CN110964815A

CN110964815A - Breast cancer molecular typing and distant metastasis risk gene group, diagnosis product and application

Info

Publication number: CN110964815A
Application number: CN201811166148.7A
Authority: CN
Inventors: 周彤; 胡志元; 周伟庆
Original assignee: Shanghai Shanzhun Biotechnology Co ltd
Current assignee: Shanghai Shanzhun Biotechnology Co ltd
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2020-04-07
Also published as: CN111566229A; WO2020064006A2; WO2020064006A3; CN111566229B

Abstract

The invention belongs to the technical field of biology, and particularly relates to breast cancer subtype typing and remote metastasis risk assessment gene groups, and in-vitro diagnosis products and application thereof.

Description

Breast cancer molecular typing and distant metastasis risk gene group, diagnosis product and application

Technical Field

Background

Breast cancer accounts for the first place of malignant tumor in women in China, and is increasing year by year at a rate of about 4% per year. 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 currently internationally recognized breast cancer molecular typing product based on tumor gene expression is PAM50 co-developed by professor Perou of university of north carolina in cooperation with Prosigna, which has been approved by the U.S. FDA. PAM50 classifies breast cancer into 4 subtypes, including Luminal A (LumA), Luminal B (Luminal B), HER2-enriched (HER2-enriched (HER2) and Basal-like (Basal-like) by detecting 55 gene expression levels, and evaluates the risk of distant metastasis of breast cancer within 10 years according to the subtypes and tumor proliferation index.

Although the concept of molecular typing of breast cancer has been widely accepted and applied to guide clinical treatment, due to the high requirements of NanoString technology, expensive equipment and reagents and no conditional development of general medical institutions, an alternative method of Immunohistochemistry (IHC) is often adopted clinically to estimate breast cancer subtypes by semi-quantitatively detecting the expression levels of ER, PR, HER2 and Ki67 proteins. Currently, the IHC method is basically adopted for breast cancer subtype typing in China, but the typing is not accurate and has about 30 percent of inconsistency with the typing based on gene expression, so that the correct formulation of a clinical treatment scheme is influenced frequently.

Recent studies have shown that immune gene expression levels in breast cancer tissues are closely related to the occurrence of distant metastasis of high-risk subtypes of breast cancer, including Luminal B (Luminal B), HER2-enriched (HER2-enriched), and Basal-like. Besides high detection technical requirements and high cost, the PAM50 does not bring immune regulation genes closely related to the prognosis and treatment effect of the breast cancer into an analysis system, so that a final typing and scoring system has certain limitation, and the molecular typing efficiency is only 70-80%.

Accordingly, there remains a need in the art for diagnostic assay products and methods that are more accurate, sensitive and/or specific for typing.

Disclosure of Invention

In one aspect, the present invention relates to a gene group (also referred to herein as prey) for molecular typing and/or assessing the risk of distant metastasis of breast cancer, comprising 72 genes, namely 66 molecular typing and risk assessment-associated genes for distant metastasis and 6 housekeeping genes, wherein

The 66 molecular typing and distant metastasis risk assessment related genes comprise:

(1) proliferation-related genes ASPM, AURKA, BIRC5, CCNB1, CDC20, CDK1, CENPU, CEP55, MELK, MKI67, NEK2, PRC1, PTTG1, RRM2, TOP2A, TPX2, TYMS, UBE2C and ZWINT,

(2) immune-related genes APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC,

(3) basal cell-associated genes ACTR3B, CDH3, EGFR, FOXC1, KRT14, KRT17, KRT5, MIA, MYC, PHGDH, and SFRP1,

(4) estrogen receptor related genes BAG1, BCL2, BLVRA, CD68, ESR1, FOXA1, GSTM1, MAPT, MDM2, MLPH, NAT1, PGR, SCUBE2 and SLC39A6,

(5) HER2 related genes ERBB2, FGFR4 and GRB7,

(6) invasion-associated genes CTSL2 and MMP 11;

the 6 housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3a1, and TFRC.

In one embodiment, the information for the genes in the gene populations of the invention is also found in table 1.

In another aspect, the invention relates to the use of said gene cluster for molecular typing of breast cancer and/or for assessing the risk of distant metastasis.

In a further aspect, the invention also relates to the use of said gene cluster for the preparation of a diagnostic product for molecular typing of breast cancer and/or for assessing the risk of distant metastasis.

In a further aspect, the invention also relates to a diagnostic product for molecular typing of breast cancer and/or assessing the risk of distant metastasis, comprising a reagent related to the detection of the expression level of a gene in the gene population of the invention. In a preferred embodiment, the diagnostic product is in the form of an in vitro diagnostic product, preferably in the form of a diagnostic kit. In a further preferred embodiment, the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

In further embodiments, the agent is a probe or primer.

In a preferred embodiment of the present invention, the breast cancer includes luminal a, luminal B, HER2-enriched, basal cell, and Immune-enhanced (also referred to herein as Immuno).

