Marker for predicting breast cancer neoadjuvant chemotherapy adverse reaction and application thereof

Abstract

The invention discloses a marker for predicting the adverse reaction of breast cancer neoadjuvant chemotherapy in the field of biomedical detection, the marker is a site rs2322718, and the marker can predict the adverse reaction of breast cancer neoadjuvant chemotherapy so as to provide certain reference for accurate prevention and treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical testing, and particularly relates to a marker for predicting adverse reactions to neoadjuvant chemotherapy for breast cancer and an application thereof. Background Art

[0002] Breast cancer (BC) is one of the most common malignancies in women. Chemotherapy is a common treatment option in clinical practice, with neoadjuvant chemotherapy (NAC) playing a key role. This treatment aims to shrink the tumor and preemptively eliminate potential metastatic cells, thereby facilitating subsequent surgical and radiotherapy treatments. Studies have shown that patients can achieve a pathological complete response (pCR) after NAC. Patients achieving a pCR are more sensitive to chemotherapy, and achieving a pCR after neoadjuvant chemotherapy correlates with disease-free survival (DFS) and overall survival (OS). Patients achieving a pCR have longer disease-free and overall survival rates, leading to the widespread use of pCR as a criterion for evaluating the short-term efficacy of NAC. However, for some patients, NAC does not result in a favorable prognosis and may even lead to severe adverse reactions, including gastrointestinal reactions, bone marrow suppression, headaches, and dizziness, which impact not only their quality of life but also their overall survival. Therefore, it is very important to find prognostic markers for neoadjuvant chemotherapy of breast cancer.

[0003] Currently, there is a lack of clinically recognized markers related to the prognosis of breast cancer NAC. It is generally believed that the main reason why the same treatment regimen has different efficacy in different patients is individual differences. Genetic variation (SNP) plays an important role in individual differences in drug efficacy. According to Nature, SNP is the main cause of abnormal splicing. Different splicing methods allow the same gene to produce multiple different mature mRNAs, forming proteins with different functions, which in turn affects gene expression. More and more studies have shown that variable splicing also plays a very important role in the occurrence and development of cancer. Abnormal splicing patterns are often observed and are believed to contribute to carcinogenesis, dedifferentiation and metastasis.

[0004] Researchers have discovered the presence of disease-specific transcripts in breast cancer tissue. Using RNA sequencing, Aversa et al. discovered 12 novel alternatively spliced transcripts in breast cancer tissue and found that these abnormal alternative splicing patterns are closely associated with breast cancer development. These studies suggest that genetic variation can influence splicing, leading to differential expression of the same gene between individuals. However, there are currently no reports on whether genetic variation associated with alternative splicing in innate immune genes affects adverse reactions and prognosis in breast cancer patients receiving NAC. Summary of the Invention

[0005] The present invention aims to provide a marker for predicting adverse reactions to neoadjuvant chemotherapy for breast cancer, thereby predicting the adverse reactions to neoadjuvant chemotherapy for breast cancer and providing a certain reference for the precise prevention and treatment of breast cancer.

[0006] In this scheme, a marker for predicting adverse reactions to neoadjuvant chemotherapy for breast cancer is located at the rs2322718 site.

[0007] Furthermore, the genotype of the locus rs2322718 was GG or TT.

[0008] Furthermore, the neoadjuvant chemotherapy is ET regimen neoadjuvant chemotherapy.

[0009] On the other hand, the applicant found through adverse reaction analysis that breast cancer patients carrying the GG genotype at rs2322718 had a significantly reduced risk of bone marrow suppression (OR=0.361, 95% CI=0.155-0.843, P=0.019). Therefore, the applicant requests protection for the application of the site rs2322718 in predicting adverse reactions to neoadjuvant chemotherapy for breast cancer.

[0010] Furthermore, the adverse reaction of neoadjuvant chemotherapy for breast cancer is the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy.

[0011] Furthermore, when predicting the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy, the genotype of the rs2322718 locus of the breast cancer patient is detected. When the GG genotype is detected, the patient has a lower risk of bone marrow suppression after neoadjuvant chemotherapy, and when the TT genotype is detected, the patient has a higher risk of bone marrow suppression after neoadjuvant chemotherapy.

