WO2020064006A2 - Gene group used for breast cancer molecular typing and distant metastasis risk, diagnostic product, and application - Google Patents

Abstract

Disclosed are a gene group used for breast cancer subtyping and distant metastasis risk evaluation, an in vitro diagnostic product, and an application.

Description

Breast cancer molecular typing and distant metastasis risk gene group and diagnostic products and applications

This application claims the priority of Chinese Patent Application No. 201811166148.7, entitled "Molecular Typing and Breast Cancer Risk Genomes and Diagnostic Products and Applications", filed on September 30, 2018, the content of which is incorporated by reference in its entirety Join this article.

Technical field

The invention belongs to the field of biotechnology, and particularly relates to breast cancer subtypes and distant metastasis risk gene groups, and in vitro diagnostic products and applications thereof.

Background technique

Breast cancer accounts for the first incidence of malignant tumors in women in China, and it is increasing year by year at a rate of about 4%. Breast cancer is a highly heterogeneous tumor, and its pathological molecular mechanism is very complicated, often involving mutations of multiple genes in many types of somatic cells. Breast cancer classification according to the pathological characteristics of the tumor, improving the accuracy of clinical diagnosis and prognosis prediction of breast cancer, is the basis of accurate treatment after breast cancer surgery, and also the key to improving the survival rate of breast cancer patients. At present, the accurate treatment of breast cancer after surgery at home and abroad still faces great challenges. For patients undergoing subsequent treatment, how to choose a targeted treatment plan and improve treatment efficiency requires new technical means to help doctors and patients make accurate judgments. How to accurately classify breast cancer patients, and then guide clinical treatment for effective treatment, while avoiding ineffective or harmful treatment is the main objective of the present invention.

The internationally recognized breast cancer molecular typing product based on tumor gene expression is the PAM50 (Prosigna) jointly developed by Professor Perou of the University of North Carolina and NanoString Corporation, which has been approved by the US FDA. By detecting the expression levels of 55 genes, PAM50 divides breast cancer into four subtypes, lumenal A (Luminal A), lumenal B (Luminal B), and HER2 enriched type ( HER2-enriched (HER2 for short) and Basal-like (Basal), and the risk of distant metastasis in breast cancer within 10 years was evaluated based on the subtype and tumor proliferation index.

Summary of the Invention

In one aspect, the present invention relates to a group of gene groups, including 66 molecular typing and genes related to distant metastasis risk assessment, and the use of the gene group in molecular diagnosis of breast cancer. The gene population can be used for molecular typing of breast cancer and / or assessing its risk of distant metastasis.

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

(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 related 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 related genes CTSL2 and MMP11.

In one embodiment, the gene population further includes a housekeeping gene. Preferably, the housekeeping genes include at least one of the following (eg, 1, 2, 3, 4, 5, or 6), preferably at least 3, and most preferably all 6: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC.

In a specific aspect, the present invention relates to a gene group (also referred to herein as Precitype) for molecular typing of breast cancer and / or assessing its risk of distant metastasis, which includes 72 Genes, that is, 66 molecular typing and distant metastasis risk assessment related genes and 6 housekeeping genes, of which

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

(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 related 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 related genes CTSL2 and MMP11;

The six housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC.

For information on genes in the gene group of the present invention, see Table 1 in one embodiment.

In another aspect, the present invention relates to the use of the genetic population in performing breast cancer molecular typing and / or assessing the risk of distant metastases.

In yet another aspect, the invention also relates to the use of the gene population in the preparation of a diagnostic product for molecularly typing breast cancer and / or assessing the risk of distant metastases. In a further aspect, the invention also relates to a method of molecularly typing breast cancer and / or assessing the risk of distant metastases, said method being performed by using a diagnostic product of the invention.

In a further aspect, the present invention also relates to a diagnostic product for molecularly typing breast cancer and / or assessing the risk of distant metastases, which comprises a reagent for detecting the expression level of a gene in the gene group of the present 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 second-generation sequencing reagent.

In a further embodiment, the reagent is a probe or a primer.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that according to the expression level of 66 genes in breast cancer tissue, breast cancer is divided into luminal A (Lum A), luminal B (Lum B), basal cell type (Basal), and HER2 enriched type (HER2 ) And immune-enhanced (Imm). A, 1951 European and American cases; B, 824 Chinese cases. Invasion-related genes (ie, CTSL2 and MMP11) have fewer genes and have fewer gene expression regions in the heat map, so they are not shown in Figure 1.

Figure 2 shows that the risk of distant metastasis is different for each subtype of breast cancer. Among them, the risk of distant metastasis of luminal type A was significantly lower than the other four subtypes, and the risk of distant metastasis of immune-enhanced type was significantly lower than that of luminal type B, basal cell type and HER2 enriched type. The risk of distant metastasis within 5 years after surgery in European and American cases. Lumen B is less than basal cell type (Basal), while basal cell type (Basal) is less than HER2 enriched type (HER2); 10 years after surgery There is no significant difference between the three. In Chinese patients, the lumen B type was smaller than the basal cell type and the HER2 enriched type within 3 years of surgery. After 10 years of surgery, the HER2 enriched type had the worst prognosis. A, 1951 European and American cases; B, 824 Chinese cases.

Figure 3 shows that the immune index has an important effect on the prognosis of breast cancer. Among them, for the high-risk breast cancer subtypes of lumen B (Lum B), basal cell type (Basal) and HER2 enriched type (HER2), strong immune function can significantly reduce the risk of distant metastasis of breast cancer in patients ; For low-risk breast cancer subtype lumen A, the strength of immune function has no significant effect on the prognosis. A, 1951 European and American cases; B, 824 Chinese cases.

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

FIG. 5 shows the second-generation sequencing (NGS) -based gene expression detection of the present invention, and its sensitivity and detection throughput are better than the existing gene chip, quantitative PCR, and Nanostring technologies.

Figure 6 shows the results of reproducible experiments, which show that the RNA (left) extracted from paraffin sections and the RNA (right) extracted from fresh tissue have excellent correlation.

detailed description

General definitions and terms

The present invention will be described in further detail below, it being understood that the terms are intended to describe the purpose, not to limit the present invention.

Unless stated otherwise, the 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 definition provided in this application shall prevail. When a certain amount, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical value and a preferred lower numerical value, it should be understood that it is equivalent to specifically revealing that the Any range combined with any lower range limit or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) 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 (for example, within a 95% confidence interval for the mean) or within ± 10 of a specified value %, Or a wider range.

