CN117269510A - Molecular marker for diagnosing breast cancer, kit and application thereof - Google Patents
Molecular marker for diagnosing breast cancer, kit and application thereof Download PDFInfo
- Publication number
- CN117269510A CN117269510A CN202311433078.8A CN202311433078A CN117269510A CN 117269510 A CN117269510 A CN 117269510A CN 202311433078 A CN202311433078 A CN 202311433078A CN 117269510 A CN117269510 A CN 117269510A
- Authority
- CN
- China
- Prior art keywords
- pbs
- proteintech
- 5min
- shaking
- washed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 26
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 26
- 239000003147 molecular marker Substances 0.000 title claims abstract description 20
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims abstract description 31
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims abstract description 31
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 claims abstract description 29
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 claims abstract description 29
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims abstract description 12
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims abstract description 8
- 208000033878 Tertiary Lymphoid Structures Diseases 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000013399 early diagnosis Methods 0.000 abstract description 3
- 210000002966 serum Anatomy 0.000 description 32
- 210000001519 tissue Anatomy 0.000 description 31
- 238000003125 immunofluorescent labeling Methods 0.000 description 30
- 230000000875 corresponding effect Effects 0.000 description 28
- 101100381525 Mus musculus Bcl6 gene Proteins 0.000 description 19
- 230000000903 blocking effect Effects 0.000 description 17
- 238000003384 imaging method Methods 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 239000012188 paraffin wax Substances 0.000 description 17
- 238000000701 chemical imaging Methods 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 16
- 238000010166 immunofluorescence Methods 0.000 description 14
- 238000011534 incubation Methods 0.000 description 10
- 238000003556 assay Methods 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000003511 endothelial effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 210000000264 venule Anatomy 0.000 description 3
- 239000012099 Alexa Fluor family Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 210000000285 follicular dendritic cell Anatomy 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000007477 logistic regression Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101150106774 9 gene Proteins 0.000 description 1
- 102100021631 B-cell lymphoma 6 protein Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 101000971234 Homo sapiens B-cell lymphoma 6 protein Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000001280 germinal center Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011309 routine diagnosis Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70517—CD8
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a molecular marker for diagnosing breast cancer, a kit and application thereof, and relates to the technical field of biomedical detection. The molecular markers for diagnosing breast cancer are CD20, CD8 and CD23. The invention adopts CD20, CD8 and CD23 as a combination for early diagnosis of TLS in breast cancer for the first time, has higher sensitivity and specificity, is beneficial to further improving the accuracy of diagnosis of TLS, and further provides more valuable clues for individual and accurate treatment of tumors.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a molecular marker for diagnosing breast cancer, a kit and application thereof.
Background
Breast Cancer (BC) has become the most frequently diagnosed malignancy worldwide, with a large amount of life being taken away every day, and its expensive treatment cost also places a great economic burden on patients. BC is a disease with a strong heterogeneity, which can be divided into multiple subtypes, with different histological types and biological characteristics, thus possessing unique clinical behavior and therapeutic sensitivity.
Ectopic lymphoid organs that develop in non-lymphoid tissues at sites of chronic inflammation, including tumors, are called tertiary lymphoid structures (Tertiary lymphoid structure, TLS), and the structural features of mature TLS are the presence of B-cell enriched regions. There is increasing evidence that in most types of solid tumors, the presence of TLS is closely related to reducing the risk of recurrence and enhancing the efficacy of immune checkpoint blocking therapy (Immune checkpoint blockade, ICB). Although immunotherapy has increased patient survival in various solid tumors or hematological malignancies in recent years, most BC subtypes have little response to ICB, which is currently only approved for use in the treatment of PD-L1 positive Triple negative breast cancer (Triple-negative breast cancer, TNBC) patients. In general, BC is not considered an immunogenic disease, lack of infiltration of T lymphocytes, poor immunogenicity, and immunosuppressive tumor microenvironment are the leading causes of poor BC immunotherapy. A recent study assessed the presence of TLS in BC by the 9 gene TLS profile and indicated that the presence of TLS was positively correlated with early tumor TNM staging, while BC patients with high TLS exhibited better prognosis, but the assessment of TLS in this study relied on methods not amenable to routine diagnosis.
