Marker LAMP3 for predicting breast cancer immunotherapy effect and application thereof

Abstract

The invention discloses a marker LAMP3 for predicting the breast cancer immunotherapy effect and application thereof, and belongs to the technical field of tumors. Through bioinformatics analysis of gene expression of triple-negative breast cancer compared with other types of breast cancer, the gene LAMP3 which is highly expressed in breast cancer and closely related to prognosis is screened out, the gene LAMP3 can be used as a novel breast tumor molecular marker, and the high expression of the gene LAMP3 predicts poor prognosis of tumor. The expression quantity of the LAMP3 in the breast cancer is higher than that of para-carcinoma cells, and the LAMP3 is related to immune cold tumors and is also in negative correlation with a biomarker of BrCa immunotherapy, so that a new target spot is provided for diagnosis, prediction and treatment of the breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumors, and specifically relates to a marker LAMP3 for predicting the immunotherapy effect of breast cancer and an application thereof. Background Art

[0002] Breast cancer (BC) is a heterogeneous malignant tumor. It accounts for 32% of all new cancers in women and has become one of the most common cancers affecting women worldwide. Based on molecular and histological evidence, breast cancer BC can be divided into 4 categories: Luminal-A, Luminal-B and human epidermal growth factor 2 positive HER 2+ and triple-negative breast cancer (TNBC) - , P.R. - , HER 2- ). Different types of breast cancer vary greatly in treatment options and prognostic results. For example, tissue resection, radiotherapy or chemotherapy can generally be used for breast cancer, and endocrine therapy can also be used for estrogen or progesterone receptor-positive types. However, for triple-negative breast cancer (TNBC), which has fewer therapeutic targets, most patients still face poor prognosis and risk of recurrence after using chemotherapy or targeted therapy.

[0003] In recent years, research on tumor immunotherapy has made breakthrough progress and has brought good news to patients with multiple malignant tumors. Tumor immunotherapy, in simple terms, is to achieve passive or active immunity to tumors by activating the patient's immune system. It is mainly divided into two directions: PD-L1 checkpoint inhibitors and CTLA4adoptive cell therapy. Immunotherapy has become one of the main treatment strategies for BrCa. Immune checkpoint inhibitors combined with chemotherapy have achieved significant results in both early and advanced triple-negative breast cancer, and new hope has been seen in the treatment and even cure of breast cancer.

[0004] However, in many cases immunotherapy does not show good efficacy, and a large number of studies have shown that the tumor immune microenvironment TIME is closely related to the effect of immunotherapy. The tumor microenvironment is composed of a variety of cellular and non-cellular components in the tumor tissue, including tumor cells, immune cells, stromal cells, cytokines and vascular networks. The infiltration level of immune cells in the microenvironment TIICs is a key factor in the poor prognosis of tumor patients during immunotherapy. According to the characteristics of TIME, tumors can be simply divided into hot tumors and cold tumors. Hot tumors are characterized by high T cell infiltration and molecular features of immune activation, while cold tumors show molecular features of T cell infiltration and immune rejection. In general, immune hot tumors are more sensitive to immunotherapy, so it is particularly important to discover biomarkers related to tumor microenvironment characteristics for evaluating immunotherapy. Unfortunately, there is currently no optimal biomarker for identifying breast cancer tumor microenvironment.

[0005] Lysosomal-associated membrane protein LAMP3 is a member of the lysosomal-associated membrane protein family LAMP. In this family, LAMP1 has been shown to play an important role in melanoma, non-small cell lung cancer, and Merkel cell carcinoma MCC. LAMP2 has also been reported to regulate pathways related to breast cancer and prostate cancer, participate in the apoptosis of immunogenic cells, and serve as a potential marker for a variety of cancers. Unlike the widespread expression of LAMP1 and LAMP2, LAMP3 is only overexpressed in lung tissue and a variety of primary cancer tissues such as esophageal cancer, colon cancer, fallopian tube cancer, ovarian cancer, breast cancer, and liver cancer. In recent years, more and more studies have shown that LAMP3 is highly expressed in breast cancer and cervical cancer and is associated with prognostic results. However, the mechanism of action of LAMP3 in breast cancer remains unclear, and its potential as a tumor marker remains to be developed. Summary of the invention

[0006] Technical issues solved:

