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WO2018214845A1 - Modèle, méthode de diagnostic et application de celui-ci - Google Patents

Modèle, méthode de diagnostic et application de celui-ci Download PDF

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Publication number
WO2018214845A1
WO2018214845A1 PCT/CN2018/087661 CN2018087661W WO2018214845A1 WO 2018214845 A1 WO2018214845 A1 WO 2018214845A1 CN 2018087661 W CN2018087661 W CN 2018087661W WO 2018214845 A1 WO2018214845 A1 WO 2018214845A1
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Prior art keywords
imprinted
imprinted gene
genes
gene
copy number
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English (en)
Chinese (zh)
Inventor
成彤
周宁
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Lisen Imprinting Diagnostics Inc
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Lisen Imprinting Diagnostics Inc
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Priority claimed from CN201810468315.7A external-priority patent/CN108929851A/zh
Application filed by Lisen Imprinting Diagnostics Inc filed Critical Lisen Imprinting Diagnostics Inc
Priority to US16/615,755 priority Critical patent/US11734818B2/en
Priority to EP18806916.5A priority patent/EP3633045B1/fr
Priority to JP2020514319A priority patent/JP2020520675A/ja
Priority to ES18806916T priority patent/ES2947579T3/es
Publication of WO2018214845A1 publication Critical patent/WO2018214845A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • This document relates to the field of biotechnology, such as the field of genetic diagnosis, such as a model and diagnostic method and its applications.
  • Cancer is caused by complex genetic and epigenetic changes that accumulate over time at the cellular level, ultimately leading to uncontrolled cell division.
  • 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.
  • the benign and malignant lesions and the judgment of cancer typing or cancer stage are largely determined by the morphology of the cells observed on the histopathological sections stained with hematoxylin and eosin.
  • this method has its inherent limitations.
  • the method cannot observe changes in the molecular level of tumors, and molecular changes can provide a more accurate basis for pre-diagnosis and diagnosis.
  • cell morphology diagnosis is subjective judgment, which itself can cause inaccurate diagnosis. The errors caused by such subjective judgments have a great impact on the diagnosis of early cancer, especially the sensitivity and accuracy of early diagnosis of cancer is life-critical for patients.
  • some malignant tumor cells are morphologically very different from benign tumor cells.
  • Genomic imprinting is a way of epigenetic gene regulation, expressed as that for a particular gene, only alleles from a particular parent can be expressed, while another allele appears as a gene silence.
  • This article provides a model and diagnostic method and its application.
  • An embodiment of the present invention provides a model which is an imprinted gene grading model for calculating imprinted genes by calculating the amount of imprinted gene expression, imprinted gene deletion expression amount, and imprinted gene copy number abnormal expression amount in a tumor. Grading.
  • 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 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 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 amount of imprinted gene expression, the amount of imprinted gene deletion, and the amount of abnormal expression of the imprinted gene copy number.
  • the imprinting gene Z1-Z16 the imprinting gene Z1 is Gnas
  • the imprinting gene Z2 is Igf2
  • the imprinting gene Z3 is Peg10
  • the imprinting gene Z4 is Igf2r
  • the imprinting The gene Z5 is Mest
  • the imprinting gene Z6 is Plagl1
  • the imprinting gene Z7 is Cdkn1c
  • the imprinting gene Z8 is Dcn
  • the imprinting gene Z9 is Dlk1
  • the imprinting gene Z10 is Gatm
  • the imprinting gene Z11 In the case of Grb10, the imprinted gene Z12 is Peg3, the imprinted gene Z13 is Sgce, the imprinted gene Z14 is Slc38a4, the imprinted gene Z15 is Diras3, and the imprinted gene Z16 is Snrpn/Snurf.
  • the method for calculating the imprinted gene is: calculating a combination of imprinted genes, wherein the imprinted gene is a combination of sixteen imprinted genes of Z1-Z16;
  • the inventors found that by calculating the LOI, CNV, and TE of the imprinted gene Z2, the sensitivity for detection of thyroid cancer can reach 71.4%, and the detection of thyroid cancer by calculating the LOI, CNV, and TE of the imprinted gene Z3.
  • the sensitivity can reach 78.6%.
  • the sensitivity for detection of thyroid cancer can reach 78.6%.
