Application of PIK3CA mutation accompanied with 5-LOX high expression as a prognostic marker for luminal type breast cancer

Abstract

The application discloses application of PIK3CA mutation accompanied by 5-LOX high expression as a prognosis marker of luminal type breast cancer. The application directly observes the expression of PIK3CA mutation accompanied by 5-LOX in tumors by adopting rolling circle nucleic acid amplification technology and gene probe to detect PIK3CA mutation in tissue samples and by dyeing 5-LOX through immunohistochemical experiment. In addition, the application improves the tumor immune microenvironment by synergistic targeted therapy and immunotherapy, and improves the curative effect of immunotherapy on endocrine-resistant breast cancer. The application takes PIK3CA mutation accompanied by 5-LOX high expression as a new prognosis marker of luminal type breast cancer, and has wide application prospect.

Description

Technical Field

[0001] This application relates to the field of tumor prognostic biomarkers, and in particular to the application of PIK3CA mutation with high 5-LOX expression as a prognostic biomarker for Luminal breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women, ranking first in incidence and second in mortality among female cancers worldwide, making it a major threat to women's physical and mental health. As one of the body's important hormone-targeting organs, over 70% of malignant tumors in the breast are hormone-dependent, characterized by the expression of estrogen receptor (ER) and / or progesterone receptor (PR), termed Luminal breast cancer. This type of breast cancer is relatively sensitive to estrogen suppression therapy and has indications for endocrine therapy; however, nearly 50% of patients develop endocrine resistance, and once recurrence and metastasis occur, the disease progresses rapidly, lacking effective treatment options. In recent years, with the development of biological and molecular biological theories and technologies, it has been recognized that gene mutations play a crucial role in the occurrence and development of breast cancer. Generally, the histological state of the tumor determines its treatment; increasing understanding of tumor biology can increase the available targeted therapies, providing patients with more treatment opportunities. Therefore, in-depth research at the molecular level into the complexity of Luminal breast cancer, and the search for new treatment strategies and targets, is a direction for the field of breast cancer treatment.

[0003] PIK3CA mutations are among the most common gene mutations in breast cancer, with a positive rate exceeding 30%, and are closely related to the development and progression of breast cancer. PIK3CA is a key member of the PI3Ks lipokinase family, and its encoded protein p110α is the catalytic subunit of PI3Ks. Functional and genetic studies have shown that the PIK3CA gene is mutated in many different types of solid tumors. The vast majority of mutations are located in the helical and kinase regions of its protein, with the most common mutations being H1047R on exon 20 and E545K on exon 9. PIK3CA mutations can lead to enhanced PI3Ks catalytic activity and abnormal activation of the PI3K / AKT pathway, thereby contributing to tumorigenesis.

[0004] Mounting evidence suggests that intracellular genetic alterations in tumor cells can drive the formation of a specific tumor microenvironment. While immunotherapy has provided clinical benefits to patients with various cancers, the vast majority of breast cancer patients, especially those with Luminal breast cancer, have been previously considered to have weak immunogenicity due to their mismatch repair deficiencies and low levels of somatic mutations. Therefore, research on their tumor immune microenvironment and immunotherapy is limited. Thus, investigating the formation mechanism of the PIK3CA-mutated tumor immunosuppressive microenvironment and identifying key targets holds promise for improving the efficacy of immunotherapy. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, this application provides an application of PIK3CA mutation accompanied by high expression of 5-LOX as a prognostic marker for Luminal breast cancer.

[0006] In the first aspect, this application provides a use of PIK3CA mutation accompanied by high expression of 5-LOX as a prognostic marker for breast cancer, which is achieved by the following technical solution.

[0007] Application of PIK3CA mutation with high 5-LOX expression as a prognostic biomarker for Luminal breast cancer.

[0008] Secondly, this application provides a second use of PIK3CA mutation accompanied by high 5-LOX expression as a prognostic marker for breast cancer, which is achieved by the following technical solution.

[0009] Application of a reagent for detecting PIK3CA mutations accompanied by high 5-LOX expression in the preparation of products for prognostic assessment or prediction of immunotherapy efficacy in Luminal breast cancer.

[0010] Furthermore, rolling circle nucleic acid amplification technology and gene probes were used to detect PIK3CA mutations; immunohistochemical staining was used to detect 5-LOX expression levels.