The term "optional" or "optionally present" means that the event or situation described later may or may not occur, and the description includes the occurrence or non-occurrence of the event or situation. For example, when a group is described as being optionally substituted, it may be unsubstituted or substituted, such as with one or more substituents independently selected from those described herein. It should be understood by those skilled in the art that the meaning of the kind and number of substituents may be optionally selected and combined, so long as the formed compound is stable.

The expression "comprising" or similar expressions "including," "containing," and "having" are open-ended, and do not exclude additional unrecited elements, steps, or ingredients. 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 ingredients, plus elements, steps or ingredients that are optionally present and do not substantially affect the basic and new characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

In this text, the expression "at least one (species)" or similar expression "one (species) or multiple (species)" means 1 (species), 2 (species), 3 (species), 4 ( Species), 5 (species), 6 (species), 7 (species), 8 (species), 9 (species) or more (species).

The term "breast cancer" originates from ducts and acinar epithelium at all levels of the breast, and gradually develops from glandular epithelium to atypical hyperplasia. It is divided into carcinoma in situ (non-invasive cancer), early invasive cancer to invasive cancer. In a preferred embodiment, the breast cancer is invasive breast cancer.

The term "risk of recurrence" refers to the likelihood of a breast cancer patient's tumor recurrence within a specified number of years, including, but not limited to, localized breast cancer recurrence, regional breast cancer recurrence, and distant metastasis. As used herein, "risk of relapse" preferably indicates the possibility of distant metastasis. Therefore, in this article, the assessment of "Risk of Recurrence Score" (RRS) can be expressed as "distant recurrence risk score" or "distant metastasis risk score".

Breast cancer recurrence risk assessment can be used to assess the possibility of local recurrence or distant metastases in breast cancer patients. In this article, breast cancer can be classified into low risk, medium risk, and high risk according to the recurrence risk score. Chemotherapy is generally considered to be low risk, while chemotherapy at high risk reduces the risk of recurrence or distant metastases. In contrast, the medium risk requires the clinician to make judgments based on the actual clinical situation. Such a "medium risk" has less guiding significance in clinical treatment. Therefore, the fewer cases of medium risk, the more efficient the assessment will be. In the examples herein, the risk of breast cancer recurrence is preferably assessed for the possibility of distant metastases.

When molecularly typing breast cancer, you may also encounter situations that have characteristics similar to normal breast tissue and are therefore called Normal-like. Without being bound by any theory, it is thought that it may be caused by "contamination" of normal breast tissue mixed in tumor tissue samples, so it is usually excluded in common practice, such as when performing molecular typing or assessing risk .

Mixed is a breast cancer case that cannot be distinguished by the current molecular typing method, and it is not a specific tumor type. Due to its complex characteristics, such cases cannot guide the choice of treatment options based on their typing characteristics. Therefore, when typing breast cancer, the fewer such cases, the higher the typing efficiency.

Detection of gene expression levels described herein can be achieved, for example, by detecting RNA transcripts, or by proteomics methods, for example. The term "RNA transcript" refers to total RNA, that is, coding or non-coding RNA, including RNA directly from tissue or peripheral blood samples, and also indirectly from tissue or blood samples after cell lysis. Total RNA includes tRNA, mRNA, and rRNA, where mRNA includes mRNA transcribed from the target gene, and mRNA from other non-target genes.

The term "polypeptide" refers to a compound consisting of amino acids connected by peptide bonds, including the full length or amino acid fragments of a polypeptide. The amount of the polypeptide encoded by the gene can be standardized with respect to the amount of total protein in the sample or the amount of the polypeptide encoded by the housekeeping gene.

Herein, RNA transcripts can be detected and quantified, for example, by methods of hybridization, amplification or sequencing. For example, hybridize RNA transcripts with probes or primers.

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

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

The term "(hybridization) probe" refers to a nucleic acid sequence including at least 5 nucleotides, for example, containing 5 to 100 nucleotides, which can interact with the expression product of the target gene or the expansion of the expression product under specified conditions. The gain products hybridize to form a complex. The hybridization probe may further include a label for detection. The label includes, but is not limited to, a label for fluorescent quantitative PCR or fluorescent in situ hybridization. In a preferred embodiment, the label may be FAM, HEX, VIC, Cy5, or the like. In another preferred embodiment, the label may be biotin, digoxin and the like.

The detection of the expression level of a gene described herein may employ assay methods known in the art, including, but not limited to, a method of detecting the amount of an RNA transcript of the gene or the amount of a polypeptide encoded by the gene.

The RNA transcript of a gene can be converted into cDNA complementary thereto by methods known in the art, and the amount of RNA transcript can be obtained by measuring the amount of complementary cDNA. The amount of a gene's RNA transcript or its complementary cDNA can be normalized to the amount of total RNA or total cDNA in a sample or to a set of housekeeping genes' RNA transcripts or its complementary cDNA.

Herein, RNA transcripts can be detected and quantified by methods such as hybridization, amplification or sequencing, including but not limited to methods for hybridizing RNA transcripts with probes or primers, by polymerase chain reaction (PCR) -based A method of detecting the amount of RNA transcript or its corresponding cDNA product by various quantitative PCR techniques or sequencing techniques. The quantitative PCR technology includes, but is not limited to, fluorescent quantitative PCR, real-time PCR, or semi-quantitative PCR technology. The sequencing technology includes, but is not limited to, Sanger sequencing, second-generation sequencing, third-generation sequencing, and single-cell sequencing. Preferably, the sequencing technology is second-generation sequencing, more preferably targeted RNA-seq technology.

Herein, the amount of polypeptide can be detected by, for example, proteomics or reagents. Preferably, the reagent is an antibody, an antibody fragment or an affinity protein.

Gene group of the present invention

The invention relates to a group of gene groups, which includes 66 genes for molecular typing and distant metastasis risk assessment. The 66 molecular typing and distant metastasis risk assessment related genes include:

(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 related 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 related genes CTSL2 and MMP11.

In one embodiment, the gene population of the present invention further includes a housekeeping gene. Preferably, the housekeeping gene includes at least one of the following (eg, 1, 2, 3, 4, 5, or 6), preferably at least 3, and most preferably 6: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC.

In a specific embodiment of the present invention, a set of gene groups is provided, which includes 72 genes, that is, 66 molecular typing and distant metastasis risk assessment related genes, and 6 housekeeping genes. This gene group is also referred to herein as Precitype. The 66 molecular typing and distant metastasis risk assessment related genes and 6 housekeeping genes are as described above.

In one embodiment, the gene group of the present invention is shown in Table 1.

