Biomarkers, kits and uses thereof for breast cancer detection

Abstract

The application discloses biomarkers, kits and application thereof for breast cancer detection. Specifically disclosed are application of biomarkers and / or substances for detecting the biomarkers in breast cancer gene detection, wherein the biomarkers include 13 gene mutation sites, namely AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1 and TP53. The application also discloses a breast cancer gene mutation detection method, which covers 173 mutation sites of 13 genes, reduces the number of detection reactions by designing a primer panel, realizes simultaneous coverage of more gene mutation sites by using fewer primers, and completes library construction through one round of PCR amplification, is short in operation time, and is high in sensitivity and can reach 0.2%.

Description

Technical Field

[0001] This invention relates to biomarkers, kits, and their applications for breast cancer detection in the field of molecular diagnostics. Background Technology

[0002] Breast carcinoma (BC) is one of the most common malignant tumors in women, accounting for 7%-10% of all malignant tumors. Its onset is often related to genetics, with a higher incidence in women aged 40-60 and around menopause. Breast cancer has become a serious threat to women's lives. In recent years, molecular targeted therapy, as a new treatment for breast cancer, has shown some efficacy and is increasingly attracting attention from the academic community. Currently, the main treatments for breast cancer include surgery, radiotherapy, chemotherapy, and endocrine therapy. With the deepening research into the genetic and molecular mechanisms of malignant tumor development, molecular targeted therapy technology targeting oncogenes has been applied in clinical medicine. Targeted therapy is highly specific, effective, and largely does not damage normal tissues; therefore, tumor targeted therapy is one of the most promising treatment options for cancer.

[0003] Endocrine therapy for breast cancer is a treatment method that inhibits the binding of estrogen to cancer cells, and most patients require endocrine therapy. Once treatment is stopped, tumor cells can bind to estrogen again, leading to disease progression. With the widespread use of endocrine therapy, drug resistance has become a major clinical problem. Primary and secondary drug resistance resulting from endocrine therapy is a major obstacle limiting the success of breast cancer treatment. Studies show that primary resistance may be related to estrogen receptor (ER) gene deletion, ER gene mutation, and co-stimulatory factor expression. Activation of the growth factor receptor pathway, activation of the cyclin kinase (CDK) signaling pathway, and alterations in the tumor microenvironment are the main mechanisms of secondary endocrine resistance. Finding and identifying effective biomarkers for predicting the efficacy of endocrine therapy is one of the urgent clinical problems to be solved. Summary of the Invention

[0004] The technical problem this invention aims to solve is how to detect endocrine resistance and target-related gene mutations in breast cancer, thereby predicting and / or monitoring the efficacy of endocrine therapy or targeted therapy regimens for breast cancer. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, this invention first provides the application of biomarkers and / or substances for detecting said biomarkers in breast cancer gene detection or in the preparation of breast cancer gene detection products. The biomarkers may include mutation sites of the following 13 genes: AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1, and TP53.

[0006] The breast cancer gene test can be a combined detection of gene mutations related to endocrine therapy resistance in breast cancer and gene mutations related to target drugs in breast cancer.

[0007] Among them, AKT1, ERBB2, ERBB3, ESR1, FBXW7, MTOR, PDGFRA, SF3B1, and TP53 are genes related to endocrine therapy resistance in breast cancer, and their detection results can be used to determine whether endocrine therapy has caused resistance in breast cancer; EGFR, KRAS, PIK3CA, and PTEN are genes related to breast cancer target drugs, and their detection results can be used to select drugs (1. Asghari A, Wall K, Gill M, Vecchio ND, Allahbakhsh F, Wu J, Deng N, Zheng WJ, Wu H, Umetani M, Maroufy VA novel group of genes that cause endocrine resistance in breast cancer identified by dynamic gene expression analysis. Oncotarget. 2022 Apr 6; 13:600-613.; 2. Katsura C, Ogunmwonyi I, Kankam HK, Saha S. Breast cancer: presentation, investigation and management. Br J Hosp Med(Lond).2022Feb 2;83(2):1-7.;3.Gong N,Wu R,Ding B,Wu W.ERBB4 promotes the progression of inflammatory breast cancer through regulating PDGFRA.Transl CancerRes.2020May;9(5):3266-3273.;4.Sukumar J,Gast K,Quiroga D,Lustberg M,WilliamsN.Triple-negative breast cancer:promising prognostic biomarkers currently in development.Expert Rev Anticancer Ther.2021Feb;21(2):135-148.;5. LevS.Targeted therapy and drug resistance in triple-negative breast cancer:theEGFR axis.Biochem Soc Trans.2020Apr 29; 48(2):657-665.; 6. Hinz N, Jücker M. Distinct functions of AKT isoforms in breast cancer: a comprehensive review. Cell Commun Signal. 2019 Nov 21; 17(1): 154.; 7. Cox DG, Hankinson SE, HunterDJ. The erbB2 / HER2 / neu Receptor polymorphism Ile655Val and breast cancer risk. Pharmacogenet Genomics. 2005Jul; 15(7):447-50.; 8. Gao pathway.J ExpClin Cancer Res. 2019 Jun 13; 38(1):256.). This invention develops a panel for detecting endocrine therapy resistance and target-drug-related mutations in breast cancer by jointly detecting mutation sites in endocrine therapy-related genes and target-drug-related genes (i.e., the primer composition of the biomarkers of this invention).

