WO2018219342A1

WO2018219342A1 - Imprinted gene graded model and diagnostic method and application

Info

Publication number: WO2018219342A1
Application number: PCT/CN2018/089494
Authority: WO
Inventors: 成彤; 周宁
Original assignee: 立森印迹诊断技术有限公司
Priority date: 2017-06-01
Filing date: 2018-06-01
Publication date: 2018-12-06

Abstract

Provided are an imprinted gene graded model and an application thereof, the model being used for detecting the benignity or malignancy of bladder tumours. The model grades the change of imprinted genes in a bladder tumour by means of calculating the amount of imprinted gene deletion expression and the amount of imprinted gene copy abnormal expression.

Description

Imprinting gene grading model and diagnosis method and application thereof

Technical field

The present disclosure relates to the field of biotechnology, such as the field of genetic diagnosis, such as an imprinted gene grading model and diagnostic method and its use, such as a model for detecting the degree of benign and malignant bladder tumors and its use.

Background technique

Bladder cancer is the most common malignant tumor of the urinary system, and the incidence rate is the first in the urinary system malignant tumor. According to the World Health Organization (WHO), there were 429,793 new cases, 165,084 deaths in 2012, 55,486 new diagnoses in China, and 26,820 deaths (World Cancer Report 2014). Bladder cancer ranks first in the incidence of malignant tumors in Chinese male genitourinary, and ranks eighth in the incidence of malignant tumors in China. The incidence of bladder cancer is increasing year by year. From 1998 to 2008, the incidence of bladder cancer in China increased by 56.69% (analysis of the incidence and trend of bladder cancer in China 2013). The recurrence rate of bladder cancer after the first surgical resection is as high as 75%, and the degree of malignancy of 10-15% of recurrent tumors increases. In the early stage of bladder cancer, after 5 years of transurethral bladder transitional cell carcinoma, the 5-year survival rate can reach 78%. In the late stage of bladder cancer, the 5-year survival rate of the patient after radical resection of the bladder does not exceed 40%. The above data indicate that bladder cancer has a high recurrence rate and good early treatment.

The production of cancer is an uncontrolled cell growth/splitting caused by epigenetic changes and genetic variations that accumulate over time. Traditional pathological diagnosis is based on the size, morphology and structural variation of cells and tissues, thereby making a benign and malignant judgment of bladder tumors. With the development and deepening of molecular biology, more and more molecular detection techniques have been applied to the detection of bladder cancer. From the analysis of the development of cancer, molecular changes (epigenetics and genetics) are much earlier than changes in cell morphology and tissue structure. Therefore, molecular biology testing is more sensitive to early detection of cancer.

Based on the above reasons, the current diagnosis of bladder cancer requires a new detection system and detection model, based on the patient biopsy sample, to analyze the molecular marker changes in the bladder cancer at the cellular level, in order to provide more accurate pre-diagnosis and diagnostic information.

Summary of the invention

The present disclosure provides an imprinted gene grading model and diagnostic method and uses thereof.

To achieve the above objectives, the present application adopts the following technical solutions:

The present disclosure provides an imprinted gene grading model for bladder tumors, which calculates the expression state of imprinted genes by calculating the total expression level of imprinted genes, the amount of imprinted gene expression, and the abnormal expression level of imprinted gene copy number in bladder tumors. Grading

Wherein, the imprinting gene is any one or a combination of at least two of Z1, Z2, Z3, Z4 or Z5, the imprinting gene Z1 is Gnas, the imprinting gene Z2 is Igf2, and the imprinting gene Z3 is Peg10 The imprinted gene Z4 is Igf2r, and the imprinted gene Z5 is Mest.

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 inventors found that by calculating the imprinted gene deletion expression amount and the abnormal expression level of imprinted gene copy number in any bladder tumor of Z1, Z2, Z3, Z4 and Z5, the sensitivity of diagnosis to bladder cancer can reach 85.0% or more. .

In one embodiment of the present document, the method for calculating the imprinted gene by the model is as follows:

Calculate any one of Z1, Z2, Z3, Z4 or Z5.

In one embodiment herein, the model calculates the imprinted gene as follows: Calculate any one of Z1, Z2, Z4 or Z5.

In one embodiment herein, the model calculates a imprinted gene as follows: Calculating a Z2 or Z4 imprinted gene.

The inventors found that the detection of a Z1 imprinted gene alone can be 85.7% sensitive to bladder cancer. A Z2 imprinted gene can be detected alone, and the sensitivity to bladder cancer can be 92.3%. A Z3 imprinted gene can be detected separately. The diagnostic sensitivity of bladder cancer can reach 85.0%. A Z4 imprinting gene can be detected alone. The sensitivity of diagnosis to bladder cancer can reach 95.0%. A Z5 imprinting gene can be detected alone, and the sensitivity of bladder cancer diagnosis can reach 87.2%.

In an embodiment herein, the method for calculating the imprinted gene is: calculating a combination of any two of the imprinted genes of Z1, Z2, Z3, Z4 or Z5, preferably calculating a combination of Z1 and Z2 imprinted genes or Z2 and A combination of Z4 imprinted genes.

The inventors have found that by calculating the amount of imprinted gene deletion and the abnormal expression level of the imprinted gene copy number of two or more imprinted genes, the sensitivity of the imprinted gene can be further improved, and the combination of the two imprinted genes of the imprinted gene is detected, and the bladder is detected. The diagnostic sensitivity of cancer can reach more than 94.9%. The combination of Z1 and Z2 is detected. When Z2 and Z4 are combined, the sensitivity of diagnosis to bladder cancer can reach 99.9% or more.