[0012] Furthermore, the site rs2322718 is used in products related to predicting the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy.

[0013] Furthermore, the product is a reagent, kit, chip, test paper capable of detecting the GG genotype and / or TT genotype of the rs2322718 site in breast cancer patients, or an assessment system that can automatically output the patient's bone marrow suppression risk based on the input genotype test results of the site rs2322718.

[0014] Furthermore, the evaluation system includes:

[0015] An input unit is provided to obtain data to be processed, wherein the data to be processed includes a genotype detection result of the rs2322718 site in the sample;

[0016] an evaluation unit, inputting the data to be processed into a bone marrow suppression evaluation model to obtain a bone marrow suppression evaluation result of the data to be processed, wherein the bone marrow suppression evaluation model is a pre-trained model, and the bone marrow suppression evaluation result includes a correlation between the genotype of the site rs2322718 and the risk of bone marrow suppression after neoadjuvant chemotherapy in breast cancer patients;

[0017] The output unit outputs the bone marrow suppression risk assessment result of the data to be processed. When the detection result is the GG genotype of the site rs2322718, the output result is that the patient has a reduced risk of bone marrow suppression; when the TT genotype is detected, the output result is that the patient has a higher risk of bone marrow suppression.

[0018] Since breast cancer patients carrying the GG genotype at rs2322718 have a significantly reduced risk of myelosuppression (OR = 0.361, 95% CI = 0.155-0.843, P = 0.019), the GG genotype at rs2322718 can be used to screen for drugs that prevent myelosuppression. Specifically, in vitro cell culture models were used to test the regulatory effect of candidate drugs on the expression level of the GG genotype at rs2322718. In addition, the TT genotype at rs2322718 can also be used to screen for therapeutic drugs that treat myelosuppression. Specifically, the TT genotype at rs2322718 is used as a target for myelosuppression therapeutic drugs to verify the therapeutic effect of candidate drugs on myelosuppression. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of different transcripts in the AS process after rs2322718 gene mutation.

[0020] Figure 2 Schematic diagram of rs2322718 mini-gene analysis.

[0021] Figure 3Figure 2 shows the expression of AS transcripts in different cell lines caused by the rs2322718 gene mutation; A is the expression level of different transcripts in the Mcf-7 cell line; B is the grayscale analysis of Figure A; C is the expression of different transcripts in the T47D cell line; D is the grayscale analysis of Figure C; * indicates P < 0.05 compared with rs2322718-T. DETAILED DESCRIPTION

[0022] The following is further described in detail through specific implementation methods:

[0023] 1. Materials and Methods

[0024] 1.1 Research subjects

[0025] Female patients who were admitted to a hospital between 2015 and 2018 and diagnosed with breast cancer by pathology were selected, and 2 ml of peripheral venous blood samples were collected before surgery and medication.

[0026] Subjects meeting the inclusion criteria:

[0027] 1) Breast cancer diagnosed by pathology;

[0028] 2) Patients were treated with ET regimen NAC for 4-6 cycles, with the initial chemotherapy dose of no less than 75 mg / m2 of epirubicin in the first cycle. 2 and docetaxel 75 mg / m 2 ;

[0029] 3) Before the start of this study, approval was obtained from the ethics committee. All subjects voluntarily participated in the investigation and signed the informed consent form.

[0030] Exclusion criteria:

[0031] 1) Patients with cancer that has metastasized to other organs or a history of other cancers;

[0032] 2) Patients with two or more malignant tumors at the same time;

[0033] 3) Male patients.

[0034] 1.2 Collection of Subject Data

[0035] Relevant data were obtained by collecting patients' electronic medical records. General demographic information included patient age and menstrual history, and clinical pathological data included disease stage, short-term efficacy (RECIST standard) and adverse reactions (CTCEA 5.0 standard).