Drawings

FIG. 1 shows the classification of breast cancer into luminal A (Lum A), luminal B (Lum B), Basal (Basal), HER2-enriched (HER2) and Immuno-enhanced (Immuno) based on the expression levels of 66 genes in breast cancer tissue. A. 1951 European and American cases; B. 824 chinese cases.

FIG. 2 shows that the risk of distant metastasis differs for each subtype of breast cancer. Wherein, the risk of lumen type A distant metastasis is significantly lower than the other four subtypes, and the risk of immune-enhanced distant metastasis is significantly lower than lumen type B, basal cell type and HER2-enriched type. Risk of distant metastasis within 5 years after the european and american case surgery, luminal B (Lum B) is less than Basal, which is less than HER2-enriched (HER 2); after 10 years of operation, the three have no significant difference. In Chinese cases, the type B of the lumen is less than the basal cell type and the HER2 enriched type within 3 years of operation, and after 10 years of operation, the HER2 enriched type has the worst prognosis. A. 1951 European and American cases; B. 824 chinese cases.

FIG. 3 shows that the immune index has a significant effect on the prognosis of breast cancer. Among the high-risk breast cancer subtypes, luminal B (Lum B), Basal cell (Basal) and HER2-enriched (HER2) have strong immune function, so that the risk of distant metastasis of the breast cancer of a patient can be remarkably reduced; for low-risk breast cancer subtype lumen type A (Lum A), the strength of the immune function has no obvious influence on prognosis. A. 1951 European and American cases; B. 824 chinese cases.

FIG. 4 shows that the risk of distant metastasis of the tumor is divided into three groups, low (0-32), moderate (33-49) and high (50-100) according to the calculated recurrence risk index. A. 1951 European and American cases; B. 824 chinese cases.

FIG. 5 shows the gene expression detection based on the second generation sequencing (NGS) of the present invention, which is superior to the existing gene chip, quantitative PCR and Nanostring technologies in sensitivity and detection throughput.

FIG. 6 is a result of a repetitive experiment showing that there is excellent correlation between RNA extracted from paraffin sections (left) and RNA extracted from fresh tissues (right).

Detailed Description

General definitions and terms

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application will control. When an amount, concentration, or other value or parameter is expressed in terms of a range, preferred range, or upper preferable numerical value and lower preferable numerical value, it is understood that any range defined by any pair of upper range limits or preferred numerical values in combination with any lower range limits or preferred numerical values is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise indicated, numerical ranges set forth herein are intended to include the endpoints of the ranges and all integers and fractions (decimal) within the range.

The terms "about" and "approximately," when used in conjunction with a numerical variable, generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within 95% confidence interval for the mean) or within ± 10% of the specified value, or more.

The terms "optional" or "optionally present" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, when a group is described as optionally substituted, it may be unsubstituted or substituted, e.g., with one or more substituents independently selected from those described herein. It will be understood by those skilled in the art that the optional inclusion of the meaning that the type and number of substituents may be arbitrarily selected and combined is also contemplated as long as the resulting compound is stable.

The expressions "comprising" or similar expressions "including", "containing" and "having" and the like which are synonymous are open-ended and do not exclude additional, unrecited elements, steps or components. The expression "consisting of …" excludes any element, step or ingredient not specified. The expression "consisting essentially of …" means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" covers the expressions "consisting essentially of …" and "consisting of …".

The detection of gene expression levels described herein can be accomplished, for example, by detecting RNA transcripts, or by detecting protein expression levels, for example, by proteomic methods. The term "RNA transcript" refers to total RNA, i.e., coding or non-coding RNA, including RNA derived directly from tissue or peripheral blood samples, and also including RNA derived indirectly from tissue or blood samples after cell lysis. Total RNA includes tRNA, mRNA, and rRNA, where mRNA includes mRNA transcribed from a target gene, as well as mRNA from other non-target genes.

In this context, RNA transcripts can be detected and quantified, for example, by hybridization, amplification or sequencing methods. For example, the RNA transcript and probe or primer hybridization.

The term "hybridization" refers to the process by which two nucleic acid fragments bind by stable and specific hydrogen bonds under appropriate conditions to form a double-helical complex.

The term "amplification primer" or "primer" refers to a nucleic acid fragment comprising 5 to 100 nucleotides, preferably 15 to 30 nucleotides, capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

The term "(hybridization) probe" refers to a nucleic acid sequence comprising at least 5 nucleotides, e.g., 5 to 100 nucleotides, that hybridizes under specified conditions to an expression product of a target gene or an amplification product of the expression product to form a complex. The hybridization probes may also include markers for detection.

The Gene group of the present invention

In response to the clinical needs and product deficiencies in the art, the present invention provides a panel of gene populations, also referred to herein as prey (Precitype). The gene group comprises 72 genes, namely 66 molecular typing and distant metastasis risk assessment related genes and 6 housekeeping genes.

