WO2017075784A1 - 肺腺癌生物标记物及其应用 - Google Patents

肺腺癌生物标记物及其应用 Download PDF

Info

Publication number
WO2017075784A1
WO2017075784A1 PCT/CN2015/093909 CN2015093909W WO2017075784A1 WO 2017075784 A1 WO2017075784 A1 WO 2017075784A1 CN 2015093909 W CN2015093909 W CN 2015093909W WO 2017075784 A1 WO2017075784 A1 WO 2017075784A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutation
lung
level
lung adenocarcinoma
gene
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.)
Ceased
Application number
PCT/CN2015/093909
Other languages
English (en)
French (fr)
Inventor
吴逵
李甫强
侯勇
朱师达
叶晓飞
梁颜
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.)
BGI Shenzhen Co Ltd
Original Assignee
BGI Shenzhen Co Ltd
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 BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to KR1020187015074A priority Critical patent/KR102086935B1/ko
Priority to JP2018522993A priority patent/JP6688884B2/ja
Priority to EP15907626.4A priority patent/EP3372686B1/en
Priority to PCT/CN2015/093909 priority patent/WO2017075784A1/zh
Priority to HK18106681.1A priority patent/HK1247242B/zh
Priority to CN201580082101.0A priority patent/CN107849569B/zh
Publication of WO2017075784A1 publication Critical patent/WO2017075784A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • 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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4748—Details p53
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to a lung adenocarcinoma biomarker and its use, and more particularly, the present invention relates to a method, a device and a kit for detecting lung adenocarcinoma in a subject in vitro, and detecting the patient in vitro A method, device and kit for the metastasis of lung adenocarcinoma.
  • next-generation sequencing technology provides a new method for the molecular mechanism research of tumorigenesis, development, invasion and metastasis, and explores the relationship between gene mutation and cancer occurrence from the cancer genome, and predicts disease. Intervention, diagnosis and treatment have become an important research strategy in the field of clinical oncology.
  • Lung cancer is one of the most malignant tumors with the fastest growth in morbidity and mortality among many cancers and the greatest threat to human health and life. In recent years, its incidence has continued to rise on a global scale. This trend is in China. Especially obvious.
  • Lung cancer mainly including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), of which non-small cell carcinoma accounts for 85%, and lung adenocarcinoma is the most common type of pathological non-small cell lung cancer, which is more than global each year. 500,000 people died of this type of cancer.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • the present invention is directed to at least one or at least one of the above mentioned problems.
  • the inventors based on the high incidence of lung adenocarcinoma in East Asian populations and the potential genetic heterogeneity among ethnic groups, comprehensive genomics analysis of East Asian populations, including genetic features of advanced lung adenocarcinoma, especially carrying metastasis In-depth study of lung adenocarcinoma.
  • the inventors used second-generation sequencing technology to conduct in-depth study of genetic mutations and screened for biomarkers including early detection of lung adenocarcinoma.
  • the inventors' findings expanded the spectrum of potential cancer-driven genes in lung adenocarcinomas.
  • Advance the auxiliary treatment and auxiliary diagnosis test of lung adenocarcinoma in particular, it can provide relevant biomarkers for the pathogenesis research, prognosis evaluation and metastasis development of lung adenocarcinoma, and is used for the effective detection of the development of lung adenocarcinoma, and Can be used to improve the cognitive understanding of metastasis development and provide guidance for the diagnosis and treatment of future lung adenocarcinoma metastasis.
  • the inventors analyzed the 335 lung adenocarcinoma primary tumor sample data, 35 lymph node metastasis cancer sample data and normal lung tissue sample data, integrated mutation frequency, gene length, gene biology function, etc., integrated analysis of mutant genes, invented Humans found 13 significant mutations in the lung adenocarcinoma samples - TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2, also in this paper These genes are called pathogenic related genes.
  • the invention provides a method for detecting the presence of lung adenocarcinoma in a subject in vitro, the method comprising: determining a level of mutation of one or more biomarkers in a first part of the subject Obtaining a first mutation level, the biomarker comprising at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2;
  • the level of mutation is compared to a first reference level, and when the first level of mutation is significantly different from the first reference level, the subject is determined to have lung adenocarcinoma, the first reference level being the organism
  • the level of mutation in the marker in the first part of a normal individual the method comprising: determining a level of mutation of one or more biomarkers in a first part of the subject Obtaining a first mutation level, the biomarker comprising at least one of the following genes
  • a device for detecting the presence of a lung adenocarcinoma in a subject the device for performing all of the above methods of detecting a lung adenocarcinoma in a subject in vitro.
  • the apparatus comprising: a first mutation detecting unit for determining a mutation level of one or more biomarkers in a first part of the subject, obtaining a first mutation level, the biomarker comprising At least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; a first determining unit, coupled to the first mutation detecting unit, for The first mutation level is compared to a first reference level, and when the first mutation level is significantly different from the first reference level, the subject is determined to have lung adenocarcinoma, the first reference level The level of mutation of the biomarker in the first part of a normal individual.
  • the present invention provides a kit for detecting a biological sample as a lung adenocarcinoma sample, the kit comprising: an reagent for detecting a mutation level of a biomarker in the biological sample,
  • the biomarker comprises at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; and indicating the level of mutation of the biomarker and lung adenocarcinoma turn off
  • a first instruction of the system optionally, the first specification comprising first reference level information, the first reference level being a level of mutation of the biomarker in a first part of a normal individual.
  • the present invention provides a method for detecting the presence of lung adenocarcinoma in a subject in vitro, the method comprising: detecting a mutation level of one or more biomarkers in a first part of the subject Obtaining a first mutation level, detecting a mutation level of the same biomarker in the second part of the subject, obtaining a second mutation level, the biomarker comprising at least one of the following genes: TP53, EGFR , LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2, said second site being a normal site; comparing said first mutation level and said second mutation level when said When the first mutation level is significantly different from the second mutation level, the subject is determined to have lung adenocarcinoma.
  • a device for detecting the presence of a lung adenocarcinoma in a subject the device for performing all of the above methods of the present invention for detecting the presence of lung adenocarcinoma in a subject in vitro.
  • the apparatus comprising: a second mutation detecting unit for detecting a mutation level of one or more biomarkers in a first part of the subject, obtaining a first mutation level, and for detecting the subject The level of mutation of the same biomarker in the second site, obtaining a second mutation level, the biomarker comprising at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF , FLT1, RHPN2, GLI3 and MRC2, the second part is a normal part; a second determining unit is connected to the second mutation detecting unit for comparing the first mutation level and the second mutation level, When the first mutation level is significantly different from the second mutation level, the subject is determined to have lung adenocarcinoma.
  • the three genes RHPN2, GLI3 and MRC2 are the first three genes driven by the inventors to have high correlation in lung adenocarcinoma.
  • the driving gene is an important gene related to the development of cancer. These three highly correlated lung adenocarcinoma drive genes have not been reported and can be used for the detection or diagnosis of lung adenocarcinoma.
  • the invention provides a panel of lung adenocarcinoma driver genes consisting of the genes RHPN2, GLI3 and MRC2.
  • the present invention provides the use of the above-described lung adenocarcinoma driver gene of the present invention for the treatment of lung adenocarcinoma, and/or for the preparation of a medicament for treating lung adenocarcinoma.
  • the present invention provides a method for detecting the presence of lung adenocarcinoma in a subject in vitro, the method comprising: detecting whether a lung adenocarcinoma driving gene in a lung tissue or a lung cell of a subject has a mutation
  • the lung adenocarcinoma driving gene is composed of genes RHPN2, GLI3 and MRC2; when there is a mutation in the lung adenocarcinoma driving gene, it is determined that the subject has lung adenocarcinoma.
  • the present invention provides a device for detecting the presence of lung adenocarcinoma in a subject, the device being used for Performing all or part of the steps of the method for detecting lung adenocarcinoma in a subject in vitro, wherein the apparatus comprises: a third mutation detecting unit for detecting lung tissue in the subject or lung gland in the lung cell Whether the cancer driving gene is mutated, the lung adenocarcinoma driving gene is composed of the genes RHPN2, GLI3 and MRC2; and the third determining unit is connected to the mutation detecting unit for the mutation of the lung adenocarcinoma driving gene At the time, the subject is determined to have lung adenocarcinoma.
  • the present invention provides a kit for determining a biological sample as a lung adenocarcinoma sample, the kit comprising: an agent for detecting a mutation in a lung adenocarcinoma driving gene in the biological sample,
  • the lung adenocarcinoma driver gene consists of the genes RHPN2, GLI3 and MRC2; and a second instruction indicating a relationship between a lung adenocarcinoma-driven gene mutation and a lung adenocarcinoma.
  • metastasis is the most critical event in the development of the disease, but there is currently no systematic study of the differences in mutation patterns between patients with primary lung adenocarcinoma carrying metastases and those without metastasis.
  • the inventors compared the lung adenocarcinoma and lymph node adenocarcinoma samples that had been diagnosed with lymph node metastasis or distant metastasis, and analyzed the Fisher's exact test to find that the TP53 mutant gene is the only one that carries metastatic lung adenocarcinoma.
  • the significantly enriched gene suggests that this gene is not only a tumor suppressor gene significantly associated with tumorigenesis, but also has a driving role in the development of tumor metastasis.
  • the mutation rate of the TP53 gene can be used as a biomarker for detecting whether lung adenocarcinoma metastasizes and treats prognosis.
  • the present invention provides a method for detecting whether a lung adenocarcinoma of a patient has metastasis in vitro, the method comprising: detecting a mutation level of a TP53 gene in a lung tissue or a lung cell of the patient, and obtaining a mutation of the TP53 gene Level, the patient has lung adenocarcinoma; comparing the TP53 gene mutation level with a second reference level, determining that the patient's lung adenocarcinoma occurs when the gene mutation level is significantly different from the second reference level Metastasis, the second reference level is the level of mutation of the TP53 gene in lung tissue or lung cells of a lung adenocarcinoma patient who has not metastasized.
  • the present invention provides a device for detecting whether a lung adenocarcinoma of a patient has metastasized, the device for performing all or part of steps of a method for detecting whether a lung adenocarcinoma of a patient has metastasized in an aspect of the present invention
  • the device comprises: a TP53 gene detecting unit for detecting a mutation level of a TP53 gene in a lung tissue or a lung cell of the patient, obtaining a mutation level of the TP53 gene, the patient having a lung adenocarcinoma; a fourth determining unit, and a
  • the TP53 gene mutation detecting unit is connected to compare the TP53 gene mutation level with a second reference level, and when the gene mutation level is significantly different from the second reference level, determining the patient lung adenocarcinoma metastasis
  • the second reference level is the level of mutation of the TP53 gene in lung tissue or lung cells of a lung aden
  • the present invention provides a kit for detecting whether a biological sample is a transferred lung adenocarcinoma sample, the kit comprising: a reagent for detecting a mutation level of a TP53 gene in the biological sample And a third instruction indicating a relationship between the level of mutation of the TP53 gene and the metastatic lung adenocarcinoma, the third instruction kit A second reference level is included, the second reference level being the level of mutation of the TP53 gene in lung tissue or lung cells of a lung adenocarcinoma patient who has not metastasized.
  • the inventors compared data from lung adenocarcinoma samples of 335 Chinese patients with lung adenocarcinoma by Kaplan-Meier survival analysis and found that among wild-type individuals, they carried seven genes including TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2. Mutations in at least one gene suggest a significantly shortened survival.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes. Mutations in any of the above genes can be used as biomarkers for predicting survival in clinical practice.
  • the present invention provides a method for detecting a prognosis effect of a patient with lung adenocarcinoma, the method comprising: detecting whether at least one of the following gene mutations is present in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis: TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2; patients with lung adenocarcinomas in which at least one of the genes are mutated have a poor prognosis.
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the present invention provides a device for detecting a prognosis effect of a patient with lung adenocarcinoma, the device for performing all or part of the steps of the method for detecting the prognosis of a patient with lung adenocarcinoma according to an aspect of the present invention described above,
  • the device comprises: a fourth mutation detecting unit for detecting presence or absence of at least one gene mutation in lung tissue or lung cells of a patient with lung adenocarcinoma prognosis: TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2;
  • the determining unit is connected to the fourth mutation detecting unit, and is used for determining that there is a poor prognosis effect of the lung adenocarcinoma patient in which at least one of the genes has a mutation.
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the present invention provides a kit for detecting a prognosis of a patient with lung adenocarcinoma, the kit comprising: at least one of the following in the lung tissue or lung cell for detecting a prognosis of a lung adenocarcinoma
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the inventors have also discovered a novel lung adenocarcinoma driver gene, IQGAP3, which is expressed at high levels in lung adenocarcinoma samples.
  • IQGAP3 lung adenocarcinoma driver gene
  • the inventors found that the highly expressed IQGAP3 gene was significantly associated with shortened overall survival and disease-free survival, suggesting a high correlation with poor prognosis of lung adenocarcinoma.
  • the IQGAP3 gene can be used as a biomarker for accurate detection and prognosis evaluation of lung adenocarcinoma.
  • the present invention provides a method for detecting a prognosis effect of a lung adenocarcinoma patient, the method comprising: detecting an expression level of an IQGAP3 gene in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis; The expression level is compared with a third reference level, and when the expression level is significantly different from the third reference level, the prognosis effect of the lung adenocarcinoma patient is determined to be poor, and the third reference level is the prognosis of the IQGAP3 gene.
  • the expression level of lung tissue or lung cells in patients with good lung adenocarcinoma is compared with a third reference level, and when the expression level is significantly different from the third reference level, the prognosis effect of the lung adenocarcinoma patient is determined to be poor, and the third reference level is the prognosis of the IQGAP3 gene.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the poor prognosis results indicate that the survival of wild-type individual patients is significantly shorter than the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • the present invention provides a device for detecting a prognosis effect of a patient with lung adenocarcinoma, the device for performing all or part of steps of a method for detecting a prognosis of a patient with a lung adenocarcinoma according to an aspect of the present invention, including a gene expression level detecting unit for detecting an expression level of an IQGAP3 gene in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis; a sixth determining unit, connected to the gene expression level detecting unit, for expressing the expression Levels are compared with a third reference level, and when the expression level is significantly different from the third reference level, it is determined that the prognosis effect of the lung adenocarcinoma patient is poor, and the third reference level is that the IQGAP3 gene has a good prognosis effect.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the poor prognosis results indicate that the survival of wild-type individual patients is significantly shorter than the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • the present invention provides a kit for detecting the prognosis of a patient with lung adenocarcinoma, the kit comprising: the expression of IQGAP3 gene in lung tissue or lung cells of a patient for detecting lung adenocarcinoma prognosis a horizontal reagent; and a fifth instruction indicating a relationship between the expression level and a prognosis effect of a lung adenocarcinoma patient, the fifth specification comprising third reference level information, wherein the third reference level is a lung with a good prognosis of the IQGAP3 gene Expression levels in lung tissue or lung cells of patients with adenocarcinoma.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the so-called prognostic effect includes a significantly longer survival period than a wild-type individual patient compared to the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • Figure 1 shows a projection of a lung adenocarcinoma-associated gene in a population or sample of different lung adenocarcinomas in one embodiment of the present invention. Variable frequency.
  • Figure 2 is a graph showing the types of mutations and mutation positions of various lung adenocarcinoma-associated genes in one embodiment of the present invention; wherein, Figure 2A shows the types of mutations and mutation positions appearing on the genes TP53, EGFR and LRP1B, and Figure 2B shows the genes.
  • Figure 2C shows the types of mutations and mutation positions on the genes BRAF, GLI3, FLT1, MRC2 and SMAD2
  • Figure 2D shows the genes APC, KEAP1, ATF7IP
  • Figure 2E show the types of mutations and locations of mutations that occur on genes ERBB2 and TERT.
  • Figure 3 shows the association of lung adenocarcinoma-associated gene mutations with clinical characterization in one embodiment of the invention.
  • Figure 4 is a graph showing the effect of seven gene mutations associated with lung adenocarcinoma on survival and survival in one embodiment of the present invention.
  • Figure 5 shows the expression levels of the IQGAP3 gene in normal tissues, primary lung adenocarcinoma, and metastatic lung adenocarcinoma samples in one embodiment of the present invention.
  • Figure 6 shows Kaplan-Meier survival curves of IQGAP3 gene expression levels in lung adenocarcinoma patients in one embodiment of the invention.
  • Figure 7 shows a test procedure for determining lung adenocarcinoma-associated biomarkers in one embodiment of the invention.
  • first, second, and the like, used herein are used for the purpose of description only and are not to be understood as indicating or implying a relative importance, implicitly indicating the number of the indicated technical features, or having a sequential relationship. Thus, features defining “first” or “second” may include one or more of the features either explicitly or implicitly. In the description of the present invention, "a plurality” means two or more unless otherwise stated.
  • connection shall be understood broadly, and may be, for example, a fixed connection, a detachable connection, or an integral one, unless explicitly stated or defined otherwise.
  • Connection it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, which can be the internal connection of the two elements.
  • intermediate medium which can be the internal connection of the two elements.
  • mutation refers to somatic mutation, somatic cell mutation, unless otherwise explicitly stated or defined. Mutation refers to mutations in somatic cells other than sex cells.
  • a method for detecting the presence of lung adenocarcinoma in a subject in vitro comprising: determining a mutation level of one or more biomarkers in a first part of a subject, obtaining At a first mutation level, the biomarker comprises at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; Comparing with the first reference level, when the first mutation level is significantly different from the first reference level, determining that the subject has lung adenocarcinoma, the first reference level is the biomarker The level of mutation in the first part of a normal individual.
  • the first portion referred to may be at least one of lung cells, lung tissue, and plasma free nucleic acids.
  • the level of mutation referred to includes whether there is a mutation in the gene and/or the number of mutations present and/or the type of mutation present. According to an embodiment of the invention, the detection of the level of mutation utilizes a second generation sequencing technique.
  • the sequencing data is aligned with a reference sequence, and the mutation on the target gene is identified based on the alignment result, including identifying whether a mutation is present on the target gene, the number of mutations present, and/or the type of mutation.
