CN111105842A

CN111105842A - Grading model for detecting benign and malignant degrees of lymphoma and lymphatic metastatic cancer and application thereof

Info

Publication number: CN111105842A
Application number: CN201811270491.6A
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: 2018-10-29
Filing date: 2018-10-29
Publication date: 2020-05-05
Anticipated expiration: 2038-10-29
Also published as: CN111105842B

Abstract

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

Description

Grading model for detecting benign and malignant degrees of lymphoma and lymphatic metastatic cancer 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, and relates to a hierarchical model for detecting the benign and malignant degrees of lymphoma and lymphatic metastatic carcinoma and application thereof, in particular to a hierarchical model for detecting the benign and malignant degrees of lymphoma and lymphatic metastatic carcinoma by a group of imprinted genes and a device formed by the hierarchical model.

Background

Lymphoma is a malignant tumor originating from the lymphohematopoietic system, mainly caused by viral infection, immunodeficiency, ionizing radiation, chemical drugs and the like, and is better in young and middle-aged years of 20-40 years old. In 2012, there are 38.6 new cases, 20.0 ten thousand deaths, about 8.8 ten thousand new cases and 5.2 ten thousand deaths of Chinese newly diagnosed cases, and in recent years, the malignant lymphoma is still growing at a rate of 4% per year. Lymphoma can occur in any part of the human body, and early symptoms are only painless lymph node enlargement, are easy to be ignored by patients and are easy to be confused with reactive lymph node hyperplasia caused by infection. Since lymphoma is a large group of tumors with high heterogeneity, it can be classified into hundreds of types according to the classification criteria of WHO, and there are great differences in pathological manifestations and prognosis among the types, so that it is difficult to diagnose and treat lymphoma. Lymphoma can be generally divided into highly aggressive, moderately aggressive and indolent lymphoma, the 5-year survival rate of highly aggressive lymphoma is usually less than 30%, while the 5-year survival rate of some types of indolent lymphoma can reach 90%, and if some moderately aggressive lymphoma can be discovered and treated early, the survival period can reach 5-10 years or even longer, so that the development of a malignant lymphoma early detection technology with higher accuracy is of great significance.

In addition to lymphoma, lymph node enlargement may also be caused by cancer metastasis, which occurs in a variety of cancers such as lung cancer, breast cancer, esophageal cancer, thyroid cancer, colorectal cancer, and bladder cancer. At present, the pathological judgment of whether cancer cells metastasized exist in lymph nodes is usually based on morphological observation, but the number of cancer cells in lymph nodes is small in the early stage of cancer metastasis, and the cancer cells may be omitted through morphological observation, so that a technology for accurately identifying whether cancer cells metastasized exist in lymph nodes is developed, a doctor can be helped to determine the surgical resection range and the lymph node cleaning range, and the selection of a subsequent treatment scheme is known, and the technology has great significance for reducing the recurrence rate after surgery and prolonging the life cycle.

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 of malignancy and malignancy of lymphoma according to 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 malignant lymphoma. 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, the current diagnosis of malignant lymphoma requires a new detection system and detection model, which can analyze the molecular marker changes existing on the cell level of malignant lymphoma based on the biopsy sample of the patient, so as to provide 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 lymphoma and lymphatic metastatic cancer 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 lymphoma, which grades the expression state of imprinted genes by calculating the changes in the lymphoma of the total expression amount of the imprinted genes, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount;

wherein the imprinted gene is any one of Z1, Z8, Z11 or Z16 or a combination of at least two of Z1, Z8, Z11 and Z16, the imprinted gene Z1 is Gnas, the imprinted gene Z8 is Dcn, the imprinted gene Z11 is Grb10, and the imprinted gene Z16 is Snrpn/Snurf.

In the present invention, the inventors have found that diagnosis of malignant lymphoma and lymphatic metastatic cancer can be achieved by calculating the imprinted gene deletion expression amount and imprinted gene copy number abnormal expression amount of any one imprinted gene among Z1, Z8, Z11 and Z16 in lymphoma.

According to the imprinted gene ranking model for lymphoma according to the present invention, if only one imprinted gene is detected in the preliminary test, any one of Z1, Z8 or Z16, preferably Z1, can be detected.

In the invention, the inventor finds that the diagnosis sensitivity can reach more than 57.4 percent by calculating the imprinted gene deletion expression quantity and the imprinted gene copy number abnormal expression quantity of any imprinted gene in Z1, Z8 or Z16 in lymphoma; if a Z1 imprinted gene is detected alone, the diagnostic sensitivity to malignant lymphoma can reach 66.1%, if a Z8 imprinted gene is detected alone, the diagnostic sensitivity to malignant lymphoma can reach 57.4%, and if a Z16 imprinted gene is detected alone, the diagnostic sensitivity to malignant lymphoma can reach 62.0%.

According to the present invention, a imprinted gene classification model for lymphoma, if a combination of two imprinted genes of an imprinted gene is detected, the method for calculating the imprinted gene by the model is: any two of Z1, Z8, or Z16 are calculated, preferably a combination of Z1 and Z8, and a combination of Z8 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 malignant lymphoma can be up to 76.0% or more by detecting the combination of any two imprinted genes among imprinted genes Z1, Z8, and Z16, that the diagnostic sensitivity for malignant lymphoma can be up to 85.2% when detecting the combination of Z1 and Z8, and that the diagnostic sensitivity for malignant lymphoma can be up to 84.4% or more when detecting the combination of Z8 and Z16.

According to the present invention, a imprinted gene grading model for lymphoma, the imprinted gene further comprising any one of or a combination of at least two of Z4, Z5, Z6, Z11 or Z13; the imprinted gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z11 is Grb10, and the imprinted gene Z13 is Sgce.

According to the invention, the imprinting gene grading model for lymphoma comprises the following steps: the combinations of the imprinted genes were calculated, the combinations of eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 were calculated.

