CN109971848B

CN109971848B - Grading model for detecting benign and malignant degrees of esophagus tumor and/or stomach tumor and application thereof

Info

Publication number: CN109971848B
Application number: CN201811548276.8A
Authority: CN
Inventors: 成彤; 周宁
Original assignee: Lisen Imprinting Diagnosis Technology Wuxi Co ltd
Current assignee: Lisen Imprinting Diagnosis Technology Wuxi Co ltd
Priority date: 2017-12-27
Filing date: 2018-12-18
Publication date: 2024-02-09
Anticipated expiration: 2038-12-18
Also published as: CN109971848A

Abstract

The invention relates to a grading model for detecting benign and malignant degree of esophagus tumor and/or stomach tumor and application thereof, wherein the model grades the change of imprinting gene in tumor by calculating imprinting gene deletion expression quantity, imprinting gene copy number abnormal expression quantity and imprinting gene total expression quantity.

Description

Grading model for detecting benign and malignant degrees of esophagus tumor and/or stomach tumor and application thereof

Technical Field

The invention relates to the field of biotechnology, relates to the field of gene diagnosis, relates to a grading model and application thereof, relates to a grading model for detecting benign and malignant degrees of esophagus tumor and/or stomach tumor and application thereof, and in particular relates to a grading model of a group of imprinting genes in detecting benign and malignant degrees of esophagus tumor and/or stomach tumor and a device and application thereof.

Background

Gastric cancer and esophageal cancer are two common digestive tract tumors, and the number of newly increased gastric cancer cases is 95.2 ten thousand, the number of deaths is 72.3 ten thousand, the number of newly increased esophageal cancer cases is 45.6 ten thousand, and the number of deaths is 40 ten thousand worldwide in 2012. The stomach cancer and esophagus cancer are a high incidence area in China, and the new cases and death numbers of the stomach cancer and esophagus cancer account for about 50% of the world each year. The 5-year survival rate of early gastric cancer and esophageal cancer exceeds 90%, while the 5-year survival rate of middle-late gastric cancer and esophageal cancer is less than 30%, so that the early diagnosis of gastric cancer and esophageal cancer has very important significance for saving the life of patients. However, stomach cancer and esophagus cancer usually have no obvious symptoms in early stages, the detection rate in China is lower than 10%, and more than half of patients are in middle and late stages when stomach cancer or esophagus cancer is found.

It has been recognized that intraepithelial neoplasia is a precancerous lesion of the stomach and can be classified into a low-grade intraepithelial neoplasia and a high-grade intraepithelial neoplasia according to the degree of hyperplasia. The likelihood of progression of low grade intraepithelial neoplasia to gastric cancer is about 20%, while the likelihood of progression of high grade intraepithelial neoplasia to gastric cancer is greater than 70%. At present, the pathological distinction between low-grade and high-grade intraepithelial neoplasia is mainly judged subjectively by pathologists according to morphology. Although there have been many studies attempting to eliminate the subjectivity of classification using mucous histochemistry, immunocytochemistry to detect differentiation or cell proliferation markers, and oncogene products and morphometric analysis, there has been no significant advantage over traditional morphological analysis to date. Similarly, current diagnosis of esophageal cancer is also mainly dependent on imaging and histomorphology, and the accuracy of diagnosis is greatly dependent on experience and subjective judgment of doctors, so that misdiagnosis is easily caused.

CN 106609037A discloses a detection kit for diagnosing gastric cancer patients based on a plurality of genes, wherein the kit comprises primers for diagnosing gastric cancer, and the kit can be used for diagnosing gastric cancer, but the detection is not accurate enough, and the specific stage of gastric cancer cannot be distinguished.

Traditional pathology makes a judgment on the benign or malignant diagnosis of cells based on the relationship of cell size, morphology, wettability and surrounding cell tissue. It has a great limitation in the discovery of early changes in cells (cancers), and thus a method for diagnosing cancer at the cellular molecular level has become a research hotspot at one time. With the continuous intensive research in the field of molecular biology, more and more molecular detection techniques are being applied to cancer diagnosis.

Cancer is the generation of uncontrolled cell growth/division 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, so that the judgment of benign and malignant tumors of esophagus and stomach is made. With the development and penetration of molecular biology, more and more molecular detection techniques are applied to the detection of esophageal cancer and gastric cancer. From the analysis of the progression of cancer, changes in molecular level (epigenetic and genetic) are far ahead of variations in cell morphology and tissue structure. Molecular biological assays are therefore more sensitive to early detection of cancer.

Genomic imprinting is one way of gene regulation in epigenetics. It is characterized in that by methylating alleles from specific parents, one gene is expressed while the other is put into a gene silencing state. This kind of gene is called a blot (marker) gene. A print deletion is an epigenetic change in which the print gene demethylation causes the activation of the silent state allele and the initiation of gene expression. Numerous studies have shown that this phenomenon (print loss) is common to all types of cancer and occurs earlier in time than changes in cell and tissue morphology. Meanwhile, in healthy cells, the proportion of imprinting missing is extremely low, in sharp contrast to cancer cells. Therefore, the methylation state of the imprinted gene can be used as a pathological marker, and the abnormal state of the cells can be analyzed by a specific molecular detection technology.

For the above reasons, new detection systems and detection models are needed for the current diagnosis of esophageal and gastric cancers, and the changes of molecular markers existing on the cell level of esophageal and gastric cancers are analyzed based on patient biopsy samples, so that more accurate prognosis and diagnosis information is provided.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a grading model for detecting benign and malignant degrees of esophagus tumor and/or stomach tumor and application thereof, wherein the detecting device and the model are used for intuitively observing the change of imprinting (trace) genes of the esophagus tumor and/or stomach tumor at an early stage under the cell and tissue level so as to judge the benign and malignant degrees of the esophagus tumor and/or the stomach tumor.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a imprinted gene classification model for esophageal tumor and/or gastric tumor, which classifies the expression state of imprinted genes by calculating the variation of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level, and the total expression level of imprinted genes in esophageal tumor and/or gastric tumor;

wherein the imprinting gene is any one or the combination of at least two of Z1, Z11 or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z11 is Grb10, and the imprinting gene Z16 is Snrpn/Snurf.

In the present invention, the inventors found that by calculating the amount of the imprinting gene deletion expression and the amount of the imprinting gene copy number abnormal expression of any one of the imprinting genes Z1, Z11 and Z16 in esophageal tumor and/or gastric tumor, the diagnostic sensitivity to esophageal cancer can be up to 90.4% or more and the diagnostic sensitivity to gastric cancer can be up to 67.6% or more.

According to the present invention, if only one imprinting gene is detected in the preliminary detection, any one of Z1, Z11 and Z16 can be detected, and the most preferable imprinting gene for detecting esophageal cancer is Z1 and the most preferable imprinting gene for detecting gastric cancer is Z11.

In the invention, the inventor finds that the diagnosis sensitivity of the single Z1 imprinting gene to esophagus cancer can reach 96.3 percent, the single Z11 imprinting gene to esophagus cancer can reach 92.5 percent, and the single Z16 imprinting gene to esophagus cancer can reach 90.4 percent;

in the invention, the inventor finds that the diagnostic sensitivity of the single detection of one Z1 imprinting gene to gastric cancer can reach 80.6%, the diagnostic sensitivity of the single detection of one Z11 imprinting gene to gastric cancer can reach 88.9%, and the diagnostic sensitivity of the single detection of one Z16 imprinting gene to gastric cancer can reach 67.6%.

According to the present invention, if a combination of two imprinted genes of imprinted genes is detected, the combination may be any two of Z1, Z11 and Z16, for example, the combination may be a combination of Z1 and Z11, a combination of Z1 and Z16 or a combination of Z11 and Z16.

In the invention, the inventor finds that sensitivity can be further improved by calculating the imprinting gene deletion expression quantity and imprinting gene copy number abnormal expression quantity of two or more imprinting genes, the diagnosis sensitivity to esophageal cancer can reach more than 98.1% by detecting the combination of the two imprinting genes of the imprinting genes, the diagnosis sensitivity to esophageal cancer can reach 98.1% by detecting the combination of Z1 and Z16, the diagnosis sensitivity to esophageal cancer can reach 99.2% by detecting the combination of Z1 and Z11, and the diagnosis sensitivity to esophageal cancer can reach 99.2% by detecting the combination of Z11 and Z16;

In the present invention, the inventors found that sensitivity can be further improved by calculating the imprinting gene deletion expression level and imprinting gene copy number abnormality expression level of two or more imprinting genes, that the diagnosis sensitivity to gastric cancer can be 94.1% or more by detecting the combination of the two imprinting genes of the imprinting genes, that the diagnosis sensitivity to gastric cancer can be 97.1% by detecting the combination of Z1 and Z16, that the diagnosis sensitivity to gastric cancer can be 97.2% by detecting the combination of Z1 and Z11, and that the diagnosis sensitivity to gastric cancer can be 94.1% by detecting the combination of Z11 and Z16.

According to the invention, the imprinted gene further comprises any one or a combination of at least two of Z3, Z4, Z5, Z6, Z8, Z10 or Z13; the marking gene Z3 is Peg10, the marking gene Z4 is Igf2r, the marking gene Z5 is test, the marking gene Z6 is Plagl1, the marking gene Z8 is Dcn, the marking gene Z10 is Gatm, and the marking gene Z13 is Sgce.

In the invention, the inventor finds that on the basis of using the Z1, Z11 and Z16 genes for detection, the Z3, Z4, Z5, Z6, Z8, Z10 and Z13 genes are additionally used for joint diagnosis, so that not only is the detection accuracy improved, but also false positives can be further avoided by adding other probes for auxiliary diagnosis, the detection accuracy can be further improved, and therefore, the accurate classification and judgment of all esophagus and stomach tumor samples can be realized.

