CN108441559B - Application of immune-related gene group as marker in preparation of product for evaluating distant metastasis risk of high-proliferative breast cancer - Google Patents
Application of immune-related gene group as marker in preparation of product for evaluating distant metastasis risk of high-proliferative breast cancer Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a group of gene groups capable of evaluating the distant metastasis risk of high-proliferative breast cancer; discloses the application of the reagent for detecting the gene expression level of the gene groups in the preparation of in vitro diagnostic products for evaluating the risk of distant metastasis of the high proliferative breast cancer; the in vitro diagnostic product comprises a quantitative PCR detection kit; the invention also discloses a method for evaluating the distant metastasis risk of the high proliferative breast cancer by using the detection kit. The method disclosed by the invention has higher accuracy in evaluating the risk of distant metastasis of the high proliferative breast cancer, and can predict the response of the breast cancer patients to future immune checkpoint inhibitors.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a quantitative PCR and analysis technology for evaluating breast cancer immune genomes. The immune index is calculated by detecting the expression of immune related genes in a group of breast cancer tissues, so that the method helps to evaluate the distant metastasis risk of the high-proliferation breast cancer, and can be used as the curative effect prediction of new auxiliary, auxiliary and targeted treatment of the breast cancer and the concomitant diagnosis of immunotherapy, including but not limited to PD1 inhibitor, PDL1 inhibitor and CTLA4 inhibitor.
Background
The breast cancer accounts for the first malignant tumor of women in China, the breast cancer increases year by year at a speed of about 4% per year, and according to statistics of Chinese tumor registration annual reports, the number of invasive breast cancer diseases in 2015 is 27.24 thousands, and 6.95 thousands of cases of death.
At present, the precise treatment of breast cancer after operation at home and abroad still faces a great challenge. For the patients who are treated after the operation, how to select a treatment scheme with pertinence to improve the treatment efficiency needs a new technical means to help doctors and patients to make accurate judgment. It is a major goal of the present invention to accurately classify breast cancer patients to guide the clinic to effective treatment while avoiding ineffective or harmful treatments.
The molecular biological typing of breast cancer based on gene expression profiles and molecular biological characteristics, which appears in recent years, can better reflect the biological behavior of tumors, and has important clinical treatment guiding significance. The currently internationally recognized breast cancer molecular typing product based on tumor gene expression is PAM50 developed by Perou of the university of North Carolina in cooperation with Proigna and approved by the U.S. FDA. PAM50 classifies breast cancer into 4 subtypes, including luminal A (LuminalA), luminal B (LuminalB), HER2-enriched (HER2-enriched) and Basal-like, by detecting the expression levels of 55 genes, and assesses the risk of distant metastasis within 10 years of breast cancer according to the subtypes and tumor proliferation index. However, the PAM50 breast cancer gene expression molecule typing method does not incorporate immune regulation genes, which are closely related to breast cancer prognosis and treatment effect, into an analysis system thereof, so that a final typing scoring system has certain limitations, and particularly, results cannot be matched with and guide a tumor immunotherapy scheme which is increasingly valued at present. Several recent studies have shown that immune gene expression levels in breast cancer tissues are closely related to the development of distant metastases of hyperproliferative breast cancer, including luminal B (luminelb), HER2-enriched (HER2-enriched), and Basal-like. The high expression level of immune genes in the tissues of these subtype breast cancers can increase the sensitivity to adjuvant therapy and reduce the risk of distant metastasis, and a common feature of these subtype breast cancers is that tumors often exhibit strong proliferation. The expression level of immune genes has no correlation with the distant metastasis of low-risk subtype breast cancer, such as luminal A (LuminalA), and the subtype is characterized by low tumor proliferation. The recently emerging tumor immunotherapy, including PD1 inhibitors, PDL1 inhibitors and CTLA4 inhibitors, is widely used for the treatment of various solid tumors. Among these, the PD1 inhibitor, Keytruda, is in clinical trials for the treatment of multiple breast cancers. The action mechanism of the treatment is to block PDL1 expressed by tumor cells and PD1 expressed by immune cells by using antibodies, so that the immune cells can recognize and kill tumors. The two preconditions of the function are provided, firstly, enough PDL1 is expressed by tumor cells, and the detection is widely developed clinically at present; secondly, the immune cells in the tumor tissue have enough activity, and the Tumor Infiltrating Lymphocyte (TIL) technology is often adopted in the current clinical research to reflect the immune function in the tumor tissue. Overall, the higher the TIL count, the better the neoadjuvant, targeting and immune checkpoint (PD1) inhibitors of hyperproliferative breast cancer are. However, the prior TIL clinical research does not consider the interaction between the proliferation of tumor cells and the immune system, the stability and repeatability of manual counting are not ideal, and the activity of immune cells cannot be reflected by single counting. The expression of immune gene originated from immune cell can reflect the activity of immune function, and the detection result is stable and reliable. Therefore, the expression level of immune-related genes in tumor tissues combined with tumor proliferation can be used as a reliable concomitant diagnostic index for novel adjuvant, targeting and PD1 inhibitor therapy.