Table 1 Genetic groups of breast cancer molecular typing and / or distant metastasis risk

Housekeeping genes are used to normalize and correct target gene expression levels. Inclusion criteria for housekeeping genes that can be considered are: 1. Stable expression in tissues, whose expression level is not affected or less affected by pathological conditions or drug treatment; 2. Expression levels should not be too high to avoid expression data ( (For example, obtained through second-generation sequencing), the proportion of data obtained is too high, which affects the accuracy of data detection and interpretation of other genes. Therefore, housekeeping genes may be included in the gene group of the present invention. Preferably, the housekeeping genes considered include at least one of GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC, such as 1, 2, 3, 4, 5, or 6, preferably at least 3, and most preferably all 6 . In one embodiment, the housekeeping genes considered include GAPDH, GUSB, MRPL19, PSMC4, SF3A1, TFRC, or any combination thereof.

In another embodiment, the present invention also relates to a group of immune-related genes comprising 17 genes: APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ, and PTPRC.

The genes in the immune-related gene group are also shown in the related information in Table 1.

In a specific embodiment, the gene population of the present invention can be used for molecular typing (subtyping) of breast cancer and / or assessing its risk of distant metastasis.

In a specific embodiment, the subtypes of breast cancer include luminal A, luminal B, HER2-enriched, basal cell-like, and immune-enhanced.

Diagnostic product of the invention

The invention relates to a diagnostic product for molecularly typing breast cancer and / or assessing its risk of distant metastasis, which comprises an agent for detecting the expression level of genes in the gene group of the invention. The gene population is as described above.

Specifically, the gene group of the present invention may include 66 genes related to molecular typing and distant metastasis risk assessment. The 66 molecular typing and distant metastasis risk assessment related genes include:

(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 related 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 related genes CTSL2 and MMP11.

In one embodiment, the gene population of the present invention further includes a housekeeping gene. Preferably, the housekeeping gene includes at least one of the following (eg, 1, 2, 3, 4, 5, or 6), preferably at least 3, and most preferably 6: GAPDH, GUSB, MRPL19, PSMC4, SF3A1 and TFRC.

In one embodiment, the gene group is 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 subtype and / or distant metastasis risk assessment.

In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed by 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 includes a total RNA extraction reagent, a reverse transcription reagent, and / or a second-generation sequencing reagent.

The total RNA extraction reagent may be a conventional total RNA extraction reagent in the art. Examples include, but are not limited to, Qiagen 73504, RNA Storm CD201, Invitrogen and ABI AM1975.

The reverse transcription reagent may be a conventional reverse transcription reagent 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.

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

Targeted RNA Index Kit A-96Indices(384Samples)(RT-402-1001)。二代测序为本领域常规的二代测序，例如为靶向RNA-seq技术。因此，二代测序试剂还可以包括可供构建靶向RNA-seq的文库Illumina定制的试剂，例如

Targeted RNA Custom Panel Kit(96 Samples)(RT-102-1001)。 The second-generation sequencing reagent may be a reagent commonly used in the art, as long as it can meet the requirements for performing second-generation sequencing on the obtained sequence. Second-generation sequencing reagents can be commercially available products, examples of which include, but are not limited to, Illumina

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

Targeted RNA Index Kit A-96 Indicators (384 Samples) (RT-402-1001). Next-generation sequencing is conventional second-generation sequencing in the art, such as targeted RNA-seq technology. Therefore, next-generation sequencing reagents can also include Illumina custom-made reagents that can be used to build libraries that target RNA-seq, such as

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

In a preferred embodiment, the reagent is a probe or a primer.

In an alternative embodiment, the reagent is a reagent that detects the amount of the polypeptide encoded by the gene, and preferably, the reagent 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 synthetic oligonucleotide fragment (preferably having high specificity) as long as it is complementary to a partial sequence of a gene in the gene group of the present invention, and the gene therein is amplified. Primers can be synthetic. More preferably, the sequences of the primers are as shown in SEQ ID No. 1-SEQ ID No. 132 and SEQ ID NO. 133-SEQ ID NO. 144. The specific protocol of the primers can be seen in Table 2.

Therefore, an embodiment of the present invention also relates to a diagnostic product (preferably an in vitro diagnostic product, especially a kit), which comprises a primer whose sequence is as described in SEQ ID NO.1-SEQ ID NO.132. In one embodiment, the diagnostic product further comprises a primer, and the sequence of the primer is as described in SEQ ID NO. 133-SEQ ID NO. 144.

In a preferred embodiment, the diagnostic product of the present invention (preferably an in vitro diagnostic product, especially a kit) comprises a primer whose sequence is shown in SEQ ID NO.1-SEQ ID NO.144 (see also Table 2 ).

The diagnostic product (preferably in the form of a kit) of the present invention also preferably includes a device for extracting a test sample from a subject; for example, a device for extracting tissue or blood from a subject, preferably any blood that can be used for blood collection Needles, syringes, etc. The subject is a mammal, preferably a human, especially a female with breast cancer.

In one embodiment, the primers can be used to detect the expression level of the gene population.

Method and application of the invention

In yet another aspect, the present invention also relates to a method for determining a breast cancer molecular typing and / or distant metastatic risk in a subject, the method comprising

(1) provide a sample of the subject,

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

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

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

The subject for use in the method of the invention is a mammal, preferably a human, especially a female with breast cancer.

The method of providing a sample in step (1) and the sample used are not particularly limited, as long as the expression level of genes in a gene group can be obtained therefrom, for example, total RNA of a 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, especially a paraffin tissue sample. In a preferred embodiment, the sample is a tumor tissue sample or a tissue sample containing tumor cells, particularly a breast tumor tissue sample or a tissue sample containing breast tumor cells.

In step (2), the expression level of a gene in the gene group of the present invention is determined. This gene group is as described above. The gene group can also be described in Table 1. The method of the present invention, for example, step (2), can be performed by a reagent for detecting a gene expression level in the gene group. In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed by the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA. In an alternative embodiment, the reagent is a reagent that detects the amount of the polypeptide encoded by the gene, and preferably, the reagent is an antibody, an antibody fragment, or an affinity protein. In another embodiment, the reagent is a probe or a primer, especially a primer. More preferably, the sequence of the primer is as described in SEQ ID NO.1-SEQ ID NO.132. In one embodiment, a primer is also used, and the sequence of the primer is as described in SEQ ID NO. 133-SEQ ID NO. 144. The specific protocol of the primers can also be shown in Table 2.

The method of the present invention, such as step (2), can be performed by a diagnostic product (especially a diagnostic kit) of the present invention.

In a preferred embodiment, step (2) may include

(2-1) extracting total RNA from 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 step (2-2).