  • Sequencing can be performed by using an existing sequencing platform, and corresponding library preparation can be performed according to the selected sequencing platform.
  • the optional sequencing platforms include, but are not limited to, CG (Complete Genomics) CGA, Illumina/Solexa, Life Technologies/Ion Torrent, and Roche 454, preparation of single-ended or double-end sequencing libraries based on the selected sequencing platform.
  • the comparison can be performed by using software such as SOAP (Short Oligonucleotide Analysis Package), BWA, etc., and the embodiment does not limit this.
  • SOAP Short Oligonucleotide Analysis Package
  • BWA Short Oligonucleotide Analysis Package
  • each reading in the sequencing data is allowed to be allowed at most.
  • h base mismatch h is preferably 1 or 2. If more than h bases in a reads are mismatched, it is considered that the reads cannot be compared to the reference sequence.
  • the comparison result includes the comparison of each read segment with the reference sequence, including whether the read segment can compare the reference sequence, the position of the reference sequence on the read alignment, how many reads at a certain point are aligned, and the comparison The base type of the corresponding position of the read of a certain site, and the like.
  • the reference sequence referred to is a known sequence, which may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the nucleic acid sample is from a human, its genome reference
  • the sequence also known as the reference genome
  • various mutations can be based on the obtained comparison results, using corresponding known software or program identification judgments, for example, SNP identification can be implemented by software such as SOAPsnp, GATK, etc. The default parameter settings are made.
  • the so-called first reference level can be determined when detecting whether a subject has lung adenocarcinoma, for example, when determining the mutation level of one or more biomarkers in the lung tissue or lung cells of the subject, simultaneously detecting The level of mutation of the biomarker in the first part of a plurality of normal individuals; the first reference level can also be pre-determined, saved or recorded for later use. According to one embodiment of the invention, the first reference level is the average level of mutation of the biomarker in the lung tissue or lung cells of the plurality of normal individuals, pre-determined for storage.
  • the so-called significant difference may be significant, substantial difference, for example, the first reference level is shown as no mutation, and the first mutation level is shown as the presence of a mutation, and for example, the first reference level is shown as a no-sense mutation,
  • the first mutation level is shown as having a missense mutation, and for example, the first reference level is shown as N mutations, and the first mutation level is shown as 1.5N, 2N or more than 2N mutations; the significant difference may also be referred to as The statistical difference is significant.
  • the above detection method of this embodiment of the present invention is based on the discovery of a high incidence of lung adenocarcinoma in East Asian populations and potential genetic heterogeneity among ethnic groups, and comprehensive genomic analysis of East Asian populations, including The genetic characteristics of lung adenocarcinoma, especially those with metastatic lung adenocarcinoma, were obtained.
  • the inventors used second-generation sequencing technology to further study genetic mutations and screened for biomarkers including early detection of lung adenocarcinoma.
  • the inventors' findings expanded the spectrum of potential cancer-driven genes in lung adenocarcinoma and actively promoted lung adenocarcinoma.
  • Auxiliary therapy and auxiliary diagnostic test can provide relevant biomarkers for the pathogenesis research, prognosis evaluation and metastasis development of lung adenocarcinoma, for the effective detection of the development of lung adenocarcinoma, and can be used to improve Transfer the discipline of cognitive development and provide guidance for the future diagnosis and treatment of lung adenocarcinoma metastasis.
  • the inventors analyzed the 335 lung adenocarcinoma primary tumor sample data, 35 lymph node metastasis cancer sample data and normal lung tissue sample data, comprehensive mutation rate, gene length, gene biology function, etc., integrated analysis of mutant genes, invention
  • the above 13 significant mutations in the lung adenocarcinoma samples were found to be statistically significant - TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2, in this paper
  • These genes are also referred to as pathogenicity related genes.
  • the TP53 gene has a statistically significant mutation in 44% of Chinese lung adenocarcinoma, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma; the inventors found that the EGFR gene is in 39% of Chinese lungs.
  • EGFR gene mutation rate is higher than KRAS, and shows EGFR major mutation At the Leu858Arg and exon19del sites, a sensitive site for tyrosine kinase inhibitors can be used as a biomarker for the diagnosis and targeted therapy of lung adenocarcinoma; the inventors found that the LRP1B gene is in 19% of Chinese lungs.
  • a statistically significant mutation in adenocarcinoma suggests a biomarker for the diagnosis of lung adenocarcinoma; the invention found that the KRAS gene has a statistically significant mutation in 11% of Chinese lung adenocarcinoma, suggesting that As a biomarker for the diagnosis of lung adenocarcinoma; the inventors found that the PTPRD gene is present in 7% of Chinese lung adenocarcinoma Significant statistically significant mutations suggest that it can be used as a biomarker for the diagnosis of lung adenocarcinoma; the inventors found that the STK11 gene has a statistically significant mutation in 4% of Chinese lung adenocarcinoma, suggesting that it can be used as a lung A biomarker for the diagnosis of adenocarcinoma; the inventors found that the PIK3CA gene has a statistically significant mutation in 5% Chinese lung adenocarcinoma, suggesting that it can be used as a biomarker for the diagnosis of lung
  • a significant mutation in the meaning of the study suggests that it can be used as a biomarker for the diagnosis of lung adenocarcinoma; the inventors found that the BRAF gene has a statistically significant mutation in 4% of Chinese lung adenocarcinoma, suggesting that it can be used as a lung adenocarcinoma A biomarker for diagnosis; the inventors found that the GLI3 gene has a statistically significant mutation in 4% of Chinese lung adenocarcinoma, suggesting that it can be used as a lung gland A biomarker for diagnosis; the inventors found that the FLT1 gene has a statistically significant mutation in 3% Chinese lung adenocarcinoma, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma; MRC2, alias uPARAP, Endo180 or CD280, found that the MRC2 gene has a statistically significant mutation in 2% Chinese lung adenocarcinoma, suggesting that it can be used as a
  • the use of the so-called biomarker for predicting the likelihood of pathogenesis of lung adenocarcinoma can be used to assist in the determination of lung adenocarcinoma as an early stage of assisted lung adenocarcinoma. diagnosis method.
  • the so-called biomarker includes any one, two, three of the genes TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2.
  • the so-called first mutation level corresponds to the first reference level and the level of mutation comprising a plurality of genes.
  • a device for detecting the presence of lung adenocarcinoma in a subject which device can be used to perform the above-described embodiment of the present invention or an in vitro test for the presence of a lung gland in a subject All or part of the steps of the method of cancer, the apparatus comprising: a first mutation detecting unit for determining a mutation level of one or more biomarkers in the first part of the subject, obtaining a first mutation level
  • the biomarker comprises at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; a first determining unit, and the first mutation a detection unit coupled for comparing the first mutation level to a first reference level, and determining that the subject has lung adenocarcinoma when the first mutation level is significantly different from the first reference level
  • the first reference level is a
  • the first portion referred to may be at least one of lung cells, lung tissue, and plasma free nucleic acids.
  • the above description of the technical features and advantages of the method for detecting lung adenocarcinoma in a subject in vitro in an embodiment of the present invention is equally applicable to the detection of the presence of lung adenocarcinoma in a subject in this embodiment of the present invention. Set, no longer repeat here.
  • the implementation manner of each step of the method in any of the above embodiments or embodiments may be implemented by using connected subunits, for example, the so-called first mutation detecting unit includes a sequencer. The unit obtains the sequencing data, and the comparison subunit is used to align the sequencing data with the reference sequence, obtain the alignment result, and the like.
  • a kit for detecting a biological sample as a lung adenocarcinoma sample comprising: an reagent for detecting a mutation level of a biomarker in the biological sample, the biomarker And at least one of the following genes: TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; and indicating a relationship between the level of mutation of the biomarker and lung adenocarcinoma
  • the first specification includes first reference level information, the first reference level being a level of mutation of the biomarker in a portion of a normal individual that is from the same source as the biological sample.
  • the so-called biological sample may be or be derived from at least one of lung cells, lung tissue, and plasma free nucleic acids.
  • the above description of the technical features and advantages of the method for detecting lung adenocarcinoma in an in vitro test subject in an embodiment or any embodiment of the present invention is equally applicable to the test kit of this embodiment of the present invention, and is no longer Narration.
  • the first instruction includes comparing the first mutation level to a first reference level, and when the first mutation level is significantly different from the first reference level, determining that the subject has lung adenocarcinoma Instructions.
  • a method for detecting the presence of lung adenocarcinoma in a subject in vitro comprising: detecting a mutation level of one or more biomarkers in a first part of the subject, obtaining a first mutation level, detecting a level of mutation of the same biomarker in a second part of the subject, to obtain a second mutation level, the biomarker comprising at least one of the following genes: TP53, EGFR, LRP1B , KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2, said second site being a normal site; comparing said first mutation level and said second mutation level, said first When the level of mutation is significantly different from the level of the second mutation, the subject is determined to have lung adenocarcinoma.
  • the first portion referred to may be at least one of lung cells, lung tissue, and plasma free nucleic acid, and the second portion is referred to as a known normal or known non-lesional site of the subject.
  • the above description of the technical features and advantages of the method for detecting lung adenocarcinoma in a subject in vitro according to an embodiment or any embodiment of the present invention is equally applicable to the presence of a lung gland in an in vitro test subject in this embodiment of the present invention. The method of cancer is not repeated here.
  • a device for detecting presence of lung adenocarcinoma in a subject the device for performing all or part of the steps of the method for detecting lung adenocarcinoma in a subject in vitro in the above embodiment
  • the device includes: a second mutation detecting unit for detecting a mutation level of one or more biomarkers in the first part of the subject, Obtaining a first mutation level, and detecting a level of mutation of the same biomarker in a second part of the subject, obtaining a second mutation level, the biomarker comprising at least one of the following genes: TP53, EGFR , LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2; a second determining unit coupled to said second mutation detecting unit for comparing said first mutation level with said A second mutation level, the subject is determined to have lung adenocarcinoma when the first
  • the first portion referred to may be at least one of lung cells, lung tissue, and plasma free nucleic acid
  • the second portion is referred to as the subject's known normal or confirmed to be non- The site of the lesion.
  • the so-called second mutation detecting unit includes a sequencing subunit to obtain The data is sequenced such that the aligned subunits are used to align the sequencing data with the reference sequence to obtain alignment results and the like.
  • a set of lung adenocarcinoma driver genes consisting of genes RHPN2, GLI3 and MRC2.
  • the combination of the three genes RHPN2, GLI3 and MRC2 is the first three genes that the inventors found to have high correlation in lung adenocarcinoma.
  • the driving gene is an important gene related to the development of cancer.
  • the lung adenocarcinoma driver gene of the above embodiment for treating lung adenocarcinoma, and/or for preparing a medicament for treating lung adenocarcinoma.
  • the three genes RHPN2, GLI3 and MRC2 are the first three genes driven by the inventors to have high correlation in lung adenocarcinoma.
  • the driving gene is an important gene related to the development of cancer. These three highly correlated lung adenocarcinoma drive genes have not been reported and can be used for the detection or diagnosis of lung adenocarcinoma.
  • a method for detecting the presence of lung adenocarcinoma in a subject in vitro comprising: detecting whether a lung adenocarcinoma driving gene in a lung tissue or a lung cell of a subject has a mutation,
  • the lung adenocarcinoma driving gene is composed of genes RHPN2, GLI3 and MRC2; when there is a mutation in the lung adenocarcinoma driving gene, it is determined that the subject has lung adenocarcinoma.
  • the detecting whether the lung adenocarcinoma driving gene in the lung tissue or the lung cell of the subject is mutated comprises: detecting whether the RHPN2 gene has a V73M mutation.
  • a device for detecting the presence of lung adenocarcinoma in a subject the device for performing the method of in vitro detection of a lung adenocarcinoma in a subject in the above embodiment or any of the embodiments.
  • the apparatus comprises: a third mutation detecting unit for detecting whether a lung adenocarcinoma driving gene in the lung tissue or the lung cell of the subject is mutated, and the lung adenocarcinoma driving gene is caused by the gene RHPN2, GLI3 and MRC2 are composed; a third determining unit is connected to the mutation detecting unit for determining that the subject has lung adenocarcinoma when there is a mutation in the lung adenocarcinoma driving gene.
  • the three genes RHPN2, GLI3 and MRC2 are the first three genes driven by the inventors to have high correlation in lung adenocarcinoma.
  • the driving gene is an important gene related to the development of cancer. These three highly correlated lung adenocarcinoma drive genes have not been reported and can be used for the detection or diagnosis of lung adenocarcinoma.
  • the detecting whether the lung adenocarcinoma driving gene in the lung tissue or the lung cell of the subject is mutated comprises: detecting whether the RHPN2 gene has a V73M mutation.
  • a kit for determining a biological sample as a lung adenocarcinoma sample comprising: an agent for detecting a mutation in a lung adenocarcinoma driving gene in the biological sample,
  • the lung adenocarcinoma driver gene consists of the genes RHPN2, GLI3 and MRC2; and a second instruction indicating the relationship between lung adenocarcinoma-driven gene mutation and lung adenocarcinoma.
  • the so-called second instruction comprises determining information about the presence of lung adenocarcinoma in the subject when the lung adenocarcinoma driver gene is mutated.
  • metastasis is the most critical event in the development of the disease, but there is currently no systematic study on the difference in the pattern of mutations in patients with primary lung adenocarcinoma who carry metastases and do not carry metastases.
  • the inventors compared the lung adenocarcinoma and lymph node adenocarcinoma samples that had been diagnosed with lymph node metastasis or distant metastasis, and analyzed the Fisher's exact test to find that the TP53 mutant gene is the only one that carries metastatic lung adenocarcinoma.
  • the significantly enriched gene (P ⁇ 0.05), see Figure 3, suggests that this gene is not only a tumor suppressor gene that is significantly associated with tumorigenesis, but also has a driving role in tumor metastasis development.
  • the mutation rate of the TP53 gene can be used as a biomarker for detecting whether lung adenocarcinoma metastasizes and treats prognosis.
  • a method for detecting whether a lung adenocarcinoma of a patient has metastasized in vitro comprising: detecting a mutation level of a TP53 gene in a lung tissue or a lung cell of the patient, and obtaining a mutation level of the TP53 gene, The patient has lung adenocarcinoma; comparing the TP53 gene mutation level with a second reference level, when the gene mutation level is significantly different from the second reference level, determining that the patient's lung adenocarcinoma metastasizes, The second reference level is the level of mutation of the TP53 gene in the lung tissue or lung cells of a lung adenocarcinoma patient who has not metastasized.
  • the level of mutation referred to includes the presence or absence of a mutation in the TP53 gene and/or the number of mutations present and/or the type of mutation present.
  • the detection of the level of mutation utilizes a second generation sequencing technique. Extracting nucleic acid in one or more lung tissue samples or lung cell samples, performing sequencing library preparation on the extracted nucleic acid by using sequencing technology, and performing on-machine sequencing on the library to obtain the offline data, ie, sequencing data; The sequencing data is analyzed, including aligning the sequencing data with a reference sequence, and identifying mutations on the target gene based on the alignment results, including identifying whether a mutation is present on the target gene, the number of mutations present, and/or the type of mutation.
  • Sequencing can be performed by using an existing sequencing platform, and corresponding library preparation can be performed according to the selected sequencing platform.
  • the optional sequencing platforms include, but are not limited to, CG (Complete Genomics) CGA, Illumina/Solexa, Life Technologies/Ion Torrent, and Roche 454, preparation of single-ended or double-end sequencing libraries based on the selected sequencing platform.
  • the comparison can be performed by using software such as SOAP (Short Oligonucleotide Analysis Package), BWA, etc., and the embodiment does not limit this. In the comparison process, according to the setting of the comparison parameter, for example, each reading in the sequencing data is allowed to be allowed at most.
  • h base mismatch h is preferably 1 or 2.
  • the comparison result includes the comparison of each read segment with the reference sequence, including whether the read segment can compare the reference sequence, the position of the reference sequence on the read alignment, how many reads at a certain point are aligned, and the comparison The base type of the corresponding position of the read of a certain site, and the like.
  • the reference sequence referred to is a known sequence, which may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the nucleic acid sample is from a human, its genome reference The sequence (also known as the reference genome) can be selected from the HG19 provided by the NCBI database.
  • various mutations can be based on the obtained comparison results, using corresponding known software or program identification judgments, for example, SNP identification can be implemented by software such as SOAPsnp, GATK, etc. The default parameter settings are made.
  • the so-called second reference level can be determined when detecting whether a subject has lung adenocarcinoma, for example, when determining the level of mutation of one or more biomarkers in a lung tissue or a lung cell sample of a patient with a lung adenocarcinoma, Simultaneously detecting the level of mutation of the same biomarker in the lung tissue or lung cells of a plurality of lung adenocarcinoma patients who have not metastasized; the second reference level can also be determined in advance, saved or recorded for use.
  • the second reference level is the average level of mutation of the biomarker in the lung tissue or lung cells of the plurality of non-metastatic lung adenocarcinoma patients, pre-determined for storage.
  • the second reference level is shown as no mutation
  • the TP53 gene mutation level is shown as the presence of a mutation
  • the second reference level is, for example, a no-sense mutation
  • the TP53 gene mutation level is shown to have a missense mutation, and for example, the second reference level shows N mutations, and the TP53 gene mutation level shows 1.5N, 2N or more than 2N mutations; the significant difference can also be referred to as Statistical difference
  • the difference is significant.
  • a device for detecting whether a lung adenocarcinoma of a patient has metastasized the device for performing all or part of a method for detecting whether a lung adenocarcinoma of a patient has metastasized in an aspect of the present invention
  • the device comprises: a TP53 gene detecting unit for detecting a mutation level of a TP53 gene in a lung tissue or a lung cell of the patient, obtaining a level of mutation of the TP53 gene, the patient having lung adenocarcinoma; a fourth determining unit, and the TP53 a gene mutation detecting unit is connected to compare the TP53 gene mutation level with a second reference level, and when the gene mutation level is significantly different from the second reference level, determining that the patient's lung adenocarcinoma metastasizes
  • the second reference level is the level of mutation in the lung tissue or lung cells of the TP53 gene in lung adenocarcinoma patients
  • kits for detecting whether a biological sample is a transferred lung adenocarcinoma sample comprising: an reagent for detecting a mutation level of a TP53 gene in the biological sample; A third specification indicating the relationship between the level of mutation of the TP53 gene and the metastatic lung adenocarcinoma, the third specification comprising second reference level information, wherein the second reference level is a lung of the TP53 gene that has not metastasized The level of mutation in lung tissue or lung cells of patients with adenocarcinoma.
  • the third instruction includes comparing the TP53 gene mutation level with a second reference level, and determining information or operation of the patient's lung adenocarcinoma metastasis when the gene mutation level is significantly different from the second reference level. Instructions.
