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WO2023065609A1 - 确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用 - Google Patents

确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用 Download PDF

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WO2023065609A1
WO2023065609A1 PCT/CN2022/085212 CN2022085212W WO2023065609A1 WO 2023065609 A1 WO2023065609 A1 WO 2023065609A1 CN 2022085212 W CN2022085212 W CN 2022085212W WO 2023065609 A1 WO2023065609 A1 WO 2023065609A1
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gastric cancer
early
gastric
lamc2
risk
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PCT/CN2022/085212
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English (en)
French (fr)
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李梢
张鹏
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清华大学
李梢
张鹏
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Priority to JP2024524380A priority Critical patent/JP7575835B1/ja
Publication of WO2023065609A1 publication Critical patent/WO2023065609A1/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
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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  • the present invention relates to a combination of markers and an assay system for assisting in determining the risk of very early occurrence of gastric cancer and other gastrointestinal tumors and assessing the risk of progression of gastric precancerous lesions such as low-grade dysplasia.
  • gastric cancer Early diagnosis of gastric cancer is of great significance. As a major subtype of gastric cancer, intestinal type gastric cancer will undergo a series of precancerous lesions such as dysplasia. Assessing the risk of progression from gastric precancerous lesions to gastric cancer and achieving very early diagnosis of gastric cancer is of great significance for the prevention and treatment of gastric cancer.
  • gastritis cancer takes a long time, and it is difficult to define the key transformation point of gastritis cancer transformation, that is, the "cancer initiation point", which is the key difficulty in the early diagnosis of gastric cancer.
  • the inventor established the world's first gastritis cancer transformation single-cell atlas, and discovered a group of very early gastric cancer cells that appeared in the low-grade dysplasia stage of the stomach and had a high risk of canceration, and used these cells as a marker for the diagnosis of very early gastric cancer [1][2].
  • Tumors in other organs of the digestive system such as the intestine, esophagus, and pancreas, also undergo a series of precancerous lesions.
  • a series of precancerous lesions Taking the molecular characteristics of very early gastric cancer cells as the starting point, it is expected to establish a common method for the early diagnosis of digestive system cancer, which is also of great significance to the prevention and treatment of digestive system cancer.
  • the present invention provides a reagent for detecting the expression level of a very early gastric cancer cell marker combination (including KLK10, KRT7 and LAMC2) in the preparation for evaluating the risk of low-grade dysplasia progressing to gastric cancer and determining the risk of very early gastric cancer.
  • the marker combination includes KRT7, KLK10 and LAMC2.
  • the cells marked by the above molecular combination at the stage of low-grade dysplasia are defined as the very early stage of gastric cancer cells, and the stage at which the very early cells of gastric cancer appear is defined as the very early stage of gastric cancer.
  • This stage is the critical time point when low-grade dysplasia and other gastric precancerous lesions transform into gastric cancer, which is also the starting point of canceration.
  • the content of very early gastric cancer cells in the sample is quantified, and then used as an important basis for assessing the risk of low-grade dysplasia progressing to gastric cancer and the early diagnosis of gastric cancer.
  • the present invention further prepares related monoclonal antibodies, which can more specifically detect the expression of marker molecules from clinical samples.
  • the present invention has developed a set of kits for detecting and evaluating the content of very early gastric cancer cells from clinical tissue samples, and developed a system for accurately determining the risk of very early gastric cancer, which is expected to be helpful for gastric cancer, etc. It provides an effective basis for patients with systemic tumors to take relevant treatment measures or decision-making, and has a good prospect for clinical application.
  • a reagent for detecting the expression level of a combination of markers for use in the preparation of a product for determining the diagnosis of very early gastric cancer, wherein the components of the combination of markers include KLK10, KRT7, and LAMC2.
  • the combinations include: 1) KLK10 and LAMC2; 2) KRT7 and LAMC2; 3) KRT7, KLK10 and LAMC2.
  • the provided reagents include self-prepared KLK10 and LAMC2 monoclonal antibodies.
  • the site sequence of the KLK10 monoclonal antibody is:
  • the site sequence of the monoclonal antibody to LAMC2 is:
  • an immunohistochemical detection kit which is used for immunostaining the protein expression level of one or more combinations of the three protein molecules KLK10, LAMC2, and KRT7, and then determining the very early stage cells of gastric cancer Content, auxiliary diagnosis of very early gastric cancer.
  • a combination of the three molecules KLK10, LAMC2 and KRT7 as a feature of a very early stage cell population of gastric cancer and for determining the risk of progression of low grade dysplasia to gastric cancer.
  • the combination of three kinds of molecules KLK10, LAMC2 and KRT7 is used to determine the characteristics of changes from precancerous lesions to early cancers in tissues such as pancreas, intestines and esophagus to determine early pancreatic cancer, intestinal cancer, etc. and use in esophageal cancer.
  • the present invention provides the use of correlating the expression levels of the three molecules KLK10, LAMC2 and KRT7 with the survival risk of postoperative recurrence of gastric cancer and using this correlation to determine the risk of postoperative recurrence of gastric cancer.
  • a detection kit is provided; the detection kit can be used for immunohistochemical (IHC) staining of the above-mentioned protein molecule to obtain its expression in the gastric tissue to be tested, and then to determine the expression of the protein molecule to be tested.
  • IHC immunohistochemical
  • the content of early stage cells of gastric precancerous lesions and early cancer cells can also be used to detect the expression of the above protein molecules in blood by enzyme-linked immunosorbent assay (ELISA).
  • a system and method for determining gastric precancerous lesions, early gastric cancer, early pancreatic cancer, early bowel cancer, and early esophageal cancer and/or determining the recurrence risk of gastric cancer after surgery are provided.
  • the system determines the number of gastric precancerous lesion cells and early cancer cells in gastric tissue, and then determines the risk of gastric precancerous lesions and/or gastric cancer, and/or determines the number of gastric precancerous lesion cells and/or gastric cancer. risk of gastric cancer recurrence after surgery.