  • the sensitivity for detection of thyroid cancer can be achieved. It can reach 64.3%.
  • the sensitivity for detection of thyroid cancer can reach 57.1%.
  • the sensitivity to detection of thyroid cancer can be reached. 58.3%, by calculating the LOI, CNV and TE of the imprinted gene Z9, the sensitivity for detection of thyroid cancer can reach 64.3%.
  • the sensitivity for detection of thyroid cancer can reach 71.4%.
  • the sensitivity for detection of thyroid cancer can reach 71.4%, by calculating the imprinting base.
  • Z12 of LOI, CNV, and TE the detection sensitivity for thyroid cancer can reach 69.2%;
  • the inventors have discovered that sensitivity can be further improved by calculating LOI, CNV, and TE of two or more imprinted genes, in a particular embodiment, detecting a combination of two imprinted genes of the imprinted gene,
  • the sensitivity of diagnosis for thyroid cancer can reach 92.9%.
  • the combination of Z3 and Z4 the sensitivity of diagnosis to thyroid cancer can reach 92.9%.
  • the sensitivity to diagnosis of thyroid cancer can be When the combination of Z4 and Z9 is detected, the sensitivity of diagnosis to thyroid cancer can reach 92.9% or more.
  • An embodiment of the present invention provides the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number 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 hematoxylin staining of cells, and there is no label in the nucleus, and the imprinted gene is not expressed; wherein b is a hematoxylin staining, a red/brown mark is present in the nucleus, and the imprinted gene is present; 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 hematoxylin staining of the cells, there are more than two red/brown marks in the nucleus, and the imprinting gene copy number variation .
  • the imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount are divided into five different grades for at least 1200 cells in the region where the probe is most positive for each probe. Counting was performed, and five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the sixteen imprinted genes of Z1-Z16 were separately divided.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 are:
  • the imprinted gene of the imprinted gene Z1 has an expression loss of less than 15% and/or the abnormal expression of the imprinted gene of the imprinted gene Z1 is less than 1.5%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 15-25% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 1.5-2.5%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 25-30% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 2.5-4%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 30-35% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 4-6%;
  • Grade IV the imprinted gene deletion expression amount of the imprinted gene Z1 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is greater than 6%;
  • five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of Z2 and Z12 are:
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of less than 1%;
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 1-2%;
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • Grade III the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 25-35% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 3-5%;
  • Grade IV the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount greater than 35% and/or the imprinted gene Z2 and Z12 imprinted gene copy number abnormal expression amount is greater than 5%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z12 are independent of each other.
  • five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z3, Z4, Z9, Z10, and Z11 are:
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression of less than 15% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have an abnormal expression number of the imprinted gene that is less than 1%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene expression level of 15-20% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z4, Z9, Z10 and Z11 The amount is 1-2.5%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression of 20-30% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z4, Z9, Z10 and Z11 The amount is 2.5-4%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression of 30-35% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have abnormal expression of the imprinted gene copy number. The amount is 4-6%;
  • Grade IV the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene expression level greater than 35% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have an abnormal expression number of the imprinted gene greater than 6%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z3, Z4, Z9, Z10 and Z11 are independent of each other.
  • five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z5, Z6, Z8, Z13, and Z16 are:
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of less than 15% and/or the imprinted genes Z5, Z6, Z8, Z13 and Z16 have an abnormal expression number of the imprinted gene that is less than 1%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression amount of 15-20% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 1-2.5%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of 20-25% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 2.5-4%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of 25-35% and/or an abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 4-6%;
  • Grade IV the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene expression level greater than 35% and/or the imprinted genes Z5, Z6, Z8, Z13 and Z16 have an abnormal expression number of the imprinted gene greater than 6%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z5, Z6, Z8, Z13 and Z16 are independent of each other.
  • five different levels of imprinted gene deletion expression and imprinted copy number anomalous expression for Z7, Z14, and Z15 are:
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted genes Z7, Z14 and Z15 have an abnormal expression amount of the imprinted gene copy number of less than 0.5%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 0.5-1%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 1-2%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount greater than 25% and/or the imprinted genes Z7, Z14 and Z15 have an abnormal expression level of the imprinted gene copy number greater than 3%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z7, Z14 and Z15 are independent of each other.
