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WO2019105381A1 - Modèle de classification pour la détection de degrés bénins et malins de tumeurs du sein et application associée - Google Patents

Modèle de classification pour la détection de degrés bénins et malins de tumeurs du sein et application associée Download PDF

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WO2019105381A1
WO2019105381A1 PCT/CN2018/117891 CN2018117891W WO2019105381A1 WO 2019105381 A1 WO2019105381 A1 WO 2019105381A1 CN 2018117891 W CN2018117891 W CN 2018117891W WO 2019105381 A1 WO2019105381 A1 WO 2019105381A1
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imprinted
gene
imprinted gene
genes
copy number
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成彤
周宁
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Lisen Imprinting Diagnostics Inc
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Lisen Imprinting Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present disclosure relates to the field of biotechnology, such as the field of genetic diagnosis, such as a hierarchical model and its application, such as a hierarchical model for detecting the degree of benign and malignant breast tumors and its application, such as a group of imprinted genes in detecting benign and malignant breast tumors.
  • a hierarchical model of the degree and its composition such as a hierarchical model of the degree and its composition.
  • Breast cancer is one of the major malignant tumors that threaten human health and life. About 1.4 million people worldwide are diagnosed with breast cancer every year, and its incidence is still growing at a rate of 3-4% per year, and about 500,000 people die from the disease. Breast cancer accounts for 32% of all cancers in women and 15% of deaths, making it the second leading cause of death among women.
  • CN 106995837 A discloses an early diagnosis kit for breast cancer comprising a capture probe, an amplification probe and a labeling probe for detecting a target gene mRNA by using three molecular markers in combination: breast cancer screening gene, breast Cancer CTC marker gene and exclusion gene to achieve early diagnosis of breast cancer.
  • CN 1898563 A discloses a method for diagnosing breast cancer, specifically expressing three BRC-related genes, namely A5657, B9769 and C7965, by detecting the expression of a differential BRC-related gene between the three BRC cells and normal cells. Level to diagnose breast cancer.
  • the prior art detection methods are complicated to operate, and it is not possible to accurately distinguish which stage of breast tumor development.
  • 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. It has great limitations on the discovery of early changes in cells (cancer), so the method of cancer diagnosis at the cellular level has once become a research hotspot. With the continuous research in the field of molecular biology, more and more molecular detection techniques are applied to cancer diagnosis.
  • the production of cancer is an uncontrolled cell growth/splitting caused by epigenetic changes and genetic variations that accumulate over time.
  • Traditional pathological diagnosis is based on the size, morphology and structural variation of cells and tissues, thereby making a benign and malignant judgment of breast tumors.
  • molecular detection techniques With the development and deepening of molecular biology, more and more molecular detection techniques have been applied to the detection of breast cancer. From the analysis of the development of cancer, molecular changes (epigenetics and genetics) are much earlier than changes in cell morphology and tissue structure. Therefore, molecular biology testing is more sensitive to early detection of cancer.
  • the current diagnosis of breast cancer requires a new detection system and detection model, based on the patient biopsy sample, to analyze the molecular markers present at the cell level of breast cancer, in order to provide more accurate pre-diagnosis and diagnostic information.
  • the present disclosure provides an imprinted gene grading model and diagnostic method and uses thereof.
  • the present disclosure provides an imprinted gene grading model for breast tumors, which is obtained by calculating a total expression amount of an imprinted gene, an imprinted gene deletion expression amount, and an imprinted gene copy number abnormal expression amount in breast cancer.
  • the change ranks the expression status of the imprinted gene;
  • the imprinting gene is any one or a combination of at least two of Z1, Z3, Z8, Z11 or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z3 is Peg10, and the imprinting gene Z8 is Dcn
  • the imprinted gene Z11 is Grb10, and the imprinted gene Z16 is Snrpn/Snurf.
  • the imprinted (trace) deletion refers to the activation (demethylation) of the allele in the imprinted (spot) gene that was originally in a silent state, and is the most common and early epigenetic change in cancer, and This property can be used as a pathological marker.
  • the proportion of imprinted deletions is very low, and the imprinted gene and the imprinted gene are simultaneously a concept, indicating the same meaning, and can be replaced.
  • the inventors found that by calculating the imprinted gene deletion expression amount and the abnormal expression level of the imprinted gene copy number of any one of Z1, Z3, Z8, Z11 and Z16 in the breast tumor, the sensitivity of diagnosis to breast cancer can reach 80.9% or more. .
  • any one of Z1, Z3, Z8, Z11, and Z16 can be detected if a preliminary detection detects only one imprinted gene.
  • any one of Z1, Z8 or Z16 can be detected if a preliminary detection detects only one imprinted gene.
  • the Z8 or Z11 imprinting gene can be detected if the initial detection detects only one imprinted gene.
  • the inventors found that if a Z1 imprinted gene is detected alone, the sensitivity of diagnosis to breast cancer can reach 91.3%. If a Z3 imprinted gene is detected alone, the sensitivity for diagnosis of breast cancer can reach 80.9%. If a Z8 imprinted gene is detected alone, the sensitivity of diagnosis to breast cancer can reach 97.8%. If a Z11 imprinted gene is detected alone, the sensitivity of diagnosis to breast cancer can reach 97.8%. If a Z16 imprinted gene is detected separately, The diagnostic sensitivity of breast cancer can reach 88.6%.