The extraction in step (2-1) can be performed by a conventional method in the art, and preferably, a total RNA of fresh frozen tissue or paraffin-embedded tissue of a subject is detected by using an RNA extraction kit. In a more preferred embodiment, extraction can be performed using Roche's RNA extraction kit (product number: RocheCatalogNumber # 3270289001) or Qiagen's RNA extraction kit (Qiagen, Rnease, FFPE, Kitalog ##, 504). .

In an exemplary embodiment, a method for constructing a library may include the following steps:

The extracted total RNA was reverse transcribed to generate cDNAs of the genes described in Table 1. The ends were filled and phosphorylated at the 5 ′ end. 30 μl of DNA, 45 μl of purified water, 10 μl of T4 DNA ligase buffer with 10 mM ATP, 4 μl of 10 mM dNTP Mix, 5 μl of T4 DNA polymerase, 1 μl of Klenow enzyme, and 5 μl of T4 ligase were mixed. Incubate at 20 ° C for 30 minutes (reagent is Illumina sample preparation kit PE-102-1001). After incubation, use QIAGEN QIAquick PCR purification kit (part # 28104) to purify DNA. End suspension A: Dissolve the product from the previous step in 32 μl of buffer, add 5 μl of Klenow buffer, 10 μl of 1 mM dATP, and 3 μl of Klenow Εχο, and keep at 37 ° C for 30 minutes (reagent is Illumina sample preparation kit). The products were ligated by QIAGEN MinElute PCR purification kit (part # 28004): DNA was dissolved in 10 μl buffer, DNA ligase buffer 2 × 25 μl, PE adapter Oligo Mix 10 μl, DNA ligase 5 μl, and kept at 20 ° C for 15 minutes ( The reagent was Illumina sample preparation kit PE-102-1001). After warming, the DNA was purified using QIAGEN QIAquick PCR purification kit (part # 28104) to obtain a library.

Step (2-3) can be completed by RNA sequencing. The sequencing method may be a conventional RNA-seq sequencing method for determining a gene expression level in the art. Illumina NextSeq / MiSeq / MiniSeq / iSeq series sequencers are preferably used for next-generation sequencing. The primers in the kit were used to amplify the genes shown in Table 1. Depending on the library prepared in step (2-2), the obtained gene sequence could be sequenced twice. Preferably, the secondary sequencing is a targeted RNA-seq technology, and double-ended sequencing is performed with an Illumina NextSeq / MiSeq / MiniSeq / iSeq sequencer. Such a process can be done automatically by the instrument itself.

In an embodiment of the present invention, step (3) may be completed by performing statistical analysis on the obtained sequencing results. Breast cancer typing and risk prediction can optionally be performed according to the single sample prediction method (SSP (Single Sample Predictor) and Parker) pioneered by Hu et al. The gene expression data of the obtained sequencing results are analyzed to obtain a subtype of a single sample, and the risk of distant metastasis can be calculated.

For the method, use, and product of the present invention, as an exemplary embodiment, the following procedures can be adopted.

First collected 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 for gene screening And optimization of gene combinations for breast cancer molecular typing and prognosis assessment. In addition to the gene expression data, the clinical information of each corresponding case, including case classification, lymph node metastasis, ER / PR detection, distant metastasis and time of occurrence, and treatment plan were collected and analyzed.

All gene chip data need to undergo standardization (RMA), batch correction (Combat), and the same gene combination before analysis for analysis, in order to eliminate gene expression caused by differences in technical platforms and operating procedures of each research cohort. The difference in data lays the foundation for the next genetic screening.

For the gene expression data of 2034 breast cancer cases that have been standardized, batch-corrected and merged with similar genes, the self-developed analysis software EPIG was used to calculate the close relationship with breast cancer metastasis through unsupervised clustering and paired correlation analysis. Relevant and statistically significant gene expression profiles, and based on this to obtain meaningful genes in the expression profile. Among them, the two most relevant genes are the cell cycle and immune response genes, respectively.

To confirm the stability of the discovered cell cycle and immune response-related genes in predicting disease relapse, stability analysis was further performed. The specific method is to randomly extract 1017 chip data (50%) from 2034 gene chip expression data, calculate expression profiles and related genes, and repeat the extraction and calculation 1000 times.

The results showed that 63 cell cycle-related genes and 121 immune response genes consistently appeared in the calculation results 1000 times. Combining the 19 cell cycle-related genes and 17 immune response genes that are most closely related to relapse-free survival, combined with ER, PR, HER2, and basal cell characteristic genes, a combination of 66 genes can be determined. Considering the housekeeping genes on this basis, a combination of 72 genes can be determined (see Table 1).

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

Accordingly, the present invention also provides the application of the gene population of the present invention to molecular typing of breast cancer and / or assessing the risk of distant metastasis.

The invention also provides the application of the gene group of the invention in the preparation of a product for molecularly typing breast cancer and / or assessing its risk of distant metastasis. In a preferred embodiment, the product is in the form of a detection kit.

The invention further provides the application of the reagent for detecting the gene expression level in the gene group of the invention in preparing an in vitro diagnostic product for molecularly typing breast cancer and / or assessing the risk of distant metastasis. In a preferred embodiment, the product is in the form of a detection kit. In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed by the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA. In an alternative embodiment, the reagent is a reagent that detects the amount of the polypeptide encoded by the gene, and preferably, the reagent is an antibody, an antibody fragment, or an affinity protein. In another embodiment, the reagent is a probe or a primer, especially a primer. More preferably, the sequences of the primers are as shown in SEQ ID No. 1-SEQ ID No. 132 and SEQ ID NO. 133-SEQ ID NO. 144. The specific protocol of the primers can also be shown in Table 2.

The subtypes of breast cancer include lumen A, lumen B, HER2 enriched, basal cell, and immune-enhanced.

The detection sample used in the present invention is preferably a tissue from a test object (subject object), as long as the 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, and more preferably a tissue sample, such as a paraffin tissue sample. In a preferred embodiment, the test sample is a tissue with a high content of tumor cells.

The present invention also relates to a group of proliferation-related genes including ASPM, AURKA, BIRC5, CCNB1, CDC20, CDK1, CENPU, CEP55, MELK, MKI67, NEK2, PRC1, PTTG1, RRM2, TOP2A, TPX2, TYMS, UBE2C, and ZWINT ( (See also the relevant information in Table 1).

The present invention also relates to a group of immune-related genes, including APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC ( (See also the relevant information in Table 1).

Accordingly, the present invention also relates to a combination of a proliferation-related gene and an immune-related gene, wherein the proliferation-related gene and the immune-related gene are as described above.