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

In a second aspect, the present invention provides an imprinted gene grading model for lymphatic metastatic cancer, which grades the expression state of imprinted genes by calculating the change of the copy number abnormal expression amount of the imprinted genes in lymphatic metastatic cancer;

In the present invention, the lymphatic metastasis cancer includes thyroid cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, bladder cancer, prostate cancer, and skin cancer.

In the present invention, the inventors have found that diagnosis of malignant lymphoma and lymphatic metastatic cancer can be achieved by calculating the copy number abnormal expression amount of a imprinted gene in lymphatic metastatic cancer of any one imprinted gene among Z1, Z8, Z11 and Z16.

According to the imprinted gene grading model of lymphatic metastatic cancer of the present invention, if only one imprinted gene is detected in the preliminary test, any one of Z1, Z11 or Z16, preferably Z16, can be detected.

In the invention, the inventor finds that the diagnosis sensitivity can reach over 86.4 percent by calculating the copy number abnormal expression quantity of any imprinted gene in Z1, Z11 or Z16 in the lymph metastasis cancer; if a Z1 imprinted gene is detected independently, the diagnostic sensitivity to cancer lymphatic metastasis can reach 87.1%, if a Z11 imprinted gene is detected independently, the diagnostic sensitivity to cancer lymphatic metastasis can reach 86.4%, and if a Z16 imprinted gene is detected independently, the diagnostic sensitivity to cancer lymphatic metastasis can reach 91.3%.

According to the present invention, a imprinted gene classification model for lymphatic metastatic cancer, if a combination of two imprinted genes of an imprinted gene is detected, the method for calculating the imprinted gene by the model is: any two of Z1, Z11, or Z16 are calculated, preferably a combination of Z1 and Z16, and a combination of Z11 and Z16.

In the present invention, the inventors found that the sensitivity can be further improved by calculating the copy number abnormal expression level of two or more imprinted genes, and that the diagnostic sensitivity for lymph metastasis cancer can be up to 95.5% or more by detecting the combination of any two imprinted genes among imprinted genes Z1, Z11, and Z16, and the diagnostic sensitivity for lymph metastasis cancer can be up to 99.0% or more by detecting the combination of Z1 and Z6, and the combination of Z11 and Z16. .

According to the present invention, a imprinted gene grading model for lymphatic metastatic cancer, the imprinted gene further comprising any one or a combination of at least two of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, 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 Z5 is Mest, the imprinting gene Z6 is Plagl1, the imprinting gene Z8 is Dcn, the imprinting gene Z9 is Dlk1, the imprinting gene Z10 is Gatm, 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.

According to the invention, the imprinted gene grading model aiming at the lymph metastasis cancer comprises a method for calculating imprinted genes by the model: calculating a combination of imprinted genes, calculating a combination of fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16.

In the invention, the inventor finds that the combined diagnosis is carried out by adding Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14 and Z15 genes on the basis of using Z1, Z11 and Z16 genes for detection, 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 the lymphatic metastasis cancer samples.

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.

According to the invention, 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 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, a imprinted gene grading model for lymphoma, the total imprinted gene expression amount, the imprinted gene deletion expression amount, and the imprinted gene copy number abnormal expression amount 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 the five different grades are classified into the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the imprinted gene total expression amount of eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16, respectively.

According to the present invention, the five different grades of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z1, Z11, Z13, and Z16 are:

level 0: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is less than 16%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is less than 1.5%, or the total expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is less than 20%;

stage I: the imprinted gene deletion expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 16-20%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 1.5-2.5% or the total expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 20-30% or the combination of at least two of the above;

II stage: the imprinted gene deletion expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 2.5-5% or the total expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 30-40%, or the combination of at least two of the above;

grade III: the imprinted gene deletion expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 25-30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 5-7% or the total expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is 40-50% or the combination of at least two of the above;

stage IV: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is more than 30%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is more than 7% or the total expression amount of the imprinted genes Z1, Z11, Z13 and Z16 is more than 50%;

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, Z11, Z13 and Z16 are independent from each other.

According to the present invention, the five different grades of the imprinted gene deletion expression amount, imprinted gene copy number abnormal expression amount, and total expression amount for Z4, Z5, Z6, and Z8 are:

level 0: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is less than 8%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is less than 0.5%, or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is less than 15%;

stage I: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 8-15%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 0.5-1.5% or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 15-20% or the combination of at least two of the above;

II stage: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 15-20%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 1.5-2.5% or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 20-30% or the combination of at least two of the above;

grade III: the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 20-25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 2.5-4% or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is 30-40%, or the combination of at least two of the above;

stage IV: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 4% or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 40%;

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 and Z8 are independent from each other.

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

According to the invention, the three different grades for the abnormal expression level of the copy number of the imprinted gene of Z1 and Z16 are:

level 0: the copy number abnormal expression quantity of the imprinted genes Z1 and Z16 is less than 5%;

stage I: the copy number abnormal expression quantity of the imprinted genes Z1 and Z16 is 5-10%;

II stage: the copy number abnormal expression quantity of the imprinted genes Z1 and Z16 is more than 10%;

in the present invention, the copy number abnormal expression levels of the imprinted genes Z1 and Z16 are independent of each other.

According to the invention, the three different grades for the abnormal expression level of the copy number of the imprinted gene aiming at Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are as follows:

level 0: the copy number abnormal expression quantity of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is less than 2%;

stage I: the copy number abnormal expression quantity of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is 2-5%;

II stage: the copy number abnormal expression quantity of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is more than 5%;

in the present invention, the copy number abnormal expression levels of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are independent of each other.