  • An embodiment of the present disclosure provides an apparatus, including the following units:
  • sampling unit obtaining a sample to be tested
  • Probe design unit design specific primers according to the imprinted gene sequence
  • the analysis unit calculates the imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount, and the model is used to thereby obtain the level of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount. Determine the degree of benign and malignant tumors.
  • 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 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 amount of imprinted gene expression, the amount of imprinted gene deletion, and the amount of abnormal expression of the imprinted gene copy number.
  • the detection device is used for early and direct observation of changes in the imprinted (spot) genes of various types of tumors at the single cell and tissue level to determine the benign and malignant degree of the tumor, and to provide the most favorable treatment opportunity for patients with early 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 in the present disclosure includes paraffin sections and/or puncture of the tissue.
  • Biopsy cells are tableted and/or exfoliated cells obtained by natural shedding and mechanical methods.
  • the specific operation step of the paraffin section of the tissue is to obtain a sample of a human tumor tissue, which is fixed in time with 10% neutral formalin or other fixing method, embedded in paraffin, cut into 10 ⁇ m thick, and used with a belt.
  • the positively charged slides are made into tissue sheets; since only 10 ⁇ m is thick, some of the microscopically seen nuclei are incomplete, so some false negative gene deletions occur.
  • the other fixing manner includes alcohol type fixing, and the specific steps of the alcohol type fixing are as follows:
  • the sample to be tested is placed in the specimen antiseptic fixing solution described in Patent ZL200710024048.6, and fixed at room temperature for more than 24 hours.
  • the inventors have found that different fixing methods have different grading standards, and each index will have a top 20% fluctuation.
  • the specific operation step of the puncture biopsy cell tableting is to obtain human cells, and the method can be fixed in time by using 10% neutral formalin or other fixing methods.
  • the specific operation steps of the exfoliated cells obtained by the natural shedding and mechanical methods are to obtain human cells, and the cells can be fixed in time by using 10% neutral formalin or other fixing methods.
  • the puncture takes a small amount of damage to the patient, the sampling process is simple, and the puncture cells can be positioned compared to the circulation characteristics of the blood, and the puncture of the cells as a test sample has its special advantages.
  • the sample to be tested is a biopsy puncture cell.
  • the imprinting gene is Z1-Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z2 is Igf2, the imprinting gene Z3 is Peg10, and the imprinting gene Z4 is Igf2r.
  • the imprinted gene Z5 is Mest
  • the imprinted gene Z6 is Plagl
  • 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 imprinted gene Z14 is Slc38a4
  • the imprinted gene Z15 is Diras3
  • the imprinted gene Z16 is Snrpn/Snurf.
  • the design probe is based on the imprinting gene Z1-Z16, ie Gnas, Igf2, Peg10, Igf2r, Mest, Plagl, Cdkn1c, Dcn, Dlk1, Gatm, Grb10, Peg3, Sgce, Slc38a4, Diras3 Designed with Snrpn/Snurf, a sequence was selected as a probe within the internal 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 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 variation in each nucleus was determined, and the imprint was calculated.
  • the 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 tumor malignancy 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 determining the degree of benign and malignant tumors is divided into benign, malignant potential, early malignant tumor, metaphase malignant tumor, and advanced malignant tumor.
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 imprinted gene deletion expression and imprinted gene copy number abnormal expression level are 0, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14,
  • the imprinted gene deletion expression of no more than one imprinted gene in Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14,
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z15 and Z16 is in any of the first order, it is a benign tumor.
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and In Z16
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade III, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes is grade III, and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes is grade IV, and is an advanced malignant tumor.
  • the tumor is a conventional tumor in the art, and the disclosure is selected from, but not limited to, any one or a combination of at least two of a thyroid tumor, a lung tumor, or a brain tumor.
  • An embodiment of the present disclosure provides a detection method, including the following steps:
  • the model is used to calculate the level of imprinted gene deletion expression and imprinted gene copy number abnormal expression level to determine the degree of benign and malignant tumor. .
  • 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 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 amount of imprinted gene expression, the amount of imprinted gene deletion, and the amount of abnormal expression of the imprinted gene copy number.
  • the detection device is used for early and direct observation of changes in the imprinted (spot) genes of various types of tumors at the single cell and tissue level to determine the benign and malignant degree of the tumor, and provides the most favorable treatment opportunity for the early tumor patients.
