CN111100930A

CN111100930A - Grading model for detecting benign and malignant degree of pancreatic tumor and application thereof

Info

Publication number: CN111100930A
Application number: CN201811259835.3A
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: 2018-10-26
Filing date: 2018-10-26
Publication date: 2020-05-05
Anticipated expiration: 2038-10-26
Also published as: WO2020082642A1; CN111100930B

Abstract

The invention relates to a grading model for detecting pancreatic tumors and application thereof, wherein the model grades the change of imprinted genes in the pancreatic tumors by calculating the deletion expression quantity of the imprinted genes, the copy number abnormal expression quantity of the imprinted genes and the total expression quantity of the imprinted genes. The detection model and the device of the invention represent the expression of the imprinting deletion on the tissue and cell samples of the pancreatic tumor patients by an intuitive method, objectively, intuitively, early and accurately detect the change of the imprinting (imprinting) gene by a method for marking the imprinting gene in situ, can provide a quantitative model and make great contribution to the diagnosis of the pancreatic tumor.

Description

Grading model for detecting benign and malignant degree of pancreatic tumor and application thereof

Technical Field

The invention relates to the field of biotechnology, relates to the field of gene diagnosis, relates to a hierarchical model and application thereof, relates to a hierarchical model for detecting the benign and malignant degree of pancreatic tumors and application thereof, and particularly relates to a hierarchical model of a group of imprinted genes in detecting the benign and malignant degree of pancreatic tumors and a device formed by the hierarchical model.

Background

Pancreatic cancer is a malignant tumor with high malignancy, and the 5-year survival rate is less than 5%, which is one of the most advanced and worst prognosis malignant tumors. About 33.8 million people worldwide are diagnosed with pancreatic cancer and die 33.0 million each year, with 6.6 million new cases and 6.4 million deaths per year in China, and the incidence of the disease is also increasing year by year.

Early diagnosis of pancreatic cancer is more difficult, and 80% of pancreatic cancer patients are diagnosed at an advanced stage and cannot be operated. Early stage pancreatic cancer has symptoms similar to pancreatitis, is atypical and is often easily overlooked by patients. Because the special anatomical site of pancreas brings the difficulty for the ultrasonic examination, the pancreas also does not have the pipeline that directly accesss to in vitro simultaneously, can't inspect through the scope. The current pancreatic cancer tumor markers also have the problems of low sensitivity and specificity. Therefore, a more sensitive and accurate detection means is needed to improve the early diagnosis rate of pancreatic cancer.

The diagnosis of benign and malignant cells by traditional pathology is based on the relationship between the size, morphology, infiltration and surrounding cell tissues. It has great limitation to the discovery of early changes of cells (cancer), so the cancer diagnosis method at cellular molecular level has once been the focus of research. With the continuous and intensive research in the field of molecular biology, more and more molecular detection techniques are applied to cancer diagnosis.

The development of cancer is uncontrolled cell growth/division due to epigenetic changes and genetic variations that accumulate over time. Traditional pathological diagnosis makes judgment on the malignancy and malignancy of pancreatic tumors according to the variation in size, morphology and structure of cells and tissues. With the development and progress of molecular biology, more and more molecular detection techniques are applied to the detection of pancreatic cancer. From the analysis of the cancer progression, changes at the molecular level (epigenetics and genetics) are much earlier than the variations in cell morphology and tissue structure. Molecular biological detection is therefore more sensitive to the detection of early stages of cancer.

Genomic imprinting is a means of gene regulation in epigenetics. It is characterized by that it utilizes methylation of allele from specific parent to make a certain gene only have one allele expression, and another gene can be trapped in gene silencing state. This type of gene is called a blot (marker) gene. An imprinted deletion is an epigenetic change in the imprinted gene that demethylates resulting in activation of the silent state allele and initiation of gene expression. Numerous studies have shown that this phenomenon (loss of imprinting) is prevalent in various types of cancer and occurs at a time earlier than the morphological changes in cells and tissues. Meanwhile, in healthy cells, the percentage of blotting loss is extremely low, in 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 pancreatic cancer diagnosis at present, which can analyze the molecular marker changes of pancreatic cancer existing on the cellular level based on the biopsy samples of patients, thereby providing more accurate prognosis and diagnosis information.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a grading model for detecting the benign and malignant degree of the pancreatic tumor and application thereof.

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

in a first aspect, the present invention provides an imprinted gene grading model for pancreatic tumors, which grades the expression state of imprinted genes by calculating the changes in total expression amount of imprinted genes, deletion expression amount of imprinted genes, and copy number abnormal expression amount of imprinted genes in pancreatic cancer;

the imprinting gene is any one or a combination of at least two of Z1, Z5, Z10, Z11 or Z16, the imprinting gene Z1 is Gnas, the imprinting gene Z5 is Mest, the imprinting gene Z10 is Gatm, the imprinting gene Z11 is Grb10, and the imprinting gene Z16 is Snrpn/Snurf.

In the present invention, the inventors found that the diagnostic sensitivity for pancreatic cancer can be up to 86.7% or more by calculating the imprinted gene deletion expression level and the imprinted gene copy number abnormal expression level of any one of the imprinted genes Z1, Z5, Z10, Z11, and Z16 in pancreatic tumors.

According to the present invention, if only one imprinted gene is preliminarily detected, any one of Z1, Z5, Z10, Z11 and Z16, preferably any one of Z1, Z11 or Z16, and more preferably Z11 or Z16 can be detected.

In the invention, the inventor finds that if a Z1 imprinted gene is detected independently, the diagnostic sensitivity to pancreatic cancer can reach 88.9%, if a Z5 imprinted gene is detected independently, the diagnostic sensitivity to pancreatic cancer can reach 86.7%, if a Z10 imprinted gene is detected independently, the diagnostic sensitivity to pancreatic cancer can reach 86.7%, if a Z11 imprinted gene is detected independently, the diagnostic sensitivity to pancreatic cancer can reach 91.1%, and if a Z16 imprinted gene is detected independently, the diagnostic sensitivity to pancreatic cancer can reach 91.1%.

