CN115125296A - 瑞戈非尼药敏标记物及其相关试剂的应用 - Google Patents

瑞戈非尼药敏标记物及其相关试剂的应用 Download PDF

Info

Publication number
CN115125296A
CN115125296A CN202210636264.0A CN202210636264A CN115125296A CN 115125296 A CN115125296 A CN 115125296A CN 202210636264 A CN202210636264 A CN 202210636264A CN 115125296 A CN115125296 A CN 115125296A
Authority
CN
China
Prior art keywords
regorafenib
drug
marker
liver cancer
drug sensitivity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210636264.0A
Other languages
English (en)
Other versions
CN115125296B (zh
Inventor
张宁
杨慧
程景辉
庄昊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University First Hospital
Original Assignee
Peking University First Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University First Hospital filed Critical Peking University First Hospital
Priority to CN202210636264.0A priority Critical patent/CN115125296B/zh
Publication of CN115125296A publication Critical patent/CN115125296A/zh
Application granted granted Critical
Publication of CN115125296B publication Critical patent/CN115125296B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • G01N33/5011—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 for testing antineoplastic activity
    • 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
    • G01N33/5067—Liver 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Theoretical Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Evolutionary Biology (AREA)
  • Medical Informatics (AREA)
  • Gastroenterology & Hepatology (AREA)

Abstract

本发明公开了一种瑞戈非尼药敏标记物及其相关试剂应用。所述瑞戈非尼药敏标记物为瑞戈非尼耐药标记物或者瑞戈非尼敏感标记物，所述瑞戈非尼耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86一组标记物中任一种或几种，所述瑞戈非尼敏感标记物为TMPRSS6、ENTPD8、CCDC159中任意一种或几种。上述瑞戈非尼药敏标记物可用于预测瑞戈非尼的药效。通过对肝癌患者组织转录组测序，或通过PCR、基因芯片、组织芯片、NanoString技术、免疫组化的方法检测相关分子的表达，可为瑞戈非尼的用药与肝癌的精准治疗提供线索。