Disclosure of Invention
The invention aims to solve the technical problem of evaluating the immune index of tumor tissues aiming at the prior high-proliferative breast cancer (high-recurrence risk type breast cancer), and provides a group of genes related to evaluation of immune function and a detection kit thereof so as to more accurately predict the distant metastasis risk of high-recurrence risk subtype breast cancer and guide clinical treatment.
The invention provides a group of genes for evaluating the immune correlation of high proliferative breast cancer, which comprises 17 immune correlation genes participating in different immune functions, 7 proliferation correlation genes and 4 housekeeping genes. Wherein, immune related genes APOBEC3, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC, proliferation related genes AURKA, BIRC5, CCNB1, CDC20, MKI67, TOP2A and UBE2C, housekeeping genes ACTB, GAPDH, MRPL19 and RPLP 0. The genes groups related to the immunity of the high proliferative breast cancer are shown in the table 1.
TABLE 1 breast cancer immune-related genes
The invention also provides a method for evaluating the distant metastasis risk of the high-proliferative breast cancer by using the immune related gene group as a marker.
The invention also provides application of the reagent for detecting the gene expression level of the immune related gene group in preparing an in vitro diagnosis product for evaluating the risk of distant metastasis of the high proliferative breast cancer.
The invention also provides an in vitro diagnostic product for evaluating the risk of distant metastasis of the high proliferative breast cancer, wherein the in vitro diagnostic reagent comprises a reagent for specifically detecting the gene expression level of the immune related gene group.
Wherein the in vitro diagnostic product comprises a detection kit.
The invention provides a detection kit for evaluating a high proliferative breast cancer immune related gene group, which comprises: primers and probes for amplifying the immune-related gene group of the invention.
The primer and the probe are conventional in the art, and preferably are synthetic oligonucleotide fragments with high gene specificity, as long as the primer and the probe can be complementary with partial sequences of genes in the immune-related gene group in the tissue for evaluating the hyperproliferative breast cancer and can amplify the genes of the immune-related gene group. Preferably, the sequences of the primers and the probes are shown in Table 2, wherein the primers comprise a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQ ID NO. 1-28, and the sequence of the reverse primer is shown as SEQ ID NO. 29-56; the sequence of the probe is shown in SEQ ID NO. 57-84. The preparation method of the primer and the probe is a conventional preparation method in the field, and preferably, the primer and the probe are synthesized artificially.
The detection kit of the present invention preferably further comprises: total RNA extraction reagent, reverse transcription reagent and/or quantitative PCR reagent.
Wherein, the total RNA extraction reagent is a total RNA extraction reagent which is conventional in the field.
Wherein the reverse transcription reagent is a reverse transcription reagent conventional in the art, preferably comprising a dNTP solution and/or an RNA reverse transcriptase.
Wherein, the quantitative PCR reagent is a reagent which is conventionally used in the field as long as the requirement of quantitative PCR on the obtained sequence can be met. The quantitative PCR reagent is preferably commercially available. The quantitative PCR is a quantitative PCR which is conventional in the field, and preferably, is a Taqman-real-time fluorescence quantitative PCR technology. The PCR reagents preferably further include reagents for constructing a library for quantitative PCR.
The detection kit of the invention preferably further comprises an apparatus for extracting a detection sample from the body of a detection object or a tumor patient; more preferably, the device further comprises a device for extracting tissue or blood from the body of the detected object or the tumor patient, and the device is preferably any blood extracting needle, syringe and the like which can be used for blood extraction.