According to the invention, the method for calculating the imprinting gene by the model comprises the following steps: combinations of ten genes of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 were calculated.

In the invention, the imprinted gene is deleted, two red/brown marks exist in the nucleus after the cells are subjected to hematoxylin staining, the imprinted gene copy number abnormality is that more than two red/brown marks exist in the nucleus after the cells are subjected to hematoxylin staining, and the copy number abnormality is caused by abnormal gene replication of cancer cells, so that the gene appears as triploid or even higher polyploid during expression.

In the invention, the hematoxylin-stained marker is selected from red or brown, and the stained marker with other colors can be used for calculating the total expression quantity of the imprinted gene, the deletion expression quantity of the imprinted gene and the abnormal expression quantity of the imprinted gene copy number.

In the present invention, the imprinting gene and the imprinting gene are the same concept, and the same meaning is expressed, and substitution is possible.

Preferably, the formulas for calculating the total expression amount of the imprinted gene, the deletion expression amount of the imprinted gene, and the copy number abnormal expression amount of the imprinted gene are as follows:

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

total expression of normal imprinted genes = b/(b+c+d) ×100%;

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

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

wherein a is a cell nucleus in which no marker exists in the cell nucleus and the imprinted gene is not expressed after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; and d is a cell nucleus with more than two red/brown marks in the cell nucleus and abnormal imprinted gene copy number after the cell is subjected to hematoxylin staining.

In the invention, the hematoxylin-stained marker is selected from red or brown, and the stained marker with other colors can be used for calculating the deletion expression level of the imprinted gene, the abnormal expression level of the copy number of the imprinted gene and the total expression level of the imprinted gene.

In the invention, the probe is subjected to in situ hybridization and a Hemotoxy (hematoxylin) cell nucleus staining amplification signal, the existence of imprinting genes, the deletion of imprinting genes or the abnormality of copy numbers in each cell nucleus are judged under a 40-multiplied or 60-multiplied microscope, and the benign and malignant tumor degree of the sample is judged by calculating the expression quantity of the imprinting genes, the abnormal expression quantity of the copy numbers of the imprinting genes and the total expression quantity of the imprinting genes; since the sections were only 10 μm, approximately 20% of the nuclei seen under the microscope were incomplete nuclei, i.e. there was a possibility of partial false negatives.

According to the present invention, the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and imprinted gene total expression level are classified into five different levels.

According to the present invention, at least 1200 cells were counted by each probe in the region where the sample expression was most positive, and five different grades were respectively divided for the imprinted gene deletion expression level, the imprinted gene copy number abnormality expression level, and the imprinted gene total expression level of ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, and Z16.

Preferably, the five different grades of the deletion expression level of the imprinting gene, the abnormal expression level of the copy number of the imprinting gene and the total expression level for Z1 are:

level 0: any one or a combination of at least two of a imprinting gene deletion expression level of less than 16% of the imprinting gene Z1, a imprinting gene copy number abnormal expression level of less than 2.5% of the imprinting gene Z1, or a total expression level of less than 35% of the imprinting gene Z1;

stage I: the imprinting gene Z1 has a imprinting gene deletion expression level of 16-20%, the imprinting gene copy number abnormal expression level of 2.5-4% of the imprinting gene Z1 or the total expression level of 35-40% of the imprinting gene Z1, or a combination of at least two of them;

Stage II: the imprinting gene Z1 has an imprinting gene deletion expression level of 20-25%, an imprinting gene copy number abnormal expression level of 4-6.5% or a total expression level of 40-50% of the imprinting gene Z1, or a combination of at least two of them;

class III: the imprinting gene Z1 has an imprinting gene deletion expression level of 25-30%, the imprinting gene copy number abnormal expression level of 6.5-8% or the total expression level of the imprinting gene Z1 of 50-65%, or a combination of at least two of them;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z1 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z1 being more than 8%, or the total expression level of the imprinting gene Z1 being more than 65%.

Preferably, the five different grades of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level for Z3 and Z13 are:

level 0: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z3 and Z13 being less than 10%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z3 and Z13 being less than 1%, or the total expression amount of the imprinted genes Z3 and Z13 being less than 25%;

Stage I: the imprinted gene deletion expression amount of the imprinted genes Z3 and Z13 is 10-15%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z3 and Z13 is 1-2%, or the total expression amount of the imprinted genes Z3 and Z13 is any one or a combination of at least two of 25-30%;

stage II: the imprinted gene deletion expression amount of the imprinted genes Z3 and Z13 is 15-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z3 and Z13 is 2-3%, or the total expression amount of the imprinted genes Z3 and Z13 is any one or a combination of at least two of 30-45%;

class III: the imprinted gene deletion expression amount of the imprinted genes Z3 and Z13 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z3 and Z13 is 3-5%, or the total expression amount of the imprinted genes Z3 and Z13 is any one or a combination of at least two of 45-55%;

grade IV: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z3 and Z13 being more than 30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z3 and Z13 being more than 5%, or the total expression amount of the imprinted genes Z3 and Z13 being more than 55%;

in the present invention, the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level of the imprinted genes Z3 and Z13 are independent of each other.

Preferably, the five different grades of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level, and the total expression level for Z4, Z5, Z6, Z10, and Z11 are divided into:

level 0: the imprinted gene deletion expression level of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is less than 16%, the imprinted gene copy number abnormal expression level of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is less than 1.5%, or the total expression level of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is less than 30% or a combination of at least two;

stage I: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 16-20%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 1.5-2.5%, or the total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is any one or a combination of at least two of 30-40%;

stage II: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 2.5-4%, or the total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is any one of 40-50% or a combination of at least two;

Class III: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is 4-6%, or the total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is any one or a combination of at least two of 50-65%;

grade IV: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is more than 30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is more than 6%, or the total expression amount of the imprinted genes Z4, Z5, Z6, Z10 and Z11 is more than 65% or a combination of at least two;

in the present invention, the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level of the imprinted genes Z4, Z5, Z6, Z10 and Z11 are independent of each other.

Preferably, when used for detecting esophageal tumors, the five different grades of the deletion expression level of the imprinting gene, the abnormal expression level of the copy number of the imprinting gene and the total expression level of the imprinting gene for Z16 are:

level 0: any one or a combination of at least two of a imprinting gene deletion expression level of the imprinting gene Z16 of less than 10%, a imprinting gene copy number abnormal expression level of the imprinting gene Z16 of less than 1%, or a total expression level of the imprinting gene Z16 of less than 25%;

Stage I: the imprinting gene Z16 has a imprinting gene deletion expression level of 10-15%, the imprinting gene Z16 has an imprinting gene copy number abnormal expression level of 1-2%, or the imprinting gene Z16 has a total expression level of 25-30%, or a combination of at least two thereof;

stage II: the imprinting gene Z16 has a imprinting gene deletion expression level of 15-25%, the imprinting gene Z16 has an imprinting gene copy number abnormal expression level of 2-4% or the imprinting gene Z16 has a total expression level of 30-40% or a combination of at least two of them;

class III: the imprinting gene Z16 has an imprinting gene deletion expression level of 25-30%, an imprinting gene copy number abnormal expression level of 4-6% of the imprinting gene Z16, or a total expression level of 40-55% of the imprinting gene Z16, or a combination of at least two thereof;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 6%, or the total expression level of the imprinting gene Z16 being more than 55%;

preferably, when used for detecting gastric tumor, the five different grades of the deletion expression amount of the imprinting gene, the abnormal expression amount of the copy number of the imprinting gene and the total expression amount of the imprinting gene for Z16 are divided into:

Level 0: any one or a combination of at least two of a imprinting gene deletion expression level of the imprinting gene Z16 of less than 15%, a imprinting gene copy number abnormal expression level of the imprinting gene Z16 of less than 1.5%, or a total expression level of the imprinting gene Z16 of less than 30%;

stage I: the imprinting gene Z16 has a imprinting gene deletion expression level of 15-20%, the imprinting gene Z16 has an imprinting gene copy number abnormal expression level of 1.5-4% or the imprinting gene Z16 has a total expression level of 30-40% or a combination of at least two of them;

stage II: the imprinting gene Z16 has an imprinting gene deletion expression level of 20-25%, an imprinting gene copy number abnormal expression level of 4-7% of the imprinting gene Z16, or a total expression level of 40-50% of the imprinting gene Z16, or a combination of at least two thereof;

class III: the imprinting gene Z16 has an imprinting gene deletion expression level of 25-30%, an imprinting gene copy number abnormal expression level of 7-10% of the imprinting gene Z16 or a total expression level of 50-60% of the imprinting gene Z16, or a combination of at least two of them;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 10%, or the total expression level of the imprinting gene Z16 being more than 60%.

Due to the specificity of the imprinted gene Z16 expressed in gastric cancer, most of the stages III and IV of Z16 appear in early and mid stages of gastric cancer, and 90% of late gastric cancer samples Z16 are expressed less.

