R EVIEW

PD-L1 testing by immunohistochemistry in

immuno-oncology
Semir Vranic 1∗ and Zoran Gatalica2

Immunotherapy, based on immune checkpoint inhibitors (ICIs) targeting the programmed cell death ligand 1 (PD-L1) and/or
programmed death receptor 1 (PD-1), has substantially improved the outcomes of patients with various cancers. However, only ∼30%
of patients benefit from ICIs. Tumor PD-L1 expression, assessed by immunohistochemistry (IHC), is the most widely validated and used
predictive biomarker to guide the selection of patients for ICIs. PD-L1 assessment may be challenging due to the necessity of different
companion diagnostic assays for required specific ICIs and a relatively high level of inter-assay variability in terms of performance and
cutoff levels. In this review, we discuss the role of PD-L1 IHC as a predictive test in immunotherapy (immuno-oncology), highlight the
complexity of the PD-L1 testing landscape, discuss various preanalytical, analytical, and clinical issues that are associated with PD-L1
assays, and provide some insights into optimization of PD-L1 as a predictive biomarker in immuno-oncology.
Keywords: Cancer, immunotherapy, immune checkpoint inhibitors (ICIs), predictive biomarkers, programmed cell death ligand 1
(PD-L1), immunohistochemistry (IHC).

Introduction functions (heart, atherosclerosis, and hypertension), neuroin-

Immunotherapy, based on the use of immune checkpoint flammation, and obesity have not been fully characterized but
inhibitors (ICIs), has recently revolutionized the treatment are actively investigated [6].
and outcome of several cancer types. ICIs therapy targeting Therefore, development of reliable predictive biomarkers
programmed death receptor 1 (PD-1), programmed cell death for ICIs would represent an essential selection tool. This review
ligand 1 (PD-L1), and CTLA-4 have become the standard of care summarizes the status of approved and emerging predictive
for several common malignancies. Anti-PD-1/PD-L1 treatment biomarkers for ICIs, focusing on PD-L1 expression and its quan-
modalities include two monoclonal antibodies against PD-1 tification using immunohistochemistry (IHC). We highlight a
receptor (nivolumab and pembrolizumab) and three against complex PD-L1 testing landscape, highlighting preanalytical,
PD-L1 (atezolizumab, durvalumab, and avelumab), all of which analytical, and clinical issues that are associated with PD-L1
were approved by Food and Drug Administration (FDA) [1]. assays. We briefly cover regulatory issues and provide some
These drugs have been approved with different indications insights into optimization of PD-L1 as a predictive biomarker in
as either monotherapy or combinatorial therapy with other immuno-oncology.
modalities, such as radiation therapy, chemotherapy, or
other ICIs. In addition, two agents are available targeting
another checkpoint regulator CTLA-4: Tremelimumab and The role of the PD-1/PD-L1 axis in cancer
ipilimumab [2, 3]. surveillance and suppression
Despite the remarkable efficacy of the ICIs, many patients Programmed death ligand 1 (PD-L1, CD274), one of two ligands
(∼70%) do not respond well or develop resistance to these for the PD-1 receptor (the other is PD-L2, CD273), interacts
drugs. The response rate to ICIs varies between 15% and 30% with the PD-1 receptor on naïve T-lymphocytes inhibiting T-cell
in most solid tumors and 45%–60% in microsatellite instability activation [7]. PD-L1 expression in a tumor is a sign of an inhi-
high (MSI-H) cancers and malignant melanoma [4]. The use bition of the anti-tumoral activity of the immune system and a
of ICIs is associated with potentially significant toxicity and predictor of a favorable response to the therapeutic monoclonal
side effects. Thus, in non-small cell lung carcinoma (NSCLC) antibodies designed to break this inhibition and elicit antitu-
treated with ICIs, the most common adverse effects are those moral activity [8].
related to endocrine, gastrointestinal, and dermatologic sites; in PD-L1 is a transmembrane receptor that interacts with
malignant melanoma, the most common adverse effect sites are PD-1 and B7.1, causing immune system suppression. PD-1
dermatologic, hepatic, and endocrine [4, 5]. Long-term adverse is overexpressed on T-lymphocytes following their activa-
effects in cancer survivors on immune system, cardiovascular tion and sustained during chronic stimulations, such as

1 College of Medicine, QU Health, Qatar University, Doha, Qatar; 2 Department of Pathology, University of Oklahoma College of Medicine, Oklahoma City, OK, United States.

∗ Correspondence to Semir Vranic: [email protected]

DOI: 10.17305/bjbms.2022.7953
© 2022 Vranic and Gatalica. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

Biomolecules and Biomedicine, 2023, Vol. 23, No. 1, 15–25 15 www.biomolbiomed.com

in chronic infections/inflammation or cancer [9]. The PD- neoantigens) that may be presented by major histocompatibil-
1/PD-L1 interactions block T-lymphocyte activation, cytokine ity complex I (MHC-I) on the cell membrane of neoplastic cells.
production, and cytolytic activity, causing functional down- These neoantigens may be recognized by the immune cells (IC)
regulation or exhaustion of T-lymphocytes [9]. B7.1 receptor is (T-cells) as non-self (“immunogenic antigens”), triggering and
overexpressed on antigen-presenting cells (APCs) and activated provoking an immune reaction [19–21]. Notably, only a minor-
T-lymphocytes. PD-L1 binding to B7.1 on T-lymphocytes and/or ity of these neoantigens (2–5 out of several hundred) become
APCs inhibits the immune responses, including inhibition of immunogenic, resulting in a T-cell response [19]. Consequently,
T-lymphocyte activation and cytokine production [10]. the greater the TMB, the higher the likelihood of producing
Within the tumor, expression of PD-L1 may be observed in potentially immunogenic neoantigens and immune reactions.
infiltrating immune and neoplastic cells [11, 12]. The previous TMB is defined as the number of mutations in the can-
studies revealed that PD-L1 expression on tumor cells (TC) is cer cells. The TMB is measured by some high-throughput
associated with the downregulation of the immune system, fol- (NGS-based) assays, such as whole-genome or whole-exome
lowed by immune evasion [9]. Therefore, interruption of the sequencing (WGS or WES) and is reported as the number of
PD-L1/PD-1 inhibitory axis represents an attractive therapeutic mutations per megabase (Mutations/Mb) [22]. Both assays
target to reactivate the T-cell response that is suppressed by the explore a wide range of mutations within cancer cells. Multiple
upregulation of PD-L1 in the tumor. WGS/WES platforms are currently available for the TMB
Currently, PD-L1 immunohistochemical (IHC) assays have assessment employing both non-synonymous and synonymous
the most FDA approvals as a companion diagnostic (CDx) for exonic mutations in the TMB estimation [23, 24]. Currently,
immunotherapy with immune checkpoint inhibitors in specific only the FoundationOne CDxTM test (Foundation Medicine,
tumor types [1]; other immunotherapy predictive biomarker Cambridge, MA, USA) is the FDA-approved assay, which
exist and will be briefly discussed in the next paragraph. includes TMB as part of its comprehensive genomic profiling
panel. The Memorial Sloan Kettering Cancer Center MSK-
IMPACT (Integrated Mutation Profiling of Actionable Cancer
Targets, the panel of 468 genes) also received FDA authorization
Predictive biomar k ers of response to immune
in 2017, but TMB as a predictive biomarker for ICIs is not
chec k point inhibitors included in the approval [25].
Numerous biomarkers are evaluated for the prediction of Previous studies have provided solid evidence that the
response to ICIs and three are currently approved. These tumors with a TMB ≥10 mut/Mb (TMB-high) are more
are PD-L1 expression, tumor mutational burden (TMB) and likely to have a favorable response to immune checkpoint
DNA mismatch repair deficiency [(dMMR) and microsatellite inhibitors (particularly pembrolizumab) [22]. This has been
instability-high (MSI-H)]. PD-L1 protein expression is tested by confirmed in several common cancers, such as NSCLC, urothe-
IHC. Tumor DNA-mismatch repair (MMR) protein deficiency is lial carcinoma, malignant melanoma, and small cell lung
tested by IHC and tumor DNA microsatellite instability is tested carcinoma (SCLC) [19]. In two recent comprehensive pan-
using either PCR- or NGS-based assays. TMB is assessed using a cancer studies exploring up to 27 different cancer types, high
large panel next-generation sequencing assay (NGS, currently TMB correlated well with the response to immune checkpoint
only FoundationOne CDxTM assay) has been approved by FDA inhibitors [26, 27]. It is noteworthy that TMB depends on
for this purpose [13]. cancer pathogenesis and may substantially vary between
Presence of tumor-infiltrating lymphocytes (TIL) was tra- and within the same/similar histologic cancer types. A good
ditionally associated with MSI-H colorectal cancers (CRC) [14] example is a Merkel cell carcinoma (MCC), a highly aggressive
which are frequently histopathologically analyzed in various neuroendocrine cutaneous neoplasm. The etiology of MCC
tumors. However, it has not been formally approved as ICI ther- is strongly associated with two important risk factors—UV
apy biomarker. In some cancers, such as breast cancer, there exposure and Merkel cell polyoma virus (MCPyV) positivity.
have been substantial efforts to standardize the assessment of Each of these risk factors causes a distinct MCC genotype
TIL as proposed by the International TIL Working Group [15]. A and phenotype [28]. Thus, UV-related MCC usually exhibits
routine assessment of TIL has also been incorporated in the fifth a high TMB in contrast to MCPyV-associated MCC with a low
edition of the WHO Breast Tumors Classification [16]. Similar TMB [29].
efforts to standardize TIL assessment have also been made in Excluding cancers with mutations in MMR or polymerase
other solid tumors [17]. genes, the highest TMB is observed in malignant melanoma,
Other predictive biomarkers are also being intensely squamous cell carcinomas of the skin, and NSCLC [30, 31]. In
explored, such as PD-1, IFN-y pathway genes, IL-8, CD39+/CD8+ this regard, there have been substantial efforts to harmonize
TIL, T-cell repertoire clonality, etc. (reviewed in [18]). None and standardize TMB assessment and reporting [23, 24]. The
of these novel biomarkers has been approved as a predictive TMB Harmonization Consortium has recently gathered the key
biomarker in immuno-oncology. stakeholders involved in developing NGS assays reporting their
initial results in harmonization and standardization of TMB
Tumor mutational burden (TMB) assessment in cancer [23]. Hopefully, these results will con-
All cancers accumulate mutations (albeit at different rates), tribute to the standardized approach in assessing TMB across
resulting in a production of novel peptides/proteins (also called cancers.