The present invention also relates to the immune-related genes (APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC) or all The proliferation-related genes (ASPM, AURKA, BIRC5, CCNB1, CDC20, CDK1, CENPU, CEP55, MELK, MKI67, NEK2, PRC1, PTTG1, RRM2, TOP2A, TPX2, TYMS, UBE2C, and ZWINT) or a combination of the two, Or the use of related reagents for detecting the immune-related genes, the proliferation-related genes, or a combination thereof in the preparation of in vitro diagnostic products for molecular typing of breast cancer and / or assessment of the risk of distant metastasis of breast cancer.

The invention also relates to the use of the immune-related genes or the proliferation-related genes or a combination thereof in performing molecular typing of breast cancer and / or assessing the risk of distant metastases. In one embodiment, the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed by the gene. In yet another embodiment, the reagent is a reagent that detects the amount of cDNA complementary to the mRNA. In an alternative embodiment, the reagent is a reagent that detects the amount of the polypeptide encoded by the gene, and preferably, the reagent is an antibody, an antibody fragment, or an affinity protein. In another embodiment, the reagent is a probe or a primer, especially a primer. More preferably, the sequences of the primers can be shown in Table 2.

Accordingly, the present invention also relates to a method for determining a breast cancer molecular typing and / or distant metastatic risk in a subject. The steps of the method are described above, wherein the corresponding gene group is the immune-related gene, the proliferation-related gene, or a combination thereof.

In the scheme of the present application, a new breast cancer subtype (immunoenhancement type) can be determined, and different effects and effects on distant metastasis of breast cancer can be shown in different subtypes.

The scheme of the present application can also be enumerated as follows.

A gene group for molecular typing of breast cancer and / or assessing the risk of distant metastasis, characterized in that the gene group comprises 66 genes related to molecular typing and risk assessment for distant metastasis, wherein:

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

(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 related 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 related genes CTSL2 and MMP11.

2. The gene group according to item 1, wherein

The gene group also includes a housekeeping gene;

Preferably, the housekeeping gene includes at least one of the following (such as 1, 2, 3, 4, 5, 6), preferably at least 3, and most preferably 6: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC.

3. A gene group for molecular typing of breast cancer and / or assessing its risk of distant metastasis, characterized by:

The gene group includes 66 genes related to molecular typing and distant metastasis risk assessment, and 6 housekeeping genes, of which,

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

(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 related 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 related genes CTSL2 and MMP11;

The six housekeeping genes include: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC.

4. Use of the genetic group according to any one of 1-3 in performing molecular typing of breast cancer and / or assessing its risk of distant metastasis.

5. Use of the genetic group of any one of items 1-3 in the preparation of a diagnostic product for molecularly typing breast cancer and / or assessing its risk of distant metastasis.

6. Use of a reagent for detecting the expression level of a gene in the gene group according to any one of items 1-3 in the preparation of a diagnostic product for molecularly typing breast cancer and / or assessing the risk of its distant metastasis.

7. A diagnostic product for molecularly typing breast cancer and / or assessing its risk of distant metastasis, comprising a reagent for detecting the expression level of a gene in the gene group according to any one of items 1-3.

8. The application or diagnostic product of item 6 or 7, characterized in that the diagnostic product is in the form of an in vitro diagnostic product, preferably in the form of a diagnostic kit.

9. The application or diagnostic product according to any one of items 6 to 8, wherein the reagent is a reagent that detects the amount of RNA, particularly mRNA, transcribed by the gene.

10. The application or diagnostic product according to any one of items 6 to 9, wherein the reagent is a reagent that detects an amount of cDNA complementary to the mRNA.

11. The application or diagnostic product according to any one of items 6 to 10, wherein the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent, and / or a second-generation sequencing reagent.

12. The application or diagnostic product according to any one of items 6 to 11, wherein the reagent is a reagent for detecting the amount of the polypeptide encoded by the gene, and preferably, the reagent is an antibody, an antibody fragment, or an affinity protein.

13. The application or diagnostic product according to any one of items 6 to 12, wherein the reagent is a probe or a primer, preferably a primer.

14. The application or diagnostic product of item 13, characterized in that the sequence of the primer is as shown in SEQ ID NO.1-SEQ ID NO.144.

15. A set of primers for molecular typing of breast cancer and / or assessing the risk of distant metastasis, wherein the sequence of said primers is shown in SEQ ID NO.1-SEQ ID NO.132.

16. The primer set of item 15, further comprising a primer having a sequence shown in SEQ ID NO. 133-SEQ ID NO. 144.

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

18. Use of the primer set of any of 15-17 in the production of a product for molecularly typing breast cancer and / or assessing its risk of distant metastasis.

19. The gene group, application, diagnostic product or primer set according to any one of items 1 to 18, wherein the breast cancer includes lumen A type, lumen B type, HER2 enriched type, basal cell type, and immunity Enhanced.

Beneficial effect

Compared with the existing technology (such as PAM50 technology), the scheme of the present invention introduces immune regulation genes into the molecular typing of breast cancer for the first time, and can further strengthen the rationality of molecular typing of breast cancer by considering the expression levels of other genes And improve the ability to guide the clinical treatment of breast cancer, and molecularly type breast cancer into lumen A, lumen B, HER2 enriched, basal cell, and immune-enhanced. A more accurate distinction can also be made between these types of transfer risks. For example, the risk of distant metastasis of luminal type A is significantly lower than the other four subtypes, while the risk of distant metastasis of immune-enhanced type is significantly lower than that of luminal type B, basal cell type and HER2 enriched type. Furthermore, the prognosis of breast cancer with different types can be judged more accurately. For example, for luminal B, basal cell, and HER2-enriched types in high-risk breast cancer subtypes, strong immune function can significantly reduce the risk of distant breast cancer metastasis.

For subtypes other than immune-enhanced, the immune genome can be further subdivided. In addition, the second-generation sequencing is used to detect gene expression and is used for molecular typing. The gene group and corresponding products of the present invention can increase detection sensitivity, improve detection capability and efficiency, and greatly reduce detection costs. Compared with the currently commonly used IHC technology, the present invention is more accurate for molecular typing of breast cancer, can calculate the risk of distant metastasis, predict the risk of distant metastasis, and can guide clinical treatment more accurately. Moreover, the products and methods of the present invention have high reliability, high sensitivity, and high repeatability. For example, in one aspect, the products and methods of the present invention can significantly reduce the proportion of types that cannot be typed, thereby providing more accurate and efficient typing methods and products. In another aspect, the products and methods of the present invention can also significantly reduce the so-called "medium risk" ratio. And this type of "medium risk" is difficult to judge in clinical treatment, and the guidance is poor.

Examples

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions specified in the following examples were selected according to conventional methods and conditions, or according to product specifications. The reagents and instruments used in the examples herein are all commercially available.