  • a method for detecting a prognosis effect of a lung adenocarcinoma patient comprising: detecting whether at least one of the following gene mutations exists in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis: TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2; patients with lung adenocarcinoma who are determined to have at least one mutation in the gene have poor prognosis.
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the inventors compared data from lung adenocarcinoma samples of 335 Chinese patients with lung adenocarcinoma by Kaplan-Meier survival analysis and found that among wild-type individuals, they carried seven genes including TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2. Mutations in at least one gene suggest a significantly shortened survival, see Figure 4.
  • the so-called wild The individual patient is a lung adenocarcinoma patient who does not have a mutation on any of the above genes. Mutations in any of the above genes can be used as biomarkers for predicting survival in clinical practice.
  • the detection of mutations can utilize second generation sequencing techniques. Extracting nucleic acid samples from lung tissue samples or lung cell samples of one or more lung adenocarcinoma prognosis patients, sequencing the extracted nucleic acid by sequencing technology, and sequencing the library on the machine to obtain the off-machine data Sequencing the data; then, analyzing the sequencing data, including aligning the sequencing data with a reference sequence, and identifying mutations in the target gene based on the alignment results. Sequencing can be performed by using an existing sequencing platform, and corresponding library preparation can be performed according to the selected sequencing platform.
  • the optional sequencing platforms include, but are not limited to, CG (Complete Genomics) CGA, Illumina/Solexa, Life Technologies/Ion Torrent, and Roche 454, preparation of single-ended or double-end sequencing libraries based on the selected sequencing platform.
  • the comparison can be performed by using software such as SOAP (Short Oligonucleotide Analysis Package), BWA, etc., and the embodiment does not limit this.
  • SOAP Short Oligonucleotide Analysis Package
  • BWA Short Oligonucleotide Analysis Package
  • each reading in the sequencing data is allowed to be allowed at most.
  • h base mismatch h is preferably 1 or 2. If more than h bases in a reads are mismatched, it is considered that the reads cannot be compared to the reference sequence.
  • the comparison result includes the comparison of each read segment with the reference sequence, including whether the read segment can compare the reference sequence, the position of the reference sequence on the read alignment, how many reads at a certain point are aligned, and the comparison The base type of the corresponding position of the read of a certain site, and the like.
  • the reference sequence referred to is a known sequence, which may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the nucleic acid sample is from a human, its genome reference
  • the sequence also known as the reference genome
  • various mutations can be based on the obtained comparison results, using corresponding known software or program identification judgments, for example, SNP identification can be implemented by software such as SOAPsnp, GATK, etc. The default parameter settings are made.
  • the so-called lung adenocarcinoma patient having the presence of at least one mutation in the gene has a poor prognosis, wherein the mutation is a missense mutation.
  • a device for detecting a prognosis effect of a lung adenocarcinoma patient the device for performing all or part of the steps of the method for detecting a prognosis of a lung adenocarcinoma patient according to an aspect of the present invention
  • the device The fourth mutation detecting unit is configured to detect whether at least one of the following gene mutations exists in lung tissue or lung cells of a patient with lung adenocarcinoma prognosis: TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2; fifth determining unit
  • the fourth mutation detecting unit is connected to determine that there is a poor prognosis of the lung adenocarcinoma patient in which at least one of the genes has a mutation.
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the above description of the technical features and advantages of the method for detecting the prognosis of a patient with lung adenocarcinoma in an embodiment of the present invention is the same The device in this embodiment is applicable, and details are not described herein again.
  • a kit for detecting a prognosis effect of a lung adenocarcinoma patient comprising: detecting whether at least one of the following genes is present in a lung tissue or a lung cell of a patient prognosis of a lung adenocarcinoma Mutant reagents: TP53, LRP1B, STK11, KEAP1, BRAF, MET and MRC2; and a fourth specification indicating poor prognosis of patients with lung adenocarcinoma in which at least one of said genes is mutated.
  • the poor prognosis described includes a significant reduction in survival compared to wild-type individual patients with prognosis.
  • the so-called wild-type individual patient is a lung adenocarcinoma patient in which no mutation is present on any of the above genes.
  • the above description of the technical features and advantages of the method for detecting the prognosis of a patient with lung adenocarcinoma in an embodiment of the present invention is equally applicable to the test kit in this embodiment, and will not be described herein.
  • a method for detecting a prognosis effect of a lung adenocarcinoma patient comprising: detecting a level of expression of an IQGAP3 gene in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis; Comparing with the third reference level, when the expression level is significantly different from the third reference level, it is determined that the prognosis effect of the lung adenocarcinoma patient is poor, and the third reference level is that the IQGAP3 gene has a good prognosis effect. Expression levels in lung tissue or lung cells of patients with lung adenocarcinoma.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the poor prognosis results indicate that the survival of wild-type individual patients is significantly shorter than the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • the gene IQGAP3 is a novel lung adenocarcinoma that was discovered by the inventors and is highly expressed in lung adenocarcinoma samples. The inventors found that the highly expressed IQGAP3 gene was significantly associated with shortened overall survival and disease-free survival, see Figures 5 and 6, suggesting a high correlation with poor prognosis of lung adenocarcinoma.
  • the IQGAP3 gene can be used as a biomarker for accurate detection and prognosis evaluation of lung adenocarcinoma.
  • detection of the level of expression of the gene can utilize second generation sequencing techniques. Extracting RNA from one or more lung tissue samples or lung cell samples, sequencing the extracted RNA by sequencing technology, and sequencing the sequencing library to obtain the offline data, ie, sequencing data; The sequencing data is analyzed, including aligning the sequencing data with a reference sequence, and calculating the expression level of the gene based on the alignment result. Sequencing can be performed by using an existing sequencing platform, and corresponding library preparation can be performed according to the selected sequencing platform.
  • the optional sequencing platforms include, but are not limited to, CG (Complete Genomics) CGA, Illumina/Solexa, Life Technologies/Ion Torrent, and Roche 454, preparation of single-ended or double-end sequencing libraries based on the selected sequencing platform.
  • Comparable This method is not limited by the software of SOAP (Short Oligonucleotide Analysis Package), BWA, etc.
  • each read in the sequencing data is allowed to be allowed at most.
  • h base mismatch h is preferably 1 or 2. If more than h bases in a reads are mismatched, it is considered that the reads cannot be compared to the reference sequence.
  • the comparison result includes the comparison of each read segment with the reference sequence, including whether the read segment can compare the reference sequence, the position of the reference sequence on the read alignment, how many reads at a certain point are aligned, and the comparison The base type of the corresponding position of the read of a certain site, and the like.
  • the reference sequence referred to is a known sequence, which may be any reference template in the biological category to which the target individual belongs, such as a published genome assembly sequence of the same biological category, if the nucleic acid sample is from a human, its genome reference
  • the sequence also known as the reference genome
  • the gene expression level is calculated using the RPKM method: Wherein C represents the number of reads aligned to the target gene, N represents the total number of reads aligned to all genes, and L represents the exon length of the target gene.
  • the so-called third reference level can be determined when detecting the prognostic effect of lung adenocarcinoma patients, for example, when detecting the expression level of IQGAP3 gene in lung tissue or lung cell samples of prognostic lung adenocarcinoma patients, simultaneous detection of IQGAP3 gene is more
  • the expression level in lung tissue or lung cell samples of patients with good prognosis is good; the third reference level can also be pre-determined, saved or recorded for use.
  • the third reference level is an average expression level of the IQGAP3 gene in lung tissue or lung cell samples of a plurality of lung adenocarcinoma patients with good prognosis, and is pre-determined for preservation.
  • the third reference level shows that the IQGAP3 gene expression level is K, and the measured expression level shows 1.5K, 2K or more than 2K; the significant difference is said. It can also mean that the statistical difference is significant.
  • An apparatus for detecting a prognosis effect of a lung adenocarcinoma patient comprising: gene expression a horizontal detection unit for detecting the expression level of the IQGAP3 gene in the lung tissue or the lung cell of the lung adenocarcinoma prognosis patient; a sixth determining unit, connected to the gene expression level detecting unit, for using the expression level Comparing the three reference levels, when the expression level is significantly different from the third reference level, determining that the lung adenocarcinoma patient has a poor prognosis effect, and the third reference level is a lung gland with a good prognosis effect of the IQGAP3 gene Expression levels in lung tissue or lung cells of cancer patients.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the poor prognosis results indicate that the survival of wild-type individual patients is significantly shorter than the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • a kit for detecting a prognosis effect of a lung adenocarcinoma patient comprising: an agent for detecting an expression level of an IQGAP3 gene in a lung tissue or a lung cell of a patient with a lung adenocarcinoma prognosis And a fifth specification indicating a relationship between the expression level and a prognosis effect of a lung adenocarcinoma patient, the fifth specification comprising third reference level information, wherein the third reference level is a lung adenocarcinoma patient with a good prognosis effect of the IQGAP3 gene Expression levels in lung tissue or lung cells.
  • the so-called prognosis is poor and the prognosis is good, and the survival time of the prognosis is relatively short and relatively long.
  • the so-called prognostic effect includes a significantly longer survival period than a wild-type individual patient compared to the prognosis.
  • the so-called wild type individual patient is a lung adenocarcinoma patient whose IQGAP3 gene expression level is not significantly high expression.
  • the third specification includes comparing the expression level to a third reference level, and when the expression level is significantly different from the third reference level, determining that the prognosis effect of the lung adenocarcinoma patient is poor or Operation instructions.
  • the reagents, instruments, or software involved in the following examples are conventional commercial products or open source, such as purchasing a sequencing library preparation kit from Illumina, and building a library according to the kit instructions.
  • Fig. 7 The inventors have found that the technical routes for the utilization of biomarkers related to various types of lung adenocarcinoma are summarized as shown in Fig. 7, that is, the application of modern molecular biology techniques to the early and metastatic lung adenocarcinoma of a large number of Chinese people.
  • Sample extraction including DNA and RNA
  • library construction fragment disruption, end repair, add-on, whole genome amplification, exon capture
  • transcriptome amplification and high-throughput sequencing platform Sequencing, using biological information software algorithm to process the sequencing data, the whole genome sequencing results, transcriptome data, exon data, etc. of early tumors, metastases, etc. were compared and found to be closely related to the onset and prognosis of lung adenocarcinoma.
  • the associated gene is an effective targeting biomarker for lung adenocarcinoma in the Chinese population (east Asian population).
  • lung adenocarcinoma Through the hospital, the patient's informed consent form was signed to collect clinical specimens of lung adenocarcinoma.
  • the patient's tumor sample if there is metastasis, take lymph node metastasis tissue
  • the sample is analyzed by pathologist Specific pathological typing, TNM staging, and tumor cell structure were identified as lung adenocarcinoma (primary cancer or lymph node metastasis lung adenocarcinoma).
  • tissue sample Immediately after obtaining the tissue sample, it was transferred to liquid nitrogen for quick freezing and stored at -80 °C for long-term scientific research.
  • Tissue genomic DNA extraction was performed according to Qiagen's QIAamp DNA Mini Kit instructions. Tissue total RNA is extracted by Life Technologies The reagent is completed. The mass and concentration of the above extracted DNA/RNA samples were obtained by agarose gel electrophoresis or Agilent 2100 analysis. Microsatellite mass spectrometry was performed on 21 common SNP loci to ensure that the DNA and RNA samples in the tumor were from the same patient.
  • the whole genome sequencing process includes: 2-3ug genomic DNA samples, which are randomly interrupted into a fragment of about 500bp on average using the Covarias ultrasound system, and then constructed according to the Illumina Paired-End standard protocol, using T4 DNA polymerase, Klenow large fragment enzyme, T4 polynucleotide kinase and nucleotide phosphate complete end-repair, and the repaired DNA fragment is phosphorylated using Klenow enzyme (3'-5'exo) 3' end, using T4 DNA ligase plus The PE index linker (the linker is methylated at the 5' end cytosine) completes library construction. After each of the above steps, purification was performed using Qiagen's QIAquick PCR Purification Kit.
  • the whole exon sequencing process includes: 3ug genomic DNA is interrupted by the above method, and the PE index linker is used to capture and enrich the exon according to the SureSelect target full-exon 50Mb kit. A region fragment was constructed to construct a 200 bp Illumina Paired-End sequencing library.
  • the transcriptome sequencing process consisted of 20 ug RNA samples, which were treated with RNase-free DNase I (New England BioLabs) for 30 minutes at 37 °C to remove DNA.
  • the mRNA of the poly A tail was picked up with oligo (dT), and the cDNA strand was synthesized using random primers and transcriptase (Invitrogen), and cDNA duplex was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).
  • the cDNA library was constructed according to the manufacturer's instructions.
  • the depth of sequencing of the whole genome was as follows: the primary tumor sample contained at least 50X coverage and the adjacent tissue had 30X coverage.
  • C represents the number of reads to the gene in question
  • N represents the total number of reads aligned to all genes
  • L represents the exon length of the test gene
  • the inventors analyzed and compared the data of the above 335 primary lung adenocarcinomas, 35 lymph node metastases, and normal tissues or cell samples, and comprehensively analyzed the mutation frequency, gene length, and gene biology functions to determine the integrated analysis of the mutant genes.
  • the following lung adenocarcinoma-associated biomarkers The following lung adenocarcinoma-associated biomarkers.
  • the inventors detected these lung adenocarcinoma-associated biomarkers for additional lung adenocarcinoma samples, and the results were 90% consistent with the clinical identification of the samples.
  • These genes were validated and can be used as lung adenocarcinoma pathogenicity detection, lung adenocarcinoma. Whether to transfer the detection marker such as.
  • Fig. 1 and Fig. 2A-C the inventors identified 13 significant mutations in the lung adenocarcinoma samples by the above experiments and data analysis. These genes are referred to herein as pathogenicity related genes. . These genes include TP53, EGFR, LRP1B, KRAS, PTPRD, STK11, SMAD2, PIK3CA, BRAF, FLT1, RHPN2, GLI3 and MRC2.
  • Figure 2 shows the types of mutations that occur on each gene.
  • TP53 gene the inventor statistical analysis found that TP53 gene has a statistically significant mutation in 44% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2A, suggesting that it can be used as a diagnosis of lung adenocarcinoma Biomarker.
  • the EGFR gene has a statistically significant mutation in 39% of Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2A, and this gene mutation in the history of no smoking or female patients Commonly, compared with Caucasian data, the mutation rate of EGFR gene is higher than that of KRAS.
  • the results show that EGFR mainly mutates to Leu858Arg and exon19del sites, which is a sensitive site for tyrosine kinase inhibitors.
  • the LRP1B gene the inventor's statistical analysis found that the LRP1B gene has a statistically significant mutation in 19% of Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2A, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the KRAS gene the inventor's statistical analysis found that the KRAS gene has a statistically significant mutation in 11% of Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2B, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the PTPRD gene the inventor's statistical analysis found that the PTPRD gene has a statistically significant mutation in 7% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2B, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the PIK3CA gene the inventor's statistical analysis found that the PIK3CA gene has a statistically significant mutation in 5% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2B, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the RHPN2 gene the inventor's statistical analysis found that the RHPN2 gene has a statistically significant mutation in 5% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2B, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the STK11 gene the inventor's statistical analysis found that the STK11 gene has a statistically significant mutation in 4% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2B, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the BRAF gene the inventor's statistical analysis found that the BRAF gene has a statistically significant mutation in 4% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2C, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the GLI3 gene the inventor's statistical analysis found that the GLI3 gene has a statistically significant mutation in 4% of Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2C, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the FLT1 gene the inventor's statistical analysis found that the FLT1 gene has a statistically significant mutation in 3% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2C, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • the MRC2 gene alias uPARAP, Endo180 or CD280, the statistical analysis of the inventors found that the MRC2 gene has a statistically significant mutation in 2% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2C, suggesting that it can be used as a lung gland.
  • the SMAD2 gene the inventor's statistical analysis found that the SMAD2 gene has a statistically significant mutation in 2% Chinese lung adenocarcinoma, as shown in Figure 1 and Figure 2C, suggesting that it can be used as a biomarker for the diagnosis of lung adenocarcinoma. Things.
  • genes that did not reach statistically significant mutations may be functionally carcinogenic, including APC, KEAP1, ATF7IP, ITIH5, IQGAP3, MET, ERBB2 and TERT. , as shown in Figure 1, Figure 2D and Figure 2E.
  • the present invention firstly found three driving genes with high correlation in lung adenocarcinoma, namely RHPN2, GLI3 and MRC2, wherein the novel high frequency mutation site V73M of RHPN2 gene can be used as a potential hot spot mutation. It acts to provide a new set of biomarkers for the diagnosis and treatment of lung adenocarcinoma in East Asian populations.
  • TP53 gene as a biomarker for detecting lung adenocarcinoma metastasis
  • metastasis is the most critical event in the development of the disease, but there is currently no systematic study on the difference in the pattern of mutations in patients with primary lung adenocarcinoma who carry metastases and do not carry metastases.
  • the present invention compares lung adenocarcinoma and lymph node adenocarcinoma samples that have been diagnosed with lymph node metastasis or distant metastasis, and the inventors have found that the TP53 gene is the only one that carries metastases by Fisher's exact test.
  • a significant (P ⁇ 0.05) enriched gene in adenocarcinoma suggests that this gene is not only a tumor suppressor gene that is significantly associated with tumorigenesis, but also occurs in tumor metastasis. Development has a driving role.
  • the inventors propose that the TP53 gene can be used as a biomarker for detecting whether lung adenocarcinoma metastasizes and treats prognosis.
  • the inventors compared the population of 335 Chinese patients with lung adenocarcinoma by Kaplan-Meier survival analysis and found that any of the seven genes including TP53 or LRP1B or STK11 or KEAP1 or BRAF or MET or MRC2 were compared with wild-type individuals. Mutations in the gene all suggest a significantly shortened survival.
  • the present invention proposes that any gene comprising the above gene can be used as a biomarker for predicting the length of survival in clinical practice. ( Figure 4)
  • IQGAP3 a novel driving gene, which is expressed at high levels in tumor samples, as shown in Figure 5.
  • the highly expressed IQGAP3 gene was significantly associated with shortened overall survival and disease-free survival, as shown in Figure 6, suggesting a high correlation with poor prognosis of lung adenocarcinoma.
  • the IQGAP3 gene provides a precise biomarker for the assessment of the diagnosis and prognosis of lung adenocarcinoma.