  • the system also determines the risk of other digestive system tumors such as colon cancer, esophageal cancer, and pancreatic cancer based on the expression of cell marker proteins in the tissue or blood of the examinee.
  • the detection kit is used to immunohistochemically stain the expression levels of KLK10, LAMC2 and KRT7, the very early cell markers of gastric cancer, so as to obtain their expression in the tissue to be tested, and/or to obtain the expression level of the tissue to be tested by enzyme-linked immunosorbent assay. Measure the content of very early gastric cancer cell markers in blood samples of patients;
  • the very early gastric cancer cell counting and stratification device is used to determine the proportion of positive cells of the very early gastric cancer cell markers KLK10, LAMC2 and KRT7 in the tissue sample to be tested and the degree of stratification (Grade), and/or determine by calculating the average value The overall expression levels of KLK10, LAMC2 and KRT7 in the serum of the blood sample to be tested.
  • KLK10, LAMC2 and KRT7 expression levels so as to obtain the reagents for their expression in the tissue to be tested and/or blood in the preparation of compositions for determining early gastric precancerous lesions and/or early gastric cancer and other early tumors of the digestive system Applications.
  • Fig. 1 is a schematic diagram of a system for assessing the risk of progression of gastric precancerous lesions and determining the risk of early gastric cancer based on a molecular marker monoclonal antibody combination according to an embodiment of the present invention.
  • Fig. 2 is a box-whisker diagram based on the expression of gastric cancer very early cell marker molecular combinations in very early gastric cancer cells according to an embodiment of the present invention, showing different outcome patient groups (low-grade dysplasia patients progress to gastric cancer group, referred to as the progress group ; Low-grade dysplasia regression to non-low-grade dysplasia group, referred to as the regression group; low-grade dysplasia maintenance group, referred to as the maintenance group).
  • Fig. 3 is a ROC curve diagram for distinguishing patients in the progression group and the regression group according to the combination of marker molecules of very early gastric cancer cells and gastric cancer very early cells according to the present invention.
  • Figure 4A shows the correlation of the overall predictive performance of the molecular combination of gastric cancer very early cell markers with the time window for progression from low-grade dysplasia to gastric cancer.
  • Figure 4B is a graph of the relationship between the predictive performance of the very early gastric cancer cell marker molecular combination on the risk of cancer progression and the progression time in the case of H.
  • Figure 5 uses the COX proportional hazards model to analyze the association of molecular combinations of very early gastric cancer cell markers, clinicopathological parameters, and the risk of progression from low-grade dysplasia to gastric cancer.
  • Fig. 1 is a schematic diagram of a gastric cancer risk assessment and very early diagnosis system based on a combination of very early gastric cancer cell markers according to an embodiment of the present invention.
  • a detection kit based on three molecules includes monoclonal antibody reagents for detecting the expression levels of KLK10, LAMC2 and KRT7 molecules, as well as blank solution, dilution solution and antigen retrieval solution.
  • the monoclonal antibody reagent is an antibody for immunohistochemical detection method, as shown in Table 1 in detail.
  • the monoclonal antibody reagent can also be an antibody for Western Blot or ELISA detection method.
  • an immunohistochemical detection method for the expression levels of the above three molecules KLK10, LAMC2 and KRT7 in gastric tissue is provided.
  • users in addition to the above-mentioned reagents contained in the kit, users can prepare or purchase the following reagents by themselves:
  • the present invention In order to verify the value of the present invention in assisting the evaluation of the risk of gastric cancer in patients with low-grade gastric dysplasia, the diagnosis of digestive system tumors such as very early gastric cancer, and the prediction of the recurrence risk of gastric cancer, the present invention conducts multi-angle retrospective sequential cases collected clinically. verify.
  • the inventors retrospectively screened a total of 324 low-grade dysplasia sequential cohort patients from three independent medical centers: Peking Union Medical College Hospital, Wangjing Hospital, China Academy of Chinese Medical Sciences, and Yijishan Hospital, Anhui.
  • the conditions for screening and enrolling patients are: 1) two or more gastroscopy records; 2) the baseline diagnosis is low-grade intraepithelial neoplasia; 3) patients do not suffer from gastric cancer, gastric ulcer, and other concomitant diseases such as tumors.
  • the patients were divided into progressive (the end point pathology was gastric cancer or high-grade intraepithelial neoplasia), maintenance (the end point pathology was low-grade intraepithelial neoplasia), and regression (the end point pathology was intestinal metaplasia). or atrophic gastritis, etc.) in three groups, including 107 cases in the progression group, 41 cases in the maintenance group, and 175 cases in the regression group, with an average follow-up monitoring time of 18 months (3-80 months).
  • center 1 the number of cases in center 1, center 2 and center 3 were 110, 114 and 100 respectively; the average ages were 53, 56 and 63 respectively; the male to female ratios were 2.24, 1.71 and 1.04; The proportion of patients with Helicobacter pylori infection was 0.33, 0.21 and 0.32; the average follow-up time was 17.2 months, 21 months and 16.5 months.
  • the test kit includes:
  • Reagent A blocking solution, which is 10% goat serum
  • Reagent B diluted ready-to-use anti-KLK10 primary antibody
  • Reagent C diluted ready-to-use primary anti-LAMC2 antibody
  • Reagent D diluted ready-to-use anti-KRT7 primary antibody
  • Reagent G anti-goat biotinylated secondary antibody
  • Reagent H streptavidin-labeled HRP
  • Reagent K 20 times concentrated DAB chromogenic solution.