Example 1: Screening of genes associated with breast cancer subtypes and distant metastasis risk

Methods: 14 breast cancer cohort studies were analyzed by the EPIG gene expression profiling program (see Zhou, Zhou et al., 2006. Health, Perspective 114 (4), 553-559; Zhou, Zhou et al., 2007. BMC Bioinformatics 8,427). Gene expression of 1951 breast cancer tumors with complete clinical information in 2034 cases (GSE3494, GSE6532, GSE1456, GSE9195, GSE2034, GSE5327, GSE7390, GSE11121, GSE2603, GSE7378, GSE8193, GSE12093, and E-TABM-158), screened Immune-related genes and cell cycle genes that are closely related to the risk of distant metastasis of breast cancer, combined with the reported ER, PR and subtype typing related genes, calculate and optimize the risk of typing and distant metastasis in each group of genes Genes with a high contribution rate.

Results: A total of 66 genes and 6 housekeeping genes related to breast cancer subtype and distant metastasis risk were screened out, that is, 72 gene test combinations. The gene list is shown in Table 1.

Example 2: Using the screened breast cancer subtypes and distant metastasis risk-related gene groups to perform molecular typing and distant metastasis risk assessment of breast cancer

Experimental method: 72 gene test combinations were used. Among them, 66 breast cancer subtypes and distant metastasis risk-related genes (proliferation-related genes ASPM, AURKA, BIRC5, CCNB1, CDC20, CDK1, CENPU, CEP55, MELK, MKI67, NEK2, PRC1, PTTG1, RRM2, TOP2A , TPX2, TYMS, UBE2C and ZWINT, immune related genes APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC, Basal cell related genes ACTR3B, CDH3, EGFR, FOXC1, KRT14, KRT17, KRT5, MIA, MYC, PHGDH and SFRP1, estrogen receptor related genes BAG1, BCL2, BLVRA, CD68, ESR1, FOXA1, GSTM1, MPT2, MDM2, MLPH, NAT1, PGR, SCUBE2 and SLC39A6, HER2-related genes ERBB2, FGFR4 and GRB7, and invasion-related genes CTSL2 and MMP11) were used for molecular typing. Six internal reference genes (including GAPDH, GUSB, MRPL19, PSMC4, SF3A1 and TFRC) can be used as internal standards. In calculating the distant metastasis risk index, all 66 genes except Table 6 reference genes in Table 1 were used. Molecular typing (subtyping) and distant metastatic risk assessment of data from 1951 European and American breast cancer patients in the European and American breast cancer public databases (Figure 1A), and molecular typing of 824 Chinese breast cancer tumor samples (Subtype typing) and distant metastatic risk assessment (Figure 1B), and compare the two.

Experimental results:

1. Molecular typing of breast cancer

As mentioned above, the 66 breast cancer molecular genotyping groups shown in Table 1 were used to molecularly type the above breast cancer cases to divide breast cancer tumors into 5 subtypes (Figure 1), and their treatment can be given Different suggestions:

1) Lumenal A

Patients with luminal A tumors have a low mutation rate of p53 gene and a good prognosis. They are not sensitive to chemotherapy and are suitable for endocrine therapy. Therefore, it has guiding significance for clinical endocrine therapy.

2) Lumenal B

Lumen B is a tumor that is sensitive to endocrine therapy, but for HER2-positive patients, tamoxifen treatment is less effective than lumen A, and aromatase inhibitors are better. For patients with HER2-positive lumen type B tumors, molecular targeted therapy can be performed.

3) HER2-enriched

Patients with HER2-enriched tumors have a high mutation rate of p53 gene and relatively poor tumor differentiation. This type is relatively sensitive to targeted molecular therapy but has a poor prognosis. HER2-enriched tumors are widely treated with Herceptin combined with systemic chemotherapy.

4) Basal-like

Basal cell tumors are the most invasive tumors, namely triple negative tumors (ER-, HER2-, PR-). Basal cell tumor patients are not sensitive to current breast cancer treatment options and generally have a poor clinical prognosis; however, if the tumor tissues have strong immune-related gene expression, the prognosis is relatively good.

5) Immune-enhanced

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

By calculating the number and time of distant metastases in different subtypes, the Kaplan-Meier survival curve can be drawn to obtain the corresponding metastatic risk. As shown in Figure 2, the distant transfer risks of the five subtypes are different. Luminous type A has the lowest risk of distant metastasis, which is a low-risk subtype. For 5-year distant metastatic risk, luminal type B is lower than the HER2 enriched type and basal cell type. It is said that the lumen B type, HER2 enriched type and basal cell type are not significantly different, and they are all high-risk subtypes, while the immune-enhancing type is between the two groups and belongs to intermediate-risk subtypes.

2.Impact of immune index on risk of distant metastasis in different subtypes

Calculate the immune index based on the expression levels of 17 immune-related genes APOBEC3G, CCL5, CCR2, CD2, CD3D, CD52, CD53, CORO1A, CXCL9, GZMA, GZMK, HLA-DMA, HLA-DQA1, IL2RG, LCK, LYZ and PTPRC. Each subtype can be further divided into two groups based on the immune index, that is, the strong immune group and the weak immune group, and the difference in metastatic risk between the two groups is observed. Among them, Lumenal B, Basal-like and HER2-enriched subtypes, the risk of distant metastasis was significantly lower in patients with strong immunity (P <0.05). Immunely weak cases; Lumenal A, immunologically weak cases and distantly metastatic cases showed no significant difference in distant metastatic risk (Figure 3).

The immune index can be obtained by averaging the expression levels of the 17 immune genes:

3.Distant risk assessment

The Cox model is used to calculate the risk of distant metastasis of the tumor. The occurrence and time of distant metastasis are used as the observation endpoint. Corresponding coefficients are determined based on the relative risk of the tumor subtype, immune index, and proliferation index for distant metastasis. Methods as below:

Calculation of distant metastasis risk score (Risk of Recurrence Score, RRS): 0-100

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

RRS = 0.2x Basal + 0.4x HER2-0.2x Immune-0.1x LumA + 0.2x LumB + 0.3x proliferation index-0.1x immune index

Among them, "Basal" represents the pearson correlation coefficient between the tumor and the basal cell tumor;

"HER2" represents the pearson correlation coefficient between the tumor and the HER2-enriched tumor;

"LumA" represents the pearson correlation coefficient between this tumor and lumen type A tumor;

"LumB" represents the pearson correlation coefficient between the tumor and luminal type B tumor;

"Immune" represents the pearson correlation coefficient between the tumor and the immune-enhanced tumor.