In a third aspect, the present invention provides an apparatus for detecting the benign and malignant degree of lymphoma and the lymphatic metastasis degree of cancer, 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 judges the benign or malignant degree of lymphoma and the degree of cancer lymphatic metastasis by calculating the total expression level of imprinted genes, the deletion expression level of imprinted genes, and the copy number abnormal expression level of imprinted genes through the imprinted gene classification model according to the first aspect and/or the second 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 genes of the lymphoma and the cancer metastasis lymph nodes at the early stage under the cell and tissue level so as to judge the benign and malignant degree of the tumor and the degree of cancer cell metastasis in the lymph nodes, and provides the most favorable treatment opportunity for early stage lymphoma patients and early stage cancer metastasis patients.

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 lymph node biopsy sample.

The specific operation steps of the paraffin section of the tissue are that a human tumor or lymph node tissue sample is obtained, fixed by 10 percent neutral formalin in time, embedded by paraffin, cut into a thickness of 10 mu m, and made into a tissue slice by a slide with positive charges; 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 lymph node puncture biopsy sample are that human lymph node cells are obtained through puncture and are fixed by 10% neutral formalin in time.

In the invention, because the lymph node aspiration biopsy has little harm to a patient and a simple sampling process, compared with the circulation characteristic of blood, the lymph node aspiration biopsy can also be positioned, and has special advantages as an experimental sample.

Preferably, the sample to be tested is a lymph node aspiration 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 lymph nodes, 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 and the cancer cell metastasis degree in lymph nodes are 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 imprinted gene classification model for lymphoma is classified into five different grades for the imprinted gene deletion expression amount, the imprinted gene copy number abnormal expression amount, and the total expression amount.

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, imprinted gene copy number abnormal expression amount, and total expression amount of the eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13, 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, Z11, Z13 and Z16 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 Z4, Z5, Z6 and Z8 are:

Preferably, the benign and malignant degree of the lymphoma is divided into benign tumor, malignant potential lymphoma, early stage malignant lymphoma, intermediate stage malignant lymphoma and advanced stage malignant lymphoma;

preferably, as a result of the determination of the malignancy and malignancy of lymphoma, when both the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 are less than class I or the imprinted gene deletion expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is class I and the imprinted gene copy number abnormal expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is class I, the lymphoma is a benign tumor;

preferably, the judging of the malignancy or malignancy of lymphoma is carried out in any case where the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have an imprinting gene deletion expression level of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 of class I, the imprinting gene copy number abnormal expression level of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class I or the imprinting gene deletion expression level of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class II and the imprinting gene copy number abnormal expression level of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class II;

preferably, the judging of the malignancy or malignancy of lymphoma is carried out in any case where the imprinting genes of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have a deletion expression level of class II, the imprinting genes of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have an abnormal expression level of copy number of class II, or the imprinting genes of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have a deletion expression level of class III and the imprinting genes of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have an abnormal expression level III, and the imprinting genes have an abnormal expression level III;

preferably, the judging of the malignancy or malignancy of lymphoma is carried out in any case where the imprinting genes of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have a deletion expression level of class III, the imprinting genes of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have an abnormal expression level of class III, or the imprinting genes of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have a deletion expression level of class IV, and the imprinting genes of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 have an abnormal expression level IV, and the imprinting genes have an abnormal expression level IV, and the lymphoma is considered to be malignant;

preferably, the advanced lymphoma is determined as a result of the determination of the malignancy or malignancy of the lymphoma if the loss expression level of at least 2 imprinted genes among imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is IV grade, or the copy number abnormal expression level of at least 2 imprinted genes among imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is IV grade.

According to the present invention, the imprinted gene grading model for lymphatic metastatic cancer, the imprinted gene copy number abnormal expression amount is divided into three different grades.

The three different grades are such that at least 1200 cells are counted in the region where the sample is most positively expressed, and the imprinted gene copy number abnormal expression amounts of fifteen imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 are divided respectively.

The three different grades of the abnormal expression level of the copy number of the imprinted gene aiming at Z1 and Z16 are:

The three different grades for the segregation of the copy number aberrant expression levels of imprinted genes for Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are:

Preferably, the degree of metastasis of cancer cells in the lymphatic metastatic cancer is judged as non-metastasis, mild metastasis and severe metastasis.

Preferably, the judging of the degree of metastasis of cancer cells of the lymphatic metastatic cancer is that the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 have abnormal expression levels of imprinted gene copy number less than level I or that the imprinted genes of not more than 1 imprinted genes among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 have abnormal expression levels of imprinted gene copy number I and are not metastatic;

preferably, the judging of the degree of metastasis of cancer cells of the lymphatic metastatic cancer is carried out in a case where the abnormal expression level of the copy number of the imprinted gene of at least 2 imprinted genes among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class I or the abnormal expression level of the copy number of the imprinted gene of not more than 1 imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class II, and the case is light metastasis;

preferably, as a result of the determination of the degree of metastasis of cancer cells in the lymphatic metastatic cancer, when the copy number abnormal expression level of the imprinted gene of at least 2 imprinted genes among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class II, the cancer cells are highly metastatic.

In a fourth aspect, the present invention provides a model according to the first and/or second aspect or a device according to the third aspect for use in the manufacture of a medicament or kit for the detection and/or treatment of lymphoma and/or lymphoid metastatic cancer.

