Biomarker and detection kit for diagnosing and treating breast cancer

Abstract

The invention discloses a biomarker for diagnosing and treating breast cancer. The biomarker is low-density neutrophil. The invention discloses application of a reagent for detecting the proportion of low-density neutrophil in mononuclear cells in preparation of a product for early diagnosis and treatment of breast cancer and / or prognosis of breast cancer, and a kit for early diagnosis and treatment of breast cancer and / or prognosis of breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to a biomarker for diagnosing and treating breast cancer and a detection kit. Background Art

[0002] According to the latest global cancer burden estimates released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2022, breast cancer remains the main cancer among women, accounting for approximately 23.8% of newly diagnosed female cancer cases and 15.4% of newly diagnosed female cancer deaths, ranking first. The pathogenesis of breast cancer is complex and has significant molecular heterogeneity, resulting in significant differences in the metastatic tendency, treatment response, and prognosis of different patients. Although current surgeries, radiotherapy, and chemotherapy can all improve the cure rate of breast cancer, the probability of recurrence and metastasis after 5 - 10 years is still relatively high. The occurrence of lymph node metastasis and distant tumor metastasis in breast cancer patients is the main cause of death in breast cancer patients. However, the most common tumor markers such as CEA, CA15 - 3, or CA125 are not ideal biomarkers for early diagnosis of breast cancer due to their low sensitivity and specificity. Therefore, it is crucial to find non - invasive, highly sensitive, specific, and easily accessible biological indicators.

[0003] Low - density neutrophils (LDNs) are a subset of neutrophils that include mature and immature states. They exist in the peripheral blood mononuclear cell (PBMC) layer, are distinguished from normal - density granulocytes (NDGs) by different buoyant densities, and can be distinguished from monocytes in PBMCs by different surface markers (monocytes highly express CD14 and lowly express CD15) due to their high expression of CD15 and low expression of CD14.

[0004] As a subset of neutrophils, LDNs possess functions related to neutrophil - associated phagocytosis, chemotaxis, and cytokine secretion. They can secrete higher levels of interleukin (IL - 6, IL - 8), tumor necrosis factor α (TNF - α), and type I interferon (IFN) than normal - density granulocytes, showing a strong pro - inflammatory effect or immunosuppressive effect.

[0005] Studies have shown that the neutrophil population in the tumor microenvironment exhibits heterogeneous phenotypes and functional diversity, playing dual roles in promoting or suppressing tumors. LDNs exhibit strong immunosuppressive effects and pro-tumor activities, including promoting tumor angiogenesis, invasion, and metastasis through various factors such as hepatocyte growth factor (HGF), oncostatin M, reactive oxygen species (ROS), reactive nitrogen species (RNS), matrix metalloproteinases (MMPs), and neutrophil elastase (NE). Their proportion increases significantly with cancer progression. However, current research on LDNs mainly focuses on various autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.) and infectious diseases (acquired immunodeficiency syndrome, tuberculosis, etc.). There is no report on the application of LDNs as biomarkers for the early diagnosis, treatment, and prediction of breast cancer. Summary of the Invention

[0006] The object of the present invention is to provide a biomarker to provide some minimally invasive, highly sensitive, highly specific, and easily accessible biological indicators for the early diagnosis, treatment, or prognosis of breast cancer.

[0007] According to the first aspect of the object of the present invention, there is provided the use of a reagent for detecting the proportion of low-density neutrophils in mononuclear cells in the preparation of a product for the early diagnosis and treatment of breast cancer and / or the prognosis of breast cancer.

[0008] As an optional embodiment, the reagent includes antibodies capable of detecting the expression levels of CD14 and CD15.

[0009] As an optional embodiment, the antibodies include PE-Cy7-CD45, PE-CD14, and APC-CD15.

[0010] According to the second aspect of the object of the present invention, there is provided a kit for the early diagnosis and treatment of breast cancer and / or the prognosis of breast cancer, the kit including a reagent for detecting the proportion of low-density neutrophils in mononuclear cells.

[0011] As an optional embodiment, the reagent determines the proportion of the low-density neutrophils in mononuclear cells by detecting the expression levels of CD14 and CD15.

[0012] As an optional embodiment, the expression levels of CD14 and CD15 are detected by flow cytometry.

[0013] As an optional embodiment, the antibodies for detecting the expression levels of CD14 and CD15 include PE-Cy7-CD45, PE-CD14, and APC-CD15.

