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WO2018218737A1 - 一种印记基因在膀胱肿瘤中的分级模型及其组成的系统 - Google Patents

一种印记基因在膀胱肿瘤中的分级模型及其组成的系统 Download PDF

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WO2018218737A1
WO2018218737A1 PCT/CN2017/092365 CN2017092365W WO2018218737A1 WO 2018218737 A1 WO2018218737 A1 WO 2018218737A1 CN 2017092365 W CN2017092365 W CN 2017092365W WO 2018218737 A1 WO2018218737 A1 WO 2018218737A1
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imprinted
imprinted gene
genes
expression amount
gene
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成彤
周宁
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Lisen Imprinting Diagnostics Wuxi Co Ltd
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Lisen Imprinting Diagnostics Wuxi Co Ltd
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    • 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
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present application relates to the field of biotechnology, and relates to the field of gene diagnosis, and in particular to a hierarchical model of a imprinted gene in a bladder tumor and a system thereof.
  • Bladder cancer is the most common malignant tumor of the urinary system, and the incidence rate is the first in the urinary system malignant tumor. According to the World Health Organization (WHO), there were 429,793 new cases, 165,084 deaths in 2012, 55,486 new diagnoses in China, and 26,820 deaths (World Cancer Report 2014). Bladder cancer ranks first in the incidence of malignant tumors in Chinese male genitourinary, and ranks eighth in the incidence of malignant tumors in China. The incidence of bladder cancer is increasing year by year. From 1998 to 2008, the incidence of bladder cancer in China increased by 56.69% (analysis of the incidence and trend of bladder cancer in China 2013).
  • WHO World Health Organization
  • the recurrence rate of bladder cancer after the first surgical resection is as high as 75%, and the degree of malignancy of 10-15% of recurrent tumors increases.
  • the 5-year survival rate can reach 78%.
  • the 5-year survival rate of the patient after radical resection of the bladder does not exceed 40%.
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylation Alleles from a particular parent cause one gene to have only one allele to be expressed, while the other is in a state of gene silencing. This type of gene is called a blot (remember) gene. Deletion of the blot is an epigenetic change in which the allelic gene of the imprinted gene results in a silenced allele being activated and beginning to express the gene. Numerous studies have shown that this phenomenon (missing of blots) is ubiquitous in various types of cancer and occurs earlier than cell and tissue morphological changes. At the same time, in healthy cells, the proportion of imprinted deletions is extremely low, in sharp contrast to cancer cells. Therefore, the methylation status of the imprinted gene can be used as a pathological marker to analyze the abnormal state of the cell by a specific molecular detection technique.
  • the current diagnosis of bladder cancer requires a new detection system and detection model, based on the patient biopsy sample, to analyze the molecular marker changes in the bladder cancer at the cellular level, in order to provide more accurate pre-diagnosis and diagnostic information.
  • the present application provides a hierarchical model of a imprinted gene in a bladder tumor and a system thereof, which are used for early visual observation of the bladder at the cell and tissue level.
  • the change of the imprinted (marked) gene of cancer determines the benign and malignant degree of bladder cancer.
  • the present application provides an imprinted gene grading model in a bladder tumor, which model changes the amount of imprinted gene expression, imprinted gene deletion expression, and imprinted gene copy number abnormal expression in bladder cancer.
  • the expression status of the imprinted gene is graded;
  • the imprinted gene is any one or a combination of at least two of T1, T2, T3, T4, T5 or T6, the imprinted gene T1 is Gnas, the imprinted gene T2 is Igf2, and the imprinted gene T3 In the case of Peg10, the imprinted gene T4 is Igf2r, the imprinted gene T5 is Mest, and the imprinted gene T6 is Plagl1.
  • the inventors found that by calculating the imprinted gene deletion expression amount and the abnormal expression amount of the imprinted gene copy number in any bladder tumor of T1-T6, the sensitivity of diagnosis to bladder cancer can reach 69% or more.
  • any one of T1-T6 can be detected, and it is preferable to detect any one of T1-T5, further preferably any one of T1, T2, T3, T4 or T5, and further preferably It is T1, T2, T3 or T5, and most preferably T2.
  • a single T6 imprinted gene can be diagnosed with a sensitivity of 69.2% for bladder cancer, and a T4 imprinted gene can be detected alone.
  • the sensitivity of diagnosis to bladder cancer can reach 84.6%, and a T1 is detected separately.
  • the imprinting gene of any one of T3 or T5 can be diagnosed with a sensitivity of 87.2% for bladder cancer.
  • a T2 imprinted gene can be detected alone, and the sensitivity for diagnosis of bladder cancer can reach 89.7%.
  • the combination may be a combination of T1 and T2, a combination of T1 and T3, a combination of T1 and T4, a combination of T1 and T5, a combination of T1 and T6.
  • a combination of T5 and T6 is preferably a combination of T2 and T3, a combination of T2 and T5.
  • the inventors have found that by calculating the amount of the imprinted gene deletion expression of the two or more imprinted genes and the abnormal expression amount of the imprinted gene copy number, it is possible to further improve the combination of the two imprinted genes of the imprinted gene for bladder cancer.
  • the diagnostic sensitivity can reach more than 80%.
  • the combination of T2 and T3 can be detected.
  • the sensitivity of diagnosis to bladder cancer can reach more than 97.4%.
  • the imprinted gene grading model is most preferably a combination of detection T1-T6 genes.
  • the imprinted gene is deleted, after the cells are subjected to hematoxylin staining, there are two red/brown marks in the nucleus, and the imprinted gene copy number is abnormal after the cells are subjected to hematoxylin staining. There are more than two red/brown markers in the nucleus, which are due to abnormal gene replication of cancer cells, resulting in the expression of this gene as triploid or even higher polyploid.
  • the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the imprinted gene and the imprinted gene are simultaneously a concept, indicating the same meaning, and can be replaced.
  • the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell; and b is a red/brown mark in the nucleus after the hematoxylin staining of the cell, and the imprinting gene exists.
  • the nucleus; the c is a hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; and the d is a hematoxylin staining of the cells, and there are more than two red/brown marks in the nucleus.
  • imprinted gene copy number abnormal cell nucleus is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell
  • b is a red/brown mark in the nucleus after the hematoxylin staining of the cell,
  • the imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount are divided into five different grades, which are imprinted gene deletion expression amount and imprinted gene copy number of six imprinted genes for T1-T6.
  • the abnormal expression levels are divided into five different levels.