[0036] 1.2.1 Criteria for efficacy evaluation

[0037] Table 1 RECIST criteria

[0038]

[0039] 1.2.2 Adverse reaction criteria

[0040] Table 2 Common Adverse Events Criteria Evaluation Criteria Version 5.0 (CTCAE 5.0) (Myelosuppression)

[0041]

[0042] Table 3 Common Adverse Events Criteria Evaluation Criteria Version 5.0 (CTCAE5.0) (Gastrointestinal Toxicity)

[0043]

[0044] Toxicity reaction indexes of grade 1-4 were recorded as adverse reactions.

[0045] 1.3 SNP selection

[0046] 449 innate immune genes were searched in the InnateDB and Immport databases. The 449 innate immune genes obtained in the previous step were retrieved in the CancerSplicingQTL database, resulting in a total of 7431 SNPs. After removing duplicates, a total of 4388 SNPs were identified. 1167 loci with a MAF greater than or equal to 0.1 were screened. Linkage disequilibrium analysis was performed using Haploview, resulting in 921 loci with linkage relationships, representing 131 linkage blocks. The HGVS numbers for these 921 loci were queried in the Vannoport database for functional prediction. A total of 646 loci were identified with HGVS numbers. Alternative splicing function prediction was performed using HSF software, and 223 loci were identified as potentially functional. The previous linkage disequilibrium analysis results were imported into the locus list through LD analysis, ultimately identifying 17 functional loci in innate immune genes with potential alternative splicing. The rs2322718 locus is shown in Table 4 below.

[0047] Table 4. Candidate SNPs

[0048]

[0049] 1.4 Collection of blood samples and isolation and purification of genomic DNA

[0050] 1.4.1 Collection of blood samples

[0051] After the subjects signed the informed consent form, 2 ml of peripheral blood was collected using an EDTA vacutainer. The tubes were gently inverted and mixed five times, and then stored in a -20°C freezer within 1 hour. If not used for an extended period, the tubes should be transferred to a -80°C freezer for long-term storage. Repeated freezing and thawing should be avoided during storage.

[0052] 1.4.2 Main reagents and instruments for the experiment

[0053] 1.4.2.1 Main experimental reagents

[0054]

[0055] 1.4.2.2 Main experimental instruments

[0056]

[0057]

[0058] 1.4.3 Genomic DNA Isolation and Purification Steps

[0059] A blood genomic DNA extraction kit was used to isolate and purify DNA from peripheral blood samples. The specific steps were as follows: (1) blood samples were taken out of the −20°C refrigerator and thawed in a 4°C refrigerator overnight;

[0060] (2) Lysis of red blood cells, the specific contents are as follows:

[0061]

[0062] (3) Lysis of leukocytes and proteins, the specific contents are as follows:

[0063]

[0064] (4) DNA separation, the details are as follows:

[0065]

[0066]

[0067] (5) Washing and desalting

[0068] Prepare 70% ethanol and add 1 ml of ethanol to the DNA pellet. Invert the tube 50 times to wash the DNA thoroughly. Centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, and repeat the previous step for 2 washes.

[0069] (6) Dissolving DNA. The specific contents are as follows:

[0070]

[0071] (7) DNA solution concentration determination and quantitative dilution, the specific contents are as follows:

[0072]

[0073] 1.5 Genotyping

[0074] The selected SNP was rs2322718. The OpenArray chip used for genotyping was designed and synthesized by Thermo Fisher Scientific. The synthesized sequence is as follows:

[0075]

[0076]

[0077] 1.5.1 Main experimental reagents for genotyping

[0078] 1.5.1.1 Main experimental reagents

[0079]

[0080] 1.5.1.2 Main experimental instruments

[0081]

[0082] 1.5.2 Specific steps of genotyping

[0083] 1.5.2.1 Taqman OpenArray Chip Typing

[0084] (1) Sample preparation:

[0085] 1) Prepare the DNA sample, vortex, and centrifuge at 1000 rpm for 1 minute. Set aside. Equilibrate the Taqman OpenArray chip at room temperature half an hour in advance.

