CN113125751A - Method for predicting PD-L1 level in breast cancer tumor tissue by peripheral blood PD-1/PD-L1 - Google Patents
Method for predicting PD-L1 level in breast cancer tumor tissue by peripheral blood PD-1/PD-L1 Download PDFInfo
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Abstract
The application belongs to the technical field of tumor diagnosis and treatment, and provides a method for predicting PD-L1 level in breast cancer tumor tissue by peripheral blood PD-1/PD-L1 and application of a corresponding reagent, which comprises the steps of detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes, and predicting PD-L1 level in the tumor tissue based on the expression level of PD-1 and/or PD-L1 in the circulating T lymphocytes. The results of the studies of the present application indicate that the clinical pathology of breast cancer patients may affect the level of PD-L1 positive T lymphocytes in the peripheral blood; the detection of PD-1/PD-L1 positive lymphocytes in peripheral blood has the capability of evaluating the expression property of PD-L1 in breast cancer tissues, and the result shows that the level of PD-1/PD-L1 on the surfaces of the lymphocytes in a blood sample is expected to replace pathological tissues clinically to become an index for evaluating the state of PD-L1.
Description
Technical Field
The application belongs to the technical field of tumor diagnosis and treatment, and particularly provides a method for predicting PD-L1 level in breast cancer tumor tissue by using peripheral blood PD-1/PD-L1, which is characterized by comprising the steps of detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes and predicting the PD-L1 level in the tumor tissue based on the expression level of PD-1 and/or PD-L1 in the circulating T lymphocytes.
Background
In recent years, the incidence of breast cancer has been on the rise worldwide, and new therapeutic means have been continuously sought. The PD-1 and PD-L1 inhibitors have taken a major breakthrough as immunotherapies, are primarily approved for the treatment of non-small cell lung cancer, melanoma and the like, and can improve median progression-free survival and overall survival of patients and gradually apply other solid or hematological tumors. Because of the better responsiveness of triple negative breast cancer to anti-PD-1/PD-L1 drugs, the PD-1/PD-L1 inhibitor-related clinical trials focused more on this molecularly typed tumor. The proportion of PD-L1 positive patients in this subtype of tumor measured by pathological immunohistochemistry is 38-78%, and this difference may be related to the race of the selected population, past treatment experience, and metastasis. In addition, the research shows that the expression level of PD-1/PD-L1 is not only related to the clinical pathological characteristics such as tumor diameter, lymph node metastasis condition, prognosis and the like, but also can be used for predicting the response of a patient to a PD-1/PD-L1 inhibitor.
At present, clinicians often assess whether to use PD-1 blocking drugs by expression of PD-1/PD-L1 in pre-treatment tumor specimens. In the case of breast cancer, the number of PD-L1 positive cells in the peritumoral infiltrating immune cells is more than or equal to 1 percent as an indication for using the Atizumab. Neglecting the fact that the change in the level of PD-1/PD-L1 in tumor tissue during treatment changes the responsiveness of the tumor to the drug, repeated tissue biopsies are often required to adjust the drug at any time. Repeated tissue biopsies are more traumatic, so researchers are constantly searching for new biomarkers with predictive effects. Peripheral blood is of great interest in its convenient means of acquisition and low cost. There are studies that indicate that the level of PD-L1 positive circulating T lymphocytes in lung cancer correlates with prognosis. Similar studies have been performed by researchers in breast cancer, and the proportion of LAG3 and PD-1 double positive T lymphocytes in breast cancer TIL was found to correlate with molecular typing, whereas such T lymphocytes in PBL were not meaningful. In addition, no statistical difference in the number of CD4+ T lymphocytes, which express both PD-1 and CTLA-4, was found in normal breast versus primary invasive breast cancer Peripheral Blood Mononuclear Cells (PBMC), but there was a difference between the two groups of people in TIL.
