Received: 24 July 2020 Revised: 23 September 2020 Accepted: 12 October 2020

DOI: 10.1002/dc.24646

ORIGINAL ARTICLE

PD-L1 expression in NSCLC: Role of cell blocks and

concordance between samples

Andrea Ambrosini-Spaltro MD, PhD1 | Alessandra Dubini MD1 |

Federica Pieri MD1 | Claudia Ravaglia MD2 | Angelo Delmonte MD, PhD3 |
Venerino Poletti MD2,4

1
Pathology Unit, Morgagni-Pierantoni
Hospital, Forlì, Italy
2
Department of Thoracic Diseases, Morgagni-
Pierantoni Hospital, Forlì, Italy
3
Thoracic Oncology Unit, Istituto Scientifico
Romagnolo per lo Studio e la Cura dei Tumori
(IRST), IRCCS, Meldola, Italy
4
Department of Respiratory Diseases and
Allergy, Aarhus University Hospital, Aarhus,
Denmark
Correspondence
Andrea Ambrosini-Spaltro, Pathology Unit,
Morgagni-Pierantoni Hospital, Via Carlo
Forlanini 34, 47121 Forlì, Italy.
Email: andrea.ambrosinispaltro@auslromagna.
it; andreaambrosini@yahoo.it
Abstract
Background: PD-L1 immunohistochemistry is currently performed in patients with
advanced non-small cell lung carcinoma (NSCLC) to identify responders to immune
checkpoint inhibitors. Cell blocks from fine needle aspiration of NSCLC are fre-
quently used for diagnostic purposes. The aims of the study are to analyze: (a) the
distribution of PD-L1 in cell blocks, in comparison to biopsies and surgical specimens;
(b) the concordance of PD-L1 in specimens of the same patients.
Methods: PD-L1 analyses conducted in NSCLCs were retrieved. Cell blocks were
prepared with the self-clotting method. PD-L1 was performed with Dako 22C3 on
the Ventana BenchMark ULTRA platform. Results were divided by tumor proportion
score (TPS) in 3 categories: <1%; 1% to 49%; ≥50%.
Results: A total of 483 samples from 456 patients was collected: 120 cell blocks
(24.8%), 307 endoscopic or transthoracic biopsies (63.6%), 56 surgical specimens
(11.6%). TPS was: <1% in 230 samples (47.8%), 1% to -49% in 136 (28.3%) and ≥ 50%
in 115 (23.9%); in two samples material was insufficient. Statistics did not reveal signifi-
cant differences in PD-L1 expression among the various materials (χ2 = 2.905;
P = .574). In 50 samples from 25 patients, PD-L1 was carried out in two samples of the
same patients, with moderate agreement (concordance rate: 68.0%, k = 0.469).
Conclusion: (a) PD-L1 is similarly distributed in the different materials; (b) PD-L1
shows moderate concordance in different samples of the same patients. PD-L1 may
be routinely tested in cell blocks, but interpreted with caution and repeated when-
ever possible.

KEYWORDS

cell block, concordance, NSCLC, PD-L1, sample

1 | I N T RO DU CT I O N proportion score (TPS). Thus, the determination of PD-L1 TPS is of

paramount importance.
Immunotherapy is one of the treatment options for advanced non- Most of the validation studies have performed PD-L1
small cell lung carcinoma (NSCLC).1 Selection of patients candidate to analysis on biopsies and surgical specimens,2,3 but in routine prac-
immunotherapy, in particular pembrolizumab,2,3 and durvalumab,4 is tice many NSCLCs are diagnosed at advanced stages in small histo-
mainly dependent on programmed death ligand-1 (PD-L1) expression logical biopsies or cytological samples, that is, in transbronchial
in neoplastic cells, evaluated immunohistochemically by tumor needle aspiration (TBNA) or endobronchial/endoesophageal

Diagnostic Cytopathology. 2020;1–8. wileyonlinelibrary.com/journal/dc © 2020 Wiley Periodicals LLC 1

