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\begin{document}

\title{Intra-patient comparison of atopic dermatitis skin transcriptome shows
differences between tape-strips and biopsies}



\author[1]{Ester Del Duca}%
\author[1]{Helen He}%
\author[1]{Ying Liu}%
\author[1]{Angel D. Pagan}%
\author[1]{Eden David}%
\author[1]{Julia Cheng}%
\author[1]{Britta Carroll}%
\author[1]{Yael Renert-Yuval}%
\author[1]{Jonathan Bar}%
\author[1]{Yeriel  Estrada}%
\author[2]{Catherine Maari}%
\author[2]{Etienne Saint-Cyr Proulx}%
\author[3]{James Krueger}%
\author[2]{Robert Bissonnette}%
\author[1]{Emma Guttman-Yassky}%
\affil[1]{Icahn School of Medicine at Mount Sinai}%
\affil[2]{Innovaderm Research}%
\affil[3]{The Rockefeller University}%


\vspace{-1em}



  
  \date{June 10, 2024}


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\textbf{Journal: Allergy}

\textbf{Title: Intra-patient comparison of atopic dermatitis skin
transcriptome shows differences between tape-strips and biopsies}

Ester Del Duca,\textsuperscript{1,2,}* Helen He,\textsuperscript{1}*
Ying Liu\textsuperscript{1}, Angel D. Pagan\textsuperscript{1}, Eden
Davis\textsuperscript{1}, Julia Cheng\textsuperscript{1}, Britta
Carroll\textsuperscript{1}, Yael Renert-Yuval\textsuperscript{1,3},
Jonathan Bar\textsuperscript{1}, Yeriel D. Estrada\textsuperscript{1},
Catherine Maari,\textsuperscript{4} Etienne Saint-Cyr
Proulx\textsuperscript{4}, James G. Krueger\textsuperscript{3}, Robert
Bissonnette\textsuperscript{4,}**, Emma
Guttman-Yassky\textsuperscript{1} **

*,** these authors equally contributed to the work

1. Department of Dermatology, and Laboratory of Inflammatory Skin
Diseases, Icahn School of Medicine at Mount Sinai, New York, USA