Description

瑞戈非尼药敏标记物及其相关试剂的应用
技术领域
本发明属于生物医学技术领域，尤其涉及肿瘤领域，具体为一种瑞戈非尼药敏标记物及其相关试剂应用。
背景技术
原发性肝癌在我国目前常见恶性肿瘤中，发病率排第四位，死亡率排第二位，严重威胁我国人民生命和健康。肝癌早期患者(巴塞罗纳0期A期)，手术切除和肝移植等治疗可以有效地提高肝癌患者的治愈率和生存预后。然而由于肝癌起病隐匿，早期症状缺乏特异性，很多患者被诊断为肝癌时，就已经处于中期或晚期。长期以来，由于药物治疗方案少，而且临床有效率低，中晚期肝癌普遍显示预后差、生存率低。
瑞戈非尼是一种口服的多靶点激酶抑制剂，可抑制VEGFR-1，2，3、TIE-2、BRAF、KIT、RET、PDGFR和FGFR，其结构与索拉非尼相似。瑞戈非尼获得FDA批准用于治疗接受过索拉非尼治疗的肝细胞癌患者。研究表明，索拉非尼-瑞戈非尼序贯治疗可以有限延长患者总体生存时间。瑞戈非尼作为肝癌治疗的二线药物，尽管一定程度可以提升肝癌的治疗效果，但其临床有效率有限，仅为11％，而且大部分人群并不适用，出现了很多无效治疗现象。因此，亟需补充瑞戈非尼的药敏分子分型方案，提高肝癌治疗疗效。
发明内容
为了克服现有技术的问题，本发明的第一个目的在于提供一种瑞戈非尼药敏标记物，采用其可对瑞戈非尼耐药及敏感人群进行提示，为肝癌精准治疗提供依据。本发明的第二个目的提出瑞戈非尼药敏标记物在非治疗目的的瑞戈非尼用药研究中的应用。本发明的第三个目的是提供该瑞戈非尼药敏标记物相关试剂的一些应用，该标记物的相关试剂包括：检测其表达的定量试剂、抑制剂、促进剂等，这些相关试剂的应用能够填补肝癌精准诊疗领域的空白。
本发明的目的是这样实现的：
本发明的第一方面：发现一组基因耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86的高表达与瑞戈非尼耐药相关。发现基因TMPRSS6、ENTPD8、CCDC159对瑞戈非尼作用敏感。因此这些基因表达能够作为指导原发性肝癌治疗的潜在药敏生物标记物。即本发明提供了一种瑞戈非尼药敏标记物，为瑞戈非尼耐药标记物或者瑞戈非尼敏感标记物；所述瑞戈非尼耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86的一组标记物中任意一种或几种；所述瑞戈非尼敏感标记物为TMPRSS6、ENTPD8、CCDC159中任意一种或几种。所述瑞戈非尼药敏标记物为RNA或其逆转录的cDNA，或蛋白。
具体筛选过程为：
本发明收集了多例原发性肝癌患者的手术标本，多点取样，通过三维培养的方法，成功建立涵盖数百个位点的肝癌类器官生物库。团队通过苏木精-伊红(Hematoxylin-Eosin，HE)染色，证实类器官保留了患者的病理学特征。通过对类器官和对应的患者标本的免疫荧光、免疫组化染色，发现肝癌类器官与临床上肝癌患者的分子标记物也是一致的。
选取近百个位点，进行组织和对应类器官的外显子、转录组测序，分析了组织与对应类器官的基因组特征。从体细胞突变、拷贝数变异和转录组相似性等多维度验证了类器官生物库高度保留组织样本的分子特征。此外，团队建立的大规模肝癌组织和类器官队列与以往肝癌研究一致，具有肝癌的高发突变。另外，本研究的肝癌数据反映了不同程度的肿瘤间(inter-tumor)和肿瘤内异质性(intra-tumor heterogeneity)，为研究肝癌药敏异质性提供了依据。
为了研究肝癌靶向药物瑞戈非尼的药敏分子分型，对多个类器官位点进行了转录组测序，通过Lasso模型及生信分析，解析出瑞戈非尼的敏感标记物为TMPRSS6、ENTPD8、CCDC159，耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86。其训练集AUC为0.82。之后，本发明通过同样的方法，收集了多个肝癌标本进行类器官培养、药敏测试和转录组测序，进行分子分型研究，得到瑞戈非尼的验证集为0.80。
本发明的第二方面：提供瑞戈非尼药敏标记物在非治疗目的的瑞戈非尼用药研究中的应用。包括在一些药效方面的基础性研究应用、对瑞戈非尼改进研究的药效测定方面的应用。
本发明的第三方面：提供瑞戈非尼药敏标记物表达量的定量试剂在制备瑞戈非尼药敏检测试剂的应用，所述药敏检测为耐药性检测或药物敏感性检测。
检测的样本为肝癌组织样本，所述定量试剂为转录组测序试剂、PCR、基因芯片、组织芯片、NanoString技术、免疫组化试剂中任意一种。
在这方便本发明提供了一种瑞戈非尼药敏评估试剂盒，包含检测所述瑞戈非尼药敏标记物表达量的定量试剂，所述药敏评估为耐药性评估或药物敏感性评估。
另外，本发明还提供了一种原发性肝癌的瑞戈非尼药敏分析***，包括：
靶点表达量检测装置：用于检测样本中所述瑞戈非尼药敏标记物的表达量；所述样本为患者肝癌组织；
药敏分析装置：基于瑞戈非尼药敏性标记物的表达量，确定肝癌的用药效果；
结果输出装置：用于输出药敏分析装置分析得到的结果，所述药敏分析为耐药性分析或敏感性分析。
本发明的第四方面：提供一种治疗肝癌的药物组合物，瑞戈非尼及所述瑞戈非尼药敏标记物的抑制剂或促进剂。
一种方案为：该药物组合物包含瑞戈非尼及所述瑞戈非尼药敏标记物的抑制剂，所述瑞戈非尼药敏标记物为瑞戈非尼耐药标记物。该药物组合物通过抑制瑞戈非尼耐药基因的表达来增进瑞戈非尼对特定患者的药效。
另一种方案为：该药物组合物包含瑞戈非尼及机所述瑞戈非尼药敏标记物的促进剂，所述瑞戈非尼药敏标记物为瑞戈非尼敏感标记物。该药物组合物通过促进瑞戈非尼敏感基因的表达来增进瑞戈非尼对特定患者的药效。
本发明的优点和有益效果是：
确定了一组基因(RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86)与瑞戈非尼耐药相关；确定了TMPRSS6、ENTPD8、CCDC159基因与瑞戈非尼敏感相关。这些标记物可用于预测瑞戈非尼的药效。通过对肝癌患者的组织测序，或通过RT-qPCR、免疫组化的方法检测相关分子的表达，可为瑞戈非尼的用药与肝癌的精准治疗提供线索。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为肝细胞肝癌(hepatocellular carcinoma，HCC)患者的类器官保留患者的病理特征和组织标记物。其中，图1A.HCC患者不同位点的HE及免疫组化染色；图1B.HCC患者类器官的显微镜下状态和HE染色；图1C.类器官的免疫荧光染色；图1D.类器官的免疫组化染色。