(5) HER2 related genes ERBB2, FGFR4 and GRB7,

(6) invasion-associated genes CTSL2 and MMP 11;

the 6 housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3a1, and TFRC.

In one embodiment, the gene populations are as shown in table 1.

TABLE 1 genetic groups for molecular typing of breast cancer and/or risk of distant metastasis

In another embodiment, the invention also relates to a set of immune-related gene populations comprising 17 genes: APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, cor 1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC.

The genes in this immune-related gene group are also shown in the relevant information in table 1.

In a particular embodiment, the gene populations of the invention can be used for molecular typing (subtyping) of breast cancer and/or for assessing the risk of distant metastasis thereof.

Diagnostic product of the invention

The present invention relates to a diagnostic product for molecular typing of breast cancer and/or assessing the risk of distant metastasis thereof, comprising an agent for detecting the expression level of genes in the gene population of the invention.

Specifically, the gene group of the present invention includes 72 genes, namely 66 molecular typing and distant metastasis risk assessment-associated genes and 6 housekeeping genes, among which

(5) HER2 related genes ERBB2, FGFR4 and GRB7,

(6) invasion-associated genes CTSL2 and MMP 11;

the 6 housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3a1, and TFRC.

In one embodiment, the gene populations are as shown in table 1.

In one embodiment, the diagnostic product is an in vitro diagnostic product. In a specific embodiment, the diagnostic product is a diagnostic kit.

In one embodiment, the diagnostic product is used for breast cancer subtyping and/or distant metastasis risk assessment thereof.

In one embodiment, the agent is an agent that detects the amount of RNA, particularly mRNA, transcribed from the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA.

In a preferred embodiment, the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

The total RNA extraction reagent can be a total RNA extraction reagent which is conventional in the field. Examples include, but are not limited to Qiagen 73504, RNA storm CD201, Invitrogen and ABI AM 1975.

The reverse transcription reagent may be a reverse transcription reagent conventional in the art, and preferably includes a dNTP solution and/or an RNA reverse transcriptase. Examples of reverse transcription reagents include, but are not limited to, NEB M0368L, Thermo K1622, ABI 4366596.

The second-generation sequencing reagent may be a reagent conventionally used in the art as long as it can satisfy the requirement of second-generation sequencing of the resulting sequence. The second-generation sequencing reagent may be a commercially available product, examples of which include, but are not limited to, Illumina Inc

Reagent Kit v3(150cycle)(MS-102-3001)、

Targeted RNA Index KitA-96Indices (384Samples) (RT-402-1001). Secondary sequencing is a technique conventional in the art, such as targeted RNA-seq technology. Thus, the secondary sequencing reagents may also include reagents that can be tailored for constructing libraries of targeting RNA-seq, e.g., Illumina

Targeted RNA Custom Panel Kit(96Samples)(RT-102-1001)。

In a preferred embodiment, the agent is a probe or primer. In alternative embodiments, the agent is an agent that detects the amount of a polypeptide encoded by the gene, preferably the agent is an antibody, an antibody fragment, or an affinity protein.

In a preferred embodiment, the reagent is a primer. In one embodiment, the primer is a highly specific synthetic oligonucleotide fragment, so long as it is complementary to a partial sequence of a gene in the gene group of the present invention and can amplify the gene therein. The primers may be artificially synthesized. More preferably, the sequence of the primer is shown in SEQ ID NO.1-SEQ ID NO. 144. The specific scheme of the primers can be seen in table 2. Thus, an embodiment of the invention also relates to a diagnostic product (preferably an in vitro diagnostic product, in particular a kit) comprising primers whose sequences are shown in SEQ ID No.1 to SEQ ID No.144 (see also table 2).

The diagnostic product of the invention (preferably in the form of a kit) also preferably comprises means for taking a test sample from a subject; such as a device for extracting tissue or blood from a subject, preferably any blood sampling needle, syringe, etc. that can be used for blood sampling. The subject is a mammal, preferably a human, in particular a female suffering from breast cancer.

Methods and applications of the invention

In a further aspect, the present invention also relates to a method for determining the molecular typing of breast cancer and/or the risk of distant metastasis in a subject, said method comprising

(1) Providing a sample of the subject object,

(2) determining the expression level of a gene in the gene group of the present invention in the sample,

(3) determining the risk of molecular typing of breast cancer and/or distant metastasis in said subject.

The methods of the invention may be used for diagnostic or non-diagnostic purposes.

The subject for use in the method of the invention is a mammal, preferably a human, in particular a female suffering from breast cancer.

The sample used in step (1) is not particularly limited as long as the expression level of the genes in the gene group can be obtained therefrom, and for example, total RNA of the subject can be extracted from the sample. The sample is preferably a sample of tissue, blood, plasma, body fluid or a combination thereof, preferably a tissue sample, in particular a paraffin tissue sample. In a preferred embodiment, the sample is a tumor tissue sample or a tissue sample comprising tumor cells.