In an embodiment herein, the imprinting gene further comprises any one or a combination of at least two of Z6, Z8, Z9, Z10, Z11, Z15 or Z16;

Wherein, the imprinting gene Z6 is Plagl, the imprinting gene Z8 is Dcn, the imprinting gene Z9 is Dlk1, the imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, and the imprinting gene Z15 is Diras3. The imprinted gene Z16 is Snrpn/Snurf.

The inventors found that the combined diagnosis of Z6, Z8, Z9, Z10, Z11, Z15 and Z16 genes based on the detection of Z1, Z2, Z3, Z4 and Z5 genes not only helps to increase the accuracy of detection, but also The addition of other probe-assisted diagnostics can further avoid the occurrence of false positives, and can further improve the detection accuracy, thereby enabling accurate classification and judgment of all bladder samples.

In one embodiment, the model calculates the imprinted gene by calculating a combination of imprinted genes of a combination of twelve imprinted genes of Z1-Z6, Z8-Z11, and Z15-Z16.

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 an embodiment of the present invention, the formula for calculating the expression level of the imprinted gene, the amount of the imprinted gene, and the abnormal amount of the imprinted gene copy number are as follows:

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 cell nucleus in which there is no label in the nucleus and no imprinted gene is expressed after the hematoxylin staining of the cell; and b is a red/brown mark in the nucleus after the hematoxylin staining of the cell, and the imprinting gene exists. The nucleus; the c is a hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the nucleus of the imprinted gene is deleted; and the d is a hematoxylin staining of the cells, and there are more than two red/brown marks in the nucleus. , imprinted gene copy number abnormal cell nucleus.

In one embodiment, the hematoxylin-stained label is selected from, but not limited to, red or brown, and staining with other colors can also be used for imprinting gene expression, imprinted gene deletion expression, and imprinted gene copy number abnormal expression. Calculation.

In one embodiment herein, the probe is amplified by in situ hybridization, and Hemotoxy (hematoxylin) nuclei are used to amplify the signal, and under the 40× or 60× microscope, the presence of the imprinted gene in each nucleus, the imprinted gene is deleted, or The copy number abnormality is determined by calculating the gene expression level of the imprinted gene, the amount of the imprinted gene deletion gene, and the gene expression amount of the imprinted gene copy number abnormality. Since the section is only 10 μm, 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 imprinted gene deletion gene expression amount, the imprinted gene copy number abnormal gene expression amount, and the total expression amount are divided into five different grades.

In one embodiment herein, the five different grades count at least 1200 cells in the region where the probe is most positive for each probe, for Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9 The imprinted gene deletion gene expression amount, the imprinted gene copy number abnormal gene expression amount, and the total expression amount of the twelve imprinted genes of Z10, Z11, Z15, and Z16 were respectively divided into five different grades.

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, Z2, Z3, and Z4 are:

Grade 0: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression amount of the imprinted gene of less than 0.5%;

Grade I: The imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is 10-20% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is 0.5- 1.5%;

Grade II: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene expression level of 20-25% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression number of the imprinted gene of 1.5- 3.2%;

Grade III: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene expression level of 25-30% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression level of the imprinted gene copy number of 3.2- 5%;

Grade IV: The imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is greater than 5%.

The imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 are independent of each other.

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 and Z6 are:

Grade 0: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene Z5 and Z6 imprinted gene copy number abnormal expression amount is less than 0.5%;

Grade I: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted genes Z5 and Z6 have an imprinted gene copy number abnormal expression amount of 0.5-1.3%;

Grade II: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 15-21% and/or the imprinted genes Z5 and Z6 have an imprinted gene copy number abnormal expression amount of 1.3-2.5%;

Grade III: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 21-30% and/or the imprinted genes Z5 and Z6 have an abnormal expression amount of the imprinted gene copy number of 2.5-4%;

Grade IV: The imprinted gene deletion expression amount of the imprinted genes Z5 and Z6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z5 and Z6 is greater than 4%.

The imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z5 and Z6 are independent of each other.

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 Z8, Z9, Z10, Z11, Z15, and Z16 are:

Grade 0: the imprinted gene Z8, Z9, Z10, Z11, Z15 and Z16 have an imprinted gene deletion expression of less than 10% and/or the imprinted gene Z8, Z9, Z10, Z11, Z15 and Z16 imprinted gene copy number The abnormal expression amount is less than 0.5%;

Grade I: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 10-15% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 0.5-1.3%;

Grade II: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 15-21% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 1.3-2.5%;

Grade III: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 21-25% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 2.5-4%;

Grade IV: the imprinted gene deletion expression amount of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 is greater than 25% and/or the imprinted gene copy number of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level is greater than 4%.

The imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are independent of each other.

In one embodiment herein, the present disclosure provides a device for detecting the degree of benign and malignant bladder tumors, which employs the model, comprising the following elements:

(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 analysis unit calculates the total expression amount of the imprinted gene, the expression amount of the imprinted gene deletion gene, and the abnormal gene expression amount of the imprinted gene copy number, and the abnormality of the gene expression amount and the imprinted gene copy number of the imprinted gene by the model The level of gene expression is used to determine the degree of benign and malignant bladder tumors.

The present disclosure provides a method of detecting the degree of benign and malignant bladder tumors, 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 analysis unit calculates the level of the imprinted gene expression, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount, and the level of the abnormal expression level of the imprinted gene and the imprinted gene copy number by the model. To 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 labeled nuclei in the nucleus. The abnormal copy number of the imprinted gene is that after the cells are subjected to hematoxylin staining, there are more than two red/brown marks in the nucleus. 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 detection system of the invention is used for early and intuitive observation of changes in the imprinted (trace) genes of various types of tumors at the cellular and tissue levels to determine the benign and malignant degree of the tumor, and to provide the most favorable therapeutic opportunity for early oncology patients.