Preferably, the benign and malignant degree of the lymphoma is judged to be benign tumor, malignant potential lymphoma, early malignant lymphoma, intermediate malignant lymphoma and advanced malignant lymphoma;

preferably, the judging of the malignancy or malignancy of lymphoma is carried out by judging that the imprinting gene deletion expression level of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class I, the imprinting gene copy number abnormal expression level of at least 2 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class I or the imprinting gene copy number abnormal expression level of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class II and the imprinting gene copy number abnormal expression level of no more than 1 of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is class II, and judging that the lymphoma is malignant;

Preferably, the degree of metastasis in the lymphatic metastatic cancer is judged as non-metastasis, mild metastasis and severe metastasis;

preferably, as a result of the determination of the degree of metastasis in the lymphatic metastatic cancer, the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 have imprinted gene copy number abnormal expression amounts less than class I or imprinted gene copy number abnormal expression amounts of not more than 1 imprinted gene among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 are class I, and are non-metastatic;

preferably, as a result of the determination of the degree of metastasis in the lymphatic metastatic cancer, mild metastasis is obtained if the abnormal expression level of the copy number of the imprinted gene of at least 2 imprinted genes among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class I or the abnormal expression level of the copy number of the imprinted genes of not more than 1 imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class II;

preferably, as a result of the determination of the degree of metastasis in the lymphatic metastatic cancer, the degree of metastasis is determined to be severe metastasis if the abnormal expression level of copy number of the imprinted gene of at least 2 imprinted genes among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is level II.

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

(1) the detection model and the device of the invention express the expression of the imprinting deletion on the samples of patients with lymphoma and cancer lymphatic metastasis 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 lymphoma and cancer lymphatic metastasis;

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

(3) the method can accurately distinguish benign lymphoproliferation from early malignant lymphoma, definitely grades the malignancy degree of the lymphoma by combined detection of imprinting genes, greatly improves early and definite diagnosis of the malignant lymphoma, has simple material taking process, can be used for early general investigation and cancer postoperative follow-up visit, particularly for tracking follow-up visit of suspected relapsing patients, can strive for time, and makes great contribution to saving the lives of the patients;

(4) the detection device can obtain whether cancer cells have lymph metastasis or not and judge the metastasis range by lymph node puncture before the operation of a cancer patient, help doctors determine the range of surgical resection and lymph node cleaning and the selection of a postoperative treatment scheme, and greatly reduce postoperative recurrence;

(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 for silencing, rejecting and rearranging the gene of the discovered lymphoma-related imprinted gene deletion site.

Drawings

FIG. 1 is a pathological section of malignant lymphoma with hematoxylin-stained nuclei according to the present invention, wherein a is that after hematoxylin-staining of cells, no marker is present in the nuclei and no imprinted gene is expressed; 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) shows the expression states of 8 genes in a pathological section of grade 0 lymphoma, FIG. 2(b) shows the expression states of 8 genes in a pathological section of grade I lymphoma, FIG. 2(c) shows the expression states of 8 genes in a pathological section of grade II lymphoma, FIG. 2(d) shows the expression states of 8 genes in a pathological section of grade III lymphoma, and FIG. 2(e) shows the expression states of 8 genes in a pathological section of grade IV lymphoma;

fig. 3(a) is the intensity of imprinting deletion of imprinted genes Z1, Z8, and Z16 for lymphoma, fig. 3(b) is the intensity of imprinting gene Z1, Z8, and Z16 for lymphoma, fig. 3(c) is the intensity of total expression amount of imprinted genes Z1, Z8, and Z16 for lymphoma, fig. 3(d) is the intensity of imprinting deletion of imprinted genes Z4, Z5, Z6, Z11, and Z13 for lymphoma, fig. 3(e) is the intensity of imprinting deletion of imprinted genes Z4, Z5, Z6, Z11, and Z13 for lymphoma, fig. 3(f) is the intensity of total expression amount of imprinted genes Z4, Z5, Z6, Z11, and Z13 for lymphoma, wherein LOI is the copy number of imprinted genes, v is the copy number of imprinted genes, and TE is the total expression amount of imprinted genes;

FIG. 4(a) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z1, FIG. 4(b) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z8, FIG. 4(c) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z16, FIG. 4(d) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z4, FIG. 4(e) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z5, FIG. 4(f) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z6, FIG. 4(g) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z11, FIG. 4(h) is intensities of imprinting deletion, copy number abnormality and total expression of imprinting gene Z13, wherein LOI is expression of imprinting deletion gene, CNV is the gene expression quantity of the imprinting gene with abnormal copy number, and TE is the total expression quantity of the imprinting gene;

FIG. 5(a) is a distribution range and classification standard of the imprinted gene Z1 applied to 77 lymphoma pathological sections, the distribution range and classification standard of the imprinted deletion, copy number abnormality and total expression amount, FIG. 5(b) is a distribution range and classification standard of the imprinted gene Z8 applied to 77 lymphoma pathological sections, the distribution range and classification standard of the imprinted deletion, copy number abnormality and total expression amount, FIG. 5(c) is a distribution range and classification standard of the imprinted gene Z16 applied to 77 lymphoma pathological sections, FIG. 5(d) is a distribution range and classification standard of the imprinted gene Z4 applied to 77 lymphoma pathological sections, the distribution range and classification standard of the imprinted deletion, copy number abnormality and total expression amount, FIG. 5(e) is a distribution range and classification standard of the imprinted gene Z5 applied to 77 lymphoma pathological sections, and FIG. 5(f) is a distribution range and classification standard of the imprinted gene Z6 applied to 77 lymphoma pathological sections, distribution ranges and classification standards of the imprinting deletion, copy number abnormality and total expression, fig. 5(g) shows the distribution ranges and classification standards of the imprinting gene Z11 applied to 77 lymphoma pathological sections, and fig. 5(h) shows the distribution ranges and classification standards of the imprinting deletion, copy number abnormality and total expression, where LOI is the imprinting gene deletion expression, CNV is the gene expression of the imprinting gene copy number abnormality, and TE is the total expression of the imprinting gene, when the imprinting gene Z13 is applied to 77 lymphoma pathological sections;

FIG. 6(a) shows the expression levels of 15 genes in a sample of punctured cells of a lymph node near non-metastatic breast cancer, FIG. 6(b) shows the expression levels of 15 genes in a sample of punctured cells of a lymph node near mildly metastatic breast cancer, and FIG. 6(c) shows the expression levels of 15 genes in a sample of punctured cells of a lymph node near heavily metastatic breast cancer;