[0086] 2) Importing sample layout information: Samples included in the study were placed in a 96-well plate. A table was created using Excel. The samples were transferred to the 96-well plate, sealed with aluminum foil tape, and stored at -80°C. Sample Tracker software was used to import the sample information into a 384-well export layout file for sample transfer.

[0087] (2) PCR reaction system configuration: Add 2.5 μl of TaqMan OpenArray Genotyping Master Mix to the sample plate according to the exported file, then use a pipette to draw 2.5 μl of DNA sample into the corresponding sample well, centrifuge at 1000 rpm for 1 min, and place it into the sample slot of the automatic sampler;

[0088] (3) Preparation of the chip: After the chip has been balanced for 30 minutes, remove the cap of the prepared oil-containing syringe and replace it with a gun tip. Place the Taqman OpenArray chip in the chip slot of the automatic sample injector. Turn on the automatic sample injector and inject the DNA sample onto the OpenArray chip. After the injection is completed, remove the OpenArray chip and seal the plate. Use the prepared syringe to inject oil into the chip. It is best to complete it within 60 seconds. The injection process should be continuous and not too fast to avoid affecting subsequent experiments. Wipe the outside of the OpenArray chip clean with alcohol.

[0089] (4) PCR reaction: Tear off the protective film on the surface of the chip and place the sealed OpenArray plate into the QuantStudio TM For the 12K Flex System, set the following reaction conditions:

[0090]

[0091] (5) Genotyping: After the reaction is completed, TM The results were read by 12K Flex software and typing was performed based on fluorescence intensity.

[0092] Biological function analysis of site 1.6

[0093] 1.6.1 Transcript Prediction

[0094] We analyzed variable splicing SNPs in the CancerSplicingQTL database to predict which variable splicing events might result from positive SNPs. The Ensembl genome database (https: / / www.ensembl.org / index.html / ), a commonly used source of eukaryotic reference genomes, automatically annotates human genes, including those from humans, mice, zebrafish, pigs, and rats, to identify splicing-capable isomers. We identified transcripts involved in splicing events based on annotations from the Ensembl database (GRCh37) and TCGA SpliceSeq analysis. We then used the UCSC Genome Browser (http: / / genome.ucsc.edu / ) to create transcript diagrams based on Ensembl gene loci.

[0095] 1.6.2 Mini gene experiment

[0096] 1.6.2.1 Main reagents for the experiment

[0097]

[0098]

[0099] 1.6.2.2 Main experimental instruments

[0100]

[0101] 1.6.2.3 Selection of cell lines and vectors

[0102] This study used two human breast cancer cell lines, Mcf-7 and T47D, purchased from Wuhan Pronose Biotechnology Co., Ltd. and tested for short tandem repeat (STR) typing. The vector used was pGL3-promoter from Promega, USA.

[0103] 1.6.2.4 Construction of target plasmid

[0104] Plasmids were constructed at Wuhan Qingke Biotechnology Co., Ltd.: a 500 bp sequence fragment centered on the predicted binding peak start sequence chr8:27400270 containing rs2322718 was downloaded from the UCSC database. This was used as a template to synthesize the target fragments containing rs2322718-T and rs2322718-G, respectively. The fragments were cloned into the vector pGL3-Promoter through the 5'Kpnl restriction site and finally verified by sequencing.

[0105] 1.6.2.5 Plasmid transformation

[0106] (1) Preparation of culture medium and culture plates:

[0107] The specific contents are as follows:

[0108]

[0109] (2) Conversion:

[0110] The specific contents are as follows:

[0111]

[0112]

[0113] 1.6.2.6 Plasmid extraction

[0114] The plasmid product number is OMEGA (D6950-01). Prepare the mixture according to the instructions, dilute the HBC Buffer, add the vial of RNase A to Solution I, and store at 2-8°C. Add 10ml of isopropanol to dilute the HBC Buffer. Add 60ml of anhydrous ethanol to dilute the DNA Wash Buffer, and store at room temperature after dilution. The specific steps are as follows:

[0115]