[0011] Furthermore, the nucleic acid sequence of the gene probe is shown in SEQ ID NO.1.

[0012] Thirdly, this application provides a kit for assessing the prognosis of lumbar breast cancer, which is achieved using the following technical solution.

[0013] A kit for assessing the prognosis of luminal breast cancer, comprising reagents for rolling circle nucleic acid amplification technology, gene probes, and immunohistochemical staining reagents.

[0014] Fourthly, this application provides a third use of PIK3CA mutation accompanied by high 5-LOX expression as a prognostic marker for breast cancer, which is achieved by the following technical solution.

[0015] Application of a reagent that inhibits the PI3K signaling pathway and / or reduces 5-LOX expression levels in the preparation of products for the treatment of lumbar breast cancer.

[0016] Furthermore, the reagent is selected from one or a combination of two or more of PI3K inhibitors, LTB4 antagonists, and anti-PD-1 mAbs.

[0017] Furthermore, the PI3K inhibitor is Alpelisib; the anti-PD-1mAbs is Pembrolizumab; and the LTB4 antagonist is LY255283.

[0018] Fifthly, this application provides a drug for treating lumens breast cancer, which is achieved by the following technical solution.

[0019] A medicine for treating lumens breast cancer, said medicine comprising one or a combination of two or more of PI3K inhibitors, LTB4 antagonists, and anti-PD-1mAbs.

[0020] This application has the following beneficial effects.

[0021] This invention, through cell and animal experiments, revealed that the arachidonic acid metabolic pathway is significantly activated in patients with PIK3CA-mutated Luminal breast cancer, with the LOX pathway showing the most significant changes. Upregulation of the 5-LOX pathway and LTB4 secretion lead to high infiltration of MDSCs in the tumor microenvironment and affect the function of tumor-killing T cells. Targeted therapy against the PI3K / 5-LOX / LTB4 axis can reverse the inhibitory microenvironment and enhance the anti-tumor activity of immunotherapy. This invention suggests that PIK3CA mutation accompanied by high 5-LOX expression can serve as a novel prognostic biomarker for tumors. Detection of PIK3CA mutation with high 5-LOX expression can be used for early diagnosis, molecular subtyping, and prognostic assessment of Luminal breast cancer, and may also be a potential therapeutic target for clinical treatment of Luminal breast cancer. Attached Figure Description

[0022] Figure 1 This is a diagram illustrating the PIK3CA gene mutation status in the pan-cancer analysis of this invention;

[0023] Figure 2 This is a graph showing the relationship between the detection of PIK3CA mutation and 5-LOX expression in tissue samples and their impact on prognosis, based on the present invention.

[0024] Figure 3 This is a graph showing the relationship between PIK3CA mutation and 5-LOX expression and the infiltration level of MDSCs in the immune microenvironment.

[0025] Figure 4 This is a diagram showing the relationship between the PIK3CA mutation and the activation of 5-LOX expression through the PI3K / AKT / STAT3 pathway in this invention.

[0026] Figure 5 This is a diagram illustrating how 5-LOX, in accordance with the present invention, affects the recruitment and immunosuppressive function of MDSCs by regulating LTB4 secretion;

[0027] Figure 6 This is a diagram illustrating the synergistic effect of targeted therapy and immunotherapy in enhancing anti-tumor efficacy against PIK3CA / MDSCs infiltration, as described in this invention.

[0028] Figure 7 This is a graph showing the results of the present invention's targeted therapy against the PIK3CA / 5-LOX / LTB4 axis effectively inhibiting MDSC infiltration. Detailed Implementation

[0029] The present patent application will be further described below with reference to the embodiments.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used in the following preparation examples and examples are commercially available unless otherwise specified.

[0031] I. Experimental Methods

[0032] 1. Collection and organization of the TCGA database

[0033] Download Java and install the bioinformatics toolkit. Download data on breast cancer somatic mutations, DNA copy number variations, mRNA and microRNA expression, protein enrichment, phosphorylated protein enrichment, and clinical follow-up information. Screen for Luminal breast cancer patients, extract patient gene mutation data, and classify patients into wild-type and mutant patients based on PIK3CA gene mutation status. Use bioinformatics tools to plot and analyze changes in downstream pathways.