[0008] In the above applications, the mutation site of AKT1 can be: chr14:g.105246551C>T;

[0009] The mutation sites of EGFR may be: chr7:g.55242466_55242480del15, chr7:g.55259446A>T, chr7:g.55259448delC, chr7:g.55259485C>A, chr7:g.55259485C>G, chr7:g.55259485C>T, chr7:g.55259515T>G, and chr7:g.55259524T>A;

[0010] The mutation sites of ERBB2 may be: chr17:g.37868205G>C, chr17:g.37868208C>T, chr17:g.37880219T>A, chr17:g.37880220T>C, chr17:g.37880261G>C, chr17:g.37880261G>T, chr17:g.3788 1000G>C、chr17:g.37881009_37881014、chr17:g.37881332G>A、chr17:g.37881616C>T , chr17:g.37881003-37881011dupGGCTCCCCA, chr17:g.37880984-37880995dupATACGTG ATGGC and chr17:g.37880257-37880257C>G;

[0011] The mutation sites of ERBB3 may be: chr12:g.56478851C>G, chr12:g.56478854G>C, chr12:g.56478854G>A, chr12:g.56481922G>A, chr12:g.56482341G>T, chr12:g.56482607C>T and chr12:g.56492633A>G;

[0012] The mutation sites of ESR1 may be: chr6:g.152332832G>C, chr6:g.152332868G>A, chr6:g.152415537T>C, chr6:g.152419922T>A, chr6:g.152419923A>G, chr6:g.152419923A>C, and chr6:g.152419926A>G;

[0013] The mutation sites of FBXW7 can be: chr4:g.153245393C>A and chr4:g.153245446G>A;

[0014] The mutation sites of the KRAS can be: chr12:g.25398281C>T, chr12:g.25398282C>A, chr12:g.25398284C>G, chr12:g.25398284C>T, chr12:g.25398284C>A, chr12:g.25398284_25398285delCCinsAA, chr12:g.25398285C>G, chr12:g.25398285C>A, and chr12:g.25398285C>T;

[0015] The mutation sites of MTOR can be: chr1:g.11184573G>A, chr1:g.11184573G>T, chr1:g.11188164G>T and chr1:g.11190804C>T;

[0016] The mutation sites of PDGFRA can be: chr4:g.55141036T>A and chr4:g.55152093A>T;

[0017] The mutation sites of PIK3CA can be: chr3:g.178921553T>G, chr3:g.178927980T>C, chr3:g.178928079G>A, chr3:g.178936082G>A, chr3:g.178936091G>C, chr3:g.178936091G>A, chr3:g.178936092A>C, chr3:g.178936092A>G, chr3:g.17893 6094C>A, chr3:g.178936095A>G, chr3:g.178936095A>C, chr3:g.178938934G>A, chr3:g.178952072A>G, chr3:g .178952074G>C, chr3:g.178952084C>T, chr3:g.178952085A>G, chr3:g.178952085A>T and chr3:g.178952090G>C;

[0018] The mutation sites of PTEN can be: chr10:g.89692904C>G, chr10:g.89692905G>A and chr10:g.89717674_89717676delACG;

[0019] The mutation site of SF3B1 may be: chr2:g.198266834T>C;

[0020] The mutation sites of the said TP53 can be: chr17:g.7577081T>C, chr17:g.7577082C>T, chr17:g.7577085C>T, chr17:g.7577090C>G, chr17:g.7577094G>A, chr17:g.7577094G>C, chr17:g.7577099C>A, chr17:g.7577099C>T, chr17:g.7577099C>G, chr17:g.7577102C>T, chr17:g.7577105G>C, chr17:g.7577105G>A, chr17:g.7577106G>C, chr17:g.7577106G>A, chr17:g.7577106G>T, chr17:g.7577108C>A, chr17:g.7577114C>T, chr17:g.7577118C>G, chr17:g.7577118C>A, chr17:g.7577120C>T, chr17:g.7577120C>A, chr17:g.7577120C>G, chr17:g.7577121G>A, chr17:g.7577124C>G, chr17:g.7577124C>T, chr17:g.7577141C>T, chr17:g.7577141C>A, chr17:g.7577142C>T, chr17:g.7577153C>A, chr17:g.7577509C>T, chr17:g.7577532G>A, chr17:g.7577534C>G, chr17:g.7577535C>T, chr17:g.7577535C>A, chr17:g.7577538C>T, chr17:g.7577538C>A, chr17:g.7577539G>A, chr17:g.7577545T>C, chr17:g.7577547C>T, chr17:g.7577547C>A, chr17:g.7577548C>T, chr17:g.7577548C>A, chr17:g.7577550C>T, chr17:g.7577550C>A, chr17:g.7577551C>A, chr17:g.7577551C>T, chr17:g.7577556C>A, chr17:g.7577556C>T, chr17:g.7577559G>A, chr17:g.7577563T>C, chr17:g.7577568C>A, chr17:g.7577568C>T, chr17:g.7577570C>G, chr17:g.7577580T>C, chr17:g.7578190T>C, chr17:g.7578191A>G, chr17:g.7578203C>T, chr17:g. 7578208T>C, chr17:g.7578211C>T, chr17:g.7578211C>A, chr17:g.7578260C>T, chr17:g.7578265A>G, chr17:g.7578268A>C, chr17:g. 7578271T>C, chr17:g.7578272G>A, chr17:g.7578279_7578281delAGG, chr17:g.7578280G>A, chr17:g.7578391_7578408del18, chr17: g.7578394T>C、chr17:g.7578394T>A、chr17:g.7578395G>A、chr17:g.7578403C>A、chr17:g.7578403C>T、chr17:g.7578406C>T、chr17:g .7578406C>A, chr17:g.7578407G>A, chr17:g.7578413C>G, chr17:g.7578413C>T, chr17:g.7578416C>A, chr17:g.7578457C>A, chr17:g .7578457C>T、chr17:g.7578461C>A、chr17:g.7578463C>G、chr17:g.7578469C>A、chr17:g.7578475G>A、chr17:g.7578478G>T、chr17:g .7578479G>A, chr17:g.7578479G>T, chr17:g.7578496A>G, ​​chr17:g.7578508C>T, chr17:g.7578509A>G, chr17:g.7578517G>A, chr17:g .7578525G>C, chr17:g.7578526C>A, chr17:g.7578526C>T, chr17:g.7578535T>C, chr17:g.7578536T>C and chr17:g.7577139-7577139C>G. .