  • the method for calculating the imprinted gene is: if detecting a combination of two imprinted genes of the imprinted gene, the combination may be any two of Z1, Z3, Z8, Z11 and Z16, preferably It is a combination of Z1 and Z8, a combination of Z8 and Z11, a combination of Z1 and Z16 or a combination of Z11 and Z16.
  • the inventors have found that by calculating the total expression amount of two or more imprinted genes, the amount of imprinted gene deletion and the abnormal expression level of imprinted gene copy number, the sensitivity can be further improved, and the imprinting genes Z1, Z3, Z8, Z11 and Z16 are detected.
  • the combination of any two imprinted genes can be more than 97.6% sensitive for the diagnosis of breast cancer.
  • the diagnostic sensitivity of breast cancer can reach more than 99.0%.
  • the imprinting gene further comprises any one or a combination of at least two of Z5, Z9, Z10 or Z13; wherein the imprinting gene Z5 is Mest and the imprinting gene Z9 is Dlk1.
  • the imprinted gene Z10 is Gatm, and the imprinted gene Z13 is Sgce.
  • the model calculates the imprinted gene by calculating a combination of imprinted genes and calculating a combination of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 genes.
  • the imprinted gene is deleted after the cells are subjected to hematoxylin staining, and 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 two or more red/brown marks in the nucleus.
  • 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 formula for calculating the total expression amount of the imprinted gene, the expression amount of the imprinted gene, and the abnormal expression amount of the imprinted gene are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell; and b is a red/brown mark in the nucleus after the hematoxylin staining of the cell, and the imprinting gene exists.
  • the nucleus; the c is a hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; and the d is a hematoxylin staining of the cells, and there are more than two red/brown marks in the nucleus.
  • imprinted gene copy number abnormal cell nucleus is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell
  • b is a red/brown mark in the nucleus after the hematoxylin staining of the cell,
  • the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for imprinting gene expression, imprinted gene deletion expression, and imprinted gene copy number abnormal expression. Calculation.
  • the probe is amplified by in situ hybridization, and Hemotoxy (hematoxylin) nuclei are used to amplify the signal, and under the 40 ⁇ or 60 ⁇ microscope, the presence of the imprinted gene in each nucleus, the imprinted gene is deleted, or The copy number is abnormal, and the degree of benign and malignant tumors of the sample is determined by calculating the gene expression amount of the imprinted gene deletion gene expression and the imprinted gene copy number abnormality. Since the section is only 10 ⁇ m, about 20% of the nuclei seen under the microscope are incomplete nuclei, which means that there is a possibility of partial false negatives.
  • the total expression amount of the imprinted gene, the amount of the imprinted gene and the abnormal expression of the imprinted gene are divided into five different levels, and each probe has at least 1200 in the region where the sample is most positively expressed.
  • the cells were counted, and the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total imprinted gene expression amount of the nine imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 were respectively performed. Five different levels of division.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of Z1 are:
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is less than 15%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is less than 1%, or the total expression amount of the imprinted gene Z1 is less than 20% One or a combination of at least two;
  • 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 1-2%, or the total expression amount of the imprinted gene Z1 is 20 Any one of -30% or a combination of at least two;
  • 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 2-3%, or the total expression amount of the imprinted gene Z1 is 30 Any one of -40% or a combination of at least two;
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is 3-5%, or the total expression level of the imprinted gene Z1 is 40%. -50% of any one or a combination of at least two;
  • 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 5%, or the total expression amount of the imprinted gene Z1 is greater than 50% One or a combination of at least two.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount for Z3, Z5, Z9, Z10, and Z13 are:
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene deletion expression of less than 10%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene of less than 0.5%. Or the total expression level of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is less than 15% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 10-15%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene copy number.
  • 0.5-1% or a total expression amount of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is any one of 15-20% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 15-20%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression level of the imprinted gene copy number. 1-2.5% or a total expression amount of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is any one of 20-30% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 20-25%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have abnormal expression levels of imprinted gene copy number. 2.5-4% or a total expression amount of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is any one of 30-40% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level greater than 25%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene greater than 4%. Or the total expression amount of the imprinted genes Z3, Z5, Z9, Z10 and Z13 is greater than 40% 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 Z3, Z5, Z9, Z10, and Z13 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount for Z8 and Z11 are:
  • the imprinted gene deletion expression amount of the imprinted genes Z8 and Z11 is less than 10%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z8 and Z11 is less than 1% or the total expression amount of the imprinted genes Z8 and Z11 Any one of less than 15% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 10-15%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 1-2% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 15-20% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 15-20%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 2-3% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 20-30% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 20-25%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 3-5% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 30-40% or a combination of at least two;
  • Grade IV the imprinted gene deletion expression amount of the imprinted genes Z8 and Z11 is greater than 25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z8 and Z11 is greater than 5% or the total expression amount of the imprinted genes Z8 and Z11 Any one of more than 40% 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 Z8 and Z11 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of Z16 are:
  • 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%, or the imprinted gene Z16 has a total expression amount of less than 20%.