[0007] This application aims at the existing technology for triple-negative breast cancer TNBC, which has fewer therapeutic targets. Most patients still face poor prognosis and risk of recurrence after chemotherapy or targeted therapy. There is currently no optimal biomarker for identifying the tumor microenvironment of breast cancer, the mechanism of action of other biomarkers in breast cancer is still unclear, and their potential as tumor markers has yet to be developed. The application provides a marker LAMP3 for predicting the effect of immunotherapy for breast cancer and its application, seeks a new combination of markers with better sensitivity and specificity for diagnosing breast cancer, and uses it in the preparation of a detection kit for identifying breast cancer cells and predicting breast cancer. The biomarker LAMP3 of this application can be used as a new molecular marker for diagnosis and prognosis of breast cancer, and its high expression indicates a poor prognosis of the tumor.

[0008] Technical solution:

[0009] To achieve the above objectives, this application is implemented through the following technical solutions:

[0010] LAMP3, a marker for predicting the efficacy of immunotherapy in breast cancer.

[0011] The present application also discloses the use of LAMP3, a marker for predicting the effect of breast cancer immunotherapy, in the preparation of a detection kit for identifying breast cancer cells and predicting breast cancer.

[0012] Principle explanation: This application uses the BrCa cohorts of TCGA and GEO databases to analyze the expression and immunological characteristics of LAMP3 in BrCa. LAMP3 can be used as a new breast cancer biomarker to identify tumor immune hot microenvironment and predict the therapeutic effect of immune checkpoint blockade. In addition, internal cohorts and immunotherapy BrCa cohorts were used to further verify the predictive effect of LAMP3. Overall, the study showed that the high expression of LAMP3 is closely related to the hot tumor microenvironment and can be used to identify BrCa that is sensitive to immunotherapy.

[0013] Beneficial effects:

[0014] The present application provides a marker LAMP3 for predicting the effect of immunotherapy for breast cancer and its application, which has the following beneficial effects compared with the prior art:

[0015] 1. The LAMP3 of the present invention can significantly inhibit the proliferation of breast cancer cells;

[0016] 2. The present invention provides a new target for the diagnosis, prediction and treatment of breast cancer;

[0017] 3. This application proposes a highly effective biomarker LAMP3 related to breast cancer. A large sample of breast cancer patient RNA-seq data and clinical information were obtained from the TCGA public database. The functional role of LAMP3 in breast cancer was predicted through gene enrichment analysis, and its intrinsic association with the tumor immune microenvironment was explored. At the same time, combined with animal experiments such as immunohistochemistry, the relationship between LAMP3 and multiple immune checkpoint gene markers was revealed, and immune infiltrating cells were highly correlated with LAMP3 expression, indicating that LAMP3 can become a biomarker of immunotherapy sensitivity;

[0018] 4. This application provides a new basis and method for the prognosis assessment kit for early diagnosis and treatment of breast cancer individuals. The present invention innovatively explores the relationship between LAMP3 and the tumor immune microenvironment, discovers its correlation with the immune-hot tumor microenvironment, verifies that high expression of LAMP3 is associated with a good prognosis of breast cancer, and provides a strong basis for the development of early treatment of breast cancer individuals and the evaluation of prognosis results, and provides a reliable reference for the timely adjustment of treatment plans for breast cancer patients. At the same time, TNBC shows the greatest prospect in the application of immunotherapy. In the present invention, LAMP3 is used as a new target to assist doctors in providing targeted treatment options for breast cancer patients.

[0019] 5. The present invention detected the correlation between LAMP3 and other breast cancer biomarkers, and experimental data analysis found that it was positively correlated with PD-L1 and CLAT4, confirming that high expression of LAMP3 is associated with a good prognosis of breast cancer, and the prediction is more accurate and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the expression of LAMP3 in breast cancer and adjacent tissues;

[0021] Figure 2 Representative images showing the expression of LAMP3 in tumor and paratumor tissues by anti-LAMP3 staining;

[0022] Figure 3 This is a diagram showing the high expression of LAMP3 in triple-negative cells;

[0023] Figure 4 This is an analysis of the prognostic value of LAMP3 in BrCa from the perspective of OS;

[0024] Figure 5 The prognostic value analysis diagram of LAMP3 in BrCa from the aspect of PFS;