2 AMBROSINI-SPALTRO ET AL.
ultrasound-guided transbronchial needle aspiration (EBUS/
EUS-TBNA).
The need to determine PD-L1 expression in NSCLC has raised
the question if cytological material can still be used also for this analy-
sis. Immunocytochemical analysis of PD-L1 in cytological smears have
provided reliable results, especially in cases of high (≥50%) PD-L1
5
expression. In cell blocks, only a few studies have been conducted so
6
far, with encouraging results. Furthermore, PD-L1 expression has
shown considerable variation in different specimens of the same
patient,7 and in different areas of the same specimen,8 highlighting its
spatial and temporal heterogeneity in tissue samples.
By examining PD-L1 analyses in NSCLC diagnosed in different
materials from a single institution, the aims of this study are to ana-
lyze: (a) the distribution of PD-L1 in cell blocks, in comparison to biop-
sies and surgical specimens; (b) the concordance of PD-L1 expression
in specimens obtained by the same patients.
2 | MATERIALS AND METHODS
2.1 | Sample selection and cell block preparation
We retrospectively collected data from our records of all PD-L1
immunohistochemical analyses conducted in NSCLC from June 2018
to April 2020 in our Institution. This was a retrospective study
and only used de-identified patient data. Cases were diagnosed
according to the latest WHO classification of lung tumors.9 Additional
immunohistochemistry (IHC) for TTF1 (8G7G3/1), p40 (BC28), pan-
cytokeratins (AE1/AE3/PCK26), or neuroendocrine markers, such as
chromogranin (LK2H10), synaptophysin (SP11), was performed in
10
poorly differentiated tumors to better define the histotype.
All cases were formalin-fixed paraffin-embedded (FFPE) samples.
Biopsies and surgical specimens were fixed in buffered formalin for
12 to 48 hours. In cytological specimens, both cell blocks and smears
with the air-dried method were prepared. Cell blocks were made using
the so-called tissue coagulum clot method.11 Fine needle aspiration
(FNA) material for cell block preparation was expelled entirely into a
slide allowed to “self-clot,” as similarly described by Shi et al.12 The
air-dried clot takes typically about 2 to 3 minutes to form depending
on the amount of the expelled aspirate. The clot material was scraped
with a blade from the glass slide in a formalin tube by a specialized
interventional pulmonologist, transferred to the Pathology Unit,
where it was fixed in neutral formalin, and processed as conventional
biopsies.
2.2 | PD-L1 immunohistochemistry
PD-L1 immunohistochemistry was routinely carried out in all stage
IIIB/IV NSCLCs diagnosed in our Institution. We used a laboratory-
developed test (LDT) with Dako 22C3 anti-PD-L1 primary antibody
(Agilent, CA) in the Ventana Benchmark ULTRA platform (Ventana
Medical Systems, AZ). Sections with 4 μm thickness were mounted on
positively charged glass slides. EZ Prep solution (Ventana Medical Sys-
tems) was used for paraffin removal and Reaction Buffer to rinse
slides between staining steps. Antigen retrieval was performed by Cell
Conditioning 1 (CC1) (pH 8.0) antigen retrieval solution (Ventana
Medical Systems) for 64 minutes at 95 C. Specimens were incubated
with primary mouse anti-PD-L1 monoclonal antibody using a concen-
tration of 1:25 for 64 minutes at 37 C, followed by OptiView DAB
IHC Detection Kit, as follows: OptiView HQ Universal Linker for
12 minutes and OptiView HRP Multimer for 12 minutes. The slides
were then counterstained with hematoxylin and coverslipped.
2.3 | PD-L1 scoring
Cases were considered adequate for interpretation if at least
100 tumor cells were detectable. Expression of PD-L1 in tumor-
associated macrophages and/or immune cells was used as internal
control. Slides were viewed by a qualified pathologist alone, with col-
legial discussion only for difficult and problematic cases. Based on the
Tumor Proportion Score (TSP), PD-L1 was scored in three groups:
(a) tumors with less than 1% PD-L1-positive cells (TPS < 1%), (b) those
with 1% to 49% PD-L1-positive cells (TPS:1-49%, or low PD-L1 expres-
sion), and (c) those with at least 50% PD-L1-positive cells (TPS≥50%,
or high PD-L1 expression).
2.4 | Statistical analyses
Cases were grouped in cross tables by type of material, diagnosis, and
PD-L1 score. Pearson chi-square (χ2) was calculated to assess statisti-
cal differences within different groups. In patients with two paired
PD-L1 analyses of different samples, we also evaluated the concor-
dance rate and Cohen's kappa. K values were interpreted as follows:
values ≤0 as indicating no agreement, 0.01 to 0.20 as none to slight,
0.21 to 0.40 as fair, 0.41 to 0.60 as moderate, 0.61 to 0.80 as sub-
stantial, and 0.81 to 1.00 as almost perfect agreement.13 Statistical
significance (P) was fixed at .05, with a 2-tailed hypothesis. Statistical
analyses were calculated using Microsoft Excel 2020 (Microsoft,
Redmond, Washington) and SPSS Version 25 (IBM, Armonk, New York).
3 | RE SU LT S
3.1 | Clinicopathological features
Samples investigated for PD-L1 were 483 from 456 patients (277 M,
179 F), with ages varying from 34 to 89 (mean: 70.6; SD: 9.7)
(Table 1). Samples consisted of 120 cell blocks (24.8%), 307 biopsies
(63.6%), 56 surgical specimens (11.6%). Cell blocks were obtained
from FNA of primary mass (22), FNA of hilar/mediastinal metastatic
lymph nodes (96), or FNA of distant metastasis (2). Biopsies were
endobronchial/transbronchial (186, of whom 9 cryobiopsies), pleural
(27), CT-guided (71), pericardial (2), from the thoracic wall (7), or
AMBROSINI-SPALTRO ET AL. 3

