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In situ Tumor PD-L1 mRNA expression is associated with increased TILs and
better outcome in breast carcinomas.
Kurt A. Schalper1, Vamsidhar Velcheti3, Daniel Carvajal1, Hallie Wimberly1, Jason
Brown1, Lajos Pusztai2 and David L. Rimm1
1Department of Pathology, Yale School of Medicine 2Department of Medical Oncology, Yale School of Medicine 3Department of Solid Tumor Oncology, Cleveland Clinic
Running head: PD-L1 mRNA positivity and outcome in breast cancer.
Key words: PD-L1 mRNA, in situ, breast cancer, inflammation, survival.
Corresponding author:
Kurt Schalper, MD, PhD
Department of Pathology, BML112
Yale University School of Medicine
310 Cedar St, PO Box 208023
New Haven, CT 06520-8023
e-mail: [email protected]
203-737-4205
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Abstract:
Purpose: Blockade of the PD-1/PD-L1 axis emerged as a promising new therapeutic
option for cancer that has resulted in lasting responses in metastatic renal, lung
carcinomas and melanomas. Tumor PD-L1 protein expression may predict response to
drugs targeting this pathway. Measurement of PD-L1 protein is limited by the lack of
standardized immunohistochemistry methods and variable performance of antibodies.
Our goal was to correlate PD-L1 mRNA expression with clinical variables in primary
breast carcinomas.
Methods: The fluorescent RNAscope paired-primer assay was used to quantify in situ
PD-L1 mRNA levels in 636 stage I-III breast carcinomas on two sets of tissue
microarrays (YTMA128 [n=238)] and YTMA201 [n=398]). Tumor infiltrating lymphocytes
(TILs) were assessed by hematoxylin-eosin stain and quantitative fluorescence.
Results: On YTMA128 and YTMA201, 55.7% and 59.5% of cases showed PD-L1
mRNA expression, respectively. Higher PD-L1 mRNA expression was significantly
associated with increased TILs (P=0.04) but not with other clinical variables. Elevated
TILs (scores 2&3+) occurred in 16.5% on YTMA128 and 14.8% on YTMA201 and was
associated with estrogen receptor-negative status (P=0.01 on YTMA128 and 0.0001 on
YTMA201). PD-L1 mRNA expression was associated with longer recurrence free
survival (log-rank P=0.01) which remained significant in multivariate analysis including
age, tumor size, histological grade, nodal metastasis, hormone receptor, HER2 status
and extent of TILs (HR=0.268 [CI=0.099-0.721], P=0.009).
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Conclusions: PD-L1 mRNA expression is identified in nearly 60% of breast tumors and
it is associated with increased TILs and improved recurrence free survival. These
observations support the evaluation of PD-1/PD-L1 targeted therapies in breast cancer.
Statement of Translational relevance.
The presence of tumor infiltrating lymphocytes in breast cancer is associated with better
prognosis and higher response to preoperative chemotherapy. Unfortunately, the
prognostic and predictive value of tumor immune infiltrates is significant but modest. In
most instances the immune system mounts only a partially effective anti-tumor
response. Expression of Programmed death ligand-1 (PD-L1) is a key mechanism of
tumor immune evasion. PD-L1 is expressed in various human cancers and tumor PD-L1
protein positivity using immunohistochemistry predicted response to anti-PD1
monoclonal antibody therapy. The literature suggests the different immunohistochemical
methods yield discordant results that hinder progress in this field. Herein we describe a
reproducible, antibody-independent and compartment-specific method of measuring
PD-L1 mRNA in formalin-fixed paraffin embedded tissue samples and demonstrate the
prognostic value of this marker in breast cancer. Further evaluation of PD-L1 mRNA in
a prospective clinical trial may help select patients for immunostimulatory drugs
targeting the PD-1/PD-L1 pathway.
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Introduction
Breast cancers harbor a large number of genomic alterations that can result in mutated
proteins. These mutations not only contribute to the malignant transformation but also
lead to neoantigens that may serve as targets for a local immune response which could
exert some control on tumor growth (1). Indeed, the presence of lymphocytes in the
tumor microenvironment (TILs) and gene expression signatures representative of these
cells have long been associated with (slightly but significantly) better prognosis,
particularly among high grade and estrogen receptor (ER) negative tumors (2-5).
