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2017, Volume 33, Number 3, Page(s) 192-197
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DOI: 10.5146/tjpath.2017.01395 |
Programmed Death Ligand 1 (PD-L1) Expression in Malignant Mesenchymal Tumors |
Kemal KÖSEMEHMETOĞLU1, Ece ÖZOĞUL1, Berrin BABAOĞLU1, Gaye GÜLER TEZEL1,2, Gökhan GEDİKOĞLU1 |
1Department of Pathology, Research and Application Center, Hacettepe University, Faculty of Medicine, ANKARA, TURKEY 2Department of Molecular Pathology, Research and Application Center, Hacettepe University, Faculty of Medicine, ANKARA, TURKEY |
Keywords: Sarcoma, Immunotherapy, Immunohistochemistry, Quantitative PCR |
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Objective: Programmed death ligand 1 (PD-L1) found on tumor cells has recently been reported to have a key role in the development and
dissemination of many tumors, such as lung and breast carcinomas. In this study, we retrospectively analyzed PD-L1 expression among different
types of sarcomas.
Material and Method: Tissue microarrays of 3-4 mm diameter were composed from paraffin blocks of 222 various sarcomas. Slides prepared
from microarrays were stained for PD-L1 antibody (Cell Signaling, E1L3N®) using Leica Bond Autostainer. Any membranous staining over 5% of
the cells was regarded as positive. Quantitative real-time PCR with TaqMan gene expression assays for PDL1 was performed using whole sections
from FFPE tissue of PD-L1 positive cases, by normalizing absolute values to β-actin. Relative expression level of mRNA of PDL1 was calculated
and scored using Log102(threshold cycle of b-actin - threshold cycle of PDL1).
Results: Immunohistochemically, PD-L1 expression was present in 34 of 222 (15%) sarcomas. 5/13 (39%) undifferentiated pleomorphic sarcomas,
6/18 (33%) malignant peripheral nerve sheath tumors, 5/16 (31%) dedifferentiated liposarcomas, 4/19 (21%) rhabdomyosarcomas, 2/16 (13%)
epithelioid sarcomas, 2/15 (13%) leiomyosarcomas, 3/26 (12%) synovial sarcomas, 1/18 (6%) myxoid liposarcoma, 1/2 (50%) extraskeletal
myxoid chondrosarcoma, 1/3 (33%) alveolar soft part sarcoma, 1/3 (33%) parachordoma/myoepithelioma, 1/5 (20%) pleomorphic liposarcoma,
1/7 (14%) angiosarcoma, 1/8 (13%) Ewing sarcoma showed PD-L1 expression. Cases of solitary fibrous tumor/hemangiopericytoma (18),
desmoplastic round cell tumor (14), Ewing-like sarcoma (6), epithelioid hemangioendothelioma (5), clear cell sarcoma (4), myxofibrosarcoma
(4), low grade fibromyxoid sarcoma (2) were all negative. Tumor-infiltrating hematopoietic cells were positive for PD-L1 in 32 cases (15%)
with only 2 cases overlapping with PD-L1 staining in tumoral cells. Sixteen of 34 (47%) immunohistochemically PD-L1 positive cases showed
significant but low-level PD-L1 mRNA overexpression.
Conclusion: We have shown PD-L1 expression in a subset of sarcomas, both at the protein and mRNA level. High-grade pleomorphic sarcomas
tend to show more frequent PD-L1 expression. Clinical trials are necessary to further assess the effect of anti PD-L1 drugs on sarcomas showing
PD-L1 expression. |
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PD-L1, CD274 or B7 homolog 1 (B7-H1) is a 40 kD type 1
transmembrane protein found on activated T cells, B cells
and myeloid lineage members plays an important role in
regulation of cellular immune response 1. Activation of
PD-1/PD-L1 complex leads to inhibition of antigen specific
T cell proliferation and induces apoptosis of these antigen
specific T cells. It is hypothesized that PD-L1 expression in
neoplastic cells enhances its disguise ability to escape from
the immune system. Many studies have shown increased
expression of PD-L1 ligand in various cancers arising from
the lung, skin, ovary, cervix, esophagus, breast, bladder,
brain, bone, kidney, and liver 2-7.