  • 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 1-2% or the imprinted gene Z16 has a total expression of 20%. Any one of -30% or a combination of at least two;
  • 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 2-3% or the imprinted gene Z16 has a total expression of 30%. Any one of -40% or a combination of at least two;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 25-30%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 3-5% or the imprinted gene Z16 has a total expression of 40%. -50% of any one or a combination of at least two;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z16 is greater than 30%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z16 is greater than 5%, or the total expression amount of the imprinted gene Z16 is greater than 50% One or a combination of at least two.
  • the imprinted gene Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 have abnormal expression and copy number abnormal expression. There is a 20% up and down fluctuation in the grading criteria for the amount and total expression, which is also within the scope of the present application.
  • the present disclosure provides a device for detecting the degree of benign and malignant breast tumors, which employs the above model, comprising the following elements:
  • sampling unit obtaining a sample to be tested
  • Probe design unit design specific primers according to the imprinted gene sequence
  • the analysis unit calculates the total expression amount of the imprinted gene, the amount of the imprinted gene, and the abnormal expression amount of the imprinted gene, and the abnormal expression level of the imprinted gene and the abnormal copy number of the imprinted gene are obtained by the imprinted gene grading model.
  • the level of expression is used to determine the degree of benign and malignant breast tumors.
  • the imprinted gene is deleted after the cells are subjected to hematoxylin staining, and there are two red/brown labeled nuclei in the nucleus.
  • the abnormal copy number of the imprinted gene is that after the cells are subjected to hematoxylin staining, there are more than two red/brown marks in the nucleus.
  • 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 total expression of imprinted gene, amount of imprinted gene deletion, and abnormal expression level of imprinted gene copy number.
  • the detection device is used for early and intuitive observation of changes in the imprinted genes of breast tumors at the cellular and tissue levels to determine the degree of benign and malignant tumors, and to provide the most favorable treatment opportunity for patients with early breast tumors.
  • the present disclosure provides a method of detecting the degree of benign and malignant breast tumors, using the model or the device, comprising the steps of:
  • the analysis unit calculates the amount of expression of the imprinted gene, the abnormal expression level of the imprinted gene copy number, and the total expression amount, and the abnormal expression level of the imprinted gene and the imprinted gene copy number are expressed by the imprinted gene grading model.
  • the grade to diagnose the degree of benign and malignant breast tumors.
  • the sample to be tested described in step (1) is derived from human tissues and/or cells.
  • the sample to be tested is feasible as long as the RNA is subjected to a timely and fixed treatment, and can be selected by a person skilled in the art as needed, and is not particularly limited herein.
  • the sample to be tested according to the present invention includes a paraffin section of the tissue and a biopsy sample. Any one or a combination of at least two.
  • the specific operation procedure of the paraffin section of the tissue is to obtain a human tumor tissue sample, which is fixed in 10% neutral formalin in time, embedded in paraffin, cut into 10 ⁇ m thick, and made into a tissue film with a positively charged slide; Only 10 ⁇ m thick, so some of the microscopically seen nuclei are incomplete, so some false negative gene deletions will occur.
  • the specific operation procedure of the puncture biopsy sample is to obtain human cells by puncture, and timely fix with 10% neutral formalin.
  • the needle biopsy has less damage to the patient, the sampling process is simple. Compared with the circulation characteristics of the blood, the needle biopsy can be positioned.
  • the needle biopsy has special advantages as an experimental sample.
  • the sample to be tested is a needle biopsy sample.
  • the imprinting genes are Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z3 is Peg10, and the imprinting gene Z5 is Mest, the imprinting gene Z8 is Dcn, the imprinting gene Z9 is Dlk1, the imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, the imprinting gene Z13 is Sgce, and the imprinting gene Z16 is Snrpn/Snurf.
  • the imprinted genes Z1 (Gnas), Z3 (Peg10), Z5 (Mest), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce), Z16 (Snrpn/Snurf) There are different degrees of expression in normal tumor cell tissues, and the expression and imprinting state will change significantly when malignant lesions occur.
  • the design probe is designed according to the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16, namely Gnas, Peg10, Mest, Dcn, Dlk1, Gatm, Grb10, Sgce and Snrpn/Snurf. Specifically, a sequence was selected as a probe in the inner loop of each gene, and the specific probe was designed by Advanced Cell Diagnostics.
  • the in situ hybridization employs an RNAscope in situ hybridization method.
  • the RNAscope in situ hybridization method uses a single or multi-channel colorimetric kit or a single or multi-channel fluorescent kit, preferably a single channel red/brown color kit or multiple channels. Fluorescent kit.
  • the multi-channel coloring kit or the multi-channel fluorescent kit comprises two or more channels of coloring kits or fluorescent kits, and the two-channel coloring kit or multi-channel fluorescent kit can use two The combined expression of imprinted gene probes or imprinted genes and other genes is even the combined expression of multiple imprinted genes and non-imprinted genes.
