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PD-L1 expression and CD8 positive lymphocytes in human neoplasms: A tissue microarray study on 11,838 tumor samples



Programmed death ligand 1 (PD-L1) is the target of immune checkpoint inhibitor therapies in a growing number of tumor types, but a unanimous picture on PD-L1 expression across cancer types is lacking.


We analyzed immunohistochemical PD-L1 expression in 11,838 samples from 118 human tumor types and its relationship with tumor infiltrating CD8 positive lymphocytes.


At a cut-off level of 10% positive tumor cells, PD-L1 positivity was seen in 85 of 118 (72%) tumor types, including thymoma (100% positive), Hodgkin’s lymphoma (93%), anaplastic thyroid carcinoma (76%), Kaposi sarcoma (71%), sarcomatoid urothelial carcinoma (71%), and squamous cell carcinoma of the penis (67%), cervix (65%), floor of the mouth (61%), the lung (53%), and pharynx (50%). In immune cells, PD-L1 positivity was detectable in 103 (87%) tumor types, including tumors of haematopoetic and lymphoid tissues (75% to 100%), Warthin tumors of the parotid glands (95%) and Merkel cell carcinoma (82%). PD-L1 positivity in tumor cells was significantly correlated with the number of intratumoral CD8 positive lymphocytes across all tumor types as well as in individual tumor types, including serous carcinoma of the ovary, invasive breast carcinoma of no special type, intestinal gastric adenocarcinoma, and liposarcoma (p< 0.0001 each).


PD-L1 expression in tumor and inflammatory cells is found in a wide range of human tumor types. Higher rates of tumor infiltrating CD8 positive lymphocytes in PD-L1 positive than in PD-L1 negative cancers suggest that the antitumor immune response may trigger tumoral PD-L1 expression.


Immune checkpoint inhibitor (CPI) therapies targeting the programmed death 1/programmed death ligand 1 (PD-L1) pathway are increasingly employed in a growing number of tumor types [1]. However, not all patients react favorably to these drugs. PD-L1 immunohistochemistry is often applied to select patients with high likelihood to respond favorably to checkpoint inhibitors but criteria for “PD-L1 positivity” vary between tumor types and sometimes also between drugs. The proportion of PD-L1 positive tumor cells (tumor proportion score, TPS), the percentage of positive immune cells (immune cell score; ICS) or the combination of both (combined positivity score; CPS) are applied at different thresholds to define positive cases [2]. The significant role of PD-L1 for the immune microenvironment of tumors is illustrated by associations between PD-L1 expression in tumor cells and elevated numbers of intratumoral CD8 positive cytotoxic T-lymphocytes which were found in several tumor types [3, 4, 5, 6].

More than 2,800 studies have analyzed cancers of various types for PD-L1 expression by immunohistochemistry. For most tumor types, however, the reported frequencies of PD-L1 positivity vary quite considerably. For example, the reported rate of PD-L1 positivity ranges from 0–92% in prostate cancer [7, 8], 1.7%–75% in breast cancer [9, 10], 5.5–89% in colorectal cancer [11, 12], 22–68% in head & neck squamous cell carcinomas [13, 14], 5.2–65% in stomach cancer [15, 16], 3.9–63% in small cell lung cancer [17, 18], 3.1–82% in liver cell carcinomas [19, 20], 17–72% in malignant mesothelioma [21, 22], 10–92% in malignant melanoma [23, 24], 0–100% in chondrosarcoma [24, 25], 0–100% in liposarcoma [24, 26], and 7–100% in angiosarcoma [19, 27]. Technical factors, staining protocols, antibodies used, definitions of thresholds to determine positivity, as well as a possible selection bias with respect to the analyzed tumors have been proposed as causes for these discrepancies. To better understand the relative importance of PD-L1 expression in different tumor types and its relationship with T-lymphocyte counts, a comprehensive study analyzing large numbers of tumors of different kinds under highly standardized conditions is required.

This study was designed to collect comparable data on the rate of PD-L1 expression in a broad range of different tissues using the same predefined scoring criteria. For this purpose, more than 14,800 tissue samples with preexisting data on intratumoral CD8 positive lymphocytes from 118 different tumor types and subtypes as well as 76 non-neoplastic tissue types were evaluated by immunohistochemistry in a tissue microarray (TMA) format.