Vranic and Gatalica

PD-L1 and immunotherapy 16 www.biomolbiomed.com
Microsatellite instability (MSI) A poor predictive value of PD-L1 expression by IHC has also been
DNA MMR machinery in healthy cells is responsible for reported in malignant melanoma, hepatocellular carcinoma,
correcting some errors during DNA replication. The defects in and renal cell carcinoma (reviewed in [5]).
MMR can lead to MSI-H status, which has been demonstrated
in at least 14 different cancers at a varying frequency (overall Currently available anti-PD-L1 diagnostic antibodies
prevalence 2%–4%), most notably in colorectal, gastric, and We summarized in Tables 1–3 currently available, approved,
endometrial cancers [19, 32]. MSI-H or dMMR cancers are char- and commercially available anti-PD-L1 diagnostic antibodies.
acterized by the accumulation of errors in genetic sequences Although different antibodies against PD-1/PD-L1 are cur-
that are usually repeated (these are called microsatellites). rently available, very few have been approved by the FDA as
Defects in MMR genes (MLH1, MSH2, MSH6, and PMS2) either companion diagnostic (CDx) or complementary assays
can be hereditary (Hereditary non-polyposis CRC or Lynch (Tables 1–3). A CDx assay is defined as “an in vitro diagnostic
syndrome, OMIM#120435) or sporadic (typically caused by device (IVD) or an imaging tool that provides information that
hypermethylation of MLH1 gene promoter region) [33]. The is essential for the safe and effective use of a corresponding
dMMR cancers are usually “immunogenic,” exhibiting higher therapeutic product” (List of Cleared or Approved Compan-
levels of immune cell reaction with higher TIL density than ion Diagnostic Devices (In Vitro and Imaging Tools): Food and
MMR proficient cancers [34]. Consequently, dMMR cancers are Drug Administration; available from [1]). In contrast, a comple-
more sensitive to ICIs [19]. mentary diagnostics test provides additional information about
Based on the study of Le et al. [35] that revealed the how a drug might be used, which is distinct from CDx tests,
predictive value of MSI-H to ICI pembrolizumab irrespective which are essential for a drug’s safe and effective use. Most
of tumor histology (12 different tumor types were assessed), of the available PD-L1 assays have been developed as predic-
the MSI-H has been recognized and approved by FDA in tive biomarkers for particular ICIs, each exploring distinct IHC
2017 as the first tumor type-agnostic biomarker in the cancer platforms, PD-L1 staining patterns, and scoring systems (algo-
immunotherapy [36]. In addition, both sporadic and hereditary rithms) (Tables 1, 2, and 4) [45, 46].
MSI-H CRC tends to express PD-L1 more frequently than MSS PD-L1 expression can be seen in cancer and IC infiltrating
CRC [37, 38]. invasive cancer (both in intra- and peritumoral stroma) [37, 45].
However, the assessment of IC includes only mononuclear infil-
trate (lymphocytes, macrophages, and dendritic cells), while
PD-L1 expression as a predictive biomar k er plasma cells and neutrophils should be ignored.
Multiple studies across the cancers have provided solid evi- The staining pattern of PD-L1 differs between cancer and
dence about a positive correlation between PD-L1 expression by IC. The neoplastic cells typically exhibit a linear membranous
IHC and response to ICIs [39, 40]. Taube et al. [41] demonstrated PD-L1 pattern (Figure 1), while the IC have granular and punc-
that PD-L1 expression on TC was a predictive biomarker for tate PD-L1 expression (Figure 2). This distinction is particularly
anti-PD-1 drug Nivolumab in 41 patients with advanced solid relevant in cancers when assessing exclusively IC for PD-L1
tumors, including 16 melanoma, 12 NSCLC, 6 CRC, 5 RCC, and 2 (e.g., triple-negative breast cancer). Although all the available
patients with castration-resistant prostate carcinoma. In their clones exhibit similar subcellular patterns of PD-L1 expression
study, PD-L1 positivity was defined as ≥5% positive TC with in cancer and IC, respectively (Figures 3 and 4), significant dif-
membranous PD-L1 expression using two anti-PD-L1 antibodies ferences in measurements exist [47].
(5H1 and M3 clones). The study revealed that PD-L1 expression The following paragraphs summarize the most relevant
by cancer cells correlated well with an objective response with information on the approved CDx for PD-L1 testing.
clinical benefit, while TIL PD-L1 expression was not associated
Clones available from Roche Tissue Diagnostics, Tucson, AZ
with objective clinical response [41].
(formerly, Ventana Medical Systems, Inc.): SP142 and SP263
In another study, Carbognin et al. [42] explored the corre-
assays
lation between PD-L1 expression and tumor response to three Both assays are monoclonal rabbit antibodies recommended
different ICIs, including pembrolizumab, nivolumab, and ate- and optimized for use on the Ventana BenchMark Ultra instru-
zolizumab. The study explored ∼1500 patients from 20 clinical ment. SP142 has been approved as a CDx for multiple malignan-
trials [42]. They also found that PD-L1 expression in cancer cies (NSCLC, urothelial carcinoma, and triple-negative breast
cells in melanoma and NSCLC patients was associated with a carcinoma); however, each of the provided indications has dif-
higher therapeutic response to ICIs. The positive impact of PD- ferent cutoffs and scoring (interpretation) systems (Tables 1, 2,
L1 expression was evident regardless of the treatment approach. and 4). In contrast, SP263 has only been approved as a comple-
In addition, they also showed that the cutoff value of 5% of TC mentary assay for predicting the urothelial (bladder) carcinoma
with PD-L1 expression was a better predictor of response to ICIs response to durvalumab (ICI against PD-L1) [48] (Table 3).
than the cutoff of 1% of positive cells [42].
In some cancers, such as SCLC, PD-L1, expression was Clones available from Agilent DAKO Products: 22C3 pharmDx
not predictive of response to ICIs, as reported in multi- assay, 28-8 pharmDx assay, and 73-10 assay
ple clinical trials [43, 44]. Consequently, ICIs atezolizumab, Both 22C3 pharmDx and 28-8 pharmDx are FDA-approved
pembrolizumab, and durvalumab have been approved in a diagnostic PD-L1 assays for multiple malignancies (Tables 1
biomarker-agnostic fashion for patients with this cancer [1]. and 2). In particular, the 22C3 clone has been utilized as a