Preferably, when used for detecting esophageal tumors, the five different grades of the deletion expression level of the imprinting gene, the copy number abnormal expression level of the imprinting gene and the total expression level of the imprinting gene for Z8 are:

level 0: any one or a combination of at least two of a imprinting gene deletion expression level of less than 16% of the imprinting gene Z8, a imprinting gene copy number abnormal expression level of less than 2.5% of the imprinting gene Z8, or a total expression level of less than 35% of the imprinting gene Z8;

stage I: the imprinting gene Z8 has a imprinting gene deletion expression level of 16-20%, the imprinting gene copy number abnormal expression level of 2.5-4% or the total expression level of the imprinting gene Z8 is 35-40% or a combination of at least two of them;

stage II: the imprinting gene Z8 has an imprinting gene deletion expression level of 20-25%, an imprinting gene copy number abnormal expression level of 4-6.5% or a total expression level of 40-50% of the imprinting gene Z8, or a combination of at least two of them;

Class III: the imprinting gene Z8 has an imprinting gene deletion expression level of 25-30%, an imprinting gene copy number abnormal expression level of 6.5-8% or a total expression level of 50-65% of the imprinting gene Z8, or a combination of at least two of them;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z8 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z8 being more than 8%, or the total expression level of the imprinting gene Z8 being more than 65%;

preferably, when used for detecting gastric tumor, the five different grades of the deletion expression amount of the imprinting gene, the copy number abnormality expression amount of the imprinting gene and the total expression amount for Z8 are:

level 0: any one or a combination of at least two of a imprinting gene deletion expression level of less than 10% of the imprinting gene Z8, a imprinting gene copy number abnormal expression level of less than 1% of the imprinting gene Z8, or a total expression level of less than 25% of the imprinting gene Z8;

stage I: the imprinting gene Z8 has a imprinting gene deletion expression level of 10-15%, the imprinting gene copy number abnormal expression level of 1-2% of the imprinting gene Z8 or the total expression level of 25-30% of the imprinting gene Z8, or a combination of at least two of them;

Stage II: the imprinting gene Z8 has a imprinting gene deletion expression level of 15-25%, the imprinting gene copy number abnormal expression level of 2-3% of the imprinting gene Z8 or the total expression level of 30-45% of the imprinting gene Z8, or a combination of at least two of them;

class III: the imprinting gene Z8 has an imprinting gene deletion expression level of 25-30%, an imprinting gene copy number abnormal expression level of 3-5% or a total expression level of 45-55% of the imprinting gene Z8, or a combination of at least two of them;

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z8 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z8 being more than 5%, or the total expression level of the imprinting gene Z8 being more than 55%.

In the present invention, the inventors found that the imprinted genes Z8 and Z16 are relatively specific, and that the classification of the amount of imprinted gene deletion expression, the amount of imprinted gene copy number abnormal expression and the total amount of expression in esophageal tumor or gastric tumor is different, so that imprinted genes Z8 and Z16 are classified separately according to the two cases of esophageal tumor or gastric tumor.

In a second aspect, the present invention provides a device for detecting the benign and malignant extent of esophageal and/or gastric tumors, comprising the following units:

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

(2) Probe design unit: designing a specific primer according to the imprinting gene sequence;

(3) And a detection unit: performing in situ hybridization on the probe in the step (2) and a sample to be detected;

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

wherein the analysis unit calculates the total expression amount of the imprinted gene, the abnormal expression amount of the imprinted gene deletion and the abnormal expression amount of the imprinted gene copy number, and judges the benign and malignant degree of the esophageal tumor and/or the gastric tumor by the grades of the imprinted gene deletion expression amount, the abnormal expression amount of the imprinted gene copy number and the total expression amount of the imprinted gene through the imprinted gene grading model of the first aspect.

In the invention, the imprinted gene is deleted, two red/brown marked cell nuclei exist in the cell nuclei after the cells are subjected to hematoxylin staining, the imprinted gene copy number abnormality is the condition that more than two red/brown marked cell nuclei exist in the cell nuclei after the cells are subjected to hematoxylin staining, and the copy number abnormality is caused by abnormal gene replication of cancer cells, so that the gene appears as triploid or even higher polyploid when expressed.

The detection system is used for intuitively observing the change of the marking genes of esophagus and stomach tumors at the early stage under the cellular and tissue level so as to judge the benign and malignant degree of the tumors, and provides the most favorable treatment opportunity for early-stage esophagus and stomach tumor patients.

According to the invention, the sample to be tested in step (1) is derived from human tissue and/or cells.

In the invention, the sample to be detected can be selected according to the needs of a person skilled in the art as long as the RNA is subjected to timely fixation, and the sample to be detected comprises any one or a combination of at least two of paraffin sections of tissues, esophageal mucosa exfoliated cells or gastroscope biopsy samples.

The paraffin section of the tissue comprises the specific operation steps of obtaining a human tumor tissue sample, fixing the tissue sample by 10% neutral formalin in time, embedding the tissue sample by paraffin, cutting the tissue sample into 10 mu m thick, and preparing a tissue sheet by using a positively charged slide; since only 10 μm thick, a part of the nuclei are incomplete under the microscope, so that a partially false negative gene deletion occurs.

The specific operation steps of the esophageal mucosa exfoliated cells are that the mucosa exfoliated cells are obtained through esophageal trabecular examination, and the esophageal mucosa exfoliated cells are timely fixed by 10% neutral formalin.

The gastroscope biopsy sample comprises the specific operation steps of obtaining human cells of esophagus or stomach under a gastroscope, and fixing the human cells by 10% neutral formalin in time.

In the invention, the esophageal mucosa exfoliated cells are similar to the gastroscope biopsy sample in acquisition principle, the operation mode of sample detection is similar to the gastroscope biopsy sample, and the result is similar.

In the invention, since the esophageal mucosa exfoliated cells and the gastroscope biopsy have little harm to patients, the sampling process is simple, compared with the blood circulation characteristic, the esophageal mucosa exfoliated cells and the gastroscope biopsy can be positioned, and the esophageal mucosa exfoliated cells and the gastroscope biopsy have special advantages as experimental samples.

Preferably, the sample to be detected is esophageal mucosa exfoliated cells and/or a gastroscope biopsy sample.

Preferably, the imprinting genes are Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is test, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, the imprinting gene Z13 is Sgce, and the imprinting gene Z16 is Snrpn/Snurf.

In the invention, the imprinting genes Z1 (Gnas), Z3 (Peg 10), Z4 (Igf 2 r), Z5 (test), Z6 (Plagl 1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb 10), Z13 (Sgce), and Z16 (Snrpn/Snurf) are expressed in normal tumor cell tissues to different degrees, and the expression quantity and imprinting state are obviously changed when malignant lesions occur.

In the present invention, the design probes were designed based on the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16, that is, gnas, peg10, igf2r, test, plagl1, dcn, gatm, grb10, sgce and Snrpn/Snurf, specifically, a sequence was selected as a probe within the intron of each gene, and a specific probe was designed by Advanced CellDiagnostics company.

Preferably, the in situ hybridization employs an RNAscope in situ hybridization method.

Preferably, the RNAscope in situ hybridization method uses a single-channel or multi-channel chromogenic kit or a single-channel or multi-channel fluorescent kit, preferably a single-channel red/brown chromogenic kit or a multi-channel fluorescent kit.

In the invention, the multi-channel coloring kit or the multi-channel fluorescent kit comprises two or more than two channels of coloring kits or fluorescent kits, and the two channels of coloring kits or the multi-channel fluorescent kits can use two imprinting gene probes or the joint expression of imprinting genes and other genes or even the comprehensive expression of a plurality of imprinting genes and non-imprinting genes.

According to the present invention, the formulas for calculating the total expression amount of the imprinted gene, the deletion expression amount of the imprinted gene and the copy number abnormal expression amount of the imprinted gene in the model are as follows:

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

total expression of normal imprinted genes = b/(b+c+d) ×100%;

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

In the invention, the probe is used for judging whether the imprinting gene exists, the imprinting gene is missing or the copy number is abnormal in each cell nucleus through in-situ hybridization and a Hemotoxy (hematoxylin) cell nucleus staining amplification signal under a 40-multiplied or 60-multiplied microscope, and the tumor benign and malignant degree of the sample is judged through calculating the total imprinting gene expression quantity, the imprinting gene missing gene expression quantity and the gene expression quantity with abnormal imprinting gene copy number. Since the sections were only 10 microns, approximately 20% of the nuclei seen under the microscope were incomplete nuclei, i.e. there was a possibility of partial false negatives.

According to the present invention, the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and total expression level are classified into five different levels.

The five different grades are that at least 1200 cells are counted in a region where each probe of the sample expresses most positive, and the imprinted gene deletion expression amount, the imprinted gene copy number abnormality expression amount and the imprinted gene total expression amount of ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are respectively divided.

Grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 6%, or the total expression level of the imprinting gene Z16 being more than 55%.

In the present invention, the inventors found that the imprinted genes Z8 and Z16 are relatively specific, and that the classification of the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and total expression level in esophageal tumor or gastric tumor is different, so that imprinted genes Z8 and Z16 are classified separately according to the two cases of esophageal tumor or gastric tumor.

In the invention, the imprinting gene Z16 is uniformly expressed in stomach tumor tissue interstitium and epithelial cells, but the expression is obviously different in early gastric cancer before clinical stage II and middle and late gastric cancer of clinical stage III-IV.

Preferably, the judging of benign and malignant degree of esophageal tumor is classified into benign tumor, esophageal cancer potential, early esophageal cancer, mid-stage esophageal cancer and late-stage esophageal cancer.

Preferably, the result of the determination of the degree of benign and malignant esophagus is that the amount of defective expression of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 and the amount of abnormal expression of the imprinted gene copy number are both less than level I, and that any one of the cases in which no more than 1 of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of defective expression of the imprinted genes of level I and no more than 1 of the imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of abnormal expression of the imprinted gene copy number of level I is benign tumor;

Preferably, the result of the determination of the degree of benign and malignant esophagus is that the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I or that the imprinted gene deletion expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II and the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level II, and is esophageal cancer potential;

preferably, the early stage esophageal cancer is a case where the result of the determination of the degree of benign and malignant esophagus is that the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class II, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z6, Z10, Z11, Z13 and Z16 is class II, or the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, and the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III;

Preferably, the result of the determination of the degree of benign and malignant esophagus is that the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III or that the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV and the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of class IV;

preferably, the result of the determination of the degree of benign and malignant esophagus is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level or that the imprinting gene copy number abnormality expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV-level, and it is advanced esophageal cancer.