2.Materials and methods

2.1Experimental subjects

Tissue Microarrays (TMAs). The normal tissue TMA was composed of 8 samples from 8 different donors for each of 76 different normal tissue types (608 samples on one slide). The cancer TMAs contained a total of 14,897 primary tumors from 118 tumor types and subtypes. The composition of both normal and cancer TMAs is described in detail in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany, the Institute of Pathology, Clinical Center Osnabrueck, Germany, and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. The TMA manufacturing process was described earlier in detail [28, 29]. In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a cancer containing donor block in an empty recipient paraffin block. The density of CD8+ cells, as measured by IHC analysis and automated counting of CD8+ tumor infiltrating immune cells (cells/mm2), was available from an earlier study [30]. The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

2.2Immunohistochemistry (IHC)

Freshly cut TMA sections were immunostained on one day and in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced retrieval for 5 minutes in an autoclave at 121C in pH 9 Dako Target Retrieveal SolutionTM (Agilent, CA, USA; #S2367). Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking SolutionTM (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific for PD-L1 protein (rabbit recombinant, MS Validated Antibodies, Hamburg, Germany, clone MSVA-711R, cat.# 2083-711-R-1) was applied at 37C for 60 minutes at a dilution of 1:150. Bound antibody was then visualized using the EnVision KitTM (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. Slide scoring, including and distinction of tumor and immune cells and estimation of the fraction of stained tumor and immune cells, was performed manually by experienced pathologists using brightfield microscopy. Membranous PD-L1 staining of the cancer cells and immune cells was evaluated separately. In cancer cells, 10% of PD-L1 positive cells was considered PD-L1 positive. In immune cells, PD-L1 staining was grouped into negative (no staining), few positive (few cells stained), and many positive (many cells stained) cells.

2.3Antibody comparison

To evaluate the impact of antibody selection on PD-L1 immunohistochemistry data, staining properties of MSVA-711R, Cell Signaling Technology E1L3N, Roche SP142, and Roche SP263 were compared in normal tissues with known physiological PD-L1 expression as detailed in Supplementary Fig. S1. Immunohistochemistry protocols and automated staining systems were employed as recommended by the antibody vendors and are listed in Supplementary Table S1. To determine the sensitivity and specificity of each antibody, consensus sets of unequivocally PD-L1 positive and unequivocally PD-L1 negative tissue samples were identified from a tissue microarray with 352 high grade muscle invasive urinary bladder cancers. Consecutive sections were taken from the TMA and stained with the 4 antibodies. For maximal standardization of the PD-L1 status calling, neural network and digital image analysis were used as described in the Supplementary Methods. For MSVA-711R, the consensus set contained 96 cancers that were consistently positive with E1L3N, SP142, and SP263, and 188 cancers that were consistently negative with E1L3N, SP142, and SP263. For E1L3N, the consensus set contained cancers that were consistently positive (n= 93) or consistently negative (n= 199) with MSVA-711R, SP142, and SP263. For SP142, the consensus set contained cancers that were consistently positive (n= 102) or consistently negative (n= 200) with MSVA-711R, E1L3N, and SP263. For SP263, the consensus set contained cancers that were consistently positive (n= 98) or consistently negative (n= 192) with MSVA-711R, E1L3N, and SP142.


Statistical calculations were performed with JMP® software 14 (SAS Institute Inc., NC, USA) [31] and R version 3.6.1 (The R foundation) [32, 33]. The Pearson’s correlation coefficient was used to measure the relationship between PD-L1 intensities and densities. ANOVA test was performed to search for associations between PD-L1 expression and CD8+ cell density.


3.1Technical issue

A total of 11,838 (79.6%) of 14,879 tumor samples were interpretable in the TMA analysis. The remaining 3,059 (20.4%) samples were not analyzable due to the lack of unequivocal tumor cells or loss of the tissue spot during the technical procedures. On the normal tissue TMA, sufficient numbers of samples were always interpretable for each tissue to determine PD-L1 expression.

3.2Antibody comparison

Table 1

Sensitivity and specificity of 4 anti-PD-L1 antibodies. Consensus set: Tumors with unequivocal presence or absence of PD-L1 expression that were used to determine specificity and sensitivity (antibody performance) for each of the indicated anti-PD-L1 antibodies

Consensus set resultPD-L1 positive (n) 96 93 102 98
PD-L1 negative (n) 188 199 200 192
Antibody performanceTrue positive (n)92929292
True negative (n)187187187187
False positive (n)112135
False negative (n)41106
Sensitivity 0.958 0.989 0.902 0.939
Specificity 0.995 0.940 0.935 0.974

Figure 1.