2. Department of Dermatology, University of Magna Graecia, Catanzaro,
Italy

3. Laboratory for Investigative Dermatology, The Rockefeller University,
New York, New York USA

4. Innovaderm Research Inc., Montreal Quebec, Canada

Ester Del Duca: https://orcid.org/0000-0001-7948-8536

Helen He: https://orcid.org/0000-0002-4752-4667

Ying Liu: https://orcid.org/0000-0001-6208-0489

Angel D. Pagan: https://orcid.org/0000-0002-4913-6705

Eden Davis: https://orcid.org/0000-0003-4557-6436

Julia Cheng: https://orcid.org/0009-0006-7909-9550

Britta Carroll: https://orcid.org/0009-0003-0154-283X

Yael Renert-Yuval: https://orcid.org/0000-0002-2876-3048

Jonathan Bar: https://orcid.org/0000-0001-7366-9416

Yeriel D. Estrada: https://orcid.org/0000-0002-5172-6747

Catherine Maari : https://orcid.org/0000-0002-9719-0810

Etienne Saint-Cyr Proulx: https://orcid.org/0000-0002-3346-3891

James Krueger: https://orcid.org/0000-0002-3775-1778

Robert Bissonnette: https://orcid.org/0000-0001-5927-6587

Emma Guttman-Yassky: https://orcid.org/0000-0002-9363-324X

\textbf{Corresponding author:}

Emma Guttman-Yassky, MD, PhD

Department of Dermatology and Laboratory of Inflammatory Skin Diseases

Icahn School of Medicine at Mount Sinai

5 East 98th Street, New York, NY, 10029

Email: emma.guttman@mountsinai.org

\textbf{Funding sources: none}

\textbf{Conflicts of Interest:} \textbf{EGY} is an employee of Mount
Sinai and has received research funds (grants paid to the institution)
from: Abbvie, Amgen, Asana BioSciences, AstraZeneca, Akari, AnaptysBio,
Boehringer-Ingelheim, Eli Lilly, Galderma, Ichnos Sciences, Innovaderm
Research, Janssen, Kiniksa, Kyowa Kirin, Leo Pharma, Novartis, Pfizer,
RAPT Therapeutics, Regeneron Pharmaceuticals, Sienna Biopharma, UCB,
Union Therapeutics; and is a consultant for Abbvie, Almirall, Amgen,
Arena Pharmaceuticals, Asana BioSciences, AstraZeneca, Aslan
Pharmaceuticals, Bristol Myers Squibb, Boehringer-Ingelheim, Cara
Therapeutics, Castle Biosciences, DBV Technologies, DS Biopharma, Eli
Lilly, Evommune, Galderma, Genzyme Corporation, GSK, Ichnos Sciences,
Incyte, Janssen, Kyowa Kirin, Leo Pharma, Novan, Novartis, RAPT
Therapeutics, Sanofi, Sato Pharmaceutical, Siolta, Union Therapeutics,
and Ventyx. \textbf{RB} is an Advisory Board Member, Consultant, Speaker
and/or Investigator for and receives honoraria and/or grant from AbbVie,
Arcutis, Arena Pharma, Aristea, Asana BioSciences, Bellus Health,
Bluefin Biomedicine, Boehringer-Ingelheim, CARA, Dermavant, Eli Lilly,
EMD Serono, Evidera, Galderma, GSK, Inmagene Bio, Incyte, Kiniksa, Kyowa
Kirin, LEO Pharma, Novan, Pfizer, Ralexar, RAPT, Regeneron, Respivant,
Sanofi-Genzyme, Sienna and Target RWE. He is also an employee and
shareholder of Innovaderm Research. \textbf{ESCP} has served as an
advisory board member, consultant, speaker and/or investigator and
received honoraria and/or grants from AbbVie, Arcutis, Arena Pharma,
Aristea, Asana BioSciences, Bellus Health, Boehringer Ingelheim,
Bristol-Myers Squibb, CARA, Celgene, Dermavant, Eli Lilly, Galderma,
GlaxoSmithKline, Incyte, Kiniksa, LEO Pharma, Neokera, Pfizer,
Regeneron, Sanofi-Genzyme, Sienna, Target RWE, and UCB Pharma. All other
authors have no conflict of interest within the scope of the submitted
work.

\textbf{--}

\textbf{IRB status:} Approved by both the Icahn School of Medicine at
Mount Sinai Institutional Review Board and the Ethics Board for clinical
activities at Innovaderm

\textbf{Reprint requests:} None

\textbf{Author Contribution: EGY} and \textbf{RB} : conceptualization of
the study, editing of the draft and review; \textbf{EDD} and\textbf{HH}
: performed the experiments related to the study, critically analyzed
and interpreted the data, drafted and reviewed the
manuscript;\textbf{HH, JB} and \textbf{BC} performed the statistical
analyses;\textbf{YED, CM, ESCP} participated to the data collection and
reviewing of the paper; \textbf{YL, EDD} performed the experiments,
contributed to the analysis and interpretation of data and to drafting
and reviewing the manuscript \textbf{YRY, ED, ADP} , \textbf{JC} and
\textbf{JGK} : contributed to the analysis and interpretation of data
and to drafting and reviewing the manuscript

\textbf{Manuscript word count:} 3140/3500

\textbf{Abstract word count:} 227/250

\textbf{References:} 124

\textbf{Figures: 5}

\textbf{Supplementary figures:} 1

\textbf{Tables: 1}

\textbf{Supplementary tables} :1

\textbf{Keywords:} Atopic dermatitis; biopsies; immune; pruritus;
tape-strips

\textbf{Abbreviations}

AD: Atopic dermatitis

CCL: CC chemokine ligand

CXCL: C-X-C Motif Chemokine Ligand

DC: Dendritic cell

DEG: Differentially expressed gene

FDR: False discovery rate

IDEC: Inflammatory dendritic epidermal cell

IGA: Investigator's Global Assessment

IL: Interleukin

LC: Langerhans cell

Th: T-helper

TSS: Total Skin Score

\textbf{ABSTRACT}

\textbf{Background}

Our knowledge of etiopathogenesis of atopic dermatitis (AD) is largely
derived from skin biopsies, which are associated with pain, scarring and
infection. In contrast, tape-stripping is a minimally invasive,
nonscarring technique to collect skin samples.

\textbf{Methods}

To construct a global AD skin transcriptomic profile comparing
tape-strips to whole skin biopsies, we performed RNA-seq on tape-strips
and biopsies taken from the lesional skin of 20 moderate-to-severe AD
patients and the skin of 20 controls. Differentially expressed genes
(DEGs) were defined by fold-change (FCH)[?]2.0 and false discovery
rate\textless{}0.05.

\textbf{Results}

We detected 4,104 (2,513 Up; 1,591 Down) and 1,273 (546 Up; 727 Down)
DEGs in AD versus controls, in tape-strips and biopsies, respectively.
Although both techniques captured dysregulation of key immune genes,
tape-strips showed higher FCHs for innate immunity (IL-1B, IL-8),
dendritic cell (ITGAX/CD11C, FCER1A), Th2 (IL-13, CCL17, TNFRSF4/OX40),
and Th17 (CCL20, CXCL1) products, while biopsies showed higher
up-regulation of Th22 associated genes (IL-22, S100As) and dermal
cytokines (IFN-\selectlanguage{greek}γ, \selectlanguage{english}CCL26). Itch-related genes (IL-31, TRPV3) were
preferentially captured by tape-strips. Epidermal barrier abnormalities
were detected in both techniques, with terminal differentiation defects
(FLG2, PSORS1C2) better represented by tape-strips and epidermal
hyperplasia changes (KRT16, MKI67) better detected by biopsies.

