WO2021213323A1 - Procédé non diagnostique de détection d'une mutation du gène pd-l1 chez un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes dans le sang périphérique - Google Patents

Procédé non diagnostique de détection d'une mutation du gène pd-l1 chez un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes dans le sang périphérique Download PDF

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WO2021213323A1
WO2021213323A1 PCT/CN2021/088118 CN2021088118W WO2021213323A1 WO 2021213323 A1 WO2021213323 A1 WO 2021213323A1 CN 2021088118 W CN2021088118 W CN 2021088118W WO 2021213323 A1 WO2021213323 A1 WO 2021213323A1
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peripheral blood
add
tumor cells
colorectal cancer
solution
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PCT/CN2021/088118
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English (en)
Chinese (zh)
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李�浩
高德海
李胜
马莹
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山东第一医科大学(山东省医学科学院)
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Publication of WO2021213323A1 publication Critical patent/WO2021213323A1/fr

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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
    • G01N33/57484—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
    • 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
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the present invention relates to a new method for detecting PD-L1 gene mutation, in particular to a method for PDL-1 gene mutation in patients with advanced or recurrent colorectal cancer whose tissue samples cannot be obtained through peripheral blood detection.
  • Colorectal cancer is one of the most common malignant tumors in the world, and the mortality rate in 2018 ranked fourth among malignant tumors. In the United States, colorectal cancer is the third most common malignant tumor. Domestically, China's annual epidemiological data in 2018: the incidence and mortality of colorectal cancer are both the fifth, with 376,300 new cases per year, and 25% of them have metastasized when they are diagnosed. Colorectal cancer is mostly asymptomatic in the early stage, and most of the symptoms appear in the middle and late stages with metastasis. Despite continuous innovations in surgical procedures and chemotherapy, the survival rate of patients with advanced colorectal cancer is still very poor. Due to the lack of better prevention methods for colorectal cancer, the most effective way to improve the prognosis of colorectal cancer and increase the survival rate is to diagnose and treat early colorectal cancer and precancerous lesions.
  • Immunotherapy with PD-1/PD-L1 as the immune target has brought a new dawn for the treatment of colorectal cancer.
  • Tumor cells can bind to PD-1 on the surface of immune cells and T cells through PD-L1 on their surface, and conduct inhibitory signals to make T cells The inability to recognize tumor cells and send out attack signals to tumor cells leads to immune escape of tumor cells.
  • CTC circulating tumor cells
  • the efficacy of PD-1 or PD-L1 immune preparations is mostly related to the immunohistochemical expression level of PD-L1 in tumor tissues, suggesting that the expression level of PD-L1 may be a biomarker predicting the efficacy of PD-1 immunotherapy; current colorectal
  • the detection of PD-L1 in cancer circulating tumor cells has not been reported at home and abroad. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 has important value for the prognosis of colorectal cancer and the evaluation of immunotherapy efficacy.
  • CTC circulating tumor cells
  • Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
  • This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
  • Circulating tumor cell detection and identification of core diagnostic technology, and further registration and identification of diagnostic kits to include PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
  • the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
  • the present invention provides a method for detecting PD-L1 gene mutations in patients with colorectal cancer through peripheral blood circulating tumor cells.
  • a detection method for non-diagnostic purposes using a membrane filtration device to separate and obtain unobtainable tissue specimens.
  • the CTC in the peripheral blood of patients with posterior colorectal cancer was further used to detect the expression of PD-L1 of CTC by immunohistochemistry.
  • a kit for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with colorectal cancer including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, PD-LI (human) primary antibody, goat Anti-human IgG/HRP 100 ⁇ L, 0.1% Triton X-100 100 ⁇ L, 0.3% H 2 O 2 100 ⁇ L, Reagent A, Reagent B, 6 ⁇ PBS buffer 60 mL.
  • the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
  • the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • the staining solution A is DAB staining solution
  • the staining solution B is hematoxylin staining solution.
  • the reagent A is composed of ethanol and formaldehyde in a volume ratio of 3:1; the reagent B is xylene.
  • a method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of colorectal cancer by using the above kit for non-diagnostic purposes including the following steps:
  • Peripheral blood sample pretreatment Dilute the collected peripheral blood sample 10 times with diluent, add paraformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, and fix the final concentration to 0.25%;
  • the membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid cylinder, and an iron stand.
  • the iron stand is provided with a base, a stand and a bracket.
  • the blood sample container is set on the upper part of the iron stand through the bracket.
  • Below the blood sample container is a filter, the filter is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
  • the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
  • the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
  • the filter membrane is made of a hydrophobic material, and the filter holes with a diameter of 8 micrometers are evenly spread on it.
  • the detection method provided by the present invention can detect the expression of PD-L1 in patients with colorectal cancer without puncture biopsy to obtain tissue samples. This technology is minimally invasive and can be detected in real time.
  • the method provided by the present invention can avoid false positive results caused by edge effects that may occur during the staining process, has good stability, reduces cell loss, and improves detection accuracy.
  • Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention.
  • FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
  • FIG. 3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
  • Figure 4 is an image of circulating tumor cells isolated from peripheral blood of patients with colorectal cancer; the arrow is CTM.
  • Component content 6 ⁇ PBS buffer 60mL Diluent 45mL Decolorizing liquid 1mL Staining solution A 0.5mL Staining Solution B 1mL PD-L1 (human) primary antibody 100 ⁇ L Goat anti-human IgG/HRP 100 ⁇ L 0.1% Triton X-100 100 ⁇ L 0.3% H 2 O 2 100 ⁇ L Reagent A 0.5mL Reagent B 1mL
  • the membrane filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
  • the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter.
  • the filter hole 10 is trapped on the filter membrane 7. What needs to be explained here is that the diluent plays the role of de-adhesion and dispersion. The laurate and poloxamer are used together to ensure that blood cells and CTC do not adhere and are fully dispersed in the diluent to effectively pass through the filter membrane. 7 was detained.
  • xylene + neutral resin is used for sealing, which needs to be fixed and dewatered and maintain neutrality. Therefore, reagent A (ethanol + formaldehyde) and reagent B (xylene) are used together.
  • Figure 4 is an image of circulating tumor cells isolated from the peripheral blood of a colorectal cancer patient.
  • the nucleus is large and the shape of the nucleus is irregular; the ratio of nucleus to cytoplasm is high.
  • the detected circulating tumor cells were confirmed by immunohistochemistry for the expression of PD-L1 and compared with the results of PD-L1 of colorectal cancer specimens to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells , To guide the targeted therapy of colorectal cancer and provide new ideas for targeted therapy of colorectal cancer.

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Abstract

La présente invention concerne un procédé non diagnostique de détection d'une mutation du gène PD-L1 chez un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes dans le sang périphérique, qui appartient au domaine de la biologie moléculaire. Le procédé de détection consiste principalement à prélever du sang périphérique, à traiter le sang périphérique, à filtrer et enrichir les cellules tumorales circulantes, et à faire appel à un procédé immunohistochimique pour détecter l'expression du gène PD-L1 des cellules tumorales circulantes. Le procédé de détection selon la présente invention peut être utilisé pour détecter l'expression de PD-L1 chez un patient atteint d'un cancer colorectal sans obtenir un échantillon de tissu au moyen d'une biopsie à l'aiguille. La technique à effraction minimale peut réaliser une détection en temps réel. De plus, le procédé peut éviter un résultat faux positif provoqué par un effet de bord qui peut être généré dans un procédé de coloration, a une bonne stabilité, réduit la perte de cellule, et améliore la précision de détection.
PCT/CN2021/088118 2020-04-21 2021-04-19 Procédé non diagnostique de détection d'une mutation du gène pd-l1 chez un patient atteint d'un cancer colorectal au moyen de cellules tumorales circulantes dans le sang périphérique WO2021213323A1 (fr)

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CN202010315331.XA CN111521790A (zh) 2020-04-21 2020-04-21 一种通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因突变的非诊断目的方法

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2022098619A1 (fr) * 2020-11-06 2022-05-12 The Regents Of The University Of Michigan Compositions et procédés pour améliorer une thérapie cancéreuse

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CN111521790A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因突变的非诊断目的方法
CN111638341A (zh) * 2020-07-01 2020-09-08 山东凯歌智能机器有限公司 一种检测小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法

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CN106198984A (zh) * 2016-08-22 2016-12-07 上海立闻生物科技有限公司 非小细胞肺癌患者外周血循环肿瘤细胞pdl1基因的检测方法
CN111521790A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因突变的非诊断目的方法

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CN105510602A (zh) * 2016-01-28 2016-04-20 山东省肿瘤防治研究院 一种晚期结直肠癌患者外周血循环肿瘤细胞vegf的检测方法
CN105572388A (zh) * 2016-01-28 2016-05-11 山东省肿瘤防治研究院 一种晚期结直肠癌患者外周血循环肿瘤细胞egfr的检测方法
CN106198984A (zh) * 2016-08-22 2016-12-07 上海立闻生物科技有限公司 非小细胞肺癌患者外周血循环肿瘤细胞pdl1基因的检测方法
CN111521790A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种通过外周血循环肿瘤细胞检测结直肠癌患者pd-l1基因突变的非诊断目的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022098619A1 (fr) * 2020-11-06 2022-05-12 The Regents Of The University Of Michigan Compositions et procédés pour améliorer une thérapie cancéreuse

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