[0116] 1.6.2.7 Transfection of Plasmids into Breast Cancer Cell Lines

[0117] (1) Cell recovery and culture

[0118] The culture medium used for the T47D and Mcf-7 cell lines is the same. Prior to the experiment, sterilize the cells in a UV-clean hood for 30 minutes and preheat a water bath (37°C). Remove the cryovial from the liquid nitrogen tank and quickly thaw in a water bath. Transfer the cells to a 15ml centrifuge tube in a clean hood and add 10x PBS. Centrifuge at 1000 rpm for 3 minutes and discard the supernatant. Add 5ml of prepared complete culture medium (10% FBS, 1% double-antibody; if cell growth is slow, increase the FBS concentration appropriately). Transfer the cell suspension into a T25 cell culture flask, label with the cell name and date, and culture in a cell incubator (37°C, 5% CO2). Mcf-7 cells have poor adherence, so the resting time may be increased. When the cells in the flask are approximately 80% confluent, plate the cells.

[0119] (2) Cell passage

[0120] When the cells cover more than 80% of the bottom of the culture flask, sterilize the clean bench with ultraviolet light for half an hour in advance and prepare DMEM complete medium, sterile PBS buffer, trypsin digestion solution-EDTA, 15ml centrifuge tubes, sterile pipettes, etc. Pour out the medium in the culture flask, add 2ml of sterile PBS to rinse gently, aspirate the PBS, add 1ml of trypsin digestion solution-EDTA, so that the trypsin digestion solution covers the bottom of the flask, digest for 3 minutes, add 4ml of complete medium, use a sterile pipette to blow the bottom of the flask until the cells fall off, transfer the culture medium containing the cells to a centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the supernatant, add the amount of culture medium required for the corresponding subculture ratio to resuspend the cells, and then transfer to a new culture flask. Place the culture flask containing the cells in the incubator for culture, with a subculture ratio of 1:2 or 1:3.

[0121] (3) Cell plating

[0122] The experimental groups in Mcf-7 and T47D cell lines were divided into the following groups: empty pGL3-promoter group, rs2322718-T group, and rs2322718-G group. Each group had 3 replicate wells and the experiment was repeated 3 times. 24-well plates were used for plating. The cultured cell culture flasks were removed and the cells were digested with trypsin. The cells were resuspended in cell culture medium according to the relevant cell passage procedures and the cell concentration was adjusted to 2-8×10 4 Each well was evenly inoculated into a 24-well plate, 500 μl per well. The 24-well plate was placed in a cell culture incubator and transfected when the cells reached about 70% fusion.

[0123] (4) Cell transfection

[0124] When using Lipo3000 as a transfection reagent, it is not recommended to use cells that have been passaged too many times or have just recovered for transfection experiments. 2 hours before transfection, replace the culture medium with fresh complete culture medium. Take out an EP tube and dilute 1μl of Lipofectamine3000 with 25μl Opti-MEM. In another EP tube, dilute 0.5μg DNA with 25μl Opti-MEM, then add 1μl P3000 and mix well. Then add the DNA / P3000 diluted in the previous step to the diluted mixture, incubate at room temperature for 10-15 minutes, add it to the target cells containing 500μl culture medium (culture medium without antibiotics) and shake gently. Place it in a cell culture incubator and transfect for about 6 hours. Change the medium according to the condition of the cells. If there are not many dead cells, you do not need to change the medium. Set aside for use 48 hours after transfection.

[0125] (5) Reverse transcription of RN to prepare cDNA

[0126] After transfection, the cells were gently scraped and collected with a scraper to form a homogenate, which was then transferred into a centrifuge tube. The total RNA of the cells was extracted using the Trizol method:

[0127]

[0128] Reverse transcribe the extracted RNA using the TOYOBO Reverse Transcription Kit (FSQ-101). Dilute the sample to a concentration of 300 ng / μL with enzyme-free sterile water. Prepare the reverse transcription reaction on ice according to the table below. Reverse transcription conditions are as follows: 37°C for 15 minutes (reverse transcription reaction), 85°C for 5 seconds (reverse transcriptase inactivation reaction), and 4°C forever.