\textbf{Conclusions}

Tape-strips and biopsies capture overlapping but distinct features of
the AD molecular signature, suggesting their respective utility for
monitoring specific AD-related immune, itch, and barrier abnormalities
in clinical trials and longitudinal studies.

\textbf{INTRODUCTION}

Atopic dermatitis (AD) is a common inflammatory skin disease affecting
up to 10\% of adults and 20\% of children
worldwide.\textsuperscript{1,2} Our knowledge of AD pathomechanisms is
largely derived from molecular studies in skin biopsies that demonstrate
primarily Th2/Th22 inflammation with variable Th1/Th17 skewing, as well
as epidermal barrier dysregulation, including epidermal hyperplasia and
abnormalities in terminal differentiation, tight junctions, and lipid
biosynthesis/metabolism.\textsuperscript{3-12} While skin biopsies were
instrumental to deciphering disease characteristics, they are associated
with pain, scarring, and infection risk, restricting their use in
large-scale clinical trials and longitudinal studies, which are critical
for adults and even more in children.\textsuperscript{13-15} Tape-strips
are a minimally invasive, nonscarring approach to collect skin samples
to the level of the upper stratum granulosum.\textsuperscript{16} Recent
studies have demonstrated significant utility of tape-strips in
profiling AD skin in both adults and
children,\textsuperscript{6,16-30}and in monitoring disease activity in
response to treatment.\textsuperscript{31-34}

Comprehensive molecular intra-patient comparison of tape-strips to
full-thickness biopsies is currently lacking. Moreover, there is no
detailed comparison of immune, barrier, and itch-related markers between
the two techniques. \textsuperscript{16,35,36} Several studies described
differences between tape-strips and biopsies using a limited panel of
markers. Andersson et al. analyzed only 17 immune markers in AD skin and
noted that CCL17 and markers of innate immunity were significantly
up-regulated and correlated with disease activity in tape-strips but not
in same-cohort biopsies.\textsuperscript{36} Simonsen et al. assessed 10
cytokines in AD lesions and noted greater up-regulation in the innate
immunity marker IL-1B in tape-strips, while dysregulation of Th2 markers
and IFN-\selectlanguage{greek}γ \selectlanguage{english}were only detected in biopsies.\textsuperscript{35} Of note,
this study measured mRNA expression in biopsies while proteomics was
performed in tape-strips. Kim et al. measured mRNA of 5 epidermal
differentiation markers in tape-strips, reporting positive correlations
with protein levels in biopsies.\textsuperscript{16} Utilizing RNAseq,
S\selectlanguage{ngerman}ølberg et al. reported that structural genes like keratins were higher
in biopsies while immune markers IL-8 and IL-36A were higher in
tape-strips.\textsuperscript{18} Dyjack et al. analyzed tape-strips from
non-lesional AD skin, noting better representation of cornification
genes in tape-strips compared to biopsies.\textsuperscript{37} These
studies enzymatically separated the epidermis and dermis in biopsies,
potentially overlooking important pathogenic features within the entire
AD skin transcriptome.

Herein, we present a global RNA-seq intra-patient profiling of
tape-strips and whole-skin biopsies in moderate-to-severe AD as compared
to healthy subjects. Our data highlight features that are better
captured by tape-strips, including Th2 polarization, itch pathway
activation, and terminal differentiation impairment, whereas Th22
changes and epidermal hyperplasia are more pronounced in biopsies. These
findings suggest that tape-strips and biopsies each preferentially
detect unique disease characteristics, which should be considered when
selecting skin sampling approaches in clinical trials or longitudinal
studies.

\textbf{METHODS}

\textbf{Study population and characteristics}

We enrolled 20 adults with moderate-to-severe AD (15 females and 5
males, mean age 31 years) and 20 healthy volunteers (11 females and 9
males, mean age 40.3 years) under institutional review board-approved
protocols (\textbf{Table 1} ). Clinical severity scores were measured
using a 5-grade AD Investigator's Global Assessment (IGA) score (mean
score 2.6, \textbf{Table 1} ). Exclusion criteria included
immunodeficiency, use of biologics within the previous 12 weeks, use of
systemic steroids, use of immunosuppressants or phototherapy within the
previous 4 weeks, and use of any topical medication within the previous
2 weeks.