[0021] The reference genome version number for the above mutation sites is GRCh37 or hg19.

[0022] Furthermore, the mutation site is a mutation site targeting genes related to endocrine therapy for breast cancer and / or genes related to targeted drugs for breast cancer.

[0023] Furthermore, the breast cancer endocrine therapy-related gene may be a breast cancer endocrine therapy resistance-related gene.

[0024] In the above applications, the substance may be a primer composition for detecting the biomarker.

[0025] In the above applications, the primer composition may include 87 single-stranded DNAs with nucleotide sequences of SEQ ID No. 5-SEQ ID No. 91 (i.e., the primer composition may include the primers shown in SEQ ID No. 5-SEQ ID No. 91).

[0026] Among them, the primers shown in SEQ ID No. 5-SEQ ID No. 42 are upstream primers F2 of the biomarker (gene marker) of the present invention; the primers shown in SEQ ID No. 43-SEQ ID No. 80 are downstream inner primers R2 of the biomarker of the present invention; the primer shown in SEQ ID No. 81 is downstream outer primer R1 (i.e., reverse universal primer R1) of the biomarker of the present invention; and the primers shown in SEQ ID No. 82-SEQ ID No. 91 are barcode primers F1.

[0027] The primer composition is also within the scope of protection of this invention.

[0028] The present invention also provides reagents or kits for detecting gene mutations in breast cancer, wherein the reagents or kits may contain the primer composition.

[0029] Furthermore, the kit may also include Taq DNA polymerase, dNTPs, PCR buffer, and Mg2+ required for PCR amplification. 2+ One or more of them.

[0030] The present invention also provides a method for detecting gene mutations in breast cancer, the method comprising the following steps:

[0031] A1) Extract nucleic acid from the sample to be tested;

[0032] A2) Constructing a DNA sequencing library: Using the nucleic acid as a template, perform PCR amplification using the primer composition to obtain the PCR amplification product, which is the DNA sequencing library;

[0033] A3) High-throughput sequencing is performed using the DNA sequencing library, and the gene mutation status in the sample to be tested is analyzed based on the sequencing results. The genes may be AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1, and TP53. The method may be a non-disease diagnosis and treatment method.

[0034] The primer composition may include the primers shown in SEQ ID No. 5-SEQ ID No. 91.

[0035] The detection of breast cancer gene mutations described in this article can be used to detect gene mutations related to endocrine therapy resistance in breast cancer and gene mutations related to breast cancer target drugs.

[0036] In the above method, the PCR amplification system may include: primer R1, primer composition F2, and primer composition R2, wherein the molar ratio of R1, F2, and R2 is 10:(1-5):(1-5), wherein primer R1 may be a single-stranded DNA with the nucleotide sequence SEQ ID No. 81, primer composition F2 may be a composition composed of 38 single-stranded DNAs with nucleotide sequences SEQ ID No. 5-SEQ ID No. 42, and primer composition R2 may be a composition composed of 38 single-stranded DNAs with nucleotide sequences SEQ ID No. 43-SEQ ID No. 80.

[0037] In the above method, the molar ratio of R1, F2 and R2 can be 10:5:5.

[0038] Furthermore, the nucleic acid mentioned in A) may be ctDNA, cDNA, gDNA, or RNA.

[0039] The sample to be tested can be a blood sample (such as a plasma sample), a tissue sample (such as an FFPE tissue sample), or a puncture sample.

[0040] The present invention also provides any of the following applications of the biomarker and / or the primer composition:

[0041] B1) Used for the detection or monitoring of the efficacy of endocrine therapy for breast cancer, or for the preparation of products used for the detection or monitoring of the efficacy of endocrine therapy for breast cancer;

[0042] B2) Used to guide precision endocrine therapy for patients with advanced refractory breast cancer or to prepare products for guiding precision endocrine therapy for patients with advanced refractory breast cancer;

[0043] B3) Used for testing endocrine therapy resistance in breast cancer or for preparing products for testing endocrine therapy resistance in breast cancer;

[0044] B4) Targeted therapy regimens used to provide or evaluate treatment for breast cancer.

[0045] The primer composition of the present invention may include the following primers:

[0046] Barcode primer F1: Sequencing adapter 1 + Barcode sequence + Universal sequence 1;

[0047] Upstream primer F2: Universal sequence 1 + molecular tag + upstream specific primer sequence;

[0048] Downstream outer primer R1: sequencing adapter 2 + universal sequence 2;

[0049] Downstream inner primer R2: universal sequence 2 + downstream specific primer sequence.

