WO2018214845A1

WO2018214845A1 - Model, diagnostic method, and application thereof

Info

Publication number: WO2018214845A1
Application number: PCT/CN2018/087661
Authority: WO
Inventors: 成彤; 周宁
Original assignee: 立森印迹诊断技术有限公司
Priority date: 2017-05-22
Filing date: 2018-05-21
Publication date: 2018-11-29

Abstract

A model and an application thereof, the model being used for detecting a benign and malignant degree of a tumor. The model grades a change of an imprinted gene in a tumor by calculating a deletion expression quantity of the imprinted gene and a copy number abnormal expression quantity of the imprinted gene. The detection model and device in the present invention presents the expression of loss of imprinting in a sample of a tumor patient in a direct way for the first time; based on an imprinted gene in situ labeling method, a change of an imprinted gene is detected objectively, directly, early, and precisely, and a quantitative model can be provided, to make a great contribution to molecular pathology diagnosis.

Description

A model and diagnostic method and its application

Technical field

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.

Background technique

According to international cancer statistics, more than 8 million people worldwide die from cancer each year. Therefore, early detection, diagnosis, treatment is particularly important. 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.

At present, 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. However, this method has its inherent limitations. First, 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. Second, 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. Finally, some malignant tumor cells are morphologically very different from benign tumor cells.

From the perspective of genetics, the development of tumors is a multi-gene process. Epigenetic modification is of great significance for the occurrence, diagnosis and treatment of tumors, and it has also been applied clinically. A large number of scientific studies have confirmed the correlation between imprinted state gene re-expression and cell carcinogenesis.

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.

Summary of the invention

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. In contrast, in healthy cell assays, 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.

In one embodiment, the imprinting gene 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 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, and 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.

In an embodiment of the present invention, 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;

In some embodiments, 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%. By calculating the LOI, CNV and TE of the imprinted gene Z4, the sensitivity for detection of thyroid cancer can reach 78.6%. By measuring the LOI, CNV and TE of the imprinted gene Z5, the sensitivity for detection of thyroid cancer can be achieved. It can reach 64.3%. By calculating the LOI, CNV and TE of the imprinted gene Z6, the sensitivity for detection of thyroid cancer can reach 57.1%. By calculating the LOI, CNV and TE of the imprinted gene Z8, 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%. By calculating the LOI, CNV and TE of the imprinted gene Z10, the sensitivity for detection of thyroid cancer can reach 71.4%. By calculating the LOI, CNV and TE of the imprinted gene Z11, 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%;

In some embodiments, 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%. When detecting the combination of Z3 and Z4, the sensitivity of diagnosis to thyroid cancer can reach 92.9%. When detecting the combination of Z3 and Z10, 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:

Total expression = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression level = b / (b + c + d) × 100%;

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%;

Wherein, 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 .

In one embodiment herein, 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.

In one embodiment herein, the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 are:

Grade 0: 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%;

Level I: 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%;

Level II: 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%;

Grade III: 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%;

In one embodiment of the present invention, five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of Z2 and Z12 are:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

In the present disclosure, 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.

In one embodiment of the present invention, 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:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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.

In one embodiment of the present invention, 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:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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.

In one embodiment herein, five different levels of imprinted gene deletion expression and imprinted copy number anomalous expression for Z7, Z14, and Z15 are:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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%;

Grade IV: 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:

(1) sampling unit: obtaining a sample to be tested;

(2) Probe design unit: design specific primers according to the imprinted gene sequence;

(3) detecting unit: in situ hybridization of the probe of step (2) with the sample to be tested;

(4) Analysis unit: microscopic imaging analysis of the expression of imprinted genes;

Wherein, 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 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.

In an embodiment herein, 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.

In an embodiment of the present invention, 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 μm thick, and used with a belt. The positively charged slides are made into tissue sheets; since only 10 μm is thick, some of the microscopically seen nuclei are incomplete, so some false negative gene deletions occur.

In an embodiment of the present invention, 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.

In an embodiment of the present invention, 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.

Since 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.