Landscapes

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

Abstract

提供了一种肺腺癌相关生物标记物及其应用。还提供一种体外检测受试者存在肺腺癌的方法，包括：检测受试者的第一部位中的一种或者多种生物标记物上的突变水平，获得第一突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；将第一突变水平与第一参考水平比较，当第一突变水平与第一参考水平具有显著差异时，判定受试者患有肺腺癌，第一参考水平为生物标记物在正常个体的第一部位中的突变水平。

Description

肺腺癌生物标记物及其应用 技术领域
本发明涉及生物医学领域，具体的，本发明涉及肺腺癌生物标记物及其应用，更具体的，本发明涉及体外检测受试者存在肺腺癌的方法、装置及试剂盒，体外检测患者肺腺癌是否发生转移的方法、装置和试剂盒。
背景技术
随着人类基因组计划的完成，新一代测序技术的发展，为肿瘤发生、发展、侵袭转移的分子机理研究提供了新方法，从癌症基因组中探究基因突变与癌症发生的关联性，对疾病预测、干预、诊断治疗，已成为临床肿瘤医学领域的重要研究策略。肺癌，是众多癌种中发病率和死亡率增长最快、对人类健康和生命威胁最大的恶性肿瘤之一，近年来其发病率在全球范围内仍呈持续上升的趋势，这一趋势在中国尤其明显。肺癌，主要包括非小细胞肺癌(NSCLC)和小细胞肺癌(SCLC)，其中，非小细胞癌占85％，而肺腺癌是最常见的一种病理型非小细胞肺癌，每年全球约超过50万人死于该种癌症。
目前外科治疗手术虽然已经有较大进步，基于基因变异的肺腺癌的分子亚型、靶向治疗、检测诊断对临床的辅助作用仍然有限，主要是因为：(1)缺乏有效的早期诊断手段，多数肺癌患者确诊时已出现癌转移；(2)多数病人缺乏有效的已知靶向基因；(3)肿瘤的异质性、复杂性；(4)恶性转移的机制不明。
现有研究以肺腺癌基因组为研究点，已鉴定出很多潜在的癌症驱动基因，包括针对被激活的致癌基因的靶向治疗，如EGFR、ERBB2、BRAF等，以及针对异位或融合基因如ALK、ROS1、RET基因的靶向治疗。然而，这些研究大多是以欧洲或北美地区患者组织进行研究筛查得到，而且收集的样本主要来源于患病早期。已筛查的这些基因标记物因种族差异，对东亚人群的肺腺癌特异性低。而且晚期肺腺癌并携带转移时，缺乏系统研究，大大增加了诊断与治疗的难度。
发明内容
本发明旨在至少解决上述现有问题至少之一或者至少提供一种商业选择。
发明人基于发现东亚人群肺腺癌的高发病率和种族人群间存在的潜在遗传异质性，对东亚人群中进行综合的基因组学分析，包括对晚期肺腺癌的基因特征，特别是携带转移肺腺癌进行深入研究。发明人利用二代测序技术，深入研究遗传突变，筛选出包括针对肺腺癌早期检测的生物标记物，发明人的发现扩大了肺腺癌中潜在的癌症驱动基因频谱，积极 推进肺腺癌的辅助治疗和辅助诊断检测，具体地，能够为肺腺癌的发病研究、预后效果评估、转移发生发展提供相关的生物标记物，用于肺腺癌发生发展的有效检测，而且能够用于改善在转移发展的学科认知和为未来肺腺癌转移诊断和治疗提供辅助指导。
发明人通过分析335个肺腺癌原发肿瘤样本数据、35个淋巴结转移癌样本数据以及正常肺组织样本数据，综合突变频率、基因长度、基因生物学功能等，进行突变基因的整合分析，发明人发现13个在肺腺癌样本中具有统计学意义的显著突变基因——TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，在本文中也将这些基因称为致病相关基因。
发明人发现，这些基因在肺腺癌群体特别是东亚人群中具有相当比例的突变率，提示这些突变基因与肺腺癌致病的关联性，具有潜在的用于检测诊断或者辅助检测诊断肺腺癌发病的作用。将这些基因作为肺腺癌生物标记物，将所称生物标记物用于预测肺腺癌致病可能性的检测，可以用于辅助确定肺腺癌，作为辅助肺腺癌早期诊断方法。
依据本发明的第一方面，本发明提供一种体外检测受试者存在肺腺癌的方法，该方法包括：测定受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，所述第一参考水平为所述生物标记物在正常个体的第一部位中的突变水平。
依据本发明的第二方面，本发明提供一种检测受试者存在肺腺癌的装置，该装置用以实施上述本发明一方面的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第一突变检测单元，用于测定所述受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；第一判定单元，与所述第一突变检测单元相连，用于将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，所述第一参考水平为所述生物标记物在正常个体的第一部位中的突变水平。
依据本发明的第三方面，本发明提供一种检测生物样品为肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的生物标记物的突变水平的试剂，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；以及指示所述生物标记物的突变水平与肺腺癌关 系的第一说明书，任选的，所述第一说明书包含第一参考水平信息，所述第一参考水平是所述生物标记物在正常个体的第一部位中的突变水平。
依据本发明的第四方面，本发明提供一种体外检测受试者存在肺腺癌的方法，该方法包括：检测受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，检测所述受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，所述第二部位为正常部位；比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。
依据本发明的第五方面，本发明提供一种检测受试者存在肺腺癌的装置，该装置用以实施上述本发明一方面的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第二突变检测单元，用于检测受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，以及用于检测受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，所述第二部位为正常部位；第二判定单元，与所述第二突变检测单元相连，用于比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。
RHPN2，GLI3和MRC2这三个基因，是发明人首次发现在肺腺癌中存在高关联性的三个驱动基因。驱动基因为与癌症发生发展相关的重要基因。这三个未见报道过的高关联性的肺腺癌驱动基因，能够用于肺腺癌的检测诊断或者辅助检测诊断。
依据本发明的第六方面，本发明提供一组肺腺癌驱动基因，其由基因RHPN2，GLI3和MRC2组成。
依据本发明的第七方面，本发明提供上述本发明一方面的肺腺癌驱动基因在治疗肺腺癌、和/或制备治疗肺腺癌药物中的用途。
依据本发明的第八方面，本发明提供一种体外检测受试者存在肺腺癌的方法，该方法包括：检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。
依据本发明的第九方面，本发明提供一种检测受试者存在肺腺癌的装置，该装置用以 实施上述本发明一方面的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第三突变检测单元，用于检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；第三判定单元，与所述突变检测单元相连，用于当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。
依据本发明的第十方面，本发明提供一种确定生物样品为肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的肺腺癌驱动基因上的突变的试剂，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；以及指示肺腺癌驱动基因突变与肺腺癌关系的第二说明书。
在肺腺癌中，转移是疾病发展的最关键事件，但目前对原发肺腺癌携带转移和不携带转移的患者之间突变模式的差异，还缺乏系统研究。发明人通过比较已确诊为伴淋巴结转移或远端转移的肺腺癌和无转移的肺腺癌样品进行比较分析，通过Fisher’s exact test检验，发现TP53突变基因是唯一一个在携带转移的肺腺癌中显著性富集的基因(P<0.05)，提示该基因不仅是与肿瘤发生显著相关的一个抑癌基因，并对肿瘤转移发生发展有驱动作用。TP53基因的突变率可以作为一种检测肺腺癌是否发生转移和治疗预后的生物标记物。
依据本发明的第十一方面，本发明提供一种体外检测患者肺腺癌是否发生转移的方法，该方法包括：检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
依据本发明的第十二方面，本发明提供一种检测患者肺腺癌是否发生转移的装置，该装置用以实施本发明一方面的检测患者肺腺癌是否发生转移的方法的全部或部分步骤，该装置包括：TP53基因检测单元，用于检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；第四判定单元，与所述TP53基因突变检测单元相连，用于比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
依据本发明的第十三方面，本发明提供一种检测生物样品是否为转移的肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的TP53基因的突变水平的试剂；以及指示所述TP53基因的突变水平与所述转移的肺腺癌的关系的第三说明书，所述第三说明书包 含第二参考水平信息，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
发明人通过Kaplan-Meier生存分析，比较335名中国肺腺癌患者的肺腺癌样品数据，发现相对野生型个体患者，携带TP53、LRP1B、STK11、KEAP1、BRAF、MET和MRC2七个基因中的至少一个的基因的突变提示显著缩短的生存期。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。上述基因中的任何一个的突变，均可以辅助作为临床实践中的预测生存期长短的生物标记物。
依据本发明的第十四方面，本发明提供一种检测肺腺癌患者预后效果的方法，该方法包括：检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；确定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。
依据本发明的第十五方面，本发明提供一种检测肺腺癌患者预后效果的装置，该装置用以实施上述本发明一方面的检测肺腺癌患者预后效果的方法的全部或部分步骤，该装置包括：第四突变检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；第五判定单元，与所述第四突变检测单元相连，用于判定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。
依据本发明的第十六方面，本发明提供一种检测肺腺癌患者预后效果的试剂盒，该试剂盒包括：用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变的试剂：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；以及指示存在至少一个所述基因存在突变的肺腺癌患者的预后效果差的第四说明书。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。
发明人还发现一个新型的肺腺癌驱动基因IQGAP3，该基因在肺腺癌样本中高水平表达。发明人发现，高表达的IQGAP3基因与缩短的总生存率和无疾病生存期显著相关，提示其与肺腺癌预后差具有高度关联性。IQGAP3基因可作为肺腺癌精准检测诊断和预后效果评估的生物标记物。
依据本发明的第十七方面，本发明提供一种检测肺腺癌患者预后效果的方法，该方法包括：检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称预后效果差指，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。
依据本发明的第十八方面，本发明提供一种检测肺腺癌患者预后效果的装置，该装置用以实施本发明一方面的检测肺腺癌患者预后效果的方法的全部或部分步骤，包括：基因表达水平检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；第六判定单元，与所述基因表达水平检测单元相连，用以将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称预后效果差指，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。
依据本发明的第十九方面，本发明提供一种检测肺腺癌患者预后效果的试剂盒，该试剂盒包括：用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平的试剂；以及指示所述表达水平与肺腺癌患者预后效果关系的第五说明书，所述第五说明书包含第三参考水平信息，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称的预后效果好包括指相较于预后的野生型个体患者的，具有显著长的生存期。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示本发明的一个实施例中的肺腺癌相关的基因在不同肺腺癌人群或样本中的突 变频率。
图2显示本发明的一个实施例中的各个肺腺癌相关基因上出现的突变类型以及突变位置；其中，图2A显示基因TP53、EGFR和LRP1B上出现的突变类型和突变位置，图2B显示基因KRAS、PTPRD、PIK3CA、RHPN2和STK11上出现的突变类型和突变位置，图2C显示基因BRAF、GLI3、FLT1、MRC2和SMAD2上出现的突变类型和突变位置，图2D显示基因APC、KEAP1、ATF7IP、ITIH5、IQGAP3和MET上出现的突变类型和突变位置，图2E显示基因ERBB2和TERT上出现的突变类型和突变位置。
图3显示本发明的一个实施例中的肺腺癌相关基因突变与临床表征的关联。
图4显示本发明的一个实施例中的肺腺癌相关的7个基因突变对生存率和生存期的影响。
图5显示本发明的一个实施例中的IQGAP3基因在正常组织、原发肺腺癌、转移肺腺癌样本中的表达水平。
图6显示本发明的一个实施例中的肺腺癌患者IQGAP3基因表达水平的Kaplan-Meier生存曲线。
图7显示本发明的一个实施例中的确定肺腺癌相关生物标记物的试验流程。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
所述实施方式在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
在本文中，所使用的术语“第一”、“第二”等仅用于描述目的，而不能理解为指示或暗示相对重要性、隐含指明所指示的技术特征的数量或者具有顺序关系。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者多个该特征。在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中，除非另有明确的规定和限定，术语“顺序连接”、“相连”、“连接”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本文中，除非另有明确的规定或限定，所称的“突变”均指体细胞突变，体细胞突 变是指除性细胞外的体细胞发生的突变。
根据本发明的一个实施方式提供的一种体外检测受试者存在肺腺癌的方法，该方法包括：测定受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，所述第一参考水平为所述生物标记物在正常个体的第一部位中的突变水平。
该实施方式中，所称的第一部位可以是肺细胞、肺组织和血浆游离核酸中的至少一种。所称的突变水平包括基因上是否存在突变和/或存在突变的数量和/或存在突变的类型。根据本发明的实施例，突变水平的检测利用二代测序技术。提取肺组织或者肺细胞中的核酸，利用测序技术对提取的核酸进行测序文库(library)制备，以及对文库进行上机测序，获得下机数据即测序数据；接着，对测序数据进行分析，包括将测序数据与参考序列比对，根据比对结果识别目标基因上的突变，包括识别目标基因上是否存在突变、存在的突变的数目和/或突变类型。其中，测序可利用现有测序平台进行，可依据所选择的测序平台进行相应的文库制备，可选用的测序平台包括但不限于CG(Complete Genomics)CGA、Illumina/Solexa、Life Technologies/Ion Torrent和Roche 454，依据所选测序平台进行单端或双末端测序文库的制备。比对可以利用SOAP(Short Oligonucleotide Analysis Package)，BWA等软件进行，本实施方式对此不作限制，比对过程中，根据比对参数的设置，例如设置测序数据中的每条读段最多允许有h个碱基错配(mismatch)，h优选为1或2，若一条reads中有超过h个碱基发生错配，则视为该条reads无法比对到参考序列。比对结果包含各条读段与参考序列的比对情况，包括读段是否能够比对上参考序列、读段比对上参考序列的位置、某一位点多少读段比对上、比对上某位点的读段的相应位置的碱基类型等。所称的参考序列是已知序列，可以是预先获得的目标个体所属生物类别中的任意的参考模板，例如，同一生物类别的已公开的基因组组装序列，若核酸样本为来自人类，其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。各种突变类型，包括SNP、CNV和InDel至少之一，各种突变可以基于获得的比对结果、利用相应的已知软件或程序识别判断，例如SNP的识别可利用SOAPsnp、GATK等软件依照软件默认参数设置进行。
所称的第一参考水平可以在检测受试者是否存在肺腺癌时测定，例如在测定受试者的肺组织或者肺细胞中的一种或者多种生物标记物的突变水平时，同时检测生物标记物在多个正常个体的第一部位中的突变水平；第一参考水平也可以预先测定，保存或记录备用。 根据本发明的一个实施例，第一参考水平为生物标记物在多个正常个体肺组织或者肺细胞中的平均突变水平，预先测定保存备用。
所称的显著差异，可以是明显的、有实质性的差异，例如第一参考水平显示为无突变、而第一突变水平显示为存在突变，再例如第一参考水平显示为无错义突变、而第一突变水平显示为存在错义突变，又例如第一参考水平显示为N个突变、而第一突变水平显示为1.5N、2N或者2N以上个突变；所称的显著差异，也可以指统计学上的差异具有显著性。
上述本发明这一实施方式的检测方法是发明人基于发现东亚人群肺腺癌的高发病率和种族人群间存在的潜在遗传异质性，对东亚人群中进行综合的基因组学分析，包括对晚期肺腺癌的基因特征，特别是携带转移肺腺癌进行深入研究而获得的。发明人利用二代测序技术，深入研究遗传突变，筛选出包括针对肺腺癌早期检测的生物标记物，发明人的发现扩大了肺腺癌中潜在的癌症驱动基因频谱，积极推进肺腺癌的辅助治疗和辅助诊断检测，具体地，能够为肺腺癌的发病研究、预后效果评估、转移发生发展提供相关的生物标记物，用于肺腺癌发生发展的有效检测，而且能够用于改善在转移发展的学科认知和为未来肺腺癌转移诊断和治疗提供辅助指导。
发明人通过分析335个肺腺癌原发肿瘤样本数据、35个淋巴结转移癌样本数据以及正常肺组织样本数据，综合突变率、基因长度、基因生物学功能等，进行突变基因的整合分析，发明人发现以上13个在肺腺癌样本中具有统计学意义的显著突变基因——TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，在本文中也将这些基因称为致病相关基因。
