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ISSN: 1556-9527 (print), 1556-9535 (electronic)

Cutan Ocul Toxicol, 2015; 34(4): 276–281

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15569527.2014.963598

RESEARCH ARTICLE

A comparative study of histopathological findings in skin biopsies

from patients with psoriasis before and after treatment with acitretin,
methotrexate and phototherapy
Seyma Ozkanli1, Ebru Zemheri1, Ayse Serap Karadag2, Ozge Akbulak2, Tulay Zenginkinet1, Ilkin Zindanci2,
Serap Gunes Bilgili3, and Necmettin Akdeniz2
1
Department of Pathology and 2Department of Dermatology, Istanbul Medeniyet University, School of Medicine, SB Goztepe Training and Research
Hospital, Istanbul, Turkey, and 3Department of Dermatology, Yuzuncu Yil University, School of Medicine, Van, Turkey

Abstract Keywords
Introduction: Psoriasis is a chronic and inflammatory skin disease. Few studies in the literature Histopathology, psoriasis, treatment
evaluate the responses to the treatment histopathologically.
Objectives: In this study, we evaluated and compared skin biopsies taken from patients with History
psoriasis before and after phototherapy and therapy with acitretin and methotrexate.
Material and methods: We included 64 patients with a diagnosis of psoriasis vulgaris in our Received 8 August 2014
study. We performed phototherapy on 33 patients (51.6%), while 19 patients (29.7%) were Revised 25 August 2014
treated with methotrexate and 12 patients (18.8%) were treated with acitretin. Accepted 5 September 2014
Results: All of the patients had chronic plaque psoriasis, and they had skin lesions on more than Published online 29 September 2014
10% of their total body surface area and a score of PASI of 7.2–21.8 (average: 12.2). The
histopathological parameter scores were similar in the initial evaluations of the pre-treatment
treatment groups. When the biopsy specimens of all cases were evaluated together, a
significant decrease was observed in terms of parakeratosis, Munro’s microabscesses, regular
acanthosis, pustules of Kogoj, lymphocyte infiltration in the papillary dermis, loss of the
granular layer, spongiosis, suprapapillary thinning, vascularity in the papillary dermis and
neutrophile infiltration in the papillary dermis.
Conclusion: We found in our study that conventional treatment modalities provided
histopathologically significant recovery in psoriasis, but they did not have an effect on some
histopathological findings. To our knowledge, it is one of the few studies to assess these
parameters in psoriasis under the continuous effect of acitretin, methotrexate and photother-
apy for three months. There is a need for studies with larger series to examine the
histopathological effects of these treatment modalities in terms of immunopathology.

Introduction blood vessels are increased in number and size, and a-mixed
leukocytic infiltrate is seen both in the dermis and in the
Psoriasis is a chronic and inflammatory skin disease;
epidermis. The pathological hallmarks of psoriatic lesions are
T-cell-mediated that presents sharply circumscribed ery-
neutrophilic granulocytes that transmigrate through the epi-
thematous-squamous plaques that affect 2–3% of the popu-
dermis and Munro’s microabscesses underneath the stratum
lation. Psoriasis is characterized by marked changes in the
corneum1.
tissue architecture and simultaneous activation of different
The psoriasis area severity index (PASI) is used to determine
cell types including epidermal keratinocytes, vascular elem-
the severity of illness2–5. PASI is the most common method for
ents and leukocytes1,2.
evaluating the prevalence and severity of the disease in
Histopathological examination is important for psoriasis
psoriasis patients and their response to the treatment.
diagnosis and differential diagnosis. The psoriatic lesion is
However, it is a subjective method and can show differences
characterized by epidermal proliferation, acanthosis with
depending on the person who performs it. When used alone, it is
elongation of epidermal rete-ridges, marked hyperkeratosis,
criticized for its lack of reliability6. This makes objective
loss of the granular layer and parakeratosis, while the dermal
histopathological evaluations more important.
Treatment of the disease can vary based on the clinical
type, severity, duration and previous treatments of the disease;
Address for correspondence: Ayse Serap Karadag, MD, Department of as well as the localization, prevalence and severity of the
Dermatology, Istanbul Medeniyet University, School of Medicine, SB
Goztepe Training and Research Hospital, Istanbul, Turkey. E-mail: lesions. There are many local and systemic treatments. For
karadagaserap@gmail.com patients who have not benefited from local treatments,
DOI: 10.3109/15569527.2014.963598 Histopathological findings in skin biopsies with psoriasis 277

