A grading model for detecting tumor benignity and malignancy and application thereof

Abstract

The present application relates to the technical field of biology, to the field of genetic diagnosis, and in particular to a grading model for detecting the benign and malignant degree of tumor and application thereof. The present application provides a grading model for detecting the benign and malignant degree of tumor and application thereof, which is used for observing the change of imprinting genes of tumor at single cell and tissue levels, so as to judge the benign and malignant degree of tumor. Meanwhile, the change of expression of imprinting genes and non-imprinting genes can be combined, so that the cancer type and the benign and malignant degree can be more accurately judged.

Description

[0001] Priority information

[0002] This application claims priority and benefit to patent application 202111342507.1 filed with the China National Intellectual Property Administration on November 12, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, specifically to the field of gene diagnostics, and to a grading model for detecting the benign or malignant degree of tumors and its applications. Background Technology

[0004] Malignant tumors are one of the major threats to human health, with 14 million new cancer cases worldwide each year and 8.2 million deaths annually. Unlimited proliferation and metastasis are key characteristics of cancer cells, and most cancer patients ultimately die from complications such as organ failure caused by the widespread and rapid metastasis of cancer cells. Early-stage cancer lesions are small, surgical trauma is minimal, and the number of cancer cells entering the circulatory system is relatively small, preventing the formation of stable metastatic lesions. Therefore, timely and effective treatment in the early stages is crucial for preventing recurrence and prolonging life. Current cancer diagnostic criteria rely on the cellular and tissue morphology of tumors; however, morphological observation requires a high level of experience from pathologists, and early-stage cancers often lack typical morphological changes, making them prone to missed diagnoses. Since changes at the molecular biological level, such as epigenetics and genetics, occur earlier than morphological changes during cancer development, they can be detected more sensitively in early-stage cancers. Therefore, molecular markers are of great significance for early cancer diagnosis.

[0005] Genomic imprinting is a form of gene regulation in epigenetics, characterized by the methylation of alleles from a specific parent, resulting in the expression of only one allele of a gene while the other remains silent. These genes are called imprinted genes. De-imprinting is an epigenetic alteration where demethylation of an imprinted gene activates the silent allele, leading to gene expression. Numerous studies have shown that de-imprinting is prevalent in various cancers and occurs earlier than changes in cell and tissue morphology. Meanwhile, the proportion of de-imprinted genes is extremely low in healthy cells, in stark contrast to cancer cells. Therefore, the methylation status of imprinted genes can serve as a pathological marker, allowing for the analysis of abnormal cellular states using specific molecular detection techniques.

[0006] Because imprinted genes function in multiple ways, including cell signaling, cell cycle regulation, intracellular and extracellular transport, and extracellular matrix formation, their roles and expression levels vary significantly across different cancers. This results in varying sensitivities and specificities, playing a crucial role in understanding tumor invasion, metastasis, and prognosis. Furthermore, combinations of different imprinted genes can differentiate between tumor types.

[0007] For the reasons mentioned above, there are currently no highly sensitive diagnostic biomarkers for early cancer diagnosis. It is necessary to develop new diagnostic biomarkers, diagnostic models, and diagnostic devices, and to analyze changes in molecular markers present in tumors at the cellular level based on patient biopsy samples, so as to provide more accurate pre-diagnosis and diagnostic information and guide the selection of treatment options. Summary of the Invention

[0008] This invention aims to at least partially address one of the technical problems in related technologies. To this end, this invention provides a grading model for detecting the benign or malignant degree of tumors and its application. This detection model is used to visually observe changes in imprinted genes in tumors at the single-cell and tissue levels in the early stages, thereby determining the benign or malignant degree of the tumor. Furthermore, by combining changes in the expression of imprinted and non-imprinted genes, the benign or malignant degree of the tumor can be determined more accurately, and cancer types can be further differentiated.

[0009] Imprinted genes undergo epigenetic changes such as DNA demethylation and histone acetylation in the earliest stages of cancer. This reactivates normally silenced genes in a pair of alleles, resulting in the expression of both alleles, a phenomenon known as deletion of imprinting. During cancer development, some imprinted and non-imprinted genes may also experience gene amplification, resulting in three or more copies, a phenomenon called copy number aberration. The phenomena of deletion of imprinted genes and copy number aberrations in both imprinted and non-imprinted genes can be used for the early molecular diagnosis of cancer.

[0010] Therefore, in a first aspect, the present invention provides a gene expression status grading model for tumors. According to an embodiment of the present invention, the model includes grading the expression status of imprinted genes by calculating the changes in total expression level, monoallelic expression level, biallelic expression level, and multiallelic expression level of imprinted genes in tumors;

[0011] The imprinted genes include Z16, Z19 and Z21, wherein imprinted gene Z16 is SNRPN / SNURF, imprinted gene Z19 is HM13 and imprinted gene Z21 is NHP2L1.

[0012] According to an embodiment of the present invention, the imprinted genes further include any one or more of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28.

[0013] Among them, the imprinted gene Z1 is GNAS, the imprinted gene Z3 is PEG10, 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 Z11 is GRB10, the imprinted gene Z12 is PEG3, the imprinted gene Z13 is SGCE, the imprinted gene Z14 is SLC38A4, the imprinted gene Z18 is GATA3, the imprinted gene Z20 is KCNQ1, the imprinted gene Z22 is PON2, and the imprinted gene Z28 is ZIC1.

[0014] According to an embodiment of the present invention, the formulas for calculating the total expression level, monoalleic expression level, biallelic expression level, and multiallelic expression level of imprinted genes are as follows:

[0015] Total expression level = (b+c+d) / (a+b+c+d);

[0016] Single allele expression level = b / (b+c+d);

[0017] Biallelic expression level = c / (b+c+d);

[0018] Multiple allele expression level = d / (b+c+d);

[0019] Wherein, a represents the number of cells in which there is no marker in the nucleus and the imprinted gene is not expressed; b represents the number of cells in which there is one imprinted gene marker in the nucleus; c represents the number of cells in which there are two imprinted gene markers in the nucleus; and d represents the number of cells in which there are more than two imprinted gene markers in the nucleus.

[0020] According to an embodiment of the present invention, the five different levels for the biallelic expression level of the imprinted gene Z16 are as follows:

[0021] Level 0: The biallelic expression level of the imprinted gene Z16 is less than 15%;

[0022] Level I: The biallelic expression level of the imprinted gene Z16 is 15-20%;

[0023] Level II: The biallelic expression level of the imprinted gene Z16 is 20-26%;

[0024] Level III: The biallelic expression level of the imprinted gene Z16 is 26-30%;

[0025] Level IV: The biallelic expression level of the imprinted gene Z16 is greater than 30%.

[0026] The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z16 are as follows:

[0027] Level 0: The expression level of multiple alleles of the imprinted gene Z16 is less than 1.4%;

[0028] Level I: The expression levels of multiple alleles of the imprinted gene Z16 are 1.4-3.4%;

[0029] Level II: The expression levels of multiple alleles of the imprinted gene Z16 are 3.4-5.5%;

[0030] Level III: The expression levels of multiple alleles of the imprinted gene Z16 are 5.5-9%;

[0031] Level IV: The expression level of multiple alleles of the imprinted gene Z16 is greater than 9%.

[0032] The two different levels for classifying the total expression level of the imprinted gene Z16 are:

[0033] Level 0: The total expression level of the imprinted gene Z16 is less than 17%;

[0034] Level I: The total expression level of the imprinted gene Z16 is greater than or equal to 17%.

[0035] According to an embodiment of the present invention, the five different levels for the biallelic expression level of the imprinted gene Z19 are as follows:

[0036] Level 0: The biallelic expression level of the imprinted gene Z19 is less than 12%;

[0037] Level I: The biallelic expression level of the imprinted gene Z19 is 12-20%;

[0038] Level II: The biallelic expression level of the imprinted gene Z19 is 20-26%;

[0039] Level III: The biallelic expression level of the imprinted gene Z19 is 26-30%;

[0040] Level IV: The biallelic expression level of the imprinted gene Z19 is greater than 30%.

[0041] The five different levels for classifying the expression levels of multiple alleles in the imprinted gene Z19 are as follows:

[0042] Level 0: The expression level of multiple alleles of the imprinted gene Z19 is less than 1.4%;

[0043] Level I: The expression levels of multiple alleles of the imprinted gene Z19 are 1.4-3.4%;

[0044] Level II: The expression levels of multiple alleles of the imprinted gene Z19 are 3.4-6.3%;

[0045] Level III: The expression levels of multiple alleles of the imprinted gene Z19 are 6.3-9%;

[0046] Level IV: The expression level of multiple alleles of the imprinted gene Z19 is greater than 9%.

[0047] The two different levels for classifying the total expression level of the imprinted gene Z19 are:

[0048] Level 0: The total expression level of the imprinted gene Z19 is less than 11%;

[0049] Level I: The total expression level of the imprinted gene Z19 is greater than or equal to 11%.

[0050] According to an embodiment of the present invention, the five different levels for the biallelic expression level of the imprinted gene Z21 are as follows:

[0051] Level 0: The biallelic expression level of the imprinted gene Z21 is less than 12%;

[0052] Level I: The biallelic expression level of the imprinted gene Z21 is 12-16%;

[0053] Level II: The biallelic expression level of the imprinted gene Z21 is 16-20%;

[0054] Level III: The biallelic expression level of the imprinted gene Z21 is 20-25%;

[0055] Level IV: The biallelic expression level of the imprinted gene Z21 is greater than 25%.

[0056] The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z21 are as follows:

[0057] Level 0: The expression level of multiple alleles of the imprinted gene Z21 is less than 1.3%;

[0058] Level I: The expression levels of multiple alleles of the imprinted gene Z21 are 1.3-3.2%;

[0059] Level II: The expression levels of multiple alleles of the imprinted gene Z21 are 3.4-4.9%;

[0060] Level III: The expression levels of multiple alleles of the imprinted gene Z21 are 4.9-6.2%;

[0061] Level IV: The expression levels of multiple alleles of the imprinted gene Z21 are greater than 6.2%.

[0062] The two different levels for classifying the total expression level of the imprinted gene Z21 are:

[0063] Level 0: The total expression level of the imprinted gene Z21 is less than 14%;

[0064] Level I: The total expression level of the imprinted gene Z21 is greater than or equal to 14%.

[0065] According to an embodiment of the present invention, the positivity of imprinted genes Z16, Z19 and Z21 is classified as negative, positive potential, low positivity, moderate positivity and high positivity;

[0066] The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is 0, or the total expression level of the imprinted gene is I and both the biallelic expression level and the multiallelic expression level are 0, then the imprinted gene is negative.

[0067] The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level I and neither reaches level II, then it is considered to have imprinted gene positivity potential.

[0068] The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level II and neither reaches level III, then the imprinted gene is considered to be low-grade positive.

[0069] The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level III and neither reaches level IV, then the imprinted gene is moderately positive.

[0070] The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I and at least one of the biallelic expression level and the multi-allelic expression level is level IV, then the imprinted gene is highly positive.

[0071] Among them, the positivity of imprinted genes Z16, Z19 and Z21 was independent.