Preferably, the judging of the benign and malignant degree of the gastric tumor is classified into benign gastric tumor, gastric cancer potential, early gastric cancer, mid gastric cancer and late gastric cancer.

Preferably, the result of the determination of the degree of benign and malignant gastric tumor is that the amount of defective imprinted gene expression and the amount of abnormal imprinted gene copy number expression of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are both less than level I, and that any one of the cases in which no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of defective imprinted gene expression of level I and no more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of abnormal imprinted gene copy number expression of level I is benign tumor;

preferably, the result of the judgment of the degree of benign or malignant stomach tumor is that the imprinting gene deletion expression amount of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I, the imprinting gene deletion expression amount of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I or more, the imprinting gene copy number abnormality expression amount of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I and the imprinting gene copy number abnormality expression amount of at least 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I and the imprinting gene copy number abnormality expression amount of at least 2 imprinting genes or more, or the imprinting gene copy number abnormality expression amount of at least one of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z11 and Z13 is level II, the imprinting gene deletion expression amount of at least 1, Z3, Z4, Z11 and Z13 is level II;

Preferably, the early gastric cancer is obtained when the result of judging the degree of benign and malignant gastric tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level II, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z10, Z11 and Z13 is level II or the imprinting gene deletion expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level III and the imprinting gene copy number abnormal expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is any one of level III;

preferably, the result of the determination of the degree of benign and malignant gastric tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class III or the imprinting gene deletion expression level of no more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class IV and the imprinting gene copy number abnormal expression level of no more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is any one of class IV;

Preferably, the result of the determination of the degree of benign or malignant stomach tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV-level or the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV-level, and the stomach cancer is advanced stage.

In a third aspect, the present invention provides a imprinted gene fractionation model as described in the first aspect and/or a device as described in the second aspect for the preparation of a medicament or kit for esophageal and gastric tumor detection and/or treatment.

Preferably, the benign and malignant degree of esophageal tumor is judged to be classified into benign tumor, esophageal cancer potential, early esophageal cancer, mid-stage esophageal cancer and late-stage esophageal cancer.

Preferably, the result of the determination of the degree of benign and malignant esophageal tumors is that the amount of defective imprinted gene expression and the amount of abnormal imprinted gene copy number expression of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are both less than level I, and that any one of the cases in which no more than 1 of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of defective imprinted gene expression of level I and no more than 1 of imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 has the amount of abnormal imprinted gene copy number expression of level I is benign tumor;

Preferably, the esophageal cancer potential is determined in a case where the result of the determination of the degree of benign and malignant tumor of the esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z10, Z11, Z16 is level I, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z8, Z10, Z11, Z13 and Z16 is level I, or that the imprinting gene deletion expression level of no more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II;

preferably, the early esophageal cancer is obtained when the result of judging the degree of benign and malignant esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z8, Z10, Z11, Z13 and Z16 is level II, or the imprinting gene deletion expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level III, and the imprinting gene copy number abnormal expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level III;

Preferably, the esophageal cancer is obtained when the result of judging the degree of benign or malignant tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, or the imprinting gene deletion expression level of no more than 1 imprinting genes among imprinting genes Z1, Z3, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of class IV;

preferably, the result of the determination of the degree of benign or malignant esophageal tumor is that the amount of the imprinting gene deletion expression of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV or that the amount of the imprinting gene copy number abnormality expression of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV.

In a fourth aspect, the present invention provides a marker for diagnosis of advanced gastric cancer, wherein the marker is a imprinting gene Z1 and a imprinting gene Z11;

wherein the imprinting gene Z1 is Gnas, and the imprinting gene Z11 is Grb10;

according to the invention, the advanced gastric cancer is gastric cancer with TNM grade of which is greater than T2N0M 0.

In the invention, the imprinting gene Z16 is uniformly expressed in stomach tumor tissue interstitium and epithelial cells, but the expression is obviously different in early gastric cancer before clinical stage II and middle and late gastric cancer of clinical stage III-IV. In early gastric cancer, particularly in intraepithelial neoplasia stage, 75% of the imprinted deletion, copy number abnormality and total expression amount of sample imprinted gene Z16 reach class I or more, and as the cancer worsens, 90% of the imprinted deletion, copy number abnormality and total expression amount of intermediate and late gastric cancer sample imprinted gene Z16 reach class 0. Therefore, the imprinting gene Z16 is combined with imprinting genes Z1 and Z11, is a good diagnostic marker for diagnosing early gastric cancer, particularly for the transition process from low-grade intraepithelial neoplasia to gastric cancer, but the imprinting gene Z16 is not very useful in late gastric cancer, and only Z1 and Z11 are needed for diagnosis.

Compared with the prior art, the invention has the following beneficial effects:

(1) The detection model and the system of the invention show the appearance of imprinting deficiency on the sample of esophagus and stomach tumor patients in an intuitive way, detect the change of imprinting (trace) genes objectively, intuitively and early and accurately by the method of marking imprinting genes in situ, provide a quantized model and make great contribution to diagnosis of esophagus and stomach tumor;

(2) The detection device can obtain the benign and malignant degree of esophagus and stomach tumor and the judgment of the invasion range of cancer cells by using a gastroscope biopsy sample before the operation of the esophagus and stomach tumor patient, thereby providing basis for the operation and the accurate treatment, and being a revolutionary breakthrough in the cell molecular field for diagnosing esophagus and stomach tumor;

(3) The invention can accurately judge the grade of esophagus and stomach tumor, can clearly classify the early lesion of esophagus and stomach tumor, namely the malignancy degree of intraepithelial neoplasia through the combination detection of imprinting genes, can particularly accurately distinguish early stomach cancer and middle and late stomach cancer, greatly improves the early and clear diagnosis of esophagus cancer and stomach cancer, is particularly used for early general investigation and follow-up visit after cancer operation, particularly for follow-up visit of suspected recurrent patients, can strive for time, and makes great contribution to saving the life of patients;

(4) The detection method is different from an immunohistochemical method, false positive and other negative effects are reduced, and moreover, the detection method can be used for guiding later treatment and medication through a target medicament or technical method for silencing, removing and rearranging the gene by the found marked gene deletion sites related to esophagus and stomach tumors.

Drawings

FIG. 1 is a pathological section of gastric cancer of hematoxylin stained nuclei according to the present invention, wherein a is that after the cells are hematoxylin stained, no marker exists in the nuclei and the imprinted gene is not expressed; the step b is that after the cells are subjected to hematoxylin staining, a red/brown mark exists in the nuclei of the cells, and a mark gene exists; the step c is that after the cells are subjected to hematoxylin staining, two red/brown marks exist in the nuclei of the cells, and the imprinted genes are deleted; the step d is that after the cells are subjected to hematoxylin staining, more than two red/brown marks exist in the nuclei of the cells, and the number of copies of the imprinted genes is abnormal;

FIG. 2 (a) shows the expression state of 10 genes in the pathological section of the grade 0 gastric tumor, FIG. 2 (b) shows the expression state of 10 genes in the pathological section of the grade I gastric cancer, FIG. 2 (c) shows the expression state of 10 genes in the pathological section of the grade II gastric cancer, FIG. 2 (d) shows the expression state of 10 genes in the pathological section of the grade III gastric cancer, and FIG. 2 (e) shows the expression state of 10 genes in the pathological section of the grade IV gastric cancer;

Fig. 3 (a) shows the intensity of imprinting deletion of imprinting genes Z1, Z11 and Z16 against gastric cancer, fig. 3 (b) shows the intensity of imprinting genes Z1, Z11 and Z16 against gastric cancer with abnormal copy numbers, fig. 3 (c) shows the intensity of total expression amount of imprinting genes Z1, Z11 and Z16 against gastric cancer, fig. 3 (d) shows the intensity of imprinting deletion of imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against gastric cancer, fig. 3 (e) shows the intensity of imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against gastric cancer with abnormal copy numbers, fig. 3 (f) shows the intensity of total expression amount of imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against gastric cancer with LOI being the expression amount of imprinting gene deletion, CNV being the gene expression amount of imprinting with abnormal copy numbers, TE being the total expression amount of imprinting gene;

FIG. 4 (a) is the intensity of the imprinting gene Z1 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (b) is the intensity of the imprinting gene Z11 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (c) is the intensity of the imprinting gene Z16 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (d) is the intensity of the imprinting gene Z3 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (e) is the intensity of the imprinting gene Z4 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (f) is the intensity of the imprinting gene Z5 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (g) is the intensity of the imprinting gene Z6 imprinting, the intensity of the copy number abnormality and the total amount of expression, FIG. 4 (h) is the intensity of the imprinting gene Z8 imprinting, the copy number abnormality and the total amount of expression, FIG. 4 (i) is the intensity of the imprinting gene Z13 imprinting, the copy number abnormality and the total amount of expression, wherein the gene I is the gene expression of the imprinting gene is the imprinting, the imprinting gene V is the imprinting;

FIG. 5 (a) shows the distribution ranges and classification criteria of the imprinting gene Z1 for 37 cases of gastric tumor pathological sections, the imprinting gene Z11 for 37 cases of gastric tumor pathological sections, the imprinting gene Z16 for 37 cases of gastric tumor pathological sections, the imprinting gene Z3 for 37 cases of gastric tumor pathological sections, the imprinting gene Z4 for 37 cases of gastric tumor pathological sections, FIG. 5 (f) shows the distribution ranges and classification criteria of the imprinting gene Z5 for 37 cases of gastric tumor pathological sections, the imprinting gene Z6 for 37 cases of gastric tumor pathological sections, the imprinting gene Z8 for 37 cases of gastric tumor pathological sections, the imprinting gene Z10 for 37 cases of gastric tumor pathological sections, the imprinting gene Z13 for the LOI for the amount of imprinting gene expression, CNV is the gene expression quantity with abnormal copy number of the imprinted gene, TE is the total expression quantity of the imprinted gene;