Disclosure of Invention
In conclusion, if the detection of certain indexes in peripheral blood can be used for replacing histopathological detection, the compliance of a patient in the treatment process is increased, the clinical diagnosis and treatment work is simplified, and the state of the PD-L1 of the patient can be timely mastered when a tumor tissue specimen is inconvenient to obtain. The present application evaluated the feasibility of PD-1/PD-L1 positive circulating T lymphocyte levels to replace pathological tissue immunohistochemistry for assessment of patient PD-L1 expression status.
In one aspect, the present application provides a method for predicting the level of PD-L1 in breast cancer tumor tissue with peripheral blood PD-1/PD-L1, characterized by comprising the steps of detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes, and predicting the level of PD-L1 in tumor tissue based on the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes.
Further, the method is a non-diagnostic method.
Further, the tumor is breast cancer.
Further, the detection detects the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes by flow cytometry.
Furthermore, the detection sample source is Luminal A type and HER-2 overexpression objects in Luminal typing.
Further, the level of PD-1 on the surface of circulating T lymphocytes was examined.
In another aspect, the application provides the use of an agent for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes in the preparation of a kit for detecting the level of PD-L1 in tumor tissue.
Further, the tumor is breast cancer, preferably breast cancer of patients with Luminal A type and HER-2 overexpression in Luminal typing.
Further, the reagent for detecting the expression level of PD-1 and/or PD-L1 in the circulating T lymphocytes is a reagent for flow cytometry.
Further, the reagent for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes comprises a PD-1 and/or PD-L1 antibody, preferably a PD-1 monoclonal antibody.
Methods for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes can use methods known or studied in the art, including but not limited to flow cytometry, ELISA, immunoblotting, etc., and the specific procedures and reagents used for these methods are described in the corresponding tool books and specifications, and can be known and selected as required by those skilled in the art. For example, monoclonal or polyclonal antibodies can be obtained commercially or made in house.
The methods of the present application include non-breast cancer diagnostic uses that can be used to assess the status of PD-L1 in tumor tissue of breast cancer patients, to guide clinical medication, and to adjust treatment regimens; the method can also be used for clinical scientific research including but not limited to research on drug development direction, breast cancer prognosis research, development of accurate treatment, scientific research data collection and the like.
Drawings
FIG. 1 is a graph of the expression level of PD-1/PD-L1 in peripheral blood of patients in the cohort;
FIG. 2 is an example of the expression level of PD-L1 in tumor tissue: A. low expression, b. high expression;
FIG. 3 is a ROC curve of peripheral blood PD-1/PD-L1 versus the expression level of PD-L1 in tissues;
FIGS. 4A-D are ROC curves of peripheral blood PD-1/PD-L1 and PD-L1 expression in tissues for different molecular subtypes of breast cancer.
Detailed Description
Example 1 basic research information
The study population is as follows:
the primary early-mid breast cancer cases of the primary diagnosis in Beijing century Tan Hospital are diagnosed from 11 months to 2019 in 2018, no treatment is received, no autoimmune diseases exist, ECOG is larger than 2 points, and no important organ dysfunction such as heart, brain, kidney and the like exists. Peripheral blood is collected before treatment is started, and a tissue sample is obtained by means of local excision or needle biopsy, and then standardized treatment is carried out. All patients signed written informed consent and the study was approved by the hospital ethics committee. Immunohistochemical analysis:
taking a paraffin specimen of tumor tissue of a patient. The high expression of Ki-67 is defined as that the Ki-67 index is more than or equal to 14 percent, and the routine molecular classification of breast cancer diseases is carried out: luminal A type (HER-2 negative, ER positive, Ki-67 low expression), Luminal B type (HER-2 negative, ER positive, Ki-67 high expression or HER-2 positive, ER positive, any Ki-67), trilobular type (HER-2 negative, ER negative, any Ki-67), HER-2 overexpression type (HER-2 positive, ER negative, any Ki-67).
The labeling of PD-L1 expression in the breast tumor microenvironment was performed using an anti-PD-L1 polyclonal antibody (SP142, diluted with 1:200, Roche LTD. shanghai.) and a secondary antibody kit (PV-6000, Beijing zhangshanjnqiao biotechnology co., LTD.) and the detection of the extent of PD-L1 expression was performed using an automated immunohistochemistry apparatus in an EnVision two-step method. The immunohistochemical results were interpreted by skilled physicians. Positive coloration was observed by the appearance of yellow to tan granules in the cytoplasm or cell membrane. Defining the number of PD-L1 positive cells more than or equal to 1% in Tumor Infiltrating Lymphocytes (TIL) around the tumor as tissue PD-L1 expression positive[16]。
Flow cytometry analysis:
taking 6ml of venous blood of a patient, collecting a blood sample by using an EDTA-K2 anticoagulation tube, and detecting the expression degree of PD-L1 and PD-1 on the surface of circulating T lymphocytes by using a Cytomics FC500 type flow cytometer. 50 mu L of whole blood is added into a flow tube, 20 mu L of each of the PD-1-PerCP/Cy5.5 monoclonal antibody and the PD-L1-PE/Cy7 monoclonal antibody are sequentially added, and the mixture is evenly stirred and incubated for 15min at room temperature in a dark place. Adding hemolytic agent 1mL (hemolysin: distilled water is 1: 9), shaking, mixing, and incubating at room temperature for 12min in dark. Adding PBS, shaking, mixing, centrifuging at 1500r/min for 5min, and removing supernatant. The cells were then resuspended in 500. mu.L PBS and tested on the machine, the data collected and analyzed by CXP software, CD3+Lymphocyte setAnd repeating the experiment for 4 times, and judging the result according to the negative control and the isotype control.
And (3) data analysis:
the data were analyzed using SPSS23.0 and MedCalc software. Mann-Whitney Utest was used to analyze the relationship of age, lymph node metastasis, ER, PR, HER-2 and Ki-67 expression to the level of PD-1/PD-L1 positive T lymphocytes in the blood; Jonckheere-Terpstra test was used to analyze the relationship between tumor size, cTNM staging, histological grading and the level of PD-1/PD-L1 positive T lymphocytes in peripheral blood; typing was analyzed by Kruskal-Wallis test analysis as a function of the level of PD-1/PD-L1 positive T lymphocytes in peripheral blood. At x degree2Testing and analyzing the relation between age, lymph node metastasis, ER, PR, HER-2 and Ki-67 expression states, molecular typing and PD-L1 expression states in a tumor microenvironment; Mann-Whitney Utest was used to analyze the relationship between tumor size, cTNM staging, histological grading and PD-L1 expression status in the tumor microenvironment. The correlation of peripheral blood PD-1/PD-L1 positive lymphocyte levels with PD-1/PD-L1 expression in tissues was analyzed by a Spearmascorelation test. Tissue PD-L1 accuracy and surrogate effect was reflected in tissue and peripheral blood PD-1/PD-L1 positive lymphocyte levels and ROC curves were plotted, Area Under Curve (AUC) was evaluated and optimal cut-off was derived. And (3) converting the variable into a two-classification variable by taking a cut-off value of the proportion of PD-1/PD-L1 positive T lymphocytes in blood as a node, evaluating the relationship between the two variables and PD-L1 expression positivity in a tumor microenvironment by using univariate two-classification Logistic regression, and calculating the Odds Ratio (ORs) and 95% Confidence Interval (CIs) after rough and age factor correction.
Example 2 results and analysis
Expression of PD-1/PD-L1 in blood and tissue samples:
among 83 cases meeting the inclusion condition, 77 cases were subjected to the measurement of the expression levels of PD-1 and PD-L1 on the surface of peripheral blood T lymphocytes, and 73 cases were subjected to immunohistochemical staining of tissues PD-1 and PD-L1. The total number of 68 cases has matched tissues and detection results of PD-1/PD-L1 level of peripheral blood, wherein the 68 cases comprise 18 Luminal A cases (26.5%), 33 Luminal B cases (48.5%), 11 Sanyizi cases (16.2%) and 6 HER-2 overexpression cases (8.8%).