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

For patients in the low-risk group, adjuvant chemotherapy is not recommended if the lymph node test is negative.

Example 3: Primer and Probe Design and Laboratory Validation to Evaluate Immune-Related Genes in High-risk Breast Cancer Subtypes

Experimental method: Targeted RNA-seq technology based on Illumina mid-throughput second-generation sequencing platform MiSeq was used to detect fresh tumor tissue or paraffin-embedded tumor tissue of 300 Chinese breast cancer patients. The expression levels of 72 genes including genes were used as the basis for further breast cancer typing.

Experimental results:

1. 72 pairs of primers for detecting 72 genes screened in Example 1 are shown in Table 2.

Table 2: Genes of the gene group of the present invention and their amplification primers

2. Establishment of second-generation sequencing database. The raw data of all next-generation sequencing of fresh tumor tissue or paraffin-embedded tumor tissue of 300 Chinese breast cancer patients described in Example 3 will be uploaded to a web-based data storage and analysis database. This method uses Java software to develop and apply many J2EE (Java Enterprise Edition) components and modes. It can 1) directly input data from Illumina NextSeq / MiSeq / MiniSeq / iSeq instruments; 2) it can display the entered data in a flexible manner. Data, and can be indexed according to different needs, such as gene correlation, samples or experimental groups; 3) calculation of gene expression profiles after normalization of housekeeping genes; 4) analysis of specific element details; 5) , Output data in different formats, such as XML, excel and text formats; 6), can be safely managed to ensure data privacy protection.

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

Obtain postoperative paraffin samples from Chinese breast cancer patients through partner hospitals, extract RNA in accordance with the previous steps, perform quality inspection, build a library by reverse transcription, use the Illumina NextSeq / MiSeq / MiniSeq / iSeq platform for RNA sequencing, and detect 72 gene expression levels The subtype classification and distant metastasis risk (RRS, also referred to as distant recurrence risk or Precitype Risk Score (PRS) in this paper) were calculated, and 300 samples were finally obtained. As mentioned above, Mixed is not included in the statistical results of this embodiment.

Table 3. Molecular genotyping of 72 genes and detection of distant metastatic risk in 300 Chinese breast cancer cases

Example 4: Analysis method of second-generation sequencing test kit for assessing breast cancer molecular typing and distant metastasis risk gene groups

Step 1: Take the tumor or paraffin-embedded tissue of the test object, and use the method in the test kit to obtain the region of the test object that contains high tumor cells as the original material.

Step 2: Extract total RNA from the tissue. You can use RNA extraction kits produced by Roche to extract RNA from tissues (product number is RocheCatalogNumber # 3270289001) or Qiagen's RNA extraction kits to extract (QiagenRNeaseFFFF kit, CatalogNumber # 73504).

Step 3: The obtained RNA is made into a library for sequencing. The RNA of the obtained tissue is made into a library that can be used for targeted RNA-seq second-generation sequencing. The method for preparing the library includes the following steps:

The RNA of the extracted tissue was generated under the guidance of a specific primer using reverse transcriptase to generate cDNAs of a variety of genes of interest (72 genes described in Table 1). The ends were filled and phosphorylated at the 5 ′ end. 30 μl of DNA, 45 μl of purified water, 10 μl of T4 DNA ligase buffer with 10 mM ATP, 4 μl of 10 mM dNTP Mix, 5 μl of T4 DNA polymerase, 1 μl of Klenow enzyme, 5 μl of Τ4 After mixing, incubate at 20 ° C for 30 minutes (reagents from Illumina sample preparation kit PE-102-1001). After incubation, the DNA was purified using QIAGEN QIAquick PCR purification kit (part # 28104). End suspension A: Dissolve the product from the previous step in 32 μl of buffer, add 5 μl of Klenow buffer, 10 μl of 1 mM dATP, and 3 μl of Klenow Εχο-3 μl, and keep at 37 ° C for 30 minutes (reagent from Illumina sample preparation kit). MinElute PCR purification kit (part # 28004) ligation: DNA was dissolved in 10 μl buffer, DNA ligase buffer 2 × 25 μl, PE adapter Adapter Oligo Mix 10 μl, DNA ligase 5 μl, and kept at 20 ° C for 15 minutes (reagent is Illumina sample Prepare the kit PE-102-1001). After warming, use the QIAGEN QIAquick PCR purification kit (part # 28104) to purify the DNA to obtain the library.

Step 4: Use the primer sequences in Table 2 of Example 3 to perform the next-generation sequencing on the resulting DNA library using NextSeq / MiSeq / MiniSeq / iSeq. Double-ended sequencing was performed with an Illumina NextSeq / MiSeq / MiniSeq / iSeq sequencer. This process is done automatically by the instrument itself (Illumina).

Step 5: Statistical analysis of results. The obtained sequencing results were analyzed statistically, and breast cancer typing and risk prediction were performed according to the single sample prediction method (SSP (Single Sample Predictor) or Parker) proposed by Hu et al. The gene expression data of the obtained sequencing results were analyzed.

Example 5: Sensitivity

The detection method of the present invention is the best in both sensitivity and copy number detection capabilities. The experimental research of the present invention shows that the sensitivity of the second-generation sequencing to determine the gene expression profile is much higher than that of the gene chip method, and at the same time, it is better than the quantitative PCR and Nanostring method in the detection throughput (Figure 5).

Example 6: Repeatability

The detection method of the present invention has high repeatability. Correlation coefficients were higher than 0.97 in 7 repeated experiments with paraffin tissue RNA. With 15 replicates of fresh frozen tissue, the correlation coefficient was higher than 0.99 (Figure 6).

Comparative example

In the following comparative examples, the scheme of the gene group used in the present invention is compared with part of the prior art. As described above, when performing typing comparisons, normal cell (like) types are not considered.

Comparative Example 1: Comparison of the scheme of the present invention with respect to the prior art

CN 104293910A discloses a group of gene groups, which are composed of 60 genes (Comparative I). It can be seen from the comparison between the scheme of the gene group of the present invention and the scheme of the present invention that a new immune-enhancing type is introduced, so that the subtype of breast cancer can be classified more accurately. Moreover, these types of transfer risks can be more accurately distinguished. As mentioned above, the risk of distant metastasis of luminal type A is significantly lower than the other four subtypes, while the risk of distant metastasis of immune-enhanced type is significantly lower than that of luminal type B, basal cell type and HER2 enriched type. Furthermore, the prognosis of breast cancer with different types can be judged more accurately. For lumen B, basal cell, and HER2-enriched types in high-risk breast cancer subtypes, strong immune function can significantly reduce the risk of distant metastasis of breast cancer.