Numerous attempts have been made in the past to exploit adoptive immunotherapy in
breast cancer (e.g. vaccines, cytokines) that have met with limited success (6). It is
increasingly recognized that inhibition of TIL activity in the tumor microenvironment
limits the extent of antitumor immune response (7).
Recent evidence highlights the pivotal role of the PD-1 (programmed cell death-
1) receptor pathway in maintaining an immunosuppressive tumor
microenvironment. PD-1 is a member of the B7-CD28 family of T cell co-regulatory
receptors and contributes inhibitory signals that mediate the physiological immune
tolerance and limit the inflammatory response to infections (8-9). PD-1 is expressed in
various immune cell types and its activation attenuates T cells function, survival and
expansion (10-11). The PD-1 ligand, PD-L1 is expressed on activated T cells, B cells,
dendritic cells, and macrophages, in addition to some immune-privileged non-
hematopoietic tissues (e.g. retina and placenta)(12-13). Tumors from diverse locations
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can express PD-L1 including breast, ovarian, gastric, pancreatic, lung and renal cell
carcinomas (14-18). PD-L1 expression by tumor cells is believed to mediate the
inhibition of local immune responses, thus shielding the tumor from T cell-mediated
killing. In support of this notion, early phase trials using monoclonal antibodies targeting
PD-1 or PD-L1 have shown substantive and durable clinical responses in patients with
refractory solid tumors, including melanoma, renal and non-small cell lung carcinomas
(19-20).
PD-L1 protein has been reported not be expressed in normal breast but to be
increased in nearly half of breast cancers, particularly in hormone receptor negative and
high-grade, proliferative tumors (14, 21). In addition, high PD-L1 protein expression in
TILs from breast cancer specimens was observed in large, high grade, HER2-positive
tumors. The presence of regulatory T cells (Tregs), tumor PD-L1 expression and PD-1
positive TILs was associated with high histological grade, ER negativity and prominent
lymphocytic infiltrates (22). Preliminary data show that tumor PD-L1 protein expression
using immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue
may predict response to drugs targeting the PD-1/PD-L1 pathway (19). However, the
PD-L1 threshold for expression is not well defined and is subject to assay and
interpretative subjectivity. In addition, the specificity and reproducibility of most
commercially available anti PD-L1 antibodies has not been thoroughly assessed and
limitations of some widely used antibodies have been communicated (23). Therefore, it
is not surprising that some authors have found association of tumor PD-L1 protein
expression with adverse outcome (24-28), while other found no association with
outcome or association with longer survival using comparable methods (24, 29-32). The
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use of alternative methods to accurately assess PD-L1 status in biological and clinical
samples could help overcome such limitations.
We report herein a novel antibody-independent and tissue compartment specific
assay for in situ PD-L1 mRNA measurement in tumor FFPE tissues using the
RNAscope assay coupled to quantitative fluorescence (AQUA®). We show that PD-L1
mRNA positivity is associated with increased tumor infiltrating lymphocytes and longer
event free survival in breast cancer.
Methods
Patient cohorts, Tissue Microarrays (TMAs) and control preparations. Two previously reported (33-34) retrospective stage I-III breast cancer collections from
Yale University represented in TMA format were used in this study, termed YTMA128
(N=238) and YTMA201 (N=398). Clinico-pathological information from patients in both
cohorts was collected from clinical records and pathology reports. The major clinico-
pathological characteristics and available treatment information of the cohorts is
presented in supplemental Table S1. Due to the lack of extensive follow-up and limited
events on the newer cohort, YTMA128, survival analysis was performed only on
YTMA201. Tissue specimens were included in a TMA format as described (35-36).
Briefly, representative tumor areas were selected in Hematoxylin/Eosin stained
preparations by a pathologist and 0.6-mm cores were obtained using a needle and
arrayed in a recipient block. A control TMA termed YTMA245 was constructed for
reagents titration, PD-L1 mRNA assay validation and reproducibility assessment. This
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index TMA contained FFPE samples from carcinomas, human placenta and
parental/PD-L1-transfected Mel624 cells. Culture conditions and cell-line TMA
construction have been reported elsewhere (35-36).
In situ mRNA hybridization.