[0072] According to an embodiment of the present invention, the degree of benignity or malignancy of a tumor is classified into benign tumor, cancer potential, early-stage cancer, intermediate-stage cancer, and late-stage cancer.

[0073] The results for determining the benign or malignant nature of a tumor are as follows: if the positivity of all three imprinted genes Z16, Z19, and Z21 is negative, or if no more than one of the three imprinted genes Z16, Z19, and Z21 has a positive potential, then it is a benign tumor.

[0074] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are positive, the tumor is considered to have positive potential; or if no more than one imprinted gene is positive, the tumor is considered to have low positivity.

[0075] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 show a low degree of positivity, or if no more than one imprinted gene shows a moderate degree of positivity, then it is considered an early-stage cancer.

[0076] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are moderately positive, or if no more than one imprinted gene is highly positive, then it is considered an intermediate-stage cancer.

[0077] The result for determining the benign or malignant nature of a tumor is that at least two of the imprinted genes Z16, Z19, and Z21 are highly positive, indicating advanced cancer.

[0078] In clinical applications, tumors that are benign or have cancerous potential are considered benign samples; tumors that are early-stage, mid-stage, or late-stage cancers are considered malignant samples.

[0079] The combination of imprinted genes Z16, Z19 and Z21 can be further combined with imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22 and Z28 to improve the accuracy of tumor benign and malignant diagnosis.

[0080] According to an embodiment of the present invention, the result of determining the benign or malignant nature of a tumor is that the positive positivity of imprinted genes Z16, Z19, and Z21 is all negative or positive potential, or the positive positivity of no more than one imprinted gene among imprinted genes Z16, Z19, and Z21 is low positive and the positive positivity of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is all negative. The result of determining the benign or malignant nature of the tumor is a benign tumor.

[0081] The results for determining the benign or malignant nature of a tumor are as follows: if no more than one of the imprinted genes Z16, Z19, and Z21 is low-positive and at least one of the genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is positive; or if at least two of the imprinted genes Z16, Z19, and Z21 are low-positive; or if at least one of the imprinted genes Z16, Z19, and Z21 is moderately positive, the tumor is classified as malignant.

[0082] According to an embodiment of the present invention, the tumor is selected from thyroid cancer, lung cancer, bladder cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, intestinal cancer, stomach cancer, esophageal cancer, pancreatic cancer, and liver cancer.

[0083] According to an embodiment of the present invention, the model further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in the total expression level, monoallelic expression level, bialelic expression level and multiallelic expression level of imprinted genes in tumors, as well as the expression level of non-imprinted genes BRAF and / or MYC.

[0084] When the positivity of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 is low positive or above, and the other two imprinted genes are negative or positive potential, and the non-imprinted genes BRAF and MYC are both negative, the tumor malignancy assessment result is a benign tumor.

[0085] When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes BRAF and MYC is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is classified as malignant.

[0086] The statement that the non-imprinted genes BRAF and MYC are negative means that the expression levels of the non-imprinted genes BRAF and MYC in the tested sample are no different from those in the normal sample.

[0087] The positive result for non-imprinted genes BRAF and MYC means that the expression levels of non-imprinted genes BRAF and MYC in the tested sample are higher than those in the normal sample.

[0088] Adding non-imprinted genes BRAF and / or MYC to the imprinted gene combination can improve the detection accuracy of the model. In the embodiments of this paper, the combination of imprinted genes Z16, Z19 and Z21 has a sensitivity of 96% and a specificity of 91% for the identification of thyroid tumors.

[0089] According to an embodiment of the present invention, the model further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in the total expression level, monoallelic expression level, bialelic expression level and multiallelic expression level of imprinted genes in tumors, as well as the expression level of non-imprinted genes HER2 and / or MYC.

[0090] When the positivity of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 is low positive or above, and the other two imprinted genes are negative or positive potential, and the non-imprinted genes HER2 and MYC are both negative, the tumor malignancy assessment result is a benign tumor.

[0091] When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes HER2 and MYC is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is judged as malignant.

[0092] The statement that the non-imprinted genes HER2 and MYC are negative means that the expression levels of the non-imprinted genes HER2 and MYC in the tested sample are no different from those in the normal sample.

[0093] The positive result for non-imprinted genes HER2 and MYC means that the expression levels of non-imprinted genes HER2 and MYC in the tested sample are higher than those in the normal sample.

[0094] Adding non-imprinted genes HER2 and / or MYC to the imprinted gene combination can improve the detection accuracy of the model. In the embodiments of this paper, the combination of imprinted genes Z16, Z19 and Z21 has a sensitivity of 98% and a specificity of 95% for the detection of lung tumors.

[0095] According to an embodiment of the present invention, the model further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in the total expression level, monoallelic expression level, bialelic expression level and multiallelic expression level of imprinted genes in tumors, as well as the expression level of non-imprinted genes GRP and / or SYP.

[0096] When the combination of imprinted genes Z16, Z19 and Z21 indicates that the tumor is early-stage cancer or above and at least one of the non-imprinted genes GRP and SYP is positive, the tumor type is determined to be small cell lung cancer.

[0097] When the combination of imprinted genes Z16, Z19, and Z21 indicates early-stage cancer or higher and both non-imprinted genes GRP and SYP are negative, the tumor type is determined to be non-small cell lung cancer.

[0098] The statement that the non-imprinted genes GRP and SYP are negative means that the expression levels of the non-imprinted genes GRP and SYP in the test sample are no different from those in the normal sample.

[0099] The positive result for non-imprinted genes GRP and SYP means that the expression levels of non-imprinted genes GRP and SYP in the test sample are higher than those in the normal sample.

[0100] Adding non-imprinted genes GRP and / or SYP to the imprinted gene combination can further differentiate the tumor type when the tumor is determined to be malignant. In the embodiments of this paper, when the combination of imprinted genes Z16, Z19 and Z21 determines the tumor to be early cancer or above, the addition of non-imprinted genes GRP and SYP further improves the accuracy of differentiating between small cell lung cancer and non-small cell lung cancer to 100%.

[0101] According to an embodiment of the present invention, the model further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in the total expression level, monoallelic expression level, bialic expression level and multiallelic expression level of imprinted genes in tumors, as well as the expression level of non-imprinted genes CDKN2A and / or MYC.

[0102] When the positivity of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 is low positive or above, and the other two imprinted genes are negative or positive potential, and the non-imprinted genes CDKN2A and MYC are both negative, the tumor malignancy assessment result is a benign tumor.

[0103] When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes CDKN2A and MYC is positive, or at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is judged as malignant.

[0104] The statement that the non-imprinted genes CDKN2A and MYC are negative means that the expression levels of the non-imprinted genes CDKN2A and MYC in the tested sample are no different from those in the normal sample.

[0105] The positive result for non-imprinted genes CDKN2A and MYC means that the expression levels of non-imprinted genes CDKN2A and MYC in the tested sample are higher than those in the normal sample.

[0106] Adding non-imprinted genes CDKN2A and / or MYC to the imprinted gene combination can improve the detection accuracy of the model. In the embodiments of this paper, the combination of imprinted genes Z16, Z19 and Z21 has a sensitivity of 96% and a specificity of 97% for the detection of bladder tumors.

[0107] According to an embodiment of the present invention, the model further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in the total expression level, monoallelic expression level, bialelic expression level and multiallelic expression level of imprinted genes in tumors, as well as the expression level of non-imprinted genes HER2 and / or BRAF.

[0108] When the positivity of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 is low positive or above, and the other two imprinted genes are negative or positive potential, and the non-imprinted genes HER2 and BRAF are negative, the tumor malignancy assessment result is a benign tumor.

[0109] When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes HER2 and BRAF is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is classified as malignant.

[0110] The statement that the non-imprinted genes HER2 and BRAF are negative means that the expression levels of the non-imprinted genes HER2 and BRAF in the tested sample are no different from those in the normal sample.

[0111] The positive result for non-imprinted genes HER2 and BRAF means that the expression levels of non-imprinted genes HER2 and BRAF in the tested sample are higher than those in the normal sample.

[0112] Adding non-imprinted genes HER2 and / or BRAF to the imprinted gene combination can improve the detection accuracy of the model. In the embodiments of this paper, the combination of imprinted genes Z16, Z19 and Z21 has a sensitivity of 94% and a specificity of 96% for the detection of breast tumors.

[0113] A second aspect of the present invention provides the use of the grading model as described in the first aspect in tumor detection and / or treatment.

[0114] A third aspect of the present invention provides a tumor detection method, the method comprising using the grading model described in the first aspect to determine the tumor type and / or benign or malignant nature of the sample to be tested.

[0115] According to an embodiment of the present invention, the sample to be tested is derived from a patient who has or is suspected of having the following types of cancer: thyroid cancer, lung cancer, bladder cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, esophageal cancer, pancreatic cancer, and liver cancer.

[0116] The fourth aspect of the present invention provides a tumor monitoring method, the method comprising using the grading model described in the first aspect to determine the benign or malignant degree of a patient's tumor before and after medication, so as to monitor the development of the patient's tumor.

[0117] In this invention, "only one of the imprinted genes Z16, Z19, and Z21 has a low degree of positivity or higher" means that any one of the three imprinted genes Z16, Z19, and Z21 has a low degree of positivity, moderate degree of positivity, or high degree of positivity, while the other two imprinted genes have a negative degree of positivity or positive potential. For example, imprinted gene Z16 has a low degree of positivity, moderate degree of positivity, or high degree of positivity, while imprinted genes Z19 and Z21 have a negative degree of positivity or positive potential.

[0118] In this invention, "positive potential or less" refers to negative and positive potential.

[0119] In this invention, the imprinted gene Z21 is SNU13, also known as "NHP2L1", and the two are equivalent.

[0120] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0121] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0122] Figure 1 The images show cell nuclei with four different expression states of imprinted genes in lung tissue sections, where a represents cell nuclei without any markers and no imprinted gene expression; b represents cell nuclei with one imprinted gene marker; c represents cell nuclei with two imprinted gene markers; and d represents cell nuclei with more than two imprinted gene markers.