Although TLS has been found to exist in many tumors, the markers used to define and characterize TLS in different studies have varied, which complicates subsequent comparative studies of TLS, while current methods of identifying TLS are not readily implemented in routine clinical work. Thus, exploring standardized measurement methods for TLS helps to exploit the diagnostic and potential prognostic value of TLS in different disease environments.
Disclosure of Invention
Therefore, the invention aims to provide a molecular marker for diagnosing breast cancer, a kit and application thereof, and the molecular marker can be used for early diagnosis of breast cancer TLS and has higher sensitivity and specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a molecular marker for diagnosing breast cancer, which is CD20, CD8 and CD23.
The invention provides application of the molecular marker in preparation of a product for diagnosing early breast cancer.
The invention provides application of the molecular marker in preparation of products for analyzing tertiary lymphoid structures.
The invention also provides a kit for diagnosing tertiary lymphoid structure, comprising: anti-CD 20 antibodies, anti-CD 8 antibodies, anti-CD 23 antibodies, and immunofluorescent secondary antibodies.
Preferably, the immunofluorescent secondary antibodies are 488-labeled goat anti-mouse IgG (h+l) and 594-labeled goat anti-mouse IgG (h+l).
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts CD20, CD8 and CD23 as a combination for early diagnosis of TLS in breast cancer for the first time, has higher sensitivity and specificity, is beneficial to further improving the accuracy of diagnosis of TLS, and further provides more valuable clues for individual and accurate treatment of tumors.
Drawings
FIGS. 1-3 show multiplex immunofluorescence results, wherein FIG. 1 shows CD20 immunofluorescence positive results in multiplex immunofluorescence of example 1, and fluorescence is green at a corresponding wavelength 647; FIG. 2 shows the positive result of CD23 immunofluorescence in multiplex immunofluorescence of example 1, with fluorescence red at wavelength 488; FIG. 3 shows the negative result of CD8 immunofluorescence in multiplex immunofluorescence of example 1, corresponding to wavelength 594, fluorescence gray;
FIG. 4 is an image of TLS positive H & E stained sections;
FIG. 5 is an image of a TLS positive CD23 marker immunohistochemical staining section;
FIG. 6 is a ROC curve of identification of TLS by single immunofluorescent markers of CD20, CD8, bcl6, CD 23; AUC (CD 20) =0.665 in the figure; AUC (Bcl 6) =0.675; AUC (CD 8) =0.722; AUC (CD 23) =0.798;
FIG. 7 is a ROC curve of two immunofluorescent marker identification TLS; AUC (CD 20& Bcl 6) =0.708 in the figure; AUC (CD 20& CD 8) =0.751; AUC (CD 20& CD 23) =0.841; AUC (Bcl 6& CD 8) =0.733; AUC (Bcl 6& CD 23) =0.816; AUC (CD 8& CD 23) =0.850;
FIG. 8 is a ROC curve of three immunofluorescent marker identification TLS; AUC in the figure (CD 20& Bcl6& CD 8) =0.755; AUC (CD 20& Bcl6& CD 23) =0.839; AUC (CD 20& CD8& CD 23) =0.854; AUC (Bcl 6& CD8& CD 23) =0.852; AUC (All)
＝0.859。
Detailed Description
The invention provides a molecular marker for diagnosing breast cancer, which is CD20, CD8 and CD23.
The invention provides application of the molecular marker in preparation of a product for diagnosing early breast cancer.
The invention provides application of the molecular marker in preparation of products for analyzing tertiary lymphoid structures.
The invention also provides a kit for diagnosing tertiary lymphoid structure, comprising: anti-CD 20 antibodies, anti-CD 8 antibodies, anti-CD 23 antibodies, and immunofluorescent secondary antibodies.