In situ detection of PD-L1 transcripts in FFPE TMA samples was performed using the
RNAscope assay with custom-designed in situ hybridization probes (Advanced Cell
Diagnostics, CA, USA) coupled to automated quantitative fluorescence (QIF) detection
as described (33-35). Briefly, 5µm sections were deparaffinized, boiled with pre-
amplification reagent for 15min and submitted to protease digestion followed by
hybridization for 2h with target probes to human PD-L1 mRNA, Ubiquitin C (UbC) as a
positive control or the bacterial gene DapB mRNA as a negative control. Hybridization
signals were detected with Cy5-tyramide. Preparations were then incubated with a
wide-spectrum rabbit anti-cow cytokeratin antibody (clone Z0622 1:100, DAKO Corp,
Carpinteria, CA) in BSA/tris-buffered saline for 1h followed by detection with a
secondary Alexa-546 conjugated goat-anti-rabbit antibody (1:100, Molecular Probes,
Eugene, OR). Slides were mounted using ProlongGold plus 4,6-diamidino-2-
phenylindole (DAPI) to highlight nuclei. Assay specificity was assessed measuring the
signal in positive and negative control samples. Reproducibility was assessed by
staining control preparations on different days using the same protocol and determining
the linear regression coefficients (R2) between such runs.
Automated quantitative fluorescence (QIF).
QIF using the AQUA® method enables objective and sensitive measurement of targets
within user-defined tissue compartments (33-35, 37-38). Briefly, the QIF score of mRNA
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signal in the tumor was calculated by dividing the target mRNA pixel intensities by the
area of the tumor compartment defined by the cytokeratin positivity. Scores were
normalized to the exposure time and bit depth at which the images were captured,
allowing scores collected at different exposure times to be comparable. The
experimenters visually evaluated all acquired histospots and cases with staining
artifacts and/or presence of less than 2% tumor were excluded. As shown in
supplemental Fig. S3, PD-L1 protein was measured using QIF with the validated mouse
monoclonal antibody clone 5h1 as recently reported (35). Detailed description of PD-L1
protein staining is provided in the supplemental methods.
Evaluation of Tumor Infiltrating Lymphocytes.
The scoring of TILs was performed in Hematoxilin/Eosin stained TMA preparations
independently by 2 pathologists (K.A.S. and D.C.) using a four-tiered scale based on
the visual estimation of the amount of lymphocytes in each histospot, as described (39).
A score of 0 indicated virtual absence of TILs, 1+=low TILs (<30%), 2+=moderate (30-
60%), and 3+=marked increase in the lymphocytic infiltrate (>60%). Cases that could
not be appropriately evaluated for technical reasons (e.g. bad staining, low tumor area,
etc) were designated as not evaluable. Spots with discordance in TILs category
between pathologists were reviewed jointly and a single consensus category was
established. In addition, the signals of different TILs subtypes were simultaneously
measured using QIF with a multiplexed immunofluorescence protocol (Fig.S4). Detailed
description of the protocol and reagents is provided in the supplemental methods.
Determination of PD-L1 mRNA positivity.
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The cutoff for PD-L1 mRNA positivity was defined as the noise threshold of the system.
This was determined by using the average QIF score of DapB (negative control
bacterial gene) in situ hybridization in a serial section slide stained in the same batch as
the experimental samples. PD-L1 mRNA scores between experiments were mean-
normalized using the DapB scores from each independent run as reference. Cases with
PD-L1 mRNA signal above the DapB were considered as positive and cases with
scores equal to or lower were considered as negative. Cases with very low UbC
(positive control) QIF scores (<60) were excluded from further analysis to rule out the
possibility of false negative technical results.
Statistical analysis.
Patient characteristics were compared using t-test for continuous variables and chi-
square test for categorical variables. Recurrence free survival (RFS) and disease
specific survival (DSS) functions were compared using Kaplan-Meier estimates and
statistical significance was determined using the log-rank test. A multivariate Cox
proportional hazards model including age, tumor size, lymph node status, estrogen
receptor and HER2 status as co-variates was built. All statistical analyses were
performed using JMP® Pro software (version 9.0.0, 2010, SAS Institute Inc.).
Results
PD-L1 mRNA assay validation.