Wherein the test sample is only required to extract total RNA from the test sample. The test sample is preferably one or more of a tissue sample, blood, plasma and body fluid, more preferably a tissue sample, more preferably a paraffin tissue sample or a fresh tissue sample from a needle biopsy, preferably a tissue with a high tumor cell content.
The invention also provides primers for detecting the immune correlation, the proliferation correlation and the housekeeping gene group expression, wherein the primers comprise a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQIDNO.1-28, and the sequence of the reverse primer is shown as SEQIDNO.29-56.
The invention also provides a probe set for detecting the expression of the immune related gene group, wherein the sequence of the probe is shown as SEQ ID NO. 57-84, and the probe specifically hybridizes with the genes in the immune related gene group shown in the table 1 to detect the genes.
The invention also provides application of the primer or the probe set in preparation of products for evaluating the risk of distant metastasis of the high proliferative breast cancer.
In the present invention, the hyperproliferative breast cancer mainly includes luminal B type, HER2-enriched type and basal cell type.
The invention also provides a using method of the detection kit, which comprises the following steps:
(1) extracting total RNA (totalRNA) of a detection object by using the detection kit;
(2) reverse transcribing the purified total RNA to cDNA, and preparing a DNA sequencing library for quantitative PCR;
(3) and (3) carrying out real-time quantitative PCR detection on the DNA sequencing library obtained in the step (2).
Wherein, the extraction method in the step (1) is a conventional method in the field, and preferably, total RNA of Fresh Frozen tissue (Fresh Frozen) or paraffin embedded tissue (FFPE) of a detection object is extracted by using a detection kit. More preferably, the RNA is extracted using an RNA extraction kit manufactured by Roche (product No. Roche cache Number #3270289001) or an RNA extraction kit manufactured by Qiangen RNA kit (Qiagen RNease FFPE kit, Catalog Number # 73504).
Wherein, the method for constructing the DNA sequencing library in the step (2) is a library construction method which is conventional in the field, and the method for constructing the library preferably comprises the following steps:
the concentration of the specific primer working solution of 28 genes is 10 μ M, 0.4 μ l (final concentration is 200nM) of reverse primer solution of each 28 genes is mixed, 2 μ l (10 is dilution multiple) of 10 XRT-PCR buffer solution, 4 μ l (final concentration is 500nM) of dNTP mixed solution, 1 μ l (10 units) of RNase inhibitor, 1 μ l (4 units) of reverse transcriptase are added, 50ng of RNA template extracted in the step (1) is added, and the rest volume is filled with water. The amplification system was mixed well and incubated at 37 ℃ for 60 minutes.
Wherein, the quantitative PCR detection method in the step (3) is a conventional method in the field, and preferably utilizes Taqman-real-time fluorescence quantitative PCR. The 17 genes are respectively subjected to fluorescent quantitative PCR detection, and one gene is subjected to PCR reaction. The reaction system is prepared as follows: 200nM each of the forward and reverse specific primers, 2. mu.l of 10 XPCR buffer, 1.6. mu.l (200nM) of dNTP mix, 0. mu.l (0.5 unit) of DNA polymerase, 100nM of Taqman fluorescent probe, 0.8. mu.l of reverse transcription product (corresponding to 2ng of the reverse transcription product of the RNA template), and the remaining volume made up by water. The amplification reaction was carried out at 95 ℃ for 15 minutes, 95 ℃ for 10-15 seconds, 60 ℃ for 10-15 seconds, 72 ℃ for 10-15 seconds for 30 cycles, and 72 ℃ for 7 minutes. After the amplification reaction was completed, the Δ Ct value of each gene was recorded and represents the expression level of each gene.