In an embodiment of the present invention, the sample to be tested is a biopsy puncture cell.

In one embodiment, 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, and 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, and the imprinted gene Z16 is Snrpn/Snurf.

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), Z16 (Snrpn/Snurf) have different degrees of expression in normal tumor cell tissues, and malignancy occurs. When the lesion is present, both the expression level and the imprint state change significantly.

In one embodiment herein, 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.

In one embodiment herein, the in situ hybridization employs an RNAscope in situ hybridization method.

In one embodiment herein, 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.

In one embodiment herein, 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.

In an embodiment of the present invention, 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, When 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.

In an embodiment of the present invention, 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 And 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 When the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z16 is in any of the second order, it is judged to be malignant potential.

In an embodiment of the present invention, 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 abnormal expression level of the imprinted gene imprinting gene of no more than one imprinted gene is grade III, which is an early malignant tumor.

In an embodiment of the present invention, 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 abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z16 is grade IV, which is a metaphase malignant tumor.

In an embodiment of the present invention, 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.

In one embodiment herein, 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:

(1) Obtaining a sample to be tested;

(2) designing specific primers based on the imprinted gene sequence;

(3) performing in situ hybridization of the probe of step (2) with the sample to be tested;

(4) Microscopic imaging analysis of the expression status of the imprinted gene;

Among them, by calculating the expression level of imprinted gene, the amount of imprinted gene deletion and the abnormal expression level of imprinted gene copy number, 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 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 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.

In one embodiment herein, the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount are divided into five different levels.

In one embodiment herein, 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.

Grade I: 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%;

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%.

In one embodiment herein, five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z2 and Z12 are:

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.

In one embodiment herein, five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z3, Z4, Z9, Z10, and Z11 are:

Grade II: 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%;

In one embodiment herein, 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:

In one embodiment herein, five different levels of imprinted gene deletion expression and imprinted gene copy number abnormal expression levels for Z7, Z14, and Z15 are:

In the examples herein, the sensitivity of each tumor to different imprinted genes is different, and each index of different tumors may have a top and bottom fluctuation of 20%.

In one embodiment herein, the use of the model or device of the invention in the manufacture of a medicament for tumor detection and/or treatment is provided.

In an embodiment of the present invention, 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.

In an embodiment of the present invention, 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 In the Z16, 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.

In an embodiment of the present invention, 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 And 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 When the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z16 is in any of the second order, it is judged to be malignant potential.

In an embodiment of the present invention, 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 abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z16 is grade III, which is an early malignant tumor.

In an embodiment of the present invention, 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 abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z16 is grade IV, which is a metaphase malignant tumor.

In an embodiment of the present invention, 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.

Compared with the related art, in the embodiments of the present invention, 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.

Other aspects will be apparent upon reading and understanding the drawings and detailed description.

DRAWINGS

The drawings are used to provide a further understanding of the technical solutions herein, and are to be considered as a part of the specification, and are used to explain the technical solutions herein, and do not constitute a limitation of the technical solutions herein.

1 is a diagram showing the relationship between imprinted gene deletion and cancer in the embodiment of the present disclosure;

2 is a pathological section of thyroid cancer in which a hematoxylin-stained nuclei according to an embodiment of the present disclosure, wherein 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;

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;

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. 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, and Fig. 4(g) was applied to the imprinting gene Z8. In the pathological section of thyroid tumor, the distribution range and grading standard of imprinting deletion and copy number abnormality, 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, and Fig. 4(j) is the imprinting gene Z11 applied to 17 cases of thyroid tumor pathological section. In the distribution range and grading standard of imprinting deletion and copy number abnormality, 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. l) For the imprinted gene Z13 applied to 17 cases of thyroid tumor pathological sections, the distribution range and grading standard of imprinting deletion and copy number abnormality, 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.

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.

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.

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.

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.

detailed description

The embodiments herein will be described in detail below with reference to the accompanying drawings.

The development of tumor is a multi-gene process. Epigenetic modification is of great significance for the occurrence, diagnosis and treatment of tumors, and it has also been applied clinically. A large number of scientific studies have confirmed the correlation between imprinted state gene re-expression and cell carcinogenesis.

