Expression of PD-L1 in triple-negative breast cancer based on different immunohistochemical antibodies

This study sought to evaluate the immunohistochemical expression of PD-L1 using several
monoclonal antibodies in TNBC. We performed IHC under the same conditions with different
antibody clones and found differences in PD-L1 expression in both tumor cells and
immune cells (Table 7).

Table 7. Multivariate analysis of triple negative breast cancer survival

The high concordance rate among the three readers in the interpretation of PD-L1 yielded
acceptable inter-reader reproducibility with a kappa value 0.610 in both cancer cells
and immune cells. Especially in cancer cells, the concordance rate was highest at
a 1 % cut-off value and lowest at a 10 % cut-off value. This is likely because the
reader tends to interpret as positive in 1 % cut-off even in slight expression and
increase concordance rate, and tends to have more likely subjective interpretation
in 10 % cut-off value. However, improvement in the response rate from 35 % at a 1 %
cut-off value to 44 % at a 5 % cut-off value was seen in a nivolumab phase I study
targeting malignant melanoma, indicating that the higher 5 % cut-off value was more
reasonable 38]. Therefore, further response-based studies of TNBC are necessary.

Variation in PD-L1 expression rates previously observed in a study of various cancers
has been attributed to differences in cut-off values, antibodies, and study populations
39], 40], and this phenomenon is also commonly seen with other antibodies 41]. In lung cancer characterized with PD-L1 expression, expression rates varied from
24 to 60 % using the same 5 % cut-off value 15], 16], 42], and from 21 to 95 % according to different cut-off values of 1, 10 and 50 % 13], 17], 18], 43].

PD-L1 expression in TNBC was present at 19 % (clone 5H1 with a 5 % cut-off) 44] and 64–80 % (clone E1L3N, with a 1 % cut-off) 45] in previous studies. In our study, the expression rate was 11.5–14.7 % with a 1 %
cut-off value and 11.0–14.7 % with a 5 % cut-off value, which was similar to the previous
report of 19 %. We presume that differences in expression rate are due primarily to
different characteristics of the PD-L1 antibody clones, because all samples were stained
using the same immunohistochemical conditions.

PD-L1 showed membranous expression in this study, in agreement with one previous study
in TNBC 44], but the other TNBC study reported both cytoplasmic and membranous expression of
PD-L1 45]. PD-L1 expression has been reported in the cell membrane 17], 26]–28] or membrane and cytoplasm 13], 16], 18] in other cancers. We performed IHC using an automatic IHC staining device, but the
previous research in TNBC that evaluated cytoplasm expression had no clear description
of staining, making it difficult to compare our findings directly with previous data
45]. We found expression of PD-L1 in 19.3–37.6 % of cancer cells and immune cells, depending
on the antibody clone. Although our expression rate was different than a previous
study with 93 % immune cell expression in TNBC, it was in concordance with the previous
report in terms of positive immune cell expression 45]. PD-L1 expression in immune cells has been reported in other cancers 16], 19], 25], 28]. There are differences between oncogene-driven PD-L1 expression and inflammation-driven
PD-L1 expression. While oncogene-driven PD-L1 expression is constitutive and diffuse,
inflammation-driven PD-L1 expression is limited to sites of IFN?-mediated immunologic
attack 46]. In addition, inflammation-driven PD-L1 expression is related to immune infiltrates,
while oncogene-driven PD-L1 expression is not 28], 47]. Thus, further study to know which expression between two expressions is more related
to the expression of PD-L1 on immune cell in TNBC.

In this study, the concordance rate between PD-L1 (28-8) and PD-L1 (E1L3N) was high
in both cancer cells and immune cells while PD-L1 (SP142) showed low concordance rates
with the other two antibodies. Previous studies reported poor concordance (kappa value:
0.124–0.340) between PD-L1 (E1L3N) and PD-L1 (SP142) in lung cancer 48], but a high concordance rate between PD-L1 (E1L3N) and PD-L1 (SP142) (more than 85 %)
in malignant melanoma 49]. Therefore, concordance seems to vary according to cancer type; further study on
this topic is needed.

PD-L1 positivity in immune cells (28-8 clone) was an independent prognostic factor
in our study, in contrast to previous studies showing good prognosis 50]–53] or poor prognosis 54], 55] with PD-L1 expression in cancer cells. Since the prognostic and predictive significance
of PD-L1 expression in immune cells such as macrophages and lymphocytes was not associated
with tumor-related PD-L1 expression 16], further study of the biologic implications of PD-L1 in immune cells during TNBC
is needed.

In breast cancer, tumor infiltrating lymphocytes (TILs) have been linked with good
prognosis 56], as have stromal TIL 57]. Therefore, tumor immunity may relate to prognosis in TNBC. PD-L1 expression was
significantly higher in inflammatory type cancer cells and immune cells with all tested
antibodies. Therefore, we assume that PD-L1 expression in stromal immune cells is
related to prognosis, and further research is needed to better elucidate this relationship.

Clinical trials targeting PD-L1 are underway 17], 58]–60] and good responses have been reported 61], 62]. In TNBC, which has no effective therapy, PD-L1-targeting agents may play an important
role and strong biomarkers that can predict treatment response are needed. Accordingly,
staining of PD-L1 monoclonal antibodies may act as a biomarker for PD-L1-targeting
agents, but clinical trials evaluating TNBC response to PD-L1-targeting agents using
monoclonal antibody staining will be needed to validate this strategy.

The limitation of this study was the potential difference in results between TMA and
the whole cancer tissue section. Breast cancer also shows intracancer heterogeneity,
like other types of cancer, and the expression of PD-L1 may cause differences between
TMA and the whole cancer tissue section. PD-L1 expression in lung cancer showed a
high discordance rate between TMA samples and whole tissue sections 48]. In a previous study, the results of immunohistochemical staining of ER in breast
cancer were different in 5.5 % between TMA and the whole cancer tissue section. Furthermore,
when the number of cores was greater than one, this difference decreased to 1.4 %
63]. In this study, two core extractions per case were performed to reduce this bias.