[0050] Barcode sequences are used to distinguish different samples, separating data from different samples during subsequent analysis. Upstream-specific and downstream-specific primer sequences are primer sequences designed to amplify specific target regions, based on the sequences of gene markers. Molecular tags are short, randomized or specific nucleotide sequences, typically designed as completely random nucleotide chains (e.g., NNNNNN), partially degenerate nucleotide chains (e.g., NNNRNYN), or fixed nucleotide chains (when template molecules are limited). Molecular tags are used to label template molecules during library construction. Universal sequence 1 and universal sequence 2 are two different specific nucleic acid sequences, which can be varied as needed. Sequencing adapter 1 and sequencing adapter 2 are adapter sequences required for sequencing, determined by the sequencing platform; for example, A and P adapters for the Ion Torrent platform.

[0051] The sequences were combined to form upstream and downstream primers. The primer sequences were dissolved after synthesis to prepare the DNA amplification mixture. Universal sequence 1 is 5'-GGCATACGTCCTCGTCTA-3' (SEQ ID No. 1); universal sequence 2 is 5'-CCTCTCTATGGGCAGTCGGTGAT-3' (SEQ ID No. 2); sequencing adapter 1 is 5'-CCATCTCATCCC TGCGTGTCTCCGACTCAG-3' (SEQ ID No. 3); sequencing adapter 2 is 5'-CCACTACGCCTCCGCTTT-3' (SEQ ID No. 4).

[0052] The molecular tag in the upstream primer includes random sequences and specific sequences. In one embodiment of the present invention, the molecular tag is: NNNNACNNNNTG, where AC and TG are specific sequences and the others are random sequences, where N is randomly selected from A, T, G or C.

[0053] Furthermore, the PCR amplification system described in A2) includes: DNA amplification primer mixture 1, barcode primer solution, template DNA, ddH2O, and KAPA HiFi HotStart ReadyMix;

[0054] The DNA amplification primer mixture 1 is formed by mixing the upstream primer F2 solution (primer sequences are shown in Table 2, and the primers in Table 2 are mixed in an equimolar ratio), the downstream inner primer R2 solution (primer sequences are shown in Table 3, and the primers in Table 3 are mixed in an equimolar ratio), and the downstream outer primer, i.e., the reverse universal primer R1 solution (primer sequences are shown in Table 4), in a certain proportion. The initial concentration of primers R1, F2, and R2 is 50 μM, and the volume ratio of R1:F2:R2 is 10:(1-5):(1-5). In one embodiment of the present invention, the volume ratio of R1:F2:R2 is 10:5:5.

[0055] The Barcode primer F1 solution (primer sequences are shown in Table 5, and each primer in Table 5 is mixed in an equimolar ratio) was diluted with TE buffer at a volume ratio of 1:1 for use. The concentration of Barcode primer F1 after mixing was 50 μM, which is the Barcode primer solution.

[0056] In one embodiment of the present invention, the PCR amplification system (30 μL) described in A2) is: 15 μL of KAPA HiFiHotStart ReadyMix (KAPA KK2602), 12 μL of DNA amplification primer mixture, 1 μL of barcode primer solution, 50 ng of template DNA, and ddH2O to make up to 30 μL.

[0057] In one embodiment of the present invention, the PCR amplification conditions described in A2) are: 98℃ for 30s; 98℃ for 10s, 64℃ for 2min, 62℃ for 2min, 60℃ for 4min, 72℃ for 30s; 2 cycles; 98℃ for 10s, 72℃ for 2min; 23 cycles; 72℃ for 10min.

[0058] In one embodiment of the present invention, the high-throughput sequencing described in A3) can be performed using the Ion Torrent platform for next-generation sequencing (NGS): based on the concentration of the target fragment (approximately 260 bp) in the quality control results, the samples are mixed in equal amounts, and the mixed products are quantified using Qubit 3.0. It is recommended that the amount of data required for each sample to be processed is 500M, and should not be less than 300M.

[0059] The A2) also includes a purification step for the DNA sequencing library.

[0060] The mutation information described in this invention adopts the naming rules of the Human Genome Variation Society (HGVS), and the version number of the reference genome is GRCh37 / hg19.

[0061] The prefix "p." indicates a human protein reference sequence; the prefix "c." indicates a human cDNA reference sequence; and the prefix "g." indicates a human genome reference sequence. Substitution (>): One nucleotide is replaced by another nucleotide, indicated by ">". Deletion (del): One or more nucleotides are removed, described by "del". Duplication (dup): One or more nucleotide copies are inserted directly downstream of the original sequence, indicated by "dup". Insertion (ins): One or more nucleotides are inserted into the sequence, and the inserted sequence is not a copy of the upstream sequence. For example: chr14:g.105246551C>T indicates that compared to the genome reference sequence, the C at position 105246551 is replaced by T. c.49G>A indicates that compared to the cDNA reference sequence, the G at position 49 is replaced by A. chr7:g.55259448delC indicates that the C at position 55259448 is deleted compared to the genome reference sequence. c.34_35delGGinsTT indicates that, compared to the cDNA reference sequence, positions 34-35 have been missing GG and replaced with TT. Those skilled in the art can determine the meaning of these codes without error.

[0062] This invention relates to the detection of 13 genes and 173 mutation hotspots related to breast cancer. It uses the Illumina next-generation sequencing platform to detect mutations in ctDNA samples from breast cancer patients, clarifying the differences in gene mutation spectra before and after endocrine therapy, further seeking biomarkers that can effectively predict the efficacy of endocrine therapy, and exploring the clinical value of using ctDNA NGS (next-generation sequencing) detection to guide precision endocrine therapy for patients with advanced refractory breast cancer.

