in situ tumor pd-l1 mrna expression is associated with ......2014/03/19 · wide-spectrum rabbit...

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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

Research. on January 5, 2021. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 19, 2014; DOI: 10.1158/1078-0432.CCR-13-2702

http://clincancerres.aacrjournals.org/

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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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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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