WO2020011195A1 - 用于检测肺肿瘤良恶性程度的分级模型及其应用 - Google Patents

用于检测肺肿瘤良恶性程度的分级模型及其应用 Download PDF

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WO2020011195A1
WO2020011195A1 PCT/CN2019/095415 CN2019095415W WO2020011195A1 WO 2020011195 A1 WO2020011195 A1 WO 2020011195A1 CN 2019095415 W CN2019095415 W CN 2019095415W WO 2020011195 A1 WO2020011195 A1 WO 2020011195A1
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imprinted
imprinted gene
gene
expression
genes
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PCT/CN2019/095415
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French (fr)
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成彤
周宁
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立森印迹诊断技术有限公司
李星
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Priority to AU2019302917A priority Critical patent/AU2019302917A1/en
Priority to JP2021500945A priority patent/JP2022516392A/ja
Priority to US17/260,074 priority patent/US20210285055A1/en
Priority to CA3110258A priority patent/CA3110258A1/en
Priority to EP19833075.5A priority patent/EP3822369A4/en
Publication of WO2020011195A1 publication Critical patent/WO2020011195A1/zh

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    • C12Q1/6841—In situ hybridisation
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/10—Ploidy or copy number detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation

Definitions

  • the present disclosure relates to the field of biotechnology, such as the field of genetic diagnosis, such as a hierarchical model and its applications, such as a hierarchical model for detecting the benign and malignant degree of lung tumors, and their applications, such as a set of imprinted genes that detect benign and malignant lung tumors Hierarchical model of degree and its constituent devices.
  • Lung cancer is the malignant tumor with the highest morbidity and mortality in the world. According to World Health Organization (WHO) statistics, in 2012 there were 1.82 million new cases worldwide, 1.59 million deaths, 733,000 new diagnoses in China, and 601,000 deaths (World Cancer Report 2014), which accounted for male morbidity and mortality. The first place is the second place in female morbidity and the first place in mortality.
  • the survival rate of patients with lung cancer is closely related to the degree of cancer progression.
  • the 5-year survival rate of patients with stage I lung cancer can reach 70-90%, while the 5-year survival rate of patients with stage IV lung cancer does not exceed 10%.
  • Early treatment is the key to saving the lives of patients with lung cancer.
  • Cancer is caused by uncontrolled cell growth / division resulting from epigenetic changes and genetic mutations that accumulate over time.
  • Traditional pathological diagnosis is based on cell, tissue size, morphological, and structural variations to make benign and malignant judgments of lung tumors.
  • molecular detection techniques are applied to the detection of lung cancer. From the analysis of the development of cancer, the changes at the molecular level (epigenetics and genetics) predate the changes in cell morphology and tissue structure. So molecular biology tests are more sensitive to early detection of cancer.
  • the current diagnosis of lung cancer requires a new detection system and detection model. Based on patient biopsy samples, analysis of molecular marker changes at the cellular level of lung cancer is provided to provide more accurate pre-diagnosis and diagnostic information.
  • the present disclosure provides an imprinted gene grading model and diagnostic method, and applications thereof.
  • the present disclosure provides an imprinted gene grading model for lung tumors.
  • the model calculates imprinted genes by calculating changes in imprinted gene deletion expression, imprinted gene copy number abnormal expression, and total expression of imprinted genes in lung tumors. Classification of expression status;
  • the imprint gene is any one or a combination of at least two of Z1, Z11 or Z16, the imprint gene Z1 is Gnas, the imprint gene Z11 is Grb10, and the imprint gene Z16 is Snrpn / Snurf.
  • the absence of the imprint (trace) means that the allele that was originally silent in the imprint (trace) gene is activated (demethylated), which is the most common and earliest epigenetic change in cancer, and This feature can be used as a pathological marker. Comparatively speaking, in the detection of healthy cells, the proportion of deletion of imprinting is very low.
  • the imprinted gene and imprinted gene have the same concept, which means the same meaning, and can be replaced.
  • the inventors have found that by calculating the expression levels of the imprinted genes in any of Z1, Z11, and Z16 in lung tumors, the abnormal expression of imprinted gene copy numbers, and the total expression of imprinted genes, the diagnostic sensitivity to lung tumors can be reached. 73.7% or more.
  • any one of the imprinted genes in Z1, Z11, and Z16 can be detected.
  • any one of the imprinted genes in Z1 or Z16 can be detected.
  • Z1 can be detected if only one imprinted gene is detected in the preliminary detection.
  • the inventors found that if a single Z1 imprinted gene is detected alone, the diagnostic sensitivity to lung tumors can reach 86.0%, and if a single Z11 imprinted gene is detected alone, the diagnostic sensitivity to lung tumors can reach 73.7% By detecting a Z16 imprinted gene alone, the diagnostic sensitivity to lung tumors can reach 78.9%.
  • the method for calculating the imprint gene by the model is: if a combination of two imprint genes of the imprint gene is detected, the combination may be any two of Z1, Z11, and Z16, preferably Z1 and Z16 The combination of Z1 and Z11.
  • the inventors have found that by calculating the expression levels of two or more imprinted gene deletion expressions, imprinted gene copy number abnormal expressions, and total imprinted expression levels, the sensitivity can be further improved, and the combination of Z1 and Z16 can be detected.
  • the diagnostic sensitivity to lung cancer can reach 93.0%, and the combination of the two imprinted genes that detect the imprinted genes Z11 and Z16 can reach more than 91.2%.
  • the imprint gene further includes any one or a combination of at least two of Z3, Z4, Z5, Z6, Z8, Z10, or Z13; wherein the imprint gene Z3 is Peg10, and the imprint Gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, the imprinted gene Z10 is Gatm, and the imprinted gene Z13 is Sgce.
  • the method for calculating the imprint gene by the model is: calculating a combination of imprint genes, and the imprint genes are a combination of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 genes. .
  • the deletion of the imprinted gene is the presence of two red / brown markers in the nucleus of the cell after hematoxylin staining
  • the abnormal copy number of the imprinted gene is the presence of more than two red / brown markers in the nucleus of the cell after hematoxylin staining.
  • the abnormal copy number is due to the abnormal gene replication of cancer cells, which results in the expression of this gene as a triploid or even higher polyploid.
  • the formula for calculating the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number is as follows:
  • a means that there is no marker in the nucleus of the cell after hematoxylin staining, and that the imprinted gene is not expressed; and b means that there is a red / brown marker in the nucleus of the cell after hematoxylin staining, and the imprinted gene exists.
  • the nucleus; c is the hematoxylin staining of the cell, there are two red / brown marks in the nucleus of the cell, and the nucleus of the gene deletion is imprinted;
  • d is the hematoxylin staining of the cell, there are more than two red / brown marks in the nuclei of the cell , Imprinted nuclei with abnormal gene copy number.
  • the marker after hematoxylin staining is selected from, but not limited to, red or brown. Marking with other colors can also be used for imprinting gene deletion expression amount, imprinting gene copy number abnormal expression amount, and imprinting gene total expression. Calculation of the amount.
  • the probe is amplified by in situ hybridization and Hemotoxy (hematoxylin) nuclear staining to amplify the signal.
  • Hemotoxy hematoxylin
  • the degree of benign and malignant tumors in this sample is determined by calculating the expression of the imprinted gene, the abnormal expression of the imprinted copy number, and the total expression of the imprinted gene. Because the section is only 10 ⁇ m, the nucleus seen under the microscope is about 20% are incomplete nuclei, which means there is a possibility of partial false negatives.
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and imprinted gene total expression amount are divided into five different grades, and each probe is used to express at least 1200 in the most positive expression region of the sample. The number of cells was counted, and the imprinted gene deletion expression amount, imprinted copy number abnormal expression amount score, and total imprinted gene expression of ten imprinted genes for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 were total. The quantity is divided into five different levels.
  • the five different levels of the imprinted gene deletion expression, imprinted gene copy number abnormal expression, and total expression for Z1, Z3, Z11, and Z16 are:
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression is less than 10%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is less than 1% or the imprinted
  • the total expression of genes Z1, Z3, Z11 and Z16 is less than any one or at least two of 20%;
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression amount is 10-20%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is 1-3% Or the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or at least two of 20-30%;
  • the imprinted gene Z1, Z3, Z11, and Z16 imprinted gene deletion expression amount is 20-25%
  • the imprinted gene Z1, Z3, Z11, and Z16 imprinted gene copy number abnormal expression amount is 3-5%
  • the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or at least two of 30-40%;
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression level is 25-30%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is 5-7% Or the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or at least two of 40-50%;
  • Class IV The imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression level is greater than 30%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is greater than 7% or the imprinted gene
  • the total expression of genes Z1, Z3, Z11 and Z16 is greater than any one or at least two of them;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z1, Z3, Z11 and Z16 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z4, Z5, Z6, Z10, and Z13 are:
  • the imprinted genes of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are expressed less than 10%, and the imprinted genes of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are expressed less than 1%. Or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is less than any one or at least two of them;
  • Class I The expression levels of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are 10-15%, and the expression levels of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are abnormal. 1-2% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or at least two of 15-20%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 have an expression of 15-20% of the imprinted genes, and the imprinted gene Z4, Z5, Z6, Z10, and Z13 have an abnormal expression of the imprinted gene copy number of 2-4% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or at least two of 20-30%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene expression levels are 20-25%, the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene copy number abnormal expression levels are 4-6% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or at least two of 30-40%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 has an imprinted gene deletion expression level greater than 25%, and the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene copy number abnormal expression levels are greater than 6%. Or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is greater than any one or at least two of 40%;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z13 are independent of each other.
  • the five different levels of Z8 imprinted gene deletion expression, imprinted gene copy number abnormal expression amount, and total expression amount division are:
  • any of the imprinted gene deletion expression level of the imprinted gene Z8 is less than 20%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z8 is less than 2%, or the total expression amount of the imprinted gene Z8 is less than 15% One or at least two;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 20-25%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 2-4%, or the total expression level of the imprinted gene Z8 is 15 Any one or at least two of -20%;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 25-30%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 4-7%, or the total expression level of the imprinted gene Z8 is 20 Any one or at least two of -30%;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 30-35%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 7-10%, or the total expression level of the imprinted gene Z8 is 30 Any one or at least two of -40%;
  • Level IV any of the imprinted gene deletion expression level of the imprinted gene Z8 is greater than 35%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is greater than 10%, or the total expression level of the imprinted gene Z8 is greater than 40% One or at least two.