  • pancreatic cancer tissues The expression of combined markers in pancreatic cancer tissues was detected using the above kits:
  • Tissue embedding Pancreatic cancer tissue specimens were fixed with 10% neutral formalin for 2 hours, washed repeatedly with running water to remove the fixative, placed in 75% alcohol overnight, and then dehydrated using gradient alcohol, 75% alcohol 1h, 85% alcohol for 1h, 95% alcohol for 1h, absolute ethanol twice, 1.5h each time, then soak in xylene for 1.5h, immerse in wax in a 60°C oven for 1h for embedding, cool and store at 4°C for later use;
  • Paraffin section Trim the wax block, adjust the slicer (SLEE paraffin slicer CUT5062) to set the slice thickness to 3-4 ⁇ m, slice continuously, place it in warm water at 60°C to float and flatten, and spread it on a carrier coated with cationic resin. on the slide;
  • Dewaxing Dewax the slices in a container filled with xylene for 3 times (i.e. xylene I, II, III), 10 min each time;
  • Antigen retrieval add 1000ml of citric acid buffer into the pressure cooker, immerse the slide rack with slices in the buffer, repair under high temperature and high pressure for 2 minutes and 45 seconds, wash with TBS 3 times, 2 minutes each time;
  • the present inventors evaluated the cancer risk prediction of gastric low-grade dysplasia by very early gastric cancer cell markers from three aspects.
  • the inventors overall assessed the distribution of marker expression levels in three different groups. By quantifying the markers into four stages of negative (0), weak positive (1), positive (2) and strong positive (3), the inventors found that the marker molecules of the progression group, whether in the overall situation or in each independent center, The expression levels were significantly higher than those in the regression group (Fig. 2, p ⁇ 0.01), suggesting that this marker has the value of distinguishing the risk of progression of low-grade dysplasia.
  • the inventors evaluated the markers for predicting the risk of progression of low-grade dysplasia and for the diagnostic performance of very early gastric cancer.
  • the markers can predict the risk of progression of low-grade dysplasia (and very early gastric cancer)
  • the AUC value was 0.87, and the overall prediction accuracy was 84%.
  • the above values are significantly better than the existing clinical indicators such as the patient's age, gender, and H.p infection (p ⁇ 0.001), further verifying that the marker molecules have the ability to predict the cancer risk of low-grade dysplasia and determine the risk of early gastric cancer.
  • the inventors used the COX proportional hazards model to assess the association of marker expression with the risk of progression of low-grade dysplasia, and to assess the coupling of this association with clinicopathological parameters.
  • the results of univariate and multivariate analysis showed that the high expression of markers was an important risk factor for the progression of low-grade dysplasia (p ⁇ 0.05).