[0034] 2. Collection and organization of the cBioPortal database

[0035] Visit https: / / www.cbioportal.org / . cBioPortal's gene datasets can be accessed or downloaded via the interactive interface. Select the Luminal breast cancer dataset and query for PIK3CA mutations within it. Group patients into mutant and wild-type groups, view and interpret the results, and create a visualization.

[0036] 3. Clinical Sample Collection

[0037] This application collected tissue samples from 140 primary breast cancer patients (including 62 cases of Luminal breast cancer) from January 2001 to December 2008. All of these patients were female, with a median age of 57 years (range 37-88 years). Clinical and pathological indicators such as patient gender, age, clinical stage, tumor size, molecular subtype, recurrence and metastasis, and overall survival were also collected.

[0038] 4. RCA-FISH detection of PIK3CA mutations

[0039] Breast cancer tissue samples were deparaffinized with xylene and treated with a gradient of ethanol, ultimately hydrating them. The sections were boiled in water for 20 minutes, and an appropriate amount of proteinase K reaction solution was added according to the tissue area, incubating at 37°C for 20 minutes. The slides were then immersed twice in a 2×SSC staining tank, dehydrated with gradient ethanol, and air-dried. Restriction endonuclease reaction solution was added, and the mixture was incubated at 37°C for 1 hour to expose the double-stranded DNA ends near the target gene. Exonuclease reaction solution was added, and the mixture was incubated at 37°C for 40 minutes to expose the single-stranded DNA. Target gene probe binding circularization reaction solution was added, and the mixture was incubated at 37°C for 1 hour, allowing the probe to form a closed circular DNA at the target gene site. The slides were immersed twice in a 2×SSC staining tank to wash away excess probe, dehydrated with gradient ethanol, and air-dried. Signal amplification reaction solution was added, and the mixture was incubated at 37°C for 1 hour, allowing the closed circular DNA to self-replicate and amplify the target gene signal. Fluorescent probe reaction solution was added, and the mixture was incubated at 37°C for 15 minutes, allowing the fluorescent probe to bind to the repetitive sequences on the extensively replicated closed circular DNA. Immerse the slide once in a 2×SSC staining bath to wash away excess probe, dehydrate with graded ethanol, and air dry. Add a DAPI-containing anti-quenching mounting medium, mount the slide, observe the fluorescence signal under a fluorescence microscope, and perform subsequent data analysis to determine the number of characteristic insertion fragments of the PIK3CA mutation.

[0040] The gene primer sequences are as follows:

[0041] PIK3CA E545K :5'-AGCAGGAGAAAGATTTCTGCGAATAGCCATCCACTCCATTCTTCTGCGAATAGCCATCCACTCCATCTCTCTCTGAAATCACTA-3'(SEQ ID NO.1)

[0042] PIK3CA H1047R :5'-ATGAAATACTCCAAAGCCTCCCTCGCATCAATACCGATCATCTCTTCCCCTCGCATCAATACCGATCATCGTGCATCATTCATTTGTTTC-3'(SEQ ID NO.2)