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 processed in a timely manner, and can be selected by a person skilled in the art as needed, and is not particularly limited herein. In one embodiment, the sample to be tested includes tissue paraffin. A combination of any one or at least two of a section, an endoscopic screening sample, a urine exfoliated cell smear, or a bladder lavage cell smear.

In an embodiment of the present invention, the specific operation step of the paraffin section of the tissue is to obtain a human tumor tissue sample, which is fixed in 10% neutral formalin in time, embedded in paraffin, cut into 10 μm thick, and used with positively charged glass. The piece is made into a tissue piece; since it is only 10 μm thick, some of the cells seen under the microscope are incomplete nuclei, so some false negative gene deletions occur.

In an embodiment of the present invention, the specific operation procedure of the endoscope screening sample is to obtain suspicious tissue under a cystoscope, embedded in paraffin, cut into 10 μm thick, and made into a sheet with a positively charged slide.

Because the cystoscopy biopsy has less damage to the patient, the sampling process is simple. Compared with the circulation characteristics of the blood, the cystoscopy biopsy can also be located. The cystoscopy biopsy has its special advantages as an experimental sample.

Urine exfoliated cell smear and bladder lavage cell smear have less damage to the patient, and the sampling process is simple, which has its special advantages as an experimental sample.

In one embodiment, the imprinting genes are Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16, the imprinting gene Z1 is Gnas, and the imprinting gene Z2 is Igf2, the imprinted gene Z3 is Peg10, the imprinted gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, and the imprinted gene Z9 is Dlk1, The imprinted gene Z10 is Gatm, the imprinted gene Z11 is Grb10, 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), Z8 (Dcn), Z9 (Dlk1), Z10 (Gatm), Z11 (Grb10), Z15 (Diras3), Z16 (Snrpn/Snurf) have different degrees of expression in normal tumor cell tissues, and the expression and imprinting state will change significantly when malignant lesions occur.

In an embodiment herein, the design probe is based on the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16, ie Gnas, Igf2, Peg10, Igf2r, Mest , Plagl1, Dcn, Dlk1, Gatm, Grb10, Diras3 and Snrpn/Snurf were designed, specifically selecting a sequence as a probe in the inner 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.

In an embodiment of the present invention, the multi-channel color-developing 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 The fluorescent kit can use two imprinted gene probes or a combination of imprinted genes and other genes to express even multiple imprinted genes and non-imprinted genes.

In one embodiment herein, the degree of benign and malignant bladder tumors to be judged is classified into benign, bladder cancer potential, early bladder cancer, metaphase bladder cancer, and advanced bladder cancer.

In one embodiment of the present invention, the result of determining the degree of benign and malignant bladder tumor is the imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The abnormal expression level of the copy number of the imprinted gene and the imprinted gene are less than the imprint of no more than one imprinted gene in the class I and/or the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The amount of gene deletion expression is I grade and the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is In either case of grade I, it is a benign tumor.

In one embodiment herein, the result of determining the degree of benign and malignant bladder tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The gene-imprinted gene deletion expression level is I, and the imprinted gene copy number abnormal expression amount of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The amount of imprinted gene deletion of the imprinted gene of class I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is class II and the imprinting gene Z1 If the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the second level, it is judged as Bladder cancer potential.

In one embodiment herein, the result of determining the degree of benign and malignant bladder tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The gene-imprinted gene deletion expression level is II, and the imprinted gene copy number abnormal expression amount of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene deletion level of the imprinted gene of class II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is grade III and the imprinting gene Z1 The abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the third grades, and is early Bladder Cancer.

In one embodiment herein, the result of determining the degree of benign and malignant bladder tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The gene-imprinted gene deletion expression level is III, and the imprinted gene copy number abnormal expression amount of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene with no more than one imprinted gene in the class III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is expressed in the IV level and the imprinted gene Z1. The abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the IV grades, and is the medium term Bladder Cancer.

In one embodiment, the result of determining the degree of benign and malignant bladder tumor is at least two imprinting genes of imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The amount of imprinted gene deletion expressed in the IV level or the imprinted gene Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is at least 2 imprinted genes. Grade IV is advanced bladder cancer.

The present disclosure provides a model or device described for the detection of a bladder tumor.

The present disclosure provides the use of the model described in the manufacture of a medicament for the detection and/or treatment of bladder tumors.

The present disclosure provides the use of the device described in the manufacture of a device for the detection and/or treatment of bladder tumors.

In an embodiment herein, the result of determining the degree of benign and malignant bladder tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The gene-imprinted gene deletion expression level is I, and the imprinted gene copy number abnormal expression amount of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The amount of imprinted gene deletion of the imprinted gene of class I or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is class II and the imprinting gene Z1 If the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the second level, it is judged as Bladder cancer potential.

In one embodiment herein, the result of determining the degree of benign and malignant bladder tumor is at least 2 imprints of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The gene-imprinted gene deletion expression level is II, and the imprinted gene copy number abnormal expression amount of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene deletion level of the imprinted gene of class II or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is grade III and the imprinting gene Z1 The abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the third grade, and is early Bladder Cancer.

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 bladder tumor patient sample in an intuitive manner, and the method for in situ labeling of the imprinted gene is objective and intuitive. In the early days, changes in the imprinted (trace) genes were accurately detected, and quantitative models were provided to make a significant contribution to the diagnosis of molecular pathology.