[0123] Figure 2 shows the expression status of BAE, MAE, and TE of imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z16, Z18, Z19, Z20, Z21, Z22, and Z28 in benign and malignant lung tumors. Figure 2(a) shows the expression status of BAE, MAE, and TE of imprinted gene Z1 in benign and malignant lung tumors; Figure 2(b) shows the expression status of BAE, MAE, and TE of imprinted gene Z3 in benign and malignant lung tumors; Figure 2(c) shows the expression status of BAE, MAE, and TE of imprinted gene Z5 in benign and malignant lung tumors; and Figure 2(d) shows the expression status of BAE of imprinted gene Z6 in benign and malignant lung tumors. Figure 2(e) shows the expression status of BAE, MAE, and TE of imprinted gene Z8 in benign and malignant lung tumors; Figure 2(f) shows the expression status of BAE, MAE, and TE of imprinted gene Z9 in benign and malignant lung tumors; Figure 2(g) shows the expression status of BAE, MAE, and TE of imprinted gene Z10 in benign and malignant lung tumors; Figure 2(h) shows the expression status of BAE, MAE, and TE of imprinted gene Z11 in benign and malignant lung tumors; Figure 2(i) shows the expression status of BAE, MAE, and TE of imprinted gene Z12 in benign and malignant lung tumors; Figure 2(j) shows the expression status of BAE, MAE, and TE of imprinted gene Z13 in benign and malignant lung tumors. The expression status of TE is shown in Figure 2(k), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z14 in benign and malignant lung tumors; Figure 2(l), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z16 in benign and malignant lung tumors; Figure 2(m), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z18 in benign and malignant lung tumors; Figure 2(n), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z19 in benign and malignant lung tumors; Figure 2(o), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z20 in benign and malignant lung tumors; and Figure 2(p), which illustrates the expression status of BAE, MAE, and TE of imprinted gene Z21 in benign and malignant lung tumors. The expression status is shown in Figure 2(q), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z22 in benign and malignant lung tumors; Figure 2(r), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z28 in benign and malignant lung tumors; Figure 2(s), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z2 in benign and malignant lung tumors; Figure 2(t), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z4 in benign and malignant lung tumors; Figure 2(u), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z15 in benign and malignant lung tumors; and Figure 2(v), which illustrates the expression status of BAE, MAE, and TE of imprinted genes Z17 in benign and malignant lung tumors.Figure 2(w) shows the expression status of BAE, MAE, and TE of imprinted gene Z23 in benign and malignant lung tumors; Figure 2(x) shows the expression status of BAE, MAE, and TE of imprinted gene Z24 in benign and malignant lung tumors; Figure 2(y) shows the expression status of BAE, MAE, and TE of imprinted gene Z25 in benign and malignant lung tumors; Figure 2(z) shows the expression status of BAE, MAE, and TE of imprinted gene Z26 in benign and malignant lung tumors; and Figure 2(aa) shows the expression status of BAE, MAE, and TE of imprinted gene Z27 in benign and malignant lung tumors.

[0124] Figure 3 shows the ROC curves of imprinted genes in twelve types of tumors. Figure 3(a) shows the ROC curve of imprinted gene Z1, Figure 3(b) shows the ROC curve of imprinted gene Z11, Figure 3(c) shows the ROC curve of imprinted gene Z16, Figure 3(d) shows the ROC curve of imprinted gene Z19, Figure 3(e) shows the ROC curve of imprinted gene Z21, Figure 3(f) shows the ROC curve of imprinted gene Z3, Figure 3(g) shows the ROC curve of imprinted gene Z5, Figure 3(h) shows the ROC curve of imprinted gene Z6, Figure 3(i) shows the ROC curve of imprinted gene Z8, Figure 3(j) shows the ROC curve of imprinted gene Z9, Figure 3(k) shows the ROC curve of imprinted gene Z10, Figure 3(l) shows the ROC curve of imprinted gene Z12, and Figure 3(m) shows the ROC curve of imprinted gene Z13. Figure 3(n) shows the ROC curve of imprinted gene Z14, Figure 3(o) shows the ROC curve of imprinted gene Z18, Figure 3(p) shows the ROC curve of imprinted gene Z20, Figure 3(q) shows the ROC curve of imprinted gene Z22, Figure 3(r) shows the ROC curve of imprinted gene Z28, Figure 3(s) shows the ROC curve of imprinted gene Z2, Figure 3(t) shows the ROC curve of imprinted gene Z4, Figure 3(u) shows the ROC curve of imprinted gene Z15, Figure 3(v) shows the ROC curve of imprinted gene Z17, Figure 3(w) shows the ROC curve of imprinted gene Z23, Figure 3(x) shows the ROC curve of imprinted gene Z24, Figure 3(y) shows the ROC curve of imprinted gene Z25, Figure 3(z) shows the ROC curve of imprinted gene Z26, and Figure 3(aa) shows the ROC curve of imprinted gene Z27.

[0125] Figure 4 is a schematic diagram of the imprinted gene grading method. Figure 4(a) shows the grading method of BAE, MAE and TE for each imprinted gene, Figure 4(b) shows the grading method of the positivity of imprinted genes, and Figure 4(c) shows the method of predicting the benign or malignant nature of tumors through multiple gene combinations.

[0126] Figure 5 shows the expression status of imprinted genes Z1, Z8, Z11, Z13, Z16, Z19, and Z21 in sentinel lymph node samples of breast cancer with and without lymph node metastasis, representing BAE, MAE, and TE. Figure 5(a) shows the expression status of imprinted gene Z1 in sentinel lymph node samples with and without lymph node metastasis, Figure 5(b) shows the expression status of imprinted gene Z8 in sentinel lymph node samples with and without lymph node metastasis, and Figure 5(c) shows the expression status of imprinted gene Z11 in sentinel lymph node samples with and without lymph node metastasis, representing BAE, MAE, and TE. Figure 5(d) shows the expression status of BAE, MAE, and TE of imprinted gene Z13 in sentinel lymph node samples of breast cancer with and without lymph node metastasis; Figure 5(e) shows the expression status of BAE, MAE, and TE of imprinted gene Z16 in sentinel lymph node samples of breast cancer with and without lymph node metastasis; Figure 5(f) shows the expression status of BAE, MAE, and TE of imprinted gene Z19 in sentinel lymph node samples of breast cancer with and without lymph node metastasis; Figure 5(g) shows the expression status of BAE, MAE, and TE of imprinted gene Z21 in sentinel lymph node samples of breast cancer with and without lymph node metastasis.

[0127] Figure 6 The study showed the multi-allelic expression levels of the BRAF gene, the multi-allelic expression level of the MYC gene, and the proportion of BRAF gene mutation-positive cells in benign and malignant thyroid tumors.

[0128] Figure 7 The expression status of CDKN2A, HER2, and MYC genes in benign and malignant lung tumors was shown.

[0129] Figure 8 The proportion of GRP and SYP gene-positive cells in non-small cell lung cancer and small cell lung cancer is shown.

[0130] Figure 9 The expression levels of CDKN2A and MYC genes in benign and malignant bladder tumors were shown.

[0131] Figure 10 This study shows the multi-allelic expression status of HER2 and BRAF genes in benign and malignant breast tumors. Detailed Implementation

[0132] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0133] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0134] According to one embodiment of the present invention, the expression status of imprinted genes is detected by in situ hybridization. Probes targeting the intron sequences of imprinted genes are designed and hybridized with nascent RNA expressing the imprinted gene to reveal the number of alleles expressing the imprinted gene within the cell nucleus. Since pre-RNA or nascent RNA containing exons and introns is first formed during gene transcription, and then the introns are cleaved and rapidly degraded, probes targeting introns can only detect pre-RNA or nascent RNA, and will not detect mRNA containing only exons. Because the intron cleavage of pre-RNA or nascent RNA occurs simultaneously with transcription, the signal markers of pre-RNA or nascent RNA actually indicate the spatial sites of imprinted genes being transcribed within the cell nucleus, while the spatial sites of imprinted genes that have not undergone transcription are not detected because they do not produce pre-RNA or nascent RNA. Using RNA in situ hybridization, when an imprinted gene is not expressed or only one allele is expressed under normal conditions, no marker or only one marker can be detected in the cell nucleus. In cancer, when two or more alleles of an imprinted gene are abnormally expressed, two or more markers can be detected in the cell nucleus. Therefore, based on the proportion of cells expressing imprinted genes and the proportion of cells with abnormal imprinted gene expression in a tumor sample, the benign or malignant nature of the tumor can be graded. Similarly, when non-imprinted genes in cancer exhibit copy number abnormalities, the specific RNA in situ hybridization method of this invention can detect three or more markers. The proportion of cells with abnormal non-imprinted gene expression in a tumor sample can also assist in determining the benign or malignant nature of the tumor using imprinted genes. Methods for detecting the expression status of imprinted and non-imprinted genes include, but are not limited to, the methods described above; other methods available in the art are also included within the scope of this invention.

[0135] According to one embodiment of the present invention, the in situ hybridization is performed using the RNAscope in situ hybridization method;

[0136] According to one embodiment of the present invention, the RNAscope in situ hybridization method uses a single-channel or multi-channel chromogenic kit or a single-channel or multi-channel fluorescence kit, preferably a single-channel red / brown chromogenic kit or a multi-channel fluorescence kit.

[0137] According to one embodiment of the present invention, based on the number of alleles expressed by the imprinted gene in the cell nucleus, the expression status of the imprinted gene is classified as imprinted gene non-expression, imprinted gene monoallelic expression, imprinted gene bialic expression, or imprinted gene polyallelic expression.

[0138] The absence of imprinted gene expression means that there is no imprinted gene marker in the cell nucleus of the sample.

[0139] The imprinted gene monoalleic expression means that there is one imprinted gene marker in the cell nucleus of the sample;

[0140] The biallelic expression of the imprinted gene means that there are two imprinted gene markers in the cell nucleus of the sample.

[0141] The imprinted gene multi-allelic expression means that there are more than two imprinted gene markers in the cell nucleus of the sample.

[0142] According to one embodiment of the present invention, the total expression level (TE), single-allelic expression level (SAE), bi-allelic expression level (BAE), and multi-allelic expression level (MAE) are calculated by the following formula:

[0143] TE = (b+c+d) / (a+b+c+d);

[0144] SAE = b / (b+c+d);

[0145] BAE = c / (b+c+d);

[0146] MAE = d / (b+c+d);

[0147] Wherein, a) refers to cell nuclei in which no marker is present in the nucleus after hematoxylin staining, and the imprinted gene is not expressed; b) refers to cell nuclei in which one red / brown marker is present in the nucleus after hematoxylin staining, and only one allele of the imprinted gene is expressed; c) refers to cell nuclei in which two red / brown markers are present in the nucleus after hematoxylin staining, and two alleles of the imprinted gene are expressed; and d refers to cell nuclei in which more than two red / brown markers are present in the nucleus after hematoxylin staining, and more than two alleles of the imprinted gene are expressed.

[0148] According to one embodiment of the present invention, the combination of imprinted genes Z16, Z19, and Z21 is used to detect whether a tumor has undergone lymph node metastasis.

[0149] The Z3 gene was added to the combination of imprinted genes Z16, Z19, and Z21 to detect early small cell carcinoma, including small cell lung cancer, small cell bladder cancer, and small cell cervical cancer.

[0150] According to one embodiment of the present invention, based on the combination of any one or more of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, the non-imprinted genes BRAF and / or MYC can also be added for the diagnosis of thyroid cancer.

[0151] BRAF and MYC were detected using in situ hybridization. Probes targeting the introns of the BRAF and MYC genes were designed and hybridized with the nascent RNA of gene expression to show the number of alleles of gene expression in the cell nucleus.

[0152] For the BRAF gene, probes targeting the V600E mutation site can also be designed to hybridize with mRNA and show the mRNA expression level of the mutated gene.

[0153] According to one embodiment of the present invention, based on the combination of any one or more of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, the non-imprinted genes HER2 and / or MYC can also be added for the diagnosis of lung cancer.

[0154] HER2 and MYC were detected using in situ hybridization. Probes targeting the introns of the HER2 and MYC genes were designed and hybridized with the nascent RNA of gene expression to show the number of alleles of gene expression in the cell nucleus.

[0155] According to one embodiment of the present invention, based on the combination of any one or more of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, the non-imprinted genes GRP and / or SYP can also be added for the diagnosis of small cell lung cancer.