In step (2), the expression level of the gene in the gene group of the present invention is measured. The gene groups are as described above, and see also the description of table 1.

In a preferred embodiment, step (2) may comprise

(2-1) extracting total RNA in the sample;

(2-2) converting the optionally purified total RNA into cDNA, and then preparing it into a library that can be used for secondary sequencing;

(2-3) sequencing the library obtained in the step (2-2).

The extraction of step (2-1) can be performed by a method conventional in the art, and total RNA of fresh frozen tissue or paraffin-embedded tissue of the test subject is preferably extracted using an RNA extraction kit. In a more preferred embodiment, the extraction can be performed using an RNA extraction kit from Roche (product Number Roche cache Number #3270289001) or an RNA extraction kit from Qiangen (Qiagen RNease FFPE kit, cache Number # 73504).

In a preferred embodiment, the library construction method may comprise the steps of:

the extracted total RNA was reverse transcribed to generate cDNA of 72 genes as described in Table 1. After the ends were filled in and phosphorylated at the 5' end, 30. mu.l of DNA, 45. mu.l of pure water, 10. mu.l of T4DNA ligase buffer containing 10mM ATP, 4. mu.l of buffer containing 10mM dNTPmix, 5. mu.l of T4DNA polymerase, 1. mu.l of Klenow enzyme, and 5. mu.l of T4 ligase were mixed, and the mixture was incubated at 20 ℃ for 30 minutes (reagents Illumina sample preparation kit PE-102-1001), and after the incubation, DNA was purified using QIAGEN QIAquick PCR purification kit (part # 28104). End suspension A the product of the previous step was dissolved in 32. mu.l buffer, 5. mu.l of Klenow buffer, 1mM dATP 10. mu.l, Klenow Ae χ -3. mu.l were added, and the mixture was held at 37 ℃ for 30 minutes (reagent Illumina sample preparation kit). The product was ligated by QIAGEN MinElute PCR purification kit (part # 28004): the DNA was dissolved in 10. mu.l of buffer, 2X 25. mu.l of DNA ligase buffer, 10. mu.l of PE Adapter Oligo Mix, and 5. mu.l of DNA ligase were added, and the mixture was kept at 20 ℃ for 15 minutes (reagent: Illumina sample preparation kit PE-102-1001), and after incubation, the DNA was purified by QIAGEN QIAquick PCR purification kit (part #28104), to obtain a library.

Step (2-3) can be accomplished by RNA sequencing. The sequencing method may be an RNA-seq sequencing method for determining the expression level of a gene, which is conventional in the art. Secondary sequencing is preferably performed using an Illumina NextSeq/MiSeq/MiniSeq/iSeq series sequencer. And (3) amplifying the genes shown in the table 1 by using primers in the kit, and performing secondary sequencing on the obtained gene sequences according to the difference of the libraries prepared in the step (2-2). Preferably, the secondary sequencing is a targeted RNA-seq technique, paired end sequencing with an Illumina NextSeq/MiSeq/MiniSeq/iSeq sequencer. Such a process may be automated by the instrument itself.

In one embodiment of the present invention, step (3) can be performed by subjecting the obtained sequencing results to statistical analysis. Breast cancer typing and risk prediction can optionally be performed according to Hu et al, pioneered single Sample prediction SSP (Single Sample predictor) and Parker et al optimized methods. Analyzing the obtained sequencing result gene expression data to obtain subtype classification of a single sample, and calculating the distant metastasis risk.

For the methods, uses and products of the present invention, as exemplary embodiments, the following procedures may be employed.

Firstly, 2034 Affymetrix U133 gene chip data (GSE3494, GSE6532, GSE1456, GSE9195, GSE2034, GSE5327, GSE7390, GSE11121, GSE2603, GSE7378, GSE8193, GSE12093 and E-TABM-158) in 14 breast cancer cohort studies are collected for gene screening and optimization of gene combinations for breast cancer molecular typing and prognosis evaluation. In addition to gene expression data, clinical information for each corresponding case, including case grade, lymph node metastasis, ER/PR detection, distant metastasis and time of occurrence, and treatment regimen, were collected and analyzed.

All gene chip data need to be subjected to standardization (RMA), batch correction (Combat) and identical gene combination before being combined and analyzed so as to eliminate gene expression data difference caused by difference of technical platforms and operation flows of each research queue, and a foundation is laid for next gene screening.

For 2034 cases of breast cancer with standardized, batch-corrected and similar gene combination, the gene expression data is analyzed by adopting the independently developed analysis software EPIG and through unsupervised clustering and pairing correlation analysis, the gene expression profile which is closely related to breast cancer metastasis and has statistical significance is calculated, and the significant genes in the expression profile are obtained on the basis of the gene expression profile. Of these, the two most relevant groups of genes are associated with the cell cycle and immune response genes, respectively.