  • the imprinted gene deletion expression amount and the imprinted gene copy for T1, T2, T3 and T4 are:
  • the imprinted gene T1, T2, T3 and T4 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted genes T1, T2, T3 and T4 have an imprinted gene copy number abnormal expression amount of less than 0.5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 10-20% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 0.5- 1.5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 20-25% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 1.5- 3%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 25-30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 3- 5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3, and T4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, and T4 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T5 and T6 are:
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 10-16% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 0.5-1.3%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 16-21% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 1.3-2.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 21-30% and/or the imprinted genes T5 and T6 have an imprinted gene copy number abnormal expression amount of 2.5-4%;
  • the imprinted gene deletion expression amount of the imprinted genes T5 and T6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 is greater than 4%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 are independent of each other.
  • the present application provides a system for detecting the degree of benign and malignant bladder tumors, comprising the following units:
  • sampling unit obtaining a sample to be tested
  • Probe design unit design specific primers according to the imprinted gene sequence
  • the analysis unit calculates the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number, and the abnormal expression of the imprinted gene and the imprinted gene copy number are expressed by the model described in the first aspect.
  • the level of the amount to determine the degree of benign and malignant bladder tumors.
  • the imprinted gene is deleted after the cells are subjected to hematoxylin staining, and two red/brown labeled nuclei are present in the nucleus, and the imprinted gene copy number abnormality is that after the cells are subjected to hematoxylin staining, there are two or more nuclei in the cell. Red/brown labeled nuclei, which are due to abnormal genetic replication of cancer cells, resulting in the expression of this gene as a triploid or even higher polyploid.
  • the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the detection system described in the present application is for early and intuitive observation of changes in the imprinted (trace) genes of various types of tumors at the cellular and tissue levels to determine the benign and malignant degree of the tumor, and is an early tumor patient. Provide the most beneficial treatment opportunities.
  • the sample to be tested described in the step (1) is derived from human tissues and/or cells.
  • the sample to be tested is feasible as long as the RNA is processed in a timely manner, and those skilled in the art can select according to the needs, and the sample to be tested includes the paraffin section of the tissue. Any one or a combination of at least two of an endoscopic screening sample, a urine exfoliated cell smear, or a bladder irrigation fluid cell smear.
  • the specific operation procedure of the paraffin section of the tissue is 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 made into a tissue film with a positively charged slide; Only 10 ⁇ m thick, so some of the microscopically seen nuclei are incomplete, so some false negative gene deletions will occur.
  • the specific operation procedure of the endoscope screening sample is to obtain suspicious tissue under the cystoscope, embedded in paraffin, cut into 10 ⁇ m thick, and obtained by using a positively charged slide to form a film.
  • the sampling process is simple, and the cystoscopy biopsy can be positioned compared with the blood circulation characteristics, and the cystoscopy biopsy has its special advantage as an experimental sample.
  • the urine smear cell smear and the bladder lavage fluid cell smear have less damage to the patient, and the sampling process is simple, and has special advantages as an experimental sample.
  • the sample to be tested is any one or a combination of at least two of an endoscopic screening sample, a urine exfoliated cell smear, and/or a bladder irrigation fluid cell smear.
  • the imprinted gene is T1-T6, the imprinted gene T1 is Gnas, the imprinted gene T2 is Igf2, the imprinted gene T3 is Peg10, the imprinted gene T4 is Igf2r, and the imprinted gene T5 is Mest, the imprinted gene T6 is Plagl.
  • the imprinted genes T1 (Gnas), T2 (Igf2), T3 (Peg10), T4 (Igf2r), T5 (Mest), T6 (Plagl1) have different degrees of expression in normal tumor cell tissues, Expression when malignant lesions occur Both the quantity and the imprint status change significantly.
  • the designed probe is designed according to the imprinting genes T1-T6, namely Gnas, Igf2, Peg10, Igf2r, Mest and Plagl, and specifically selects a sequence as a probe in the inner loop of each gene, specifically
  • the selected gene sequence and the location of the specific gene are as follows:
  • T4 Hs-IGF2R: chr6: 160059099-160060546;
  • the in situ hybridization employs an RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single-channel or multi-channel colorimetric kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel red/brown color kit or a multi-channel fluorescent kit. .
  • the multi-channel coloring kit or the multi-channel fluorescent kit comprises two or more channels of coloring kits or fluorescent kits, and the two-channel coloring kit or multi-channel fluorescent reagent
  • the cassette can use two imprinted gene probes or a combination of imprinted genes and other genes to express even multiple imprinted genes and non-imprinted genes.
  • the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy in the model are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell; and b is a red/brown mark in the nucleus after the hematoxylin staining of the cell, and the imprinting gene exists.
  • the nucleus; the c is a hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; and the d is a hematoxylin staining of the cells, and there are more than two red/brown marks in the nucleus.
  • imprinted gene copy number abnormal cell nucleus is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell
  • b is a red/brown mark in the nucleus after the hematoxylin staining of the cell,
  • the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for calculation of imprinted gene expression amount, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount.
  • the probe is amplified by in situ hybridization and Hemotoxy (hematoxylin) nuclear staining, and the presence of imprinted genes, imprinted gene deletions or copy number abnormalities in each nucleus is determined under a 40 ⁇ or 60 ⁇ microscope.
  • the degree of benign and malignant tumors of the sample was determined by calculating the amount of imprinted gene expression, the amount of imprinted gene deletion gene expression, and the gene expression amount of the imprinted gene copy number abnormality. Since 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 partial false negatives.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount are divided into five different grades.
  • the five different grades are respectively divided into an imprinted gene deletion expression amount and an imprinted gene copy number abnormal expression amount for the six imprinted genes of T1-T6.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T1, T2, T3 and T4 are:
  • the imprinted genes of the imprinted genes T1, T2, T3 and T4 are less than 10% and/or Or the imprinted gene T1, T2, T3 and T4 have an abnormal expression amount of the imprinted gene of less than 0.5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 10-20% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 0.5- 1.5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 20-25% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 1.5- 3%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3 and T4 is 25-30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 is 3- 5%;
  • the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3, and T4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, and T4 is greater than 5%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3 and T4 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for T5 and T6 are:
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is less than 0.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 10-16% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 0.5-1.3%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 16-21% and/or the imprinted gene T5 and T6 imprinted gene copy number abnormal expression amount is 1.3-2.5%;
  • the imprinted gene T5 and T6 have an imprinted gene deletion expression amount of 21-30% and/or the imprinted genes T5 and T6 have an imprinted gene copy number abnormal expression amount of 2.5-4%;
  • the imprinted gene deletion expression amount of the imprinted genes T5 and T6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 is greater than 4%.