Vranic and Gatalica

PD-L1 and immunotherapy 17 www.biomolbiomed.com
Table 1. The list of cleared and approved companion diagnostic PD-L1 tests by cancer type (Source: Food and Drug Administration, [1])

Tumor type Antibody clone (Manufacturer) Scoring algorithms FDA-approved drugs

Non-small cell lung VENTANA SP142 Ventana Medical TC and IC Score TECENTRIQ (atezolizumab)
carcinoma (NSCLC) Systems, Inc.*
28-8 pharmDx Dako North America, Inc. TC expression (%) OPDIVO (nivolumab) combined
with YERVOY (ipilimumab)
22C3 pharmDx Dako North America, Inc. Tumor Proportion Score (TPS) KEYTRUDA (pembrolizumab)
Libtayo (cemiplimab-rwlc)
Gastric/gastroesophageal 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
junction carcinoma (GEJ) Libtayo (cemiplimab-rwlc)
Cervical carcinoma 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
Libtayo (cemiplimab-rwlc)
Urothelial carcinoma 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
(bladder) Libtayo (cemiplimab-rwlc)
VENTANA SP142 Ventana Medical TC and IC score TECENTRIQ (atezolizumab)
Systems, Inc.*
Head and neck 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
squamous cell carcinoma Libtayo (cemiplimab-rwlc)
(HNSCC)
Esophageal squamous 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
cell carcinoma (ESCC) Libtayo (cemiplimab-rwlc)
Triple-negative breast 22C3 pharmDx Dako North America, Inc. Combined positive score (CPS) KEYTRUDA (pembrolizumab)
carcinoma (TNBC) Libtayo (cemiplimab-rwlc)
VENTANA SP142 Ventana Medical IC score TECENTRIQ (atezolizumab)**
Systems, Inc.*

TNBC: Triple-negative breast carcinoma; NSCLC: Non-small cell lung carcinoma; GEJ: Gastroesophageal junction adenocarcinoma; HNSCC: Head and neck
squamous cell carcinoma; ESCC: Esophageal squamous cell carcinoma; FDA: Food and Drug Administration; IC: Immune cells; TC: Tumor cells; PD-L1:
Programmed cell death ligand 1. * Now Roche Tissue Diagnostics (Tucson, AZ, USA). **Withdrawn voluntarily by Genentech in August 2021.

Table 2. Summary of the associated scoring algorithms’ cutoffs and detection platforms for the approved companion diagnostic PD-L1 tests

Antibody (clone) Scoring algorithms’ cutoff (tumor type) Detection system/platform

Ventana PD-L1 SP142 ≥5% IC (UC) ≥1% IC (TNBC) ≥50% TC or ≥10% IC (NSCLC) OptiView Detection and Amplification
Assay Benchmark ULTRA
Dako PD-L1 IHC 28-8 ≥1% TC (NSCLC) EnVision Flex-Autostainer Link 48
pharmDx Assay
Dako PD-L1 IHC 22C3 TPS ≥ 1% (NSCLC) CPS ≥ 10 (UC) CPS ≥ 1 (Gastric/GEJ carcinoma) CPS ≥ 1 EnVision Flex-Autostainer Link 48
pharmDx Assay (cervical carcinoma) CPS ≥ 10 (ESCC) CPS ≥ 1 (HNSCC) CPS ≥ 10 (TNBC)

UC: Urothelial carcinoma; TNBC: Triple-negative breast carcinoma; NSCLC: Non-small cell lung carcinoma; ESCC: Esophageal squamous cell carcinoma;
HNSCC: Head and neck squamous cell carcinoma; GEJ: Gastroesophageal junction carcinoma; PD-L1: Programmed cell death ligand 1; IC: Immune cells;
TC: Tumor cells.

Table 3. Overview of the complementary and other available diagnostic PD-L1 tests

Antibody (clone/manufacturer) Scoring algorithms’ cutoff (tumor type/drug)

Ventana PD-L1 SP263 Assay* ≥25% of tumor cells exhibit membrane staining; or, ICP > 1% and IC+ ≥ 25%; or, ICP = 1% and IC+ = 100% (UC)
(Durvalumab)
73-10 (Dako Agilent) Not established yet
E3L1N (Cell Signaling) Not established yet

PD-L1: Programmed cell death ligand 1. *Complementary assay (FDA)

Vranic and Gatalica

PD-L1 and immunotherapy 18 www.biomolbiomed.com
Table 4. Definitions of the currently used scoring systems (algorithms) for the immunohistochemical assessment of PD-L1 expression in cancer

Scoring algorithm Interpretation

Tumor Proportion Score (TPS) The percentage of viable tumor cells showing partial or complete membrane staining relative to all viable tumor
cells present in the sample (positive and negative).
Combined Positive Score (CPS) Number of PD-L1-positive cells (Tumor cells, lymphocytes, and macrophages) divided by the total number of
viable tumor cells in the assessed area, multiplied by 100.
Tumor cells (TC) Score The percentage of PD-L1-positive tumor cells at any intensity.
Immune cells (IC) Score The proportion of tumor area occupied by PD-L1-positive immune (mononuclear) cells at any intensity.

PD-L1: Programmed cell death ligand 1.

Figure 1. A morphology (Image A; magnification 40×) of poorly differentiated pulmonary non-small cell lung cancer with a marked nuclear atypia
with diffuse and strong membranous PD-L1 expression (Image B; SP142 clone, magnification 20×). PD-L1: Programmed cell death ligand 1.

Figure 2. A high-grade triple-negative breast cancer case with metaplastic and pleomorphic features (Image A, magnification 20×) with PD-L1
expression in the tumor-infiltrating lymphocytes (SP142 clone) [99, 100]. The tumor cells were devoid of PD-L1 expression (Image B, magnification
20×). PD-L1: Programmed cell death ligand 1.

predictive biomarker for several cancers, including NSCLC, other anti-PD-L1 antibodies and confirmed predictive value
urothelial (bladder) carcinoma, cervical carcinoma, gastroe- in NSCLC [49–51], the 73-10 clone has not yet been approved
sophageal/gastric (GEJ) carcinoma, esophageal squamous cell by FDA (Table 3). The threshold for positivity and scoring
carcinoma (ESCC), triple-negative breast carcinoma, and head algorithm is also to be determined for this antibody.
and neck squamous cell carcinoma (HNSCC) (Tables 1 and 2).
Similar to the SP142 assay, the 22C3 clone also has different Scoring algorithms
cutoff and cancer-specific scoring algorithms (Tables 3 and 4). Four different scoring systems (algorithms) have been proposed
The 28-8 pharmDx assay has been primarily utilized for NSCLC and validated for the PD-L1 assessment and quantification by
(Tables 1 and 2). IHC so far. These include the TC score, IC score, tumor pro-
The third clone is 73-10. It has initially been developed portion score (TPS), and combined positive score (CPS) (their
for clinical trials exploring the anti-PD-L1 agent avelumab. definitions are summarized in Table 4). Each scoring algorithm
Despite good analytical performance and concordance with has been designed and approved for the specific ICIs (Table 1).

Vranic and Gatalica

PD-L1 and immunotherapy 19 www.biomolbiomed.com
Figure 3. There is an excellent concordance between the four clones in assessing PD-L1 expression in metastatic soft tissue neoplasm (dedifferen-
tiated liposarcoma) (magnification 10×). PD-L1: Programmed cell death ligand 1.

Figure 4. A case of diffuse large B-cell lymphoma (DLBCL) exhibiting a discordant expression (in both intensity and percentage) of PD-L1 with the
lowest expression with the 22C3 and the highest with SP263 clone (magnification 10×). PD-L1: Programmed cell death ligand 1.