发明人发现，这些基因在肺腺癌群体特别是东亚人群中具有相当比例的突变率，参见图1以及图2，提示这些突变基因与肺腺癌致病的关联性，具有潜在的用于检测诊断或者辅助检测诊断肺腺癌发病的作用。例如，发明人发现TP53基因在44％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现EGFR基因在39％中国人肺腺癌中具有统计学意义的显著性突变，且此基因突变在无吸烟史或女性患者中更常见，与白种人数据相比，EGFR基因的突变率比KRAS更高，而且显示EGFR主要突变于Leu858Arg和exon19del位点，为酪氨酸激酶抑制剂治疗敏感性位点，作为提示可以作为肺腺癌诊断及靶向治疗的一种生物标记物；发明人发现LRP1B基因在19％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明发现KRAS基因在11％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现PTPRD基因在7％中国人肺腺癌中具 有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现STK11基因在4％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现PIK3CA基因在5％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现RHPN2基因在5％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现SMAD2基因在2％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现BRAF基因在4％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现GLI3基因在4％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；发明人发现FLT1基因在3％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物；MRC2，别名uPARAP、Endo180或CD280，发现人发现MRC2基因在2％中国人肺腺癌中具有统计学意义的显著性突变，提示可以作为肺腺癌诊断的一种生物标记物。将这些基因中的一个或者组合作为肺腺癌生物标记物，将所称生物标记物用于预测肺腺癌致病可能性的检测，可以用于辅助确定肺腺癌，作为辅助肺腺癌早期诊断方法。
根据本发明的实施例，所称的生物标记物包括基因TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2中的任意一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个、十二个或者全部十三个。所称的第一突变水平和第一参考水平相应的包含多个基因的突变水平。
根据本发明的一个实施方式提供的一种检测受试者存在肺腺癌的装置，该装置可以用以实施上述本发明的一个实施方式或者任一实施例中的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第一突变检测单元，用于测定所述受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；第一判定单元，与所述第一突变检测单元相连，用于将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，所述第一参考水平为所述生物标记物在正常个体第一部位中的突变水平。该实施方式中，所称的第一部位可以是肺细胞、肺组织和血浆游离核酸中的至少一种。上述对本发明一个实施方式中的体外检测受试者存在肺腺癌的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的检测受试者存在肺腺癌的装 置，在此不再赘述。本领域技术人员可以理解，上述任一实施方式或者实施例中的方法的各步骤的实现执行方式，可以借助相连接的子单元来实施，例如，使所称的第一突变检测单元包含测序子单元来获得测序数据，使包括比对子单元用用以将测序数据与参考序列比对，获得比对结果等。
根据本发明的一个实施方式提供的一种检测生物样品为肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的生物标记物的突变水平的试剂，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；以及指示所述生物标记物的突变水平与肺腺癌关系的第一说明书，所述第一说明书包含第一参考水平信息，所述第一参考水平是所述生物标记物在正常个体的与所述生物样品来源相同的部位中的突变水平。该实施方式中，所称的生物样品可以是或者来自肺细胞、肺组织和血浆游离核酸中的至少一种。上述对本发明一个实施方式或者任一实施例中的体外检测受试者存在肺腺癌的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的检测试剂盒，在此不再赘述。所称第一说明书包含比较所述第一突变水平与第一参考水平，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌的指示。
根据本发明的一个实施方式提供的一种体外检测受试者存在肺腺癌的方法，该方法包括：检测受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，检测所述受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，所述第二部位为正常部位；比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。所称的受试者的其它正常部位，包括该受试者的其它正常组织、细胞和/或体液样品，例如受试者的血液样本。该实施方式中，所称的第一部位可以是肺细胞、肺组织和血浆游离核酸中的至少一种，所称的第二部位为受试者的已知正常的或者已知非病变的部位。上述对本发明一个实施方式或者任一实施例中的体外检测受试者存在肺腺癌的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的体外检测受试者存在肺腺癌的方法，在此不再赘述。
根据本发明的一个实施方式提供的一种检测受试者存在肺腺癌的装置，该装置用以实施上述实施方式中的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第二突变检测单元，用于检测受试者的第一部位中的一种或者多种生物标记物的突变水平， 获得第一突变水平，以及用于检测受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；第二判定单元，与所述第二突变检测单元相连，用于比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。该实施方式中，所称的第一部位可以是肺细胞、肺组织和血浆游离核酸中的至少一种，所称的第二部位为受试者的已知正常的或者说为已确认为非病变的部位。上述对本发明一个实施方式或者任一实施例中的体外检测受试者存在肺腺癌的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的检测受试者存在肺腺癌的装置，在此不再赘述。本领域技术人员可以理解，上述任一具体实施方式中的各步骤的实现或执行方式，可以借助相连接的子单元来实现，例如，使所称的第二突变检测单元包含测序子单元来获得测序数据，使包括比对子单元用用以将测序数据与参考序列比对，获得比对结果等。
根据本发明的另一个实施方式提供的一组肺腺癌驱动基因，其由基因RHPN2，GLI3和MRC2组成。RHPN2，GLI3和MRC2这三个基因的组合，是发明人首次发现在肺腺癌中存在高关联性的三个驱动基因。驱动基因为与癌症发生发展相关的重要基因。这三个未见报道过的高关联性的肺腺癌驱动基因，能够用于肺腺癌的检测诊断或者辅助检测诊断。
根据本发明的另一个实施方式，提供上述实施方式中的肺腺癌驱动基因在治疗肺腺癌、和/或制备治疗肺腺癌药物中的用途。RHPN2，GLI3和MRC2这三个基因，是发明人首次发现在肺腺癌中存在高关联性的三个驱动基因。驱动基因为与癌症发生发展相关的重要基因。这三个未见报道过的高关联性的肺腺癌驱动基因，能够用于肺腺癌的检测诊断或者辅助检测诊断。
根据本发明的另一个实施方式提供的一种体外检测受试者存在肺腺癌的方法，该方法包括：检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。
根据本发明的一个实施例，所述检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，包括：检测RHPN2基因是否存在V73M突变。发明人发现RHPN2基因上的V73M突变只高频存在在肺腺癌人群中，需要说明的是，RHPN2基因的V73M突变是未报道过的，可作为潜在的肺腺癌相关热点突变，可作为东亚人群肺腺癌诊断和治疗的新的生物标记物。
根据本发明的另一个实施方式提供的一种检测受试者存在肺腺癌的装置，该装置用以实施上述实施方式或者任一实施例中的体外检测受试者存在肺腺癌的方法的全部或部分步骤，该装置包括：第三突变检测单元，用于检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；第三判定单元，与所述突变检测单元相连，用于当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。RHPN2，GLI3和MRC2这三个基因，是发明人首次发现在肺腺癌中存在高关联性的三个驱动基因。驱动基因为与癌症发生发展相关的重要基因。这三个未见报道过的高关联性的肺腺癌驱动基因，能够用于肺腺癌的检测诊断或者辅助检测诊断。
根据本发明的一个实施例，所述检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，包括：检测RHPN2基因是否存在V73M突变。发明人发现RHPN2基因上的V73M突变只高频存在在肺腺癌人群中，需要说明的是，RHPN2基因的V73M突变是未报道过的，可作为潜在的肺腺癌相关热点突变，可作为东亚人群肺腺癌诊断和治疗的新的生物标记物。
根据本发明的另一个实施方式提供的一种确定生物样品为肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的肺腺癌驱动基因上的突变的试剂，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；以及指示肺腺癌驱动基因突变与肺腺癌关系的第二说明书。所称第二说明书包含当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌的信息。
在肺腺癌中，转移是疾病发展的最关键事件，但目前对原发肺腺癌携带转移和不携带转移的患者之前突变模式的差异，还缺乏系统研究。发明人通过比较已确诊为伴淋巴结转移或远端转移的肺腺癌和无转移的肺腺癌样品进行比较分析，通过Fisher’s exact test检验，发现TP53突变基因是唯一一个在携带转移的肺腺癌中显著性富集的基因(P<0.05)，参见图3，提示该基因不仅是与肿瘤发生显著相关的一个抑癌基因，并对肿瘤转移发生发展有驱动作用。TP53基因的突变率可以作为一种检测肺腺癌是否发生转移和治疗预后的生物标记物。
根据本发明的又一个实施方式提供的一种体外检测患者肺腺癌是否发生转移的方法，该方法包括：检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
该实施方式中，所称的突变水平包括TP53基因上是否存在突变和/或存在突变的数量和/或存在突变的类型。根据本发明的实施例，突变水平的检测利用二代测序技术。提取一个或多个肺组织样本或者肺细胞样本中的核酸，利用测序技术对提取的核酸进行测序文库(library)制备，以及对文库进行上机测序，获得下机数据即测序数据；接着，对测序数据进行分析，包括将测序数据与参考序列比对，根据比对结果识别目标基因上的突变，包括识别目标基因上是否存在突变、存在的突变的数目和/或突变类型。其中，测序可利用现有测序平台进行，可依据所选择的测序平台进行相应的文库制备，可选用的测序平台包括但不限于CG(Complete Genomics)CGA、Illumina/Solexa、Life Technologies/Ion Torrent和Roche 454，依据所选测序平台进行单端或双末端测序文库的制备。比对可以利用SOAP(Short Oligonucleotide Analysis Package)，BWA等软件进行，本实施方式对此不作限制，比对过程中，根据比对参数的设置，例如设置测序数据中的每条读段最多允许有h个碱基错配(mismatch)，h优选为1或2，若一条reads中有超过h个碱基发生错配，则视为该条reads无法比对到参考序列。比对结果包含各条读段与参考序列的比对情况，包括读段是否能够比对上参考序列、读段比对上参考序列的位置、某一位点多少读段比对上、比对上某位点的读段的相应位置的碱基类型等。所称的参考序列是已知序列，可以是预先获得的目标个体所属生物类别中的任意的参考模板，例如，同一生物类别的已公开的基因组组装序列，若核酸样本为来自人类，其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。各种突变类型，包括SNP、CNV和InDel至少之一，各种突变可以基于获得的比对结果、利用相应的已知软件或程序识别判断，例如SNP的识别可利用SOAPsnp、GATK等软件依照软件默认参数设置进行。
所称的第二参考水平可以在检测受试者是否存在肺腺癌时测定，例如在测定肺腺癌患者的肺组织或者肺细胞样本中的一种或者多种生物标记物的突变水平时，同时检测相同生物标记物在多个未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平；第二参考水平也可以预先测定，保存或记录备用。根据本发明的一个实施例，第二参考水平为生物标记物在多个未发生转移的肺腺癌患者的肺组织或者肺细胞中的平均突变水平，预先测定保存备用。
所称的显著差异，可以是明显的、有实质性的差异，例如第二参考水平显示为无突变、而TP53基因突变水平显示为存在突变，再例如第二参考水平显示为无错义突变、而TP53基因突变水平显示为存在错义突变，又例如第二参考水平显示为N个突变、而TP53基因突变水平显示为1.5N、2N或者2N以上个突变；所称的显著差异，也可以指统计学上的差 异具有显著性。
根据本发明的又一个实施方式提供的一种检测患者肺腺癌是否发生转移的装置，该装置用以实施本发明一方面的检测患者肺腺癌是否发生转移的方法的全部或部分步骤，该装置包括：TP53基因检测单元，用于检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；第四判定单元，与所述TP53基因突变检测单元相连，用于比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。上述对本发明一个实施方式或者任一实施例中的检测患者肺腺癌是否发生转移的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的装置，在此不再赘述。本领域技术人员可以理解，可以通过使这一实施方式中的装置中的功能单元包含对应的相连的功能子单元，以实现上述实施例中的检测患者肺腺癌是否发生转移的方法中的步骤中的具体实现或执行方式，例如，通过使TP53基因检测单元包含测序子单元，用于获得测序数据等。
根据本发明的又一个实施方式提供的一种检测生物样品是否为转移的肺腺癌样品的试剂盒，该试剂盒包括：用于检测所述生物样品中的TP53基因的突变水平的试剂；以及指示所述TP53基因的突变水平与所述转移的肺腺癌的关系的第三说明书，所述第三说明书包含第二参考水平信息，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。所称第三说明书包含比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，判定所述患者肺腺癌发生转移的信息或操作指示。上述对本发明一个实施方式或者任一实施例中的检测患者肺腺癌是否发生转移的方法的技术特征和优点的描述，同样适用本发明这一实施方式中的检测试剂盒，在此不再赘述。
根据本发明的再一个实施方式提供的一种检测肺腺癌患者预后效果的方法，该方法包括：检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；确定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。
发明人通过Kaplan-Meier生存分析，比较335名中国肺腺癌患者的肺腺癌样品数据，发现相对野生型个体患者，携带TP53、LRP1B、STK11、KEAP1、BRAF、MET和MRC2七个基因中的至少一个的基因的突变提示显著缩短的生存期，参见图4。这里，所称的野 生型个体患者为上述任一基因上不存在突变的肺腺癌患者。上述基因中的任何一个的突变，均可以辅助作为临床实践中的预测生存期长短的生物标记物。
该实施方式中，突变的检测可以利用二代测序技术。提取一个或多个肺腺癌预后患者的肺组织样本或者肺细胞样本中的核酸，利用测序技术对提取的核酸进行测序文库(library)制备，以及对文库进行上机测序，获得下机数据即测序数据；接着，对测序数据进行分析，包括将测序数据与参考序列比对，根据比对结果识别目标基因上的突变。其中，测序可利用现有测序平台进行，可依据所选择的测序平台进行相应的文库制备，可选用的测序平台包括但不限于CG(Complete Genomics)CGA、Illumina/Solexa、Life Technologies/Ion Torrent和Roche 454，依据所选测序平台进行单端或双末端测序文库的制备。比对可以利用SOAP(Short Oligonucleotide Analysis Package)，BWA等软件进行，本实施方式对此不作限制，比对过程中，根据比对参数的设置，例如设置测序数据中的每条读段最多允许有h个碱基错配(mismatch)，h优选为1或2，若一条reads中有超过h个碱基发生错配，则视为该条reads无法比对到参考序列。比对结果包含各条读段与参考序列的比对情况，包括读段是否能够比对上参考序列、读段比对上参考序列的位置、某一位点多少读段比对上、比对上某位点的读段的相应位置的碱基类型等。所称的参考序列是已知序列，可以是预先获得的目标个体所属生物类别中的任意的参考模板，例如，同一生物类别的已公开的基因组组装序列，若核酸样本为来自人类，其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。各种突变类型，包括SNP、CNV和InDel至少之一，各种突变可以基于获得的比对结果、利用相应的已知软件或程序识别判断，例如SNP的识别可利用SOAPsnp、GATK等软件依照软件默认参数设置进行。
根据本发明的实施例，所称的确定存在至少一个所述基因存在突变的肺腺癌患者预后效果差，其中的突变为错义突变。
根据本发明的再一个实施方式提供的一种检测肺腺癌患者预后效果的装置，该装置用以实施上述本发明一方面的检测肺腺癌患者预后效果的方法的全部或部分步骤，该装置包括：第四突变检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；第五判定单元，与所述第四突变检测单元相连，用于判定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。上述对本发明一个实施方式中的检测肺腺癌患者预后效果的方法的技术特征和优点的描述，同样 适用这一实施方式中的装置，在此不再赘述。本领域技术人员可以理解，可以通过使该实施方式中的装置的功能单元包含相连的功能子单元，以实施上述实施例中的检测肺腺癌患者预后效果的方法中的步骤的具体实现方式。
根据本发明的再一个实施方式提供的一种检测肺腺癌患者预后效果的试剂盒，该试剂盒包括：用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变的试剂：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；以及指示存在至少一个所述基因存在突变的肺腺癌患者的预后效果差的第四说明书。所称的预后效果差包括，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为上述任一基因上不存在突变的肺腺癌患者。上述对本发明一个实施方式中的检测肺腺癌患者预后效果的方法的技术特征和优点的描述，同样适用这一实施方式中的检测试剂盒，在此不再赘述。