[0043] 5. Immunohistochemistry

[0044] Baked paraffin tissue sections or tissue microarrays in a 75°C oven for at least 2 hours. Then, sequentially immerse the paraffin sections in xylene I, xylene II, and xylene III (30 min / time). Immerse the paraffin sections in anhydrous ethanol (twice), 95% ethanol (once), 85% ethanol (once), and 75% ethanol (once), 5 minutes each time. Immerse the sections in 3% H2O2 at room temperature, protected from light, for at least 15 minutes, and wash with PBS buffer for 5 min × 3 times. Completely immerse the tissue sections in antigen retrieval solution using an autoclave. Start timing after the autoclave valve begins to rotate; stop heating after 2-3 minutes and allow to cool naturally to room temperature. Draw circles around the tissue on a glass slide using a hydrophobic pen, add goat serum to completely cover the tissue, and incubate in a humidified chamber at room temperature for 15 minutes to prevent drying. Incubate with primary antibody: Add the prepared primary antibody to a glass slide (approximately 100 μL / slide) to completely cover the tissue section, and incubate overnight at 4°C in a humidified chamber. Rewarming: Place the humidified chamber at room temperature and rewarm for at least 30 minutes. The rewarming time can be adjusted within 1-2 hours as needed. Wash with PBS for 5 minutes each time (3 times). Blot off excess moisture from the paraffin sections, add the enhancer to completely cover the tissue area, and incubate in a humidified chamber at room temperature for 30 minutes. Wash with PBS for 5 minutes each time (3 times). Add rabbit universal secondary antibody, incubate at room temperature for 15 minutes, and then wash with PBS for 5 minutes each time (3 times). Add DAB working solution to the tissue sections and develop the color for about 3-5 minutes. Stop the development with DW water. Stain the tissue sections with hematoxylin solution for about 3-5 minutes, and then stop the development with DW water. Place the sections in hydrochloric acid alcohol to develop red color, then in ammonia water to develop blue color, and stop the development with DW water. Immerse the tissue sections in 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene solution for 15 minutes three times in sequence. After drying in a fume hood, seal the tissue sections with neutral resin and coverslips, and observe the staining under a microscope.

[0045] Immunohistochemical result interpretation: Under double-blind conditions, the staining results were independently interpreted by two pathologists, and the average score of the two physicians was taken. Staining intensity was rated from weakest to strongest as follows: 0 points - no staining, 1 point - light yellow, 2 points - brown. Tumor cell proportion scores were as follows: no positive tumor cells 0 points, <10% positive tumor cells 1 point, <50% positive tumor cells 2 points, <75% positive tumor cells 3 points, ≥75% positive tumor cells 4 points. The formula for calculating the percentage of positive tumor cells in the tissue section was: percentage of positive tumor cells × staining intensity. For 5-LOX staining, positive cells were counted in 5 fields at 200× magnification, and the total number of positive cells was calculated.

[0046] 6. Real-time quantitative PCR

[0047] (1) Extraction of total RNA:

[0048] Collect cells into 1.5 ml EP tubes, add 1 ml of Trizol lysis buffer, and repeatedly lyse the cells using a pipette. Incubate on ice for 5 min. Add 200 μl of chloroform, vortex vigorously for 30 s, incubate on ice for 5 min, and then centrifuge at 13000 rpm for 15 min at 4 °C. Carefully aspirate the colorless supernatant, avoiding the white membrane layer, and transfer the clear supernatant to a new enzyme-free EP tube. Add an equal volume of pre-chilled isopropanol solution, gently invert to mix, and incubate on ice. Let stand for 10 min, then centrifuge at 13000 rpm for 10 min at 4℃; discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water), wash the RNA precipitate at 7500 rpm for 5 min for 3 times at 4℃, carefully aspirate the upper layer, and air dry the liquid in the EP tube; after the bottom RNA precipitate becomes colorless and transparent, dissolve it with an appropriate volume of DEPC water, and use a spectrophotometer to detect the RNA concentration and purity. Pure RNA samples have A260 / A280 > 2.0 and 260 / 230 > 2.0;

[0049] (2) Reverse transcription reaction:

[0050] Prepare the reverse transcription reaction system and set up the PCR instrument according to the conditions of "37℃, 15 min; 85℃, 5 s; 4℃, infinite cycling". The specific reaction system is shown below:

[0051]

[0052] (3) Real-time PCR:

[0053] 1) Prepare the PCR reaction system as follows:

[0054]

[0055] Primer sequence

[0056]

[0057] 2) The setup for the real-time PCR procedure is as follows:

[0058]

[0059] (4) Results Analysis:

[0060] After the reaction was completed, the dissolution curve and amplification curve were confirmed using 2... -ΔCT The relative expression level of the target gene is calculated using this method.

[0061] 7. Western blot assay

[0062] Collect 2×10 respectively 6 Cells were collected, and 0.2 mL of cell lysis buffer containing protease inhibitors was added. The cells were centrifuged at 14000g for 15 minutes at 4°C, and the supernatant was collected to determine protein concentration. 40 μg of cell lysate was denatured at 100°C for 10 minutes. The denatured protein was then added to a 10% SDS-PAGE gel. Electrophoresis was performed at 60V for the stacking gel and 100V for the separating gel for 2 hours. After electrophoresis, the cells were transferred to a membrane, blocked at room temperature for 1 hour, and incubated overnight at 4°C with monoclonal antibody (1:1000). The membrane was washed with TBST, and HRP-labeled secondary antibody (1:2000) was added. Chemiluminescence imaging was then performed. The cumulative optical density of the protein was detected using Quantity One software, and the protein expression level was calculated.