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

DRAWINGS

1 is a pathological section of bladder cancer stained with hematoxylin-stained nuclei of the present invention, wherein the a is a hematoxylin staining of cells, and there is no label in the nucleus, and the imprinted gene is not expressed; and b is a hematoxylin staining of the cells. There is a red/brown mark in the nucleus, and the imprinted gene is present; the c is the hematoxylin staining of the cells, there are two red/brown marks in the nucleus, and the imprinted gene is deleted; the d is the cell nucleus after hematoxylin staining There are more than two red/brown marks in memory, and the imprinted gene copy number is abnormal;

Fig. 2(a) shows the expression status of 12 imprinted genes in the pathological section of grade 0 bladder cancer, and Fig. 2(b) shows the expression status of 12 imprinted genes in the pathological section of grade I bladder cancer, Fig. 2(c) is The expression status of 12 imprinted genes in the pathological sections of grade II bladder cancer, Fig. 2(d) shows the expression status of 12 imprinted genes in the pathological sections of grade III bladder cancer, and Fig. 2(e) shows the pathology of grade IV bladder cancer. The expression status of 12 imprinted genes in the section;

Figure 3 (a) shows the intensity of imprinted deletions of bladder cancer Z1, Z2, Z3, Z4 and Z5 for bladder cancer, and Figure 3 (b) shows the abnormal copy number of bladder cancer Z1, Z2, Z3, Z4 and Z5 for bladder cancer. The intensity of Figure 3(c) shows the intensity of imprinted genes Z6, Z8, Z9, Z10, Z11, Z15 and Z16 for bladder cancer, and Figure 3(d) shows the imprinted genes Z6, Z8, Z9, Z10, Z11. The intensity of copy number abnormality of bladder cancer in Z15 and Z16, wherein LOI is the expression level of imprinted gene deletion gene, and CNV is the gene expression amount of imprinted gene copy number abnormality;

Figure 4 (a) shows the intensity of the imprinted gene Z1 imprint deletion and copy number abnormality, Figure 4 (b) shows the intensity of the imprinted gene Z2 imprint deletion and copy number abnormality, and Figure 4 (c) shows the imprinted gene Z3 imprint deletion and copy number. The intensity of the abnormality, Fig. 4(d) shows the intensity of the imprinted gene Z4 imprint deletion and copy number abnormality, Fig. 4(e) shows the intensity of the imprinted gene Z5 imprint deletion and copy number abnormality, and Fig. 4(f) shows the imprinted gene Z6 imprint The intensity of deletion and copy number anomalies, Figure 4(g) shows the intensity of the imprinted Z8 imprint deletion and copy number anomaly, and Figure 4(h) shows the intensity of the imprinted gene Z9 imprint deletion and copy number anomaly, Figure 4(i) is The imprinting gene Z10 imprinted deletion and copy number abnormality intensity, Figure 4 (j) is the intensity of imprinting gene Z11 imprint deletion and copy number abnormality, Figure 4 (k) is the imprinting gene Z15 imprint deletion and copy number abnormality intensity, Figure 4 (1) The intensity of the imprinting gene Z16 imprinting deletion and copy number abnormality, wherein LOI is the imprinting gene deletion gene expression amount, and CNV is the gene expression amount of the imprinted gene copy number abnormality;

Fig. 5(a) shows the distribution range and grading standard of imprinting gene and Zigma in 62 cases of bladder cancer pathological sections, and Fig. 5(b) shows that imprinting gene Z2 is used in 62 cases of bladder cancer pathological sections. , the distribution range and grading standard of imprinting deletion and copy number abnormality, Fig. 5(c) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 62 pathological sections of bladder cancer with imprinting gene Z3, Fig. 5(d) The imprinting gene Z4 was applied to the pathological sections of 62 cases of bladder cancer, the distribution range and grading standard of imprinting deletion and copy number abnormality, and Fig. 5(e) was the imprinting gene Z5 applied to 62 cases of bladder cancer pathological sections, imprinting deletion and The distribution range and grading standard of copy number abnormality, Fig. 5(f) shows the distribution range and grading standard of imprinting deletion and copy number abnormality in 62 pathological sections of bladder cancer, and the imprinting gene is shown in Fig. 5(g) Z8 was applied to the pathological sections of 62 cases of bladder cancer, the distribution range and grading standard of imprinting deletion and copy number abnormality. Figure 5(h) was applied to the pathological section of 62 cases of bladder cancer with imprinting gene Z9, with imprinting deletion and copy number abnormality. distributed Range and grading criteria, Figure 5 (i) is the distribution range and grading standard of imprinted gene Z10 in 62 cases of bladder cancer pathological sections, imprinting deletion and copy number abnormality, Fig. 5 (j) is the imprinting gene Z11 applied to 62 cases In the pathological section of bladder cancer, the distribution range and grading standard of imprinting deletion and copy number abnormality, Fig. 5(k) is the distribution range and grading standard of imprinting deletion and copy number abnormality in 62 pathological sections of bladder cancer with imprinting gene Z15. Fig. 5(l) shows the distribution range and grading criteria of imprinting deletion and copy number abnormality in 62 pathological sections of bladder cancer, in which imprinted gene Z16 is the imprinting gene deletion gene expression, and CNV is the imprinted gene copy number. Abnormal gene expression.

detailed description

The technical solutions of the present application are further described in the following with reference to the accompanying drawings, and the present invention is not limited to the scope of the embodiments.

Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by methylation of alleles from a particular parent, such that one gene has only one allele and the other is in a state of gene silencing. This type of gene is called a blot (remember) gene. Deletion of the blot is an epigenetic change in which the allelic gene of the imprinted gene results in a silenced allele being activated and beginning to express the gene. Numerous studies have shown that this phenomenon (missing of blots) is ubiquitous in various types of cancer and occurs earlier than cell and tissue morphological changes. At the same time, in healthy cells, the proportion of imprinted deletions is extremely low, in sharp contrast to cancer cells. Therefore, the methylation status of the imprinted gene can be used as a pathological marker to analyze the abnormal state of the cell by a specific molecular detection technique.

