cancers

Review
Tumor Infiltrating Lymphocytes (TILS) and PD-L1 Expression in
Breast Cancer: A Review of Current Evidence and Prognostic
Implications from Pathologist’s Perspective
Giuseppe Angelico 1, * , Giuseppe Broggi 1 , Giordana Tinnirello 1 , Lidia Puzzo 1 , Giada Maria Vecchio 1 ,
Lucia Salvatorelli 1 , Lorenzo Memeo 2 , Angela Santoro 3 , Jessica Farina 1 , Antonino Mulé 3 , Gaetano Magro 1
and Rosario Caltabiano 1

1 Department of Medical, Surgical Sciences and Advanced Technologies G.F. Ingrassia, Anatomic Pathology,
University of Catania, 95123 Catania, Italy; [email protected] (G.B.);
[email protected] (G.T.); [email protected] (L.P.); [email protected] (G.M.V.);
[email protected] (L.S.); [email protected] (J.F.); [email protected] (G.M.);
[email protected] (R.C.)
2 Department of Experimental Oncology, Mediterranean Institute of Oncology, 95029 Catania, Italy;
[email protected]
3 Pathology Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy;
[email protected] (A.S.); [email protected] (A.M.)
* Correspondence: [email protected]; Tel.: +39-09-5378-2030

Simple Summary: The aim of our study is to provide a wide perspective on the available literature
data on the immune landscape of breast cancers, focusing on TILs and PD-L1 expression across differ-
ent breast cancer subtypes. Moreover, treatment options such as immunotherapy and chemotherapy
in adjuvant and neoadjuvant settings are discussed, along with the most relevant cut-offs and scores
Citation: Angelico, G.; Broggi, G.; for TILs and PD-L1 pathological assessment.
Tinnirello, G.; Puzzo, L.; Vecchio,
G.M.; Salvatorelli, L.; Memeo, L.; Abstract: With the rise of novel immunotherapies able to stimulate the antitumor immune response,
Santoro, A.; Farina, J.; Mulé, A.; et al.
increasing literature concerning the immunogenicity of breast cancer has been published in recent
Tumor Infiltrating Lymphocytes
years. Numerous clinical studies have been conducted in order to identify novel biomarkers that
(TILS) and PD-L1 Expression in
could reflect the immunogenicity of BC and predict response to immunotherapy. In this regard, TILs
Breast Cancer: A Review of Current
Evidence and Prognostic
have emerged as an important immunological biomarker related to the antitumor immune response
Implications from Pathologist’s in BC. TILs are more frequently observed in triple-negative breast cancer and HER2+ subtypes,
Perspective. Cancers 2023, 15, 4479. where increased TIL levels have been linked to a better response to neoadjuvant chemotherapy and
https://doi.org/10.3390/ improved survival. PD-L1 is a type 1 transmembrane protein ligand expressed on T lymphocytes, B
cancers15184479 lymphocytes, and antigen-presenting cells and is considered a key inhibitory checkpoint involved
in cancer immune regulation. PD-L1 immunohistochemical expression in breast cancer is observed
Academic Editors: Fabio Puglisi and
Hamid Band
in about 10–30% of cases and is extremely variable based on tumor stage and molecular subtypes.
Briefly, TNBC shows the highest percentage of PD-L1 positivity, followed by HER2+ tumors. On
Received: 5 June 2023 the other hand, PD-L1 is rarely expressed (0–10% of cases) in hormone-receptor-positive BC. The
Revised: 5 September 2023
prognostic role of PD-L1 expression in BC is still controversial since different immunohistochemistry
Accepted: 6 September 2023
(IHC) clones, cut-off points, and scoring systems have been utilized across published studies. In
Published: 8 September 2023
the present paper, an extensive review of the current knowledge of the immune landscape of BC is
provided. TILS and PD-L1 expression across different BC subtypes are discussed, providing a guide
for their pathological assessment and reporting.
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland. Keywords: breast cancer; TILs; PD-L1; immunotherapy; chemotherapy; triple-negative breast cancer;
This article is an open access article HER2; luminal breast cancer; CPS
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Cancers 2023, 15, 4479. https://doi.org/10.3390/cancers15184479 https://www.mdpi.com/journal/cancers