[0063] Currently, there are many next-generation sequencing (NGS) testing panels on the market for breast cancer targeted therapy, but few for mutation detection related to endocrine therapy. This invention's breast cancer endocrine and targeted drug-related mutation detection panel specifically targets the combined detection of mutation sites in both endocrine and targeted drugs. Targeted therapy can significantly improve the prognosis of patients carrying driver genes, and its combination with endocrine therapy can effectively reverse endocrine resistance and improve patient survival rates. This product has enormous clinical testing demand and high potential market value.

[0064] There are many endocrine and targeted drug-related mutated genes in breast cancer. This invention designs primers for 13 of these genes (AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1, and TP53) to detect 173 hotspot mutations. Based on this, biomarkers and kits for breast cancer gene detection have been developed.

[0065] This invention, based on extensive and in-depth research, identifies differences in gene mutation spectra before and after endocrine therapy by detecting mutations in ctDNA samples from breast cancer patients. It then identifies, screens, and analyzes biomarkers for breast cancer gene detection, targeting genes related to endocrine therapy and breast cancer target drugs. The final biomarkers identified 173 hotspot mutation sites across 13 genes. Based on this, a method for detecting breast cancer gene mutations was constructed. Compared to existing technologies, this invention's method covers 173 mutation sites across 13 genes. By designing primer panels for the biomarkers, the number of detection reactions is reduced, allowing for simultaneous coverage of more genes and / or multiple mutation sites with a smaller number of primers, significantly reducing the cost of clinical testing. Furthermore, this method is simple, requiring only one round of PCR amplification to complete library construction, minimizing manual operation time. It also boasts high sensitivity, reaching 0.2%. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] Example 1: Obtaining biomarkers for breast cancer gene detection

[0069] Through extensive and in-depth research, the inventors have identified the differences in gene mutation spectra before and after endocrine therapy by detecting mutations in ctDNA samples from breast cancer patients. They have also searched for, screened, and analyzed biomarkers for breast cancer gene detection, targeting genes related to endocrine therapy and drug targets.

[0070] The obtained biomarkers consist of 173 mutation sites in 13 genes (AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1, and TP53), as detailed in Table 1.

[0071] Table 1. Biomarkers for breast cancer gene detection

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] The mutation information in Table 1 is described using the naming conventions of the Human Genome Variation Society (HGVS), with the reference genome version number being GRCh37 or hg19.

[0078] The prefix "p." indicates a human protein reference sequence; the prefix "c." indicates a human cDNA reference sequence; and the prefix "g." indicates a human genome reference sequence.

[0079] In the "Information on Mutation Sites on the Genome," the part before the colon is the human chromosome number, such as chr1, chr2, chr3, chr4, chr6, chr7, chr10, chr12, chr14, and chr17, representing human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 14, and 17, respectively. The "g." followed by the number indicates the location on the genome, and the rest indicates the variant type. The meaning of the variant type is the same as that in HGVS nomenclature, such as: Substitution (>): One nucleotide is replaced by another nucleotide, indicated by ">". Deletion (del): One or more nucleotides are removed, described by "del". Duplication (dup): One or more nucleotide copies are directly inserted downstream of the original sequence, indicated by "dup". Insertion (ins): One or more nucleotides are inserted into the sequence, and the inserted sequence is not a copy of the upstream sequence. For example: chr14:g.105246551C>T indicates that compared to the genomic reference sequence, the C at position 105246551 has been replaced by T. c.49G>A indicates that compared to the cDNA reference sequence, the G at position 49 has been replaced by A. chr7:g.55259448delC indicates that compared to the genomic reference sequence, the C at position 55259448 has been deleted. c.34_35delGGinsTT indicates that compared to the cDNA reference sequence, the GG at positions 34-35 has been deleted and replaced by TT.

[0080] Among them, AKT1, ERBB2, ERBB3, ESR1, FBXW7, MTOR, PDGFRA, SF3B1, and TP53 are genes related to resistance to endocrine therapy in breast cancer, and their test results can be used to determine whether endocrine therapy has caused resistance in breast cancer; EGFR, KRAS, PIK3CA, and PTEN are genes related to targeted drugs in breast cancer, and medication can be selected based on their test results.

[0081] Example 2: Detection Method for Breast Cancer Gene Mutations

[0082] This embodiment combines the detection of mutation sites in endocrine therapy resistance-related genes and drug-targeted genes to develop a panel for detecting endocrine and drug-targeted mutations in breast cancer (i.e., the primer composition of the biomarker of this invention).

[0083] 1. Nucleic acid extraction

[0084] Circulating tumor DNA (ctDNA) was extracted using a nucleic acid extraction and purification kit (QIAamp Circulating Nucleic Acid Kit, QIAGEN). ctDNA is a gene fragment released into the peripheral circulation system due to apoptosis or rupture of solid tumor cells. It is cell-free extracellular DNA, mainly found in fluids such as blood, cerebrospinal fluid, and synovial fluid, and possesses characteristics consistent with the primary tumor tissue. ctDNA originates from genomic mutations in tumor cells, rarely producing false positives, and its short half-life accurately reflects the current status of the tumor.

[0085] The extracted ctDNA sample was analyzed for its concentration and used as template DNA for subsequent library construction.