PD-L1 immunostaining of normal cells using MSVA-711R. The panels show a membranous PD-L1 positivity of Corpus luteum cells in the ovary (A), macrophages in colon epithelium (B), small (littoral) blood vessels in the spleen (C), a fraction of crypt epithelial cells and macrophages of the tonsil (D), dendritic cells and macrophages in a lymph node (E), surface membranes of the syncytiotrophoblast in the placenta (F), alveolar macrophages in the lung (G) and of a fraction of epithelial cells in the adenohypophysis.

PD-L1 immunostaining of normal cells using MSVA-711R. The panels show a membranous PD-L1 positivity of Corpus luteum cells in the ovary (A), macrophages in colon epithelium (B), small (littoral) blood vessels in the spleen (C), a fraction of crypt epithelial cells and macrophages of the tonsil (D), dendritic cells and macrophages in a lymph node (E), surface membranes of the syncytiotrophoblast in the placenta (F), alveolar macrophages in the lung (G) and of a fraction of epithelial cells in the adenohypophysis.

Representative images of our comparison of 4 anti-PD-L1 antibodies are shown in Supplementary Fig. S1. All antibodies showed the expected staining in normal tonsil epithelium, placenta, corpus luteum of the ovary, macrophages, and blood vessels. The comparatively low staining intensity observed with SP142 is in line with many earlier reports (reviewed in [34]). The results of the consensus set testing and the calculated sensitivity and specificity of each of the 4 antibodies are shown in Table 1. All antibodies proved to be highly specific and sensitive, with comparable performance.

3.3PD-L1 staining pattern in normal tissue

A moderate to strong membranous PD-L1 immunostaining was found in alveolar macrophages of the lung, macrophages in the endometrium of the pregnant uterus and of the gastrointestinal tract, corpus luteum cells of the ovary, surface cell layers of the syncytiotrophoblast and chorion cells of the placenta, thymic epithelial cells, a fraction of squamous epithelial cells of the tonsil crypts as well as in dendritic cells and macrophages of lymphoid tissues. A weak to moderate PD-L1 staining was also observed in a fraction of epithelial cells of the adenohypophysis and in venous sinuses in the spleen (littoral cells). In addition, weak staining was found in fibrils of the anterior lobe of the pituitary gland. Representative images of PD-L1 positive normal tissues are shown in Fig. 1. PD-L1 staining was absent in epithelial cells of adrenal gland, thyroid gland, parathyroid gland, breast, respiratory epithelium, gastrointestinal tract, esophagus, gallbladder, pancreas, liver, cervix, endometrium, fallopian tube, epididymis, kidney, urinary bladder, prostate, seminal vesicle, testis, skin, as well as in muscle cells, fat, aorta, cerebellum, and the cerebrum.

3.4PD-L1 in neoplastic tissue

Figure 2.

PD-L1 immunostaining in cancer using MSVA-711R. The panels show a strong, predominantly membranous PD-L1 immunostaining of tumor cells in an epitheloid malignant mesothelioma (A), a muscle-invasive urothelial carcinoma (B), a squamous cell carcinoma of the oral cavity (C), and an anaplastic thyroid cancer (D). A papillary carcinoma of the thyroid shows a membranous staining of both cancer cells (strong intensity) and macrophages (moderate intensity) (E). Cases of seminoma (F), colorectal adenocarcinoma (G), and a Merkel cell carcinoma of the skin (H) do not show tumor cell staining but contain macrophages with intense PD-L1 positivity.

PD-L1 immunostaining in cancer using MSVA-711R. The panels show a strong, predominantly membranous PD-L1 immunostaining of tumor cells in an epitheloid malignant mesothelioma (A), a muscle-invasive urothelial carcinoma (B), a squamous cell carcinoma of the oral cavity (C), and an anaplastic thyroid cancer (D). A papillary carcinoma of the thyroid shows a membranous staining of both cancer cells (strong intensity) and macrophages (moderate intensity) (E). Cases of seminoma (F), colorectal adenocarcinoma (G), and a Merkel cell carcinoma of the skin (H) do not show tumor cell staining but contain macrophages with intense PD-L1 positivity.