FIG. 6 (a) is the expression state of 10 genes in the pathological section of a grade 0 esophageal tumor, FIG. 6 (b) is the expression state of 10 genes in the pathological section of a grade I esophageal cancer, FIG. 6 (c) is the expression state of 10 genes in the pathological section of a grade II esophageal cancer, FIG. 6 (d) is the expression state of 10 genes in the pathological section of a grade III esophageal cancer, and FIG. 6 (e) is the expression state of 10 genes in the pathological section of a grade IV esophageal cancer;

FIG. 7 (a) shows the intensities of the imprinting deletion of the imprinting genes Z1, Z11 and Z16 against esophageal cancer, FIG. 7 (b) shows the intensities of the imprinting genes Z1, Z11 and Z16 against esophageal cancer with abnormal copy numbers, FIG. 7 (c) shows the intensities of the total expression amounts of the imprinting genes Z1, Z11 and Z16 against esophageal cancer, FIG. 7 (d) shows the intensities of the imprinting deletion of the imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against esophageal cancer, FIG. 7 (e) shows the intensities of the imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against esophageal cancer with abnormal copy numbers, FIG. 7 (f) shows the intensities of the total expression amounts of the imprinting genes Z3, Z4, Z5, Z6, Z8, Z10 and Z13 against esophageal cancer with LOI being the expression amounts of the imprinting genes with abnormal copy numbers, CNV being the total expression amounts of TE genes;

wherein FIG. 8 (a) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z1, FIG. 8 (b) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z11, FIG. 8 (c) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z16, FIG. 8 (d) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z3, FIG. 8 (e) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z4, FIG. 8 (f) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z5, FIG. 8 (g) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z6, FIG. 8 (h) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z8, FIG. 8 (i) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z10, FIG. 8 (j) shows the intensities of the imprinting deletion, the copy number abnormality and the total expression amount of the imprinting gene Z13, wherein LOI shows the gene expression amount of the imprinting gene deletion, CNV shows the gene expression amount of the imprinting gene copy number abnormality, TE shows the total expression amount of the imprinting gene;

FIG. 9 (a) shows the distribution ranges and classification criteria of the imprinting gene Z1 for 57 cases of esophageal tumor pathological sections, FIG. 9 (b) shows the distribution ranges and classification criteria of the imprinting gene Z11 for 57 cases of esophageal tumor pathological sections, FIG. 9 (c) shows the distribution ranges and classification criteria of the imprinting gene Z16 for 57 cases of esophageal tumor pathological sections, FIG. 9 (d) shows the distribution ranges and classification criteria of the imprinting gene Z3 for 57 cases of esophageal tumor pathological sections, FIG. 9 (e) shows the distribution ranges and classification criteria of the imprinting gene Z4 for 57 cases of esophageal tumor pathological sections, FIG. 9 (f) shows the distribution ranges and classification criteria of the imprinting gene Z5 for 57 cases of esophageal tumor pathological sections, the imprinting gene Z6 for 57 cases of esophageal tumor pathological sections, the imprinting gene Z8 for 57 cases of esophageal tumor pathological sections, the imprinting gene Z10 for 57 cases of esophageal tumor pathological sections, the imprinting gene Z13 for 57 cases of esophageal tumor pathological sections, LOI is the gene expression level of the imprinting gene deletion, CNV is the gene expression level of the imprinting gene copy number abnormality, and TE is the total gene expression level of the imprinting gene.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below by the specific embodiments in combination with the accompanying drawings, but the invention is not limited to the examples.

Example 1 imprinted Gene analysis of gastric tumors

The detection method of the imprinting gene comprises the following steps:

(1) Obtaining tissue cell slices (10 microns) of gastric tumors, fixing the tissue cell slices in 10% neutral formalin solution for 24 hours for preventing RNA degradation, embedding paraffin (FFPE), removing the slide glass by positive charges, and baking the slices for more than 3 hours in a 40 ℃ oven;

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

(3) Designing a probe: designing a specific primer according to the imprinting gene sequence;

the design probes were designed based on the imprinted genes Z1 (Gnas), Z3 (Peg 10), Z4 (Igf 2 r), Z5 (test), Z6 (Plagl 1), Z8 (Dcn), Z10 (Gatm), Z11 (Grb 10), Z13 (Sgce) and Z16 (Snrpn/Snuf), with a specific sequence selected as a probe within the introns of each gene, and the specific probes were designed by Advanced CellDiagnostics company.

(4) Carrying out RNA SCope in situ hybridization on the probe in the step (3) and a sample to be detected through a kit;

(5) Signal amplification and hematoxylin staining, and analyzing the expression condition of the imprinted gene by using a microscope for imaging;

the formula for calculating the total expression quantity of the imprinting genes, the deletion expression quantity of the imprinting genes and the abnormal expression quantity of the copy number of the imprinting genes in the model is as follows:

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

total expression of normal imprinted genes = b/(b+c+d) ×100%;

imprinted gene deleted gene expression amount (LOI) =c/(b+c+d) ×100%;

as shown in fig. 1, a, b, c, d, a is a cell nucleus with no marker in the cell nucleus and no expressed imprinted gene after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; and d is a cell nucleus with more than two red/brown marks in the cell nucleus and abnormal imprinted gene copy number after the cell is subjected to hematoxylin staining.

From FIGS. 2 (a) -2 (e), it can be seen that the proportion of cells with a loss of imprint (two signal points in the nucleus) and an abnormality in copy number (three or more signal points in the nucleus) gradually increases with the increase in malignancy in the samples from grade 0 to grade IV.

Example 2 imprinted Gene analysis of gastroscopic biopsy samples

The gastroscope biopsy sample is obtained by taking out suspicious lesion tissues under a gastroscope, fixing the suspicious lesion tissues for more than 24 hours by using 10% neutral formalin solution, and other detection methods are the same as in the example 1.

As can be seen from fig. 3 (a) -3 (f), the intensity and status of the sensitivity of each of the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, Z16 to gastric cancer or the absence of imprinting corresponding to gastric cancer expression are different.

Specifically, the sensitivity of each imprinting gene to gastric cancer is as shown in fig. 4 (a) -4 (j), and as can be seen from fig. 4 (a) -4 (c), imprinting deletion and increase in expression amount of imprinting gene Z1 begin to occur in the stage of malignant potential, rapidly rise to a very high level in the progress of early-to-late gastric cancer, and abnormality in copy number of imprinting gene Z1 begin to occur in the stage of malignant potential, rapidly rise in the early-and-middle gastric cancer, and slow down in the rising rate in the stage of late gastric cancer; the imprinting deletion of the imprinting gene Z11 begins to appear in the malignant potential stage, rises rapidly in the early gastric cancer stage, slows down the rising speed in the middle gastric cancer stage, and continues to rise to a higher level in the late gastric cancer stage, and the copy number abnormality and the expression quantity increase of the imprinting gene Z11 begin to appear in the malignant potential stage and rise to a higher level gradually in the early-to-late gastric cancer development process; the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z16 rapidly rise in malignant potential and early gastric cancer stage, but obviously decline in middle and late gastric cancer stage;

As can be seen from fig. 4 (d) -4 (j), the imprinting deletion, copy number abnormality, and expression amount increase of the imprinting gene Z3 start to occur in the early gastric cancer stage, continue to rise in the mid-gastric cancer, and slow down the rising rate in the late gastric cancer; the imprinting deletion of the imprinting gene Z4 begins to appear in the early gastric cancer stage, rises rapidly in the middle gastric cancer stage, and slowly rises in the late gastric cancer stage, the copy number abnormality of the imprinting gene Z4 rises rapidly in the early gastric cancer stage, and rises slowly in the middle and late gastric cancer stages, the expression quantity of the imprinting gene Z4 increases to begin to appear in the malignant potential stage, rises rapidly in the early gastric cancer stage, does not rise obviously in the middle gastric cancer stage, and continues to rise in the late gastric cancer stage; the imprinting deletion of the imprinting gene Z5 rapidly rises in the early gastric cancer stage, the rising speed of the imprinting gene Z5 is slowed down in the middle gastric cancer stage, the imprinting gene Z5 continuously rises to a higher level until the late gastric cancer stage, the copy number abnormality of the imprinting gene Z5 begins to appear in the early gastric cancer stage, the imprinting gene Z5 rapidly rises in the middle gastric cancer stage, the rising speed of the imprinting gene Z5 is slowed down in the late gastric cancer stage, the expression quantity of the imprinting gene Z5 begins to appear in the early gastric cancer stage, and the imprinting gene Z5 gradually rises to a higher level in the middle gastric cancer stage and the late gastric cancer stage; the imprinting deletion of the imprinting gene Z6 starts to appear in the early gastric cancer stage, continues to rise in the middle gastric cancer stage, slows down the rising speed of the imprinting gene Z6 until the late gastric cancer stage, starts to appear in the early gastric cancer stage, slows down the rising speed of the imprinting gene Z6 until the late gastric cancer stage, continues to rise, and increases the expression quantity of the imprinting gene Z6 starts to appear in the early gastric cancer stage, and slowly rises in the middle and late gastric cancers; the imprinting deletion and the increase in the expression amount of the imprinting gene Z8 start to appear in early gastric cancer, slowly rise in mid-gastric cancer, rise at a rate of rise in late gastric cancer stage, but the level is still not high, and the copy number abnormality of the imprinting gene Z8 rises rapidly in early gastric cancer stage, but does not continue to rise in mid-and late gastric cancer; imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z10 begin to occur in the malignant potential stage, and gradually rise to higher levels in the early-to-late gastric cancer development process; the imprinting deletion and the increase in the expression amount of the imprinting gene Z13 begin to appear in the malignant potential stage, gradually rise in the early-stage and middle-stage gastric cancer, slow down the rising speed in the late-stage gastric cancer, begin to appear in the malignant potential stage, slow down the rising speed in the early-stage gastric cancer, increase the rising speed in the middle-stage gastric cancer, and slow down the rising speed in the late-stage gastric cancer.