The median percentage of PD-1 positive T lymphocytes in blood was 15.2% (3.6% -41.2%); the median percentage of PD-L1 positive T lymphocytes was 0.7% (0.0% -6.5%) (fig. 1A, 1B).
PD-L1 expression in peritumoral TILs with some heterogeneity in pathological immunohistochemical results (fig. 2A, 2B) 24 of 73 (32.9%) demonstrated positive PD-L1 expression in immunohistochemistry of tissues.
The relation between the expression level of T lymphocyte PD-1/PD-L1 in blood samples and the expression condition of PD-L1 in tissue samples and clinical pathological parameters:
analysis of 77 blood samples revealed that PD-1/PD-L1 expression in peripheral blood was independent of clinical pathology such as age, tumor size, clinical stage, histological grade, estrogen receptor status, Ki-67 expression level and molecular typing (p > 0.05, Table 1). While the median positive rate of peripheral blood T lymphocytes PD-L1 in cases with lymph node metastasis was 1.4% significantly higher than in patients without lymph node metastasis (0.6%, p ═ 0.005, table 1); the median proportion of PD-L1 positive cells in the blood of HER-2 positive cases was 0.5% significantly lower than that of HER-2 negative cases (0.9%, p ═ 0.034, table 1), with statistically significant results. The level of PD-1 positive lymphocytes in the blood was shown to be independent of both (lymph node metastasis: p 0.816; nature of HER-2: p 0.432, table 1).
Age, tumor size, lymph node metastasis, clinical staging, histological grade, estrogen-receptor properties, HER-2 properties, Ki-67 expression levels and molecular typing were found to be independent of patient PD-L1 expression status in 73 tissue specimens (p > 0.05, table 2).
TABLE 1 relationship of lymphocyte surface PD-1/PD-L1 expression levels in blood of breast cancer patients to clinicopathological parameters
TABLE 2 relationship of PD-L1 Properties in breast cancer patient tissues to clinicopathological parameters
Analysis of the consistency of the expression level of PD-1/PD-L1 on the surface of peripheral blood lymphocytes and in peritumoral tissues:
the correlation coefficient of the peripheral blood lymphocyte surface PD-1 expression rate and the PD-L1 expression condition in a tumor tissue microenvironment is 0.24(p is 0.046), and the correlation coefficient of the peripheral blood lymphocyte surface PD-L1 expression rate and the PD-L1 expression condition in the tumor tissue microenvironment is 0.258(p is 0.034).
ROC plots were plotted for PD-1/PD-L1 positive T lymphocyte expression levels in blood and PD-L1 expression status in mammary tissue, respectively (fig. 3), with peripheral blood PD-1AUC values of 0.650 (95% CI 0.525-0.762; p 0.037; sensitivity 76.19%; specificity 61.70%), and peripheral blood PD-L1 AUC values of 0.661 (95% CI 0.536-0.771p 0.033; sensitivity 57.14%; specificity 78.72%).
The optimal critical value for the proportion of PD-1 positive T lymphocytes in the blood was 14.6%, and the optimal critical value for the proportion of PD-L1 positive T lymphocytes was 1.1%. Univariate Logistic regression analysis showed that after age factor correction, the PD-L1 positive rate was 4.420 times higher in the proportion of PD-1 positive T lymphocytes in blood than 14.6% (p 0.005, table 3) than in the tumor tissue microenvironment of the population below this value, and the PD-L1 positive rate was 4.755 times higher in the proportion of PD-L1 positive T lymphocytes in blood than 1.1% of the tumor tissue microenvironment of the population below 1.1% (p 0.007, table 3).
TABLE 3 correlation between PD-1/PD-L1 positive T lymphocyte ratio in blood and PD-L1 expression in tumor tissue microenvironment
Analysis of the concordance of the expression level of PD-1/PD-L1 on the surface of peripheral blood lymphocytes in the molecular typing subgroup with the expression of PD-L1 in peritumoral tissues
Molecular typing subgroup analysis found 0.862 (95% CI: 0.619-0.976; p < 0.001; cut-off value: 3.917; sensitivity 100.00%; specificity 76.92%) and 0.731 (95% CI: 0.474-0.908; p: 0.128; sensitivity 60.00%; specificity 84.62%, FIG. 4A) of PD-L1 AUC values in Luminal type A blood; PD-1AUC value in luminel type B blood was 0.600 (95% CI ═ 0.415-0.766; p ═ 0.317; sensitivity 80.00%; specificity 52.17%), PD-L1 AUC value was 0.652 (95% CI ═ 0.467-0.809; p ═ 0.209; sensitivity 70.00%; specificity 73.91%, fig. 4B); triparental PD-1AUC values of 0.714 (95% CI: 0.378-0.933; p: 0.313; sensitivity 50.00%; specificity 100.00%), PD-L1 AUC values of 0.750 (95% CI: 0.412-0.950; p: 0.175; sensitivity 75.00%; specificity 85.71%, fig. 4C); PD-1AUC values in HER-2 overexpressing blood were 1.000 (95% CI: 0.541-1.000; p < 0.001; sensitivity 100.00%; specificity 100.00%), PD-L1 AUC values were 0.500 (95% CI: 0.118-0.882; p: 1.000; sensitivity 100.00%; specificity 25.00%, fig. 4D).
From the results of this study, the expression of PD-L1 in tumor tissues and the proportion of PD-1 positive T lymphocytes in peripheral blood were independent of the clinical pathological variables of patients, and the expression of PD-L1 on the surface of circulating T lymphocytes was correlated with the lymph node metastasis status and the nature of HER-2. This is not exactly the same conclusion as drawn by other studies. Because the atelizumab is the only PD-1/PD-L1 inhibitor approved to be applied to the breast cancer, SP142 corresponding to the atelizumab is selected for interpretation of the expression condition of PD-L1 in tissues, and the result is more referential. The different groups of people may also produce some differences in the results. Previous studies have shown that metastasis and treatment will result in changes in the expression levels of PD-1/PD-L1 and sensitivity to immunotherapy. To exclude this interference, all patients in our cohort were early-to-mid untreated patients without distant metastasis. In addition, the positive expression rate of PD-L1 was different among human species. Subgroup analysis of Japanese patients from the Impassion130 trial showed 38% of PD-L1 positive cases in triple negative breast cancer in the Asian population, with 36% of the results being in substantial agreement.
In the correlation and consistency assessment analysis, the percentage of PD-1/PD-L1 positive T lymphocytes in peripheral circulation is related to the expression level of the tumor-surrounding immune cells PD-L1, and the immunohistochemical result can be predicted to a certain extent. The analysis of different molecular typing cases shows that the level of PD-1 on the surfaces of Luminal A-type and HER-2 overexpression circulating T lymphocytes has the best prediction effect on tissue PD-L1 expression.
Claims (10)
1. A method for predicting PD-L1 levels in breast cancer tumor tissue with peripheral blood PD-1/PD-L1, comprising the steps of detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes, and predicting PD-L1 levels in tumor tissue based on the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes.
2. The method of claim 1, which is a non-diagnostic method.
3. The method of claim 1 or 2, wherein the tumor is breast cancer.
4. The method of any one of claims 1-3, wherein the detecting detects the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes by flow cytometry.
5. The method of any one of claims 1-4, wherein the sample source tested is Luminal type A and HER-2 overexpressing subjects in Luminal typing.
6. The method of claim 5, wherein the level of circulating T lymphocyte surface PD-1 is detected.
7. Application of a reagent for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes in preparing a kit for detecting the level of PD-L1 in tumor tissues.
8. Use according to claim 7, wherein the tumor is a breast cancer, preferably a breast cancer in a patient with lumineal type a and HER-2 overexpression in luminel typing.
9. The use according to claim 7 or 8, wherein the reagent for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes is a reagent for flow cytometry.
10. The use according to any one of claims 7 to 9, wherein the agent for detecting the expression level of PD-1 and/or PD-L1 in circulating T lymphocytes comprises a PD-1 and/or PD-L1 antibody, preferably a PD-1 monoclonal antibody.
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