Abstract
Background/Aim: The clinicopathological and prognostic significances of programmed death ligand 1 (PD-L1) expression in triple-negative breast carcinoma (TNBC) are still unclear. We investigated whether PD-L1 expression is associated with clinicopathological characteristics and outcomes of TNBC patients. Materials and Methods: We performed immunostaining for PD-L1 (SP142) in 83 TNBCs. Staining proportion of ≥1% was regarded as positive PD-L1 expression. Results: Positive intratumoral (IT) PD-L1 expression (19/83; 22.9%) was inversely associated with lymphovascular invasion (LVI) and distant metastasis, and was significantly associated with better disease-free survival for TNBC patients. Positive stromal PD-L1 expression (44/83; 53.0%) also correlated inversely with LVI. Conclusion: Positive IT PD-L1 expression was associated with favorable outcomes in TNBC. In addition, positive IT and stromal PD-L1 were inversely associated with LVI and distant metastasis of TNBC.
Triple-negative breast carcinoma (TNBC) is a subtype of breast carcinoma that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (1, 2). TNBC accounts for approximately 15% of all breast carcinomas (3). Compared to other subtypes, TNBC tends to have worse prognosis, and is often associated with a higher histological grade and more frequent rates of relapse and distant metastasis (4-6). There is no approved targeted therapy for TNBC. Therapeutic options for TNBC patients are limited to cytotoxic chemotherapy.
Although breast carcinoma is not traditionally considered strongly antigenic, some TNBCs show increased tumor-infiltrating lymphocytes (TILs) (7, 8). Several ongoing clinical trials are using programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy in combination with chemotherapy. Thus far, these studies have shown mixed overall responses (9-12). PD-1 is expressed in activated T-lymphocytes, and PD-L1 is expressed on both the tumor cells and immune cells of the tumor microenvironment (13). PD-L1, which belongs to the B7 family, binds PD-1 and CD80 as counter receptors to offer negative signals that control and suppress cytotoxic T-lymphocyte responses in both autoimmune responses and evasion of tumor immunity (14-17). When PD-1 binds to PD-L1, PD-1 suppresses the immune functions of T-lymphocytes by inhibiting expression of their transcription factors, which leads to apoptosis of T-lymphocytes and potentiates tumor progression.
The prognostic implications of PD-L1 expression vary according to primary sites and histological subtypes of malignancies. Positive PD-L1 expression has been reported to be associated with worse prognosis in patients with non-small cell lung carcinoma, esophageal carcinoma, gastric carcinoma, renal cell carcinoma, and malignant melanoma (18-21). In contrast, significant associations have also been reported between PD-L1 positivity and better outcomes in patients with breast carcinoma (22-27). Antibodies to PD-L1, including atezolizumab and durvalumab, have been approved by the United States (US) Food and Drug Administration (FDA) to treat certain solid tumors, such as advanced urothelial carcinoma and non-small cell lung carcinoma, and are under development to treat other malignancies (28).
Studies that have investigated whether PD-L1 expression has favorable or adverse prognostic significance in breast carcinoma have reported conflicting findings. Some studies have shown a positive correlation between PD-L1 expression and favorable prognosis (29-33). Others have found a negative or non-significant correlation between PD-L1 expression and prognosis (34-38). The reason for these controversies may be that many studies interpreted cell types differently (tumor cells or immune cells), used different cut-offs to determine positivity, or used different clones. Currently, the College of American Pathologists (CAP) recommends the use of FDA-approved PD-L1 SP142 antibody (Ventana Medical Systems, Oro Valley, AZ, USA) and regards ≥1% expression as positive in TILs and not in tumor cells in TNBC, unlike other tumors (39).
The purpose of this study was to determine the prevalence of PD-L1 positivity in TNBC and examine the relationship between clinicopathological characteristics and outcomes in a Korean population. In addition, we reviewed the previously published literature regarding the prognostic significance of PD-L1 expression in TNBC.
Patients and Methods
Patient selection. Following approval (2020-02-007) by the Institutional Review Board of Kangbuk Samsung Hospital (Seoul, Republic of Korea), the records of 83 cases of TNBC diagnosed from 2008 to 2013 were retrieved from the pathology archives. All patients underwent segmental mastectomies with axillary lymph node dissection. None of the patients underwent neoadjuvant systemic chemotherapy. All available hematoxylin and eosin (H&E)-stained slides were reviewed by two board-certified pathologists specialized in breast pathology to evaluate nuclear grade, histological grade, presence of lymphovascular invasion (LVI), pathological tumor (pT) stage, and pathological node stage (pN). The development of locoregional recurrence and distant metastasis as well as survival data were retrieved from the electronic medical records. Overall survival (OS) was defined as the period from the date of initial diagnosis to the date of last contact. Disease-free survival (DFS) was defined as the period from the date of initial diagnosis to the date of first disease recurrence, which include locoregional recurrence and metastasis to distant organs.
