not permitted.

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means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
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© 2016 EDIZIONI MINERVA MEDICA Giornale Italiano di Dermatologia e Venereologia 2018 June;153(3):316-25
Online version at http://www.minervamedica.it DOI: 10.23736/S0392-0488.16.05427-4

ORIGINAL ARTICLE

Biomolecular index of therapeutic efficacy

in psoriasis treated with anti-TNF-α agents
Luca BIANCHI  1, Gaetana COSTANZA 2, Elena CAMPIONE  1, 3 *, Manuela RUZZETTI  1,
Alessandro DI STEFANI  1, Laura DILUVIO  3, Emiliano GIARDINA 2, Raffaella CASCELLA 2,
Paola CORDIALI-FEI  4, Claudio BONIFATI  4, Andrea CHIRICOZZI  5,
Giuseppe NOVELLI  2, Fabrizio ENSOLI  4, Augusto ORLANDI  2, 3

1Department of Dermatology, University of Rome Tor Vergata, Rome Italy; 2Department of Biomedicine and Prevention, University

of Rome Tor Vergata, Rome Italy; 3Polyclinic of University Tor Vergata of Rome, Rome Italy; 4Department of Clinical Pathology and
Microbiology, and Clinical Dermatology, San Gallicano Institute, Rome Italy; 5Department of Dermatology, University of Pisa, Pisa Italy
*Corresponding author: Elena Campione, Department of Dermatology, University of Rome Tor Vergata, Viale Oxford 81, 00133 Rome, Italy.
E-mail: campioneelena@hotmail.com

A B S TRA C T
BACKGROUND: Clinical or quality of life assessments are currently available for psoriasis severity evaluation and therapeutic response. Labo-
ratory scores focused to measure and follow treatment efficacy are lacking at present.
METHODS: A microscopic and biomolecular score was designed to monitor skin disease severity and clinical response to anti-psoriatic treat-
ments. A susceptibility gene analysis on cellular retinoic acid binding protein-II (CRABP-II), acting on keratinocyte differentiation, was also
performed. A Molecular Index of Therapeutic Efficacy (MITE) was defined by assembling morphometric/semiquantitative measurement of epi-
dermal thickness, immunohistochemical Ki-67, keratin 17 and CRABP-II expression of lesional and non-lesional psoriatic skin biopsies before
and after anti-tumor necrosis factor (TNF) α therapies. A 0-12 MITE score was correlated with Psoriasis Area and Severity Index (PASI) and
Psoriasis Disability Index (PDI) scores and inflammation. Three CRABP-II SNPs were analyzed by TaqMan assay.
RESULTS: All parameters were highly expressed in psoriatic lesions and reduced after 12 weeks of anti-TNF-α treatments. MITE score strongly
correlated with PASI and PDI values either before or after therapies (P<0.001 and P<0.001, respectively). Conversely, MITE values did not
change after treatments of non-responder patients. CRABP-II did not result in a psoriatic susceptibility gene for the SNPs probes analyzed.
CONCLUSIONS: MITE score variations corresponded to the patients’ clinical improvement following anti-TNF-α treatments, with significant
statistical correlation among MITE, PASI and PDI scores. If confirmed in a larger series and/or in different hyperproliferative and inflamma-
tory dermatoses, MITE score could be proposed as additional monitoring system to evaluate treatment protocols in skin disorders and targeted
biomolecular pathways supporting clinical efficacy.
(Cite this article as: Bianchi L, Costanza G, Campione E, Ruzzetti M, Di Stefani A, Diluvio L, et al. Biomolecular index of therapeutic efficacy in
psoriasis treated with anti-TNF-α agents. G Ital Dermatol Venereol 2108;153:316-25. DOI: 10.23736/S0392-0488.16.05427-4)
Key words: Psoriasis - Patient outcome assessment - Inflammation - Therapeutics.