  • the formula for calculating the total expression amount of the imprinted gene, the amount of the imprinted gene, and the abnormal expression amount of the imprinted gene in the model are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell; and b is a red/brown mark in the nucleus after the hematoxylin staining of the cell, and the imprinting gene exists.
  • the nucleus; the c is a hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; and the d is a hematoxylin staining of the cells, and there are more than two red/brown marks in the nucleus.
  • imprinted gene copy number abnormal cell nucleus is a cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell
  • b is a red/brown mark in the nucleus after the hematoxylin staining of the cell,
  • the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for calculation of the total expression level of the imprinted gene, the amount of imprinted gene deletion, and the abnormal expression level of the imprinted gene copy number.
  • the probe was amplified by in situ hybridization, and Hemotoxy (hematoxylin) nuclei were used to amplify the signal, and under the 40 ⁇ or 60 ⁇ microscope, the presence of imprinted genes, imprinted gene deletion or copy number abnormality in each nucleus was determined, and the imprint was calculated.
  • the total gene expression level, the amount of the imprinted gene deletion gene, and the gene expression amount of the imprinted gene copy number abnormality were used to determine the degree of benign and malignant tumors of the sample. Since the section is only 10 microns, about 20% of the nuclei seen under the microscope are incomplete nuclei, which means that there is a possibility of partial false negatives.
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount are divided into five different levels.
  • the five different grades count at least 1200 cells in the region where the probe is most positive for each probe, and nine imprinted genes for Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16
  • the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount were separately classified.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount for Z1 are:
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is less than 15%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is less than 1%, or the total expression amount of the imprinted gene Z1 is less than 20% One or a combination of at least two;
  • 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 1-2%, or the total expression amount of the imprinted gene Z1 is 20 Any one of -30% or a combination of at least two;
  • 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 2-3%, or the total expression amount of the imprinted gene Z1 is 30 Any one of -40% or a combination of at least two;
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is 3-5%, or the total expression level of the imprinted gene Z1 is 40%. -50% of any one or a combination of at least two;
  • 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 5%, or the total expression amount of the imprinted gene Z1 is greater than 50% One or a combination of at least two.
  • the five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount for Z3, Z5, Z9, Z10, and Z13 are:
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene deletion expression of less than 10%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene of less than 0.5%. Or the total expression level of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is less than 15% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 10-15%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene copy number.
  • 0.5-1% or a total expression amount of the imprinted genes Z3, Z5, Z9, Z10 and Z13 is any one of 15-20% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 15-20%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression level of the imprinted gene copy number. 1-2.5% or a total expression amount of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is any one of 20-30% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level of 20-25%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have abnormal expression levels of imprinted gene copy number. 2.5-4% or a total expression amount of the imprinting genes Z3, Z5, Z9, Z10 and Z13 is any one of 30-40% or a combination of at least two;
  • the imprinted gene Z3, Z5, Z9, Z10 and Z13 have an imprinted gene expression level greater than 25%, and the imprinted genes Z3, Z5, Z9, Z10 and Z13 have an abnormal expression number of the imprinted gene greater than 4%. Or the total expression amount of the imprinted genes Z3, Z5, Z9, Z10 and Z13 is greater than 40% 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 Z3, Z5, Z9, Z10, and Z13 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount for Z8 and Z11 are:
  • the imprinted gene deletion expression amount of the imprinted genes Z8 and Z11 is less than 10%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z8 and Z11 is less than 1% or the total expression amount of the imprinted genes Z8 and Z11 Any one of less than 15% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 10-15%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 1-2% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 15-20% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 15-20%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 2-3% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 20-30% or a combination of at least two;
  • the imprinted gene Z8 and Z11 have an imprinted gene deletion expression of 20-25%, the imprinted genes Z8 and Z11 have an imprinted gene copy number abnormal expression of 3-5% or the imprinted genes Z8 and Z11
  • the total expression amount is any one of 30-40% or a combination of at least two;
  • Grade IV the imprinted gene deletion expression amount of the imprinted genes Z8 and Z11 is greater than 25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z8 and Z11 is greater than 5% or the total expression amount of the imprinted genes Z8 and Z11 Any one of more than 40% 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 Z8 and Z11 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of Z16 are:
  • 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%, or the imprinted gene Z16 has a total expression amount of less than 20%.
  • 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 1-2% or the imprinted gene Z16 has a total expression of 20%. Any one of -30% or a combination of at least two;
  • 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 2-3% or the imprinted gene Z16 has a total expression of 30%. Any one of -40% or a combination of at least two;
  • the imprinted gene Z16 has an imprinted gene deletion expression of 25-30%, the imprinted gene Z16 has an imprinted gene copy number abnormal expression of 3-5% or the imprinted gene Z16 has a total expression of 40%. -50% of any one or a combination of at least two;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z16 is greater than 30%, the imprinted gene copy number abnormal expression amount of the imprinted gene Z16 is greater than 5%, or the total expression amount of the imprinted gene Z16 is greater than 50% One or a combination of at least two.