The anti-CD 20 antibody is one anti-CD 20,1:300 60271-1-Ig, proteintech; the anti-CD 8 antibody is a primary anti-CD 8,1:300 66868-1-Ig, proteintech; the anti-CD 23 antibody is an anti-CD 23,1:300 60208-1-Ig, proteintech.
The immunofluorescence secondary antibody is anti-mouse 594,00013-4, proteintech,1:300; anti-mouse 488,SA00013-1, proteintech,1:500; anti-rabbit 594,SA00013-4, proteintech,1:300.
the method for using the kit for multiplex immunofluorescence in the invention is preferably as follows: paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Then, primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added, incubated at room temperature for 1h, and the slides were washed 3 times with shaking in PBS on a destaining shaker for 5min each. After the sections are slightly dried, corresponding fluorescent secondary antibodies (anti-mouse 594, CL594-66868, proteintech, 1:300) are dripped into the water-blocking rings to cover the tissues, and the tissues are incubated for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Finally, primary antibody (CD 23,1:300, 60208-1-Ig, proteintech) was added and incubated overnight at 4 ℃. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Another fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) was then added to cover the tissue and incubated for 60min in the dark at RT. The slide is put in PBS and washed for 3 times on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
According to the invention, the multiplex immunofluorescence results of the three markers CD20 and CD8 combined CD23 are counted, the prediction probability formed by establishing a logistic regression model is used as a unique analysis index, and an ROC curve is established, and the result shows that the AUC value of the markers CD20 and CD8 combined CD23 is 0.854, so that the markers are adopted for detection, have higher sensitivity and specificity, help to further improve the accuracy of diagnosis TLS, and further provide more valuable clues for the individuation accurate treatment of tumors.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Multiplex immunofluorescent staining of CD20, CD8 and CD23 as molecular markers
1. Sample collection
Collecting tissue specimens of 175 breast cancer patients collected and treated by breast surgery in a tumor hospital in Yunnan province in 2017, 6-2023 and 3, and carrying out pathological section. 175 cases of formalin-fixed, paraffin-embedded breast cancer specimens were sectioned into 4 μm tissue sections and subjected to immunohistochemical staining. These tissues were all H & E stained sections, on which specific tumor areas with morphological representation were marked, and then sectioned again. All sections were dewaxed with xylene and ethanol, warmed with citric acid for antigen retrieval and 3% hydrogen peroxide solution to block endogenous peroxidase.
2. Multiplex immunofluorescence
Paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Then, primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added, incubated at room temperature for 1h, and the slides were washed 3 times with shaking in PBS on a destaining shaker for 5min each. After the sections are slightly dried, corresponding fluorescent secondary antibodies (anti-mouse 594, CL594-66868, proteintech, 1:300) are dripped into the water-blocking rings to cover the tissues, and the tissues are incubated for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Finally, primary antibody (CD 23,1:300, 60208-2-Ig, proteintech) was added and incubated overnight at 4 ℃. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Another fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) was then added to cover the tissue and incubated for 60min in the dark at RT. The slide is put in PBS and washed for 3 times on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Multiplex immunofluorescence was performed using a TLS positive paraffin section as an example, and the imaging results are shown in fig. 1-3.
As can be seen in fig. 1-3, TLS is seen under the mirror to exist in or around lymphatic aggregates with high endothelial venules (high endothelial venules, HEV).