[0014] It is understandable that flow cytometry can be used to measure the expression of surface markers to distinguish monocytes and low-density neutrophils in PBMCs. Monocytes highly express CD14 and lowly express CD15, while LDNs highly express CD15 and lowly express CD14. Therefore, in the present invention, CD14 and CD15 (CD14 - CD15 + ) are selected as markers. However, the surface markers of LDNs also include CD11b, CD66b, CD16, CD10, etc. Therefore, CD11b, CD66b, CD116, and CD14 (CD11b + CD66b + CD116 + CD14 - ) can also be selected to measure the expression of surface markers.

[0015] The beneficial effects of the present invention are as follows:

[0016] 1. Through research, it is found in the present invention that compared with the healthy control group, the proportion of peripheral blood LDNs in PBMCs of breast cancer patients is significantly increased. Therefore, the proportion of peripheral blood low-density neutrophils (LDNs) in mononuclear cells (PBMCs) provided in the present invention can be used as a marker for the early diagnosis of breast cancer, providing certain application value for the early diagnosis of breast cancer.

[0017] 2. Through research, it is found in the present invention that the proportion of peripheral blood LDNs in PBMCs can effectively predict breast cancer. The optimal diagnostic threshold is 11.18%, the diagnostic sensitivity is 77.94%, the specificity is 94.83%, and the accuracy is 83.00%, indicating that the proportion of peripheral blood LDNs in PBMCs can be used as a breast cancer diagnostic marker.

[0018] 3. Through research, it is found in the present invention that the proportion of peripheral blood LDNs in PBMCs of breast cancer patients is related to lymph node metastasis. The proportion of peripheral blood LDNs in PBMCs of patients with lymph node metastasis is significantly increased, which can be used as a reference index for the treatment and prognosis of breast cancer. Description of the Drawings

[0019] Figure 1 is a flow cytometry diagram for detecting the proportion of peripheral blood LDNs in PBMCs using flow cytometry (FCM) in an example of the present invention; among them, A is to gate PBMCs according to FSC-A and SSC-A, B is to gate single cells according to SSC-A and SSC-H, C is to gate the CD45 + cell population, D is to gate the CD14 - CD15 + cell population, and the gating sequence is A→B→C→D.

[0020] Figure 2 It is a statistical chart showing the proportion of peripheral blood LDNs in PBMCs in the control group and the disease group in the examples of the present invention.

[0021] Figure 3 It is an ROC curve graph for ROC curve analysis of the proportion level of peripheral blood LDNs in PBMCs in the examples of the present invention.

[0022] Figure 4 It is a statistical chart showing the proportion of peripheral blood LDNs in PBMCs in the non-lymph node metastasis group and the lymph node metastasis group in the examples of the present invention. Detailed implementation manners

[0023] To better understand the technical content of the present invention, specific embodiments are given and described in conjunction with the accompanying drawings as follows.

[0024] In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to cover all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those concepts and embodiments described in more detail below, can be implemented in any of many ways.

[0025] Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0026] Example 1

[0027] 1. Case selection

[0028] (1) Diagnostic criteria

[0029] The diagnosis of primary breast cancer was confirmed based on postoperative pathology, and the pathological TNM staging was according to the staging criteria of the 8th edition of the American Joint Committee on Cancer (AJCC).

[0030] (2) Grouping design

[0031] Healthy control group and breast cancer group.

[0032] (3) Case data

[0033] A total of 136 breast cancer patients were collected in this study from September 2024 to April 2025, aged 24 - 70 years old, with a median age of 50 years.

[0034] Inclusion criteria for breast cancer patients: 1) Meeting the clinical breast cancer diagnostic criteria and confirmed by postoperative pathological histology; 2) Not receiving any form of anti-tumor treatment before surgery; 3) Primary breast tumor; 4) Female; 5) Aged 20 - 70 years old.

[0035] Exclusion criteria: 1) History of combined other tumors or malignant tumors; 2) Severe complications occurred during the operation; 3) Combined with autoimmune diseases; 4) Combined with other severe diseases, such as grade 3 hypertension, severe cardiopulmonary insufficiency, etc.

[0036] There were 58 cases in the healthy physical examination control group, aged 23 - 67 years old, with a median age of 43 years old.

[0037] Inclusion criteria for healthy physical examination subjects: 1) Aged 20 - 70 years old; 2) Female.

[0038] Exclusion criteria: 1) History of combined other tumors or malignant tumors; 2) Associated with autoimmune diseases; 3) Combined with other severe diseases, such as grade 3 hypertension, severe cardiopulmonary insufficiency, etc.