P soriasis is a chronic inflammatory skin disease

characterized by altered epidermal architecture
with reduced keratinocyte differentiation and increased
proliferation.1 A complex cytokines and chemokines
network participates in induction and maintenance of
or other proprietary information of the Publisher.
proved by the successful response to anti-TNF-α agents
in clearing psoriatic plaques and recovering the pro-
gression of psoriatic arthritis.6 Clinical evaluations are
currently proposed to monitor the response to specific
therapies. Psoriasis Area and Severity Index (PASI) and

the psoriatic plaque.2, 3 Tumor necrosis factor (TNF) several questionnaires on the patients’ quality of life
α, secreted by innate or adaptive immune system cells, (QoL) are the most employed methods for clinical and
sustains the clinical manifestations of psoriasis at both therapeutic scoring.7, 8 Possible soluble biomarkers or
skin and joint level.4, 5 Its relevance has been clinically laboratory parameters associated with psoriasis sever-

316 Giornale Italiano di Dermatologia e Venereologia June 2018

not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
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BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY BIANCHI
ity or systemic inflammation or response to treatment
with TNF-inhibitors are also investigated but none of
them translated as a score into the clinical practice.9 In
the past, multiparameters analyzed by flow cytometry
and immunohistochemistry have tried to assess prolif-
eration and differentiation simultaneously in some hy-
perproliferative skin disorders including psoriasis.10, 11
We believe that the definition of a unique biomolecular
score comprehensive of epidermal proliferative and dif-
ferentiative parameters could reinforce the clinical as-
sessment of severity of the disease and the response to
the different therapies. We designed a semiquantitative
assay, named Molecular Index of Therapeutic Efficacy
(MITE), based on the measurement of epidermal thick-
ness, and semiquantitative evaluation of the expres-
sion of selected proliferative and psoriatic markers in
lesional and non-lesional skin biopsies, before and after
anti-TNF-α therapies, in an open multicenter prospec-
tive study on 59 plaque-type psoriatic patients treated
with three anti-TNF-α biologics (etanercept, adalimum-
ab, infliximab).12, 13 Although in previous studies differ-
ent key protein levels have been already analyzed and
correlated to clinical scores, we tried to combine into a
unique index microscopic and biomolecular parameters
altered in skin psoriasis.14, 15 To validate our proposal,
lesional MITE has been compared to lymphocyte accu-
mulation and PASI and Psoriasis Disability Index (PDI)
scores at baseline and after 12 weeks of three different
anti-TNF-α treatments. Cellular retinol and retinoic acid
binding proteins critically regulate retinol and its ac-
tive metabolite all-trans retinoic acid (atRA)-mediated
transcriptional activity.16 Retinoic acid binding protein
II (CRABP-II) mediates epidermal hyperproliferation
acting on keratinocyte differentiation and CRABP-II
mRNA level is dramatically increased in psoriatic le-
sions.17 Consequently, we also investigated the possible
role of CRABP-II as susceptibility psoriatic gene. This
gene maps on chromosome 1q21.3 and clusters into
PSORS4, a well-known psoriasis susceptibility locus,
part of keratinocyte differentiation-related genes.18
Materials and methods
Ethics Committee approval
or other proprietary information of the Publisher.
This study has been approved by the Ethics Com-
mittee at our institution (Comitato Etico Tor Vergata;
chairperson: Sergio Chimenti; protocol no. 20745; date
Vol. 153 - No. 3 Giornale Italiano di Dermatologia e Venereologia 317
of approval: 25th March 2005) and has been performed
in accordance with the ethical standards as laid down in
the 1964 Declaration of Helsinki and its later amend-
ments or comparable ethical standards.
Informed consent was obtained from all individual
participants included in the research study.
Study population
Fifty-nine patients (19 female and 40 male patients,
mean age 46.3±12.3 years) affected by moderate-to-
severe plaque-type psoriasis were randomly enrolled in
an open, prospective, observational study at the Depart-
ment of Dermatology, University of Rome Tor Vergata,
and at San Gallicano Dermatological Institute, Rome,
Italy. Approval of the study was obtained from institu-
tional Ethical Committees and Public Health Ministry
in accordance with the Declaration of Helsinki. Patients
were randomly assigned to each agent, namely etaner-
cept, adalimumab and infliximab, as in real life setting.
Patients’ clinical characteristics are listed in Table I. The
clinical response to the biologic was measured by PASI
and Psoriasis Disability Index (PDI) at baseline and at 12
weeks.19 All patients eligible for biologic therapy did not
receive any systemic treatment for at least three months
or any topical therapy for at least 2 weeks prior the study
entry. All patients signed a written informed consent.
Analysis and quantification of MITE parameters on skin
lesions and their comparison with clinical scores were
performed to monitor the clinical counterpart of anti-
TNF-α effects. An age- and sex-matched control group
Table I.—General demographic and clinical characteristics of en-
rolled psoriatic patients at baseline.