Comparative example 1
Immunofluorescent staining of CD20 as molecular marker
Sample selection was the same as in example 1, immunofluorescence staining procedure as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, after 1h the slide is placed in PBS and washed 3 times with shaking on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 2
Immunofluorescent staining of CD8 as molecular marker
Sample selection was the same as in example 1, immunofluorescence staining procedure as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 8,1:300, 66868-1-Ig, proteintech), incubating overnight at 4℃and washing the slides in PBS with shaking on a destaining shaker 3 times for 5min each. After the sections are slightly dried, corresponding fluorescent secondary antibodies (anti-mouse 594, CL594-66868, proteintech, 1:300) are dripped into the water-blocking rings to cover the tissues, and the tissues are incubated for 60 minutes in a dark place at RT. The slide is put in PBS and washed for 3 times on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 3
Immunofluorescent staining of CD23 as molecular marker
Sample selection was the same as in example 1, immunofluorescence staining procedure as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 23,1:300, 60208-2-Ig, proteintech) were incubated overnight at 4 ℃. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. The tissue was then covered with a fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) and incubated for 60min at RT in the absence of light. The slide is put in PBS and washed for 3 times on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 4
Immunofluorescent staining of Bcl6 as molecular marker
Sample selection was the same as in example 1, immunofluorescence staining procedure as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (Bcl 6,1:300, 66340-1-Ig, proteintech) were incubated overnight at 4 ℃. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. The tissue was then covered with a fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) and incubated for 60min at RT in the absence of light. The slide is put in PBS and washed for 3 times on a decolorizing shaker for 5min each time, and then the slide is sealed after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 5
Multiplex immunofluorescent staining of CD20 and Bcl6 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Primary antibodies (Bcl 6,1:300, 66340-1-Ig, proteontech) were added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 488, SA00013-1, proteintech, 1:500) to cover tissues in the water-blocking ring after the slices are slightly dried, and incubating for 60min in a dark place at RT. Placing the slide in PBS, shaking and washing on a decolorizing shaker for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 6
Multiplex immunofluorescent staining of CD20 and CD8 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Primary antibody (CD 8,1:300, 66868-1-Ig, proteontech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-rabbit 594, SA00013-4, proteintech, 1:300) into the water-blocking ring to cover tissues after the sections are slightly dried, and incubating for 60 minutes in a dark place at RT. Placing the slide in PBS, shaking and washing on a decolorizing shaker for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 7
Multiplex immunofluorescent staining of CD20 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (Bcl 6,1:300, 66340-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Primary antibody (CD 23,1:300, 60208-2-Ig, proteontech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 488, SA00013-1, proteintech, 1:500) to cover tissues in the water-blocking ring after the slices are slightly dried, and incubating for 60min in a dark place at RT. The slide is put into PBS and is rocked on a decolorizing shakerWashing for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 8
Multiplex immunofluorescent staining of Bcl6 and CD8 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (Bcl 6,1:300, 66340-1-Ig, proteintech) was incubated for 1h at room temperature. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 594, SA00013-3, proteintech, 1:300) into the water-blocking ring to cover tissues after the slices are slightly dried, and incubating for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. After the sections were slightly dried, another primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added dropwise to the water-resistant ring and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping the corresponding other fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) into the water-blocking ring to cover the tissue after the slice is slightly dried, and incubating for 60min in a dark place at RT. Placing the slide in PBS, shaking and washing on a decolorizing shaker for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 9
Multiplex immunofluorescent staining of Bcl6 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (Bcl 6,1:300, 66340-1-Ig, proteintech) was incubated for 1h at room temperature. After 1h, the slides were washed 3 times with shaking in PBS on a decolorizing shaker for 5min each. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 488, SA00013-1, proteintech, 1:500) to cover tissues in the water-blocking ring after the slices are slightly dried, and incubating for 60min in a dark place at RT. Subsequently, primary antibody (CD 23,1:300, 60208-2-Ig, proteintech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 594, SA00013-3, proteintech, 1:500) to cover tissues in the water-blocking ring after the sections are slightly dried, and incubating for 60 minutes in a dark place at RT. Placing the slide in PBS, shaking and washing on a decolorizing shaker for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 10
Multiplex immunofluorescent staining of CD8 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 8,1:300, 66868-1-Ig, proteintech) was incubated for 1h at room temperature. After 1h, the slides were washed 3 times with shaking in PBS on a decolorizing shaker for 5min each. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 488, SA00013-1, proteintech, 1:500) to cover tissues in the water-blocking ring after the slices are slightly dried, and incubating for 60min in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. After the sections were slightly dried, another primary antibody (CD 23,1:300, 60208-2-Ig, proteintech) was added dropwise to the water-resistant ring and incubated overnight at 4 ℃.