[0025] Figure 6 The prognostic value analysis diagram of LAMP3 in BrCa from the perspective of DFS;

[0026] Figure 7 It is a graph of the protein of the present application and different subtypes of breast cancer and TMN clinical stages, wherein A is a graph of negative correlation between LAMP3 and ER sites of breast cancer; B is a graph of negative correlation between LAMP3 and PR sites of breast cancer; C is a graph of negative correlation between LAMP3 and ER sites of breast cancer; D is a graph of LAMP3 and four molecular subtypes of breast cancer; E is a graph of LAMP3 and T stage of breast cancer; F is a graph of LAMP3 and N stage of breast cancer; G is a graph of LAMP3 and M stage of breast cancer; H is a graph of LAMP3 and four clinical stages of breast cancer; I is a graph of LAMP3 and the age of breast cancer patients;

[0027] Figure 8 This is a graph of LAMP3-related gene screening and Pearson correlation analysis;

[0028] Fig. 9 is the heat map of the top 50 positively correlated genes with LAMP3;

[0029] Fig.10 is the heat map of the top 50 negatively correlated genes with LAMP3;

[0030] Fig.11 is the GO pathway enrichment map of genes related to LAMP3;

[0031] Fig.12 is the KEGG pathway enrichment map of genes related to LAMP3;

[0032] Fig.13 is the correlation diagram between LAMP3 and BrCa immune tumor microenvironment immunomodulators (chemokines, immunostimulants, MHC and receptors);

[0033] Fig.14 This is the correlation diagram of LAMP3 and common immune infiltration genes;

[0034] Fig.15 This is a graph showing the correlation between LAMP3 and immune cell infiltration levels;

[0035] Fig.16 is the correlation diagram between LAMP3 expression and ips_ctla4_neg_pd1_pos;

[0036] Fig.17 is the correlation diagram between LAMP3 expression and ips_ctla4_pos_pd1_pos;

[0037] Fig.18 is the correlation diagram between LAMP3 expression and ips_ctla4_pos_pd1_neg;

[0038] Fig.19 is the correlation diagram between LAMP3 expression and ips_ctla4_neg_pd1_neg;

[0039] Fig. 20 is a graph showing the changes in common genes in BrCa in different LAMP3 groups;

[0040] Fig.21 is the correlation diagram between LAMP3 and TMB score;

[0041] Fig. 22 is the correlation diagram between LAMP3 and T cell score;

[0042] Fig.23 This is a diagram of the predicted effect of LAMP3 in immunotherapy of the present application, wherein A is a PCR validation diagram and B is a ROC curve diagram. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

[0044] Example 1

[0045] A marker LAMP3 for predicting the efficacy of immunotherapy for breast cancer and its application. LAMP3 was screened out through association omics. The results of bioinformatics analysis showed that the expression of LAMP3 in triple-negative breast cancer tissues was higher than that in non-triple-negative breast cancer cell lines and breast cancer adjacent tissues. In various tumor tissues, high expression of LAMP3 was correlated with good tumor prognosis. A marker LAMP3 for predicting the efficacy of immunotherapy for breast cancer and its application. The marker LAMP3 was screened through association omics, and its steps included data collection, genotyping, quality control and data cleaning, association analysis, model selection, and result verification, and finally LAMP3-related genes were screened out.

[0046] The LAMP3 detection primers are shown in primer library ID156627583c1, primer library ID156627583c2, and primer library ID156627583c3, respectively. The forward primer sequence of primer library ID156627583c1 is shown in SEQ ID NO.1: 5'-GCGTCCCTGGCCGTAATTT-3', and the reverse primer sequence is shown in SEQ ID NO.2:

[0047] 5'-TGCTTGCTTAGCTGGTTGCT-3'; the forward primer sequence of primer library ID156627583c2 is as SEQ ID NO.3: 5'-AGCAAGCACCTCACCAAACTT-3', and the reverse primer sequence is as SEQ ID NO.4: 5'-TGTAGTCGCTGGGGTAGTTGT-3'; primer library

[0048] The forward primer sequence of ID156627583c3 is as shown in SEQ ID NO.5: 5'

[0049] -ACTACCCCAGCGACTACAAAA-3', reverse primer sequence is as SEQ ID NO.6:5'

[0050] -CTAGGGCCGACTGTAACTTCA-3'.