phototherapy is used in the first stage that is followed by The pre-treatment and post-treatment PASI values for the
conventional treatment methods such as acitretin, methotrex- patients were recorded and the averages for the groups were
ate and cyclosporine. However, a few studies in the literature calculated. The age, gender and PASI of the patients are
evaluate the responses to the treatment histopathologically. shown in Table 1.
In this study, we evaluated and compared skin biopsies
taken from patients with psoriasis before and after photother- Acitretin treatment
apy and therapy with acitretin and methotrexate.
Acitretin treatment was initiated at 10–25 mg/day in 1st
month, and increased to 20–35 mg per day in 2nd month
Materials and methods (average 0.25–0.40 mg/kg/d). They received this therapy
average six months. We performed punch biopsy before
We included 64 patients with a diagnosis of psoriasis vulgaris
treatment and after three months.
in our study. We performed phototherapy (narrowband
ultraviolet B, NB-UVB) on 33 patients (51.6%), while 19
Methotrexate treatment
patients (29.7%) were treated with methotrexate and 12
patients (18.8%) were treated with acitretin. The 64 patients in Methotrexate treatment was initiated at 10–15 mg/w at three
the study had chronic plaque psoriasis, and they had skin months. A pre-treatment biopsy was conducted on this group
lesions on more than 10% of their total body surface area and before treatment and after three months. They received
a score of PASI of 7.2–21.8 (average: 12.2) (Table 1). In the methotrexate therapy average 6–8 months.
third group treated with acitretin, we included patients whose
average PASI values were not statistically different. Age,
Phototherapy treatment
gender and severity of psoriasis were registered. The study
received local ethics committee approval with number of NB-UVB treatment started according to skin types (all
IMU/2014-0057, and written informed consent was obtained patient’s skin type 3 or 4) with 0.2–0.3 J/cm2 and increased
from all participating individuals. 20% by erythema response. All patients were treated three
The inclusion criteria were a clinical diagnosis of chronic days a week, and on the 30th clinical treatment, control
plaque psoriasis (i.e. lasting at least six months), age 418 biopsies of the patients were taken. For consistent delivery of
years, and the absence of systemic anti-psoriatic treatment for phototherapy, all patients were treated as inpatients in the
at least two months before inclusion in the study. The study same therapy cabin. Increasing (sub-erythogenic) doses of
group was selected from male and non-pregnant female UVB were delivered each day for an average treatment of
patients with psoriasis vulgaris. Females of childbearing age 27 days (the cumulative exposure to 2 UVB averaged
were included if they were using at least two separate and 4.7 J/cm2 per patient).
effective methods of birth control and had a negative serum For all patients, biopsies were taken typically from the
pregnancy test one week before the initiation of therapy in the abdominal or back region. The biopsies were taken from the
acitretin and methotrexate groups. same anatomic localization in the pre-treatment and post-
Patients who used vitamin supplements or who had any of treatment periods. A 3 mm punch biopsy material was used
the following problems were excluded from this study: for the biopsies, and two independent pathologists who were
sensitivity or allergy to parabens, recent history of psychiatric not informed about the treatment method evaluated all biopsy
disorders, pregnancy, cigarette smoking and previously materials. In both the pre-treatment and post-treatment
known diabetes mellitus. periods, the biopsy specimens of all patients were stained
with hematoxylene eosin and evaluated histopathologically.
Table 1. Histopathological criteria with significant recovery in all cases. Histopathological evaluation was carried out according to
12 criteria. For six of the criteria – parakeratosis, regular
Pre-treatment Post-treatment p acanthosis, Munro’s microabscesses, pustules of Kogoj,
Parakeratosisa 2.0 0.0 50.001 lymphocytes in the papillary dermis and neutrophils in the
Loss of granular layerb 77.4 16.1 50.001 papillary dermis – the histopathological changes were graded
Regular acanthosis* 2.5 1.0 50.001 between 0 and 3: 0 as none, 1 as mild, 2 as moderate and 3 as
Munro’smicroabscessa 2.0 0.0 50.001
severe. As the variability was high, these criteria were graded
Thinning in supra 80.6 24.2 50.001
papillary dermisb in four levels. The other six criteria – the loss of the granular
Spongiosisb 46.8 21.0 0.002 layer, suprapapillary thinning, spongiosis, dilated tortuous
Pustules of Kogoja 0.0 0.0 50.001 vessels in the papillary dermis, basal vacuolar degeneration
Dilated tortuous vessels 93.5 64.5 50.001
in the papillary dermisb
and melanophages in the papillary dermis – are graded as 0
Lymphocytes in the 2.0 1.0 50.001 for none and 1 for existing. As the variability of these criteria
papillary dermisa was low, they were graded as two levels.
Neutrophils in the 0.0 0.0 0.003 Statistical analyses were performed with SPSS-11 package
papillary dermisa
Melanophages in the 8.1 19.4 0.065 program (Chicago, IL). In normal distribution numeric
superficial dermis b variables, One-way ANOVA test was used while we used
b
Basal vacuolar degeneration 9.7 12.9 0.774 Kruskal–Wallis and Mann–Whitney U-test in parametric
PASIa 12.2 2.7 50.001 comparisons. Spearman correlation analysis was used in
a
Wilcoxon test (median values), bMcNemar’s test (values are the rate of correlation analysis and significance level was taken as
pathological findings) p50.05. p50.05.
278 S. Ozkanli et al. Cutan Ocul Toxicol, 2015; 34(4): 276–281