[0129]

[0130] (6) Real-Time PCR reaction

[0131] According to the instructions, take 5 μl of the solution, 0.4 μl of each Mix primer, and 1 μl of the cDNA sample, add water to make up to 10 μl of the system, and perform real-time quantitative PCR amplification on each sample. The reaction conditions are as follows:

[0132]

[0133] The preparation of PCR reaction system is as follows:

[0134]

[0135] After the PCR reaction was completed, the melting curve was observed and the Ct value of each sample was obtained. -△△CtThe expression level of the transcript was determined by the method. The primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. The specific sequences are as follows:

[0136]

[0137] (7) Agarose gel electrophoresis

[0138] To verify the effects of genetic variation on different transcripts, all experiments were repeated three times. The specific steps are as follows:

[0139]

[0140]

[0141] 1.7 Analytical methods

[0142] SPSS 29.0 was used for statistical analysis. t-test was used for comparison of continuous data between groups, and X-test was used for categorical data. 2 Unconditional logistic regression was used to analyze the association between candidate SNPs and adverse reactions (bone marrow suppression).

[0143] 2. Results

[0144] 2.1 Estimating Sample Size Power

[0145] Based on a literature review, the pCR rate for breast cancer was 20%, the expected OR was 2, the required α was 0.05 (two-sided test), and the β was 0.10. The sample size was calculated as n = 278. A total of 553 BC patients were included, which was a sufficient sample size. The sample size calculation formula is as follows:

[0146]

[0147] 2.2 Basic Information of Research Subjects

[0148] This study enrolled 553 breast cancer patients undergoing NAC with the ET regimen (mean age: 50.20 ± 9.739 years). Of these, 264 (47.7%) were postmenopausal and 289 (52.3%) were premenopausal. Myelosuppression was reported in 258 (46.7%) patients, and gastrointestinal toxicity was reported in 15 (2.7%). The response rate (pCR + CR + PR) after NAC was 81.1% (Table 5).

[0149] Table 5. Basic information of the research subjects

[0150]

[0151] Note: Pathological complete response (pCR); complete response (CR); partial response (PR); no response (SD); progressive disease (PD)

[0152] 2.3 Association analysis between rs2322718 and myelosuppression after NAC in breast cancer

[0153] Results showed that rs2322718 was associated with myelosuppression after neoadjuvant chemotherapy for breast cancer. After adjustment for age and menopausal status, individuals carrying the rs2322718 GG genotype had a significantly lower risk of myelosuppression by approximately 63.9% (OR = 0.361, 95% CI = 0.155-0.843, P = 0.019) compared with wild-type homozygotes (TT). A recessive model (OR = 0.354, 95% CI = 0.153-0.819, P = 0.015) also confirmed that the mutation at this site had a protective effect against myelosuppression after neoadjuvant chemotherapy (Table 6).

[0154] Table 6. Association of rs2322718 with myelosuppression after NAC in breast cancer

[0155]

[0156] 2.4 Study on the biological mechanism of SNP affecting the prognosis of breast cancer after neoadjuvant chemotherapy

[0157] 2.4.1 Acquisition of transcripts

[0158] Positive SNPs were queried from the Cancersplicing database and it was found that the rs2322718G allele promoted the alternative splicing of PTK2B exon 35. The splicing mode was AT, that is, after the gene underwent AS, a new isoform exon 35 was formed, and the splicing position was different at chr8:27315811. The results are shown in Table 7.

[0159] Table 7 Spliced exons and splicing patterns of positive sites

[0160]

[0161] Note: AT: Alternate terminator, variable terminator.

[0162] According to the annotations of the RefSeq gene database and the Ensembl database (GRCh37), the RS number and the splicing region were input into the restriction conditions, and the transcripts involved in the splicing event were analyzed in combination with TCGA SpliceSeq. Then, the UCSC Genome Browser (http: / / genome.ucsc.edu / ) was used to draw a schematic diagram of the PTK2B transcript according to the Ensembl gene track. The results are shown in the figure. Figure 1As shown in Figure 2, it was found that the gene was spliced after mutation, with the rs2322718-G allele corresponding to the transcript ENST00000346049 (PTK2B_V1) and the rs2322718-T allele corresponding to the transcript ENST00000420218 (PTK2B_V2).