The using method of the detection kit of the invention preferably further comprises the step (4) of carrying out statistical analysis on the obtained detection result, carrying out immune index calculation according to an autonomously developed and optimized calculation method, and thus helping to predict the distant metastasis risk of different subtype breast cancers. And (3) calculating a Proliferation index (PS) and an Immune index (Immune Score, IS) of the patient according to the delta Ct of each gene calculated in the step (3), and judging the distant metastasis risk of the high-Proliferation breast cancer patient according to the Immune index (such as the distant metastasis recurrence risk of 10 years). Specific calculation and risk assessment criteria are as follows:
proliferation gene score ═ (AURKA + BIRC5+ CCNB1+ CDC20+ MKI67+ TOP2A + UBE2C)/7
Proliferation index (PS) ═ 36x (proliferation gene score +1.35), score 0-100; slow proliferation rate of 0-49 and fast proliferation rate of 50-100
Antigen presentation gene score (CD52+ CORO1A + CD53+ PTPRC + HLA-DMA + HLA-DQA1+ LYZ)/7
Tumor killing gene score (apobecc 3G + CCL5+ CCR2+ CD2+ CD3D + CXCL9+ GZMA + GZMK + IL2RG + LCK)/10
Immune gene score-0.44 x antigen presentation gene score +0.56x tumor killing gene score
Immune Index (IS) ═ 33x (immune gene score +1.35), score 0-100; weak immunity of 0-42 and strong immunity of 43-100
Definition of risk of relapse: IS less than 42 suggests that hyperproliferative breast cancer IS poorly immune and has a high risk of distant metastasis; IS greater than or equal to 42 suggests that hyperproliferative breast cancer IS immune-robust and has a low risk of distant metastasis.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: compared with the prior art, the method disclosed by the invention has the advantages that the immune function in the high-proliferation breast cancer is evaluated for the first time, and the evaluation accuracy of the risk evaluation of the breast cancer is further enhanced by the aid of the Basal-like model, the HER2 enriched model (HER2-enriched model) and the luminal B model (LuminalB), and the response of the breast cancer patients to future immune checkpoint inhibitors is predicted.
Drawings
FIG. 1 the risk of distant metastasis in breast cancer patients with strong tumor cell proliferation is closely related to the immune index. The immunity is strong, and the risk of distant metastasis is relatively low; the immunity is weak and the risk of distant metastasis is relatively high.
FIG. 2 the risk of distant metastasis in breast cancer patients with weak tumor cell proliferation is not immune-related. The risk of distant metastasis is relatively low, whether it is immune-strong or immune-weak.
FIG. 3 Single cell sequencing shows the expression of 17 immune-related genes in peripheral blood in different types of immune cells and the expression level of 7 proliferation genes in different types of immune cells.
FIG. 4 Single cell sequencing shows the expression levels of 17 immune-related genes and 7 proliferative genes in tumor infiltrating lymphocytes (T cells vs. monocytes, T _ cell + Mac + M; B cells, B _ cell) and tumor cells (Breast cancer Group1, BC _ Group 1; Breast cancer Group2, BC _ Group 2; Breast cancer Group3, BC _ Group 3; Breast cancer sample 2, BC 02; Breast cancer sample 5, BC 05).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples, in which specific conditions are not specified, were selected according to conventional methods and conditions or according to the commercial instructions.
Example 1 screening for evaluation of the immune-related Gene group of hyperproliferative Breast cancer
The experimental method comprises the following steps:
2034 cases of breast cancer tumor gene expression and clinical variables in 14 breast cancer cohort studies are analyzed by an EPIG gene expression profiling program, and a gene group closely related to the distant metastasis risk of breast cancer is screened. Firstly, a gene specific expression profile is formed through clustering analysis calculation, then the correlation between each gene and the specific expression profile is calculated, candidate genes are selected, and the correlation between the candidate genes and the distant metastasis risk of the breast cancer is analyzed. As a result, the immune related gene and the cell cycle gene are most closely related to the far metastasis risk of the breast cancer. To further test the selected immune-related genes, the present invention randomly selected half of each sample (1017) for the same process analysis and repeated 1000 times, and retained the genes present in all 1000 tests.
The experimental results are as follows: 151 immune-related genes related to the distant metastasis risk of breast cancer are obtained by co-screening, and a test combination comprising 17 immune-related genes, 7 proliferation-related genes and 28 genes of 4 housekeeping genes is finally determined according to the contribution rate and different functions of each gene, and the gene list is shown in table 1.
Example 2 primer and probe design for assessing genes involved in the immunity of hyperproliferative breast cancer.
Primers and probes of 17 immune-related genes and 4 housekeeping genes are designed and optimized by adopting a fluorescent quantitative PCR technology, the expression levels of 28 genes obtained by screening in example 1 in fresh tumor tissues or paraffin-embedded tumor tissues of 100 cases of Chinese breast cancer patients are respectively detected, and the genes and the primers obtained by screening in example 1 are shown in Table 2.
TABLE 2 Breast cancer immune-related Gene primer and Probe sequences
Example 3 distant metastasis risk assessment of hyperproliferative breast cancer using immune index.
