肺癌临床辅助诊断的标记物只有蛋白标记物， 为下列 几种：
( 1 ) CEA: Gold和 Freeman在 1965年首先在大肠癌中发现 CEA， 它 是一种糖蛋白， 分子量为 180KD, 半衰期为 3-4天， 是胚胎发展过程产生 的抗原， 出生后显著下降； 其特异性不强， 但可用于肿瘤发展的监测、 疗 效判断和预后估计， 对于早期诊断并不敏感； 肺癌病人虽有升高， 但存在 较高的假阴性和假阳性， 不适于在肺癌筛查诊断中单独使用；
( 2 ) CA125: Bast在 1981首先发现 CA125这种大分子糖蛋白， 其半 衰期约 4. 8天， 主要应用于卵巢癌， 同时在肺癌中也有上升；
( 3 ) CYFRA21-1 ( CYFRA211 ) ： CYFRA211是细胞角蛋白 19的水溶性 片段， 半衰期约 4天；
虽然目前以上蛋白标记物已经用于肺癌的辅助诊断， 但其诊断效率低 下，达不到临床需求。因此，开发新的诊断标记物和诊断方法已迫不及待。
IDH1是一种位于细胞质和过氧化物酶体中的 NADP+依赖的异柠檬酸脱 氢酶， 可催化异柠檬酸进行氧化脱羧反应为 a -酮戊二酸。 本发明的发明 人在以往的研究中发现， 肺鳞癌和肺腺癌患者的 IDH1 水平明显高于正常 人群及肺良性肿瘤患者， 能够作为肺癌的新的诊断标记物。
发明公开
本发明的目的是提供一种辅助诊断非小细胞肺癌患者的试剂盒。 本发明提供的辅助诊断非小细胞肺癌患者的试剂盒， 包括用于检测蛋 白标志物 IDH1 的产品、 用于检测蛋白标志物 CA125 的产品、 用于检测蛋 白标志物 CYFRA21-1的产品和记载有如下函数式的载体： P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι xl代表蛋白标志物 IDH1的浓度， χ2代表蛋白标志物 CA125的浓度， χ3 代表蛋白标志物 CYFRA21-1的浓度。
所述蛋白标志物 IDH1具体可为 GENBANK ACCESSION NO. CAG46496. 1所示 的蛋白质（序列表的序列 1 ; GENBANK ACCESSION NO. CAG46496. 1； 01 : 49456351； linear PRI 16- OCT- 2008) 。 所述蛋白标志物 CA125具体可为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质 （GENBANK ACCESSION NO.
AAL65133. 2； 01 : 24419041； linear PRI 29- OCT- 2002 ) 。 所述蛋白标志物 CYFRA21-1具体可为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质 （序 列表的序列 2; 01 : 24234699； linear PRI 06- MAY- 2012 ) 。
所述蛋白标志物 IDH1的浓度的单位具体为 U/L， 所述蛋白标志物 CA125 的浓度的单位具体为 U/mL， 所述蛋白标志物 CYFRA21-1的浓度的单位具体 为 ng/mL。
所述载体上还可记载有如下诊断标准： 当 P 0. 846时待测患者为候选 的非小细胞肺癌患者， 当？<0. 846时待测患者为候选的非非小细胞肺癌患 者。
本发明还保护用于检测蛋白标志物 IDH1 的产品、 用于检测蛋白标志 物 CA125的产品、 用于检测蛋白标志物 CYFRA21-1的产品和记载有函数式 和诊断标准的载体在制备辅助诊断非小细胞肺癌患者的试剂盒中的应用： 所述函数式如下：
P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度、 单位为 U/L， x2代表蛋白标志物 CA125 的浓度、单位为 U/mL， x3代表蛋白标志物 CYFRA21-1的浓度、单位为 ng/mL; 所述诊断标准如下：
当 P 0. 846时待测患者为候选的非小细胞肺癌患者， 当？<0. 846时待 测患者为候选的非非小细胞肺癌患者。
所述蛋白标志物 IDH1具体可为 GENBANK ACCESSION NO. CAG46496. 1所示 的蛋白质（序列表的序列 1 ; GENBANK ACCESSION NO. CAG46496. 1； 01 : 49456351； linear PRI 16- OCT- 2008) 。 所述蛋白标志物 CA125具体可为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质 （GENBANK ACCESSION NO.