If a cut-off level of 10% positive PD-L1 tumor cells was applied, PD-L1 positivity was observed in 1,691 (14.3%) of 11,838 analyzable tumors. PD-L1 positivity was seen in cases from 85 of 118 (72%) tumor types. At least 50% PD-L1 positive cases were found in 10 (8.5%) tumor types, including thymoma (100%), Hodgkin lymphoma (93%), anaplastic thyroid carcinoma (76.3%), Kaposi sarcoma (71.4%), sarcomatous urothelial carcinoma (70.8%), as well as in squamous cell carcinomas of the penis (66.7%), cervix (64.5%), floor of the mouth (60.5%), lung (52.5%), and the pharynx (50.0%). PD-L1 was absent in tumor cells of all analyzed cases in 33 (28%) tumor categories, including non-Hodgkin lymphomas, germ cell tumors of the testis, mucinous carcinoma of the ovary, as well as tubular and mucinous carcinoma of the breast. Representative images of PD-L1 positive tumors are shown in Fig. 2. The staining in cancer cells was easy to identify in cases with a high number of positive tumor cells. In cases with few PD-L1 positive cells it was often difficult to decide whether positivity was caused by tumor cells or macrophages. In questionable cases, such cells were rather considered immune cells than tumor cells. In immune cells, PD-L1 staining was found in 3,630 (30.7%) cancers, including 15.3% cancers with few and 15.4% cancers with many positive stained immune cells. These positive cases were distributed among 103 of 118 tumor types (87.3%). The highest rates of PD-L1 positive immune cells were seen in tumors of haematopoetic and lymphoid systems (75% to 100%), seminoma (75.8%), Warthin tumors of the parotid gland (95%), and Merkel cell carcinoma (82.2%). A detailed description of the immunostaining results in tumors is given in Table 2 and Fig. 3.

3.5PD-L1 and CD8 expression

Data on intratumoral CD8+ cell density was available for 5,500 (36.9%) of the tumors for which PD-L1 data were collected. Across all tumor entities, the intratumoral CD8+ cell density was significantly higher in tumors with PD-L1 positive tumor cells (612.2 ± 22.9) than in PD-L1 negative tumors (254.2 ± 7.1; p< 0.0001). In a separate analysis of individual tumor categories, the relationship between PD-L1 expression in cancer cells and the density of CD8+ cells reached significance in 10 of 33 analyzed tumor types/subtypes. Tumor entities with a significant association of PD-L1 positivity and a high density of CD8+ cells included serous carcinoma of the ovary, invasive breast carcinoma of no special type, adenocarcinoma of the colon, clear cell renal cell carcinoma, intestinal gastric adenocarcinoma, and liposarcoma (p< 0.0001 each, Table 3).

Table 2

PD-L1 in human tumor cells and immune cells

PD-L1 in tumor cellsPD-L1 in immune cells
Tumor entityOn TMA (n)Analyzable (n)Negative (%)Positive (%)Negative (%)Few (%)Many (%)
Tumors of the skin
 Basal cell carcinoma886895.64.467.68.823.5
 Benign nevus2926100.
 Squamous cell carcinoma of the skin908355.444.657.818.124.1
 Malignant melanoma463987.212.866.717.915.4
 Merkel cell carcinoma464597.82.217.828.953.3
 Basal cell adenoma of the salivary gland1513100.00.0100.00.00.0
Tumors of the lung, pleura and thymus
 Adenocarcinoma of the lung1969958.641.447.517.235.4
 Squamous cell carcinoma of the lung804047.552.565.012.522.5
 Small cell carcinoma of the lung161693.86.331.325.043.8
 Mesothelioma, epitheloid393387.912.
 Mesothelioma, other types767164.835.
Tumors of the female genital tract
 Squamous cell carcinoma of the vagina302965.534.565.524.110.3
 Squamous cell carcinoma of the vulva807758.441.651.924.723.4
 Squamous cell carcinoma of the cervix807635.564.543.419.736.8
 Endometrioid endometrial carcinoma18614694.55.577.49.613.0
 Endometrial serous carcinoma322391.38.765.213.021.7
 Carcinosarcoma of the uterus483797.32.778.45.416.2
 Endometrial carcinoma, high grade, G313785.714.314.342.942.9
 Endometrial clear cell carcinoma84100.
 Endometrioid carcinoma of the ovary735384.915.173.618.97.5
 Serous carcinoma of the ovary50939884.215.858.016.125.9
 Mucinous carcinoma of the ovary7048100.
 Clear cell carcinoma of the ovary504077.522.572.517.510.0
 Carcinosarcoma of the ovary473783.816.
Tumors of the breast
 Invasive breast carcinoma of no special type1345112094.65.479.77.113.1
 Lobular carcinoma of the breast25119999.
 Medullary carcinoma of the breast11966.733.30.00.0100.0
 Tubular carcinoma of the breast94100.00.0100.00.00.0
 Mucinous carcinoma of the breast3624100.00.087.512.50.0
Tumors of the digestive system
 Adenomatous polyp, low-grade dysplasia504397.72.351.225.623.3
 Adenomatous polyp, high-grade dysplasia504695.74.323.923.952.2
 Adenocarcinoma of the colon1882140896.23.852.037.410.6
 Gastric adenocarcinoma, diffuse type17613097.72.390.06.23.8
 Gastric adenocarcinoma, intestinal type17413176.323.748.124.427.5
 Gastric adenocarcinoma, mixed type625384.915.
 Adenocarcinoma of the esophagus836090.010.046.728.325.0
 Squamous cell carcinoma of the esophagus764854.245.833.331.335.4
 Squamous cell carcinoma of the anal canal898463.136.947.626.226.2
 Hepatocellular carcinoma504897.
 Ductal adenocarcinoma of the pancreas61244889.110.979.916.14.0
 Pancreatic/Ampullary adenocarcinoma896188.511.563.924.611.5
 Acinar cell carcinoma of the pancreas1611100.00.0100.00.00.0
 Gastrointestinal stromal tumor (GIST)504575.624.473.320.06.7
Tumors of the urinary system
 Non-invasive papillary urothelial carcinoma, pTa G2 low grade17714899.30.787.26.86.1
 Non-invasive papillary urothelial carcinoma, pTa G2 high grade14112899.20.889.14.76.3
 Non-invasive papillary urothelial carcinoma, pTa G318715093.36.764.017.318.7
 Urothelial carcinoma, pT2-4 G3120693670.829.267.516.116.3
 Small cell neuroendocrine carcinoma of the bladder2020100.