Cancers 2023, 15, 4479 2 of 14

1. Introduction
Breast cancer (BC) exhibits a wide morphological and molecular spectrum of neo-
plasms with different clinical behavior, prognosis, and response to treatments [1].
On the basis of gene expression patterns, the following molecular subtypes of BC
have been identified: (i) luminal-like tumors (the most common subtype of BCs, showing
high genetic expression of the estrogen receptor (ER) and lacking significant expression
of Erb-B2); (ii) HER2-enriched tumors (characterized by overexpression of Erb-B2 and
showing low genetic expression of ER); (iii) basal-like tumors (characterized by expression
of genes found in the basal cells of the terminal duct lobular units) [2]. However, the most
widely accepted classification of BC is based on the immunohistochemical expression of
ER, PR, HER-2, and KI-67, which represent reliable surrogate markers for the molecular
classification [2,3]. On this basis, the following subtypes of BC can be identified: (i) luminal
A tumors: characterized by high expression of ER and PR, HER2 negativity, and low
expression of Ki-67 (less than 20%). This BC subtype shows the best prognosis and a high
response to hormone therapy.
(ii) Luminal B tumors: characterized by high expression of ER, low expression/negativity
of PR, and high Ki67 (greater than 20%). This subtype shows a worse prognosis compared
with Luminal A and may benefit from chemotherapy.
(iii) Triple-negative tumors: characterized by negative immunohistochemical staining
for ER, PR, and HER2 and high Ki67. TNBC represents the most aggressive BC subtype,
usually presenting in advanced stages and characterized by early relapse and distant
metastases. By immunohistochemistry, tumors expressing basal-cell markers such as
cytokeratins 5/6, CK17, and CK14 are categorized as basal-like [2,3].
With the rise of novel immunotherapies able to stimulate the antitumor immune
response, increasing literature concerning the immunogenicity of BC has been published
in recent years [4–7]. Recent RNA sequencing studies demonstrated the existence of
three transcriptome-based subtypes of BC corresponding to different immune categories:
immune high, medium, and low [8].
Immune-high tumors are characterized by the highest expression of tumor-infiltrating
lymphocytes (TILS) as well as PDL1 [8]. Triple-negative breast cancer (TNBC) and HER2+
tumors are frequently included in the immune-high category and represent potential re-
sponders to immunotherapies [8]. On the other hand, immune medium and immune low
tumors show little to no immune cell infiltration and are unresponsive to immunother-
apies [8]. Interestingly, estrogen receptor (ER) and progesterone receptor (PR)-positive
tumors usually fall into the immune medium and low groups [9]. In recent years, numerous
clinical studies have been conducted in order to identify novel biomarkers that could reflect
the immunogenicity of BC and predict response to immunotherapy [10]. In this regard, TILs
have emerged as an important immunological biomarker related to the antitumor immune
response in BC [4,5]. TILs are more frequently observed in triple-negative breast cancer
and HER2+ subtypes, where increased TIL levels have been linked to a better response to
neoadjuvant chemotherapy and improved survival [4,5]. PD-L1 is a type 1 transmembrane
protein ligand expressed on T lymphocytes, B lymphocytes, and antigen-presenting cells
and is considered a key inhibitory checkpoint involved in cancer immune regulation [6].
The PD-1/PD-L1 pathway plays a crucial immunoregulatory role by suppressing the im-
mune system in both physiological and pathological conditions, including cancer. PD-L1
ligand specifically binds to the PD-1 receptor expressed on T-lymphocytes, leading to a
decrease in the immune response [6–10].
PD-L1 ligand is expressed by several inflammatory cells, including activated T-
lymphocytes, B-lymphocytes, macrophages, and dendritic cells [6–10]. Moreover, tumor
cells can also express PD-L1 as an “adaptive immune mechanism” to remain undetected
by the immune system [6–10]. Additionally, PD-L1 exerts pro-tumorigenic activity by
promoting tumor growth and survival [6–10].
PD-L1 immunohistochemical expression in breast cancer is observed in about 10–30%
of cases and is extremely variable based on tumor stage and molecular subtypes [11]. Briefly,
 TNBC shows the highest percentage of PD-L1 positivity, followed by HER2+ tumors [11].
On the other hand, PD-L1 is rarely expressed in hormone-receptor-positive BC [11,12].
Cancers 2023, 15, 4479 The prognostic role of PD-L1 expression in BC is still controversial since different 3 of 14