Characteristics Value
N. 59 (100)
Male 40 (67.8)
Female 19 (32.2)
Treatment
Etanercept 29 (49.1)
Infliximab 15 (25.45)
Adalimumab 15 (25.45)
Mean age, years 46.3±1.4 (25-57)
PASI at baseline 11.25±1.2 (0-34.2)
PDI at baseline 23.9±2.7 (0-34)
MITE at baseline 11±0.47 (0-12)
Data are expressed and number (percentage) or as mean±SEM (range).
PASI: Psoriasis Area and Severity Index; PDI: Psoriasis Disability Index; MITE:
Molecular Index of Therapeutic Efficacy.
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
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BIANCHI BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY
included 20 healthy untreated subjects who were under-
going an Institutional health surveillance program.
Treatments and skin biopsy
Etanercept and adalimumab were administered subcu-
taneously, whereas infliximab intravenously, following
the protocol procedures and dosages in accordance with
the international guidelines.19 Only chronic stable plaques
were selected for the biopsies. At baseline, before starting
the treatment, 3-mm punch skin biopsies were obtained
from lesional and non-lesional areas for morphological
and immunohistochemical evaluation. In order to inves-
tigate therapy-induced reversal of the proposed skin pa-
rameters, punch biopsy samples were collected in all the
59 patients in remaining lesions at 12 weeks of therapy.
Microscopic and immunohistochemical study
Biopsies were fixed in 10% formalin for 24 hours,
paraffin embedded and four-μm thick serial sections
stained with hematoxylin and eosin for histopathologi-
cal examination or employed for immunohistochemis-
try.20 For the latter, serial 4 μm thick sections were in-
cubated with polyclonal rabbit anti-CRABP-II (1:300,
1 hour; Bethyl Laboratories, Montgomery, TX, USA),
followed by biotin-labelled goat anti-rabbit secondary
antibody and streptavidin-horseradish peroxidase conju-
gated (Ylem,1:100).21 Serial slides were also incubated
with monoclonal mouse anti-K17 (1:50; Cell Marque
Corporation, Rocklin, CA, USA), followed by revelation
with Ultravision LP Detection System (Thermo Scien-
tific, Waltham, MA, USA). Immunostaining with mouse
anti-Ki-67 (clone 30-9) and rabbit anti-CD3 lymphocyte
monoclonal antibodies (clone 2GV6) was performed by
using an Automated Immunohistochemistry Staining
(Ventana Medical Systems, Roche Holding, Basel, Swit-
zerland), according to manufacturer’s instructions, and
positive and negative controls.22 As control, anonymous
control skin sections from paraffin block archive of surgi-
cally excided non-neoplastic biopsies were also investi-
gated (N.=13; mean age 43.5±0.75 years; range 24-58).
or other proprietary information of the Publisher.
Evaluation of MITE, clinical scores and inflammation
A new score system, MITE, was based on morpho-
metric and semiquantitative evaluation of epidermal pa-
318 Giornale Italiano di Dermatologia e Venereologia June 2018
rameters. A 0-3 value was attributed to each of the items
investigated, with a final 0-12 MITE score. Four catego-
ries of MITE degrees severity (0-3; 3.1-6; 6.1-9; 9.1-12),
respectively absent/minimal, mild, moderate, severe,
were arbitrarily obtained and calculated, as summarized
in the Table II. Epidermal thickness was quantified us-
ing arbitrary score units: 0 for 0-152 μm, 1 for 153-313
μm, 2 for 314-396 μm, 3 for 396-540 μm.15 Score units
(0-3) for immunohistochemical evaluation of CRABP-
II were arbitrarily defined as reported.23 Ki-67 expres-
sion was evaluated as the percentage of positive epider-
mal nuclei as follows: 0 for 0-10%, 1 for 11-20%, 2 for
21-30%, 3 for >30%.24 K17 expression was graded as
reported with modifications: 0 for no staining or focal
<5% total, 1 for skip areas between positive staining, 2
for confluent horizontally plus <40% positive epidermis,
3 for confluent horizontally plus >40% positive epider-
mis.25 Finally, CD3+ lymphocytes were quantified and
results expressed for epidermal (positive cells per mm)
and dermal compartment (positive cells per mm2), re-
spectively.26 For comparisons, four arbitrary categories
(0-3) were obtained considering mean values. Blinded
evaluations were performed by two independent re-
searchers at 200× magnification in at least 10 randomly
selected fields, with interobserver variability <5%.27 In
order to correlate with MITE, PASI and PDI scores were
converted in four categories of severity for each patient:
PASI as absent (0-0.99 = 0), mild (1-10 = 1), moderate
(11-20 = 2), severe (21-72 = 3);9 for PDI: absent-mild
(0-15 = 0); mild-moderate (16-23 = 1); moderate-severe
(24-33 = 2) and very severe (33-45 = 3). PDI and PASI
evaluations were performed by three trained clinical ob-
servers, with interobserver variability <5%.
Table II.—Categories and detailed description used for MITE
scores (0-12).
Score MITE category Details
0-3 Absent/minimal Overall expression of absent or focal biomarkers/
Skin morphologically similar to normal
3.1-6 Mild Overall expression of biomarkers mild/Skin
morphologically shows mild psoriatic typical
alterations
6.1-9 Moderate Overall expression of biomarkers moderate/
Skin morphologically shows the typical psoriatic
alterations
9.1-12 Severe Overall expression of biomarkers is high and
marked/Skin morphologically shows severe
psoriatic alterations
MITE: Molecular Index of Therapeutic Efficacy.
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
or other proprietary information of the Publisher.