\textbf{Tape strip collection, biopsy collection, RNA extraction, and
RNA-seq}

Twenty consecutive large D-Squame tape-strips (CuDerm, Dallas, Texas)
were taken from the lesional skin of AD patients and from the skin of
healthy controls. The first tape-strip was discarded, and remaining
samples were frozen at --80\textsuperscript{o}C as previously
described.\textsuperscript{17,38} Four millimeters skin biopsies were
taken from the lesional skin of AD patients and from the skin of
controls (same subjects as tape-strips). RNA was extracted using an
miRNAeasy Mini Kit (Qiagen, Hilden, Germany) as previously
described.\textsuperscript{39} In tape-strips, RNA AmpliSeq libraries
were constructed with the Ion AmpliSeq Transcriptome Human Gene
Expression Kit by using 5 ng of RNA per sample. RNA-seq libraries were
pooled and sequenced on the Ion S5 XL system sequencer with Ion 550
Chips (Thermo Fisher, Waltham, Mass). In biopsies, libraries were
generated using TruSeq Stranded mRNA Library Prep kit (Illumina). Next
generation sequencing was performed on Illumina NovaSeq6000 (Illumina
Inc., 100 cycles, single-read sequencing).

\textbf{Statistical analyses}

Statistical analyses were performed by using the statistical language R
(www.R-project.org). The quality of samples was assessed using FastQC.
Each sample was aligned to the human reference genome by using STAR
(open-source aligner).\textsuperscript{40} Mapped sequencing reads were
assigned to genomic features using the featureCounts function. Counts
were transformed to log scale by voom-transform and fit in a linear
model.\textsuperscript{41} Fold-changes (FCHs) were estimated, and
hypothesis testing was conducted by using contrasts under the general
framework for linear models in the limma package. P-values were adjusted
for multiple hypotheses by using the Benjamini-Hochberg procedure,
controlling for false discovery rate (FDR). Genes with FCH\selectlanguage{english}[?]2 and
FDR\textless{}0.05 were considered DEGs.

\textbf{RESULTS}

Using RNA-seq, we provided a global transcriptomic profile of
tape-strips and biopsies taken from the lesional skin of
moderate-to-severe AD patients, and from healthy control skin (n=20
each) (\textbf{Figure 1A} ). Sample recovery rates were 19/20 (95\%) in
AD and 19/20 (95\%) in controls for tape-strips and 100\% of AD and
controls for biopsies.

\textbf{Tape-strips and biopsies capture key AD-related immune
alterations}

Using FCH\selectlanguage{english}[?]2 and FDR\textless{}0.05 criteria, tape-strips and biopsies,
respectively, detected 4,104 (2,513 Up; 1,591 Down) and 1,273 (546 Up;
727 Down) DEGs in AD versus controls (\textbf{Figure 1B} ). The common
phenotype between tape-strip and biopsy groups comprised of 536 DEGs
(310 Up; 226 Down).\textsuperscript{42} The tape-strip group had 3,568
unique DEGs (2,203 Up; 1,365 Down), whereas biopsies had 737 unique DEGs
(230 Up; 292 Down) as depicted in the Venn diagram in \textbf{Figure 1B}
.

Using a previously published immune-data
set,\textsuperscript{17,29,42-46} we identified inflammatory genes that
were differentially expressed in AD versus controls in at least one
comparison (\textbf{Figure 2, Table E1} ). The immune-related DEGs with
the highest FCHs are shown in a heatmap in \textbf{Figure 2A-B} and a
visual word cloud of representative immune markers, where the size of
the letters represents the magnitude of the FCHs in AD versus healthy
skin, is depicted in \textbf{Figure 2C-D} . Both tape-strips and
biopsies showed significant up-regulation in key products involved in
general inflammation (MMP12), innate immunity (IL-6), T-cells/T-cell
activation (CD2, CD3D/E/G, CD4, CD5, CD28, ICOS, GZMA), dendritic cell
(DC) activation and migration (FCER1G, ITGAM/CD11B, ITGAX/CD11C, CD80,
CXCR4), regulatory markers (IL-10, CTLA4, FOXP3, CD274), Th2 (IL-13,
CCL13, CCL17, CCL18, CCL22, IL-4R, TNFRSF4/OX40, OSM, CCR4), Th1
(IL-12B, CXCL9, CXCL10, MX1, OASL), Th17 (CCL20, CXCL1, IL-19, IL-20,
IL-36A, PI3), and antimicrobial peptides (CAMP/LL37) (\textbf{Figure 2,
Table E1} ). Both techniques also demonstrated significant
down-regulation of negative regulators IL-34, IL-37, IL-1F10. These
markers all notably showed greater FCHs in tape-strips than in biopsies,
particularly for select markers of T-cells/T-cell activation (CD3D,
CD3E, GZMA), negative regulation (CD274, IL-34), DC activation and
migration (FCER1G, CD80, ITGAM/CD11B, ITGAX/CD11C, CXCR4), and Th2
(IL-13, CCL17, TNFRSF4/OX40, CCR4, OSM), where the FCHs between AD and
controls were at least 5-fold in tape-strips compared to biopsies
(\textbf{Figure 2A and C} ). Moreover, multiple additional genes
involved in innate immunity or general immune activation (IL-1B, IL-8,
PDE4A, PDE4B, PTPRC, NLRP3, TLR4), tissue resident memory T-cells
(CD69), DCs (CD83, CD86, CD1A, CD1C, CD1E, FCER1A, CD207/langerin,
IL-3RA, IRF4),\textsuperscript{9,47,48} macrophages (RNASE1, MS4A4A,
F13A1, CECR1), Th1/Th2 (CCL3), and Th17 (CXCL2, CXCL3, GPR183, TGM2,
DUSP4) were uniquely up-regulated in tape-strips, but not biopsies
(\textbf{Figure 2, E1,Table E1} ). Conversely, the Th1-defining cytokine
IFN-\selectlanguage{greek}γ, \selectlanguage{english}CCL26, a Th2 chemokine, and CCL19, a chemokine involved in T-cell
trafficking, were uniquely significantly up-regulated in biopsies. Th22
and Th17/Th22-related genes (IL-22, IL-26, S100A7, S100A8, S100A9,
DEFB4B, and LCN2) showed higher or unique up-regulation in biopsies
compared to tape-strips. Collagens (COL6A5, COL6A6, TNC) implicated in
driving AD inflammation were also uniquely up-regulated in biopsies
(\textbf{Figure E1, Table E1} ).\textsuperscript{9}