As shown in supplemental Fig.S1A, PD-L1 mRNA signal was higher in PD-L1-
transfected Mel624 cells than in parental cells and was recognized as multiple relatively
small dots with predominant perinuclear cellular distribution (supplemental Fig.S1A,
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red fluorescence channel). The PD-L1 mRNA QIF score was significantly higher than
DapB (negative control) in FFPE preparations from PD-L1 Mel624 transfectants, but not
in parental Mel624 cells (P<0.001, supplemental Fig.S1A). In samples from human
placenta, PD-L1 mRNA signal was located predominantly in the trophoblastic cell
compartment characterized by intense cytokeratin positivity (supplemental Fig.S1B,
left panel, green fluorescence channel). In contrast, the UbC mRNA signal was
evenly distributed within the epithelial and mesenchymal areas of the chorionic villi
(supplemental Fig.S1B, central panels). As expected, the DapB mRNA signal was
faint throughout the placental tissue, indicating a low background signal (Fig.S1B, right
panels).
In situ PD-L1 mRNA expression in breast cancer.
In breast cancer samples, PD-L1 mRNA signal showed a similar dotted
hybridization pattern and was predominantly located within the tumor (cytokeratin-
positive) compartment (Fig.1A, left panel). As expected, consecutive serial section
samples hybridized with UbC and DapB mRNA probes showed high and low signal,
respectively (Fig.1A, middle and right panels). The cytokeratin signal and staining
pattern were comparable between the serial section specimens.
In YTMA128, 178 spots (75%) were informative for all three mRNA targets and
only 2 cases (0.8%) showed extremely low UbC mRNA scores and were excluded from
the analysis. Ninety-eight cases (55.7%) showed positive PD-L1 mRNA signal (Fig.1B).
In YTMA201, 358 cases (89.5%) were informative and 22 cases (5.5%) were excluded
for low UbC scores. In this cohort, 201 cases (59.5%) showed PD-L1 mRNA levels
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above the detection threshold (Fig.1C). Although the serial section reproducibility of PD-
L1 mRNA assay on YTMA245 was high (R2=0.73, supplemental Fig.S2A), the
regression between measurements in 2 cores from the same tumor block (inter-core
regression) on YTMA201 was considerably lower (R2=0.2, supplemental Fig.S2B),
suggesting that PD-L1 mRNA is a rather heterogeneous marker in breast cancer. Serial
section regression of UbC was comparable to that of PD-L1 mRNA (R2=0.81,
supplemental Fig.S2C), but the inter-core regression was higher using the same
experimental conditions (R2=0.48, supplemental Fig.S2D).
Consistent with previous findings in non-small cell lung cancer (35), breast tumor
cells showed a predominant membranous-like PD-L1 protein staining pattern
(supplemental Fig.S3A) and the levels of PD-L1 mRNA showed a positive non-linear
relationship with PD-L1 protein in both breast cancer collections (regression coefficient
[R] of 0.2 on YTMA128 P=0.01 and 0.16 on YTMA201 P=0.003, supplemental Fig.S3C-
D).
Characterization of lymphocytic infiltrates in breast cancer.
Representative pictures of breast tumors cases showing different levels of TILs
(scores 0-3+, see methods) are depicted in Fig.2A. Both YTMA128 and YTMA201
showed a high proportion of cases with low TILs (scores 0&1+) of 75.6% and 77.7%,
respectively (Fig.2B and C). The number of cases with more prominent lymphocytic
infiltrates (scores 2&3+) was comparably low in both cohorts (16.8% on YTMA128 and
14.8% on YTMA201, Fig.2B and C). Cases with elevated TILs showed significantly
higher PD-L1 mRNA levels in both YTMA128 and YTMA201 (P<0.01, Fig.2D and E).
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The overall concordance for categorical TILs status determination between pathologists
was 93% in YTMA128 (κ=0.77) and 95% in YTMA201 (κ=0.83).
Characterization of TILs subpopulations using multiplexed QIF revealed that
cases from YTMA128 showing PD-L1 mRNA expression had 14.3% more CD3 signal,
21% more CD20 signal and 8.8% higher CD8 signal than PD-L1 mRNA negative tumors
(supplemental Fig.S4C). Similarly, PD-L1 mRNA expressing samples from YTMA201
showed 18.3% higher CD3 signal, 19.2% more CD20 and only 2.6% change in CD8
fluorescence (Fig.S4D).