[0039] All the research subjects were from Jiangsu Cancer Hospital. This research protocol conforms to the ethical standards of human trials and has been approved by the Ethics Committee. The subjects were informed before the trial and gave written consent.

[0040] 2. Main reagents, consumables and instruments

[0041] PE-Cy7-CD45 (Jiangxi Saiji CEGER), PE Mouse Anti-Human CD14 (BD Pharmingen TM ), APC Mouse Anti-Human CD15 (BD Pharmingen TM ), human lymphocyte separation medium (Absin Biosciences), and LDNs were examined using a NovoCyte flow cytometer.

[0042] 3. Experimental methods and techniques

[0043] (1) Sample collection and isolation of peripheral blood mononuclear cells (PBMCs)

[0044] 1) The research subjects collected 2 - 4 mL of peripheral blood using EDTA anticoagulant in the early morning on an empty stomach, and immediately inverted and mixed gently to prevent blood clotting;

[0045] 2) Add an equal volume of RPMI-1640 culture medium to dilute the whole blood;

[0046] 3) Take an appropriate amount of Ficoll separation medium and place it at the bottom of a 15 mL centrifuge tube, avoiding the separation medium sticking to the upper tube wall;

[0047] 3) Slowly add 4 - 6 mL of diluted blood to the surface of the Ficoll solution, and pay attention to gentle and slow operation throughout the process to prevent breaking the Ficoll separation medium layer;

[0048] 4) Centrifuge in a horizontal centrifuge at 20°C and 2000 rpm for 20 min. After centrifugation, the contents in the tube can be divided into five layers from top to bottom, namely the plasma and platelet layer, the mononuclear cell buffy coat layer (PBMCs layer), the Ficoll lymphocyte separation solution layer, the granulocyte layer, and the erythrocyte layer;

[0049] 5) Prepare a new 15 mL centrifuge tube in advance and add 5 mL of PBS buffer for standby. Carefully aspirate the buffy coat layer using a sterile pipette, transfer it to the prepared 15 mL centrifuge tube, and mix well;

[0050] 6) Centrifuge at 400 g / min for 10 min, discard the supernatant, and retain the cell layer;

[0051] 7) Consider whether erythrocyte lysis is required according to the erythrocyte situation: After centrifugation in step 6, if the erythrocyte layer can be seen at the bottom of the centrifuge tube, add 1 mL of 1× erythrocyte lysis buffer, lyse the erythrocytes at room temperature for 5 min, centrifuge at 400 g / min for 10 min, and discard the supernatant;

[0052] 8) Add 10 mL of PBS solution, pipette and mix well, centrifuge at 2000 rpm for 10 min, discard the supernatant, and obtain PBMCs;

[0053] 9) Take 1 mL of PBS to resuspend the PBMCs, pipette and mix well, take a drop and count it on a Neubauer counting chamber.

[0054] (2) Detection of the proportion of LDNs in PBMCs in peripheral blood by flow cytometry (FCM)

[0055] 1) Take 1 flow tube for each patient, add 100 μL of the corresponding resuspended PBMCs, with the cell number approximately 1×10 6 cells, and label the patient number. Add 10 μL of each of the flow antibodies PE-Cy7-CD45, PE-CD14, and APC-CD15 to each tube, mix well by oscillation, and incubate in the dark at room temperature for 15 - 20 min;

[0056] 2) At the same time, set up blank (Blank) tubes and single-positive control tubes for each single tube in step 1) to adjust the voltage and fluorescence compensation: Take 4 flow tubes, add 100 μL of the corresponding resuspended PBMCs to each, with the cell number approximately 1×10 6 cells, and label them as Blank tube (without adding any antibody), CD45 + tube (only add 10 μL of the flow antibody PE-Cy7-CD45), CD14 + tube (only add 10 μL of the flow antibody PE-CD14), and CD15 + tube (only add 10 μL of the flow antibody APC-CD15);

[0057] 3) Add 2 mL of PBS to each tube to wash the cells, centrifuge at 4°C and 400 g / min for 5 minutes, and discard the supernatant;

[0058] 4) Repeat the above step 3;

[0059] 5) Add 400 μL of PBS to each tube to resuspend the cells, mix well and then load onto the flow cytometer for detection. The phenotype of LDNs in the PBMCs layer cells was identified as CD14 - CD15 + , circle the PBMCs according to the forward scatter (FSC) pattern and side scatter (SSC) pattern, then circle single cells from the PBMCs gate according to SSC-A and SSC-H, then circle the CD45 + cell population, and finally circle the LDNs cell population from the CD45 + cell population, and calculate the proportion of LDNs in PBMCs.