\textbf{Tape-strips preferentially captured up-regulation of
itch-related gene products}

Gene products associated with pruritus and
nociception\textsuperscript{49-55} that were dysregulated in either
tape-strips or biopsies are shown in \textbf{Figure 3} . IL-31 and OSM
drive pruritus via interactions with the IL-31RA and OSMR heterodimer on
sensory neural cells.\textsuperscript{56-61} IL-31, OSM, and OSMR were
either uniquely upregulated or showed higher FCHs in AD versus controls
in tape-strips compared to biopsies. STAT3, which regulates IL-31
signaling showed a similar trend.\textsuperscript{56}

Transient receptor potential (TRP) channels (TRPV2, TRPV3,
TRPM2)\textsuperscript{55} demonstrated greater up-regulation in
tape-strips compared to biopsies, while TRPV6 showed greater
down-regulation in tape-strips \textbf{(Figure 3)} . TRPV3 activation
was shown to induce a pruritogenic pathway involving SERPINE1,
PLAUR/u-PAR, and TLR2,\textsuperscript{62,63} which were either uniquely
(PLAUR/u-PAR, TLR2) or markedly more (SERPINE1) increased in tape-strips
than in biopsies (\textbf{Figure 3, E1, Table E1)} . Other genes with
higher up-regulation in tape-strips than biopsies include cathepsins
(CTSS, CTSL, CTSB), serine protease inhibitor SERPINB1, CGRP receptor
component RAMP1,\textsuperscript{54} and phospholipase PLCB3, a neuronal
marker required for histamine and serotonin-mediated pruritus
(\textbf{Figure 3, Table E1}
).\textsuperscript{51-53}Histamine-dependent itch markers (AOC1, HDC)
and PTGER2, which is involved in vasodilation and cytokine secretion by
endothelial cells,\textsuperscript{49,50} were only significantly
up-regulated in tape-strips, while the histamine receptor HRH3 was
up-regulated only in biopsies.\textsuperscript{64} The kallikreins KLK6
and KLK9, which are enriched or restricted to the stratum granulosum,
were comparably up-regulated in tape-strips and biopsies, while KLK7,
which is highest in the basal layer, was only up-regulated in
biopsies.\textsuperscript{65}

\textbf{Epidermal barrier alterations in tape-strips compared to
biopsies}

We next evaluated DEGs between AD and controls in either tape-strips or
biopsies among a previously published epidermal barrier
gene-subse\textsuperscript{4,11,30,66} (\textbf{Figure 4)} . While
terminal differentiation (FLG2, SCEL, PSORS1C2, LCE5A) and keratin
(KRT77, KRT79) products were significantly decreased in both techniques,
greater decreases were observed in tape-strips. AKR1C3, which is
expressed by differentiated keratinocytes, was the most down-regulated
gene unique to tape-strips (\textbf{Figure E1} ). Significant decreases
in gap junction genes (GJB3, GJB5) were only seen in tape-strips
(\textbf{Figure 4, Table E1} ). Tight junction components like claudins
and cadherins (CLDN8, CDH12)\textsuperscript{67} were comparably
suppressed in tape-strips and biopsies, while CLDN1 was uniquely
significantly decreased in biopsies. Markers of epidermal
hyperplasia/proliferation (KRT6A, KRT6B, KRT16, KRT17, MKI67, AKR1B10,
AKR1B15) and cornification (SPRR1A, SPRR2C, SPRR3) showed higher or
unique increases in biopsies.

There was considerable variability in lipid biosynthesis and metabolism
genes. PNPLA3, GAL, FA2H, FABP7, DGAT2, ACER1, ORMDL3, and DHCR7 were
significantly decreased in both, with slightly greater FCHs in
tape-strips, and DEGS2 and SPTLC3 were significantly down-regulated only
in tape-strips. However, several other lipid-related genes (ELOVL3,
FAR2, FADS2, ACOX2) were uniquely down-regulated in biopsies. A few
genes (AGPAT3, FADS1, ELOVL5, SOAT1, LPIN1, PPARG) were decreased in
biopsies, but increased in lesional tape-strips.