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T5 and T6 are independent of each other.
  • the judging degree of benign and malignant bladder tumor is divided into benign bladder tumor, bladder cancer potential, early bladder cancer, metaphase bladder cancer and advanced bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are all less than grade I, and the imprinted gene T1
  • the imprinted gene deletion expression of no more than 1 imprinted gene in T2, T3, T4, T5 and T6 is no more than 1 imprinted gene in class I and imprinted genes T1, T2, T3, T4, T5 and T6
  • the imprinted gene copy number abnormal expression level is I or the imprinted genes T1, T2, T3, T4, T5 and T6 of the two imprinted genes have an imprinted gene deletion expression level of I and the imprinted gene copies of the two imprinted genes If the number of abnormal expressions is any of the 0 grades, it is a benign tumor.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression level of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is grade I and the two imprinted genes
  • One of the imprinted gene copy number abnormal expression levels is grade I
  • the imprinted gene deletion expression level of at least three imprinted genes in the imprinted genes T1, T2, T3, T4, T5, and T6 is grade I and the imprinted genes T1, T2.
  • the imprinted gene copy number of at least 2 imprinted genes of T3, T4, T5 and T6 is abnormally expressed in the level I or the imprinted gene of the imprinted genes T1, T2, T3, T4, T5 and T6 is not more than one imprinted gene. Both the amount and the amount of the imprinted gene deletion expression were in any of the second grades, and it was judged to be the bladder cancer potential.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both
  • the level of imprinted gene deletion and the amount of imprinted gene deletion in the class II or imprinted genes T1, T2, T3, T4, T5 and T6 are both grade III, which is early bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumor is imprinted genes T1, T2, T3,
  • the imprinted gene deletion expression level and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in T4, T5 and T6 are all grade III or no more than one imprinted gene in the imprinted genes T1, T2, T3, T4, T5 and T6.
  • the imprinted gene deletion expression level and the imprinted gene deletion expression level are both grade IV, which is a metaphase bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both Grade IV is advanced bladder cancer.
  • the present application provides a model of the first aspect or the system of the second aspect, for use in the manufacture of a medicament for the detection and/or treatment of bladder cancer.
  • the determining the degree of benign and malignant bladder tumors is classified into benign, bladder cancer potential, early bladder cancer, metaphase bladder cancer, and advanced bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are all less than grade I, and the imprinted gene T1
  • the imprinted gene deletion expression of no more than 1 imprinted gene in T2, T3, T4, T5 and T6 is no more than 1 imprinted gene in class I and imprinted genes T1, T2, T3, T4, T5 and T6
  • the imprinted gene copy number abnormal expression level is I or the imprinted genes T1, T2, T3, T4, T5 and T6 of the two imprinted genes have an imprinted gene deletion expression level of I and the imprinted gene copies of the two imprinted genes If the number of abnormal expressions is any of the 0 grades, it is a benign tumor.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression level of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is grade I and the two imprinted genes One of the imprinted gene copy number abnormal expression levels is grade I, and the imprinted gene deletion expression level of at least three imprinted genes in the imprinted genes T1, T2, T3, T4, T5, and T6 is grade I and the imprinted genes T1, T2.
  • the imprinted gene copy number of at least 2 imprinted genes of T3, T4, T5 and T6 is abnormally expressed in the level I or the imprinted gene deletion table of no more than 1 imprinted gene in the imprinted genes T1, T2, T3, T4, T5 and T6 If both the amount of expression and the amount of deletion of the imprinted gene are in the second grade, it is judged to be the potential of bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both
  • the level of imprinted gene deletion and the amount of imprinted gene deletion in the class II or imprinted genes T1, T2, T3, T4, T5 and T6 are both grade III, which is early bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of no more than one imprinted gene of the class III or imprinted genes T1, T2, T3, T4, T5 and T6 are all grade IV, and are metaphase bladder cancer.
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both Grade IV is advanced bladder cancer.
  • the detection model and system described in the present application express the performance of the imprinted defect in the sample of the bladder tumor patient in an intuitive manner, and are objectively, intuitively, early, and accurately detected by the method of in situ labeling of the imprinted gene.
  • Imprinted (trace) gene changes and can provide quantitative models that make a significant contribution to the diagnosis of bladder tumors;
  • the detection system of the present application can determine the degree of benign and malignant bladder tumor before surgery of bladder tumor patients, thereby providing a basis for surgery and precise treatment, which is a revolutionary breakthrough in the diagnosis of bladder tumors in the field of cell molecules;
  • This application can accurately determine the type of bladder tumor.
  • T2 probe alone the sensitivity of detection of samples above the malignant potential level is 89.7%; if combined detection of T2 and T5, T2 and T3 probes, The sensitivity of sample detection above the level of malignant potential can be increased to 97.4%; if combined detection
  • the T1-T6 gene greatly improves the early diagnosis and early diagnosis of bladder cancer, especially in the early screening and postoperative follow-up of cancer, especially for the follow-up of Achilles tendon in patients with suspected recurrence, which can win time and save lives for patients. Make a significant contribution;
  • the detection method of the present application is different from the immunohistochemical method, which reduces false positives and other negative effects, and not only that, through the discovery of the bladder tumor-associated imprinted gene deletion site, the targeting of the gene silencing, rejection, rearrangement A drug or technical method that can be used to guide later treatment and medication.
  • 1 is a pathological section of thyroid cancer in which a hematoxylin-stained nuclei according to an embodiment of the present invention, wherein the a is a cell nucleus in which no stain is present in the nucleus after the cells are subjected to hematoxylin staining, and the b is a cell.
  • the c is the hematoxylin staining of the cell, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted;
  • the cells with hematoxylin there are more than two red/brown markers in the nucleus, and the nuclei with abnormal copy number of the imprinted gene are printed;
  • Fig. 2 shows the expression status of six genes in the pathological sections of bladder cancer with different malignant degrees in the embodiment of the present invention, wherein Fig. 2(a) shows the expression status of six genes in the pathological section of grade 0 bladder cancer, Fig. 2 (Fig. 2 b) is the expression status of 6 genes in the pathological section of grade I bladder cancer, Figure 2 (c) is the expression status of 6 genes in the pathological section of grade II bladder cancer, and Figure 2 (d) is the grade III bladder cancer.