[0060] 4. Statistical analysis

[0061] Data analysis was performed using SPSS 20.0 and GraphPad Prism v9.4.1 software. After the Kolmogorov-Smirnov test, it was judged that the continuous variables in this article were skewed distributions. Measurement data were expressed as the interquartile range [M(Q1,Q3)], and the Mann-Whitney U test was used for comparison between groups. The diagnostic efficacy of LDNs for breast cancer was analyzed by the ROC curve. P<0.05 indicated a statistically significant difference.

[0062] Example 2

[0063] [The proportion of LDNs in PBMCs of breast cancer patients was significantly higher than that of the healthy control group

[0064] The proportion of LDNs in PBMCs of the disease group and the control group was detected by FCM. As Figure 1 shown, the gating order was A→B→C→D. A was to gate PBMCs according to FSC-A and SSC-A, B was to gate single cells according to SSC-A and SSC-H, C was to gate the CD45 + cell population, D was to gate the CD14 - CD15 + cell population, and LDNs was Figure 1 the cell population in the Q1 region of D (CD14 - CD15 +) Detection results: The proportion of LDNs in PBMCs in the control group was 5.02 (0.88, 8.38)%, and the proportion of LDNs in PBMCs in the disease group was 21.19 (12.71, 31.72)%.

[0065] As Figure 2 shown, by statistically analyzing the proportion level of LDNs in PBMCs in each group of samples, it was found that the proportion of peripheral blood LDNs in PBMCs in breast cancer patients was significantly higher than that in the healthy control group (P < 0.0001); this indicates that the proportion level of peripheral blood LDNs in PBMCs has statistical significance with breast cancer and has certain diagnostic value for breast cancer.

[0066] Example 3

[0067] [ROC curve to determine the optimal cut-off value for LDNs to judge the occurrence of breast cancer]

[0068] As Figure 3 shown, with healthy people as the control group and breast cancer patients as the disease group, a ROC curve analysis was performed on the proportion level of peripheral blood LDNs in PBMCs. The AUC value was 0.871; the optimal diagnostic threshold was 11.18%, the diagnostic sensitivity was 77.94%, the specificity was 94.83%, and the accuracy was 83.00%.

[0069] From the above results, it can be seen that the proportion of peripheral blood LDNs in PBMCs can be a potential marker for differentiating and diagnosing healthy people from breast cancer.

[0070] Example 4

[0071] [The proportion of peripheral blood LDNs in PBMCs in breast cancer patients with lymph node metastasis is significantly increased]

[0072] As Figure 4 shown, the proportion of peripheral blood LDNs in PBMCs in patients with lymph node metastasis group and the detection results in the group without lymph node metastasis: the proportion of LDNs in PBMCs in the group without lymph node metastasis was 18.72 (11.99, 29.16)%, and the proportion of LDNs in PBMCs in the group with lymph node metastasis was 21.91 (13.63, 36.47)%. The proportion of peripheral blood LDNs in PBMCs in breast cancer patients with lymph node metastasis was significantly increased (P < 0.05). This indicates that the proportion of peripheral blood LDNs in PBMCs can be used as a reference index for the treatment and prognosis of breast cancer.

[0073] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Those of ordinary skill in the art to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to what is defined by the claims.

Claims

1. Use of a reagent for detecting the proportion of low-density neutrophils in mononuclear cells in the preparation of a product for early diagnosis, treatment and / or prognosis of breast cancer.

2. The use according to claim 1, characterized in that, The reagent comprises antibodies capable of detecting the expression levels of CD14 and CD15.

3. The use according to claim 2, characterized in that, The antibodies comprise PE-Cy7-CD45, PE-CD14 and APC-CD15.

4. A kit for early diagnosis and treatment of breast cancer and / or prognosis of breast cancer, characterized in that, The kit comprises a reagent for detecting the proportion of low-density neutrophils in mononuclear cells.

5. The kit according to claim 4, characterized in that, The reagent determines the proportion of the low-density neutrophils in mononuclear cells by detecting the expression levels of CD14 and CD15.

6. The kit according to claim 5, wherein The expression levels of CD14 and CD15 are detected by flow cytometry.

7. The kit according to claim 6, wherein Antibodies for detecting the expression levels of CD14 and CD15 comprise PE-Cy7-CD45, PE-CD14 and APC-CD15.

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