[0086] 2. Sequencing library construction

[0087] 2-1. Design of primer panels (primer combinations)

[0088] By designing primer panels for biomarkers, the reaction number for detection can be reduced, significantly lowering the cost of clinical testing. A one-step multiplex PCR method is used to construct sequencing libraries; this method is a rapid and efficient library construction approach for detecting gene mutations in various sample types, involving the following primers:

[0089] Barcode primers, F1: Sequencing adapter 1 + Barcode sequence + Universal sequence 1;

[0090] Upstream primer, F2: Universal sequence 1 + molecular tag + upstream specific primer sequence;

[0091] Downstream outer primer, R1: sequencing adapter 2 + universal sequence 2;

[0092] Downstream inner primer, R2: universal sequence 2 + downstream specific primer sequence;

[0093] Barcode sequences are used to distinguish different samples, separating data from different samples during subsequent analysis. Upstream-specific and downstream-specific primer sequences are primer sequences designed to amplify specific target regions, based on the sequences of gene markers. Molecular tags are short, randomized or specific nucleotide sequences, typically designed as completely random nucleotide chains (e.g., NNNNNN), partially degenerate nucleotide chains (e.g., NNNRNYN), or fixed nucleotide chains (when template molecules are limited). Molecular tags are used to label template molecules during library construction. Universal sequence 1 and universal sequence 2 are two different specific nucleic acid sequences, which can be varied as needed. Sequencing adapter 1 and sequencing adapter 2 are adapter sequences required for sequencing, determined by the sequencing platform; for example, A and P adapters for the Ion Torrent platform.

[0094] The individual sequences were combined to form upstream and downstream primers. The primer sequences were dissolved after synthesis to prepare the DNA amplification mixture.

[0095] The universal sequence 1 is: 5'-GGCATACGTCCTCGTCTA-3' (SEQ ID No. 1);

[0096] The universal sequence 2 is: 5'-CCTCTCTATGGGCAGTCGGTGAT-3' (SEQ ID No. 2);

[0097] Sequencing adapter 1 is: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3' (SEQ ID No. 3);

[0098] Sequencing adapter 2 is: 5'-CCACTACGCCTCCGCTTT-3' (SEQ ID No. 4).

[0099] In this embodiment, the DNA amplification primer mixture 1 consists of the upstream primer F2 solution of the gene marker (the solute is a variety of single-stranded DNAs listed in Table 2 (upstream primer F2), with each single-stranded DNA having a concentration of 50 μM) (primer sequences are shown in Table 2, and the primers in Table 2 are mixed in equimolar ratios), the downstream inner primer R2 solution of the gene marker (the solute is 38 single-stranded DNAs listed in Table 3 (downstream inner primer R2), with each single-stranded DNA having a concentration of 50 μM), and the downstream outer primer, i.e., the reverse universal primer R1 solution (…). The solute is a single-stranded DNA (reverse universal primer R1) listed in Table 4, with a concentration of 50 μM. The solute is mixed in a certain proportion to form the solution. The concentrations of the reverse universal primer R1 solution (R1), the upstream primer F2 solution (F2), and the downstream inner primer R2 solution (R2) are all 50 μM. The mixing ratio of these three solutions is R1:F2:R2 (volume ratio) = 10:(1-5):(1-5). In this embodiment, the volume ratio of R1:F2:R2 is selected as 10:5:5.

[0100] The molecular tag in the upstream primer includes random sequences and specific sequences. In this embodiment, the molecular tag is: NNNNACNNNNTG, where AC and TG are specific sequences and the others are random sequences, where N is randomly selected from A, T, G or C.

[0101] The Barcode primer F1 solution (solutes are the 10 single-stranded DNAs listed in Table 5, each with a concentration of 100 μM) was diluted with TE buffer at a volume ratio of 1:1 before use. The concentration of Barcode primer F1 after mixing was 50 μM, which is the Barcode primer solution.

[0102] For sequencing on the Ion Torrent platform, sequencing adapter 1 and sequencing adapter 2 are A and P adapters, respectively.

[0103] The sequence information of 173 mutation sites in the above 13 genes was screened through a large number of experiments, and the preferred primer panels (primer combinations) are shown in Tables 2-5.

[0104] Table 2. Upstream primer F2 sequence of gene markers

[0105]

[0106]

[0107] Table 3. Downstream primer R2 sequences of gene markers

[0108]

[0109]

[0110] Table 4. Downstream outer primer, i.e., reverse universal primer R1 sequence

[0111]

[0112] Table 5. Barcode primer F1 sequence

[0113]

[0114] 2-2. Construction of DNA sequencing libraries

[0115] Prepare the primer panel according to the specified ratio. The starting template DNA for library construction can be 5-50 ng, with a minimum of 5 ng. In this example, the template DNA is 50 ng. Different samples use different barcodes. The PCR amplification reaction system and reaction conditions are as follows:

[0116] Table 6. PCR amplification reaction system

[0117] Components content KAPA HiFi HotStart ReadyMix (KAPA KK2602) 15μL DNA amplification primer mixture 1 2μL Barcode primer solution 1μL Template DNA 50ng <![CDATA[ddH2O]]> To make up to 30μL Total volume 30μL

[0118] Table 7. PCR amplification reaction conditions

[0119]

[0120] The DNA sequencing library solution was obtained after the above PCR amplification.

[0121] 2-3. DNA sequencing library purification

[0122] (1) After PCR, add 33 μL of AM Pure XP magnetic bead reagent to the reaction system (30 μL) of each sample, that is, the volume ratio of DNA sequencing library solution to magnetic bead reagent is 1:1.1, vortex to mix, and let stand at room temperature for 5 min.

[0123] (2) The magnetic rack adsorbs the liquid until it becomes clear. After about 5 minutes, the supernatant is discarded.

[0124] (3) Add 200 μL of freshly prepared 80% ethanol to each, incubate for 30 s, and discard the supernatant;

[0125] (4) Add 200 μL of freshly prepared 80% ethanol again, incubate for 30 seconds, discard the supernatant, remove all ethanol, and air dry on a magnetic rack for 3 minutes until the ethanol has completely evaporated.