FIG. 7(a) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z1 was applied, FIG. 7(b) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z2 was applied, FIG. 7(c) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z3 was applied, FIG. 7(d) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z4 was applied, FIG. 7(e) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z5 was applied, FIG. 7(f) is a distribution range and a classification standard of copy number abnormality in a lymph node puncture sample of 42 cancer patients to which the imprinted gene Z6 was applied, FIG. 7(g) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z8 was applied, FIG. 7(h) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z9 was applied, FIG. 7(i) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z10 was applied, FIG. 7(j) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z11 was applied, FIG. 7(k) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z12 was applied, FIG. 7(l) is a distribution range and a classification standard of copy number abnormality in the lymph node puncture sample of 42 cancer patients to which the imprinted gene Z13 was applied, FIG. 7(m) is the distribution range and classification standard of copy number abnormality of the imprinted gene Z14 applied to the lymph node puncture sample of 42 cancer patients, FIG. 7(n) is the distribution range and classification standard of copy number abnormality of the imprinted gene Z15 applied to the lymph node puncture sample of 42 cancer patients, and FIG. 7(o) is the distribution range and classification standard of copy number abnormality of the imprinted gene Z16 applied to the lymph node puncture sample of 42 cancer patients, wherein CNV is the gene expression level of copy number abnormality 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 imprinting Gene analysis of malignant lymphoma

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

(1) obtaining a tissue cell section (10 microns) of malignant lymphoma, 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, the 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 are designed according to imprinted genes Z1(Gnas), Z4(Igf2r), Z5(Mest), Z6(Plagl1), Z8(Dcn), Z11(Grb10), Z13(Sgce) and Z16(Snrpn/Snurf), and specifically, a sequence is selected from the internal rotor of each gene as a probe, and the specific probe is designed by Advanced Cell 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 lymph node biopsy samples from lymphoma patients

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

As can be seen from fig. 3(a) -3 (f), the response sensitivity of each gene Z1, Z4, Z5, Z6, Z8, Z11, Z13, Z16 to malignant lymphoma or the intensity and state of absence of imprinting corresponding to malignant lymphoma expression are different.

The sensitivity of each imprinted gene to malignant lymphoma is shown in fig. 4(a) -4 (h), and as can be seen from fig. 4(a) -4 (c), the imprinting deletion, copy number abnormality and expression increase of imprinted gene Z1 rapidly increase in the malignant potential stage, and the increase rate is reduced in the early and middle stages of malignant lymphoma, but rapidly increases to a high level in the late stage of malignant lymphoma; the imprinting deletion and copy number abnormality of the imprinting gene Z8 begin to appear at a malignant potential stage, gradually rise from an early stage to a middle-stage malignant lymphoma stage, the imprinting deletion in the late-stage malignant lymphoma continues to increase, the copy number abnormality does not increase any more, the expression quantity of the imprinting gene Z8 increases and begins to appear at the malignant potential stage, the imprinting deletion and copy number abnormality is maintained stable at the early and middle-stage malignant lymphoma stages, and the imprinting deletion and copy number abnormality rapidly rises to a higher level at the late-stage malignant lymphoma stage; the imprinting deletion, copy number abnormality and expression quantity increase of the imprinting gene Z16 rapidly increase in malignant potential and early malignant lymphoma stage, are maintained stable in the middle malignant lymphoma stage, and rapidly increase to a high level in the late malignant lymphoma stage;

as can be seen from fig. 4(d) -fig. 4(h), the imprinting deletion of the imprinting gene Z4 appeared in small amounts at the beginning of the malignant potential stage, slowly increased at the early and middle stages of malignant lymphoma, and rapidly increased at the late stage of malignant lymphoma, and the abnormal copy number and increased expression amount of the imprinting gene Z4 appeared at the beginning of the malignant potential stage, but did not significantly increase during the development of malignant lymphoma; the imprinting deletion, copy number abnormality and expression increase of the imprinting gene Z5 begin to appear in the malignant potential stage, but do not obviously increase in the development process of malignant lymphoma; the copy number abnormality of the imprinting gene Z6 appears a little at the beginning of the malignant potential stage, slowly rises in the early and middle stages of malignant lymphoma, and rapidly rises to the late stage of malignant lymphoma, and the imprinting deletion and the expression increase of the imprinting gene Z6 appear at the beginning of the malignant potential stage, but do not obviously rise in the development process of the malignant lymphoma; the imprinting deletion, copy number abnormality and expression increase of the imprinting gene Z11 slightly appear at the malignant potential stage, slowly increase at the early and middle stage malignant lymphoma stages, and increase speed at the late stage malignant lymphoma stage; the imprinting deletion of the imprinting gene Z13 begins to appear at the malignant potential stage, slowly rises at the malignant lymphoma stage in the early and middle stages, the rising speed is accelerated to the advanced malignant lymphoma stage, but the level is still low, the copy number abnormality of the imprinting gene Z13 gradually rises at the malignant potential and early malignant lymphoma stages, the rising speed is not obviously increased in the intermediate and advanced malignant lymphoma, the expression level of the imprinting gene Z13 is increased at the malignant potential stage, the rising speed is slowly increased at the malignant lymphoma stage in the early and middle stages, and the rising speed is accelerated to the advanced malignant lymphoma stage, but the level is still not high.

Example 377 imprinted gene analysis of lymphoma samples

The tissue from 77 patients with malignant lymphoma was obtained by biopsy of lymph node, and the procedure was the same as in example 1.

As can be seen from FIGS. 5(a) -5 (h), the ratios of the loss of print and copy number abnormality of 8 probes in 77 lymphoma tissue samples showed a low-to-high distribution, and according to the distribution trend of different probes, we calculated the grading standard shown by the dotted line in the figure, and the loss of print, copy number abnormality and total expression of each probe were graded from low to high respectively into 5 grades.