The detection model and system of the present disclosure express the impression of the imprinted defect on the sample of the bladder tumor patient in an intuitive manner, and objectively, intuitively, earlyly and accurately detect the imprint by the method of in situ labeling of the imprinted gene. Gene changes and can provide quantitative models that make a significant contribution to the diagnosis of bladder tumors;

The disclosed detection method device can determine the degree of benign and malignant bladder tumor before surgery of a bladder tumor patient, thereby providing a basis for surgery and precise treatment, which is a revolutionary breakthrough in the diagnosis of bladder tumor in the field of cell molecules;

The present disclosure can accurately determine the type of bladder tumor, and the combination detection of imprinted genes can greatly improve the early diagnosis of bladder cancer, and the diagnosis is made, especially in early screening and postoperative follow-up of cancer, especially for patients with suspected recurrence. Tracking follow-up can gain time and make a significant contribution to saving the lives of patients;

The present disclosure can accurately detect the true benign and malignant tissues of the adjacent tissues in the operation of bladder cancer, guide the selection of the scope of surgery, reduce the recurrence caused by the incomplete resection of the cancer tissue, and make a positive contribution to the prognosis and survival of the patient;

The disclosed detection method is different from the immunohistochemical method, which reduces false positives and other negative effects, and not only the targeted drug or technology that causes silencing, rejection, and rearrangement of the gene by the discovery of a bladder tumor-associated imprinted gene deletion site. The method can be used to guide the later treatment and medication.

Example 1 Imprinted Gene Analysis of Bladder Cancer

This embodiment provides a method for detecting a bladder tumor using an imprinted gene, comprising the following steps:

(1) Obtaining histological cell sections (10 μm) of bladder cancer and placing them in a 10% neutral formalin solution to prevent RNA degradation for 24 hours, 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 imprinting genes Z1 (Gnas), Z2 (Igf2), Z3 (Peg10), Z4 (Igf2r), Z5 (Mest), Z6 (Plagl1), Z8 (Dcn), Z9 (Dlk1), Z10. (Gatm), Z11 (Grb10), Z15 (Diras3) and Z16 (Snrpn/Snurf) were designed to select a sequence as a probe in the internal loop of each gene. The specific probe was designed by Advanced Cell Diagnostics. ;

(4) performing RNA SCope in situ hybridization of the probe of step (3) and the sample to be tested by 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. 1 , wherein a is a cell nucleus in which no hemoglobin is stained in the nucleus and the imprinted gene is not expressed; and b is a hematoxylin staining of the cell. 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 more than two red/brown markers in the nucleus, and the nuclei with abnormal copy number of the imprinted gene are shown, and the results are shown in Fig. 2(a) - Fig. 2(e).

As can be seen from Fig. 2(a) - Fig. 2(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 Imprinted Gene Analysis of Cystic Biopsy Samples

Suspected lesions were taken under cystoscopy, fixed in 10% neutral formalin solution for 24 h, embedded in paraffin (FFPE), and cut into 10 μm thick sections. The other detection methods were the same as in Example 1. The results are shown in Figure 3 (a). ) - Figure 3 (d) and Figure 4 (a) - Figure 4 (1).

It can be seen from Fig. 3(a)-Fig. 3(d) that the sensitivity of each gene of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15, Z16 to bladder cancer or It is said that the intensity and state of the imprint deletion corresponding to the expression of bladder cancer are different.

The specific sensitivity of each imprinted gene to bladder cancer is shown in Fig. 4(a) - Fig. 4(1):

As can be seen from Fig. 4(a), the imprinting of the imprinted gene Z1 begins to appear in the malignant potential stage, gradually increases to a very high level with the development of bladder cancer, and the copy number abnormality of the imprinted gene Z1 begins to appear in the malignant potential stage. , rapidly rising in early bladder cancer, and continues to increase to a high level with the development of bladder cancer;

As can be seen from Fig. 4(b), the imprinted deletion and copy number abnormality of the imprinted gene Z2 reached a high level in the malignant potential stage, and maintained high sensitivity in early to late stage bladder cancer;

As can be seen from Fig. 4(c), the imprinting deletion and copy number abnormality of the imprinted gene Z3 began to appear in the malignant potential stage, and gradually increased to a high level with the development of bladder cancer;

As can be seen from Figure 4(d), the imprinted deletion of the imprinted gene Z4 is markedly elevated during the malignant potential phase, continues to rise to a high level in early bladder cancer, and remains stable in advanced bladder cancer, imprinted with the gene Z4. The copy number abnormality increased significantly during the malignant potential stage and gradually increased to a high level in early to late stage bladder cancer;

As can be seen from Fig. 4(e), the imprinted deletion of the imprinted gene Z5 rapidly increased in the stage of malignant potential and early stage bladder cancer, and remained stable in the metaphase to late stage bladder cancer. The copy number abnormality of the imprinted gene Z5 began to appear in the malignant potential stage. , gradually rising to a higher level as the bladder cancer progresses;

As can be seen from Fig. 4(f), the imprinted deletion and copy number abnormality of the imprinted gene Z6 rapidly increased to a high level in early bladder cancer, and remained stable in metaphase to advanced bladder cancer;

As can be seen from Fig. 4(g), the imprinted deletion and copy number abnormality of the imprinted gene Z8 began to appear in the early stage of bladder cancer, but did not continue to rise in the metaphase to late stage of bladder cancer;

As can be seen from Fig. 4(h), the imprinted deletion of the imprinted gene Z9 rapidly increased in the early stage of bladder cancer, but remained stable in the metaphase to late stage of bladder cancer, and the copy number of the imprinted gene Z9 was abnormal in the malignant potential and early stage of bladder cancer. Gradually rising, but remained stable during the mid- to late-stage bladder cancer phase;