TPS (%) is defined as the percentage of viable cancer cells TC score (%) implies the number of PD-L1-positive cancer
with partial or complete membrane expression (≥1+) relative to cells divided by the total number of cancer cells.
all viable cancer cells present in the entire sample (positive and IC score (%) is expressed as the total number of PD-L1-
negative). positive mononuclear cells (lymphocytes and macrophages) at
CPS is calculated as the number of PD-L1 positive cells (both any intensity within the tumor area. The tumor area includes
cancer and IC) divided by the total number of viable cancer cells intra- and peritumoral stroma. The expression of PD-L1 in
multiplied by 100. cancer cells is not considered for the IC score.

Vranic and Gatalica

PD-L1 and immunotherapy 20 www.biomolbiomed.com
 Multiple studies have evaluated the concordance (inter- recognized an unmet need to harmonize the design and utility
assay heterogeneity) between the different PD-L1 clones in of various CDx in a clinical setting [46]. Other researchers also
common cancers revealing highly variable results [52–63]. recognized this problem, highlighting the need for PD-L1 IHC
For instance, in the IMpassion130 trial, 46% of TNBC samples standardization and harmonization in clinical practice [73–75].
were positive with SP142 clone (Ventana); when another Recent technology advances (digital pathology and deep learn-
assay (22C3, Agilent) was utilized on the same samples, the ing/artificial intelligence/AI/) with computer-assisted PD-L1
positivity significantly increased (∼80%) [64]. However, a assessment and scoring may be good solutions to overcome
recent comprehensive systematic review of Prince et al. [65] the shortcomings related to manual PD-L1 evaluation [76, 77].
revealed an excellent concordance between 28-8, 22C3, and A recent, multi-institutional study explored the utility of the
SP263 clones in assessing PD-L1 expression in cancer cells AI-assisted method in the evaluation of PD-L1 IC expression
among the common cancer subtypes (e.g., NSCLC, HNSCC, in breast cancer (SP142 clone) [78]. The proposed AI tool
and urothelial carcinoma); in contrast, SP142 clone stained substantially improved the accuracy and concordance in PD-L1
substantially fewer cancer cells than the three other clones interpretation, contributing to a better PD-L1 standardization in
in these cancers. Notably, the concordance between the clinical practice [78]. Several studies analyzing the AI assistance
four clones was substantially lower for PD-L1 assessment in PD-L1 assessment in NSCLC [79–83] and HNSCC [84] have
in IC [65]. been recently published.
Similar findings were reported in another systematic review In addition, intratumoral PD-L1 expression is very complex
with a meta-analysis conducted by Torlakovic et al. [66]. The and dynamic. PD-L1 status may be genetically upregulated
study explored the diagnostic accuracy of the laboratory- in cancer cells, e.g., via PD-L1 (CD274) gene amplification (a
developed PD-L1 assays. It also revealed that the Ventana SP142 good example is Hodgkin lymphoma) [68]. In addition, PD-L1
assay’s analytical sensitivity was significantly lower than the expression is tightly regulated at different molecular levels,
three other FDA-approved PD-L1 assays in NSCLC and some including transcriptional, posttranscriptional, and protein
other cancers (22C3 pharmDx, 28-8 pharmDx, and Ventana levels [85]. Furthermore, the presence of PD-L1 positive TC
SP263 assays). The authors of the meta-analysis concluded or IC may differ in different parts of the tumor as well as it
that “fit-for-purpose” PD-L1 laboratory-developed assays may differ between primary and metastatic sites (e.g., NSCLC
(particularly in referral and expert-led laboratories) might with different microenvironment in the primary and metastatic
be comparable with the PD-L1 FDA-approved assays (CDx and sites /brain/) [86, 87]. Consequently, single slide PD-L1 IHC
complementary assays) when both types of assays are compared may be an insufficient method to fully reflect the dynamic PD-
with an appropriately designated reference standard [66]. L1 expression in cancer [71]. Davis and Patel [88] assessed the
In line with these findings are recent recommendations predictive utility of PD-L1 expression based on all ICIs approved
from the Canadian Association of Pathologists-Association by the FDA through 2019. They found a low predictive value
Canadienne Des Pathologistes (CAP-ACP) regarding the fit- (∼29%) of PD-L1 positivity, while in the remaining cases, PD-
for-purpose PD-L1 assay development and optimization for L1 expression was either not predictive (53%) or not tested
selecting the patients in I-O [67]. Figures 3 and 4 illustrate (18%) [88].
the performance of four (SP142, SP263, 22C3, 28-8) anti-PD-L1 Broad and comprehensive reviews on this important topic
assays [68]. have already been published by Nimmagadda [89] and Cottrell
and Taube [8]. A recent study on the predictive value of PD-1
Practical considerations, potential pitfalls and refinements in IHC (coupled with image analysis) in patients undergoing treat-
immunohistochemistry assessments of immune checkpoint ment with PD-1 inhibitors (nivolumab and pembrolizumab) for
blockade NSCLC patients showed that PD-1 density is a better predic-
As shown above, several cancers are currently routinely tested tive biomarker for durable clinical benefit in these two NSCLC
for PD-L1 expression using IHC [69]. However, this method is cohorts treated with PD-1 blockade than PD-L1 score [90]. It
not absolute regarding the predictive utility of PD-L1 expression is important to note that presence of PD-1-positive TILs was
of responsiveness to ICIs, particularly monotherapy [69, 70]. It observed in many cancer types beyond NSCLC and was gen-
is also well known that some patients whose cancers exhibit PD- erally associated with the increased number of mutations in
L1 expression may not have a therapeutic response to ICIs, while TC [37].
some patients with negative PD-L1 test may still be responsive From the practical point of view, it is essential to highlight
to ICIs [39]. These facts represent potential caveats predicting that PD-L1 expression in TC is considered positive regardless
therapeutic response through PD-L1 assessment [70–72]. of the completeness of the membranous staining; however, in
Several important limitations associated with PD-L1 IHC gland-forming cancers (e.g., adenocarcinomas), staining con-
testing should be mentioned: a) Sensitivity of the commercially fined exclusively to the luminal border is considered negative.
available assays (CDx), as demonstrated in several studies; On the other hand, both membranous and cytoplasmic expres-
b) Various issues related to IHC assay and methodology; sions in IC are considered positive. Intratumoral macrophages
and c) Tissue sampling and preparation. Recently published may also overexpress PD-L1, including macrophages within
recommendations of pharmaceutical and in vitro CDx industries glandular lumens; however, this staining should not be counted
on one side and the Personalized Health Care Committee as PD-L1 positivity. Similar to other IHC stains, intracellular
of the College of American Pathologists (CAP) on the other pigments in some cancers (e.g., melanin, hemosiderin, and