TABLE 1 Clinicopathological features of patients and cases 3.2 | PD-L1 analysis in all cases
examined

N. (%) Samples adequate for PD-L1 were 478 out of 483 (99.0%), specifically

Patients 456 303/307 (98.7%) biopsies and 119/120 (99.2%) cell blocks. In two
cases (2 small biopsies), the material was insufficient to analyze
Gender M 277 (60.7)
PD-L1, while in 3 cases (1 cell block, 2 biopsies), we evaluated PD-L1
F 179 (39.3)
even if the neoplastic cells were less than 100. The total number of
Cases 483
PD-L1 analyses was 481.
Type of material Cell block 120 (24.8)
The time between material preparation and PD-L1 analysis was within
Biopsy 307 (63.6)
1 month in 439 cases, within 2 to 12 months in 23, more than 1 year in
Surgical Specimen 56 (11.6)
15, more than 3 years in 5; in 1 case the information was not available.
Histotype Adenocarcinoma 348 (72.0) TPS was: <1% in 230 (47.8%) samples, 1-49% in 136 (28.3%)
Squamous cell carcinoma 112 (23.2) and ≥ 50% in 115 (23.9%) (Figures 1, 2). Cell blocks showed slightly
NSCLC-NOS 15 (3.1) more cases with high PD-L1 expression (29.2%), in comparison to
Other 8 (1.7) biopsies (22.0%) and surgical specimens (23.2%), while biopsies and
surgical specimens exhibited more similar results (Table 2, Figure 3).
Note: Percentages of cases are expressed in parentheses (NSCLC-NOS:
non-small cell lung carcinoma, not otherwise specified). However, statistical analysis did not reveal any significant differences
in PD-L1 expression among the three various materials (χ2
[4] = 2.905; P = .574). The analysis also did not show any significant
metastatic organs (1 from the liver, 6 from the bone, 2 from the skin, difference in PD-L1 expression in the various diagnoses (χ2
5 from a distant lymph node). Surgical specimens were mainly lung [4] = 8.229; P = .222).
resections (49), and surgical removals of metastatic nodules:
adrenal (3), cerebral (1), bone (2), lymph node (1).
Considering all 483 samples, histotypes were: 348 (72.0%) adeno- 3.3 | PD-L1 analysis in paired cases
carcinomas, 112 (23.2%) squamous cell carcinomas, 15 (3.1%) NSCLCs
not otherwise specified (NSCLC-NOS), 3 (0.6%) adenosquamous carci- In 50 samples from 25 patients, PD-L1 was conducted in two materials
nomas, 3 (0.6%) NSCLCs with sarcomatoid features (spindle and/or of the same patient. The two samples were obtained in a lapse time var-
giant cell carcinoma without any other differentiation), 1 (0.2%) iable from 0 to 16 months (mean: 3.9 ± 4.1; in 9 patients within the
NSCLC possible large cell neuroendocrine carcinoma, 1 (0.2%) high- same month, in 16 patients in more than 1 month) and were composed
grade mucoepidermoid carcinoma. of 13 cell blocks, 32 biopsies, and 5 surgical specimens. The two paired