  • the present disclosure provides a device for detecting the benign and malignant degree of lung tumors, which adopts the above model and includes the following units:
  • sampling unit obtain the sample to be tested
  • Probe design unit design specific primers based on the imprinted gene sequence
  • Detection unit performing in situ hybridization of the probe of step (2) and the sample to be tested;
  • the analysis unit calculates the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the imprinted gene total expression amount, and then uses the model described in the first aspect to determine the imprinted gene deletion expression amount and imprinted gene copy number abnormality.
  • the level of expression and the total expression of imprinted genes to determine the benign and malignant degree of lung tumors.
  • the deletion of the imprinted gene refers to the presence of two red / brown labeled nuclei in the nucleus of the cell after hematoxylin staining, and the abnormal copy number of the imprinted gene indicates the presence of more than two red / brown labeled nuclei in the nucleus of the cell after hematoxylin staining.
  • the abnormal copy number is due to abnormal gene replication by cancer cells, which results in the expression of this gene as a triploid or even higher polyploid.
  • the markers after hematoxylin staining are selected from, but not limited to, red or brown, and staining markers with other colors can also be used to calculate the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene copy number.
  • the detection device is used for early and intuitive observation of changes in the imprinted (trace) genes of lung tumors at the level of cells and tissues to judge the benign and malignant levels of lung tumors, and to provide the most favorable treatment opportunities for patients with early lung tumors.
  • the present disclosure provides a method for detecting the benign and malignant degree of a lung tumor, using the model or the device, including the following steps:
  • the analysis unit calculates the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount through the model, so as to determine the level of the imprinted gene deletion expression amount and imprinted gene copy number abnormal expression amount through the model. Diagnosis of benign and malignant lung tumors.
  • the test sample described in step (1) is from human tissue and / or cells.
  • the sample to be tested is feasible as long as the RNA is processed in a timely manner. Those skilled in the art can select according to the needs, which is not particularly limited here.
  • the sample to be tested includes paraffin sections of tissues and lung puncture samples. Any one or a combination of at least one of a bronchoscopic biopsy sample, a lung lavage fluid smear, or a sputum cell smear.
  • the specific operation steps of the paraffin section of the tissue are to obtain a human tumor tissue sample, which is fixed in 10% neutral formalin in time, embedded in paraffin, cut into 10 ⁇ m thick, and a tissue piece is made from a positively charged glass slide; because Only 10 ⁇ m thick, so part of the nucleus seen under the microscope, so some false negative gene deletions will occur.
  • the specific operation steps of the lung puncture sample are to obtain human cells through puncture, and it can be fixed with 10% neutral formalin in time.
  • the specific operation steps of the bronchoscopic biopsy sample are to obtain suspicious tissue cells under the bronchoscope, and fix them with 10% neutral formalin in time.
  • the bronchoscopy biopsy is less harmful to the patient and the sampling process is simple, compared with the circulation characteristics of the blood, the bronchoscopy biopsy can also be positioned.
  • the bronchoscopy biopsy has its special advantages as an experimental sample.
  • the sputum cell smear is harmless to the patient, the sampling process is simple, and it has its special advantages as an experimental sample.
  • the test sample is any one or a combination of at least two of a lung puncture sample, a bronchoscopy biopsy sample, a lung washing fluid smear, or a sputum cell smear.
  • the imprint genes are Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the imprint gene Z1 is Gnas
  • the imprint gene Z3 is Peg10
  • the imprint gene Z4 is Igf2r
  • the imprint gene Z5 is Mest
  • the imprint gene Z6 is Plagl1
  • the imprint gene Z8 is Dcn
  • the imprint gene Z10 is Gatm
  • the imprint gene Z11 is Grb10
  • the imprint gene Z13 is Sgce
  • the imprinted gene Z16 is Snrpn / Snurf.
  • the imprinted genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce), Z16 (Snrpn / Snurf) is expressed to varying degrees in normal tumor cell tissues. When malignant lesions occur, the expression level and imprinting status will change significantly.
  • the design probe is based on the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, that is, Gnas, Peg10, Igf2r, Mest, Plagl1, Dcn, Gatm, Grb10, Sgce, and Snrpn / Designed by Snurf, a specific sequence was selected as a probe in the inner rotor of each gene, and the specific probe was designed by Advanced Cell Diagnostics.
  • the in situ hybridization uses an RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single-channel or multi-channel color rendering kit or a single-channel or multi-channel fluorescence kit, preferably a single-channel red / brown coloring kit or a multi-channel Fluorescent kit.
  • the multi-channel color rendering kit or multi-channel fluorescence kit includes two-channel or more than two-channel color rendering kits or fluorescent kits.
  • the two-channel color rendering kit or multi-channel fluorescence kit can use two Imprinted gene probes or the combined expression of imprinted genes and other genes or even the combined expression of multiple imprinted and non-imprinted genes.
  • the formula for calculating the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene number in the model is as follows:
  • a means that there is no marker in the nucleus of the cell after hematoxylin staining, and that the imprinted gene is not expressed; and b means that there is a red / brown marker in the nucleus of the cell after hematoxylin staining, and the imprinted gene exists.
  • the nucleus; c is the hematoxylin staining of the cell, there are two red / brown marks in the nucleus of the cell, and the nucleus of the gene deletion is imprinted;
  • d is the hematoxylin staining of the cell, there are more than two red / brown marks in the nucleus of the cell , Imprinted nuclei with abnormal gene copy number.
  • the marker after hematoxylin staining is selected from, but not limited to, red or brown. Staining markers with other colors can also be used to calculate the total expression level of the imprinted gene, the expression level of the imprinted gene deletion, and the abnormal expression amount of the imprinted gene copy number.
  • the probe was amplified by in situ hybridization, and the signal was amplified by Hemotoxy (hematoxylin) cell nuclear staining. Under a 40 ⁇ or 60 ⁇ microscope, the presence of imprinted genes, deletion of imprinted genes, or abnormal copy number in each nucleus was determined. The total gene expression level, the expression level of the imprinted gene, and the expression level of the imprinted gene copy number were abnormal to determine the benign and malignant degree of the tumor in the sample. Because the section is only 10 microns, about 20% of the nuclei seen under the microscope are incomplete nuclei, which means that there is a possibility of some false negatives.
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount are divided into five different grades, and the five different grades are the most positive expressions of each probe in the sample. Area counts at least 1200 cells, and the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and ten imprinted genes for Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16, and The total expression levels of imprinted genes were divided separately.
  • the five different levels of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z1, Z3, Z11, and Z16 are:
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression is less than 10%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is less than 1% or the imprinted
  • the total expression of genes Z1, Z3, Z11 and Z16 is less than any one of the genes or a combination of at least two of them;
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression amount is 10-20%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is 1-3% Or the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or a combination of at least two of 20-30%;
  • the imprinted gene Z1, Z3, Z11, and Z16 imprinted gene deletion expression amount is 20-25%
  • the imprinted gene Z1, Z3, Z11, and Z16 imprinted gene copy number abnormal expression amount is 3-5%
  • the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or a combination of at least two of 30-40%;
  • the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression level is 25-30%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is 5-7% Or the total expression of the imprinted genes Z1, Z3, Z11 and Z16 is any one or a combination of at least two of 40-50%;
  • Class IV The imprinted gene Z1, Z3, Z11 and Z16 imprinted gene deletion expression level is greater than 30%, the imprinted gene Z1, Z3, Z11 and Z16 imprinted gene copy number abnormal expression amount is greater than 7% or the imprinted gene
  • the total expression of genes Z1, Z3, Z11, and Z16 is greater than any one of 50% or a combination of at least two of them;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z1, Z3, Z11 and Z16 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z4, Z5, Z6, Z10, and Z13 are:
  • the imprinted genes of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are expressed less than 10%, and the imprinted genes of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are expressed less than 1%. Or the total expression of the imprinted genes Z4, Z5, Z6, Z10, and Z13 is less than any one or a combination of at least two of them;
  • Class I The expression levels of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are 10-15%, and the expression levels of the imprinted genes Z4, Z5, Z6, Z10, and Z13 are abnormal. 1-2% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or a combination of at least two of 15-20%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 have an expression of 15-20% of the imprinted genes, and the imprinted gene Z4, Z5, Z6, Z10, and Z13 have an abnormal expression of the imprinted gene copy number of 2-4% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or a combination of at least two of 20-30%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene expression levels are 20-25%, the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene copy number abnormal expression levels are 4-6% or the total expression of the imprinted genes Z4, Z5, Z6, Z10 and Z13 is any one or a combination of at least two of 30-40%;
  • the imprinted gene Z4, Z5, Z6, Z10, and Z13 has an imprinted gene deletion expression level greater than 25%, and the imprinted gene Z4, Z5, Z6, Z10, and Z13 imprinted gene copy number abnormal expression levels are greater than 6%. Or the total expression of the imprinted genes Z4, Z5, Z6, Z10, and Z13 is greater than any one or a combination of at least two of them;
  • the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount and total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z13 are independent of each other.
  • the five different levels of Z8 imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount division are:
  • any of the imprinted gene deletion expression level of the imprinted gene Z8 is less than 20%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z8 is less than 2%, or the total expression amount of the imprinted gene Z8 is less than 15% One or a combination of at least two;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 20-25%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 2-4%, or the total expression level of the imprinted gene Z8 is 15 Any one of -20% or a combination of at least two of them;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 25-30%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 4-7%, or the total expression level of the imprinted gene Z8 is 20 Any one or a combination of at least two of -30%;
  • the imprinted gene deletion expression level of the imprinted gene Z8 is 30-35%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is 7-10%, or the total expression level of the imprinted gene Z8 is 30 Any one or a combination of at least two of -40%;
  • Level IV any of the imprinted gene deletion expression level of the imprinted gene Z8 is greater than 35%, the imprinted gene copy number abnormal expression level of the imprinted gene Z8 is greater than 10%, or the total expression level of the imprinted gene Z8 is greater than 40% One or a combination of at least two.
  • the judging degree of benign and malignant lung tumors is divided into benign tumors, lung cancer potential, early lung cancer, intermediate lung cancer, and advanced lung cancer;
  • the result of judging the benign and malignant degree of lung tumor is the imprinted gene deletion expression amount and imprinted gene copy of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16.
  • the number of abnormal expression levels is less than level I or the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed one imprinted gene.
  • Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed the imprinted gene copy number of the imprinted gene with an abnormal expression level of level I, which is a benign tumor;
  • the result of judging the benign and malignant degree of the lung tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level I, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene deletion is level II and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the level II, and it is judged that the potential of lung cancer;
  • the result of judging the benign and malignant degree of the lung tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II,
  • the imprinted gene copy number of at least two imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is the abnormal expression level of class II or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, and the expression level of imprinted genes is class III and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the III cases, which is early lung cancer;
  • the result of judging the benign and malignant degree of the lung tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade III, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class III or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene is level IV and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene that does not exceed 1 imprinted gene is any of the grade IV, and it is intermediate-stage lung cancer;
  • the result of judging the benign and malignant degree of lung tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade IV or imprinted.
  • the abnormal expression level of the copy number of the imprinted gene of at least two imprinted genes among the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV, which is advanced lung cancer.
  • the present disclosure provides a model or a device for lung tumor detection.
  • the present disclosure provides a use of the model or the device for preparing a medicine or a device for treating lung cancer.
  • the benign and malignant degree of lung tumors are divided into benign tumors, lung cancer potential, early lung cancer, intermediate lung cancer, and advanced lung cancer;
  • the results of benign and malignant lung tumors were that the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 had less than the expression level of imprinted genes and the abnormal expression of imprinted gene copy numbers were less than grade I or imprinted.
  • the expression level of the imprinted gene deletion is not more than one and the imprinted genes Z1, Z3, Z4, Z5, Z6, In Z8, Z10, Z11, Z13, and Z16, the abnormal expression level of the copy number of the imprinted gene of no more than one imprinted gene is Grade I, which is a benign tumor;
  • At least two of the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have a level I expression of imprinted genes and the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have no more than one imprinted gene with an expression level of class II and imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1 If the imprinted gene copy number abnormal expression level of each imprinted gene is in any of grade II, it is judged as lung cancer potential;
  • the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 were expressed at a level of class II, and the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class II or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have no more than one imprinted gene with an expression level of imprinted gene class III and imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1
  • the abnormal expression level of the imprinted gene copy number of each imprinted gene is any of grade III, which is early lung cancer;
  • the imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 were expressed at a level of III, and the imprinted gene Z1 , Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class III or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 not more than 1 imprinted gene has an expression level of imprinted gene deletion level IV and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 do not exceed 1
  • the abnormal expression level of the imprinted gene copy number of each imprinted gene is any one of grade IV, which is metastatic lung cancer;
  • At least two of the imprinted genes in the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 were expressed at a level of IV or imprinted genes Z1.
  • Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 have at least two imprinted gene imprinted gene copy number abnormal expression levels of grade IV, which is advanced lung cancer.
  • FIG. 1 is a pathological section of lung cancer with hematoxylin-stained nuclei of the present disclosure, where a is the hematoxylin-stained cell, there is no marker in the nucleus, and the imprinted gene is not expressed; and b is the nuclei of the cell after hematoxylin-stained There is a red / brown marker in the presence of imprinted genes; c refers to the presence of two red / brown markers in the nucleus of the cell after hematoxylin staining, and d refers to the memory of the nucleus in the cell after hematoxylin staining. Abnormal gene copy number imprinted on more than two red / brown marks;
  • Fig. 2 (a) is the expression status of 10 genes in the pathological section of a grade 0 lung tumor
  • Fig. 2 (b) is the expression status of 10 genes in the pathological section of a class I lung cancer
  • Fig. 2 (c) is a class II lung cancer
  • the expression status of 10 genes in the pathological section of Fig. 2 (d) is the expression status of 10 genes in the pathological section of class III lung cancer
  • Fig. 2 (e) is the expression status of 10 genes in the pathological section of class IV lung cancer ;
  • Figure 3 (a) is the intensity of the imprinted genes Z1, Z11, and Z16 on lung cancer
  • Figure 3 (b) is the intensity of the imprinted genes Z1, Z11, and Z16 on lung cancer copy number abnormalities
  • Figure 3 (c) is the imprint.
  • Figure 3 (d) is the intensity of the imprinting of the genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13 on lung cancer.
  • Figure 3 (e) is The imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13 have abnormal copy number intensity on lung cancer.
  • Figure 3 (f) shows the total expression of the imprinted genes Z3, Z4, Z5, Z6, Z8, Z10, and Z13 on lung cancer.
  • the intensity of the quantity, where LOI is the expression level of the imprinted gene deletion
  • CNV is the expression level of the imprinted gene with an abnormal copy number
  • TE is the total expression level of the imprinted gene;
  • Figure 4 (a) is the intensity of the imprinted gene Z1 signature deletion, copy number abnormality, and the total expression level.
  • Figure 4 (b) is the intensity of the imprinted gene Z11 signature deletion, copy number abnormality, and the total expression level.
  • Figure 4 (c) is The imprinting gene Z16 imprint deletion, copy number abnormality, and total expression intensity.
  • Figure 4 (d) is the imprinting gene Z3 imprint deletion, copy number abnormality, and total expression intensity.
  • Figure 4 (e) is the imprinting gene Z4 imprint deletion, Copy number abnormality and the intensity of total expression
  • Figure 4 (f) is the intensity of the imprinted gene Z5 imprint, abnormal copy number and the total expression intensity
  • Figure 4 (g) is the imprinted gene Z6 imprint, the copy number is abnormal, and the total expression.
  • Figure 4 (h) is the intensity of the imprinted gene Z8 signature deletion, copy number abnormality, and the total expression level
  • Figure 4 (i) is the intensity of the imprinted gene Z10 signature deletion, copy number abnormality, and the total expression level
  • Figure 4 (j) is the intensity of the imprinted gene Z13 imprint deletion, copy number abnormality, and the total expression level
  • LOI is the imprinted gene deletion gene expression level
  • CNV is the imprinted gene copy number abnormal gene expression level
  • TE is the imprinted gene total expression level.
  • Figure 5 (a) shows the distribution range and grading standard of the imprinted gene Z1 in 61 cases of lung cancer pathological sections.
  • Figure 5 (b) shows the imprinted gene Z11 in 61 cases of lung cancer pathological sections. Distribution range and grading standard of deletion and copy number abnormalities.
  • Figure 5 (c) shows the distribution range and grading standard of imprinted gene Z16 in 61 cases of lung cancer pathological sections.
  • Figure 5 (d) shows the imprinting.
  • Gene Z3 was applied to 61 cases of lung cancer pathological sections, and the distribution range and grading standard of imprinted deletion and copy number abnormalities were shown in Figure 5 (e).
  • Figure 5 (e) shows the distribution of imprinted gene Z4 in 61 cases of lung cancer pathological sections and the distribution of imprinted deletion and copy number abnormalities.
  • Figure 5 (f) shows the distribution range and grading criteria of the imprinted gene Z5 in 61 cases of lung cancer pathological sections.
  • Figure 5 (g) shows the imprinted gene Z6 in 61 cases of lung cancer.
  • Figure 5 (h) shows the distribution range and grading standard of imprint deletion and copy number abnormality in 61 cases of lung cancer pathological section.
  • Figure 5 (h) shows the distribution range and grading standard of imprint deletion and copy number abnormality in 61 cases of lung cancer pathological section.
  • Figure 5 (i) shows the distribution range and grading standard of the imprinted gene Z10 in 61 cases of lung cancer pathological sections
  • Figure 5 (j) shows the imprinted gene Z13 in 61 cases of lung cancer pathological sections.
  • the distribution range and grading criteria of imprinted deletions and copy number abnormalities where LOI is the expression level of imprinted gene deletion genes, CNV is the expression level of imprinted gene copy number abnormalities, and TE is the total expression level of imprinted genes.
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylating alleles from a particular parent so that only one allele is expressed in one gene and the other is in a state of gene silencing. This type of gene is called an imprinted gene. Blot deletion is an epigenetic change in which the demethylation of a blot gene causes the silenced allele to be activated and start gene expression. A large number of studies have shown that this phenomenon (missing imprinting) is common in various types of cancer and occurs earlier than changes in cell and tissue morphology. At the same time, in healthy cells, the proportion of missing blots is extremely low, in sharp contrast to cancer cells. Therefore, the methylation status of imprinted genes can be used as a pathological marker to analyze the abnormal state of cells through specific molecular detection techniques.
  • the detection model and device described in the present disclosure express the performance of the lack of imprints on the samples of patients with lung tumors in an intuitive way.
  • the imprints can be detected objectively, intuitively, early, and accurately.
  • Gene changes, and can provide a quantified model, making a huge contribution to the diagnosis of lung tumors;
  • the detection device of the present disclosure can determine the benign and malignant degree of lung tumors through minimally invasive or non-invasive biopsy before surgery for patients with lung tumors, thereby providing a basis for surgery and precise treatment, which is a revolutionary breakthrough in the diagnosis of lung tumors in the field of cell molecules;
  • the present disclosure can accurately diagnose the type of lung tumor.
  • the combined detection of imprinted genes greatly improves the early and clear diagnosis of lung cancer, especially for early screening and postoperative follow-up of cancer, especially for follow-up follow-up of patients with suspected recurrence. Can gain time and make a significant contribution to saving patients' lives;
  • the detection method of the present disclosure is different from the immunohistochemical method, and reduces false positives and other negative effects. Not only that, the targeted drug or technology that causes the gene to be silenced, eliminated, or rearranged through the deletion site of the lung tumor-related imprinted gene. Methods can be used to guide the later treatment and medication.
  • the method for detecting an imprinted gene includes the following steps:
  • Design probe design specific primers based on the imprinted gene sequence
  • the design probe is based on the imprinted genes Z1 (Gnas), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce) and Z16 (Snrpn / Snurf) were designed, and a specific sequence was selected as a probe in the inner rotator of each gene.
  • the specific probe was designed by Advanced Cell Diagnostics.
  • FIG. 1 a, b, c, and d are shown in FIG. 1, where a is a cell nucleus after hematoxylin staining, and there is no marker in the nucleus, and imprinted genes are not expressed; b is a cell after hematoxylin staining, There is a red / brown mark in the nucleus, and the nucleus of the gene is imprinted; c is the red / brown mark in the nucleus, and the nucleus of the gene is missing, and d is the hematoxylin. After staining, there were more than two red / brown markers in the nucleus, marking the nucleus with abnormal gene copy number.
  • the bronchoscopy biopsy sample is obtained by removing suspicious diseased tissue under a bronchoscope, and fixing it in a 10% neutral formalin solution for more than 24 hours.