Vranic and Gatalica

PD-L1 and immunotherapy 21 www.biomolbiomed.com
anthracotic pigment) can occasionally make the interpretation Conflicts of interest: The authors declare no conflicts of
of PD-L1 staining difficult [91]. interest.
Other essential aspects relevant when considering the pre-
Funding: The authors received no specific funding for this
dictive value of PD-L1 testing include inter- and intra-tumoral
work.
heterogeneity with variable effects on PD-L1 expression [54].
Sample details (primary vs. metastatic cancer, sample age, sam- Submitted: 21 July 2022
ple type/small/core biopsy vs. large/surgical/ biopsy, naïve vs. Accepted: 6 August 2022
treated samples) and various preanalytical variables [e.g., time Published online: 7 August 2022
of collection relative to treatment testing/”age” of the specimen,
time to fixation, type of fixative, fixation time (cold ischemia
time), decalcification for bone specimens] may have a signif- References
icant impact on PD-L1 expression and interpretation [5]. In [1] List of cleared or approved companion diagnostic devices (in vitro
particular, bone samples may not be suitable for PD-L1 assess- and imaging tools): Food and Drug Administration; 2022 Available
from: https://www.fda.gov/medical-devices/in-vitro-diagnostics/
ment as currently approved CDx, such as SP142 and SP263, are list-cleared-or-approved-companion-diagnostic-devices-in-vitro-
only validated for non-decalcified samples fixed in 10% neutral- and-imaging-tools.
buffered formalin. Similarly, CDx for PD-L1 is not validated on [2] Gong J, Chehrazi-Raffle A, Reddi S, Salgia R. Development of PD-
1 and PD-L1 inhibitors as a form of cancer immunotherapy: a
cytology samples, including cellblocks, despite recent studies comprehensive review of registration trials and future considera-
that confirmed an excellent concordance between biopsy and tions. J Immunother Cancer 2018;6(1):8. http://dx.doi.org/10.1186/
cytology samples in some cancers such as NSCLC [92, 93]. s40425-018-0316-z.
[3] Wei SC, Duffy CR, Allison JP. Fundamental mechanisms of immune
Mansour et al. [94] also demonstrated an excellent PD-L1 checkpoint blockade therapy. Cancer Discov 2018;8(9):1069–86.
concordance between cytology and histology (biopsy) samples https://doi.org/10.1158/2159-8290.CD-18-0367.
in NSCLC. Their comprehensive literature survey, based [4] Das S, Johnson DB. Immune-related adverse events and anti-tumor
efficacy of immune checkpoint inhibitors. J Immunother Cancer
on 25 published studies with ∼1700 paired cytopatholo- 2019;7(1):306. https://doi.org/10.1186/s40425-019-0805-8.
gy/histopathology samples, showed the median (range) concor- [5] Doroshow DB, Bhalla S, Beasley MB, Sholl LM, Kerr KM, Gnjatic
dance between 81% and 85% (62%–100%) at a threshold of 1% for S, et al. PD-L1 as a biomarker of response to immune-checkpoint
inhibitors. Nat Rev Clin Oncol 2021;18(6):345–62. https://doi.
a positive PD-L1 staining and 89% (67%–100%) at the threshold org/10.1038/s41571-021-00473-5.
of 50% [94]. However, they also found significant variations [6] Johnson DB, Nebhan CA, Moslehi JJ, Balko JM. Immune-
between laboratories, stressing the importance of optimization checkpoint inhibitors: long-term implications of toxicity. Nat
Rev Clin Oncol 2022;19(4):254–67. https://doi.org/10.1038/
and quality assurance in PD-L1 testing [94]. s41571-022-00600-w.
Like all other IHC stains, a lab that performs PD-L1 staining [7] Patsoukis N, Wang Q, Strauss L, Boussiotis VA. Revisiting the PD-
must be continuously involved in external quality assurance of 1 pathway. Sci Adv 2020;6(38):eabd2712. https://doi.org/10.1126/
sciadv.abd2712.
the assay (e.g., NordiQC, UK NEQAS, and CAP) [95]. [8] Cottrell TR, Taube JM. PD-L1 and emerging biomarkers in immune
Several studies also explored the effects of chemotherapy on checkpoint blockade therapy. Cancer J 2018;24(1):41–6. https://doi.
PD-L1 expression in cancers [96–98], reporting the upregulated org/10.1097/PPO.0000000000000301.
[9] Blank C, Mackensen A. Contribution of the PD-L1/PD-1 pathway to
effects of cytotoxic therapy on PD-L1 expression. In lung cancer, T-cell exhaustion: an update on implications for chronic infections
this upregulation of PD-L1 was associated with an adverse and tumor evasion. Cancer Immunol Immunother 2007;56(5):739–45.
clinical outcome [96], while in some other cancers (e.g., ESCC), https://doi.org/10.1007/s00262-006-0272-1.
[10] Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Pro-
PD-L1 activation following chemotherapy had no impact on grammed death-1 ligand 1 interacts specifically with the B7-1 costim-
patients’ outcomes [98]. ulatory molecule to inhibit T cell responses. Immunity 2007;27(1):
111–22. https://doi.org/10.1016/j.immuni.2007.05.016.
[11] Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7
family, co-stimulates T-cell proliferation and interleukin-10 secre-
Conclusion tion. Nat Med 1999;5(12):1365–9. https://doi.org/10.1038/70932.
Immunotherapy based on immune checkpoint inhibitors has [12] Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon
MS, et al. Predictive correlates of response to the anti-PD-L1 anti-
substantially improved the outcome and prognosis of numer- body MPDL3280A in cancer patients. Nature 2014;515(7528):563–7.
ous common malignancies. Several predictive biomarkers have https://doi.org/10.1038/nature14011.
been validated and approved, including PD-L1 testing by IHC as [13] FDA approves foundation medicine’s FoundationOne CDx™ , the
first and only comprehensive genomic profiling test for all solid
the most widely used predictive biomarker. Despite its evident tumors incorporating multiple companion diagnostics; 2017. Avail-
clinical utility and CDx status by FDA for several cancers, the able from: https://www.foundationmedicine.com/press-releases/f2
predictive value of PD-L1 expression remains low across can- b20698-10bd-4ac9-a5e5-c80c398a57b5.
[14] Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor-infiltrating
cers. In addition, PD-L1 testing and interpretation are not easy lymphocytes are a marker for microsatellite instability in
to perform due to the unique IHC assays and interpretations colorectal carcinoma. Cancer 2001;91(12):2417–22. https://doi.
for each immune checkpoint inhibitor and various preanalyt- org/10.1002/1097-0142.
[15] Salgado R, Denkert C, Demaria S, Sirtaine N, Klauschen F, Pruneri
ical issues common for all IHC assays. Additional efforts from G, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in
industry, stakeholders/regulatory bodies/, academia, and clin- breast cancer: recommendations by an International TILs Working
icians are necessary to harmonize and improve the overall PD- Group 2014. Ann Oncol 2015;26(2):259–71. https://doi.org/10.1093/
annonc/mdu450.
L1 IHC performance and provide novel predictive biomarkers to [16] WHO Classification of Tumours Editorial Board. Breast Tumours. 5th
immune checkpoint inhibitors. ed. Lyon: IARC; 2019.

