2.1Study Design
This study was designed as a cross-sectional study. Circulating phenotypic lymphocytes and cytokines were detected in the pre-operative blood and PD-L1 expression was evaluated in the biopsy or surgical tissues. The NAT treatment was also collected from electrical medicine records.
2.2Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of Institutional Review Board of Beijing Shijitan Hospital, Capital Medical University (sjtkyll-1x-2018(56)) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants included in the study.
2.3Patients
134 cases of operable breast cancer patients were recruited at Beijing Shijitan Hospital between November 2018 and November 2020. Patients did not have any invasions to skin or chest wall. Patients did not have any diagnosis of inflammatory BC, autoimmune diseases, heart, brain, kidney, or other vital organs insufficiency. Patients should have the ECOG> 2. Among patients with negative NAT, peripheral blood samples were collected preoperatively and among patients with NAT, peripheral blood was collected after NAT and before surgery. Tumor tissues were obtained through biopsy or surgery.
2.4Immunohistochemistry (IHC) Staining And Scoring
The positive threshold of ER and PR detection in IHC was set as 1% tumor cell staining. Positive expression of HER-2 was defined as +++ in IHC tests or positive in situ hybridization (ISH) test; negative expression was defined as -, + in IHC test or negative ISH test. Ki-67 index was detected by IHC on 4μm-thick formalin fixed paraffin-embedded sections. Hot-spot area was determined under low-power field and the index ≥14% was defined as high expression. Molecular subtypes were defined as Luminal A (HER-2 negative, ER positive, Ki-67 low expression), Luminal B (HER-2 negative, ER positive, Ki-67 high expression), TNBC (HER-2 negative, ER negative, Ki-67 arbitrary), HER-2 overexpression/HR negative (HER-2 positive, ER negative, Ki-67 arbitrary) and HER-2 overexpression/HR positive (HER-2 positive, ER positive, Ki-67 arbitrary).
Monoclonal antibodies to PD-L1 (rabbit anti-human, #SP142) were purchased from Roche Shanghai Co. Ltd. Second antibodies were purchased from Beijing Zhongshanjinqiao biotechnology Co Ltd. The EnVision two-step method was used to detect the expression of PD-L1 on the immune cells in TME. Two pathologists interpreted the IHC staining of immune cells on the whole section. Tumor-infiltrating immune cells with brown staining accounting for more than 1% tumor area was determined as positive expression of PD-L1 in TME[13, 14].
2.5Flow Cytometry Tests And Scoring
6ml venous blood was collected into EDTA-K2 anticoagulation tube (Becton, Dickinson and Company) and three-color flow cytometric analysis was performed to determine cell phenotypes by Cytomics FC500 flow cytometer (Beckman-Coulter). The monoclonal antibodies of CD3-FITC (A07746, Beckman-Coulter), CD4-PE (A07751, Beckman-Coulter), CD8-PC5 (A07758, Beckman-Coulter), CD127-PC7 (A64618, Beckman-Coulter), CD28-PE (IM2071u, Beckman-Coulter) and CD25-PC5 (IM2646, Beckman-Coulter) were added into the flow tube. Subtype lymphocytes were defined as percentage of total circulating T lymphocytes (CD3+ cells). 5000 lymphocytes were gated to calculate the percentage of positive T lymphocyte subtypes by a CXP analysis software (Beckman-Coulter).
2.6Cytokine Detection
Peripheral cytokines, including interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, IL-12P70, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IFN-α, were detected via a multi-cytokine detection kit (Cellgene Biotech Co, LTD, China). The detection followed the protocol recommended by the manufacturer. 20μl capture microsphere solution, 25μl plasma sample or diluted standard sample, and 25μl fluorescence detection solution were added into a tube for a 2.5-hour incubation. 1ml PBS was then added and centrifugated at 200g for 5min. With supernatant discarded, the precipitant was resuspended in 100ml PBS. The detection process was conducted in the BD LSRFortessa flow cytometer (BD Biosciences, USA), and the results were analyzed by the BD FACSDivaTM (BD Biosciences, USA) software. The cytokine levels were determined based on a logistic curve-fitting equation generated with the serial diluted standard sample.
2.7Variables
Tumor size was categorized by the diameter, ≤2cm (T1), ≤5cm (T2) and >5cm (T3). Clinical TNM (cTNM) stage was classified as Ⅰ (T1N0M0), Ⅱ (T0~1N1M0, T2N0~1M0, T3N0M0) and Ⅲ (T0~2N2M0, T3N1~2M0, T4N0~3M0, T0~4N3M0) stage. Histological grade was defined by the scores estimated according to the glandular duct formation, nuclear pleomorphism and mitotic ability. The score range of grade Ⅰ, Ⅱ and Ⅲ was 3 to 5, 6 to 7 and 8 to 9, respectively.
2.8Statistical Analysis
All statistical analyses were performed using the SPSS software (version 23.0). The difference between peripheral blood lymphocyte subtypes, cytokines and TME PD-L1 expression was conducted by Mann–Whitney U test. The association between the peripheral lymphocyte subtypes, cytokines and the positive levels of TME PD-L1 expression rate was estimated by Spearman correlation tests. The receiver operative character (ROC) curve was illustrated between the peripheral lymphocyte subtypes, cytokines and TME PD-L1 expression, and the validity was estimated by the area under the curve (AUC). Logistical Regression Model was used to control confounding factors such as tumor grade, tumor TNM stage and age, and then the influence of peripheral blood related cell subtypes and cytokines on TME PD-L1 positive expression was further analyzed. TNF-α was set as categorical variable in the multivariate Logistic analysis with cut-off value in the ROC analysis. All analyses were two-sided and p<0.05 was considered statistically significant.