[0051] Through bioinformatics analysis of LAMP3 expression in breast cancer, the specific process is as follows:

[0052] Get breast cancer data from TCGA database

[0053] Specimen collection: This application uses a group of breast cancer cohorts from the TCGA dataset to compare the mRNA levels of LAMP3 in cancer tissues and adjacent tissues. Survival data were downloaded, including three endpoints, including overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS). In order to explore the correlation between LAMP3 expression and clinical pathological parameters, this application combined the open database TCGA and GEO joint cohorts, and selected breast cancer subtypes and pathological stages, including ER, PR, HER2, clinical T, N, M stages, and age. Table 1 LAMP3 primer sequence data source:

[0054] https: / / www.genelibs.com / gen / command / search / gen / detail / HGNC:14582

[0055] Figure 1 The present invention uses a set of standardized RNA-seq data of BrCa samples from the TCGA database, and then inputs the data into the Graphpad Prism8 software. After software analysis, a box plot is automatically generated, and the graphic coordinates and colors are manually adjusted. The mRNA levels of LAMP3 in cancer and adjacent tissues are compared. Data analysis and histochemical detection found that the expression level of LAMP3 in breast cancer is higher than that in adjacent cancer cells.

[0056] Figure 2The present invention uses an immunohistochemical method, and the clinical cohort BrCa (Cat. HBreD050Bc01) tissue chip (TMA) was provided by Outdo BioTech (Shanghai, China). The HBreD050Bc01 cohort contains 40 tumor samples and 10 adjacent tumor samples. Specific clinical pathological characteristics were provided by Outdo BioTech. The use of TMA was ethically approved by the Clinical Research Ethics Committee of Outdo Biotech (Shanghai). The above TMA was stained with immunohistochemistry (IHC). The primary antibodies used in the study are as follows: anti-LAMP3 (1:1000 dilution, 12632-1-AP, ProteinTech, Wuhan, China) and anti-PD-L1 (Ready-to-use, Cat.17952-1-AP, GT2280, Gene Technology, Shanghai). Antibody staining was counterstained with DAB and hematoxylin. All stained sections were independently evaluated by two pathologists. For semi-quantitative evaluation of LAMP3 and PD-L1 staining, the immune response score (IRS) was used (Cai et al., 2021). Briefly, the percentage of positively stained cells was divided into 0-4: 0 (<5%), 1 (6-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%). The staining intensity was divided into 0-3: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). IRS is equal to the percentage of positive cells multiplied by the staining intensity. The results showed that most cells in BrCa samples were stained with anti-LAMP3, while most cells in adjacent tumor samples were not stained.

[0057] Figure 3 The present invention uses a set of standardized RNA-seq data from TNBC samples in the TCGA database, and then inputs the data into the Graphpad Prism8 software. After software analysis, a box plot is automatically generated, and the coordinates and colors of the graph are manually adjusted. The results show that the expression level of LAMP3 in triple-negative breast cancer is much higher than that in adjacent cancer cells.

[0058] Figure 4-Figure 6 The present invention uses the TCGA data set to download survival data including three endpoints: overall survival (OS), progression-free survival (PFS) and disease-specific survival (DSS). The data are analyzed by Graphpad Prism8 software. The prognostic value of the categorical variable is evaluated by the log-rank test. Finally, the survival curve is obtained, and the coordinates and colors of the graph are manually adjusted. At the same time, the results show that breast cancer patients with high expression of LAMP3 mRNA have poor OS, PFS and DSS, confirming that high expression of LAMP3 in BrCa is associated with a good prognosis;

[0059] Figure 7The present invention combines the open database TCGA and GEO joint cohort to obtain standardized RNA-seq data of BrCa samples, and uses Graphpad Prism8 software to obtain a box plot. The present invention analyzes the relationship between LAMP3 expression and each clinical stage data, and finds that the expression of LAMP3 is high in ER and PR positive, but the expression of HER2 is not statistically significant. In the clinical pathological stage, the expression level of LAMP3 decreases with the increase of T, N, and M stages, which indicates that the expression of LAMP3 decreases with the occurrence and development of the tumor, which is related to a good prognosis.