Table 2, continued

PD-L1 in tumor cellsPD-L1 in immune cells
Tumor entityOn TMA (n)Analyzable (n)Negative (%)Positive (%)Negative (%)Few (%)Many (%)
 Sarcomatoid urothelial carcinoma252429.270.891.74.24.2
 Clear cell renal cell carcinoma85766595.
 Papillary renal cell carcinoma25519984.415.685.48.06.5
 Clear cell (tubulo) papillary renal cell carcinoma2115100.
 Chromophobe renal cell carcinoma13110085.015.0100.00.00.0
Tumors of the male genital organs
 Adenocarcinoma of the prostate, Gleason 3 + 38370100.
 Adenocarcinoma of the prostate, Gleason 4 + 4806797.03.086.610.43.0
 Adenocarcinoma of the prostate, Gleason 5 + 5856892.67.469.111.819.1
 Adenocarcinoma of the prostate (recurrence)25821096.73.394.33.32.4
 Small cell neuroendocrine carcinoma of the prostate1917100.00.052.935.311.8
 Embryonal carcinoma of the testis5035100.00.014.322.962.9
 Yolk sac tumor5027100.00.025.925.948.1
 Squamous cell carcinoma of the penis807533.366.733.322.744.0
Tumors of endocrine organs
 Adenoma of the thyroid gland1139984.815.
 Papillary thyroid carcinoma39134569.031.078.813.08.1
 Follicular thyroid carcinoma15413067.732.397.71.50.8
 Medullary thyroid carcinoma1119084.415.694.44.41.1
 Anaplastic thyroid carcinoma453823.776.357.915.826.3
 Adrenal cortical adenoma5042100.
 Adrenal cortical carcinoma262592.
 Appendix, neuroendocrine tumor (NET)221492.
 Colorectal, neuroendocrine tumor (NET)1212100.
 Ileum, neuroendocrine tumor (NET)4947100.
 Lung, neuroendocrine tumor (NET)191894.45.6100.00.00.0
 Pancreas, neuroendocrine tumor (NET)974989.810.
 Colorectal, neuroendocrine carcinoma (NEC)121291.78.350.033.316.7
 Gallbladder, neuroendocrine carcinoma (NEC)44100.
 Pancreas, neuroendocrine carcinoma (NEC)1412100.
Tumors of haemotopoetic and lymphoid tissues
 Hodgkin Lymphoma103437.
 Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL)5049100.
 Diffuse large B cell lymphoma (DLBCL)11410980.719.317.410.172.5
 Follicular lymphoma8886100.
 T-cell Non Hodgkin lymphoma242483.316.716.74.279.2
 Mantle cell lymphoma1818100.00.05.616.777.8
 Marginal zone lymphoma1616100.
 Diffuse large B-cell lymphoma (DLBCL) in the testis161687.512.512.512.575.0
 Burkitt lymphoma54100.
Tumors of soft tissue and bone
 Tendosynovial giant cell tumor4545100.00.0100.00.00.0
 Granular cell tumor534897.92.1100.00.00.0
 Malignant peripheral nerve sheath tumor (MPNST)131291.78.391.78.30.0
 Dermatofibrosarcoma protuberans2116100.00.0100.00.00.0
 Kaposi sarcoma8728.671.471.40.028.6