According to the invention, the method for calculating the imprinted gene by the model comprises the following steps: if a combination of two imprinted genes of the imprinted genes is detected, the combination may be any two of Z1, Z5, Z10, Z11 and Z16, preferably a combination of Z1 and Z10, a combination of Z5 and Z11, a combination of Z10 and Z16 or a combination of Z11 and Z16.

In the present invention, the inventors found that sensitivity can be further improved by calculating the total expression amount of two or more imprinted genes, the deletion expression amount of imprinted genes, and the copy number abnormal expression amount of imprinted genes, that the diagnostic sensitivity for pancreatic cancer can be 93.3% or more by detecting the combination of any two imprinted genes among imprinted genes Z1, Z5, Z10, Z11, and Z16, that the diagnostic sensitivity for pancreatic cancer can be 97.8% or more by detecting the combination of Z1 and Z10, and the combination of Z11 and Z16, and that the diagnostic sensitivity for pancreatic cancer can be 99.0% or more by detecting the combination of Z5 and Z11, and the combination of Z10 and Z16.

According to the invention, the imprinted gene further comprises any one of or a combination of at least two of Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14 or Z15; the imprinting gene Z2 is Igf2, the imprinting gene Z3 is Peg10, the imprinting gene Z4 is Igf2r, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the imprinting gene Z9 is Dlk1, the imprinting gene Z12 is Peg3, the imprinting gene Z13 is Sgce, the imprinting gene Z14 is Slc38a4, and the imprinting gene Z15 is Diras 3.

In the invention, the inventor finds that the combined diagnosis is carried out by adding Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14 and Z15 genes on the basis of the detection of the Z1, Z5, Z10, Z11 and Z16 genes, which not only is beneficial to increasing the detection accuracy, but also can further avoid the occurrence of false positive by adding other probe-assisted diagnoses, can further improve the detection accuracy, and can realize the accurate classification and judgment of all pancreatic tumor samples.

According to the invention, the method for calculating the imprinted gene by the model comprises the following steps: the combinations of imprinted genes were calculated, the combinations of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 genes were calculated.

In the invention, the imprinting gene deletion is that two red/brown markers exist in a cell nucleus after hematoxylin staining is carried out on a cell, the imprinting gene copy number abnormality is that more than two red/brown markers exist in the cell nucleus after hematoxylin staining is carried out on the cell, and the copy number abnormality is the condition that the gene is expressed as a triploid or even a higher polyploid due to abnormal gene replication of a cancer cell.

In the present invention, the imprinted gene and the imprinted gene are simultaneously a concept, which means the same meaning, and can be substituted.

Preferably, the formula for calculating the total expression level of the imprinted gene, the deletion expression level of the imprinted gene, and the copy number abnormal expression level of the imprinted gene is as follows:

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

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

the expression Level (LOI) of the imprinted gene-deleted gene (c/(b + c + d) × 100%;

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

wherein a is cell nucleus without marker and without expressed marking gene after hematoxylin staining; b is the cell nucleus with a red/brown mark and the gene existence is marked after the cell is stained by hematoxylin; c, after hematoxylin staining is carried out on the cells, two red/brown marks exist in cell nuclei, and the cell nuclei with gene deletion are marked; and d is the nucleus with more than two red/brown marks and abnormal copy number of the marked gene after the cell is subjected to hematoxylin staining.

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

In the invention, the existence of imprinted gene, imprinted gene deletion or copy number abnormality in each cell nucleus is judged by a probe through in situ hybridization and Hemotoxy (hematoxylin) cell nucleus staining amplification signals under a microscope of 40X or 60X, and the tumor benign and malignant degree of the sample is judged by calculating the imprinted gene deletion gene expression quantity and the imprinted gene copy number abnormal gene expression quantity. Since the section is only 10 μm, about 20% of the nuclei seen under the microscope are incomplete nuclei, i.e., there is a possibility of partial false negatives.

According to the present invention, the total imprinted gene expression level, the imprinted gene deletion expression level, and the imprinted gene copy number abnormal expression level are classified into five different grades, at least 1200 cells are counted in a region where a sample is most positively expressed by each probe, and fifteen different grades are classified into the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level, and the imprinted gene total expression level for the fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16, respectively.

According to the invention, the five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z1 and Z11 are:

level 0: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z1 and Z11 is less than 15%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z11 is less than 1.5%, or the total expression amount of the imprinted genes Z1 and Z11 is less than 25%;

stage I: any one or a combination of at least two of the imprinted gene deletion expression quantity of the imprinted genes Z1 and Z11 is 15-20%, the imprinted gene copy number abnormal expression quantity of the imprinted genes Z1 and Z11 is 1.5-3%, or the total expression quantity of the imprinted genes Z1 and Z11 is 25-35%;

II stage: the imprinted gene deletion expression amount of the imprinted genes Z1 and Z11 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z11 is 3-5%, or the total expression amount of the imprinted genes Z1 and Z11 is 35-45%, or the combination of at least two of the two;

grade III: the imprinted gene deletion expression amount of the imprinted genes Z1 and Z11 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1 and Z11 is 5-8%, or the total expression amount of the imprinted genes Z1 and Z11 is 45-55%, or the combination of at least two of the two;

stage IV: any one or the combination of at least two of the imprinted gene deletion expression quantity of the imprinted genes Z1 and Z11 is more than 30 percent, the imprinted gene copy number abnormal expression quantity of the imprinted genes Z1 and Z11 is more than 8 percent, or the total expression quantity of the imprinted genes Z1 and Z11 is more than 55 percent;

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 Z1 and Z11 are independent of each other.