Breast cancer tumor tissue was taken and RNA was extracted from tumor cells (Roche Catalog Number #3270289001 or Qiangen Catalog Number # 73504). The expression levels of the immune-related genes and proliferative genes screened in example 2 were examined, the immune index was calculated and the risk of distant metastasis of hyperproliferative breast cancer was assessed.
The experimental results are as follows:
1. evaluation of breast cancer proliferation value-added according to proliferation index
First, proliferation indexes were calculated based on the expression of 7 proliferation-related genes, and breast cancer cases were divided into two groups, i.e., fast proliferation and slow proliferation (FIG. 1).
FIG. 1 shows that the breast cancer is divided into two groups according to the breast cancer proliferation index, and the proliferation is fast and slow. The rate of distant metastasis of breast cancer that proliferates rapidly is significantly higher than that of breast cancer that proliferates slowly.
2. Distant metastasis risk prediction for breast cancer based on immune index
The effect of the immune index on the occurrence of distant metastasis was assessed in both fast and slow proliferating breast cancers, respectively, based on their proliferation. The immune index has a significant impact on the risk of distant metastasis of rapidly proliferating breast cancer. The risk of distant metastasis is about 20% lower in the group of cases with a strong immune index (istrong) than in the group of cases with a weak immune index (iweak) by 10 years. It is expected that the therapeutic effect of PD1 or PDL1 inhibitors will be better in the more immune group of cases among the rapidly increasing breast cancers. While the immune index had no effect on the development of distant metastasis of slow-proliferating breast cancer (fig. 2).
FIG. 2 is a graph of the effect of immune index on the development of distant metastasis in both rapidly (upper) and slowly (lower) proliferating breast cancer.
Example 4 a panel of quantitative breast cancer immune index gene populations and the use of PCR detection kits.
Step 1: taking a tumor or paraffin embedded tissue of a detection object, and acquiring a region containing high tumor cells of the detection object as an original material by using a method in the detection kit.
Step 2: total RNA was extracted from the tissue.
RNA in tissues can be extracted using an RNA extraction kit manufactured by Roche (product Number: Roche cache Number #3270289001) or an RNA extraction kit manufactured by Qiangen RNA corporation (Qiagen RNease FFPE kit, cache Number # 73504).
And step 3: and (3) establishing a cDNA library.
The construction method preferably comprises the steps of:
the concentration of the specific primer working solution of 28 genes was 10. mu.M, 0.4. mu.l (final concentration of 200nM) of the reverse primer solution was mixed for each 28 genes, 2. mu.l (10 is dilution factor) of 10 XTR-PCR buffer, 4. mu.l (final concentration of 500nM) of dNTP mixture, 1. mu.l (10 units) of RNase inhibitor, 1. mu.l (4 units) of reverse transcriptase, 50ng of RNA template extracted in step 2 in this example, and the remaining volume was made up with water. The amplification system was mixed well and incubated at 37 ℃ for 60 minutes.
And 4, step 4: the gene expression level was detected by quantitative PCR.
The quantitative PCR method is a method which is conventional in the field, and is Taqman-real-time fluorescence quantitative PCR. The 17 genes are respectively subjected to fluorescent quantitative PCR detection, and one gene is subjected to PCR reaction.
Wherein, the ABI7500Taqman reaction system is prepared as follows: 200nM each of the forward and reverse specific primers, 2. mu.l of 10 XPCR buffer, 1.6. mu.l (200nM) of dNTP mix, 0. mu.l (0.5 unit) of DNA polymerase, 100nM of Taqman fluorescent probe, 0.8. mu.l of reverse transcription product (corresponding to 2ng of the reverse transcription product of the RNA template), and the remaining volume made up by water.
The amplification reaction was performed at 95 ℃ for 15 minutes, 95 ℃ for 10 seconds, 60 ℃ for 10 seconds, and 72 ℃ for 10 seconds for 30 cycles, and 72 ℃ for 7 minutes. After the amplification reaction was completed, the Ct value of each gene was recorded and represents the expression level of each gene.
And 5: and (6) analyzing results. Based on the expression levels of 17 immune-related genes, an immune index was calculated.