In addition, using the method described in Example 2 of the present invention, 1951 breast cancer tumors with complete clinical information in 2034 cases were subjected to typing analysis and distant metastasis risk assessment. The scheme of the present invention (such as 66 in Table 1) Molecular typing and risk of distant metastasis and 6 housekeeping genes) were compared with comparison I. The results are shown in Tables 4 and 5. For a detailed description of the results, please refer to Comparative Example 2.

Table 4

table 5

As can be seen from the above table, in addition to the above advantages, compared with the prior art, the scheme of the present invention can significantly reduce the proportion of Mixed cases. Compared with the solution of the prior art, the solution of the present invention provides a more complete analysis system, for example, the types of typing are increased. At the same time, more accurate typing can be achieved. It also significantly reduced the number of Mixed cases. The effect of the scheme of the present invention is obviously better than that of the prior art, and the proportion of medium risk is significantly reduced.

Comparative Example 2: Impact of related gene selection

1. Impact on molecular typing

In order to compare the role of gene group selection more clearly, the scheme of the present invention (66 molecular typing and distant metastatic risk and 6 housekeeping genes described in Table 1) is compared with comparison II and comparison III below. For a more detailed analysis of gene selection. For the method, see Comparative Example 1. The results are shown in Table 6.

Comparison II (59 genes): The 20 proliferation-related genes in the above comparison I were replaced with 19 proliferation-related genes used in the scheme of the present invention.

Comparison III (77 genes): The above comparison I was added to the 17 immune-related genes used in the present invention.

Table 6

Program Mixed ratio Solution of the invention 3.73% Contrast I (60 genes) 14.65% Contrast II (59 genes) 12.70% Contrast III (77 genes) 9.43%

As can be seen from the above table, compared with the prior art, the scheme of the present invention can significantly reduce the proportion of Mixed cases. From the differences between the protocol of the present invention and Comparative II or the difference between Comparative I and Comparative III shown in the table above, it can be known that the protocol of the present invention introduces immune regulatory genes into the molecular typing of breast cancer for the first time, making the mixed case The proportion is significantly reduced.

From the difference between comparison I and comparison II, it can be known that only changing the proliferation-related genes (replacement of the proliferation-related genes in the prior art scheme with the proliferation-related genes in the scheme of the present invention) can reduce the proportion of Mixed cases, and the typing method Improved efficiency. It can be known from the difference between the scheme of the present invention and the comparison III that after the introduction of immune-related genes, the change of the proliferation-related genes has a particularly significant effect on the efficiency of the typing method. The combination of the proliferation-related genes and the immune-related genes contained in the scheme of the present invention has better effect than the prior art.

2. Impact on distant transfer risk assessment

In accordance with the method of Example 2, the protocol of the present invention (66 molecular typing and distant metastatic risk and 6 housekeeping genes described in Table 1) is compared with Comparative I, Comparative II, and Comparative III below. The distant metastasis risk score was calculated based on the gene expression level in the 1951 breast cancers described above, and the risk of distant metastasis was divided into three groups: low risk, medium risk, and high risk. The results are shown in Table 7, where Shows the proportion of "medium risk" types that are less instructive in clinical treatment in the total sample.

Table 7

Program Risk ratio Solution of the invention 16.50% Contrast I (60 genes) 25.83% Contrast II (59 genes) 23.58% Contrast III (77 genes) 21.22%

As can be seen from the above table, the effect of the scheme of the present invention is significantly better than that of the prior art, and the proportion of risks is significantly reduced.

Although the invention has been illustrated and described with typical embodiments, the invention is not limited to the details. Since various possible modifications and substitutions do not depart from the spirit of the present invention, those skilled in the art can use the variations and equivalents of the present invention that can be conceived by routine experimentation, so all these variations and equivalents fall within the scope of the appended claims. Within the spirit and scope of the invention as defined.

Claims (16)

A genetic group for molecular typing of breast cancer and / or assessing its risk of distant metastasis, characterized in that: The gene group includes 66 genes related to molecular typing and distant metastasis risk assessment, of which, The 66 molecular typing and distant metastasis risk assessment related genes include: (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 related 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 related genes CTSL2 and MMP11. The gene group according to claim 1, wherein: The gene group also includes a housekeeping gene; Preferably, the housekeeping gene includes at least one, preferably at least 3, and most preferably 6 of the following: GAPDH, GUSB, MRPL19, PSMC4, SF3A1, and TFRC. The use of the gene group according to claim 1 or 2 for performing breast cancer molecular typing and / or assessing the risk of distant metastasis. The use of the gene group according to claim 1 or 2 in the preparation of a diagnostic product for molecularly typing breast cancer and / or assessing the risk of distant metastasis. Use of an agent for detecting the expression level of a gene in the gene group according to claim 1 or 2 for preparing a diagnostic product for molecularly typing breast cancer and / or assessing the risk of distant metastasis. A diagnostic product for molecularly typing breast cancer and / or assessing its risk of distant metastasis, comprising a reagent for detecting the expression level of a gene in the gene group according to claim 1 or 2. The application or diagnostic product according to claim 5 or 6, wherein the diagnostic product is in the form of an in vitro diagnostic product, preferably in the form of a diagnostic kit. The application or diagnostic product according to any one of claims 5 to 7, wherein the reagent is a reagent for detecting the amount of RNA, particularly mRNA, transcribed by the gene. The application or diagnostic product according to any one of claims 5 to 8, wherein the reagent is a reagent for detecting the amount of cDNA complementary to the mRNA. The application or diagnostic product according to any one of claims 5-9, wherein the diagnostic product further comprises a total RNA extraction reagent, a reverse transcription reagent, and / or a second-generation sequencing reagent. The application or diagnostic product according to any one of claims 5 to 10, wherein the reagent is a reagent for detecting an amount of a polypeptide encoded by the gene, and preferably the reagent is an antibody, an antibody fragment, or an affinity Sexual protein. The application or diagnostic product according to any one of claims 5 to 11, wherein the reagent is a probe or a primer, preferably a primer. The application or diagnostic product according to claim 12, characterized in that the sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO.144. A set of primers for molecular typing of breast cancer and / or assessing the risk of distant metastasis, wherein the sequence of said primers is shown in SEQ ID NO.1-SEQ ID NO.144. Use of a primer set according to claim 14 for the production of a product for molecularly typing breast cancer and / or assessing its risk of distant metastasis. The gene group, application, diagnostic product or primer set according to any one of claims 1 to 15, wherein the typing of breast cancer comprises a lumen A type, a lumen B type, a HER2 enriched type, and a basal Cellular and immune-enhanced.

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