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. A revolutionary breakthrough in the field of diagnosis of cancer;

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.

Example 1 Imprinted Gene Analysis of Thyroid Cancer

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:

(1) Obtaining 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;

(2) Dewaxing according to the sample processing method of RNASCope, blocking endogenous peroxidase activity in the sample, enhancing permeability and exposing RNA molecules;

(3) 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.

(4) performing RNASCope in situ hybridization of the probe of step (3) and the sample to be tested through a kit;

(5) Signal amplification and hematoxylin staining, and microscopic imaging analysis of the expression of imprinted genes;

The formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy in the model are as follows:

Total expression = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression level = b / (b + c + d) × 100%;

Imprinted gene deletion gene expression level (LOI) = c / (b + c + d) × 100%;

Gene expression amount (CNV) of imprinted gene copy number abnormality = d / (b + c + d) × 100%;

Wherein, 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. There is a red/brown mark in the nucleus to imprint the nucleus of the gene; the c is the hematoxylin staining of the cell, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; the d is 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).

As can be seen from Fig. 3(a) - Fig. 3(e), in the samples from grade 0 to grade IV, the imprint is missing (two signal points in the nucleus) and the copy number is abnormal (three or more signal points in the nucleus) The proportion of cells gradually increases as the degree of malignancy increases.

Example 2 Sensitivity of Z1-Z16 imprinted gene to thyroid cancer

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).

As can be seen from Fig. 4(a)-Fig. 4(m), the ratio of imprinted deletion and copy number abnormality of each probe in 17 thyroid tumor tissue samples showed a low to high distribution, according to the distribution trend of different probes. We calculated the grading standard shown by the dotted line in the figure. We can divide the missing and copy number anomalies of each probe into five grades from low to high, and the specific grades are as follows:

As can be seen from Fig. 4(a), for the imprinted gene Z1, 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;

As can be seen from Fig. 4(b), for the imprinted gene Z2, 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;

As can be seen from Fig. 4(c), for the imprinted gene Z3, 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;

As can be seen from Fig. 4(d), for the imprinted gene Z4, 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;

As can be seen from Fig. 4(e), for the imprinted gene Z5, 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;

As can be seen from Fig. 4(f), for the imprinted gene Z6, 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;

As can be seen from Fig. 4(g), for the imprinted gene Z8, 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;

As can be seen from Fig. 4(h), for the imprinted gene Z9, 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;

As can be seen from Fig. 4(i), for the imprinted gene Z10, 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;

As can be seen from Fig. 4(j), for the imprinted gene Z11, 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;

As can be seen from Fig. 4(k), for the imprinted gene Z12, 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;

As can be seen from Fig. 4(l), for the imprinted gene Z13, 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;

As can be seen from Fig. 4(m), for the imprinted gene Z16, 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.

A comprehensive analysis of the samples from these 17 thyroid tumors can be drawn:

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 When 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. Gene imprinted gene deletion expression level II, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 at least 2 imprinted genes 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.

Example 3 Imprinted Gene Analysis of Skin 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.

As can be seen from Fig. 5(a)-Fig. 5(b), Fig. 5(a) is a benign black sputum, and 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.

Example 4 Imprinted Gene Analysis of Lung Cancer

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).

As can be seen from Fig. 6(a)-Fig. 6(b), Fig. 6(a) is a benign lung tumor, and Fig. 6(b) is a lung cancer. In 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.

Example 5 Imprinted Gene Analysis of Bladder Cancer

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).

As can be seen from Fig. 7(a)-Fig. 7(b), Fig. 7(a) is a benign bladder tumor, and Fig. 7(b) is a bladder cancer. In 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.

Example 6 Imprinted Gene Analysis of Pancreatic Cancer

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).

As can be seen from Fig. 8(a) - Fig. 8(b), Fig. 8(a) is a benign pancreatic tumor, and Fig. 8(b) is a pancreatic cancer. In 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.

In summary, 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.

