PD-L1 Assessment in Needle Core Biopsies of Non-Small Cell Lung Cancer: Interpathologist Agreement and Potential Associated Histopathological Features
Ezgi HACIHASANOGLU, Buket BAMBUL SIGIRCI, Gamze USUL, Taha Cumhan SAVLI
1Department of Pathology, 1Yeditepe University, School of Medicine, ÝSTANBUL, TURKEY
2University of Health Sciences, Sisli Hamidiye Etfal Training Hospital, ÝSTANBUL, TURKEY
3Basaksehir Cam and Sakura City Hospital, ÝSTANBUL, TURKEY
4Istanbul Medipol University, School of Medicine, ÝSTANBUL, TURKEY
Keywords: Programmed death ligand 1, Lung, Interobserver, Biopsy, Needle
Immune checkpoint inhibitors are used in the treatment of non-small cell lung cancer (NSCLC). Programmed cell death-ligand
1 (PD-L1) immunohistochemistry (IHC) assessed by pathologists is subject to interobserver variability. In advanced/metastatic disease
and inoperable patients, PD-L1 assessment relies on biopsy specimens, commonly needle core biopsies (NCB). We aimed to determine the
interobserver agreement for PD-L1 tumor proportion score (TPS) in NSCLC NCBs and identify histopathological features that may be related
to interobserver variability.
Material and Methods: Sixty NSCLC NCBs with PD-L1 IHC were evaluated independently by four pathologists from different institutions.
PD-L1 TPS was evaluated in three categories: no/low expression (<1%), intermediate expression (1%–49%), and high expression (≥50%).
Histological tumor type, necrosis, tumor-infiltrating lymphocytes, tumor length/percentage in the biopsy, and crush/squeeze artifact was
Results: The statistical analysis of the three PD-L1 TPS categories demonstrated moderate agreement (Fleiss Kappa 0.477) in the no/low
category, fair agreement (Fleiss Kappa 0.390) in the intermediate category, and almost perfect agreement (Fleiss Kappa 0.952) in the high
category. A significant correlation (p=0.003) was found between the crush/squeeze artifact in NCB and rate of discordant TPS categories. There
was no significant correlation between pathologists’ agreement in the TPS categories and histological tumor type, tumor length, tumor ratio,
necrosis, and tumor-infiltrating lymphocytes.
Conclusion: Our results demonstrated moderate agreement among pathologists for the PD-L1 TPS 1% cut-off in NSCLC NCB, which is lower
than that reported in resection materials. The presence of crush/squeeze artifact in NCBs is significantly related to the rate of discordant TPS
categories, suggesting that PD-L1 assessment of pulmonary NCBs requires an awareness of this artifact.
Lung cancer is the leading cause of death among cancers1
. The treatment of non-small cell lung cancer (NSCLC)
has significantly advanced over the last two decades, contributing
to an improvement in survival rates. Through the
use of biomarkers-based treatment approaches, lung cancer
patients have been able to receive personalized treatment.
Recently, research on immune checkpoint inhibitors
has made significant contributions to the development of
novel treatment approaches for advanced-stage lung cancer.
The evidence suggests that immunotherapy is superior
to cytotoxic chemotherapy in a subset of patients, as well as
the possibility of combining immunotherapy and chemotherapy2
Programmed cell death-ligand 1 (PD-L1) immunohistochemistry
(IHC) is the first Food and Drug Administration
(FDA)-approved companion diagnostic test for immune
checkpoint inhibitors. The FDA approved the 22C3 assay
as a companion diagnostic test for pembrolizumab, an
immune checkpoint inhibitor. PD-L1 IHC is evaluated
and scored by pathologists. Different scoring methods are
employed in different organ tumors to examine the level
of PD-L1 staining in tumor cells and/or inflammatory cells
within the tumor stroma. To determine PD-L1 expression
in NSCLC, the Tumor Proportion Score (TPS) is used,
which is a percentage of viable tumor cells that stain partially
or completely by PD-L1 at any intensity (3).
PD-L1 assessment is subject to variability between pathologists.
This variability impacts the therapeutic outcome.
The interobserver concordance of TPS in NSCLC has been
investigated in a number of studies4-11. The majority
of these studies used resection material or tissue microarrays
(TMA), while only one study included needle core
biopsy (NCB) specimens. To the extent of our knowledge,
no study has been published using only NCB materials4-7,10. Image-guided transthoracic NCB is a routinely
used diagnostic tool in lung masses, and the assessment of
PD-L1 in advanced or metastatic disease and in inoperable
patients is based on the least invasive biopsies. Therefore, it
is imperative that NCB specimens be scored accurately in
order to guide treatment decisions.