Results Table 2. Histopathological criteria with significant recovery in all

groups.
Pre-treatment histopathological evaluations were almost
similar in all three treatment groups, and there were no Pre-treatment Post-treatment p
significantly statistical differences (Table 1). Based on the a
Parakeratosis
cases, there were no differences among the groups in terms of NB-UVB 2.0 0.0 50.001
the histopathological evaluations. In the evaluations of the Methotrexate 2.0 0.0 0.004
Acitretin 2.0 0.0 0.012
cases after the treatment, parakeratosis, Munro’s abscesses, Loss of granular layerb
regular acanthosis, pustules of Kogoj, lymphocytes in the NB-UVB 75.0 12.5 50.001
papillary dermis, loss of the granular layer, spongiosis, Methotrexate 78.9 26.3 0.002
suprapapillary thinning, a decrease in dilated vessels in the Acitretin 81.8 9.1 0.008
Regular acanthosisa
papillary dermis (p50.001) and a decrease in the neutrophil NB-UVB 2 0.5 50.001
infiltration in the papillary dermis (p ¼ 0.003) were observed. Methotrexate 3.0 2.0 0.004
However, there was no difference compared to melanophages Acitretin 2.0 1.0 0.007
and basal vacuolar degeneration. Munro’s microabscessesa
NB-UVB 1.0 0.0 50.001
When evaluation was conducted on the treatment groups in Methotrexate 2.0 0.0 0.001
terms of changes in the histopathological criteria, it was Acitretin 2.0 0.0 0.005
observed that parakeratosis, Munro’s microabscesses, regular Thinning in suprapapillary
dermisb
acanthosis and loss of the granular layer significantly
NB-UVB 87.5 15.6 50.001
decreased. In addition to these findings, pustules of Kogoj, Methotrexate 73.7 36.8 0.065
lymphocytes in the papillary dermis, neutrophils in the Acitretin 72.7 27.3 0.063
papillary dermis, spongiosis, thinning in the suprapapillary Spongiosisb
NB-UVB 50.0 12.5 0.002
dermis and dermal papillary vascularization significantly Methotrexate 47.4 31.6 0.549
decreased in the phototherapy group (Table 2), while the pus- Acitretin 36.4 27.3 1.000
tules of Kogoj decreased in the methotrexate group (Table 2). Pustules of Kogoja
There was no significant difference among treatment NB-UVB 0.0 0.0 0.001
Methotrexate 1.0 0.0 0.007
groups in terms of recovery scores obtained by the difference Acitretin 0.5 0.0 0.084
of the arithmetical sum of numerical values belonging to Dilated tortuous vessels
the pre-treatment and post-treatment criteria (p ¼ 0.440; in the papillary dermisb
Kruskal–Wallis test) (Figures 1–3). NB-UVB 96.9 59.4 0.002
Methotrexate 94.7 73.7 0.125
Acitretin 81.8 63.6 0.625
Lymphocytes in the
Discussion papillary dermisa
NB-UVB 2.0 1.0 0.000
In our study, the biopsy specimens of all cases were evaluated Methotrexate 2.0 2.0 0.073
together, a significant decrease was observed in terms of Acitretin 1.5 1.0 0.408
Neutrophils in the