In order to confirm the stability of the found genes related to cell cycle and immune response in predicting disease recurrence, further stability analysis was performed. Specifically, 1017 chip data (50%) are randomly extracted from 2034 gene chip expression data, expression profile and related gene calculation are performed, and the extraction and calculation are repeated 1000 times.

The results showed that 63 cell cycle-associated genes and 121 immune response genes consistently appeared stably among the 1000 calculations. Among them, 19 cell cycle-associated genes and 17 immune response genes, which are most closely related to relapse-free survival, have been widely reported for molecular typing of breast cancer and 6 housekeeping genes, and finally, a 72-gene combination was determined (see table 1).

The detection method of the present invention can be used for diagnostic purposes or non-diagnostic purposes.

Accordingly, the invention also provides the use of the gene cluster of the invention for molecular typing of breast cancer and/or for assessing the risk of distant metastasis thereof.

The invention also provides application of the gene group in preparing a product for carrying out molecular typing on the breast cancer and/or evaluating the distant metastasis risk of the breast cancer. In a preferred embodiment, the product is in the form of a test kit.

The invention further provides application of the reagent for detecting the gene expression level in the gene group in preparing an in vitro diagnosis product for carrying out molecular typing on the breast cancer and/or evaluating the distant metastasis risk of the breast cancer. In a preferred embodiment, the product is in the form of a test kit. In one embodiment, the agent is an agent that detects the amount of RNA, particularly mRNA, transcribed from the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA. In alternative embodiments, the agent is an agent that detects the amount of a polypeptide encoded by the gene, preferably the agent is an antibody, an antibody fragment, or an affinity protein. In another embodiment, the agent is a probe or primer, in particular a primer. More preferably, the sequence of the primer is shown in SEQ ID NO.1-SEQ ID NO. 144. The specific scheme of the primers can also be seen in table 2.

The subtypes of breast cancer include luminal a, luminal B, HER2-enriched, basal cell, and immunopotentiating.

The test sample used in the present invention is preferably a tissue derived from a test object (subject), as long as total RNA of the test object can be extracted 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, such as a paraffin tissue sample. In a preferred embodiment, the test sample is a tissue having a high tumor cell content.

The invention also relates to a panel of immune-related genes comprising APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, cor 1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC (see also the relevant information in table 1).

The invention also relates to the application of the immune related gene (APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC) or a related reagent for detecting the immune related gene in the preparation of in vitro diagnostic products for molecular typing of breast cancer and/or evaluating the distant metastasis risk of the breast cancer. The invention also relates to the application of the immune related gene in molecular typing of breast cancer and/or evaluating the risk of distant metastasis. In one embodiment, the agent is an agent that detects the amount of RNA, particularly mRNA, transcribed from the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA. In alternative embodiments, the agent is an agent that detects the amount of a polypeptide encoded by the gene, preferably the agent is an antibody, an antibody fragment, or an affinity protein. In another embodiment, the agent is a probe or primer, in particular a primer. More preferably, the sequences of the primers can be found in table 2.

In the present application, by introducing 17 immune genes, a new breast cancer subtype (immunopotentiation) can be determined and also different effects and influences on distant metastasis of breast cancer can be shown in different subtypes.

Advantageous effects

Compared with the prior art (such as PAM50 technology), the invention introduces the immune regulation gene into breast cancer molecular typing for the first time, further enhances the rationality and accuracy of breast cancer molecular typing, improves the capability of guiding clinical treatment of breast cancer, and classifies the breast cancer molecules into a lumen A type, a lumen B type, HER2 enriched type, a basal cell type and an immune enhancement type. For subtypes other than immune-enhanced, further subdivision by the immune genome may be made. Furthermore, gene expression was examined by using next generation sequencing and used for molecular typing. The gene group and the corresponding product thereof can increase the sensitivity of detection, improve the detection capability and efficiency and greatly reduce the detection cost. Compared with the currently common IHC technology, the accuracy of breast cancer molecular typing is improved by more than 20%, the distant metastasis risk can be calculated, the distant metastasis risk can be predicted, and the clinical treatment can be guided more accurately. Moreover, the products and methods of the present invention have high reliability, high sensitivity and high repeatability.

Examples

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. Reagents and instruments used in the examples herein are all commercially available.

Example 1: screening of Gene groups associated with evaluation of Breast cancer subtype typing and distant metastasis Risk

The method comprises the following steps: the expression level of 1951 breast cancer tumor genes with complete clinical information in 2034 cases (GSE3494, GSE6532, GSE1456, GSE9195, GSE2034, GSE5327, GSE7390, GSE11121, GSE2603, GSE7378, GSE8193, GSE12093 and E-TABM-158) was analyzed by EPIG gene expression profiling program (refer to Zhou, Chou et al,2006, environ health Perspectrum 114 (4); 553-; Chou, Zhou et al,2007, BMC Bioinformatics 8,427), immune-related genes closely related to the risk of distant metastasis of breast cancer and cell cycle genes were screened, and genes related to reported ER, PR and subtype typing were combined, and genes having a large contribution rate to typing and distant metastasis risk were calculated and preferred in each group of genes.