Among the histopathological factors that may affect interobserver
agreement of PD-L1 scoring, only the histological
type of the tumor was investigated in a previous study8. Factors that may complicate the evaluation of PD-L1 in
NSCLC NCBs, such as tumor necrosis, tumor-infiltrating
lymphocytes, tumor length and percentage in the biopsy,
and effect of crush/squeeze artifact have not been evaluated
The purpose of this study was to determine the interobserver
agreement for PD-L1 TPS in NCB specimens of NSCLC
and identify histopathological features of the tumor that
may be related to interobserver variability.
|Study Design and Case Selection
A search was conducted in the electronic database of a single
center for patients diagnosed with NSCLC diagnosed
through a NCB specimen. Cases with available PD-L1
immunohistochemistry (IHC) studies were documented.
The study included 60 cases with at least 100 viable tumor
cells on Hematoxylin-Eosin (HE) stained slides and PD-L1
positive tumor cells ranging from 0% to 100%. Representative
HE slides and PD-L1 IHC slides of the cases were evaluated
independently and blinded to the diagnosis by four
pathologists from different institutions (EH, BBS, GU, TCS).
This study has been approved by the Ethics Committee
for Non-Interventional Clinical Studies (decision number:
202211Y0307, date: 14/11/2022). This study protocol is in
accordance with the Declaration of Helsinki of the World
PD-L1 IHC Staining and Scoring
The PD-L1 IHC 22C3 pharmDx IHC assay (Agilent Technologies/
Dako, Carpinteria, California, USA) was performed
in all cases. All tissue samples underwent fixation in 10% neutral buffered formalin for 6-24 hours. Four-
μm thick formalin-fixed, paraffin-embedded tissue sections
were dried at 60°C for 30 minutes. PD-L1 IHC 22C3
pharmDx IHC assays were performed using an EnVision
FLEX visualization system (Agilent, Santa Clara, USA) and
an Autostainer Link 48 system (Dako). In accordance with
the instructions of the manufacturer, positive and negative
controls were used. All cases were stained within 6 months
The Tumor Proportion Score (TPS) is defined as the percentage
of viable tumor cells with partial or complete PD-L1
membranous staining at any intensity (≥ 1+) relative to all
viable tumor cells in the sample3. The IHC results of
60 cases were evaluated by four pathologists and TPS was
assigned by each. TPS was categorized into three expression
levels: no/low expression (<1%), intermediate expression
(1–49%) and high expression (≥50%) (Figure 1).
Click Here to Zoom
|Figure 1: A-C) PD-L1 expression levels in tumor cells in NSCLC cases (x100). A) No/low expression (<1%) - Tumor cells are negative
for PD-L1, while some tumor-associated immune cells show positive staining. B) Intermediate expression (1–49%) - A small proportion
(1-10%) of the tumor cells show partial/complete membrane staining with PD-L1. C) High expression (≥50%) - Almost all tumor cells
show membrane staining with PD-L1 at varying intensities.
A comprehensive examination of histopathological features
that may be associated with interobserver variability
was performed. The histological type of tumor was documented.
Presence of necrosis was determined. Extent of
tumor-infiltrating lymphocytes was evaluated as mildmoderate
(<50%) or extensive (≥50%). Tumor length in the
biopsy specimen was measured in each case. The percentage
of tumor in the biopsy specimen was evaluated as <50%
or ≥50%. Presence of crush/squeeze artifact was evaluated.
The descriptive statistics are expressed in terms of number
(n) and percentage (%) for the variables in the study.
Fleiss’s Kappa analysis was used to calculate the agreement
between pathologists. Agreement for 1% and 50%
cut-off values were assessed. Evaluation was done according
to Landis and Koch’s methodology, according to the
following: kappa value of 0-0.2 indicated slight agreement,
0.2-0.4 fair agreement, 0.4-0.6 moderate agreement, 0.6-
0.8 substantial agreement, and 0.8-1 almost perfect agreement12. Statistical analysis of quantitative independent
data was conducted using the Mann-Whitney U test, and
a qualitative analysis of independent data was conducted
using the Chi-square test. The statistical significance level
was taken as 5% in the calculations. The statistical analysis
was carried out using IBM SPSS Statistics (version 26).
|PD-L1 TPS Evaluation by Pathologists
shows the number of cases scored by each pathologist
as low, moderate, and high TPS (Table I
). In 31 cases, four pathologists scored within the same TPS category. In
29 cases, at least one pathologist scored at a different TPS
Click Here to Zoom
|Table I: Number of cases scored by each pathologist as low,
moderate, and high TPS.