EXAMPLE 3 imprinting Gene analysis of 37 gastric tumor samples

37 gastric tumor patients were obtained from tissues including gastroscopic biopsies (10 microns) and tested in the same manner as in example 1.

As can be seen from FIGS. 5 (a) -5 (j), the ratios of the imprinting deletion and the copy number abnormality of the ten probes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 in 37 gastric tumor tissue samples are distributed from low to high, and according to the distribution trend of different probes, we calculate the grading standard shown by the dotted line in the graph, and can divide the imprinting deletion and the copy number abnormality of each probe from low to high into 5 grades.

The specific classification is as follows:

as can be seen from fig. 5 (a), for the imprinted gene Z1, the imprinted gene deletion expression level is less than 16%, the imprinted gene copy number abnormal expression level is less than 2.5% or the combination of at least two of the imprinted gene total expression levels is 0 level, the imprinted gene deletion expression level is 16-20%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene total expression levels is 35-40% is I level, the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 4-6.5% or the combination of at least two of the imprinted gene total expression levels is 40-50% is II level, the imprinted gene copy number abnormal expression level is 25-30%, the imprinted gene copy number abnormal expression level is 6.5-8% or the combination of at least two of the imprinted gene total expression levels is 50-65%, the gene deletion expression level is more than 30%, the imprinted gene copy number abnormal expression level is greater than 8% or the combination of at least two of the imprinted gene total expression levels is 65% or the combination of at least two of the imprinted gene is 65 level;

As can be seen from fig. 5 (b), for the above-mentioned imprinted gene Z11, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the amount of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprint gene is 65%, the amount of the one or combination of the imprinted gene is greater than 65%.

As can be seen from fig. 5 (c), for the imprinted gene Z16, any one or a combination of at least two of the imprinted gene deletion expression level of less than 15%, the imprinted gene copy number abnormal expression level of less than 1.5%, or the imprinted gene total expression level of less than 30% is 0 level, any one or a combination of at least two of the imprinted gene deletion expression level of 15 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 4%, or the imprinted gene total expression level of 30 to 40% is I level, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 4 to 7%, or the imprinted gene total expression level of 40 to 50% is II level, the imprinted gene deletion expression level of 7 to 30%, or the imprinted gene copy number abnormal expression level of 50 to 60% is III level, the any one or a combination of at least two of the imprinted gene deletion expression levels of more than 30%, the imprinted gene copy number abnormal expression level of more than 10%, or the imprinted gene total expression level of more than 60% is IV level of any one or a combination of at least two of the imprinted gene expression levels of more than 60%;

As can be seen from fig. 5 (d), for the imprinted gene Z3, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 25% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 25 to 30% is level I, the imprinted gene deletion expression level of 15 to 25%, the imprinted gene copy number abnormal expression level of 2 to 3%, or the imprinted gene total expression level of 30 to 45% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 3 to 5%, or the imprinted gene total expression level of 45 to 55% is level III, the imprinted gene deletion expression level of more than 30%, the gene copy number abnormal expression level of more than 5%, or the imprinted gene total expression level of more than 55% is level IV, or the imprinted gene total expression level of any one or a combination of at least 55% is level IV;

as can be seen from fig. 5 (e), for the above-mentioned imprinted gene Z4, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the amount of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprint gene is 65%, the amount of the one or combination of the imprinted gene is greater than 65%.

As can be seen from fig. 5 (f), for the imprinted gene Z5, the imprinted gene deletion expression level is less than 16%, the imprinted gene copy number abnormal expression level is less than 1.5% or the combination of at least two of the imprinted gene total expression levels is 0 level, the imprinted gene deletion expression level is 16-20%, the imprinted gene copy number abnormal expression level is 1.5-2.5% or the combination of at least two of the imprinted gene total expression levels is 30-40%, the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene total expression levels is 40-50%, the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 4-6% or the combination of at least two of the imprinted gene total expression levels is 50-65%, the gene deletion expression level is more than 30%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene copy number abnormal expression levels is 65% or the combination of at least two of the imprinted gene is 65 level II;

as can be seen from fig. 5 (g), for the imprinted gene Z6, any one or a combination of at least two of the imprinted gene deletion expression level of less than 16%, the imprinted gene copy number abnormal expression level of less than 1.5% or the imprinted gene total expression level of less than 30% is 0 level, any one or a combination of at least two of the imprinted gene deletion expression level of 16 to 20%, the imprinted gene copy number abnormal expression level of 1.5 to 2.5% or the imprinted gene total expression level of 30 to 40% is I level, the imprinted gene deletion expression level of 20 to 25%, the imprinted gene copy number abnormal expression level of 2.5 to 4% or the imprinted gene total expression level of 40 to 50% is II level, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 4 to 6% or the imprinted gene total expression level of 50 to 65% is III level, the combination of any one or a combination of at least two of the imprinted gene deletion expression levels of more than 30%, the imprinted gene copy number abnormal expression level of more than 6% or the imprinting gene total expression level of more than 65% is IV level of at least one of the imprinted gene or a combination of at least two of the imprinted gene;

As can be seen from fig. 5 (h), for the imprinted gene Z8, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 25% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 25 to 30% is level I, the imprinted gene deletion expression level of 15 to 25%, the imprinted gene copy number abnormal expression level of 2 to 3%, or the imprinted gene total expression level of 30 to 45% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 3 to 5%, or the imprinted gene total expression level of 45 to 55% is level III, the imprinted gene deletion expression level of more than 30%, the gene copy number abnormal expression level of more than 5%, or the imprinted gene total expression level of more than 55% is level IV, or the imprinted gene total expression level of any one or a combination of at least 55% is level IV;

as can be seen from fig. 5 (I), for the imprinted gene Z10, the imprinted gene deletion expression level is less than 16%, the imprinted gene copy number abnormal expression level is less than 1.5% or the combination of at least two of the imprinted gene total expression levels is 0 level, the imprinted gene deletion expression level is 16-20%, the imprinted gene copy number abnormal expression level is 1.5-2.5% or the combination of at least two of the imprinted gene total expression levels is 30-40%, the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene total expression levels is 40-50%, the imprinted gene deletion expression level is 25-30%, the imprinted gene copy number abnormal expression level is 4-6% or the combination of at least two of the imprinted gene total expression levels is 50-65%, the gene deletion expression level is more than 30%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene copy number abnormal expression levels is 65% or the combination of at least two of the imprinted gene is 65 level II;

As can be seen from FIG. 5 (j), for the above-mentioned imprinted gene Z13, the amount of the imprinted gene deletion expression is less than 10%, the amount of the imprinted gene copy number abnormal expression is less than 1% or the amount of the imprinted gene total expression is less than 25%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 10 to 15%, the amount of the imprinted gene copy number abnormal expression is 1 to 2% or the amount of the imprinted gene total expression is 25 to 30%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 15 to 25%, the amount of the imprinted gene copy number abnormal expression is 2 to 3% or the amount of the imprinted gene total expression is 30 to 45%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 3 to 5% or the amount of the imprinted gene total expression is 45 to 55%, the amount of the imprinted gene deletion expression is greater than 30%, the amount of the imprinted gene copy number abnormal expression is greater than 5% or the amount of the imprint gene total expression is greater than 55%, and the amount of the imprinted gene is at least 55%.

From a comprehensive analysis of these 37 samples of gastric tumors, it can be derived that:

judging the benign and malignant degree of the gastric tumor is divided into benign tumor, gastric cancer potential, early gastric cancer, middle gastric cancer and advanced gastric cancer:

The result of the judgment of the benign and malignant degree of the gastric tumor is that the imprinting gene deletion expression amount and the imprinting gene copy number abnormal expression amount of imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are both smaller than the I level, and that the imprinting gene deletion expression amount of not more than 1 imprinting gene in the imprinting genes Z1, Z3, Z4, Z6, Z8, Z10, Z8, Z10, Z13 and Z16 is the I level and that the imprinting gene copy number abnormal expression amount of not more than 1 imprinting gene in the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of the I level is a benign tumor;

the result of the determination of the degree of benign malignancy of the gastric tumor is that the imprinting gene deletion expression amount of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I, the imprinting gene copy number abnormal expression amount of at least 2 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I, the imprinting gene deletion expression amount of not more than 1 imprinting genes among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level I and the imprinting gene copy number abnormal expression amount of not more than 1 imprinting genes among the imprinting genes Z16 is level I, or the gastric cancer gene copy number abnormal expression amount of at least one of the imprinting genes Z1, Z3, Z5, Z6, Z8, Z4, Z5, Z11 and Z13 is level II, or the imprinting gene copy number of not more than 1 among the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10 and Z13 is level II;

The result of the determination of the degree of benign and malignant gastric tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level II, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level II or the imprinting gene deletion expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is level III and the imprinting gene copy number abnormal expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is any one of level III;

the result of the determination of the degree of benign malignancy of gastric tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class III or that the imprinting gene deletion expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is class IV and the imprinting gene copy number abnormal expression level of not more than 1 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is any one of class IV;

The result of judging the benign and malignant degree of the gastric tumor is that the imprinting gene deletion expression quantity of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV grade or the imprinting gene copy number abnormal expression quantity of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV grade, and the gastric cancer is advanced stage.

EXAMPLE 4 imprinted Gene analysis of esophageal tumors

Tissue cell sections (10 μm) of esophageal tumors were obtained and examined in the same manner as in example 1.