The applicant claims that the detailed method of the present application is described by the above embodiments, but the present application is not limited to the above detailed methods, that is, it does not mean that the present application must rely on the above detailed methods to implement. It should be apparent to those skilled in the art that any modification of the present application, the equivalent replacement of each raw material of the product of the present application, the addition of an auxiliary component, the selection of a specific manner, and the like, are all within the scope of protection and disclosure of the present application.

Claims

A model for classifying the expression state of an imprinted gene by calculating a change in the total expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal expression amount of the imprinted gene in the tumor.
The model according to claim 1, wherein said imprinted genes Z1-Z16, said imprinted gene Z1 is Gnas, said imprinted gene Z2 is Igf2, said imprinted gene Z3 is Peg10, and said imprinted 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, and 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, and the imprinted gene Z16 is Snrpn/Snurf.
The model according to claim 1 or 2, wherein the method of calculating the imprinted gene is: calculating a combination of imprinted genes of a combination of sixteen imprinted genes of Z1-Z16.
The model according to any one of claims 1 to 3, wherein 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 are as follows:

Total expression = (b + c + d) / (a + b + c + d) × 100%;

Normal imprinted gene expression level = b / (b + c + d) × 100%;

Imprinted gene deletion expression amount = c / (b + c + d) × 100%;

Imprinted gene copy number abnormal expression amount = d / (b + c + d) × 100%;

Wherein, 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 is abnormal. .
The model according to any one of claims 1 to 4, wherein 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 levels;
The model according to claim 5, wherein said five different levels are the imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount of the sixteen imprinted genes for Z1-Z16, respectively. Five different levels of division;
The model according to claim 5 or 6, wherein the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1 are:

Grade 0: the imprinted gene Z1 has an imprinted gene deletion expression amount of less than 15% and/or the imprinted gene Z1 has an imprinted gene copy number abnormal expression amount of less than 1.5%;

Grade I: 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%;

Level II: 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%;

Grade III: 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%.
The model according to any one of claims 5 to 7, wherein the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z2 and Z12 are:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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 deletion expression amount of the imprinted genes Z2 and Z12 is greater than 35% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z2 and Z12 is greater than 5%.
The model according to any one of claims 5-8, wherein the 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:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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 model according to any one of claims 5 to 9, wherein 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:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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 model according to any one of claims 5 to 10, wherein the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z7, Z14 and Z15 are:

Grade 0: 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%;

Grade I: 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%;

Grade II: 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%;

Grade III: 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%;

Grade IV: The imprinted gene deletion expression amount of the imprinted genes Z7, Z14 and Z15 is greater than 25% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z7, Z14 and Z15 is greater than 3%.
A device for detecting the degree of benign and malignant tumors, using the model according to any one of claims 1-11, comprising the following units:

(1) sampling unit: obtaining a sample to be tested;

(2) Probe design unit: design specific primers according to the imprinted gene sequence;

(3) detecting unit: in situ hybridization of the probe of step (2) with the sample to be tested;

(4) Analysis unit: microscopic imaging analysis of the expression status of the imprinted gene;

Wherein, the analyzing unit calculates the amount of expression of the imprinted gene by using the model according to any one of claims 1 to 11 by calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number. The level of abnormal expression of the imprinted gene copy number is used to determine the degree of benign and malignant tumors.
A method for detecting the degree of benign and malignant tumors, using the model according to any one of claims 1-11, comprising the steps of:

(1) Obtaining a sample to be tested;

(2) designing specific primers based on the imprinted gene sequence;

(3) performing in situ hybridization of the probe of step (2) with the sample to be tested;

(4) Microscopic imaging analysis of the expression status of the imprinted gene;