图2为类器官保留患者的外显子、转录组特征。其中，图2A、图2B.外显子测序揭示类器官保留了患者的突变特征。图2C、图2D.转录组测序显示类器官保留了患者的转录组特征。
图3为类器官药敏测试。图3A瑞戈非尼的药敏曲线，图3B，对376例类器官的7种肝癌靶向药物测试。
图4为瑞戈非尼药敏分子分型。图4A.瑞戈非尼的药敏分子分型。图4B.瑞戈非尼训练集和验证集的AUC。图4C.瑞戈非尼耐药靶点互作网络图。
图5为瑞戈非尼分子分型在100例肝癌组织中的验证。
具体实施方式
下面结合实验数据，进一步说明本发明的技术方案。
实施例1
瑞戈非尼药敏标记物的筛选：
本实施例通过3D培养的方法，成功建立了涵盖376个位点的肝癌类器官生物库。以肝细胞肝癌(Hepatocellular carcinoma,HCC)患者的手术标本为例，发明人通过HE染色的方法，证实类器官保留了患者的病理学特征(图1A、1B)。通过对类器官和对应的患者标本进行免疫荧光、免疫组化染色，发现肝细胞癌患者及其类器官高中HCC诊断标记物AFP、hepatocyte阳性(图1C、1D)，而胆管癌标记物EPCAM阴性，与临床上肝癌患者的分子标记物也是一致的。说明本实施例建立的类器官体系很好的保留了患者的病理特征。
本实施例选取99个位点，进行组织和对应类器官的外显子、转录组测序，分析了组织与对应类器官的基因组特征。从体细胞突变、拷贝数变异和转录组相似性等多维度验证了类器官生物库高度保留组织样本的分子特征。团队建立的大规模肝癌组织和类器官队列与以往肝癌研究一致，具有肝癌的高发突变(图2A-B)。在转录组层面，类器官和对应患者的组织也具有高度的相关性(图2C-D)。
本实施例对376个类器官进行肝癌一线药物索拉非尼、仑伐替尼，常用的二/三线药物瑞戈非尼、阿帕替尼、贝伐单抗，以及针对胆管癌FGFR2融合的药物Pemigatinib、胆管癌IDH1突变的药物Ivosidenib的药敏测试。本实施例绘制了一线药物瑞戈非尼的药敏曲线(图3A)和所有靶向药物的热图(图3B)。
为了研究肝癌靶向药物瑞戈非尼的药敏分子分型，本实施例对106个类器官位点进行了转录组测序，通过Lasso模型及生信分析，瑞戈非尼的敏感标记物为TMPRSS6、ENTPD8、CCDC159，耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86(图4A)。其训练集AUC为0.82(图4B)。之后，随后通过同样的方法，收集了106个肝癌标本进行类器官培养、药敏测试和转录组测序，进行分子分型研究，得到瑞戈非尼的验证集为0.80(图4B)。并揭示了瑞戈非尼耐药靶点互作网络图(图4C)。
本实施例在100例肝癌患者的组织中验证了此药敏分子分型的有效性。在肝癌组织中，瑞戈非尼的药敏分子分型AUC为0.79(图5)。
实施例2
瑞戈非尼标记物在非治疗目的的瑞戈非尼用药研究中的应用。本实施例中为对瑞戈非尼活性的研究：选取特定肝癌组织，将不同批次的瑞戈非尼与之作用，测定实施例1中筛选的瑞戈非尼药敏分子(比如RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86)中的一种或几种的定量表达，由此判断各个批次瑞戈非尼的生物活性。
实施例3
本实施例提供一种RT-qPCR瑞戈非尼药敏评估试剂盒，包含检测实施例1中所筛选出的瑞戈非尼药敏标记物(不同试剂盒可以为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86、TMPRSS6、ENTPD8、CCDC159中任意一种)表达量的定量试剂。该试剂盒包含瑞戈非尼药敏标记物的RNA的上下游引物。利用本发明的试剂盒结合常用的RNA提取试剂以及反转录试剂，可以特异性地检测样本中瑞戈非尼药敏标记物的表达量，有助于提前判断肝癌患者对瑞戈非尼的敏感性。可以在肝癌病人进行手术或者病理穿刺取得肝癌组织后即刻测定，在使用瑞戈非尼前即可评估药物疗效，为患者提供个性化精准的治疗方案。
实施例4
本实施例提供一种RT-qPCR瑞戈非尼药敏评估试剂盒，包含检测实施例1中所筛选出的瑞戈非尼敏感耐药标记物(RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86中任意一种)表达量的定量试剂。该试剂盒包含瑞戈非尼敏感耐药标记物的RNA的上下游引物。利用本发明的试剂盒结合常用的RNA提取试剂以及反转录试剂，可以特异性地检测样本中瑞戈非尼敏感耐药标记物的表达量，有助于提前判断肝癌患者对瑞戈非尼的耐药性。可以在肝癌病人进行手术或者病理穿刺取得肝癌组织后即刻测定，在使用瑞戈非尼前即可评估药物疗效，为患者提供个性化精准的治疗方案。
实施例5
本实施例提供一种原发性肝癌的瑞戈非尼耐药分析***，包括：
RT-qPCR靶点表达量检测装置：用于检测样本中瑞戈非尼耐药标记物(RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86中任意一种)的表达量；所述样本为患者肝癌组织；所述靶点表达量检测装置采用的试剂为RT-qPCR定量试剂，包括该基因上下游引物；
耐药分析装置：基于瑞戈非尼耐药标记物RPL39等的表达量，确定肝癌的用药效果；
结果输出装置：用于输出耐药分析装置分析得到的结果。
在患者使用瑞戈非尼前即可评估药物疗效，为患者提供个性化精准的治疗方案。
实施例6
一种治疗肝癌的药物组合物，包含瑞戈非尼及瑞戈非尼耐药标记物(RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86中任意一种或几种)的抑制剂。该药物组合物可以为注射制剂，在肝癌患者治疗中使用，能够降低患者的瑞戈非尼的耐药性。
最后应说明的是，以上仅用以说明本发明的技术方案而非限制，尽管参照较佳布置方案对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (10)