Interpathologist Agreement of PD-L1 TPS
Comparison of the TPS values determined by four pathologists
showed moderate agreement with Fleiss Kappa of
0.576 (Table II). The statistical analysis of the three TPS
categories [no/low expression (<1%), intermediate expression
(1–49%) or high expression (≥50%)] demonstrated
moderate agreement (Fleiss Kappa 0.477) in the “no/low”
category, fair agreement (Fleiss Kappa 0.390) in the intermediate
category, and almost perfect agreement (Fleiss
Kappa 0.952) in the high category (Table III).
The histopathological examination revealed 37 cases of
adenocarcinoma, 22 cases of squamous cell carcinoma, and
1 case of NSCLC, not otherwise specified (NOS). Tumor
length range in the biopsy specimen was 1-41 millimeters,
with a mean of 8.07 millimeters. Tumor ratio in the biopsy
specimen was <50% in 31 cases and ≥50% in 29 cases.
Necrosis was present in 16 cases. Tumor-infiltrating lymphocytes
were ≥50% in 10 cases. A crush/squeeze artifact
was present in 20 cases. Table IV illustrates the histopathological
features of the cases.
Relationship Between PD-L1 TPS Interpathologist
Agreement and Histopathological Parameters
In order to investigate the relationship between TPS concordance
and histopathological parameters, the study cases
were divided into two groups: cases evaluated in the same
TPS category by four pathologists (compatible group) and
cases evaluated in a different TPS category by at least one
pathologist (noncompatible group). As a result, 31 cases fell
within the compatible group and 29 were within the noncompatible
group. In terms of histological type, tumor size,
tumor ratio, necrosis, and tumor-infiltrating lymphocytes, no statistically significant differences were found between
the two groups. Compared to the compatible group, the
noncompatible group had a significantly greater incidence
of crush/squeeze artifacts (p<0.05) (Figure 2). Table V
demonstrates the relationship between TPS concordance
among pathologists and histopathological parameters.
Click Here to Zoom
|Table V: Relationship between TPS concordance among pathologists and histopathological parameters.
Click Here to Zoom
|Figure 2: A-D) Examples of crush/squeeze artifact in PD-L1 immunohistochemistry studies (x100). A) The image illustrates some PDL1
staining cells in which the cell type (tumor cell/immune cell/stroma cell) cannot be determined clearly (circle). B) The details of the
cell structure and the staining of PD-L1 cannot be clearly assessed due to the crush/squeeze artifact in this example. C) The tumor cells
overlap with the immune cells at the edge of the biopsy specimen, making the identification of the correct cell types more challenging
in PD-L1 staining (arrow). D) The area that shows staining with PD-L1 in this example contains crush artifacts that make it difficult to
determine whether PD-L1 staining tumor cells are present among the immune cells (circle).
The purpose of this study was to evaluate the agreement
between pathologists for PD-L1 TPS in NSCLC needle core
biopsy specimens. The results of this study demonstrated moderate agreement between four pathologists in PD-L1
TPS in tru-cut biopsy samples of NSCLCs. There was
almost perfect agreement among pathologists for the 50%
cut-off, but only fair agreement for the 1% cut-off, which
is used to determine therapy eligibility. As a secondary
objective, this study attempted to identify histopathological
features that might be associated with interobserver variability.
A statistically significant correlation did not exist
between interpathologist agreement on PD-L1 scores and
histological type, tumor length, tumor rate, necrosis and
tumor-infiltrating lymphocytes. The crush/squeeze artifact,
however, was found to significantly affect the interobserver
Prior studies investigating the interobserver agreement of
TPS in NSCLC reported an overall substantial to almost perfect agreement, with only a few of the studies reporting
moderate agreement4-11 (Table VI). Compared to the
previous studies, lower interobserver agreement regarding
1% cut-off was observed in our study. Considering that
this study was conducted with four pathologists working
in four different centers, it can be clearly seen how critical
interpathologist compatibility in PDL1 scoring is in guiding
Click Here to Zoom
|Table VI: Summary of prior studies on interobserver agreement of TPS in NSCLC.