[0063] 8. ELISA experiment

[0064] Dilute the antibody to a concentration of 1-10 μg / ml using coating buffer, add 0.1 ml to each well of a polystyrene plate, and incubate overnight at 4°C. The next day, discard the solution in the wells and rinse with personal detergent for 3 min × 3 times. Add 0.1 ml of the sample to be tested to each antibody-coated well and incubate at 37°C for 1 h. Add the diluted enzyme-labeled antibody and incubate at 37°C for 1 h. Add 0.1 ml of the prepared TMB substrate solution to each well and incubate at 37°C for 30 min. Add 0.05 ml of stop solution and measure the OD value at 450 nm using a microplate reader.

[0065] 9. Transwell Experiment

[0066] Add approximately 500 μl of tumor cells or complete culture medium containing exogenous stimulating factors to the lower chamber of the small chamber; add 2 × 10⁻⁶ mcg of tumor cells to the upper chamber of the small chamber. 4 One MDSC (myeloid-derived immunosuppressive cell) cell, approximately 200 μl in volume, was co-cultured in an incubator for 24 to 48 hours. The cells in the upper chamber were gently wiped away with a cotton swab, fixed with methanol for 30 minutes, and the chamber was removed. The cells were then stained with 0.1% crystal violet for 20 minutes, and the staining was terminated with ddH2O. After drying, the membrane was carefully removed, preserved with a mounting medium, and observed under a microscope. The cells were counted and photographed.

[0067] 10. CFSE Experiment

[0068] Dissolve 500 μg of CFSE lyophilized powder in 90 μl of DMSO solution to obtain CFSE stock solution (10 mM). Dilute the stock solution to 10 μM using serum-free empty culture medium. Resuspend the cells to be tested in empty culture medium (1 × 10⁻⁶ mM). 7Mix the cell suspension with an equal volume of 10 μM CFSE (final concentration 5 μM) for 15 min (5% CO2, 37℃). Incubate the cells in a cell culture incubator for 15 min, gently shaking to mix every 5 min. Add 5 times the volume of complete culture medium to stop staining. After centrifugation and washing twice, resuspend the cells and plate them in the dark. After culturing for 72 h, collect the cells to be tested and directly analyze them. Analyze the experimental data.

[0069] 11. Mouse xenograft model

[0070] Digested tumor cells were mixed, resuspended into a single-cell suspension, and the final cell concentration was 1×10⁻⁶. 7 / ml. After anesthetizing the mice, the cells were disinfected with 75% ethanol, and 100 μl of the cell suspension was injected into the fat pad of the mice. After inoculation, the mice were kept alive after recovery, and the tumor growth and mouse condition were closely observed and recorded. Tumor tissue was collected: the mice were sacrificed 3 weeks later, and the tumor tissue was used for subsequent experiments.

[0071] 12. Statistical Analysis

[0072] Data analysis was performed using SPSS 22.0, R language version 4.1.0, and GraphPad Prism 6.0. For statistical description, the mean and standard deviation were used for normally distributed continuous variables; the median and percentiles were used for non-normally distributed continuous variables. For two normally distributed groups, Student's t-test was used; for two non-normally distributed groups, the annular sum test was used; for multiple data samples, analysis of variance was used for normally distributed samples, and the annular sum test was used for non-normally distributed samples. Kaplan-Meier analysis was used to analyze patient survival outcomes. Pearson correlation coefficient analysis was used to analyze the correlation between two indicators. A p-value < 0.05 was considered statistically significant.

[0073] II. Experimental Results

[0074] 1. In previous studies, pan-cancer analysis revealed that PIK3CA is the gene most frequently mutated in somatic cells. Cancers with high PIK3CA mutation activation rates include breast cancer, squamous cell carcinoma of the lung, colorectal cancer, and squamous cell carcinoma of the head and neck. Figure 1 A). Data from breast cancer patient samples were downloaded from the TCGA database, and 697 patients with Luminal breast cancer were identified. A waterfall plot shows all breast cancer patients and common mutated genes in the TCGA cohort. The results show that PIK3CA is one of the most common mutated genes in breast cancer. Figure 1 B), its mutation frequency is higher in Luminal breast cancer, at approximately 42%. Figure 1 C).