1.一种瑞戈非尼药敏标记物，其特征在于：所述瑞戈非尼药敏标记物为瑞戈非尼耐药标记物或者瑞戈非尼敏感标记物；瑞戈非尼耐药标记物为RPL39、KIF9、GPC2、NKPD1、PNMAL1、ATP1A3、OSR2、ADCYAP1R1、WDR86一组标记物中任一种或几种，所述瑞戈非尼敏感标记物为TMPRSS6、ENTPD8、CCDC159中任意一种或几种。
2.根据权利要求1所述的瑞戈非尼药敏标记物，其特征在于：为RNA或其逆转录的cDNA，或蛋白。
3.权利要求1所述瑞戈非尼药敏标记物在非治疗目的瑞戈非尼用药研究中的应用。
4.检测权利要求1中所述瑞戈非尼药敏标记物表达量的定量试剂在制备瑞戈非尼药敏检测试剂的应用，所述药敏检测为耐药性检测或药物敏感性检测。
5.根据权利要求4所述的应用，检测的样本为肝癌组织样本，所述定量试剂为转录组测序试剂、PCR、基因芯片、组织芯片、NanoString技术、免疫组化试剂中任意一种。
6.一种瑞戈非尼药敏评估试剂盒，其特征在于：包含检测权利要求1中所述瑞戈非尼药敏标记物表达量的定量试剂，所述药敏评估为耐药性评估或药物敏感性评估。
7.一种原发性肝癌的瑞戈非尼药敏分析***，其特征在于，包括：
靶点表达量检测装置：用于检测样本中权利要求1所述瑞戈非尼药敏标记物的表达量；所述样本为患者肝癌组织；
药敏分析装置：基于瑞戈非尼药敏性标记物的表达量，确定肝癌的用药效果；
结果输出装置：用于输出药敏分析装置分析得到的结果，所述药敏分析为耐药性分析或敏感性分析。
8.一种治疗肝癌的药物组合物，其特征在于：包含瑞戈非尼及权利要求1所述瑞戈非尼药敏标记物的抑制剂或促进剂。
9.根据权利要求8所述的治疗肝癌的药物组合物，其特征在于：包含瑞戈非尼及权利要求1所述瑞戈非尼药敏标记物的抑制剂，所述瑞戈非尼药敏标记物为瑞戈非尼耐药标记物。
10.根据权利要求8所述的治疗肝癌的药物组合物，其特征在于：包含瑞戈非尼及权利要求1所述瑞戈非尼药敏标记物的促进剂，所述瑞戈非尼药敏标记物为瑞戈非尼敏感标记物。
CN202210636264.0A 2022-06-07 2022-06-07 瑞戈非尼药敏标记物及其相关试剂的应用 Active CN115125296B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210636264.0A CN115125296B (zh) 2022-06-07 2022-06-07 瑞戈非尼药敏标记物及其相关试剂的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210636264.0A CN115125296B (zh) 2022-06-07 2022-06-07 瑞戈非尼药敏标记物及其相关试剂的应用

