WO2019214063A1 - Procédé de détection de cellules tumorales circulantes - Google Patents

Procédé de détection de cellules tumorales circulantes Download PDF

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Publication number
WO2019214063A1
WO2019214063A1 PCT/CN2018/095808 CN2018095808W WO2019214063A1 WO 2019214063 A1 WO2019214063 A1 WO 2019214063A1 CN 2018095808 W CN2018095808 W CN 2018095808W WO 2019214063 A1 WO2019214063 A1 WO 2019214063A1
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Prior art keywords
cells
pcr
sequence
tumor cells
circulating tumor
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PCT/CN2018/095808
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English (en)
Chinese (zh)
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孙奋勇
潘秋辉
江赐忠
吴棋
黄楠
崔中奇
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上海市第十人民医院
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Publication of WO2019214063A1 publication Critical patent/WO2019214063A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

Definitions

  • the invention relates to the technical field of cell detection, in particular to a method for efficiently detecting rare cells in a body fluid, in particular to a method for accurately detecting circulating tumor cells from blood, and the invention relates to a method for using tumors from tumors.
  • IMS immunomagnetic beads separation
  • ISET isolation by size of epithelial tumor cells
  • Peripheral blood suggests a better prognosis; the number of CTCs in metastatic prostate cancer and breast cancer Less than 5 / 7.5mL peripheral blood suggests that the patient has a better prognosis.
  • Elaine et al. established a low-cost, high-throughput CTCs enrichment device that significantly enhances CTCs by microcontacting EpCAM antibodies onto nanoporous silica substrates combined with microfluidic technology. The efficiency of the acquisition.
  • this type of technology is limited to markers.
  • epithelial-derived tumor cells may not express or underexpress CK and EpCAM under exogenous stimulation or self-variation, resulting in inefficient capture of CTCs.
  • CTCs in the same patient do not express consistent markers, so false positive or false negative results may occur.
  • the immunomagnetic bead positive enrichment method has many disadvantages such as cumbersome operation, inconvenient use and missed detection, and microfluidic chip technology exists. The disadvantages of high cost and high technical difficulty.
  • a first object of the present invention is to provide a method for detecting circulating tumor cells or other non-humoral rare cells from body fluids in view of the deficiencies of the prior art circulating tumor cell detection technique.
  • a second object of the present invention is to provide a kit for detecting circulating tumor cells.
  • the circulating tumor cells include circulating tumor cells with or without epithelial-mesenchymal transition.
  • the specifically recognized marker in step d is a marker of epithelial cells, a tumor-specific marker or an interstitial cell marker.
  • the reverse transcription and amplification primers of the tumor cell-specific marker are used to detect circulating tumor cells in a body fluid.
  • the permeation reagent is a nonionic surfactant.
  • the nonionic surfactant is Triton-X 100.
  • the technical solution adopted by the present invention is: a detection system for circulating tumor cells, the detection system being capable of performing the above method, and/or capable of processing a sample to be detected using the above kit.
  • the detection system is an automation system.
  • the method of the present invention can prevent cell rupture and RNA degradation by adding a fixed reagent as soon as possible after collecting blood.
  • Figure 1 is a circulating tumor cell detection strategy.
  • Figure 4 is the result of tumor cell recovery experiments.
  • Non-humoral rare nuclear cells circulating tumor cells having epithelial origin or no epithelial origin and circulating vascular endothelial cells, tumor stem cells, stem cells, fetal cells in blood, and some rare cells such as immune cells.
  • GSS Gene Specific Sequence
  • a nucleated cell which contains immune cells, endothelial cells, fibroblasts, and circulating tumor cells, undergoes mesenchymal-epithelial conversion of circulating tumor cells.
  • the sample is collected, and after the reagent is fixed or permeabilized, the expression of the protein, the transcription of the RNA, and the like are fixed, and the sample can be stored at 4 ° C or stored at -20 ° C for a long time.
  • Pre-treatment of the sample includes:
  • the number of PCR manifolds can be three or more, depending on the expected number of circulating tumor cells, and a 24-well PCR reaction tube is commonly used.
  • one or more epithelial and/or tumor-specific marker-encoding gene transcription products are subjected to targeted reverse transcription, mainly considering the presence of various types of rare cells in the body fluid, and the circulating tumor cells also have various forms.
  • targeted reverse transcription mainly considering the presence of various types of rare cells in the body fluid, and the circulating tumor cells also have various forms.
  • the simultaneous reverse transcription of a variety of marker gene transcription products can facilitate the identification of subsequent cell types.
  • the cells subjected to reverse transcription were collected and added again to a PCR 8-tube, each well containing 12 ⁇ M of different coding strands (GSS+ID2+G_F), and a Taq enzyme mixture was added for extension reaction.
  • GSS is a sequence capable of specifically recognizing a downstream of a cDNA molecule
  • ID2 is a recognition sequence specific to each tube, consisting of two or more nucleic acids, and is arranged into 16 or more unique sequences by alignment, corresponding to Each well contains cells.
  • cDNA purification refers to the process of removing proteins, RNA, various ions and impurities.
  • techniques and kits available for DNA purification on the market.
  • the present invention uses magnetic bead purification in a manner that is primarily considered to facilitate automated operation.
  • the method adopts the “non-enrichment” method to minimize the loss of circulating tumor cells, and the combined PCR pre-amplification and high-throughput sequencing technologies are greatly improved.
  • the sensitivity of the detection theoretically ensures that every circulating cell can be detected.
  • combined with a variety of epithelial cells and tumor-specific indicators through bioinformatics analysis can distinguish various rare cells in body fluids, including epithelial cells, tumor cells, epithelial-mesenchymal transition tumor cells, tumor stem cells.
  • the method of the present invention can serve as a gold standard to fill the gap in the field of standard solutions.
  • the sequence obtained by sequencing is subjected to quality control analysis to delete low-quality sequence data. Then, only the sequences containing ID1 and ID2 were retained, and the ID1, ID2, and linker sequences on these sequences were removed, and then the sequence was compared with the human reference genome by HiSat2 software to calculate the expression level of the tumor marker gene.
  • the number of circulating cells is obtained by counting the combination of ID1 + ID2. Tumor cells can be typed based on the detection of different tumor marker genes by these cells.
  • the recovery rate of the tumor cells within 30 cells after addition was 100%, and the addition of 50 cells lost 2 cells. It is fully demonstrated that the method of the present invention has excellent specific recovery ability for tumor cells, can effectively remove background interference of blood cells, and has a reliable recovery rate for low cell numbers.
  • GSS_R sequence (3'-5'): CGACGAAACCCTCAAATT (SEQ ID NO: 2)
  • liver cancer To summarize the important pathological features of liver cancer including tumor size, portal vein tumor thrombus, Child-Pugh, cirrhosis and lymph node metastasis.
  • the relationship between pathological features and CTCs is shown in Table 6.
  • GSS_R sequence (3'-5'): TCCCTTGAGTTACGTATT (SEQ ID NO: 20)
  • GSS_F sequence (3'-5'): CCACCTCCACTACTGACT (SEQ ID NO: 39)
  • GSS_F sequence (3'-5'): CGGTCGTAGGCGCTCACT (SEQ ID NO: 55)
  • the tumors were divided into two groups according to the presence or absence of tumor invasion.
  • the proportion of epithelial CTCs detected in peripheral blood of patients with capsule invasion was smaller than that of patients without capsule invasion (14.5% vs 21.4%), while the proportion of interstitial CTCs was greater than that of patients without capsule invasion. (46.6% vs 30.4%).