  • 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 in the present disclosure includes a paraffin section of the tissue and a biopsy cell. Exfoliated cells obtained by tableting or natural shedding and mechanical methods.
  • the specific operation step of the paraffin section of the tissue is to obtain a sample of a human tumor tissue, which is fixed in time with 10% neutral formalin or other fixing method, embedded in paraffin, cut into 10 ⁇ m thick, and used with a belt.
  • the positively charged slides are made into tissue sheets; since only 10 ⁇ m is thick, some of the microscopically seen nuclei are incomplete, so some false negative gene deletions occur.
  • the other fixing manner includes alcohol type fixing, and the specific steps of the alcohol type fixing are as follows:
  • the sample to be tested is placed in the specimen antiseptic fixing solution described in Patent ZL200710024048.6, and fixed at room temperature for more than 24 hours.
  • the inventors have found that different fixing methods have different grading standards, and each index will have a top 20% fluctuation.
  • the specific operation step of the puncture biopsy cell tableting is to obtain human cells, and the method can be fixed in time by using 10% neutral formalin or other fixing methods.
  • the specific operation steps of the exfoliated cells obtained by the natural shedding and mechanical methods are to obtain human cells, and the cells can be fixed in time by using 10% neutral formalin or other fixing methods.
  • the puncture takes a small amount of damage to the patient, the sampling process is simple, and the puncture cells can be positioned compared to the circulation characteristics of the blood, and the puncture of the cells as a test sample has its special advantages.
  • the sample to be tested is a biopsy puncture cell.
  • the imprinting gene is Z1-Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z2 is Igf2, the imprinting gene Z3 is Peg10, and the imprinting gene Z4 is Igf2r.
  • the imprinted gene Z5 is Mest
  • the imprinted gene Z6 is Plagl
  • 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 imprinted gene Z14 is Slc38a4
  • the imprinted gene Z15 is Diras3
  • the imprinted gene Z16 is Snrpn/Snurf.
  • the design probe is based on the imprinting gene Z1-Z16, ie Gnas, Igf2, Peg10, Igf2r, Mest, Plagl, Cdkn1c, Dcn, Dlk1, Gatm, Grb10, Peg3, Sgce, Slc38a4, Diras3 Designed with Snrpn/Snurf, a sequence was selected as a probe within the internal 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 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 variation in each nucleus was determined, and the imprint was calculated.
  • the 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 tumor malignancy 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 and the imprinted gene copy number abnormal expression amount are divided into five different levels.
  • the five different levels are at least 1200 cells counted in the region where the probe is most positive for each probe, and the imprinted gene deletion expression for the sixteen imprinted genes of Z1-Z16 And the abnormal expression level of the imprinted gene copy number was separately divided.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 are:
  • the imprinted gene of the imprinted gene Z1 has an expression loss of less than 15% and/or the abnormal expression of the imprinted gene of the imprinted gene Z1 is less than 1.5%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 15-25% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 1.5-2.5%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 25-30% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 2.5-4%;
  • the imprinted gene Z1 has an imprinted gene deletion expression amount of 30-35% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of 4-6%;
  • the imprinted gene deletion expression amount of the imprinted gene Z1 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted gene Z1 is greater than 6%.
  • five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z2 and Z12 are:
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of less than 15% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of less than 1%;
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 1-2%;
  • the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • Grade III the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount of 25-35% and/or the imprinted genes Z2 and Z12 have an imprinted gene copy number abnormal expression amount of 3-5%;
  • Grade IV the imprinted gene Z2 and Z12 have an imprinted gene deletion expression amount greater than 35% and/or the imprinted gene Z2 and Z12 imprinted gene copy number abnormal expression amount is greater than 5%;
  • the imprinted gene deletion expression amount of the imprinted genes Z2 and Z12 and the abnormal expression amount of the imprinted gene copy number are independent of each other.