Publications (2)

Publication Number Publication Date
CN115125296A true CN115125296A (zh) 2022-09-30
CN115125296B CN115125296B (zh) 2023-04-07

Family

ID=83378795

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210636264.0A Active CN115125296B (zh) 2022-06-07 2022-06-07 瑞戈非尼药敏标记物及其相关试剂的应用

Country Status (1)

Country Link
CN (1) CN115125296B (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130230511A1 (en) * 2012-02-03 2013-09-05 Board Of Regents, The University Of Texas System Biomarkers for response to tyrosine kinase pathway inhibitors in cancer
US20150267258A1 (en) * 2012-05-31 2015-09-24 Bayer Pharma Aktiengesellschaft Biomarkers for determining effective response of treatments of hepatocellular carcinoma (hcc) patients
CN106574306A (zh) * 2014-07-23 2017-04-19 Cbs生物科学公司 用于预测对靶向抗癌药物的接受性的方法
US20170369949A1 (en) * 2016-06-22 2017-12-28 Heinz-Josef Lenz Polymorphism biomarkers predict clinical outcomes of cancer patients receiving regorafenib
WO2019229259A1 (en) * 2018-05-31 2019-12-05 The Institute Of Cancer Research: Royal Cancer Hospital Materials and methods for monitoring the development of resistance of cancers to treatment
CN110904232A (zh) * 2019-12-12 2020-03-24 上海东方肝胆外科医院 评估索拉非尼疗效的分子标记及其检测试剂盒
CN111718902A (zh) * 2020-06-10 2020-09-29 南京大学 敲减SphK2的小干扰RNA在制备逆转肝癌对瑞戈非尼耐药药物中的应用
CN112824540A (zh) * 2019-11-21 2021-05-21 上海市肿瘤研究所 Snx5作为肝癌预后的生物学标志物及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130230511A1 (en) * 2012-02-03 2013-09-05 Board Of Regents, The University Of Texas System Biomarkers for response to tyrosine kinase pathway inhibitors in cancer
US20150267258A1 (en) * 2012-05-31 2015-09-24 Bayer Pharma Aktiengesellschaft Biomarkers for determining effective response of treatments of hepatocellular carcinoma (hcc) patients
CN106574306A (zh) * 2014-07-23 2017-04-19 Cbs生物科学公司 用于预测对靶向抗癌药物的接受性的方法
US20170369949A1 (en) * 2016-06-22 2017-12-28 Heinz-Josef Lenz Polymorphism biomarkers predict clinical outcomes of cancer patients receiving regorafenib
WO2019229259A1 (en) * 2018-05-31 2019-12-05 The Institute Of Cancer Research: Royal Cancer Hospital Materials and methods for monitoring the development of resistance of cancers to treatment
CN112824540A (zh) * 2019-11-21 2021-05-21 上海市肿瘤研究所 Snx5作为肝癌预后的生物学标志物及其应用
CN110904232A (zh) * 2019-12-12 2020-03-24 上海东方肝胆外科医院 评估索拉非尼疗效的分子标记及其检测试剂盒
CN111718902A (zh) * 2020-06-10 2020-09-29 南京大学 敲减SphK2的小干扰RNA在制备逆转肝癌对瑞戈非尼耐药药物中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANYONG ZHUO等: "The distinct responsiveness of cytokeratin 19-positive hepatocellular carcinoma to regorafenib", 《CELL DEATH DIS》 *
吕晓雯等: "瑞戈非尼治疗肝细胞癌的机制及临床效果", 《临床肝胆病杂志》 *