F I G U R E 1 Cell block with PD-L1

TPS ≥ 50%. A cell block from fine-
needle aspiration (FNA) of a
mediastinal lymph node revealed
highly atypical cells (A), with
vacuolated cytoplasm (A, arrow),
immunoreactive for TTF1 (B),
consistent with the localization of
pulmonary adenocarcinoma.
Immunohistochemical analysis for
PD-L1 showed diffuse
immunoreactivity (C), with membrane
staining (D) in more than 50% of
neoplastic cells (TPS≥50%) (TPS:
tumor proportion score)
4 AMBROSINI-SPALTRO ET AL.

F I G U R E 2 Cell block with PD-L1

TPS < 50%. A cell block obtained
from fine-needle aspiration (FNA) of a
bronchial mass depicted neoplastic
cells (A), with focal keratinization (A,
arrow), immunoreactive for p40 (B),
consistent with squamous cell
carcinoma. Immunohistochemical
analysis for PD-L1 showed focal
immunoreactivity (C), with membrane
staining (D) in less than 50% of
neoplastic cells (TPS < 50%) (TPS:
tumor proportion score)

TABLE 2 PD-L1 TPS scores in different types of material and diagnoses; statistical comparisons showed no significant differences within
groups

Type of Material χ2 = 2.905 P = .574 Diagnosis χ2 = 8.229 P = .222

PD-L1 TPS Total Cell Block Biopsy Surgical Specimen Adenocarcinoma Squamous cell carcinoma NSCLC-NOS Other
<1% 230 (47.8) 56 (46.7) 147 (48.2) 27 (48.2) 172 (49.6) 51 (45.9) 5 (33.3) 2 (25.0)
1–49% 136 (28.3) 29 (24.2) 91 (29.8) 16 (28.6) 90 (25.9) 37 (33.3) 7 (46.7) 2 (25.0)
≥50% 115 (23.9) 35 (29.2) 67 (22.0) 13 (23.2) 85 (24.5) 23 (20.7) 3 (20.0) 4 (50.0)
Total 481 120 305 56 347 111 15 8

Note: Percentages of cases are expressed in parentheses (TPS: tumor proportion score; χ : Pearson chi-squared; NSCLC-NOS: non-small cell lung
2

carcinoma, not otherwise specified).

materials were both from the primary site in 12 patients, and from the
primary and metastatic sites in 13 patients.
PD-L1 showed a concordance rate of 68.0% and k = 0.469
(P = .001), revealing moderate agreement (Table 3). Paired cases in
which one of them was a cell block were only 26 cases from
13 patients, with concordance rate: 53.8%, k = 0.310 and low statisti-
cal significance (P = .079).

4 | DI SCU SSION

This study shows that:

F I G U R E 3 Distribution of PD-L1 TPS among different materials.