C
B
K17 (AU) Ki-67 + nuclei (%)

A
H&E

D
E. thickness (μm)

NS

Vol. 153 - No. 3

NLS
NS

LS (T0)

chromogen (original magnification: 100×).

LS (T12)
Ki-67 + cells (%)
BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY

etanercept

H&E: hematoxylin and eosin stain; AU: arbitrary units.

NS
NLS
©

NLS

# P<0.007 by ANOVA; *P<0.007 by t-test, § P<0.005 by ANOVA and t-test.

infliximab

LS (T0)
LS (T12)
LS (T0)

K17 expression (AU)

adalimumab

Giornale Italiano di Dermatologia e Venereologia

NS
NLS
COPYRIGHT 2018 EDIZIONI MINERVA MEDICA

LS (T0)
LS (T12)

LS (T12)

319
ment. D) Bar graph showing the increased of parameters in lesional skin (LS) at baseline (T0), compared to control normal (NS) and non-lesional
psoriatic skin. After 12 weeks of treatment (T12), all values are drastically decreased compared to the baseline. Diaminobenzidine is used as a
sentative images of epidermal thickness in H&E-stained, Ki-67 and K17 in non-lesional skin, lesional skin at baseline and after 12 weeks of treat-
Figure 1.—Evaluation of anti-TNF-α changes on epidermal thickness, proliferation and differentiation in normal and psoriatic skin. A-C) Repre-
BIANCHI
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
COPYRIGHT 2018 EDIZIONI MINERVA MEDICA
BIANCHI BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY
Genetic analysis
To investigate the potential role of CRABP-II as sus-
ceptibility gene, 400 patients with psoriasis (includ-
ing those enrolled in the present study) and 490 non-
psoriatic controls were recruited in a wide cohort of
blood donors originating from the same geographical
areas. Written consent was obtained from all patients
and the study was approved by the local Ethics Com-
mittees. Genomic DNA was extracted from peripheral
blood using standard procedures. International HapMap
Project has been used to perform an in-silico analysis
to select SNPs (www.hapmap.org) and to tag the high-
est proportion of genetic variability.28 We selected three
different SNPs. (rs4661213 T/C; rs12041913 G/A and
rs124006221 A/G) located in CRABP-II gene (1q21.3).
Genotyping was performed by TaqMan assays (Applied
Biosystems, Foster City, CA, USA). Fast real-time
polymerase chain reactions (PCR; Applied Biosystems)
were interpreted by Sequence Detection System v. 2.1
software (Applied Biosystems), according to the manu-
facturer’s instructions. Each plate contained three posi-
tive control samples, previously confirmed by direct
sequencing, and the negative control.
Statistical analysis
Immunohistochemical data were analyzed using one-
way ANOVA test, followed by a Bonferroni post-hoc test
or Student’s t-test. Results of semiquantitative evalua-
tion were represented as the mean±SEM. Correlations
between indexes and single clinical-pathological and
biological features were performed using Spearman cor-
relation rank test.24 In general, P<0.05 was considered
statistically significant. SPSS v. 16.0 (SPSS Inc., Chica-
go, IL, USA) was used for statistical analyses. Statistical
genetic analysis was performed by standard 2×2 table and
Fisher’s exact text, considering allele, genotype and hap-
lotype frequencies of all tested variations. Odds Ratios
(OR) were calculated; allele and haplotype analysis in
unrelated samples by using the UNPHASED software.28
Results
or other proprietary information of the Publisher.
Characteristics of enrolled patients and treatments
Demographic characteristics and mean values of
PASI, PDI and MITE scores, measured at baseline,
320 Giornale Italiano di Dermatologia e Venereologia June 2018
together with the biologics prescribed, etanercept
(49.2%), infliximab (25.4%) and adalimumab (25.4%)
at baseline, are reported in Table I.
Epidermal thickness, Ki-67 and K17 expression
At baseline (Figure 1A), lesional epidermal thick-
ness was greater than non-lesional and control coun-
terparts (Figure 1D; P<0.007), whereas after 12 weeks
of anti-TFN-α treatment, epidermal thickness was re-
duced and similar to non-lesional skin (P<0.0005; Fig-
ure 1A). Infliximab treatment resulted somehow more
effective. Concerning proliferative index, as reported
in Figure 1B, the percentage Ki-67+ keratinocyte nu-
clei in suprabasal layers of the lesional epidermis at
baseline was greatly increased compared to control and
to non-lesional skin (Figure 1D; P<0.007). After 12
weeks of treatment, the percentage of lesional Ki-67+
nuclei had strongly decreased compared to baseline in
a similar manner comparing the different TNF-α drugs
(Figure 1D; P<0.0005). We also evaluated the K17 ex-
pression (Figure 1C), absent in normal epidermis but