As can be seen from Fig. 4(i), the imprinted deletion and copy number abnormality of the imprinted gene Z10 began to appear in the malignant potential stage, gradually increased with the progress of bladder cancer, and reached a higher level in the late stage of bladder cancer;

It can be seen from Fig. 4(j) that the imprinting of the imprinted gene Z11 begins to appear in the early stage of bladder cancer, and remains stable in the advanced bladder cancer. The copy number abnormality of the imprinted gene Z11 begins to appear in the malignant potential stage in the early bladder. The cancer stage continues to rise and remains stable in advanced bladder cancer;

As can be seen from Fig. 4(k), the imprinting of the imprinted gene Z15 begins to appear in the early stage of bladder cancer, and remains stable in the advanced bladder cancer. The copy number abnormality of the imprinted gene Z15 begins to appear in the malignant potential stage in the early bladder. The cancer stage continues to rise and remains stable in advanced bladder cancer;

As can be seen from Fig. 4(l), the imprinted deletion of the imprinted gene Z16 increased significantly in early bladder cancer, and gradually increased to a higher level in the late stage of bladder cancer, and the copy number abnormality of the imprinted gene Z16 began to appear in the malignant potential stage. With the progress of bladder cancer, it gradually rises to a higher level.

Example 3 Imprinted Gene Analysis of 62 Cases of Bladder Tumors

The tissues of 62 patients with bladder cancer were obtained including cystoscopy biopsy samples (10 micrometers). The other detection methods were the same as those in Example 1, and the results were shown in Fig. 5(a)-Fig. 5(1).

As can be seen from Fig. 5(a)-Fig. 5(1), the ratio of imprinted deletion and copy number abnormality of 10 probes in 62 bladder tumor tissue samples showed a low to high distribution, according to the distribution of different probes. Trend, we calculated the grading standard shown by the dashed line in the figure, and can divide the imprint missing and copy number anomalies of each probe into five grades from low to high.

As can be seen from Fig. 5(a), for the imprinted gene Z1, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3.2% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression level of imprinted gene copy number is 3.2-5%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 5%. Grade IV;

As can be seen from Fig. 5(b), for the imprinted gene Z2, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3.2% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression level of imprinted gene copy number is 3.2-5%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 5%. Grade IV;

As can be seen from Fig. 5(c), for the imprinted gene Z3, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3.2% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression level of imprinted gene copy number is 3.2-5%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 5%. Grade IV;

As can be seen from Fig. 5(d), for the imprinted gene Z4, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 20% and/or imprinted gene copy number abnormal expression level is 0.5-1.5% for grade I, imprinted gene deletion expression level is 20-25% and/or imprinted gene copy number abnormal expression level is 1.5-3.2% for grade II, The amount of imprinted gene deletion is 25-30% and/or the abnormal expression level of imprinted gene copy number is 3.2-5%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 5%. Grade IV;

As can be seen from Fig. 5(e), for the imprinted gene Z5, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The amount of imprinted gene deletion is 21-30% and/or the abnormal expression level of imprinted gene copy number is 2.5-4%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 4%. Grade IV;

As can be seen from Fig. 5(f), for the imprinted gene Z6, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The amount of imprinted gene deletion is 21-30% and/or the abnormal expression level of imprinted gene copy number is 2.5-4%, the imprinting gene deletion expression is greater than 30% and/or the imprinted gene copy number is abnormally expressed more than 4%. Grade IV;

As can be seen from Fig. 5(g), for the imprinted gene Z8, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. Grade IV;

As can be seen from Fig. 5(h), for the imprinted gene Z9, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. Grade IV;

As can be seen from Fig. 5(i), for the imprinted gene Z10, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. Grade IV;

As can be seen from Fig. 5(j), for the imprinted gene Z11, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. Grade IV;

As can be seen from Fig. 5(k), for the imprinted gene Z15, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. Grade IV;

As can be seen from Fig. 5(l), for the imprinted gene Z16, the imprinted gene deletion expression amount is less than 10% and/or the imprinted gene copy number abnormal expression amount is less than 0.5%, and the imprinted gene deletion expression amount is 10- 15% and/or imprinted gene copy number abnormal expression level is 0.5-1.3% for grade I, imprinted gene deletion expression level is 15-21% and/or imprinted gene copy number abnormal expression level is 1.3-2.5% for grade II, The imprinted gene deletion expression level is 21-25% and/or the imprinted gene copy number abnormal expression level is 2.5-4% as the grade III, the imprinted gene deletion expression amount is greater than 25% and/or the imprinted gene copy number abnormal expression amount is greater than 4%. It is level IV.

From a comprehensive analysis of the 62 bladder cancer tumors, we can conclude:

The judging degree of benign and malignant bladder tumors is classified into benign tumor, bladder cancer potential, early bladder cancer, metaphase bladder cancer and advanced bladder cancer:

The results of determining the degree of benign and malignant bladder tumors are the abnormal expression of imprinted gene deletion and imprinted gene copy number of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The amount of the imprinted gene whose expression is less than the level I and/or the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is not more than one imprinted gene. And the abnormal expression level of the imprinted gene copy number of no more than one imprinted gene in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is any one of the I grades In the case, it is a benign tumor;

The result of determining the degree of benign and malignant bladder tumor is the imprinted gene deletion expression amount of at least two imprinted genes in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. For class I, the imprinted gene copy number of at least two imprinting genes of the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is abnormally expressed in grade I or imprinted gene Z1. The imprinted gene deletion expression of no more than one imprinted gene in Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is grade II and the imprinted genes Z1, Z2, Z3, Z4, Z5 And the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the second grade, and the potential of bladder cancer is determined;