parakeratosis, Munro’s microabscesses, regular acanthosis, papillary dermisa
pustules of Kogoj, lymphocyte infiltration in the papillary NB-UVB 0.0 0.0 0.025
dermis, loss of the granular layer, spongiosis, suprapapillary Methotrexate 0.0 0.0 0.132
thinning, vascularity in the papillary dermis and neutrophile Acitretin 0.0 0.0 0.102
Melanophages in the
infiltration in the papillary dermis. On the other hand, no superficial dermisb
significant change after the treatment was seen in terms of NB-UVB 12.5 18.8 0.625
melanophages in the papillary dermis and basal vacuolar Methotrexate 5.3 26.3 0.219
degeneration. Acitretin 0.0 9.1 –
Basal vacuolar degenerationb
In psoriasis treatment, conventional treatment methods NB-UVB 15.6 9.4 0.687
such as phototherapy, acitretin, methotrexate and cyclospor- Methotrexate 5.3 21.1 0.375
ine, are used in both moderate and severe cases. The Acitretin 0.0 9.1 –
PASIa
effectiveness of these methods is evaluated clinically and
NB-UVB 11.3 3.0 0.000
according to histopathological parameters. Methotrexate 21.8 3.0 50.001
Skin irradiation with ultraviolet light in the 280–320 nm Acitretin 7.2 1.9 0.003
ranges (UVB) is one of the most common treatments for a
Wilcoxon test (median values), bMcNemar’s test (values are the rate of
extensive psoriasis and can produce long-term clinical pathological findings) p50.05.
remission7. The most efficient wavelength was found to be – Statistical evaluation could not be made.
313 nm and NB-UVB treatment was used in the studies. UVB
was anti-proliferative and cytotoxic toward T cells and
keratinocytes, but the T cells were ten-fold more sensitive. penetrate the epidermis, inhibited proliferation and induced
Photo-products formed by UVB effects stopped the progres- cytotoxicity or apoptosis in T cells to a much greater degree
sion of the cell cycle, increased prostaglandin release and than it did in keratinocytes7. In the studies, the recovery
made changes in cytokine release8–10. It primarily reversed effects of phototherapy were shown clinically and histopatho-
the infiltration of epidermal, rather than dermal and T cells. logically. Krueger et al. reported that after UVB, lesional
Studies showed that UVB, in doses that might be expected to psoriatic epidermis had markedly reduced acanthosis, the
DOI: 10.3109/15569527.2014.963598 Histopathological findings in skin biopsies with psoriasis 279

Figure 1. (A) Skin biopsies from patients with psoriasis. (B) After acitretin, decrease in parakeratosis, epidermal thickness, pustules of Kogoj, Munro’s
abscesses, infiltration of lymphocytes and dilated vessels in papillary dermis. (H&E  100).

Figure 2. (A) Skin biopsies from patient with psoriasis. (B) After methotrexate, decrease in epidermal thickness, munro’s abscesses, infiltration of
lymphocytes and dilated vessels in papillary dermis. (H&E  100).