typically expressed in the granular and upper epidermal
layers in psoriatic skin.29 Baseline K17 expression in
psoriatic lesions was marked and strong and similarly
reduced for all the three TNF-α inhibitors (Figure 1C,
D; P<0.0005), returning to values similar to non-lesion-
al and control skin.
CRABP-II expression
CRABP-II immunostaining of normal epidermis
(Figure 2A) was weak and cytoplasmatic in the basal
layer, stronger and also nuclear in the spinous and gran-
ular layers and absent in the stratum corneum. CRABP-
II immunostaining of non-lesional skin was similar to
control (Figure 2A, B). Semiquantitative evaluation
revealed that CRABP-II expression in lesional skin
was greatly increased compared to both non-lesional
skin and control (P<0.007), mainly in the spinous and
granular layer. After 12 weeks of therapy, CRABP-II
expression was strongly reduced compared to baseline
(P<0.0005, Figure 2B), whereas in non-responder pa-
tients it remained similar to baseline (not shown). Re-
duction of CRABP-II expression was similar with the
three TNF-α inhibitors, with a 50-54% mean decrease
comparing to the baseline.
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
COPYRIGHT 2018 EDIZIONI MINERVA MEDICA
BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY BIANCHI
CRABP-II
A
etanercept
CRABP-II expression (AU)
infliximab
adalimumab
NS NLS LS (T0) LS (T12)
B
Figure 2.—CRABP-II expression in normal and psoriatic skin before
and after treatment. A) Representative images of CRABP-II immu-
nostainings of normal control skin (NS), non-lesional psoriatic (NLS)
and lesional skin (LS) at baseline (T0). Baseline LS CRABP-II expres-
sion is very high and strongly reduced after 12 weeks of treatment (T12)
with anti-TNF-α drugs. B) Semiquantitative CRABP-II evaluation after
12 weeks of treatment with anti-TNF-α drugs. Diaminobenzidine is used
as a chromogen (original magnification: 100×).
# P<0.007 by ANOVA; *P<0.007 by t-test, § P<0.005 by ANOVA and
t-test. H&E: hematoxylin and eosin stain; AU: arbitrary units.
MITE scores at baseline and after therapy with TNF-α
inhibitors
Before therapy, 15.4% of biopsies investigated
showed moderate MITE Index (6.1-9), whereas 84.6%
severe MITE Index (9.1-12). After 12 weeks of therapy,
these values consistently changed: 62.9% of the samples
or other proprietary information of the Publisher.
showed an absent/minimal MITE Index (0-3), whereas
the remaining 37.1% displayed mild MITE Index (3.1-6)
(Figure 3). After 12 weeks of therapy, a consistent MITE
score reduction in lesional skin was detectable, either
Vol. 153 - No. 3 Giornale Italiano di Dermatologia e Venereologia 321
MITE category
absent/minimal
mild
moderate
% of caases
severe
Before anti-TNFα After anti-TNFα
treatment treatment
Figure 3.—Distribution of cases according MITE categories before and
after treatments. Before therapy, MITE score was high: 15.4% of cases
has a moderate MITE and 84.6% showed a severe index. No case was
present in absent/minimal or mild category. Conversely, after 12 weeks
therapy 62.9% was in absent/minimal MITE category and the 31.7% in
the wild MITE category, but nobody was in moderate or severe category.
for all the biologics considered together or for each drug
evaluated (Figure  4A). Infliximab and adalimumab in-
duced the highest response (P<0.0003), compared with
the improvement due to the etanercept (P<0.003).
Reduction of the number of skin CD3+ T lymphocytes
Immunohistochemistry revealed that the number of
CD3+ T lymphocytes at baseline in the lesional dermis
and epidermis was 260.7±2.6/mm2 and 41.1±1.7/mm,
respectively. These values were strongly reduced after
therapy with the three TNF-α inhibitors, with a mean
decrease of epidermal CD3+ T lymphocytes of around
70.8% compared to baseline values (Figure 4B). Simi-
lar findings were observed in the dermis (not shown).
A positive correlation was also documented between
MITE and inflammation as CD3+ T lymphocytes reduc-
tion rate after 12 weeks of treatment (ρ=0.872; P<0.01).
PASI and PDI and correlation with epidermal param-
eters
PASI calculated after 12 weeks of treatment (Fig-
ure  4C) showed a significant decrease compared with
the baseline, as expected, especially with infliximab and
adalimumab (P<0.002 and P<0.01, respectively). After
12 weeks of anti-TNF-α treatments, also PDI score de-
creased (Figure 4D; P<0.01) for all the 3 drugs, although
the decrease was less pronounced for etanercept (P<0.05).
not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
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BIANCHI BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY

As reported in Table III, strong positive correlations

between the selected parameters were documented, be-
LS (T0) fore and after therapy. Each parameter also showed sig-
LS (T12) nificant correlations with PASI and PDI scores, before
and after therapy (Table IV).
MITE score

Correlation between MITE, PASI and PDI

A significant positive correlation between MITE

A All drugs
Reduction rate epidermal
CD3 lymphocytes (%)
E I A
and PASI indexes was evidenced at baseline (ρ=0.76;
P<0.001) and after 12 weeks of treatment (ρ=0.88;
LS (T12)/LS (T0)
P<0.001). A positive correlation between MITE and
PDI indexes, although somehow weaker, was also

documented comparing baseline (ρ=0.69; P<0.001)

and values after 12 weeks of treatment (ρ=0.71;
P<0.001).

Table III.—Correlations among biomarkers before and after anti-

B All drugs E I A TNFα treatment.
LS (T0) Biomarker Epidermal thickness Ki-67 K17 CRABP-II
LS (T12) Before treatment
Epidermal thickness 1 0.75* 0.7* 0.9 #
Ki-67 1 0.71* 0.86 #
PASI score

K17 1 0.98 #
CRABP-II 1
After treatment
Epidermal thickness 1 0.68* 0.9 # 0.86 #
Ki-67 1 0.91 # 0.88 #
K17 1 0.95 #
CRABP-II 1
C All drugs E I A CRABP-II: retinoic acid binding protein II.
*P<0.01; # P<0.001.
LS (T0)
LS (T12)
Table IV.—Correlation among epidermal biomarkers and PASI
and PDI indexes before and after anti-TNF-α treatments.
PDI score

PASI PDI
Before treatment
Epidermal thickness 0.67* 0.48*
Ki-67 0.77 # 0.55*
K17 0.63* 0.5*
All drugs E I A CRABP-II 0.78 # 0.62*
D After treatment
Figure 4.—Distribution of MITE, CD3+ lymphocytes reduction rate, Epidermal thickness 0.7* 0.49*
PASI and PDI score after anti-TNF-α regimen. A) MITE score decrease Ki-67 0.9 # 0.63*
or other proprietary information of the Publisher.

in lesional skin (LS) after 12 weeks treatment (T12), considering all K17 0.62* 0.55*
treatments compared to baseline (T0); B) rate reduction of CD3 positive CRABP-II 0.9 # 0.6*
cells (%) after 12 weeks of treatments (T12/T0). C, D) PASI and PDI
decrease after 12 weeks of treatments (T12). *P<0.05; # P<0.01.
E: etanercept; I: infliximab; A: adalimumab. PASI: Psoriasis Area and Severity Index; PDI: Psoriasis Disability Index; CRABP-
II: retinoic acid binding protein II.
*P<0.01; **P<0.003; ***P<0.0005 (all by t-test).

322 Giornale Italiano di Dermatologia e Venereologia June 2018

not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
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BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY BIANCHI

CRABP-II gene analysis Table VI.—Haplotype CRABP-II SNPs frequency in Italian pa-
tients and controls. The CRABP-II gene was analyzed using three
The analysis of CRABP-II gene (1q21.3) performed specific TaqMan probes (rs4661213 T/C, rs12041913 G/A and
rs124006221 A/G).
using three specific TaqMan probes (rs4661213 T/C,
Frequency
rs12041913 G/A and rs124006221 A/G) and the soft- Haplotype P value
ware UNPHASED failed to reveal a significant P value Cases Controls

between the disease and the three tested variants; no T-G-A 0.58 0.52 NS
departure from Hardy-Weinberg equilibrium was de- T-G-G 0.3607 0.3814 NS
T-A-A 0.009 0.004 NS
tected (Tables V, VI). These findings suggested that, at T-A-G 0.0 0.002 NS
least for those probes and for population of considered, C-G-A 0.05 0.04 NS
CRABP-II is not a susceptibility gene for skin psoriasis. C-G-G 0.0 0.006 NS
C-A-A 0.0007 0.0 NS
NS: not statistically significant.
Discussion
In this study, we proposed a molecular index to quan-
psoriatic parameters during the course of the disease to
tify and measure epidermal psoriatic parameters during the
evaluate efficacy of the treatments but, to our knowledge,
course of biologic anti-TNF-α therapies.15, 25, 29, 30 MITE
the different tools were not collected together to realize a
score was obtained from the sum of morphometric/semi-
score as PASI or QoL calculations do clinically.10, 11, 34-36
quantitative evaluation of four epidermal morphological,
PASI is widely considered as the mainstay for assessing
proliferative and differentiative parameters: thickness, Ki-
the course of psoriasis severity.7 Nevertheless, because
67, K17 and CRABP-II expression.14, 17, 25, 31 Similarly to
of the possible PASI limitations due to clinical variants
the currently employed clinical scores, semiquantitative
or mild forms, different outcome measures have been
sums converted in MITE score were categorized into
routinely incorporated into clinical trials.37 Microscopi-
four degrees of MITE severity.32, 33 Previous studies al-
cally, plaque psoriasis is a hyperproliferative epidermal
ready compared histological and immunohistochemical
disorder characterized by spinous layer hyperplasia and
incomplete differentiation of granular and cornified
layers.38 The evaluation of the epidermal hyperplasia
Table V.—Allele and genotype association between psoriasis and has been employed to confirm the diagnosis, measure
specific CRABP-II SNPs. The CRABP-II gene was analyzed us-
ing three specific TaqMan probes (rs4661213 T/C, rs12041913 the clinical severity and follow the efficacy of thera-
G/A and rs124006221 A/G). pies.14,  39,  40 Prominent epidermal hyperplasia induces
rs4661213 P value rs12041913 P value rs12406221 P value an increased basal and suprabasal Ki-67 positivity, oth-
Allele Allele Allele erwise observed only in the basal layer of the normal
frequency frequency frequency skin.16, 38, 41 Similarly, proliferative rate has been used in
Cases Cases Cases several studies to monitor the efficacy of anti-psoriatic
T: 0.953 NS G: 0.991 NS A: 0.64 NS
C: 0.047 A: 0.009 G: 0.36
drugs.16, 25, 26, 31 It has been shown that the keratinocyte
Controls Controls Controls cell cycle time is greatly reduced in psoriasis whereas the
T: 0.959 G 0.995 A: 0.61 number of dividing cells is increased, resulting in a hyper-
C: 0.041 A: 0.005 G: 0.39 plastic epithelium.42 In addition, aberrant keratinocytes
Genotype Genotype Genotype
frequency frequency frequency differentiation occurs in psoriatic epidermis, likely for
Cases Cases Cases abnormal expression of specific keratin filaments related
TT: 0.90 NS GG: 0.98 NS AA: 0.42 NS to an accelerated cell turn-over.1, 43, 44 K17 expression is
TC: 0.10 GA: 0.02 AG: 0.44
CC: 0.0 AA: 0.0 GG: 0.14
absent in normal epidermis but is strong in psoriatic su-
Controls Controls Controls prabasal keratinocytes.45 K17 expression is considered a
or other proprietary information of the Publisher.