The slide was washed with shaking 3 times in PBS for 5min each on a decolorizing shaker, and then another fluorescent secondary antibody (anti-mouse 594,00013-3, roteintech, 1:300) was added dropwise to the water-blocking ring to cover the tissue and incubated for 60min in the absence of light at RT. Placing the slide in PBS, shaking and washing on a decolorizing shaker for 3 times and 5min each time ， And (5) sealing the sheet after drying slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 11
Multiplex immunofluorescent staining of CD20, bcl6 and CD8 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Then, primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added, incubated at room temperature for 1h, and the slides were washed 3 times with shaking in PBS on a destaining shaker for 5min each. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 594, SA00013-3, proteintech, 1:300) into the water-blocking ring to cover tissues after the slices are slightly dried, and incubating for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Finally, primary antibody (Bcl 6,1:300, 66340-1-Ig, proteintech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Then, another fluorescent secondary antibody (anti-mouse 488,SA00013-1, proteintech, 1:500) was added to cover the tissue, incubated for 60min at RT in the dark, the slides were washed 3 times with shaking on a decolorizing shaker in PBS for 5min each time, and then the slides were blocked after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 12
Multiplex immunofluorescent staining of CD20, bcl6 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Then, primary antibody (CD 23,1:300, 60208-2-Ig, proteintech) was added, incubated at room temperature for 1h, and the slides were washed 3 times with shaking in PBS on a destaining shaker for 5min each. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 594, SA00013-3, proteintech, 1:300) into the water-blocking ring to cover tissues after the slices are slightly dried, and incubating for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Finally, primary antibody (Bcl 6,1:300, 66340-1-Ig, proteintech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Then, another fluorescent secondary antibody (anti-mouse 488,SA00013-1, proteintech, 1:500) was added to cover the tissue, incubated for 60min at RT in the dark, the slides were washed 3 times with shaking on a decolorizing shaker in PBS for 5min each time, and then the slides were blocked after being dried slightly. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 13
Multiplex immunofluorescent staining of Bcl6, CD8 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (Bcl 6,1:300, 66340-1-Ig, proteintech) was incubated for 1h at room temperature. After 1h, the slides were washed 3 times with shaking in PBS on a decolorizing shaker for 5min each. And (3) dripping a corresponding fluorescent secondary antibody (anti-mouse 555,ab150118,Alexa Fluor,1:500) into the water-blocking ring to cover tissues after the slices are slightly dried, and incubating for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Subsequently, primary antibodies (CD 23,1:300, 60208-2-Ig, proteintech) were added dropwise to the water-blocking ring, incubated at room temperature for 1h, and after incubation the slides were washed 3 times with PBS on a destaining shaker for 5min each. Another secondary antibody (anti-mouse 594,00013-3, proteintech, 1:300) was then added and incubated for 60min at RT in the absence of light. The last primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added and incubated overnight at 4 ℃.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 488, SA00013-1, proteintech, 1:500) into the water-blocking ring to cover tissues after the slices are slightly dried, incubating for 60min at the dark RT, placing the glass slide in PBS, shaking and washing on a decolorizing shaker for 3 times, each time for 5min, and slightly airing and sealing the slide. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Comparative example 14
Multiplex immunofluorescent staining of CD20, bcl6, CD8 and CD23 as molecular markers
Sample selection the same as in example 1, multiplex immunofluorescent staining procedure was as follows:
paraffin sections were blocked with PBS containing 1% human serum and 1% bsa for 10min at room temperature, and after removal of the blocking solution, serum was used: pbs=1: 19 (CD 20,1:300, 60271-1-Ig, proteintech) was incubated for 1h at room temperature. Since CD20 is ready-to-use (1:300, 60271-1-Ig, proteinech) (with corresponding secondary antibody 647), no secondary antibody incubation is required, and after 1h the slide is placed in PBS and washed 3 times with shaking on a destaining shaker for 5min each. Then, primary antibody (CD 8,1:300, 66868-1-Ig, proteintech) was added, incubated at room temperature for 1h, and the slides were washed 3 times with shaking in PBS on a destaining shaker for 5min each. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 594, SA00013-3, proteintech, 1:300) to cover tissues in the water-blocking ring after the sections are slightly dried, and incubating for 60 minutes in a dark place at RT. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Primary antibody (Bcl 6,1:300, 66340-1-Ig, proteontech) was added and incubated for 1h at room temperature.