  • five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z3, Z4, Z9, Z10, and Z11 are:
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression of less than 15% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have an abnormal expression number of the imprinted gene that is less than 1%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene expression level of 15-20% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z4, Z9, Z10 and Z11 The amount is 1-2.5%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression amount of 20-30% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z3, Z4, Z9, Z10 and Z11 The amount is 2.5-4%;
  • the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene deletion expression of 30-35% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have abnormal expression of the imprinted gene copy number. The amount is 4-6%;
  • Grade IV the imprinted gene Z3, Z4, Z9, Z10 and Z11 have an imprinted gene expression level greater than 35% and/or the imprinted genes Z3, Z4, Z9, Z10 and Z11 have an abnormal expression number of the imprinted gene greater than 6%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z3, Z4, Z9, Z10 and Z11 are independent of each other.
  • the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z5, Z6, Z8, Z13, and Z16 are:
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of less than 15% and/or the imprinted genes Z5, Z6, Z8, Z13 and Z16 have an abnormal expression number of the imprinted gene that is less than 1%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression amount of 15-20% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 1-2.5%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of 20-25% and/or abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 2.5-4%;
  • the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene deletion expression of 25-35% and/or an abnormal expression of the imprinted gene copy number of the imprinted genes Z5, Z6, Z8, Z13 and Z16 The amount is 4-6%;
  • Grade IV the imprinted gene Z5, Z6, Z8, Z13 and Z16 have an imprinted gene expression level greater than 35% and/or the imprinted genes Z5, Z6, Z8, Z13 and Z16 have an abnormal expression number of the imprinted gene greater than 6%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z5, Z6, Z8, Z13 and Z16 are independent of each other.
  • five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z7, Z14, and Z15 are:
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted genes Z7, Z14 and Z15 have an abnormal expression amount of the imprinted gene copy number of less than 0.5%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 0.5-1%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 15-20% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 1-2%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount of 20-25% and/or the imprinted genes Z7, Z14 and Z15 have an imprinted gene copy number abnormal expression amount of 2-3%;
  • the imprinted gene Z7, Z14 and Z15 have an imprinted gene deletion expression amount greater than 25% and/or the imprinted genes Z7, Z14 and Z15 have an abnormal expression level of the imprinted gene copy number greater than 3%;
  • the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z7, Z14 and Z15 are independent of each other.
  • the determining the degree of benign and malignant tumors is divided into benign, malignant potential, early malignant tumor, metaphase malignant tumor, and advanced malignant tumor.
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 imprinted gene deletion expression and imprinted gene copy number abnormal expression level are 0, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14,
  • the imprinted gene deletion expression of no more than one imprinted gene in Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14,
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z15 and Z16 is in any of the first order, it is a benign tumor.
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and In Z16
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade III, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes is grade III, and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes is grade IV, and is an advanced malignant tumor.
  • each tumor to different imprinted genes is different, and each index of different tumors may have a top and bottom fluctuation of 20%.
  • the use of the model or device of the invention in the manufacture of a medicament for tumor detection and/or treatment is provided.
  • the degree of benign and malignant tumors to be judged is divided into benign, malignant potential, early malignant tumor, metaphase malignant tumor, and advanced malignant tumor.
  • the results of determining the degree of benign and malignant tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of imprinted gene deletion and imprinted gene copy number were 0, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene is in any of the first order, and is a benign tumor.
  • the result of determining the degree of benign and malignant tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15
  • the result of determining the degree of benign and malignant tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of imprinted gene copy number of at least 2 imprinted genes is grade II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the result of determining the degree of benign and malignant tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade III, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least 2 imprinted genes is grade III, and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression of no more than one imprinted gene in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the result of determining the degree of benign and malignant tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and
  • the imprinted gene deletion expression level of at least 2 imprinted genes in Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of at least two imprinted genes is grade IV, and is an advanced malignant tumor.
  • the detection model and the device are used to express the performance of the imprinted gene on the tumor patient sample in an intuitive manner, and the method for in situ labeling of the imprinted gene is objective, intuitive and early. It accurately detects changes in the imprinted (trace) gene and provides a quantitative model that makes a significant contribution to the diagnosis of molecular pathology.