  • the predictive performance of markers for the risk of cancer progression was independent of clinical indicators, while gender and Helicobacter pylori infection were not independent significant risk factors (Figure 5).
  • the inventors detected the expression of markers at different time points in the progression of low-grade dysplasia to evaluate the correlation of the predictive performance of markers with the progression time window ( Figure 4A and Figure 4B).
  • the predictive performance of markers on the risk of cancer progression is closely related to the time of progression.
  • the prediction accuracy rate is significantly increased to 88% 12 months before the onset of cancer, and 3-6 months before the onset of cancer.
  • the accuracy rate is as high as 96%, indicating that the very early markers of gastric cancer can effectively advance the early detection window of gastric cancer by 6-12 months.

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Abstract

制备特异标记胃癌极早期细胞的分子标志物（KRT7、KLK10、LAMC2）单克隆抗体，并将其应用到制备基于胃组织或血液确定胃癌或其他消化道肿瘤极早期发生风险的试剂盒中。结果表明：1）对低级别异型增生进展风险的整体预测准确率达86％，AUC值达0.87；2）准确率与低级别异型增生进展时间密切相关，在胃癌发生前6个月准确率提升至95％，可前移胃癌早诊窗口平均10个月；3）标志物对胃癌的诊断准确率超97％。标志物还可用于区分胃癌术后复发风险。

Description

确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用 技术领域
本发明涉及一种用于辅助确定胃癌及其他消化道肿瘤极早期发生风险以及评估低级别异型增生等胃癌前病变进展风险的标志物组合及其测定系统。
背景技术
胃癌早诊意义重大。肠型胃癌作为一种主要的胃癌亚型，其发生会经历异型增生等一系列癌前病变的过程。评估胃癌前病变向胃癌的进展风险、实现胃癌的极早诊断对于胃癌的防治具有重要的意义。
胃炎癌转化时程长，难以界定胃炎癌转化的关键转化点、亦即“癌变起始点”，这是胃癌极早诊断的关键难点。发明人前期通过建立国际首个胃炎癌转化单细胞图谱，发现了一群在胃低级别异型增生阶段开始出现、具有高度癌变风险的胃癌极早期细胞，并将这类细胞作为诊断极早期胃癌的标志[1][2]。发掘胃癌极早期细胞的分子特征，进而建立特异识别胃癌极早期细胞的诊断系统，有望精准评估异型增生等胃癌前病变的进展风险、实现胃癌的极早诊断。
消化系统其他器官如肠、食管以及胰腺中肿瘤的发生也会经历系列癌前病变的过程。以胃癌极早期细胞相关分子特征为切入点，有望建立消化系统癌症极早诊断的共性方式，对消化系统肿瘤防治也具有十分重要的意义。
发明内容
本发明提供了一种检测胃癌极早期细胞标志物分子组合(包括KLK10,KRT7以及LAMC2)表达水平的试剂在制备用于评估低级别异型增生向胃癌进展风险及确定胃癌极早期发生风险的应用。其中，标志物组合包含KRT7，KLK10以及LAMC2。本发明将上述分子组合在低级别异型增生阶段所标记的细胞定义为胃癌极早期细胞，并将胃癌极早期细胞出现的阶段定义为胃癌极早期阶段。该阶段是低级别异型增生等胃 癌前病变向胃癌转化的关键时点，也就是癌变起始点。通过评估胃癌极早期细胞标志物分子组合的表达水平，定量样本中胃癌极早期细胞的含量，进而作为评估低级别异型增生向胃癌进展风险评估以及胃癌极早期诊断的重要依据。
为了提高标志物分子的特异性，本发明进一步制备了相关单克隆抗体，可以更特异地从临床样本中检测出标志物分子的表达。以上述抗体为核心，本发明研制了一套用以从临床组织样本中检测评估胃癌极早期细胞含量的试剂盒，并开发了一套用以精准确定胃癌极早期发生风险的系统，有望为胃癌等消化系统肿瘤患者采取相关治疗措施或决策提供有效依据，临床应用前景良好。
根据本发明的一个方面，提供了检测标志物组合的表达水平的试剂在制备确定极早期胃癌诊断的产品中应用，其中所述标志物组合成分包括KLK10、KRT7、LAMC2。组合的方式包括：1)KLK10与LAMC2；2)KRT7与LAMC2；3)KRT7、KLK10与LAMC2。
根据本发明的另一个方面，所提供的试剂包括自主制备的KLK10和LAMC2单克隆抗体。
其中，KLK10单克隆抗体的位点序列为：
Figure PCTCN2022085212-appb-000001
LAMC2的单克隆抗体的位点序列为：
>sp|Q13753|22-1193
Figure PCTCN2022085212-appb-000002
Figure PCTCN2022085212-appb-000003
根据本发明的另一个方面，提供了一种免疫组化检测试剂盒，用于免疫染色三个蛋白分子KLK10，LAMC2，KRT7的一种或多种组合的蛋白表达水平,进而确定胃癌极早期细胞含量、辅助诊断极早期胃癌。
根据本发明的一个方面，提供了把3种分子KLK10、LAMC2和KRT7的组合作为胃癌极早期细胞群的特征并把该组合用于确定低级别异型增生向胃癌进展风险的用途。
根据本发明的另一个方面，提供了把3种分子KLK10、LAMC2和KRT7的组合在胰腺、肠和食管等组织中在从癌前病变到早癌的变化特征用作确定早期胰腺癌、肠癌和食管癌的用途。
根据本发明的又一个方面，提供了把3种分子KLK10、LAMC2和KRT7的表达水平与胃癌术后复发生存风险相关联并把这种关联用于确定胃癌术后复发风险的用途。