Tissue acquisition. The surgically resected specimens were examined macroscopically, fixed in 10% neutral buffered formalin, processed, and embedded in paraffin using a standard protocol. After the review of all available H&E-stained slides, the most representative tumor areas were carefully selected and marked on individual formalin-fixed, paraffin-embedded tissue blocks. The tumor tissue cores (2 mm in diameter) were obtained from each specimen and manually arrayed in recipient tissue microarray (TMA) paraffin blocks (40-42).
Immunohistochemical evaluation. From the TMA blocks, serial 3-μm sections were cut consecutively and adhered to charged glass slides for subsequent H&E and immunohistochemical staining. PD-L1 immunostaining was performed using the FDA-approved kit (prediluted, clone SP142, Ventana Medical Systems). Immunostaining for ER (1:200, clone SP1, LabVision Corporation, Fremont, CA, USA), PR (1:200, clone PgR 636, Dako, Glostrup, Denmark), and HER2 (1:200, clone SP3, LabVision Corporation) was also performed and confirmed that all examined cases were TNBC. To meet the definition of TNBC, tumors had to be negative for ER and PR (<1% staining) and negative for HER2 (a score of 0 or 1+ by immunostaining or 2+ with HER2/centromere 17 reference probe ratio <2.0 by silver-enhanced in situ hybridization) (42). PD-L1 expression was evaluated separately in the intratumoral and stromal TILs, according to the interpretation guidelines recommended by the CAP (39), in which ≥1% of staining proportion with any intensity was regarded as positive expression. Negative PD-L1 expression was defined as <1% staining (Figure 1).
Statistical analysis. The Pearson's chi-square test, Fisher's exact test, or linear-by-linear association test was used to examine whether PD-L1 expression status is significantly associated with clinicopathological characteristics. Univariate survival analysis was performed to examine the prognostic significance of PD-L1 expression status with respect to DFS and OS. Kaplan–Meier plot was used to display survival curves. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 23.0 (IBM Corp., Armonk, NY, USA). Statistical significance was defined as p<0.05.
Literature review. The Medline database was thoroughly searched using the PubMed retrieval service. Searches were performed in October 2019, using the terms “PD-L1”, “breast carcinoma”, and “TNBC”. The resulting 118 publications were reviewed and 29 studies were identified that examined PD-L1 expression status in TNBC. Of them, 12 studies reporting PD-L1 expression in TILs and its association with survival data were selected.
Results
Patient characteristics. Patients' ages ranged from 25-79 years (mean: 47 years). The majority (75.9%; 63/83) of the patients were diagnosed with ≥ pT2 tumors. More than half (65.1%; 54/83) of the cases were diagnosed as pT2, and 9 (10.8%) cases were pT3 (9.6%; 8/83) or pT4 (1.2%; 1/83). The remaining 20 (24.1%) cases were pT1. Approximately three-fourths (74.7%; 62/83) of the cases showed high nuclear grade. Histological grades were 2 and 3 in 27 (32.5%) and 53 (63.9%) cases, respectively. Lymph node metastases were detected in 37 (44.6%) patients. During the median follow-up time of 89 months, 20 (24.1%) patients developed distant metastases.
Clinicopathological significance of PD-L1 Expression in TNBC. Intratumoral PD-L1 expression was positive in 19 (22.9%) cases (Figure 1). Table I summarizes the associations between intratumoral PD-L1 expression status and the clinicopathological characteristics of TNBC. The intratumoral PD-L1 positivity was inversely associated with lymphovascular invasion and distant metastasis. In the majority of cases with positive intratumoral PD-L1 expression, neither lymphovascular invasion nor distant metastasis were identified. Only two of the 19 (10.5%) patients whose tumors showed intratumoral PD-L1 positivity had lymphovascular invasion (p=0.001). Similarly, only one (5.3%) patient with positive intratumoral PD-L1 expression developed distant metastasis (p=0.033). No significant association was identified between the intratumoral PD-L1 expression status and other clinicopathological characteristics, including pT, histological and nuclear grades, and lymph node metastasis.
Expression of programmed death ligand 1 (PD-L1) in triple-negative breast carcinoma.
Kaplan–Meier plots for disease-free survival (DFS) and overall survival (OS) according to intratumoral (A and B) and stromal (C and D) programmed cell death ligand 1 (PD-L1) expression in patients with triple-negative breast carcinoma.
Clinicopathological significance of programmed cell death ligand 1 (PD-L1) expression in the intratumoral tumor-infiltrating lymphocytes.
Forty-four (53.0%) cases exhibited positive stromal PD-L1 expression (Figure 1). The associations of stromal PD-L1 expression status with the clinicopathological characteristics of TNBC are shown in Table II. The stromal PD-L1 showed a positive inverse correlation with lymphovascular invasion (p=0.013) and a positive correlation with both histological grade (p=0.002) and nuclear grade (p=0.019). Consistent with the results of the intratumoral PD-L1 expression, more than two-thirds (70.5%; 31/44) of the cases showing stromal PD-L1 positivity were free of lymphovascular invasion. In contrast, most of the cases (86.4%; 38/44) with positive stromal PD-L1 expression displayed high nuclear grade, and all cases showing stromal PD-L1 positivity showed histological grade 2 (22.7%; 10/4) or 3 (77.3%; 34/44). We observed no significant relationship between stromal PD-L1 expression status and other clinicopathological characteristics, including pT, lymph node metastasis, and distant metastasis.