  • 1 is a diagram showing the relationship between imprinted gene deletion and cancer in the embodiment of the present disclosure
  • the a is a hematoxylin-stained cell, and there is no label in the nucleus, and the imprinted gene is not expressed
  • the b is a hematoxylin After staining, 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 hematoxylin staining of the cells. After that, there are more than two red/brown markers in the nucleus, and the imprinted gene copy number variation
  • FIG. 3 is a representation of the expression of 16 genes in different pathological sections of thyroid tumors according to the first embodiment of the present disclosure, wherein FIG. 3(a) shows the expression of 16 genes in the pathological section of grade 0 thyroid tumors, FIG. 3 (b) The expression of 16 genes in the pathological section of grade I thyroid cancer, Figure 3 (c) shows the expression of 16 genes in the pathological section of grade II thyroid carcinoma, and Figure 3 (d) is the grade III thyroid carcinoma. The expression of 16 genes in the pathological section, Figure 3 (e) shows the expression of 16 genes in the pathological section of grade IV thyroid cancer;
  • FIG. 4 is a distribution range and grading standard of imprinting deletion and copy number abnormality in 16 pathological sections of 17 cases of thyroid tumors according to the second embodiment of the present disclosure, wherein FIG. 4(a) is an imprinted gene Z1 applied to 17 cases of thyroid gland In the tumor pathological section, the distribution range and grading standard of imprinting deletion and copy number abnormality, Fig. 4(b) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 17 cases of thyroid tumor with imprinting gene Z2. Fig. 4(c) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 17 cases of thyroid tumor pathological sections, and Fig.
  • FIG. 4(d) is the imprinting gene Z4 applied to 17 cases of thyroid tumor pathological sections.
  • the distribution range and grading standard of imprinting deletion and copy number abnormality Fig. 4(e) is the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 17 cases of thyroid tumor with imprinting gene Z5
  • Fig. 4(f) The imprinting gene Z6 was applied to the pathological sections of 17 cases of thyroid tumors
  • the distribution range and grading standard of imprinting deletion and copy number abnormality was applied to the imprinting gene Z8.
  • the distribution range and grading standard of imprinting deletion and copy number abnormality Fig.
  • FIG. 4(h) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 17 cases of thyroid tumor with imprinting gene Z9.
  • Fig. 4(i) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 17 cases of thyroid tumor pathological section
  • Fig. 4(j) is the imprinting gene Z11 applied to 17 cases of thyroid tumor pathological section.
  • Fig. 4(k) is the distribution range and grading standard of imprinting deletion and copy number abnormality in the pathological section of 17 cases of thyroid tumor with imprinting gene Z12.
  • Figure 4 (m) is the imprinting gene Z16 applied to 17 cases of thyroid tumor pathological section, the imprint is missing And the distribution range and grading criteria of copy number anomalies.
  • FIG. 5 is a representational state of 16 genes of different malignant degrees in a skin pathological section of Example 3 of the present disclosure, wherein FIG. 5(a) shows the expression status of 16 genes in the tissue section of the black scorpion, and FIG. 5(b) is Expression status of 16 genes in pathological sections of cutaneous malignant melanoma.
  • FIG. 6 is a representational state of pathological sections of lung tumors of different malignant degrees of 16 genes of Example 4 of the present disclosure, wherein FIG. 6(a) shows the expression status of 16 genes in a tissue section of a benign lung tumor, FIG. 6(b) ) is the expression status of 16 genes in the pathological section of lung cancer.
  • FIG. 7 is a representational state of pathological sections of bladder tumors of different malignant degrees in the fifth embodiment of the present disclosure, wherein FIG. 7(a) shows the expression status of 16 genes in a tissue section of a benign bladder tumor, FIG. 7(b) ) is the expression status of 16 genes in the pathological section of bladder cancer.
  • FIG. 8 is a representational state of pathological sections of pancreatic tumors of different malignant degrees of 16 genes of Example 6 of the present disclosure, wherein FIG. 8(a) shows the expression status of 16 genes in a tissue section of a benign pancreatic tumor, FIG. 8(b) ) is the expression status of 16 genes in the pathological section of pancreatic cancer.
  • Genomic imprinting is a way of epigenetic gene regulation, expressed as that for a particular gene, only alleles from a particular parent can be expressed, while another allele appears as a gene silence.
  • the present disclosure creates a detection method and device for directly diagnosing a deletion of a mark from a biopsy cell of a patient, and can determine a degree of benign and malignant tumor before surgery, thereby providing a basis for surgery and precision treatment, which is a cell molecule.