Vranic and Gatalica

PD-L1 and immunotherapy 22 www.biomolbiomed.com
 [17] Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. [35] Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al.
Assessing tumor-infiltrating lymphocytes in solid tumors: a practi- Mismatch repair deficiency predicts response of solid tumors to PD-
cal review for pathologists and proposal for a standardized method 1 blockade. Science 2017;357(6349):409–13. https://doi.org/10.1126/
from the international immunooncology biomarkers working group: science.aan6733.
part 1: assessing the host immune response, TILs in invasive breast [36] FDA grants accelerated approval to pembrolizumab for first
carcinoma and ductal carcinoma in situ, metastatic tumor deposits tissue/site agnostic indication; 2017. Available from: https://www.
and areas for further research. Adv Anat Pathol 2017;24(5):235–51. fda.gov/drugs/resources-information-approved-drugs/fda-grants-
https://doi.org/10.1097/PAP.0000000000000162. accelerated-approval-pembrolizumab-first-tissuesite-agnostic-
[18] Bai R, Lv Z, Xu D, Cui J. Predictive biomarkers for cancer immunother- indication.
apy with immune checkpoint inhibitors. Biomark Res 2020;8:34. [37] Gatalica Z, Snyder C, Maney T, Ghazalpour A, Holterman DA, Xiao
https://doi.org/10.1186/s40364-020-00209-0. N, et al. Programmed cell death 1 (PD-1) and its ligand (PD-L1) in
[19] Shum B, Larkin J, Turajlic S. Predictive biomarkers for response to common cancers and their correlation with molecular cancer type.
immune checkpoint inhibition. Semin Cancer Biol 2022 Feb;79:4–17. Cancer Epidemiol Biomarkers Prev 2014;23(12):2965–70. https://doi.
https://doi.org/10.1016/j.semcancer.2021.03.036. org/10.1158/1055-9965.EPI-14-0654.
[20] Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, [38] An Y, Zhou J, Lin G, Wu H, Cong L, Li Y, et al. Clinicopathological and
et al. Checkpoint blockade cancer immunotherapy targets tumour- molecular characteristics of colorectal signet ring cell carcinoma: a
specific mutant antigens. Nature 2014;515(7528):577–81. https://doi. review. Pathol Oncol Res 2021;27:1609859. https://doi.org/10.3389/
org/10.1038/nature13988. pore.2021.1609859.
[21] Vranic S. Microsatellite instability status predicts response to [39] Daud AI, Wolchok JD, Robert C, Hwu WJ, Weber JS, Ribas A,
anti-PD-1/PD-L1 therapy regardless the histotype: a comment on et al. Programmed death-ligand 1 expression and response
recent advances. Bosn J Basic Med Sci 2017;17(3):274–5. https://doi. to the anti-programmed death 1 antibody pembrolizumab in
org/10.17305/bjbms.2017.2366. melanoma. J Clin Oncol 2016;34(34):4102–9. https://doi.org/10.1200/
[22] Fusco MJ, West HJ, Walko CM. Tumor mutation burden and can- JCO.2016.67.2477.
cer treatment. JAMA Oncol 2021;7(2):316. https://doi.org/10.1001/ [40] Gandhi L, Rodriguez-Abreu D, Gadgeel S, Esteban E, Felip E, De Ange-
jamaoncol.2020.6371. lis F, et al. Pembrolizumab plus chemotherapy in metastatic non-
[23] Merino DM, McShane LM, Fabrizio D, Funari V, Chen SJ, White JR, small-cell lung cancer. N Engl J Med 2018;378(22):2078–92. https://
et al. Establishing guidelines to harmonize tumor mutational burden doi.org/10.1056/NEJMoa1801005.
(TMB): in silico assessment of variation in TMB quantification across [41] Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, et al. Asso-
diagnostic platforms: phase I of the friends of cancer research TMB ciation of PD-1, PD-1 ligands, and other features of the tumor
harmonization project. J Immunother Cancer 2020;8(1):e000147. immune microenvironment with response to anti-PD-1 therapy. Clin
https://doi.org/10.1136/jitc-2019-000147. Cancer Res 2014;20(19):5064–74. https://doi.org/10.1158/1078-0432.
[24] Vega DM, Yee LM, McShane LM, Williams PM, Chen L, Vilimas T, CCR-13-3271.
et al. Aligning tumor mutational burden (TMB) quantification across [42] Carbognin L, Pilotto S, Milella M, Vaccaro V, Brunelli M, Calio A, et al.
diagnostic platforms: phase II of the friends of cancer research TMB Differential activity of nivolumab, pembrolizumab and MPDL3280A
harmonization project. Ann Oncol 2021;32(12):1626–36. https://doi. according to the tumor expression of programmed death-ligand-
org/10.1016/j.annonc.2021.09.016. 1 (PD-L1): sensitivity analysis of trials in melanoma, lung and
[25] FDA authorizes MSK-IMPACT test for analyzing patient tumors; 2017. genitourinary cancers. PLoS One. 2015;10(6):e0130142. https://doi.
Available from: https://www.mskcc.org/news/fda-authorizes-msk- org/10.1371/journal.pone.0130142.
impact-test-analyzing-patient-tumors. [43] Horn L, Mansfield AS, Szczesna A, Havel L, Krzakowski M, Hochmair
[26] Yarchoan M, Hopkins A, Jaffee EM. Tumor mutational burden and MJ, et al. First-line atezolizumab plus chemotherapy in extensive-
response rate to PD-1 inhibition. N Engl J Med 2017;377(25):2500–1. stage small-cell lung cancer. N Engl J Med 2018;379(23):2220–9.
https://doi.org/10.1056/NEJMc1713444. https://doi.org/10.1056/NEJMoa1809064.
[27] Valero C, Lee M, Hoen D, Wang J, Nadeem Z, Patel N, et al. The asso- [44] Chung HC, Piha-Paul SA, Lopez-Martin J, Schellens JHM, Kao S,
ciation between tumor mutational burden and prognosis is depen- Miller WH, Jr.,et al. Pembrolizumab after two or more lines of pre-
dent on treatment context. Nat Genet 2021;53(1):11–5. https://doi. vious therapy in patients with recurrent or metastatic SCLC: results
org/10.1038/s41588-020-00752-4. from the KEYNOTE-028 and KEYNOTE-158 studies. J Thorac Oncol
[28] Silk AW, Barker CA, Bhatia S, Bollin KB, Chandra S, Eroglu Z, 2020;15(4):618–27. https://doi.org/10.1016/j.jtho.2019.12.109.
et al. Society for Immunotherapy of Cancer (SITC) clinical practice [45] Paver EC, Cooper WA, Colebatch AJ, Ferguson PM, Hill SK, Lum
guideline on immunotherapy for the treatment of nonmelanoma T, et al. Programmed death ligand-1 (PD-L1) as a predictive marker
skin cancer. J Immunother Cancer 2022;10(7):e004434. https://doi. for immunotherapy in solid tumours: a guide to immunohistochem-
org/10.1136/jitc-2021-004434. istry implementation and interpretation. Pathology 2021;53(2):141–
[29] Knepper TC, Montesion M, Russell JS, Sokol ES, Frampton GM, 56. https://doi.org/10.1016/j.pathol.2020.10.007.
Miller VA, et al. The genomic landscape of Merkel cell carcinoma [46] Willis JE, Eyerer F, Walk EE, Vasalos P, Bradshaw G, Yohe SL,
and clinicogenomic biomarkers of response to immune checkpoint et al. Companion diagnostics. Arch Pathol Lab Med 2022. https://doi.
inhibitor therapy. Clin Cancer Res 2019;25(19):5961–71. https://doi. org/10.5858/arpa.2021-0151-CP.
org/10.1158/1078-0432.CCR-18-4159. [47] Rimm DL, Han G, Taube JM, Yi ES, Bridge JA, Flieder DB, et al. A
[30] Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. prospective, multi-institutional, pathologist-based assessment of 4
Analysis of 100,000 human cancer genomes reveals the landscape immunohistochemistry assays for PD-L1 expression in non-small cell
of tumor mutational burden. Genome Med 2017;9(1):34. https://doi. lung cancer. JAMA Oncol 2017;3(8):1051–8. https://doi.org/10.1001/
org/10.1186/s13073-017-0424-2. jamaoncol.2017.0013.
[31] Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, [48] VENTANA PD-L1 (SP263) Assay; Available from: https://www.
Biankin AV, et al. Signatures of mutational processes in human accessdata.fda.gov/cdrh_docs/pdf16/p160046c.pdf.
cancer. Nature 2013;500(7463):415–21. https://doi.org/10.1038/ [49] Grote HJ, Feng Z, Schlichting M, Helwig C, Ruisi M, Jin H, et al. Pro-
nature12477. grammed death-ligand 1 immunohistochemistry assay comparison
[32] Hause RJ, Pritchard CC, Shendure J, Salipante SJ. Classification studies in NSCLC: characterization of the 73-10 assay. J Thorac Oncol
and characterization of microsatellite instability across 18 can- 2020;15(8):1306–16. https://doi.org/10.1016/j.jtho.2020.04.013.
cer types. Nat Med 2016;22(11):1342–50. https://doi.org/10.1038/ [50] Heo YJ, Kim B, Kim H, Kim S, Jang MS, Kim KM. PD-L1 expression
nm.4191. in paired biopsies and surgical specimens in gastric adenocarcinoma:
[33] Dudley JC, Lin MT, Le DT, Eshleman JR. Microsatellite instability as a digital image analysis study. Pathol Res Pract 2021;218:153338.
a biomarker for PD-1 blockade. Clin Cancer Res 2016;22(4):813–20. https://doi.org/10.1016/j.prp.2020.153338.
https://doi.org/10.1158/1078-0432.CCR-15-1678. [51] Park K, Ozguroglu M, Vansteenkiste J, Spigel D, Yang JCH, Ishii H,
[34] Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring et al. Avelumab versus docetaxel in patients with platinum-treated
AD, et al. PD-1 blockade in tumors with mismatch-repair defi- advanced NSCLC: 2-year follow-up from the JAVELIN lung 200 phase
ciency. N Engl J Med 2015;372(26):2509–20. https://doi.org/10.1056/ 3 trial. J Thorac Oncol 2021;16(8):1369–78. https://doi.org/10.1016/j.
NEJMoa1500596. jtho.2021.03.009.