According to the invention, the five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are:

level 0: any one or a combination of at least two of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16, wherein the imprinted gene deletion expression amount is less than 11%, and the imprinted gene copy number abnormal expression amount of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12 and Z12 is less than 1.5%, or the total expression amount of the imprinted genes Z12, Z12 and Z12 is less than 20%;

stage I: the imprinted gene deletion expression amount of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 is 11-15%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12 and Z12 is 1.5-2.5% or the total expression amount of the imprinted genes Z12, Z12 and Z12 is 20-30% or a combination of at least two of them;

II stage: any one or a combination of at least two of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 with an imprinted gene deletion expression amount of 15-20%, the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12 and Z12 with an imprinted gene copy number abnormal expression amount of 2.5-4% or the total expression amount of the imprinted genes Z12, Z12 and Z12 is 30-40%;

grade III: the imprinted gene deletion expression amount of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12 and Z12 is 4-6% or the total expression amount of the imprinted genes Z12, Z12 and Z12 is 40-50% or a combination of at least two of the two;

stage IV: any one or a combination of at least two of the imprinted gene deletion expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 25%, the imprinted gene copy number abnormal expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 6% or the total expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 50%;

in the present invention, the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are independent of the imprinted gene deletion expression level, the imprinted gene copy number abnormal expression level and the total expression level.

In a second aspect, the present invention provides an apparatus for detecting the benign and malignant degree of a pancreatic tumor, comprising the following units:

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

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

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

(4) an analysis unit: analyzing the expression condition of the imprinted gene by microscope imaging;

wherein the analysis unit determines the degree of malignancy and well-being of the pancreatic tumor by calculating the total expression level of the imprinted gene, the deletion expression level of the imprinted gene, and the copy number abnormal expression level of the imprinted gene through the model of the first aspect.

In the invention, the imprinting gene deletion is the condition that after hematoxylin staining is carried out on cells, two red/brown marked cell nucleuses exist in the cell nucleuses, the imprinting gene copy number abnormality is the condition that after hematoxylin staining is carried out on the cells, more than two red/brown marked cell nucleuses exist in the cell nucleuses, and the copy number abnormality is the condition that the cancer cells abnormally carry out gene replication, so that the gene is expressed as triploid or even higher polyploid.

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

The detection device is used for visually observing the change of the imprinting gene of the pancreatic tumor at an early stage under the cell and tissue level so as to judge the benign and malignant degree of the tumor, and provides the most favorable treatment opportunity for patients with early-stage pancreatic tumor.

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

In the present invention, the sample to be tested is feasible as long as the RNA is fixed in time, and can be selected by a person skilled in the art according to the need, and is not particularly limited herein, and the sample to be tested includes any one or a combination of at least two of a paraffin section of a tissue and a biopsy sample obtained by needle biopsy.

The specific operation steps of the paraffin section of the tissue are that a human tumor tissue sample is obtained, the human tumor tissue sample is fixed by 10 percent neutral formalin in time, the paraffin is embedded, the tissue sample is cut into a thickness of 10 mu m, and a slide with positive charges is used for preparing a tissue slice; since only 10 μm thick, a part of nuclei were found to be incomplete under a microscope, and thus, gene deletion in a partial false negative was observed.

The specific operation steps of the biopsy sample include that human body cells are obtained through puncture, and the human body cells are fixed by 10% neutral formalin in time.

In the invention, the puncture biopsy has little harm to patients, the sampling process is simple, compared with the circulation characteristic of blood, the puncture biopsy can also be positioned, and the puncture biopsy has special advantages as an experimental sample.

Preferably, the sample to be tested is a needle biopsy sample.

Preferably, the imprinted gene is Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, the imprinted gene Z1 is Gnas, the imprinted gene Z2 is Igf2, the imprinted gene Z3 is Peg10, the imprinted gene Z4 is Igf2r, the imprinted gene Z r is Mest, the imprinted gene Z r is Plagl r, the imprinted gene Z r is Dcn, the imprinted gene Z r is Dlk r, the imprinted gene Z r is Gatm, the imprinted gene Z r is Grb r, the imprinted gene Z r is Peg r, the imprinted gene Z r is Sgce, the imprinted gene Z r is slsc 38a, the imprinted gene Z r is snrpf r, and the imprinted gene Z r is snrpf r.

In the invention, the imprinted genes Z1(Gnas), Z2(Igf2), Z3(Peg10), Z4(Igf2r), Z5(Mest), Z6(Plagl1), Z8(Dcn), Z9(Dlk1), Z10(Gatm), Z11(Grb10), Z12(Peg3), Z13(Sgce), Z14(Slc38a4), Z15(Diras3) and Z16(Snrpn/Snurf) have different degrees of expression in normal tumor cell tissues, and the expression amount and the imprinting state can be obviously changed in the case of malignant lesion.

In the present invention, the designed probe is designed based on imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16, i.e., Gnas, Igf2, Peg10, Igf2r, Mest, Plagl1, Dcn, Dlk1, Gatm, Grb10, Peg3, Sgce, Slc38a4, Diras3 and Snrpn/Snurf, specifically, a sequence is selected as a probe in the intron of each gene, and a specific probe is designed by Advanced Diagnostics.

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

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

The multichannel chromogenic kit or the multichannel fluorescence kit comprises two channels or a chromogenic kit or a fluorescence kit with more than two channels, and the two-channel chromogenic kit or the two-channel fluorescence kit can use two imprinted gene probes or the joint expression of an imprinted gene and other genes or even the comprehensive expression of a plurality of imprinted genes and non-imprinted genes.

According to the present invention, the formula for calculating the total expression level of the imprinted gene, the deletion expression level of the imprinted gene, and the copy number abnormal expression level of the imprinted gene in the model is as follows:

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

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

In the invention, the existence of imprinted gene, imprinted gene deletion or copy number abnormality in each cell nucleus is judged by a probe through in situ hybridization and Hemotoxy (hematoxylin) cell nucleus staining amplification signals under a microscope of 40X or 60X, and the tumor benign and malignant degree of the sample is judged by calculating the total expression quantity of the imprinted gene, the expression quantity of the imprinted gene deletion gene and the gene expression quantity of the imprinted gene copy number abnormality. Since the section is only 10 μm, approximately 20% of the nuclei seen under the microscope are incomplete, i.e., there is a possibility of partial false negatives.

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

The five different grades were such that at least 1200 cells were counted in the region where each probe was most positively expressed in the sample, and the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount of fifteen imprinted genes for Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 were divided, respectively.