  • the present disclosure can accurately determine the type of tumor, fills in the limitations of current tissue morphological diagnosis, can achieve early accurate diagnosis, and provides assistance for later targeted therapy;
  • the present disclosure is the first to detect the expression of imprinted genes at a single cell and tissue level, and to qualitatively, quantitatively and spatially localize the expression of imprinted genes at the cellular level, indicating the relationship between the loss of organ tissue imprinting and the stage of tumor development. ;
  • the disclosed detection method is different from the immunohistochemical method, and reduces false positives and other negative effects, and not only the targeted drugs or technical methods for silencing, knocking out, rearranging, and rearrangement of the gene by the discovery of a tumor-associated imprinted gene deletion site, Can be used to guide later treatment and medication.
  • This embodiment provides a method for detecting thyroid cancer by using an imprinted gene, and the relationship is as shown in FIG. 1.
  • the detecting method includes the following steps:
  • tissue cell sections (10 micrometers) of thyroid cancer, and fixing them in a fixing solution described in Patent ZL200710024048.6 to prevent RNA degradation, the fixation time is 24 hours, and paraffin embedding (FFPE), The slide needs to be loaded with a positive charge, and the slice is baked in an oven at 40 ° C for more than 3 hours;
  • FFPE paraffin embedding
  • Design probe design specific primers according to the imprinted gene sequence
  • the design probe is based on the imprinted 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) were 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.
  • 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 two or more red/brown markers in the nucleus, and the nuclei with abnormal copy number of the imprinted gene are printed, and the results are shown in Fig. 3(a) to Fig. 3(e).
  • the tissue (10 micrometers) of 17 patients with thyroid tumors was obtained.
  • the fixation and detection methods were the same as those in Example 1.
  • the test results are shown in Fig. 4(a) to Fig. 4(m).
  • 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 the imprinted gene deletion expression amount is 15- 25% and/or imprinted gene copy number abnormal expression level is 1.5-2.5% for grade I, imprinted gene deletion expression level is 25-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2-3% for grade II, The imprinted gene deletion expression level is 25-35% and/or the imprinted gene copy number abnormal expression level is 3-5% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% as 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II,
  • the amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 4-6% to grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 4-6% to grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 4-6% to grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-30% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II, The imprinted gene deletion expression amount is 30-35% and/or the imprinted gene copy number abnormal expression level is 4-6% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2-3% for grade II, The imprinted gene deletion expression level is 25-35% and/or the imprinted gene copy number abnormal expression level is 3-5% to grade III, the imprinted gene deletion expression amount is greater than 35% and/or the imprinted gene copy number abnormal expression amount is greater than 5%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% is 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II,
  • the amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 4-6% to grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 6%.
  • Grade IV is
  • the imprinted gene deletion expression amount is less than 15% and/or the imprinted gene copy number abnormal expression amount is less than 1% as 0 level, and the imprinted gene deletion expression amount is 15- 20% and/or imprinted gene copy number abnormal expression level is 1-2.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 2.5-4% for grade II,
  • the amount of imprinted gene deletion is 25-35% and/or the abnormal expression level of imprinted gene copy number is 4-6% to grade III, the amount of imprinted gene deletion is greater than 35% and/or the abnormal expression of imprinted gene copy number is greater than 6%. It is level IV.
  • the result of determining the degree of benign and malignant thyroid tumor is the deletion expression of the imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression levels of the amount and the imprinted gene copy number are all 0, and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 do not exceed
  • the imprinted gene deletion expression level of one imprinted gene is grade I, and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 do not exceed
  • the abnormal expression level of the imprinted gene copy number of one imprinted gene is in any of the first order, it is judged to be a benign thyroid tumor.
  • the result of determining the degree of benign and malignant thyroid tumor is at least two of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the imprinted gene of the imprinted gene has a deletion level of at least 2 imprints of the imprinting gene Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of the imprinted gene copy number of the gene is I, and the imprinting gene Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 does not exceed 1
  • the imprinted gene is not expressed in the imprinted gene level I or in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number of the imprinted gene is any one of the second grade, and it is the malignant potential of the thyroid tumor.
  • the result of determining the degree of benign and malignant thyroid tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of the imprinted gene copy number is Grade II, and no more than one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the imprinted gene of the imprinted gene has a deletion level of III or no more than one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of the imprinted gene copy number of the imprinted gene is grade III, which is early thyroid cancer.