根据本发明的一个实施方式提供的一种检测肺腺癌患者预后效果的方法，该方法包括：检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称预后效果差指，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。
基因IQGAP3是发明人发现的一个新型的肺腺癌驱动，该基因在肺腺癌样本中高水平表达。发明人发现，高表达的IQGAP3基因与缩短的总生存率和无疾病生存期显著相关，参见图5和图6，提示其与肺腺癌预后差具有高度关联性。IQGAP3基因可作为肺腺癌精准检测诊断和预后效果评估的生物标记物。
在该实施方式中，基因的表达水平的检测可以利用第二代测序技术。提取一个或多个肺组织样本或者肺细胞样本中的RNA，利用测序技术对提取的RNA进行测序文库(library)制备，以及对测序文库进行上机测序，获得下机数据即测序数据；接着，对测序数据进行分析，包括将测序数据与参考序列比对，根据比对结果计算基因的表达水平。其中，测序可利用现有测序平台进行，可依据所选择的测序平台进行相应的文库制备，可选用的测序平台包括但不限于CG(Complete Genomics)CGA、Illumina/Solexa、Life Technologies/Ion Torrent和Roche 454，依据所选测序平台进行单端或双末端测序文库的制备。比对可以利 用SOAP(Short Oligonucleotide Analysis Package)，BWA等软件进行，本实施方式对此不作限制，比对过程中，根据比对参数的设置，例如设置测序数据中的每条读段(reads)最多允许有h个碱基错配(mismatch)，h优选为1或2，若一条reads中有超过h个碱基发生错配，则视为该条reads无法比对到参考序列。比对结果包含各条读段与参考序列的比对情况，包括读段是否能够比对上参考序列、读段比对上参考序列的位置、某一位点多少读段比对上、比对上某位点的读段的相应位置的碱基类型等。所称的参考序列是已知序列，可以是预先获得的目标个体所属生物类别中的任意的参考模板，例如，同一生物类别的已公开的基因组组装序列，若核酸样本为来自人类，其基因组参考序列(也称为参考基因组)可选择NCBI数据库提供的HG19。
根据本发明的一个实施例，使用RPKM方法计算基因表达水平：
Figure PCTCN2015093909-appb-000001
其中，C代表比对到目标基因的reads数目，N代表比对到所有基因的总的reads数，L代表目标基因的外显子长度。
所称的第三参考水平可以在检测肺腺癌患者预后效果时测定，例如在测定预后的肺腺癌患者的肺组织或者肺细胞样本中的IQGAP3基因的表达水平时，同时检测IQGAP3基因在多个预后效果好的肺腺癌患者的肺组织或者肺细胞样本中的表达水平；第三参考水平也可以预先测定，保存或记录备用。根据本发明的一个实施例，第三参考水平为IQGAP3基因在多个预后效果好的肺腺癌患者的肺组织或者肺细胞样本中的平均表达水平，预先测定保存备用。
所称的显著差异，可以是明显的、有实质性的差异，例如第三参考水平显示IQGAP3基因表达水平为K、而所测表达水平显示为1.5K、2K或者大于2K；所称的显著差异，也可以指统计学上的差异具有显著性。
根据本发明的一个实施方式提供的一种检测肺腺癌患者预后效果的装置，该装置用以实施本发明一方面的检测肺腺癌患者预后效果的方法的全部或部分步骤，包括：基因表达水平检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；第六判定单元，与所述基因表达水平检测单元相连，用以将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称预后效果差指，相较于预后的野生型个体患者的生存期显著缩短。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。上 述对本发明一个实施方式或是任一实施例中的检测肺腺癌患者预后效果的方法的技术特征和优点的描述，同样适用这一实施方式中的装置，在此不再赘述。本领域技术人员可以理解，可以通过使该实施方式中的装置的功能单元包含相应功能的子单元，来实施上述方法步骤中的各种具体实施方式。
根据本发明的一个实施方式提供的一种检测肺腺癌患者预后效果的试剂盒，该试剂盒包括：用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平的试剂；以及指示所述表达水平与肺腺癌患者预后效果关系的第五说明书，所述第五说明书包含第三参考水平信息，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。这里，所称的预后效果差和预后效果好，是以预后的生存期相对短和相对长来划分的。所称的预后效果好包括指相较于预后的野生型个体患者的，具有显著长的生存期。这里，所称的野生型个体患者为IQGAP3基因表达水平非为显著高表达的肺腺癌患者。所称的第三说明书包含将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差的信息或者操作指示。上述对本发明一个实施方式中的检测肺腺癌患者预后效果的方法的技术特征和优点的描述，同样适用这一实施方式中的试剂盒，在此不再赘述。
下面详细描述本发明的实施例，下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
除另有交待，以下实施例中涉及的试剂、仪器或者软件，都是常规市售产品或者开源的，比如从Illumina公司购买测序文库制备试剂盒，依照试剂盒说明书进行建库等。
实施例
发明人发现各种/各类肺腺癌相关的生物标记物利用的技术线路，汇总起来如图7所示，即应用现代分子生物学技术，对大量中国人的肺腺癌早期和发生转移的样本进行提取(包括DNA和RNA)、文库构建(片段打断、末端修复、加接头、全基因组扩增技术、外显子捕获技术)，转录组扩增建库技术，使用高通量测序平台测序，利用生物信息软件算法对测序数据进行处理，将早期瘤、转移瘤等的全基因组测序结果、转录组数据、外显子数据等进行比对分析，发现与肺腺癌发病、预后有密切关联的基因，为中国人群(东亚人群)肺腺癌有效的靶向生物标记物。
一、样品的收集与核酸提取
通过医院，经与患者签订知情同意书，收集肺腺癌临床标本。通过外科手术，取得患者的肿瘤样本(若有转移，则取淋巴结转移组织)和临近正常组织，样品经病理医生分析 具体的病理分型、TNM分期以及肿瘤细胞结构，确定为肺腺癌(原发癌或淋巴结转移肺腺癌)。获得组织样本后，立刻转至液氮速冻，并保存在-80℃以备长期科研使用。
组织基因组DNA提取是按Qiagen公司的QIAamp DNA Mini Kit使用说明实现。组织总RNA的提取是按Life Technologies公司
Figure PCTCN2015093909-appb-000002
试剂完成。上述提取的DNA/RNA样品质量和浓度通过琼脂糖凝胶电泳或Agilent 2100分析检测获得。并利用21对常见SNP位点进行微卫星质谱验证，确保肿瘤中的DNA和RNA样本来源于同一个患者。
二、全基因组、转录组、外显子测序
1、全基因组测序过程包括：2-3ug基因组DNA样品，使用Covarias超声系统，被随机打断成平均500bp左右的断片段序列，然后依据Illumina Paired-End标准流程进行文库构建，使用T4DNA聚合酶、Klenow大片段酶、T4多聚核苷酸激酶和核苷酸磷酸盐完成末端修复，修复后的DNA片段使用Klenow酶(3’-5’exo)3’端磷酸化，用T4DNA连接酶加上PE index接头(接头是在5’端胞嘧啶甲基化合成)，完成文库构建。上述每个步骤完成后，均使用Qiagen公司QIAquick PCR Purification Kit进行纯化。
2、全外显子测序过程包括：3ug基因组DNA按上述方法进行打断，加上PE index接头，按SureSelect靶向富集系统Sureselect人全外显子50Mb试剂盒说明捕获和富集外显子区域片段，构建200bp Illumina Paired-End测序文库。
3、转录组测序过程包括：20ugRNA样本，经RNase-free DNase I(New England BioLabs)37℃处理30分钟以去除DNA。用oligo(dT)调取多聚A尾的mRNA，用随机引物和转录酶(Invitrogen公司)合成cDNA一链，使用RNase H(Invitrogen)和DNA polymerase I(New England BioLabs).合成cDNA二链。根据厂家说明书完成cDNA文库构建。
4、上机
上述文库构建完成后，均是通过Hiseq2000测序平台进行上机测序，其中全基因组高深度测序得测序深度为：肿瘤原发癌样品含有至少50X的覆盖度，癌旁组织有30X的覆盖度。
三、信息数据的处理
1、数据过滤
上述文库测序完成后，1)使用cutadapt软件(v1.8)(http://cutadapt.readthedocs.org/en/latest/index.html)对原始的下机数据进行初步过滤，包括去除包含接头序列的读段(reads)，去除低质量数据，包括去除>10％的Ns序列即未知碱基的含量大于10％的读段，以及大于50％的碱基q值小于5的读段。以下所有序列分析基于过滤后的数据。
2、全基因组和外显子数据分析
1)使用BWA软件(v0.5.9)(http://bio-bwa.sourceforge.net/)对高质量PE reads与UCSC数据库上hg19参考序列(ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz)进行比对得到SAM格式(http://samtools.github.io/hts-specs/SAMv1.pdf)的比对结果。
2)使用samtools软件(v0.1.18)(http://samtools.sourceforge.net/)将SAM格式的比对结果转换成BAM格式并按照坐标进行排序。
3)使用Picard软件(v1.54)(http://picard.sourceforge.net/)去除因PCR实验产生的重复序列。
4)使用软件Genome Analysis Toolkit(GATK)(v1.0.6076)(https://www.broadinstitute.org/gatk/)对indel附近的序列进行局部重排。
完成基本分析后，1)使用MutTect软件(v1.1.4)(http://www.broadinstitute.org/cancer/cga/mutect)点突变。2)使用Platypus软件(v0.7.9.1)(http://www.well.ox.ac.uk/platypus)进行成对分析(早期肿瘤组织与正常组织、转移癌组织和正常组织的体突变)得到体细胞的插入缺失。3)通过SegSeq算法(http://www.broadinstitute.org/cancer/cga/segseq)进行成对分析(早期肿瘤组织与正常组织、转移癌组织和正常组织的体突变)得到体细胞的拷贝数变异。4)使用CREST软件(v1.0)(www.stjuderesearch.org/site/lab/zhang)进行成对分析(早期肿瘤组织与正常组织、转移癌组织和正常组织的体突变)得到体细胞的结构变异。上述变异检测均是通过ANNOVAR软件(2011Oct02)(http://annovar.openbioinformatics.org/en/latest/)进行注释。
肺腺癌显著性突变基因鉴定：基于统计学程序进行分析，构建驱动基因预测模型，计算每个检测到的突变意义，利用该方法评估突变相关性和功能影响。同时考虑背景和样本突变的分数和统计检验，使用Benjamini-Hochberg方法确定错误检错率(FDR)，筛选出FDR q值小于0.1的基因，然后用MutSigCV软件(v1.4)(http://www.broadinstitute.org/cancer/cga/mutsig)预测显著性突变的基因。
3、转录组数据分析
数据比对：下载UCSC数据库上hg19参考序列(ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/chromFa.tar.gz)和转录组数据(ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz)，过滤后的数据使用SOAP2软件(v2.21)(http://soap.genomics.org.cn/soapaligner.html)进行比对，允许每条read可以有少于5个错配。使用RPKM方法计算基因表达水平：
Figure PCTCN2015093909-appb-000003
其中，C代表比对到所在基因的reads数目，N代表比对到所有基因的总的reads数，L代表检测基因的外显子长度。
四、结果
发明人通过分析比较以上335个肺腺癌原发肿瘤、35个淋巴结转移癌以及正常组织或细胞样本的数据，综合突变频率、基因长度、基因生物学功能等，进行突变基因的整合分析，确定以下肺腺癌相关生物标记物。而且，发明人将这些肺腺癌相关生物标记物检测另外的肺腺癌样本，检测结果90％与样本的临床鉴定结果一致，这些基因得到验证，能够作为肺腺癌致病检测、肺腺癌是否转移等的检测标记物。
1、肺腺癌致病相关的驱动突变基因
如图1和图2A-C所示，发明人通过以上实验及数据分析，确定13个在肺腺癌样本中具有统计学意义的显著突变基因，在本文中将这些基因称为致病相关基因。这些基因包括TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2。图2显示各个基因上出现的突变类型。
这些基因在中国人肺腺癌群体中具有相当比例的突变率，提示这组突变基因与肺腺癌致病的关联性，具有潜在的用于检测诊断肺腺癌发病的作用。这13个基因中的任一个或者任意组合都可以用于预测肺腺癌致病可能性检测，可以作为肺腺癌早期诊断方法。
其中，TP53基因，发明人统计分析发现TP53基因在44％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2A所示，提示其可以作为肺腺癌诊断的一种生物标记物。
EGFR基因，本发明统计分析发现EGFR基因在39％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2A所示，且此基因突变在无吸烟史或女性患者中更常见，与白种人数据相比，EGFR基因的突变率比KRAS更高，结果显示，EGFR主要突变于Leu858Arg和exon19del位点，为酪氨酸激酶抑制剂治疗敏感性位点，作为提示可以作为肺腺癌诊断及靶向治疗的一种生物标记物。
LRP1B基因，发明人统计分析发现LRP1B基因在19％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2A所示，提示其可以作为肺腺癌诊断的一种生物标记物。
KRAS基因，发明人统计分析发现KRAS基因在11％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2B所示，提示其可以作为肺腺癌诊断的一种生物标记物。
PTPRD基因，发明人统计分析发现PTPRD基因在7％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2B所示，提示其可以作为肺腺癌诊断的一种生物标记物。
PIK3CA基因，发明人统计分析发现PIK3CA基因在5％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2B所示，提示其可以作为肺腺癌诊断的一种生物标记物。
RHPN2基因，发明人统计分析发现RHPN2基因在5％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2B所示，提示其可以作为肺腺癌诊断的一种生物标记物。
STK11基因，发明人统计分析发现STK11基因在4％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2B所示，提示其可以作为肺腺癌诊断的一种生物标记物。
BRAF基因，发明人统计分析发现BRAF基因在4％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2C所示，提示其可以作为肺腺癌诊断的一种生物标记物。
GLI3基因，发明人统计分析发现GLI3基因在4％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2C所示，提示其可以作为肺腺癌诊断的一种生物标记物。
FLT1基因，发明人统计分析发现FLT1基因在3％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2C所示，提示其可以作为肺腺癌诊断的一种生物标记物。
MRC2基因，别名uPARAP、Endo180或CD280，发明人统计分析发现MRC2基因在2％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2C所示，提示其可以作为肺腺癌诊断的一种生物标记物。
SMAD2基因，发明人统计分析发现SMAD2基因在2％中国人肺腺癌中具有统计学意义的显著性突变，如图1和图2C所示，提示其可以作为肺腺癌诊断的一种生物标记物。
除了发现以上显著突变的基因，发明人同时还发现未达统计学意义显著性突变的基因，但可能在功能上致癌的突变基因，包括APC，KEAP1，ATF7IP，ITIH5，IQGAP3，MET，ERBB2和TERT，如图1、图2D和图2E所示。
2、新型肺腺癌驱动突变基因
上述基因中，本发明首次发现在肺腺癌中存在高关联性的3个驱动基因，分别是RHPN2，GLI3和MRC2，其中RHPN2基因新型高频突变位点V73M，可作为潜在的热点突变，可作用于为东亚人群肺腺癌诊断和治疗提供一组新的生物标记物。
3、TP53基因作为检测肺腺癌转移生物标记物
在肺腺癌中，转移是疾病发展的最关键事件，但目前对原发肺腺癌携带转移和不携带转移的患者之前突变模式的差异，还缺乏系统研究。本发明通过比较已确诊为伴淋巴结转移或远端转移的肺腺癌和无转移的肺腺癌样品进行比较分析，本发明人通过Fisher’s exact test检验，发现TP53基因是唯一一个在携带转移的肺腺癌中显著性(P<0.05)富集的基因，如图3所示，提示该基因不仅是与肿瘤发生显著相关的一个抑癌基因，并对肿瘤转移发生 发展有驱动作用。发明人提出TP53基因可以作为一种检测肺腺癌是否发生转移和治疗预后的生物标记物。
4、突变基因与临床表征关联性
通过Fisher’s exact test检验，发明人发现EGFR基因突变显著地与非吸烟患者相关，而LRP1B，KRAS，PTPRD，GLI3和KEAP1基因突变显著地与吸烟患者相关。类似的，通过Fisher’s exact test检验，发明人发现EGFR基因突变显著地与女性患者相关，而LRP1B，APC，KRAS，PTPRD，KEAP1，ITIH5，FLT1和STK11基因突变显著地与男性患者相关。类似的，通过Fisher’s exact test检验，发明人还发现TP53，GLI3和ITIH5基因突变显著地与年龄大于60岁的患者相关，如图3所示。
5、肺腺癌临床实践预后诊断生物标记物
发明人通过Kaplan-Meier生存分析，比较335名中国肺腺癌患者队列人群数据，发现相对野生型个体患者，携带TP53或LRP1B或STK11或KEAP1或BRAF或MET或MRC2等7个基因中的任意一个基因的突变，均提示显著缩短的生存期，本发明提出，包含上述基因的任何一个基因，均可以作为临床实践中的预测生存期长短的生物标记物。(附图4)
6、新型肺腺癌预后效果检测标记物
基于测序数据，结合体细胞突变、拷贝数变异、结构变异和基因见融合等研究分析，发明人发现一个新型的驱动基因IQGAP3，该基因在肿瘤样本中高水平表达，如图5所示。高表达的IQGAP3基因与缩短的总生存率和无疾病生存期显著相关，如图6所示，提示其与肺腺癌预后差具有高度关联性。IQGAP3基因可作为为肺腺癌诊断和预后效果评估提供一种精准检测生物标记物。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、、“一些示例”或“具体实施方式”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (21)