The diagram shows percentages of cases by PD-L1 TPS in different
materials: cell blocks, biopsies and surgical specimens. Cases with
TPS < 1% are highlighted in blue, those with TPS 1% to 49% in red,
and those with TPS≥50% in green (TPS: tumor proportion score)
a. Frequencies and distribution of PD-L1 values obtained from cell
blocks are comparable to those obtained from biopsies and surgical
specimens.
b. Concordance of PD-L1 results in paired specimens from the same
patient is moderate.
AMBROSINI-SPALTRO ET AL.
T A B L E 3 Comparison between PD-L1 TPS scores in different specimens of the same patients (50 cases form 25 patients; TPS: tumor
proportion score)
First specimen
Second specimen PD-L1 <1% 1–49%
TPS
<1% 11 1 1
1–49% 2 2
≥50% 1 1
Total 14 4 7
Determining PD-L1 is of paramount importance in the treatment
of NSCLC, since its expression selects patients candidate to immuno-
therapy.1 NSCLC is frequently diagnosed with cytological specimens
by TBNA, an extremely useful tool to limit surgical and invasive proce-
dures especially in fragile patients.14 Immunocytochemistry for PD-L1
on cytological smears has provided excellent results, but it is more
challenging. Cytological smears are usually prepared with alcohol-
based fixative, display more dispersed cells and are more difficult to
evaluate in a membrane staining such as PD-L1.6 Cell blocks, instead,
are processed in formalin-based fixatives, are more easily interpreted,
and immunohistochemical analyses on them are more similar to their
15
histopathological counterparts.
In our study, the distribution of PD-L1 expression was similar in
the three types of material: cell blocks, biopsies, and surgical speci-
mens, thus making reasonable that cell blocks have correctly identified
and scored the cases. Cases with high PD-L1 expression was even
slightly higher in cell blocks (29.2%) than in biopsies (22.0%) and surgi-
cal specimens (23.2%). These differences were not significant, with
cell blocks, biopsies and surgical specimens showing similar distribu-
tion of PD-L1. Wang et al. also reported a slightly greater percentage
of cases with high PD-L1 expression in cell blocks (42%), in compari-
16
son to small biopsies (36%) and surgical resections (29%). Heyman
found comparable results with high PD-L1 expression in 35% of cell
17
blocks, in 22% of biopsies, and in 26% of surgical specimens. Torous
et al. observed a similar distribution of cases, with no significant dif-
ferences of cases with high PD-L1 expression between cell blocks
(35.1%) and surgical pathology group (34.8%, consisting of biopsies
and surgical specimens).18 Dong detected lower values with the PD-
L1 28-8 assay, with high PD-L1 positivity in 20.5% of cell blocks and
19
27.7% of surgical specimens. Vigliar et al. observed fewer cases with
high expression of PD-L1 in cell blocks (4.9%) in comparison to biop-
20
sies (28.7%) and surgical specimens (15.7%) ; however, cell blocks
represented a limited number of their study (41 cell blocks). In cell
21
blocks only, Biswas reported 14 (32%) cases with high PD-L1
expression, Noll 14 (36.8%),22 and Stoy 2 (10%)23 in a limited number
of cases. We summarized in Table 4 all the literature regarding PD-L1
expression in cell blocks. Because our study and the previous litera-
ture reported similar distribution of PD-L1 expression in the different
materials, it is reasonable that cell blocks, biopsies, and surgical speci-
mens may be valid alternatives to assess PD-L1 in NSCLC.
5
Concordance rate (%) kappa P
≥50% Total 68.0 0.469 .001
13
2 6
4 6
25
We also calculated the concordance of PD-L1 expression in
25 patients in which two paired samples were analyzed for PD-L1: the
agreement was moderate (k = 0.469), with a concordance rate of 68.0%
(P = .001). In 13 patients with paired cases in which one of them was a
cell block, the agreement was lower (concordance rate: 53.8%;
k = 0.310), but the high P-value (P = .079) made such analysis not signif-
icant, probably due to the limited number of cases. Among the all
25 paired cases examined, 12 paired cases were both from the primary
sites, and 13 were from the primary and metastatic sites. Similar ana-
lyses compared PD-L1 values in the primary and metastatic sites, most
of them on surgical specimens only. Saito7 described very low values of
agreement using the Dako PD-L1 22C3 between the primary and meta-
static NCSLC (concordance rate: 28.6%). Kim instead observed higher
concordance between the primary and metastatic NCSLC (concordance
rate: 75.2%). Intermediate results were found by Xu24 (concordance
rate: 62.5%; k = 0.497) and Luo25 (concordance rate: 61%; k = 0.39).
These intermediate values were similar to those calculated in our series,
although we considered only a limited number of cases (50 cases form
25 patients), independently from type, site, and time. By examining pri-
mary cancer at initial diagnosis and metastasis at recurrence in resected
NSCLCs, Kim detected 78.4% concordance (k = 0.374).26 Other studies,
conducted in surgical specimens only, showed considerable variation in
PD-L1 immunoreactivity in different patterns of the same tumor.8 Vari-
ations reported by us and by the literature highlight spatial (and proba-
bly temporal) heterogeneity of PD-L1 expression in NSCLC, making it
advisable to re-test PD-L1 whenever possible.
A technical limitation of our study is the absence of external con-
trol in our PD-L1 reactions. However, we evaluated internal positivity
of PD-L1 in immune cells of adjacent native tissue. In pulmonary
parenchyma, alveolar macrophages are consistently stained by PD-L1,
serving as a positive internal control.27 PD-L1 is also expressed in
dendritic cells of nonneoplastic lymph node parenchyma, as well as in
other immune cells.28 Another possible technical limitation is the time
between the preparation of cell blocks and the immunohistochemical
analysis. Blocks should ideally be less than 3 years old to perform
PD-L1 analysis.29 Some studies have even evidenced to assess PD-L1
carefully in tissue blocks older than 1 year.30 In our series, blocks were
older than 3 years in 5 cases and older than 1 year in 15 cases; how-
ever, the limited number of such cases should not have significantly
impacted the final results.
6 AMBROSINI-SPALTRO ET AL.