  • the imprinted gene Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 have abnormal expression and copy number abnormal expression. There is a 20% up and down fluctuation in the grading criteria for the amount and total expression, which is also within the scope of the present application.
  • the determining the degree of benign and malignant breast tumors is classified into benign tumors, breast cancer potential, early breast cancer, metaphase breast cancer, and advanced breast cancer.
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 have abnormal expression levels of imprinted genes and abnormal copy number of imprinted genes.
  • the amount of expression of the imprinted gene whose expression amount is less than the level I or the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is not more than one imprinted gene is grade I and the imprinted genes Z1, Z3,
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade I, which is a benign tumor;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade I, imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade I or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinting gene in Z13 and Z16 is expressed in the level II and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16
  • the abnormal expression level of the gene copy number is in any of the second grade, which is the potential of breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is II, imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade II or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11
  • the imprinting gene with no more than one imprinted gene in Z13 and Z16 has a deletion level of III and the imprint of no more than one imprinted gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the grade III, it is early breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III, imprinted gene
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinted gene in Z13 and Z16 is expressed in the IV level and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the IV grades, it is a metaphase breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is IV or imprinted gene Z1
  • the abnormal expression level of the imprinted gene copy number of at least two imprinting genes in Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 is Grade IV, and is advanced breast cancer.
  • the present disclosure provides the imprinted gene grading model or the device for breast tumor detection.
  • the present disclosure provides the use of the model or device described for the preparation of a medicament or device for treating breast cancer.
  • the degree of benign and malignant diagnosis of breast tumors is classified into benign tumors, breast cancer potential, early breast cancer, metaphase breast cancer, and advanced breast cancer;
  • the results of the diagnosis of the degree of benign and malignant breast tumors are that the imprinted gene Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 have less than the level I or the abnormal expression of the imprinted gene copy number.
  • the imprinted gene of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 has an imprinted gene deletion level of I and the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z13 and Z16 is grade I, which is a benign tumor;
  • the result of the diagnosis of the degree of benign and malignant breast tumors is that the imprinted gene of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 has a deletion level of I, and the imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade I or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinting gene in Z13 and Z16 is expressed in the level II and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16
  • the abnormal expression level of the gene copy number is in any of the second grade, which is the potential of breast cancer;
  • the result of the diagnosis of the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade II, imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade II or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11
  • the imprinting gene with no more than one imprinted gene in Z13 and Z16 has a deletion level of III and the imprint of no more than one imprinted gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the grade III, it is early breast cancer;
  • the result of the diagnosis of the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III, imprinted gene
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinted gene in Z13 and Z16 is expressed in the IV level and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the IV grades, it is a metaphase breast cancer;
  • the result of the diagnosis of the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is IV or imprinted gene Z1
  • the abnormal expression level of the imprinted gene copy number of at least two imprinting genes in Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 is Grade IV, and is advanced breast cancer.
  • the detection model and the device are used to visually express the performance of the imprinted gene on the breast tumor patient sample, and the method for in situ labeling of the imprinted gene is objective and intuitive.
  • changes in the imprinted (trace) genes were accurately detected, and quantitative models were provided to make a significant contribution to the diagnosis of molecular pathology.
  • 1 is a pathological section of a breast cancer of a hematoxylin-stained nuclei according to the present invention, wherein the a is a hematoxylin-stained cell, and there is no label in the nucleus, and the imprinted gene is not expressed; and the b is a hematoxylin staining of the cell.
  • the imprinted gene There is a red/brown mark in the nucleus, and the imprinted gene is present; the c is the hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the imprinted gene is deleted; the d is the cell nucleus after hematoxylin staining There are more than two red/brown marks in memory, and the imprinted gene copy number is abnormal;
  • Fig. 2(a) shows the expression status of 9 genes in the pathological section of grade 0 breast tumor
  • Fig. 2(b) shows the expression status of 9 genes in the pathological section of grade I breast cancer
  • Fig. 2(c) is level II.
  • Fig. 2(d) shows the expression status of 9 genes in the pathological section of grade III breast cancer
  • Fig. 2(e) shows 9 cases in the pathological section of grade IV breast cancer.
  • Figure 3 (a) shows the intensity of imprinted deletions of breast cancer genes Z1, Z3, Z8, Z11 and Z16
  • Figure 3 (b) shows abnormal copy number of breast cancer by imprinting genes Z1, Z3, Z8, Z11 and Z16
  • the intensity of Figure 3(c) is the intensity of total expression of breast cancer genes Z1, Z3, Z8, Z11 and Z16
  • Figure 3 (d) is the imprint of imprinted genes Z5, Z9, Z10 and Z13 on breast cancer.