The specific classification is as follows:

as can be seen from FIG. 5(a), with respect to the imprinted gene Z1, any one or at least two of the imprinted gene deletion expression amount of less than 16%, 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 class 0, any one or at least two of the imprinted gene deletion expression amount of 16 to 20%, 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 class I, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 2.5 to 5%, or the imprinted gene total expression amount of 30 to 40% is class II, any one or 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 7%, or the imprinted gene total expression amount of 40 to 50% is class III, 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 7 percent or the total expression quantity of the imprinted gene is more than 50 percent is grade IV;

as can be seen from FIG. 5(b), for the imprinted gene Z8, any one or at least two of the imprinted gene deletion expression amount of less than 16%, 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 class 0, any one or at least two of the imprinted gene deletion expression amount of 16 to 20%, 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 class I, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 2.5 to 5%, or the imprinted gene total expression amount of 30 to 40% is class II, any one or 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 7%, or the imprinted gene total expression amount of 40 to 50% is class III, 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 7 percent or the total expression quantity of the imprinted gene is more than 50 percent is grade IV;

as can be seen from FIG. 5(c), for the imprinted gene Z11, any one or at least two of the imprinted gene deletion expression amount of less than 16%, 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 class 0, any one or at least two of the imprinted gene deletion expression amount of 16 to 20%, 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 class I, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, the imprinted gene copy number abnormal expression amount of 2.5 to 5%, or the imprinted gene total expression amount of 30 to 40% is class II, any one or 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 7%, or the imprinted gene total expression amount of 40 to 50% is class III, 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 7 percent or the total expression quantity of the imprinted gene is more than 50 percent is grade IV;

as can be seen from FIG. 5(d), for the imprinted gene Z4, any one or at least two of the imprinted gene deletion expression amount of less than 8%, the imprinted gene copy number abnormal expression amount of less than 0.5%, or the imprinted gene total expression amount of less than 15% is class 0, any one or at least two of the imprinted gene deletion expression amount of 8 to 15%, the imprinted gene copy number abnormal expression amount of 0.5 to 1.5%, or the imprinted gene total expression amount of 15 to 20% is class I, any one or 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 2.5%, or the imprinted gene total expression amount of 20 to 30% is class II, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, 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 class III, 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 4 percent or the total expression quantity of the imprinted gene is more than 40 percent, and the IV grade is obtained;

as can be seen from FIG. 5(e), for the imprinted gene Z5, any one or at least two of the imprinted gene deletion expression amount of less than 8%, the imprinted gene copy number abnormal expression amount of less than 0.5%, or the imprinted gene total expression amount of less than 15% is class 0, any one or at least two of the imprinted gene deletion expression amount of 8 to 15%, the imprinted gene copy number abnormal expression amount of 0.5 to 1.5%, or the imprinted gene total expression amount of 15 to 20% is class I, any one or 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 2.5%, or the imprinted gene total expression amount of 20 to 30% is class II, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, 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 class III, 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 4 percent or the total expression quantity of the imprinted gene is more than 40 percent, and the IV grade is obtained;

as can be seen from FIG. 5(f), for the imprinted gene Z6, any one or at least two of the imprinted gene deletion expression amount of less than 8%, the imprinted gene copy number abnormal expression amount of less than 0.5%, or the imprinted gene total expression amount of less than 15% is class 0, any one or at least two of the imprinted gene deletion expression amount of 8 to 15%, the imprinted gene copy number abnormal expression amount of 0.5 to 1.5%, or the imprinted gene total expression amount of 15 to 20% is class I, any one or 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 2.5%, or the imprinted gene total expression amount of 20 to 30% is class II, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, 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 class III, 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 4 percent or the total expression quantity of the imprinted gene is more than 40 percent, and the IV grade is obtained;

as can be seen from FIG. 5(g), for the imprinted gene Z11, any one or at least two of less than 8% of the deletion expression amount of the imprinted gene, less than 0.5% of the copy number abnormal expression amount of the imprinted gene, or less than 15% of the total expression amount of the imprinted gene is class 0, any one or at least two of 8 to 15% of the deletion expression amount of the imprinted gene, 0.5 to 1.5% of the copy number abnormal expression amount of the imprinted gene, or 15 to 20% of the total expression amount of the imprinted gene is class I, any one or at least two of 15 to 20% 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 II, any one or at least two of 20 to 25% 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 III, 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 4 percent or the total expression quantity of the imprinted gene is more than 40 percent, and the IV grade is obtained;

as can be seen from FIG. 5(h), for the imprinted gene Z13, any one or at least two of the imprinted gene deletion expression amount of less than 8%, the imprinted gene copy number abnormal expression amount of less than 0.5%, or the imprinted gene total expression amount of less than 15% is class 0, any one or at least two of the imprinted gene deletion expression amount of 8 to 15%, the imprinted gene copy number abnormal expression amount of 0.5 to 1.5%, or the imprinted gene total expression amount of 15 to 20% is class I, any one or 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 2.5%, or the imprinted gene total expression amount of 20 to 30% is class II, any one or at least two of the imprinted gene deletion expression amount of 20 to 25%, 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 class III, the IV grade is 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 4 percent or the total expression quantity of the imprinted gene is more than 40 percent.