AAL65133. 2； 01 : 24419041； linear PRI 29- OCT- 2002 ) 。 所述蛋白标志物 CYFRA21-1具体可为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质 （序 列表的序列 2; 01 : 24234699； linear PRI 06- MAY- 2012 ) 。
以上任一所述用于检测蛋白标志物 IDH1的产品具体可为购自武汉优 尔生科技有限公司的 IDH1酶联免疫吸附测定试剂盒 (ELISA Kit ; 其中包括 IDH1标准品）。
以上任一所述用于检测蛋白标志物 CYFRA21-1的产品具体可为购自罗 氏诊断产品 （上海） 有限公司的 CYFRA21-1定量测定试剂盒。
以上任一所述用于检测蛋白标志物 CA125的产品具体可为罗氏诊断产 品 （上海） 有限公司的 CA125定量测定试剂盒。
本发明还保护一种辅助诊断非小细胞肺癌患者的方法， 包括如下步骤:
( 1 ) 分别检测待测患者外周静脉血中蛋白标志物 IDH1的浓度、 蛋白 标志物 CA125的浓度和蛋白标志物 CYFRA21-1的的浓度；
( 2 ) 采用如下函数式进行辅助诊断：
P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度、 单位为 U/L， x2代表蛋白标志物 CA125 的浓度、单位为 U/mL， x3代表蛋白标志物 CYFRA21-1的浓度、单位为 ng/mL; 当 P 0. 846时待测患者为候选的非小细胞肺癌患者， 当？<0. 846时待测患 者为候选的非非小细胞肺癌患者。
所述蛋白标志物 IDH1具体可为 GENBANK ACCESSION NO. CAG46496. 1所示 的蛋白质（序列表的序列 1 ; GENBANK ACCESSION NO. CAG46496. 1； 01 : 49456351； linear PRI 16- OCT- 2008) 。 所述蛋白标志物 CA125具体可为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质 （GENBANK ACCESSION NO.
AAL65133. 2； 01 : 24419041； linear PRI 29- OCT- 2002 ) 。 所述蛋白标志物 CYFRA21-1具体可为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质 （序 列表的序列 2; 01 : 24234699； linear PRI 06- MAY- 2012 ) 。
用于检测所述蛋白标志物 IDH1的浓度的产品具体可为购自武汉优尔 生科技有限公司的 IDH1酶联免疫吸附测定试剂盒（ELISA Kit ; 其中包括 IDH1标准品）。
用于检测所述蛋白标志物 CYFRA21-1的浓度的产品具体可为购自罗氏 诊断产品 （上海） 有限公司的 CYFRA21-1定量测定试剂盒。
用于检测所述蛋白标志物 CA125的浓度的产品具体可为罗氏诊断产品 (上海） 有限公司的 CA125定量测定试剂盒。
附图说明
图 1为 NSCLC诊断模型的 R0C曲线， 纵坐标为灵敏性， 横坐标为特异 性。
实施发明的最佳方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施 例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试 验材料， 如无特殊说明， 均为自常规生化试剂商店购买得到的。 以下实施 例中的定量试验， 均设置三次重复实验， 结果取平均值。 灵敏性： 诊断为 阳性的癌症患者数目占总癌症患者数目的百分比。 特异性： 诊断为阴性的 健康人数目占总健康人群数目的百分比。 实施例 1、 本发明方法的建立
一、 模型的建立