immunohistochemistry (IHC) clones, cut-off points, and scoring systems have been uti-
lized across published studies [12,13].
TNBCThe showsprognostic
the highestrole percentage
of PD-L1 expression
of PD-L1 in BC was demonstrated
positivity, followed by HER2+ for thetumors
first time by
[11].
On the other
Muenst et al.hand,
in 2015 PD-L1
[14]. isInrarely expressed
this study, in hormone-receptor-positive
positive staining (both membranousBC and [11,12].
cytoplas-
mic)The for prognostic
PD-L1 was role observedof PD-L1
in 152 expression
out of 650inBC BCpatients,
is still controversial
and a significant sincecorrelation
different
immunohistochemistry
was observed between(IHC) PDL-1clones, cut-off
positivity and points,
several and scoring systems have
clinicopathological been utilized
parameters (large
across published studies [12,13].
tumor size, lymph node involvement, tumor grade, ER negativity, HER2-positive tumors,
andThe highprognostic
Ki67 index) role of PD-L1 expression in BC was demonstrated for the first time by
[14].
Muenst et al. in
Despite 2015
this [14]. In this
association with study,
worse positive staining (both
clinico-pathologic membranous
features, severaland cytoplas-
studies high-
mic)
lightedfor that
PD-L1 PD-L1was expression
observed incould 152 out of 650
predict BC patients,
a better response andto achemotherapy
significant correlation
and a bet-
was observed between
ter prognosis, mainly in PDL-1 positivity
the TNBC and [11,13].
subtype several Moreover,
clinicopathological
a significantparameters
correlation (large
be-
tumor size, lymph node involvement, tumor grade,
tween PD-L1 positivity and TIL scores has been documented [11,13]. ER negativity, HER2-positive tumors,
and high Ki67
In the index)paper,
present [14]. an extensive review of the current knowledge of the immune
Despite this association
landscape of BC is provided. with worse clinico-pathologic
Moreover, TILS and PD-L1 expressionfeatures, severalacrossstudies
different high-
BC
lighted that PD-L1 expression could predict a better response
subtypes are discussed, providing a guide for their pathological assessment and report-to chemotherapy and a better
prognosis,
ing. mainly in the TNBC subtype [11,13]. Moreover, a significant correlation between
PD-L1 positivity and TIL scores has been documented [11,13].
In the present paper,
2. Tumor-Infiltrating an extensive
Lymphocytes in review of the current knowledge of the immune
Breast Cancer
landscape of BC is provided. Moreover, TILS and PD-L1 expression across different BC
Increasing scientific evidence suggests the prognostic and predictive role of TILs in
subtypes are discussed, providing a guide for their pathological assessment and reporting.
breast cancer [15,16]. In this regard, the presence of TILs within a tumor is strictly related
2.toTumor-Infiltrating
the anti-tumor host immune response
Lymphocytes in Breast[15,16].
Cancer
TILs are constituted by all mononuclear cells (lymphocytes, plasma cells, monocytes,
Increasing scientific evidence suggests the prognostic and predictive role of TILs in
and NK-T cells) dispersed in the tumor stroma (stromal TILs) or located within the tumor
breast cancer [15,16]. In this regard, the presence of TILs within a tumor is strictly related
(intratumoral TILs) [15–17].
to the anti-tumor host immune response [15,16].
Based
TILs areon their phenotype,
constituted TILs can be classified
by all mononuclear as CD8+ T cells,
cells (lymphocytes, plasma CD8+ tissue-resident
cells, monocytes,
and NK-T cells) dispersed in the tumor stroma (stromal TILs) or located within thehelper
memory T cells, CD4+ T helper cells, CD4+ regulatory T cells, CD4+ follicular tumorT
cells, and tumor-infiltrating
(intratumoral TILs) [15–17]. B cells. However, the specific role and clinical significance of
eachBased
TIL subpopulation are still uncertain [15–18].
on their phenotype, TILs can be classified as CD8+ T cells, CD8+ tissue-resident
memory T cells, to
According CD4+the recommendations
T helper cells, CD4+ for assessment
regulatory Tofcells,TILs CD4+
in breast cancer proposed
follicular helper T
by the “International Working Group for TILs in Breast Cancer”,
cells, and tumor-infiltrating B cells. However, the specific role and clinical significance the pathological assess-of
ment of TILs should include only
each TIL subpopulation are still uncertain [15–18]. stromal TILs [19]. TILs evaluation is performed on he-
matoxylin
According andtoeosin (H&E)-stained sections
the recommendations by evaluating
for assessment of TILs inthebreast
ratiocancer
between the intra-
proposed by
tumoral stromal area containing lymphocytes and plasma cells
the “International Working Group for TILs in Breast Cancer”, the pathological assessment and the total intratumoral
ofstromal area [19].
TILs should According
include to theTILs
only stromal percentages
[19]. TILs ofevaluation
stromal TILs, three different
is performed groups can
on hematoxylin
be identified:
and low TILs (0–10%
eosin (H&E)-stained sectionsimmune cells inthe
by evaluating stromal tissue within
ratio between the tumor), interme-
the intratumoral stromal
diate
area TILs (11–40%),
containing and high
lymphocytes andTILs (>40%)
plasma (Figure
cells and the 1).total
[19] intratumoral stromal area [19].
AccordingFollowing
to the the above-mentioned
percentages of stromal recommendations,
TILs, three differentthe prognostic
groups and predictive
can be identified: low
role of TILs has been investigated by several studies and clinical
TILs (0–10% immune cells in stromal tissue within the tumor), intermediate TILs (11–40%), trials, mainly in triple-
negative
and high TILsand HER-2-positive
(>40%) (Figure 1). breast
[19] cancer patients (Table 1) [17,18,20].

Figure 1. Hematoxylin and eosin (H&E)-stained sections illustrating breast carcinomas with different
TIL distributions in our series. (A) Invasive breast carcinoma with low TILs (0–10%) in stromal
(H&E, ×20). (B) Another case showing increased TILs in tumor stroma, categorized as intermediate
(11–40%) (H&E, ×20). (C) Invasive carcinoma of the breast showing high TILs (>40%) in tumor
stroma (H&E, ×20).
Cancers 2023, 15, 4479 4 of 14

Following the above-mentioned recommendations, the prognostic and predictive role

of TILs has been investigated by several studies and clinical trials, mainly in triple-negative
and HER-2-positive breast cancer patients (Table 1) [17,18,20].