  1. 一组肺腺癌驱动基因，其由基因RHPN2，GLI3和MRC2组成。
  2. 权利要求1的肺腺癌驱动基因在治疗肺腺癌、和/或制备治疗肺腺癌药物中的用途。
  3. 一种体外检测受试者存在肺腺癌的方法，其特征在于，包括：
    检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；
    当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。
  4. 权利要求3的方法，其特征在于，所述检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，包括：
    检测RHPN2基因是否存在V73M突变。
  5. 一种检测受试者存在肺腺癌的装置，其特征在于，包括：
    第三突变检测单元，用于检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；
    第三判定单元，与所述突变检测单元相连，用于当所述肺腺癌驱动基因都存在突变时，确定所述受试者存在肺腺癌。
  6. 权利要求5的装置，其特征在于，所述检测受试者的肺组织或者肺细胞中的肺腺癌驱动基因是否都存在突变，包括：
    检测RHPN2基因是否存在V73M突变。
  7. 一种确定生物样品为肺腺癌样品的试剂盒，其特征在于，包括：
    用于检测所述生物样品中的肺腺癌驱动基因上的突变的试剂，所述肺腺癌驱动基因由基因RHPN2，GLI3和MRC2组成；以及
    指示肺腺癌驱动基因突变与肺腺癌关系的第二说明书。
  8. 一种体外检测受试者存在肺腺癌的方法，其特征在于，包括：
    检测受试者的第一部位中的一种或者多种生物标记物上的突变水平，获得第一突变水平，
    所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；
    将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，
    所述第一参考水平为所述生物标记物在正常个体中的第一部位的突变水平。
  9. 一种检测受试者存在肺腺癌的装置，其特征在于，包括：
    第一突变检测单元，用于测定所述受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，
    所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；
    第一判定单元，与所述第一突变检测单元相连，用于将所述第一突变水平与第一参考水平比较，当所述第一突变水平与所述第一参考水平具有显著差异时，判定所述受试者患有肺腺癌，
    所述第一参考水平为所述生物标记物在正常个体中的第一部位的突变水平。
  10. 一种确定生物样品为肺腺癌样品的试剂盒，其特征在于，包括：
    用于检测所述生物样品中的生物标记物的突变水平的试剂，所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2；以及
    指示所述生物标记物的突变水平与肺腺癌关系的第一说明书，任选的，所述第一说明书包含第一参考水平信息，所述第一参考水平是所述生物标记物在正常个体中的与所述生物样品来源相同的部位中的突变水平。
  11. 一种体外检测受试者存在肺腺癌的方法，其特征在于，包括：
    检测受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，
    检测所述受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，
    所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，所述第二部位为正常部位；
    比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。
  12. 一种检测受试者存在肺腺癌的装置，其特征在于，包括：
    第二突变检测单元，用于检测受试者的第一部位中的一种或者多种生物标记物的突变水平，获得第一突变水平，以及
    用于检测受试者的第二部位中的相同所述生物标记物的突变水平，获得第二突变水平，
    所述生物标记物包括以下至少一种基因：TP53，EGFR，LRP1B，KRAS，PTPRD，STK11，SMAD2，PIK3CA，BRAF，FLT1，RHPN2，GLI3和MRC2，所述第二部位 为正常部位；
    第二判定单元，与所述第二突变检测单元相连，用于比较所述第一突变水平和所述第二突变水平，当所述第一突变水平与所述第二突变水平具有显著差异时，判定所述受试者患有肺腺癌。
  13. 一种体外检测患者肺腺癌是否发生转移的方法，其特征在于，包括：
    检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；
    比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，
    所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
  14. 一种检测患者肺腺癌是否发生转移的装置，其特征在于，包括：
    TP53基因检测单元，用于检测患者的肺组织或者肺细胞中的TP53基因的突变水平，获得TP53基因突变水平，所述患者患有肺腺癌；
    第四判定单元，与所述TP53基因突变检测单元相连，用于比较所述TP53基因突变水平与第二参考水平，当所述基因突变水平与所述第二参考水平具有显著差异时，确定所述患者肺腺癌发生转移，
    所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
  15. 一种检测生物样品是否为转移的肺腺癌样品的试剂盒，其特征在于，包括：
    用于检测所述生物样品中的TP53基因的突变水平的试剂；以及
    指示所述TP53基因的突变水平与所述转移的肺腺癌的关系的第三说明书，所述第三说明书包含第二参考水平信息，所述第二参考水平为TP53基因在未发生转移的肺腺癌患者的肺组织或者肺细胞中的突变水平。
  16. 一种检测肺腺癌患者预后效果的方法，其特征在于，包括：
    检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；
    确定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。
  17. 一种检测肺腺癌患者预后效果的装置，其特征在于，包括：
    第四突变检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至 少一个基因存在突变：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；
    第五判定单元，与所述第四突变检测单元相连，用于判定存在至少一个所述基因存在突变的肺腺癌患者预后效果差。
  18. 一种检测肺腺癌患者预后效果的试剂盒，其特征在于，包括：
    用于检测肺腺癌预后患者的肺组织或者肺细胞中是否存在以下至少一个基因存在突变的试剂：TP53，LRP1B，STK11，KEAP1，BRAF，MET和MRC2；以及
    指示存在至少一个所述基因存在突变的肺腺癌患者的预后效果差的第四说明书。
  19. 一种检测肺腺癌患者预后效果的方法，其特征在于，包括：
    检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；
    将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，
    所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。
  20. 一种检测肺腺癌患者预后效果的装置，其特征在于，包括：
    基因表达水平检测单元，用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平；
    第六判定单元，与所述基因表达水平检测单元相连，用以将所述表达水平与第三参考水平比较，当所述表达水平与所述第三参考水平具有显著差异时，判定所述肺腺癌患者的预后效果差，
    所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。
  21. 一种检测肺腺癌患者预后效果的试剂盒，其特征在于，包括：
    用于检测肺腺癌预后患者的肺组织或者肺细胞中的IQGAP3基因的表达水平的试剂；以及
    指示所述表达水平与肺腺癌患者预后效果关系的第五说明书，所述第五说明书包含第三参考水平信息，所述第三参考水平为IQGAP3基因在预后效果好的肺腺癌患者的肺组织或者肺细胞中的表达水平。
PCT/CN2015/093909 2015-11-05 2015-11-05 肺腺癌生物标记物及其应用 Ceased WO2017075784A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
KR1020187015074A KR102086935B1 (ko) 2015-11-05 2015-11-05 폐 선암종을 검출하기 위한 생체마커 및 그의 사용
JP2018522993A JP6688884B2 (ja) 2015-11-05 2015-11-05 肺腺癌を検出するためのバイオマーカー及びその使用
EP15907626.4A EP3372686B1 (en) 2015-11-05 2015-11-05 Biomarker for detection of lung adenocarcinoma and use thereof
PCT/CN2015/093909 WO2017075784A1 (zh) 2015-11-05 2015-11-05 肺腺癌生物标记物及其应用
HK18106681.1A HK1247242B (zh) 2015-11-05 肺腺癌生物标记物及其应用
CN201580082101.0A CN107849569B (zh) 2015-11-05 2015-11-05 肺腺癌生物标记物及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/093909 WO2017075784A1 (zh) 2015-11-05 2015-11-05 肺腺癌生物标记物及其应用

