Background
Programmed cell death ligand 1 (PD-L1, also known as B7-H1 or CD274) is believed to mediate local immune evasion in many types of cancer by binding to programmed cell death 1 (PD-1), its co-stimulatory receptor on T cells, to induce saturation of activated anti-tumor T cells [
1]. Recently, PD-1 and PD-L1 have been shown to be promising targets for the treatment of different tumor types [
2]. In particular, triple negative breast cancer (TNBC) comprises 10–15% of all breast cancer cases and usually exhibits a poorer clinical prognosis than non-TNBC, as it appears to be an aggressive subtype of breast cancer and lacks therapeutic targets [
3]. As previous studies showed that TNBC had more frequently PD-L1 expression [
4,
5], anti-PD-L1/anti-PD-1 therapy has become a promising therapeutic strategy for TNBC, and several trials have shown that anti-PD-1 therapy was effective for breast cancer, and particularly TNBC [
6,
7].
PD-L1 protein expression in tumor cells and infiltrating immune cells has been used as a biomarker to predict the responses of TNBC patients to anti-PD-L1/anti-PD-1 therapy [
8]. However, certain patients with negative PD-L1 expression have been observed to respond to PD-1/PD-L1-blockade therapy [
9]. The reason for this finding may be the dynamic nature of PD-L1 expression during the progression of breast cancer [
10], as shown in a previous study demonstrating PD-L1 status conversion from negative in the primary tumor (PT) to positive in lung metastasis in 1 of 12 TNBC patients [
11]. Therefore, exclusion of patients whose PTs exhibit negative PD-L1 expression from anti-PD-L1/anti-PD-1 therapy might omit potential responders.
Lymph nodes are the initial and the most frequent sites of breast cancer metastasis [
12]; thus, lymph node metastasis (LNM) formation is a crucial step in breast cancer progression. Half of the primary TNBC exhibit lymph node involvement, and these patients have poorer prognoses than patients without lymph node involvement [
13]. In terms of the important role of the PD-1/PD-L1 axis in immune system evasion [
14], we hypothesized that PD-L1 expression would be more frequent and stronger in LNMs than in PTs.
Here, we aimed to elucidate the differences in PD-L1 expression between PTs and paired LNMs by examining the PD-L1 statuses of 101 node-positive TNBC patients’ PTs and synchronous axillary LNMs. In addition, we assessed the association between PD-L1 expression and the clinicopathological features as well as the prognosis of node-positive TNBC patients.
Discussion
This study revealed differences in PD-L1 expression between LNMs and paired PTs in both tumor cells and infiltrating immune cells or nodal lymphocytes in node-positive TNBC. Furthermore, the presence of PD-L1-positive tumor cells was significantly associated with a high score of TIL. PD-L1 expression was also associated with worse DFS, and the PT-/LNM+ and PT+/LNM+ groups had similar DFS rates. The results of this study suggest that testing of only PT specimens might result in exclusion of a potentially responsive subgroup of PT-/LNM+ patients from receiving anti-PD-L1/anti-PD-1 therapy.
PD-L1 expression in breast cancer has been frequently evaluated in recent studies, most of which have used tissue microarrays (TMAs) due to their large sample sizes and including a variety of breast cancer subtypes [
4,
20]. Considering the spatial heterogeneity [
9] of PD-L1 expression, we selected representative slides for evaluation by IHC, rather than using TMAs, and scored tumor cells and lymphocytes separately in PTs and paired LNMs. In our study, PD-L1 expression was detected in 38.61% (39/101) of the PTs of node-positive TNBC patients, with 31 (30.69%) exhibiting PD-L1-positive infiltrating lymphocytes, and 26 (25.74%) possessing positive tumor cells. PD-L1 expression was significantly associated with poorer survival. These results are relatively consistent with those of two recent studies showing that PD-L1 expression is a poor prognostic marker in breast cancer patients [
4,
20]. One of these studies specifically reported the detection of PD-L1 expression in 59% of all TNBC cells; this rate was higher than that observed in the current study. However, another large-scale study using TMAs has shown that PD-L1 is expressed in 19% of basal-like tumors in association with improved disease-specific survival [
16]. Other methods have also been used to evaluate PD-L1 expression. For example, one study measured PD-L1 expression using DNA microarrays, which revealed positive expression in 38% of basal tumors [
5]. Another study using in situ mRNA hybridization coupled with TMAs detected PD-L1 mRNA expression in nearly 60% of breast cancer cells [
21]. These two studies both demonstrated that PD-L1 expression is a good prognostic indicator. To date, however, no standardized assays have been developed for evaluation of tumor PD-L1 expression, as there are no specific anti-PD-L1 monoclonal antibodies available for use in IHC, no set criterion for a PD-L1 “positive” tumor, and no standard methods.
Differences in the levels of molecular markers, including ER, PR, HER2 [
22] and epithelial-to-mesenchymal transition-related markers [
23], between primary breast cancers and both lymph nodes and distant metastases have been frequently demonstrated in previous studies. These results suggest that making treatment decisions solely based on the expression of these molecular markers in PTs may result in the inappropriate use of hormone and targeted therapies in cancer patients. The heterogeneity of PD-L1 status has also been reported in clear cell renal cell carcinoma [
24] and bladder cancer [
25]. Our findings demonstrated that the paired LNMs (59.41%) more commonly and strongly exhibited PD-L1 expression than the PTs (38.61%) did, with 20.79% of the node-positive TNBC patients demonstrating negative-to-positive conversion of their PD-L1 status. Moreover, our results revealed that PT-/LNM+ patients showed worse DFS than the PT-/LNM- group and showed similar DFS with the PT+/LNM+ group. Thus, PD-L1 negativity in a PT may be not sufficient to exclude a node-positive TNBC patient from receiving anti-PD-L1 therapy. We postulate that measurement of PD-L1 expression in LNMs could improve the selection of patients for treatment by identifying an increased number of potential responders.
The discordance detected in PD-L1 expression between PTs and paired LNMs reflects the dynamic nature of this protein. Many hypotheses could explain this expression difference. First, many studies have demonstrated that PD-L1 expression is upregulated in tumor cells stimulated by inflammatory cytokines, and particularly interferons (IFNs) produced by infiltrating immune cells [
1,
26]. In addition, one study has indicated that basal-like breast cancer cells have the capacity to evade the immune system via upregulation of PD-1 ligands adapted to IFN-c, which is secreted by T helper cells [
10]. Thus, the enriched infiltrating T cells in lymph nodes may drive PD-L1 expression to induce adaptive immune resistance during infiltration of tumor cells [
27]. Second, loss of PTEN expression is a mechanism that could regulate PD-L1 expression in TNBC patients [
19], as has previously been described in glioma patients [
28]. Clonal selection may be an additional mechanism that promotes discordance in PD-L1 expression between PTs and LNMs [
29].
Further studies using a larger cohort of patients are warranted to confirm the differences in PD-L1 expression between PTs and LNMs in node-positive TNBC patients. Factors associated with the induction of local PD-L1 expression and conversion in LNMs should also be identified. Furthermore, tumor infiltrating immune cells and nodal lymphocyte subsets within tumor microenvironments in PTs and LNMs should be analyzed.
Acknowledgments
We thank Lei Dong, and Weige Wang for their excellent technical assistance.