2.1. TILs in Triple Negative Breast Cancer

TNBC is a breast cancer subtype lacking expression of ERs, HER2, and PRs and
characterized by a poor prognosis [21]. TNBC shows higher levels of TILs, a higher
tumor mutation burden (TMB), and high PD-L1 expression [21]. Increasing literature data
indicate that high levels of TILs in TNBC are significantly related to a better response to
chemotherapy as well as a better prognosis [22]. TNBC represents the most immunogenic
BC subtype [8]. The high immune cell infiltrate observed in these tumors is thought to
represent an adaptive mechanism of the immune system to prevent tumor growth and
metastatic spread [8,22]. Therefore, in TNBC patients, an increase in TILs levels, which
represent the anti-tumor host immune response against cancer cells, has been related to
improved response to chemotherapy and longer survival [8,22]. Moreover, chemotherapy
treatments may also boost the antitumor immune response [8,22].
Concerning the prognostic role of TILs in early TNBC, two studies, including 2148 patients
treated with adjuvant chemotherapy and 906 women treated with neoadjuvant chemotherapy,
respectively, demonstrated the clinical utility of TIL evaluation [4,23].
Briefly, high TILs have been demonstrated to predict responses to adjuvant and
neoadjuvant chemotherapy with anthracycline; moreover, each 10% increment in TILs was
significantly related to longer disease-free survival and overall survival [4,23]. On the other
hand, low-TILs are more frequently detected in patients with older ages, larger tumor sizes,
and lymph node metastases [4,23].
Based on these findings, the World Health Organization (WHO) classification of
tumors (5th edition) strongly suggests TIL assessment in TNBC and HER2+ subtypes as a
prognostic biomarker [24].
Several scientific studies concerning the predictive role of TILs for immune therapy or
combined immune therapy/chemotherapy in TNBC are emerging [19,21]. Results of previ-
ous studies in early-stage TNBC suggest that high TILs predict response to neoadjuvant
immune therapy alone or in combination with chemotherapy [19,21].
Similar predictive roles of TILs for immune checkpoint inhibitor therapy have also
emerged in advanced/metastatic TNBC [25].

2.2. TILs in HER2+ Breast Cancer

HER2-positive breast cancer accounts for approximately 15–20% of all breast carcino-
mas and is considered a biologically aggressive subtype [26,27]. Due to their low mutational
burden, HER2-positive BCs are generally considered “cold” tumors [27]. However, recent
studies have started to explore immunotherapeutic approaches to target HER2-positive
tumors both in neoadjuvant and adjuvant settings [20,28,29]. Regarding the latter, the
results of the FIN-HER study and ShortHER trial showed that high TIL levels were related
to longer overall survival and an improved response to trastuzumab compared with low
TILs [20,29]. However, in contrast to these data, Perez et al. reported that breast tumors
with high TILs showed a worse response when combining trastuzumab with chemother-
apy than those treated with chemotherapy alone [30]. Moreover, the analysis from the
N9831 trial demonstrated that high TILs predicted a lack of response to trastuzumab [31].
Therefore, given the conflicting results concerning the role of TILs in predicting response to
adjuvant chemotherapy, further studies on larger cohorts are still needed to understand
TILs biological role in HER-2 breast tumors.
TILs evaluation in a post-NAD setting has also been associated with breast cancer
prognosis [5,32,33]. According to the results of three recent meta-analyses, high TILs are
significantly related to a better response to neoadjuvant chemotherapy plus trastuzumab,
regardless of the type of neoadjuvant regimen [22,30,31]. Moreover, a statistically significant
correlation between high TILs and improved prognosis has also emerged [5,23,32].
Cancers 2023, 15, 4479 5 of 14

Concerning advanced/metastatic HER-2-positive breast tumors, the prognostic and

predictive role of TILs is still controversial. According to a retrospective analysis of the
patients enrolled in the CLEOPATRA trial, high TILs were associated with increased OS; on
the contrary, TIL count showed no significant prognostic or predictive value in the analysis
of the MA.31 phase 3 trial [33,34].
Lastly, controversial data from the metastatic setting have also emerged when evaluat-
ing the prognostic and predictive role of TILs in patients receiving immunotherapy [35,36].
Therefore, further studies are needed to investigate the interactions between the immune
system and tumor cells in HER2+ breast cancer.

2.3. TILs in Hormone-Receptor+/HER2− Breast Cancer

The prognostic and predictive role of TILs in hormone-receptor-positive (HR+) and
HER-2-negative breast cancer subtypes (luminal A and luminal B tumors) is still poorly
established.
In this regard, HR+/HER2− BC is associated with a low TIL count and a lower
TMB [13]. Moreover, ER expression has been related to decreased MHC class II expression
on lymphocytes, suppression of interferon-γ (IFN-γ) signaling, and decreased activity of
CD8+T-cells [15,37]. However, given the wide morphological and biological heterogeneity
of luminal A and luminal B tumors, several studies have tried to identify “immunogenic”
subgroups. Briefly, it has been shown that TIL positivity is more frequently detected in
luminal B subtypes [15,22].
Concerning the predictive and prognostic role of TILs in early HR+/HER2− BC,
conflicting results have been highlighted: a significant association between high TILs and a
worse prognosis has emerged in some studies, while other authors failed to demonstrate
the prognostic significance of TILs [22,37]. Moreover, TILs have been associated with a
poor response to aromatase inhibitor therapy in HR+/HER2− BC [22,37].
Regarding TIL subpopulations, limited literature data are still available; however, a
recent retrospective analysis of 563 patients with early HR+/HER2− BC documented that
high CD8+ sTILs were more frequently detected in patients with PIK3CA-mutated tumors
and that they were related to a higher risk of recurrence [38].
Based on these preliminary findings, routine assessment of TILs in HR+/HER2− BC
is still not recommended and cannot be considered a prognostic or predictive biomarker.

Table 1. Prognostic and predictive roles of TILs in different breast cancer subtypes.

Pathological
BC Subtypes TILS Levels Prognostic Role Predictive Role References
Assessment
Yes to adjuvant Bianchini, 2021 [21]
High
TNBC Yes and neoadjuvant Recommended Stanton, 2016 [22]
(>40%)
chemotherapy Denkert, 2018 [4]
Yes to neoadjuvant Nuciforo, 2017 [28]
High
HER2+ Yes chemotherapy + Recommended Loi, 2014 [20]
(>40%)
immunotherapy Dieci, 2019 [29]
El Bairi, 2021 [15]
Low/intermediate Not fully
HR+ Yes Not recommended Stanton, 2016 [22]
(0–10%/11–40%) established
Valenza, 2023 [37]
Abbreviations: BC—breast cancer; TNBC—triple-negative breast cancer; HR+—hormone receptor positive
breast cancer.

3. PD-L1 Pathway, General Considerations

PD-L1 is a type 1 transmembrane protein ligand generally expressed on T lymphocytes,
B lymphocytes, and antigen-presenting cells; it is involved in the immune regulation of sev-
eral physiological and pathological conditions, including pregnancy, antigen presentation
to T lymphocytes, tissue and organ transplants, infectious diseases, and cancer [6].
 Cancers 2023, 15, 4479 6 of 14

R PEER REVIEW Following the binding of PD-L1 with the PD-1 or B7.1 (CD80) receptors, a suppressive 8 o
signal is transmitted to T lymphocytes, leading to a decrease in the immune response
(Figure 2) [6,11,12].

Figure 2. PD-L1-mediated immune escape.

Figure 2. PD-L1-mediated immune escape.

Moreover, the intracellular signals transmitted by PD-L1 promote neoplastic cell

Tumor-infiltrating lymphocytes,
proliferation recruited
and inhibit pro-apoptotic by tumor
signals mediated antigens,
by interferons (Figurerelease
2) [6,11,12].cytoki
In the last few years, several immunotherapeutic molecules capable of inhibiting the
including IFN-γ, which increases the expression of PD-L1. Following the binding of
PD-1/PD-L1 axis have been shown to improve the immunological response by inducing T
L1 to the PD-1 receptor, a suppressive
cells to recognize signal
and suppress cancer cellsis transmitted to T-cells; moreover, an a
[13].
Based on these findings, several monoclonal antibodies targeting the PD-1 receptor and
apoptotic signal is transmitted to tumor cells, leading to tumor survival and T
PD-L1, so-called immune checkpoint inhibitors, have been successfully utilized in clinical
dysfunction. practice; these include Pembrolizumab and Nivolumab (targeting the PD-1 receptor),
Atezolizumab, Avelumab, and Durvalumab (inhibiting the PD-L1 ligand) [13].
In clinical practice, the immunohistochemical evaluation of PD-L1 expression in
neoplastic tissues is the gold standard method for selecting patients eligible for immune
checkpoint inhibitor therapy. In this regard, several immunohistochemical assays have
been developed for PD-L1 evaluation. The most common PD-L1 assays used in clinical
trials include SP142, 28-8, 22C3, and SP263, each of which has been validated with specific
platforms [12,13,39].
Accordingly, different immunohistochemical scoring systems have been proposed for
quantifying PD-L1 expression in different neoplastic tissues: (i) tumor proportion score
(TPS), which evaluates the percentage of positive PD-L1 neoplastic cells among all viable
tumor cells; (ii) combined proportion score (CPS), namely the ratio between all PD-L1
positive neoplastic cells and inflammatory cells and the total number of viable tumor cells
multiplied by 100; (iii) immune cell score (IC), which takes into account the percentage
of the area occupied by PD-L1-positive immune cells relative to the whole tumor area
(Table 2) [12,13,39].
Cancers 2023, 15, 4479 7 of 14

Table 2. FDA-approved PD-L1 assays in clinical practice.

ICI PD-L1 Assay PD-L1 Score Setting Therapy References

Unresectable/metastatic Pembrolizumab plus
CPS ≥ 10 Cortes, 2022 [40]
TNBC chemotherapy
Pembrolizumab plus
22C3 chemotherapy
Pembrolizumab as neoadjuvant
(pharmDx)
CPS ≥ 1/regardless high-risk early-stage
treatment, and then Downs-Canner, 2023 [41]
of PDL1 status (NAD/AD)
continued as a single
agent as adjuvant
therapy
Unresectable/metastatic Atezolizumab plus
SP142 IC score ≥ 1 Emens, 2021 [42]
Atezolizumab TNBC nab-paclitaxel
(Ventana)
Regardless of IC NAD Atezolizumab Mittendorf, 2020 [43]
Abbreviations: ICI—immune checkpoint inhibitor; TNBC—triple-negative breast cancer; NAD—neoadjuvant
chemotherapy; AD—adjuvant chemotherapy; CPS—combined proportion score; IC—immune cell score.

Tumor-infiltrating lymphocytes, recruited by tumor antigens, release cytokines, in-

cluding IFN-γ, which increases the expression of PD-L1. Following the binding of PD-L1 to
the PD-1 receptor, a suppressive signal is transmitted to T-cells; moreover, an anti-apoptotic
signal is transmitted to tumor cells, leading to tumor survival and T-cell dysfunction.

Temporal and Spatial Heterogeneity of TILs and PD-L1 Expression during Metastatic Progression
Recent studies have demonstrated extensive discrepancies in TIL count and PD-L1
expression among primary tumors and their paired metastases [44]. Biomarker analyses
based on the clinical trial IMPassion130 documented a highly heterogeneous PD-L1 expres-
sion between primary tumors and their paired metastases [42]. Higher PD-L1 concordance
has been documented in synchronous tumor samples compared with metachronous ones;
additionally, a greater clinical benefit with immunotherapy was observed when PD-L1
status was evaluated in metastatic sites [11,45].
The temporal evolution of TILS has been reported by several studies, which docu-
mented lower TIL counts at metastatic biopsies compared with primary tumors [11,45].
A recent meta-analysis on this topic demonstrated a significant decrease in PD-L1
expression at metastatic sites [45]. This reported discordance of PD-L1 expression was
bi-directional since 50% of patients with PD-L1 positive primary tumors showed PD-
L1 negative metastatic biopsies; conversely, about 30% of patients with PD-L1 negative
primary tumors were PD-L1 positive in their metastatic biopsies [45].
However, to date, little is known about the optimal metastatic site for PD-L1 evaluation
since the few available studies demonstrated a significant difference in PD-L1 expression
between different metastatic sites from the same patient [11,42,45]. Further studies on
this topic are needed to clarify this reported discordance; however, based on the current
literature evidence, PD-L1 assessment in metastatic bioptic samples should be assessed
when possible to achieve a more accurate treatment strategy.

4. PD-L1 and Immunotherapy in Breast Cancer Subtypes

Immune checkpoint inhibitors (ICI) have recently emerged as a novel immunother-
apeutic approach capable of improving the anti-cancer immune response by targeting
specific immunologic receptors on the surface of T-lymphocytes [6,7]. These latter recep-
tors are immunological checkpoints composed of inhibitory and stimulatory pathways
that maintain a balance between pro-inflammatory and anti-inflammatory signals [6,7,10].
Therefore, ICIs have been specifically developed to target the most relevant immune in-
hibitory receptors, including CTLA-4, PD-1, and PD-L1 [6,7,10]. Other antibodies are still in
clinical development to target additional immune checkpoints such as B7H3, CD39, CD73,
the adenosine A2A receptor, CD47, LAG-3, and TIM-3 [6,7,10].
Cancers 2023, 15, 4479 8 of 14

To date, the major clinical benefits of ICIs in breast cancer are restricted to the inhibition
of the PD1/PD-L1 pathway [6,7,10]. The combination of PD1/PD-L1 blockade with CTLA-4
inhibition has been investigated in a single-arm pilot study where a response rate of 43% was
observed in patients with metastatic TNBC; on the other hand, patients with HR+ breast cancer
showed no significant responses [46]. With the increasing use of ICIs in BC treatment, several
adverse events related to the enhanced immune response induced by these therapies [47].
Adverse events related to ICIs are the result of an autoimmune response that can affect any
organ of the body [47]. The most common manifestations include dermatitis, diarrhea, colitis,
and endocrine dysfunctions mainly affecting the thyroid, hypophysis, and adrenal glands [47].
Pneumonitis and myocarditis represent rare but potentially fatal adverse events [47]. Moreover,
combination therapies (ICIs plus chemotherapy; dual immunotherapy) are associated with a
higher incidence of adverse events [47].

4.1. Hormone-Receptor Positive/HER2 Negative Breast Cancer

In the HR-positive and HER2-negative BC subgroups, PD-L1 immunoreactivity is
documented in up to 9% of luminal A and 42% of luminal B subtypes [48]. A significant
decrease in PD-L1 expression is observed in metastatic tumors, among which 0–1% and
10–12% positive rates are reported in luminal A and luminal B patients, respectively [48].
Given the wide heterogeneity of PD-L1 and TILs in this BC subgroup, only a few studies
have considered an immunotherapeutic approach in patients with PD-L1+/ER+/HER2−
BC [49]. Promising data have been highlighted by the KEYNOTE-028 and I-SPY2 trials,
in which an improved pathological complete response was observed in PD-L1-positive
tumors [49,50]. However, results are still preliminary, and significant clinical benefits of
immunotherapy have not been reported yet.
Despite the fact that invasive lobular breast carcinoma (ILC) represents the second
most common BC subtype, only a few data points are currently available on the immune
landscape of this tumor. Interestingly, in the KEYNOTE-028 trial, two patients with PD-L1-
positive and ER-positive BC showed a pathological response to pembrolizumab [51].
Moreover, studies based on transcriptomic profiling demonstrated the existence of an
immune-related ILC subtype characterized by a high immune infiltrate and the expression
of immune-related genes [52]. In this regard, the GELATO trial represents the first clinical
trial to investigate the benefits of immune checkpoint inhibitors in patients with ILC [53].
In this study, four partial responses and two stable diseases were observed in patients
with metastatic ILC treated with carboplatin and atezolizumab [53]. However, most
responders (4/6 patients) had triple-negative ILC; therefore, the antitumor activity of
immune checkpoint inhibitors, especially in patients with HR+ ILC, is still a matter of
debate [53]. Additional trials specifically designed for ILC patients are needed to establish
the role of immunotherapy in this BC subtype.

4.2. HER2 Positive Breast Cancer

In early-stage HER2+ BC tumors, PD-L1 immunoreactivity is documented in around
30% of cases, whereas a significant decrease in PD-L1 expression (9–10%) is observed in
metastatic tumors [54,55].
However, the prognostic significance of PD-L1 in HER2+ BC is still controversial since
some studies documented poor outcomes in metastatic HER2+/PD-L1+tumors, whereas
other authors documented improved survival in patients with high levels of PD-1/PD-L1
expression [13,55].
Some studies are currently investigating the clinical benefits of immunotherapy with
anti-PD-1/PD-L1 antibodies combined with trastuzumab. In this regard, preliminary
results from the phase II randomized KATE2 trial indicate an improved OS in PD-L1-
positive patients affected by locally advanced or metastatic BC treated with the combination
of atezolizumab and ado-trastuzumab emtansine (T-DM1) [56]. Moreover, the phase Ib/II
PANACEA trial documented a 15% response rate in HER2+ advanced BC patients treated
with the combination of pembrolizumab and trastuzumab [36].
Cancers 2023, 15, 4479 9 of 14

4.3. Triple-Negative Breast Cancer

TNBC encompasses a wide morphological and molecular spectrum of neoplasms, as
recent RNA sequencing studies demonstrated the existence of several transcriptome-based
subtypes of TNBC (luminal androgen receptor, immunomodulatory, basal-like immune-
suppressed, and mesenchymal-like) [8]. In this regard, only the immunomodulatory
subtype would benefit from immunotherapy.
In this scenario, PD-L1 expression in tumor and immune cells, evaluated by immuno-
histochemistry, has become a crucial step for selecting potential responders to immunother-
apy (Table 3) [12].

Table 3. Therapeutic approach in advanced TNBC based on PD-L1 IHC.

FDA-Approved
PD-L1 Status Therapy Relevant Clinical Trials
PD-L1 Scores
CPS < 10
(22C3)
PD-L1-negative No Immunotherapy
IC < 1
(SP142)
CPS < 10 Atezolizumab plus
PD-L1-positive IMpassion130 [42]
IC score ≥ 1% Nab-paclitaxel
Pembrolizumab/Atezolizumab
Keynote-355 [40]
CPS ≥ 10 plus chemotherapy
PD-L1-positive Keynote-522 [41]
IC score ≥ 1% (Nab.paclitaxel or
IMpassion130 [42]
Carbo/Gem or paclitaxel)
Pembrolizumab plus
CPS ≥ 10 chemotherapy Keynote-355 [40]
PD-L1-positive
IC score < 1% (Nab.paclitaxel or Keynote-522 [41]
Carbo/Gem or paclitaxel)
Abbreviations: CPS—combined proportion score; IC—immune cell score.

In early-stage TNBC tumors, PD-L1 immunoreactivity is documented in around 45 to

55% of cases; metastatic patients show higher PD-L1 staining percentages (around 35% of
cases) compared with other BC subtypes [12]. Moreover, a significant increase in PD-L1
expression following immunotherapy has been documented in the PCD4989g trial [57].
As far as prognosis is concerned, several studies demonstrated better overall survival
in PD-L1-positive patients; moreover, BRCA1 gene mutations and expression of cytotoxic
T-lymphocyte antigen 4 (CTLA-4) are more frequently detected in PD-L1+ TNBC [58].

5. PD-L1 Assays and Immunohistochemical Scores: Results from Clinical Trials

Several PD-L1 IHC assays are currently approved for predicting the response to anti-
PD1 and anti-PD-L1 immunotherapy [12,13,39]. Validated PD-L1 clones for TNBC include
SP142, 28-8, SP263, and 22C3, each linked to different therapies [12,13,39].
In TNBC, clinical trials proposed several PD-L1 IHC clones, staining platforms, scoring
systems, and cut-offs [12,13,39]. Based on the results of the Keynote-355 trial, the PD-1
inhibitor Pembrolizumab, in combination with chemotherapy, has been approved by the
FDA for patients with unresectable/metastatic TNBC showing a PD-L1 CPS ≥ 10 [40].
Following the Keynote-522 trial results, neoadjuvant pembrolizumab in combination with
chemotherapy has been approved for high-risk early-stage TNBC as treatment; following
surgery, monotherapy with pembrolizumab is approved as adjuvant therapy [41]. In the
Keynote-522 trial, tumor samples were considered PD-L1-positive based on a CPS ≥ 1;
however, a better pathological complete response in patients treated with pembrolizumab
was observed regardless of the PD-L1 immunohistochemical expression [41].
Based on the results of the IMpassion130 trial, the Ventana PD-L1 SP142 test (IC score ≥ 1%)
has been approved by the FDA for the selection of advanced TNBC or mTNBC suitable for
combination therapy with Atezolizumab plus nab-paclitaxel [42]. Based on the same cohort, a
Cancers 2023, 15, 4479 10 of 14

post hoc harmonization study attempted to evaluate the inter-assay variability of three PD-L1
assays, namely the Ventana SP142 and SP263 and the Dako 22C3 [59]. IC score (≥1%) was
utilized for SP142 and SP263 assays, while CPS score (≥1) was evaluated for the 22C3 assay [59].
Interestingly, 22C3 and SP263 clones individuated a higher rate of PD-L1 positive patients (81%
and 75%, respectively) compared with the SP142 clone (46%) [59]. Despite the lower number of
PD-L1-positive tumors detected, patients that were only positive for the SP-142 assay showed
higher progression-free survival (4.2 months) compared with patients that were only positive
for the 22C3 assay or SP-263 clones [59].
In early TNBC, the Impassion031 trial demonstrated that the clinical benefits of neoad-
juvant atezolizumab were independent from the PD-L1 IC (SP142) status [43].

Relevant PD-L1 Scoring Methods

In TNBC, IC and CPS have been specifically validated for the selection of patients
eligible for atezolizumab and pembrolizumab therapies, respectively (Table 4) [12,13,39].

Table 4. PD-L1 scoring methods.

PD-L1 Scoring Methods Assay Scoring Method ICI

All PD-L1-positive immune cells
IC Ventana (SP142) located intratumorally or in a small Atezolizumab
peritumoral stromal rim
The number of PD-L1 positive tumor
cells and intratumoral immune cells
CPS DAKO (22C3) Pembrolizumab
divided by the total number of viable
tumor cells, multiplied by 100

Immune Cell Score.

The IC is specifically developed for the VENTANA PD-L1 (SP142) assay. It takes
into account all PD-L1-positive immune cells (lymphocytes, macrophages, granulocytes,
dendritic cells, and plasma cells) located intratumorally or in a small peritumoral stromal
rim [12,13,39]. Briefly, immune cells are scored as the percentage of the area occupied by all
PD-L1-positive immune cells relative to the whole tumor area [12,13,39]. A PD-L1 IC score
of ≥1% is considered adequate for atezolizumab therapy [12,13,39].
Combined Positive Score.
CPS is specifically developed for the 22C3 (pharmDx) assay [12,13,39]. CPS is the
number of PD-L1-positive tumor cells and intratumoral immune cells divided by the
total number of viable tumor cells, multiplied by 100 [12,13,39]. PD-L1 CPS score ≥ 10 is
considered adequate for pembrolizumab combination therapy in inoperable/metastatic
TNBC patients [12,13,39].

6. Limitations of PD-L1 IHC

To date, the main limitations of PD-L1 IHC testing are related to the presence of several
immunohistochemical assays for PD-L1 evaluation paired with different immunohisto-
chemical scoring systems. Despite several harmonization studies that attempted to evaluate
the inter-assay variability of different PD-L1 assays, the presence of several PD-L1 IHC
clones, staining platforms, scoring systems, and cut-offs has generated confusion among
pathologists and oncologists. Moreover, the temporal and spatial heterogeneity of TILs and
PD-L1 expression among primary tumors and their paired metastases may also account for
the suboptimal response rates to PD-L1 inhibitors.
Therefore, future research should focus on the interchangeability of the IHC assays in
order to provide definitive guidelines for PD-L1 assessment in breast cancer. The optimal
anatomical site for PD-L1 testing in metastatic tumors should also be clarified.
Cancers 2023, 15, 4479 11 of 14

7. Conclusions
Although immune checkpoint inhibitors have undoubtedly represented a major step
forward in the therapy of many malignancies, including breast cancer, it is widely accepted
that not all patients may undergo immunotherapy; accordingly, the accurate selection of
breast tumors that may benefit from these relatively novel therapeutic approaches is one of
the most debated topics of BC oncological research [60–62]. In this regard, the study of TILs
and the immunohistochemical evaluation of PD-L1 immunoreactivity among different BC
subtypes are actually considered the most reliable markers capable of predicting response
to immunotherapy. Although it has been shown that PD-L1-positive BCs frequently exhibit
high levels of TILs and a lack of expression of ER, PR, and HER2, and that patients affected
by TNBC are the ones who may benefit the most from immunotherapy, promising results
have been obtained in some trials also for patients with HR+ and/or HER2+ BC [63].
Accordingly, further studies are needed to validate these preliminary data, but it seems
likely that in the future, the use of immunotherapy could also be extended to the other
subtypes of BC.
This review also highlights that the predictive and prognostic role of TILs in BC is still
controversial and under investigation. In this regard, despite the correlation between TILs and
better prognosis/response to chemotherapy in TNBC, a worse outcome has been documented
in other BC subtypes exhibiting high TILS. Some studies indicated a worse prognosis and
poor response to aromatase inhibitors in HR+/HER2− BC; moreover, a worse response to
trastuzumab has been documented in HER2+ tumors exhibiting high TILs.

Author Contributions: Conceptualization: G.A. and R.C.; methodology, G.T. and J.F.; software, G.B.
and A.M.; validation, L.P., G.M.V. and L.S.; formal analysis, L.M.; investigation, A.S. and A.M.;
resources, G.M.; data curation, R.C.; writing—original draft preparation, G.A.; writing—review and
editing, G.T. and G.B.; visualization, L.P.; supervision, G.M.; project administration, G.M.V.; funding
acquisition, L.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data can be shared up on request.
Conflicts of Interest: The authors declare no conflict of interest.

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