Example 5 Single cell sequencing technology confirmed that immune-related gene expression originated from tumor infiltrating lymphocytes but not tumor cells
When screening immune related genes, the influence size of the immune related genes on the distant metastasis of the high-proliferation breast cancer and the expression of the immune related genes in different types of immune cells are comprehensively considered. The single cell sequencing result shows that 17 immune related genes are differentially expressed in 8 different immune cells in peripheral blood, and the expression of the immune related genes can be mainly divided into high expression of antigen presenting cells, high expression of tumor killer cells and low expression of all immune cells. The proliferative gene expression level showed that all peripheral blood immune cells were in a non-proliferative state (fig. 3). The single cell sequencing results of the tumor infiltrating lymphocytes and the simple tumor cells separated from the breast tumor tissue show that 17 immune-related genes are only expressed in the tumor infiltrating lymphocytes (T cell group and B cells) and are not expressed in the tumor cells, which indicates that the immune-related genes in the tumor tissue are derived from the tumor infiltrating lymphocytes but not from the tumor cells. The proliferative gene was expressed only in a part of lymphocytes and tumor cells, indicating that only a part of lymphocytes and tumor cells were in an actively proliferating state (FIG. 4). At present, the prognosis and treatment effect of breast cancer are generally predicted by adopting tumor infiltration lymphocyte count internationally, and the defect is that the lymphocytes which are actively proliferated and have immunological activity and are in dormancy and have no immunological activity can not be distinguished, and meanwhile, the interaction between the tumor proliferation activity and an immune system is not considered, so that the prediction effect is limited. The immune gene and the expression of the proliferation gene are combined, so that the immune function activity of tumor tissues and the influence of the immune function activity on breast cancer recurrence can be more accurately evaluated.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope and spirit of the invention as disclosed in the appended claims, and all such modifications and variations fall within the scope of the invention as defined in the claims.
Sequence listing
<110> Haiman Shanghai accurate Biotech Co., Ltd
SHANGHAI SHANZHUN BIOTECHNOLOGY Co.,Ltd.
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caaggagaac cagcctgaaa 20
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
taacaccatc agcagggaaa g 21
<210> 27
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
gacgcttcgt tatgggaaga ta 22
<210> 28
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
tctggaatta gggagggtga g 21
<210> 29
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
ccactctctt caaatccctc tg 22
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
gacagccaac attccactct 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
gacagccaac attccactct 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
gcagatggat cagccaagaa 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
aatacacctg gcctcgaaag 20
<210> 34
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
gatgtactcc cgaacccatt t 21
<210> 35
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
gcctgaaagt agagccatag tc 22
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
aggaggatgt tgggaagttg 20
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
agggaaggta cagttggtaa tg 22
<210> 38
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
gtcctgagag tccagtttgt atc 23
<210> 39
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
cagcaggatg aagaacgaca ta 22
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
catggagaga tagtgcagga aag 23
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
ggtgcactgg agagaagaaa 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
cgagggtctc cgcatttatt 20
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
tttgtatgag gcgggacaag 20
<210> 44
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
cccagagact tctaccctaa ga 22
<210> 45
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
gctccacctt gcagtcataa a 21
<210> 46
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
ggcagctgca ggaataaga 19
<210> 47
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
gaggagcaca cagaggtatt ag 22
<210> 48
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
gtagccatcc attcccaatc t 21
<210> 49
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
gctttgccct gtcacaaata c 21
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
cgaaccttgc ctccagatta t 21
<210> 51
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
gctgtctcta ctttccagga tg 22
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
tcccgaccca gtaggtattt 20
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
ggatcttggc ttcctctaca tc 22
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 54
ctgcactgga gttcccataa a 21
<210> 55
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 55
gggccagttg tgatggataa 20
<210> 56
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 56
gggaaggcag aaatcccttt at 22
<210> 57
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 57
aggctccacc atgttcaggg attt 24
<210> 58
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 58
tcctgaaatc ttatctggcc gggc 24
<210> 59
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 59