[0075] 2. This application used rolling circle amplification (RCA) and gene probes to detect PIK3CA mutations in 62 Luminal breast cancer tissue samples. Based on probe luminescence, the samples were divided into a wild-type group and a PIK3CA mutant group. Figure 2 A). Immunohistochemical staining of tissue samples was then performed to detect 5-LOX, resulting in high expression of 5-LOX in PIK3CA-mutated tumors. Figure 2 A). To further investigate the relationship between PIK3CA mutation and 5-LOX expression, this application used Pearson correlation coefficients on 62 Luminal breast cancer tissues, combined with clinical case data and survival information, to conduct statistical analysis. The results showed that patients with PIK3CA mutation and high 5-LOX expression had significantly shorter survival times than other groups. Figure 2 B).

[0076] 3. This application further analyzed the relationship between PIK3CA mutation status and 5-LOX expression level and the infiltration level of MDSCs in the immune microenvironment. To explore the relationship between PIK3CA mutation and 5-LOX expression and MDSC infiltration, this application used immunohistochemical experiments to stain and statistically analyze 62 Luminal breast cancer tissues. Figure 3 A). The results showed that patients with high MDSC infiltration had shorter survival ( Figure 3 A), and the chi-square test results showed that MDSCs had significantly high infiltration in the PIK3CA mutant group and the 5-LOX high expression group. Figure 3 BC).

[0077] 4. This application compares breast cancer EO771 WT EO771 E545K and EO771 H1047R The transcriptional level of ALOX5 in cells was determined by qPCR experiments, showing that EO771 E545K and EO771 H1047R The transcriptional level of ALOX5 in cells was significantly increased. Figure 4 A). This application then used Western blotting to detect changes in 5-LOX expression, AKT and STAT3 proteins, and phosphorylated proteins. The results showed that EO771 E545K and EO771 H1047R Cellular P-AKT (S473, S308) and P-STAT3 (S705, S727) levels were significantly elevated, and this change could be reversed by PIK3CA inhibitors. Figure 4B); In breast cancer cell lines MCF7 (E545K) and T47D (H1047R), the use of PIK3CA inhibitors (Alpelisib or CH5132799) revealed decreased 5-LOX expression and significantly reduced AKT and STAT3 phosphorylation levels. Figure 4 C), while the use of the PI3K activator 740-YP in wild-type PIK3CA MCF10A and EO771 cells increased the expression of 5-LOX and the phosphorylation levels of AKT and STAT3. Figure 4 D). To further explore the activation mode of the AKT / STAT3 pathway, this application used 740-YP on EO771. WT EO771 E545K and EO771 H1047R Cells were pretreated for 0, 30, 60, and 90 minutes, and the activation of the pathway was observed. Western blot analysis showed that the activation of the pathway increased with time in EO771 cells. WT In cells, P-AKT and P-STAT3 are activated sequentially, while EO771 E545K and EO771 H1047R This change was not observed in the cells. Figure 4 The above results indicate that the PIK3CA mutation increases 5-LOX expression by affecting the transcriptional level of the ALOX5 gene through the AKT / STAT3 pathway.