根据本发明的又一个方面，提供了一种检测试剂盒；该检测试剂盒既可用于免疫组化(IHC)染色上述蛋白分子获取其在待测胃组织中的 表达情况，进而确定待测组织中的胃癌前病变早期阶段细胞以及早癌细胞含量，也可以用于通过酶联免疫吸附测定(ELISA)检测上述蛋白分子在血液中的表达情况。
根据本发明的又一方面，提供了一种确定胃癌前病变、早期胃癌、早期胰腺癌、早期肠癌以及早期食管癌和/或确定胃癌术后复发风险的系统和方法。该系统依据组织或血液中细胞标志蛋白的表达情况，确定胃组织中胃癌前病变细胞以及早癌细胞数量，进而确定被检测者罹患胃癌前病变和/或胃癌的风险，和/或确定被检测者胃癌术后复发的风险。该系统还依据被检测者组织或血液中细胞标志蛋白的表达情况，确定被检测者罹患肠癌、食管癌、胰腺癌等消化系统其他肿瘤的风险。
根据本发明的一个实施例的上述系统包括：
检测试剂盒，用于分别免疫组化染色胃癌极早期细胞标志KLK10、LAMC2和KRT7的表达水平从而获取其在待测组织中的表达情况，和/或用于通过酶联免疫吸附测定检测获取待测患者血液样本中胃癌极早期细胞标志的含量；
胃癌极早期细胞计数及分层装置，用于确定待测组织样本中的胃癌极早期细胞标志KLK10、LAMC2和KRT7阳性细胞比例分数以及分层程度(Grade)，和/或通过计算平均值而确定待测血液样本的血清中KLK10、LAMC2和KRT7的总体表达水平。
根据本发明的一个进一步的方面，上述系统根据胃癌极早期细胞比例占整个待测组织中细胞总数的比例，进行表达水平的分层化处理，包括阴性(占比＝0％，Grade 0),低度(占比<5％,Grade 1),中度(5％<占比<30％,Grade 2),高度(占比>30％,Grade 3)，进而确定患者向胃癌进展的风险指数和/或胃癌术后复发风险指数；和/或根据食管、胰腺以及肠道组织中胃癌极早期细胞标志物分子的表达水平，利用上述的分层化处理方式，来确定罹患食管癌、胰腺癌以及肠癌的风险指数；和/或利用胃癌极早期细胞的标志物分子在血清中的总体表达水平来确定待测患者罹患消化系统肿瘤的风险指数。
根据本发明的又一个方面，提供了测定3种胃癌极早期细胞标志
KLK10、LAMC2和KRT7的表达水平从而获取其在待测组织和/或血液中的表达情况的试剂在制备用于确定早期胃癌前病变和/或早期胃癌及其他消化系统早期肿瘤中的组合物中的应用。
附图说明
图1是根据本发明的一个实施例的基于分子标志单克隆抗体组合的胃癌前病变进展风险评估以及确定胃癌极早发生风险系统的原理图。
图2是根据本发明的一个实施例的基于胃癌极早期细胞胃癌极早期细胞标志物分子组合的表达的箱线图，表示不同结局患者组(低级别异型增生患者进展为胃癌组，简称进展组；低级别异型增生回退到非低级别异型增生组，简称为回退组；低级别异型增生维持组，简称为维持组)中标志物分子表达的情况。*表示p-value≤0.05。
图3是根据本发明的胃癌极早期细胞胃癌极早期细胞标志物分子组合用以进行区分进展组和回退组患者的ROC曲线图。
图4A是胃癌极早期细胞胃癌极早期细胞标志物分子组合的整体预测性能随低级别异型增生向胃癌进展时间窗的关联。图4B是以幽门螺杆菌感染情况分组，胃癌极早期细胞胃癌极早期细胞标志物分子组合在幽门螺杆菌阳性和阴性情况下对癌变进展风险的预测性能与进展时间的关系图。
图5是利用COX比例风险模型，分析胃癌极早期细胞标志物分子组合、临床病理参数与低级别异型增生向胃癌进展风险的关联。
具体实施方式
图1是根据本发明的一个实施例的基于胃癌极早期细胞标志物组合的胃癌发生风险评估及极早期诊断系统的原理图。
具体地，根据本发明的第一个方面，提供了一种基于三种分子的检测试剂盒。该试剂盒包括检测KLK10、LAMC2和KRT7分子表达水平的单克隆抗体试剂，还包括空白液、稀释液、抗原修复液。在本发明的一个优选实施方案中，所述单克隆抗体试剂为免疫组化检测方法用抗体，具体如表1所示。根据本发明的另外的实施例，所述单克隆抗体试剂也可以为Western Blot或ELISA检测方法用抗体。
表1.待测分子所用免疫组化检测抗体列表及配制浓度比
Figure PCTCN2022085212-appb-000004
根据本发明的第二个方面，提供了一种胃组织中上述三种分子KLK10、LAMC2和KRT7的表达水平的免疫组化检测方法。在根据本发明的该检测方法的一个实施例中，除试剂盒中包含的上述试剂外，用户可自行配制或购买以下试剂：
(1)蒸馏水或去离子水；
(2)3％H 2O 2；
(3)二甲苯；
(4)75％、85％、95％酒精及无水乙醇；
(5)10mM TBS溶液(pH7.2～7.4)：三羟基氨基甲烷1.21g，氯化钠7.6g，加蒸馏水800mL，浓盐酸调pH值至7.2～7.4，最后定容至1000mL；
(6)10mM pH6.0柠檬酸缓冲液：柠檬酸0.38g，柠檬酸三钠2.45g，加蒸馏水900mL，浓盐酸调pH值至6.0，最后定容至1000mL；
(7)苏木精溶液；
(8)中性树脂。
实施例
为了验证本发明在辅助评估胃低级别异型增生患者胃癌发生风险、极早期胃癌等消化系统肿瘤诊断、胃癌复发风险预测的价值，本发明对临床采集的多中心、回顾性序贯病例进行多角度验证。
胃癌极早期细胞标志物组合确定胃癌极早期发生风险中的应用
实施例1
标志物验证患者人群入组情况以及临床特征分析
首先，本发明人从北京协和医院、中国中医科学院望京医院以及安徽弋矶山医院三个独立的医学中心回顾性筛选低级别异型增生序贯队列患者共计324例患者。患者筛选入组的条件为：1)有两次及以上胃镜检查记录；2)基线诊断为低级别上皮内瘤变；3)患者未罹患胃癌、胃溃疡、以及其他肿瘤等伴随疾病。依据病人最终内镜诊断结果，将病人分为进展(终点病理为胃癌或高级别上皮内瘤变)、维持(终点病理为低级别上皮内瘤变)和回退(终点病理为肠上皮化生或萎缩性胃炎等)三组，其中进展组107例，维持组41例，回退组175例，平均随访监测时间为18个月(3-80个月)。
通过分析各组患者的基线临床特征，发现：1)总体上，入组病人平均年龄为59岁，男女性别比为1.59，幽门螺杆菌感染病人比例为0.28，平均病理记录为2.2次，各临床指标中不存在明显的关联；2)在各组别中，进展组、维持组、回退组的平均年龄分别为63岁、55岁和56岁；男女性别比分别为2.29、1.92和1.24。幽门螺杆菌感染病人比例为0.21、0.27和0.33；平均追踪时间为15.7个月、15.9个月和18.9个月。各中心中，中心一、中心二和中心三的病例数分别为110例、114例和100例；平均年龄分别为53岁、56岁和63岁；男女性别比分别为2.24、1.71和1.04；幽门螺杆菌感染病人比例为0.33、0.21和0.32；平均追踪时间为17.2个月、21个月和16.5个月。
胃癌极早期组合标志物表达的检测
首先，运用根据本发明的免疫组化检测试剂盒(20)获得基于3个蛋白分KLK10，LAMC2，KRT7的组合标志物在324例病理标本中的表达。该试剂盒利用免疫组化(IHC)来衡量组合标志物的表达水平。采用10％福尔马林缓冲液固定石蜡包埋手术样本，组织切片为4微米/张。本实施例中的试剂盒包括：
(1)试剂A：封闭液，为10％山羊血清；
(2)试剂B：已稀释的即用型抗KLK10一抗；
(3)试剂C：已稀释的即用型抗LAMC2一抗；
(4)试剂D:已稀释的即用型抗KRT7一抗；
(4)试剂G：抗山羊生物素化二抗；
(5)试剂H：链亲和素标记的HRP；
(6)试剂I：20倍浓缩DAB底物溶液；
(7)试剂J：20倍浓缩DAB底物缓冲溶液；
(8)试剂K：20倍浓缩DAB显色溶液。
除试剂盒中包含的上述试剂，还自行配制了以下试剂：
(1)蒸馏水或去离子水；
(2)3％H 2O 2；
(3)二甲苯；
(4)75％、85％、95％酒精及无水乙醇；
(5)10mM TBS溶液(pH7.2～7.4)：三羟基氨基甲烷1.21g，氯化钠7.6g，加蒸馏水800mL，浓盐酸调pH值至7.2～7.4，最后定容至1000mL；
(6)10mM pH6.0柠檬酸缓冲液：柠檬酸0.38g，柠檬酸三钠2.45g，加蒸馏水900mL，浓盐酸调pH值至6.0，最后定容至1000mL；
(7)苏木精溶液；
(8)中性树脂。
利用上述试剂盒检测了胰腺癌组织中组合标志物的表达：
(1)组织包埋：10％的中性福尔马林固定胰腺癌组织标本2h，用流水反复冲洗以去除固定液，将标本放置入75％酒精过夜，然后采用酒精梯度脱水，75％酒精1h，85％酒精1h，95％酒精1h，无水乙醇2次，每次1.5h，然后置于二甲苯中浸泡1.5h，60℃烘箱中浸蜡1h包埋，冷却后4℃保存备用；
(2)石蜡切片：修整蜡块，调整切片机(SLEE石蜡切片机CUT5062)将切片厚度设为3～4μm，连续切片，置于60℃温水漂浮展平，平铺于涂有阳离子树脂的载玻片上；
(3)烤片：将待做切片置于切片架上，于60℃恒温烤箱中至少烤1h；
(4)脱蜡：切片放入盛有二甲苯的容器中脱蜡3次(即二甲苯Ⅰ、Ⅱ、Ⅲ)，每次10min；
(5)水化：切片经下行酒精水化，无水乙醇5min，95％乙醇2次(每次2min)，85％乙醇2min；75％乙醇2min，蒸馏水冲洗1分钟；
(6)抗原修复：高压锅中加入柠檬酸缓冲液1000ml，将装有切片的切片架浸入缓冲液中，高温高压修复2分45秒，用TBS洗涤3次，每次2min；
(7)3％H 2 O 2滴加在切片上，室温静置15min，TBS洗涤3次，每次2min；
(8)封闭：滴加试剂A于切片上，需完全覆盖组织切片，室温孵育10min后吸干液体，无需冲洗；
(9)加一抗：不同的切片分别滴加试剂B(抗KLK10一抗)、试剂C(抗LAMC2一抗)、试剂D(抗KRT7一抗)，需完全覆盖组织切片，37℃湿盒孵育2hr或4℃过夜；
(10)洗涤：TBS-T洗涤(3×5min)；
(11)加二抗：试剂G(滴加生物素化二抗)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(12)洗涤：TBS洗涤3次，每次5min；
(13)加HRP-SA：滴加试剂H(链亲和素标记的HRP)，需完全覆盖组织切片，37℃湿盒中孵育30min；
(14)洗涤：TBS洗涤3次，每次5min；
(15)制备DAB显色液：需现用现配，以染一张切片为例，取2.5ul试剂I加入到50ul蒸馏水中并混匀，再分别向上述液体中加入2.5ul试剂J及2.5ul试剂K，混匀；
(16)显色：滴加上述DAB显色液于切片上，需完全覆盖组织切片，显微镜下观察显色，蒸馏水冲洗终止显色；
(17)复染：苏木精复染3min，盐酸酒精分化；
(18)封片：75％酒精浸泡2min，85％酒精浸泡2min，95％酒精浸泡2min，无水乙醇浸泡2min，然后置于二甲苯中浸泡15min，更换二甲苯后再浸泡15min，中性树脂封片；
(19)结果判读：显微镜下观察染色的胰腺癌组织切片，阳性结果成棕黄色颗粒样染色，随机选择了5个高倍视野(10*40)计数阳性细胞个数。把阳性细胞数比例0％，1-5％、6-30％及31-100％分别判定为0、1、2、3分。每张切片阳性细胞的着色强度按无着色、淡黄色、棕黄色及棕褐色分别判读为0、1、2、3分。
胃癌极早期细胞标志物组合对胃低级别异型增生的癌变风险预测
本发明人从三个方面评估了胃癌极早期细胞标志对胃低级别异型增生的癌变风险预测。
首先，本发明人总体评估了标志物表达水平在三个不同组别中的分布情况。通过将标志物定量为阴性(0)、弱阳性(1)、阳性(2)以及强 阳性(3)四个阶段，本发明人发现无论是总体情况还是各个独立中心，进展组的标志物分子表达水平均显著高于回退组(图2，p<0.01)，提示该标志物具有区分低级别异型增生进展风险的价值。
进而，发明人评估了标志物对于预测低级别异型增生进展风险的预测以及对极早期胃癌的诊断性能。通过绘制ROC曲线图(图3)，并与现有临床病理指标(年龄、性别、幽门螺杆菌感染等)的比较分析，确定了标志物对于低级别异型增生进展风险(及极早期胃癌)预测AUC值为0.87，总体预测准确率为84％。上述数值均显著优于患者年龄、性别、H.p感染情况等现有临床指标(p<0.001)，进一步验证了标志物分子具有预测低级别异型增生癌变风险以及确定胃癌极早发生风险的能力。
进一步地，本发明人利用COX比例风险模型，评估标志物表达与低级别异型增生进展风险的关联，并评估该关联与临床病理参数的耦合情况。单因素和多因素分析的结果均表明了，标志物的高表达是低级别异型增生癌变进展的重要危险因素(p<0.05)。同时，标志物对于癌变进展风险的预测性能独立于临床指标，而性别与幽门螺杆菌感染等均不是独立的显著风险因素(图5)。
最后，本发明人检测了标志物在低级别异型增生进展不同时间点的表达，以评估标志物的预测性能随进展时间窗的关联(图4A和图4B)。在总体患者人群中，标志物对癌变进展风险的预测性能与进展时间密切相关，在癌症发生前12个月，预测准确率大幅提升至88％，在癌症发生前3-6个月时，预测准确率高达96％，表明胃癌极早期标志物可有效前移胃癌早诊窗口6-12个月。以幽门螺杆菌感染情况分组，确定了在幽门螺杆菌阴性组中标志物可平均前移的窗口期为12个月，准确率达93％，表明该标志物在幽门螺杆菌阴性人群中可进一步延长胃癌早诊窗口。对于3个月内的极早期胃癌，标志物分子在总体样本中的预测准确率达97％，在幽门螺杆菌阳性的组别中，标志物分子筛选预测出了100％的极早期胃癌患者，彰显出该标志物对于极早期胃癌的诊断潜力。
参考文献:
1.Zhang P,Yang M,Zhang Y,et al.Dissecting the single-cell transcriptome network underlying gastric premalignant lesions and early gastric cancer.Cell Rep.2019；27(6):1934-1947.e5.
2.李梢,张鹏.一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用.中国专利，ZL202080001970.7

Claims (9)

  1. 检测一种胃癌极早期细胞标志物分子组合在来自被检测者的胃组织或血清样品中的表达水平的试剂在制备用于确定胃低级别异型增生向胃癌进展的风险和/或用于确定胃癌极早期发生风险的产品上的用途，其特征在于：
    所述极早期细胞胃癌极早期细胞标志物分子组合包含从下列胃癌极早期细胞标志物分子组合中选出的一种：
    1)KLK10与LAMC2；
    2)KRT7与LAMC2；
    3)KRT7、KLK10与LAMC2。
  2. 根据权利要求1所述的用途，其中将所述极早期细胞胃癌极早期细胞标志物分子组合在低级别异型增生样本中所标记的细胞定义为胃癌极早期细胞，并将胃癌极早期细胞出现的阶段定义为胃癌极早期阶段，通过评估所述胃癌极早期细胞标志物分子组合的表达水平定量待测样本中胃癌极早期细胞的含量。
  3. 根据权利要求1或2所述的用途，其特征在于：
    所述试剂包括KLK10和LAMC2单克隆抗体，其中，KLK10单克隆抗体的位点序列为：
    AEAALLPQNDTRLDPEAYGSPCARGSQPWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNK
    PLWARVGDDHLLLLQGEQLRRTTRSVVHPKYHQGSGPILPRRTDEHDLMLLKLARPVVLG
    PRVRALQLPYRCAQPGDQCQVAGWGTTAARRVKYNKGLTCSSITILSPKECEVFYPGVVT
    NNMICAGLDRGQDPCQSDSGGPLVCDETLQGILSWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN，
    LAMC2单克隆抗体的位点序列为：
    >sp|Q13753|22-1193
    TSRREVCDCNGKSRQCIFDRELHRQTGNGFRCLNCNDNTDGIHCEKCKNGFYRHRERDRC
    LPCNCNSKGSLSARCDNSGRCSCKPGVTGARCDRCLPGFHMLTDAGCTQDQRLLDSKCDC
    DPAGIAGPCDAGRCVCKPAVTGERCDRCRSGYYNLDGGNPEGCTQCFCYGHSASCRSSAE
    YSVHKITSTFHQDVDGWKAVQRNGSPAKLQWSQRHQDVFSSAQ RLDPVYFVAPAKFLGNQ
    QVSYGQSLSFDYRVDRGGRHPSAHDVILEGAGLRITAPLMPLGKTLPCGLTKTYTFRLNE
    HPSNNWSPQLSYFEYRRLLRNLTALRIRATYGEYSTGYIDNVTLISARPVSGAPAPWVEQ
    CICPVGYKGQFCQDCASGYKRDSARLGPFGTCIPCNCQGGGACDPDTGDCYSGDENPDIE
    CADCPIGFYNDPHDPRSCKPCPCHNGFSCSVMPETEEVVCNNCPPGVTGARCELCADGYF
    GDPFGEHGPVRPCQPCQCNNNVDPSASGNCDRLTGRCLKCIHNTAGIYCDQCKAGYFGDP
    LAPNPADKCRACNCNPMGSEPVGCRSDGTCVCKPGFGGPNCEHGAFSCPACYNQVKIQMD
    QFMQQLQRMEALISKAQGGDGVVPDTELEGRMQQAEQALQDILRDAQISEGASRSLGLQL
    AKVRSQENSYQSRLDDLKMTVERVRALGSQYQNRVRDTHRLITQMQLSLAESEASLGNTN
    IPASDHYVGPNGFKSLAQEATRLAESHVESASNMEQLTRETEDYSKQALSLVRKALHEGV
    GSGSGSPDGAVVQGLVEKLEKTKSLAQQLTREATQAEIEADRSYQHSLRLLDSVSRLQGV
    SDQSFQVEEAKRIKQKADSLSSLVTRHMDEFKRTQKNLGNWKEEAQQLLQNGKSGREKSD
    QLLSRANLAKSRAQEALSMGNATFYEVESILKNLREFDLQVDNRKAEAEEAMKRLSYISQ
    KVSDASDKTQQAERALGSAAADAQRAKNGAGEALEISSEIEQEIGSLNLEANVTADGALA
    MEKGLASLKSEMREVEGELERKELEFDTNMDAVQMVITEAQKVDTRAKNAGVTIQDTLNT
    LDGLLHLMDQPLSVDEEGLVLLEQKLSRAKTQINSQLRPMMSELEERARQQRGHLHLLET
    SIDGILADVKNLENIRDNLPPGCYNTQALEQQ。
  4. 一种检测试剂盒，其特征在于：
    该检测试剂盒用于免疫组化染色一种胃癌极早期细胞标志物分子组合从而获取一种胃癌极早期细胞标志物分子组合在待测胃组织样品中的表达情况，和/或用于通过酶联免疫吸附测定检测胃癌极早期细胞标志物分子组合在血液样品中的表达情况，
    其中：
    所述胃癌极早期细胞标志物分子组合包含从下列胃癌极早期细胞标志物分子组合中选出的一种：
    1)KLK10与LAMC2；
    2)KRT7与LAMC2；
    3)KRT7、KLK10与LAMC2。
  5. 根据权利要求4所述的检测试剂盒，其特征在于：
    所述检测试剂盒包括KLK10和LAMC2单克隆抗体，其中，KLK10单克隆抗体的位点序列为：
    AEAALLPQNDTRLDPEAYGSPCARGSQPWQVSLFNGLSFHCAGVLVDQSWVLTAAHCGNK
    PLWARVGDDHLLLLQGEQLRRTTRSVVHPKYHQGSGPILPRRTDEHDLMLLKLARPVVLG
    PRVRALQLPYRCAQPGDQCQVAGWGTTAARRVKYNKGLTCSSITILSPKECEVFYPGVVT
    NNMICAGLDRGQDPCQSDSGGPLVCDETLQGILSWGVYPCGSAQHPAVYTQICKYMSWINKVIRSN，
    其中LAMC2的单克隆抗体的位点序列为：
    >sp|Q13753|22-1193
    TSRREVCDCNGKSRQCIFDRELHRQTGNGFRCLNCNDNTDGIHCEKCKNGFYRHRERDRC
    LPCNCNSKGSLSARCDNSGRCSCKPGVTGARCDRCLPGFHMLTDAGCTQDQRLLDSKCDC
    DPAGIAGPCDAGRCVCKPAVTGERCDRCRSGYYNLDGGNPEGCTQCFCYGHSASCRSSAE
    YSVHKITSTFHQDVDGWKAVQRNGSPAKLQWSQRHQDVFSSAQRLDPVYFVAPAKFLGNQ
    QVSYGQSLSFDYRVDRGGRHPSAHDVILEGAGLRITAPLMPLGKTLPCGLTKTYTFRLNE
    HPSNNWSPQLSYFEYRRLLRNLTALRIRATYGEYSTGYIDNVTLISARPVSGAPAPWVEQ
    CICPVGYKGQFCQDCASGYKRDSARLGPFGTCIPCNCQGGGACDPDTGDCYSGDENPDIE
    CADCPIGFYNDPHDPRSCKPCPCHNGFSCSVMPETEEVVCNNCPPGVTGARCELCADGYF
    GDPFGEHGPVRPCQPCQCNNNVDPSASGNCDRLTGRCLKCIHNTAGIYCDQCKAGYFGDP
    LAPNPADKCRACNCNPMGSEPVGCRSDGTCVCKPGFGGPNCEHGAFSCPACYNQVKIQMD
    QFMQQLQRMEALISKAQGGDGVVPDTELEGRMQQAEQALQDILRDAQISEGASRSLGLQL
    AKVRSQENSYQSRLDDLKMTVERVRALGSQYQNRVRDTHRLITQMQLSLAESEASLGNTN
    IPASDHYVGPNGFKSLAQEATRLAESHVESASNMEQLTRETEDYSKQALSLVRKALHEGV
    GSGSGSPDGAVVQGLVEKLEKTKSLAQQLTREATQAEIEADRSYQHSLRLLDSVSRLQGV
    SDQSFQVEEAKRIKQKADSLSSLVTRHMDEFKRTQKNLGNWKE EAQQLLQNGKSGREKSD
    QLLSRANLAKSRAQEALSMGNATFYEVESILKNLREFDLQVDNRKAEAEEAMKRLSYISQ
    KVSDASDKTQQAERALGSAAADAQRAKNGAGEALEISSEIEQEIGSLNLEANVTADGALA
    MEKGLASLKSEMREVEGELERKELEFDTNMDAVQMVITEAQKVDTRAKNAGVTIQDTLNT
    LDGLLHLMDQPLSVDEEGLVLLEQKLSRAKTQINSQLRPMMSELEERARQQRGHLHLLET
    SIDGILADVKNLENIRDNLPPGCYNTQALEQQ。
  6. 一种用于确定病变的风险的系统，所述病变为胃癌前病变、早期胃癌、胃癌术后复发中的一种，
    其中：
    该系统依据来自被检测者的待测胃组织样品中胃癌极早期细胞标志物分子组合的表达情况，确定待测胃组织样品中的胃癌前病变细胞和/或早癌细胞数量，进而确定被检测者罹患癌前病变和/或发生胃癌术后复发的风险，
    上述系统包括：
    检测试剂盒(20)，用于免疫组化染色所述胃癌极早期细胞标志物分子组合的表达水平(11)从而获取其在待测胃组织样品中的表达情况，和/或用于通过酶联免疫吸附测定检测获取待测患者血液样品中所述胃癌极早期细胞标志物分子组合的含量(11)；
    计数及分层装置(30)，用于确定待测胃组织样品中的所述胃癌极早期细胞标志物分子组合阳性细胞比例分数以及分层程度(31)，和/或通过计算平均值而确定待测血液样品的血清中所述胃癌极早期细胞标志物分子组合的总体表达水平，
    其中：
    所述胃癌极早期细胞标志物分子组合包含从下列胃癌极早期细胞标志物分子组合中选出的一种：
    1)KLK10与LAMC2；
    2)KRT7与LAMC2；
    3)KRT7、KLK10与LAMC2。
  7. 根据权利要求6所述的系统，其特征在于：
    计数及分层装置(30)根据胃癌极早期细胞比例占整个待测组织样品中细胞总数的比例，进行表达水平的分层化处理，包括阴性(占比＝0％，Grade 0),低度(占比<5％,Grade 1),中度(5％<占比<30％,Grade 2),高度(占比>30％,Grade 3)，进而确定患者向胃癌进展的风险指数和/或胃癌术后复发风险指数；和/或利用胃癌极早期细胞的标志物分子在血清中的总体表达水平来确定待测患者罹患消化系统肿瘤的风险指数。
  8. 一种用于确定病变的风险的系统，所述病变为早期胰腺癌、早期肠癌以及早期食管癌中的一种，
    其中：
    该系统依据来自被检测者的组织或血液样品中细胞标志蛋白的表达情况，确定被检测者罹患肠癌、食管癌、胰腺癌等消化系统其他肿瘤的风险,
    上述系统包括：
    检测试剂盒(20)，用于通过酶联免疫吸附测定检测获取血液样品中所述胃癌极早期细胞标志物分子组合的含量；
    计数及分层装置(30)，用于通过计算平均值而确定血液样品的血清中所述胃癌极早期细胞标志物分子组合的总体表达水平(33)，
    其中：
    所述胃癌极早期细胞标志物分子组合包含从下列胃癌极早期细胞标志物分子组合中选出的一种：
    1)KLK10与LAMC2；
    2)KRT7与LAMC2；
    3)KRT7、KLK10与LAMC2。
  9. 根据权利要求8所述的系统，其特征在于：
    计数及分层装置(30)根据来自被检测者的食管、胰腺和/或肠道组织或血液样品中胃癌极早期细胞标志物分子的表达水平确定被检测者罹患食管癌、胰腺癌以及肠癌的风险指数。
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