From FIGS. 6 (a) -6 (e), it can be seen that the proportion of cells with a loss of imprint (two signal points in the nucleus) and an abnormality in copy number (three or more signal points in the nucleus) gradually increases with the increase in malignancy in the samples from grade 0 to grade IV.

EXAMPLE 5 imprinted Gene analysis of gastroscopic biopsy samples of the esophagus

The gastroscope biopsy sample of the esophagus is obtained by taking out suspicious lesion tissues of the esophagus under a gastroscope, fixing the suspicious lesion tissues for more than 24 hours by using 10% neutral formalin solution, and other detection methods are the same as in example 1.

As can be seen from FIGS. 7 (a) -7 (f), the sensitivity of each of the genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13, Z16 to esophageal cancer or the intensity and status of the absence of an imprint corresponding to the expression of esophageal cancer are different.

Specifically, the sensitivity of each imprinting gene to esophageal cancer is as shown in fig. 8 (a) -8 (j), and as can be seen from fig. 8 (a) -8 (c), imprinting deletion of imprinting gene Z1 starts to appear in the malignant potential stage, does not rise significantly in early esophageal cancer, rises gradually to a very high level in middle and late esophageal cancer, the copy number abnormality of imprinting gene Z1 rises gradually in the malignant potential to late esophageal cancer stage, and the expression level of imprinting gene Z1 rises slowly in the esophageal cancer development process; the imprinting deletion of the imprinting gene Z11 begins to appear in the malignant potential stage, does not rise obviously in the early-stage and middle-stage esophageal cancer stage, rises obviously in the late-stage esophageal cancer stage, the copy number abnormality of the imprinting gene Z11 rises rapidly in the malignant potential stage, the rising speed gradually slows down in the development process of early-stage to late-stage esophageal cancer, and the expression quantity of the imprinting gene Z11 increases obviously in the malignant potential to middle-stage esophageal cancer stage, but rises obviously in the late-stage esophageal cancer stage; the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z16 begin to appear in the malignant potential stage, rapidly rise in the early and middle stage esophageal cancer stage, and slow down in the rising speed of the late stage esophageal cancer stage;

From fig. 8 (d) -8 (j), it can be seen that the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z3 started to rise in the middle stage of esophageal cancer, continued to rise in the late stage of esophageal cancer, but the level was still not high; the imprinting deletion of the imprinting gene Z4 starts to appear in the middle-stage esophageal cancer stage, but the ascending speed is slowed down in the late-stage esophageal cancer stage, the copy number abnormality and the expression quantity increase of the imprinting gene start to appear in the middle-stage esophageal cancer stage, and the imprinting gene continues to rise in the late-stage esophageal cancer stage; the imprinting deletion, copy number abnormality and expression amount increase of the imprinting gene Z5 start to rise in the middle stage of esophageal cancer and continue to rise in the late stage of esophageal cancer, but the level is still not high; the imprinting deletion of the imprinting gene Z6 begins to appear in the middle-stage esophageal cancer stage, and increases in an accelerated manner in the late-stage esophageal cancer, but the level is still not high, the copy number abnormality and the expression quantity increase of the imprinting gene Z6 begin to appear in the malignant potential stage, and the level is still not high from the late-stage esophageal cancer stage along with the slow increase of the development of the esophageal cancer; the imprinting deletion and copy number abnormality of the imprinting gene Z8 begin to appear in the malignant potential stage, rapidly rise in early and middle stage esophageal cancer, slow down the rising speed of the late stage esophageal cancer stage, and the increase of the expression quantity of the imprinting gene Z8 begins to appear in the malignant potential stage, but does not rise obviously in the development process of esophageal cancer; the imprinting deletion of the imprinting gene Z10 begins to appear in the middle-stage esophageal cancer stage, does not continue to rise in the late-stage esophageal cancer stage, the copy number abnormality of the imprinting gene Z10 begins to appear in the middle-stage esophageal cancer stage, continues to rise in the late-stage esophageal cancer stage, but does not have a high level, and the increase of the expression level of the imprinting gene Z10 begins to appear in the malignant potential stage, but does not rise significantly in the esophageal cancer development process; the imprinting deletion of the imprinting gene Z13 starts to appear in early esophageal cancer, gradually rises in middle and late esophageal cancer stages, but at a low level, the copy number abnormality and the increase in expression amount of the imprinting gene Z13 rise rapidly in the middle esophageal cancer stage, and the rising speed in the late esophageal cancer stage is slowed down.

EXAMPLE 6 imprinting Gene analysis of 57 esophageal tumor samples

A gastroscopic biopsy (10 microns) of the tissue of 57 esophageal tumor patients, including esophagus, was obtained and tested as in example 1.

As can be seen from FIGS. 9 (a) -9 (j), the ratios of the imprinting deletion and copy number abnormality of the ten probes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 in the 57 esophageal tumor tissue samples are distributed from low to high, and according to the distribution trend of different probes, we calculate the grading standard shown by the dotted line in the graph, and can divide the imprinting deletion and copy number abnormality of each probe from low to high into 5 grades.

The specific classification is as follows:

as can be seen from fig. 9 (a), for the imprinted gene Z1, the imprinted gene deletion expression level is less than 16%, the imprinted gene copy number abnormal expression level is less than 2.5% or the combination of at least two of the imprinted gene total expression levels is 0 level, the imprinted gene deletion expression level is 16-20%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene total expression levels is 35-40% is I level, the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 4-6.5% or the combination of at least two of the imprinted gene total expression levels is 40-50% is II level, the imprinted gene copy number abnormal expression level is 25-30%, the imprinted gene copy number abnormal expression level is 6.5-8% or the combination of at least two of the imprinted gene total expression levels is 50-65%, the gene deletion expression level is more than 30%, the imprinted gene copy number abnormal expression level is greater than 8% or the combination of at least two of the imprinted gene total expression levels is 65% or the combination of at least two of the imprinted gene is 65 level;

As can be seen from fig. 9 (b), for the above-mentioned imprinted gene Z11, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, the amount of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is 30%, the amount of the gene is greater than 30%, the amount of the imprinted gene copy number is 65%, the amount of the one or the combination of the imprinted gene is greater than 65%.

As can be seen from fig. 9 (c), for the imprinted gene Z16, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 25% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 25 to 30% is level I, the imprinted gene deletion expression level of 15 to 25%, the imprinted gene copy number abnormal expression level of 2 to 4%, or the imprinted gene total expression level of 30 to 40% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 4 to 6%, or the imprinted gene total expression level of 40 to 55% is level III, the imprinted gene deletion expression level of more than 30%, the gene copy number abnormal expression level of more than 6%, or the imprinted gene total expression level of more than 55% is level IV, or the imprinted gene total expression level of any one or a combination of at least two of more than 55%;

As can be seen from fig. 9 (d), for the imprinted gene Z3, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1%, or the imprinted gene total expression level of less than 25% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2%, or the imprinted gene total expression level of 25 to 30% is level I, the imprinted gene deletion expression level of 15 to 25%, the imprinted gene copy number abnormal expression level of 2 to 3%, or the imprinted gene total expression level of 30 to 45% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 3 to 5%, or the imprinted gene total expression level of 45 to 55% is level III, the imprinted gene deletion expression level of more than 30%, the gene copy number abnormal expression level of more than 5%, or the imprinted gene total expression level of more than 55% is level IV, or the imprinted gene total expression level of any one or a combination of at least 55% is level IV;

as can be seen from fig. 9 (e), for the above-mentioned imprinted gene Z4, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the amount of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprint gene is 65%, the amount of the one or combination of the imprinted gene is greater than 65%.

As can be seen from fig. 9 (f), for the above-mentioned imprinted gene Z5, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the amount of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprint gene is 65%, the amount of the one or combination of the imprinted gene is greater than 65%.

As can be seen from fig. 9 (g), for the above-mentioned imprinted gene Z6, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the combination of at least two of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprinting gene is 65%, the amount is the IV;

As can be seen from fig. 9 (h), for the imprinted gene Z8, the imprinted gene deletion expression level is less than 16%, the imprinted gene copy number abnormal expression level is less than 2.5% or the combination of at least two of the imprinted gene total expression levels is 0 level, the imprinted gene deletion expression level is 16-20%, the imprinted gene copy number abnormal expression level is 2.5-4% or the combination of at least two of the imprinted gene total expression levels is 35-40% is I level, the imprinted gene deletion expression level is 20-25%, the imprinted gene copy number abnormal expression level is 4-6.5% or the combination of at least two of the imprinted gene total expression levels is 40-50% is II level, the imprinted gene copy number abnormal expression level is 25-30%, the imprinted gene copy number abnormal expression level is 6.5-8% or the combination of at least two of the imprinted gene total expression levels is 50-65%, the gene deletion expression level is more than 30%, the imprinted gene copy number abnormal expression level is greater than 8% or the combination of at least two of the imprinted gene total expression levels is 65% or the combination of at least two of the imprinted gene is 65 level;

as can be seen from fig. 9 (I), for the above-mentioned imprinted gene Z10, the amount of the imprinted gene deletion expression is less than 16%, the amount of the imprinted gene copy number abnormal expression is less than 1.5% or the amount of the imprinted gene total expression is less than 30%, any one or combination of at least two of the imprinted gene deletion expression is 0 level, the amount of the imprinted gene deletion expression is 16 to 20%, the amount of the imprinted gene copy number abnormal expression is 1.5 to 2.5% or the amount of the imprinted gene total expression is 30 to 40%, any one or combination of at least two of the imprinted gene copy number abnormal expression is 1.5 to 2.5%, or the amount of the imprinted gene total expression is 30 to 40%, the amount of the imprinted gene deletion expression is 20 to 25%, the amount of the imprinted gene copy number abnormal expression is 2.5 to 4%, or the combination of at least two of the imprinted gene copy number abnormal expression is 40 to 50%, or the combination of at least two of the imprinted gene copy number abnormal expression is 25 to 30%, the amount of the imprinted gene copy number abnormal expression is 4 to 6%, or the combination of at least two of the imprinted gene copy number is 50 to 65%, the amount of the imprinted gene is greater than 30%, the amount of the imprinting gene is 65%, the IV;

As can be seen from fig. 9 (j), for the above-mentioned imprinted gene Z13, any one or a combination of at least two of the imprinted gene deletion expression level of less than 10%, the imprinted gene copy number abnormal expression level of less than 1% or the imprinted gene total expression level of less than 25% is level 0, any one or a combination of at least two of the imprinted gene deletion expression level of 10 to 15%, the imprinted gene copy number abnormal expression level of 1 to 2% or the imprinted gene total expression level of 25 to 30% is level I, the imprinted gene deletion expression level of 15 to 25%, the imprinted gene copy number abnormal expression level of 2 to 3% or the imprinted gene total expression level of 30 to 45% is level II, the imprinted gene deletion expression level of 25 to 30%, the imprinted gene copy number abnormal expression level of 3 to 5% or the imprinted gene total expression level of 45 to 55% is level III, the imprinted gene deletion expression level of more than 30%, the imprinted gene copy number abnormal expression level of more than 5% or the random gene expression level of more than 55% is level IV.

From a comprehensive analysis of samples of these 57 esophageal tumors, it can be derived that:

judging benign and malignant degrees of esophageal tumors are classified into benign tumors, esophageal cancer potential, early-stage esophageal cancer, medium-stage esophageal cancer and advanced-stage esophageal cancer:

The result of judging the benign and malignant degree of the esophageal tumor is that the abnormal expression amount of the imprinting gene deletion and the abnormal expression amount of the imprinting gene copy number of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 are less than the I level, and the abnormal expression amount of the imprinting gene copy number of the imprinting genes of which not more than 1 imprinting genes in the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is the I level and the abnormal expression amount of the imprinting gene copy number of the imprinting genes of which not more than 1 imprinting genes in the imprinting genes Z1, Z3, Z4, Z6, Z8, Z10, Z11, Z13 and Z16 is the I level is the benign tumor;

the result of the determination of the degree of benign or malignant esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level I or the imprinting gene deletion expression level of not more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II and the imprinting gene copy number abnormal expression level of not more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is any one of level II, and the esophageal tumor potential;

The result of the determination of the degree of benign or malignant esophageal tumor is that the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level II or any one of the imprinted gene copy number abnormal expression levels of not more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is level III and the imprinted gene copy number abnormal expression level of not more than 1 imprinted gene among imprinted genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is early stage carcinoma;

the result of the determination of the degree of benign or malignant esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class III or any one of the imprinting gene copy number abnormal expression levels of not more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is class IV and the imprinting gene copy number abnormal expression level of not more than 1 imprinting gene among imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is mid-stage esophageal cancer;

The result of judging the benign and malignant degree of the esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV grade or the imprinting gene copy number abnormal expression level of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV grade, and the esophageal cancer is advanced stage.

In summary, the detection model and the detection system of the invention represent the representation of imprinting deficiency on the samples of patients with esophagus and stomach tumors in an intuitive way, detect the change of imprinting (trace) genes objectively, intuitively, early and accurately by the method of marking imprinting genes in situ, and can provide a quantized model to make great contribution to diagnosis of esophagus and stomach tumors.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A device for detecting benign and malignant extent of esophageal tumor and/or gastric tumor, comprising the following units:

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

(2) Probe design unit: designing a specific probe according to the intron sequence of the imprinted gene;

wherein the imprinting gene comprises a combination of Z1, Z11 and Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z11 is Grb10, the imprinting gene Z16 is Snrpn/Snurf,

the imprinted gene further comprises any one or a combination of at least two of Z3, Z4, Z5, Z6, Z8, Z10 or Z13; wherein the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z5 is test, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the imprinting gene Z10 is Gatm, the imprinting gene Z13 is Sgce,

the analysis unit judges the benign and malignant degree of the esophagus tumor and/or the stomach tumor by calculating the abnormal expression quantity of the imprinting gene deletion expression quantity and the abnormal expression quantity of the imprinting gene copy number through the imprinting gene grading model of the esophagus tumor and/or the stomach tumor so as to judge the benign and malignant degree of the esophagus tumor and/or the stomach tumor through the grade of the abnormal expression quantity of the imprinting gene deletion expression quantity and the abnormal expression quantity of the imprinting gene copy number,

The imprinted gene grading model grades the expression state of imprinted genes by calculating the variation of the imprinted gene deletion expression quantity, the imprinted gene copy number abnormal expression quantity and the total expression quantity of imprinted genes in esophagus tumors and/or stomach tumors;

any one or at least two of the imprinted genes Z1, Z11 and Z16 and Z3, Z4, Z5, Z6, Z8, Z10 or Z13 are detected by in situ hybridization by using a sequence selected from introns of each gene as a probe,

the formulas for calculating the total expression quantity of the imprinting genes, the deletion expression quantity of the imprinting genes and the copy number abnormal expression quantity of the imprinting genes are as follows:

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

total expression of normal imprinted genes = b/(b+c+d) ×100%;

imprinted gene deleted gene expression amount = c/(b+c+d) ×100%;

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

wherein a is a cell nucleus in which no marker exists in the cell nucleus and the imprinted gene is not expressed after the cell is subjected to hematoxylin staining; b is a cell nucleus with a red/brown mark in the cell nucleus and a marking gene after the cell is subjected to hematoxylin staining; c is a cell nucleus with two red/brown marks in the cell nucleus and marking the gene deletion after the cell is subjected to hematoxylin staining; the d is the cell nucleus with more than two red/brown marks in the cell nucleus after the cell is subjected to hematoxylin staining and the marked gene copy number is abnormal,

The deletion expression level of the imprinting gene, the abnormal copy number expression level of the imprinting gene and the total expression level of the imprinting gene are divided into five different levels;

the five different grades are five different grades respectively divided into a imprinted gene deletion expression amount, an imprinted gene copy number abnormal expression amount and an imprinted gene total expression amount for ten imprinted genes of Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16;

for detecting esophageal and/or gastric tumors, the five different grades of the marked gene deletion expression level, the marked gene copy number abnormal expression level and the total expression level for Z1 are:

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z1 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z1 being more than 8%, or the total expression level of the imprinting gene Z1 being more than 65%;

for detection of esophageal and/or gastric tumors, the five different grades of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level for Z3 and Z13 are:

for detecting esophageal and/or gastric tumors, the five different grades of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level for Z4, Z5, Z6, Z10 and Z11 are divided into:

when used for detecting esophageal tumors, the five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity of the imprinting gene aiming at Z16 are as follows:

for detecting gastric tumors, the five different grades of the marked gene deletion expression level, the marked gene copy number abnormal expression level and the total expression level for Z16 are:

grade IV: any one or a combination of at least two of the imprinting gene deletion expression level of the imprinting gene Z16 being more than 30%, the imprinting gene copy number abnormal expression level of the imprinting gene Z16 being more than 10%, or the total expression level of the imprinting gene Z16 being more than 60%;

when used for detecting esophageal tumors, the five different grades of the deletion expression quantity of the imprinting gene, the abnormal expression quantity of the copy number of the imprinting gene and the total expression quantity of the imprinting gene aiming at Z8 are as follows:

for detecting gastric tumors, the five different grades of the marked gene deletion expression quantity, the marked gene copy number abnormal expression quantity and the total expression quantity aiming at Z8 are divided into:

2. The device of claim 1, wherein the sample to be tested in step (1) is derived from human tissue and/or cells.

3. The device of claim 2, wherein the sample to be tested is a paraffin section of tissue, esophageal mucosa exfoliated cells, or a gastroscopic biopsy sample.

4. The device according to claim 2, wherein the sample to be tested is esophageal mucosa exfoliated cells and/or a gastroscopic biopsy sample.

5. The apparatus of claim 1, wherein said in situ hybridization employs an RNAscope in situ hybridization method.

6. The device of claim 5, wherein the RNAscope in situ hybridization method uses a single-channel or multi-channel chromogenic kit or a single-channel or multi-channel fluorogenic kit.

7. The device of claim 5, wherein the RNAscope in situ hybridization method uses a single channel red/brown color kit or a multichannel fluorescent kit.

8. The device of any one of claims 1-7, wherein the judging the benign malignancy of esophageal tumor is classified into benign tumor, esophageal cancer potential, early esophageal cancer, mid-stage esophageal cancer, and late-stage esophageal cancer;

The result of judging the benign and malignant degree of the esophageal tumor is that the imprinting gene deletion expression level of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV grade or the imprinting gene copy number abnormal expression level of at least 2 imprinting genes in imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11, Z13 and Z16 is IV grade, and the esophageal cancer is advanced stage;

the judging stomach tumor benign and malignant degree is divided into benign stomach tumor, stomach cancer potential, early stomach cancer, middle stomach cancer and advanced stomach cancer;

9. Use of a device according to any one of claims 1-8 for the preparation of a medicament or kit for esophageal and/or gastric tumor detection.

10. The use according to claim 9, wherein the determination of benign and malignant tumor of esophagus is classified into benign tumor, potential of esophagus cancer, early esophagus cancer, middle esophagus cancer and late esophagus cancer.

11. The use according to claim 9, wherein the determination of the degree of benign or malignant gastric tumor is carried out by determining that the number of copies of at least 2 of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV or that the number of copies of at least 2 of the imprinting genes Z1, Z3, Z4, Z5, Z6, Z8, Z10, Z11 and Z13 is IV.

12. The use according to claim 11, wherein the advanced gastric cancer is gastric cancer with a TNM grade greater than T2N0M 0.