Clinico-pathological associations of PD-L1 mRNA status and TILs breast
carcinomas.
In both YTMA128 and YTMA201, PD-L1 mRNA expression was significantly
associated with the presence of elevated TILs (scores 2&3+, P=0.04, Table 1), but not
with age, tumor size, lymph node positivity, histological grade, estrogen receptor
positivity and HER2 status. The presence of elevated TILs was significantly associated
with ER negative status in both YTMA128 and YTMA201 (P=0.01 and P=0.001,
respectively, Table 2). The presence of prominent lymphocytic infiltrates was also
significantly associated with younger age at diagnosis in YTMA128 (P=0.006, Table 2).
The latter association was also apparent in YTMA201, but did not reach statistical
significance (P=0.08, Table 2).
Association of PD-L1 mRNA status and TILs with survival in breast cancer
patients.
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In patients from YTMA201 with a relatively prolonged follow-up (median follow-
up= 139 months, supplemental Table S1), expression of PD-L1 mRNA in the tumor
compartment was significantly associated with longer recurrence free survival (log rank
P=0.01, Fig.3A). The presence of elevated TILs in the TMA spot was also associated
with longer recurrence free survival, although without reaching statistical significance
(log rank P=0.07, Fig.3B). Concordant with PD-L1 mRNA, elevated levels of PD-L1
protein were also significantly associated with longer recurrence free survival in
YTMA201 (Wimberly et al., 2013, SABCS December 2013, manuscript in preparation).
The presence of increased TILs was not significantly associated with the risk of
recurrence in multivariate analysis (HR=0.765 [CI=0.188-2.548], P=0.67). In the
multivariate model, PD-L1 mRNA expression in breast tumors from YTMA201 was
significantly associated with lower risk of recurrence (HR=0.268 [CI=0.099-0.721],
P=0.009, Table 3) and this association was independent from age, tumor size, lymph
node status, histological grade, ER status, HER2 status and the presence of low or high
TILs. As expected, larger tumor size (>2 cm) and tumor involvement of lymph nodes
were significantly associated with higher recurrence risk (HR=2.872 [CI=0.865-6.919],
P=0.02 and HR=5.305 [CI=1.961-14.878], P=0.001, respectively, Table 3). In addition,
PD-L1 mRNA expression or the presence of elevated TILs showed a mild, non-
statistically significant association with longer disease specific survival ([DSS]log rank
P=0.16 and P=0.06, respectively, Fig.3C-D).
Using the online biomarker validation tool SurvExpress (40 [see Fig. S5 and
supplemental methods]) containing information from 30 published breast cancer
mRNA datasets, we identified 6 cohorts including PD-L1 mRNA records. Of them, only
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1 had recurrence annotations and increased PD-L1 mRNA was significantly associated
with longer recurrence free survival (Wang-Richardson GSE19615, P=0.02;
supplemental Fig.S5A-B). The other five datasets had only overall survival data and
high PD-L1 mRNA was marginally significantly associated with longer overall survival in
one of them (Kao-Huang GSE20685, P=0.05; Fig.S5C-D). In the other 4 cohorts
elevated PD-L1 mRNA trended, but was not significantly associated with overall survival
(TCGA, Ma-Sgroi GSE1378, Miller-Bergh GSE3494 and Enerly-Yakhini GSE19536; not
shown).
Discussion
Recent studies suggest that determination of PD-L1 status in tumor samples could help
select patients for novel anti PD-1/PD-L1 monoclonal antibody therapies (19). However,
accurate determination of PD-L1 protein levels in FFPE tumor samples is limited by the
absence of validated assays, reliable antibodies and interpretative uncertainties (e.g.
cutoff for positivity, marker heterogeneity). We describe herein a reproducible, antibody-
independent assay for in situ PD-L1 mRNA measurement. A major strength of this
method relies in the simultaneous measurement of a negative control indicator (DapB)
that helps identifying the assay detection threshold; and a positive control probe (UbC)
used to exclude inadequate samples. Our data demonstrate that in situ PD-L1 mRNA
expression can be measured in FFPE breast tumor samples and in TMAs. We also
observed substantial variability between cores in PD-L1 expression, which will need to
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be taken into consideration when interpreting results from a single core biopsy of a large
tumor. A limitation of our results is the lack of mRNA assessment by RT-PCR or array-
based methods to establish concordance and quantification across mRNA
measurement platforms. An advantage of the RNAscope technique is that it provides in
situ measurement and can quantify mRNA in the epithelial cells in the TMA spot. Since
all other mRNA measurement methods grind the sample to produce the mRNA, from
these methods the cellular source of the mRNA cannot be determined. However, we
could validate the prognostic effect of PD-L1 mRNA expression in publicly available
breast cancer mRNA datasets (Fig.S5). Overall and despite the methodological
differences, the results are consistent with our findings measuring PD-L1 mRNA in situ.