  • the intensity of deletion, Figure 3 (e) is the intensity of copy number abnormalities of imprinted genes Z5, Z9, Z10 and Z13 for breast cancer
  • Figure 3 (f) shows the total expression of imprinted genes Z5, Z9, Z10 and Z13 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 quantity of the imprinted gene copy number abnormality
  • TE is the total expression level of the imprinted gene;
  • Fig. 4(a) shows the intensity of the imprinting gene Z1 imprinting deletion, copy number abnormality and total expression amount
  • Fig. 4(b) shows the intensity of the imprinting gene Z3 imprinting deletion, copy number abnormality and total expression amount
  • FIG. 4(c) is The imprinting gene Z8 imprinted deletion, copy number abnormality and total expression intensity
  • Figure 4 (d) is the imprinting gene Z11 imprint deletion, copy number abnormality and total expression intensity
  • Figure 4 (e) is the imprinted gene Z16 imprint missing
  • the intensity of copy number abnormality and total expression level
  • Figure 4 (f) is the imprinting gene Z5 imprint deletion, copy number abnormality and total expression level intensity
  • Figure 4 (g) is the imprinted gene Z9 imprint deletion, copy number abnormality and total expression
  • Figure 4 (h) is the intensity of the imprinting gene Z10 imprint deletion, copy number abnormality and total expression amount
  • Figure 4 (i) is the intensity of the imprinting gene Z13 imprint deletion, copy number abnormality and total expression amount
  • 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 level of the imprinted gene
  • Fig. 5(a) shows the distribution range and grading standard of imprinting gene and Z1 in 80 cases of breast cancer pathological sections, and the imprinting gene Z3 is applied to 80 cases of breast cancer pathological sections.
  • the distribution range and grading standard of imprinting deletion and copy number abnormality Fig. 5(c) is the distribution range and grading standard of imprinting deletion and copy number abnormality in 80 cases of breast cancer pathological section, which is imprinted gene Z8, Fig. 5(d)
  • the imprinting gene Z11 was applied to 80 cases of breast cancer pathological sections, the distribution range and grading standard of imprinting deletion and copy number abnormality, and Fig.
  • FIG. 5(e) was the imprinting gene Z16 applied to 80 cases of breast cancer pathological sections, imprinting deletion and The distribution range and grading standard of copy number abnormality
  • Fig. 5(f) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 80 cases of breast cancer pathological section
  • the imprinting gene is shown in Fig. 5(g) Z9 was applied to the pathological sections of 80 cases of breast cancer, the distribution range and grading standard of imprinting deletion and copy number abnormality.
  • Figure 5(h) was applied to the pathological section of 80 cases of breast cancer with imprinting gene Z10, with imprinting deletion and copy number abnormality. Minute Cloth range and grading standard, Fig.
  • 5(i) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 80 cases of breast cancer pathological sections, in which imprinting gene Z13 is the imprinting gene deletion gene expression amount, CNV.
  • imprinting gene Z13 is the imprinting gene deletion gene expression amount, CNV.
  • TE is the total expression level of the imprinted gene;
  • 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. Numerous studies have shown that this phenomenon (missing of blots) is ubiquitous in various types of cancer and occurs earlier than cell and tissue morphological changes. At the same time, in healthy cells, the proportion of imprinted deletions is extremely low, in sharp contrast to cancer cells. Therefore, the methylation status of the imprinted gene can be used as a pathological marker to analyze the abnormal state of the cell by a specific molecular detection technique.
  • the detection model and device of the present disclosure express the impression of the imprinted defect on the sample of the breast tumor patient in an intuitive manner, and objectively, intuitively, earlyly and accurately detect the imprint by the method of in situ labeling of the imprinted gene. ) genetic changes and can provide quantitative models that make a significant contribution to the diagnosis of breast tumors;
  • the disclosed detection device can determine the degree of benign and malignant breast tumors by puncture cells before surgery in a breast tumor patient, thereby providing a basis for surgery and precise treatment, which is a revolutionary breakthrough in the diagnosis of breast tumors in the field of cell molecules;
  • the present disclosure can detect early breast cancer very sensitively, and the combined detection of imprinted genes can detect the onset of canceration in patients with precancerous lesions of the breast such as intraductal papilloma, atypical ductal hyperplasia, atypical lobular hyperplasia and the like.
  • the sample, and the breast puncture has less damage to the patient, and the process of taking the material is simple. It is used in early screening and post-operative follow-up of cancer, especially for follow-up and follow-up of suspected relapsed patients, which can gain time and make a significant contribution to saving the patient's life;
  • the present disclosure can accurately detect the benign and malignant tissues of the adjacent tissues, and has an important guiding significance for accurately determining the scope of surgical resection of breast cancer, and greatly reduces the postoperative recurrence of breast cancer.
  • the disclosed detection method is different from the immunohistochemical method, which reduces false positives and other negative effects, and not only the targeted drugs or techniques for silencing, culling, and rearrangement of the gene by the discovery of a breast tumor-associated imprinted gene deletion site.
  • the method can be used to guide the later treatment and medication.
  • the method for detecting the imprinted gene comprises the following steps:
  • FFPE paraffin embedding
  • Design probe design specific primers according to the imprinted gene sequence
  • the design probe is based on the imprinting genes Z1 (Gnas), Z3 (Peg10), Z5 (Mest), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 (Grb10), Z13 (Sgce), and Z16.
  • Z1 Gas
  • Z3 Peg10
  • Z5 Mest
  • Z8 Dcn
  • Z9 Dlk1
  • Z10 Gaatm
  • Z11 Grb10
  • Z13 Sgce
  • Z16 Z16.