NSCLC患者血浆样品 943例， 健康人 （正常对照） 血浆样品共 479例， 取自中国医学科学院肿瘤医院胸外科就诊并经病理确诊的肺癌患者及参 加癌症普查的正常人群。 本实验经伦理委员会同意， 且受试者均被知情告 知病签署知情同意书。血浆样本均在手术前获得， 患者此前并未接受化疗、 放疗、 介入等治疗。
数据处理使用 curve expert 1. 4处理标准曲线； GraphPad Pri sm 5. 01 处理 Column统计图； SPSS 13. 0进行数据拟合， 建立数学模型； SAS 9. 2进 行数学模型检验； MedCalc 9. 6. 2. 0版本计算模型 ROC曲线及 cutoff值； Adobe i l lustrator CS3进行图片处理。
R0C曲线 ( receiver operat ing characteri st ic curve , 受试者工作 曲线） 以灵敏性为纵坐标 （Y轴） ， 以特异性为横坐标 （X轴） ， 当给试验 不同临界值 （cutoff ) 时， 即可产生相应的坐标点， 连成线所产生的曲线 即 ROC曲线。 ROC曲线能比较不同肿瘤标志物的诊断效率， 曲线下面积（AUC) 越大者诊断效率越高。
通过 SPSS软件的 Logistic二项回归回归分析数据， 并且使用 SAS进行 模型检验， 最终确定了一个模型， 用于 NSCLC的诊断。 将该模型的计算结 果绘制 R0C曲线（见图 1)，曲线下面积为 0.896(95%置信区间：0.871-0.918， p<0.0001) ， 与对 NSCLC诊断效率最高的标记物—— IDH1 (AUC为 0.817) 相比， 具有显著性差异 （/ 0.0001) 。
二、 模型的具体描述
步骤一中， 通过对大量临床样本进行各个蛋白标志物的检测， 进行大 量数据模拟和模型建立， 得到用于辅助诊断非小细胞肺癌的函数式如下：
p = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度（单位为 U/L)， χ2代表蛋白标志物 CA125 的浓度（单位为 U/mL)，x3代表蛋白标志物 CYFRA21-1的浓度（单位为 ng/mL)。
基于上述函数式辅助诊断非小细胞肺癌的方法如下：
(1) 取待测患者空腹状态下的外周静脉血， 室温静置 lh， 然后 3000 r/min离心 15min， 取上清， 分装 1.5mL EP管， -80°C冰箱保存。
(2) 检测步骤 （1) 得到的样本中的蛋白标志物 IDH1 浓度、 蛋白标 志物 CA125浓度和蛋白标志物 CYFRA211浓度，然后通过上述函数式计算 p 值， 当 P 0.846时待测患者为候选的非小细胞肺癌患者， 当？<0.846时 待测患者为候选为非非小细胞肺癌患者。 实施例 2、 本发明方法的验证
用于本实施例的患者为 150例临床确诊的非小细胞肺癌患者 （外周静 脉血均在手术前获得， 患者此前并未接受化疗、 放疗、 介入等治疗） 和 50 例健康人 （非非小细胞肺癌患者） ， 均为知情同意的志愿者。
1、 取待测患者空腹状态下的 5ml 外周静脉血， 室温静置 lh， 然后
3000r/min离心 15min， 取上清， 分装于两个 1.5mL EP管， -80°C冰箱保 存。
2、 在室温中溶解步骤 1的样本 3h， 然后 3000 r/min离心 5min。
3、 检测步骤 2得到的样本 （上清） 中的蛋白标志物 IDH1浓度 （单位 为 U/L) 、 蛋白标志物 CA125浓度 （单位为 U/mL) 、 蛋白标志物 CYFRA211 浓度 （单位为 ng/mL) 和蛋白标志物 CEA浓度 （单位为 ng/mL ) 。
所述蛋白标志物 IDH1具体可为 GENBANK ACCESSION NO. CAG46496. 1所示 的蛋白质（序列表的序列 1 ; GENBANK ACCESSION NO. CAG46496. 1； 01 : 49456351； linear PRI 16- OCT- 2008) 。 所述蛋白标志物 CA125具体可为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质 （GENBANK ACCESSION NO.