[0156] GRP and SYP were detected using in situ hybridization. Probes targeting GRP and SYP gene mRNA were designed and hybridized with the mRNA to show the mRNA expression level of the genes.

[0157] According to one embodiment of the present invention, based on the combination of any one or more of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, the non-imprinted genes CDKN2A and MYC can be added for the diagnosis of bladder cancer. CDKN2A and MYC are detected by in situ hybridization. Probes targeting the introns of the CDKN2A and MYC genes are designed and hybridized with the nascent RNA of gene expression to show the number of alleles of gene expression in the cell nucleus.

[0158] According to one embodiment of the present invention, based on the combination of any one or more of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, the non-imprinted genes HER2 and / or BRAF can also be added for the diagnosis of breast cancer;

[0159] HER2 and BRAF were detected using in situ hybridization. Probes targeting the introns of the HER2 and BRAF genes were designed and hybridized with the nascent RNA of gene expression to show the number of alleles of gene expression in the cell nucleus.

[0160] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0161] Example 1: Analysis of imprinted gene expression status in tissue sections of benign and malignant lung tumors

[0162] (1) Ten cases of benign lung tumor tissue and 20 cases of malignant lung tumor tissue were obtained and fixed in 10% neutral formalin solution to prevent RNA degradation for 24 hours. The tissues were embedded in paraffin and cut into 10 μm thick sections, which were then adhered to glass slides. The glass slides were positively charged to prevent detachment and the sections were baked in an oven at 40°C for more than 3 hours.

[0163] (2) Dewaxing was performed according to the RNAscope sample processing method to block the endogenous peroxidase activity in the sample, enhance permeability and expose RNA molecules.

[0164] (3) Probe design: Specific probes were designed based on the intron sequences of the imprinted genes Z1 (GNAS), Z3 (PEG10), Z5 (MEST), Z6 (PLAGL1), Z8 (DCN), Z9 (DLK1), Z10 (GATM), Z11 (GRB10), Z12 (PEG3), Z13 (SGCE), Z14 (SLC38A4), Z16 (SNRPN / SNURF), Z18 (GATA3), Z19 (HM13), Z20 (KCNQ1), Z21 (SNU13), Z22 (PON2), Z28 (ZIC1), Z2 (IGF2), Z4 (IGF2R), Z15 (DIRAS3), Z17 (ALDH1L1), Z23 (RASGRF1), Z24 (RB1), Z25 (TFPI2), Z26 (TRAPPC9), and Z27 (UBE3A).

[0165] (4) Perform RNAscope in situ hybridization with the probe from step (3) and the sample using a kit;

[0166] (5) Signal amplification and hematoxylin staining were performed, and the expression of imprinted genes was analyzed by microscopic imaging.

[0167] In this embodiment, the expression status of imprinted genes includes no expression of imprinted genes, monoallelic expression of imprinted genes, biallelic expression of imprinted genes, or polyallelic expression of imprinted genes. Specifically, no expression of imprinted genes means that there are no imprinted gene markers in the cell nuclei of the sample; monoallelic expression of imprinted genes means that there is one imprinted gene marker in the cell nuclei of the sample; biallelic expression of imprinted genes means that there are two imprinted gene markers in the cell nuclei of the sample; and polyallelic expression of imprinted genes means that there are more than two imprinted gene markers in the cell nuclei of the sample.

[0168] Observe the results of in situ hybridization under a microscope, such as Figure 1 As shown, 1200-2000 cells were counted, and the total expression (TE), single-allelic expression (SAE), bi-allelic expression (BAE), and multi-allelic expression (MAE) were calculated using the following formulas:

[0169] TE = (b+c+d) / (a+b+c+d);

[0170] SAE = b / (b+c+d);

[0171] BAE = c / (b+c+d);

[0172] MAE = d / (b+c+d);

[0173] Where a represents the number of cells without an imprinted gene marker in the nucleus, b represents the number of cells with one imprinted gene marker in the nucleus, c represents the number of cells with two imprinted gene markers in the nucleus, and d represents the number of cells with more than two imprinted gene markers in the nucleus.

[0174] The quantitative results are shown in Figure 2 (Figure 2(a)-(aa)). The TE, BAE, and MAE of imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z16, Z18, Z19, Z20, Z21, Z22, and Z28 showed significant differences in benign and malignant lung tumors, while the TE, BAE, and MAE of imprinted genes Z2, Z4, Z15, Z17, Z23, Z24, Z25, Z26, and Z27 showed relatively small differences in benign and malignant lung tumors.

[0175] Example 2: Analysis of the expression status of 27 imprinted genes in twelve types of tumor tissue sections and establishment of a benign / malignant grading model.

[0176] Ten benign tumor tissues and 20 malignant tumor tissues were obtained from the thyroid, bladder, prostate, skin, breast, cervix, intestine, stomach, esophagus, pancreas, and liver. The tissues were fixed, embedded, sectioned, and subjected to RNAscope in situ hybridization in the same manner as in Example 1.

[0177] The BAE, MAE, and TE of the imprinted genes were quantified according to the method in Example 1. The quantitative data of eleven types of tumors in this example and the data of lung tumor tissue samples in Example 1 were combined. ROC curves were plotted based on the BAE, MAE, and TE data of 120 benign tumor samples and 240 malignant tumor samples for each gene to analyze the ability of each gene to distinguish between benign and malignant tumors. The results are shown in Figure 3 (Figure 3(a)-(aa)). The area under the curve (AUC) of BAE, MAE, and TE for each gene is shown in Table 1.

[0178] Table 1

[0179]

[0180]

[0181] The AUCs of BAE, MAE, and TE for the five genes Z1, Z11, Z16, Z19, and Z21 are all greater than 0.7, indicating that Z1, Z11, Z16, Z19, and Z21 have a good ability to distinguish between benign and malignant tumors. The AUCs of BAE, MAE, and TE for the thirteen genes Z3, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 are between 0.6 and 0.7, indicating that these genes have some ability to distinguish between benign and malignant tumors. The AUCs of BAE, MAE, and TE for the nine genes Z2, Z4, Z15, Z17, Z23, Z24, Z25, Z26, and Z27 are all less than 0.6, indicating that these genes can hardly distinguish between benign and malignant tumors.

[0182] First, five genes—Z1, Z11, Z16, Z19, and Z21—with good ability to distinguish between benign and malignant tumors were selected to construct a tumor benign / malignant differentiation model. Four points on the ROC curve were selected as thresholds from threshold_1 to threshold_4, and the BAE and MAE of each gene were divided into five levels. The specific grading method is as follows:

[0183] Level 0: BAE or MAE is less than threshold_1;

[0184] Level I: BAE or MAE is greater than or equal to threshold_1 and less than threshold_2;

[0185] Level II: BAE or MAE is greater than or equal to threshold_2 and less than threshold_3;

[0186] Level III: BAE or MAE is greater than or equal to threshold_3 and less than threshold_4;

[0187] Level IV: BAE or MAE is greater than or equal to threshold_4.

[0188] The TE of each gene is divided into two levels: 0 and I. The specific grading method is as follows:

[0189] Level 0: TE is less than threshold_1;

[0190] Level I: TE is greater than or equal to threshold_1.

[0191] The specific methods for selecting the thresholds for the five genes Z1, Z11, Z16, Z19, and Z21 are as follows:

[0192] For the BAE of the five genes Z1, Z11, Z16, Z19 and Z21, the thresholds corresponding to the sensitivity of 90%, 60%, 30% and 10% were selected on the ROC curve and were used as threshold_1, threshold_2, threshold_3 and threshold_4, respectively.

[0193] For the MAE of the five genes Z1, Z11, Z16, Z19 and Z21, the thresholds corresponding to the sensitivity of 95%, 80%, 70% and 50% were selected on the ROC curve and used as threshold_1, threshold_2, threshold_3 and threshold_4, respectively.

[0194] For the TE of the five genes Z1, Z11, Z16, Z19 and Z21, the threshold corresponding to a sensitivity of 99% was selected as threshold_1 on the ROC curve.

[0195] The thresholds for the five genes Z1, Z11, Z16, Z19 and Z21 are shown in Table 2.

[0196] Table 2

[0197]

[0198] Based on the BAE, MAE, and TE grades of each gene, the positivity rate of each gene is divided into five levels: negative, positive potential, low positivity, moderate positivity, and high positivity. The specific grading method is as follows:

[0199] The result for determining the degree of positivity of imprinted genes is that the TE of the imprinted gene is level 0, or the TE of the imprinted gene is level I and both BAE and MAE are level 0, then the imprinted gene is negative.

[0200] The result of judging the degree of imprint gene positivity is that the imprint gene TE is grade I and at least one of BAE and MAE is grade I and neither reaches grade II, then it is an imprint gene positivity potential.

[0201] The result for judging the degree of positivity of imprinted genes is that the TE of the imprinted gene is grade I and at least one of BAE and MAE is grade II and neither reaches grade III, then the imprinted gene is considered to be low-grade positive.

[0202] The result for determining the degree of positivity of imprinted genes is that the TE of the imprinted gene is grade I and at least one of BAE and MAE is grade III and neither reaches grade IV, then the imprinted gene is moderately positive.

[0203] The result for determining the degree of positivity of imprinted genes is that the TE of the imprinted gene is grade I and at least one of BAE and MAE is grade IV, then the imprinted gene is highly positivity.

[0204] Two or more genes can be randomly selected from the five genes Z1, Z11, Z16, Z19, and Z21 for combination. Based on the degree of positivity of different genes, tumors are classified into five grades: benign tumors, tumors with cancer potential, early-stage cancer, intermediate-stage cancer, and late-stage cancer. The specific combination method is as follows:

[0205] The result for determining the benign or malignant nature of a tumor is that all imprinted genes in the combination are negative, or no more than one imprinted gene in the combination has a positive potential; then it is a benign tumor.

[0206] The result for determining the benign or malignant nature of a tumor is that the combination of at least two imprinted genes has a positive positivity level, or the combination of no more than one imprinted gene has a low positivity level, in which case it is considered to have cancer potential.

[0207] The result for determining the benign or malignant nature of a tumor is that at least two imprinted genes in the combination are low-grade positive, or no more than one imprinted gene is moderately positive, then it is early-stage cancer.

[0208] The results for determining the benign or malignant nature of a tumor are as follows: if at least two imprinted genes in the combination are moderately positive, or if no more than one imprinted gene is highly positive, then it is considered an intermediate-stage cancer.

[0209] The result for determining the benign or malignant nature of a tumor is that at least two imprinted genes in the combination are highly positive, which indicates advanced cancer.

[0210] The grading methods for BAE, MAE, and TE of genes, the grading methods for gene positivity, and the gene combination methods are shown in Figure 4. Figures 4(a)-4(c) As shown in the figure.

[0211] Two or more genes were randomly selected from the five genes Z1, Z11, Z16, Z19 and Z21 for combination. Based on the above model, 120 benign tumor samples and 240 malignant tumor samples were divided into five grades, of which benign tumors and cancer potential were predicted as benign, and early cancer, intermediate cancer and late cancer were predicted as malignant. The sensitivity and specificity of single genes and different gene combinations were evaluated, and the results are shown in Table 3.