[0126] (5) Add 23 μL of water to each product for elution, vortex mix, and let stand at room temperature for 5 min.

[0127] (6) After the magnetic rack adsorbs the liquid until it is clear, about 5 minutes later, aspirate 20 μL of supernatant (i.e. the purified DNA sequencing library) into a new centrifuge tube and discard the magnetic beads;

[0128] (7) Use Qubit 3.0 to determine the concentration of the purified product, and use Agilent 2200 to quality control the purified product and label it.

[0129] (8) If the target fragment percentage is below 60% after the purified product is tested by Agilent 2200, a second purification can be performed, with the same steps as (1)-(7) above.

[0130] After purification, the construction of the DNA sequencing library is complete.

[0131] 2-4. Sequencing

[0132] Quality control standard: After purification, the product is tested by Agilent 2200 and the target fragment accounts for more than 60% and can be used for subsequent sequencing.

[0133] After quantifying the constructed sequencing library, perform next-generation sequencing (NGS) using the Ion Torrent platform: Based on the concentration of the target fragment (approximately 260 bp) in the quality control results, mix equal amounts of samples. Quantify the mixed product using Qubit 3.0. It is recommended that each sample require 500M of data for sequencing, and no less than 300M.

[0134] 2-5. Bioinformatics Analysis

[0135] The sequencing library obtained by the one-step method was sequenced, and the mutation status of the detected genes was obtained after data processing and bioinformatics analysis of the sequencing results.

[0136] After the sequencing data is processed, the quality control of the sample data is first performed. Then, the raw BAM data is converted into FASTQ files. The FASTQ files are compared with the human reference genome, and molecular tags are extracted. For each site, reads are clustered according to molecular tags to form families (reads must have at least 2 reads with the same molecular tags, and 80% of reads supporting the same base type are considered to be in the same family). The number of ref (Reference: wild-type base sequence at the site in the reference genome) and alt (Alternative: mutant base sequence at the site after mutation) families at each site is counted, and the corresponding family depth (number of families) is obtained for each. Then, each site is quality controlled according to the site quality control standards (pass or fail). Based on the number of ref and alt families at each site, the mutation frequency is calculated as follows: Mutation frequency = alt family depth / (alt family depth + ref family depth).

[0137] Example 3: Validation of the accuracy of breast cancer gene detection methods

[0138] Two commercially available human gDNA standards with known results, HD701 (Horizon Discovery Corporation, catalog number HD701) and HD734 (Horizon Discovery Corporation, catalog number HD734), were selected and tested according to the method in Example 2. The mutation frequency of the mutation sites was obtained, and the accuracy of the test results was examined. The results are shown in Tables 8 and 9.

[0139] Table 8. HD701 Test Results

[0140]

[0141] Table 9. HD734 Test Results

[0142]

[0143]

[0144] The results showed that the mutation frequency of the mutation sites obtained by the breast cancer gene detection method developed based on the biomarkers of this invention was basically close to the theoretical mutation frequency when detecting commercially available gDNA standards with known results. This demonstrates that the biomarkers and detection method of this invention have high accuracy in detecting mutations in breast cancer endocrine and drug-targeted genes (AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, MTOR, PDGFRA, PIK3CA, PTEN, SF3B1, and TP53).

[0145] Example 4: Sensitivity Experiment of Breast Cancer Gene Detection Method

[0146] One standard sample HD734 was diluted at 50% and 25% of the theoretical mutation frequency, respectively. Then, library construction and sequencing were performed according to the method in Example 2. Based on the sequencing results, the mutation frequency of the mutation site was obtained, and the results are shown in Table 10.

[0147] Table 10. HD734 Test Results

[0148]

[0149] The results show that this experiment can detect mutations with a frequency as low as 0.2%.

[0150] Example 5: Comparative Experiment of Detection Methods

[0151] Eight human breast cancer samples (tissue samples) were selected, ctDNA was extracted, and a library was constructed and sequenced using the probe capture method. The detection frequency is shown in the table below as "Detection Frequency of Comparison Method". The remaining ctDNA was detected according to the method in Example 2, and the mutation frequency of the mutation sites was obtained (see Table 11). It can be seen that the detected mutation sites and frequencies are consistent with those of the comparison method.

[0152] Table 11. Clinical Sample Test Results

[0153]

[0154]

[0155] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of a substance for detecting biomarkers in the preparation of a product for detecting resistance to endocrine therapy in breast cancer, characterized in that, The biomarker consists of mutation sites in the following nine genes: AKT1, ERBB2, ERBB3, ESR1, FBXW7, MTOR, PDGFRA, SF3B1, and TP53. The mutation site of AKT1 is: chr14:g.105246551C>T; The mutation sites of ERBB2 are: chr17:g.37868205G>C, chr17:g.37868208C>T, chr17:g.37880219T>A, chr17:g.37880220T>C, chr17:g.37880261G>C, chr17:g.37880261G>T, chr17:g.37881000G>C, chr17:g.37 881009_37881014, chr17:g.37881332G>A, chr17:g.37881616C>T, chr17:g.37881003-37881011d upGGCTCCCCA, chr17:g.37880984-37880995dupATACGTGATGGC and chr17:g.37880257-37880257C>G; The mutation sites of ERBB3 are: chr12:g.56478851C>G, chr12:g.56478854G>C, chr12:g.56478854G>A, chr12:g.56481922G>A, chr12:g.56482341G>T, chr12:g.56482607C>T and chr12:g.56492633A>G; The mutation sites of ESR1 are: chr6:g.152332832G>C, chr6:g.152332868G>A, chr6:g.152415537T>C, chr6:g.152419922T>A, chr6:g.152419923A>G, chr6:g.152419923A>C and chr6:g.152419926A>G; The mutation sites of the FBXW7 are: chr4:g.153245393C>A and chr4:g.153245446G>A; The mutation sites of the MTOR are: chr1:g.11184573G>A, chr1:g.11184573G>T, chr1:g.11188164G>T and chr1:g.11190804C>T; The mutation sites of the PDGFRA are: chr4:g.55141036T>A and chr4:g.55152093A>T; The mutation site of SF3B1 is: chr2:g.198266834T>C; The mutation sites of the said TP53 are: chr17:g.7577081T>C, chr17:g.7577082C>T, chr17:g.7577085C>T, chr17:g.7577090C>G, chr17:g.7577094G>A, chr17:g.7577094G>C, chr17:g.7577099C>A, chr17:g.7577099C>T, chr17:g.7577099C>G, chr17:g.7577102C>T, chr17:g.7577105G>C, chr17:g.7577105G>A, chr17:g.7577106G>C, chr17:g.7577106G>A, chr17:g.7577106G>T, chr17:g.7577108C>A, chr17:g.7577114C>T, chr17:g.7577118C>G, chr17:g.7577118C>A, chr17:g.7577120C>T, chr17:g.7577120C>A, chr17:g.7577120C>G, chr17:g.7577121G>A, chr17:g.7577124C>G, chr17:g.7577124C>T, chr17:g.7577141C>T, chr17:g.7577141C>A, chr17:g.7577142C>T, chr17:g.7577153C>A, chr17:g.7577509C>T, chr17:g.7577532G>A, chr17:g.7577534C>G, chr17:g.7577535C>T, chr17:g.7577535C>A, chr17:g.7577538C>T, chr17:g.7577538C>A, chr17:g.7577539G>A, chr17:g.7577545T>C, chr17:g.7577547C>T, chr17:g.7577547C>A, chr17:g.7577548C>T, chr17:g.7577548C>A, chr17:g.7577550C>T, chr17:g.7577550C>A, chr17:g.7577551C>A, chr17:g.7577551C>T, chr17:g.7577556C>A, chr17:g.7577556C>T, chr17:g.7577559G>A, chr17:g.7577563T>C, chr17:g.7577568C>A, chr17:g.7577568C>T, chr17:g.7577570C>G, chr17:g.7577580T>C, chr17:g.7578190T>C, chr17:g.7578191A>G, chr17:g.7578203C>T, chr17:g.7578208T>C, chr17:g.7578211C>T, chr17:g.7578211C>A, chr17:g.7578260C>T, chr17:g.7578265A>G, chr17:g.7578268A>C, chr17:g.7578271T>C, chr17:g.7578272G>A, chr17:g.7578279_7578281delAGG, chr17:g.7578280G>A, chr17:g.7578391_7578408del18, chr17:g.7578394T>C, chr17:g.7578394T>A, chr17:g.7578395G>A, chr17:g.7578403C>A, chr17:g.7578403C>T, chr17:g.7578406C>T, chr17:g.7578406C>A, chr17:g.7578407G>A, chr17:g.7578413C>G, chr17:g.7578413C>T, chr17:g.7578416C>A, chr17:g.7578457C>A, chr17:g.7578457C>T, chr17:g.7578461C>A, chr17:g.7578463C>G, chr17:g.7578469C>A, chr17:g.7578475G>A, chr17:g.7578478G>T, chr17:g.7578479G>A, chr17:g.7578479G>T, chr17:g.7578496A>G, chr17:g.7578508C>T, chr17:g.7578509A>G, chr17:g.7578517G>A, chr17:g.7578525G>C, chr17:g.7578526C>A, chr17:g.7578526C>T, chr17:g.7578535T>C, chr17:g.7578536T>C and chr17:g.7577139-7577139C>G;. The substance is a primer composition, which consists of 87 single-stranded DNAs with nucleotide sequences of SEQ ID No. 5-SEQ ID No.

91.

2. The application according to claim 1, characterized in that, The detection method for the application includes the following steps: A1) Extract nucleic acid from the sample to be tested; A2) Constructing a DNA sequencing library: Using the nucleic acid as a template, PCR amplification is performed using the primer composition described in claim 1, and the PCR amplification product is the DNA sequencing library. A3) High-throughput sequencing was performed using the DNA sequencing library. Based on the sequencing results, the mutation status of endocrine therapy resistance-related genes in the sample was analyzed. The endocrine therapy resistance-related genes in breast cancer are AKT1, ERBB2, ERBB3, ESR1, FBXW7, MTOR, PDGFRA, SF3B1, and TP53.

3. The application according to claim 2, characterized in that, The PCR amplification system includes primer R1, primer composition F2, and primer composition R2, wherein the molar ratio of R1, F2, and R2 is 10:1-5:1-5. Primer R1 is a single-stranded DNA with the nucleotide sequence SEQ ID No.

81. Primer composition F2 is composed of 38 single-stranded DNAs with nucleotide sequences SEQ ID No. 5-SEQ ID No.

42. Primer composition R2 is composed of 38 single-stranded DNAs with nucleotide sequences SEQ ID No. 43-SEQ ID No.

80.

4. The application according to claim 3, characterized in that, The molar ratio of R1, F2 and R2 is 10:5:5.

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