AAL65133. 2； 01 : 24419041； linear PRI 29- OCT- 2002 ) 。 所述蛋白标志物 CYFRA21-1具体可为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质 （序 列表的序列 2; 01 : 24234699； linear PRI 06- MAY- 2012 )。 蛋白标志物 CEA 具体可为 GENBANK ACCESSION NO. CAE75559. 1所示的蛋白质 （序列表的序列 3； GENBANK ACCESSION NO. CAE75559. 1； 01 : 38522495； linear PAT 25- NOV- 2003) 。
采用购自武汉优尔生科技有限公司的 IDH1酶联免疫吸附测定试剂盒 (ELISA Kit ; 其中包括 IDH1标准品； 产品目录号为 E97839Hu 96T)并按试 剂盒的说明检测蛋白标志物 IDH1的浓度。
采用购自罗氏诊断产品 （上海） 有限公司的 CYFRA21-1定量测定试剂 盒 （产品目录号为 11820966122 ) 并按试剂盒的说明检测蛋白标志物 CYFRA21-1的浓度。
采用罗氏诊断产品 （上海） 有限公司的 CEA定量测定试剂盒并按试剂 盒 （产品目录号为 11731629322 ) 的说明检测蛋白标志物 CEA的浓度。
采用罗氏诊断产品 （上海） 有限公司的 CA125定量测定试剂盒 （产品 目录号为 11776223822 )并按试剂盒的说明检测蛋白标志物 CA125的浓度。
4、 将步骤 3得到的蛋白标志物 IDH1浓度、 蛋白标志物 CA125浓度和蛋 白标志物 CYFRA21-1浓度代入实施例 1的函数式， 得到 p值， 根据实施例 1的 标准判断患者是否为非小细胞肺癌患者。
步骤 3和步骤 4的结果见表 1和表 2 (性别中， 1代表男性， 2代表女性）。 表 1和表 2中， 将根据各个单个蛋白标志物的诊断结果作为采用实施例 1的函数式进行诊断的结果的对照。 单独采用蛋白标志物 IDH1时， 按照常 规阈值， 当浓度 2. 19时待测患者为候选的非小细胞肺癌患者， 当浓度 < 2. 19时待测患者为候选为非非小细胞肺癌患者。 单独采用蛋白标志物 CA125时， 按照常规阈值， 当浓度 35时待测患者为候选的非小细胞肺癌 患者， 当浓度 <35时待测患者为候选为非非小细胞肺癌患者。 单独采用蛋 白标志物 CYFRA21-1时， 按照常规阈值， 当浓度 3.3时待测患者为候选的 非小细胞肺癌患者， 当浓度 <3.3时待测患者为候选为非非小细胞肺癌患 者。 单独采用蛋白标志物 CEA时， 按照常规阈值， 当浓度 5时待测患者为 候选的非小细胞肺癌患者， 当浓度 <5时待测患者为候选为非非小细胞肺 癌患者。
表 1确诊非小细胞肺癌患者患者（+代表诊断阳性， -代表诊断阴性)
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330 2 65 2. 72 0. 774517 7. 39 3. 92 + 0. 50531
331 2 59 2. 69 6. 771228 + 13. 7 2. 77 0. 99893 +
332 1 51 13. 63 + 2. 111876 11. 78 3. 79 + 0. 86305 +
333 2 70 1. 55 0. 430524 8. 56 1. 86 0. 20324
334 1 57 0. 974 0. 69933 10. 21 1. 78 0. 27509
335 1 57 8. 57 + -0. 58369 11. 82 2. 96
336 1 57 6. 97 + 0. 231979 1. 47 0. 902 0. 06595
337 1 63 5. 55 + 1. 075089 19. 07 1. 65 0. 52181
338 1 58 12. 65 + 2. 407226 + 5. 1 1. 95 0. 66741
339 2 70 3. 33 0. 681601 38. 16 + 1. 72 0. 76061
340 1 67 3. 02 1. 161651 11. 85 3. 59 + 0. 65542
341 1 66 1. 76 0. 642938 9. 49 1. 41 0. 21681
342 2 58 1. 03 0. 364447 9. 66 3. 04 0. 32350
343 2 68 1. 98 1. 414274 10. 21 2. 55 0. 56501
344 2 54 1. 15 0. 463013 7. 6 1. 61
345 2 43 0. 817 1. 344979 18. 12 2. 07 0. 63312
346 2 65 4. 78 0. 745316 23. 11 2. 27 0. 58566
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347 2 49 0. 957 1. 207906 9. 65 1. 45 0. 35484
348 2 57 1. 19 1. 695267 7. 42 1. 44 0. 4468O C5
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349 2 53 1. 35 -0. 28141 11. 56 1. 72