The result of determining the degree of benign and malignant bladder tumor is the imprinted gene deletion expression amount of at least two imprinted genes in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. For class II, the abnormal expression level of imprinted copy number of at least two imprinted genes of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is class II or imprinted gene Z1 No more than one imprinted gene in Z2, Z3, Z4, Z4, Z6, Z8, Z9, Z10, Z11, Z15, Z16, and the imprinted gene has a deletion level of III and the imprinted genes Z1, Z2, Z3, Z4, Z5 , in the Z6, Z8, Z9, Z9, Z9, Z10, Z8, Z9, Z10, Z11, Z15, and Z16, the abnormal expression level of the imprinted gene copy number of any one of the imprinted genes is any one of the grade III, and is an early bladder cancer;

The result of determining the degree of benign and malignant bladder tumor is the imprinted gene deletion expression amount of at least two imprinted genes in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. For class III, the abnormal expression level of the imprinted gene copy number of at least two imprinting genes of the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is grade III or imprinted gene Z1 The imprinting gene of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is not more than one imprinted gene, and the imprinted gene is expressed in grade IV and the imprinted genes Z1, Z2, Z3, Z4, Z5 And the abnormal expression level of the imprinted gene copy number of not more than one imprinted gene in Z6, Z8, Z9, Z10, Z11, Z15, and Z16 is any one of the IV grades, and is a metaphase bladder cancer;

The result of determining the degree of benign and malignant bladder tumor is that the imprinted gene deletion expression amount of at least two imprinted genes in the imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is The abnormal expression level of imprinted gene copy number of at least 2 imprinted genes of class IV or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is grade IV, which is late bladder 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. The imprinting (trace) gene changes and can provide a quantitative model that makes a significant contribution to the diagnosis of bladder 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

An imprinting gene grading model for classifying the expression state of imprinted genes by calculating changes in total expression of imprinted genes, expression of imprinted gene deletions, and abnormal expression of imprinted gene copies in bladder tumors;

Wherein, the imprinting gene is any one or a combination of at least two of Z1, Z2, Z3, Z4 or Z5, the imprinting gene Z1 is Gnas, the imprinting gene Z2 is Igf2, and the imprinting gene Z3 is Peg10 The imprinted gene Z4 is Igf2r, and the imprinted gene Z5 is Mest.
The model according to claim 1, wherein said model calculates a imprinted gene as follows:

It is preferred to calculate any one of Z1, Z2, Z3, Z4 or Z5, and it is preferable to calculate any one of Z1, Z2, Z4 or Z5, and further preferably to calculate Z2 or Z4.
The model according to claim 1 or 2, wherein the model calculates the imprinted gene by calculating a combination of any two of the imprinted genes of Z1, Z2, Z3, Z4 or Z5, preferably for calculating the Z1 and Z2 imprints Combination of genes or a combination of Z2 and Z4 imprinted genes.
The model according to any one of claims 1 to 3, wherein the imprinted gene further comprises any one or a combination of at least two of Z6, Z8, Z9, Z10, Z11, Z15 or Z16; The imprinted gene Z6 is Plagl, the imprinted gene Z8 is Dcn, the imprinted gene Z9 is Dlk1, the imprinted gene Z10 is Gatm, the imprinted gene Z11 is Grb10, and the imprinted gene Z15 is Diras3, the imprint The gene Z16 is Snrpn/Snurf.
The model according to any one of claims 1 to 4, wherein the model calculates a imprinted gene by calculating a combination of imprinted genes of Z1-Z6, Z8-Z11, Z15-Z16 A combination of twelve imprinted genes.
The model according to any one of claims 1 to 5, 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 classified into five different levels.
The model according to claim 7, wherein said five different grades are for twelve imprinting genes for Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount were respectively divided into five different levels.
The model according to claim 7 or 8, wherein the five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount for Z1, Z2, Z3 and Z4 are:

Grade 0: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression amount of the imprinted gene of less than 0.5%;

Grade I: The imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is 10-20% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is 0.5- 1.5%;

Grade II: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene expression level of 20-25% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression number of the imprinted gene of 1.5- 3.2%;

Grade III: the imprinted gene Z1, Z2, Z3 and Z4 have an imprinted gene expression level of 25-30% and/or the imprinted genes Z1, Z2, Z3 and Z4 have an abnormal expression level of the imprinted gene copy number of 3.2- 5%;

Grade IV: The imprinted gene deletion expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3 and Z4 is greater than 5%.
The model according to any one of claims 7 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 and Z6 are:

Grade 0: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of less than 10% and/or the imprinted gene Z5 and Z6 imprinted gene copy number abnormal expression amount is less than 0.5%;

Grade I: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 10-15% and/or the imprinted genes Z5 and Z6 have an imprinted gene copy number abnormal expression amount of 0.5-1.3%;

Grade II: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 15-21% and/or the imprinted genes Z5 and Z6 have an imprinted gene copy number abnormal expression amount of 1.3-2.5%;

Grade III: the imprinted gene Z5 and Z6 have an imprinted gene deletion expression amount of 21-30% and/or the imprinted genes Z5 and Z6 have an abnormal expression amount of the imprinted gene copy number of 2.5-4%;

Grade IV: The imprinted gene deletion expression amount of the imprinted genes Z5 and Z6 is greater than 30% and/or the imprinted gene copy number abnormal expression amount of the imprinted genes Z5 and Z6 is greater than 4%.
The model according to any one of claims 7 to 10, wherein five different levels of the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of Z8, Z9, Z10, Z11, Z15 and Z16 are divided. for:

Grade 0: the imprinted gene Z8, Z9, Z10, Z11, Z15 and Z16 have an imprinted gene deletion expression of less than 10% and/or the imprinted gene Z8, Z9, Z10, Z11, Z15 and Z16 imprinted gene copy number The abnormal expression amount is less than 0.5%;

Grade I: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 10-15% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 0.5-1.3%;

Grade II: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 15-21% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 1.3-2.5%;

Grade III: The imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 are deleted in an amount of 21-25% and/or the imprinted genes of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of copy number is 2.5-4%;

Grade IV: the imprinted gene deletion expression amount of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 is greater than 25% and/or the imprinted gene copy number of the imprinted genes Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level is greater than 4%.
A device for detecting the degree of benign and malignant bladder 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 bladder tumors.
A method for detecting the degree of benign and malignant bladder 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 in the step (1) is a paraffin section of the tissue, an endoscopic screening sample, a urine exfoliated cell smear or a bladder lavage cell smear. Any one or a combination of at least two.
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 bladder tumor to be judged is classified into a benign bladder tumor, a bladder cancer potential, early bladder cancer, metaphase bladder cancer, and advanced bladder cancer.
The method according to claim 18, wherein the result of determining the degree of benign and malignant bladder tumors is the deletion of the imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 Abnormal expression levels of expression and imprinted gene copy number are less than 1 level and/or no more than 1 imprinted gene in imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene deletion expression level is I grade and the imprinted gene copy number is abnormally expressed in no more than one imprinted gene in the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 If the amount is in any of the grade I, it is a benign bladder tumor.
The method according to claim 18 or 19, wherein said results of determining the degree of benign and malignant bladder tumors are imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. The imprinted gene deletion expression level of at least two imprinted genes is grade I, and the imprints of at least two imprinted genes of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of the gene copy number is I or the imprinted gene has no more than one imprinted gene in the imprinted genes Z1, Z2, Z3, Z9, Z10, Z11, Z15 and Z16. The abnormal expression level of the imprinted gene copy number of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is one of the II levels. In this case, it is judged as the potential of bladder cancer.
The method according to any one of claims 18 to 20, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinting gene deletion expression level of at least two imprinting genes in Z15 and Z16 is class II, and at least two imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene copy number abnormal expression level of the imprinted gene is II or imprinted gene Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 with no more than one imprinted gene imprinted gene deleted The abnormal expression level of the imprinted gene copy number of the imprinted gene of class III and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is not more than one imprinted gene. In either case, it is early bladder cancer.
The method according to any one of claims 18 to 21, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinting gene deletion expression level of at least two imprinting genes in Z15 and Z16 is III, and at least two imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 Imprinted gene copy number abnormal expression level of the imprinted gene of class III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 with no more than one imprinted gene The abnormal expression level of the imprinted gene copy number of the imprinted gene of class IV and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is not more than one imprinted gene. In either case, it is a mid-stage bladder cancer.
The method according to any one of claims 18 to 22, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinted gene deletion expression level of at least 2 imprinted genes in Z15 and Z16 is IV level or at least 2 imprints of imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of the imprinted gene copy number of the gene is grade IV, which is advanced bladder cancer.
Use of a model according to any of claims 1-11 or a device according to claim 12 in the manufacture of a medicament and/or device for the detection and/or treatment of bladder tumors.
The use according to claim 24, wherein the degree of benign and malignant bladder tumor to be judged is classified into benign bladder tumor, bladder cancer potential, early bladder cancer, metaphase bladder cancer, and advanced bladder cancer.
The use according to claim 24 or 25, wherein the result of determining the degree of benign and malignant bladder tumor is an imprint of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The amount of gene deletion expression and the abnormal expression of the imprinted gene copy number are less than 1 level and/or no more than 1 of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16. Imprinted gene imprinted gene deletion expression level I and imprinted gene Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 imprinted gene copy number of no more than 1 imprinted gene If the abnormal expression level is any of the grade I, it is a benign bladder tumor.
The use according to any one of claims 24 to 26, wherein the result of determining the degree of benign and malignant bladder tumor is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinting gene deletion expression level of at least two imprinting genes in Z15 and Z16 is class I, at least two of imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 Imprinted gene imprinting gene abnormal expression level of imprinted gene is 1 or imprinted gene Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 in which no more than one imprinted gene is deleted. The abnormal expression level of the imprinted gene copy number of the imprinted gene of class II and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is no more than one imprinted gene. In either case, it is judged to be bladder cancer potential.
The use according to any one of claims 24 to 27, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinting gene deletion expression level of at least two imprinting genes in Z15 and Z16 is class II, and at least two imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The imprinted gene copy number abnormal expression level of the imprinted gene is II or imprinted gene Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 with no more than one imprinted gene imprinted gene deleted The abnormal expression level of the imprinted gene copy number of the imprinted gene of class III and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is not more than one imprinted gene. In either case, it is early bladder cancer.
The use according to any one of claims 24 to 28, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinting gene deletion expression level of at least two imprinting genes in Z15 and Z16 is III, and at least two imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 Imprinted gene copy number abnormal expression level of the imprinted gene of class III or imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 with no more than one imprinted gene The abnormal expression level of the imprinted gene copy number of the imprinted gene of class IV and the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 is not more than one imprinted gene. In either case, it is a mid-stage bladder cancer.
The use according to any one of claims 24 to 29, wherein the result of determining the degree of benign and malignant bladder tumors is imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 The imprinted gene deletion expression level of at least 2 imprinted genes in Z15 and Z16 is IV level or at least 2 imprints of imprinting genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z15 and Z16 The abnormal expression level of the imprinted gene copy number of the gene is grade IV, which is advanced bladder cancer.