[0163] 2.4.2 Mini gene experiment

[0164] The PTK2B gene fragments containing rs2322718-G / rs2322718-T were cloned into pcDNA3.1 vectors and then transfected into Mcf-7 and T47D cell lines. Figure 2 ) and RT-PCR results showed that compared with the rs2322718-T allele, the overexpression of rs2322718-G promoted the alternative splicing of PTK2B, resulting in an increase in the expression level of PTK2B_V1 (P<0.05) and a decrease in the expression level of PTK2B_V2 (P<0.05) ( Figure 3 ).

[0165] 2.5. Conclusion

[0166] This study obtained the following conclusions through bioinformatics analysis, large-sample retrospective cohort study, and biological function experiments:

[0167] (1) rs2322718 is associated with the risk of myelosuppression after neoadjuvant chemotherapy for breast cancer. The risk of myelosuppression in individuals with the GG genotype is 36.1% of that in individuals with the TT genotype.

[0168] (2) The rs2322718-G allele mutation resulted in variable splicing, which led to an increase in the expression level of the innate immune gene PTK2B transcript ENST00000346049 (PTK2B_V1) and a decrease in the expression level of the transcript ENST00000420218 (PTK2B_V2), suggesting that this allele mutation can affect the function of the gene through variable splicing, thereby affecting the adverse reactions of chemotherapy drugs.

[0169] The above is only an embodiment of the present invention, and the common knowledge such as the specific structure and characteristics of the scheme is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A marker for predicting adverse reactions to neoadjuvant chemotherapy for breast cancer, characterized by: The marker is site rs2322718.

2. The marker according to claim 1, characterized in that: The genotype of locus rs2322718 was GG or TT.

3. The marker according to claim 2, characterized in that: The neoadjuvant chemotherapy is ET regimen neoadjuvant chemotherapy.

4. Use of the marker according to any one of claims 1 to 3 in predicting adverse reactions to neoadjuvant chemotherapy for breast cancer.

5. The use according to claim 4, characterized in that: The adverse reaction of neoadjuvant chemotherapy for breast cancer is the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy.

6. The use according to claim 5, characterized in that: When predicting the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy, the genotype of the rs2322718 locus of the breast cancer patient is detected. When the GG genotype is detected, the patient has a lower risk of bone marrow suppression after neoadjuvant chemotherapy. When the TT genotype is detected, the patient has a higher risk of bone marrow suppression after neoadjuvant chemotherapy.

7. The use according to claim 5, characterized in that: Application of locus rs2322718 in predicting the risk of bone marrow suppression in breast cancer patients after neoadjuvant chemotherapy.

8. The use according to claim 7, characterized in that: The product is a reagent, kit, chip, test paper capable of detecting the GG genotype and / or TT genotype of the rs2322718 locus in breast cancer patients, or an assessment system capable of automatically outputting the patient's risk of bone marrow suppression based on the input genotype test results of the locus rs2322718. The assessment system includes: An input unit is provided to obtain data to be processed, wherein the data to be processed includes a genotype detection result of the rs2322718 site in the sample; an evaluation unit, inputting the data to be processed into a bone marrow suppression evaluation model to obtain a bone marrow suppression evaluation result of the data to be processed, wherein the bone marrow suppression evaluation model is a pre-trained model, and the bone marrow suppression evaluation result includes a correlation between the genotype of the site rs2322718 and the risk of bone marrow suppression after neoadjuvant chemotherapy in breast cancer patients; The output unit outputs the bone marrow suppression risk assessment result of the data to be processed. When the detection result is the GG genotype of the site rs2322718, the output result is that the patient has a reduced risk of bone marrow suppression; when the TT genotype is detected, the output result is that the patient has a higher risk of bone marrow suppression.

9. Use of the marker according to claim 2 in screening drugs for preventing myelosuppression.

10. Use of the marker according to claim 2 in screening drugs for treating myelosuppression.

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