  • the other detection methods are the same as those in Example 1.
  • Figs. 4 (a)-4 (j) Specific sensitivities of each imprinted gene to lung cancer are shown in Figs. 4 (a)-4 (j).
  • the imprint of the imprinted gene Z1 is missing, the copy number is abnormal, and The increase in expression levels began to appear at the stage of malignant potential, and rose rapidly in early lung cancer, and continued to increase to a high level with the development of lung cancer;
  • the imprint of the imprinted gene Z11 and the copy number abnormality began to rise at the stage of malignant potential, with the development of lung cancer Reaching high levels in advanced lung cancer, the increase in the expression level of the imprinted gene Z11 began to appear in early lung cancer, and the development of aged lung cancer reached a higher level in advanced lung cancer; imprinting of the imprinted gene Z16, copy number abnormalities, and expression
  • the increase in volume began to rise in the malignant potential stage, reached a high level in the middle stage of lung cancer, and continued to be maintained in advanced lung cancer;
  • the copy number abnormality of imprinted gene Z4 began to appear in the stage of malignant potential, in the early to middle stage There is no significant increase in lung cancer, and it continues to increase in advanced lung cancer, but the sensitivity is still not high; the deletion of the imprinted gene Z5 and the increase in expression levels begin to appear in the malignant potential stage, maintain stability in early lung cancer, and reach the stage of intermediate and advanced lung cancer.
  • copy number abnormalities of the imprinted gene Z5 begin to appear in the early stage of lung cancer
  • the deletion of the imprinted gene Z6 gradually increases in malignant potential and early lung cancer, maintains stability in intermediate-stage lung cancer, and rapidly rises to higher levels in the advanced stage of lung cancer.
  • the abnormal copy number of the imprinted gene Z6 began to appear in early lung cancer, remained stable in the middle stage of lung cancer, and quickly rose to a higher level in the advanced stage of lung cancer.
  • the increase in the expression of the imprinted gene Z6 quickly rose to a higher level in the malignant potential stage.
  • the deletion of the imprinted gene Z8 begins to appear in the stage of malignant potential, and it does not rise significantly in the early stage of lung cancer. It rapidly rises to a higher level in the middle stage of lung cancer and maintains stability in advanced lung cancer.
  • the copy number abnormality of the imprinted gene Z8 began to appear in the stage of malignant potential, and it quickly rose to a very high level in the early and middle stages of lung cancer, and remained stable in the advanced stage of lung cancer.
  • the increased expression of the imprinted gene Z8 began to appear in the early stage of lung cancer.
  • Tissues (10 micrometers) of 61 lung cancer patients were obtained including bronchoscopy biopsy samples, and the detection method was the same as in Example 1.
  • the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 20%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression is 10-20%, the imprinted gene copy number abnormal expression is 1-3%, or the total imprinted gene expression is 20-30%
  • the species is class I. Any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 3-5%, or the total imprinted gene expression levels are 30-40%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 5-7%, or the total imprinted gene expression level is 40-50% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 7%, or the total imprinted gene expression levels greater than 50% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 20%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression is 10-20%, the imprinted gene copy number abnormal expression is 1-3%, or the total imprinted gene expression is 20-30%
  • the species is class I. Any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 3-5%, or the total imprinted gene expression levels are 30-40%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 5-7%, or the total imprinted gene expression level is 40-50% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 7%, or the total imprinted gene expression levels greater than 50% are grade IV;
  • the expression level of the imprinted gene deletion is less than 10%, the abnormal expression amount of the imprinted copy number is less than 1%, or the total expression level of the imprinted gene is less than 20%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression is 10-20%, the imprinted gene copy number abnormal expression is 1-3%, or the total imprinted gene expression is 20-30%
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 20-25%, the imprinted gene copy number abnormal expression level is 3-5%, or the total imprinted gene expression level is 30-40%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 5-7%, or the total imprinted gene expression level is 40-50% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 7%, or the total imprinted gene expression levels greater than 50% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 20%. Or at least two of them are grade 0, any one or at least two of the imprinted gene deletion expression is 10-20%, the imprinted gene copy number abnormal expression is 1-3%, or the total imprinted gene expression is 20-30%
  • the species is class I. Any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 3-5%, or the total imprinted gene expression levels are 30-40%.
  • any one or at least two of the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 5-7%, or the total imprinted gene expression level is 40-50% is level III, imprinted Any one or at least two of the gene deletion expression levels greater than 30%, the imprinted gene copy number abnormal expression levels greater than 7%, or the total imprinted gene expression levels greater than 50% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression amount is 1-2%, or total imprinted gene expression amount is 15-20%.
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 15-20%, the imprinted gene copy number abnormal expression levels are 2-4%, or the total imprinted gene expression levels are 20-30% are II.
  • any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 4-6%, or the total imprinted gene expression levels are 30-40% are level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 6%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression amount is 1-2%, or total imprinted gene expression amount is 15-20%.
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 15-20%, the imprinted gene copy number abnormal expression levels are 2-4%, or the total imprinted gene expression levels are 20-30% are II.
  • any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 4-6%, or the total imprinted gene expression levels are 30-40% are level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 6%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any one of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression amount is 1-2%, or total imprinted gene expression amount is 15-20%.
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 15-20%, the imprinted gene copy number abnormal expression levels are 2-4%, or the total imprinted gene expression levels are 20-30% are II.
  • any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 4-6%, or the total imprinted gene expression levels are 30-40% are level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 6%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 20%, the imprinted gene copy number abnormal expression is less than 2%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 20-25%, imprinted gene copy number abnormal expression amount is 2-4%, or total imprinted gene expression amount is 15-20%
  • the species is class I, and any one or at least two of the imprinted gene deletion expression is 25-30%, the imprinted gene copy number abnormal expression is 4-7%, or the total imprinted gene expression is 20-30% is II Grade, any one or at least two of the imprinted gene deletion expression is 30-35%, the imprinted gene copy number abnormal expression is 7-10%, or the total imprinted gene expression is 30-40% is grade III, imprinted Any one or at least two of the gene deletion expression levels greater than 35%, the imprinted gene copy number abnormal expression levels greater than 10%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression amount is 1-2%, or total imprinted gene expression amount is 15-20%.
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 15-20%, the imprinted gene copy number abnormal expression levels are 2-4%, or the total imprinted gene expression levels are 20-30% are II.
  • any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 4-6%, or the total imprinted gene expression levels are 30-40% are level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 6%, or the total imprinted gene expression levels greater than 40% are grade IV;
  • any one of the imprinted gene deletion expression is less than 10%, the imprinted gene copy number abnormal expression is less than 1%, or the total imprinted gene expression is less than 15%. Or at least two of them are grade 0, any one or at least two of imprinted gene deletion expression is 10-15%, imprinted gene copy number abnormal expression amount is 1-2%, or total imprinted gene expression amount is 15-20%.
  • the species is class I, and any one or at least two of the imprinted gene expression levels are 15-20%, the imprinted gene copy number abnormal expression levels are 2-4%, or the total imprinted gene expression levels are 20-30% are II.
  • any one or at least two of the imprinted gene deletion expression levels are 20-25%, the imprinted gene copy number abnormal expression levels are 4-6%, or the total imprinted gene expression levels are 30-40% are level III, imprinted Any one or at least two of the gene deletion expression levels greater than 25%, the imprinted gene copy number abnormal expression levels greater than 6%, or the total imprinted gene expression levels greater than 40% are grade IV.
  • the judgement of the benign and malignant degree of lung tumor is divided into benign tumor, lung cancer potential, early lung cancer, intermediate lung cancer and advanced lung cancer:
  • the results of judging the benign and malignant degree of lung tumors are that the imprinted gene deletion expression amount and imprinted gene copy number abnormal expression amount of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 are less than 1 Class 1 or imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16.
  • the expression level of the imprinted gene deletion is no more than 1 and the imprinted genes Z1, Z3, Z4, Z5.
  • the abnormal expression level of the copy number of the imprinted gene of not more than 1 imprinted gene in Z6, Z8, Z10, Z11, Z13, and Z16 is Grade I, and it is a benign tumor;
  • the result of judging the benign and malignant degree of the lung tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is level I, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class I or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene deletion is level II and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the level II, and it is judged that the potential of lung cancer;
  • the result of judging the benign and malignant degree of the lung tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade II,
  • the imprinted gene copy number of at least two imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is the abnormal expression level of class II or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, and the expression level of imprinted genes is class III and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene of no more than 1 imprinted gene is any of the III cases, which is early lung cancer;
  • the result of judging the benign and malignant degree of the lung tumor is that the imprinted gene deletion expression level of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade III, Imprinted gene Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 have at least two imprinted gene copy number abnormal expression levels of class III or imprinted genes Z1, Z3, Z4, Z5, Z6 , Z8, Z10, Z11, Z13, and Z16 are not more than one of the imprinted genes, the expression level of the imprinted gene is level IV and the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16
  • the abnormal expression level of the copy number of the imprinted gene that does not exceed 1 imprinted gene is any of the grade IV, and it is intermediate-stage lung cancer;
  • the result of judging the benign and malignant degree of lung tumor is that the expression level of the imprinted gene deletion of at least two imprinted genes among the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16 is grade IV or imprinted.
  • the abnormal expression level of the copy number of the imprinted gene of at least two imprinted genes among the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is grade IV, which is advanced lung cancer.
  • the detection model and system described in the present disclosure express the performance of the lack of imprints on samples of patients with lung tumors in an intuitive way.
  • the method of in situ labeling of imprinted genes it is objective, intuitive, early and accurate. Detects changes in imprinted (trace) genes and can provide a quantified model, making a significant contribution to the diagnosis of lung tumors.