As a result: co-screening yielded 66 genes and 6 housekeeping genes, 72 gene test combinations, associated with breast cancer subtype typing and risk of distant metastasis. The gene list is shown in Table 1.

Example 2: molecular typing and remote metastasis risk assessment of breast cancer by using the screened breast cancer subtype typing and remote metastasis risk related gene group

The experimental method comprises the following steps: the combination was tested using 72 genes, wherein 66 breast cancer subtype typing and distant metastasis risk associated gene groups (proliferation associated genes ASPM, AURKA, BIRC, CCNB, CDC, CDK, CENPU, CEP, MELK, MKI, NEK, PRC, PTTG, RRM, TOP2, TPX, TYMS, UBE2 and zwit, immune associated genes APOBEC3, CCL, CCR, CD3, CD, cor 1, CXCL, GZMA, GZMK, HLA-DMA, HLA-DQA, IL2, LCK, LYZ and PTPRC, basal cell associated genes ACTR3, CDH, EGFR, FOXC, KRT, MIA, MYC, PHGDH and SFRP, estrogen receptor associated genes BAG, BCL, bla, CD, ESR, FOXA, GSTM, MAPT, plm, MLPH, pgnat, SLC, scslc and SFRP, and the related genes included the internal standard genes sigb, gfrp, tprd, and tfc. All 66 genes except the 6 reference genes in table 1 were used in calculating the distant metastasis risk index. The data of 1951 European and American breast cancer patients in the European and American breast cancer public database were subjected to molecular typing (subtyping) and distant metastasis risk assessment (FIG. 1A), and 824 Chinese breast cancer tumor samples were subjected to molecular typing (subtyping) and distant metastasis risk assessment (FIG. 1B), and the two were compared.

The experimental results are as follows:

1. molecular typing of breast cancer

As described above, the above breast cancer cases were molecularly typed using the 66 breast cancer molecular typing gene groups shown in table 1, breast cancer tumors were classified into 5 subtypes (fig. 1), and different suggestions for their treatment could be given:

1) lumen A type (Luminal A)

The p53 gene mutation rate of the patient with the luminal A tumor is very low, the prognosis is good, and the patient is insensitive to chemotherapy and suitable for endocrine treatment, so the method has guiding significance for clinical endocrine treatment.

2) Lumen B type (Luminal B)

Luminal B belongs to endocrine treatment sensitive tumors, but for HER2 positive patients, treatment with tamoxifen is less effective than luminal a, while aromatase inhibitors are more effective. For patients with HER2 positive luminal B tumor, molecular targeted therapy can be performed.

3) HER2 enriched type (HER2-enriched)

The mutation rate of the p53 gene of a HER2 enriched tumor patient is high, the tumor differentiation is relatively poor, and the tumor is relatively sensitive to targeted molecule treatment and has poor prognosis. HER2-enriched tumors were extensively treated with herceptin in combination with systemic chemotherapy.

4) Basal cell type (basic-like)

Basal cell type tumors are the most invasive, i.e. triple negative tumors (ER-, HER2-, PR-). Patients with basal cell type tumors are not sensitive to the current breast cancer treatment regimen, and generally have poor clinical prognosis; however, if the tumor tissue has strong expression of the immune-related gene, the prognosis is relatively good.

5) Immunity enhancement type (Immune-enhanced)

This type has a slightly worse prognosis than luminal a, but better than the other three subtypes. Immune genes are highly expressed and are therefore relatively sensitive to various adjuvant therapies.

Corresponding metastasis risks can be obtained by calculating the number and time of distant metastasis of different subtypes and drawing a Kaplan-Meier survival curve. As shown in fig. 2, the five subtypes have different distant metastasis risks, and luminal a has the lowest distant metastasis risk and moderate risk; luminal B is lower than HER2-enriched and basal cell types for distant metastasis risk of 5 years; for the transfer risk at a distance of 10 years, the luminal B type, the HER2 enriched type and the basal cell type have no significant difference and all belong to high-risk subtypes, while the Immune-enhanced type (Immune-enhanced) is between two groups and belongs to medium-risk subtypes.

2. Effect of immune index on the risk of distant metastasis of different subtypes

Immune indices were calculated from the expression levels of 17 immune-related genes APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, cor 1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC, each subtype was further divided into two groups, i.e., a group that is immune strong and a group that is immune weak, according to the immune indices, and differences in metastatic risk between the two groups were observed. Among them, among Luminal B (Luminal B), Basal-like (Basal-like) and HER2-enriched (HER2-enriched) subtypes, the distant metastasis risk of the cases with strong immunity is significantly lower (P < 0.05) than the cases with weak immunity; while Luminal a (Luminal a), the distant metastasis risk was not significantly different in the cases with strong immunity and in the cases with weak immunity (fig. 3).