  • the expression status of six genes in the pathological section Fig. 2(e) shows the expression status of six genes in the pathological section of grade IV bladder cancer;
  • FIG. 3 is a response sensitivity of six genes to bladder cancer according to an embodiment of the present invention, wherein FIG. 3(a) shows the intensity of deletion of 6 genes for bladder cancer, and FIG. 3(b) shows 6 genes for bladder.
  • the intensity of the copy number abnormality of the cancer wherein the LOI is the expression level of the imprinted gene deletion gene, and the CNV is the gene expression amount of the imprinted gene copy number abnormality;
  • FIG. 4 is a response sensitivity of six genes to bladder cancer according to an embodiment of the present invention, wherein FIG. 4(a) is The intensity of T1 imprinting deletion and copy number abnormality of imprinted gene, Fig. 4(b) is the intensity of T2 imprinting deletion and copy number abnormality of imprinted gene, and Fig. 4(c) is the intensity of imprinting gene T3 imprint deletion and copy number abnormality, Fig. 4 (d) The intensity of T4 imprinting deletion and copy number abnormality of imprinted gene, Fig. 4(e) is the intensity of T5 imprinting deletion and copy number abnormality of imprinted gene, and Fig. 4(f) is the imprinting gene T6 imprint deletion and copy number abnormality. Intensity, LOI is the expression level of the imprinted gene deletion gene, and CNV is the gene expression amount of the imprinted gene copy number abnormality;
  • FIG. 5 is a distribution range and grading standard of imprinting deletion and copy number abnormality in 44 pathological sections of bladder cancer according to an embodiment of the present invention, wherein FIG. 5(a) is an imprinted gene T1 applied to 44 cases of bladder.
  • FIG. 5(b) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 44 pathological sections of bladder cancer with imprinting gene T2.
  • Fig. 5(c) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 44 pathological sections of bladder cancer
  • Fig. 5(d) shows that imprinted gene T4 is applied to 44 cases of bladder cancer pathological sections.
  • Fig. 5(e) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 44 pathological sections of bladder cancer with imprinting gene T5
  • Fig. 5(f) The imprinting gene T6 was applied to the pathological sections of 44 cases of bladder cancer.
  • the distribution range and grading standard of imprinting deletion and copy number abnormality LOI is the imprinting gene deletion gene expression level
  • CNV is the base of imprinting gene copy number abnormality. Due to the amount of expression.
  • the method for detecting the imprinted gene comprises the following steps:
  • Design probe design specific primers according to the imprinted gene sequence
  • the designed probe is designed according to the imprinting genes T1 (Gnas), T2 (Igf2), T3 (Peg10), T4 (Igf2r), T5 (Mest) and T6 (Plagl1), specifically in the inner loop of each gene. Select a sequence as a probe, and select the specific gene sequence and the location of the specific gene as follows:
  • T4 Hs-IGF2R: chr6: 160059099-160060546;
  • the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy in the model are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a, b, c, and d are as shown in FIG. 1 , wherein a is a cell nucleus in which no hemoglobin is stained in the nucleus and the imprinted gene is not expressed; and b is a hematoxylin staining of the cell.
  • the c is the hematoxylin staining of the cell, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted;
  • the d is the hematoxylin After staining, there are more than two red/brown markers in the nucleus, and the nuclei with abnormal copy number of the imprinted gene are imprinted.
  • the cystoscopic biopsy sample was obtained by taking a suspicious lesion under a cystoscope, fixed in a 10% neutral formalin solution for 24 hours, embedded in paraffin (FFPE), and cut into 10 micron thick sections. Other detection methods are the same as in the examples. 1.
  • each imprinted gene to bladder cancer is shown in Fig. 4(a)-Fig. 4(f).
  • the deletion of the imprinted gene T2 and the abnormal copy number appear in the earliest stage of bladder cancer, and are most obvious in the malignant potential stage, but In the late stage of bladder cancer, T2 imprinting deletion and copy number abnormality no longer increased significantly; imprinted gene T5 showed obvious imprinting deletion and copy number abnormality in the early stage of bladder cancer lesion, and T5 imprinted deletion in late case
  • the increase in copy number abnormality was limited; the imprinting deletion of the imprinted gene T3 began to increase in the early stage of bladder cancer, and the imprinted deletion and copy number abnormalities were significantly increased in the intermediate cases, but slightly decreased in the advanced cases; the imprinted gene T4 was missing.
  • Tissues of 44 patients with bladder cancer including cystoscopy biopsy samples (10 micron), were obtained in the same manner as in Example 1.
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression of imprinted gene copy number is 3-5% to grade III, the imprinted gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed by more than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II, Imprinted gene deletion expression level is 25-30% and/or imprinted gene copy number abnormal expression level is 3-5% to grade III, imprinted gene deficiency The expression loss is greater than 30% and/or the abnormal expression level of the imprinted gene copy number is greater than 5% is IV;
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II,
  • the amount of imprinted gene deletion is 25-30% and/or the abnormal expression of imprinted gene copy number is 3-5% to grade III, the imprinted gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed by more than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression of imprinted gene copy number is 3-5% to grade III, the imprinted gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed by more than 5%. Grade IV;
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 16% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 16-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II,
  • the amount of imprinted gene deletion is 21-30% and/or the abnormal expression level of imprinted gene copy number is 2.5-4%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 4%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 16% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 16-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, Imprinted gene deletion The expression level is 21-30% and/or the abnormal expression level of the imprinted gene copy number is 2.5-4% for the grade III, the imprinted gene deletion expression amount is greater than 30% and/or the imprinted gene copy number abnormal expression amount is greater than 4% for the grade IV. .
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are all less than grade I, and the imprinted genes T1, T2.
  • Imprinted gene deletion of no more than 1 imprinted gene in T3, T4, T5 and T6 is a copy of imprinted gene of class I and no more than 1 imprinted gene in imprinted genes T1, T2, T3, T4, T5 and T6
  • the number of abnormal gene expression levels of the imprinted genes of class I or imprinted genes T1, T2, T3, T4, T5 and T6 is I level and the imprinted gene copy number of the two imprinted genes is abnormally expressed. If the amount is in any of the 0 grades, it is a benign tumor;
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount of the two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is I level and one of the two imprinted genes
  • the abnormal expression level of the imprinted gene copy number is grade I
  • the imprinted gene deletion expression level of at least 3 imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 is grade I and the imprinted genes T1, T2, T3, T4
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of T5 and T6 is the imprinted gene deletion expression amount and imprint of no more than one imprinted gene of class I or imprinted genes T1, T2, T3, T4, T5 and T6 If the gene deletion expression level is any of the second grade, it is judged to be bladder cancer potential;
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both grade II or The imprinted gene deletion expression amount and the imprinted gene deletion expression amount of the imprinted genes T1, T2, T3, T4, T5 and T6 are both grade III, which is early bladder cancer;
  • the result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are
  • the imprinted gene deletion expression amount and the imprinted gene deletion expression amount of each of the class III or imprinted genes T1, T2, T3, T4, T5 and T6 are all grade IV, which is a metaphase bladder cancer;
  • the result of determining the degree of benign and malignant bladder tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of at least two imprinted genes in the imprinted genes T1, T2, T3, T4, T5 and T6 are both IV. It is advanced bladder cancer.
  • the detection model and system described in the present application express the impression of the imprinted defect on the sample of the bladder tumor patient in an intuitive manner, and the method of in situ labeling of the imprinted gene is objective, intuitive, early, and accurate. Changes in the imprinted (trace) gene are detected and quantitative models can be provided to make a significant contribution to the diagnosis of bladder tumors.

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Abstract

本申请公开了一种印记基因在膀胱肿瘤中的分级模型及其组成的系统，所述模型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对印记基因在肿瘤中的变化进行分级。

Description

一种印记基因在膀胱肿瘤中的分级模型及其组成的系统 技术领域
本申请涉及生物技术领域，涉及基因诊断领域，具体涉及一种印记基因在膀胱肿瘤中的分级模型及其组成的系统。
背景技术
膀胱癌是泌尿系统最常见的恶性肿瘤，发病率居泌尿系统恶性肿瘤的首位。据世界卫生组织(WHO)统计，2012年全球新增病例429,793例，死亡165,084例，中国新诊断55,486例，死亡26,820例(World Cancer Report 2014)。膀胱癌居中国男性泌尿生殖系恶性肿瘤发病率第一位，居中国恶性肿瘤发病率第八位。膀胱癌发病率呈逐年上升态势，1998至2008年间，中国膀胱癌发病率增长56.69％(中国膀胱癌发病率现状及流行趋势分析2013)。膀胱癌在第一次手术切除后复发率高达75％，并且10-15％的复发肿瘤恶性程度增加。膀胱癌早期，在经尿道膀胱移行细胞癌电切术治疗后，5年存活率可达78％。而膀胱癌晚期，病人接受膀胱根治性切除后，其5年存活率不超过40％。以上数据表明，膀胱癌存在复发率高，早期治疗效果好的特点。
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，从而做出膀胱肿瘤良恶性判断。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膀胱癌症的检测。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。
基于上述原因，目前的膀胱癌诊断需要新的检测系统和检测模型，基于患者活检样本，解析膀胱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。
发明内容
针对现有技术的不足及实际的需求，本申请提供了一种印记基因在膀胱肿瘤中的分级模型及其组成的系统，该检测系统和模型是用于细胞和组织水平下早期直观地观察膀胱癌的印记(迹)基因的变化从而判断膀胱癌的良恶性及恶性程度。
为达到上述目的，本申请采用以下技术方案：
第一方面，本申请提供了一种在膀胱肿瘤中的印记基因分级模型，所述模型通过计算印记基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在膀胱癌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T1、T2、T3、T4、T5或T6中的任意一个或至少两个的组合，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
本申请中，发明人发现通过计算T1-T6中任意一个印记基因在膀胱肿瘤中的印记基因缺失表达量和印记基因拷贝数异常表达量，对膀胱癌的诊断敏感度可以达到69％以上。
根据本申请，若只检测一个印记基因，可以检测T1-T6中的任意一个，优选检测T1-T5中的任意一个，进一步优选为T1、T2、T3、T4或T5中的任意一个，再优选为T1、T2、T3或T5中的任意一个，最优选为T2。
本申请中，发明人发现，单独检测一个T6印记基因，对膀胱癌的诊断敏感度可以达到69.2％，单独检测一个T4印记基因，对膀胱癌的诊断敏感度可以达到84.6％，单独检测一个T1、T3或T5中的任意一个印记基因，对膀胱癌的诊断敏感度可以达到87.2％，单独检测一个T2印记基因，对膀胱癌的诊断敏感度可以达到89.7％。
根据本申请，若检测印记基因的两个印记基因的组合，所述组合可以是T1和T2的组合，T1和T3的组合，T1和T4的组合，T1和T5的组合，T1和T6的组合，T2和T3的组合，T2和T4的组合，T2和T5的组合，T2和T6的组合，T3和T4的组合，T3和T5的组合，T3和T6的组合，T4和T5的组合，T4和T6的组合，T5和T6的组合，优选为T2和T3的组合，T2和T5的组合。
本申请中，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和印记基因拷贝数异常表达量可以进一步提高，检测印记基因的两个印记基因的组合，对膀胱癌的诊断敏感度可以达到80％以上，检测T2和T3的组合，T2和T5的组合时，对膀胱癌的诊断敏感度可以达到97.4％以上。
根据本申请，所述印记基因分级模型最优选为检测T1-T6基因的组合。
本申请中，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。
本申请中，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迹基因表达量、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。
本申请中，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。
优选地，所述计算印记基因表达量、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。
优选地，所述印记基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级为针对T1-T6的六个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分的五个不同的等级。
优选地，所述针对T1、T2、T3和T4的印记基因缺失表达量和印记基因拷 贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量小于10％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为10-20％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为1.5-3％；
III级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为25-30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为3-5％；
IV级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量大于30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因T1、T2、T3和T4的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，所述针对T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T5和T6的印记基因缺失表达量为10-16％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.3％；
II级：所述印记基因T5和T6的印记基因缺失表达量为16-21％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.3-2.5％；
III级：所述印记基因T5和T6的印记基因缺失表达量为21-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-4％；
IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于4％。
本申请中，所述印记基因T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
第二方面，本申请提供了一种用于检测膀胱肿瘤良恶性程度的系统，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状态；
其中，所述分析单元通过计算印记基因表达量、印记基因缺失表达量和印记基因拷贝数异常表达量，通过第一方面所述的模型，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判断膀胱肿瘤的良恶性程度。
本申请中，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。
本申请中，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迹基因表达量、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。
本申请所述检测系统是用于细胞和组织水平下早期直观地观察各类型肿瘤的印记(迹)基因的变化从而判断肿瘤的良恶性及恶性程度，为早期肿瘤患者 提供最有利的治疗机会。
根据本申请，步骤(1)所述的待测样本来自于人的组织和/或细胞。
本申请中，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，本申请所述待测样本包括组织的石蜡切片、内窥镜筛查样本、尿液脱落细胞涂片或膀胱冲洗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。
所述内窥镜筛查样本具体操作步骤为在膀胱镜下获取可疑组织，石蜡包埋，切成10μm厚，用带正电荷的玻片制成片子即得。
本申请中，由于膀胱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，膀胱镜活检还能定位，膀胱镜活检作为实验样本有其特殊的优势。
本申请中，尿液脱落细胞涂片和膀胱冲洗液细胞涂片对病人伤害小，取样过程简单，作为实验样本有其特殊的优势。
优选地，所述待测样本为内窥镜筛查样本、尿液脱落细胞涂片和/或膀胱冲洗液细胞涂片中的任意一种或至少两种的组合。
优选地，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
本申请中，所述印记基因T1(Gnas)，T2(Igf2)，T3(Peg10)，T4(Igf2r)，T5(Mest)，T6(Plagl1)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。
本申请中，所述设计探针是根据印记基因T1-T6，即Gnas，Igf2，Peg10，Igf2r，Mest和Plagl1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，具体选择的基因序列和具体基因的位置如下：
T1(Hs-GNAS)：chr6：143968979-143985134；
T2(Hs-IGF2)：chr11：2133666-2135366；
T3(Hs-PEG10)：chr7：94656592-94663333；
T4(Hs-IGF2R)：chr6：160059099-160060546；
T5(Hs-Mest)：chr7：130492340-130495367；
T6(Hs-PLAGL1)：chr6：143968979-143985134。
优选地，所述原位杂交采用RNAscope原位杂交方法。
优选地，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。
本申请中，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。
根据本申请，所述模型中的计算印记基因表达量、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。
本申请中，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迹基因表达量、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。
本申请中，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。
优选地，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级。
优选地，所述五个不同的等级为针对T1-T6的六个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分。
优选地，所述针对T1、T2、T3和T4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量小于10％和/ 或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为10-20％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为0.5-1.5％；
II级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为1.5-3％；
III级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为25-30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为3-5％；
IV级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量大于30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量大于5％。
本申请中，所述印记基因T1、T2、T3和T4的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，所述针对T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
0级：所述印记基因T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
I级：所述印记基因T5和T6的印记基因缺失表达量为10-16％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.3％；
II级：所述印记基因T5和T6的印记基因缺失表达量为16-21％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.3-2.5％；
III级：所述印记基因T5和T6的印记基因缺失表达量为21-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-4％；
IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于4％。
本申请中，所述印记基因T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量是相互独立的。
优选地，所述判断膀胱肿瘤的良恶性程度分为良性膀胱肿瘤、膀胱癌潜能、早期膀胱癌、中期膀胱癌和晚期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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，印记基因T1、T2、T3、T4、T5和T6中的不超过1个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因T1、T2、T3、T4、T5和T6中的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为膀胱癌潜能。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为II级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、 T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为III级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为IV级，则为晚期膀胱癌。
第三方面，本申请提供一种如第一方面所述的模型或如第二方面所述的系统用于制备膀胱癌检测和/或治疗的药物。
优选地，所述判断膀胱肿瘤的良恶性程度分为良性、膀胱癌潜能、早期膀胱癌、中期膀胱癌和晚期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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，印记基因T1、T2、T3、T4、T5和T6中的不超过1个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因T1、T2、T3、T4、T5和T6中的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表 达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为膀胱癌潜能。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为II级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为III级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期膀胱癌。
优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为IV级，则为晚期膀胱癌。
与现有技术相比，本申请具有如下有益效果：
(1)本申请所述检测模型和系统，以直观的方法表现了印记缺失在膀胱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，为膀胱肿瘤的诊断做出巨大贡献；
(2)本申请检测方系统，可以在膀胱肿瘤病人手术前得出膀胱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膀胱肿瘤的革命性突破；
(3)本申请可以精确的判断膀胱肿瘤的类型，单独检测T2探针时，对恶性潜能以上等级的样本检测敏感度为89.7％；若联合检测T2和T5、T2和T3探针时，对恶性潜能以上等级的样本检测敏感度可以增加到97.4％；若联合检测 T1-T6基因，极大地提高了对膀胱癌的早期，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踵随访，可以争取时间，为挽救病人生命做出重大贡献；
(4)本申请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，通过发现的膀胱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。
附图说明
图1是本发明实施例的苏木素染色细胞核的甲状腺癌的病理切片，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；
图2本发明实施例的6个基因在膀胱癌不同恶性程度的病理切片中的表达状态，其中，图2(a)为0级膀胱癌的病理切片中6个基因的表达状态，图2(b)为I级膀胱癌的病理切片中6个基因的表达状态，图2(c)为II级膀胱癌的病理切片中6个基因的表达状态，图2(d)为III级膀胱癌的病理切片中6个基因的表达状态，图2(e)为IV级膀胱癌的病理切片中6个基因的表达状态；
图3为本发明实施例的6个基因对膀胱癌的反应敏感性，其中，图3(a)为6个基因对膀胱癌的印记缺失的强度，图3(b)为6个基因对膀胱癌的拷贝数异常的强度，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；
图4为本发明实施例的6个基因对膀胱癌的反应敏感性，其中图4(a)为 印记基因T1印记缺失和拷贝数异常的强度，图4(b)为印记基因T2印记缺失和拷贝数异常的强度，图4(c)为印记基因T3印记缺失和拷贝数异常的强度，图4(d)为印记基因T4印记缺失和拷贝数异常的强度，图4(e)为印记基因T5印记缺失和拷贝数异常的强度，图4(f)为印记基因T6印记缺失和拷贝数异常的强度，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；
图5为本发明实施例的6个基因应用于44例膀胱癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，其中，图5(a)为印记基因T1应用于44例膀胱癌病理切片中，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T2应用于44例膀胱癌病理切片中，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T3应用于44例膀胱癌病理切片中，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T4应用于44例膀胱癌病理切片中，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T5应用于44例膀胱癌病理切片中，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T6应用于44例膀胱癌病理切片中，印记缺失和拷贝数异常的分布范围和分级标准，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。
具体实施方式
为更进一步阐述本申请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申请的技术方案，但本申请并非局限在实施例范围内。
实施例1 膀胱癌的印记基因分析
所述的印记基因的检测方法，包括如下步骤：
(1)获取膀胱癌的组织细胞，切成10微米厚，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T1(Gnas)、T2(Igf2)、T3(Peg10)、T4(Igf2r)、T5(Mest)和T6(Plagl1)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，具体选择的基因序列和具体基因的位置如下：
T1(Hs-GNAS)：chr6：143968979-143985134；
T2(Hs-IGF2)：chr11：2133666-2135366；
T3(Hs-PEG10)：chr7：94656592-94663333；
T4(Hs-IGF2R)：chr6：160059099-160060546；
T5(Hs-Mest)：chr7：130492340-130495367；
T6(Hs-PLAGL1)：chr6：143968979-143985134。
(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状态；
所述模型中的计算印记基因表达量、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
所述膀胱镜活检样本是，在膀胱镜下取出可疑病变组织，10％中性福尔马林溶液固定24h，石蜡包埋(FFPE)，切成10微米厚的切片，其他检测方法同实施例1。
从图3(a)-图3(b)可以看出，T1，T2，T3，T4，T5，T6每个基因对膀胱癌的反应敏感性或者说对应于膀胱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膀胱癌的敏感度如图4(a)-图4(f)，印记基因T2的缺失和拷贝数异常出现在膀胱癌病变的最早期，在恶性潜能阶段最为明显，但是，在膀胱癌晚期的病例，T2的印记缺失和拷贝数异常不再明显增加；印记基因T5在膀胱癌病变的早期即出现明显的印记缺失和拷贝数异常，在晚期病例中T5的印记缺失和拷贝数异常增加幅度有限；印记基因T3的印记缺失现象在膀胱癌早期开始上升，在中期病例中印记缺失和拷贝数异常都显著增加，但在晚期病例中有轻微下降；印记基因T4的印记缺失和拷贝数异常在恶性潜能阶段比较明显，在中晚期病例中印记缺失增加较为迅速，拷贝数异常增加较为缓慢； 印记基因T1在膀胱癌早期病变中印记缺失现象开始出现，但拷贝数异常比例较少，随癌症恶性程度的增高，印记缺失和拷贝数异常都有显著增加；印记基因T6在膀胱癌早期病变中较不敏感，随癌症恶性程度的增高，中晚期癌症中印记缺失和拷贝数异常都有显著增加。
实施例3
获取44例膀胱癌病人的组织包括膀胱镜活检样本(10微米)，检测方法同实施例1。
从图5(a)-图5(f)可以看出，44例膀胱肿瘤组织样本中6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和拷贝数异常分别从低到高分成5个等级。
具体等级分型如下：
从图5(a)可以看出，对于所述印记基因T1，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-20％和/或印记基因拷贝数异常表达量为0.5-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(b)可以看出，对于所述印记基因T2，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-20％和/或印记基因拷贝数异常表达量为0.5-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺 失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(c)可以看出，对于所述印记基因T3，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-20％和/或印记基因拷贝数异常表达量为0.5-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(d)可以看出，对于所述印记基因T4，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-20％和/或印记基因拷贝数异常表达量为0.5-1.5％为I级，印记基因缺失表达量为20-25％和/或印记基因拷贝数异常表达量为1.5-3％为II级，印记基因缺失表达量为25-30％和/或印记基因拷贝数异常表达量为3-5％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于5％为IV级；
从图5(e)可以看出，对于所述印记基因T5，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-16％和/或印记基因拷贝数异常表达量为0.5-1.3％为I级，印记基因缺失表达量为16-21％和/或印记基因拷贝数异常表达量为1.3-2.5％为II级，印记基因缺失表达量为21-30％和/或印记基因拷贝数异常表达量为2.5-4％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于4％为IV级；
从图5(f)可以看出，对于所述印记基因T6，印记基因缺失表达量小于10％和/或印记基因拷贝数异常表达量小于0.5％为0级，印记基因缺失表达量为10-16％和/或印记基因拷贝数异常表达量为0.5-1.3％为I级，印记基因缺失表达量为16-21％和/或印记基因拷贝数异常表达量为1.3-2.5％为II级，印记基因缺失 表达量为21-30％和/或印记基因拷贝数异常表达量为2.5-4％为III级，印记基因缺失表达量大于30％和/或印记基因拷贝数异常表达量大于4％为IV级。
从这44个膀胱癌肿瘤的样本综合分析可以得出：
所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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，印记基因T1、T2、T3、T4、T5和T6中的不超过1个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因T1、T2、T3、T4、T5和T6中的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为膀胱癌潜能；
所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为II级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期膀胱癌；
所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量 均为III级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期膀胱癌；
所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为IV级，则为晚期膀胱癌。
综上所述，本申请所述检测模型和系统，以直观的方法表现了印记缺失在膀胱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，为膀胱肿瘤的诊断做出巨大贡献。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请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，对本申请的任何改进，对本申请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，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种在膀胱肿瘤中的印记基因分级模型，其通过计算印记基因的表达量、印记基因缺失表达量和印记基因拷贝数异常表达量在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T1、T2、T3、T4、T5或T6中的任意一个或至少两个的组合，所述印记基因T1为Gnas，所述印记基因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1。
  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T1-T5中的任意一个印记基因。
  3. 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T1-T6中的任意两个印记基因的组合。
  4. 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T1-T6的六个印记基因的组合。
  5. 根据权利要求2所述的模型，其中，所述模型计算印记基因的方法如下：计算T1、T2、T3或T5中的任意一个印记基因，优选地，计算印记基因T2。
  6. 根据权利要求3所述的模型，其中，所述模型计算印记基因的方法如下：计算印记基因T2和T3的组合，或者计算印记基因T2和T5的组合。
  7. 根据权利要求1-6中任一项所述的模型，其中，所述计算印记基因的表达量、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。
  8. 根据权利要求1-7中任一项所述的模型，其中，所述印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级为针对T1-T6的六个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分的五个不同的等级；
    优选地，所述针对T1、T2、T3和T4的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量小于10％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为10-20％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为1.5-3％；
    III级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为25-30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为3-5％；
    IV级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量大于30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量大于5％。
    优选地，所述针对T5和T6的印记基因缺失表达量和印记基因拷贝数异常 表达量划分的五个不同的等级为：
    0级：所述印记基因T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T5和T6的印记基因缺失表达量为10-16％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.3％；
    II级：所述印记基因T5和T6的印记基因缺失表达量为16-21％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.3-2.5％；
    III级：所述印记基因T5和T6的印记基因缺失表达量为21-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-4％；
    IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于4％。
  9. 一种用于检测膀胱肿瘤良恶性程度的系统，其包括如下单元：
    (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状态；
    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判断膀胱肿瘤的良恶性程度。
  10. 根据权利要求9所述的系统，其中，步骤(1)所述的待测样本为内窥镜筛查样本、尿液脱落细胞涂片或膀胱冲洗液细胞涂片中的任意一种或至少两种的组合；
    优选地，所述印记基因为T1-T6，所述印记基因T1为Gnas，所述印记基 因T2为Igf2，所述印记基因T3为Peg10，所述印记基因T4为Igf2r，所述印记基因T5为Mest，所述印记基因T6为Plagl1；
    优选地，所述原位杂交采用RNAscope原位杂交方法；
    优选地，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。
  11. 根据权利要求9或10所述的系统，其中，所述模型中的计算印记基因的表达量、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。
  12. 根据权利要求9-11中任一项所述的系统，其中，所述印记基因的表达量、印记基因缺失表达量和印记基因拷贝数异常表达量分成五个不同的等级；
    优选地，所述五个不同的等级为针对T1-T6的六个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量分别进行划分；
    优选地，所述针对T1、T2、T3和T4的印记基因缺失表达量和印记基因拷 贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量小于10％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为10-20％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为0.5-1.5％；
    II级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为20-25％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为1.5-3％；
    III级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量为25-30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量为3-5％；
    IV级：所述印记基因T1、T2、T3和T4的印记基因缺失表达量大于30％和/或所述印记基因T1、T2、T3和T4的印记基因拷贝数异常表达量大于5％；
    优选地，所述针对T5和T6的印记基因缺失表达量和印记基因拷贝数异常表达量划分的五个不同的等级为：
    0级：所述印记基因T5和T6的印记基因缺失表达量小于10％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量小于0.5％；
    I级：所述印记基因T5和T6的印记基因缺失表达量为10-16％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为0.5-1.3％；
    II级：所述印记基因T5和T6的印记基因缺失表达量为16-21％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为1.3-2.5％；
    III级：所述印记基因T5和T6的印记基因缺失表达量为21-30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量为2.5-4％；
    IV级：所述印记基因T5和T6的印记基因缺失表达量大于30％和/或所述印记基因T5和T6的印记基因拷贝数异常表达量大于4％。
  13. 根据权利要求9-12中任一项所述的系统，其中，所述判断膀胱肿瘤的良恶性程度分为良性、膀胱癌潜能、早期膀胱癌、中期膀胱癌和晚期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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，印记基因T1、T2、T3、T4、T5和T6中的不超过1个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因T1、T2、T3、T4、T5和T6中的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为膀胱癌潜能；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为II级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异 常表达量均为III级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为IV级，则为晚期膀胱癌。
  14. 一种如权利要求1-8中任一项所述的模型或如权利要求9-13中任一项所述的系统在制备膀胱肿瘤检测和/或治疗的药物中的用途。
  15. 根据权利要求14所述的用途，其中，所述膀胱肿瘤检测为判断膀胱肿瘤的良恶性程度，所述膀胱肿瘤的良恶性程度分为良性、膀胱癌潜能、早期膀胱癌、中期膀胱癌和晚期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6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，印记基因T1、T2、T3、T4、T5和T6中的不超过1个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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级或印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因的印记基因拷贝数异常表达量均为0级中的任意一种情况，则为良性肿瘤；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中的2个印记基因的印记基因缺失表达量为I级且所述2个印记基因之一的印记基因拷贝数异常表达量为I级，印记基因T1、T2、T3、T4、T5和T6中的至少3个印记基因的印记基因缺失表达量为I级和印记基因T1、T2、T3、T4、T5和T6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T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为II级中的任意一种情况，则判断为膀胱癌潜能；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为II级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为III级，则为早期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为III级或印记基因T1、T2、T3、T4、T5和T6中不超过1个印记基因的印记基因缺失表达量和印记基因缺失表达量均为IV级，则为中期膀胱癌；
    优选地，所述判断膀胱肿瘤的良恶性程度的结果为印记基因T1、T2、T3、T4、T5和T6中至少2个印记基因的印记基因缺失表达量和印记基因拷贝数异常表达量均为IV级，则为晚期膀胱癌。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103890587A (zh) * 2011-08-31 2014-06-25 昂科赛特公司 用于治疗和诊断癌症的方法和组合物
CN105229169A (zh) * 2013-01-31 2016-01-06 飞纳生物技术单人有限责任公司 用于诊断膀胱癌的非侵入性诊断方法
CN105378110A (zh) * 2013-04-17 2016-03-02 生命技术公司 与癌症相关的基因融合体和基因变异体

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL108879A (en) * 1994-03-07 2000-08-31 Yissum Res Dev Co Diagnostic assay for malignancies using the H19 gene and kit
WO2004076690A2 (de) * 2003-02-19 2004-09-10 Universit�T Duisburg-Essen Verwendung einer genveränderung im humanen gnas-gen zur vorhersage von erkrankungsrisiken, krankheitsverläufen und zur vorhersage des ansprechens auf krankheitstherapien
ES2304306B1 (es) * 2007-03-20 2009-07-07 Indas Biotech, S.L.U. Metodo de diagnostico y/o pronostico de cancer vesical.
CA2696545C (en) * 2010-03-15 2019-08-06 Queen's University At Kingston Methods, probe sets, and kits for detection of deletion of tumor suppressor genes by fluorescence in situ hybridization
EP2873740A1 (en) * 2013-11-19 2015-05-20 Oncodiag Methods for the surveillance, diagnosis and screening of bladder cancer
US10023922B2 (en) * 2015-03-23 2018-07-17 Whitehead Institute For Biomedical Research Reporter of genomic methylation and uses thereof
WO2019041689A1 (zh) * 2017-08-31 2019-03-07 立森印迹诊断技术（无锡）有限公司 用于结直肠肿瘤的印记基因分级模型及其组成的系统

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103890587A (zh) * 2011-08-31 2014-06-25 昂科赛特公司 用于治疗和诊断癌症的方法和组合物
CN105229169A (zh) * 2013-01-31 2016-01-06 飞纳生物技术单人有限责任公司 用于诊断膀胱癌的非侵入性诊断方法
CN105378110A (zh) * 2013-04-17 2016-03-02 生命技术公司 与癌症相关的基因融合体和基因变异体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHANG, LIN ET AL.: "Expression Changes of Paternally Expressed Gene 10 in Hepatocellular Carcinoma Tissue", SHANDONG MEDICAL JOURNAL, 30 November 2016 (2016-11-30) *
YANG, LIXIN ET AL.: "PLAGLI Gene Expression and Methylation Level and Correlation with SDHB Mutations in Benign and Malignant Pheochromocytoma", CHINA ONCOLOGY, vol. 24, no. 8, 31 August 2014 (2014-08-31), pages 589 - 593 *

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