The majority of the studies in the literature used resection
samples or tissue microarrays (TMA), and only one included
NCB specimens. To our knowledge, no study using only
NCB materials has been published. There is a critical need
to ensure high agreement between pathologists in PD-L1
evaluation in NCBs. This is because PD-L1 expression is
assessed in the least invasive biopsy samples in the setting of advanced and metastatic disease, and this information
guides treatment decisions. The only study in the literature
that included NCB specimens investigated the interobserver
reproducibility in a total of 107 NSCLC cases, consisting
of 66 NCB specimens, 22 resection materials, and 19
endobronchial ultrasound-guided transbronchial needle
aspirates (EBUS-TBNAs)8. The authors reported higher
agreement in resection and NCB specimens in comparison
to EBUS-TBNA, but did not disclose a statistical comparison
between resection materials and NCB specimens (kappa
for NCB: 0.776, kappa for resection: 0.716)8.
Considering the histopathological parameters that may be
associated with high interpathologist variability, the results
of our study have shown that there is no significant correlation
between the pathologists’ agreement in TPS categories
and the histological type of tumor, tumor length,
tumor ratio in the biopsy specimen, presence of necrosis,
and tumor-infiltrating lymphocytes. There was, however,
a significant correlation between the presence of a crush/
squeeze artifact in the biopsy and the rate of discordant TPS
categories. In the literature, there is only one study that has
investigated the relationship between NSCLC histological type and PD-L1 TPS interobserver agreement8. This
study indicated that interobserver agreement was influenced
by histologic type, with the squamous cell carcinoma
group showing slightly higher agreement than adenocarcinoma
group8. Our study is the first study to examine the
effects of tumor necrosis, tumor-infiltrating lymphocytes,
tumor length and percentage, and crush/squeeze artifact
on interobserver variability.
A crush/squeeze artifact is typically observed at the periphery
of the specimens, as a result of damage from crushing
or squeezing by forceps or other surgical instruments13.
A crush/squeeze artifact presents difficulties in the assessment
of cell types and the distinction of membranous
staining from cytoplasmic staining. Membranous staining
of other cell types, such as macrophages, stromal cells and
necrotic tumor cells, and granular and cytoplasmic staining
in tumor cells may be challenging to interpret in areas
of crush/squeeze artifact. This may result in an incorrect
assessment of the percentage of PD-L1 stained tumor cells.
According to a recent study conducted on gastric, gastroesophageal
junction and esophageal mucosal biopsy specimens,
crush artifact was among the features associated with
higher pathologist disagreements in PD-L1 scores14. To
our knowledge, no study has been published examining the
relationship between crush artifact in NCB specimens from
NSCLC and interpathologist agreement on PD-L1 scores.
A limitation of our study may be the small number of cases
and participants. However, all our cases were sourced
from and processed at one center, whereas the participating
pathologists were affiliated with different institutions.
The cases were representative of real clinical practice, and
tissue processing and immunohistochemical staining followed
the same standard procedure. Our study also has a
limitation in that no relationship was established between
the results and prognosis or treatment response. In order to
provide a more comprehensive understanding of this issue,
we are accelerating our studies on this subject in order to
acquire data on treatment responses and prognoses.
In conclusion, our results indicate an overall moderate
agreement between pathologists for PD-L1 TPS in
NSCLC needle core biopsy materials. Significant correlation
between the presence of a crush/squeeze artifact in
the biopsy and the rate of discordant TPS categories was
observed, indicating that PD-L1 assessment of needle core
biopsies requires awareness of this artifact and meticulous
Conflict of Interest
The authors declare no conflict of interest.
This study received no funding.
Concept: EH, BBS, Design: EH, BBS, GU, TCS, Data collection or
processing: EH, BBS, GU, TCS, Analysis or Interpretation: EH, BBS,
GU, TCS, Literature search: EH, BBS, GU, TCS, Writing: EH, TCS,
Approval: EH, BBS, GU, TCS.
1) Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022.
CA Cancer J Clin. 2022;72:7-33.
2) Doroshow DB, Sanmamed MF, Hastings K, Politi K, Rimm DL,
Chen L, Melero I, Schalper KA, Herbst RS. Immunotherapy in
non-small cell lung cancer: Facts and hopes. Clin Cancer Res.