tcctgaaatc ttatctggcc gggc 24
<210> 60
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 60
acggattaca ccttcccact tgct 24
<210> 61
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 61
cgtctggctg tgctacgaag tgaa 24
<210> 62
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 62
agcagtcgtc tttgtcaccc gaaa 24
<210> 63
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 63
tgatgcaagc aagaaacact gggc 24
<210> 64
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 64
cccaccaaat tccagcttca accc 24
<210> 65
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 65
acttgttccg agcccagttt cctc 24
<210> 66
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 66
tcttcctcct actcaccatc agcct 25
<210> 67
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 67
ttccttgata gagcccatgc agcc 24
<210> 68
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 68
aaagtaccgg attgagctgt caccc 25
<210> 69
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 69
agtcagctct tctccatcct accaca 26
<210> 70
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 70
ccctttgttg tgcgagggtg ttt 23
<210> 71
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 71
ccacaaagcc tggaatctac accct 25
<210> 72
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 72
tgggcaggat gtgagaaatc tgagc 25
<210> 73
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 73
tacctcacct tcctcccttc tgct 24
<210> 74
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 74
caagcgccat gttgaagcca tcat 24
<210> 75
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 75
aagttcctca agggccagga cttt 24
<210> 76
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 76
tccagggcaa ggtctttgaa aggt 24
<210> 77
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 77
tccagaaagg caaagccaaa tgcc 24
<210> 78
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 78
agtgaaagta gccacgagaa ttgtgct 27
<210> 79
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 79
tgggctatgg gtgaggttcc aatg 24
<210> 80
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 80
ttgaggaaga gcaagcagtc agacc 25
<210> 81
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 81
agacgcccac caagaaggaa catc 24
<210> 82
<211> 25
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 82
ccaaaccagc aggaggtgat gagaa 25
<210> 83
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 83
atggttccca catcaaaggc ttgc 24
<210> 84
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 84
aggatcgtcc tgtggatctg gcta 24
Claims (5)
1. The application of an immune related gene group as a marker in the preparation of a product for evaluating the distant metastasis risk of the high proliferative breast cancer is characterized in that the immune related gene group is a high proliferative breast cancer immune related gene group;
the high proliferative breast cancer immune-related gene group is immune-related genes APOBEC3, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC, proliferation-related genes AURKA, BIRC5, CCNB1, CDC20, MKI67, TOP2A and UBE2C, and housekeeping genes ACTB, GAPDH, MRPL19 and RP LP 0;
the hyperproliferative breast cancer is luminal B, HER2-enriched and basal cell;
the method for evaluating the risk of distant metastasis of the high proliferative breast cancer comprises the following steps:
calculating delta Ct of each related gene by using a fluorescent quantitative PCR method, calculating a proliferation index PS and an immune index IS of a patient, and judging the distant metastasis risk of the high-proliferation breast cancer patient according to the immune index; the specific calculation and risk assessment criteria are:
proliferation gene score ═ (AURKA + BIRC5+ CCNB1+ CDC20+ MKI67+ TOP2A + UBE 2C)/7;
proliferation index (PS) ═ 36x (proliferation gene score +1.35), score 0-100; the proliferation is slow by 0-49 and fast by 50-100;
antigen presentation gene score ═ (CD52+ cor 1A + CD53+ PTPRC + HLA-DMA + HLA-DQA1+ LYZ)/7;
tumor killing gene score ═ (apobecc 3G + CCL5+ CCR2+ CD2+ CD3D + CXCL9+ GZMA + GZMK + IL2RG + LCK)/10;
immune gene score-0.44 x antigen presentation gene score +0.56x tumor killing gene score;
immune Index (IS) ═ 33x (immune gene score +1.35), score 0-100;
wherein IS less than 42 suggests that hyperproliferative breast cancer IS poorly immune and has a high risk of distant metastasis; IS greater than or equal to 42 suggests that hyperproliferative breast cancer IS immune-robust and has a low risk of distant metastasis.
2. An in vitro diagnostic product for assessing the risk of distant metastasis of hyperproliferative breast cancer, comprising reagents for specifically detecting the gene expression level of the immune-related gene population of claim 1.
3. The in vitro diagnostic product for assessing the risk of distant metastasis of hyperproliferative breast cancer according to claim 2, wherein said in vitro diagnostic product is a test kit.
4. The in vitro diagnostic product for assessing the risk of distant metastasis of hyperproliferative breast cancer according to claim 3, wherein said test kit comprises primers and probes for amplifying the immune-related gene population according to claim 1.
5. The in vitro diagnostic product for evaluating the risk of distant metastasis of hyperproliferative breast cancer according to claim 4, wherein the primers comprise a forward primer and a reverse primer, the sequence of the forward primer is shown as SEQ ID No. 1-28, and the sequence of the reverse primer is shown as SEQ ID No. 29-56; the sequence of the probe is shown in SEQ ID NO. 57-84.
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