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Abstract

本公开提供一种用于检测肺肿瘤良恶性程度的分级模型及其应用。该模型通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量对印记基因在肺肿瘤中的变化进行分级。

Description

[根据细则37.2由ISA制定的发明名称]　用于检测肺肿瘤良恶性程度的分级模型及其应用 技术领域
本公开涉及生物技术领域，例如基因诊断领域，例如一种分级模型及其应用，例如一种用于检测肺肿瘤良恶性程度的分级模型及其应用，例如一组印记基因在检测肺肿瘤良恶性程度中的分级模型及其组成的装置。
背景技术
肺癌是全球发病率和死亡率最高的恶性肿瘤。据世界卫生组织(WHO)统计，2012年全球新增病例182万例，死亡159万例，中国新诊断73.3万例，死亡61.0万例(World Cancer Report 2014)，占男性发病率和死亡率的第一位，女性发病率的第二位和死亡率的第一位。肺癌患者的生存率与癌症的进展程度密切相关，I期肺癌患者的5年存活率可达70-90％，而IV期肺癌患者的5年存活率不超过10％，因此肺癌的早期诊断和早期治疗是挽救肺癌患者生命的关键。近年来，低剂量螺旋CT、正电子发射计算机断层显像(PET-CT)、荧光纤维支气管镜、经支气管腔内超声(EBUS)等多种新技术的应用，将肺癌的早期诊断率从不足30％提高到了55％，但仍有大量的患者在诊断为肺癌时已经是晚期，因此急需一种更敏感、更准确的肺癌早期检测方法。
癌症的产生是随时间推移而累积的表观遗传改变和基因上的变异所导致的不受控制的细胞生长/分裂。传统病理学诊断根据细胞和组织的大小，形态和结构上的变异，从而做出肺肿瘤良恶性判断。随着分子生物学的发展与深入，越来越多的分子检测技术被应用于肺癌症的检测。从癌症的发展过程分析，分子层面的改变(表观遗传学和基因学)远早于细胞形态和组织结构的变异。所以分子生物学检测对癌症早期的检测更敏感。
基于上述原因，目前的肺癌诊断需要新的检测系统和检测模型，基于患者活检样本，解析肺癌在细胞层面上存在的分子标记物变化，以此提供更精确的预诊和诊断信息。
公开内容
本公开提供了一种印记基因分级模型和诊断方法及其应用。
为达到上述目的，本公开采用以下技术方案：
本公开提供了一种用于肺肿瘤的印记基因分级模型，所述模型通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因的总表达量在肺肿瘤中的变化对印记基因的表达状态进行分级；
其中，所述印记基因为Z1、Z11或Z16中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。
所述印记(迹)缺失是指印记(迹)基因中原先处在沉寂状态的等位基因被激活(去甲基化)，是癌症中最常见和最早期就发生的表观遗传改变，并且这个特性可以用作病理标记。相对而言，在健康细胞检测中，印迹缺失比例很低，所述印记基因与印迹基因同时一个概念，表示同一个意思，可以进行替换。
发明人发现通过计算Z1、Z11和Z16中任意一个印记基因在肺肿瘤中的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因的总表达量，对肺肿瘤的诊断敏感度可以达到73.7％以上。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1、Z11和Z16中的任意一个印记基因。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1或Z16中的任意一个印记基因。
在本文一实施例中，若初步检测只检测一个印记基因，可以检测Z1。
在本文一实施例中，发明人发现，若单独检测一个Z1印记基因，对肺肿瘤的诊断敏感度可以达到86.0％，若单独检测一个Z11印记基因，对肺肿瘤的诊断敏感度可以达到73.7％，单独检测一个Z16印记基因，对肺肿瘤的诊断敏感度可以达到78.9％。
在本文一实施例中，所述模型计算印记基因的方法为：若检测印记基因的两个印记基因的组合，所述组合可以是Z1、Z11和Z16中的任意两个，优选为Z1和Z16的组合，Z1和Z11的组合。
发明人发现通过计算两个或两个以上的印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因的总表达量可以进一步提高敏感度，检测Z1和Z16的组合，Z1和Z11的组合时，对肺癌的诊断敏感度可以达到93.0％以上，检测印记基因Z11和Z16的两个印记基因的组合，对肺癌的诊断敏感度可以达到91.2％以上。
在本文一实施例中，所述印记基因还包括Z3、Z4、Z5、Z6、Z8、Z10或Z13中的任意一个或至少两个的组合；其中，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z13为Sgce。
发明人发现在使用Z1、Z11和Z16基因检测的基础上再增加Z3、Z4、Z5、Z6、Z8、Z10、Z13基因进行联合诊断，不仅有助于增加检测的准确度，而且增加其他探针辅助诊断可以进一步避免假阳性的出现，能够将检测准确度进一步提高，从而能够实现所有肺肿瘤样本的精确分级和判断。
在本文一实施例中，所述模型计算印记基因的方法为：计算印记基因的组合，所述印记基因为Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16基因 的组合。
所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。
在本文一实施例中，所述计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。
在本文一实施例中，所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量的计算。
在本文一实施例中，将探针通过原位杂交，和Hemotoxy(苏木精)细胞核染色扩增信号，在40×或60×显微镜下，判断每一个细胞核内印记基因存在、印记基因缺失或拷贝数异常，通过计算印记基因缺失基因表达量、印记基因拷贝 数异常表达量和印记基因总表达量来判定该样本的肿瘤良恶性程度；由于切片仅为10μm，所以在显微镜下所见细胞核大约有20％为不完整细胞核，也就是说有部分假阴性的可能性存在。
在本文一实施例中，所述印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分成五个不同的等级，通过每个探针在样本表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量分和印记基因总表达量分别进行划分的五个不同的等级。
在本文一实施例中，所述针对Z1、Z3、Z11和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量小于10％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1、Z3、Z11和Z16的总表达量小于20％中的任意一种或至少两种；
I级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为10-20％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为1-3％或所述印记基因Z1、Z3、Z11和Z16的总表达量为20-30％中的任意一种或至少两种；
II级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1、Z3、Z11和Z16的总表达量为30-40％中的任意一种或至少两种；
III级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为5-7％或所述印记基因Z1、Z3、Z11和Z16的总表达量为40-50％中的任意一种或至少两种；
IV级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量大于30％、 所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量大于7％或所述印记基因Z1、Z3、Z11和Z16的总表达量大于50％中的任意一种或至少两种；
所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
在本文一实施例中，所述针对Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量小于10％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量小于1％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量小于15％中的任意一种或至少两种；
I级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为10-15％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为15-20％中的任意一种或至少两种；
II级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为15-20％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为2-4％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为20-30％中的任意一种或至少两种；
III级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为20-25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为4-6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为30-40％中的任意一种或至少两种；
IV级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量大于 25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量大于6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量大于40％中的任意一种或至少两种；
所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
在本文一实施例中，所述针对Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z8的印记基因缺失表达量小于20％、所述印记基因Z8的印记基因拷贝数异常表达量小于2％或所述印记基因Z8的总表达量小于15％中的任意一种或至少两种；
I级：所述印记基因Z8的印记基因缺失表达量为20-25％、所述印记基因Z8的印记基因拷贝数异常表达量为2-4％或所述印记基因Z8的总表达量为15-20％中的任意一种或至少两种；
II级：所述印记基因Z8的印记基因缺失表达量为25-30％、所述印记基因Z8的印记基因拷贝数异常表达量为4-7％或所述印记基因Z8的总表达量为20-30％中的任意一种或至少两种；
III级：所述印记基因Z8的印记基因缺失表达量为30-35％、所述印记基因Z8的印记基因拷贝数异常表达量为7-10％或所述印记基因Z8的总表达量为30-40％中的任意一种或至少两种；
IV级：所述印记基因Z8的印记基因缺失表达量大于35％、所述印记基因Z8的印记基因拷贝数异常表达量大于10％或所述印记基因Z8的总表达量大于40％中的任意一种或至少两种。
在本文一实施例中，本公开提供了一种检测肺肿瘤良恶性程度的装置，其 采用上述模型，包括如下单元：
(1)取样单元：获取待测样本；
(2)探针设计单元：根据印记基因序列设计特异性引物；
(3)检测单元：将步骤(2)的探针与待测样本进行原位杂交；
(4)分析单元：显微镜成像分析印记基因的表达情况；
其中，所述分析单元通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量，通过第一方面所述的模型，从而通过印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量的等级来判断肺肿瘤的良恶性程度。
所述印记基因缺失为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记的细胞核，所述印记基因拷贝数异常为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记的细胞核，所述拷贝数异常是由于癌细胞异常地进行基因复制，导致这个基因表达时呈现为三倍体甚至更高的多倍体的情况。
所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。
所述检测装置是用于细胞和组织水平下早期直观地观察肺肿瘤的印记(迹)基因的变化从而判断肺肿瘤的良恶性及恶性程度，为早期肺肿瘤患者提供最有利的治疗机会。
在本文一实施例中，本公开提供一种检测肺肿瘤良恶性程度的方法，采用所述的模型或所述的装置，包括如下步骤：
(1)获取待测样本；
(2)根据印记基因序列设计特异性引物；
(3)将步骤(2)的探针与待测样本进行原位杂交；
(4)显微镜成像分析印记基因的表达情况，从而诊断肺肿瘤的良恶性程度；
其中，所述分析单元通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量，通过所述的模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来诊断肺肿瘤的良恶性程度。
在本文一实施例中，步骤(1)所述的待测样本来自于人的组织和/或细胞。
所述待测样本只要RNA经过及时固定的处理都是可行的，本领域技术人员可以根据需要进行选择，在此不做特殊限定，本公开所述待测样本包括组织的石蜡切片、肺穿刺样本、支气管镜活检样本、肺冲洗液细胞涂片或痰液细胞涂片中的任意一种或至少两种的组合。
所述组织的石蜡切片具体操作步骤为获取人体肿瘤组织样本，及时用10％中性福尔马林固定，石蜡包埋，切成10μm厚，用带正电荷的玻片制成组织片子；因为只有10μm厚，因此显微镜下看见的有一部分为不完整的细胞核，所以会出现部分假阴性的基因缺失。
所述肺穿刺样本具体操作步骤为通过穿刺获取人体细胞，及时用10％中性福尔马林固定即可。
所述支气管镜活检样本具体操作步骤为在支气管镜下获取可疑组织细胞，及时用10％中性福尔马林固定。
由于支气管镜活检对病人伤害小，取样过程简单，相较于血液的循环特性，支气管镜活检还能定位，支气管镜活检作为实验样本有其特殊的优势。
痰液细胞涂片对病人无伤害，取样过程简单，作为实验样本有其特殊的优 势。
在本文一实施例中，所述待测样本为肺穿刺样本、支气管镜活检样本、肺冲洗液细胞涂片或痰液细胞涂片中的任意一种或至少两种的组合。
在本文一实施例中，所述印记基因为Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16，所述印记基因Z1为Gnas，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z11为Grb10，所述印记基因Z13为Sgce，所述印记基因Z16为Snrpn/Snurf。
所述印记基因Z1(Gnas)，Z3(Peg10)，Z4(Igf2r)，Z5(Mest)，Z6(Plagl1)，Z8(Dcn)，Z10(Gatm)，Z11(Grb10)，Z13(Sgce)，Z16(Snrpn/Snurf)在正常肿瘤细胞组织内有不同程度的表达，在发生恶性病变时，表达量和印记状态都会发生明显变化。