Figure 3. (A) Skin biopsies from patient with psoriasis. (B) After phototherapy, decrease in epidermal thickness, Munro’s abscesses, infiltration of
lymphocytes and dilated vessels in papillary dermis. (H&E  100).
280 S. Ozkanli et al.
granular layer was typically restored and parakeratosis in the
stratum corneum was resolved, although this layer was thicker
than normal. Parakeratosis was not observed in the stratum
corneum of psoriatic lesions after phototherapy. Of note,
however, were the numerous mononuclear inflammatory cells
that remained present in the dermis of UVB-treated psoriatic
lesions. The latter finding parallels the known inability of
these doses of UVB to penetrate the dermis7. Coven et al.’s
study showed that epidermal acanthosis and epidermal
hyperplasia clearly recovered after NB-UVB treatment11.
In our study, a significant recovery was observed in ten
parameters in phototherapy treatment, which is compatible
with the literature. However, there was no significant change
in terms of melanophages in the superficial dermis on in the
basal vacuolar degeneration. Because of the effectiveness of
UVB on epidermis and the increase in epidermal thickness in
psoriasis, we think that the reason of this situation was the
wavelength that did not sufficiently reach dermis and basal
layer.
Methotrexate is a clinically proven medicine for chronic
plaque, localized and generalized pustular, and erythrodermic
forms of psoriasis. It has even been proven to be effective with
psoriatic arthritis2,3. The action mechanism of methotrexate is
immuno-suppressant effects, including the inhibition of T and
B lymphocytes, the suppression of pro-inflammatory cyto-
kines and the inhibition of the chemotaxis of neutrophils and
monocytes. In addition, methotrexate decreases DNA synthe-
sis and induces apoptosis in keratinocytes12,13. As the
pathogenesis of psoriasis involves an aberrant T-cell response,
the immune system is a possible target for the antipsoriatic
effects of methotrexate12–15. Eskiçırak et al. reported that
histopathological criteria, such as papillomatosis, paraker-
atosis, thickness of the epidermis, Munro’s abscesses, the
number of capillaries and the degree of lymphatic infiltration,
improved after methotrexate treatment16. When the biopsy
specimens of methotrexate-treated patients were analyzed, we
observed that, in accordance with the literature, there was
significant recovery in terms of parakeratosis, loss of the
granular layer, regular acanthosis, Munro’s microabscesses
and pustules of Kogoj. The median value of the histopatho-
logical scores decreased from 12 to 6 after methotrexate
treatment (p50.001); that is, it was observed to be effective
for recovery based on the histopathological findings. These
findings were similar to our findings.
Acitretin is a drug with proven efficacy in the treatment of
plaque, pustular, palmoplantar, guttate and erythrodermic
psoriasis3. One study reported that etretinate produced a 44%
decrease in epidermal thickness and a 62% reduction in
keratinocyte proliferation. The stratum corneum in resolving
psoriatic lesions was unusually thin, which was probably
caused by retinoid-induced shedding of corneocytes, and the
CD3+, CD8+ and CD25+ T-Iymphocyte subsets were
reduced by 50–65% in lesional tissue. Thus, etretinate
shows unexpected anti-inflammatory and pro-differentiation
actions in psoriasis17.
In biopsy evaluations of the acitretin-treated patients, due
to the mechanism that affects keratinocyte proliferation,
parakeratosis, loss of the granular layer and a decrease in
regular acanthosis were pointed out. In addition, due to
the anti-inflammatory effects, a significant recovery was
Cutan Ocul Toxicol, 2015; 34(4): 276–281
observed in Munro’s microabscesses. The median value of the
histopathological scores of the patients in this group
decreased from 12.5 to 4.
When our findings were examined, it was observed that
significant recovery was provided by phototherapy for 10
of 12 histopathological criteria, while methotrexate led
to significant recovery for five criteria and acitretin led to
significant recovery for four criteria (Table 2). However,
when evaluated in terms of histopathological recovery scores,
no significant statistical differences were found among the
three treatment groups.
Conclusion
In conclusion, we found in our study that conventional
treatment modalities provided histopathologically significant
recovery in psoriasis, but they did not have an effect on some
histopathological findings. Moreover, although there was a
difference in terms of clinical recovery rates, there were no
significantly statistical differences in terms of the histopatho-
logical parameters. In this case, it was seen that all the drugs
led to a histopathological recovery similar to the clinical
recovery.
Furthermore, our study showed that NB-UVB treatment,
which is slightly more reliable in terms of side effects because
of its similar effects to the histopathological parameters and
clinical recovery, could be preferable as a first step. In
addition, there were no significant changes detected with the
melanophages in the superficial dermis or the basal vacuolar
degeneration in any of the treatment groups. These two
criteria are considered to be related to non-specific psoriasis.
To our knowledge, it is one of the few studies to assess
these parameters in psoriasis under the continuous effect of
acitretin, methotrexate and phototherapy for three months.
There is a need for studies with larger series to examine the
histopathological effects of these treatment modalities in
terms of immunopathology.
Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of this
article.
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