Publications (1)

Publication Number Publication Date
WO2017075784A1 true WO2017075784A1 (zh) 2017-05-11

Family

ID=58661479

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/093909 Ceased WO2017075784A1 (zh) 2015-11-05 2015-11-05 肺腺癌生物标记物及其应用

Country Status (5)

Country Link
EP (1) EP3372686B1 (zh)
JP (1) JP6688884B2 (zh)
KR (1) KR102086935B1 (zh)
CN (1) CN107849569B (zh)
WO (1) WO2017075784A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113186287A (zh) * 2021-05-10 2021-07-30 深圳康华君泰生物科技有限公司 用于非小细胞肺癌分型的生物标志物及其应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110396537B (zh) * 2018-04-24 2023-06-20 深圳华大生命科学研究院 哮喘生物标志物及其用途
CN110592212B (zh) * 2019-08-15 2023-05-26 吴一龙 一种肺癌检测联合标志物、检测试剂盒及其用途
CN112442535A (zh) * 2019-08-27 2021-03-05 上海善准生物科技有限公司 原发性肺腺癌分子分型及生存风险基因群及诊断产品和应用
CN111273023B (zh) * 2020-02-12 2020-11-17 牡丹江医学院 一种肺腺癌肿瘤标记物蛋白及其应用
CN113046441A (zh) * 2021-04-30 2021-06-29 中山大学孙逸仙纪念医院 用于快速辅助鉴别原发性多发肺癌与肺内转移癌的标记物组合物及其应用
CN115472294B (zh) * 2022-11-14 2023-04-07 中国医学科学院肿瘤医院 预测小细胞转化肺腺癌患者转化速度的模型及其构建方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112707A1 (en) * 1997-06-20 2005-05-26 Altaba Ariel R.I. Method and compositions for inhibiting tumorigenesis
TW201311906A (zh) * 2011-07-05 2013-03-16 Univ Nat Taiwan 預測ｅｇｆｒ突變肺腺癌病患對藥物治療的反應與預後之方法
WO2014172456A1 (en) * 2013-04-17 2014-10-23 Hedgepath, Llc Treatment and prognostic monitoring of proliferation disorders using hedgehog pathway inhibitors
CN104818334A (zh) * 2015-06-02 2015-08-05 北京泱深生物信息技术有限公司 与肺腺癌转移相关的微小rna

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5688497B2 (ja) * 2005-10-04 2015-03-25 国立大学法人名古屋大学 肺腺癌患者の術後予後を予測するための方法及び組成物
US20120178111A1 (en) * 2009-09-23 2012-07-12 Diamandis Eleftherios P Methods and compositions for the detection of lung cancers
JP5858405B2 (ja) * 2010-03-30 2016-02-10 公益財団法人ヒューマンサイエンス振興財団 肺腺がんの予後予測方法、および、肺腺がんの検出キット
US20140045915A1 (en) * 2010-08-31 2014-02-13 The General Hospital Corporation Cancer-related biological materials in microvesicles
CA3064363C (en) * 2011-10-24 2022-05-17 Somalogic, Inc. Non-small cell lung cancer biomarkers and uses thereof
WO2013152989A2 (en) * 2012-04-10 2013-10-17 Eth Zurich Biomarker assay and uses thereof for diagnosis, therapy selection, and prognosis of cancer
CN113337604A (zh) * 2013-03-15 2021-09-03 莱兰斯坦福初级大学评议会 循环核酸肿瘤标志物的鉴别和用途
US20150153346A1 (en) * 2013-11-15 2015-06-04 The Regents Of The University Of Michigan Lung cancer signature

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112707A1 (en) * 1997-06-20 2005-05-26 Altaba Ariel R.I. Method and compositions for inhibiting tumorigenesis
TW201311906A (zh) * 2011-07-05 2013-03-16 Univ Nat Taiwan 預測ｅｇｆｒ突變肺腺癌病患對藥物治療的反應與預後之方法
WO2014172456A1 (en) * 2013-04-17 2014-10-23 Hedgepath, Llc Treatment and prognostic monitoring of proliferation disorders using hedgehog pathway inhibitors
CN104818334A (zh) * 2015-06-02 2015-08-05 北京泱深生物信息技术有限公司 与肺腺癌转移相关的微小rna

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3372686A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113186287A (zh) * 2021-05-10 2021-07-30 深圳康华君泰生物科技有限公司 用于非小细胞肺癌分型的生物标志物及其应用

Also Published As

Publication number Publication date
EP3372686B1 (en) 2020-05-20
EP3372686A1 (en) 2018-09-12
HK1247242A1 (zh) 2018-09-21
JP6688884B2 (ja) 2020-04-28
CN107849569B (zh) 2021-08-03
CN107849569A (zh) 2018-03-27
KR20180066248A (ko) 2018-06-18
EP3372686A4 (en) 2018-09-12
KR102086935B1 (ko) 2020-03-09
JP2018533952A (ja) 2018-11-22

Similar Documents

Publication Publication Date Title
ES2831148T3 (es) Identificación y uso de marcadores tumorales de ácido nucleico circulante
JP2025016591A (ja) まれな変異およびコピー数多型を検出するためのシステムおよび方法
ES2925014T3 (es) Identificación y uso de ácidos nucleicos circulantes
CN107849569B (zh) 肺腺癌生物标记物及其应用
JP7781105B2 (ja) 遺伝子型決定アッセイにおける一意でないバーコード
JP5963679B2 (ja) 後天的な体細胞性再編成に基づく診断方法
TW201840853A (zh) 使用核酸片段之診斷應用
Ledgerwood et al. The degree of intratumor mutational heterogeneity varies by primary tumor sub-site
CN107750279A (zh) 核酸分析系统和方法
JP2019504618A5 (zh)
CN105779434A (zh) 试剂盒及其用途
WO2016095093A1 (zh) 肿瘤筛查方法、目标区域变异检测方法和装置
US20230002831A1 (en) Methods and compositions for analyses of cancer
CN105780129A (zh) 目标区域测序文库构建方法
CN105779435A (zh) 试剂盒及其用途
CN105779432A (zh) 试剂盒及其用途
Del Portillo et al. Evaluation of mutational testing of preneoplastic Barrett's mucosa by next-generation sequencing of formalin-fixed, paraffin-embedded endoscopic samples for detection of concurrent dysplasia and adenocarcinoma in Barrett's esophagus
CN118932064B (zh) 一种用于检测非小细胞肺癌mrd的探针组及其应用
CN117441027A (zh) Heatrich-BS：用于亚硫酸氢盐测序的富含CpG的区域的热富集
CN111201572A (zh) 用于提高精确度的癌症患者的综合基因组转录组肿瘤-正常样基因组套分析
HK1247242B (zh) 肺腺癌生物标记物及其应用
KR102954537B1 (ko) 조직 특이적 메틸화 마커
HK40080902A (zh) 可用於胃癌her2伴随诊断的基因甲基化标记物或其组合和应用
WO2022107136A1 (en) Methods and biomarkers for diagnostics, disease monitoring, personalized drug discovery and targeted therapy in glioma patients
KR20250175126A (ko) 변이체 대립유전자 빈도 분석을 통한 암 진단 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15907626

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2018522993

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20187015074

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2015907626

Country of ref document: EP