所述设计探针是根据印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16，即Gnas，Peg10，Igf2r，Mest，Plagl1，Dcn，Gatm，Grb10，Sgce和Snrpn/Snurf进行设计的，具体在每个基因的内旋子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计。
在本文一实施例中，所述原位杂交采用RNAscope原位杂交方法。
在本文一实施例中，所述RNAscope原位杂交方法使用单通道或多通道的呈色试剂盒或者单通道或多通道的荧光试剂盒，优选为单通道红色/棕色呈色试剂盒或多通道的荧光试剂盒。
所述多通道呈色试剂盒或多通道荧光试剂盒包括两通道或两通道以上的呈色试剂盒或荧光试剂盒，所述两通道的呈色试剂盒或多通道的荧光试剂盒可以使用两个印记基因探针或印记基因和其他基因的联合表达甚至多个印记基因和 非印记基因的综合表达。
在本文一实施例中，所述模型中的计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。
所述苏木素染色后的标记选自但不限于红色或棕色，用其他颜色进行染色标记也可用于印迹基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算。
将探针通过原位杂交，和Hemotoxy(苏木精)细胞核染色扩增信号，在40×或60×显微镜下，判断每一个细胞核内印记基因存在、印记基因缺失或拷贝数异常，通过计算印记基因总表达量、印记基因缺失基因表达量和印记基因拷贝数异常的基因表达量来判定该样本的肿瘤良恶性程度。由于切片仅为10微米，所以在显微镜下所见细胞核大约有20％为不完整细胞核，也就是说有部分假阴性的可能性存在。
在本文一实施例中，所述印记基因缺失表达量、印记基因拷贝数异常表达 量和总表达量分成五个不同的等级，所述五个不同的等级为在样本每个探针表达最阳性的区域对至少1200个细胞进行计数，针对Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分别进行划分。
所述针对Z1、Z3、Z11和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量小于10％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1、Z3、Z11和Z16的总表达量小于20％中的任意一种或至少两种的组合；
I级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为10-20％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为1-3％或所述印记基因Z1、Z3、Z11和Z16的总表达量为20-30％中的任意一种或至少两种的组合；
II级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1、Z3、Z11和Z16的总表达量为30-40％中的任意一种或至少两种的组合；
III级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为5-7％或所述印记基因Z1、Z3、Z11和Z16的总表达量为40-50％中的任意一种或至少两种的组合；
IV级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量大于30％、 所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量大于7％或所述印记基因Z1、Z3、Z11和Z16的总表达量大于50％中的任意一种或至少两种的组合；
所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
所述针对Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量小于10％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量小于1％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量小于15％中的任意一种或至少两种的组合；
I级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为10-15％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为15-20％中的任意一种或至少两种的组合；
II级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为15-20％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为2-4％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为20-30％中的任意一种或至少两种的组合；
III级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为20-25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为4-6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为30-40％中的任意一种或至少两种的组合；
IV级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量大于25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量大于6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量大于40％中的任意一种或至少两种的组合；
所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量是相互独立的。
所述针对Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
0级：所述印记基因Z8的印记基因缺失表达量小于20％、所述印记基因Z8的印记基因拷贝数异常表达量小于2％或所述印记基因Z8的总表达量小于15％中的任意一种或至少两种的组合；
I级：所述印记基因Z8的印记基因缺失表达量为20-25％、所述印记基因Z8的印记基因拷贝数异常表达量为2-4％或所述印记基因Z8的总表达量为15-20％中的任意一种或至少两种的组合；
II级：所述印记基因Z8的印记基因缺失表达量为25-30％、所述印记基因Z8的印记基因拷贝数异常表达量为4-7％或所述印记基因Z8的总表达量为20-30％中的任意一种或至少两种的组合；
III级：所述印记基因Z8的印记基因缺失表达量为30-35％、所述印记基因Z8的印记基因拷贝数异常表达量为7-10％或所述印记基因Z8的总表达量为30-40％中的任意一种或至少两种的组合；
IV级：所述印记基因Z8的印记基因缺失表达量大于35％、所述印记基因Z8的印记基因拷贝数异常表达量大于10％或所述印记基因Z8的总表达量大于40％中的任意一种或至少两种的组合。
在本文一实施例中，所述判断肺肿瘤的良恶性程度分为良性肿瘤、肺癌潜能、早期肺癌、中期肺癌和晚期肺癌；
在本文一实施例中，所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为肺癌潜能；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则 为早期肺癌；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期肺癌；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
在本文一实施例中，本公开提供一种所述的模型或所述的装置用于肺肿瘤检测。
在本文一实施例中，本公开提供一种所述的模型或所述的装置用于制备治疗肺癌的药物或器械的用途。
在本文一实施例中，肺肿瘤的良恶性程度分为良性肿瘤、肺癌潜能、早期肺癌、中期肺癌和晚期肺癌；
肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z3、Z4、Z5、Z6、 Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；
肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为肺癌潜能；
肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期肺癌；
肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且 印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期肺癌；
肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
与相关技术相比，本文实施例中，利用所述检测模型和装置，以直观的方法表现了印记基因在膀胱肿瘤病人样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为分子病理学的诊断做出巨大贡献。
附图说明
图1是本公开苏木素染色细胞核的肺癌的病理切片，其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常；
图2(a)为0级肺肿瘤的病理切片中10个基因的表达状态，图2(b)为I级肺癌的病理切片中10个基因的表达状态，图2(c)为II级肺癌的病理切片中10个基因的表达状态，图2(d)为III级肺癌的病理切片中10个基因的表达状态，图2(e)为IV级肺癌的病理切片中10个基因的表达状态；
图3(a)为印记基因Z1、Z11和Z16对肺癌的印记缺失的强度，图3(b) 为印记基因Z1、Z11和Z16对肺癌的拷贝数异常的强度，图3(c)为印记基因Z1、Z11和Z16对肺癌的总表达量的强度，图3(d)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对肺癌的印记缺失的强度，图3(e)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对肺癌的拷贝数异常的强度，图3(f)为印记基因Z3、Z4、Z5、Z6、Z8、Z10和Z13对肺癌的总表达量的强度，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量；
图4(a)为印记基因Z1印记缺失、拷贝数异常和总表达量的强度，图4(b)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图4(c)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图4(d)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图4(e)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图4(f)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图4(g)为印记基因Z6印记缺失、拷贝数异常和总表达量的强度，图4(h)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图4(i)为印记基因Z10印记缺失、拷贝数异常和总表达量的强度，图4(j)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量；
图5(a)为印记基因Z1应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(b)为印记基因Z11应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(c)为印记基因Z16应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(d)为印记基因Z3应用于61例肺癌病理切片中，印记缺失和拷贝数异常 的分布范围和分级标准，图5(e)为印记基因Z4应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(f)为印记基因Z5应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(g)为印记基因Z6应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(h)为印记基因Z8应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(i)为印记基因Z10应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，图5(j)为印记基因Z13应用于61例肺癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，其中，LOI为印记基因缺失基因表达量，CNV为印记基因拷贝数异常的基因表达量，TE为印记基因总表达量。
具体实施方式
为更进一步阐述本公开所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本公开的技术方案，但本公开并非局限在实施例范围内。
基因组印记是表观遗传学中基因调控的一种方式。其特点是，通过甲基化来自特定亲代的等位基因，使某个基因只有一个等位基因表达，而另一个则陷入基因沉默状态。该种类的基因，被称为印迹(记)基因。印迹缺失是印迹基因去甲基化导致沉默状态的等位基因被激活并且开始基因表达的一种表观遗传改变。大量研究表明，该现象(印迹缺失)普遍存在于各类癌症并且发生时间早于细胞和组织形态改变。与此同时，在健康细胞中，印迹缺失比例极低，与癌细胞成鲜明对比。所以，印迹基因的甲基化状态可以作为病理标记，通过特定分子检测技术，对细胞异常状态进行分析。
本公开所述检测模型和装置以直观的方法表现了印记缺失在肺肿瘤病人的 样本上的表现，通过对印记基因原位标记的方法，客观，直观，早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为肺肿瘤的诊断做出巨大贡献；
本公开检测装置可以在肺肿瘤病人手术前通过微创或无创的活检得出肺肿瘤良恶性程度的判断，从而为手术及精准治疗提供依据，这是细胞分子领域诊断肺肿瘤的革命性突破；
本公开可以精确诊断肺肿瘤的类型，通过印记基因的组合检测极大地提高了对肺癌的早期、明确诊断，特别是用在早期普查和癌症术后随访，尤其是对于疑似复发病人的跟踪随访，可以争取时间，为挽救病人生命做出重大贡献；
本公开检测方法区别于免疫组化方法，减少了假阳性和其他负面作用，不仅如此，通过发现的肺肿瘤相关印记基因缺失位点的致该基因沉默、剔除、重排的靶向药物或技术方法，可用于指导后期的治疗和用药。
实施例1肺癌的印记基因分析
所述的印记基因的检测方法，包括如下步骤：
(1)获取肺癌的组织细胞切片(10微米)，放入10％中性福尔马林溶液中进行固定，以防RNA降解，固定时间为24小时，石蜡包埋(FFPE)，所述玻片需要用正电荷脱载玻片，所述切片在40℃烤箱烘烤3h以上；
(2)按照RNASCope的样品处理方法进行脱蜡处理，封闭样本中内源性过氧化物酶活性，增强通透性并暴露出RNA分子；
(3)设计探针：根据印记基因序列设计特异性引物；
所述设计探针是根据印记基因Z1(Gnas)、Z3(Peg10)、Z4(Igf2r)、Z5(Mest)、Z6(Plagl1)、Z8(Dcn)、Z10(Gatm)、Z11(Grb10)、Z13(Sgce)和Z16 (Snrpn/Snurf)进行设计的，具体在每个基因的内旋子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计。
(4)将步骤(3)的探针与待测样本通过试剂盒进行RNA SCope原位杂交；
(5)信号扩增和苏木精染色，用显微镜成像分析印记基因的表达情况；
所述模型中的计算印记基因总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常印记基因表达量＝b/(b+c+d)×100％；
印记基因缺失基因表达量(LOI)＝c/(b+c+d)×100％；
印记基因拷贝数异常的基因表达量(CNV)＝d/(b+c+d)×100％；
其中，a、b、c、d如图1所示，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。
从图2(a)-图2(e)可以看出，从0级到IV级的样本中，印记缺失(细胞核内有两个信号点)和拷贝数异常(细胞核内有三个或以上信号点)的细胞比例随恶性程度的增加而逐渐增加。
实施例2支气管镜活检样本的印记基因分析
所述支气管镜活检样本是，在支气管镜下取出可疑病变组织，10％中性福尔马林溶液固定24h以上，其他检测方法同实施例1。
从图3(a)-图3(f)可以看出，Z1，Z3，Z4，Z5，Z6，Z8，Z10，Z11， Z13，Z16每个基因对肺癌的反应敏感性或者说对应于肺癌表达的印记缺失的强度和状态是不同的。
具体每个印记基因对肺癌的敏感度如图4(a)-图4(j)，从图4(a)-图4(c)可以看出，印记基因Z1的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段开始出现，在早期肺癌中迅速上升，随肺癌的发展继续增加到很高的水平；印记基因Z11的印记缺失和拷贝数异常在恶性潜能阶段开始上升，随肺癌的发展到晚期肺癌中达到很高的水平，印记基因Z11的表达量增加在早期肺癌中开始出现，岁肺癌的发展到晚期肺癌中达到较高的水平；印记基因Z16的印记缺失、拷贝数异常和表达量增加在恶性潜能阶段开始上升，到中期肺癌阶段达到很高的水平，在晚期肺癌中继续维持；
从图4(d)-图4(j)可以看出，印记基因Z3的印记缺失在恶性潜能到中期肺癌阶段缓慢增加，到晚期肺癌中上升速度加快，达到较高的水平，印记基因Z3的拷贝数异常在恶性潜能阶段开始出现，随肺癌的发展而逐渐增加到较高的水平，印记基因Z3的表达量增加在恶性潜能阶段开始出现，在早期到晚期肺癌的发展过程中缓慢上升；印记基因Z4的印记缺失和表达量增加在恶性潜能阶段开始出现，在早期肺癌中维持稳定，到中晚期肺癌阶段又继续上升，印记基因Z4的拷贝数异常在恶性潜能阶段开始出现，在早期到中期肺癌中没有明显上升，在晚期肺癌中继续上升，但是敏感度仍然不高；印记基因Z5的印记缺失和表达量增加在恶性潜能阶段开始出现，在早期肺癌中维持稳定，到中晚期肺癌阶段又继续上升到较高水平，印记基因Z5的拷贝数异常在早期肺癌阶段开始出现，在中期肺癌中没有明显上升，到晚期肺癌阶段继续上升；印记基因Z6的印记缺失在恶性潜能和早期肺癌中逐渐上升，在中期肺癌中维持稳定，到晚期肺癌阶段又快速上升到较高水平，印记基因Z6的拷贝数异常在早期肺癌中开始出 现，在中期肺癌中维持稳定，到晚期肺癌阶段又快速上升到较高水平，印记基因Z6的表达量增加在恶性潜能阶段迅速上升到较高水平，在早期到晚期肺癌阶段继续缓慢上升；印记基因Z8的印记缺失在恶性潜能阶段开始出现，在早期肺癌阶段没有明显上升，在中期肺癌阶段迅速上升到较高水平，在晚期肺癌中维持稳定，印记基因Z8的拷贝数异常在恶性潜能阶段开始出现，在早期和中期肺癌阶段迅速上升到很高的水平，并在晚期肺癌阶段维持稳定，印记基因Z8的表达量增加在早期肺癌阶段开始出现，在中期和晚期肺癌中逐渐上升到较高水平；印记基因Z10的印记缺失和表达量增加在早期肺癌阶段开始出现，随肺癌的进展缓慢上升，到晚期肺癌阶段灵敏度仍然不高，印记基因Z10的拷贝数异常在恶性潜能阶段开始出现，随肺癌的进展缓慢上升，到晚期肺癌阶段灵敏度仍然不高；印记基因Z13的印记缺失和拷贝数异常在早期肺癌阶段开始出现，在中期到晚期肺癌阶段继续上升到较高水平，印记基因Z13的表达量增加在恶性潜能阶段开始出现，在早期肺癌阶段没有明显上升，在中期到晚期肺癌中继续上升到较高水平。
实施例3 61例肺肿瘤样本的印记基因分析
获取61例肺癌病人的组织(10微米)包括支气管镜活检样本，检测方法同实施例1。
从图5(a)-图5(j)可以看出，61例肺肿瘤组织样本中10个探针的印记缺失和拷贝数异常的比例呈现从低到高的分布，根据不同探针的分布趋势，我们计算得到了图中虚线所示的分级标准，可以将每个探针的印记缺失、拷贝数异常和总表达量分别从低到高分成5个等级。
具体的分级如下：
从图5(a)可以看出，对于所述印记基因Z1，印记基因缺失表达量小于10％、 印记基因拷贝数异常表达量小于1％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为10-20％、印记基因拷贝数异常表达量为1-3％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；
从图5(b)可以看出，对于所述印记基因Z11，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为10-20％、印记基因拷贝数异常表达量为1-3％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；
从图5(c)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为10-20％、印记基因拷贝数异常表达量为1-3％或印记基因总表达量为20-30％中的任意一种或至少两种为I 级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；
从图5(d)可以看出，对于所述印记基因Z3，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于20％中的任意一种或至少两种为0级，印记基因缺失表达量为10-20％、印记基因拷贝数异常表达量为1-3％或印记基因总表达量为20-30％中的任意一种或至少两种为I级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为3-5％或印记基因总表达量为30-40％中的任意一种或至少两种为II级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为5-7％或印记基因总表达量为40-50％中的任意一种或至少两种为III级，印记基因缺失表达量大于30％、印记基因拷贝数异常表达量大于7％或印记基因总表达量大于50％中的任意一种或至少两种为IV级；
从图5(e)可以看出，对于所述印记基因Z4，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为4-6％或印记基因总表达量为30-40％中 的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于6％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(f)可以看出，对于所述印记基因Z5，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为4-6％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于6％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(g)可以看出，对于所述印记基因Z6，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为4-6％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于6％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(h)可以看出，对于所述印记基因Z8，印记基因缺失表达量小于20％、印记基因拷贝数异常表达量小于2％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为25-30％、印记基因拷贝数异常表达量为4-7％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为30-35％、印记基因拷贝数异常表达量为7-10％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于35％、印记基因拷贝数异常表达量大于10％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(i)可以看出，对于所述印记基因Z10，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异常表达量为1-2％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为4-6％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于6％或印记基因总表达量大于40％中的任意一种或至少两种为IV级；
从图5(j)可以看出，对于所述印记基因Z13，印记基因缺失表达量小于10％、印记基因拷贝数异常表达量小于1％或印记基因总表达量小于15％中的任意一种或至少两种为0级，印记基因缺失表达量为10-15％、印记基因拷贝数异 常表达量为1-2％或印记基因总表达量为15-20％中的任意一种或至少两种为I级，印记基因缺失表达量为15-20％、印记基因拷贝数异常表达量为2-4％或印记基因总表达量为20-30％中的任意一种或至少两种为II级，印记基因缺失表达量为20-25％、印记基因拷贝数异常表达量为4-6％或印记基因总表达量为30-40％中的任意一种或至少两种为III级，印记基因缺失表达量大于25％、印记基因拷贝数异常表达量大于6％或印记基因总表达量大于40％中的任意一种或至少两种为IV级。
从这61个肺肿瘤的样本综合分析可以得出：
所述判断肺肿瘤的良恶性程度分为良性肿瘤、肺癌潜能、早期肺癌、中期肺癌和晚期肺癌：
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则 判断为肺癌潜能；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期肺癌；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期肺癌；
所述判断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
综上所述，本公开所述检测模型和系统，以直观的方法表现了印记缺失在肺肿瘤病人的样本上的表现，通过对印记基因原位标记的方法，客观，直观， 早期，精确地检测出印记(迹)基因的变化，并可以提供量化的模型，为肺肿瘤的诊断做出巨大贡献。
申请人声明，本公开通过上述实施例来说明本公开的详细方法，但本公开并不局限于上述详细方法，即不意味着本公开必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本公开的任何改进，对本公开产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本公开的保护范围和公开范围之内。

Claims (20)

  1. 一种用于肺肿瘤的印记基因分级模型，其通过计算印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因的总表达量在肺肿瘤中的变化对印记基因的表达状态进行分级；
    其中，所述印记基因为Z1、Z11或Z16中的任意一个或至少两个的组合，所述印记基因Z1为Gnas，所述印记基因Z11为Grb10，所述印记基因Z16为Snrpn/Snurf。
  2. 根据权利要求1所述的模型，其中，所述模型计算印记基因的方法如下：
    计算Z1、Z11或Z16中的任意一个，优选为Z1或Z16，进一步优选为Z1。
  3. 根据权利要求1或2所述的模型，其中，所述模型计算印记基因的方法为：计算Z1、Z11或Z16中的任意两个印记基因的组合，优选为Z1和Z16的组合或Z1和Z11的组合。
  4. 根据权利要求1-3中任一项所述的模型，其中，所述印记基因还包括Z3、Z4、Z5、Z6、Z8、Z10或Z13中的任意一个或至少两个的组合；其中，所述印记基因Z3为Peg10，所述印记基因Z4为Igf2r，所述印记基因Z5为Mest，所述印记基因Z6为Plagl1，所述印记基因Z8为Dcn，所述印记基因Z10为Gatm，所述印记基因Z13为Sgce。
  5. 根据权利要求1-4中任一项所述的模型，其中，所述模型计算印记基因的方法为：计算印记基因的组合，计算Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的组合。
  6. 根据权利要求1-5中任一项所述的模型，其中，所述计算印记基因的总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的公式如下：
    总表达量＝(b+c+d)/(a+b+c+d)×100％；
    正常印记基因表达量＝b/(b+c+d)×100％；
    印记基因缺失基因表达量＝c/(b+c+d)×100％；
    印记基因拷贝数异常的基因表达量＝d/(b+c+d)×100％；
    其中，所述a为将细胞进行苏木素染色后，细胞核内不存在标记，印记基因没有表达的细胞核；所述b为将细胞进行苏木素染色后，细胞核内存在一个红色/棕色标记，印记基因存在的细胞核；所述c为将细胞进行苏木素染色后，细胞核内存在两个红色/棕色标记，印记基因缺失的细胞核；所述d为将细胞进行苏木素染色后，细胞核内存在两个以上红色/棕色标记，印记基因拷贝数异常的细胞核。
  7. 根据权利要求1-6中任一项所述的模型，其中，所述印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分成五个不同的等级。
  8. 根据权利要求7所述的模型，其中，所述五个不同的等级为针对Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的十个印记基因的印记基因缺失表达量、印记基因拷贝数异常表达量和印记基因总表达量分别进行划分的五个不同的等级；
    所述针对Z1、Z3、Z11和Z16的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
    0级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量小于10％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量小于1％或所述印记基因Z1、Z3、Z11和Z16的总表达量小于20％中的任意一种或至少两种的组合；
    I级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为10-20％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为1-3％或所述 印记基因Z1、Z3、Z11和Z16的总表达量为20-30％中的任意一种或至少两种的组合；
    II级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为3-5％或所述印记基因Z1、Z3、Z11和Z16的总表达量为30-40％中的任意一种或至少两种的组合；
    III级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量为5-7％或所述印记基因Z1、Z3、Z11和Z16的总表达量为40-50％中的任意一种或至少两种的组合；
    IV级：所述印记基因Z1、Z3、Z11和Z16的印记基因缺失表达量大于30％、所述印记基因Z1、Z3、Z11和Z16的印记基因拷贝数异常表达量大于7％或所述印记基因Z1、Z3、Z11和Z16的总表达量大于50％中的任意一种或至少两种的组合；
    所述针对Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
    0级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量小于10％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量小于1％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量小于15％中的任意一种或至少两种的组合；
    I级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为10-15％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为1-2％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为15-20％中的任意一种 或至少两种的组合；
    II级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为15-20％和、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为2-4％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为20-30％中的任意一种或至少两种的组合；
    III级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量为20-25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量为4-6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量为30-40％中的任意一种或至少两种的组合；
    IV级：所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因缺失表达量大于25％、所述印记基因Z4、Z5、Z6、Z10和Z13的印记基因拷贝数异常表达量大于6％或所述印记基因Z4、Z5、Z6、Z10和Z13的总表达量大于40％中的任意一种或至少两种的组合；
    所述针对Z8的印记基因缺失表达量、印记基因拷贝数异常表达量和总表达量划分的五个不同的等级为：
    0级：所述印记基因Z8的印记基因缺失表达量小于20％、所述印记基因Z8的印记基因拷贝数异常表达量小于2％或所述印记基因Z8的总表达量小于15％中的任意一种或至少两种的组合；
    I级：所述印记基因Z8的印记基因缺失表达量为20-25％、所述印记基因Z8的印记基因拷贝数异常表达量为2-4％或所述印记基因Z8的总表达量为15-20％中的任意一种或至少两种的组合；
    II级：所述印记基因Z8的印记基因缺失表达量为25-30％、所述印记基因Z8的印记基因拷贝数异常表达量为4-7％或所述印记基因Z8的总表达量为 20-30％中的任意一种或至少两种的组合；
    III级：所述印记基因Z8的印记基因缺失表达量为30-35％、所述印记基因Z8的印记基因拷贝数异常表达量为7-10％或所述印记基因Z8的总表达量为30-40％中的任意一种或至少两种的组合；
    IV级：所述印记基因Z8的印记基因缺失表达量大于35％、所述印记基因Z8的印记基因拷贝数异常表达量大于10％或所述印记基因Z8的总表达量大于40％中的任意一种或至少两种的组合。
  9. 一种用于检测肺肿瘤良恶性程度的装置，采用如权利要求1-8中任一项所述的模型，包括如下单元：
    (1)取样单元：获取待测样本；
    (2)探针设计单元：根据印记基因序列设计特异性引物；
    (3)检测单元：将步骤(2)的探针与待测样本进行原位杂交；
    (4)分析单元：显微镜成像分析印记基因的表达情况；
    其中，所述分析单元通过计算印记基因缺失表达量和印记基因拷贝数异常表达量，通过权利要求1-8中任一项所述的模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来诊断肺肿瘤的良恶性程度。
  10. 一种检测肺肿瘤良恶性程度的方法，采用如权利要求1-8中任一项所述的模型或如权利要求9所述的装置，包括如下步骤：
    (1)获取待测样本；
    (2)根据印记基因序列设计特异性引物；
    (3)将步骤(2)的探针与待测样本进行原位杂交；
    (4)显微镜成像分析印记基因的表达情况，从而诊断肺肿瘤的良恶性程度；
    其中，所述分析单元通过计算印记基因缺失表达量、印记基因拷贝数异常 表达量和总表达量，通过权利要求1-8中任一项所述的模型，从而通过印记基因缺失表达量和印记基因拷贝数异常表达量的等级来诊断肺肿瘤的良恶性程度。
  11. 根据权利要求10所述的方法，其中，步骤(1)所述的待测样本来自于人的组织和/或细胞。
  12. 根据权利要求10或11所述的方法，其中，所述待测样本为组织的石蜡切片、肺穿刺样本、支气管镜活检样本、肺冲洗液细胞涂片或痰液细胞涂片中的任意一种或至少两种的组合。
  13. 根据权利要求10-12中任一项所述的方法，其中，所述原位杂交采用RNAscope原位杂交方法。
  14. 根据权利要求10-13中任一项所述的方法，其中，所述RNAscope原位杂交方法使用单通道或多通道的呈色试剂盒或者单通道或多通道的荧光试剂盒，优选为单通道红色/棕色呈色试剂盒或多通道的荧光试剂盒。
  15. 根据权利要求10-14中任一项所述的方法，其中，所述判断肺肿瘤的良恶性程度分为良性肿瘤、肺癌潜能、早期肺癌、中期肺癌和晚期肺癌。
  16. 根据权利要求10-15中任一项所述的方法，其中，所述诊断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；
    所述诊断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I 级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为肺癌潜能；
    所述诊断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期肺癌；
    所述诊断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期肺癌；
    所述诊断肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、 Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
  17. 一种如权利要求1-8中任一项所述的模型和/或如权利要求9所述的装置用于肺肿瘤检测。
  18. 一种如权利要求1-8中任一项所述的模型和/或如权利要求9所述的装置用于制备治疗肺肿瘤的药物或器械的用途。
  19. 根据权利要求17或18所述的用途，其中，肺肿瘤的良恶性程度分为良性肿瘤、肺癌潜能、早期肺癌、中期肺癌和晚期肺癌。
  20. 根据权利要求17-19中任一项所述的用途，其中，肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的印记基因缺失表达量和印记基因拷贝数异常表达量均小于I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因缺失表达量为I级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为I级，则为良性肿瘤；
    肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为I级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为I级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为II级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为II级中的任意一种情况，则判断为肺 癌潜能；
    肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为II级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为II级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为III级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为III级中的任意一种情况，则为早期肺癌；
    肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的至少2个印记基因的印记基因缺失表达量为III级，印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16的至少2个印记基因的印记基因拷贝数异常表达量为III级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中不超过1个印记基因的印记基因缺失表达量为IV级且印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中的不超过1个印记基因的印记基因拷贝数异常表达量为IV级中的任意一种情况，则为中期肺癌；
    肺肿瘤的良恶性程度的结果为印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因缺失表达量为IV级或印记基因Z1、Z3、Z4、Z5、Z6、Z8、Z10、Z11、Z13和Z16中至少2个印记基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (en) * 1994-03-07 1995-09-14 Rapaport, Erich Sensitive cancer test
WO2007097741A1 (en) * 2006-02-17 2007-08-30 The Trustees Of Columbia University In The City Of New York Method of diagnosing intrauterine growth restriction
WO2016154330A1 (en) * 2015-03-23 2016-09-29 Whitehead Institute For Biomedical Research Reporter of genomic methylation and uses thereof

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE235190T1 (de) * 1996-12-30 2003-04-15 Univ Johns Hopkins Med Bestimmung der krebsanfälligkeit eines organs oder gewebes durch bestimmung seiner zellprägung
US6087164A (en) * 1997-10-03 2000-07-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Methods and compositions for inducing tumor-specific cytotoxicity
DE60117542T2 (de) * 2000-05-22 2006-12-07 The Johns Hopkins University Testverfahren zur bestimmung von gen-imprinting und methylierte cpg inseln
US8150627B2 (en) * 2003-05-15 2012-04-03 Illumina, Inc. Methods and compositions for diagnosing lung cancer with specific DNA methylation patterns
WO2006105642A1 (en) * 2005-04-05 2006-10-12 British Columbia Cancer Agency Biomarkers for the detection of lung cancer and uses thereof
US20070026424A1 (en) * 2005-04-15 2007-02-01 Powell Charles A Gene profiles correlating with histology and prognosis
WO2009105549A2 (en) * 2008-02-19 2009-08-27 Oncomethylome Sciences Sa Detection and prognosis of lung cancer
EP2233590A1 (en) * 2009-01-28 2010-09-29 AIT Austrian Institute of Technology GmbH Methylation assay
CN103146801B (zh) * 2011-12-07 2015-04-22 上海市普陀区中心医院 一种筛选抗肠癌药物的方法
WO2014093872A1 (en) * 2012-12-13 2014-06-19 Baylor Research Institute Blood transcriptional signatures of active pulmonary tuberculosis and sarcoidosis
CN107723352A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种循环肿瘤dna肝癌驱动基因高通量检测方法
WO2018214845A1 (zh) * 2017-05-22 2018-11-29 立森印迹诊断技术有限公司 一种模型和诊断方法及其应用
WO2018219342A1 (zh) * 2017-06-01 2018-12-06 立森印迹诊断技术有限公司 一种印记基因分级模型和诊断方法及其应用
CN107619867A (zh) * 2017-10-18 2018-01-23 广州漫瑞生物信息技术有限公司 用于同时检测肺癌多种基因突变类型的序列组合和探针
WO2019129144A1 (zh) * 2017-12-27 2019-07-04 立森印迹诊断技术有限公司 一种用于检测食道肿瘤和/或胃肿瘤良恶性程度的分级模型及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (en) * 1994-03-07 1995-09-14 Rapaport, Erich Sensitive cancer test
WO2007097741A1 (en) * 2006-02-17 2007-08-30 The Trustees Of Columbia University In The City Of New York Method of diagnosing intrauterine growth restriction
WO2016154330A1 (en) * 2015-03-23 2016-09-29 Whitehead Institute For Biomedical Research Reporter of genomic methylation and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3822369A4 *

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