The five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z1 and Z11 are:

The five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are:

According to the invention, the benign and malignant degree of the pancreatic tumor is divided into benign tumor, pancreatic cancer potential, early pancreatic cancer, metaphase pancreatic cancer and late pancreatic cancer;

preferably, the judging result of the malignancy and malignancy of the pancreatic tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, and Z11 are both less than the I-grade, or the imprinted gene deletion expression amount of no more than 1 imprinted genes among the imprinted genes Z11, Z36;

preferably, the judging of the degree of malignancy of pancreatic tumor results in any one of the case where the imprinted gene deletion expression amount of at least 2 of the imprinted genes Z, Z and Z is class I, the imprinted gene copy number abnormal expression amount of at least 2 of the imprinted genes Z, Z and Z is class I, or the imprinted gene copy number abnormal expression amount of no more than 1 of the imprinted genes Z, Z and Z is class II, pancreatic cancer potential;

preferably, the judging of the degree of malignancy of pancreatic tumor results in any one of the case where the imprinted gene deletion expression level of at least 2 of the imprinted genes Z, Z and Z is class II, the imprinted gene copy number abnormal expression level of at least 2 of the imprinted genes Z, Z and Z is class II, or the imprinted gene deletion expression level of no more than 1 of the imprinted genes Z, Z and Z is class III, and the imprinted gene copy number abnormal expression level of no more than 1 of the imprinted genes Z, Z and Z is class III, early stage pancreatic cancer;

preferably, the judging of the degree of malignancy of pancreatic tumor results in any one of the cases where the imprinted gene deletion expression level of at least 2 of the imprinted genes Z, Z and Z is class III, the imprinted gene copy number abnormal expression level of at least 2 of the imprinted genes Z, Z and Z is class III, or the imprinted gene copy number abnormal expression level of no more than 1 of the imprinted genes Z, Z and Z is class IV, and the imprinted gene copy number abnormal expression level of no more than 1 of the imprinted genes Z, Z and Z is class IV, then the cancer is a metaphase pancreatic cancer;

preferably, the judging result of the malignancy and malignancy of pancreatic tumor is that the advanced pancreatic cancer is obtained if the loss expression level of imprinted genes of at least 2 imprinted genes selected from the group consisting of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV or the copy number abnormal expression level of imprinted genes of at least 2 imprinted genes selected from the group consisting of imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 is grade IV.

In a third aspect, the present invention provides a model according to the first aspect or a device according to the second aspect for use in the preparation of a medicament or kit for pancreatic tumour detection and/or treatment.

According to the invention, the benign and malignant degree of the pancreatic tumor is judged to be benign tumor, pancreatic cancer potential, early pancreatic cancer, metaphase pancreatic cancer and late pancreatic cancer;

according to the present invention, as a result of the determination of the degree of malignancy and malignancy of the pancreatic tumor, if the amount of expression of the imprinted gene deletion and the amount of abnormal expression of the imprinted gene copy number of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, and Z11 are both less than the I-grade, or if the amount of expression of the imprinted gene deletion of no more than 1 imprinted genes among the imprinted genes Z11, and Z11 are not more than the amount of the benign tumor copy number of the tumor is;

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

(1) the detection model and the device of the invention represent the expression of the loss of the mark on the sample of the pancreatic tumor patient by an intuitive method, and the change of the mark gene can be objectively, intuitively and accurately detected at early stage by the method for marking the mark gene in situ, and a quantitative model can be provided, thereby making great contribution to the diagnosis of the pancreatic tumor;

(2) the detection device can judge the benign and malignant degree of the pancreatic tumor by puncturing cells before the operation of the pancreatic tumor patient, thereby providing a basis for the operation and the accurate treatment, which is a revolutionary breakthrough of diagnosing the pancreatic tumor in the field of cell molecules;

(3) the early pancreatic cancer can be detected sensitively, samples which start to become cancerous can be detected from patients with precancerous lesions of pancreas such as pancreatic intraepithelial neoplasia, chronic pancreatitis, intraductal papillary mucinous tumor (IPMN), mucinous cystic tumor and the like through the combined detection of imprinted genes, the injury of pancreatic puncture to the patients is small, the material taking process is simple, the early general investigation and the follow-up visit after cancer operation are used, especially the follow-up visit of suspected recurring patients, the time can be won, and the great contribution is made to the saving of the lives of the patients;

(4) the technology of the invention can be used for detecting the tumor of the exocrine pancreatic part and detecting the benign and malignant of the neuroendocrine tumor of the pancreatic islet, has important guiding significance for accurately judging the type of the pancreatic cancer and selecting a treatment method, and greatly reduces the postoperative recurrence of the pancreatic cancer.

(5) The detection method is different from an immunohistochemical method, reduces false positive and other negative effects, and can be used for guiding later treatment and medication through a targeted medicine or a technical method which is found to cause the gene to be silenced, knocked out and rearranged at the pancreatic tumor-related imprinted gene deletion site.

Drawings

FIG. 1 is a pathological section of a pancreatic cancer with a cell nucleus stained with hematoxylin according to the present invention, wherein a indicates that no marker is present in the cell nucleus and no imprinted gene is expressed after the cell is stained with hematoxylin; b is that after the cells are stained by hematoxylin, a red/brown mark exists in the cell nucleus, and the imprinting gene exists; c, after hematoxylin staining is carried out on the cells, two red/brown marks exist in cell nuclei, and the imprinting genes are deleted; d is that after the cells are stained by hematoxylin, more than two red/brown marks exist in cell nuclei, and the copy number of the imprinting gene is abnormal;

FIG. 2(a) is the expression states of 15 genes in a pathological section of a grade 0 pancreatic tumor, FIG. 2(b) is the expression states of 15 genes in a pathological section of a grade I pancreatic cancer, FIG. 2(c) is the expression states of 15 genes in a pathological section of a grade II pancreatic cancer, FIG. 2(d) is the expression states of 15 genes in a pathological section of a grade III pancreatic cancer, and FIG. 2(e) is the expression states of 15 genes in a pathological section of a grade IV pancreatic cancer;