Also Published As

Publication number Publication date
CN115125296B (zh) 2023-04-07

Similar Documents

Publication Publication Date Title
JP6246845B2 (ja) 遺伝子発現を用いた前立腺癌の予後を定量化する方法
DK2582847T3 (en) METHODS AND MATERIALS TO ASSESS loss of heterozygosity
CN108315416A (zh) 基于高通量测序技术确定肺癌基因突变位点的引物、试剂盒及方法
Yang et al. Technical validation of a next-generation sequencing assay for detecting clinically relevant levels of breast cancer–related single-nucleotide variants and copy number variants using simulated cell-free DNA
AU2009234444A1 (en) Methods, agents and kits for the detection of cancer
AU2012345789A1 (en) Methods of treating breast cancer with taxane therapy
CN106755344A (zh) 用于胰腺癌临床预后诊断的分子标记物及其应用
Munoz et al. Molecular profiling and the reclassification of cancer: divide and conquer
WO2020191521A1 (zh) 核酸序列、rna目标区域测序文库的构建方法及应用
CN114990215A (zh) microRNA生物标志物在肺癌诊断或预测预后中的应用
CN110004229A (zh) 多基因作为egfr单克隆抗体类药物耐药标志物的应用
CN106755343A (zh) 胰腺癌预后诊断分子标记物
US20220136065A1 (en) High-grade serous ovarian carcinoma (hgsoc)
CN109852698B (zh) 检测环指蛋白32表达水平的试剂的应用和试剂盒
CN115125296B (zh) 瑞戈非尼药敏标记物及其相关试剂的应用
CN115125303B (zh) 阿帕替尼药敏标记物及其相关试剂的应用
CN114959035B (zh) 索拉非尼耐药分子标记物及其相关试剂的应用
CN114959034B (zh) 仑伐替尼药敏标记物及其相关试剂的应用
JP2022547520A (ja) 肺がんリスクについて検査するためのキットおよび方法
CN104975096B (zh) 非小细胞肺癌的一种诊断标志物
RU2818360C1 (ru) Способ создания таргетной панели для исследования геномных регионов для выявления терапевтических биомаркеров ингибиторов иммунных контрольных точек (ИКТ)
CN115831378A (zh) 一种胆管癌化疗联合免疫治疗疗效预测的模型及其应用
CN117512116A (zh) 一种用于胆管癌检测的生物标志物及其应用
Patel Gene-Level Germline Investigation of Breast Cancer Subtypes and Mortality Among Women of European and African Ancestry
EP4185716A1 (en) Method for in vitro diagnosis of head and neck cancer and related kit

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant