WO2019218338A1 - 通过活检细胞样本诊断癌症的方法 - Google Patents

通过活检细胞样本诊断癌症的方法 Download PDF

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WO2019218338A1
WO2019218338A1 PCT/CN2018/087470 CN2018087470W WO2019218338A1 WO 2019218338 A1 WO2019218338 A1 WO 2019218338A1 CN 2018087470 W CN2018087470 W CN 2018087470W WO 2019218338 A1 WO2019218338 A1 WO 2019218338A1
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imprinted
gene
imprinted gene
expression
copy number
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PCT/CN2018/087470
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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
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Priority to EP18918659.6A priority Critical patent/EP3795693A4/en
Priority to PCT/CN2018/087470 priority patent/WO2019218338A1/zh
Priority to JP2021514458A priority patent/JP2021524758A/ja
Priority to CN201880093393.1A priority patent/CN112313345B/zh
Priority to CN202410101683.3A priority patent/CN118064580A/zh
Publication of WO2019218338A1 publication Critical patent/WO2019218338A1/zh
Priority to US16/950,053 priority patent/US20210071261A1/en
Priority to JP2023204052A priority patent/JP2024028824A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present application relates to the field of biotechnology, and relates to the field of genetic diagnosis, and in particular to a method for diagnosing cancer by biopsy cell samples.
  • Cancer is one of the major diseases that threaten human health. About 14 million cancer patients are added globally each year, 8.2 million patients die of cancer, and these two numbers are still growing year by year. Cancer has a gradual process of development. Early cancers have a low degree of malignancy. If you get timely surgery, you can usually achieve a high 5-year survival rate. Advanced cancer is highly malignant, prone to metastasis, and cannot be treated surgically. Radiotherapy and chemotherapy are also difficult to obtain good results, so the 5-year survival rate is very low. The key to cancer treatment is early detection, but many cancers usually have no symptoms at an early stage, or the symptoms are atypical, which may lead to missed diagnosis and misdiagnosis.
  • Imaging techniques such as ultrasound and CT can detect solid tumors at an early stage, but it is difficult to judge the benign and malignant tumors. Therefore, a suspicious site sampling is required for biopsy.
  • the commonly used biopsy sampling methods include fine needle aspiration, thick needle aspiration, endoscopic guided tissue biopsy, and brush cell examination of the bronchus, esophagus, oral cavity, and cervix.
  • Fine needle aspiration is a sampling method used to detect lumps and masses. It is directly pierced by a large mass that is accessible to the body surface, or is deep or small under the guidance of ultrasound and CT. The lump was taken for puncture sampling. Fine needle aspiration is widely used for tumor sampling in the thyroid, breast, lymph nodes, parotid gland, pancreas, liver, lung, prostate, ovary and other parts.
  • the pathological diagnosis of benign and malignant cells by traditional pathology is based on the relationship between cell size, morphology, invasiveness and peripheral cellular tissues. Because the sample volume obtained by fine needle aspiration is small, it is difficult to reflect the histological morphology, and the degree of cell atypia of early cancer is also low. Therefore, it is difficult to achieve high accuracy in puncture cytological pathological diagnosis, and it is difficult for pathologists. Experience dependence is very high, and some cancers cannot be diagnosed even in the middle and late stages.
  • the method of sampling the thick needle puncture and the fine needle puncture is similar.
  • the difference is that the thick needle is used for the thick needle, so that a larger tissue strip can be obtained, which can provide certain histological information.
  • the sampling amount is still small relative to the whole tumor, some histological features, especially the invasion relationship of cancer cells to surrounding tissues, are difficult to judge, so there are still some limitations.
  • Endoscopically guided biopsy is a method for sampling suspicious lesions under endoscopy guided by endoscopy, such as endoscopy, colonoscopy, cystoscopy, hysteroscopy, and otolaryngoscopy. It is commonly used in esophageal cancer, gastric cancer, colorectal cancer, and bladder. Diagnosis of cancer, endometrial cancer, and nasopharyngeal cancer. Tissue biopsies have obtained a large sample size, but some cases of early cancer patients are still unable to be diagnosed due to low tissue atypia.
  • Brush cell examination usually uses a brush to take cells in the bronchi, mouth, esophagus, and cervix for examination.
  • the sample size obtained by this method is also small, and it is difficult to reflect the histological morphology, so the pathological diagnosis accuracy is also low.
  • Urine exfoliated cell detection is a routine test for the diagnosis of urinary cancer, but it is mainly based on the morphology of the cells. The accuracy is low, and it is still necessary to rely on cystoscopy and biopsy for diagnosis, which brings great pain to patients.
  • BRAF for papillary thyroid carcinoma
  • BRAC for breast cancer
  • CEA for lung cancer
  • PSA for prostate cancer
  • BRAF mutations can only be used for the detection of papillary thyroid carcinoma, and it is impossible to detect follicular tumors
  • PSA has high sensitivity but low specificity, resulting in higher False positive.
  • detection techniques for detecting tumor antigens and in situ hybridization in urine but the sensitivity and specificity are not ideal.
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylation of alleles from a particular parent, such that one gene has only one allele and 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.
  • 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 in situ detection of the imprinted gene is sensitive and effective in complementing the morphological pathology, making the morphological pathological uncertainty region no longer exist.
  • imprinted gene detection does not require tissue morphology and uses a small sample size. It is a cancer detection method that is very suitable for biopsy samples.
  • Detection of cancers such as central nervous system tumors, parotid malignant tumors, malignant lymphomas, and leukemias can provide more accurate pre-diagnosis and diagnostic information, completely avoiding problems that cannot be diagnosed due to insufficient cell or tissue abnormalities.
  • Increasing the early diagnosis rate of cancer is of great significance for improving the quality of life of patients and prolonging their survival.
  • the present application provides a method for diagnosing cancer by biopsy cell samples, which can be judged by visually observing changes in the imprinted (spot) genes of cancer at the early stage of biopsy cell level. The degree of benign and malignant and malignant cancer.
  • the present application provides a method for detecting cancer by biopsy samples by calculating imprinted gene deletion gene expression (LOI), imprinted gene copy number abnormal gene expression (CNV), and total expression (TE). Changes in the tumor classify the expression status of the imprinted gene;
  • LOI imprinted gene deletion gene expression
  • CNV imprinted gene copy number abnormal gene expression
  • TE total expression
  • the imprinted gene is Z1 and/or Z16
  • the imprinted gene Z1 is Gnas
  • the imprinted gene Z16 is Snrpn/Snurf.
  • the inventors found that by calculating the LOI, CNV and TE of the imprinted gene Z1, the sensitivity for diagnosis of thyroid cancer can reach 99.2%, the sensitivity to breast cancer can reach 99.8%, and the sensitivity to pancreatic cancer can be reached. 90.0%, the sensitivity to lung cancer can reach 99.8%, and the sensitivity to urinary cancer can reach 89.0%.
  • the inventors found that by calculating the LOI, CNV and TE of the imprinted gene Z16, the sensitivity for diagnosis of thyroid cancer can reach 99.8%, the sensitivity to breast cancer can reach 91.7%, and the sensitivity to pancreatic cancer can reach 90.0%.
  • the sensitivity to lung cancer can reach 90.0%, and the sensitivity to urinary cancer can reach 89.0%.
  • the inventors have found that the sensitivity can be further improved by calculating the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the two imprinted genes of Z1 and Z16, using a combination of Z1 and Z16 for thyroid cancer, breast cancer,
  • the diagnostic sensitivity of pancreatic cancer, lung cancer, and urinary cancer can reach 99.9% or more.
  • the imprinting gene further comprises any one or a combination of at least two of Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14 or Z15;
  • the imprinted gene Z2 is Igf2
  • the imprinted gene Z3 is Peg10
  • the imprinted gene is Igf2r at Z4
  • the imprinted gene Z5 is Mest
  • the imprinted gene Z6 is Plagl1
  • the imprinted gene Z7 is Cdkn1c.
  • the imprinted gene Z8 is Dcn
  • the imprinted gene Z9 is Dlk1
  • the imprinted gene Z10 is Gatm
  • the imprinted gene Z11 is Grb10
  • the imprinted gene Z12 is Peg3
  • the imprinted gene Z13 is Sgce
  • the imprint The gene Z14 is Slc38a4
  • the imprinting gene Z15 is Diras3.
  • the inventors found that the Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14 or Z15 genes are further combined based on the detection of the Z1 and Z16 genes. Diagnosis, which helps to increase the accuracy of the detection, the inventors found that the imprinted genes Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14 or Z15 are different due to the detection of tumors. Different sensitivity can be used in combination with other imprinted genes to help diagnose the degree of benign and malignant cancer. The combination of 16 imprinting genes Z1-Z16 can fully realize the accurate diagnosis of tumor.
  • the formula for calculating the expression level of the imprinted gene deletion gene and the expression amount of the imprinted gene copy number abnormal gene is as follows:
  • Imprinted gene deletion gene expression amount c / (b + c + d) ⁇ 100%;
  • the gene expression level of the imprinted gene copy number abnormality d / (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 is deleted, after the cells are subjected to hematoxylin staining, there are two red/brown marks in the nucleus, and the abnormal copy number of the imprinted gene is that after the cells are subjected to hematoxylin staining, there are more than two red in the nucleus/ Brown marker, the copy number abnormality is caused by 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 imprinted gene deletion gene expression amount, the imprinted gene copy number abnormal gene expression amount, and the total expression amount are divided into five different levels.
  • the five different grades are five of the imprinted gene deletion gene expression amount, the imprinted gene copy number abnormal gene expression amount, and the total expression amount of the sixteen imprinted genes for Z1-Z16, respectively. Different levels.
  • the five different levels of the imprinted gene deletion gene expression amount, the imprinted gene copy number abnormal gene expression amount, and the total expression amount for Z1 and Z16 are:
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of less than 15%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of less than 2% or the total expression of the imprinted genes Z1 and Z16 Any one of less than 25% or a combination of at least two;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of 15-20%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of 2-4% or the imprinted genes Z1 and Z16
  • the total expression amount is any one of 25-30% or a combination of at least two;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of 20-25%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of 4-8% or the imprinted genes Z1 and Z16
  • the total expression amount is any one of 30-40% or a combination of at least two;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 25-35%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 8-12% or the imprinted genes Z1 and Z16
  • the total expression amount is any one of 40-50% or a combination of at least two;
  • Grade IV the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is greater than 35%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is greater than 12% or the total expression amount of the imprinted genes Z1 and Z16 Any one of more than 50% or a combination of at least two.
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • each imprinted gene is different for different tumors, and each index of different tumors may have a top and bottom fluctuation of 20%.
  • the tumor comprises a thyroid tumor, a breast tumor, a pancreatic tumor, a lung tumor, a liver tumor, a colorectal tumor, a bladder tumor, a prostate tumor, a stomach tumor, an esophageal tumor, a nasopharyngeal tumor, an oral tumor, an ovary Any one or a combination of at least two of a tumor, an endometrial tumor, a cervical tumor, a urinary system tumor, a central nervous system tumor, a salivary gland tumor, a lymphoma, or a leukemia.
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of less than 15%, the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of less than 1.5%, or the total expression amount of the imprinted gene Z1 is less than 40%;
  • Grade I the imprinted gene deletion expression amount of the imprinted gene Z1 is 15-20%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is 1.5-4%, or the total expression amount of the imprinted gene Z1 is 40 -45%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 20-30%, the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 4-8% or the imprinted gene Z1 has a total expression amount of 45%. -60%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 30-40%, the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 8-15% or the imprinted gene Z1 has a total expression amount of 60%. -65%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount greater than 40%, the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount greater than 15%, or the total expression amount of the imprinted gene Z1 is greater than 65%;
  • the imprinted gene Z16 has an imprinted gene deletion expression amount of less than 15%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression amount of less than 1.5%, or the total expression amount of the imprinted gene Z16 is less than 30%;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 15-20%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 1.5-4% or the imprinted gene Z16 has a total expression of 30%. -35%;
  • the imprinted gene Z16 has an imprinted gene deletion expression amount of 20-30%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression amount of 4-8% or the imprinted gene Z16 has a total expression amount of 35 -50%;
  • the imprinted gene Z16 has an imprinted gene deletion expression amount of 30-40%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression amount of 8-15% or the imprinted gene Z16 has a total expression amount of 50%. -55%;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z16 is greater than 40%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z16 is greater than 15%, or the total expression amount of the imprinted gene Z16 is greater than 55%.
  • the sensitivity of combined diagnosis of Z1 and Z13 can be increased to over 99.9%.
  • the sensitivity of combined diagnosis of Z11 and Z16 can be increased to over 99.9%.
  • Sensitivity of combined diagnosis of Z13 and Z16 The degree can be increased to over 99.9%.
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of less than 15%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of less than 1% or the total expression of the imprinted genes Z1 and Z16 Less than 25%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 15-20%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 1-3% or the imprinted genes Z1 and Z16 The total expression amount is 25-30%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of 20-25%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of 3-7% or the imprinted genes Z1 and Z16 The total expression amount is 30-40%;
  • Grade III the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 25-30%, and the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 7-10% or the imprinted genes Z1 and Z16 The total expression amount is 40-50%;
  • Grade IV the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is greater than 30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is greater than 10% or the total expression amount of the imprinted genes Z1 and Z16 More than 50%.
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • the sensitivity of combined diagnosis of Z1 and Z10 can be increased to over 99.9%.
  • the sensitivity of combined diagnosis of Z1 and Z11 can be increased to over 99.9%.
  • the sensitivity of combined diagnosis of Z1 and Z13 can be increased to over 99.9%.
  • Combined diagnosis of Z1 and Z16 The sensitivity can be increased to over 99.9%.
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of less than 15%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of less than 2% or the total expression of the imprinted genes Z1 and Z16 Less than 20%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of 15-20%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of 2-4% or the imprinted genes Z1 and Z16 The total expression amount is 20-30%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of 20-25%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of 4-8% or the imprinted genes Z1 and Z16 The total expression amount is 30-40%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 25-30%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 8-12% or the imprinted genes Z1 and Z16 The total expression amount is 40-50%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression of more than 30%, the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression of more than 12% or the total expression of the imprinted genes Z1 and Z16 More than 50%.
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • the sensitivity of combined diagnosis of Z1 and Z16 can be increased to over 99.9%.
  • the sensitivity of combined diagnosis of Z10 and Z16 can be increased to over 99.9%.
  • Sensitivity of combined diagnosis of Z11 and Z16 The degree can be increased to over 99.9%.
  • the five different grades of the deletion gene expression of the imprinting gene Z1, the abnormal expression level of the imprinted gene copy number, and the total expression amount are:
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of less than 15%, the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of less than 2%, or the total expression amount of the imprinted gene Z1 is less than 30%;
  • Grade I the imprinted gene deletion expression amount of the imprinted gene Z1 is 15-20%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is 2-4%, or the total expression amount of the imprinted gene Z1 is 30 -40%;
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is 4-8% or the total expression amount of the imprinted gene Z1 is 40 -50%;
  • Grade III the imprinted gene of the imprinted gene Z1 is expressed in an amount of 25-30%, the imprinted gene of the imprinted gene Z1 is abnormally expressed by 8-12%, or the total expression of the imprinted gene Z1 is 50. -60%;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z1 is greater than 30%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is greater than 12% or the total expression amount of the imprinted gene Z1 is greater than 60%;
  • the imprinted gene Z16 has an imprinted gene deletion expression amount of less than 10%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression amount of less than 1%, and the total expression amount of the imprinted gene Z16 is less than 25%;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 10-15%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 1-2% or the imprinted gene Z16 has a total expression of 25%. -30%;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 15-20%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 2-5% or the imprinted gene Z16 has a total expression of 30%. -40%;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 20-25%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 5-8% or the imprinted gene Z16 has a total expression of 40%. -50%;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z16 is greater than 25%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z16 is greater than 8%, or the total expression amount of the imprinted gene Z16 is greater than 50%.
  • the inventors found that the combined diagnosis of the imprinted genes Z3, Z8, Z11 and Z1, Z16 can improve the diagnostic sensitivity to lung cancer, and the sensitivity of the combined diagnosis of Z1 and Z3 can reach 99.9% or more, and the combination of Z1 and Z8
  • the sensitivity of diagnosis can reach more than 99.9%
  • the sensitivity of Z1 and Z11 combined diagnosis can reach more than 99.9%
  • the sensitivity of Z1 and Z16 combined diagnosis can reach more than 99.9%
  • the sensitivity of Z3 and Z16 combined diagnosis can reach 99.9%. the above.
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of less than 17% and/or the imprinted genes Z1 and Z16 have an abnormal expression amount of the imprinted gene copy number of less than 2%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 17-20% and/or the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 3-7%;
  • the imprinted gene Z1 and Z16 have an imprinted gene deletion expression amount of 25-30% and/or the imprinted genes Z1 and Z16 have an imprinted gene copy number abnormal expression amount of 7-12%;
  • the imprinted gene deletion expression amount of the imprinted genes Z1 and Z16 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z16 is greater than 12%.
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of the imprinted genes Z1 and Z16 are independent of each other.
  • the inventors found that the combined diagnosis of the imprinted genes Z2, Z3, Z4 and Z1, Z16 can improve the diagnostic sensitivity to urinary cancer, and the sensitivity of the combined diagnosis of Z1 and Z3 can reach more than 94.3%, Z1 and The sensitivity of Z4 combined diagnosis can reach more than 94.3%, the sensitivity of Z1 and Z16 combined diagnosis can reach more than 99.9%, the sensitivity of Z2 and Z16 combined diagnosis can reach more than 99.9%, and the sensitivity of Z3 and Z16 combined diagnosis can reach More than 99.9%.
  • the method of diagnosing cancer comprises the steps of:
  • the analysis unit calculates the amount of the imprinted gene deletion gene, the imprinted gene copy number abnormal gene expression amount, and the total imprinted gene expression amount, thereby imprinting the gene deletion gene expression amount, imprinting gene copy number abnormal gene expression amount, and imprinting gene.
  • the level of total expression is used to determine the degree of benign and malignant tumors.
  • the sample to be tested described in the step (1) is a human tissue and/or a cell.
  • the sample to be tested comprises any one or a combination of at least two of a biopsy cell sample, a biopsy cell sample, an exfoliated cell sample, a blood sample, or a brush sample.
  • the specific sampling operation step of the biopsy sample is to obtain a sample of human tumor tissue cells by puncture, sampling clamp, brush, or to obtain exfoliated cells in urine, sputum, feces and pleural effusion, and timely use 10% neutral formalin or other fixation method is adhered to the slide after fixation. Because the biopsy has less damage to the patient, the urine, sputum and feces are completely non-invasive, and the sampling process is simple. Compared with the circulation characteristics of the blood, the biopsy sample can be positioned, which has its special advantages as an experimental sample.
  • the specific sampling operation step of the blood sample is to remove the red blood cells by using the red blood cell lysate after obtaining the peripheral blood, and then fixed in 10% neutral formalin or other fixing method, and then adhered to the slide.
  • the needle biopsy cell sample comprises a fine needle and/or a needle biopsy cell sample, preferably a fine needle of the thyroid, breast, pancreas, lung, liver, prostate, ovary, lymph node or parotid gland and/or Any one or a combination of at least two of the needle needle biopsy cell samples.
  • the biopsy cell sample comprises any one or a combination of at least two of a gastroscope, enteroscopy, cystoscopy, hysteroscopy or otolaryngoscope biopsy cell sample.
  • the exfoliated cell sample comprises any one or a combination of at least two of urine, sputum, feces, or pleural and abdominal effusion cell samples.
  • the brush sample comprises any one or a combination of at least two of a bronchial, esophageal, oral or cervix brush sample.
  • the in situ hybridization employs an RNAscope in situ hybridization method using a single or multi-channel colorimetric kit or a single or multi-channel fluorescent kit, preferably a single Channel red/brown coloring kit or multi-channel fluorescent kit;
  • the inventors judge the degree of benign and malignant tumors by the ranks of LOI, CNV and TE by calculating LOI, CNV and TE in at least 100 cells expressing the imprinted gene, and the degree of benign and malignant tumors to be judged It is benign, cancerous, early cancer, metaphase cancer and advanced cancer.
  • the result of judging the degree of benign and malignant tumors is that the imprinted gene Z1 and Z16 imprinted gene deletion gene expression and the imprinted gene copy number abnormal gene expression are less than the imprinting gene of only one imprinting gene in the imprinting gene Z1 or Z16.
  • the case where the expression level of the deletion is I or the copy number of only one of the imprinted genes Z1 or Z16 is one of the grade I is a benign tumor;
  • the result of judging the degree of benign and malignant tumors is that the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 is I, and the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is expressed as the grade I.
  • the imprinted gene deletion gene of one imprinted gene in imprinting genes Z1 and Z16 is of level I and has one copying gene.
  • the copy number of the imprinted gene is one level, and only one gene of imprinting gene Z1 or Z16. If the imprinted gene deletion gene expression level is II or the imprinted gene Z1 or Z16 has only one gene, the imprinting gene copy number abnormal gene expression level reaches any of the second level, and the cancer potential;
  • the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 is II and/or the abnormal expression of the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16
  • the imprinted gene deletion gene of one imprinted gene in imprinted genes Z1 and Z16 is of level II and has one imprinted gene.
  • the copy number of the abnormal gene is expressed at level II, and the imprinted gene Z1 or Z16 is the only one. If the expression level of the imprinted gene deletion gene of one gene is III or the expression level of the imprinted gene copy number abnormal gene of only one of the imprinted genes Z1 or Z16 reaches the level III, it is an early stage cancer;
  • the result of judging the degree of benign and malignant tumors is that the imprinting gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 is III, and the imprinting gene copy number abnormal gene expression of the two genes in the imprinting genes Z1 and Z16 is III.
  • the imprinted gene deletion gene of one of the imprinted genes Z1 and Z16 has a gene expression level of III and one copy gene has a copy number abnormal gene expression level of III, and only one of the imprinted genes Z1 or Z16 If the imprinted gene has an imprinted gene deletion level of IV or the imprinted gene Z1 or Z16 has only one gene, and the imprinted gene copy number is expressed in any of the IV levels, it is a metaphase cancer;
  • the result of judging the degree of benign and malignant tumors is that the imprinted gene of the two genes in the imprinted genes Z1 and Z16 has an imprinted gene whose expression level is IV and/or the imprinted genes Z1 and Z16 have an imprinted copy number abnormal expression of IV. Grade, it is advanced cancer.
  • the application provides a use of the method of the first aspect for use in tumor detection and/or treatment.
  • the method described in the present application expresses the imprinted cell sample in the thyroid, breast, pancreas, prostate, lymph node, lung bronchial brush cell sample, urine, sputum, feces, pleural fluid cell sample and intestine in an intuitive manner.
  • Mirror and cystoscopy biopsy on the cell sample objectively, intuitively, early, and accurately detect changes in the imprinted (spot) gene by in situ labeling of the imprinted gene, and can provide a quantitative model for thyroid cancer, Great contribution to the early diagnosis of breast cancer, pancreatic cancer, lung cancer, liver cancer, colorectal cancer, and urinary cancer;
  • the method of the present application can provide quantitative detection results, accurately determine the type of tumor, and clearly classify the degree of malignancy of the tumor, and solve the problem that traditional cells and histopathology cannot be clearly diagnosed in some cases, and fill the current
  • the limitations of histomorphological diagnosis of tissue cells provide assistance for later targeted therapies
  • This application can accurately distinguish the benign and malignant thyroid eosinophilic tumor (HCT) from the molecular level, and provides a solution for the problem that tissue histomorphology is difficult to distinguish between benign and malignant thyroid eosinophilic tumors.
  • TAA thyroid tumor
  • FTC cancer
  • This application can be simultaneously tested with immunohistochemical methods to accurately diagnose early and malignant pancreatic tumors, reducing false negatives and other negative effects;
  • the present invention can not only perform benign and malignant judgments by puncture cytology at an early stage, but also puncture lymph nodes near tumor sites to detect cancer. Whether the cells metastasize through the lymphatics;
  • This application can clearly distinguish between black sputum and cutaneous melanoma and other skin diseases, especially the differential diagnosis of melanoma in the early in situ state, and solve the problem that morphological diagnosis is difficult to distinguish;
  • This application can be used for early diagnosis of ductal adenocarcinoma of the breast in situ, and accurate differentiation of benign breast lesions;
  • This application can accurately diagnose the early malignant potential and malignant state of colon polyps, and solve the early diagnosis of intestinal cancer
  • This application does not depend on the tissue morphology, for prostate cancer, ovarian cancer, central nervous system tumors, parotid malignant tumors, malignant lymphoma biopsy samples, gastric cancer, esophageal cancer, nasopharyngeal cancer, endometrial cancer Mirror biopsy samples, lung cancer, oral cancer, esophageal cancer, cervical cancer brush samples, blood samples from leukemia can also be used to diagnose benign and malignant tumors using the technique of the present invention.
  • the present invention can diagnose leukemia with a drop of peripheral blood, and solves the problem that the bone marrow puncture trauma is currently required to be large;
  • This application can screen the urinary system cancer, lung cancer and intestinal cancer through the cells in the urine, sputum and feces.
  • the sample collection method is simple, no professional operation is required, and the patient can collect it at home.
  • Applicable to large-scale physical examination, as well as urinary system cancer surgery and drug efficacy monitoring, especially preoperative and postoperative detection of urinary exfoliated cells, can solve the problem of early detection and timely treatment of postoperative recurrence;
  • the disease-related gene-imprinted deletion site found in the detection method of the present application, the targeted drug or technical method for silencing, knocking out, rearranging the gene, can be used to guide the later treatment and medication.
  • the a is a cell nucleus in which no label is present in the nucleus and the imprinted gene is not expressed after the hematoxylin staining of the cell
  • the b is a hematoxylin After staining, there is a red/brown mark in the nucleus to imprint the nucleus of the gene
  • 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 After the cells are stained 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. 3 is a representation of the expression of 9 genes of 16 genes in puncture cell pellets of different malignant degrees of thyroid tumors according to an embodiment of the present invention, wherein FIG. 3(a) is a puncture cell tablet of a grade 0 thyroid tumor.
  • the expression status of 9 genes Figure 3 (b) shows the expression status of 9 genes in the puncture cell pellet of grade I thyroid cancer, and Figure 3 (c) shows the 9 genes in the puncture cell pellet of grade II thyroid cancer.
  • the expression status, Fig. 3(d) shows the expression status of 9 genes in the puncture cell pellet of grade III thyroid carcinoma, and Fig. 3(e) shows the expression status of 9 genes in the puncture cell pellet of grade IV thyroid cancer;
  • Figure 4 shows the distribution range and grading criteria of imprinting deletion, copy number abnormality and total expression in Z1 and Z16 genes in 177 thyroid tumor puncture cell samples.
  • Figure 4(a) shows the imprinted gene Z1 applied to 177 cases.
  • the distribution range and grading standard of imprinting deletion, copy number abnormality and total expression amount Fig. 4(b) is the imprinting gene Z16 applied to 177 thyroid tumor puncture cell samples, imprinting deletion, copy number abnormality And the distribution range and grading standard of total expression;
  • Figure 5 (a) shows the intensity of Z1, Z4, Z11, Z13, Z16 for thyroid cancer
  • Figure 5 (b) shows the intensity of Z1, Z4, Z11, Z13, Z16 for thyroid cancer
  • 5(c) is the intensity of total expression of thyroid cancer by Z1, Z4, Z11, Z13, Z16
  • Fig. 5(d) is the intensity of imprinting loss of thyroid cancer by Z2, Z3, Z5, Z6, Fig. 5 (e) The intensity of copy number abnormality of Z2, Z3, Z5 and Z6 for thyroid cancer, Fig.
  • 5(f) is the intensity of total expression of Z2, Z3, Z5 and Z6 on thyroid cancer, wherein LOI is the imprinted gene deletion gene
  • CNV is the gene expression level of the imprinted gene copy number abnormality
  • TE is the total expression level of the imprinted gene
  • Fig. 6(a) shows the intensity of the imprinting gene Z1 imprint deletion, copy number abnormality and total expression amount
  • Fig. 6(b) shows the imprinting gene Z16 imprint deletion, copy number abnormality and total expression amount intensity
  • Fig. 6(c) is The imprinting gene Z4 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 6 (d) is the imprinting gene Z11 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 6 (e) is the imprinted gene Z13 imprint deletion, The intensity of copy number abnormality and total expression amount
  • Fig. 6(f) shows the intensity of imprinted gene Z2 imprint deletion, copy number abnormality and total expression amount
  • FIG. 6(g) shows imprinted gene Z3 imprint deletion, copy number abnormality and total expression.
  • the intensity of the amount Fig. 6(h) is the intensity of the imprinting gene Z5 imprinting deletion, copy number abnormality and total expression amount
  • Fig. 6(i) is the intensity of the imprinting gene Z6 imprint deletion, copy number abnormality and total expression amount
  • the LOI is the expression level of the imprinted gene deletion gene
  • the CNV is the gene expression amount of the imprinted gene copy number abnormality
  • the TE is the total expression amount of the imprinted gene
  • Figure 7 shows the distribution range and grading criteria of Z1 and Z16 genes in 18 breast tumor puncture cell samples with imprinting deletion, copy number abnormality and total expression.
  • Figure 7(a) shows the imprinting gene Z1 applied to 18 cases. In the breast tumor puncture cell samples, the distribution range and grading standard of imprinting deletion, copy number abnormality and total expression amount
  • Fig. 7(b) is the imprinting gene Z16 applied to 18 breast tumor puncture cell samples, imprinting deletion, copy number abnormality And the distribution range and grading standard of total expression;
  • Figure 8 (a) shows the intensity of imprinted deletion of breast cancer by Z1, Z8, Z10, Z11, Z13, Z16
  • Figure 8 (b) shows the abnormal copy number of breast cancer by Z1, Z8, Z10, Z11, Z13, Z16.
  • the intensity of Figure 8, (c) is the intensity of total expression of Z1, Z8, Z10, Z11, Z13, Z16 for breast cancer
  • Figure 8 (d) is the imprint of Z3, Z4, Z5, Z6, Z9 on breast cancer
  • the intensity of deletion, Figure 8 (e) is the intensity of copy number abnormality of Z3, Z4, Z5, Z6, Z9 for breast cancer
  • Figure 8 (f) is the total expression of Z3, Z4, Z5, Z6, Z9 for breast cancer.
  • the intensity of the quantity, wherein LOI is the expression level of the imprinted gene deletion gene
  • CNV is the gene expression amount of the imprinted gene copy number abnormality
  • TE is the total expression amount of the imprinted gene
  • Fig. 9(a) shows the intensity of the imprinting gene Z1 imprinting deletion, copy number abnormality and total expression amount
  • Fig. 9(b) shows the intensity of the imprinting gene Z16 imprint deletion, copy number abnormality and total expression amount
  • Fig. 9(c) is The imprinting gene Z8 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 9 (d) is the imprinting gene Z10 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 9 (e) is the imprinted gene Z11 imprint deletion, The intensity of copy number abnormality and total expression amount
  • Fig. 9(f) shows the intensity of imprinting gene Z13 imprint deletion, copy number abnormality and total expression amount
  • Fig. 9(c) is The imprinting gene Z8 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 9 (d) is the imprinting gene Z10 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 9 (e) is the imprinted gene Z11 im
  • FIG. 9(g) shows imprinted gene Z3 imprint deletion, copy number abnormality and total expression.
  • the intensity of the amount Fig. 9(h) is the intensity of the imprinting gene Z4 imprint deletion, copy number abnormality and total expression amount
  • Fig. 9(i) shows the imprinting gene Z5 imprint deletion, copy number abnormality and total expression amount intensity
  • Fig. 9 (j) The intensity of the imprinting gene Z6 imprinting deletion, copy number abnormality and total expression amount
  • Fig. 9(i) shows imprinted gene Z4 imprint deletion, copy number abnormality and total expression amount
  • Fig. 9(i) shows the imprinting gene Z5 imprint deletion, copy number abnormality and total expression amount intensity
  • Fig. 9 (j) The intensity of the imprinting gene Z6 imprinting deletion, copy number abnormality and total expression amount
  • 9(k) is the intensity of the imprinting gene Z9 imprinting deletion, copy number abnormality and total expression amount, wherein LOI is the imprinted gene deletion gene
  • CNV is the amount of gene expression abnormality in imprinted gene copy number TE is the total amount of gene expression imprinting
  • Figure 10 shows the distribution range and grading criteria of Z1 and Z16 genes in 21 cases of pancreatic tumor puncture cell samples, imprinting deletion, copy number abnormality and total expression level.
  • Figure 10 (a) shows the imprinted gene Z1 applied to 21 cases.
  • the distribution range and grading standard of imprinting deletion, copy number abnormality and total expression amount Fig. 10(b) is the imprinting gene Z16 applied to 21 pancreatic tumor puncture cell samples, imprinting deletion, copy number abnormality And the distribution range and grading standard of total expression;
  • Figure 11 (a) shows the intensity of Z1, Z3, Z10, Z11, Z16 imprinted with pancreatic cancer
  • Figure 11 (b) shows the intensity of Z1, Z3, Z10, Z11, Z16 for pancreatic cancer.
  • 11(c) is the intensity of total expression of pancreatic cancer by Z1, Z3, Z10, Z11, Z16
  • Fig. 11(d) is the intensity of imprinting loss of pancreatic cancer by Z4, Z5, Z6, Z8, Z13
  • Fig. 11 (e) the intensity of copy number abnormality of pancreatic cancer by Z4, Z5, Z6, Z8, Z13
  • Fig. 11(f) is the intensity of total expression of pancreatic cancer by Z4, Z5, Z6, Z8, Z13, Fig.
  • FIG. 11 (g) is the intensity of Z2, Z9, Z12, Z14, Z15 imprinted with pancreatic cancer
  • Figure 11 (h) is the intensity of copy number abnormality of Z2, Z9, Z12, Z14, Z15 for pancreatic cancer
  • Figure 11 ( i) the intensity of total expression of pancreatic cancer by Z2, Z9, Z12, Z14, Z15, wherein LOI is the expression level of the imprinted gene deletion gene, CNV is the gene expression amount of the imprinted gene copy number abnormality, and TE is the imprinted gene Total expression amount;
  • Fig. 12(a) shows the intensity of the imprinting gene Z1 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12(b) shows the intensity of the imprinting gene Z16 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12(c) is The imprinting gene Z3 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 12 (d) is the imprinting gene Z10 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 12 (e) is the imprinted gene Z11 imprint deletion, The intensity of copy number abnormality and total expression level
  • Fig. 12(f) shows the intensity of imprinted gene Z4 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12(c) is The imprinting gene Z3 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 12 (d) is the imprinting gene Z10 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 12 (e) is the imprinted gene Z11 imprint deletion
  • FIG. 12(g) shows imprinted gene Z5 imprint deletion, copy number abnormality and total expression.
  • the intensity of the amount Fig. 12(h) is the intensity of the imprinting gene Z6 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12(i) shows the intensity of the imprinted gene Z8 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12 (j) The intensity of imprinting gene Z13 imprinting deletion, copy number abnormality and total expression amount
  • Fig. 12(k) is the imprinting gene Z2 imprint deletion, copy number abnormality and total expression amount intensity
  • Fig. 12(h) is the intensity of the imprinting gene Z6 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12(i) shows the intensity of the imprinted gene Z8 imprint deletion, copy number abnormality and total expression amount
  • Fig. 12 (j) The intensity of imprinting gene Z13 imprinting deletion, copy number abnormality and total expression amount
  • FIG. 12(1) is the imprinted gene Z9 imprint deletion, copy number anomaly and total expression intensity
  • Figure 12(m) is The gene Z12 imprint deletion, copy number abnormality and total expression intensity were shown.
  • Figure 12(n) shows the imprinted gene Z14 imprint deletion, copy number abnormality and total expression intensity.
  • Figure 12(o) shows the imprinted gene Z15 imprint missing. The intensity of copy number abnormality and total expression amount, wherein LOI is the expression level of the imprinted gene deletion gene, CNV is the gene expression amount of the imprinted gene copy number abnormality, and TE is the total expression amount of the imprinted gene;
  • Figure 13 shows the distribution range and grading criteria of Z1 and Z16 genes in 23 bronchial brush cells, with imprinting deletion, copy number abnormality and total expression.
  • Figure 13 (a) shows imprinting gene Z1 applied to 23 cases. In the bronchial brush cell sample, the distribution range and grading standard of imprinting deletion, copy number abnormality and total expression amount,
  • Fig. 13(b) is the imprinting gene Z16 applied to 23 bronchial brush cell samples, the imprint is missing, the copy number is abnormal. And the distribution range and grading standard of total expression;
  • Fig. 14(a) shows the intensity of imprinted deletion of lung cancer by Z1, Z3, Z8, Z13, and Z16
  • Fig. 14(b) shows the intensity of copy number abnormality of lung cancer by Z1, Z3, Z8, Z13, and Z16
  • Fig. 14 (Fig. 14 (Fig. 14) c) is the intensity of total expression of lung cancer by Z1, Z3, Z8, Z13, Z16
  • Fig. 14(d) is the intensity of imprinting loss of lung cancer by Z4, Z10, Z11, and Fig. 14(e) is Z4, Z10
  • Fig. 14(f) is the intensity of the total expression of Z4, Z10, and Z11 on lung cancer.
  • LOI is the expression of the imprinted gene deletion gene
  • CNV is the gene with abnormal imprinting gene copy number.
  • the amount of expression, TE is the total expression of the imprinted gene;
  • Fig. 15(a) shows the intensity of the imprinting gene Z1 imprint deletion, copy number abnormality and total expression amount
  • Fig. 15(b) shows the imprinting gene Z16 imprint deletion, copy number abnormality and total expression amount intensity
  • Fig. 15(c) is The imprinting gene Z3 imprinted deletion, copy number abnormality and total expression level intensity
  • Figure 15 (d) is the imprinting gene Z8 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 15 (e) is the imprinted gene Z13 imprint missing
  • Fig. 15(f) shows the intensity of imprinting gene Z4 imprint deletion, copy number abnormality and total expression amount
  • Figure 15(g) shows imprinted gene Z10 imprint deletion, copy number abnormality and total expression.
  • the intensity of the amount is the intensity of the imprinting gene Z11 imprinting deletion, copy number abnormality and total expression amount, wherein LOI is the imprinting gene deletion gene expression amount, CNV is the gene expression amount of the imprinted gene copy number abnormality, TE The total expression level of the imprinted gene;
  • Figure 16 shows the distribution range and grading criteria of imprinting deletion and copy number abnormality in 70 samples of urine exfoliated cells using Z1 and Z16 genes.
  • Figure 16 (a) shows imprinted gene Z1 applied to 70 urine exfoliated cells.
  • Fig. 16(b) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 70 samples of urine exfoliated cells in imprinted gene Z16;
  • Figure 17 (a) shows the intensity of Z1, Z2, Z3, Z10, Z16 for the deletion of the urinary system cancer
  • Figure 17 (b) shows the intensity of the copy number abnormality of the Z1, Z2, Z3, Z10, and Z16 for urinary system cancer
  • Figure 17 (c) shows the intensity of Z4, Z5, Z6, Z8, Z9, Z15 for the deletion of the urinary system cancer
  • Figure 17 (d) for the urinary system cancer of Z4, Z5, Z6, Z8, Z9, Z15.
  • the intensity of the copy number abnormality 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;
  • Figure 18 (a) shows the intensity of the imprinted gene Z1 imprint deletion and copy number abnormality
  • Figure 18 (b) shows the intensity of the imprinted gene Z16 imprint deletion and copy number abnormality
  • Figure 18 (c) shows the imprinted gene Z2 imprint deletion and copy number.
  • the intensity of the abnormality Fig. 18(d) shows the intensity of the imprinted gene Z3 imprint deletion and copy number abnormality
  • Fig. 18(e) shows the intensity of the imprinted gene Z10 imprint deletion and copy number abnormality
  • Fig. 18(f) shows the imprinted gene Z4 imprint.
  • Fig. 18(g) shows the intensity of the imprinted gene Z5 imprint deletion and copy number abnormality
  • FIG. 18(h) shows the intensity of the imprinted gene Z6 imprint deletion and copy number abnormality
  • Fig. 18(i) is The intensity of the imprinting gene Z8 imprint deletion and copy number abnormality
  • Fig. 18(j) is the intensity of the imprinting gene Z9 imprint deletion and copy number abnormality
  • Fig. 18(k) is the intensity of the imprinting gene Z15 imprint deletion and copy number abnormality
  • the LOI is the expression level of the imprinted gene deletion gene
  • the CNV is the gene expression amount of the imprinted gene copy number abnormality
  • Figure 19 (a) shows the expression status of 16 genes in a colonoscopy biopsy sample of a benign colorectal tumor
  • Figure 19 (b) shows the expression status of 16 genes in a colonoscopy biopsy sample of a malignant colorectal tumor
  • Figure 19 ( c) the expression status of 16 genes in a colonoscopy biopsy sample for colorectal cancer
  • Figure 20 shows the expression status of 16 genes in cystoscopic biopsy samples of benign bladder tumors
  • Figure 20 shows the expression status of 16 genes in cystoscopy biopsy samples of malignant bladder tumors
  • Fig. 20(c) Expression status of 16 genes in cystoscopic biopsy samples of bladder cancer
  • Figure 21 (a) shows the expression status of 16 genes in sputum cell samples of benign lung tumors
  • Figure 21 (b) shows the expression status of 16 genes in sputum cell samples of lung cancer
  • Figure 22 (a) shows the expression status of 16 genes in puncture cell pellets of benign liver tumors
  • Figure 22 (b) shows the expression status of 16 genes in puncture cell pellets of liver cancer
  • Figure 23 (a) shows the expression status of 16 genes in puncture cell pellets of benign prostate tumors
  • Figure 23 (b) shows the expression status of 16 genes in puncture cell pellets of prostate cancer
  • Fig. 24(a) shows the expression status of 16 genes in the puncture cell pellet of lymph nodes near the untransferred benign breast tumor
  • Fig. 24(b) shows the expression of 16 genes in the puncture cell pellet of lymph nodes near the metastatic breast cancer. status
  • Fig. 25(a) shows the expression status of 16 genes in a pleural fluid cell sample of a benign lung tumor
  • Fig. 25(b) shows the expression status of 16 genes in a pleural fluid cell sample of lung cancer.
  • Figure 26 (a) shows the expression status of 16 genes in fecal exfoliated cell samples from patients with benign colorectal cancer
  • Figure 26 (b) shows the expression status of 16 genes in fecal exfoliated cell samples from patients with colorectal cancer
  • Fig. 27(a) shows the expression status of 16 genes in a blood cell sample of a healthy person
  • Fig. 27(b) shows the expression state of 16 genes in a blood cell sample of a leukemia patient.
  • the method for detecting the imprinted gene comprises the following steps:
  • Design probe design specific primers according to the imprinted gene sequence
  • the design probe is based on the imprinting genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z7 (Cdkn1c), Z8 (Dcn), Z9. (Dlk1), Z10 (Gatm), Z11 (Grb10), Z12 (Peg3), Z13 (Sgce), Z14 (Slc38a4), Z15 (Diras3), and Z16 (Snrpn/Snurf) are designed specifically for each gene. A sequence was selected as a probe in the inner loop, and the specific probe was designed by Advanced Cell Diagnostics.
  • 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. 2, 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.
  • 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.
  • a brown mark in the nucleus to imprint the nucleus of the gene There is a brown mark in the nucleus to imprint the nucleus of the gene
  • the c is the hematoxylin staining of the cell, there are two brown marks in the nucleus, and the nucleus of the imprinted gene is deleted
  • the d is the cell nucleus after hematoxylin staining
  • the nuclei with abnormal copy number of the imprinted gene are imprinted.
  • Fig. 3(a) - Fig. 3(e) the results of the test are shown in Fig. 3(a) - Fig. 3(e).
  • Fig. 3(a) - Fig. 3(e) from the samples of grade 0 to grade IV, The proportion of cells with missing imprints (two signal points in the nucleus) and abnormal copy number (three or more signal points in the nucleus) gradually increases with increasing malignancy.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1.5% and/or the total imprinted gene expression amount is less than 40%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 1.5-4% and/or the total expression of the imprinted gene is 40-45%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-30% and/or imprinted gene copy number is 4-8% and/or the total expression level of imprinted gene is 45-60% is grade II, and the imprinted gene deletion expression amount is 30-40% and/or
  • the abnormal expression level of the imprinted gene copy number is 8-15% and/or the total expression level of the imprinted gene is 60-65% for the grade III, the imprinted gene deletion expression amount is greater than 40% and/or the imprinted gene copy number abnormal expression amount is greater than 15%.
  • the total expression level of the gene and/or the imprinted gene is greater than 65% and is IV.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1.5% and/or the total imprinted gene expression amount is less than 40%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 1.5-4% and/or the total expression of the imprinted gene is 40-45%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-30% and/or imprinted gene copy number is 4-8% and/or the total expression level of imprinted gene is 45-60% is grade II, and the imprinted gene deletion expression amount is 30-40% and/or
  • the abnormal expression level of the imprinted gene copy number is 8-15% and/or the total expression level of the imprinted gene is 60-65% for the grade III, the imprinted gene deletion expression amount is greater than 40% and/or the imprinted gene copy number abnormal expression amount is greater than 15%.
  • the total expression level of the gene and/or the imprinted gene is greater than 65% and is IV.
  • each gene of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 Sensitivity to thyroid cancer or the intensity and status of imprinted deletions corresponding to thyroid cancer expression are different, and Z1, Z16, Z4, Z11, and Z13 are highly sensitive to thyroid tumors.
  • the imprinted gene deletion gene expression amount and the imprinted gene copy number abnormal gene expression amount of the imprinted genes Z1 and Z16 are both smaller than the I grade, and only one imprinted gene imprinted in the imprinted gene Z1 or Z16.
  • the case where the expression level of the gene deletion is Grade I or the copy number of only one imprinted gene in the imprinting gene Z1 or Z16 is in the class I is a benign tumor;
  • the result of judging the degree of benign and malignant thyroid tumors is that the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 is I, and the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is abnormal.
  • the imprinted gene deletion gene of one of the imprinted genes Z1 and Z16 has a level I expression level and has a copy number of the imprinted gene.
  • the gene expression level is I, and only one of the imprinted genes Z1 or Z16
  • the thyroid cancer potential is the case where the gene-imprinting gene-deficient gene expression level is II or the imprinting gene Z1 or Z16 has only one gene imprinting gene copy number abnormal gene expression level reaches the level II;
  • the result of judging the degree of benign and malignant thyroid tumors is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is II and/or the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is abnormally expressed.
  • the amount of the imprinted gene deletion gene of one imprinted gene in the imprinted genes Z1 and Z16 is class II and the copy number of the imprinted gene has an abnormal expression level of II, and only the imprinted gene Z1 or Z16 If the expression level of the imprinted gene deletion gene of one gene is III or the expression level of the imprinted gene copy number abnormal gene of only one of the imprinted genes Z1 or Z16 reaches the level III, it is early thyroid cancer. ;
  • the imprinted gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 was III, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 was
  • the imprinting gene deletion gene expression level of one of the imprinted genes of class III and imprinted genes Z1 and Z16 is grade III and the copy number of one imprinted gene is abnormally expressed at level III, and only one of imprinted genes Z1 or Z16 is If the imprinted gene has an imprinted gene deletion level of IV or the imprinted gene Z1 or Z16 has only one gene, and the imprinted gene copy number is expressed in any of the IV levels, it is a metaphase thyroid cancer;
  • the abnormal expression level of the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is imprinted with the IV gene and/or the imprinted genes Z1 and Z16 are Grade IV is advanced thyroid cancer.
  • the imprinting of the gene Z16 is rapidly increasing in the malignant potential stage and is maintained at a high level with the development of thyroid cancer; the abnormal copy number and expression of the imprinted gene Z16 increase rapidly in early thyroid cancer and remain in the advanced thyroid cancer. Very high level.
  • the imprinting deletion and copy number abnormality of the imprinted gene Z4 began to rise in early thyroid cancer, and gradually increased to a higher level in the metaphase and advanced thyroid cancer; the increase in the expression level of the imprinted gene Z4 began to appear in the malignant potential phase, with cancer
  • the development has gradually increased to a higher level in advanced thyroid cancer; the imprinting of the imprinted gene Z11 is rapidly increasing in the malignant potential stage, but it does not continue to rise in the development of early to late thyroid cancer; a copy of the imprinted gene Z11
  • the number of abnormalities increased rapidly in the stage of malignant potential, remained stable in early stage thyroid cancer, and continued to rise in the stage of advanced thyroid cancer; the increase in the expression of imprinted gene Z11 began to appear in the metaphase thyroid cancer stage, and further increased in advanced thyroid cancer.
  • the copy number abnormality of the imprinted gene Z13 rapidly increased in the malignant potential stage, and the increase in imprinting and expression increased in early thyroid cancer, but remained stable in advanced thyroid cancer.
  • the imprinting gene Z2 has an imprinted deletion and increased expression level, which starts to rise in the malignant potential stage. It rises slowly during the development of early to late thyroid cancer; the copy number abnormality of the imprinted gene Z2 begins to appear in the malignant potential stage, and there is no early stage of thyroid cancer. Significantly increased, and the thyroid cancer stage increased further in the metaphase and remained stable in advanced thyroid cancer.
  • the imprinting gene Z3 has an imprinted deletion and increased expression from the malignant potential stage, which gradually increases with the development of thyroid cancer, and the sensitivity to the advanced stage of thyroid cancer is still not high; the copy number of the imprinted gene Z3 is abnormal before the metaphase thyroid cancer. The stage is at a very low level and rapidly rises to a higher level in the advanced stage of thyroid cancer.
  • the imprinted gene Z5 has an imprinted deletion and copy number abnormality that rapidly increases in the early stage of thyroid cancer and remains stable in the middle and advanced stage thyroid cancer; the increase in the expression level of the imprinted gene Z5 begins to appear in metaphase thyroid cancer and remains stable in advanced thyroid cancer.
  • Imprinting gene Z6 deletion and expression increase began to appear in the malignant potential stage, and remained at a low level during the development of thyroid cancer; the copy number abnormality of the imprinted gene Z6 began to appear in early thyroid cancer, in the middle and advanced stage thyroid cancer Maintained at a lower level.
  • the breast puncture cell sample was obtained by puncture, and the other detection methods were the same as in Example 1.
  • the results are shown in Fig. 7(a) - Fig. 7(b), Fig. 8(a) - Fig. 8(f) and Fig. 9(a) - Fig. 9. (k) is shown.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% and/or the total imprinted gene expression amount is less than 25%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 1-3% and/or the total expression of the imprinted gene is 25-30%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-25% and/or imprinted gene copy number is 3-7% and/or the total expression level of imprinted gene is 30-40% is grade II, and the imprinted gene deletion expression amount is 25-30% and/or The abnormal expression level of the imprinted gene copy number is 7-10% and/or the total expression level of the imprinted gene is 40-50% 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 10%. And/or the total expression level of the imprinted gene is greater than 50% to the IV level.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% and/or the total imprinted gene expression amount is less than 25%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 1-3% and/or the total expression of the imprinted gene is 25-30%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-25% and/or imprinted gene copy number is 3-7% and/or the total expression level of imprinted gene is 30-40% is grade II, and the imprinted gene deletion expression amount is 25-30% and/or The abnormal expression level of the imprinted gene copy number is 7-10% and/or the total expression level of the imprinted gene is 40-50% 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 10%. And/or the total expression level of the imprinted gene is greater than 50% to the IV level.
  • the imprinted gene deletion gene expression amount and the imprinted gene copy number abnormal gene expression amount of the imprinted genes Z1 and Z16 are both smaller than the I grade, and only one imprinted gene imprinted in the imprinted gene Z1 or Z16.
  • the case where the expression level of the gene deletion is Grade I or the copy number of only one imprinted gene in the imprinting gene Z1 or Z16 is in the class I is a benign tumor;
  • the result of judging the degree of benign and malignant breast tumors is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is I, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 is I.
  • the imprinted gene deletion gene of one of the imprinted genes Z1 and Z16 has a level I expression level and has a copy number of the imprinted gene.
  • the gene expression level is I, and only one of the imprinted genes Z1 or Z16
  • the gene with the imprinted gene deletion gene expression level II or the imprinted gene Z1 or Z16 has only one gene, and the imprinting gene copy number abnormal gene expression level reaches any of the second level, which is the breast cancer potential;
  • the result of judging the degree of benign and malignant breast tumors is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is II and/or the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is abnormally expressed.
  • the amount of the imprinted gene deletion gene of one imprinted gene in the imprinted genes Z1 and Z16 is class II and the copy number of the imprinted gene has an abnormal expression level of II, and only the imprinted gene Z1 or Z16
  • one gene has an imprinted gene deletion gene expression level of III or an imprinted gene Z1 or Z16, only one of the imprinted gene copy number abnormal gene expression levels reaches the level III, it is early breast cancer ;
  • the imprinted gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 was III, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 was
  • the imprinting gene deletion gene expression level of one of the imprinted genes of class III and imprinted genes Z1 and Z16 is grade III and the copy number of one imprinted gene is abnormally expressed at level III, and only one of imprinted genes Z1 or Z16 is
  • the imprinting gene has a deletion gene expression level of IV, or only one of the imprinted genes Z1 or Z16 has an imprinted gene copy number expression level of any of the IV levels, and is a metaphase breast cancer;
  • the abnormal expression level of the imprinted gene copy number of the two genes of the imprinted genes Z1 and Z16 with the imprinted gene deletion level of the IV gene and/or the imprinted genes Z1 and Z16 is Grade IV is advanced breast cancer.
  • each gene of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 Sensitivity to breast cancer or the intensity and status of imprinted deletions corresponding to breast cancer expression are different, and Z1, Z16, Z8, Z10, Z11, and Z13 are highly sensitive to breast tumors.
  • the deletion of the imprinted gene Z1 and the abnormal copy number are rapidly increased in the malignant potential stage, and have reached high sensitivity in the early stage of breast cancer, and the sensitivity is maintained at a high level in the advanced breast cancer; the expression of the imprinted gene Z1
  • the increase in levels is not high in the malignant potential stage, but rapidly rises to a higher level in the early stage of breast cancer and is maintained at a higher level in advanced breast cancer.
  • Imprinting deletion and copy number abnormality of imprinted gene Z16 increased rapidly in early breast cancer and remained at a high level in the middle and advanced stage of breast cancer; the expression of imprinted gene Z16 increased in the early stage of breast cancer, in the middle and late stage thyroid Cancer continues to rise to very high levels.
  • Imprinting deletion and copy number abnormality of imprinted gene Z8 began to appear in the malignant potential stage, continued to increase in the early stage of breast cancer, and remained stable in the middle and advanced stage breast cancer; the expression of the imprinted gene Z8 increased in the malignant potential stage, but did not follow The development of breast cancer continues to rise.
  • the abnormal copy number and expression of imprinted gene Z10 increased rapidly in early breast cancer, but did not continue to increase in advanced breast cancer; the imprinting of imprinted gene Z10 began to appear in the mid-stage breast cancer stage, in the stage of advanced breast cancer. Slightly increased.
  • the abnormal copy number and expression of the imprinted gene Z11 began to appear in the malignant potential stage, and the rate of increase in early breast cancer slowed down, and remained stable in the middle and advanced stage of breast cancer; the imprinting of the imprinted gene Z11 began to appear in the mid-stage breast cancer. , rose to a higher level in advanced breast cancer. Imprinting deletion, copy number abnormality, and increased expression of the imprinted gene Z13 rapidly increased in the early stage of breast cancer and remained stable in advanced breast cancer. Imprinting deletion, copy number abnormality and increased expression of the imprinted gene Z3 appeared in the advanced breast cancer stage, in which the copy number abnormality level was higher, the imprinting loss and the expression level increased.
  • Imprinting deletions, copy number abnormalities, and increased expression levels of the imprinted genes Z4 and Z5 occur only in the advanced stage of breast cancer, in which the level of imprinting deletion and copy number abnormality is high, and the level of expression increase is low. Imprinting deletion, copy number abnormality, and increased expression of the imprinted gene Z6 occur only in the advanced stage of breast cancer, and the level is not high.
  • the abnormal copy number of the imprinted gene Z9 began to appear in the metaphase breast cancer stage and continued to rise in advanced breast cancer; the imprinted deletion and expression of the imprinted gene Z9 increased in the advanced breast cancer stage, but the level was not high.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 2% and/or the total imprinted gene expression amount is less than 20%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 2-4% and/or the total expression of the imprinted gene is 20-30%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-25% and/or imprinted gene copy number is 4-8% and/or the total expression level of imprinted gene is 30-40% is grade II, and the imprinted gene deletion expression amount is 25-30% and/or The abnormal expression level of the imprinted gene copy number is 8-12% and/or the total expression level of the imprinted gene is 40-50% 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 12%. And/or the total expression level of the imprinted gene is greater than 50% to the IV level.
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 2% and/or the total imprinted gene expression amount is less than 20%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 2-4% and/or the total expression of the imprinted gene is 20-30%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-25% and/or imprinted gene copy number is 4-8% and/or the total expression level of imprinted gene is 30-40% is grade II, and the imprinted gene deletion expression amount is 25-30% and/or The abnormal expression level of the imprinted gene copy number is 8-12% and/or the total expression level of the imprinted gene is 40-50% 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 12%. And/or the total expression level of the imprinted gene is greater than 50% to the IV level.
  • the imprinted gene deletion gene expression amount and the imprinted gene copy number abnormal gene expression amount of the imprinted genes Z1 and Z16 are both smaller than the I grade, and only one imprinted gene imprinted in the imprinted gene Z1 or Z16.
  • the case where the expression level of the gene deletion is Grade I or the copy number of only one imprinted gene in the imprinting gene Z1 or Z16 is in the class I is a benign tumor;
  • the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 was I, and the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 was expressed as I.
  • the imprinted gene deletion gene of one of the imprinted genes Z1 and Z16 has a level I expression level and has a copy number of the imprinted gene.
  • the gene expression level is I, and only one of the imprinted genes Z1 or Z16
  • the expression level of the imprinted gene deletion gene of the gene is Grade II or the expression level of the imprinted gene copy number abnormal gene of only one gene of the imprinted gene Z1 or Z16 reaches the level II, which is the pancreatic cancer potential;
  • the result of judging the degree of benign and malignant tumors of the pancreatic tumor is that the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 is II and/or the abnormal expression of the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16
  • the amount of the imprinted gene deletion gene of one imprinted gene in the imprinted genes Z1 and Z16 is class II and the copy number of the imprinted gene has an abnormal expression level of II, and only the imprinted gene Z1 or Z16
  • one gene has an imprinted gene deletion gene expression level of III or only one of the imprinted genes Z1 or Z16 has an imprinted gene copy number abnormal gene expression level of any of the grade III, it is early pancreatic cancer ;
  • the imprinted gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 was III, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 was
  • the imprinting gene deletion gene expression level of one of the imprinted genes of class III and imprinted genes Z1 and Z16 is grade III and the copy number of one imprinted gene is abnormally expressed at level III, and only one of imprinted genes Z1 or Z16 is If the imprinted gene has an imprinted gene deletion level of IV or the imprinted gene Z1 or Z16 has only one gene, and the imprinted gene copy number is expressed in any of the IV levels, it is a metaphase pancreatic cancer;
  • the abnormal expression level of the imprinted gene copy number of the two genes of the imprinted genes Z1 and Z16 with the imprinted gene deletion level of the IV gene and/or the imprinted genes Z1 and Z16 is Grade IV is advanced pancreatic cancer.
  • each gene of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 The sensitivity to pancreatic cancer or the intensity and state of the imprinted deletion corresponding to pancreatic cancer expression are different, and Z1, Z16, Z3, Z10, and Z11 are highly sensitive to pancreatic tumors.
  • Imprinting gene Z1 has an imprinted deletion, copy number abnormality and increased expression level from the malignant potential stage.
  • the copy number abnormality rises fastest, rises to a high level in the early stage of pancreatic cancer, and remains stable, and the imprinting loss rises. It is faster, and it rises to a high level in the mid-stage pancreatic cancer stage.
  • the increase in expression rate is slower and rises to a higher level in advanced pancreatic cancer.
  • the imprinting of the imprinted gene Z16 begins to appear in the malignant potential stage, gradually increases with the development of pancreatic cancer, and reaches a higher level in the advanced pancreatic cancer stage; the abnormal copy number of the imprinted gene Z16 begins in the early stage of pancreatic cancer, in the mid-stage pancreas The cancer stage rises rapidly and maintains a high level in the advanced stage of pancreatic cancer; the increase in the expression level of the imprinted gene Z16 begins in the early stage of pancreatic cancer, gradually increases with the development of pancreatic cancer, and reaches a higher level in the advanced stage of pancreatic cancer. .
  • the imprinting of the imprinted gene Z3 begins to appear in the malignant potential stage, and continues to rise with the development of pancreatic cancer, and the rate of increase in the stage of advanced pancreatic cancer is slowed down; the abnormal copy number of the imprinted gene Z3 begins to appear in the early stage of pancreatic cancer, in the mid-stage pancreatic cancer There was no significant increase in the stage, and the stage of advanced pancreatic cancer continued to rise to a higher level; the increase in the expression of the imprinted gene Z3 began to appear in early pancreatic cancer, but remained low in the advanced pancreatic cancer.
  • Imprinting deletion and copy number abnormality of imprinting gene Z10 began to appear in the malignant potential stage, and did not rise significantly in early and mid-stage pancreatic cancer, and continued to rise to a higher level in advanced pancreatic cancer stage; the expression of imprinted gene Z10 increased in the early stage. It begins to appear in pancreatic cancer, but is maintained at a lower level in advanced pancreatic cancer.
  • the copy number abnormality of the imprinted gene Z11 began to appear in the malignant potential stage, gradually increased in early and mid-stage pancreatic cancer, and maintained at a higher level in the advanced pancreatic cancer stage; the imprinted gene Z11 has an imprinted deletion and increased expression in early pancreatic cancer.
  • Imprinting deletion and copy number abnormality of imprinted gene Z4 began to appear in the early stage of pancreatic cancer, rapidly increased in the stage of pancreatic cancer, and maintained at a high level in the stage of advanced pancreatic cancer; the expression of imprinted gene Z4 increased in the stage of early pancreatic cancer It began to appear, and there was no significant increase in metaphase pancreatic cancer, and it continued to rise in the late stage of pancreatic cancer.
  • Imprinting deletion and copy number abnormality of imprinted gene Z5 began to appear in the malignant potential stage, imprinting loss was not obvious in early stage to advanced stage of pancreatic cancer, and copy number abnormality did not increase significantly in early to mid pancreatic cancer stage, and continued to advanced pancreatic cancer. It rose to a higher level; the increase in the expression of the imprinted gene Z5 began to appear in the mid-stage pancreatic cancer stage, and continued to rise in the advanced stage of pancreatic cancer but the level was still not high.
  • Imprinting deletion and copy number abnormality of imprinted gene Z6 began to appear in the early stage of pancreatic cancer, gradually increased with the development of pancreatic cancer, reached a higher level in advanced pancreatic cancer stage; the expression of imprinted gene Z6 increased in early pancreatic cancer It began to appear, but remained at a lower level in advanced pancreatic cancer.
  • the imprinted deletion of the imprinted gene Z8 began to appear in the early stage of pancreatic cancer, and gradually increased with the development of pancreatic cancer, reaching a higher level in the advanced pancreatic cancer stage; the abnormal copy number of the imprinted gene Z8 appeared in advanced pancreatic cancer, and the level was higher.
  • Increased expression of the imprinted gene Z8 began to appear in early pancreatic cancer, but remained low in advanced pancreatic cancer.
  • the imprinting deletion and copy number abnormality of the imprinted gene Z13 began to appear in the early stage of pancreatic cancer, and it increased rapidly in the stage of pancreatic cancer in the metaphase, and remained stable in the stage of advanced pancreatic cancer.
  • the increase in the expression of the imprinted gene Z13 began to appear in the metaphase pancreatic cancer stage, but It does not continue to rise in advanced pancreatic cancer.
  • the imprinting deletion and expression increase of the imprinted gene Z2 began to appear in the early stage of pancreatic cancer, and the abnormal copy number of the imprinted gene Z2 began to appear in the metaphase pancreatic cancer stage, but remained low in the advanced pancreatic cancer.
  • the imprinted deletion of the imprinted gene Z9 began to appear in the metaphase pancreatic cancer stage, and gradually increased with the development of pancreatic cancer, and the level of advanced pancreatic cancer was still not high; the abnormal copy number of the imprinted gene Z9 began to appear in the metaphase pancreatic cancer stage, in the advanced pancreas Cancer continues to rise, but the level is not high; the increase in the expression of the imprinted gene Z9 begins in the mid-stage pancreatic cancer stage, but does not continue to rise in advanced pancreatic cancer.
  • Imprinting deletion, copy number abnormality, and increased expression of the imprinted gene Z12 began to appear in the metaphase pancreatic cancer stage, but did not continue to rise in advanced pancreatic cancer.
  • the imprinted deletion of the imprinted gene Z14 began to rise rapidly in the metaphase pancreatic cancer stage, and remained high in the advanced pancreatic cancer stage.
  • the abnormal copy number of the imprinted gene Z14 began to appear in the metaphase pancreatic cancer stage and continued to rise in advanced pancreatic cancer. However, the level was not high; there was no significant increase in the expression level of the imprinted gene Z14 during the development of pancreatic cancer.
  • the imprinted deletion of the imprinted gene Z15 began to appear in the metaphase pancreatic cancer stage, but did not continue to rise in advanced pancreatic cancer; the copy number abnormality of the imprinted gene Z15 began to appear in advanced pancreatic cancer, and the level was not high; in the development of pancreatic cancer There was no significant increase in expression of the imprinted gene Z15 during the process.
  • Lung tumor cell samples were obtained by bronchial brush.
  • the other detection methods were the same as in Example 1.
  • the results are shown in Figures 13(a) - 13(b), 14(a) - 14(f) and 15(a) - Figure 15 (h).
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 2% and/or the total imprinted gene expression amount is less than 30%.
  • Level 0 the expression of the imprinted gene is 15-20% and/or the abnormal expression of the imprinted gene copy number is 2-4% and/or the total expression of the imprinted gene is 30-40%, and the imprinted gene is deleted.
  • the abnormal expression level of 20-25% and/or imprinted gene copy number is 4-8% and/or the total expression level of imprinted gene is 40-50% is grade II, and the imprinted gene deletion expression amount is 25-30% and/or
  • the abnormal expression level of the imprinted gene copy number is 8-12% and/or the total expression level of the imprinted gene is 50-60%, the level of the imprinted gene is more than 30%, and/or the abnormal expression of the imprinted gene is greater than 12%.
  • the total expression level of the gene and/or the imprinted gene is greater than 60% and 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 1% and/or the total imprinted gene expression amount is less than 25%.
  • the expression of the imprinted gene is 10-15% and/or the abnormal expression of the imprinted gene copy number is 1-2% and/or the total expression of the imprinted gene is 25-30%, and the imprinted gene is deleted.
  • the abnormal expression level of 15-20% and/or imprinted gene copy number is 2-5% and/or the total expression level of imprinted gene is 30-40% is grade II, and the amount of imprinted gene deletion is 20-25% and/or
  • the abnormal expression level of the imprinted gene copy number is 5-8% and/or the total expression level of the imprinted gene is 40-50%, the level of the imprinted gene is more than 25%, and/or the abnormal expression of the imprinted gene is more than 8%. And/or the total expression level of the imprinted gene is greater than 50% to the IV level.
  • the imprinted gene deletion gene expression amount and the imprinted gene copy number abnormal gene expression amount of the imprinted genes Z1 and Z16 are both smaller than the I grade, and only one imprinted gene imprinted in the imprinted gene Z1 or Z16.
  • the case where the expression level of the gene deletion is Grade I or the copy number of only one imprinted gene in the imprinting gene Z1 or Z16 is in the class I is a benign tumor;
  • the result of judging the degree of benign and malignant lung tumors is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is I, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 is I.
  • the imprinted gene deletion gene of one of the imprinted genes Z1 and Z16 has a level I expression level and has a copy number of the imprinted gene.
  • the gene expression level is I, and only one of the imprinted genes Z1 or Z16
  • the gene with the imprinted gene deletion gene expression level II or the imprinted gene Z1 or Z16 has only one gene, and the imprinted gene copy number abnormal gene expression level reaches the level II, which is the lung cancer potential;
  • the result of judging the degree of benign and malignant lung tumors is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is II and/or the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is abnormally expressed.
  • the amount of the imprinted gene deletion gene of one imprinted gene in the imprinted genes Z1 and Z16 is class II and the copy number of the imprinted gene has an abnormal expression level of II, and only the imprinted gene Z1 or Z16
  • one gene has an imprinted gene deletion gene expression level of III or only one of the imprinted gene Z1 or Z16 has an imprinted gene copy number abnormal gene expression level of any of the grade III, it is an early stage lung cancer;
  • the result of judging the degree of benign and malignant lung tumors is that the imprinted gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 is III, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 is The imprinting gene deletion gene expression level of one of the imprinted genes of class III and imprinted genes Z1 and Z16 is grade III and the copy number of one imprinted gene is abnormally expressed at level III, and only one of imprinted genes Z1 or Z16 is If the imprinted gene has an imprinted gene deletion level of IV or only one of the imprinted genes Z1 or Z16 has an imprinted gene copy number expression level of any of the IV grades, it is a metaphase lung cancer;
  • the abnormal expression level of the imprinted gene copy number of the two genes of the imprinted genes Z1 and Z16 with the imprinted gene deletion level of the IV gene and/or the imprinted genes Z1 and Z16 is Grade IV is advanced lung cancer.
  • Z1, Z16, Z3, Z8, and Z13 are highly sensitive to lung tumors.
  • the imprinting deletion and copy number abnormality of the imprinted gene Z1 increased rapidly in the malignant potential stage and remained at a high level during the development of lung cancer; the expression of the imprinted gene Z1 increased rapidly in the malignant potential stage, but in the early to late stage lung cancer It does not continue to increase.
  • the imprinted deletion of imprinted gene Z16 rapidly increased in early stage lung cancer and maintained at a high level in advanced lung cancer.
  • the abnormal copy number and expression of imprinted gene Z16 increased rapidly in metaphase lung cancer, and remained higher in advanced lung cancer. High level.
  • the imprinted deletion of the imprinted gene Z3 began to appear in the malignant potential stage, and did not increase significantly in the early and middle stages of lung cancer, and rapidly increased to a high level in advanced lung cancer; the abnormal copy number of the imprinted gene Z3 rapidly rose to very high in the mid-stage lung cancer. High levels remain stable in advanced lung cancer; increased expression of the imprinting gene Z3 occurs in metaphase lung cancer, but does not increase significantly in advanced lung cancer.
  • Imprinting deletion and copy number abnormality of imprinted gene Z8 began to appear in the malignant potential stage, and continued to rise in early stage lung cancer, which was maintained at a high level in metaphase lung cancer, but showed a certain degree of decline in advanced lung cancer; expression of imprinted gene Z8 The increase in amount occurs in early and mid-stage lung cancer, and the expression level of Z8 in late stage lung cancer returns to a lower level.
  • the imprinted deletion and copy number abnormality of the imprinted gene Z13 increased rapidly in the metaphase lung cancer and maintained a high level in advanced lung cancer; the increase in the expression level of the imprinted gene Z13 was slightly increased in advanced lung cancer.
  • the imprinting of the imprinted gene Z4 is rapidly increasing in the malignant potential stage and maintaining a high level in early to late stage lung cancer; the abnormal copy number of the imprinted gene Z4 begins to appear in early stage lung cancer, and there is no significant increase in the middle stage lung cancer to the advanced stage of lung cancer. It continued to rise to a higher level; the increase in the expression level of the imprinted gene Z4 began to appear in the malignant potential stage, but did not increase significantly with the development of lung cancer.
  • the copy number abnormality of the imprinted gene Z10 rapidly increased in the malignant potential stage and maintained a high level in the early to late stage lung cancer; the imprinted deletion of the imprinted gene Z10 rapidly increased in the metaphase lung cancer and reached a higher level in advanced lung cancer; There was no significant increase in expression of the imprinted gene Z10 during the development of lung cancer. Imprinting deletion and copy number abnormality of imprinted gene Z11 increased rapidly in the malignant potential stage, and did not increase significantly in early and middle stage lung cancer, but the stage of advanced lung cancer continued to rise to a higher level; the expression level of imprinted gene Z11 was in advanced lung cancer. Only a slight rise.
  • the imprinted gene deletion expression amount is less than 17% and/or the imprinted gene copy number abnormal expression amount is less than 2% as 0 level, and the imprinted gene deletion expression amount is 17- 20% and/or imprinted gene copy number abnormal expression level is 2-3% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 3-7% for grade II, Imprinted gene deletion expression level is 25-30% and/or imprinted gene copy number abnormal expression level is 7-12% is grade III, imprinted gene deletion expression amount is greater than 30% and/or imprinted gene copy number abnormal expression amount is greater than 12% It is level IV.
  • the imprinted gene deletion expression amount is less than 17% and/or the imprinted gene copy number abnormal expression amount is less than 2% is 0 level, and the imprinted gene deletion expression amount is 17- 20% and/or imprinted gene copy number abnormal expression level is 2-3% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 3-7% for grade II, Imprinted gene deletion expression level is 25-30% and/or imprinted gene copy number abnormal expression level is 7-12% is grade III, imprinted gene deletion expression amount is greater than 30% and/or imprinted gene copy number abnormal expression amount is greater than 12% It is level IV.
  • the imprinted gene deletion gene expression amount and the imprinted gene copy number abnormal gene expression amount of the imprinted genes Z1 and Z16 are both smaller than that of the I grade, and only one imprinted gene of the imprinted gene Z1 or Z16.
  • the imprinted gene is expressed in a level I or the imprinted gene Z1 or Z16 has only one copy of the imprinted gene.
  • the expression level of the imprinted gene deletion gene of the two genes in the imprinted genes Z1 and Z16 is I
  • the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16 is The imprinting gene deletion gene expression level of one imprinting gene in class I, imprinting genes Z1 and Z16 is class I and the copy number of one imprinting gene is abnormally expressed in grade I, and only one in imprinting gene Z1 or Z16.
  • the gene expression level of the imprinted gene deletion gene is II or the imprinting gene Z1 or Z16 has only one gene imprinting gene copy number abnormal gene expression level reaches any of the second level, which is the urinary system cancer potential;
  • the result of determining the degree of benign and malignant tumors of the urinary system is that the imprinted gene deletion gene expression level of the two genes in the imprinted genes Z1 and Z16 is II and/or the imprinted gene copy number of the two genes in the imprinted genes Z1 and Z16 is abnormal.
  • the expression level was grade II, and the imprinted gene deletion gene expression level of one imprinted gene in imprinted genes Z1 and Z16 was grade II and the copy number abnormal gene expression level of one imprinted gene was grade II, imprinted gene Z1 or Z16 When only one gene has an imprinted gene deletion gene expression level of III or only one of the imprinted genes Z1 or Z16 has an imprinted gene copy number abnormal gene expression level of any of the grade III, it is early urinary Systemic cancer
  • the result of determining the degree of benign and malignant tumors of the urinary system is that the imprinted gene deletion expression level of the two genes in the imprinted genes Z1 and Z16 is III, and the imprinted gene copy number abnormal gene expression of the two genes in the imprinted genes Z1 and Z16
  • the imprinted gene deletion gene of one of the grade III, imprinted genes Z1 and Z16 has a gene expression level of III and has one imprinted gene.
  • the copy number of the gene is III, and the imprinted gene Z1 or Z16 is the only one.
  • the imprinting gene deletion expression level of one imprinted gene or the imprinting gene expression number of only one of the imprinted genes Z1 or Z16 is any one of the IV levels, and is a metaphase urinary system cancer;
  • the result of determining the degree of benign and malignant tumors of the urinary system is that the imprinting gene of the two genes in the imprinted genes Z1 and Z16 has an imprinted gene expression level of IV and/or two genes of the imprinted genes Z1 and Z16.
  • grade IV it is advanced urinary system cancer.
  • each gene of Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 Sensitivity to urinary cancer cancer or the intensity and status of imprinted deletions corresponding to bladder cancer expression are different, and Z1, Z16, Z2, Z3, and Z10 are highly sensitive to urinary system tumors.
  • the imprinting of the imprinted gene Z1 begins to appear in the malignant potential stage, continues to rise in early urinary cancer, and maintains a high level in the middle and late stage urinary cancer; the abnormal copy number of the imprinted gene Z1 rises rapidly in the malignant potential stage. It has risen to a very high level in the urinary system cancer in the middle to late stages.
  • the imprinting of the imprinted gene Z16 begins to appear in early urinary cancer, and no longer increases significantly in the middle and late stage urinary cancer; the abnormal copy number of the imprinted gene Z16 begins in the early stage of urinary cancer, and gradually develops with the development of urinary cancer. Rise to a very high level.
  • the imprinting of the imprinted gene Z2 begins to appear in the mid-stage urinary system cancer, and reaches a high level in the advanced urinary system cancer; the abnormal copy number of the imprinted gene Z2 begins to appear in the malignant potential stage, and gradually rises to a high level with the development of urinary cancer. Level.
  • the imprinting of the imprinted gene Z3 begins to appear in the mid-stage urinary system cancer, and the advanced urinary system cancer continues to rise, but the level is not high; the copy number abnormality of the imprinted gene Z3 begins to appear in the malignant potential stage, and gradually increases with the development of urinary system cancer. To a very high level.
  • the imprinting of the imprinted gene Z10 begins to appear in the mid-stage urinary system cancer, and rapidly rises to a higher level in advanced urinary cancer; the abnormal copy number of the imprinted gene Z10 begins in the early stage of urinary cancer, with the development of urinary cancer Gradually rise to a higher level.
  • the imprinted deletion of the imprinted gene Z4 began to appear in the mid-stage urinary system cancer, and the urinary system cancer continued to rise in the late stage, but the level was not high; the copy number abnormality of the imprinted gene Z4 began to appear in the malignant potential stage, and gradually increased with the development of cancer. High level.
  • the imprinting of the imprinted gene Z5 begins to appear in the mid-stage urinary system cancer, and continues to rise in advanced cancer, but the level is not high; the copy number abnormality of the imprinted gene Z5 begins to appear in the malignant potential stage, and rapidly rises in early cancer, in the middle and late stages. Maintain a high level of cancer in the urinary system.
  • the copy number abnormality of the imprinted gene Z6 began to appear in the malignant potential stage, and rapidly rose to a higher level in the late stage of cancer; the imprinted gene Z6 imprinted with a slight increase in advanced urinary cancer.
  • Imprinting deletions and copy number abnormalities of the imprinted genes Z8 and Z9 began to appear in metaphase urinary cancer and rapidly rose to higher levels in advanced urinary cancer.
  • Abnormal copy number of the imprinted gene Z15 began to appear in metaphase urinary cancer and rapidly rose to higher levels in advanced cancer; the imprinted deletion of the imprinted gene Z15 showed a higher level of increase in advanced urinary cancer.
  • Biopsy samples were taken under colonoscopy, and other detection methods were the same as in Example 1, and the results are shown in Figs. 19(a) to 19(c).
  • Fig. 19(a) is a benign colorectal polyp
  • Fig. 19(b) is a malignant colorectal tumor
  • Fig. 19(c) is a colorectal cancer.
  • the degree of malignancy of the tumor increases, the proportion of imprinted cells and the proportion of cells with abnormal copy number gradually increase.
  • a biopsy sample was taken under a cystoscope, and other detection methods were the same as in Example 1, and the results are shown in Figs. 20(a) to 20(c).
  • Fig. 20(a) is a benign bladder tumor
  • Fig. 20(b) is a malignant bladder tumor
  • Fig. 20(c) is a bladder cancer with a tumor.
  • the cell sample was taken from the sputum of the lung tumor patient, and the other detection methods were the same as in Example 1, and the results are shown in Fig. 21 (a) - Fig. 21 (b).
  • Fig. 21(a) is a benign lung tumor
  • Fig. 21(b) is a lung cancer.
  • the proportion of imprinted cells and copy number abnormalities are abnormal.
  • the proportion of cells is gradually increasing.
  • liver puncture cell sample was obtained by puncture, and the other detection methods were the same as in Example 1, and the results are shown in Fig. 22 (a) - Fig. 22 (b).
  • Fig. 22(a) is a benign liver tumor
  • Fig. 22(b) is a liver cancer.
  • the proportion of imprinted cells and copy number abnormalities are abnormal.
  • the proportion of cells is gradually increasing.
  • a prostate biopsy sample was obtained by puncture, and other detection methods were the same as in Example 1, and the results are shown in Fig. 23(a) - Fig. 23(b).
  • Fig. 23(a) shows a benign prostate tumor
  • Fig. 23(b) shows prostate cancer.
  • the proportion of imprinted cells and copy number are marked.
  • the proportion of abnormal cells gradually increased.
  • the lymph node puncture cell sample near the breast tumor was obtained by puncture, and the other detection methods were the same as in Example 1, and the results are shown in Fig. 24(a) to Fig. 24(b).
  • Fig. 24(a) shows lymph node puncture cells in the vicinity of untransferred benign breast tumors
  • Fig. 24(b) shows lymph node puncture cells in the vicinity of metastatic breast cancer.
  • the lymph nodes near the benign tumor only the cells with individual imprinted deletions, no cancer cells with abnormal copy number were found, and there were a large number of cancer cells with abnormal imprinting and copy number abnormality in the lymph nodes near the metastatic breast cancer.
  • the pleural fluid cell sample was obtained by puncture, and the other detection methods were the same as in Example 1, and the results are shown in Fig. 25(a) - Fig. 25(b).
  • Fig. 25(a)-Fig. 25(b) shows pleural fluid cells of patients with benign lung tumors
  • Fig. 25(b) shows pleural fluid cells of lung cancer patients in pleural fluid of patients with benign lung tumors. Only cells with missing imprints, no cancer cells with abnormal copy number, and a large number of cancer cells with abnormal imprinting and copy number in the pleural fluid of lung cancer patients.
  • the cell sample was taken from the stool of a colorectal tumor patient, and the other detection methods were the same as in Example 1, and the results are shown in Fig. 26(a) to Fig. 26(b).
  • Fig. 26(a) is a benign colorectal tumor
  • Fig. 26(b) is a colorectal cancer.
  • the degree of malignancy of the tumor increases, the proportion of the missing cells is marked and The proportion of abnormal cells in copy number gradually increased.
  • Fig. 27 (a) is a blood cell of a healthy person
  • Fig. 27 (b) is a blood cell of a leukemia patient, and only a single imprint is missing in the blood of a healthy person. Cells, no cells with abnormal copy number were found, and there were a large number of cells with abnormal imprinting and copy number abnormalities in the blood of leukemia patients.
  • the method described in the present application expresses the imprinted cell sample in the thyroid, breast, pancreas, prostate, lymph node, lung bronchial brush cell sample, urine, sputum, feces, pleural effusion cell sample in an intuitive manner.
  • the performance of colonoscopy, cystoscopy biopsy cell samples through the method of in situ labeling of imprinted genes, objective, intuitive, early, accurate detection of changes in imprinted (trace) genes, and can provide a quantitative model for the thyroid gland Cancer, breast cancer, pancreatic cancer, lung cancer, liver cancer, colorectal cancer, and early diagnosis of urinary cancer have made great contributions.

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Abstract

本申请提供了一种通过活检样本检测癌症的方法，其通过计算印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总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Z1和/或Z16，所述印记基因Z1为Gnas，所述印记基因Z16为Snrpn/Snurf。本申请的方法检测了活检样本上的印记缺失，提供了量化模型，用于癌的早期诊断或筛查。

Description

[根据细则37.2由ISA制定的发明名称]　通过活检细胞样本诊断癌症的方法 技术领域
本申请涉及生物技术领域，涉及基因诊断领域，具体涉及一种通过活检细胞样本诊断癌症的方法。
背景技术
癌症是威胁人类健康的主要疾病之一，每年全球新增约1400万癌症患者，820万患者死于癌症，并且这两个数字还在逐年增长。癌症有一个逐渐发展的过程，早期癌症的恶性程度较低，如果能够得到及时的手术治疗，通常能够达到较高的5年生存率。而晚期癌症恶性程度较高，容易发生转移，无法进行手术治疗，放疗和化疗也难以得到较好的疗效，因此5年生存率非常低。癌症治疗的关键在于早期发现，但是很多癌症在早期通常没有任何症状，或者症状不典型，容易造成漏诊和误诊。超声和CT等影像学技术能够在较早期发现实体肿瘤，但难以对肿瘤的良恶性进行判断，因此需要对可疑部位取样进行活检。目前常用的活检取样方式主要有细针穿刺，粗针穿刺，内镜引导下的组织活检，以及支气管、食道、口腔、子宫颈等部位的刷细胞检查。
细针穿刺(FNA)是一种用于检测硬块和肿块的取样方法，通过空心针对体表可触及的较大肿块进行直接穿刺，或者在超声和CT的引导下对位置较深或体积较小的肿块进行穿刺取样。细针穿刺广泛应用于甲状腺、乳腺、淋巴结、腮腺、胰腺、肝脏、肺、前列腺、卵巢等部位的肿瘤取样。传统病理学对细胞的良恶性诊断是基于细胞的大小，形态，侵润性和周边细胞组织的关系来作出判断的。由于细针穿刺所获得的样本量较少，难以反映组织学形态，同时早期癌症的细胞异型性程度也较低，因此穿刺细胞学病理诊断通常难以达到较高的准确率，而且对病理医生的经验依赖性非常高，有些癌症甚至到中晚期都无法确诊。
粗针穿刺与细针穿刺的取样方法较为类似，区别在于粗针穿刺使用较粗的针，因此可以获得较大的组织条，可以提供一定的组织学信息。但是由于取样量相对于整个肿瘤来说仍然很小，一些组织学特征，尤其是癌细胞对周围组织的侵犯关系较难判断，因此仍有一定的局限性。
内镜引导下的组织活检是在胃镜、肠镜、膀胱镜、宫腔镜、耳鼻咽喉镜等内镜引导下对可疑病变组织进行取样的方法，常用于食道癌、胃癌、结直肠癌、膀胱癌、子宫内膜癌、鼻咽癌的诊断。组织活检获得的样本量较多，但仍有一部分早期癌症患者的病例由于组织异型性较低而无法明确诊断。
刷细胞检查通常使用毛刷在支气管、口腔、食道、子宫颈获取细胞进行检查。使用这种方法获取的样本量也较少，难以反映组织学形态，因此病理学诊断准确率也较低。尿液脱落细胞检测是诊断泌尿系统癌症的常规检查项目，但主要是根据细胞的形态来进行判断，准确率较低，仍然要依靠膀胱镜和活检进行确诊，给患者带来较大的痛苦。
为了避免传统病理学在对早期癌症诊断中的局限性，人们开发了多种在细胞分子水平检测肿瘤的生物标志物，如用于甲状腺乳头状癌的BRAF，用于乳腺癌的BRAC，用于肺癌的CEA，用于前列腺癌的PSA等。但是目前已有的肿瘤标志物还存在一些不足，如BRAF的突变仅能用于甲状腺乳头状癌的检测，无法检测滤泡性肿瘤；PSA的灵敏度很高，但是特异性较低，造成较高的假阳性。目前也有一些在尿液中进行肿瘤抗原检测和原位杂交的检测技术，但是灵敏度和特异性都不太理想。
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。如图1所示，印迹基因原位检测敏感而有效地补足了形态病理的不足，使得形态病理的不确定区不再存在。
基于以上原因，印迹基因检测对组织形态没有要求，使用的样本量也较少，是一种非常适用于活检 样本的癌症检测方法。通过印迹基因检测技术对甲状腺癌、乳腺癌、胰腺癌、肺癌、肝癌、结直肠癌、膀胱癌、前列腺癌、胃癌、食道癌、鼻咽癌、口腔癌、卵巢癌、子宫内膜、宫颈癌、中枢神经系统肿瘤、腮腺恶性肿瘤、恶性淋巴瘤、白血病等癌症进行检测，可以提供更精确的预诊和诊断信息，彻底避免由于细胞或组织异型性不够明显而造成无法诊断的问题，极大提高癌症的早期诊断率，对提高患者生活质量、延长生存期具有重要的意义。
发明内容
针对现有技术的不足及实际的需求，本申请提供了一种通过活检细胞样本诊断癌症的方法，该检测方法可以通过活检细胞水平下早期直观地观察癌的印记(迹)基因的变化从而判断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，本申请提供了一种通过活检样本检测癌症的方法，其通过计算印记基因缺失基因表达量(LOI)、印记基因拷贝数异常基因表达量(CNV)和总表达量(TE)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Z1和/或Z16，所述印记基因Z1为Gnas，所述印记基因Z16为Snrpn/Snurf。
本申请中，发明人发现通过计算印记基因Z1的LOI、CNV和TE，对于甲状腺癌的诊断敏感度可以达到99.2％，对乳腺癌的敏感度可以达到99.8％，对胰腺癌的敏感度可以达到90.0％，对肺癌的敏感度可以达到99.8％，对泌尿系统癌症的敏感度可以达到89.0％。
此外，发明人发现通过计算印记基因Z16的LOI、CNV和TE，对于甲状腺癌的诊断敏感度可以达到99.8％，对乳腺癌的敏感度可以达到91.7％，对胰腺癌的敏感度可以达到90.0％，对肺癌的敏感度可以达到90.0％，对泌尿系统癌症的敏感度可以达到89.0％。
根据本申请，发明人发现通过计算Z1和Z16两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可以进一步提高敏感度，使用Z1和Z16的组合，对甲状腺癌、乳腺癌、胰腺癌、肺癌、泌尿系统癌症的诊断敏感度可以达到99.9％以上。
在一些实施方式中，所述印记基因还包括Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12、Z13、Z14或Z15中的任意一个或至少两个的组合；其中，所述印记基因Z2为Igf2，印记基因Z3为Peg10，所述印记基因在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Z7为Cdkn1c，所述印记基因Z8为Dcn，所述印记基因Z9为Dlk1，所述印记基因Z10为Gatm，所述印记基因Z11为Grb10，所述印记基因Z12为Peg3，所述印记基因Z13为Sgce，所述印记基因Z14为Slc38a4，所述印记基因Z15为Diras3。
在本申请中，发明人发现在使用Z1、Z16基因检测的基础上再增加Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12、Z13、Z14或Z15基因进行联合诊断，有助于增加检测的准确度，发明人发现印记基因Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12、Z13、Z14或Z15由于检测肿瘤的不同，其敏感度不同，可以在用于和别的印记基因组合辅助确诊癌症的良恶性程度，采用Z1-Z16这16个印记基因进行组合，可以完全实现肿瘤的精确诊断。
在一些实施方式中，所述计算印记基因缺失基因表达量和印记基因拷贝数异常基因表达量的公式如下：
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。
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。
在一些实施方式中，所述印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总表达量分成五个不同的等级。
在一些实施方式中，所述五个不同的等级为针对Z1-Z16的十六个印记基因的印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总表达量分别进行划分的五个不同的等级。
在一些实施方式中，所述针对Z1和Z16的印记基因缺失基因表达量、印记基因拷贝数异常基因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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％或所述印记基因Z1和Z16的总表达量小于25％中的任意一种或至少两种的组合；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1和Z16的总表达量为25-30％中的任意一种或至少两种的组合；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1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和Z16的印记基因缺失表达量为25-35％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1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和Z16的印记基因缺失表达量大于35％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％或所述印记基因Z1和Z16的总表达量大于50％中的任意一种或至少两种的组合。
本申请中，所述印记基因Z1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。
本申请中，针对不同的肿瘤各个印记基因的敏感性不同，不同肿瘤的各个指标会有上下20％的浮动。
在一些实施方式中，所述肿瘤包括甲状腺肿瘤、乳腺肿瘤、胰腺肿瘤、肺肿瘤、肝脏肿瘤、结直肠肿瘤、膀胱肿瘤、前列腺肿瘤、胃肿瘤、食道肿瘤、鼻咽部肿瘤、口腔肿瘤、卵巢肿瘤、子宫内膜肿瘤、子宫颈肿瘤、泌尿系统肿瘤、中枢神经系统肿瘤、腮腺肿瘤、淋巴瘤或白血病中的任意一种或至少两种的组合。
在一些实施方式中，对于甲状腺肿瘤，印记基因Z1的缺失表达量、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的印记基因缺失表达量小于15％、所述印记基因Z1的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z1的总表达量小于40％；
I级：所述印记基因Z1的印记基因缺失表达量为15-20％、所述印记基因Z1的印记基因拷贝数异常表达量为1.5-4％或所述印记基因Z1的总表达量为40-45％；
II级：所述印记基因Z1的印记基因缺失表达量为20-30％、所述印记基因Z1的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1的总表达量为45-60％；
III级：所述印记基因Z1的印记基因缺失表达量为30-40％、所述印记基因Z1的印记基因拷贝数异常表达量为8-15％或所述印记基因Z1的总表达量为60-65％；
IV级：所述印记基因Z1的印记基因缺失表达量大于40％、所述印记基因Z1的印记基因拷贝数异常 表达量大于15％或所述印记基因Z1的总表达量大于65％；
对于甲状腺肿瘤，印记基因Z16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Z16的印记基因缺失表达量小于15％、所述印记基因Z16的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z16的总表达量小于30％；
I级：所述印记基因Z16的印记基因缺失表达量为15-20％、所述印记基因Z16的印记基因拷贝数异常表达量为1.5-4％或所述印记基因Z16的总表达量为30-35％；
II级：所述印记基因Z16的印记基因缺失表达量为20-30％、所述印记基因Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z16的总表达量为35-50％；
III级：所述印记基因Z16的印记基因缺失表达量为30-40％、所述印记基因Z16的印记基因拷贝数异常表达量为8-15％或所述印记基因Z16的总表达量为50-55％；
IV级：所述印记基因Z16的印记基因缺失表达量大于40％、所述印记基因Z16的印记基因拷贝数异常表达量大于15％或所述印记基因Z16的总表达量大于55％。
在一些实施方式中，发明人发现印记基因Z3、Z11、Z13与Z1、Z16的联合诊断可以提高对甲状腺癌的诊断敏感度，Z1和Z3联合诊断的敏感度可以提高到99.9％以上，Z1和Z11联合诊断的敏感度可以提高到99.9％以上，Z1和Z13联合诊断的敏感度可以提高到99.9％以上，Z11和Z16联合诊断的敏感度可以提高到99.9％以上，Z13和Z16联合诊断的敏感度可以提高到99.9％以上。
对于乳腺肿瘤，印记基因Z1和Z16的缺失表达量、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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1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和Z16的总表达量小于25％；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1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和Z16的总表达量为25-30％；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-7％或所述印记基因Z1和Z16的总表达量为30-40％；
III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为7-10％或所述印记基因Z1和Z16的总表达量为40-50％；
IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于10％或所述印记基因Z1和Z16的总表达量大于50％。
本申请中，所述印记基因Z1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。
在一些实施方式中，发明人发现印记基因Z8、Z10、Z11、Z13、Z16与Z1的联合诊断可以提高对乳腺癌的诊断敏感度，Z1和Z8联合诊断的敏感度可以提高到99.9％以上，Z1和Z10联合诊断的敏感度可以提高到99.9％以上，Z1和Z11联合诊断的敏感度可以提高到99.9％以上，Z1和Z13联合诊断的敏感度可以提高到99.9％以上，Z1和Z16联合诊断的敏感度可以提高到99.9％以上。
对于胰腺肿瘤，印记基因Z1和Z16的缺失表达量、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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％或所述印记基因Z1和Z16的总表达量小于20％；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1和Z16的总表达量为20-30％；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1和Z16的总表达量为30-40％；
III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1和Z16的总表达量为40-50％；
IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％或所述印记基因Z1和Z16的总表达量大于50％。
本申请中，所述印记基因Z1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。
在一些实施方式中，发明人发现印记基因Z5、Z10、Z11与Z1、Z16的联合诊断可以提高对胰腺癌的诊断敏感度，Z1和Z5联合诊断的敏感度可以提高到99.9％以上，Z1和Z10联合诊断的敏感度可以提高到99.9％以上，Z1和Z16联合诊断的敏感度可以提高到99.9％以上，Z10和Z16联合诊断的敏感度可以提高到99.9％以上，Z11和Z16联合诊断的敏感度可以提高到99.9％以上。
对于肺肿瘤，印记基因Z1的缺失表达量、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的印记基因缺失表达量小于15％、所述印记基因Z1的印记基因拷贝数异常表达量小于2％或所述印记基因Z1的总表达量小于30％；
I级：所述印记基因Z1的印记基因缺失表达量为15-20％、所述印记基因Z1的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1的总表达量为30-40％；
II级：所述印记基因Z1的印记基因缺失表达量为20-25％、所述印记基因Z1的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1的总表达量为40-50％；
III级：所述印记基因Z1的印记基因缺失表达量为25-30％、所述印记基因Z1的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1的总表达量为50-60％；
IV级：所述印记基因Z1的印记基因缺失表达量大于30％、所述印记基因Z1的印记基因拷贝数异常表达量大于12％或所述印记基因Z1的总表达量大于60％；
对于肺肿瘤，印记基因Z16的缺失表达量、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Z16的印记基因缺失表达量小于10％、所述印记基因Z16的印记基因拷贝数异常表达量小于1％、所述印记基因Z16的总表达量小于25％；
I级：所述印记基因Z16的印记基因缺失表达量为10-15％、所述印记基因Z16的印记基因拷贝数异常表达量为1-2％或所述印记基因Z16的总表达量为25-30％；
II级：所述印记基因Z16的印记基因缺失表达量为15-20％、所述印记基因Z16的印记基因拷贝数异常表达量为2-5％或所述印记基因Z16的总表达量为30-40％；
III级：所述印记基因Z16的印记基因缺失表达量为20-25％、所述印记基因Z16的印记基因拷贝数异常表达量为5-8％或所述印记基因Z16的总表达量为40-50％；
IV级：所述印记基因Z16的印记基因缺失表达量大于25％、所述印记基因Z16的印记基因拷贝数异常表达量大于8％或所述印记基因Z16的总表达量大于50％。
在一些实施方式中，发明人发现印记基因Z3、Z8、Z11与Z1、Z16的联合诊断可以提高对肺癌的诊断敏感度，Z1和Z3联合诊断的敏感度可以达到99.9％以上，Z1和Z8联合诊断的敏感度可以达到99.9％以上，Z1和Z11联合诊断的敏感度可以达到99.9％以上，Z1和Z16联合诊断的敏感度可以达到99.9％以上，Z3和Z16联合诊断的敏感度可以达到99.9％以上。
对于泌尿系统肿瘤，印记基因Z1和Z16的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Z1和Z16的印记基因缺失表达量小于17％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％；
I级：所述印记基因Z1和Z16的印记基因缺失表达量为17-20％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-3％；
II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-7％；
III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％和/或所述印记基因Z1和Z16的印记 基因拷贝数异常表达量为7-12％；
IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％。
本申请中，所述印记基因Z1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。
在一些实施方式中，发明人发现印记基因Z2、Z3、Z4与Z1、Z16的联合诊断可以提高对泌尿系统癌症的诊断敏感度，Z1和Z3联合诊断的敏感度可以达到94.3％以上，Z1和Z4联合诊断的敏感度可以达到94.3％以上，Z1和Z16联合诊断的敏感度可以达到99.9％以上，Z2和Z16联合诊断的敏感度可以达到99.9％以上，Z3和Z16联合诊断的敏感度可以达到99.9％以上。
根据本申请，所述诊断癌症的方法包括如下步骤：
(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状态；
其中，所述分析单元通过计算印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和印记基因总表达量，从而通过印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和印记基因总表达量的等级来判断肿瘤的良恶性程度。
根据本申请，步骤(1)所述的待测样本为人的组织和/或细胞。
在一些实施方式中，所述待测样本包括穿刺活检细胞样本、活检细胞样本、脱落细胞样本、血液样本或刷检样本中的任意一种或至少两种的组合。
本申请中，所述活检样本的具体取样操作步骤为通过穿刺、取样钳、毛刷获取人体肿瘤组织细胞样本，或在尿液、痰液、粪便和胸腹腔积液中获取脱落细胞，及时用10％中性福尔马林或其他固定方法固定后粘附到玻片上。由于活检对病人伤害小，尿液、痰液和粪便的获得完全无创，取样过程简单，相较于血液的循环特性，活检样本还能定位，作为实验样本有其特殊的优势。所述血液样本的具体取样操作步骤为获取外周血后用红细胞裂解液去除红细胞，然后及时用10％中性福尔马林或其他固定方法固定后粘附到玻片上。
在一些实施方式中，所述穿刺活检细胞样本包括细针和/或粗针穿刺活检细胞样本，优选为甲状腺、乳腺、胰腺、肺、肝脏、前列腺、卵巢、淋巴结或腮腺的细针和/或粗针穿刺活检细胞样本中的任意一种或至少两种的组合。
在一些实施方式中，所述活检细胞样本包括胃镜、肠镜、膀胱镜、宫腔镜或耳鼻咽喉镜活检细胞样本中的任意一种或至少两种的组合。
在一些实施方式中，所述脱落细胞样本包括尿液、痰液、粪便或胸腹腔积液脱落细胞样本中的任意一种或至少两种的组合。
在一些实施方式中，所述刷检样本包括支气管、食道、口腔或子宫颈的刷检样本中的任意一种或至少两种的组合。
在一些实施方式中，所述原位杂交采用RNAscope原位杂交方法，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，发明人通过在至少100个表达印记基因的细胞中计算LOI、CNV和TE，从而通过LOI、CNV和TE的等级来判断肿瘤的良恶性程度，待判断的肿瘤的良恶性程度分为良性、癌潜能、早期癌、中期癌和晚期癌。
判断肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、 印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为癌潜能；
判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期癌；
判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期癌；
判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期癌。
第二方面，本申请提供一种如第一方面所述的方法用于肿瘤检测和/或治疗中的用途。
与现有技术相比，本申请具有如下有益效果：
(1)本申请所述方法以直观的方法表现了印记缺失在甲状腺、乳腺、胰腺、前列腺、淋巴结穿刺细胞样本，肺支气管毛刷细胞样本，尿液、痰液、粪便、胸水细胞样本和肠镜、膀胱镜活检细胞样本上的表现，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，为甲状腺癌、乳腺癌、胰腺癌、肺癌、肝癌、结直肠癌、泌尿系统癌症的早期诊断做出巨大贡献；
(2)本申请方法可以提供定量的检测结果，精确的判断肿瘤的类型，对肿瘤的恶性程度进行明确地分级，解决了传统细胞和组织病理学对部分病例无法明确诊断的问题，填补了目前组织细胞形态学诊断的局限性，为后期的靶向性治疗提供帮助；
(3)本申请可以从分子层面精确地区分甲状腺嗜酸细胞肿瘤(HCT)的良恶性，为目前组织细胞形态学难以区分甲状腺嗜酸细胞肿瘤良恶性的问题提供了解决方案，对于滤泡性甲状腺瘤(FTA)和癌(FTC)的区分，也是形态学诊断的难点，本申请的方法可以在细胞层面进行区分；
(4)本申请可以与免疫组化方法同时检测，对胰腺肿瘤的良恶性进行准确的早期诊断，减少了假阴性和其他负面作用；
(5)对于乳腺肿瘤、肺肿瘤、肝肿瘤等可以通过淋巴道进行转移的肿瘤，本发明不但可以在早期通过穿刺细胞学进行良恶性判断，还可以对肿瘤位置附近的淋巴结进行穿刺，检测癌细胞是否通过淋巴道发生转移；
(6)本申请可以对黑痣和皮肤黑色素瘤以及其他皮肤病进行明确区分，特别是早期原位状态下的黑色素瘤鉴别诊断，解决形态学诊断难以区分的难题；
(7)本申请可以对乳腺的原位导管腺癌进行早期诊断，和乳腺良性病变进行准确区分；
(8)本申请可以准确诊断结肠息肉的早期恶性潜能及恶性表现状态，解决肠癌的早期诊断问题；
(9)本申请可以对胃炎、胃溃疡和早期胃癌进行明确区分，解决胃癌的早期诊断问题；
(10)本申请不依赖于组织形态，对于前列腺癌、卵巢癌、中枢神经系统肿瘤、腮腺恶性肿瘤、恶性淋巴瘤的穿刺活检样本，胃癌、食道癌、鼻咽癌、子宫内膜癌的内镜活检样本，肺癌、口腔癌、食道癌、宫颈癌的刷检样本、白血病的血液样本也可以使用本发明的技术进行肿瘤良恶性的诊断
(11)本发明可以用一滴外周血诊断白血病，解决了目前需要进行骨髓穿刺创伤较大的问题；
(12)本申请可以通过尿液、痰液、粪便中脱落的细胞对泌尿系统癌症、肺癌和肠癌进行早期筛查，样本收集方法简单，不需要专业人员操作，可由患者在家中自行收集，适用于大规模体检普查，以及泌尿 系统癌症术后和药物疗效监测，特别是尿路脱落细胞术前和术后的检测，可以解决患者术后复发早期检测、及时治疗的问题；
(13)在外科手术中癌旁组织的真正良恶性判断是病人预后及生存期的重要因素，本申请的方法可以作为至关重要的指导；
(14)本申请检测方法中发现的疾病相关基因印记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是本发明所述的印记基因原位检测方法与传统形态学诊断的差异；
图2是本发明苏木素染色细胞核的甲状腺癌穿刺细胞压片，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3为本申请实施例的16个基因中的9个基因在甲状腺肿瘤不同恶性程度的穿刺细胞压片中的表达状态，其中，图3(a)为0级甲状腺肿瘤的穿刺细胞压片中9个基因的表达状态，图3(b)为I级甲状腺癌的穿刺细胞压片中9个基因的表达状态，图3(c)为II级甲状腺癌的穿刺细胞压片中9个基因的表达状态，图3(d)为III级甲状腺癌的穿刺细胞压片中9个基因的表达状态，图3(e)为IV级甲状腺癌的穿刺细胞压片中9个基因的表达状态；
图4为Z1和Z16基因应用于177例甲状腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，其中，图4(a)为印记基因Z1应用于177例甲状腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图4(b)为印记基因Z16应用于177例甲状腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准；
图5(a)为Z1、Z4、Z11、Z13、Z16对甲状腺癌的印记缺失的强度，图5(b)为Z1、Z4、Z11、Z13、Z16对甲状腺癌的拷贝数异常的强度，图5(c)为Z1、Z4、Z11、Z13、Z16对甲状腺癌的总表达量的强度，图5(d)为Z2、Z3、Z5、Z6对甲状腺癌的印记缺失的强度，图5(e)为Z2、Z3、Z5、Z6对甲状腺癌的拷贝数异常的强度，图5(f)为Z2、Z3、Z5、Z6对甲状腺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为印记基因的总表达量；
图6(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6(b)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图6(c)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图6(d)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图6(e)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，图6(f)为印记基因Z2印记缺失、拷贝数异常和总表达量的强度，图6(g)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图6(h)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图6(i)为印记基因Z6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为印记基因的总表达量；
图7为Z1和Z16基因应用于18例乳腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，其中，图7(a)为印记基因Z1应用于18例乳腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图7(b)为印记基因Z16应用于18例乳腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准；
图8(a)为Z1、Z8、Z10、Z11、Z13、Z16对乳腺癌的印记缺失的强度，图8(b)为Z1、Z8、Z10、Z11、Z13、Z16对乳腺癌的拷贝数异常的强度，图8(c)为Z1、Z8、Z10、Z11、Z13、Z16对乳腺癌的总表达量的强度，图8(d)为Z3、Z4、Z5、Z6、Z9对乳腺癌的印记缺失的强度，图8(e)为Z3、Z4、Z5、Z6、Z9对乳腺癌的拷贝数异常的强度，图8(f)为Z3、Z4、Z5、Z6、Z9对乳腺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为印记基因的总表达量；
图9(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9(b)为印记基因Z16印记缺 失、拷贝数异常和总表达量的强度，图9(c)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图9(d)为印记基因Z10印记缺失、拷贝数异常和总表达量的强度，图9(e)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图9(f)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，图9(g)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图9(h)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图9(i)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图9(j)为印记基因Z6印记缺失、拷贝数异常和总表达量的强度，图9(k)为印记基因Z9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为印记基因的总表达量；
图10为Z1和Z16基因应用于21例胰腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，其中，图10(a)为印记基因Z1应用于21例胰腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图10(b)为印记基因Z16应用于21例胰腺肿瘤穿刺细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准；
图11(a)为Z1、Z3、Z10、Z11、Z16对胰腺癌的印记缺失的强度，图11(b)为Z1、Z3、Z10、Z11、Z16对胰腺癌的拷贝数异常的强度，图11(c)为Z1、Z3、Z10、Z11、Z16对胰腺癌的总表达量的强度，图11(d)为Z4、Z5、Z6、Z8、Z13对胰腺癌的印记缺失的强度，图11(e)为Z4、Z5、Z6、Z8、Z13对胰腺癌的拷贝数异常的强度，图11(f)为Z4、Z5、Z6、Z8、Z13对胰腺癌的总表达量的强度，图11(g)为Z2、Z9、Z12、Z14、Z15对胰腺癌的印记缺失的强度，图11(h)为Z2、Z9、Z12、Z14、Z15对胰腺癌的拷贝数异常的强度，图11(i)为Z2、Z9、Z12、Z14、Z15对胰腺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为印记基因的总表达量；
图12(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12(b)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图12(c)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图12(d)为印记基因Z10印记缺失、拷贝数异常和总表达量的强度，图12(e)为印记基因Z11印记缺失、拷贝数异常和总表达量的强度，图12(f)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度，图12(g)为印记基因Z5印记缺失、拷贝数异常和总表达量的强度，图12(h)为印记基因Z6印记缺失、拷贝数异常和总表达量的强度，图12(i)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图12(j)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，图12(k)为印记基因Z2印记缺失、拷贝数异常和总表达量的强度，图12(1)为印记基因Z9印记缺失、拷贝数异常和总表达量的强度，图12(m)为印记基因Z12印记缺失、拷贝数异常和总表达量的强度，图12(n)为印记基因Z14印记缺失、拷贝数异常和总表达量的强度，图12(o)为印记基因Z15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为印记基因的总表达量；
图13为Z1和Z16基因应用于23例支气管毛刷细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，其中，图13(a)为印记基因Z1应用于23例支气管毛刷细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准，图13(b)为印记基因Z16应用于23例支气管毛刷细胞样本中，印记缺失、拷贝数异常和总表达量的分布范围和分级标准；
图14(a)为Z1、Z3、Z8、Z13、Z16对肺癌的印记缺失的强度，图14(b)为Z1、Z3、Z8、Z13、Z16对肺癌的拷贝数异常的强度，图14(c)为Z1、Z3、Z8、Z13、Z16对肺癌的总表达量的强度，图14(d)为Z4、Z10、Z11对肺癌的印记缺失的强度，图14(e)为Z4、Z10、Z11对肺癌的拷贝数异常的强度，图14(f)为Z4、Z10、Z11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为印记基因的总表达量；
图15(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15(b)为印记基因Z16印记缺失、拷贝数异常和总表达量的强度，图15(c)为印记基因Z3印记缺失、拷贝数异常和总表达量的强度，图15(d)为印记基因Z8印记缺失、拷贝数异常和总表达量的强度，图15(e)为印记基因Z13印记缺失、拷贝数异常和总表达量的强度，图15(f)为印记基因Z4印记缺失、拷贝数异常和总表达量的强度， 图15(g)为印记基因Z10印记缺失、拷贝数异常和总表达量的强度，图15(h)为印记基因Z11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为印记基因的总表达量；
图16为Z1和Z16基因应用于70例尿液脱落细胞样本中，印记缺失和拷贝数异常的分布范围和分级标准，其中，图16(a)为印记基因Z1应用于70例尿液脱落细胞样本中，印记缺失和拷贝数异常的分布范围和分级标准，图16(b)为印记基因Z16应用于70例尿液脱落细胞样本中，印记缺失和拷贝数异常的分布范围和分级标准；
图17(a)为Z1、Z2、Z3、Z10、Z16对泌尿系统癌症的印记缺失的强度，图17(b)为Z1、Z2、Z3、Z10、Z16对泌尿系统癌症的拷贝数异常的强度，图17(c)为Z4、Z5、Z6、Z8、Z9、Z15对泌尿系统癌症的印记缺失的强度，图17(d)为Z4、Z5、Z6、Z8、Z9、Z15对泌尿系统癌症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18(a)为印记基因Z1印记缺失和拷贝数异常的强度，图18(b)为印记基因Z16印记缺失和拷贝数异常的强度，图18(c)为印记基因Z2印记缺失和拷贝数异常的强度，图18(d)为印记基因Z3印记缺失和拷贝数异常的强度，图18(e)为印记基因Z10印记缺失和拷贝数异常的强度，图18(f)为印记基因Z4印记缺失和拷贝数异常的强度，图18(g)为印记基因Z5印记缺失和拷贝数异常的强度，图18(h)为印记基因Z6印记缺失和拷贝数异常的强度，图18(i)为印记基因Z8印记缺失和拷贝数异常的强度，图18(j)为印记基因Z9印记缺失和拷贝数异常的强度，图18(k)为印记基因Z15印记缺失和拷贝数异常的强度，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19(a)为良性结直肠肿瘤的肠镜活检样本中16个基因的表达状态，图19(b)为恶性潜能结直肠肿瘤的肠镜活检样本中16个基因的表达状态，图19(c)为结直肠癌的肠镜活检样本中16个基因的表达状态；
图20(a)为良性膀胱肿瘤的膀胱镜活检样本中16个基因的表达状态，图20(b)为恶性潜能膀胱肿瘤的膀胱镜活检样本中16个基因的表达状态，图20(c)为膀胱癌的膀胱镜活检样本中16个基因的表达状态；
图21(a)为良性肺肿瘤的痰液细胞样本中16个基因的表达状态，图21(b)为肺癌的痰液细胞样本中16个基因的表达状态；
图22(a)为良性肝脏肿瘤的穿刺细胞压片中16个基因的表达状态，图22(b)为肝癌的穿刺细胞压片中16个基因的表达状态；
图23(a)为良性前列腺肿瘤的穿刺细胞压片中16个基因的表达状态，图23(b)为前列腺癌的穿刺细胞压片中16个基因的表达状态；
图24(a)为未转移的良性乳腺肿瘤附近淋巴结的穿刺细胞压片中16个基因的表达状态，图24(b)为转移的乳腺癌附近淋巴结的穿刺细胞压片中16个基因的表达状态；
图25(a)为良性肺肿瘤的胸水细胞样本中16个基因的表达状态，图25(b)为肺癌的胸水细胞样本中16个基因的表达状态。
图26(a)为良性结直肠肿瘤患者的粪便脱落细胞样本中16个基因的表达状态，图26(b)为结直肠癌患者的粪便脱落细胞样本中16个基因的表达状态；
图27(a)为健康人的血液细胞样本中16个基因的表达状态，图27(b)为白血病癌患者的血液细胞样本中16个基因的表达状态。
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 穿刺细胞的印记基因分析
所述的印记基因的检测方法，包括如下步骤：
(1)用活检穿刺针获取穿刺细胞，经10％中性福尔马林固定，以防RNA降解，固定时间为24小时，制作细胞压片；
(2)按照RNASCope的样品处理方法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)、Z2(Igf2)、Z3(Peg10)、Z4(Igf2r)、Z5(Mest)、Z6(Plagl1)、Z7(Cdkn1c)、Z8(Dcn)、Z9(Dlk1)、Z10(Gatm)、Z11(Grb10)、Z12(Peg3)、Z13(Sgce)、Z14(Slc38a4)、Z15(Diras3)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RNASCope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2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棕色标记，印记基因拷贝数异常的细胞核。
以甲状腺癌为例，其检测结果如图3(a)-图3(e)所示，从图3(a)-图3(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2 177例甲状腺穿刺细胞的印记基因分析
通过穿刺获取177例甲状腺穿刺细胞样本，其他检测方法同实施例1，结果如图4(a)-图4(b)、图5(a)-图5(f)和图6(a)-图6(i)所示。
从图4(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15％和/或印记基因拷贝数异常表达量小于1.5％和/或印记基因总表达量小于40％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为1.5-4％和/或印记基因总表达量为40-45％为I级，印记基因缺失表达量为20-30％和/或印记基因拷贝数异常表达量为4-8％和/或印记基因总表达量为45-60％为II级，印记基因缺失表达量为30-40％和/或印记基因拷贝数异常表达量为8-15％和/或印记基因总表达量为60-65％为III级，印记基因缺失表达量大于40％和/或印记基因拷贝数异常表达量大于15％和/或印记基因总表达量大于65％为IV级。
从图4(b)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于1.5％和/或印记基因总表达量小于40％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为1.5-4％和/或印记基因总表达量为40-45％为I级，印记基因缺失表达量为20-30％和/或印记基因拷贝数异常表达量为4-8％和/或印记基因总表达量为45-60％为II级，印记基因缺失表达量为30-40％和/或印记基因拷贝数异常表达量为8-15％和/或印记基因总表达量为60-65％为III级，印记基因缺失表达量大于40％和/或印记基因拷贝数异常表达量大于15％和/或印记基因总表达量大于65％为IV级。
从图5(a)-图5(f)可以看出，Z1，Z2，Z3，Z4，Z5，Z6，Z7，Z8，Z9，Z10，Z11，Z12，Z13，Z14，Z15，Z16每个基因对甲状腺癌的反应敏感性或者说对应于甲状腺癌表达的印记缺失的强度和状态是不同的，Z1、Z16、Z4、Z11、Z13对甲状腺肿瘤敏感度高。
从这177例甲状腺穿刺细胞样本综合分析可以得出：
判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有 1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为甲状腺癌潜能；
判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期甲状腺癌；
判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期甲状腺癌；
判断甲状腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期甲状腺癌。
具体每个印记基因对甲状腺癌的敏感度如图6(a)-图6(i)：
印记基因Z1的缺失、拷贝数异常和表达量增加在恶性潜能阶段迅速升高，在早期甲状腺癌阶段已经达到很高的敏感度，在中晚期甲状腺癌中敏感度维持在很高的水平；印记基因Z16的印记缺失在恶性潜能阶段迅速上升，随甲状腺癌的发展维持在很高的水平；印记基因Z16的拷贝数异常和表达量增加在早期甲状腺癌中迅速上升，在中晚期甲状腺癌中维持很高的水平。
印记基因Z4的印记缺失和拷贝数异常在早期甲状腺癌中开始上升，在中期和晚期甲状腺癌中逐渐上升到较高的水平；印记基因Z4的表达量增加在恶性潜能阶段开始出现，随着癌症的发展逐渐上升，到晚期甲状腺癌中达到较高的水平；印记基因Z11的印记缺失在恶性潜能阶段迅速上升，但是在早期到晚期甲状腺癌的发展过程中不再继续上升；印记基因Z11的拷贝数异常在恶性潜能阶段迅速上升，在早期甲状腺癌中维持稳定，到中晚期甲状腺癌阶段又继续上升；印记基因Z11的表达量增加在中期甲状腺癌阶段才开始出现，在晚期甲状腺癌中进一步上升。印记基因Z13的拷贝数异常在恶性潜能阶段迅速上升，印记缺失和表达量增加在早期甲状腺癌中开始上升，但是在中晚期甲状腺癌中都维持稳定。
印记基因Z2的印记缺失和表达量增加在恶性潜能阶段开始上升，在早期到晚期甲状腺癌的发展过程中上升比较缓慢；印记基因Z2的拷贝数异常在恶性潜能阶段开始出现，早期甲状腺癌阶段没有明显上升，到中期甲状腺癌阶段进一步上升，在晚期甲状腺癌中维持稳定。印记基因Z3的印记缺失和表达量增加从恶性潜能阶段开始出现，随甲状腺癌的发展而缓慢上升，到晚期甲状腺癌阶段敏感度仍然不高；印记基因Z3的拷贝数异常在中期甲状腺癌之前的阶段都处于很低的水平，在晚期甲状腺癌阶段迅速上升到较高的水平。印记基因Z5的印记缺失和拷贝数异常在早期甲状腺癌阶段迅速上升，在中晚期甲状腺癌中维持稳定；印记基因Z5的表达量增加在中期甲状腺癌中开始出现，在晚期甲状腺癌中维持稳定。印记基因Z6的印记缺失和表达量增加在恶性潜能阶段开始出现，在甲状腺癌发展过程中维持在较低的水平；印记基因Z6的拷贝数异常在早期甲状腺癌中开始出现，在中晚期甲状腺癌中维持在较低的水平。
实施例3 18例乳腺穿刺细胞的印记基因分析
通过穿刺获取乳腺穿刺细胞样本，其他检测方法同实施例1，结果如图7(a)-图7(b)、图8(a)-图8(f)和图9(a)-图9(k)所示。
从图7(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15％和/或印记基因拷贝数异常表达量小于1％和/或印记基因总表达量小于25％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为1-3％和/或印记基因总表达量为25-30％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3-7％和/或印记基因总表达量为30-40％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7-10％和/或印记基因总表达量为40-50％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10％和/或印记基因总表达量大于50％为IV级。
从图7(b)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于1％和/或印记基因总表达量小于25％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为1-3％和/或印记基因总表达量为25-30％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3-7％和/或印记基因总表达量为30-40％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7-10％和/或印记基因总表达量为40-50％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10％和/或印记基因总表达量大于50％为IV级。
从这18例乳腺穿刺细胞样本综合分析可以得出：
判断乳腺肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断乳腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为乳腺癌潜能；
判断乳腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期乳腺癌；
判断乳腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期乳腺癌；
判断乳腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期乳腺癌。
从图8(a)-图8(f)可以看出，Z1，Z2，Z3，Z4，Z5，Z6，Z7，Z8，Z9，Z10，Z11，Z12，Z13，Z14，Z15，Z16每个基因对乳腺癌的反应敏感性或者说对应于乳腺癌表达的印记缺失的强度和状态是不同的，Z1、Z16、Z8、Z10、Z11、Z13对乳腺肿瘤敏感度高。
具体每个印记基因对乳腺癌的敏感度如图9(a)-图9(k)：
印记基因Z1的缺失、拷贝数异常在恶性潜能阶段迅速升高，在早期乳腺癌阶段已经达到很高的敏感度，在中晚期乳腺癌中敏感度维持在很高的水平；印记基因Z1的表达量增加在恶性潜能阶段水平不高，但是在早期乳腺癌阶段迅速上升到较高水平，并且在中晚期乳腺癌中维持在较高的水平。印记基因Z16的印记缺失和拷贝数异常在早期乳腺癌中迅速上升，到中晚期乳腺癌阶段维持在很高的水平；印记基因Z16的表达量增加在早期乳腺癌阶段开始出现，在中晚期甲状腺癌中继续上升到很高的水平。
印记基因Z8的印记缺失和拷贝数异常在恶性潜能阶段开始出现，在早期乳腺癌阶段继续增加，并在中晚期乳腺癌中维持稳定；印记基因Z8的表达量增加恶性潜能阶段开始出现，但是不随乳腺癌的发展而继续上升。印记基因Z10的拷贝数异常和表达量增加在早期乳腺癌中快速上升，但是在中晚期乳腺癌中不在继续增加；印记基因Z10的印记缺失在中期乳腺癌阶段才开始出现，在晚期乳腺癌阶段略有上升。印记基因Z11的拷贝数异常和表达量增加在恶性潜能阶段开始出现，在早期乳腺癌中上升速度减缓，到中晚期乳腺癌阶段维持稳定；印记基因Z11的印记缺失在中期乳腺癌中才开始出现，在晚期乳腺癌中上升到较高的水平。印记基因Z13的印记缺失、拷贝数异常和表达量增加在早期乳腺癌阶段迅速上升，在中晚期乳腺癌中维持稳定。印记基因Z3的印记缺失、拷贝数异常和表达量增加在晚期乳腺癌阶段才出现，其中拷贝数异常水平较高，印记缺失和表达量增加水平较低。印记基因Z4和Z5的印记缺失、拷贝数异常和表达量 增加在晚期乳腺癌阶段才出现，其中印记缺失和拷贝数异常水平较高，表达量增加水平较低。印记基因Z6的印记缺失、拷贝数异常和表达量增加在晚期乳腺癌阶段才出现，并且水平不高。印记基因Z9的拷贝数异常在中期乳腺癌阶段开始出现，在晚期乳腺癌中继续上升；印记基因Z9的印记缺失和表达量增加在晚期乳腺癌阶段才出现，但是水平都不高。
实施例4 21例胰腺穿刺细胞的印记基因分析
通过穿刺获取21例胰腺穿刺细胞样本，其他检测方法同实施例1，结果如图10(a)-图10(b)、图11(a)-图11(i)和图12(a)-图12(o)所示。
从图10(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15％和/或印记基因拷贝数异常表达量小于2％和/或印记基因总表达量小于20％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为2-4％和/或印记基因总表达量为20-30％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4-8％和/或印记基因总表达量为30-40％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8-12％和/或印记基因总表达量为40-50％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12％和/或印记基因总表达量大于50％为IV级。
从图10(b)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于15％和/或印记基因拷贝数异常表达量小于2％和/或印记基因总表达量小于20％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为2-4％和/或印记基因总表达量为20-30％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4-8％和/或印记基因总表达量为30-40％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8-12％和/或印记基因总表达量为40-50％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12％和/或印记基因总表达量大于50％为IV级。
从这21例胰腺穿刺细胞样本综合分析可以得出：
判断胰腺肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断胰腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为胰腺癌潜能；
判断胰腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期胰腺癌；
判断胰腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期胰腺癌；
判断胰腺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期胰腺癌。
从图11(a)-图11(i)可以看出，Z1，Z2，Z3，Z4，Z5，Z6，Z7，Z8，Z9，Z10，Z11，Z12，Z13，Z14，Z15，Z16每个基因对胰腺癌的反应敏感性或者说对应于胰腺癌表达的印记缺失的强度和状态是不同的，Z1、Z16、Z3、Z10、Z11对胰腺肿瘤敏感度高。
具体每个印记基因对胰腺癌的敏感度如图12(a)-图12(o)：
印记基因Z1的印记缺失、拷贝数异常和表达量增加从恶性潜能阶段开始出现，其中拷贝数异常上升 速度最快，在早期胰腺癌阶段就上升到很高的水平，并维持稳定，印记缺失上升速度较快，到中期胰腺癌阶段上升到很高的水平，表达量增加上升的速度较慢，在晚期胰腺癌中上升到较高的水平。印记基因Z16的印记缺失在恶性潜能阶段开始出现，随胰腺癌的发展逐渐上升，到晚期胰腺癌阶段达到较高的水平；印记基因Z16的拷贝数异常在早期胰腺癌阶段开始出现，在中期胰腺癌阶段迅速上升，到晚期胰腺癌阶段维持在很高的水平；印记基因Z16的表达量增加在早期胰腺癌阶段开始出现，在随胰腺癌的发展逐渐上升，到晚期胰腺癌阶段达到较高水平。
印记基因Z3的印记缺失在恶性潜能阶段开始出现，随胰腺癌的发展持续上升，到中晚期胰腺癌阶段上升速度减缓；印记基因Z3的拷贝数异常在早期胰腺癌阶段开始出现，在中期胰腺癌阶段没有明显上升，到晚期胰腺癌阶段继续上升到较高的水平；印记基因Z3的表达量增加在早期胰腺癌中开始出现，但是在中晚期胰腺癌中都维持在较低的水平。印记基因Z10的印记缺失和拷贝数异常在恶性潜能阶段开始出现，在早期和中期胰腺癌中上升不明显，在晚期胰腺癌阶段继续上升到较高的水平；印记基因Z10的表达量增加在早期胰腺癌中开始出现，但是在中晚期胰腺癌中都维持在较低的水平。印记基因Z11的拷贝数异常在恶性潜能阶段开始出现，在早期和中期胰腺癌中逐渐上升，到晚期胰腺癌阶段维持在较高的水平；印记基因Z11的印记缺失和表达量增加在早期胰腺癌中开始出现，其中印记缺失水平上升较快，在中晚期胰腺癌阶段维持在较高水平，表达量增加上升较慢，在中晚期胰腺癌阶段水平仍然不高。印记基因Z4的印记缺失和拷贝数异常在早期胰腺癌阶段开始出现，在中期胰腺癌阶段迅速上升，到晚期胰腺癌阶段维持在很高的水平；印记基因Z4的表达量增加在早期胰腺癌阶段开始出现，在中期胰腺癌中没有显著上升，到晚期胰腺癌阶段又继续上升。印记基因Z5的印记缺失和拷贝数异常在恶性潜能阶段开始出现，印记缺失在早期到晚期胰腺癌阶段上升不明显，拷贝数异常在早期到中期胰腺癌阶段没有明显上升，到晚期胰腺癌中继续上升到较高水平；印记基因Z5的表达量增加在中期胰腺癌阶段才开始出现，在晚期胰腺癌阶段继续上升但水平仍然不高。印记基因Z6的印记缺失和拷贝数异常在早期胰腺癌阶段开始出现，随胰腺癌的发展而逐渐上升，到晚期胰腺癌阶段达到较高的水平；印记基因Z6的表达量增加在早期胰腺癌中开始出现，但是在中晚期胰腺癌中都维持在较低的水平。印记基因Z8的印迹缺失在早期胰腺癌阶段开始出现，随胰腺癌的发展而缓慢上升，到晚期胰腺癌阶段达到较高水平；印记基因Z8的拷贝数异常在晚期胰腺癌中出现，水平较高；印记基因Z8的表达量增加在早期胰腺癌中开始出现，但是在中晚期胰腺癌中都维持在较低的水平。印记基因Z13的印记缺失和拷贝数异常在早期胰腺癌阶段开始出现，在中期胰腺癌阶段迅速上升，晚期胰腺癌阶段维持稳定；印迹基因Z13的表达量增加在中期胰腺癌阶段开始出现，但是在晚期胰腺癌中不继续上升。印记基因Z2的印记缺失和表达量增加在早期胰腺癌阶段开始出现，印记基因Z2的拷贝数异常在中期胰腺癌阶段开始出现，但是在中晚期胰腺癌中都维持在较低的水平。印记基因Z9的印迹缺失在中期胰腺癌阶段开始出现，随胰腺癌的发展缓慢上升，到晚期胰腺癌阶段水平仍然不高；印记基因Z9的拷贝数异常在中期胰腺癌阶段开始出现，在晚期胰腺癌中继续上升，但水平不高；印记基因Z9的表达量增加在中期胰腺癌阶段开始出现，但是在晚期胰腺癌中不继续上升。印记基因Z12的印记缺失、拷贝数异常和表达量增加在中期胰腺癌阶段开始出现，但是在晚期胰腺癌中不继续上升。印记基因Z14的印迹缺失在中期胰腺癌阶段开始迅速上升，到晚期胰腺癌阶段维持在较高的水平，印记基因Z14的拷贝数异常在中期胰腺癌阶段开始出现，在晚期胰腺癌中继续上升，但水平不高；在胰腺癌的发展过程中印记基因Z14没有出现明显的表达量增加。印记基因Z15的印记缺失在中期胰腺癌阶段开始出现，但是在晚期胰腺癌中不继续上升；印记基因Z15的拷贝数异常在晚期胰腺癌中才开始出现，并且水平不高；在胰腺癌的发展过程中印记基因Z15没有出现明显的表达量增加。
实施例5 23例支气管毛刷细胞的印记基因分析
通过支气管毛刷获取肺肿瘤细胞样本，其他检测方法同实施例1，结果如图13(a)-图13(b)、图14(a)-图14(f)和图15(a)-图15(h)所示。
从图13(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15％和/或印记基因拷贝数异常表达量小于2％和/或印记基因总表达量小于30％为0级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为2-4％和/或印记基因总表达量为30-40％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4-8％和/或印记基因总表达量为40-50％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8-12％和/或印记基因总表达量为50-60％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12％和/或印记基因总表达量大于60％为IV级。
从图13(b)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％和/或印记基因拷贝数异常表达量小于1％和/或印记基因总表达量小于25％为0级，印记基因缺失表达量为10-15％和/或印记基因拷贝数异常表达量为1-2％和/或印记基因总表达量为25-30％为I级，印记基因缺失表达量为15-20％和/或印记基因拷贝数异常表达量为2-5％和/或印记基因总表达量为30-40％为II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5-8％和/或印记基因总表达量为40-50％为III级，印记基因缺失表达量大于25％和/或印记基因拷贝数异常表达量大于8％和/或印记基因总表达量大于50％为IV级。
从这23例支气管毛刷细胞样本综合分析可以得出：
判断肺肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断肺肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为肺癌潜能；
判断肺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期肺癌；
判断肺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期肺癌；
判断肺肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期肺癌。
从图14(a)-图14(f)可以看出，Z1，Z2，Z3，Z4，Z5，Z6，Z7，Z8，Z9，Z10，Z11，Z12，Z13，Z14，Z15，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，Z1、Z16、Z3、Z8、Z13对肺肿瘤敏感度高。
具体每个印记基因对肺癌的敏感度如图15(a)-图15(h)：
印记基因Z1的印记缺失和拷贝数异常在恶性潜能阶段迅速上升，在肺癌的发展过程中维持在很高的水平；印记基因Z1的表达量增加在恶性潜能阶段迅速上升，但是在早期到晚期肺癌中不继续增加。印记基因Z16的印迹缺失在早期肺癌中迅速上升，在中晚期肺癌中维持在很高的水平；印记基因Z16的拷贝数异常和表达量增加在中期肺癌中迅速上升，到晚期肺癌中维持在较高的水平。
印记基因Z3的印迹缺失在恶性潜能阶段开始出现，在早期和中期肺癌中没有明显上升，到晚期肺癌中迅速上升到很高的水平；印记基因Z3的拷贝数异常在中期肺癌中迅速上升到很高的水平，在晚期肺癌中维持稳定；印记基因Z3的表达量增加在中期肺癌中出现，但是在晚期肺癌中没有明显增加。印记基因Z8的印记缺失和拷贝数异常在恶性潜能阶段开始出现，在早期肺癌中继续上升，中期肺癌中维持在很高的水平，但是在晚期肺癌中出现一定程度的下降；印记基因Z8的表达量增加在早期和中期肺癌中出现，晚期肺癌中Z8的表达量又恢复到较低水平。印记基因Z13的印记缺失和拷贝数异常在中期肺癌中迅速上升，并在晚期肺癌中维持很高的水平；印记基因Z13的表达量增加在晚期肺癌中出现轻微的增加。印记基因Z4的印记缺失在恶性潜能阶段迅速上升，在早期到晚期肺癌中维持很高的水平；印记基因Z4的拷贝数 异常在早期肺癌中开始出现，中期肺癌中没有明显上升，到晚期肺癌阶段又继续上升到较高水平；印记基因Z4的表达量增加在恶性潜能阶段开始出现，但是随肺癌的发展没有显著增加。印记基因Z10的拷贝数异常在恶性潜能阶段迅速上升，在早期到晚期肺癌中维持较高的水平；印记基因Z10的印记缺失在中期肺癌中迅速上升，在晚期肺癌中达到较高的水平；在肺癌的发展过程中印记基因Z10没有出现明显的表达量增加。印记基因Z11的印记缺失和拷贝数异常在恶性潜能阶段迅速上升，在早期和中期肺癌中没有明显上升，但是晚期肺癌阶段又继续上升到较高的水平；印记基因Z11的表达量在晚期肺癌中才有轻微的上升。
实施例6 70例尿液脱落细胞的印记基因分析
从泌尿系统肿瘤患者的尿液中获取细胞样本，其他检测方法同实施例1，检测结果如图16(a)-图16(b)、图17(a)-图17(d)和图18(a)-图18(k)所示。
从图16(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17％和/或印记基因拷贝数异常表达量小于2％为0级，印记基因缺失表达量为17-20％和/或印记基因拷贝数异常表达量为2-3％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3-7％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7-12％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12％为IV级。
从图16(b)可以看出，对于所述印记基因Z16，印记基因缺失表达量小于17％和/或印记基因拷贝数异常表达量小于2％为0级，印记基因缺失表达量为17-20％和/或印记基因拷贝数异常表达量为2-3％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3-7％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7-12％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12％为IV级。
从这70例尿液脱落细胞样本综合分析可以得出：
判断泌尿系统肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
判断泌尿系统肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为泌尿系统癌潜能；
判断泌尿系统肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期泌尿系统癌；
判断泌尿系统肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期泌尿系统癌；
判断泌尿系统肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期泌尿系统癌。
从图17(a)-图17(d)可以看出，Z1，Z2，Z3，Z4，Z5，Z6，Z7，Z8，Z9，Z10，Z11，Z12，Z13，Z14，Z15，Z16每个基因对泌尿系统癌症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，Z1、Z16、Z2、Z3、Z10对泌尿系统肿瘤的敏感度高。
具体每个印记基因对泌尿系统癌症的敏感度如图18(a)-图18(k)：
印记基因Z1的印记缺失在恶性潜能阶段开始出现，在早期泌尿系统癌症中继续上升，在中晚期泌尿系统癌症中维持在较高的水平；印记基因Z1的拷贝数异常在恶性潜能阶段迅速上升，在中期到晚期泌尿系统癌症中进一步上升到很高水平。印记基因Z16的印记缺失在早期泌尿系统癌症中开始出现，在中晚期泌尿系统癌症中不再明显增加；印记基因Z16的拷贝数异常在早期泌尿系统癌症阶段开始出现，随泌尿系统癌症的发展逐渐上升到很高水平。
印记基因Z2的印记缺失在中期泌尿系统癌症中开始出现，晚期泌尿系统癌症中达到较高水平；印记基因Z2的拷贝数异常在恶性潜能阶段开始出现，随泌尿系统癌症的发展逐渐上升到很高水平。印记基因Z3的印记缺失在中期泌尿系统癌症中开始出现，晚期泌尿系统癌症中继续上升，但水平不高；印记基因Z3的拷贝数异常在恶性潜能阶段开始出现，随泌尿系统癌症的发展逐渐上升到很高水平。印记基因Z10的印记缺失在中期泌尿系统癌症中开始出现，在晚期泌尿系统癌症中迅速上升到较高水平；印记基因Z10的拷贝数异常在早期泌尿系统癌症阶段开始出现，随泌尿系统癌症的发展逐渐上升到较高水平。印记基因Z4的印记缺失在中期泌尿系统癌症中开始出现，晚期泌尿系统癌症中继续上升，但水平不高；印记基因Z4的拷贝数异常在恶性潜能阶段开始出现，随癌症的发展逐渐上升到较高水平。印记基因Z5的印记缺失在中期泌尿系统癌症中开始出现，晚期癌症中继续上升，但水平不高；印记基因Z5的拷贝数异常在恶性潜能阶段开始出现，在早期癌症中迅速上升，在中晚期泌尿系统癌症中维持较高的水平。印记基因Z6的拷贝数异常在恶性潜能阶段开始出现，到晚期癌症阶段才迅速上升到较高水平；印记基因Z6的印记缺失在晚期泌尿系统癌症中有轻微的上升。印记基因Z8和Z9的印记缺失和拷贝数异常在中期泌尿系统癌症中开始出现，在晚期泌尿系统癌症中迅速上升到较高的水平。印记基因Z15的拷贝数异常在中期泌尿系统癌症中开始出现，在晚期癌症中迅速上升到较高的水平；印记基因Z15的印迹缺失在晚期泌尿系统癌症中出现较高水平的增加。
实施例7 肠镜活检细胞的印记基因分析
在肠镜下获取活检样本，其他检测方法同实施例1，结果如图图19(a)-图19(c)所示。
从图19(a)-图19(c)可以看出，图19(a)为良性结直肠息肉，图19(b)为恶性潜能的结直肠肿瘤，图19(c)为结直肠癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例8 膀胱镜活检细胞的印记基因分析
在膀胱镜下获取活检样本，其他检测方法同实施例1，结果如图20(a)-图20(c)所示。
从图20(a)-图20(c)可以看出，图20(a)为良性膀胱肿瘤，图20(b)为恶性潜能的膀胱肿瘤，图20(c)为膀胱癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例9 痰液脱落细胞的印记基因分析
从肺肿瘤患者的痰液中获取细胞样本，其他检测方法同实施例1，结果如图图21(a)-图21(b)所示。
从图21(a)-图21(b)可以看出，图21(a)为良性肺肿瘤，图21(b)为肺癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例10 肝穿刺活检细胞的印记基因分析
通过穿刺获取肝穿刺细胞样本，其他检测方法同实施例1，结果如图22(a)-图22(b)所示。
从图22(a)-图22(b)可以看出，图22(a)为良性肝肿瘤，图22(b)为肝癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例11 前列腺穿刺活检细胞的印记基因分析
通过穿刺获取前列腺活检样本，其他检测方法同实施例1，结果如图23(a)-图23(b)所示。
从图23(a)-图23(b)可以看出，图23(a)为良性前列腺肿瘤，图23(b)为前列腺癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例12 淋巴结穿刺活检细胞的印记基因分析
通过穿刺获取乳腺肿瘤附近的淋巴结穿刺细胞样本，其他检测方法同实施例1，结果如图24(a)-图24(b)所示。
从图24(a)-图24(b)可以看出，图24(a)为未转移的良性乳腺肿瘤附近的淋巴结穿刺细胞，图24(b)为转移的乳腺癌附近的淋巴结穿刺细胞，在良性肿瘤附近的淋巴结中只有个别印记缺失的细胞，没有发现拷贝数异常的癌细胞，在转移的乳腺癌附近的淋巴结中存在大量印记缺失和拷贝数异常的癌细胞。
实施例13 胸水细胞的印记基因分析
通过穿刺获取胸水细胞样本，其他检测方法同实施例1，结果如图25(a)-图25(b)所示。
从图25(a)-图25(b)可以看出，图25(a)为良性肺肿瘤患者的胸水细胞，图25(b)为肺癌患者的胸水细胞，在良性肺肿瘤患者的胸水中只有个别印记缺失的细胞，没有发现拷贝数异常的癌细胞，在肺癌患者的胸水中存在大量印记缺失和拷贝数异常的癌细胞。
实施例14 粪便脱落细胞的印记基因分析
从结直肠肿瘤患者的粪便中获取细胞样本，其他检测方法同实施例1，结果如图26(a)-图26(b)所示。
从图26(a)-图26(b)可以看出，图26(a)为良性结直肠肿瘤，图26(b)为结直肠癌，随着肿瘤恶性程度的增加，印记缺失细胞比例和拷贝数异常细胞比例都逐渐上升。
实施例15 血液细胞的印记基因分析
从健康人和白血病患者的外周血中获取细胞样本，使用红细胞裂解液去除红细胞，其他检测方法同实施例1，结果如图27(a)-图27(b)所示。
从图27(a)-图27(b)可以看出，图27(a)为健康人的血液细胞，图27(b)为白血病患者的血液细胞，健康人的血液中只有个别印记缺失的细胞，没有发现拷贝数异常的细胞，白血病患者的血液中存在大量印记缺失和拷贝数异常的细胞。
综上所述，本申请所述方法以直观的方法表现了印记缺失在甲状腺、乳腺、胰腺、前列腺、淋巴结穿刺细胞样本，肺支气管毛刷细胞样本，尿液、痰液、粪便、胸水细胞样本和肠镜、膀胱镜活检细胞样本上的表现，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，为甲状腺癌、乳腺癌、胰腺癌、肺癌、肝癌、结直肠癌、泌尿系统癌症的早期诊断做出巨大贡献。
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 (11)

  1. 一种通过活检样本检测癌症的方法，其通过计算印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总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Z1和/或Z16，所述印记基因Z1为Gnas，所述印记基因Z16为Snrpn/Snurf。
  2. 根据权利要求1所述的方法，其中，所述印记基因还包括Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9、Z10、Z11、Z12、Z13、Z14或Z15中的任意一个或至少两个的组合；其中，所述印记基因Z2为Igf2，印记基因Z3为Peg10，所述印记基因在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Z7为Cdkn1c，所述印记基因Z8为Dcn，所述印记基因Z9为Dlk1，所述印记基因Z10为Gatm，所述印记基因Z11为Grb10，所述印记基因Z12为Peg3，所述印记基因Z13为Sgce，所述印记基因Z14为Slc38a4，所述印记基因Z15为Diras3。
  3. 根据权利要求1或2所述的方法，其中，所述计算印记基因缺失基因表达量和印记基因拷贝数异常基因表达量的公式如下：
    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。
  4. 根据权利要求1-3中任一项所述的方法，其中，所述印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总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Z1和Z6两个印记基因的印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和总表达量分别进行划分的五个不同的等级；
    优选地，所述针对Z1和Z16的印记基因缺失基因表达量、印记基因拷贝数异常基因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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％或所述印记基因Z1和Z16的总表达量小于25％中的任意一种或至少两种的组合；
    I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1和Z16的总表达量为25-30％中的任意一种或至少两种的组合；
    II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1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和Z16的印记基因缺失表达量为25-35％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1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和Z16的印记基因缺失表达量大于35％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％或所述印记基因Z1和Z16的总表达量大于50％中的任意一种或至少两种的组合。
  5. 根据权利要求1-4中任一项所述的方法，其中，所述肿瘤包括甲状腺肿瘤、乳腺肿瘤、胰腺肿瘤、肺肿瘤、肝脏肿瘤、结直肠肿瘤、膀胱肿瘤、前列腺肿瘤、胃肿瘤、食道肿瘤、鼻咽部肿瘤、口腔肿瘤、卵巢肿瘤、子宫内膜肿瘤、子宫颈肿瘤、泌尿系统肿瘤、中枢神经系统肿瘤、腮腺肿瘤、淋巴瘤或白血病中的任意一种或至少两种的组合；
    优选地，对于甲状腺肿瘤，印记基因Z1的缺失表达量、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的印记基因缺失表达量小于15％、所述印记基因Z1的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z1的总表达量小于40％；
    I级：所述印记基因Z1的印记基因缺失表达量为15-20％、所述印记基因Z1的印记基因拷贝数异常表达量为1.5-4％或所述印记基因Z1的总表达量为40-45％；
    II级：所述印记基因Z1的印记基因缺失表达量为20-30％、所述印记基因Z1的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1的总表达量为45-60％；
    III级：所述印记基因Z1的印记基因缺失表达量为30-40％、所述印记基因Z1的印记基因拷贝数异常表达量为8-15％或所述印记基因Z1的总表达量为60-65％；
    IV级：所述印记基因Z1的印记基因缺失表达量大于40％、所述印记基因Z1的印记基因拷贝数异常表达量大于15％或所述印记基因Z1的总表达量大于65％；
    对于甲状腺肿瘤，印记基因Z16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Z16的印记基因缺失表达量小于15％、所述印记基因Z16的印记基因拷贝数异常表达量小于1.5％或所述印记基因Z16的总表达量小于30％；
    I级：所述印记基因Z16的印记基因缺失表达量为15-20％、所述印记基因Z16的印记基因拷贝数异常表达量为1.5-4％或所述印记基因Z16的总表达量为30-35％；
    II级：所述印记基因Z16的印记基因缺失表达量为20-30％、所述印记基因Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z16的总表达量为35-50％；
    III级：所述印记基因Z16的印记基因缺失表达量为30-40％、所述印记基因Z16的印记基因拷贝数异常表达量为8-15％或所述印记基因Z16的总表达量为50-55％；
    IV级：所述印记基因Z16的印记基因缺失表达量大于40％、所述印记基因Z16的印记基因拷贝数异常表达量大于15％或所述印记基因Z16的总表达量大于55％；
    优选地，对于乳腺肿瘤，印记基因Z1和Z16的缺失表达量、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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1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和Z16的总表达量小于25％；
    I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1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和Z16的总表达量为25-30％；
    II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-7％或所述印记基因Z1和Z16的总表达量为30-40％；
    III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为7-10％或所述印记基因Z1和Z16的总表达量为40-50％；
    IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于10％或所述印记基因Z1和Z16的总表达量大于50％；
    优选地，对于胰腺肿瘤，印记基因Z1和Z16的缺失表达量、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和Z16的印记基因缺失表达量小于15％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％或所述印记基因Z1和Z16的总表达量小于20％；
    I级：所述印记基因Z1和Z16的印记基因缺失表达量为15-20％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1和Z16的总表达量为20-30％；
    II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1和Z16的总表达量为30-40％；
    III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1和Z16的总表达量为40-50％；
    IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％、所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％或所述印记基因Z1和Z16的总表达量大于50％；
  6. 根据权利要求1-5中任一项所述的方法，其中，对于肺肿瘤，印记基因Z1的缺失表达量、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的印记基因缺失表达量小于15％、所述印记基因Z1的印记基因拷贝数异常表达量小于2％或所述印记基因Z1的总表达量小于30％；
    I级：所述印记基因Z1的印记基因缺失表达量为15-20％、所述印记基因Z1的印记基因拷贝数异常表达量为2-4％或所述印记基因Z1的总表达量为30-40％；
    II级：所述印记基因Z1的印记基因缺失表达量为20-25％、所述印记基因Z1的印记基因拷贝数异常表达量为4-8％或所述印记基因Z1的总表达量为40-50％；
    III级：所述印记基因Z1的印记基因缺失表达量为25-30％、所述印记基因Z1的印记基因拷贝数异常表达量为8-12％或所述印记基因Z1的总表达量为50-60％；
    IV级：所述印记基因Z1的印记基因缺失表达量大于30％、所述印记基因Z1的印记基因拷贝数异常表达量大于12％或所述印记基因Z1的总表达量大于60％；
    对于肺肿瘤，印记基因Z16的缺失表达量、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Z16的印记基因缺失表达量小于10％、所述印记基因Z16的印记基因拷贝数异常表达量小于1％、所述印记基因Z16的总表达量小于25％；
    I级：所述印记基因Z16的印记基因缺失表达量为10-15％、所述印记基因Z16的印记基因拷贝数异常表达量为1-2％或所述印记基因Z16的总表达量为25-30％；
    II级：所述印记基因Z16的印记基因缺失表达量为15-20％、所述印记基因Z16的印记基因拷贝数异常表达量为2-5％或所述印记基因Z16的总表达量为30-40％；
    III级：所述印记基因Z16的印记基因缺失表达量为20-25％、所述印记基因Z16的印记基因拷贝数异常表达量为5-8％或所述印记基因Z16的总表达量为40-50％；
    IV级：所述印记基因Z16的印记基因缺失表达量大于25％、所述印记基因Z16的印记基因拷贝数异常表达量大于8％或所述印记基因Z16的总表达量大于50％；
    优选地，对于泌尿系统肿瘤，印记基因Z1和Z16的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Z1和Z16的印记基因缺失表达量小于17％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量小于2％；
    I级：所述印记基因Z1和Z16的印记基因缺失表达量为17-20％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量为2-3％；
    II级：所述印记基因Z1和Z16的印记基因缺失表达量为20-25％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量为3-7％；
    III级：所述印记基因Z1和Z16的印记基因缺失表达量为25-30％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量为7-12％；
    IV级：所述印记基因Z1和Z16的印记基因缺失表达量大于30％和/或所述印记基因Z1和Z16的印记基因拷贝数异常表达量大于12％。
  7. 根据权利要求1-6中任一项所述的方法，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状态；
    其中，所述分析单元通过计算印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和印记基因总表达量，从而通过印记基因缺失基因表达量、印记基因拷贝数异常基因表达量和印记基因总表达量 的等级来判断肿瘤的良恶性程度。
  8. 根据权利要求1-7中任一项所述的方法，其中，步骤(1)所述的待测样本为人的组织和/或细胞；
    优选地，所述待测样本包括穿刺活检细胞样本、活检细胞样本、脱落细胞样本、血液样本或刷检样本中的任意一种或至少两种的组合；
    优选地，所述穿刺活检细胞样本包括细针和/或粗针穿刺活检细胞样本，优选为甲状腺、乳腺、胰腺、肺、肝脏、前列腺、卵巢、淋巴结或腮腺的细针和/或粗针穿刺活检细胞样本中的任意一种或至少两种的组合；
    优选地，所述活检细胞样本包括胃镜、肠镜、膀胱镜、宫腔镜或耳鼻咽喉镜活检细胞样本中的任意一种或至少两种的组合；
    优选地，所述脱落细胞样本包括尿液、痰液、粪便或胸腹腔积液脱落细胞样本中的任意一种或至少两种的组合；
    优选地，所述刷检样本包括支气管、食道、口腔或子宫颈的刷检样本中的任意一种或至少两种的组合。
  9. 根据权利要求1-8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；
    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；
  10. 根据权利要求1-9中任一项所述的方法，其中，待判断的肿瘤的良恶性程度分为良性、癌潜能、早期癌、中期癌和晚期癌；
    优选地，判断肿瘤的良恶性程度的结果为印记基因Z1和Z16的印记基因缺失基因表达量和印记基因拷贝数异常基因表达量均小于I级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量为I级或印记基因Z1或Z16中仅有1个印记基因的拷贝数表达量为I级中的任意一种情况则为良性肿瘤；
    优选地，判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中2个基因的印记基因缺失基因表达量为I级、印记基因Z1和Z16中2个基因的印记基因拷贝数异常基因表达量为I级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为I级并有1个印记基因的拷贝数异常基因表达量为I级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为II级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到II级中的任意一种情况，则为癌潜能；
    优选地，判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失基因表达量为II级和/或印记基因Z1和Z16中的2个基因的印记基因拷贝数异常表达量为II级，印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为II级并有1个印记基因的拷贝数异常基因表达量为II级、印记基因Z1或Z16中仅有1个基因的印记基因缺失基因表达量为III级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数异常基因表达量达到III级中的任意一种情况，则为早期癌；
    优选地，判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为III级、印记基因Z1和Z16中的2个基因的印记基因拷贝数异常基因表达量为III级、印记基因Z1和Z16中有1个印记基因的印记基因缺失基因表达量为III级并有1个印记基因的拷贝数异常基因表达量为III级、印记基因Z1或Z16中仅有1个印记基因的印记基因缺失表达量IV级或印记基因Z1或Z16中仅有1个基因的印记基因拷贝数表达量为IV级中的任意一种情况，则为中期癌；
    优选地，判断肿瘤的良恶性程度的结果为印记基因Z1和Z16中的2个基因的印记基因缺失表达量为IV级和/或印记基因Z1和Z16的2个基因的印记基因拷贝数异常表达量为IV级，则为晚期癌。
  11. 一种如权利要求1-10中任一项所述的方法用于肿瘤检测和/或治疗中的用途。
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