WO2021213316A1 - Kit de détection d'une mutation du gène pd-l1 de cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer du rein, et procédé de détection - Google Patents

Kit de détection d'une mutation du gène pd-l1 de cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer du rein, et procédé de détection Download PDF

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WO2021213316A1
WO2021213316A1 PCT/CN2021/088097 CN2021088097W WO2021213316A1 WO 2021213316 A1 WO2021213316 A1 WO 2021213316A1 CN 2021088097 W CN2021088097 W CN 2021088097W WO 2021213316 A1 WO2021213316 A1 WO 2021213316A1
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peripheral blood
solution
add
ctc
filter
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PCT/CN2021/088097
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English (en)
Chinese (zh)
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邹本奎
李胜
李�浩
刘智鸿
于冰
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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 WO2021213316A1 publication Critical patent/WO2021213316A1/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
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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 invention belongs to the technical field of molecular biology, and particularly relates to a kit and a detection method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of renal cancer patients.
  • Renal cell carcinoma is one of the most common malignant tumors in the urinary system.
  • the morbidity and mortality rate account for about 2-3% of systemic malignancies, and its incidence has been on the rise in recent decades.
  • Radical surgery is an effective means to treat kidney cancer, but about 20% to 30% of kidney cancer patients have seen distant metastases when they visit a doctor. Even after radical renal cancer surgery, there are still 20%-40% of patients will have recurrence and metastasis.
  • the survival period of patients with advanced renal cell carcinoma has been greatly prolonged, but for patients with high-risk metastatic renal cell carcinoma, the effect of targeted therapy alone is still not satisfactory, and the 5-year survival rate is less than 10%.
  • immunotherapy targeting PD-1/PD-L1 has brought a new dawn to the treatment of renal cell carcinoma.
  • 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 industrial promotion 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 Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
  • 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 peripheral blood circulating tumor cell PD-L1 gene mutation in renal cancer patients for non-diagnostic purposes Detection method: Separate CTC in the peripheral blood of patients with advanced or recurrent non-kidney cancer who cannot obtain tissue samples by using a membrane filtration device, and further use immunohistochemistry to detect the expression of CTC's PD-L1.
  • a kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with renal cell carcinoma including dilution solution 50mL, decolorizing solution 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, 6 ⁇ PBS buffer 60 mL.
  • the diluent is composed of 1 mmol/L EDTA+0.1%BSA+0.1% trehalose+0.2% laureth.
  • the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • the staining solution A is a DAB staining solution
  • the staining solution B is a hematoxylin staining solution.
  • a method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of patients with renal cell carcinoma for non-diagnostic purposes of the above kit includes the following steps:
  • the specific method of the present invention for detecting the expression of PD-L1 of CTC is as follows:
  • 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.
  • a filter which 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 advanced or recurrent renal 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 obtained from peripheral blood of a renal cancer patient
  • Component content 6 ⁇ PBS buffer 60mL Diluent 45mL Decolorizing liquid 1mL Staining solution A 0.3mL Staining Solution B 1mL PD-L1 (human)-anti 100 ⁇ L Goat anti-human IgG/HRP 100 ⁇ L 0.1%Trition X-100 100 ⁇ L 0.3% H 2 O 2 100 ⁇ L
  • This technique was used to separate, obtain and identify 10 cases of renal cancer patients (10 normal samples were tested at the same time as a negative control) examples of circulating tumor cells in the peripheral blood.
  • 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.
  • Figure 4 is an image of circulating tumor cells isolated from the peripheral blood of a renal cancer patient.
  • the CTC cells have nuclear atypia, with a nuclear to cytoplasm ratio greater than 0.8, cell diameter (long end) greater than 15 ⁇ m, and hyperchromatic nuclei (due to increased chromatin in cancer cells, The granules become thicker and the nucleus is deeply stained).
  • the nucleus is larger 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 in gross specimens of renal cancer, to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells. Guide the targeted therapy of kidney cancer and provide new ideas for targeted therapy of kidney cancer.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un kit de détection d'une mutation du gène PD-L1 de cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer du rein, et un procédé de détection, qui relèvent du domaine technique de la biologie moléculaire. Le kit comprend un diluant, une solution de décoloration, une solution de coloration A, une solution de coloration B, un anticorps primaire anti-PD-L1 (humain), une IgG de chèvre anti-humaine/HRP, du Triton X-100 à 0,1 %, de l'H2O2 à 0,3 % et une solution tampon de PBS 6×. Au moyen du procédé de détection fourni par la présente invention, l'expression du PD-L1 chez un patient atteint d'un cancer du rein avancé ou récurrent peut être détectée sans prélever d'échantillon de tissu par biopsie à l'aiguille. La technologie est une technologie minimalement invasive, peut réaliser une détection en temps réel, peut éviter des résultats faux positifs provoqués par des effets de bord éventuellement générés dans le processus de coloration, a une bonne stabilité, réduit la perte de cellule et améliore la précision de détection.
PCT/CN2021/088097 2020-04-21 2021-04-19 Kit de détection d'une mutation du gène pd-l1 de cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer du rein, et procédé de détection WO2021213316A1 (fr)

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CN202010317669.9 2020-04-21
CN202010317669.9A CN111521798A (zh) 2020-04-21 2020-04-21 一种检测肾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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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118090377A (zh) * 2024-04-28 2024-05-28 北京大学第一医院(北京大学第一临床医学院) 一种用于肾穿刺组织活检光镜特殊染色的试剂盒及染色方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111562376A (zh) * 2020-04-20 2020-08-21 山东第一医科大学(山东省医学科学院) 一种检测胃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
CN111521798A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种检测肾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
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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CN105588943A (zh) * 2016-01-28 2016-05-18 山东省肿瘤防治研究院 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法
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
CN109596831A (zh) * 2019-01-14 2019-04-09 臻悦生物科技江苏有限公司 一种肺癌的多重免疫组化分析试剂盒及其使用方法和应用
CN111521798A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种检测肾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

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
CN105588943A (zh) * 2016-01-28 2016-05-18 山东省肿瘤防治研究院 一种胃癌患者外周血循环肿瘤细胞Her-2基因的检测方法
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
CN109596831A (zh) * 2019-01-14 2019-04-09 臻悦生物科技江苏有限公司 一种肺癌的多重免疫组化分析试剂盒及其使用方法和应用
CN111521798A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种检测肾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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118090377A (zh) * 2024-04-28 2024-05-28 北京大学第一医院(北京大学第一临床医学院) 一种用于肾穿刺组织活检光镜特殊染色的试剂盒及染色方法

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