Vranic and Gatalica

PD-L1 and immunotherapy 23 www.biomolbiomed.com
[52] Lee SE, Park HY, Lim SD, Han HS, Yoo YB, Kim WS. Concor- Mol Morphol 2019;27(10):699–714. https://doi.org/10.1097/
dance of programmed death-ligand 1 expression between SP142 and PAI.0000000000000800.
22C3/SP263 assays in triple-negative breast cancer. J Breast Cancer [68] Vranic S, Ghosh N, Kimbrough J, Bilalovic N, Bender R, Arguello
2020;23(3):303–13. https://doi.org/10.4048/jbc.2020.23.e37. D, et al. PD-L1 status in refractory lymphomas. PLoS One
[53] Pang JB, Castles B, Byrne DJ, Button P, Hendry S, Lakhani SR, 2016;11(11):e0166266. https://doi.org/10.1371/journal.pone.0166266.
et al. SP142 PD-L1 scoring shows high interobserver and intraob- [69] Udall M, Rizzo M, Kenny J, Doherty J, Dahm S, Robbins P, et al. PD-L1
server agreement in triple-negative breast carcinoma but over- diagnostic tests: a systematic literature review of scoring algorithms
all low percentage agreement with other PD-L1 clones SP263 and and test-validation metrics. Diagn Pathol 2018;13(1):12. https://doi.
22C3. Am J Surg Pathol 2021;45(8):1108–17. https://doi.org/10.1097/ org/10.1186/s13000-018-0689-9.
PAS.0000000000001701. [70] Ribas A, Hu-Lieskovan S. What does PD-L1 positive or negative
[54] Buttner R, Gosney JR, Skov BG, Adam J, Motoi N, Bloom KJ, et al. Pro- mean? J Exp Med 2016;213(13):2835–40. https://doi.org/10.1084/
grammed death-ligand 1 immunohistochemistry testing: a review of jem.20161462.
analytical assays and clinical implementation in non-small-cell lung [71] Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al.
cancer. J Clin Oncol 2017;35(34):3867–76. https://doi.org/10.1200/ Nivolumab in previously untreated melanoma without BRAF muta-
JCO.2017.74.7642. tion. N Engl J Med 2015;372(4):320–30. https://doi.org/10.1056/
[55] Ahn S, Woo JW, Kim H, Cho EY, Kim A, Kim JY, et al. Pro- NEJMoa1412082.
grammed death ligand 1 immunohistochemistry in triple-negative [72] Ribas A, Hamid O, Daud A, Hodi FS, Wolchok JD, Kefford R, et al.
breast vancer: evaluation of inter-pathologist concordance and inter- Association of pembrolizumab with tumor response and survival
assay variability. J Breast Cancer 2021;24(3):266–79. https://doi. among patients with advanced melanoma. JAMA. 2016;315(15):1600–
org/10.4048/jbc.2021.24.e29. 9. https://doi.org/10.1001/jama.2016.4059.
[56] Rugo HS, Loi S, Adams S, Schmid P, Schneeweiss A, Barrios CH, et al. [73] Savic Prince S, Bubendorf L. Predictive potential and need for
PD-L1 immunohistochemistry assay comparison in atezolizumab standardization of PD-L1 immunohistochemistry. Virchows Arch
plus nab-paclitaxel-treated advanced triple-negative breast cancer. 2019;474(4):475–84. https://doi.org/10.1007/s00428-018-2445-7.
J Natl Cancer Inst 2021;113(12):1733–43. https://doi.org/10.1093/jnci/ [74] Darmon-Novello M, Adam J, Lamant L, Battistella M, Ortonne
djab108. N, Balme B, et al. Harmonization of programmed death-ligand 1
[57] Marchetti A, Barberis M, Franco R, De Luca G, Pace MV, Staibano immunohistochemistry and mRNA expression scoring in metastatic
S, et al. Multicenter comparison of 22C3 pharmDx (agilent) and melanoma: a multicentre analysis. Histopathology 2022;80(7):1091–
SP263 (ventana) assays to test PD-L1 expression for NSCLC patients 101. https://doi.org/10.1111/his.14651.
to be treated with immune checkpoint inhibitors. J Thorac Oncol [75] Marletta S, Fusco N, Munari E, Luchini C, Cimadamore A, Brunelli
2017;12(11):1654–63. https://doi.org/10.1016/j.jtho.2017.07.031. M, et al. Atlas of PD-L1 for pathologists: indications, scores, diagnostic
[58] Adam J, Le Stang N, Rouquette I, Cazes A, Badoual C, Pinot-Roussel platforms and reporting systems. J Pers Med 2022;12(7):1073. https://
H, et al. Multicenter harmonization study for PD-L1 IHC testing in doi.org/10.3390/jpm12071073.
non-small-cell lung cancer. Ann Oncol 2018;29(4):953–8. https://doi. [76] Inge LJ, Dennis E. Development and applications of computer
org/10.1093/annonc/mdy014. image analysis algorithms for scoring of PD-L1 immunohistochem-
[59] Ratcliffe MJ, Sharpe A, Midha A, Barker C, Scott M, Scorer P, istry. Immunooncol Technol 2020;6:2–8. https://doi.org/10.1016/j.
et al. Agreement between programmed cell death ligand-1 diagnos- iotech.2020.04.001.
tic assays across multiple protein expression cutoffs in non-small [77] Koelzer VH, Sirinukunwattana K, Rittscher J, Mertz KD. Pre-
cell lung cancer. Clin Cancer Res 2017;23(14):3585–91. https://doi. cision immunoprofiling by image analysis and artificial intelli-
org/10.1158/1078-0432.CCR-16-2375. gence. Virchows Arch 2019;474(4):511–22. https://doi.org/10.1007/
[60] Eckstein M, Cimadamore A, Hartmann A, Lopez-Beltran A, Cheng s00428-018-2485-z.
L, Scarpelli M, et al. PD-L1 assessment in urothelial carcinoma: [78] Wang X, Wang L, Bu H, Zhang N, Yue M, Jia Z, et al. How can arti-
a practical approach. Ann Transl Med 2019;7(22):690. https://doi. ficial intelligence models assist PD-L1 expression scoring in breast
org/10.21037/atm.2019.10.24. cancer: results of multi-institutional ring studies. NPJ Breast Cancer
[61] Eckstein M, Erben P, Kriegmair MC, Worst TS, Weiss CA, Wirtz 2021;7(1):61. https://doi.org/10.1038/s41523-021-00268-y.
RM, et al. Performance of the food and drug administration/EMA- [79] Huang Z, Chen L, Lv L, Fu CC, Jin Y, Zheng Q, et al. A new AI-assisted
approved programmed cell death ligand-1 assays in urothelial car- scoring system for PD-L1 expression in NSCLC. Comput Meth-
cinoma with emphasis on therapy stratification for first-line use ods Programs Biomed 2022;221:106829. https://doi.org/10.1016/j.
of atezolizumab and pembrolizumab. Eur J Cancer 2019;106:234–43. cmpb.2022.106829.
https://doi.org/10.1016/j.ejca.2018.11.007. [80] Wu J, Liu C, Liu X, Sun W, Li L, Gao N, et al. Artificial intelligence-
[62] Zajac M, Scott M, Ratcliffe M, Scorer P, Barker C, Al-Masri H, assisted system for precision diagnosis of PD-L1 expression in non-
et al. Concordance among four commercially available, validated pro- small cell lung cancer. Mod Pathol 2022;35(3):403–11. https://doi.
grammed cell death ligand-1 assays in urothelial carcinoma. Diagn org/10.1038/s41379-021-00904-9.
Pathol 2019;14(1):99. https://doi.org/10.1186/s13000-019-0873-6. [81] Hondelink LM, Huyuk M, Postmus PE, Smit V, Blom S, von der
[63] Karnik T, Kimler BF, Fan F, Tawfik O. PD-L1 in breast cancer: com- Thusen JH, et al. Development and validation of a supervised deep
parative analysis of 3 different antibodies. Hum Pathol 2018;72:28–34. learning algorithm for automated whole-slide programmed death-
https://doi.org/10.1016/j.humpath.2017.08.010. ligand 1 tumour proportion score assessment in non-small cell lung
[64] Rugo HS, Loi S, Adams S, Schmid P, Schneeweiss A, Barrios cancer. Histopathology 2022;80(4):635–47. https://doi.org/10.1111/
CH, et al. Performance of PD-L1 immunohistochemistry (IHC) his.14571.
assays in unresectable locally advanced or metastatic triple-negative [82] Choi S, Cho SI, Ma M, Park S, Pereira S, Aum BJ, et al. Artificial
breast cancer (mTNBC): post-hoc analysis of IMpassion130. intelligence-powered programmed death ligand 1 analyser reduces
Ann Oncol 2019;30:858–9. https://doi.org/10.1093/annonc/ interobserver variation in tumour proportion score for non-small cell
mdz394.009. lung cancer with better prediction of immunotherapy response. Eur
[65] Prince EA, Sanzari JK, Pandya D, Huron D, Edwards R. Analytical J Cancer 2022;170:17–26. https://doi.org/10.1016/j.ejca.2022.04.011.
concordance of PD-L1 assays utilizing antibodies from FDA-approved [83] Sha L, Osinski BL, Ho IY, Tan TL, Willis C, Weiss H, et al. Multi-
diagnostics in advanced cancers: a systematic literature review. JCO field-of-view deep learning model predicts nonsmall cell lung cancer
Precis. Oncol 2021;5:953–73. https://doi.org/10.1200/PO.20.00412. programmed death-ligand 1 status from whole-slide hematoxylin and
[66] Torlakovic E, Lim HJ, Adam J, Barnes P, Bigras G, Chan AWH, et al. eosin images. J Pathol Inform 2019;10:24. https://doi.org/10.4103/jpi.
“Interchangeability” of PD-L1 immunohistochemistry assays: a meta- jpi_24_19.
analysis of diagnostic accuracy. Mod Pathol 2020;33(1):4–17. https:// [84] Puladi B, Ooms M, Kintsler S, Houschyar KS, Steib F, Modabber A,
doi.org/10.1038/s41379-019-0327-4. et al. Automated PD-L1 scoring using artificial intelligence in head
[67] Cheung CC, Barnes P, Bigras G, Boerner S, Butany J, Calabrese and neck squamous cell carcinoma. Cancers (Basel) 2021;13(17):4409.
F, et al. Fit-for-purpose PD-L1 biomarker testing for patient https://doi.org/10.3390/cancers13174409.
selection in immuno-oncology: guidelines for clinical laboratories [85] Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms controlling
from the Canadian Association of Pathologists-Association PD-L1 expression in cancer. Mol Cell 2019;76(3):359–70. https://doi.
Canadienne des Pathologistes (CAP-ACP). Appl Immunohistochem org/10.1016/j.molcel.2019.09.030.