FIG. 3(a) is the intensity of the imprinting deletion of imprinted genes Z1, Z5, Z10, Z11 and Z16 for pancreatic cancer, FIG. 3(b) is the intensity of the copy number abnormality of imprinted genes Z1, Z5, Z10, Z11 and Z16 for pancreatic cancer, FIG. 3(c) is the intensity of the imprinting deletion of imprinted genes Z16, Z16 and Z16 for pancreatic cancer, FIG. 3(d) is the intensity of the imprinting deletion of imprinted genes Z16, Z16 and Z16 for pancreatic cancer, FIG. 3(e) is the intensity of the imprinting deletion of imprinted genes Z16, Z16 and Z16 for pancreatic cancer, and the intensity of the imprinting deletion of imprinted genes Z16, Z16 and Z16 is the copy number abnormality, Z16, and Z16 is the expression of the imprinting deletion of the expression of the copy number abnormality, Z16 is the expression of the imprinting of the expression of the, TE is the total expression quantity of the imprinted gene;

FIG. 4(a) is the intensity of deletion of imprinting gene Z1, copy number abnormality and total expression amount, FIG. 4(b) is the intensity of deletion of imprinting gene Z5, copy number abnormality and total expression amount, FIG. 4(c) is the intensity of deletion of imprinting gene Z10, copy number abnormality and total expression amount, FIG. 4(d) is the intensity of deletion of imprinting gene Z11, copy number abnormality and total expression amount, FIG. 4(e) is the intensity of deletion of imprinting gene Z16, copy number abnormality and total expression amount, FIG. 4(f) is the intensity of deletion of imprinting gene Z2, copy number abnormality and total expression amount, FIG. 4(g) is the intensity of deletion of imprinting gene Z3, copy number abnormality and total expression amount, FIG. 4(h) is the intensity of deletion of imprinting gene Z4, copy number abnormality and total expression amount, FIG. 4(i) is the intensity of deletion of imprinting gene Z6778, copy number abnormality and total expression amount, FIG. 4(j) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z8, FIG. 4(k) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z9, FIG. 4(l) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z12, FIG. 4(m) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z13, FIG. 4(n) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z14, FIG. 4(o) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z15, wherein LOI is expression of imprinting gene deletion, CNV is expression of gene copy number abnormality, and TE is total expression of imprinting gene;

FIG. 5(a) is a graph showing the distribution range and classification standard of the imprinted gene Z1, the distribution range and classification standard of the imprinted deletion and copy number abnormality, FIG. 5(b) is a graph showing the distribution range and classification standard of the imprinted gene Z5, the imprinted deletion and copy number abnormality, the distribution range and classification standard of the imprinted gene Z10, the imprinted deletion and copy number abnormality, the distribution range and classification standard of the imprinted gene Z11, the distribution range and classification standard of the imprinted deletion and copy number abnormality, the distribution range and classification standard of the imprinted gene Z16, the distribution range and classification standard of the imprinted deletion and copy number abnormality, and the distribution range and classification standard of the imprinted gene Z2, FIG. 5(g) is a graph showing the distribution range and classification criteria of the imprinted gene Z3, the imprinted deletion and copy number abnormality, in 45 pathological sections of pancreatic cancer, in 5(h) is a graph showing the distribution range and classification criteria of the imprinted gene Z4, in 45 pathological sections of pancreatic cancer, in 5(i) is a graph showing the distribution range and classification criteria of the imprinted gene Z6, in 45 pathological sections of pancreatic cancer, in 5(j) is a graph showing the distribution range and classification criteria of the imprinted gene Z8, in 45 pathological sections of pancreatic cancer, in deletion and copy number abnormality, in 5(k) is a graph showing the distribution range and classification criteria of the imprinted gene Z9, in 45 pathological sections of pancreatic cancer, in 5(l) is a graph showing the distribution range and classification criteria of the imprinted gene Z12, in 45 pathological sections of pancreatic cancer, FIG. 5(m) is a distribution range and a classification standard of a imprinted gene Z13 for pathological sections of 45 pancreatic cancers, and FIG. 5(n) is a distribution range and a classification standard of a imprinted gene Z14 for pathological sections of 45 pancreatic cancers, and FIG. 5(o) is a distribution range and a classification standard of a imprinted gene Z15 for pathological sections of 45 pancreatic cancers, wherein LOI is an expression amount of a imprinted gene deleted gene, CNV is an expression amount of a gene with an abnormal copy number of the imprinted gene, and TE is a total expression amount of the imprinted gene;

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

Example 1 imprinted Gene analysis of pancreatic cancer

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

(1) obtaining a tissue cell section (10 microns) of pancreatic cancer, putting the tissue cell section into a 10% neutral formalin solution for fixing to prevent RNA degradation, wherein the fixing time is 24 hours, paraffin embedding (FFPE) is carried out, a slide glass needs to be removed by positive charge, and the section is baked in an oven at 40 ℃ for more than 3 hours;

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

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

the designed probes were designed based on imprinted genes Z1(Gnas), Z2(Igf2), Z3(Peg10), Z4(Igf2r), Z5(Mest), Z6(Plagl1), Z8(Dcn), Z9(Dlk1), Z10(Gatm), Z11(Grb10), Z12(Peg3), Z13(Sgce), Z14(Slc38a4), Z15(Diras3) and Z16(Snrpn/Snurf), specifically selecting a sequence within the intron of each gene as a probe, and specifically a probe designed by Advanced Diagnostics.

(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 microscope imaging;

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

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

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

wherein a, b, c and d are shown in figure 1, a is cell nucleus without marker and without expressed marking gene after hematoxylin staining of cell; b is the cell nucleus with a red/brown mark and the gene existence is marked after the cell is stained by hematoxylin; c, after hematoxylin staining is carried out on the cells, two red/brown marks exist in cell nuclei, and the cell nuclei with gene deletion are marked; and d is the nucleus with more than two red/brown marks and abnormal copy number of the marked gene after the cell is subjected to hematoxylin staining.

As can be seen from FIGS. 2(a) -2 (e), the proportion of cells with loss of imprinting (two signal points in the nucleus) and abnormal copy number (three or more signal points in the nucleus) gradually increased with the degree of malignancy in the samples from grade 0 to grade IV.

Example 2 imprinted Gene analysis of needle biopsy samples

The biopsy sample is obtained by taking out suspicious lesion tissues through puncture, fixing the suspicious lesion tissues in 10% neutral formalin solution for more than 24 hours, and performing other detection methods as in example 1.