TABLE 4 PD-L1 expression in cell blocks reported in the literature

TPS

Cell block Cases Adequate Total cases TPS TPS ≥

Author Fixation method preparation Antibody clone (N.) specimens examined TPS < 1% 1–49% 50%
Wang16 4 methods: CytoLyt HistoGel 22C3 PhamDx 371 342/371 (92) 342 97 101 144
(Hologic Inc., (28) (30) (42)
Marlborough, MA)
or alcohol followed
by 10% BNF; BNF
only, TissuFix
(Chaptec INC,
Montreal, Canada);
CytoLyt or 50%
alcohol only
Dong19 ThinPrep (HOLOGIC Agarose gel Abcam 28.8 112 79/112 (70.5) 112 a
(20.5)a
Gen-Probe, San Ventana SP142 (14.3)a
Diego, CA)
cytology test (TCT)
preservation
solution
Torous18 Formalin fixationc Plasma-thrombin 22C3 pharmDx 94 88/94 (93.6) 88 35 20 (21.3) 33
method or kit (37.2) (35.1)
HistoGel method
(Thermo Fisher
Scientific,
Waltham, MA)
Vigliar20 NBF a
22C3 (laboratory- 45 41/45 (91.1) 45 33 6 2
developed test) (80.5) (14.6) (4.9)
Biswas21 Alcohol and Plasma-thrombin 22C3 pharmDx 50 43/50 43 29 14
subsequent (86.0) (68)b (32)
formalin
Noll22 NBF Centrifugation, cell 22C3 pharmDx 41 38/41 38 9 15 14
pellet (92.7) (23.7) (39.5) (36.8)
Heymann17 NBF and/or CytoLyt Bio-Wrap (Leica 22C3 pharmDx 40 36/40 36 22 14
solution (Hologic Inc, Biosystems (90.0) (61.1)b (38.9)
Marlborough, Inc, Buffalo Grove,
Massachusetts) Illinois)
alternatively with
HistoGel specimen
processing
gel (Thermo Scientific
Richard-Allan
Scientific,
Waltham,
Massachusetts)
added to poorly
clotted specimens
Stoy23 CytoLyt solution a
Abcam 28.8 22 (90.9)a 20 14 4 2
(Hologic Inc, (70) (20) (10)
Marlborough,
Massachusetts)
Present Series NBF Self-clotting 22C3 (laboratory- 120 119/120 120 56 29 (24.2) 35
developed test) (99.2) (46.7) (29.2)

Note: Percentages are expressed in parentheses (TPS: tumor proportion score, NBF, neutral-buffered formalin).
a
Not specified.
b
Not specified in the two subclasses.
c
Cytology aspirates specimens were collected directly into a methanol-water fixative (CytoLyt; Hologic Corp, Marlborough, MA) before formalin fixation.
AMBROSINI-SPALTRO ET AL.
In conclusion, this study highlights that: (a) frequencies and distri-
bution of PD-L1 values obtained from cell blocks are comparable to
those obtained from biopsies and surgical specimens; (b) PD-L1
expression shows moderate concordance in different samples of the
same patients. PD-L1 may be routinely tested in cell blocks, but this
analysis should be interpreted with caution, especially in cases with
limited cellularity, and repeated whenever possible.
AUTHORS C ON TRIBUTIONS
AAS conceived of the study, collected the data, performed the statisti-
cal analyses and wrote the manuscript. AD and FP performed most of
the pathological diagnoses and immunohistochemical examinations.
ADM critically revised the manuscript focusing on a clinical perspective.
CR and VP performed most of the biopsies and cell blocks. VP critically
revised and corrected the manuscript, and supervised the project. All
authors discussed the results and contributed to the final manuscript.
CONF LICT OF IN TE RE ST
The authors declare they have no conflict of interest.
DATA AVAI LAB ILITY S TATEMENT
The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.
ORCID
Andrea Ambrosini-Spaltro https://orcid.org/0000-0001-7800-4229
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