The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. Another fluorescent secondary antibody (anti-mouse 488, SA00013-1, proteintech, 1:500) was then added to cover the tissue and incubated for 60min in the dark at RT. Finally, primary antibody (CD 23,1:300, 60208-2-Ig, proteintech) was added and incubated overnight at 4 ℃. The slides were washed 3 times with shaking in PBS for 5min each on a decolorizing shaker. And (3) dripping corresponding fluorescent secondary antibodies (anti-mouse 555,ab150118,Alexa Fluor,1:500) into the water-blocking ring to cover tissues after the sections are slightly dried, incubating for 60 minutes in a dark place at RT, placing the glass slide in PBS, shaking and washing on a decolorizing shaker for 3 times, 5 minutes each time, and slightly drying and sealing the glass slide. Captured with a multispectral imaging system and analyzed by imaging with an OLYMPUS OlyVIA.
Example 2
Interpretation of results
1. TLS interpretation criteria
TLS was quantified by a breast cancer pathologist who was unaware of the pathology and molecular data of all samples.
(1) The interstitial area which is less than or equal to 5mm around the invasive cancer focus in the slice is used as an interpretation area of TLS;
(2) The presence of ectopic and organized lymphoid aggregates within the interpretation region, comprising anatomically distinct but adjacent T-cell and cd20+ B-cell regions of cd4+ or cd8+;
(3) High endothelial venules exist in or around lymphoid aggregates;
(4) Immunohistochemistry showed the presence or absence of CD23 positive follicular dendritic cells (follicular dendritic cells, FDC) and BCL6 positive germinal center cells with or without concomitant center of development within the lymphocyte foci.
2. H & E and immunohistochemistry
58 of the TLS positive patients and 117 of the TLS negative patients were determined according to TLS interpretation criteria. And the result of immunohistochemical TLS was positive in the presence of 55 CD23 markers in 58 patients who were eventually judged to be TLS positive.
3. Multiplex immunofluorescence
TLS was judged as gold standard with earlier H & E staining and immunohistochemical results, and fig. 4 and 5 are H & E staining and immunohistochemical results, respectively, and are typical TLS performance.
175 Zhang Ruxian cancer pathological sections were subjected to multiplex immunofluorescent staining in the same manner as in example 1 and comparative examples 1 to 14.
4. Statistical inspection
The multi-immunofluorescence results of four markers of CD20, CD8, CD23 and Bcl6 are counted, a prediction probability formed by establishing a logistic regression model is used as a unique analysis index, an ROC curve is established, and the result shows that the AUC of the ROC curve is the highest that all the markers are detected together, but part of the ROC curve is located below a reference line, and is verified to be related to the statistical significance that the part does not have diagnosis, so that the ROC curve is not suitable for being used as an index of a diagnosis test.
ROC curves were established based on single, two, three, and four immunofluorescent markers, respectively. The specific results are shown in FIGS. 6-8.
From fig. 6-8, it can be seen that the CD20& CD8& CD23 triple assay (auc=0.854) > Bcl6& CD8& CD23 triple assay (auc=0.852) > CD8& CD23 double assay (auc=0.850) > CD20& CD23 double assay (auc=0.841) > Bcl6& CD23 double assay (auc=0.816) > CD23 assay (auc=0.798), and the balance being lower than the single CD23 assay, and thus the detection accuracy of TLS can be significantly improved by combining CD20 and CD8 with CD23 assays.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. A molecular marker for diagnosing breast cancer, wherein the molecular marker is CD20, CD8 and CD23.
2. Use of the molecular marker of claim 1 for the preparation of a product for diagnosing early stages of breast cancer.
3. Use of the molecular marker of claim 1 for the preparation of a product for analysis of tertiary lymphoid structures.
4. A kit for diagnosing tertiary lymphoid structure comprising: anti-CD 20 antibodies, anti-CD 8 antibodies, anti-CD 23 antibodies, and immunofluorescent secondary antibodies.