TT: 0.91 GG: 0.99 AA: 0.39 marker of incomplete differentiation and its reduction as
TC: 0.09 GA: 0.01 AG: 0.46 an index of efficacy of anti-psoriatic treatment.35, 37 We
CC: 0.0 AA: 0.0 GG: 0.15
also considered epidermal CRABP-II expression, since
NS: not statistically significant.
CRABP-II is an intracytoplasmic receptor that mediates

Vol. 153 - No. 3 Giornale Italiano di Dermatologia e Venereologia 323

not permitted. It is not permitted to remove, cover, overlay, obscure, block, or change any copyright notices or terms of use which the Publisher may post on the Article. It is not permitted to frame or use framing techniques to enclose any trademark, log
means which may allow access to the Article. The use of all or any part of the Article for any Commercial Use is not permitted. The creation of derivative works from the Article is not permitted. The production of reprints for personal or commercial use i
This document is protected by international copyright laws. No additional reproduction is authorized. It is permitted for personal use to download and save only one file and print only one copy of this Article. It is not permitted to make additional copies
(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
COPYRIGHT 2018 EDIZIONI MINERVA MEDICA
BIANCHI BIOMOLECULAR INDEX OF ANTI-TNF-α EFFICACY
RA intracellular trafficking and activity, and RA a crucial
effector or keratinocyte differentiation.46 Previous stud-
ies reported increased CRABP-II expression in psoriatic
skin.47, 48 Because of the increased keratinocyte turn-over
in psoriasis plaque, aberrant CRABP-II expression likely
mediates hyperproliferative epidermal condition, becom-
ing a useful marker for monitoring effects of anti-psori-
atic treatment on aberrant keratinocyte proliferation and
differentiation.48 All epidermal biomarkers were signifi-
cantly increased in psoriatic skin samples at baseline in
comparison to non-lesional skin, and their values com-
pletely reversed to similar to control skin after the dif-
ferent anti-TNF-α treatments, in parallel with the overall
restoration of normal epidermal architecture. We assem-
bled all these described parameters into MITE Index and
documented that after 12 weeks of treatment with three
anti-TNF-α inhibitors, MITE Indexes correlated with
changes of PASI and PDI scores and with the reduction of
CD3+ T lymphocyte accumulation. No significant MITE
scores differences were observed with the different anti-
TNF-α agents, adalimumab, etanercept and infliximab, in
responder patients. Nevertheless, MITE, PASI and PDI
documented a higher rate of individual failures after etan-
ercept treatment (50% of non-responders), as reported in
the literature.13,  49 MITE might facilitate the measure-
ment of disease severity focusing and collecting different
features of psoriatic keratinocytes. We also investigated
the possible role of CRABP-II as psoriasis susceptibility
gene. CRABP-II gene maps on chromosome 1q21.3, clus-
tering into the psoriasis susceptibility locus, PSORS4,
which includes several keratinocyte differentiation-re-
lated genes.18, 50 The altered CRABP-II expression might
represent a response to a psoriatic inflammatory status or
indicate an involvement of the gene products in the hy-
perproliferative phenotype. We analyzed three different
SNPs and seven different haplotypes, but no significant
associations could be highlighted, although other SNPs
on CRABP-II gene potentially associated with the disease
should be investigated, at least for the population of Cen-
tral Italy we considered.
Conclusions
or other proprietary information of the Publisher.
Our study correlates effectiveness of the anti-TNF-α
drugs to the restoration of the disease biomarkers inves-
tigated in our series of patients. Further studies would
require extending the use of this index to verify and
324 Giornale Italiano di Dermatologia e Venereologia June 2018
validate its potential role as a support to other therapeu-
tic and clinical indexes. MITE score could assist and
complete PASI and QoL evaluations in clinic trials as
additional useful screening to monitor therapeutic ap-
proaches or to better follow treatment protocols in hy-
perproliferative inflammatory skin disorders. Studies
are also needed to investigate alternative genetically de-
termined CRABP-II dependent pathways which might
influence keratinocyte differentiation in psoriasis.
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(either sporadically or systematically, either printed or electronic) of the Article for any purpose. It is not permitted to distribute the electronic copy of the article through online internet and/or intranet file sharing systems, electronic mailing or any other
©
COPYRIGHT 2018 EDIZIONI MINERVA MEDICA
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Funding.—This work was partially funded by the Italian Ministry of Health (2008).
Conflicts of interest.—The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
Acknowledgements.—The authors wish to thank M. Maccarone, President of Italian Psoriatic Patient Association (ADIPSO), for providing access to database
and questionnaires, and S. Cappelli for providing technical collaboration.
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