Table 2, continued

PD-L1 in tumor cellsPD-L1 in immune cells
Tumor entityOn TMA (n)Analyzable (n)Negative (%)Positive (%)Negative (%)Few (%)Many (%)
 Sarcoma, not otherwise specified (NOS)747062.937.
 Ewing sarcoma232095.
 Synovial sarcoma1211100.00.0100.00.00.0

Table 3

PD-L1 n human tumor cells and intratumoral CD8 positive (CD8+) cells

PD-L1 IHC in tumor cells n CD8+ cell density (mean ± SD)P values
All cancersNegative5,016254.2 ± 7.1<0.0001
Positive484612.2 ± 22.9
Mesothelioma, epitheloidNegative29261.6 ± 54.30.0864
Positive4537.9 ± 146.3
Mesothelioma, other typesNegative18231.8 ± 82.40.1312
Positive10446.7 ± 110.5
Endometrioid carcinoma of the ovaryNegative28124.0 ± 62.60.9657
Positive5117.0 ± 148.2
Serous carcinoma of the ovaryNegative279142.0 ± 24.5<0.0001
Positive43532.1 ± 62.4
Clear cell carcinoma of the ovaryNegative718.2 ± 7.70.7127
Positive211.9 ± 14.5
Carcinosarcoma of the ovaryNegative20117.0 ± 46.60.5896
Positive454.5 ± 104.2
Invasive breast carcinoma of no special typeNegative997294.6 ± 14.0<0.0001
Positive58699.0 ± 58.0
Lobular carcinoma of the breastNegative134199.7 ± 22.30.7999
Positive1134.0 ± 258.0
Medullary carcinoma of the breastNegative61470.0 ± 485.90.1396
Positive32872.1 ± 687.2
Adenocarcinoma of the colonNegative1229259.3 ± 13.7<0.0001
Positive52692.9 ± 66.8
Clear cell renal cell carcinomaNegative568435.6 ± 29.5<0.0001
Positive311164.2 ± 126.2
Papillary cell renal cell carcinomaNegative127233.9 ± 39.70.2765
Positive27337.3 ± 86.0
OncocytomaNegative5774.0 ± 18.10.3923
Positive31100.2 ± 24.5
Gastric adenocarcinoma, diffuse typeNegative69260.3 ± 50.50.7595
Positive2352.8 ± 296.5
Gastric adenocarcinoma, intestinal typeNegative61324.7 ± 62.3<0.0001
Positive151142.5 ± 125.7
Gastric adenocarcinoma, mixed typeNegative45386.4 ± 94.70.1399
Positive8751.9 ± 224.6
Ductal carcinoma of the pancreasNegative351222.2 ± 15.80.1014
Positive42301.3 ± 45.5
Pancreatic/Ampullary adenocarcinomaNegative34268.8 ± 81.10.1313
Positive4654.7 ± 236.4
Sarcomatoid urothelial carcinomaNegative7229.3 ± 342.10.2457
Positive17714.1 ± 219.5
Granular cell tumorNegative1961.9 ± 11.10.0681
Positive1158.0 ± 48.3
LeiomyosarcomaNegative3282.7 ± 28.60.1047
Positive3245.5 ± 93.3

Table 3, continued

PD-L1 IHC in tumor cells n CD8+ cell density (mean ± SD)P values
LiposarcomaNegative5688.3 ± 53.9< 0.0001
Positive101086.6 ± 127.6
Malignant peripheral nerve sheath tumor (MPNST)Negative11100.9 ± 61.30.0007
Positive11130.0 ± 203.2
MyofibrosacromaNegative1853.0 ± 272.60.0587
Positive81028.3 ± 408.9
AngiosarcomaNegative22105.6 ± 109.30.0042
Positive17610.5 ± 124.4
AngiomyolipomaNegative84178.9 ± 45.90.9519
Positive4165.9 ± 210.5
GanglioneuromaNegative932.4 ± 11.10.0339
Positive297.2 ± 23.5
Kaposi sarcomaNegative2304.3 ± 177.70.6885
Positive5393.7 ± 112.4
Sarcoma, not otherwise specified (NOS)Negative4466.8 ± 100.40.0004
Positive26677.0 ± 129.6
ParagangliomaNegative35150.6 ± 46.00.9313
Positive2167.7 ± 192.4
Primitive neuroectodermal tumor (PNET)Negative1965.7 ± 23.70.5439
Positive10.0 ± 103.5
SchwannomaNegative9881.1 ± 15.90.3795
Positive2180.4 ± 111.3
ChondrosarcomaNegative5481.4 ± 320.50.8664
Positive4397.6 ± 358.4

Figure 3.