Management of primary sarcoma is basically surgery, while
chemotherapy or radiotherapy has only marginal effect on
metastatic sarcomas. Immunotherapy with anti PD-L1 monoclonal antibodies is a promising treatment option in
the management of metastatic sarcoma. Therefore, in this
study we investigated the presence of PD-L1 expression
among sarcomas at both the protein and RNA expression
level. |
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Abstract
Introduction
Methods
Results
Disscussion
References
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Slides of 222 cases of various sarcomas were retrieved from
the archive and the diagnoses were reviewed (KK). Twentysix
synovial sarcomas, 19 rhabdomyosarcomas, 18 myxoid
liposarcomas, 18 malignant peripheral nerve sheath
tumors, 18 solitary fibrous tumor/hemangiopericytomas,
16 dedifferentiated liposarcomas, 16 epithelioid sarcomas,
15 leiomyosarcomas, 14 desmoplastic round cell tumors, 13
undifferentiated pleomorphic sarcomas, 8 Ewing sarcomas,
6 Ewing-like sarcomas, 7 angiosarcomas, 5 epithelioid
hemangioendotheliomas, 5 pleomorphic liposarcomas, 4 clear cell sarcomas, 4 myxofibrosarcomas, 3 alveolar soft
part sarcomas, 3 parachordoma/myoepiheliomas, 2 extraskeletal
myxoid chondrosarcomas, 2 low grade fibromyxoid
sarcomas were included in the study. Bone tumors, such as
osteosarcomas and chondrosarcomas, and mesenchymal
tumors with indolent clinical behavior, such as atypical
lipomatous tumor/well differentiated liposarcoma were
excluded.
Representative areas were selected and 3-4 mm diameter
tissue microarrays were composed from paraffin blocks.
Microarray slides were stained for PD-L1 antibody (Cell
Signaling, E1L3N®, 1/400) using Leica Bond Autostainer,
according to the manufacturer’s instructions. Antigen
retrieval was performed with EDTA pretreatment for 20
minutes. Any membranous staining over 5% of the cells
was regarded as positive. PD-L1 expression in tumor
infiltrating inflammatory cells was also noted.
Quantitative real-time PCR with Fast-Plus EvaGreen Master
Mix (Cat No:31020, Biotium) and gene expression assays
for CD274 was performed using whole sections from FFPE
tissue of CD274 positive cases, by normalizing absolute
values to β-actin. Total RNA was extracted using Hybrid-R
RNA purification kit (GeneAll Biotechnology Co. Ltd.)
according to the manufacturer protocol. RNA samples were
quantified using the highly sensitive Qubit quantitation
assays (Thermo Fisher Scientific). Equal amounts of
RNA were reverse transcribed using a HyperScriptTM
Reverse Transcriptase (GeneAll Biotechnology Co. Ltd.)
with oligod (T) primers according to the manufacturer’s
instruction (Table I) PCR was performed with Fast-Plus
EvaGreen gene expression master mix, cDNA, nuclease
free water, forward and reverse primers in a 20μl final
reaction volume. The amplification cycles were performed
by the Rotor Gene - Q System (Qiagen) as follows, hold
at 95°C for 3 min, followed by amplification for 40 cycles,
each cycle consisting of denaturation at 95°C for 5 s,
annealing at 55°C for 30 s. The housekeeping gene β-actin
(Table I) expression level was used as an internal control to
evaluate the integrity of each sample. Relative expression
level of mRNA of PDL1 was calculated and scored using
Log102(threshold cycle of b-actin - threshold cycle of PDL1). Relative expression
was scored on a log10 scale (0–2).
 Click Here to Zoom |
Table I: The forward and reverse primers used in the real-time
polymerase chain reaction analyses for CD274 and β-Actin
genes. |
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Abstract
Introduction
Methods
Results
Disscussion
References
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Neoplastic cells showed PD-L1 expression in 34 of 222
(15.3%) sarcomas (Table II). 5/13 (39%) undifferentiated
pleomorphic sarcomas, 6/18 (33%) malignant peripheral
nerve sheath tumors, 5/16 (31%) dedifferentiated
liposarcomas, 4/19 (21%) rabdomyosarcomas, 2/16 (13%) epithelioid sarcomas, 2/15 (13%) leiomyosarcomas, 3/26
(12%) synovial sarcomas, 1/18 (6%) myxoid liposarcoma,
1/2 (50%) extraskeletal myxoid chondrosarcoma, 1/3
(33%) alveolar soft part sarcoma, 1/3 (33%) parachordoma/
myoepithelioma, 1/5 (20%) pleomorphic liposarcoma,
1/7 (14%) angiosarcoma, and 1/8 (13%) Ewing sarcoma
showed PD-L1 expression (Figure 1). Solitary fibrous
tumor/hemangiopericytomas (n=18), desmoplastic round
cell tumors (n=14), Ewing-like sarcomas (n=6), epithelioid
hemangioendotheliomas (n=5), clear cell sarcomas (n=4),
myxofibrosarcomas (n=4), and low-grade fibromyxoid
sarcomas (n=2) were all negative.