As can be seen from fig. 3(a) -3 (f), the response sensitivity of each of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, Z16 to pancreatic cancer or the intensity and status of the deletion of the imprint corresponding to pancreatic cancer expression are different.

The sensitivity of each imprinted gene to pancreatic cancer is shown in fig. 4(a) -4 (o), and as can be seen from fig. 4(a) -4 (e), the imprinting deletion, copy number abnormality and expression increase of the imprinted gene Z1 are obviously increased in the malignant potential stage, the increase rate is slightly reduced in the early pancreatic cancer stage, rapidly increased to a high level in the middle pancreatic cancer stage, and maintained stable in the late pancreatic cancer stage; the imprinting deletion and the expression increase of the imprinting gene Z5 are rapidly increased in a malignant potential stage, are maintained stable in an early pancreatic cancer stage, are continuously increased to a high level in a middle pancreatic cancer stage, are continuously maintained in an advanced pancreatic cancer stage, the copy number abnormality of the imprinting gene Z5 is rapidly increased in the malignant potential stage, is maintained stable in the early and middle pancreatic cancer stages, and is gradually increased to a high level in the advanced pancreatic cancer stage; the imprinting deletion of the imprinting gene Z10 begins to appear in the malignant potential stage, does not obviously rise in early pancreatic cancer, gradually rises to a high level in middle and late pancreatic cancer, the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z10 begin to appear in the malignant potential stage, and gradually rises to a high level in the development process of the early pancreatic cancer to the late pancreatic cancer; the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z11 rapidly increase in a malignant potential stage, the increase speed is slowed down in an early pancreatic cancer stage, the increase speed is rapidly increased in a middle pancreatic cancer stage, and a high level is maintained in a late pancreatic cancer; the imprinting deletion and the expression quantity increase of the imprinting gene Z16 begin to appear at a malignant potential stage, gradually rise to a high level in early and middle pancreatic cancer, continue to maintain stable at a late pancreatic cancer stage, begin to appear at a malignant potential stage with abnormal copy number of the imprinting gene Z16, rapidly rise in early pancreatic cancer, slow down in the middle and late pancreatic cancer, and reach a high level;

as can be seen from fig. 4(f) -fig. 4(o), the deletion of the imprint and the copy number abnormality of the imprinted gene Z2 began to appear at the middle pancreatic cancer stage, and rapidly increased to a higher level at the advanced pancreatic cancer stage, and the increase in the expression level of the imprinted gene Z2 rapidly increased to a higher level at the middle pancreatic cancer stage, and remained stable at the advanced pancreatic cancer stage; the imprinting deletion and the expression quantity increase of the imprinting gene Z3 begin to appear at the middle pancreatic cancer stage, rapidly rise to a higher level at the late pancreatic cancer stage, the copy number abnormality of the imprinting gene Z3 begins to appear at the early pancreatic cancer stage, and is stably maintained at the middle pancreatic cancer stage, and rapidly rise to a very high level at the late pancreatic cancer stage; the imprinting deletion of the imprinting gene Z4 rapidly rises in the early pancreatic cancer stage, and continues to rise to a higher level in the middle and late pancreatic cancers, the copy number abnormality of the imprinting gene Z4 begins to appear in the middle pancreatic cancer stage, and rapidly rises to a higher level in the late pancreatic cancer stage, the expression amount increase of the imprinting gene Z4 begins to appear in the early pancreatic cancer stage, and rapidly rises to a high level in the middle pancreatic cancer stage, and continues to be maintained in the late pancreatic cancer stage; the imprinting deletion and copy number abnormality of the imprinting gene Z6 begin to appear at the imprinting deletion stage, gradually rise to a high level in the development process of pancreatic cancer from early stage to late stage, the expression quantity increase of the imprinting gene Z6 begins to appear at the early pancreatic cancer stage, and rapidly rise to a high level at the middle and late pancreatic cancer stages; the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z8 are rapidly increased in the middle pancreatic cancer stage and are stably maintained in the late pancreatic cancer stage; the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z9 are rapidly increased in the middle pancreatic cancer stage and are stably maintained in the late pancreatic cancer stage; the imprinting deletion of the imprinting gene Z12 begins to appear at the early pancreatic cancer stage, rapidly rises to a higher level at the middle pancreatic cancer stage, and is maintained to be stable at the late pancreatic cancer stage, the copy number abnormality and the expression increase of the imprinting gene Z12 begin to appear at the early pancreatic cancer stage, and gradually rises to a higher level at the middle and late pancreatic cancer stages; the imprinting deletion and the expression increase of the imprinting gene Z13 begin to appear in early pancreatic cancer stage, rapidly rise to a high level in the middle pancreatic cancer stage, and keep stable in the late pancreatic cancer stage, and the copy number abnormality of the imprinting gene Z13 begins to appear in the early pancreatic cancer stage, and gradually rises to a high level in the middle and late pancreatic cancer stages; the imprinting deletion and copy number abnormality of the imprinting gene Z14 begin to appear at the malignant potential stage, rapidly rises at the early pancreatic cancer stage, slows down the rising speed at the middle pancreatic cancer stage, rapidly rises to a higher level at the late pancreatic cancer stage, increases the expression level of the imprinting gene Z14 begin to appear at the early pancreatic cancer stage, slowly rises at the middle pancreatic cancer stage, and rapidly rises to a higher level at the late pancreatic cancer stage; the imprinting deletion of the imprinting gene Z15 begins to appear at the early pancreatic cancer stage, gradually rises to a higher level at the middle and late pancreatic cancer stages, the copy number abnormality of the imprinting gene Z15 begins to appear at the malignant potential stage, gradually rises to a higher level in the development process of the early to late pancreatic cancer, the expression amount of the imprinting gene Z15 is increased to appear at the early pancreatic cancer stage, quickly rises to a higher level at the middle pancreatic cancer stage, and is kept stable at the late pancreatic cancer stage.

Example 345 analysis of imprinted genes of pancreatic tumor samples

The tissue from 45 pancreatic cancer patients was obtained by biopsy and examined as in example 1.

As can be seen from FIGS. 5(a) -5 (o), the ratios of the imprint deletion and copy number abnormality of 15 probes in 45 pancreatic tumor tissue samples showed a low-to-high distribution, and we calculated a grading criterion as shown by the dotted line in the figure according to the distribution trend of different probes, and the imprint deletion and copy number abnormality of each probe could be graded from low to high into 5 grades, respectively.

The specific classification is as follows:

as can be seen from FIG. 5(a), with respect to the imprinted gene Z1, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 15%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 25% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 1.5 to 3%, or the imprinted gene total expression amount of 25 to 35% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 3 to 5%, or the imprinted gene total expression amount of 35 to 45% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 25 to 30%, the imprinted gene copy number abnormal expression amount of 5 to 8%, or the imprinted gene total expression amount of 45 to 55% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 30 percent, the copy number abnormal expression quantity of the imprinted gene is more than 8 percent or the total expression quantity of the imprinted gene is more than 55 percent, and the grade IV is obtained;

as can be seen from FIG. 5(b), for the imprinted gene Z5, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 15%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 25% is class 0, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 1.5 to 3%, or the imprinted gene total expression amount of 25 to 35% is class I, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 3 to 5%, or the imprinted gene total expression amount of 35 to 45% is class II, any one or a combination of at least two of the imprinted gene deletion expression amount of 25 to 30%, the imprinted gene copy number abnormal expression amount of 5 to 8%, or the imprinted gene total expression amount of 45 to 55% is class II Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 30 percent, the copy number abnormal expression quantity of the imprinted gene is more than 8 percent or the total expression quantity of the imprinted gene is more than 55 percent, and the grade IV is obtained;

as can be seen from FIG. 5(c), with respect to the imprinted gene Z10, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 15%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 25% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 1.5 to 3%, or the imprinted gene total expression amount of 25 to 35% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 3 to 5%, or the imprinted gene total expression amount of 35 to 45% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 25 to 30%, the imprinted gene copy number abnormal expression amount of 5 to 8%, or the imprinted gene total expression amount of 45 to 55% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 30 percent, the copy number abnormal expression quantity of the imprinted gene is more than 8 percent or the total expression quantity of the imprinted gene is more than 55 percent, and the grade IV is obtained;

as can be seen from FIG. 5(d), for the imprinted gene Z11, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 15%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 25% is class 0, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 1.5 to 3%, or the imprinted gene total expression amount of 25 to 35% is class I, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 3 to 5%, or the imprinted gene total expression amount of 35 to 45% is class II, any one or a combination of at least two of the imprinted gene deletion expression amount of 25 to 30%, the imprinted gene copy number abnormal expression amount of 5 to 8%, or the imprinted gene total expression amount of 45 to 55% is class II Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 30 percent, the copy number abnormal expression quantity of the imprinted gene is more than 8 percent or the total expression quantity of the imprinted gene is more than 55 percent, and the grade IV is obtained;

as can be seen from FIG. 5(e), for the imprinted gene Z16, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 15%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 25% is class 0, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 1.5 to 3%, or the imprinted gene total expression amount of 25 to 35% is class I, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 3 to 5%, or the imprinted gene total expression amount of 35 to 45% is class II, any one or a combination of at least two of the imprinted gene deletion expression amount of 25 to 30%, the imprinted gene copy number abnormal expression amount of 5 to 8%, or the imprinted gene total expression amount of 45 to 55% is class II Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 30 percent, the copy number abnormal expression quantity of the imprinted gene is more than 8 percent or the total expression quantity of the imprinted gene is more than 55 percent, and the grade IV is obtained;

as can be seen from FIG. 5(f), for the imprinted gene Z2, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(g), for the imprinted gene Z3, any one or a combination of at least two of less than 11% of the deletion expression amount of the imprinted gene, less than 1.5% of the copy number abnormal expression amount of the imprinted gene, or less than 20% of the total expression amount of the imprinted gene is class 0, any one or a combination of at least two of 11 to 15% of the deletion expression amount of the imprinted gene, 1.5 to 2.5% of the copy number abnormal expression amount of the imprinted gene, or 20 to 30% of the total expression amount of the imprinted gene is class I, any one or a combination of at least two of 15 to 20% of the deletion expression amount of the imprinted gene, 2.5 to 4% of the copy number abnormal expression amount of the imprinted gene, or 30 to 40% of the total expression amount of the imprinted gene is class II, any one or a combination of at least two of 20 to 25% of the deletion expression amount of the imprinted gene, 4 to 6% of the copy number abnormal expression amount of the imprinted gene, or 40 to 50% of the total expression amount of the imprinted gene Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(h), for the imprinted gene Z4, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(I), for the imprinted gene Z6, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(j), for the imprinted gene Z8, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(k), for the imprinted gene Z9, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(l), for the imprinted gene Z12, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(m), for the imprinted gene Z13, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(n), for the imprinted gene Z14, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade Grade III, wherein the combination of any one or at least two of the deletion expression quantity of the imprinted gene is more than 25 percent, the copy number abnormal expression quantity of the imprinted gene is more than 6 percent or the total expression quantity of the imprinted gene is more than 50 percent, and the grade IV is obtained;

as can be seen from FIG. 5(o), for the imprinted gene Z15, any one or a combination of at least two of the imprinted gene deletion expression amount of less than 11%, the imprinted gene copy number abnormal expression amount of less than 1.5%, or the imprinted gene total expression amount of less than 20% is of the 0 grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 11 to 15%, the imprinted gene copy number abnormal expression amount of 1.5 to 2.5%, or the imprinted gene total expression amount of 20 to 30% is of the I grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 15 to 20%, the imprinted gene copy number abnormal expression amount of 2.5 to 4%, or the imprinted gene total expression amount of 30 to 40% is of the II grade, any one or a combination of at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 4 to 6%, or the imprinted gene total expression amount of 40 to 50% is of the II grade And in the level III, the combination of any one or at least two of the imprinted gene deletion expression amount is more than 25%, the imprinted gene copy number abnormal expression amount is more than 6% or the imprinted gene total expression amount is more than 50% is level IV.