From the comprehensive analysis of the 77 lymphoma samples, it can be concluded that:

judging the benign and malignant degree of the lymphoma to be benign tumor, malignant potential lymphoma, early malignant lymphoma, intermediate malignant lymphoma and late malignant lymphoma;

as a result of judging the degree of malignancy and malignancy of lymphoma, when both the imprinted gene deletion expression amount and the imprinted gene copy number abnormal expression amount of the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 are less than the I-grade, or when the imprinted gene deletion expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is the I-grade and the imprinted gene copy number abnormal expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is the I-grade, the lymphoma is benign tumor;

judging whether the benign or malignant degree of the lymphoma is a malignant lymphoma if the imprinting gene deletion expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade I, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade I or the imprinting gene copy number abnormal expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade II, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade II;

judging whether the lymphoma is malignant if the result of judging the benign/malignant degree of the lymphoma is that the imprinted gene deletion expression amount of at least 2 imprinted genes among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is of class II, the imprinted gene copy number abnormal expression amount of at least 2 imprinted genes among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is of class II or the imprinted gene copy number abnormal expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is of class III and the imprinted gene copy number abnormal expression amount of no more than 1 imprinted gene among the imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 is of class III, and if the lymphoma is malignant early stage;

judging whether the benign or malignant degree of the lymphoma is malignant when the imprinting gene deletion expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade III, the imprinting gene copy number abnormal expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade III or the imprinting gene copy number abnormal expression level of at least 2 imprinting genes of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade IV, and the imprinting gene copy number abnormal expression level of no more than 1 imprinting gene of the imprinting genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is grade IV, and when the intermediate stage lymphoma is malignant;

the grade of malignancy of lymphoma was judged by determining whether the deletion expression level of imprinted genes of at least 2 imprinted genes among imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 was IV grade or the abnormal expression level of the copy number of imprinted genes of at least 2 imprinted genes among imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 was IV grade, and the lymphoma was advanced stage malignant lymphoma.

Example 4 analysis of imprinted genes of sentinel lymph nodes of Breast cancer

Sentinel lymph node samples of breast cancer patients were obtained by puncture and fixed in 10% neutral formalin for over 24 h. Probes were designed based on 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), specifically selecting a sequence as a probe within the intron of each gene, specifically a probe designed by Advanced Cell Diagnostics, Ex 1.

As can be seen from fig. 6(a) to 6(c), fig. 6(a) shows lymph node-piercing cells in the vicinity of non-metastatic breast cancer, fig. 6(b) shows lymph node-piercing cells in the vicinity of slightly metastatic breast cancer, and fig. 6(c) shows lymph node-piercing cells in the vicinity of heavily metastatic breast cancer, and only cells with a single imprint loss and no abnormal copy number were found in lymph nodes in the vicinity of non-metastatic breast cancer, while a small number of cancer cells with an abnormal copy number of the imprinted gene were present in lymph nodes in the vicinity of slightly metastatic breast cancer, and a large number of cancer cells with an abnormal copy number of the imprinted gene were present in lymph nodes in the vicinity of heavily metastatic breast cancer.

Example 542 imprinted genetic analysis of lymph node puncture in cancer patients

Lymph node puncture samples of patients with breast cancer, lung cancer and pancreatic cancer are obtained and fixed for more than 24 hours by using 10% neutral formalin solution, and other detection methods are the same as those in example 1.

As can be seen from FIGS. 7(a) to 7(o), the ratios of the loss of print and the copy number abnormality of 15 probes in the lymph node puncture samples of 42 cancer patients 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 grade the copy number abnormality of each probe from low to high into 3 grades.

As can be seen from fig. 7(a), for the imprinted gene Z1, the level 0 is the imprinted gene copy number abnormal expression level of less than 5%, the level I is the imprinted gene copy number abnormal expression level of 5-10%, and the level II is the imprinted gene copy number abnormal expression level of more than 10%;

as can be seen from fig. 7(b), for the imprinted gene Z2, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(c), for the imprinted gene Z3, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(d), for the imprinted gene Z4, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(e), for the imprinted gene Z5, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(f), for the imprinted gene Z6, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(g), for the imprinted gene Z8, the imprinted gene copy number abnormal expression level of less than 2% was class 0, the imprinted gene copy number abnormal expression level of 2-5% was class I, and the imprinted gene copy number abnormal expression level of more than 5% was class II;

as can be seen from fig. 7(h), for the imprinted gene Z9, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(I), for the imprinted gene Z10, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(j), for the imprinted gene Z11, the imprinted gene copy number abnormal expression level of less than 2% is level 0, the imprinted gene copy number abnormal expression level of 2-5% is level I, and the imprinted gene copy number abnormal expression level of more than 5% is level II;

as can be seen from fig. 7(k), for the imprinted gene Z12, the imprinted gene copy number abnormal expression level of less than 2% is level 0, the imprinted gene copy number abnormal expression level of 2-5% is level I, and the imprinted gene copy number abnormal expression level of more than 5% is level II;

as can be seen from fig. 7(l), for the imprinted gene Z13, the imprinted gene copy number abnormal expression level of less than 2% is level 0, the imprinted gene copy number abnormal expression level of 2-5% is level I, and the imprinted gene copy number abnormal expression level of more than 5% is level II;

as can be seen from fig. 7(m), for the imprinted gene Z14, the imprinted gene copy number abnormal expression level of less than 2% is level 0, the imprinted gene copy number abnormal expression level of 2-5% is level I, and the imprinted gene copy number abnormal expression level of more than 5% is level II;

as can be seen from fig. 7(n), for the imprinted gene Z15, the level 0 is the imprinted gene copy number abnormal expression level of less than 2%, the level I is the imprinted gene copy number abnormal expression level of 2-5%, and the level II is the imprinted gene copy number abnormal expression level of more than 5%;

as can be seen from fig. 7(o), for the imprinted gene Z16, the level 0 was obtained when the imprinted gene copy number abnormal expression level was less than 5%, the level I was obtained when the imprinted gene copy number abnormal expression level was 5-10%, and the level II was obtained when the imprinted gene copy number abnormal expression level was more than 10%.