PD-L1 expression in tumor infiltrating inflammatory cells
was present in 32 of 222 (14.4%) sarcomas (Figure 2A-C).
Only 4 of 34 (11.8%) PD-L1 positive sarcomas, 3 of which
were dedifferentiated liposarcomas, also had PD-L1 positive
inflammatory cells within the neoplasm (Table III).
 Click Here to Zoom |
Figure 2: A) Both tumor cells and tumor infiltrating inflammatory
cells were positive in a dedifferentiated liposarcoma (PD-L1;
x200). B-C) In 13% of cases, only inflammatory cells were positive
(PD-L1;x400). |
 Click Here to Zoom |
Table III: PD-L1 expression in tumor and accompanying inflammatory cells |
Quantitative real-time PCR revealed that 16 of 34 (47%)
cases showed significant (>1 log) but low-level PD-L1 mRNA overexpression (Figure 3). None of the cases
showed >2 log mRNA expression.
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Abstract
Introduction
Methods
Results
Disscussion
References
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With this sarcoma series investigating PD-L1 expression
with 222 cases, we have shown that a small subset (~15%)
of sarcomas express PD-L1 in neoplastic cells. The highest
PD-L1 expression among sarcomas was reported from the Republic of Korea: In one series of 105 sarcomas, PD-L1
was positive in 65% of the cases 8, and more recently,
Kim et al. found that 43% of 82 sarcomas expressed PDL1 9. This relatively high PD-L1 expression prevalence
may be possibly due to the use of different antibodies
(R&D System antibody, clone 130021 [polyclonal] and
Santa Cruz Biotechnology, clone H-130 [monoclonal]),
cut-off levels (10% cut-off and combined score above 8),
staining methods or retrieval technique 8,9. It is known
that antibodies against PD-L1 are not identical and the
use of some clones are not recommended 10. High
levels of PD-L1 expression from Korea may also indicate
the possible impact of race on differential expression of
immunomodulatory mechanisms. Similar to our results,
expression prevalence seems to be lower in the Western
population. In a comprehensive immunohistochemical
analysis of PD-L1 in 5536 cases, 59 of 986 (6%) demonstrated
PD-L1 expression over 5% of tumor cells 11. D’Angelo et
al. 12 and Matthew et al. 13 from USA reported that
12% of 50 sarcomas and 20% of 85 sarcomas were positive
for PD-L1, respectively. Similarly, Paydas et al. from
Turkey recorded a PD-L1 positivity of 29% of 65 cases 14. Although we used tissue microarrays unlike many
of the previous studies, our result of 15.3% is comparable
with previous results of D’Angelo et al., Matthew et al.,
and Paydas et al., that used DAKO antibody with 1% cutoff,
Cell Signaling antibody with 5% cut-off, and Acris
Antibody with 5% cut-off, respectively. We believe that a
relatively lower figure of 6% by Inaguma et al. is mainly
due to the absence of cases diagnosed as undifferentiated
pleomorphic sarcomas and dedifferentiated liposarcomas
in their series 11.