\textbf{Enrichment of key immune, pruritus and barrier-related pathways}

We used gene set variation analysis (GSVA) on the previously published
AD transcriptome (MADAD), and Th1-, Th2- and Th17/Th22- as well as
pruritus and barrier-related genes, to evaluate how the two approaches
compare.\textsuperscript{29,42,68} We observed similar significant
enrichment in all key analyses in both biopsies and tape-strips analyses
(\textbf{Figure 5} ).

\textbf{DISCUSSION}

We present the most comprehensive intra-patient transcriptomic
comparison of tape-strips and biopsies in moderate-to-severe AD to date.
We previously developed a framework for using tape-strips to measure
mRNA and protein in AD in both pediatric and adult populations,
including for monitoring skin activity with topical or systemic
treatment.\textsuperscript{29,30,69,70} Here, we provided insights into
the common AD phenotype detected by both tape-strips and biopsies as
well as potential differences between these approaches in detecting
specific disease features. Past reports comparing tape-strips and
biopsies in AD are limited either by a small panel of
markers,\textsuperscript{16,35,36} separation of epidermal and dermal
layers in biopsies,\textsuperscript{18,37,71} or focus on non-lesional
skin.\textsuperscript{37,71} An intra-patient, RNA-seq profiling of
active AD lesions comparing tape-strips to full-thickness biopsies, thus
capturing the full spectrum of immune, barrier, and pruritus-related
molecular abnormalities is not available.

Our study showed that both tape-strips and biopsies captured key
molecular immune abnormalities of AD, including T-cell and DC
activation, innate immunity, Th2/Th1/Th17/Th22, and attenuated negative
regulation to varying degrees. However, tape-strips and biopsies also
showed some distinct biomarker profiles, with some notable biomarkers
differentially expressed in only one of the two techniques. Overall
tape-strips outperformed biopsies for most T-helper axes, with greater
differentiation from controls for Th2 (IL-4R, IL-13, IL-31, CCL17,
TNFRSF4/OX40), Th17 (CCL20, CXCL1, CXCL2), innate immunity (IL-1B, IL-8,
TNF), some Th1-related chemokines (CXCL10, CCL3), and negative regulator
markers (IL-34, IL-37). IL-34, in particular, was shown to distinguish
AD from healthy skin with almost perfect accuracy in
tape-strips,\textsuperscript{30} a property that is likely unique to
tape-strips given the \textgreater{}35x higher fold-change between AD
and controls in tape-strips compared to biopsies. These findings suggest
that chemokines that promote Th2/Th17 differentiation and their defining
cytokines are found in high concentration in the upper epidermis,
whereas their expression may be diluted in larger biopsy samples.
Tape-strips also better captured DC-related products, including myeloid
DCs (ITGAM/CD11B, ITGAX/CD11C), LCs, and inflammatory DCs (CD1A, FCER1A,
CD207/langerin), mainly localized to the
epidermis,\textsuperscript{48,72,73} which are major sources of
chemokines. Inflammatory DCs correlate with clinical AD severity and
drive Th2 skewing,\textsuperscript{74,75} representing a potential
pathogenic cell type in AD with better detection by tape-strips.

In contrast, biopsies demonstrated higher fold-changes than tape-strips
for hyperplasia related cytokines and chemokines, including the Th22
cytokine IL-22 and the S100As that are Th17 and Th22
co-regulated,\textsuperscript{76} suggesting that biopsies may be more
ideal for evaluating hyperplasia markers. IL-22 is primarily expressed
in the dermis during inflammatory states,\textsuperscript{77,78} while
S100As are produced by suprabasal keratinocytes that are likely below
the depth of tape-stripping.\textsuperscript{9} Both promote
keratinocyte proliferation and epidermal
hyperplasia.\textsuperscript{77} IFN-\selectlanguage{greek}γ, \selectlanguage{english}the main Th1 cytokine, and
CCL26, perhaps the best biomarker of therapeutic response to dupilumab,
are additional AD biomarkers primarily localized to the
dermis\textsuperscript{78,79} and accordingly showed dysregulation only
in biopsies. Markers of an inflammatory dermal fibroblast population
that may orchestrate lymphocyte migration to secondary lymphoid organs
and Th2 immunity, including COL6A5, COL6A6, TNC, and
CCL19,\textsuperscript{9} were also predictably solely up-regulated in
biopsies. Of note, many markers that are more prominently detected by
tape-strips (IL-4R, IL-13, IL-31, TNFRSF4/OX40) or biopsies (IL-22) are
therapeutic targets for AD that are either FDA-approved or under
investigation,\textsuperscript{80-88} reinforcing that although both
techniques can detect molecular changes in AD in response to
therapy,\textsuperscript{80,82,85,86} the type of therapeutic target may
dictate the appropriate sampling technique.

Pruritus, the cardinal clinical feature of AD significantly impacting
patients' quality of life,\textsuperscript{89-91} is orchestrated by
complex cross-talk between immune cells, neurons, and
keratinocytes.\textsuperscript{51,56,63,65,88,92-105} One of the
advantages of tape-strips over biopsies was the ability to better detect
changes in the neuroimmune pathways mediating pruritus. For example, the
Th2 cytokine IL-31, perhaps the key driver of pruritus in
AD,\textsuperscript{56,88,92-95} was uniquely up-regulated in
tape-strips, with similar trends in OSM and OSMR, which are part of the
same itch pathway and directly interact with
IL-31.\textsuperscript{56}STAT3, which mediates IL-31-induced neuronal
overgrowth contributing to skin sensitivity to minimal
stimuli,\textsuperscript{93} was also dysregulated only in tape-strips.
Several transient receptor channel components (TRPV2, TRPV3, TRPM3)
showed more pronounced dysregulation in tape-strips, likely owing to
their expression by sensory neurons, as well as keratinocytes and/or
immune cells.\textsuperscript{106,107}Among these, TRPV3 is most
abundant on keratinocytes,\textsuperscript{106} and has been shown to be
increased in lesional AD,\textsuperscript{62} and promote a
pro-inflammatory cascade via the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.\textsuperscript{96} In
contrast, TRPV6, which is the only down-regulated TRP gene, with more
negative fold-changes in tape-strips than biopsies, is a pre-requisite
for keratinocyte entry into differentiation.\textsuperscript{100} As an
example of the complex intercellular interactions that drive itch,
IL-31-induced up-regulation of TRPV3 was proposed to induce keratinocyte
release of SERPINE1, which then directly activates PLAUR/u-PAR in
sensory neurons to perpetuate a cycle of itch and neuroinflammation via
the innate immune system.\textsuperscript{63,108} Accordingly, SERPINE1
and PLAUR/u-PAR were also exclusively dysregulated in tape-strips.
Overall, given the large contribution of pruritus to the burden of
AD,\textsuperscript{89-91} the ability to measure the itch signature of
AD before and after treatment through minimally invasive approaches,
such as tape-strips, which in fact outperformed biopsies, is critical.

In addition to immune/neuroimmune dysregulation, tape-strips and
biopsies also preferentially captured distinct aspects of epidermal
barrier impairment. Although markers of terminal
differentiation\textsuperscript{109-111} (FLG2, SCEL, PSORS1C2) were
decreased in both techniques, at least two-fold greater changes between
healthy and lesional AD were seen in tape-strips. In contrast, biopsies
showed more pronounced up-regulation of markers of epidermal hyperplasia
(KRT6A, KRT16, MKI67), which are primarily expressed in the basal and
immediately suprabasal layers.\textsuperscript{112}, Tight junction
components, which are variably distributed throughout the
epidermis,\textsuperscript{78} were largely attenuated in AD in both
techniques, with some differences. For example, down-regulation of
CLDN1, which is expressed at all suprabasal layers and has been
described as an AD susceptibility gene with inverse correlations to
Th2,\textsuperscript{113} was only detected in biopsies. In contrast,
the gap junction components (GJB3, GJB5), which are primarily expressed
in the granular layer,\textsuperscript{114} were only significantly
decreased in tape-strips. Lipid biosynthesis/metabolism abnormalities,
which are partly driven by Th2 in AD,\textsuperscript{115} were observed
in both tape-strips and biopsies in line with prior
reports,\textsuperscript{7,86,115-119} but also showed differences
between the two techniques. For example, genes involved in the
biosynthesis of ceramides, the main lipid constituent of the stratum
corneum (DGS2, SPTLC3),\textsuperscript{120} were only decreased in
tape-strips, while products related to phospholipid synthesis (AGPAT3,
LPIN1), which are more common in the lower
epidermis,\textsuperscript{120} were only down-regulated in biopsies.
The inclusion of lipids from the sebocytes of sebaceous glands in
biopsies but not tape strips, which also contribute to barrier
function,\textsuperscript{121} may further contribute to differences in
lipid-related genes between the two techniques. FAR2, which regulates
synthesis of wax esters exclusively produced in sebaceous
glands,\textsuperscript{122} and FADS2, which regulates production of
sapienate, the most abundant fatty acid in
sebum,\textsuperscript{123}were down-regulated only in biopsies.
Overall, tape-strips and biopsies each preferentially capture gene
alterations that complement each other to constitute the defective
barrier phenotype characteristic of AD.

Our study has some limitations. Non-lesional biopsies were not available
for this cohort, preventing a parallel intra-patient comparison of
non-lesional tissues analyzed by both approaches. Nevertheless, prior
studies demonstrated that tape-strips capture the non-lesional AD
phenotype.\textsuperscript{29,37,69,71,124} Additionally, our analyses
were performed on a primarily adult Caucasian population. Future
analysis should also include other age groups and ethnic backgrounds.