The immune index can be obtained by the mean expression level after the normalization of 17 immune genes:

3. remote metastasis risk assessment

The calculation of the distant metastasis risk of the tumor adopts a Cox model, whether the distant metastasis occurs and the occurrence time are used as observation endpoints, and corresponding coefficients are determined according to the relative risk of the subtype, the immune index and the proliferation index of the tumor on the occurrence of the distant metastasis, and the calculation method is as follows:

calculation of distant metastasis Risk Score (Risk of Recurrence Score, RRS): 0-100

0-32, low risk; 33-49, risk of stroke; 50-100, high risk;

RRS ═ 0.2x Basal +0.4x HER 2-0.2 x Immune-0.1x LumA +0.2x LumB +0.3x proliferation index-0.1 x Immune index

Wherein "Basal" represents the pearson correlation coefficient of the tumor with a Basal cell type tumor;

"HER 2" represents the pearson correlation coefficient of this tumor with HER2-enriched tumors;

"LumA" represents the pearson correlation coefficient of the tumor to luminal A tumors;

"LumB" represents the pearson correlation coefficient of the tumor with luminal B;

"Immune" represents the pearson correlation coefficient of the tumor to an immunopotentiating tumor.

As shown in FIG. 4, the risk of distant metastasis of the tumor can be classified into three groups, low risk (0-32), medium risk (33-49), and high risk (50-100), based on the calculated risk score of distant metastasis.

For the low risk group cases, adjuvant chemotherapy is not recommended if the lymph node check is negative.

Example 3: primer and probe design for evaluating high-risk breast cancer subtype immune related gene and laboratory verification

The experimental method comprises the following steps: the expression levels of 72 genes including immune related genes obtained by screening in example 1 in fresh tumor tissues or paraffin-embedded tumor tissues of 300 cases of Chinese breast cancer patients are detected by adopting a targeted RNA-seq technology based on a flux next-generation sequencing platform MiSeq in Illumina, and the expression levels are used as the basis for further breast cancer typing.

The experimental results are as follows:

1. the 72 pairs of primers for detecting the 72 genes selected in example 1 are shown in Table 2.

TABLE 2 genes of the Gene group of the present invention and amplification primers therefor

2. And (4) establishing a second-generation sequencing database. All of the 300 chinese breast cancer patients described in example 3 were subjected to secondary sequencing of fresh tumor tissue or paraffin-embedded tumor tissue and the primary data were uploaded to a web-based data storage and analysis library. This method employs Java software development and applies a number of J2EE (Java enterprise edition) components and schemas, which can 1), directly inputs data from Illumina NextSeq/MiSeq/MiniSeq/iSeq instruments; 2) the input data can be displayed in a flexible mode, and the index can be performed according to different requirements, such as gene correlation, samples or experimental groups; 3) calculating a gene expression profile after the gene is normalized by the housekeeping gene; 4) analyzing the details of the specific elements; 5) outputting data in different formats, such as XML, excel and text formats; 6) the method can safely manage and ensure the data privacy protection.

3. Results of 300 cases of subtype, proliferation index and immune index of Chinese breast cancer (Table 3).

Obtaining paraffin wax samples of Chinese breast cancer patients after operation through a cooperative hospital, extracting RNA according to the steps, performing quality inspection, performing reverse transcription to build a library, performing RNA sequencing by adopting an Illumina NextSeq/MiSeq/MiniSeq/iSeq platform, detecting 72 gene expression levels, performing subtype typing and remote transfer risk calculation, and finally obtaining 300 sample detection results.

TABLE 3.300 results of 72-gene molecular typing and distant metastasis risk detection in Chinese breast cancer cases

Example 4: analysis method of second-generation sequencing detection kit for evaluating breast cancer molecular typing and distant metastasis risk gene group

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 Inc. (product No. Roche cache Number #3270289001) or an RNA extraction kit manufactured by Qiangen Inc. (Qiagen RNease FFPE kit, cache Number # 73504).

And step 3: the resulting RNA is made into a library for sequencing. Making the RNA of the obtained tissue into a library for targeted RNA-seq technology next generation sequencing, wherein the preparation method of the library comprises the following steps:

the RNA of the extracted tissue is used for generating cDNA of various genes (such as 72 genes shown in Table 1) of interest by reverse transcriptase under the direction of specific primers. The ends were filled in and phosphorylated at the 5' end, and 30. mu.l of DNA, 45. mu.l of pure water, 10. mu.l of T4DNA ligase buffer with 10mMATP, and 4. mu.l of a buffer containing 10mM dNTP Mix, 5. mu.l of T4DNA polymerase, 1. mu.l of Klenow enzyme, and 5. mu.l of T4 ligase were mixed, incubated at 20 ℃ for 30 minutes (reagents from Illumina sample preparation kit PE-102-1001), and after incubation, DNA was purified using QIAGEN QIAquick PCR purification kit (part # 28104). End suspension A. the product of the previous step was dissolved in 32. mu.l buffer, 5. mu.l of Klenow buffer, 1mM dATP 10. mu.l, Klenow Ae χ -3. mu.l, held at 37 ℃ for 30 minutes (reagents from Illumina specimen preparation kit), and the product was ligated by QIAGEN MinElute PCR purification kit (part # 28004): the DNA was dissolved in 10. mu.l of buffer, and 2X 25. mu.l of DNA ligase buffer, 10. mu.l of PE Adapter oligo Mix, and 5. mu.l of DNA ligase were added, and the mixture was kept at 20 ℃ for 15 minutes (reagent: Illumina sample preparation kit PE-102-.

And 4, step 4: the resulting DNA library was subjected to next generation sequencing with NextSeq/MiSeq/MiniSeq/iSeq using the primer sequences in Table 2 of example 3. Paired-end sequencing was performed using an Illumina NextSeq/MiSeq/MiniSeq/iSeq sequencer. This process is done automatically by the instrument itself (Illumina).

And 5: and (5) carrying out statistical analysis on results. And carrying out statistical analysis on the obtained sequencing result, and carrying out breast cancer typing and risk prediction according to an optimized method such as a Single Sample Predictor (SSP) or Parker provided by Hu and the like. And analyzing the obtained sequencing result gene expression data.

Example 5: sensitivity of the device

The detection method of the present invention is best for both sensitivity and copy number detection capability. The experimental research of the invention shows that the Sensitivity (Sensitivity) of the gene expression profile measured by the second-generation sequencing is far higher than that of the gene chip method, and the detection flux is better than that of the quantitative PCR and Nanostring methods (figure 5).

Example 6: repeatability of

The detection method has high repeatability. In 7 repeated experiments with paraffin tissue RNA, the correlation coefficient is higher than 0.97. With 15 replicates of fresh frozen tissue, the correlation coefficient was higher than 0.99 (fig. 6).

While the invention has been illustrated and described with reference to exemplary embodiments, the invention is not intended to be limited to the details shown. Since various modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, it is intended that all such modifications and equivalents fall within the spirit and scope of the invention as defined by the appended claims, using routine experimentation.

Claims

1. A gene cluster for molecular typing of breast cancer and/or assessing the risk of distant metastasis thereof,

the gene group comprises 66 molecular typing and distant metastasis risk assessment related genes and 6 housekeeping genes, wherein,

(5) HER2 related genes ERBB2, FGFR4 and GRB7,

(6) invasion-associated genes CTSL2 and MMP 11;

the 6 housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3a1, and TFRC.

2. Use of the gene cluster of claim 1 for molecular typing of breast cancer and/or for assessing the risk of distant metastasis thereof.

3. Use of a gene population according to claim 1 for the preparation of a diagnostic product for molecular typing of breast cancer and/or for assessing the risk of distant metastasis thereof.

4. Use of an agent for detecting the expression level of a gene in the gene population according to claim 1 for the manufacture of a diagnostic product for molecular typing of breast cancer and/or for assessing the risk of distant metastasis thereof.

5. A diagnostic product for molecular typing of breast cancer and/or assessing the risk of distant metastasis thereof, comprising a reagent associated with detecting the expression level of a gene in the gene population of claim 1.

6. The use or diagnostic product according to claim 4 or 5, in the form of an in vitro diagnostic product, preferably a diagnostic kit.

7. Use or diagnostic product according to any of claims 4 to 6, wherein said agent is an agent for measuring the amount of RNA, in particular mRNA, transcribed from said gene.

8. The use or diagnostic product of any one of claims 4 to 7, wherein said reagent is a reagent for detecting the amount of cDNA complementary to said mRNA.

9. The use or diagnostic product according to any one of claims 4 to 8, wherein said diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent and/or a secondary sequencing reagent.

10. Use or diagnostic product according to any of claims 4 to 9, wherein said agent is an agent for detecting the amount of a polypeptide encoded by said gene, preferably wherein said agent is an antibody, an antibody fragment or an affinity protein.

11. The use or diagnostic product of any one of claims 4 to 10, wherein the agent is a probe or primer, preferably a primer.

12. The use or diagnostic product of claim 11, wherein the primer has the sequence shown in SEQ ID No.1 to SEQ ID No. 144.

13. A set of primers for molecular typing of breast cancer and/or assessing the risk of distant metastasis thereof, wherein the sequence of the primers is shown in SEQ ID No.1-SEQ ID No. 144.

14. Use of a primer set according to claim 13 for the preparation of a product for molecular typing and/or assessing the risk of distant metastasis of breast cancer.

15. The panel of genes, use, diagnostic product or primer set of claims 1-14, wherein said breast cancer comprises luminal a, luminal B, HER2-enriched, basal cell type and immunopotentiated.