5. The kit of claim 4, wherein the immunofluorescent secondary antibodies are 488-labeled goat anti-mouse IgG (h+l) and 594-labeled goat anti-mouse IgG (h+l).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311433078.8A CN117269510A (en) | 2023-10-31 | 2023-10-31 | Molecular marker for diagnosing breast cancer, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311433078.8A CN117269510A (en) | 2023-10-31 | 2023-10-31 | Molecular marker for diagnosing breast cancer, kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117269510A true CN117269510A (en) | 2023-12-22 |
Family
ID=89202734
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311433078.8A Pending CN117269510A (en) | 2023-10-31 | 2023-10-31 | Molecular marker for diagnosing breast cancer, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117269510A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118376480A (en) * | 2024-03-15 | 2024-07-23 | 上海阿克曼医学检验所有限公司 | Multiplex immunofluorescent staining method and kit for detecting FOXC1, DCLK1, CD8 and AR proteins |
-
2023
- 2023-10-31 CN CN202311433078.8A patent/CN117269510A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118376480A (en) * | 2024-03-15 | 2024-07-23 | 上海阿克曼医学检验所有限公司 | Multiplex immunofluorescent staining method and kit for detecting FOXC1, DCLK1, CD8 and AR proteins |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN117269510A (en) | Molecular marker for diagnosing breast cancer, kit and application thereof | |
| CN108802389B (en) | Kit for diagnosing early non-small cell lung cancer | |
| CN109266740A (en) | For pulmonary cancer diagnosis or the marker and diagnostic reagent of prognosis | |
| EP3165926A1 (en) | Method for characterization of cell specific microvesicles | |
| Cabrera et al. | Hepatocellular carcinoma immunopathogenesis: clinical evidence for global T cell defects and an immunomodulatory role for soluble CD25 (sCD25) | |
| CN111458507B (en) | Marker for evaluating sensitivity of intrahepatic bile duct cancer gemcitabine chemotherapy and application thereof | |
| CN113092761A (en) | Peripheral blood TCR marker of diffuse large B cell lymphoma and detection kit and application thereof | |
| Lu et al. | An special epithelial staining agents: folic acid receptor-mediated diagnosis (FRD) effectively and conveniently screen patients with cervical cancer | |
| Hasanbegovic et al. | Determination of the reference values of the tumor marker HE4 in female population of Canton Sarajevo | |
| CN102803968A (en) | Esophageal cancer marker | |
| Tsui et al. | Comparisons of voided urine cytology, nuclear matrix protein‐22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China | |
| CN114814224A (en) | Application of Hsp90 alpha in nipple discharge in breast cancer | |
| CN112698033A (en) | Detection method and application of blood-borne exosome HER2 | |
| CN109696547B (en) | Marker for judging colorectal cancer prognosis and application thereof | |
| RU2104526C1 (en) | Method for diagnosing t-cellular lymphomae of skin | |
| JP2001289861A (en) | Method for evaluating cancer by using tumor marker | |
| RU2530557C1 (en) | Differential diagnostic technique for malignant and benign breast pathology | |
| CN105628784B (en) | Colorectal cancer sialoprotein finger-print molecule diagnostic model method for building up | |
| CN112763712B (en) | Tumor diagnosis marker and application thereof | |
| Bhagwan Valjee et al. | Investigation of exosomal tetraspanin profile in sepsis patients as a promising diagnostic biomarker | |
| US20050106642A1 (en) | Saliva test for early diagnosis of cancers | |
| KR102518761B1 (en) | Prognosis method of pancreatic cancer and kit therefor | |
| CN115602313B (en) | Biomarker for disease curative effect and survival prognosis prediction and application thereof | |
| CN113267626B (en) | Test strip for early screening of cardia adenocarcinoma of high risk group | |
| CN111735945B (en) | Application of CICs in pancreatic tumor tissue in preparation of product for predicting pancreatic cancer prognosis survival |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |