Published OnlineFirst January 10, 2014; DOI: 10.1158/2326-6066.

CIR-13-0127

Cancer
Immunology
Research Article Research

PD-L1 Expression in Triple-Negative Breast Cancer

Elizabeth A. Mittendorf1, Anne V. Philips1, Funda Meric-Bernstam1, Na Qiao1, Yun Wu2, Susan Harrington9,
Xiaoping Su3, Ying Wang3, Ana M. Gonzalez-Angulo4, Argun Akcakanat1, Akhil Chawla1, Michael Curran5,
Patrick Hwu6, Padmanee Sharma7, Jennifer K. Litton4, Jeffrey J. Molldrem8, and Gheath Alatrash8

Abstract
Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown
clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-
negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The
Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene
in TNBC (n ¼ 120) compared with non-TNBC (n ¼ 716; P < 0.001). Breast tumor tissue microarrays were
evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1þ tumors had
greater CD8þ T-cell infiltrate than PD-L1— tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine
the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA
(shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts,
suggesting tran- scriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the
AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K
signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of
altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to
decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1
as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents
targeting the PI3K pathway may increase the antitumor adaptive immune responses. Cancer Immunol Res;
2(4); 361–70. ©2014 AACR.

Introduction therapeutic strategies are needed to improve the manage-

Triple-negative breast cancer (TNBC), which constitutes ment of patients with TNBC.
10% to 20% of all breast tumors, is characterized by a lack of There is significant heterogeneity within TNBC. A study
expression of estrogen receptor (ER), progesterone receptor analyzing gene expression profiles identified six TNBC sub-
(PR), and HER2/neu (HER2; refs. 1, 2). TNBCs are generally types, one of which was an immunomodulatory subtype
high-grade, aggressive tumors with a high rate of distant enriched for genes involved in immune cell processes
metastasis and poorer disease-specific survival than other including immune cell signaling, cytokine signaling, antigen
breast cancer subtypes (1, 3). The poor outcomes occur even processing and presentation, and signaling through core
though standard chemotherapy regimens have activity immune signal transduction pathways (2). In a meta-analysis
against these tumors. Studies evaluating chemotherapy in integrating published gene expression data with
the neoadjuvant setting have demonstrated that TNBC has clinicopathologic data, investigators developed gene
higher rates of pathologic complete response than other expression modules related to key biologic processes in breast
tumor types; however, there is a paradoxical shortening of cancer. For TNBC, only the immune response module was
progression-free and overall survival (4). Therefore, novel associated with clinical out- come (5). Loi and colleagues
recently reported a prognostic role of tumor-infiltrating
lymphocytes (TIL) in TNBC in a large prospective clinical
trial (6), and in a study looking specifically at CD8þ
Authors' Affiliations: Departments of 1Surgical Oncology, 2Pathology,
3
Bioinformatics and Computational Biology, 4Breast Medical
intratumoral lymphocytes, Liu and colleagues found that
Oncology, 5
Immunology, 6
Melanoma Medical Oncology, TNBC had higher rates of CD8 þ T-cell infiltration, which was
7
Genitourinary Medical Oncology, and 8Stem Cell Transplantation and an independent favorable prognostic factor (7). Taken together,
Cellular Therapy, The University of Texas MD Anderson Cancer these data suggest that immunotherapy may have a role in the
Center, Houston, Texas; and 9Department of Urology, The Mayo management of patients with TNBC.
Clinic, Rochester, Minnesota A promising approach to augmenting antitumor
Note: Supplementary data for this article are available at Cancer Immu- immunity is blockade of immune checkpoints. One example
nology Research Online (http://cancerimmunolres.aacrjournals.org/). is CTL- associated antigen 4 (CTLA-4), a T-cell inhibitory
Corresponding Author: Elizabeth A. Mittendorf, Department of Surgical
receptor that is expressed on activated CD8þ T cells.
Oncology, The University of Texas MD Anderson Cancer Center, 1400 CTLA-4 attenu- ates the T-cell immune response by
Pressler Street, Unit 1484, Houston, TX 77030. Phone: 713-792-2362; counteracting the activity of the T-cell costimulatory
Fax: 713-745-1462; E-mail:eamitten@mdanderson.org receptor CD28 (8, 9). Ipilimumab, a monoclonal antibody
doi: 10.1158/2326-6066.CIR-13-0127
targeting CTLA-4, has received approval from the U.S.
Food and Drug Administration for
©2014 American Association for Cancer Research.

361

Downloaded from cancerimmunolres.aacrjournals.org on February 1, 2020. © 2014 American Association for Cancer Research.
 Mittendorf et al.

the treatment of metastatic or unresectable melanoma. Pro- þ

immunohistochemistry (IHC) or 2 by IHC and negative for
grammed cell death protein 1 (PD-1) is a second immune
gene amplification by FISH. Fresh-frozen tumor samples used
checkpoint receptor that limits T-cell effector function within
tissues (10). PD-1 has two known ligands, PD-L1 and PD-L2, to isolate breast cancer cells by laser capture microdissection
which have distinct expression profiles with PD-L1 being (LCM) were obtained from Origene. Cultured breast cancer
cell lines were obtained from American Type Culture
expressed on several tumor types (11). In a small study of
Collection. Cell lines were validated by short tandem repeat
44 breast tumors, PD-L1 was shown to be expressed in 34%
(STR) DNA fingerprinting using the AmpF/STR Identifier Kit
of the tumors and the expression was associated with high-risk
clinicopathologic features, including high histologic grade according to the manufacturer's instructions (Applied
and hormone receptor–negative status (12). Recently repor- Biosystems). Cells were cultured in Dulbecco's Modified
Eagle Medium with 10% FBS, 100 U/mL penicillin, and 100
ted phase I studies evaluating antibodies targeting either PD-1
mg/mg streptomycin.
or PD-L1 have shown that these agents elicited durable,
objective responses in patients with melanoma, non–small-
cell lung cancer, and renal-cell carcinoma (13, 14). Immunohistochemistry
PD-L1 regulation is an area of active investigation. One One-millimeter cores from paraffin blocks of breast
mechanism of regulation is the induction of tumor cell surface tumors were used to generate TMAs. Before staining,
expression of PD-L1 in response to IFN-g (11). This is likely microarrays were baked overnight after which they were
a mechanism whereby tumor cells evade the antitumor deparaffinized and rehydrated. Nonspecific binding was
immune response of tumor-specific T cells. A second blocked and then the sections were incubated with primary
mechanism is through oncogenic signaling. Expression of PD- antibody. For PD-L1 staining, the primary antibody used
L1 on glioblas- tomas is increased by the deletion or silencing was 5H1, a mouse anti- human PD-L1 monoclonal antibody
of PTEN, implicating involvement of the phosphoinositide 3- previously reported by Dong and colleagues for human
kinase (PI3K) pathway (15). The PTEN/PI3K pathway is tumor staining (19, 20). The specificity of this antibody for
important in breast cancer as PTEN loss and PD-L1 was validated using a PD- L1 fusion protein and
phosphatidylinositol-4,5- bisphosphate 3-kinase (PIK3CA) PD-L1–transduced melanoma cells (positive control) and
mutations have been identi- fied in approximately 30% and nontransduced parental cells (negative control; ref. 20).
40% of primary breast tumors, respectively (16). Furthermore, Slides were stained for 60 minutes with antibody diluted at
PTEN loss is correlated with ER/PR–negative tumors (17). 1:300 with antibody diluent containing background-
Recently published data from The Cancer Genome Atlas reducing components. Slides were washed and incubated in
(TCGA) showed that basal-like tumors, the majority of which fluorescein isothiocyanate (FITC)-labeled anti- mouse
were TNBCs, showed PTEN mutation or loss in 35% of immunoglobulins and then anti-FITC horseradish per-
tumors, which also correlated with PI3K pathway activation oxidase (HRP). Slides were visualized with 3,30 —diaminoben-
(18). zidine (DAB). Consistent with the previous reports of PD-
In this study, we hypothesized that TNBCs express PD- L1 staining using the 5H1 antibody in renal cell carcinoma,
L1 and that PD-L1 expression may be regulated in part by cell surface membrane staining >5% was considered
PTEN loss. We show that 20% of TNBCs express PD-L1 positive (20). For PTEN staining, TMAs were incubated
and PTEN is deficient in almost half of the PD-L1– with primary anti- PTEN antibody (1:100; clone 6H2.1;
expressing tumors. Furthermore, we show that PTEN Dako). After washing, slides were incubated with the
knockdown results in increased cell surface PD-L1 secondary anti-mouse immunoglob- ulin G (IgG)
expression, an effect that is in part transcriptionally conjugated with HRP, and then visualized with chromogen
regulated. Increased PD-L1 expression following PTEN DAB. Any staining of PTEN was considered pos- itive. For
knockdown has functional consequences, as we show that CD8 staining, TMAs were incubated with primary anti-
activated T cells cocultured with PTEN-silenced breast CD8 antibody (1:20; LabVision). Slides were incubated
cancer cells have decreased proliferation and increased with the secondary anti-mouse IgG-biotin antibody (1:200;
apoptosis. Taken together, these data provide evidence for Vectastain Elite ABC Kit; Vector Laboratories), and then
PTEN loss as a mechanism regulating PD-L1 expression in with the avidin–biotin peroxidase complex (1:100;
TNBC and suggest that antibodies targeting PD-1 or PD-L1 Vectastain Elite ABC Kit), after which visualization was
may have utility as a novel therapeutic strategy in TNBC. conducted with chro- magen. The number of CD8 þ T cells
per 1-mm core was determined. Human tonsil tissue was
Materials and Methods used as a positive control for both PD-L1 and CD8
staining. For PD-L1 staining, irrelevant isotype-matched
Study patients, tumors and cell lines antibodies were used to control for nonspe- cific staining
TCGA RNA sequencing data were obtained from the during protocol development. Specificity of stain- ing was
Cancer Genomics Hub (CGHub; https://cghub.ucsc.edu) confirmed by preincubation of primary antibody with
and the TCGA Data Portal (https://tcga- recombinant PD-L1 antibody. For CD8 staining, omission
data.nci.nih.gov/tcga/). Tumor samples used to construct of primary antibodies was used as a negative staining
tissue microarrays (TMA) and peripheral blood control.
mononuclear cells (PBMC) were obtained through an
Institutional Review Board–approved protocol. Tumors RNA extraction and ampliftcation, cDNA synthesis, and
were considered hormone receptor–nega- tive if nuclear
staining for both the ERs and PRs was ≤5%. Tumors were reverse transcription PCR
considered HER2-negative if they were 0 or 1þ by Breast cancer cells were isolated from fresh-frozen tumor
samples by LCM and RNA was extracted, purified, and ampli-
fied as described previously (21). Before PCR, RNA was
ampli- fied using the Arcturus RiboAmp RNA Amplification
Kit (Life

362 Cancer Immunol Res; 2(4) April 2014 Cancer Immunology Research
 PD-L1 in Triple-Negative Breast Cancer

Technologies, Applied Biosystems) to generate amplified anti-

sense RNA (aRNA). cDNA was synthesized from 1 mg of aRNA Biosciences). Flow cytometry analyses were performed as
using the Roche Transcriptor First Strand cDNA Synthesis Kit described above.
(Roche Applied Science). For cultured cell lines, total cellular Apoptosis assay
RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA
was synthesized from 2 mg of RNA using the Roche Transcrip- CD4þ or CD8þ T cells isolated from PBMCs were plated
tor First Strand cDNA Synthesis Kit. Reverse transcription PCR ratio (3 105) in 96-well
with breast cancer cells at a 1:1 ×
(RT-PCR) reactions were performed on an iCycler iQ thermal plates. T cells were stimulated with anti-CD3 and anti-
cycler (Bio-Rad Laboratories). Quantitative RT-PCR (qRT- CD28 antibodies. Cells were cocultured at 37○ for 20 hours
PCR) was performed on a StepOnePlus instrument (Applied after which they were stained with APC-conjugated anti-
Biosystems). Data were analyzed as relative mRNA expression CD4 antibody and PE-con- jugated anti-CD8 antibody.
quantified with StepOnePlus software and normalized to actin Cells were washed in PBS and then resuspended in
transcription levels. Primer sequences used included: cytoker- Annexin binding buffer containing FITC-con- jugated
atin 7 (CK7; forward primer 50- Annexin V and 7-amino-actinomycin D (7-AAD) via- bility
stain (BD Biosciences). Flow cytometry analyses were
TGTGGATGCTGCCTACATGA- GC-30, reverse primer 50- performed as described above.
AGCACCACAGATGTGTCGGAGA-30), PD-L1 (forward primer
50-TATGGTGGTGCCGACTACAA-30, Statistical analysis
reverse primer 50-TGGCTCCCAGAATTACCAAG-30), and Comparison of PD-L1 between TNBC and non-TNBC sam-
actin, an endogenous control, (forward primer 50- ples in the TCGA dataset was made using a two-sample t test
TCCTGTGGCATC- CACGAAAC-30, reverse primer 50- performed in R (http://www.R-project.org). Remaining statis-
GAAGCATTTGCGGACGAT- tical analyses were performed using GraphPad Prism 5.0
30; oligonucleotides from Sigma-Aldrich). software. P < 0.05 were considered significant.

shRNA constructs and transduction Results

PTEN short hairpin RNA (shRNA) lentiviral transduction TNBC expresses PD-L1
particles (TRCN0000002746 and TRCN0000002749) and To evaluate the presence of PD-L1 in breast cancer, we
non- targeting shRNA lentiviral transduction particles used the TCGA RNA sequencing data to determine whether
[pLKO.1- puro Non-Target Control (SHC016V)] were the PD- L1 transcript was present. This analysis
obtained from Sigma-Aldrich. Transductions were demonstrated differ- ential PD-L1 expression with
performed with 1 104 cells per well in 96-well plates. ×
significantly higher levels in TNBC (n 120) as compared
Lentiviral particles were added at a multiplicity of infection ¼ non-TNBC (n 716) samples (P < 0.001; Fig. 1A).
with
of 5. After 48 hours, media was changed to fresh media Because whole tumors such as those evaluated by TCGA
with 2 mg/mL puromycin. Media was replaced every third contain cells of the microenvironment including
day with fresh puromycin-containing media until stable inflammatory cells that might express PD-L1, we next per-
clones were identified. PTEN knockdown was confirmed formed LCM to isolate breast cancer cells from primary
using Western blot analysis. tumors. Following RNA extraction, qRT-PCR confirmed
the presence of PD-L1 mRNA in all five samples with
Drug reagents transcript levels in three tumors being higher than in the
MK-2206 was provided (to F. Meric-Bernstam) by the MDA-MB-231 cell line, which has been shown to have
SU2C PI3K Dream Team Consortium. Rapamycin was baseline high PD-L1 expression (Fig. 1B; ref. 12). Two of
purchased from LC Laboratories, Inc. the three tumors (LCM1 and LCM 4) with the highest PD-
L1 mRNA levels were TNBCs.
Flow cytometry analysis Having shown the presence of PD-L1 transcripts in breast
To assess cell surface PD-L1 expression, cells were stained tumors, we next evaluated PD-L1 expression at the protein
with phycoerythrin (PE)-conjugated anti-PD-L1 antibody level. Immunohistochemical staining for PD-L1 expression
(eBioscience). Aqua LIVE/DEAD stain (Invitrogen) was used was performed on a TMA comprising 105 tumors from
to assess cell viability. Flow cytometry was performed using patients with early-stage TNBC who had not received
the Fortessa flow cytometer (BD Biosciences) and data were neoadjuvant chemotherapy. PD-L1 expression, defined as
analyzed using FlowJo software (TreeStar Inc.). >5% membra- nous staining, was identified in 20 (19%) of
the tumors. Furthermore, nine of 17 evaluable PD-L1þ
T-cell proliferation assay tumors were neg- ative for PTEN staining on IHC. Staining
CD4þ and CD8þ T cells were isolated from healthy donor for the presence of CD8þ T cells was available for 82
PBMC using negative selection kits (Miltenyi Biotech). Cells tumors. For tumors that were PD-L1þ, the average number
were labeled with carboxyfluorescein succinimidyl ester of CD8þ T cells per 1-mm core was 668, compared with
(CFSE; Invitrogen) after which 3 10 5 CD4þ or CD8þ T cells 263 for tumors that were PD-L1— (P < 0.0001; Fig. 1C and
were combined with 1 103 to 1 ×10 4breast cancer cells and D).
×
seeded into 96-well plates. × were stimulated by the
T cells Taken together, these data confirm that TNBC expresses
addition of anti-CD3 and anti-CD28 antibodies (3 mg/mL; BD PD- L1, and approximately half of the PD-L1 þ tumors did
Biosciences). After 72 hours, cells were stained with allophy- not express PTEN.
cocyanin (APC)-conjugated anti-CD4 antibody, PE-conjugated
anti-CD8 antibody and Aqua LIVE/DEAD stain (BD PD-L1 expression and PTEN status
Having demonstrated PD-L1 expression in approximately
20% of TNBC specimens, we next screened a panel of cell
lines,

www.aacrjournals.org Cancer Immunol Res; 2(4) April 2014 363

Downloaded from cancerimmunolres.aacrjournals.org on February 1, 2020. © 2014 American Association for Cancer Research.
Mittendorf et al.

A B 30 Figure 1. PD-L1 is expressed in

breast cancer. A, analysis of TCGA
12

data demonstrated higher PD-L1

20
mRNA expression in breast tissue
Log2

CT
specimens from patients with
10 TNBC (n 120)
¼ in contrast with
810

patients with non-TNBC (n ¼ 716).

Data are mean T SD of PD-L1
0
mRNA expression. Analysis was
done using a t test. Log2
6

LCM1 LCM2 LCM3 LCM4 LCM5 MDA-MB-231

–++–+–ER expression of PD-L1 is shown on

P < 0.001 the y-axis. B, PD-L1 mRNA
4

–++–+–PR expression (mean T SD) in breast

Non-TNBC TNBC cancer patient tumors was
–++–––HER2 measured using RT-PCR. RNA was
extracted from breast cancer cells
CK7
that were isolated from tumors
using LCM (1–5). Cytokeratin 7
(CK7) was used as a marker of
C D breast cancer to confirm the
H&EPTENH&E PTEN source of the extracted RNA.
MDA-MB- 231 was used as a
positive control for PD-L1. C,
representative TNBC patient
tumor tissues showing loss of
PTEN expression and high PD- L1
expression in tumor cells, and a
significant CD8þ T-cell infiltrate in
contrast with another TNBC
PD-L1 PD-L1 patient tissue (D), which shows
CD8+ T cells CD8+ T cells
high PTEN expression in breast
tumor cells, no PD-L1 expression
in tumor cells, minimal PD-L1
expression in associated
inflammatory cells, and minimal
×
intratumoral CD8þ T-cell infiltrate.
Magnification, 100.
H&E, hematoxylin and eosin.

including five TNBC cell lines, for cell surface PD-L1

expression (Fig. 2). Four of the five TNBC cell lines knockdown in the MDA-MB-231 cell line resulted in a
expressed high levels of cell-surface PD-L1; two of these PD- greater increase in PD-L1 expression than the addition of
L1–expressing TNBC cell lines (MDA-MB-468 and BT-549) IFN-g, which is known to enhance PD-L1 expression
are PTEN deficient (22). Notably, the TNBC cell line MDA- (Supplementary Fig. 1). For all three cell lines, RNA was
MB-157, which has wild-type PTEN, had low levels of PD-L1 extracted and qRT-PCR demonstrated a significant increase
expression. Our data also show PTEN expression in the MDA- in the PD-L1 mRNA transcript, suggesting that regulation
MB-231 and HS-578 TNBC cell lines, which have high levels of PD-L1 by PTEN may be in part at the level of
of cell surface PD-L1, suggesting that there are other transcription (Fig. 3G–I).
mechanisms of PD-L1 regulation in addition to PTEN.
We next sought to further investigate if PTEN regulates PD- Effects of inhibiting the PI3K pathway on PD-L1
L1 expression. We transduced the MDA-MB-157 cell line, expression
which expresses PTEN and low levels of cell surface PD-L1, Cancer cells lacking PTEN have increased levels of PI3K
with two different PTEN lentiviral shRNA vectors to generate activity. To determine whether PTEN regulation of PD-L1
stable PTEN knockdown clones (Fig. 3A). Cell surface PD-L1 in TNBC is mediated by PI3K signaling, we treated MDA-
expression was assessed using flow cytometry. As shown (Fig. MB-468 cells with either the Akt inhibitor MK-2206 or the
3D), loss of PTEN led to a significant increase in PD-L1 cell mTOR inhibitor rapamycin. MDA-MB-468 was chosen
surface expression (P < 0.0001). Results were confirmed in the because it has been rendered PTEN deficient by a deletion
MCF-7 cell line (Fig. 3B and E), which also expresses PTEN mutation at codon 70, resulting in increased PI3K signaling
and low levels of cell surface PD-L1. In addition, we showed as evidenced by higher levels of basal AKT
that PTEN knockdown could further increase PD-L1 phosphorylation (22). As shown in Fig. 4A and B,
expression in the MDA-MB-231 cell line that expresses PTEN treatment with either agent resulted in a significant decrease
and high levels of cell surface PD-L1 (Fig. 3C and F). in PD-L1 cell surface expression.
Interestingly, PTEN Because our data generated using PTEN shRNA
suggested that PTEN regulation of PD-L1 is transcriptional,
we next investigated the effect of treatment with MK-2206
or
364 Cancer Immunol Res; 2(4) April 2014 Cancer Immunology Research
 PD-L1 in Triple-Negative Breast Cancer

CD8þ (Fig. 5D) T cells that were enriched from healthy

10,000 Unstained
donor PBMCs as well as CD4 þ and CD8þ T cells in bulk
Stained
8,000 PBMCs (Supplementary Fig. S4) cocultured with PTEN-
Cell surface PD-L1 (MFI)

6,000 silenced breast cancer cells. These data confirm a functional

4,000 effect of PTEN loss that is mediated by increased PD-L1
2,000 expression.
800
600 Discussion
400
Evading antitumor immunity is a hallmark for the
200 develop- ment and progression of cancer (25). Tumors use
0 multiple mechanisms to avoid recognition by the host
MDA-MB-231 MDA-MB-157 MDA-MB-468

MDA-MB-453
immune system, including expression of the negative T-cell
regulatory molecule PD-L1 (11). In this study, we
identified PD-L1 expression in approximately 20% of
TNBC. We also showed that PTEN loss is one mechanism
– – – – – – + + + + + –ER regulating PD-L1 at the transcriptional level and that this
effect occurs via signaling through the PI3K pathway.
– – – – – – + + – – + –PR
Importantly, increased PD-L1 expression on the surface of
– – – – – + – – – + + +HER2 TNBC cells had functional consequences on T cells
including decreasing their proliferation and increasing apo-
PTEN ptosis. These observations provide the rationale for imple-
menting therapeutic strategies targeting the PD-1/PD-L1
Actin axis in TNBC and suggest a role for enhanced antitumor
Figure 2. PD-L1 and PTEN expression in cultured breast cancer cell lines. immunity when targeting the PI3K pathway in TNBC.
A panel of breast cancer cell lines was evaluated for cell-surface PD-L1 PD-L1 is not expressed on normal epithelial tissues but is
expression by flow cytometry (MFI meanTSD) and for PTEN expression expressed on many cancers including renal cell carcinoma
by Western blot analysis. Actin was used as a loading control. Data (20, 26), pancreatic cancer (27), ovarian cancer (28), gastric
show higher PD-L1 expression in four of the five TNBC cell lines cancer (29), esophageal cancer (30), and hepatocellular
evaluated in comparison with non-TNBC cell lines. MFI, median carci- noma (31). PD-L1 expression in breast cancer has
fluorescence intensity. previously been investigated by Ghebeh and colleagues
who identified PD-L1 expression in 22 (50%) of 44 tumors
rapamycin on PD-L1 transcript levels. As shown in Fig. evaluated; in 15 (34%) it was restricted to the tumor
4C, there was a significant decrease in the PD-L1 mRNA epithelium, whereas in 18 (41%) it was identified in TILs
transcripts after treatment with either agent when compared (12). Furthermore, they found that intratumoral expression
with untreated controls. These data provide additional of PD-L1 was associated with high histologic grade and
evidence that PD-L1 regulation by PTEN is in part negative hormone receptor status. Consistent with the
transcriptional via PI3K signaling. previous study, we found that approxi- mately 20% of
TNBC tumors express PD-L1. The majority (95%) of these
Functional effects of PTEN loss and PD-L1 upregulation TNBC tumors were grade 3.
PD-L1 is the ligand for the T-cell inhibitory receptor PD-1. The difference in the rates of PD-L1 expression in the
Activation of the PD-1/PD-L1 pathway decreases T-cell pro- current study (20%) and the study by Ghebeh and
liferation, survival, and cytokine production (11, 23, 24). To colleagues (34%) may be attributable to several factors.
address the functional consequences of increased PD-L1 cell One important difference is that in our study, PD-L1
surface expression that is mediated by PTEN knockdown, we expression was assessed on TMAs. Importantly, this
first measured the proliferation of activated CD4 þ and CD8þ T allowed us to evaluate a greater number of tumors.
cells isolated from healthy donor PBMC and cocultured with However, there are limitations of using a TMA. One
(i) MDA-MB-231 breast cancer cells transduced with a PTEN limitation that is relevant for the current study is that
shRNA lentiviral vector, (ii) parental cells, or (iii) cells trans- multiple cores were dislodged during the PTEN and CD8
duced with a control shRNA lentiviral vector. In staining; therefore, we only had PTEN data available for 17
experiments using PBMCs from three separate healthy of 20 PD- L1þ tumors and CD8þ T-cell data on 82
donors, coculturing activated T cells with breast cancer specimens. A second limitation of using a TMA is the
cells with increased PD-L1 cell surface expression induced small size (1 mm) of cores used to construct the TMA.
by PTEN-silencing resulted in a significant decrease in However, because of the small size, and because tumor
CD4þ T-cell proliferation compared with PBMCs cocultured tissues are known to be heterogeneous, the rate of PD-L1
with parental cells (P < 0.0001; Fig. 5A). Similarly, there was positivity identified in our study may be an under-
a significant decrease in CD8þ T-cell proliferation (P < estimation of the true frequency of PD-L1 expression.
0.005; Fig. 5B). Similar results were seen using bulk None- theless, by showing PD-L1 expression in 20% of
PBMCs from MDA-MB-231 (Supplementary Fig. S2) and TNBC, we have provided a rationale for investigating PD-
MDA-MB-157 cells (Supplementary Fig. S3). Annexin V L1/PD-1 targeting therapies in TNBC, which is known to
assays showed increased apoptosis of both CD4þ (Fig. 5C) have few therapeutic options. The recently reported phase I
and study evaluating the anti-PD-1 antibody BMS-936588
included data from a nonran- dom subset of patients
enrolled on the trial in whom a

www.aacrjournals.org Cancer Immunol Res; 2(4) April 2014 365

 Mittendorf et al.

A MDA-MB-157
B MCF7
C MDA-MB-231

Nontargeted shRNA

Nontargeted shRNA
Nontargeted shRNA
PTEN shRNA #1

PTEN shRNA #2

PTEN shRNA #1
PTEN shRNA #1
MDA-MB-157

MDA-MB-231
MCF7
PTEN PTEN
PTEN

Actin Actin Actin

DEF
Cell surface PD-L1 (MFI)

Cell surface PD-L1 (MFI)

2,000 1,500

Cell surface PD-L1 (MFI)

** ** 50,000
**
1,500 40,000 **
1,000
1,000 30,000

500 20,000
500
10,000
0 0 0
Un UntraPTENPTEN
Nontarget Un Untra PTENNontarget Un Untra PTENNontarget

G H I
2.0* 2.5 6.0
*
*
2.0
1.5

RQ value
4.0
RQ value

RQ value

1.5
1.0
1.0
2.0
0.5
0.5
0.0 0.0 0.0

Un PTEN Nontarget Un PTEN Nontarget Un PTEN Nontarget

Figure 3. PTEN downregulation increases PD-L1 cell surface expression. MDA-MB-157, MCF7, and MDA-MB-231 breast cancer cell lines were transduced
with PTEN shRNA lentiviral transduction particles. A–C, Western blot analysis performed on lysates obtained from these cells confirmed decreased PTEN
expression. Nontargeting lentiviral transduction particles were used as a control. Actin was used to show equal loading. D–F, cell surface PD-L1 expression
was evaluated by flow cytometry staining and PD-L1 MFI (mean T SD) demonstrated a significant increase in PD-L1 expression on cells, correlating with
a decrease in PTEN expression. Decreased PTEN expression in MDA-MB-231 cell line, which has the highest baseline cell surface expression of
PD-L1, resulted in a significant further increase in PD-L1 expression. G–I, RNA was extracted from MDA-MB-157, MCF7, and MDA-MB-231 cells that were
transduced with PTEN or nontargeting shRNA. Mean SD T relative quotient (RQ) values obtained from qRT-PCR demonstrated a significant increase
in the PD-L1 transcript that coincided with decreased PTEN expression. All assays were performed in triplicate. ANOVA test was performed using
Prism 5.0 software (m, P < 0.05; mm, P < 0.0001). MFI, median fluorescence intensity.

pretreatment biopsy was available to assess PD-L1 An interesting finding in our study was that in the 82
expression (14). Although this analysis included patients with TNBC in whom staining was available, there
pretreatment biopsies from only 42 of 296 patients, was a significantly greater number of intratumoral CD8þ T
investigators did make the observation that an objective cells in tumors that were PD-L1 þ when compared with PD-L1
—
response occurred in 9 (36%) of 25 patients with PD-L1– tumors. This is in contrast to a recent study of 45 oral
positive tumors. In contrast, none of the 17 patients with squamous cell carcinoma cases in which 39 cases had PD-
PD-L1–negative tumors responded. The investigators urge L1 expression, which was associated with low peritumoral
caution in interpreting these data due to the small sample CD8þ T cell infiltrate (32). Our results, however, are
size; nevertheless, their findings suggest that PD- L1 consistent with a recent report from Taube and colleagues,
expression in pretreatment tumor samples predicts a greater who identified a strong association between the expression of
likelihood of objective response. Future trials should PD-L1 in melanoma and TILs (33). In that study, 98% of PD-
incorporate biopsies to further test this hypothesis. L1þ tumors

366 Cancer Immunol Res; 2(4) April 2014 Cancer Immunology Research
 PD-L1 in Triple-Negative Breast Cancer

A C
1,500 1.5
Cell surface PD-L1 (MFI)

0 500500 Dose MK-2206 (nmol/L)

* 2448Time (h)1.0 * *

RQ value
1,000
p-S6 S235/236

1.00.85 0.66 0.5

500
Tubulin
0.0
0
Unt M Rap
Un Unt M

B
1,000
0 100Dose rapamycin (nmol/L)
Cell surface PD-L1 (MFI)

800 * 72Time (h)

600 p-S6
S235/236
400 1.0.003

200 Tubulin

Un Unt Rap

Figure 4. Inhibition of the PI3K pathway decreases PD-L1 expression. A, MDA-MB-468 breast cancer cells were treated for 48 hours with the AKT inhibitor
MK- 2206 (500 nmol/L) after which PD-L1 cell surface expression assessed by flow cytometry. B, MDA-MB-468 breast cancer cells were treated with the
mTOR inhibitor rapamycin (100 nmol/L). After 72 hours, cells were harvested and PD-L1 cell surface expression was assessed by flow cytometry. The
addition of the PI3K pathway inhibitors significantly decreased the levels of surface PD-L1 expression. Data represent PD-L1
T MFI (mean SD). Western blot
analysis showing decreased expression of p-S6 confirmed pathway inhibition by MK-2206 and rapamycin. C, RNA was extracted from additional MDA-MB-
468 breast cancer cells treated with MK-2206 or rapamycin. qRT-PCR showed a decrease in PD-L1 mRNA expression after addition of AKT and mTOR
inhibitors.
Data shown are representative of three separate experiments. All experiments were performed in triplicate. Data represent relative quotient (RQ) value
(mean T SD). ANOVA test was performed using Prism 5.0 software ( m, P < 0.01). MFI, median fluorescence intensity.

had associated TIL versus 28% of PD-L1 — tumors. Further-

more, IFN-g expression was detected at the interface of PD- A second mechanism by which tumors can drive PD-L1
L1– positive tumor cells and CD45-positive infiltrating expression is by oncogenic signaling pathways. This was
immune cells. The results are also consistent with a study first demonstrated in glioblastomas when Parsa and
from Spranger and colleagues that showed CD8 þ T cells in colleagues showed that PTEN loss was associated with
metastatic mel- anoma foci that also contained high levels increased PD- L1 expression, suggesting the involvement of
of regulatory T cells and expressed the immunosuppressive the PI3K pathway (15). Because PTEN loss is commonly
factors indoleamine- 2,3-dioxygenase (IDO) and PD-L1 seen in TNBC, we investigated the relationship between
(34). Mouse models from that study further suggested that PTEN and PD-L1 expres- sion. In approximately 50% of
PD-L1 upregulation was dependent on IFN-g (34). Other TNBC tumors included in our TMA in which there was
>5% PD-L1 expression, we identified a loss of PTEN
studies evaluating PD-L1 regulation on antigen-presenting
cells have shown that inter- leukin (IL)-6, IL-10, and the staining. Similarly, in a panel of TNBC cell lines, we found
common g-chain cytokines IL-2, IL- 7, and IL-15 upregulate that two cell lines with PTEN loss, MDA-MB-468 and BT-
549, had high cell surface PD-L1 expression. Together, these
PD-L1 expression on tumor cells, suggesting that multiple
data suggest that there are likely multiple mechanisms of
factors present in the tumor micro- environment in addition
PD- L1 regulation in TNBC.
to IFN-g may promote increased PD- L1 expression by
Using PTEN shRNA transduction particles, we generated
tumors (35, 36). These data suggest a positive feedback stable cell lines and consistently found significantly increased
loop whereby inflammatory factors produced by immune PD-L1 expression after PTEN silencing. In addition, in the
cells in the tumor microenvironment cause tumor cells to MDA-MB-468 cell line, which has PTEN loss and increased
increase cell surface expression of PD-L1 (37), a possible PI3K signaling, treatment with the AKT inhibitor MK-2206
mechanism whereby tumor cells evade the adaptive and the mTOR inhibitor rapamycin resulted in decreased cell
immune response. surface PD-L1 expression, confirming a role for PTEN loss
and

www.aacrjournals.org Cancer Immunol Res; 2(4) April 2014 367

 Mittendorf et al.

A 100 CD4+ T cells C 80

CD4+ T
HD 1 cells HD 1
HD 2
* * HD 2
*

% Annexin V–Positive
80 HD 3 HD 3
60
% Proliferation

60 * * *
40
40

20 20

0 0
MDA + + MDA + + MDA + + MDA + + MDA + + MDA + +

B 100 CD8+ T cells

D CD8+ T cells
80
HD 1
HD 1 * * * HD 2

% Annexin V–Positive
HD 2
80 HD 3 HD 3
60
% Proliferation

60 ** ** **
40
40

20 20

0 0
MDA + + MDA + + MDA + + MDA + + MDA + + MDA + +

Figure 5. Increased PD-L1 cell surface expression following PTEN knockdown inhibits T-cell proliferation and increases apoptosis. To determine the effect
of the increased PD-L1 cell surface expression following PTEN knockdown on T-cell proliferation, CD4 þ (A) or CD8þ (B) T cells were isolated from PBMCs
from healthy donors, were labeled with CFSE and cocultured with MDA-MB-231 breast cancer cells that were transduced with PTEN shRNA (i.e., increase
surface PD-L1) or negative control groups including parental MDA-MB-231 cells or MDA-MB-231 cells transduced with nontargeting shRNA. After
stimulation with anti-CD3/CD28, proliferation was measured using flow cytometry. The experiment was performed three times in triplicate and the
average percentage
T proliferation SD for each experiment is shown. CD4þ (A) or CD8þ (B) T cells were also cultured alone (unstimulated, negative control)
or stimulated with anti- CD3-CD28 in the absence of MDA-MB-231 cells (positive control). To determine the effect of increased PD-L1 cell surface
expression on apoptosis, standard Annexin V assays were performed. Anti-CD3/CD28-stimulated CD4þ (C) or CD8þ (D) T cells were cocultured with breast
cancer cells for 20 hours and then resuspended in Annexin binding buffer. Analysis was performed using flow cytometry. The experiment was repeated
three times in triplicate and the average percentage Annexin V–positive TCD4þ (C) and CD8þ (D) T cells SD for each experiment is shown. Significance
was determined using an ANOVA test ( mm , P < 0.005; m , P < 0.0001).

PI3K signaling in PD-L1 regulation. Our results confirm find-

ings from Crane and colleagues, who reported increased PD- 578T) with wild-type PTEN, highlight that multiple mechan-
L1 protein using Western blot analyses of whole-cell lysates isms may be involved in PD-L1 regulation in tumors. These
in two PTEN-mutant breast cancer cell lines when compared mechanisms may be influenced by the tumor type and other
with two PTEN wild-type cell lines (38). Furthermore, their oncogenic signaling pathways that are activated by the tumor
data demonstrated decreased PD-L1 in the BT549 cell line cell. Ongoing work in our laboratory is investigating the
following treatment with multiple inhibitors of the PI3K mechanisms by which PTEN loss and increased PI3K
pathway. How- ever, in contrast to the findings by Crane and signaling regulate PD-L1 expression.
colleagues showing regulation of PD-L1 by PTEN/PI3K In conclusion, we have shown that approximately 20% of
signaling at the translational level, our data suggest cases of TNBC express PD-L1, suggesting a potential role for
transcriptional regulation of PD-L1 by PTEN/PI3K pathway. anti-PD-L1/anti-PD1 therapy in this patient population. Fur-
This discrepancy in our data and the previous report, along thermore, we show that PTEN loss upregulates PD-L1 expres-
with our data showing high PD- L1 expression in two TNBC sion, indicating that therapeutic strategies targeting the PI3K
cell lines (MDA-MB-231 and HS- pathway may enhance adaptive immune responses against

368 Cancer Immunol Res; 2(4) April 2014 Cancer Immunology Research
 PD-L1 in Triple-Negative Breast Cancer

TNBC. Additional investigations are required to fully Administrative, technical, or material support (i.e., reporting or orga-
elucidate the mechanisms regulating PD-L1 expression in nizing data, constructing databases): E.A. Mittendorf
TNBC and to determine the extent of regulation by Study supervision: E.A. Mittendorf, G. Alatrash
PTEN/PI3K pathway.
Disclosure of Potential Conflicts of Interest Grant Support
This work was supported in part by grant R00CA133244 (to E.A. Mittendorf)
M. Curran is a consultant/advisory board member of Jounce Therapeutics and The State of Texas Grant for Rare and Aggressive Cancer through the
and Innovent. J.K. Litton has received commercial research support from Morgan Welch Inflammatory Breast Cancer Research Program (to E.A.
Bristol- Myers Squibb and Novartis. No potential conflicts of interest were Mittendorf).
disclosed by the other authors. F. Meric-Bernstam, A. Akcakanat, and A.M. Gonzalez-Angulo were supported
by a Stand Up To Cancer Dream Team Translational Cancer Research Grant, a
Authors' Contributions Program of the Entertainment Industry Foundation (SU2C-AACR-DT0209 0).
This work was also supported by Susan G. Komen for the Cure SAC10006 (to
Conception and design: E.A. Mittendorf, F. Meric-Bernstam, J.K. Litton, F. Meric- Bernstam), National Center for Research Resources Grant
J.J. Molldrem, G. Alatrash 3UL1RR024148 (to
Development of methodology: E.A. Mittendorf, A.V. Philips, N. Qiao, M. F. Meric-Bernstam, X. Su, and Y. Wu), and NIH grant T32CA009599 (A.
Curran Acquisition of data (provided animals, acquired and managed Chawla). The Flow Cytometry and Cellular Imaging core and Characterized Cell
patients, provided facilities, etc.): A.V. Philips, N. Qiao, Y. Wu, S. Harrington, Line core are supported by Cancer Center Support Grant NCI P30CA16672.
Y. Wang, The costs of publication of this article were defrayed in part by the
A.M. Gonzalez-Angulo, A. Akcakanat, A. Chawla payment of page charges. This article must therefore be hereby marked
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
computational analysis): E.A. Mittendorf, A.V. Philips, N. Qiao, Y. Wu, X. Su, this fact.
A.M. Gonzalez-Angulo, G. Alatrash
Writing, review, and/or revision of the manuscript: E.A. Mittendorf,
F. Meric-Bernstam, Y. Wu, S. Harrington, A.M. Gonzalez-Angulo, M. Curran, Received August 19, 2013; revised December 6, 2013; accepted December 31,
P. Hwu, P. Sharma, J.K. Litton, G. Alatrash 2013; published OnlineFirst January 10, 2014.

References
1. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, 13. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et
et al. Triple-negative breast cancer: clinical features and patterns of al. Safety and activity of anti-PD-L1 antibody in patients with
recurrence. Clin Cancer Res 2007;13:4429–34. advanced cancer. N Engl J Med 2012;366:2455–65.
2. Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr 14. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDer-
Y, et al. Identification of human triple-negative breast cancer mott DF, et al. Safety, activity, and immune correlates of anti-PD-1
subtypes and preclinical models for selection of targeted therapies. antibody in cancer. N Engl J Med 2012;366:2443–54.
J Clin Invest 2011;121:2750–67. 15. Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, et
3. Haffty BG, Yang Q, Reiss M, Kearney T, Higgins SA, Weidhaas J, et al. Loss of tumor suppressor PTEN function increases B7-H1
al. Locoregional relapse and distant metastasis in conservatively expression and immunoresistance in glioma. Nat Med 2007;13:84–
man- aged triple negative early-stage breast cancer. J Clin Oncol 8.
2006;24: 5652–7. 16. Gonzalez-Angulo AM, Ferrer-Lozano J, Stemke-Hale K, Sahin A, Liu
4. Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, et al. S, Barrera JA, et al. PI3K pathway mutations and PTEN levels in
Response to neoadjuvant therapy and long-term survival in patients primary and metastatic breast cancer. Mol Cancer Ther
with triple-negative breast cancer. J Clin Oncol 2008;26:1275–81. 2011;10:1093–101.
5. Desmedt C, Haibe-Kains B, Wirapati P, Buyse M, Larsimont D, Bon- 17. Saal LH, Holm K, Maurer M, Memeo L, Su T, Wang X, et al. PIK3CA
tempi G, et al. Biological processes associated with breast cancer mutations correlate with hormone receptors, node metastasis, and
clinical outcome depend on the molecular subtypes. Clin Cancer Res ERBB2, and are mutually exclusive with PTEN loss in human breast
2008;14:5158–65. carcinoma. Cancer Res 2005;65:2554–9.
6. Loi S, Sirtaine N, Piette F, Salgado R, Viale G, Van Eenoo F, et al. 18. Cancer Genome Atlas Network. Comprehensive molecular portraits
Prognostic and predictive value of tumor-infiltrating lymphocytes of human breast tumours. Nature 2012;490:61–70.
in a phase III randomized adjuvant breast cancer trial in node- 19. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the
positive breast cancer comparing the addition of docetaxel to B7 family, co-stimulates T-cell proliferation and interleukin-10
doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. J secretion. Nat Med 1999;5:1365–9.
Clin Oncol 2013; 31:860–7. 20. Thompson RH, Kuntz SM, Leibovich BC, Dong H, Lohse CM,
7. Liu S, Lachapelle J, Leung S, Gao D, Foulkes WD, Nielsen TO. CD8 þ Webster WS, et al. Tumor B7-H1 is associated with poor
lymphocyte infiltration is an independent favorable prognostic indi- prognosis in renal cell carcinoma patients with long-term follow-
cator in basal-like breast cancer. Breast Cancer Res 2012;14:R48. up. Cancer Res 2006;66: 3381–5.
8. Brunet JF, Denizot F, Luciani MF, Roux-Dosseto M, Suzan M, Mattei 21. Mittendorf EA, Alatrash G, Qiao N, Wu Y, Sukhumalchandra P, St
MG, et al. A new member of the immunoglobulin superfamily— John LS, et al. Breast cancer cell uptake of the inflammatory
CTLA- 4. Nature 1987;328:267–70. mediator neutrophil elastase triggers an anticancer adaptive
9. Krummel MF, Allison JP. CD28 and CTLA-4 have opposing effects immune response. Cancer Res 2012;72:3153–62.
on the response of T cells to stimulation. J Exp Med 1995;182: 22. Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX, et al. The
459–65. PTEN/ MMAC1/TEP tumor suppressor gene decreases cell growth
10. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD- and induces apoptosis and anoikis in breast cancer cells.
1, a novel member of the immunoglobulin gene superfamily, upon Oncogene 1999; 18:7034–45.
pro- grammed cell death. EMBO J 1992;11:3887–95. 23. Mazanet MM, Hughes CC. B7-H1 is expressed by human
11. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et endothelial cells and suppresses T cell cytokine synthesis. J
al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential Immunol 2002;169: 3581–8.
mechanism of immune evasion. Nat Med 2002;8:793–800. 24. Patsoukis N, Sari D, Boussiotis VA. PD-1 inhibits T cell proliferation
12. Ghebeh H, Mohammed S, Al-Omair A, Qattan A, Lehe C, Al-Qudaihi by upregulating p27 and p15 and suppressing Cdc25A. Cell Cycle
G, et al. The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is 2012; 11:4305–9.
expressed in breast cancer patients with infiltrating ductal 25. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
carcinoma: correlation with important high-risk prognostic factors. Cell 2011;144:646–74.
Neoplasia 2006;8:190–8. 26. Thompson RH, Dong H, Kwon ED. Implications of B7-H1 expression
in clear cell carcinoma of the kidney for prognostication and
therapy. Clin Cancer Res 2007;13:709s–15s.
www.aacrjournals.org Cancer Immunol Res; 2(4) April 2014 369
Mittendorf et al.

27. Nomi T, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, et al.

cytes in oral squamous cell carcinoma. Oral Oncol 2011;47: 1148–
Clinical significance and therapeutic potential of the programmed
53.
death-1 ligand/programmed death-1 pathway in human pancreatic
33. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL,
cancer. Clin Cancer Res 2007;13:2151–7.
et al. Colocalization of inflammatory response with B7-h1
28. Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y,
expression in human melanocytic lesions supports an adaptive
Yamaguchi K, et al. Programmed cell death 1 ligand 1 and tumor-
resistance mecha- nism of immune escape. Sci Transl Med
infiltrating CD8þ T lymphocytes are prognostic factors of human
2012;4:127ra37.
ovarian cancer. Proc Natl Acad Sci U S A 2007;104:3360–5.
34. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, et al.
29. Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. Immunohistochem-
Up- regulation of PD-L1, IDO, and T(regs) in the melanoma tumor
ical localization of programmed death-1 ligand-1 (PD-L1) in gastric
micro- environment is driven by þ CD8( ) T cells. Sci Transl Med
carcinoma and its clinical significance. Acta Histochem 2006;108:
2013;5: 200ra116.
19–24.
35. Kinter AL, Godbout EJ, McNally JP, Sereti I, Roby GA, O'Shea MA,
30. Ohigashi Y, Sho M, Yamada Y, Tsurui Y, Hamada K, Ikeda N, et al.
et al. The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-
Clinical significance of programmed death-1 ligand-1 and pro-
21 induce the expression of programmed death-1 and its
grammed death-1 ligand-2 expression in human esophageal cancer.
ligands. J Immunol 2008;181:6738–46.
Clin Cancer Res 2005;11:2947–53.
36. Wolfle SJ, Strebovsky J, Bartz H, Sahr A, Arnold C, Kaiser C, et
31. Gao Q, Wang XY, Qiu SJ, Yamato I, Sho M, Nakajima Y, et al. Over-
al. PD- L1 expression on tolerogenic APCs is controlled by STAT-
expression of PD-L1 significantly associates with tumor aggres-
3. Eur J Immunol 2011;41:413–24.
siveness and postoperative recurrence in human hepatocellular car-
37. Pardoll DM. The blockade of immune checkpoints in cancer immu-
cinoma. Clin Cancer Res 2009;15:971–9.
notherapy. Nat Rev Cancer 2012;12:252–64.
32. Cho YA, Yoon HJ, Lee JI, Hong SP, Hong SD. Relationship
38. Crane CA, Panner A, Murray JC, Wilson SP, Xu H, Chen L, et al.
between the expressions of PD-L1 and tumor-infiltrating lympho-
PI(3) kinase is associated with a mechanism of immunoresistance in
breast and prostate cancer. Oncogene 2009;28:306–12.
370 Cancer Immunol Res; 2(4) April 2014 Cancer Immunology Research
PD-L1 Expression in Triple-Negative Breast Cancer
Elizabeth A. Mittendorf, Anne V. Philips, Funda Meric-Bernstam, et al.

Cancer Immunol Res 2014;2:361-370. Published OnlineFirst January 10, 2014.

Updated version Access the most recent version of this article at:
doi:10.1158/2326-6066.CIR-13-0127
Supplementary Access the most recent supplemental material at:
Material http://cancerimmunolres.aacrjournals.org/content/suppl/2014/01/10/2326-6066.CIR-13-0127.DC1

Cited articles This article cites 38 articles, 18 of which you can access for free at:
http://cancerimmunolres.aacrjournals.org/content/2/4/361.full#ref-list-1

Citing articles This article has been cited by 49 HighWire-hosted articles. Access the articles at:
http://cancerimmunolres.aacrjournals.org/content/2/4/361.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department
Subscriptions at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, use this link
http://cancerimmunolres.aacrjournals.org/content/2/4/361.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.

Downloaded from cancerimmunolres.aacrjournals.org on February 1, 2020. © 2014 American Association for Cancer Research.