A number of smaller studies have been published that assess expression levels
of PD-L1 in breast cancer using immunohistochemistry. A study by Ghebeh et al. (14)
including 44 specimens with available frozen tissue and using the mouse monoclonal
MIH1 clone for PD-L1 IHC, showed absence of signal in normal breast and expression
of PD-L1 in 22 cases (50%). Fifteen of these cases showed signal in tumor cells with
membranous/cytoplasmic pattern and were associated with higher histologic grade and
hormone-receptor negativity. PD-L1 positive TILs were found in 18 of these cases
(41%) and were associated with larger tumors, histologic grade III, HER2 positivity and
increased inflammatory infiltrates (14). In a subsequent study from the same group and
using an expanded version of the cohort (N=68), similar associations were found (22).
Our results using mRNA measurements show that nearly 60% of breast carcinomas
express PD-L1 transcripts and this was similar in two independent cohorts. However, no
significant association with high grade/hormone receptor negativity was found. This
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difference might be due, at least in part, to the distinct properties and independent
information provided by PD-L1 protein and mRNA molecules, as reported (33).
Methodological differences might also account for this apparent inconsistency. In fact,
the MIH1 clone used in some studies failed validation using western blot and IHC in our
laboratory together with 2 other commercially available antibodies (35). The same
antibody clone was reported by others not to be suitable for FFPE samples (23).
Our results show that tumor PD-L1 mRNA expression is significantly associated with
increased local immune cell infiltrates and with longer recurrence free survival.
Moreover, the survival effect of PD-L1 mRNA was independent from other well-known
prognostic factors such as tumor size, lymph node involvement and hormone-
receptor/HER2 status; suggesting that PD-L1 mRNA is independently associated with
favorable prognosis in breast cancer. However, these results are not in agreement with
previous studies showing an adverse prognostic effect of PD-L1 expression by IHC in
various malignancies including melanoma, renal, urothelial, gastric, lung and colorectal
carcinomas (24-28).. In contrast, our observations are consistent with 3 recent studies in
metastatic melanomas, Merkel-cell carcinomas and lung non-small cell carcinomas
using the validated monoclonal antibody clone 5H1 and showing association of PD-L1
expression with TILs and longer survival (30, 31, 35). In addition, a recent study found
positive association between PD-L1 IHC expression, presence of CD8-positive TILs and
overall survival using 2 different antibodies (monoclonal clone-27A2 from MBL and
polyclonal ab82059 from Abcam) in a large TMA-based colorectal cancer cohort (32).
The biological determinants of the association between PD-L1 expression, increased
TILs and better outcome are not well understood. For instance, tumor infiltration by
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CD8-positive T lymphocytes was recently shown to be associated with Tregs, PD-L1
protein and mRNA and IDO expression in human metastatic melanomas; and induction
of these immune inhibitory pathways in the tumor microenvironment required and was
mediated by CD8-positive T cells and IFN-γ in a murine model (41). Therefore, it is
possible that rather than an indication of total immune-evasion, expression of PD-L1 by
tumor cells might reflect the presence of antigenic-induced anti-tumor immune pressure
mediated by TILs. Although partially ineffective, recruitment of TILs to the tumor
microenvironment due to preserved chemotactic signals, could still induce a partial anti-
tumor effect and explain the observed survival benefit. Further studies will be required
to clarify this in other solid tumor models and carefully weigh the effect of additional
active co-stimulatory pathways.