Ranking order of PD-L1 immunostaining in human tumors. Only staining in tumor cells is shown.

Ranking order of PD-L1 immunostaining in human tumors. Only staining in tumor cells is shown.

Figure 4.

Graphical comparison of PD-L1 data from this study (×) in comparison with the previous literature (circles). Color of circles indicates the threshold used to define PD-L1 positivity in these studies: red = 1%, blue = 5%, green = 10%, orange = 25%, grey = other threshold. A list of studies used to build the figure is given in Supplementary Table S2.

Graphical comparison of PD-L1 data from this study (×) in comparison with the previous literature (circles). Color of circles indicates the threshold used to define PD-L1 positivity in these studies: red = 1%, blue = 5%, green = 10%, orange =⩾ 25%, grey = other threshold. A list of studies used to build the figure is given in Supplementary Table S2.


The analysis of more than 14,000 tumors in a highly standardized way enabled us to define the relative importance of PD-L1 expression across 118 important human tumor entities and to define its relationship with tumor infiltrating CD8 positive lymphocytes. A Medline Search using the terms “PD-L1 + cancer + immunohistochemistry” had identified 2,887 previous publications on October 13th, 2021. Even rare tumor types such as anaplastic thyroid cancer (4 studies), osteosarcoma (11 studies) and Merkel cell cancer (10 studies) have repeatedly been analyzed (e.g., [35, 36, 37]). However, the large number of studies has not led to a unanimous picture on PD-L1 expression in cancer as the results were highly variable in most tumor entities. Data from 907 studies on 72 different tumor entities are summarized in Fig. 4. These data show that criteria for defining PD-L1 positivity, including cutoffs ranging from 1% to 50% stained tumor cells as well as scores combining staining intensity and the fraction of stained tumor cells have contributed to the wide spread of data. Significant differences also exist, however, between studies employing identical definitions. For example, at a cut-off of 5%, the positivity rates varied from 6.1 to 45.9% in colon cancer [38, 39] , between 6.7% and 48.1% in gastric [40, 41], or between 8.3% and 75% in non-small cell lung cancer [42, 43]. The high concordance of the staining results and diagnostic performance obtained by 4 different anti-PD-L1 antibodies argues against a major role of antibody properties for these discrepant data. Various previous studies have also shown that the antibodies that are most commonly used for PD-L1 analysis can result in comparable data within studies [34, 44, 45, 46], and that even the use of lab developed PD-L1 tests yield similar results as FDA approved companion diagnostics [47]. The comparison of data obtained from studies using identical antibodies also argues against a major role of antibody characteristics as drivers for the large bandwidth of PD-L1 data. For example, the antibody clone E1L3N has been used in more than 300 previous studies and resulted in PD-L1 positivity in 0–33% of clear cell renal cell carcinomas [23, 48], 0–25% of colorectal carcinomas [23, 49], 19-90% of lung adenocarcinomas [50, 51], and 0–79% of pancreas carcinomas [23, 52] at cut-off levels of 1% or 5% stained cancer cells to define positivity.

Rather underestimated causes for discrepant PD-L1 data include slide ageing and difficulties in the distinction of tumor associated macrophages from tumor cells. Others and we had earlier demonstrated that the immunostaining intensity on stored formalin-fixed tissue sections decreases over time [53, 54] and that a significant reduction of staining may already occur 2 weeks after a tissue section has been taken [55]. This may be a relevant source of discrepant staining results particularly in clinical studies, where sections are often taken long before the analysis is made. In case of PD-L1, where macrophages often express the target protein at high levels, and where low thresholds are used for defining tumor cell positivity, it appears also likely that the quantity of tissue analyzed per patient and difficulties in the distinction of PD-L1 positive macrophages from cancer cells may have contributed to interpretation difficulties. That the analysis of larger tissue fragments more often leads to the perception of PD-L1 positivity than the analysis of small portions is shown by significant differences in data derived from TMA and from large section studies. For example, in 16 studies utilizing cut-off levels of 1% or 5% to define PD-L1 positivity in lung adenocarcinomas with the E1L3N antibody, the average positivity rate was 26% for TMA analyses but 41% for conventional large section staining. While these data might suggest that relevant PD-L1 findings are missed on TMAs, it is also evident that interpretation errors – such as mistaking macrophages for tumor cells – are more likely to occur on large sections [56]. Moreover, TMA studies comparing multiple samples per tumor versus only one sample per tumor have regularly found a significant relationship between the quantity of analyzed tissue and IHC positivity rate [56, 57, 58, 59]. Only recently, it was shown that posttranslational glycolysation of the PD-L1 protein can negatively affect binding of anti-PD-L1 antibodies in formalin fixed tissue samples [60]. Therefore, it has been suggested that tissue samples should be pretreated with deglycolysing reagents to reduce the risk of false-negative PD-L1 IHC findings. In our study, such a systematic change in staining protocol would potentially result in a higher overall number of PD-L1 positive tumors. However, because all tumor types would be equally affected, the relative ranking of PD-L1 positive tumor types would not change.