Wherein, the analyzing unit calculates the amount of expression of the imprinted gene by using the model according to any one of claims 1 to 11 by calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number. The level of abnormal expression of the imprinted gene copy number is used to determine the degree of benign and malignant tumors.
The method according to claim 13, wherein the sample to be tested according to step (1) is human tissue and/or cells.
The method according to claim 13 or 14, wherein the sample to be tested according to step (1) is a needle biopsy sample;
The method according to any one of claims 13 to 15, wherein the in situ hybridization employs an RNAscope in situ hybridization method.
The method according to any one of claims 13 to 16, wherein the RNAscope in situ hybridization method uses a single-channel or multi-channel colorimetric kit or a single-channel or multi-channel fluorescent kit, preferably a single channel. Red/brown color kit or multi-channel fluorescent kit.
The method according to any one of claims 13-17, wherein the degree of benign and malignant tumors to be judged is classified into benign, malignant potential, early malignant tumor, metaphase malignant tumor and advanced malignant tumor.
The method according to claim 18, wherein 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, The imprinted gene deletion expression of no more than one imprinted gene in Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, When the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z14, Z15 and Z16 is in any of the first order, it is a benign tumor.
The method according to claim 18 or 19, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, The imprinted gene deletion expression level of at least two imprinted genes in Z13, Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z13, Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12 The imprinted gene deletion expression of no more than one imprinted gene in Z13, Z14, Z15 and Z16 is grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12 When the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z13, Z14, Z15 and Z16 is in any of the second order, it is judged to be malignant potential.
The method according to any one of claims 18 to 20, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinting gene deletion expression level of at least 2 imprinting genes in Z11, Z12, Z13, Z14, Z15 and Z16 is grade II, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of imprinting gene copy number of at least 2 imprinting genes in Z12, Z13, Z14, Z15 and Z16 is grade II, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III, which is an early malignant tumor.
The method according to any one of claims 18 to 21, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression level of at least 2 imprinted genes in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of imprinting gene copy number of at least 2 imprinting genes in Z12, Z13, Z14, Z15 and Z16 is grade III, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV, which is a metaphase malignant tumor.
The method according to any one of claims 18 to 22, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression level of at least 2 imprinted genes in Z11, Z12, Z13, Z14, Z15 and Z16 is IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of the imprinted gene copy number of at least two of the Z12, Z13, Z14, Z15 and Z16 is grade IV, and is an advanced malignant tumor.
Use of a model according to any one of claims 1 to 11 or a device according to claim 12 for the preparation of a medicament for tumor detection and/or treatment.
The use according to claim 24, wherein the degree of benign and malignant tumors to be judged is classified into benign, malignant potential, early malignant tumor, metaphase malignant tumor, and advanced malignant tumor.
The use according to claim 24 or 25, wherein 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, The imprinted gene deletion expression of no more than one imprinted gene in Z13, Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12, When the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z13, Z14, Z15 and Z16 is in any of the first order, it is a benign tumor.
The use according to any one of claims 24 to 26, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression level of at least two imprinted genes in Z11, Z12, Z13, Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of Z11, Z12, Z13, Z14, Z15 and Z16 is grade I, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10 The imprinted gene deletion expression of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10 When the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z11, Z12, Z13, Z14, Z15, and Z16 is any of the second grade, it is judged to be malignant potential.
The use according to any one of claims 24 to 27, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinting gene deletion expression level of at least 2 imprinting genes in Z11, Z12, Z13, Z14, Z15 and Z16 is grade II, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of imprinting gene copy number of at least 2 imprinting genes in Z12, Z13, Z14, Z15 and Z16 is grade II, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III, which is an early malignant tumor.
The use according to any one of claims 24 to 28, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression level of at least 2 imprinted genes in Z11, Z12, Z13, Z14, Z15 and Z16 is grade III, imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of imprinting gene copy number of at least 2 imprinting genes in Z12, Z13, Z14, Z15 and Z16 is grade III, imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV, which is a metaphase malignant tumor.
The use according to any one of claims 24 to 29, wherein the result of determining the degree of benign and malignant tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, The imprinted gene deletion expression level of at least 2 imprinted genes in Z11, Z12, Z13, Z14, Z15 and Z16 is IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11 The abnormal expression level of the imprinted gene copy number of at least two of the Z12, Z13, Z14, Z15 and Z16 is grade IV, and is an advanced malignant tumor.