350 2 55 2. 26 0. 37326 12. 4 1. 74 0. 23214
按照实施例 1的函数式及诊断方法进行结果判定， 灵敏性为 62%， 特 异性为 92%。 单独采用蛋白标志物 IDH1及其阈值进行结果判定， 灵敏性为 43. 33%， 特异性为 92%。 单独采用蛋白标志物 CA125及其阈值进行结果判 定， 灵敏性为 13. 33%， 特异性为 98%。 单独采用蛋白标志物 CYFRA21-1及 其阈值进行结果判定， 灵敏性为 55. 33%， 特异性为 82%。 单独采用蛋白标 志物 CEA及其阈值进行结果判定， 灵敏性为 25. 33%， 特异性为 72%。 综合 考虑灵敏性和特异性， 采用实施例 1的函数式和诊断方法显著优于采用几 个单独的蛋白标志物进行诊断。
工业应用
肺癌居于世界癌症死亡原因的首位， 也是危害我国人民健康的头号癌 症杀手。 因此， 寻找可用于肺癌早期诊断、 个体治疗、 预后和监测复发的 肿瘤标记物， 成为近年研究的热点。 采用本发明提供的试剂盒并按照相应 的诊断标准辅助诊断非小细胞肺癌患者，具有灵敏性高、特异性强的特点， 诊断结果可信度远远高于采用各个单独的蛋白质标记物进行诊断， 对于肺 癌的诊断和治疗具有重大价值和应用前景。

Claims

权利要求
1、 辅助诊断非小细胞肺癌患者的试剂盒， 包括用于检测蛋白标志物 IDH1 的产品、 用于检测蛋白标志物 CA125 的产品、 用于检测蛋白标志物 CYFRA21-1的产品和记载有如下函数式的载体：
P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度， x2代表蛋白标志物 CA125的浓度， x3 代表蛋白标志物 CYFRA21-1的浓度。
2、 如权利要求 1所述的试剂盒， 其特征在于： 所述蛋白标志物 IDH1为 GENBANK ACCESSION NO. CAG46496. 1所示的蛋白质； 所述蛋白标志物 CA125 为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质； 所述蛋白标志物 CYFRA21-1为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质。
3、如权利要求 1或 2所述的试剂盒， 其特征在于： 所述蛋白标志物 IDH1 的浓度的单位为 U/L， 所述蛋白标志物 CA125的浓度的单位为 U/mL， 所述蛋 白标志物 C YFRA21 - 1的浓度的单位为 ng/mL。
4、 如权利要求 3所述的试剂盒， 其特征在于： 所述载体上还记载有如 下诊断标准： 当 P 0. 846时待测患者为候选的非小细胞肺癌患者， 当？< 0. 846时待测患者为候选的非非小细胞肺癌患者。
5、 用于检测蛋白标志物 IDH1的产品、 用于检测蛋白标志物 CA125的 产品、 用于检测蛋白标志物 CYFRA21-1的产品和记载有函数式和诊断标准 的载体在制备辅助诊断非小细胞肺癌患者的试剂盒中的应用：
所述函数式如下：
P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度、 单位为 U/L， x2代表蛋白标志物 CA125 的浓度、单位为 U/mL， x3代表蛋白标志物 CYFRA21-1的浓度、单位为 ng/mL; 所述诊断标准如下： 当 P 0. 846时待测患者为候选的非小细胞肺癌患者， 当？< 0. 846时待测患者为候选的非非小细胞肺癌患者。
6、 如权利要求 5所述的应用， 其特征在于： 所述蛋白标志物 IDH1为 GENBANK ACCESSION NO. CAG46496. 1所示的蛋白质； 所述蛋白标志物 CA125 为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质； 所述蛋白标志物 CYFRA21- 1为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质。
7、 辅助诊断非小细胞肺癌患者的方法， 包括如下步骤：
( 1 ) 分别检测待测患者外周静脉血中蛋白标志物 IDH1的浓度、 蛋白 标志物 CA125的浓度和蛋白标志物 CYFRA21-1的的浓度；
( 2 ) 采用如下函数式进行辅助诊断：
P = -
1 + (0.030 * 3.177χι * 1.081χ2 * 1.690χ3)-ι
xl代表蛋白标志物 IDH1的浓度、 单位为 U/L， x2代表蛋白标志物 CA125 的浓度、单位为 U/mL， x3代表蛋白标志物 CYFRA21-1的浓度、单位为 ng/mL; 当 P 0. 846时待测患者为候选的非小细胞肺癌患者， 当？< 0. 846时待测患 者为候选的非非小细胞肺癌患者。
8、 如权利要求 7所述的方法， 其特征在于： 所述蛋白标志物 IDH1为 GENBANK ACCESSION NO. CAG46496. 1所示的蛋白质； 所述蛋白标志物 CA125 为 GENBANK ACCESSION NO. AAL65133. 2所示的蛋白质； 所述蛋白标志物 CYFRA21-1为 GENBANK ACCESSION NO. NP— 002267. 2所示的蛋白质。
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