Groups of cancers that are of special interest based on our data include cancers with very high and very low rate of PD-L1 expression in cancer cells and tumors with a particularly high density of tumor associated PD-L1 inflammatory cells. The group of tumors with highest rates of PD-L1 positivity in tumor cells includes several tumor entities already approved for treatment with CPIs, such as Hodgkin lymphoma, squamous cell carcinomas of the head and neck, urothelial cancers and malignant mesothelioma. If the response to CPIs is indeed driven by tumoral PD-L1 expression in these tumors, cancers with a comparably high PD-L1 expression such as penile carcinoma, squamous cell carcinomas of the esophagus and the anal canal or anaplastic thyroid cancer should also represent premium targets for CPIs. Evidence for clinical responses already exists for anaplastic thyroid cancer [61], squamous cell cancers of the head and neck [62, 63, 64, 65], oral cavity [66], esophagus [67, 68] and skin [69], and a clinical trial is ongoing for squamous cell carcinoma of the cervix [70].

Cancers with a very low rate of tumoral PD-L1 expression for example include prostate cancer, a tumor known for is particularly poor response to CPIs [71] but also cancers such as Merkel cell carcinoma and small cell lung cancer which are both approved for CPI therapy. It is of note, that Merkel cell carcinoma (82.2%) and small cell lung cancer (68.7%) belong to these tumor types with the highest rates of PD-L1 positive immune cells in our analysis. These findings fit well with experimental data highlighting the particularly important role of PD-L1 expressing immune cells. For example, in colon and breast cancer mice models, anti–PD-L1 treatment changed the activity of tumor macrophages from an immune-suppressive to an immune-stimulatory state with an increase in activated CD8 positive cytotoxic T cells [72]. Triple negative breast cancer is the first tumor entity where the indication for CPI atezolizumab solely depends on the presence of intratumoral PD-L1 positive immune cells and is independent of whether tumor cells express PD-L1 [73, 74].

Our data also show that an elevated density of CD8 positive intratumoral lymphocytes in PD-L1 expressing tumors is a general feature occurring across all cancer types. This observation is consistent with various reports describing associations between PD-L1 positivity in tumor cells and high numbers of tumor infiltrating lymphocytes in various individual cancer types [3, 4, 5, 6]. Studies have also demonstrated that PD-L1 positivity is statistically linked to high mutation burden and microsatellite instability [75]. Altogether, these observations are well consistent with a model suggesting that PD-L1 is one of several immune-escape mechanisms that can be activated in highly immunogenic cancer cells in response to “lymphocyte attack”.

In summary, the results of our study provide a ranking order of cancer types according to their PD-L1 expression in tumor and inflammatory cells. A consistently higher rate of tumor infiltrating CD8 positive lymphocytes in PD-L1 positive than in PD-L1 negative cancers corroborates the concept that tumoral PD-L1 expression is driven by a hostile immune environment.

Author contributions

Conception: KM, TK, RS, GS.

Interpretation or analysis of data: MK, EB, MJS, SDR, MK, CHM, NCB, TM, ML, AM, AML, DH, CF, NG, FJ, TSC, SS, EB, SM, AHM.

Preparation of the manuscript: KM, TK, GS.

Revision for important intellectual content: KM, TK, RS, GS, DH.

Supervision: KM, TK, RS, GS.

All authors agree to be accountable for the content of the work.

Supplementary data

The supplementary files are available to download from


We are grateful to Melanie Witt, Laura Behm, Inge Brandt, Maren Eisenberg, and Sünje Seekamp for excellent technical assistance.

Conflict of interest

The PD-L1 antibody clone MSVA-711R was provided by MS Validated Antibodies GmbH (owned by a family member of GS).



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