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  • Life Sciences & Earth Sciences (AREA)
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  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection de cellules tumorales circulantes, comprenant : la collecte d'un échantillon de fluide corporel humain et la fixation de celui-ci avec du formaldéhyde pour améliorer la perméabilité de la membrane cellulaire ; l'ajout de cellules à une pluralité de groupes de bandes multi-tubes de PCR, une amorce de transcription inverse portant ID1 étant ajoutée à chaque bande ; après la transcription inverse des cellules en molécules d'ADNc, la collecte des cellules, leur mélange, et leur ajout à un autre groupe de bandes multi-tubes de PCR, une amorce d'extension portant ID2 et dont l'extrémité 3' est capable d'identifier spécifiquement l'extrémité 3' d'une molécule d'ADNc étant ajoutée à chaque bande ; la réalisation d'une réaction d'extension après la complémentation de séquence ; la collecte et la lyse des cellules ; et la pré-amplification du produit d'extension à l'aide d'une amorce de PCR. Un séquençage à haut débit est réalisé pour analyser la combinaison de ID1/ID2. De plus, la transcription inverse et l'amplification par PCR pour différents marqueurs peuvent identifier avec précision des cellules tumorales circulantes et les cellules tumorales circulantes dans lesquelles l'EMT se produit, et peuvent être utilisées en combinaison avec d'autres technologies pour détecter dans la plus grande mesure des cellules tumorales circulantes en quantité extrêmement faible et d'autres cellules rares n'étant pas de fluide corporel dans un échantillon de fluide corporel.
PCT/CN2018/095808 2018-05-07 2018-07-16 Procédé de détection de cellules tumorales circulantes WO2019214063A1 (fr)

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CN2018104250659 2018-05-07
CN201810425065.9A CN110456034B (zh) 2018-05-07 2018-05-07 一种循环肿瘤细胞的检测方法

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CN114460305A (zh) * 2021-12-30 2022-05-10 江苏汇先医药技术有限公司 一种生物分子或细胞捕获芯片的质控方法

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CN111521789A (zh) * 2020-04-20 2020-08-11 山东第一医科大学(山东省医学科学院) 一种检测胰腺癌患者外周血循环肿瘤细胞ca199表达的免疫荧光试剂盒及检测方法
CN111521793A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 检测非小细胞肺癌患者外周血循环肿瘤细胞cea基因突变的免疫荧光试剂盒及检测方法
CN111521794A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 检测小细胞肺癌患者外周血循环肿瘤细胞nse基因突变的免疫荧光试剂盒及检测方法
CN111521791A (zh) * 2020-04-21 2020-08-11 山东第一医科大学(山东省医学科学院) 一种检测小细胞肺癌患者外周血循环肿瘤细胞nse基因突变的试剂盒及检测方法
CN111596058A (zh) * 2020-05-18 2020-08-28 山东第一医科大学(山东省医学科学院) 一种检测胆管癌患者外周血循环肿瘤细胞fgfr基因突变的试剂盒及检测方法
CN115197914A (zh) * 2022-05-25 2022-10-18 广州复大医疗有限公司 一种分离和检测循环肿瘤细胞的方法

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