Vranic and Gatalica

PD-L1 and immunotherapy 24 www.biomolbiomed.com
[86] Li M, Hou X, Sai K, Wu L, Chen J, Zhang B, et al. Immune [93] Jug R, Giovacchini CX, Liu B, Green CL, Clarke JM, Mahmood K,
suppressive microenvironment in brain metastatic non-small et al. EBUS-FNA cytologic-histologic correlation of PD-L1 immuno-
cell lung cancer: comprehensive immune microenvironment histochemistry in non-small cell lung cancer. J Am Soc Cytopathol
profiling of brain metastases versus paired primary lung tumors 2020;9(6):485–93. https://doi.org/10.1016/j.jasc.2020.04.003.
(GASTO 1060). Oncoimmunology 2022;11(1):2059874. https://doi. [94] Mansour MSI, Lindquist KE, Seidal T, Mager U, Mohlin R, Tran L,
org/10.1080/2162402X.2022.2059874. et al. PD-L1 testing in cytological non-small cell lung cancer speci-
[87] Batur S, Dulger O, Durak S, Yumuk PF, Caglar HB, Bozkurtlar E, mens: a comparison with biopsies and review of the literature. Acta
et al. Concordance of PD-L1 expression and CD8+ TIL intensity Cytol 2021;65(6):501–9. https://doi.org/10.1159/000517078.
between NSCLC and synchronous brain metastases. Bosn J [95] Chebib I, Mino-Kenudson M. PD-L1 immunohistochemistry: clones,
Basic Med Sci 2020;20(3):329–35. https://doi.org/10.17305/ cutoffs, and controversies. APMIS 2022;130(6):295–313. https://doi.
bjbms.2019.4474. org/10.1111/apm.13223.
[88] Davis AA, Patel VG. The role of PD-L1 expression as a [96] Guo L, Song P, Xue X, Guo C, Han L, Fang Q, et al. Variation of
predictive biomarker: an analysis of all US Food and Drug programmed death ligand 1 expression after platinum-based neoad-
Administration (FDA) approvals of immune checkpoint inhibitors. juvant chemotherapy in lung cancer. J Immunother 2019;42(6):215–
J Immunother Cancer 2019;7(1):278. https://doi.org/10.1186/ 20. https://doi.org/10.1097/CJI.0000000000000275.
s40425-019-0768-9. [97] Liang Y, Yu M, Zhou C, Zhu X. Variation of PD-L1 expres-
[89] Nimmagadda S. Quantifying PD-L1 expression to monitor immune sion in locally advanced cervical cancer following neoadjuvant
checkpoint therapy: opportunities and challenges. Cancers (Basel) chemotherapy. Diagn Pathol 2020;15(1):67. https://doi.org/10.1186/
2020;12(11):3173. https://doi.org/10.3390/cancers12113173. s13000-020-00977-1.
[90] Hummelink K, van der Noort V, Muller M, Schouten RD, Lalezari [98] Fukuoka E, Yamashita K, Tanaka T, Sawada R, Sugita Y, Ari-
F, Peters D, et al. PD-1T TILs as a predictive biomarker for clinical moto A, et al. Neoadjuvant chemotherapy increases PD-L1 expres-
benefit to PD-1 blockade in patients with advanced NSCLC. Clin Can- sion and CD8(+) tumor-infiltrating lymphocytes in esophageal squa-
cer Res 2022 Jul 19;CCR-22-0992. https://doi.org/10.1158/1078-0432. mous cell carcinoma. Anticancer Res. 2019;39(8):4539–48. https://
CCR-22-0992. doi.org/10.21873/anticanres.13631.
[91] Akhtar M, Rashid S, Al-Bozom IA. PD-L1 immunostaining: what [99] Vranic S, Stafford P, Palazzo J, Skenderi F, Swensen J, Xiu J, et al.
pathologists need to know. Diagn Pathol 2021;16(1):94. https://doi. Molecular profiling of the metaplastic spindle cell carcinoma
org/10.1186/s13000-021-01151-x. of the breast reveals potentially targetable biomarkers. Clin
[92] Chauhan A, Siegel L, Freese R, Racila E, Stewart J, 3rd,Amin K. Breast Cancer 2020;20(4):326–31 e1. https://doi.org/10.1016/j.
Performance of Ventana SP263 PD-L1 assay in endobronchial ultra- clbc.2020.02.008.
sound guided-fine-needle aspiration derived non-small-cell lung car- [100] Vranic S, Palazzo J, Swensen J, Xiu J, Florento E, Gatalica Z. Theranos-
cinoma samples. Diagn Cytopathol 2021;49(3):355–62. https://doi. tic molecular profiling of pleomorphic ductal carcinoma of the breast.
org/10.1002/dc.24654. Breast J 2019;25(1):175–6. https://doi.org/10.1111/tbj.13187.

Related articles published in BJBMS

1. Relationship between PD-L1 expression and prognostic factors in high-risk cutaneous squamous and basal cell carcinoma
Özden Yülek et al., BJBMS, 2022
2. Concordance of PD-L1 expression and CD8+ TIL intensity between NSCLC and synchronous brain metastases
Sebnem Batur et al., BJBMS, 2020
3. Targeted immunotherapy with a checkpoint inhibitor in combination with chemotherapy: A new clinical paradigm in the treatment of
triple-negative breast cancer
Farhan S. Cyprian et al., BJBMS, 2019

Vranic and Gatalica

PD-L1 and immunotherapy 25 www.biomolbiomed.com