[0078] 5. At the cellular level, small interfering RNA was used to block EO771. E545K and EO771 H1047R The expression of 5-LOX in cells was investigated to examine its effect on LTB4 production. ELISA results showed that EO771 E545K and EO771 H1047R LTB4 secretion was significantly reduced in cell supernatant. Figure 5 A), and the same results were observed in T47D and MCF7 cells. Figure 5 B) indicates that PIK3CA-mutated breast cancer cells have enhanced arachidonic acid metabolism, leading to increased LTB4 levels in the tumor immune microenvironment. To elucidate the effect of LTB4 on the immunosuppressive activity of MDSCs, this application uses in vitro cell co-culture to simulate the tumor immune microenvironment. EO771 WT EO771 E545K and EO771 H1047RMDSCs were isolated from a cell tumor model and co-cultured with T cells at a ratio of 3:1 in a cell culture chamber. After 96 hours, T cells were collected, and T cell proliferation was assessed using a CFSE assay. Changes in IFN-γ secretion levels by T cells were detected using an ELISA assay to assess the immunosuppressive effect of MDSCs on T cells. Results showed that, compared with EO771... WT Compared to the co-incubation group, EO771 E545K and EO771 H1047R Co-incubated MDSCs resulted in lower T cell proliferation levels. Figure 5 CD). The use of the LTB4 antagonist LY255283 can partially reverse T cell proliferation. Figure 5 E) and IFN-γ secretion levels ( Figure 5 F). Pretreatment of MDSCs with exogenous LTB4 and detection by live-cell imaging experiments showed that the recruitment effect of MDSCs was more significant in the co-culture system. Figure 5 G). Pretreated MDSCs were co-incubated with T cells, and the anti-tumor function of T cells was then detected. The results showed that the proliferation capacity of T cells co-incubated with LTB4-pretreated MDSCs was significantly reduced. Figure 5 H). The above results indicate that the PIK3CA mutation leads to enhanced MDSC recruitment and immunosuppressive function against T cells due to 5-LOX overexpression-dependent LTB4 secretion.

[0079] 6. To further investigate whether blocking the PIK3CA / 5-LOX pathway can exert an anti-tumor effect in PIK3CA-mutant Luminal breast cancer, this application established a PIK3CA-mutant xenograft model and administered PI3K inhibitors (Alpelisib, 20 mg / kg / day), anti-PD-1 mAbs (Pembrolizumab, 10 mg / kg / day), and LTB4 / BLT2 antagonists (LY255283, 20 mg / kg / day), alone or in combination. In vivo imaging results in small animals showed that, compared with the control group, Alpelisib, LY255283, or anti-PD-1 mAbs, the combined administration of Alpelisib, LY255283, and anti-PD-1 mAbs significantly inhibited the growth of the xenograft. Figure 6 AB); After taking tumor tissue and measuring it, the results showed that the combined treatment group had the smallest tumor mass. Figure 6CD). The therapeutic effect was particularly evident in the comparison between the Alp+LY255283 group and the Alp+anti-PD-1+LY255283 group: The total dosage of the Alp+LY255283 group (20 mg / kg / day) was only 10 mg / kg / day more than that of the Alp+anti-PD-1+LY255283 group (50 mg / kg / day), but the final therapeutic effect was significantly different. On day 21, the average fluorescence intensity of the tumor in the Alp+LY255283 group was 9.6 × 10⁻⁶. 7 The average fluorescence intensity of the tumor composed of Alp+anti-PD-1+LY255283 was 4.02×10⁻⁶. 7 The tumor volume decreased by approximately 58%. These results confirm that PI3K inhibitors or LTB4 inhibitors can significantly enhance the immunotherapy efficacy in PIK3CA-mutant tumors. Flow cytometry was used to detect the presence of immune cells in tumor tissues from different treatment groups. The results showed that, compared to the control group, the Alpelisib, LY255283, or anti-PD-1 mAbs groups exhibited reduced MDSC infiltration, with the combination group showing the lowest MDSC infiltration level. Figure 7 AB), and CD8 in the combined treatment group + T cell infiltration was highest ( Figure 7 CD). The above results indicate that the development of lumbrical breast cancer tumors is regulated by the infiltration of MDSCs in the TME, and targeted therapy against the PIK3CA / 5-LOX / LTB4 axis can effectively inhibit this infiltration and synergize with immunotherapy to improve the anti-tumor efficacy in vivo.

[0080] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. The application of a reagent for detecting PIK3CA mutations and 5-LOX protein expression levels in tissues in the preparation of prognostic assessment products for Luminal breast cancer, characterized in that: The PIK3CA mutation is the PIK3CA E545K and / or H1047R mutation.

2. The application according to claim 1, characterized in that: Rolling circle nucleic acid amplification technology and gene probes were used to detect PIK3CA mutations; immunohistochemical staining was used to detect 5-LOX protein expression levels.

3. The application according to claim 2, characterized in that: The nucleic acid sequence of the gene probe is shown in SEQ ID NO.1-2.

Images