Malignant mesenchymal tumors showing predominantly
pleomorphic morphology, such as undifferentiated
pleomorphic sarcomas, malignant peripheral nerve sheath
tumors, and dedifferentiated liposarcomas demonstrated
more PD-L1 expression (over 30% of the cases) than other
types of sarcomas. In line with this, in a study including
53 MPNSTs, 13% were shown to express PD-L1 in tumor
cells15, while in others 25% and 21% of MPNSTs were
PD-L1 positive11,14. Some studies also confirmed
a significant correlation between PD-L1 expression
and advanced clinicopathological parameters, such as
higher clinical stage, presence of distant metastasis, as
well as higher histological grade, poor differentiation of
tumor, and tumor necrosis8,9. Moreover, both PD1-
and PD-L1 positivity were reported to be independent
prognostic factors for overall survival by multivariate
analysis; cases with a PD1+/PD-L1+ phenotype had the
worst prognosis, which may benefit from PD1-based immunotherapy8,9. Besides, few low-grade sarcomas,
such as myxoid liposarcoma without a high-grade round
cell component also showed PD-L1 expression. Knowing
ththat chemotherapy and radiotherapy has very limited
effect on low grade sarcomas, anti-PD-L1 treatment may
be the best option to prevent progression in metastatic low
grade sarcomas showing PD-L1 expression. Larger series
are required to assess the prevalence of PD-L1 expression
among extraskeletal myxoid chondrosarcomas, alveolar
soft part sarcomas, and parachordoma/myoepitheliomas
as PD-L1 was positive in a significant percent of the low
number of cases. Unfortunately, various sarcoma types
including solitary fibrous tumor/hemangiopericytoma,
desmoplastic round cell tumor, Ewing-like sarcoma,
epithelioid hemangioendothelioma, clear cell sarcoma,
myxofibrosarcoma, and low-grade fibromyxoid sarcoma,
many of which are high-grade by definition, did not
express PD-L1, thus having less potential to benefit from
anti-PD-L1 therapies.
Expression of PD-L1 in tumor-infiltrating lymphocytes
and macrophages was previously documented in sarcomas in detail8,12. Inflammatory cells are present
in almost all of the sarcomas, and 30% of lymphocytes
and 58% of macrophages expressed PD-L1 (12). Paydas
et al. also recorded that 30% of cells forming tumor
microenvironment expressed PD-L114. Our result of
14.4% PD-L1 positive tumor-infiltrating inflammatory
cells seems to be lower than expected, possibly due to the
use of tissue microarrays instead of whole sections. We
found a very low level (4%) of co-expression of PD-L1 in
both tumor and tumor infiltrating inflammatory cells. In
the series of Paydas et al., 21% of the sarcomas showed PDL1
expression in both the tumor microenvironment and
tumor cells; and Kaposi sarcoma, not surprisingly, was the
most common sarcoma with 80% of the cases showing this
phenotype14. This high level of co-expression could be
attributed to the phenomenal role of the immune system
on the development and regression of this tumor.
Previously, PD-L1 expression was also demonstrated in
both mRNA and protein level in a subset of osteosarcomas7. In their study, 9 samples had high-level expression
(24%) with over 3 log fold increase, 19 samples (50%) had intermediate expression, 4 (10%) had low-level expression,
and 6 (16%) were negative. More recently, only 5 of 89
(5.6%) non-small cell lung carcinomas were shown to have
a PD-L1 gene amplification, which is associated with worse
prognosis16. In our series, immunohistochemically PDL1
positive cases showed a low to intermediate level of
PD-L1 mRNA expression by quantitative PCR. Our results
indicate that overexpression of PD-L1 protein is mainly
modulated by post-translational mechanisms rather than
at the transcriptional level, and the mechanism of PD-L1
overexpression differs among different kind of tumors.
In conclusion, we have shown PD-L1 expression in a subset
of sarcomas both on the protein and mRNA level. Highgrade
sarcomas including undifferentiated pleomorphic
sarcomas, dedifferentiated liposarcomas and MPNSTs tend
to show more frequent PD-L1 expression. Quantitative
PCR results indicate the influence of post-translational
modifications rather than transcriptional level as the
possible mechanisms of PD-L1 overexpression in sarcomas.
The results of ongoing clinical trials are necessary to further
assess the effect of anti PD-L1 drugs on sarcomas showing
PD-L1 expression.
ACKNOWLEDGEMENTS
The authors thank to Elif Nihan Çetin for her technical
support. This study was supported by the Hacettepe
University Scientific Research Unit, Grant number: THD-
2015-5202. |
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Abstract
Introduction
Methods
Results
Discussion
References
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|
1) Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL. SHP-
1 and SHP-2 associate with immunoreceptor tyrosine-based
switch motif of programmed death 1 upon primary human T cell
stimulation, but only receptor ligation prevents T cell activation.
J Immunol. 2004;173:945-54.
2) Velcheti V, Rimm DL, Schalper KA. Sarcomatoid lung
carcinomas show high levels of programmed death ligand-1 (PDL1).
J Thorac Oncol. 2013;8:803-5.
3) Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, Syrigos
KN, Sznol M, Herbst RS, Gettinger SN, Chen L, Rimm DL.
Programmed death ligand-1 expression in non-small cell lung
cancer. Lab Invest. 2014;94:107-16.
4) Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y,
Yamaguchi K, Higuchi T, Yagi H, Takakura K, Minato N,
Honjo T, Fujii S. Programmed cell death 1 ligand 1 and tumorinfiltrating
CD8+ T lymphocytes are prognostic factors of human
ovarian cancer. Proc Natl Acad Sci U S A. 2007;104:3360-5.
5) Hamanishi J, Mandai M, Matsumura N, Abiko K, Baba T, Konishi
I. PD-1/PD-L1 blockade in cancer treatment: Perspectives and
issues. Int J Clin Oncol. 2016;21:462-73
6) Thompson RH, Gillett MD, Cheville JC, Lohse CM, Dong H,
Webster WS, Krejci KG, Lobo JR, Sengupta S, Chen L, Zincke H,
Blute ML, Strome SE, Leibovich BC, Kwon ED. Costimulatory
B7-H1 in renal cell carcinoma patients: Indicator of tumor
aggressiveness and potential therapeutic target. Proc Natl Acad
Sci U S A. 2004;101:17174-9.
7) Shen JK, Cote GM, Choy E, Yang P, Harmon D, Schwab J,
Nielsen GP, Chebib I, Ferrone S, Wang X, Wang Y, Mankin H,
Hornicek FJ, Duan Z. Programmed cell death ligand 1 expression
in osteosarcoma. Cancer Immunol Res. 2014;2:690-8.
8) K im JR, Moon YJ, Kwon KS, Bae JS, Wagle S, Kim KM, Park
HS, Lee H, Moon WS, Chung MJ, Kang MJ, Jang KY. Tumor
infiltrating PD1-positive lymphocytes and the expression of PDL1
predict poor prognosis of soft tissue sarcomas. PLoS One.
2013;8:e82870.
9) K im C, Kim EK, Jung H, Chon HJ, Han JW, Shin KH, Hu H, Kim
KS, Choi YD, Kim S, Lee YH, Suh JS, Ahn JB, Chung HC, Noh
SH, Rha SY, Kim SH, Kim HS. Prognostic implications of PDL1
expression in patients with soft tissue sarcoma. BMC Cancer.
2016;16:434.
10) McLaughlin J, Han G, Schalper KA, Carvajal-Hausdorf D,
Pelekanou V, Rehman J, Velcheti V, Herbst R, LoRusso P,
Rimm DL. Quantitative assessment of the heterogeneity of
PD-L1 expression in non-small-cell lung cancer. JAMA Oncol.
2016;2:46-54.
11) Inaguma S, Wang Z, Lasota J, Sarlomo-Rikala M, McCue PA,
Ikeda H, Miettinen M. Comprehensive immunohistochemical
study of programmed cell death ligand 1 (PD-L1): Analysis
in 5536 cases revealed consistent expression in trophoblastic
tumors. Am J Surg Pathol. 2016;40:1133-42.
12) D’Angelo SP, Shoushtari AN, Agaram NP, Kuk D, Qin LX,
Carvajal RD, Dickson MA, Gounder M, Keohan ML, Schwartz
GK, Tap WD. Prevalence of tumor-infiltrating lymphocytes and
PD-L1 expression in the soft tissue sarcoma microenvironment.
Hum Pathol. 2015;46:357-65.
13) Matthew S, Mackinnon AC. Programmed Death-Ligand 1
expression in sarcomas, a clinical a pathologic study [abstract].
Modern Pathology. 2016;29 (suppl 2):22A-23A.
14) Paydas S, Bagir EK, Deveci MA, Gonlusen G. Clinical and
prognostic significance of PD-1 and PD-L1 expression in
sarcomas. Med Oncol. 2016;33:93.
15) Shurell E, Singh AS, Crompton JG, Jensen S, Li Y, Dry S, Nelson
S, Chmielowski B, Bernthal N, Federman N, Tumeh P, Eilber
FC. Characterizing the immune microenvironment of malignant
peripheral nerve sheath tumor by PD-L1 expression and
presence of CD8+ tumor infiltrating lymphocytes. Oncotarget.
2016;7:64300-8.
16) Ikeda S, Okamoto T, Okano S, Umemoto Y, Tagawa T, Morodomi
Y, Kohno M, Shimamatsu S, Kitahara H, Suzuki Y, Fujishita T,
Maehara Y. PD-L1 is upregulated by simultaneous amplification
of the PD-L1 and JAK2 genes in non-small cell lung cancer. J
Thorac Oncol. 2016;11:62-71. |
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Abstract
Introduction
Methods
Results
Discussion
References
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