  • the formula for calculating the total expression of the imprinted gene, the amount of the imprinted gene, and the abnormal expression of the imprinted gene in the model are as follows:
  • Imprinted gene deletion gene expression level (LOI) c / (b + c + d) ⁇ 100%;
  • a, b, c, and d are as shown in FIG. 1 , wherein a is a cell nucleus in which no hemoglobin is stained in the nucleus and the imprinted gene is not expressed; and b is a hematoxylin staining of the cell.
  • the c is the hematoxylin staining of the cell, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted;
  • the d is the hematoxylin After staining, there are more than two red/brown markers in the nucleus, and the nuclei with abnormal copy number of the imprinted gene are imprinted.
  • the breast biopsy sample is obtained by puncturing the suspicious lesion tissue, and the 10% neutral formalin solution is fixed for more than 24 hours.
  • the other detection methods are the same as those in the first embodiment.
  • each imprinted gene Z1 has an imprinted deletion and copy number abnormality. In the stage of malignant potential, it rises rapidly, and it rises rapidly in the early stage of breast cancer. In the middle and advanced stage of breast cancer, the rate of increase slows down and reaches a high level. The expression of imprinted gene Z1 increases gradually from the malignant potential to the development of advanced breast cancer.
  • imprinted gene Z3 imprinting deletion and copy number abnormality began to appear in the malignant potential stage, slowly rising in early breast cancer, and the rate of increase in the middle and advanced breast cancer increased, reaching a high level, imprinting gene Z3 Increased expression levels begin to appear in the malignant potential phase, remain stable during early breast cancer, and rapidly rise to higher levels in the metaphase and advanced breast cancer stages; imprinted gene Z8 imprinted deletion and copy number abnormalities are rapidly in the malignant potential phase Rising, the rate of increase in early breast cancer slows, and it rises rapidly to a high level in advanced breast cancer, and the expression of imprinted gene Z8 increases.
  • the malignant potential stage begins to appear, slowly rising in early and mid-stage breast cancer, and the rate of increase in advanced breast cancer is accelerated, reaching a higher level; imprinting deletion and copy number abnormality of imprinted gene Z11 rise rapidly in malignant potential and early stage breast cancer In the middle and late stage of breast cancer, the rate of increase slows down and reaches a very high level.
  • the increase of the expression level of the imprinted gene Z11 begins in the malignant potential stage, and rapidly rises in the early stage of breast cancer, and the rate of increase in the mid-stage breast cancer stage slows down to the late stage.
  • the stage of breast cancer has rapidly risen to a high level; the imprinting deletion, copy number abnormality and expression increase of the imprinted gene Z16 begin to appear in the malignant potential stage, and gradually rise to a high level in the development of early to late breast cancer. ;
  • the imprinted deletion and copy number abnormality of the imprinted gene Z5 began to appear in the malignant potential stage, rapidly rising in the early and middle stages of breast cancer, and the advanced breast cancer stage was maintained.
  • imprinted gene Z9 imprinted deletion, copy number abnormality and increased expression began to appear in the advanced breast cancer stage, but the level is not high; imprinted gene Z10 imprinted deletion and increased expression began to appear in the advanced breast cancer stage, but the level Not high, the copy number abnormality of the imprinted gene Z10 began to appear in the advanced breast cancer stage, reaching a higher level; the imprinting of the imprinted gene Z13 rapidly increased to a higher level in the advanced breast cancer stage, and the copy number of the imprinted gene Z13 was abnormal. It began to appear in the mid-stage breast cancer stage and rapidly increased to a higher level in the advanced breast cancer stage. The increase in the expression level of the imprinted gene Z13 began to appear in the early stage of breast cancer, and did not increase significantly in the mid-stage breast cancer stage. Fast rise to a higher level.
  • the tissues of 80 patients with breast tumors were obtained in the same manner as in Example 1.
  • any one of the imprinted gene deletion expression amount is less than 15%, the imprinted gene copy number abnormal expression amount is less than 1%, or the total imprinted gene expression amount is less than 20%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2-3%, or the imprinted gene total expression amount is 30-40%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 25-30%, the imprinted gene copy number abnormal expression amount is 3-5%, or the imprinted gene total expression amount is 40-50%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 30%, the imprinted gene copy number abnormal expression amount is greater than 5%, or the
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 1-2.5%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2.5-4%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinted gene copy number abnormal expression amount is greater than 4%,
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 1%, or the total imprinted gene expression amount is less than 15%.
  • a combination of at least two of which is level 0 an imprinted gene deletion expression amount of 10-15%, an imprinted gene copy number abnormal expression amount of 1-2%, or an imprinted gene total expression amount of 15-20% or
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 2-3%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 3-5%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinting gene copy number abnormal expression amount is greater than 5%, or the imprinted
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 1%, or the total imprinted gene expression amount is less than 15%.
  • a combination of at least two of which is level 0 an imprinted gene deletion expression amount of 10-15%, an imprinted gene copy number abnormal expression amount of 1-2%, or an imprinted gene total expression amount of 15-20% or
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 2-3%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 3-5%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinting gene copy number abnormal expression amount is greater than 5%, or the imprinting gene deletion
  • any one of the imprinted gene deletion expression amount is less than 15%, the imprinted gene copy number abnormal expression amount is less than 1.5%, or the total imprinted gene expression amount is less than 20%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 3.5-6%, or the imprinted gene total expression amount is 30-40%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 25-30%, the imprinted gene copy number abnormal expression amount is 6-9%, or the imprinted gene total expression amount is 40-50%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 30%, the imprinted gene copy number abnormal expression amount is greater than
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 1-2.5%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2.5-4%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinted gene copy number abnormal expression amount is greater than 4%
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 1-2.5%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2.5-4%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinted gene copy number abnormal expression amount is greater than
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 1-2.5%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2.5-4%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinted gene copy number abnormal expression amount is greater than 4%
  • any one of the imprinted gene deletion expression amount is less than 10%, the imprinted gene copy number abnormal expression amount is less than 0.5%, or the total imprinted gene expression amount is less than 15%.
  • the combination of at least two is class I, the imprinting gene deletion expression amount is 15-20%, the imprinted gene copy number abnormal expression amount is 1-2.5%, or the imprinted gene total expression amount is 20-30%, or at least The combination of the two is class II, the imprinting gene deletion expression amount is 20-25%, the imprinted gene copy number abnormal expression amount is 2.5-4%, or the imprinted gene total expression amount is 30-40%, or at least two
  • the combination of the species is class III, the imprinting gene deletion expression amount is greater than 25%, the imprinted gene copy number abnormal expression amount is greater than
  • the determining the degree of benign and malignant breast tumors is divided into benign tumor, breast cancer potential, early breast cancer, metaphase breast cancer and advanced breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 are all less than grade I or
  • the imprinted gene of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 has an imprinted gene deletion level of I and the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z13 and Z16 is grade I, which is a benign tumor;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade I, imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade I or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinting gene in Z13 and Z16 is expressed in the level II and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the second grade, it is judged as the potential of breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is II, imprinted gene
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is Grade II or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11
  • the imprinting gene with no more than one imprinted gene in Z13 and Z16 has a deletion level of III and the imprint of no more than one imprinted gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the grade III, it is early breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes of the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III, imprinted gene
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is grade III or imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11 , the imprinting gene of no more than one imprinted gene in Z13 and Z16 is expressed in the IV level and the imprinting of no more than one imprinting gene in the imprinting genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 If the abnormal expression level of the gene copy number is in any of the IV grades, it is a metaphase breast cancer;
  • the result of determining the degree of benign and malignant breast tumors is that the imprinted gene deletion expression level of at least two imprinted genes in the imprinted genes Z1, Z3, Z5, Z8, Z9, Z10, Z11, Z13 and Z16 is IV or imprinted gene Z1
  • the abnormal expression level of the imprinted gene copy number of at least two imprinting genes in Z3, Z5, Z8, Z9, Z10, Z11, Z13, and Z16 is Grade IV, and is advanced breast cancer.
  • the detection model and system of the present invention express the impression of the imprinted defect on the sample of the breast tumor patient in an intuitive manner, and the method of in situ labeling of the imprinted gene is objective, intuitive, early, and accurate. Changes in the imprinted (trace) gene are detected and quantitative models can be provided to make a significant contribution to the diagnosis of breast tumors.

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Abstract

L'invention concerne un modèle de classification pour la détection de tumeurs du sein et une application associée. Le modèle classifie la variation d'un gène soumis à empreinte dans des tumeurs du sein en calculant la quantité d'expression de délétion du gène soumis à empreinte, la quantité d'expression des nombres de copies anormales du gène soumis à empreinte et la quantité d'expression totale du gène soumis à empreinte. Le dispositif et le modèle de détection présentent, de manière visuelle, l'expression de la délétion soumise à empreinte dans les échantillons de tissus et de cellules de patients atteints d'une tumeur du sein, et détectent, de manière objective, visuelle, précoce et précise, la variation dans le gène soumis à empreinte (transféré) au moyen du procédé de marquage in situ du gène soumis à empreinte, et peuvent fournir des modèles quantitatifs.
PCT/CN2018/117891 2017-11-28 2018-11-28 Modèle de classification pour la détection de degrés bénins et malins de tumeurs du sein et application associée Ceased WO2019105381A1 (fr)

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CN201811281044.0A CN109837342A (zh) 2017-11-28 2018-10-30 一种用于检测乳腺肿瘤良恶性程度的分级模型及其应用

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (fr) * 1994-03-07 1995-09-14 Rapaport, Erich Test sensible utilise dans la detection des cancers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024503A1 (fr) * 1994-03-07 1995-09-14 Rapaport, Erich Test sensible utilise dans la detection des cancers

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GARCIA-MURILLAS, I. ET AL.: "An siRNA Screen Identifies the GNAS Locus as A Driver in 20q Amplified", BREAST CANCER ONCOGENE, vol. 33, no. 19, 8 November 2014 (2014-11-08), pages 2478 - 2486, XP055615446 *
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