[0060] Figure 8-Figure 12 :The related gene screening and enrichment analysis of the present invention utilized Linked Omics (https: / / www.linkedomics.org / login.php), an interactive platform for online management of TCGA data (Vasaikar et al., 2018). The present invention used Linked Omics to screen genes related to the expression of LAMP3 in BrCa. Through genome enrichment analysis (GSEA), the functional role of LAMP3 in BrCa was predicted using the association characterization platform from the perspective of biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and all parameters used the default options. The results showed that LAMP3 was associated with the immune pathway.

[0061] Fig.13 :The present invention uses the TCGA database and imports the data into origin, and uses the correlation analysis plug-in Correlation Plot for analysis to obtain a correlation analysis heat map of LAMP3 and immunomodulators (including chemokines, MHC, immunostimulatory factors and receptors) in BrCa. The results show that with the increase in LAMP3 expression level, the regulation levels of a large number of chemokines, paired receptors, immunomodulators, MHC molecules and immune chemokines are also increasing.

[0062] Fig.14 :The present invention uses the data from TCGA and imports the data into origin, and uses the correlation analysis plug-in Correlation Plot to analyze and obtain a heat map. The present invention analyzes the correlation between LAMP3 and immune infiltration-related immune marker genes and finds that they are positively correlated.

[0063] Fig.15: The IPS values ​​of BrCa patients in the present invention were downloaded from the Cancer Immunobiology Atlas (TCIA) (https: / / tcia.at / home). After downloading, the data were analyzed. The present invention used the data in the TCGA dataset and the correlation between LAMP3 and TIICs levels calculated by the TIMER algorithm. The present invention further studied the relationship between LAMP3 expression level and immune infiltration level. The results showed that the infiltration of B cells, CD8+T cells, CD4+T cells, macrophages, neutrophils and dendritic cells in BrCa was positively correlated with LAMP3 expression, indicating that high expression of LAMP3 promotes immune behavior, thereby inhibiting tumors;

[0064] Figure 16-Figure 19 : The IPS values ​​of BrCa patients in the present invention were downloaded from The Cancer Immunobiome Atlas (TCIA) (https: / / tcia.at / home), and were found to be positively correlated with some immune biomarkers;

[0065] Fig. 20 :In addition, the simple nucleotide variation data of TCGA samples processed by the present invention using the "MuTect2" software (Beroukhim et al., 2010) are from the Genomic Data Commons (GDC, https: / / portal.gdc.cancer.gov / ) website. Then a mutation frequency graph was made. The present invention found that genetic material mutations were more common in the high LAMP3 group through mutation frequency, and LAMP3 was also negatively correlated with T cell inflammation score, suggesting that it is a marker of immune-hot tumors;

[0066] Fig.21 : The present invention used the R software package "maftools" to visualize the mutation landscape and calculate the tumor mutation burden (TMB) of each sample, and found a positive correlation between LAMP3 and TMB levels.

[0067] Fig. 22 : The present invention also calculated the T cell inflammation score based on the linear combination of the expression levels and weighted coefficients of 18 genes (Ayers et al., 2017), and also found that LAMP3 expression was positively correlated with the T cell inflammation score.

[0068] Fig.23 :The present invention uses receiver operating characteristic (ROC) analysis to evaluate the specificity and sensitivity of PSME2 in predicting immunotherapy response, and calculates the area under the ROC curve (AUC). The results showed that the expression level of LAMP3 was lower in the pCR group, indicating that overexpression of LAMP3 mRNA would produce less response to immunotherapy, confirming the role of LAMP3 as a potential biomarker; ROC validation indicates high credibility and accuracy.

[0069] Example 2

[0070] Using immunohistochemistry, tissues were stained by histochemical methods to observe the expression of LAMP3 in breast cancer and adjacent tissues. The steps are as follows:

[0071] Step 1, dewaxing after tissue drying: Immunohistochemical staining is performed directly on tissue sections. First, the tissue sections are placed at 55°C for dewaxing for 30 minutes. Dewaxing and hydration of tissue sections: the tissue sections are immersed in xylene, taken out after 10 minutes, immersed three times, and then placed in 95%, 90%, 85%, 80%, and 75% anhydrous ethanol in sequence, each treatment lasting 5 minutes, and then rinsed with tap water for 1 minute and double distilled water for 1 minute;

