CN113125751B - 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 PDF

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CN113125751B
CN113125751B CN202110389918.XA CN202110389918A CN113125751B CN 113125751 B CN113125751 B CN 113125751B CN 202110389918 A CN202110389918 A CN 202110389918A CN 113125751 B CN113125751 B CN 113125751B
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宋清坤
李艳萍
周全
伍江平
吕淑贞
袁可玉
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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 using 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 research results of the present application indicate that the clinical pathology of breast cancer patients may affect the level of PD-L1 positive T lymphocytes in peripheral blood; the PD-1/PD-L1 positive lymphocyte detection in peripheral blood has the capability of evaluating the PD-L1 expression property in the breast cancer tissue, and the result shows that the PD-1/PD-L1 level on the lymphocyte surface in the blood sample is expected to replace pathological tissues clinically to become the index for PD-L1 state evaluation.

Description

Method for predicting PD-L1 level in breast cancer tumor tissue by peripheral blood PD-1/PD-L1
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 in immunotherapy, are primarily approved for the treatment of non-small cell lung cancer, melanoma and the like, can improve median progression-free survival and overall survival of patients, and gradually cover the application of other solid or hematological tumors. Because of the better responsiveness of triple negative breast cancer to anti-PD-1/PD-L1 drugs, PD-1/PD-L1 inhibitor-related clinical trials have focused more on this molecularly typed tumor. The proportion of PD-L1 positive patients in the tumor of the subtype measured by a pathological immunohistochemical method is 38-78%, and the difference can be related to the race of a selected population, the previous treatment experience, the metastasis condition and the like. 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 usually evaluate whether PD-1 blocking drugs are applied or not through the expression of PD-1/PD-L1 in tumor specimens before treatment. In breast cancer, the number of PD-L1 positive cells in peritumoral infiltrating immune cells is more than or equal to 1 percent as an indication for using the att Zhu Shankang. Neglecting the fact that the level of PD-1/PD-L1 in tumor tissue changes during treatment, which changes the responsiveness of the tumor to the drug, repeated 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 levels of PD-L1 positive circulating T lymphocytes in lung cancer correlate 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 PD-L1 state of the patient can be timely mastered when a tumor tissue specimen is inconvenient to obtain. The application evaluates the feasibility of PD-1/PD-L1 positive circulating T lymphocyte levels to replace pathological tissue immunohistochemistry for assessment of PD-L1 expression status in patients.
In one aspect, the present application provides a method for predicting PD-L1 levels in breast cancer tumor tissue from 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 the tumor tissue based on the expression level of PD-1 and/or PD-L1 in the circulating T lymphocytes.
Further, the method is a non-diagnostic method.
Further, the tumor is breast cancer.
Further, the detecting detects the expression level of PD-1 and/or PD-L1 in the circulating T lymphocytes by flow cytometry.
Furthermore, the detection sample source is Luminal type A 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 a reagent 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, and can be used to assess the status of PD-L1 in the 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 levels of PD-1/PD-L1 in peripheral blood of patients in a cohort;
FIG. 2 is an example of PD-L1 expression levels 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 in breast cancers of different molecular subtypes A.Luminal A, B.Luminal B, C.trigonal form, D.HER-2 overexpression.
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).
Breast tumor microanalysis was performed using an anti-PD-L1 polyclonal antibody (SP 142, diluted with 1The marker of PD-L1 expression in the environment, and the detection of the PD-L1 expression degree is carried out by an automatic immunohistochemical instrument by 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 cells [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 the circulating T lymphocyte by using a Cytomics FC500 type flow cytometer. 50 mu L of whole blood is added into the 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 = 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. Then 500. Mu.L PBS was added to resuspend the cells and test them on the machine, data were collected and analyzed with CXP software, CD3 + The lymphocyte is gated, the experiment is repeated for 4 times, and the result is judged according to negative control and isotype control.
And (3) data analysis:
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 the peripheral blood. At x degree 2 Checking 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. PD-1-The PD-L1 positive lymphocyte levels reflect the accuracy and surrogate effect of tissue PD-L1, and ROC curves were plotted, the area under the curve (AUC) was evaluated and the optimal cut-off value was derived. And (3) converting the variable into a two-classification variable by taking the cut-off value of the proportion of PD-1/PD-L1 positive T lymphocytes in blood as a node, evaluating the positive relation between the two variables and PD-L1 expression 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 levels 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.
Relation between expression level of T lymphocyte PD-1/PD-L1 in blood sample and expression of PD-L1 in tissue sample and clinical pathology 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). Whereas the median positive rate for peripheral blood T lymphocytes PD-L1 was 1.4% significantly higher in cases with lymph node metastasis 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-progesterone receptor properties, HER-2 properties, ki-67 expression level and molecular typing were found in 73 tissue specimens to be independent of the PD-L1 expression status of the patients (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
Figure BDA0003016220400000061
TABLE 2 relationship between PD-L1 Properties and clinicopathological parameters in breast cancer patient tissues
Figure BDA0003016220400000071
Analysis of the consistency of PD-1/PD-L1 expression levels 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 the tumor tissue microenvironment is 0.24 (p = 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 = 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 gland 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 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 in blood was 4.420 times higher for people with a PD-1 positive T lymphocyte ratio greater than 14.6% than in the tumor tissue microenvironment of people below this value (p =0.005, table 3), and the PD-L1 positive rate in blood was 4.755 times higher for people with a PD-L1 positive T lymphocyte ratio greater than 1.1% than in the tumor tissue microenvironment of people 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
Figure BDA0003016220400000081
Analysis of the concordance of the peripheral blood lymphocyte surface PD-1/PD-L1 expression levels in the molecular typing subgroup with the PD-L1 expression in the peritumoral tissues
Molecular typing subgroup analysis found a PD-1AUC value of 0.862 (95% CI =0.619-0.976 p < 0.001 cut-off value: 3.917; sensitivity 100.00%; specificity 76.92%) in Luminal type A blood, a PD-L1 AUC value of 0.731 (95% CI =0.474-0.908 p =0.128; sensitivity 60.00%; specificity 84.62%, FIG. 4A); the PD-1AUC value in Luminal type B blood was 0.600 (95% CI =0.415-0.766 p =0.317; sensitivity 80.00%; specificity 52.17%), the PD-L1 AUC value was 0.652 (95% CI =0.467-0.809; sensitivity 70.00%; specificity 73.91%, FIG. 4B); triple negative blood 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; 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 the study, the expression of PD-L1 in tumor tissues and the proportion of PD-1 positive T lymphocytes in peripheral blood are irrelevant to clinical pathological variables of patients, and the expression of PD-L1 on the surface of circulating T lymphocytes is relevant to the lymph node metastasis condition and the property of HER-2. This is not exactly the same conclusion as drawn by other studies. Since Att Zhu Shankang is now the only PD-1/PD-L1 inhibitor approved for breast cancer, SP142 corresponding thereto was selected for interpretation of PD-L1 expression in tissues, and the results were 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 PD-1/PD-L1 expression levels 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 is different among different human species. Subgroup analysis of Japanese patients in the Impassion130 experiment showed that PD-L1 positive cases in triple negative breast cancer in Asian population accounted for 38%, and the results of this experiment were 36% and essentially consistent therewith.
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 PD-L1 of tumor-surrounding immune cells, 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 (5)

1. A non-diagnostic method for predicting the level of PD-L1 in a breast cancer tumor tissue using peripheral blood PD-1, comprising the steps of detecting the level of CD3+ PD-1+ cells in peripheral blood circulating T lymphocytes, and predicting the level of PD-L1 in the tumor tissue based on the level of CD3+ PD-1+ cells in peripheral blood circulating T lymphocytes; wherein the peripheral blood source is Luminal A type and HER-2 overexpression type objects in Luminal typing; the breast cancer tumor tissue is breast cancer tumor tissue of Luminal A type and HER-2 overexpression type objects in Luminal typing.
2. The method of claim 1, wherein said detecting the level of CD3+ PD-1+ cells in circulating T lymphocytes in peripheral blood is performed by flow cytometry.
3. The application of a reagent for detecting the level of CD3+ PD-1+ cells in peripheral blood circulating T lymphocytes in preparing a kit for detecting the level of PD-L1 in tumor tissues; wherein the tumor is breast cancer of patients with Luminal A type and HER-2 overexpression in Luminal typing.
4. The use according to claim 3, wherein the reagent for detecting the level of CD3+ PD-1+ cells in T lymphocytes circulating in peripheral blood is a reagent for flow cytometry.
5. The use of claim 3, wherein the agent for detecting the level of CD3+ PD-1+ cells in T lymphocytes circulating in peripheral blood comprises a PD-1 monoclonal antibody.
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