From the comprehensive analysis of these 45 pancreatic cancer tumors samples, it can be concluded that:

the benign and malignant degree of the pancreatic tumor is divided into benign tumor, pancreatic cancer potential, early pancreatic cancer, metaphase pancreatic cancer and late pancreatic cancer;

the result of judging the benign and malignant degree of the pancreatic tumor is that the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11 and Z11 are less than I grade, or the imprinted gene deletion expression amount of no more than 1 imprinted genes in the imprinted genes Z11, Z11 and Z11 is I grade, and the imprinted genes Z11, Z11 and Z11 are no more than I grade, and the imprinted genes in the imprinted genes Z11, Z11 and Z11 are no more than 1 grade, and the benign tumor copy number of the imprinted genes is abnormal expression amount of I grade;

the judging result of the degree of malignancy of pancreatic tumor is any one of the case where the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class I, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class I, or the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class II, and the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z, Z and Z is class II, pancreatic cancer potential;

the judging result of the degree of malignancy of pancreatic tumor is any one of the cases where the imprinted gene deletion expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class II, the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class II or the imprinted gene copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z, Z and Z is class III, and the imprinted gene copy number abnormal expression level of no more than 1 imprinted gene among imprinted genes Z, Z and Z is class III, early stage pancreatic cancer;

the judging result of the degree of malignancy of pancreatic tumor is any one of the cases where the amount of deletion expression of imprinted genes of at least 2 imprinted genes among imprinted genes Z, Z and Z is class III, the amount of abnormal expression of imprinted gene copy number of at least 2 imprinted genes among imprinted genes Z, Z and Z is class III, or the amount of abnormal expression of imprinted gene copy number of at least 2 imprinted genes among imprinted genes Z, Z and Z is class IV, and the amount of abnormal expression of imprinted gene copy number of no more than 1 imprinted gene among imprinted genes Z, Z and Z is class IV, then the cancer is a metaphase pancreatic cancer;

the result of the determination of the degree of malignancy of the pancreatic tumor is that the advanced pancreatic cancer is obtained when the amount of deletion expression of at least 2 of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class IV or the amount of abnormal expression of the copy number of the imprinted genes of at least 2 of the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class IV.

In conclusion, the detection model and the system of the invention represent the expression of the loss of the imprinting on the sample of the pancreatic tumor patient in an intuitive method, objectively, intuitively, early and accurately detect the change of the imprinting gene by the method for marking the imprinting gene in situ, can provide a quantitative model, and make a great contribution to the diagnosis of the pancreatic tumor.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A imprinted gene grading model for pancreatic tumors, which grades the expression state of imprinted genes by calculating the changes in total expression amount of imprinted genes, deletion expression amount of imprinted genes, and copy number abnormal expression amount of imprinted genes in pancreatic tumors;

2. The imprinted gene ranking model of claim 1, wherein the model computes imprinted genes by:

calculating any one of Z1, Z5, Z10, Z11 or Z16, preferably any one of Z1, Z11 or Z16, and more preferably Z11 or Z16;

preferably, the method for calculating the imprinted gene by the model is as follows: any two of Z1, Z5, Z10, Z11 or Z16 are calculated, preferably a combination of Z1 and Z10, a combination of Z5 and Z11, a combination of Z10 and Z16 or a combination of Z11 and Z16.

3. The imprinted gene classification model according to claim 1 or 2, wherein the imprinted gene further comprises any one of or a combination of at least two of Z2, Z3, Z4, Z6, Z8, Z9, Z12, Z13, Z14, or Z15; the imprinted gene Z2 is Igf2, the imprinted gene Z3 is Peg10, the imprinted gene Z4 is Igf2r, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, the imprinted gene Z9 is Dlk1, the imprinted gene Z12 is Peg3, the imprinted gene Z13 is Sgce, the imprinted gene Z14 is Slc38a4, and the imprinted gene Z15 is Diras 3;

preferably, the method for calculating the imprinted gene by the model is as follows: the combination of the imprinted genes was calculated, and the combination of fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 was calculated.

4. The imprinted gene ranking model of any one of claims 1 to 3, wherein the formula for calculating the total expression level, the imprinted gene deletion expression level and the imprinted gene copy number abnormal expression level of imprinted genes is as follows:

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

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

the expression level of the imprinted gene deletion gene is c/(b + c + d) × 100%;

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

5. The imprinted gene classification model according to any one of claims 1 to 4, wherein the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and imprinted gene total expression amount are classified into five different grades;

preferably, the five different grades are five different grades divided for the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the imprinted gene total expression amount of fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16, respectively;

preferably, the five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z1 and Z11 are:

preferably, the five different grades of the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount and the total expression amount for Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are:

stage IV: any one or a combination of at least two of the imprinted gene deletion expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 25%, and the imprinted gene copy number abnormal expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 6% or the total expression amounts of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z15 and Z16 are more than 50%.

6. An apparatus for detecting the degree of malignancy and malignancy of a pancreatic tumor, comprising:

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

wherein the analyzing unit judges the degree of benign or malignant pancreatic tumor by calculating the deletion expression level of imprinted gene, the copy number abnormal expression level of imprinted gene and the total expression level through the imprinted gene classification model according to any one of claims 1 to 5.

7. The device according to claim 6, wherein the sample to be tested in step (1) is derived from human tissue and/or cells;

preferably, the sample to be tested is a needle biopsy sample;

preferably, the in situ hybridization uses an RNAscope in situ hybridization method;

8. The apparatus of claim 6 or 7, wherein the degree of malignancy of the pancreatic tumor is determined as benign tumor, pancreatic cancer potential, early pancreatic cancer, mid-stage pancreatic cancer, and late-stage pancreatic cancer;

9. Use of the model according to any one of claims 1-5 and/or the device according to any one of claims 6-8 for the preparation of a medicament or kit for the detection and/or treatment of pancreatic tumours.

10. The use of claim 9, wherein the benign or malignant extent of a pancreatic tumor is judged as benign tumor, pancreatic cancer potential, early pancreatic cancer, mid-stage pancreatic cancer, and late-stage pancreatic cancer;