From the comprehensive analysis of these 42 lymph node puncture samples, it can be concluded that:

judging the degree of cancer cell metastasis in the lymph nodes into non-metastasis, mild metastasis and severe metastasis;

judging the degree of cancer cell metastasis in lymph nodes, wherein the abnormal expression amount of the copy number of the imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 is less than grade I, or the abnormal expression amount of the copy number of the imprinted genes of no more than 1 imprinted genes of Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 is grade I, so that the lymph nodes are not metastatic;

judging the degree of cancer cell metastasis in the lymph node as a result of the abnormal expression level of the copy number of the imprinted gene of at least 2 imprinted genes among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 being of level I or the abnormal expression level of the copy number of the imprinted gene of not more than 1 imprinted gene among the imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15 and Z16 being of level II, which is a mild metastasis;

as a result of determining the degree of cancer cell metastasis in the lymph node, when the abnormal expression level of copy number of imprinted genes of at least 2 imprinted genes among imprinted genes Z1, Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, and Z16 is class II, the lymph node is a severe metastasis.

In conclusion, the detection model and the system of the invention represent the expression of imprinting deletion on the samples of lymphoma and cancer lymphatic metastasis patients in an intuitive method, detect the change of imprinting (imprinting) genes objectively, intuitively, early and accurately by a method for marking the imprinting genes in situ, provide a quantitative model and make great contribution to the diagnosis of lymphoma and cancer lymphatic metastasis.

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 lymphoma, characterized in that the model grades the expression state of imprinted genes by calculating the changes in the lymphoma of the total expression amount of the imprinted genes, the deletion expression amount of the imprinted genes, and the copy number abnormal expression amount of the imprinted genes;

2. The lymphoma imprinting gene staging model according to claim 1, wherein the model calculates imprinting genes by:

calculating any one of Z1, Z8 or Z16, preferably Z1;

preferably, the method for calculating the imprinted gene by the model is as follows: calculating any two of Z1, Z8, or Z16, preferably a combination of Z1 and Z8, a combination of Z8 and Z16;

preferably, the imprinted gene further comprises any one of Z4, Z5, Z6, Z11, or Z13, or a combination of at least two thereof; the imprinted gene Z4 is Igf2r, the imprinted gene Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z11 is Grb10, and the imprinted gene Z13 is Sgce;

preferably, the method for calculating the imprinted gene by the model is as follows: the combinations of the imprinted genes were calculated, the combinations of eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16 were calculated.

3. A imprinted gene grading model for lymphatic metastatic cancer, which is characterized in that the model grades the expression state of imprinted genes by calculating the change of the copy number abnormal expression quantity of the imprinted genes in the lymphatic metastatic cancer;

4. The imprinted gene grading model of lymphatic metastatic cancer according to claim 3, wherein the model calculates imprinted genes by the following method:

calculating any one of Z1, Z11 or Z16, preferably Z16;

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

preferably, the imprinted gene further comprises any one of or a combination of at least two of Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, 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 Z5 is Mest, the imprinted gene Z6 is Plagl1, the imprinted gene Z8 is Dcn, the imprinted gene Z9 is Dlk1, the imprinted gene Z10 is Gatm, 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.

5. The imprinted gene ranking model of any one of claims 1 to 4, 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%;

6. The imprinted gene classification model for lymphoma according to claim 1 or 2, wherein the imprinted gene deletion expression level, imprinted gene copy number abnormal expression level and imprinted gene total expression level 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 the eight imprinted genes of Z1, Z4, Z5, Z6, Z8, Z11, Z13, and Z16, respectively;

preferably, the five different grades of imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression for Z1, Z11, Z13 and Z16 are:

preferably, the five different grades of imprinted gene deletion expression, imprinted gene copy number abnormal expression and total expression for Z4, Z5, Z6 and Z8 are:

stage IV: any one or a combination of at least two of the imprinted gene deletion expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 25%, the imprinted gene copy number abnormal expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 4% or the total expression amount of the imprinted genes Z4, Z5, Z6 and Z8 is more than 40%.

7. The imprinted gene classification model for lymphatic metastasis cancer according to claim 3 or 4, characterized in that the imprinted gene copy number abnormal expression amount is classified into three different classes;

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

preferably, the three different grades of abnormal expression of copy number of imprinted genes for Z1 and Z16 are:

preferably, the three different levels of the abnormal expression level of copy number of imprinted genes for Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 are:

II stage: the copy number abnormal expression quantity of the imprinted genes Z2, Z3, Z4, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14 and Z15 is more than 5%.

8. An apparatus for detecting the benign and malignant degree of lymphoma and the lymphatic metastasis degree of cancer, comprising the following units:

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

wherein the analyzing unit judges the degree of malignancy of lymphoma and the degree of cancer lymphatic metastasis by calculating the deletion expression amount of imprinted gene, the copy number abnormal expression amount of imprinted gene and the total expression amount through the imprinted gene classification model according to any one of claims 1 to 7;

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

preferably, the sample to be detected is a lymph node puncture biopsy sample;

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

9. The apparatus of claim 8, wherein the benign or malignant degree of the lymphoma is classified as benign tumor, malignant potential lymphoma, early stage malignant lymphoma, intermediate stage malignant lymphoma, and late stage malignant lymphoma;

Preferably, the judging of the cancer cell metastasis degree of the lymphatic metastatic cancer is divided into non-metastasis, mild metastasis and severe metastasis;

10. A medicament or kit for the preparation of a medicament for the detection and/or treatment of lymphoma and/or lymphoid metastatic cancer according to the model of any one of claims 1-7 and/or the device of claim 8 or 9;

preferably, the judging result of the malignancy and malignancy of lymphoma is advanced malignant lymphoma if the loss expression level of imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is IV grade or the copy number abnormal expression level of imprinted genes of at least 2 imprinted genes of imprinted genes Z1, Z4, Z5, Z6, Z8, Z11, Z13 and Z16 is IV grade;

preferably, the degree of metastasis of cancer cells of the lymphatic metastatic cancer is judged to be non-metastasis, mild metastasis and severe metastasis;