3) Agilent Technologies. PD-L1 IHC 22C3 pharmDx Instructions
for Use 2021 [Available from: https://www.agilent.com/cs/
4) Rimm DL, Han G, Taube JM, Yi ES, Bridge JA, Flieder DB, Homer
R, West WW, Wu H, Roden AC, Fujimoto J, Yu H, Anders
R, Kowalewski A, Rivard C, Rehman J, Batenchuk C, Burns
V, Hirsch FR, Wistuba, II. A prospective, multi-institutional,
pathologist-based assessment of 4 immunohistochemistry assays
for PD-L1 expression in non-small cell lung cancer. JAMA
5) Brunnström H, Johansson A, Westbom-Fremer S, Backman M,
Djureinovic D, Patthey A, Isaksson-Mettavainio M, Gulyas M,
Micke P. PD-L1 immunohistochemistry in clinical diagnostics
of lung cancer: Inter-pathologist variability is higher than assay
variability. Mod Pathol. 2017;30:1411-21.
6) Cooper WA, Russell PA, Cherian M, Duhig EE, Godbolt D, Jessup
PJ, Khoo C, Leslie C, Mahar A, Moffat DF, Sivasubramaniam
V, Faure C, Reznichenko A, Grattan A, Fox SB. Intra- and
interobserver reproducibility assessment of PD-L1 biomarker in
non-small cell lung cancer. Clin Cancer Res. 2017;23:4569-77.
7) Scheel AH, Baenfer G, Baretton G, Dietel M, Diezko R, Henkel
T, Heukamp LC, Jasani B, Johrens K, Kirchner T, Lasitschka F,
Petersen I, Reu S, Schildhaus HU, Schirmacher P, Schwamborn
K, Sommer U, Stoss O, Tiemann M, Warth A, Weichert W,
Wolf J, Buttner R, Ruschoff J. Interlaboratory concordance of
PD-L1 immunohistochemistry for non-small-cell lung cancer.
8) Chang S, Park HK, Choi YL, Jang SJ. Interobserver reproducibility
of PD-L1 biomarker in non-small cell lung cancer: A multiinstitutional
study by 27 pathologists. J Pathol Transl Med.
9) De Marchi P, Leal LF, Duval da Silva V, da Silva ECA, Cordeiro
de Lima VC, Reis RM. PD-L1 expression by tumor proportion
score (TPS) and combined positive score (CPS) are similar in
non-small cell lung cancer (NSCLC). J Clin Pathol. 2021;74:735-
10) Butter R, Hondelink LM, van Elswijk L, Blaauwgeers JLG,
Bloemena E, Britstra R, Bulkmans N, van Gulik AL, Monkhorst
K, de Rooij MJ, Slavujevic-Letic I, Smit V, Speel EM, Thunnissen
E, von der Thusen JH, Timens W, van de Vijver MJ, Yick DCY,
Zwinderman AH, Cohen D, t Hart NA, Radonic T. The impact
of a pathologist’s personality on the interobserver variability and
diagnostic accuracy of predictive PD-L1 immunohistochemistry
in lung cancer. Lung Cancer. 2022;166:143-9.
11) Yu SL, Hsiao YJ, Cooper WA, Choi YL, Aviles-Salas A, Chou TY,
Coudry R, Raskin GA, Fox SB, Huang CC, Jeon YK, Ko YH, Ku
WH, Kwon GY, Leslie C, Lin MC, Lou PJ, Scapulatempo-Neto C,
Mendoza Ramirez S, Savelov N, Shim HS, Lara Torres CO, Cunha
IW, Zavalishina L, Chen YM. The Ring Study: an international
comparison of PD-L1 diagnostic assays and their interpretation
in non-small cell lung cancer, head and neck squamous cell
cancer and urothelial cancer. Pathology. 2023;55:19-30.
12) Landis JR, Koch GG. The measurement of observer agreement
for categorical data. Biometrics. 1977;33:159-74.
13) Taqi SA, Sami SA, Sami LB, Zaki SA. A review of artifacts in
histopathology. J Oral Maxillofac Pathol. 2018;22:279.
14) Robert ME, Ruschoff J, Jasani B, Graham RP, Badve SS,
Rodriguez-Justo M, Kodach LL, Srivastava A, Wang HL, Tang
LH, Troncone G, Rojo F, Van Treeck BJ, Pratt J, Shnitsar I,
Kumar G, Karasarides M, Anders RA. High interobserver
variability among pathologists using combined positive score to
evaluate PD-L1 expression in gastric, gastroesophageal junction
and esophageal adenocarcinoma. Mod Pathol. 2023;36:100154.
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