[0072] Step 2, antigen repair: During the sectioning process, tissue processing may cause the antigen to be partially shielded or destroyed. In order to restore the antibody reactivity of the antigen, antigen repair is required. The steps are as follows: Pour the sodium citrate antigen repair solution into the box until the slide can be immersed. The slide is placed in the box, heated to 98°C in a water bath and placed in it for 20 minutes. Then take out the box and let it cool to room temperature (15-20 minutes). Then wash it three times with a PBS shaker, each time for 5 minutes. After drawing a circle with an immunohistochemistry pen, drop the anti-peroxidase blocker on the slice to block it, and cover the tissue slice. After standing for 15 minutes, wash it three times with a PBS shaker, each time for 5 minutes.

[0073] Step 3, blocking: In order to reduce the background interference of the experiment, it is necessary to block the non-specific binding sites in the tissue sections. The steps are as follows: After washing, use a pipette to aspirate 200ul of sheep serum and drop it on the tissue sections in turn. Block for 20 minutes at room temperature in the dark, and then shake off the excess liquid;

[0074] Step 4, primary antibody incubation: add primary antibody dropwise to cover the tissue sections, and then incubate at 4°C overnight for 12 hours. The specific antibodies are: anti-LAMP3 (1:1000 dilution, Cat.12632-1-AP, ProteinTech, Wuhan, China) and anti-PD-L1 (Ready-to-use, Cat.17952-1-AP, GT2280, Genentech, Shanghai). This antibody binds to the target antigen to form an antigen-antibody complex;

[0075] Step 5: Rewarm to ambient temperature, recover the primary antibody for next use, and then record the concentration and number of uses. Wash on a PBS shaker for 3 minutes, for a total of 3 washes;

[0076] Step 6, secondary antibody incubation: Use enzyme-labeled goat anti-mouse / rabbit IgG polymer (secondary antibody) to drop on the slide, react for 20 minutes at room temperature, rinse with PBS for 5 minutes, and wash 3 times in total. The secondary antibody binds to the primary antibody to form an antigen-antibody-antibody complex;

[0077] Step 7, prepare DAB: add five drops of solution A and 2000ul of solution B in the DAB kit into a container until the solution turns orange;

[0078] Step 8, color observation: Use DAB colorimetric method to make the antigen-antibody-antibody complex generate visible staining signals, and then observe the distribution and expression of the target antigen in the tissue under a microscope. The steps are as follows: immediately observe after adding DAB colorimetric solution until the color of the tissue section changes significantly from colorless to yellow, terminate the reaction, rinse the slide with tap water for 1 minute, then stain with hematoxylin dye for 30 seconds, then rinse off the excess dye with tap water, and finally cover the section with acidic ethanol for 2-10 seconds and rinse with running water for 5 minutes;

[0079] Step 9, dehydration: put the slides into 70%, 80%, 90%, and 95% anhydrous ethanol in order, soak for 3 minutes respectively, and then put them into xylene solution and soak for 20 minutes before taking them out;

[0080] Step 10, sealing: After the slide is dried, drop a drop of neutral resin on the center of the slide, immediately cover it with a cover slip, press lightly and wait for it to dry naturally;

[0081] Step 11, result interpretation: Scanning was performed using the Aperio digital pathology slide scanner, and immunohistochemical staining results were scored using immunohistochemical staining results. For the semi-quantitative evaluation of LAMP3 and PD-L1 staining, the immunoreactivity score (IRS) was used (Cai et al, 2021). Briefly, the percentage of positively stained cells was scored as 0-4: 0 (<5%), 1 (6-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%). The staining intensity was scored as 0-3: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). IRS is equal to the percentage of positive cells multiplied by the staining intensity. Experimental verification showed that LAMP3 was highly expressed in tumor tissues.

[0082] The examples selected in the above materials are for ease of understanding, not to limit the process. People in the same technical field can easily modify the process or transfer it to other cases without creative changes. If these changes also belong to the same claims or similar technologies of the present invention, then the original intention of the invention also includes these changes.

Claims

1. LAMP3, a marker for predicting the efficacy of immunotherapy for breast cancer.

2. Use of the marker LAMP3 for predicting the immunotherapy effect of breast cancer according to claim 1 in preparing a detection kit for identifying breast cancer cells and predicting breast cancer.

Images