  • the result of determining the degree of benign and malignant thyroid tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the gene has an imprinted gene deletion level of III, and at least two imprinted genes in the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the abnormal expression level of the imprinted gene copy number is III, and no more than one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16
  • the imprinted gene of the imprinted gene has a deletion level of IV or no more than one of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of the imprinted gene copy number of the imprinted gene is grade IV, which is metaphase thyroid cancer.
  • the result of determining the degree of benign and malignant thyroid tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the gene is imprinted with a gene that is at least 2 imprinted genes in class IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16.
  • the abnormal expression level of the imprinted gene copy number is grade IV, which is advanced thyroid cancer.
  • Tissue samples of normal black sputum and skin malignant melanoma were fixed by 10% neutral formalin for more than 24 hours. Other methods were the same as in Example 1. The results are shown in Figure 5(a)-Fig. 5(b). Show.
  • Fig. 5(a) is a benign black sputum
  • Fig. 5(b) is a cutaneous malignant melanoma. Only the cells with missing marks in the black scorpion have no copy found. A few abnormal cells, there are a large number of cells with imprinted deletions and copy number abnormalities in skin malignant melanoma.
  • Tissue samples of benign lung tumors and lung cancer were obtained and fixed in 10% neutral formalin for more than 24 hours.
  • the other detection methods were the same as in Example 1, and the results were shown in Fig. 6(a)-Fig. 6(b).
  • Fig. 6(a) is a benign lung tumor
  • Fig. 6(b) is a lung cancer.
  • benign lung tumors only cells with individual imprinted deletions are found, and no copy number abnormality is found.
  • Cells, lung cancer have a large number of cells with imprinted deletions and abnormal copy number.
  • Tissue samples of benign bladder tumor and bladder cancer were obtained and fixed in 10% neutral formalin for more than 24 hours.
  • the other detection methods were the same as in Example 1, and the results were shown in Fig. 7(a)-Fig. 7(b).
  • Fig. 7(a) is a benign bladder tumor
  • Fig. 7(b) is a bladder cancer.
  • benign bladder tumors only cells with individual imprinted deletions are found, and no copy number is found.
  • Abnormal cells, bladder cancer have a large number of cells with imprinted deletions and abnormal copy number.
  • Tissue samples of benign pancreatic tumor and pancreatic cancer were obtained and fixed in 10% neutral formalin for more than 24 hours.
  • the other detection methods were the same as those in Example 1, and the results were shown in Fig. 8(a)-(8).
  • Fig. 8(a) is a benign pancreatic tumor
  • Fig. 8(b) is a pancreatic cancer.
  • the benign pancreatic tumor only cells with individual imprinted deletions are found, and no copy number is found.
  • Abnormal cells cells with a large number of imprinted deletions and copy number abnormalities in pancreatic cancer.
  • the detection model and device described in the present application express the performance of the imprinted defect on the sample of the tumor patient in an intuitive manner, and objectively, intuitively, earlyly and accurately detect the in situ labeling of the imprinted gene. Changes in the imprinted (trace) genes and can provide quantitative models that make a significant contribution to the diagnosis of tumors.

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Abstract

L'invention concerne un modèle et une application de celui-ci, le modèle étant utilisé pour détecter un degré bénin et malin d'une tumeur. Le modèle classe un changement d'un gène imprimé dans une tumeur par calcul d'une quantité d'expression de délétion du gène imprimé et d'une quantité d'expression anormale du nombre de copies du gène imprimé. Le modèle et le dispositif de détection de la présente invention présentent l'expression de perte d'empreinte dans un échantillon d'un patient atteint d'une tumeur d'une manière directe pour la première fois ; sur la base d'une méthode d'un marquage in situ de gène imprimé, un changement d'un gène imprimé est détecté de manière objective, directe, précoce et précise, et un modèle quantitatif peut être fourni, pour apporter une grande contribution au diagnostic de pathologie moléculaire.
PCT/CN2018/087661 2017-05-22 2018-05-21 Modèle, méthode de diagnostic et application de celui-ci Ceased WO2018214845A1 (fr)

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JP2020514319A JP2020520675A (ja) 2017-05-22 2018-05-21 モデル、診断方法及びその応用
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* Cited by examiner, † Cited by third party
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