Our data also show that the presence of elevated TILs was significantly
associated with hormone receptor-negative tumors and with a clear trend towards
longer survival (Fig.3B-D). Similar findings have been reported by others (3-5) and point
to the critical role of local immunity in limiting tumor progression, particularly in more
aggressive triple negative and basal like breast neoplasms. In addition, increased TILs
have also been shown to predict response to neoadjuvant chemotherapy in breast
cancer (42-43). However, our results show that the proportion of breast tumors with
prominent lymphocytic infiltration is relatively low (~15% of cases) and the prognostic
information provided by TILs is limited.
Our analysis has a number of limitations. One major limitation is that it includes
only retrospectively collected cases and that mature survival information was only
available for one cohort. A second issue is that the use of TMAs may underestimate or
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18
overestimate the mRNA markers expression due to intra-tumoral heterogeneity of
expression. Assays in the clinic always use whole slides, and examination of high
number of fields seen in a histologic section may attenuate the sampling effect of TMAs.
Given these limitations, the results of this study should be considered as hypothesis
generating. Although our results support the value of measuring PD-L1 in TMA
samples, translation of these findings into the clinical setting could certainly benefit from
using whole tissue section samples. This could better reflect the tissue distribution of
PD-L1 and its topographical relationships with tumor cells and TILs. Determination of
the amount of tumor tissue necessary to accurately measure PD-L1 in patient samples
requires further evaluation, but should likely consider the tumor size, availability of
sample material (core vs tumor resection), clinical purpose (prognostic vs predictive;
adjuvant vs neoadjuvant) and disease stage (one primary vs multiple primaries vs
disseminated disease).
One key unaddressed issue is the potential of PD-L1 mRNA, alone or in
combination with other markers such as PD-L1 protein, to predict response to anti-PD-
1/PD-L1 therapies. Ongoing studies measuring PD-L1 mRNA and protein levels using
QIF in whole tissue sections specimens from patients treated with anti-PD-1 therapy
might provide a better substrate to conclusively address the prognostic/predictive value
of these markers.
Acknowledgements:
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The authors acknowledge Lori Charette and the Yale Pathology Tissue Services for
production of the high quality TMAs used in this study. This study was supported by the
Breast Cancer Research Foundation (DLR and LP) and a Novartis/Genoptix sponsored
research agreement (DLR).
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Figure and tables.
Figure legends.
Figure 1. PD-L1 mRNA expression in breast cancer samples and distribution of
scores in two cohorts. A) Representative fluorescence microphotographs showing
PD-L1, UbC and DapB mRNA signal in consecutive sections from one PD-L1 mRNA
positive breast tumor from YTMA201 (histospot 263). PD-L1 positivity showed a micro-
dotted staining pattern (inset) and the signal was allocated predominantly in the tumor
compartment (red channel, upper panels), as evident in the pancytokeratin positive area
(green channel, lower panel). Nuclei were stained with DAPI. Bar=100 µm. B-C)
Distribution of PD-L1 mRNA scores in cases from YTMA128 (B) and YTMA201 (C). The
dashed black line indicates the DapB cutoff for positive/negative results. Values are
expressed as arbitrary units of fluorescence.
Figure 2. Presence of inflammatory infiltrates in breast carcinomas. A)
Representative microphotographs of hematoxilin/eosin preparations from breast cancer
samples showing different TILs categories (TILs scores 0-3+). B-C) Graphs showing the
number and proportion of cases in each TILs category on YTMA128 (B) and YTMA201
(C). NE= not evaluable (see methods section). D-E) PD-L1 mRNA scores in cases with
low TILs (score 0&1+) and high TILs (score 2&3+) from YTMA128 (D) and YTMA201
(E). Chart shows mean ± S.E.M. of QIF scores and the number within each bar
indicates the amount of cases in each group.
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Figure 3. PD-L1 mRNA positivity is associated with better outcome in breast
carcinomas. A-D) Kaplan-Meier graphical analysis of the recurrence free survival
(RFS) and disease specific survival (DSS) in patients with breast cancer from YTMA201
according to PD-L1 mRNA status (A and C) or the amount of TILs (B and D). The
number of subjects at risk in each group are indicated below the chart. The respective
log rank P values, hazards ratios (HR) and confidence intervals (CI) are indicated within
each chart.
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Published OnlineFirst March 19, 2014.Clin Cancer Res Kurt A Schalper, Vamsidhar Velcheti, Daniel Carvajal, et al. increased TILs and better outcome in breast carcinomas.In situ Tumor PD-L1 mRNA expression is associated with
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