In summary, this is the first global, intra-patient, full-thickness
molecular profiling of lesional skin from moderate-to-severe AD that
compares sampling by tape-strip versus biopsies. Although both capture
the main immune and barrier abnormalities of AD, tape-strips may be the
preferred technique for Th2/Th17, innate immunity, DC, pruritus, and
terminal differentiation related markers. Biopsies may be more
appropriate for evaluating genes below the granular layer, including
markers of epidermal hyperplasia, dermal cytokines (IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-22), and
inflammatory dermal populations like fibroblasts. The choice of
appropriate sampling technique should be determined considering which
specific immune or barrier features are of greatest importance to the
investigation. Still, this study highlights that tape-strips are not
only adequate but can even outperform biopsies in detecting certain AD
features, including several biomarkers that are therapeutic targets.
Thus, tape-stripping provides a minimally invasive alternative or
supplement to biopsies in clinical trials and longitudinal studies with
the potential to minimize biopsy-related complications and bolster
patient participation in adult and pediatric AD studies.

\textbf{Table 1. Demographics}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{AD (n = 20)} & \textbf{Controls (n =20)}\tabularnewline
\midrule
\endhead
\selectlanguage{ngerman}\textbf{Age, mean ± SD} & 31.0 \selectlanguage{ngerman}± 13.8 & 40.3 \selectlanguage{ngerman}± 15.7\tabularnewline
\textbf{Sex, no. (\%)} & &\tabularnewline
Female & 15 & 11\tabularnewline
Male & 5 & 9\tabularnewline
\textbf{Race, no. (\%)} & &\tabularnewline
White & 18 & 20\tabularnewline
African American & 2 & 0\tabularnewline
Asian & 0 & 0\tabularnewline
\textbf{Clinical severity} & &\tabularnewline
IGA, mean \selectlanguage{ngerman}± SD & 2.6 \selectlanguage{ngerman}± 0.6 & N/A\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURES LEGEND} :

\textbf{Figure 1. Study summary.} (A) Study design. (B) Venn diagram of
differentially expressed genes (DEGs) in tape-strip and biopsy groups by
fold-change (\textbar{}FCH\textbar{})\textgreater{}2 and false discovery
rate (FDR)\textless{}0.05. Up-regulated (\emph{red} ) and down-regulated
(\emph{blue} ) DEGs in AD versus control in each group depict shared
versus unique genes across the two techniques.

\textbf{Figure 2. Heatmap of immune-related genes.} Heatmap of the top
differentially expressed immune genes in tape-stripped (A), and biopsied
(B) AD lesional skin. Fold-change
(\textbar{}FCH\textbar{})\textgreater{}2 and false discovery rate
(FDR)\textless{}0.05. Tables show fold-changes in lesional AD versus
normal skin. World Cloud diagram of immune DEGs of tape-strips (C) and
biopsy (D) group. The size of the letters represents the magnitude of
the FCHs in AD versus healthy skin. LS, Lesional.
***FDR\textless{}0.001, **FDR\textless{}0.01, *FDR\textless{}0.05,
+FDR\textless{}0.1.

\textbf{Figure 3. Heatmap of pruritus-relates genes.} Heatmap of the
differentially expressed pruritus-related genes in tape-stripped (A),
and biopsied (B) AD lesional skin. Fold-change
(\textbar{}FCH\textbar{})\textgreater{}2 and false discovery rate
(FDR)\textless{}0.05. Tables show fold-changes in lesional AD versus
normal skin. LS, Lesional. ***FDR\textless{}0.001, **FDR\textless{}0.01,
*FDR\textless{}0.05, +FDR\textless{}0.1.

\textbf{Figure 4. Heatmap of barrier-related genes.} Heatmap of the
differentially expressed barrier-related genes in tape-stripped (A), and
biopsied (B) AD lesional skin. Fold-change
(\textbar{}FCH\textbar{})\textgreater{}2 and false discovery rate
(FDR)\textless{}0.05.Tables show fold-changes in lesional AD versus
normal skin. LS, Lesional. ***FDR\textless{}0.001, **FDR\textless{}0.01,
*FDR\textless{}0.05, +FDR\textless{}0.1.

\textbf{Figure 5. Gene-set variation analysis (GSVA).} Gene-set
variation analyses (GSVA) of immune-related and barrier-related genes.
Red bars represent means. \emph{Black symbols} : significance of
comparison to normal\emph{; red symbols:} significance of comparison
between lesional and non-lesional skin. MADAD, Meta-Analysis Derived AD
transcriptome; LS, Lesional. ***\emph{P} \textless{}0.001, **\emph{P}
\textless{}0.01, *\emph{P} \textless{}0.05, +\emph{P} \textless{}0.1.

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