[0212] Table 3

[0213] Gene or combination of genes Sensitivity Specificity Z1 80.4％ 70.8％ Z11 81.7％ 60.8％ Z16 82.1％ 80.0％ Z19 88.3％ 90.0％ Z21 82.9％ 74.2％ Z1+Z11 92.1％ 63.3％ Z1+Z16 93.8％ 75.8％ Z1+Z19 92.5％ 83.3％ Z1+Z21 92.5％ 76.7％ Z11+Z16 92.9％ 65.0％ Z11+Z19 90.8％ 68.3％ Z11+Z21 90.4％ 67.5％ Z16+Z19 94.6％ 90.0％ Z16+Z21 94.6％ 90.0％ Z19+Z21 88.3％ 92.5％ Z1+Z11+Z16 98.3％ 59.2％ Z1+Z11+Z19 98.8％ 61.7％ Z1+Z11+Z21 97.5％ 59.2％ Z1+Z16+Z19 98.8％ 75.8％ Z1+Z16+Z21 98.8％ 73.3％ Z1+Z19+Z21 96.3％ 75.8％ Z11+Z16+Z19 98.8％ 64.2％ Z11+Z16+Z21 98.3％ 64.2％ Z11+Z19+Z21 95.0％ 66.7％ Z16+Z19+Z21 98.3％ 90.0％ Z1+Z11+Z16+Z19 100.0％ 58.3％ Z1+Z11+Z16+Z21 99.2％ 56.7％ Z1+Z11+Z19+Z21 99.2％ 58.3％ Z1+Z16+Z19+Z21 99.6％ 73.3％ Z11+Z16+Z19+Z21 99.6％ 63.3％ Z1+Z11+Z16+Z19+Z21 100.0％ 55.8％

[0214] From Table 3, we can see that:

[0215] When only one of the five genes Z1, Z11, Z16, Z19 and Z21 is used, the sensitivity is no higher than 90%.

[0216] When using a combination of two genes, the sensitivity increases. The combination of Z16 and Z19 and the combination of Z16 and Z21 have the highest sensitivity, reaching 94.6%, while the combination of Z19 and Z21 has the highest specificity, reaching 92.5%.

[0217] When using combinations of three genes, the sensitivity continued to increase. Among them, the combinations of Z1+Z11+Z19, Z1+Z16+Z19, Z1+Z16+Z21, and Z11+Z16+Z19 had the highest sensitivity, reaching 98.8%, but the specificity was low, only 61.7%-75.8%. The combination of Z16+Z19+Z21 had slightly lower sensitivity, reaching 98.3%, but the highest specificity, reaching 90.0%.

[0218] When using combinations of four or five genes, the specificity decreases significantly, both falling below 75%.

[0219] Therefore, considering both sensitivity and specificity, the combination of the three genes Z16, Z19 and Z21 can achieve the best effect in distinguishing between benign and malignant tumors.

[0220] In addition to the five genes Z1, Z11, Z16, Z19, and Z21, ROC curves show that thirteen genes—Z3, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z18, Z20, Z22, and Z28—also possess some ability to differentiate between benign and malignant tumors. Adding one or more of these thirteen genes to the existing five (Z1, Z11, Z16, Z19, and Z21) can further improve sensitivity and prevent missed diagnoses. Furthermore, adjusting the grading model for Z1 and Z11 to appropriately increase specificity, as an adjunct to the combination of Z16, Z19, and Z21 genes, can also enhance sensitivity.

[0221] On the ROC curves of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28, a point is selected as the threshold_1. The BAE, MAE, and TE of these fifteen genes are each divided into two levels. The specific grading method is as follows:

[0222] Level 0: BAE, MAE, or TE is less than threshold_1;

[0223] Level I: BAE, MAE, or TE are greater than or equal to threshold_1.

[0224] Based on the BAE, MAE, and TE grades of each gene in Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28, the positivity of each gene is divided into two levels: negative and positive. The specific grading method is as follows:

[0225] The result of judging the degree of positivity of imprinted genes is that at least one of the TE, BAE or MAE of the imprinted gene is level 0, then the imprinted gene is negative.

[0226] The result for determining the degree of positivity of imprinted genes is that the TE, BAE, and MAE of the imprinted genes are all at level I, then the imprinted genes are positive.

[0227] Since the combination of the three genes Z16, Z19 and Z21 already achieves a high level of sensitivity, in order to avoid excessively increasing sensitivity and causing a decrease in specificity, the threshold corresponding to 95% specificity is selected from the ROC curves of BAE and MAE for each gene as threshold_1, and the threshold corresponding to 90% sensitivity is selected from the ROC curve of TE for each gene as threshold_1.

[0228] The thresholds for BAE, MAE, and TE for each gene of Z3, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 are shown in Table 4.

[0229] Table 4

[0230] Imprinted genes BAE MAE TE Z1 25％ 11.6％ 17％ Z3 13％ 6.0％ 5％ Z5 19％ 4.7％ 5％ Z6 19％ 5.6％ 7％ Z8 28％ 19.8％ 6％ Z9 13％ 7.2％ 5％ Z10 23％ 16.8％ 6％ Z11 25％ 11.8％ 12％ Z12 15％ 3.1％ 6％ Z13 16％ 7.4％ 7％ Z14 22％ 6.1％ 6％ Z18 26％ 18.6％ 8％ Z20 29％ 21.7％ 10％ Z22 21％ 14.0％ 6％ Z28 17％ 14.1％ 5％

[0231] Based on the combination of the three genes Z16, Z19, and Z21, and combined with the positivity rates of Z3, Z5, Z6, Z8, Z9, Z10, Z12, Z13, Z14, Z18, Z20, Z22, and Z28, the benign and malignant tumors are further divided into two grades. The specific grading method is as follows:

[0232] The results for determining the benign or malignant nature of a tumor are as follows: if the positivity of imprinted genes Z16, Z19, and Z21 is all negative or positive potential, or if no more than one of the imprinted genes Z16, Z19, and Z21 is low positive and the positivity of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is all negative, the tumor is considered benign.

[0233] The results for determining the benign or malignant nature of a tumor are as follows: if no more than one of the imprinted genes Z16, Z19, and Z21 is low-positive and at least one of the genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is positive; or if at least two of the imprinted genes Z16, Z19, and Z21 are low-positive; or if at least one of the imprinted genes Z16, Z19, and Z21 is moderately positive, the tumor is classified as malignant.

[0234] The combination of the three genes Z16, Z19, and Z21 with any one of the genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 was further combined to obtain the sensitivity and specificity for tumor diagnosis, as shown in Table 5.

[0235] Table 5

[0236] Imprinted gene combinations Sensitivity Specificity Z16+Z19+Z21 98.3％ 90.0％ Z16+Z19+Z21+Z1 100.0％ 86.7％ Z16+Z19+Z21+Z3 99.2％ 89.2％ Z16+Z19+Z21+Z5 99.2％ 88.3％ Z16+Z19+Z21+Z6 98.8％ 90.0％ Z16+Z19+Z21+Z8 98.8％ 90.0％ Z16+Z19+Z21+Z9 100.0％ 86.7％ Z16+Z19+Z21+Z10 99.2％ 88.3％ Z16+Z19+Z21+Z11 100.0％ 86.7％ Z16+Z19+Z21+Z12 98.8％ 89.2％ Z16+Z19+Z21+Z13 99.6％ 90.0％ Z16+Z19+Z21+Z14 99.2％ 86.7％ Z16+Z19+Z21+Z18 98.8％ 87.5％ Z16+Z19+Z21+Z20 98.8％ 88.3％ Z16+Z19+Z21+Z22 98.8％ 87.5％ Z16+Z19+Z21+Z28 99.2％ 87.5％

[0237] Table 5 shows that each of the imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22 and Z28 can increase the sensitivity of tumor diagnosis, but have little effect on specificity.

[0238] The ROC curves show that the imprinted genes Z2, Z4, Z15, Z17, Z23, Z24, Z25, Z26, and Z27 have almost no ability to distinguish between benign and malignant tumors, so these genes are not used in the model.

[0239] Example 3: Validation of the model for differentiating benign and malignant thyroid tumors using imprinted genes in fine-needle aspiration biopsy samples.

[0240] One hundred fine-needle aspiration samples each of benign and malignant thyroid tumors were obtained and fixed in 10% neutral formalin solution. After 24 hours, the samples were adhered to positively charged glass slides. The remaining steps were the same as in Example 1.

[0241] Based on the biallelic expression level, multiallelic expression level, and total expression level of imprinted genes in the samples, the benign and malignant tumors were graded using the model constructed in Example 2.

[0242] The five different levels of biallelic expression levels of the imprinted gene Z16 are as follows:

[0243] Level 0: The biallelic expression level of the imprinted gene Z16 is less than 15%;

[0244] Level I: The biallelic expression level of the imprinted gene Z16 is 15-20%;

[0245] Level II: The biallelic expression level of the imprinted gene Z16 is 20-26%;

[0246] Level III: The biallelic expression level of the imprinted gene Z16 is 26-30%;

[0247] Level IV: The biallelic expression level of the imprinted gene Z16 is greater than 30%.

[0248] The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z16 are as follows:

[0249] Level 0: The expression level of multiple alleles of the imprinted gene Z16 is less than 1.4%;

[0250] Level I: The expression levels of multiple alleles of the imprinted gene Z16 are 1.4-3.4%;

[0251] Level II: The expression levels of multiple alleles of the imprinted gene Z16 are 3.4-5.5%;

[0252] Level III: The expression levels of multiple alleles of the imprinted gene Z16 are 5.5-9%;

[0253] Level IV: The expression level of multiple alleles of the imprinted gene Z16 is greater than 9%.

[0254] The two different levels for classifying the total expression level of the imprinted gene Z16 are:

[0255] Level 0: The total expression level of the imprinted gene Z16 is less than 17%;

[0256] Level I: The total expression level of the imprinted gene Z16 is greater than or equal to 17%.

[0257] According to an embodiment of the present invention, the five different levels for the biallelic expression level of the imprinted gene Z19 are as follows:

[0258] Level 0: The biallelic expression level of the imprinted gene Z19 is less than 12%;

[0259] Level I: The biallelic expression level of the imprinted gene Z19 is 12-20%;

[0260] Level II: The biallelic expression level of the imprinted gene Z19 is 20-26%;

[0261] Level III: The biallelic expression level of the imprinted gene Z19 is 26-30%;

[0262] Level IV: The biallelic expression level of the imprinted gene Z19 is greater than 30%.

[0263] The five different levels for classifying the expression levels of multiple alleles in the imprinted gene Z19 are as follows:

[0264] Level 0: The expression level of multiple alleles of the imprinted gene Z19 is less than 1.4%;

[0265] Level I: The expression levels of multiple alleles of the imprinted gene Z19 are 1.4-3.4%;

[0266] Level II: The expression levels of multiple alleles of the imprinted gene Z19 are 3.4-6.3%;

[0267] Level III: The expression levels of multiple alleles of the imprinted gene Z19 are 6.3-9%;

[0268] Level IV: The expression level of multiple alleles of the imprinted gene Z19 is greater than 9%.

[0269] The two different levels for classifying the total expression level of the imprinted gene Z19 are:

[0270] Level 0: The total expression level of the imprinted gene Z19 is less than 11%;

[0271] Level I: The total expression level of the imprinted gene Z19 is greater than or equal to 11%.

[0272] According to an embodiment of the present invention, the five different levels for the biallelic expression level of the imprinted gene Z21 are as follows:

[0273] Level 0: The biallelic expression level of the imprinted gene Z21 is less than 12%;

[0274] Level I: The biallelic expression level of the imprinted gene Z21 is 12-16%;

[0275] Level II: The biallelic expression level of the imprinted gene Z21 is 16-20%;

[0276] Level III: The biallelic expression level of the imprinted gene Z21 is 20-25%;

[0277] Level IV: The biallelic expression level of the imprinted gene Z21 is greater than 25%.

[0278] The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z21 are as follows:

[0279] Level 0: The expression level of multiple alleles of the imprinted gene Z21 is less than 1.3%;

[0280] Level I: The expression levels of multiple alleles of the imprinted gene Z21 are 1.3-3.2%;

[0281] Level II: The expression levels of multiple alleles of the imprinted gene Z21 are 3.4-4.9%;

[0282] Level III: The expression levels of multiple alleles of the imprinted gene Z21 are 4.9-6.2%;

[0283] Level IV: The expression levels of multiple alleles of the imprinted gene Z21 are greater than 6.2%.

[0284] The two different levels for classifying the total expression level of the imprinted gene Z21 are:

[0285] Level 0: The total expression level of the imprinted gene Z21 is less than 14%;

[0286] Level I: The total expression level of the imprinted gene Z21 is greater than or equal to 14%.

[0287] The positivity of imprinted genes Z16, Z19, and Z21 is classified as negative, positive potential, low positivity, moderate positivity, and high positivity.

[0288] The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is 0, or the total expression level of the imprinted gene is I and both the biallelic expression level and the multiallelic expression level are 0, then the imprinted gene is negative.

[0289] The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level I and neither reaches level II, then it is considered to have imprinted gene positivity potential.

[0290] The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level II and neither reaches level III, then the imprinted gene is considered to be low-grade positive.

[0291] The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level III and neither reaches level IV, then the imprinted gene is moderately positive.

[0292] The result for determining the degree of positivity of imprinted genes is that the total expression level of imprinted genes is grade I and at least one of the biallelic expression level and the multi-allelic expression level is grade IV, which indicates that the imprinted gene is highly positive.

[0293] The degree of malignancy of tumors is classified into benign tumors, cancer potential, early-stage cancer, intermediate-stage cancer, and late-stage cancer.

[0294] The results for determining the benign or malignant nature of a tumor are as follows: if the positivity of all three imprinted genes Z16, Z19, and Z21 is negative, or if no more than one of the three imprinted genes Z16, Z19, and Z21 has a positive potential, then it is a benign tumor.

[0295] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are positive, the tumor is considered to have positive potential; or if no more than one imprinted gene is positive, the tumor is considered to have low positivity.

[0296] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 show a low degree of positivity, or if no more than one imprinted gene shows a moderate degree of positivity, then it is considered an early-stage cancer.

[0297] The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are moderately positive, or if no more than one imprinted gene is highly positive, then it is considered an intermediate-stage cancer.

[0298] The result for determining the benign or malignant nature of a tumor is that at least two of the imprinted genes Z16, Z19, and Z21 are highly positive, indicating advanced cancer.

[0299] Samples with tumor benignity or cancer potential were classified as benign, while samples with early-stage, mid-stage, or late-stage cancer were classified as malignant. The benignity or malignancy of the samples was compared with the benignity or malignancy determined by postoperative pathological diagnosis to evaluate the sensitivity and specificity of the model.

[0300] Table 6 shows the combinations of imprinted genes Z16, Z19, and Z21, and imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, and Z11.

[0301] The sensitivity and specificity of models combining Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, respectively, for detecting benign and malignant thyroid tumors.

[0302] Table 6

[0303] Imprinted gene combinations Sensitivity Specificity Z16+Z19+Z21 96％ 91％ Z16+Z19+Z21+Z1 100％ 82％ Z16+Z19+Z21+Z3 98％ 87％ Z16+Z19+Z21+Z5 97％ 89％ Z16+Z19+Z21+Z6 98％ 89％ Z16+Z19+Z21+Z8 97％ 91％ Z16+Z19+Z21+Z9 97％ 90％ Z16+Z19+Z21+Z10 98％ 89％ Z16+Z19+Z21+Z11 100％ 88％ Z16+Z19+Z21+Z12 97％ 91％ Z16+Z19+Z21+Z13 99％ 90％ Z16+Z19+Z21+Z14 97％ 91％ Z16+Z19+Z21+Z18 99％ 89％ Z16+Z19+Z21+Z20 98％ 87％ Z16+Z19+Z21+Z22 97％ 89％ Z16+Z19+Z21+Z28 100％ 85％

[0304] As shown in Table 6, when only the three imprinted genes Z16, Z19, and Z21 were detected, the model had a sensitivity of 96% and a specificity of 91% for thyroid tumors.

[0305] When the imprinted gene Z1 was added to the three genes, the model's sensitivity was 100% and its specificity was 82%.

[0306] When the imprinted gene Z3 was added to the three genes, the model's sensitivity was 98% and its specificity was 87%.

[0307] When the imprinted gene Z5 was added to the three genes, the model's sensitivity was 97% and its specificity was 89%.

[0308] When the imprinted gene Z6 was added to the three genes, the model's sensitivity was 98% and its specificity was 89%.

[0309] When the imprinted gene Z8 was added to the three genes, the model's sensitivity was 97% and its specificity was 91%.

[0310] When the imprinted gene Z9 was added to the three genes, the model's sensitivity was 97% and its specificity was 90%.

[0311] When the imprinted gene Z10 was added to the three genes, the model's sensitivity was 98% and its specificity was 89%.

[0312] When the imprinted gene Z11 was added to the three genes, the model's sensitivity was 100% and its specificity was 88%.

[0313] When the imprinted gene Z12 was added to the three genes, the model's sensitivity was 97% and its specificity was 91%.

[0314] When the imprinted gene Z13 was added to the three genes, the model's sensitivity was 99% and its specificity was 90%.

[0315] When the imprinted gene Z14 was added to the three genes, the model's sensitivity was 97% and its specificity was 91%.

[0316] When the imprinted gene Z18 was added to the three genes, the model's sensitivity was 99% and its specificity was 91%.

[0317] When the imprinted gene Z20 was added to the three genes, the model's sensitivity was 98% and its specificity was 87%.

[0318] When the imprinted gene Z22 was added to the three genes, the model's sensitivity was 97% and its specificity was 89%.

[0319] When the imprinted gene Z28 was added to the three genes, the model's sensitivity was 100% and its specificity was 85%.

[0320] Example 4: Validation of the benign and malignant imprinted gene differential model in lung biopsy samples

[0321] One hundred fine-needle aspiration samples each of benign and malignant lung tumors were obtained. The processing method was the same as in Example 3. The benignity and malignancy of the tumors were graded using the model constructed in Example 2. Samples with benign tumors and cancer potential were recorded as benign, while samples with early-stage cancer, intermediate-stage cancer, and late-stage cancer were recorded as malignant. The results were compared with the benignity and malignancy of the pathological diagnosis to evaluate the sensitivity and specificity of the model.

[0322] Table 7 shows the sensitivity and specificity of models for detecting benign and malignant lung tumors in the combination of imprinted genes Z16, Z19, and Z21, and in the combination of imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, respectively.

[0323] Table 7

[0324] Imprinted gene combinations Sensitivity Specificity Z16+Z19+Z21 98％ 95％ Z16+Z19+Z21+Z1 100％ 91％ Z16+Z19+Z21+Z3 100％ 94％ Z16+Z19+Z21+Z5 100％ 93％ Z16+Z19+Z21+Z6 99％ 94％ Z16+Z19+Z21+Z8 99％ 95％ Z16+Z19+Z21+Z9 99％ 94％ Z16+Z19+Z21+Z10 99％ 93％ Z16+Z19+Z21+Z11 100％ 95％ Z16+Z19+Z21+Z12 99％ 94％ Z16+Z19+Z21+Z13 100％ 93％ Z16+Z19+Z21+Z14 99％ 95％ Z16+Z19+Z21+Z18 100％ 92％ Z16+Z19+Z21+Z20 100％ 93％ Z16+Z19+Z21+Z22 100％ 92％ Z16+Z19+Z21+Z28 100％ 90％

[0325] As shown in Table 7, when only the three imprinted genes Z16, Z19, and Z21 are detected, the model has a sensitivity of 98% and a specificity of 95% for lung tumors.

[0326] When the imprinted gene Z1 was added to the three genes, the model's sensitivity was 100% and its specificity was 91%.

[0327] When the imprinted gene Z3 was added to the three genes, the model's sensitivity was 100% and its specificity was 94%.

[0328] When the imprinted gene Z5 was added to the three genes, the model's sensitivity was 100% and its specificity was 93%.

[0329] When the imprinted gene Z6 was added to the three genes, the model's sensitivity was 99% and its specificity was 94%.

[0330] When the imprinted gene Z8 was added to the three genes, the model's sensitivity was 99% and its specificity was 95%.

[0331] When the imprinted gene Z9 was added to the three genes, the model's sensitivity was 99% and its specificity was 94%.

[0332] When the imprinted gene Z10 was added to the three genes, the model's sensitivity was 99% and its specificity was 93%.

[0333] When the imprinted gene Z11 was added to the three genes, the model's sensitivity was 100% and its specificity was 95%.

[0334] When the imprinted gene Z12 was added to the three genes, the model's sensitivity was 99% and its specificity was 94%.

[0335] When the imprinted gene Z13 was added to the three genes, the model's sensitivity was 100% and its specificity was 93%.

[0336] When the imprinted gene Z14 was added to the three genes, the model's sensitivity was 99% and its specificity was 95%.

[0337] When the imprinted gene Z18 was added to the three genes, the model's sensitivity was 100% and its specificity was 92%.

[0338] When the imprinted gene Z20 was added to the three genes, the model's sensitivity was 100% and its specificity was 93%.

[0339] When the imprinted gene Z22 was added to the three genes, the model's sensitivity was 100% and its specificity was 92%.

[0340] When the imprinted gene Z28 was added to the three genes, the model's sensitivity was 100% and its specificity was 90%.

[0341] Example 5: Validation of the benign / malignant imprinted gene model in bladder tumors, prostate tumors, skin tumors, breast tumors, cervical tumors, intestinal tumors, gastric tumors, esophageal tumors, pancreatic tumors, and liver tumors.

[0342] One hundred benign and one hundred malignant biopsy samples were obtained from cystoscopy of bladder tumors, puncture biopsy of prostate tumors, skin tumors, breast tumors, colposcopy of cervical tumors, colonoscopy of intestinal tumors, gastroscopy of gastric tumors, gastroscopy of esophageal tumors, pancreatic tumors, and liver tumors. The processing method was the same as in Example 3. The expression status of imprinted genes Z16, Z19, Z21, Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 was detected, and the TE, BAE, and MAE of each gene were quantified.

[0343] The model constructed in Example 2 was used to classify the benign and malignant nature of tumors. Samples with benign tumors or tumors with cancer potential were classified as benign, while samples with early-stage, mid-stage, and late-stage cancer were classified as malignant. The results were compared with the benign and malignant diagnoses from pathological examinations to evaluate the sensitivity and specificity of the model.

[0344] Tables 8 and 9 show the sensitivity and specificity of the models for detecting bladder tumors, prostate tumors, skin tumors, breast tumors, cervical tumors, intestinal tumors, gastric tumors, esophageal tumors, pancreatic tumors, and liver tumors, using combinations of imprinted genes Z16, Z19, and Z21, and combinations of imprinted genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 with Z16, Z19, and Z21, respectively.

[0345] Table 8

[0346]

[0347] Table 9

[0348]

[0349] As shown in Tables 8 and 9, when detecting the three imprinted genes Z16, Z19, and Z21, the model showed the following detection rates: 96% sensitivity and 97% for bladder cancer; 94% sensitivity and 97% specificity for prostate cancer; 93% sensitivity and 92% specificity for skin cancer; 94% sensitivity and 96% specificity for breast cancer; 97% sensitivity and 93% specificity for cervical cancer; 94% sensitivity and 94% specificity for colorectal cancer; 96% sensitivity and 92% specificity for gastric cancer; 96% sensitivity and 94% specificity for esophageal cancer; 98% sensitivity and 92% specificity for pancreatic cancer; and 97% sensitivity and 90% specificity for liver cancer.

[0350] Adding the imprinted gene Z1 to the existing three genes improved the detection sensitivity to 98% and specificity to 92% for bladder cancer, 98% and specificity to 93% for prostate cancer, 100% and specificity to 89% for skin cancer, 98% and specificity to breast cancer, 99% and specificity to 93% for cervical cancer, 99% and specificity to colorectal cancer, 91% and specificity to 99% for gastric cancer, 98% and specificity to 89% for esophageal cancer, 100% and specificity to 90% for pancreatic cancer, and 100% and specificity to 86% for liver cancer.

[0351] Adding the imprinted gene Z3 to the existing three genes improved the sensitivity for bladder cancer to 100% and the specificity to 96%; the sensitivity for prostate cancer to 96% and the specificity to 94%; the sensitivity for skin cancer to 95% and the specificity to 90%; the sensitivity for breast cancer to 97% and the specificity to 94%; the sensitivity for cervical cancer to 99% and the specificity to 91%; the sensitivity for colorectal cancer to 98% and the specificity to 92%; the sensitivity for gastric cancer to 98% and the specificity to 90%; the sensitivity for esophageal cancer to 97% and the specificity to 92%; the sensitivity for pancreatic cancer to 100% and the specificity to 91%; and the sensitivity for liver cancer to 100% and the specificity to 88%.

[0352] Adding the imprinted gene Z8 to the existing three genes improved the detection sensitivity to 97% and specificity to 95% for bladder cancer, 96% and 94% for prostate cancer, 100% and 89% for skin cancer, 95% and 94% for breast cancer, 98% and 90% for cervical cancer, 99% and 92% for colorectal cancer, 97% and 90% for gastric cancer, 97% and 93% for esophageal cancer, 100% and 91% for pancreatic cancer, and 99% and 87% for liver cancer.

[0353] Adding the imprinted gene Z10 to the existing three genes improved the detection sensitivity to 98% and specificity to 94% for bladder cancer, 99% and specificity to 94% for prostate cancer, 94% and specificity to 89% for skin cancer, 99% and specificity to breast cancer, 99% and specificity to 94% for cervical cancer, 100% and specificity to colorectal cancer, 100% and specificity to 92% for gastric cancer, 90% and specificity to 97% for esophageal cancer, 100% and specificity to 91% for pancreatic cancer, and 100% and specificity to 87% for liver cancer.

[0354] Adding the imprinted gene Z11 to the existing three genes improved the detection sensitivity to 98% and specificity to 96% for bladder cancer, 97% and specificity to 95% for prostate cancer, 97% and specificity to 90% for skin cancer, 96% and specificity to 93% for breast cancer, 98% and specificity to 91% for cervical cancer, 98% and specificity to 93% for colorectal cancer, 98% and specificity to 91% for gastric cancer, 98% and specificity to 91% for esophageal cancer, 100% and specificity to 92% for pancreatic cancer, and 100% and specificity to 89% for liver cancer.

[0355] Adding the imprinted gene Z13 to the existing three genes improved the detection sensitivity to 99% and specificity to 94% for bladder cancer, 96% and 95% for prostate cancer, 100% and 90% for skin cancer, 98% and 93% for breast cancer, 99% and 91% for cervical cancer, 99% and 92% for colorectal cancer, 99% and 91% for gastric cancer, 100% and 92% for esophageal cancer, 99% and 89% for pancreatic cancer, and 100% and 87% for liver cancer.

[0356] Adding the imprinted gene Z28 to the existing three genes improved the detection sensitivity to 98% and specificity to 94% for bladder cancer, 96% and specificity to 93% for prostate cancer, 95% and specificity to 89% for skin cancer, 97% and specificity to breast cancer, 100% and specificity to 90% for cervical cancer, 97% and specificity to colorectal cancer, 98% and specificity to gastric cancer, 98% and specificity to 89% for esophageal cancer, 99% and specificity to pancreatic cancer, and 99% and specificity to liver cancer.

[0357] Example 6: Analysis of Tumor Lymph Node Metastasis

[0358] Fine-needle aspiration samples were obtained from sentinel lymph nodes of metastatic and non-metastatic breast cancer. The processing method was the same as in Example 2. The expression status of imprinted genes Z1, Z8, Z11, Z13, Z16, Z19 and Z21 was detected, and the TE, BAE and MAE of each gene were quantified.

[0359] The quantitative results are shown in Figure 5 (Figure 5(a)-(g)). The TE, BAE and MAE of the imprinted genes Z1, Z8, Z11, Z13, Z16, Z19 and Z21 were significantly different in lymph nodes with and without tumor metastasis.

[0360] Example 7: Analysis of BRAF and MYC gene expression status in benign and malignant thyroid tumors

[0361] Fine-needle aspiration samples from benign and malignant thyroid tumors were obtained and processed using the same method as in Example 2. The expression of non-imprinted genes BRAF and MYC was detected. The quantification method for BRAF and MYC genes detected using intron probes was the same as that for imprinted genes. BRAF gene mutations detected using mRNA probes were quantified as follows: cells with more than 3 signal points were considered positive cells, and 1200-2000 cells were counted to calculate the proportion of positive cells.

[0362] Quantitative results such as Figure 6 As shown, the multiple allele expression levels (MAE) of BRAF and MYC genes and the proportion of BRAF mutation-positive cells differ significantly between benign and malignant thyroid tumors.

[0363] Example 8: Analysis of CDKN2A, HER2, and MYC gene expression status in benign and malignant lung tumors

[0364] Fine-needle aspiration samples from benign and malignant lung tumors were obtained and processed using the same method as in Example 2. The expression of non-imprinted genes EGFR, HER2, and MYC was detected. The quantification methods for EGFR, HER2, and MYC genes were the same as those for imprinted genes.

[0365] Quantitative results such as Figure 7 As shown, the multiple allele expression (MAE) of EGFR, HER2, and MYC genes differs significantly between benign and malignant lung tumors.

[0366] Example 9: Analysis of GRP and SYP gene expression status in small cell lung cancer and non-small cell lung cancer

[0367] Fine-needle aspiration samples were obtained from small cell lung cancer and non-small cell lung cancer, and the processing method was the same as in Example 2. The expression of non-imprinted genes GRP and SYP was detected. GRP and SYP were quantified as follows: cells with more than two signal points were considered positive cells, and 1200-2000 cells were counted to calculate the proportion of positive cells.

[0368] Quantitative results such as Figure 8 As shown, the proportions of GRP and SYP positive cells differ significantly between small cell lung cancer and non-small cell lung cancer. Therefore, adding GRP and SYP genes to the detection of imprinted gene expression status can enable early diagnosis and subtyping of lung cancer, contributing to precision treatment.

[0369] Example 10: Analysis of CDKN2A and MYC gene expression status in bladder cancer

[0370] Biopsy samples of benign and malignant bladder tumors were obtained and processed in the same manner as in Example 2. The expression of non-imprinted genes CDKN2A and MYC was detected. The quantification methods for CDKN2A and MYC genes were the same as those for imprinted genes.

[0371] Quantitative results such as Figure 9 As shown, the multiple allele expression (MAE) of CDKN2A and MYC genes differs significantly between benign and malignant bladder tumors.

[0372] Example 11 Analysis of HER2 and BRAF gene expression status in breast cancer

[0373] Biopsy samples from benign and malignant breast tumors were obtained and processed using the same method as in Example 2. The expression of non-imprinted genes HER2 and BRAF was detected. The quantification methods for HER2 and BRAF genes were the same as for imprinted genes.

[0374] Quantitative results such as Figure 10 As shown, the multiple allele expression (MAE) of HER2 and BRAF genes differs significantly between benign and malignant breast tumors.

[0375] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0376] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An imprinted gene combination for grading gene expression status in tumors, characterized in that, The imprinted gene combination is a combination of Z16, Z19, and Z21, wherein the imprinted gene Z16 is SNRPN / SNURF, the imprinted gene Z19 is HM13, and the imprinted gene Z21 is SNU13. The imprinted genes also include any one of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28. Specifically, the imprinted gene Z1 is GNAS, the imprinted gene Z3 is PEG10, 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 Z11 is GRB10, the imprinted gene Z12 is PEG3, the imprinted gene Z13 is SGCE, the imprinted gene Z14 is SLC38A4, the imprinted gene Z18 is GATA3, the imprinted gene Z20 is KCNQ1, the imprinted gene Z22 is PON2, and the imprinted gene Z28 is ZIC1. The gene expression status grading includes classifying the expression status of imprinted genes by calculating the changes in total expression level, monoallelic expression level, biallelic expression level, and multiallelic expression level of imprinted genes in tumors. The formulas for calculating the total expression level, monoalleic expression level, biallelic expression level, and polyallelic expression level of imprinted genes are as follows: Total expression level = (b+c+d) / (a+b+c+d); Monoallelic expression level = b / (b + c + d); Biallelic expression level = c / (b+c+d); Multiple allele expression level = d / (b + c + d); Where a represents the number of cells in which there is no marker in the nucleus and the imprinted gene is not expressed; b represents the number of cells in which there is one imprinted gene marker in the nucleus; c represents the number of cells in which there are two imprinted gene markers in the nucleus; and d represents the number of cells in which there are more than two imprinted gene markers in the nucleus.

2. The imprinted gene combination according to claim 1, characterized in that, The five different levels of biallelic expression levels of the imprinted gene Z16 are as follows: Level 0: The biallelic expression level of the imprinted gene Z16 is less than 15%; Level I: The biallelic expression level of the imprinted gene Z16 is 15-20%; Level II: The biallelic expression level of the imprinted gene Z16 is 20-26%; Level III: The biallelic expression level of the imprinted gene Z16 is 26-30%; Level IV: The biallelic expression level of the imprinted gene Z16 is greater than 30%; The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z16 are as follows: Level 0: The expression level of multiple alleles of the imprinted gene Z16 is less than 1.4%; Level I: The expression levels of multiple alleles of the imprinted gene Z16 are 1.4-3.4%; Level II: The expression levels of multiple alleles of the imprinted gene Z16 are 3.4-5.5%; Level III: The expression levels of multiple alleles of the imprinted gene Z16 are 5.5-9%; Level IV: The expression level of multiple alleles of the imprinted gene Z16 is greater than 9%; The two different levels for classifying the total expression level of the imprinted gene Z16 are: Level 0: The total expression level of the imprinted gene Z16 is less than 17%; Level I: The total expression level of the imprinted gene Z16 is greater than or equal to 17%; The five different levels of biallelic expression levels of the imprinted gene Z19 are as follows: Level 0: The biallelic expression level of the imprinted gene Z19 is less than 12%; Level I: The biallelic expression level of the imprinted gene Z19 is 12-20%; Level II: The biallelic expression level of the imprinted gene Z19 is 20-26%; Level III: The biallelic expression level of the imprinted gene Z19 is 26-30%; Level IV: The biallelic expression level of the imprinted gene Z19 is greater than 30%; The five different levels for classifying the expression levels of multiple alleles in the imprinted gene Z19 are as follows: Level 0: The expression level of multiple alleles of the imprinted gene Z19 is less than 1.4%; Level I: The expression levels of multiple alleles of the imprinted gene Z19 are 1.4-3.4%; Level II: The multi-allelic expression level of the imprinted gene Z19 is 3.4-6.3%; Level III: The multi-allele expression level of the imprinted gene Z19 is 6.3-9%; Level IV: The expression levels of multiple alleles of the imprinted gene Z19 are greater than 9%; The two different levels for classifying the total expression level of the imprinted gene Z19 are: Level 0: The total expression level of the imprinted gene Z19 is less than 11%; Level I: The total expression level of the imprinted gene Z19 is greater than or equal to 11%; The five different levels of biallelic expression levels of the imprinted gene Z21 are as follows: Level 0: The biallelic expression level of the imprinted gene Z21 is less than 12%; Level I: The biallelic expression level of the imprinted gene Z21 is 12-16%; Level II: The biallelic expression level of the imprinted gene Z21 is 16-20%; Level III: The biallelic expression level of the imprinted gene Z21 is 20-25%; Level IV: The biallelic expression level of the imprinted gene Z21 is greater than 25%; The five different levels for classifying the expression levels of multiple alleles of the imprinted gene Z21 are as follows: Level 0: The expression level of multiple alleles of the imprinted gene Z21 is less than 1.3%; Level I: The multi-allelic expression level of the imprinted gene Z21 is 1.3-3.2%; Level II: The multi-allelic expression level of the imprinted gene Z21 is 3.4-4.9%; Level III: The multi-allelic expression level of the imprinted gene Z21 is 4.9-6.2%; Level IV: The expression levels of multiple alleles of the imprinted gene Z21 are greater than 6.2%; The two different levels for classifying the total expression level of the imprinted gene Z21 are: Level 0: The total expression level of the imprinted gene Z21 is less than 14%; Level I: The total expression level of the imprinted gene Z21 is greater than or equal to 14%.

3. The imprinted gene combination according to claim 2, characterized in that, The positivity of imprinted genes Z16, Z19, and Z21 is classified as negative, positive potential, low positivity, moderate positivity, and high positivity. The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is 0, or the total expression level of the imprinted gene is I and both the biallelic expression level and the multiallelic expression level are 0, then the imprinted gene is negative. The result of judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level I and neither reaches level II, then it is considered to have imprinted gene positivity potential. The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level II and neither reaches level III, then the imprinted gene is considered to be low-grade positive. The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I, and at least one of the biallelic expression level and the multi-allelic expression level is level III and neither reaches level IV, then the imprinted gene is moderately positive. The result for judging the degree of imprinted gene positivity is that the total expression level of the imprinted gene is level I and at least one of the biallelic expression level and the multi-allelic expression level is level IV, then the imprinted gene is highly positive. Among them, the positivity of imprinted genes Z16, Z19 and Z21 was independent.

4. The imprinted gene combination according to claim 3, characterized in that, The degree of malignancy of a tumor is classified into benign tumors, cancer potential, early-stage cancer, intermediate-stage cancer, and late-stage cancer. The results for determining the benign or malignant nature of a tumor are as follows: if the positivity of all three imprinted genes Z16, Z19, and Z21 is negative, or if no more than one of the three imprinted genes Z16, Z19, and Z21 has a positive potential, then it is a benign tumor. The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are positive, the tumor is considered to have positive potential; or if no more than one imprinted gene is positive, the tumor is considered to have low positivity. The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 show a low degree of positivity, or if no more than one imprinted gene shows a moderate degree of positivity, then it is considered an early-stage cancer. The results for determining the benign or malignant nature of a tumor are as follows: if at least two of the imprinted genes Z16, Z19, and Z21 are moderately positive, or if no more than one imprinted gene is highly positive, then it is considered an intermediate-stage cancer. The result for determining the benign or malignant nature of a tumor is that at least two of the imprinted genes Z16, Z19, and Z21 are highly positive, indicating advanced cancer.

5. The imprinted gene combination according to claim 3, characterized in that, The results for determining the benign or malignant nature of a tumor are as follows: if the positivity of imprinted genes Z16, Z19, and Z21 is all negative or positive potential, or if no more than one of the imprinted genes Z16, Z19, and Z21 is low positive and the positivity of Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is all negative, the tumor is considered benign. The results for determining the benign or malignant nature of a tumor are as follows: if no more than one of the imprinted genes Z16, Z19, and Z21 is low-positive and any one of the genes Z1, Z3, Z5, Z6, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z18, Z20, Z22, and Z28 is positive; or if at least two of the imprinted genes Z16, Z19, and Z21 are low-positive; or if at least one of the imprinted genes Z16, Z19, and Z21 is moderately positive, the tumor is considered malignant.

6. The imprinted gene combination according to claim 1, characterized in that, The tumors are selected from thyroid cancer, lung cancer, bladder cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, intestinal cancer, stomach cancer, esophageal cancer, pancreatic cancer, and liver cancer.

7. The imprinted gene combination according to claim 3, characterized in that, The gene expression status grading further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in total expression levels, monoallelic expression levels, bialic expression levels, and multiallelic expression levels of imprinted genes in tumors, as well as the expression levels of non-imprinted genes BRAF and / or MYC. Among them, when the positive degree of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 has a low positive degree or above, and the positive degree of the other two imprinted genes is negative or positive potential, and the non-imprinted genes BRAF and MYC are both negative, the tumor benign or malignant tumor judgment result is benign tumor. When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes BRAF and MYC is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is classified as malignant. The statement that the non-imprinted genes BRAF and MYC are negative means that the expression levels of the non-imprinted genes BRAF and MYC in the tested sample are no different from those in the normal sample. The positive result for non-imprinted genes BRAF and MYC means that the expression levels of non-imprinted genes BRAF and MYC in the tested sample are higher than those in the normal sample.

8. The imprinted gene combination according to claim 3, characterized in that, The gene expression status grading further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in total expression levels, monoallelic expression levels, bialic expression levels, and multiallelic expression levels of imprinted genes in tumors, as well as the expression levels of non-imprinted genes HER2 and / or MYC. Among them, when the positive degree of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 has a low positive degree or above, and the positive degree of the other two imprinted genes is negative or positive potential, and the non-imprinted genes HER2 and MYC are both negative, the tumor benign or malignant tumor judgment result is benign tumor. When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes HER2 and MYC is positive, or at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is judged as malignant. The statement that the non-imprinted genes HER2 and MYC are negative means that the expression levels of the non-imprinted genes HER2 and MYC in the tested sample are no different from those in the normal sample. The positive result for non-imprinted genes HER2 and MYC means that the expression levels of non-imprinted genes HER2 and MYC in the tested sample are higher than those in the normal sample.

9. The imprinted gene combination according to claim 3, characterized in that, The gene expression status grading further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in total expression levels, monoallelic expression levels, bialic expression levels, and polyallelic expression levels of imprinted genes in tumors, as well as the expression levels of non-imprinted genes GRP and / or SYP. Among them, when the combination of imprinted genes Z16, Z19 and Z21 determines the tumor malignancy as early-stage cancer or above and at least one of the non-imprinted genes GRP and SYP is positive, the tumor type is determined to be small cell carcinoma. When the combination of imprinted genes Z16, Z19 and Z21 indicates that the tumor is early-stage cancer or above, and both non-imprinted genes GRP and SYP are negative, the tumor type is determined to be non-small cell carcinoma. The statement that the non-imprinted genes GRP and SYP are negative means that the expression levels of the non-imprinted genes GRP and SYP in the test sample are no different from those in the normal sample. The positive result for non-imprinted genes GRP and SYP means that the expression levels of non-imprinted genes GRP and SYP in the test sample are higher than those in the normal sample.

10. The imprinted gene combination according to claim 3, characterized in that, The gene expression status grading further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in total expression levels, monoallelic expression levels, bialic expression levels, and multiallelic expression levels of imprinted genes in tumors, as well as the expression levels of non-imprinted genes CDKN2A and / or MYC. Among them, when the positive degree of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 has a low positive degree or above, and the positive degree of the other two imprinted genes is negative or positive potential, and the non-imprinted genes CDKN2A and MYC are both negative, the tumor benign or malignant tumor judgment result is benign tumor; When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes CDKN2A and MYC is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is classified as malignant. The statement that the non-imprinted genes CDKN2A and MYC are negative means that the expression levels of the non-imprinted genes CDKN2A and MYC in the tested sample are no different from those in the normal sample. The positive result for non-imprinted genes CDKN2A and MYC means that the expression levels of non-imprinted genes CDKN2A and MYC in the tested sample are higher than those in the normal sample.

11. The imprinted gene combination according to claim 3, characterized in that, The gene expression status grading further includes classifying the expression status of imprinted and non-imprinted genes by calculating the changes in total expression levels, monoallelic expression levels, bialic expression levels, and polyallelic expression levels of imprinted genes in tumors, as well as the expression levels of non-imprinted genes HER2 and / or BRAF. Among them, when the positive degree of imprinted genes Z16, Z19 and Z21 is positive potential or below, or when only one of the imprinted genes Z16, Z19 and Z21 has a low positive degree or above, and the positive degree of the other two imprinted genes is negative or positive potential, and the non-imprinted genes HER2 and BRAF are both negative, the tumor benign or malignant tumor is determined to be benign. When only one of the imprinted genes Z16, Z19, and Z21 is low-positive or higher, the other two imprinted genes are negative or have positive potential, and at least one of the non-imprinted genes HER2 and BRAF is positive, or when at least two of the imprinted genes Z16, Z19, and Z21 are low-positive or higher, the tumor is classified as malignant. The statement that the non-imprinted genes HER2 and BRAF are negative means that the expression levels of the non-imprinted genes HER2 and BRAF in the tested sample are no different from those in the normal sample. The positive result for non-imprinted genes HER2 and BRAF means that the expression levels of non-imprinted genes HER2 and BRAF in the tested sample are higher than those in the normal sample.

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