Clinicopathological significance of programmed cell death ligand 1 (PD-L1) expression in the stromal tumor-infiltrating lymphocytes.
Prognostic significance of PD-L1 expression in TNBC. In our TNBC study cohort, positive intratumoral PD-L1 expression was significantly associated with better DFS (p=0.036; Figure 2A). DFS for patients with intratumoral PD-L1-positive TNBC was higher than 90% during the entire observation period, whereas DFS for patients with intratumoral PD-L1-negative TNBC declined steadily (up to approximately 65%) during the first four years postoperatively. OS for patients with TNBC whose tumors were positive for PD-L1 was also higher than that for patients with intratumoral PD-L1-negative TNBC, but the difference was not statistically significant (p=0.083; Figure 2B). Similarly, there were no statistical differences in survival according to the stromal PD-L1 expression status, even though both DFS (Figure 2C) and OS (Figure 2D) for patients with stromal PD-L1-positive TNBC were higher than those for patients with negative stromal PD-L1 expression.
Previously published results regarding prognostic significance of programmed cell death ligand 1 (PD-L1) expression in triple-negative breast carcinoma patients.
Review of the literature. Table III summarizes previously published data regarding the prognostic significance of PD-L1 expression in patients with TNBC. A systematic review of the literature revealed that PD-L1 positivity can be associated with better (22-27) or worse (43-45) prognosis, or not significantly associated with patient outcome (46, 47). The most recent study (22) has documented that positive PD-L1 expression was associated with pathological complete response and higher OS. In contrast, a study by Zhu et al. (43) has reported that positive PD-L1 expression overrode the favorable prognosis associated with high TILs. In addition, Tomioka et al. (44) have suggested that positive PD-L1 expression exacerbated the worse survival associated with low TILs. Taken together, the findings of previously published studies offer no consensus on the prognostic significance of PD-L1 expression.
Discussion
A number of previous studies have reported an association between PD-L1 expression status and the clinicopathological characteristics of human malignancies (8, 48-56). However, there is no standard system for evaluating PD-L1 expression, and the authors of previous studies have applied different cut-off values for positive PD-L1 expression, and used different clones. Although definitions of PD-L1 positivity vary from ≥1% to >70% (Table III), recent guidelines have recommended that a specimen should be considered as having PD-L1 expression if it exhibits ≥1% of immune cells. In terms of PD-L1 antibodies for TNBC, SP142 (Ventana Medical Systems) is the only antibody that has been approved by the US FDA. Therefore, in this study we used the SP142 antibody with a cut-off value of ≥1% for positive PD-L1 expression.
We demonstrated that the intratumoral PD-L1 positivity was significantly associated with better DFS in TNBC. Based on the available literature, the prognostic implication of PD-L1 in TNBC is controversial, especially in terms of patient survival. Such conflicting results might be attributable to differences in sample size and stage, differences between tissue microarrays and whole sections, differences in antibodies and/or antigen retrieval, and staining procedures with varying degrees of sensitivity coupled with the lack of a standard evaluation method for immunostaining. However, recent literature suggests positive PD-L1 expression in TNBC as a favorable factor when it was evaluated in the immune cells and not in the tumor cells (22-27).
In this study, the stromal PD-L1 expression (53.0%) was higher than the intratumoral PD-L1 expression (22.8%). Interestingly, we showed that positive stromal PD-L1 expression was associated with higher nuclear and histological grades, both of which are known to be potentially worse prognostic factors. In contrast, we also observed a significant correlation between stromal PD-L1 positivity and less frequent lymphovascular invasion and distant metastasis, which is consistent with the inverse relationship between intratumoral PD-L1 positivity and lymphovascular invasion and distant metastasis. This inconsistency may reflect the intratumoral heterogeneity and complex functions of PD-L1 (57, 58). Further investigations are necessary to confirm or refute our results.
In conclusion, this study demonstrated that positive intratumoral PD-L1 expression is associated with favorable outcome in patients with TNBC. In addition, both the intratumoral and stromal PD-L1 positivities were inversely associated with lymphovascular invasion and distant metastasis of TNBC. This is the first study to examine PD-L1 expression using both the FDA-approved antibody for TNBC and the CAP guidelines for defining positivity (i.e., ≥1% of immune cells rather than tumor cells). It remains unknown whether the use of anti-PD-L1 treatment can further increase the survival benefit for patients with PD-L1-positive TNBC, and this should be investigated in future studies.
Acknowledgements
This research was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (2018R1C1B5043725).
Footnotes
Authors' Contributions
All Authors made substantial contributions to the conception and design of the study, acquisition of data, analysis and interpretation of the data, as well as to drafting the manuscript, revising the article critically for important intellectual content, and providing final approval of the version to be published.
Conflicts of Interest
None of the Authors has any conflicts of interest to declare regarding this study.
- Received February 5, 2020.
- Revision received February 14, 2020.
- Accepted February 14, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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