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To cite this article: H. El Sayed, S. Shalaby, M.R.E. Abdel-Halim, D M. Aboelfadl & N. Samir
(2019): Efficacy of Doxycycline in the Treatment of Early Stages of Mycosis Fungoides, a
Randomized Controlled Trial, Journal of Dermatological Treatment
Key words: Randomized controlled trial, Mycosis fungoides, Doxycycline, Apoptosis, Bcl-2
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Abstract: 198
Manuscript: 3445
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Number of figures: 2
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Number of tables: 4
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Authors: H. El Sayed*, MBBCh, MSc; S. Shalaby*, MD; M.R.E. Abdel-Halim*, MD, Dip
Hospital.
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Corresponding author:
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Suzan Shalaby, MD
Email:suzyandnls@gmail.com
No funding was received for this work
Abstract
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Background: Mycosis fungoides is the most common type of primary cutaneous T cell
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lymphomas. Doxycycline promoted apoptosis in different human malignant cell lines and in-vivo
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models.
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Objectives: To test for the therapeutic efficacy of doxycycline in comparison to PUVA in early
PUVA (3 weekly sessions) (n=18) for 12 weeks. The primary outcome (therapeutic efficacy)
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was defined in terms of objective response rate (ORR) which was measured according to
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PUVA (11.1%, 50% respectively, p= 0.016). The percent reduction in mSWAT, CAILS,
histopathology score and CD3 expression was significantly less in the doxycycline group
(p=0.001, p=0.001, p˂0.001, and p=0.004 respectively). Within the doxycycline group, changes
in mSWAT and CAILS showed no correlation with changes in the CD3 or Bcl-2 expression.
Gastric upset was significantly more encountered in the doxycycline group (p=0.001).
Conclusion: Doxycycline is not suitable as a sole agent in the treatment of early stages of classic
Introduction
Mycosis fungoides (MF) is the most common form of primary cutaneous T cell
lymphomas (CTCLs). The widely accepted etiopathogenic theory of MF implies that chronic
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antigenic stimulation renders T cells resistant to apoptosis. The NF-κB pathway which induces
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the antiapoptotic protein Bcl-2 was found to be activated in CTCLs and many alterations in
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apoptosis regulating genes (p53, Fas and Bcl-2) have been reported in patients with MF [1-4].
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Despite the indolent behavior of early disease stages (stages IA-IIA), prompt treatment is
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essential to prevent progression to more aggressive forms, where the median survival drops from
10-35 years in early stages to only 1.5 years in advanced stages [5,6]. Skin directed therapy is the
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standard approach for treating early stages. It includes topical steroids, phototherapy, topical
nitrogen mustard, topical bexarotene, local radiation and total skin electron beam therapy
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(TSEBT). Refractory early stage patients are candidates for combination therapies; phototherapy
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Avoiding long term side effects especially in younger patients, and in those receiving
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repeated cycles of treatment is the main concern when choosing a therapeutic modality [8]. This
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has triggered the continuous search for safer, more tolerable and effective therapies directed
affordable and generally well tolerated with a low side effect profile [10]. In addition to its
antimicrobial action, doxycycline exerts also many non-antimicrobial effects, the most important
of which are the anti-inflammatory effects exerted by vast mechanisms mostly through inhibition
Since 2002, many researchers have been shedding light also on the anti-cancer effects of
radio-sensitizing and quorum sensing inhibitory functions. Its selective toxicity on cancer cells
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researches, whether in solid malignancies or in hematological malignancies (including
leukemias, diffuse large B cell lymphoma (DLBCL), MF and Sézary Syndrome), are still
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experimental studies conducted on different human malignant cell lines or in vivo models [13-
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26].
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A recent phase II study was reported where doxycycline was used to treat canine multi-
centric B cell lymphoma [27]. The only available reports in literature where doxycycline was
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used to treat humans with lymphoma included a case of primary cutaneous CD4+ small- to
medium sized pleomorphic T cell lymphoma which achieved complete remission after receiving
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200mg doxycycline for 6 weeks and cases of ocular adnexal lymphoma where almost two thirds
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of the patients remained progression free 5 years after treatment with one or two cycles of
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Accordingly, this study aimed to test for the therapeutic efficacy of doxycycline in
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comparison to PUVA in early stages of classic MF and its effect on the apoptosis of T
This randomized controlled trial (RCT) was conducted at the Dermatology Out Patient
Clinic (OPC), Dermatology Department, Faculty of Medicine, Cairo University (Kasr Al Aini
University Hospital) during the period from January 2017 to July 2018. It was approved by the
Department Scientific and Ethical Committee Boards and informed consents were signed by all
participants.
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Recruitment and enrollment
Forty adult patients with MF presenting consecutively to the Dermatology OPC were
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assessed based on certain inclusion and exclusion criteria for their eligibility to be enrolled in the
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study. Assessment parameters included: medical history [history of relevant allergies,
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photosensitivity, medical illnesses and drug history], type of MF skin lesions [patches, plaques or
tumors], body surface area (BSA) affected (rule of 9) [30], clinical variant of MF,
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histopathological variant of MF [provided that diagnostic biopsies were taken within a maximum
of 30 days, otherwise, recent biopsies were taken to document the current histopathological
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state/variant of the disease], staging work up [complete blood count (CBC), cervical, axillary and
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inguinal lymph node ultrasound, lymph node biopsy (if indicated), chest X-ray and abdomino-
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pelvic ultrasound] and laboratory work-up [liver function tests (AST, ALT, bilirubin and
albumin), kidney function tests (urea and creatinine) and pregnancy test for females in the child
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bearing period].
Included in the study were adults (age above 18) of both sexes with early stage MF (IA-
IIA). Excluded from the study were patients with any clinical or histopathological variant of MF
other than the classic variant, advanced stages of classic MF (IIB, III or IV), pregnant or
lactating females, patients with autoimmune diseases, patients with solid or hematological
malignancies and patients with any contraindications for doxycycline, phototherapy or psoralens.
patients were enrolled in the therapeutic phase. A wash out period of at least 4 weeks from any
relevant topical or systemic treatment that can affect the course of MF was adopted. Females in
the child bearing period were stressed upon keeping a reliable method of contraception.
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Randomization
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All 36 patients were randomly allocated with 1:1 ratio, using blinded cards, into either
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one of the two parallel arms of the trial: active treatment group (18 patients) who received
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Doxycycline 100 mg oral capsules (twice daily) or a control group (18 patients) who received
according to the degree of erythema and based on the guidelines of the Phototherapy Unit,
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Hospital]. No concomitant treatment was allowed except for Vaseline topical application (if
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needed).
End of study
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At both day 0 and at EOS, all 36 randomized patients were clinically assessed by a
blinded senior investigator using standardized clinical scoring systems for MF which included:
modified severity weighted assessment tool (mSWAT) and composite assessment of index lesion
severity (CAILS) [31]. Moreover, pruritus was assessed using a visual analog scale from 0-10,
where 0=no pruritus and 10=worst imaginable pruritus and patients’ satisfaction was assessed
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at EOS.
Slides and paraffin blocks of the pre-treatment diagnostic biopsies of the 36 randomized patients
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were retrieved. At EOS, another 4mm punch biopsy was taken close to the site of the original
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biopsy. Biopsied lesions were ensured to be among the 5 lesions scored in the CAILS. Biopsies
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were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin and
stained with hematoxylin and eosin (H&E) [33]. Additional sections were obtained for
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immunohistochemical staining using rabbit monoclonal antibody against CD3 (catalog number
BSB 790-4341, Ventana, USA) and mouse monoclonal antibody against Bcl-2 (catalog number
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790-4464, Ventana, USA). Automated immunohistochemical staining was carried out using
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BenchMark XT autostainer (Ventana Medical Systems, Inc. Tucson, Arizona, USA) and
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Pre-treatment and post-treatment H&E stained slides were scored according to the
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sections were analyzed using the Leica Qwin 3.5.1 2008 Image Analyzer (LEICA Imaging
Patients were followed up at week 4 and week 8 where mSWAT, CAILS and pruritus
scoring were done. In addition, any possible side effects were assessed, reported and managed
accordingly.
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The primary treatment outcome at EOS was to compare the therapeutic efficacy of
doxycycline with PUVA. This was defined in terms of the objective response rate (ORR) and
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was measured according to changes in mSWAT from baseline (Table 1) [31].
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Secondary treatment outcomes at EOS was to compare the effect of doxycycline with
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PUVA as regards time to response (TTR) [the time from the first dose till the patient first met a
50% decrease in mSWAT] as well as regards changes in CAILS, pruritus score, DLQI score,
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H&E score of MF, CD3 count and Bcl-2 count.
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Patients with progressive disease were shifted to PUVA, patients with partial response
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continued the same dose for another 12 weeks while patients with stable disease received 400mg
daily for another 12 weeks. At the end of the extended treatment period, patients with the same
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Power calculation
A statistical power analysis was performed for sample size estimation. According to
Cohen's criteria [35], the effect size was arbitrarily selected at d=1 due to absence of prior studies
or pilots. With a two tailed alpha error probability α set at 0.05, a sample size of 36 with 18 per
group was calculated. To be able to reject the null hypothesis that the difference is zero,
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probability power was estimated at 0.8 using Mann Whitney test for independent non-parametric
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Statistical analysis
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Data were coded and entered using the statistical package SPSS (Statistical Package for
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the Social Sciences) version 25. For quantitative data, data were described using mean and
standard deviation (SD) if homogenous (normally distributed) and median and range if non-
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homogenous (non-normally distributed). Categorial data were presented using frequency (count)
groups were done using the student t-test in homogenous data and the non-parametric Mann-
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Whitney test in non-homogenous data. For comparisons of serial measurements within each
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patient, paired t-test was used in homogenous data and the non-parametric Wilcoxon signed rank
test was used in non-homogenous data [36]. For comparing categorical data, Chi square (χ2) test
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was performed. Exact test was used instead when the expected frequency is less than 5 [37].
Correlations between quantitative variables were done using Spearman correlation coefficient
intention to treat analysis (ITT)] and secondary treatment outcomes [based on per protocol
Results
Patient's flow diagram is presented in Fig. 1 [39]. At baseline, both groups were
statistically homogenous apart from gender distribution and CD3 count (Table 2).
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Results of the RCT
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Evaluation of primary treatment outcome, percent change in mSWAT and mSWAT at EOS
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Objective response rate was significantly less in the doxycycline group than in the PUVA
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group (p= 0.016) with partial response achieved in 11.1% and 50% of the cases respectively (Fig.
2). Complete response was not achieved in either group. Although the percent reduction in
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mSWAT score in the doxycycline group was significantly less than in the PUVA group
(p=0.001), the mSWAT score at EOS showed no significant difference between both groups
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The percent reduction in CAILS score in the doxycycline group was significantly less
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than in the PUVA group (p=0.001) with significantly higher CAILS score at EOS (p=0.017)
(Table 3).
The percent reduction in H&E score of MF and CD3 count in the doxycycline group was
significantly less than in the PUVA group (p˂0.001 and p=0.004 respectively) with significantly
higher H&E score and CD3 count at EOS (p=0.003 and p ˂0.001). A significant difference
existed between both groups as regards the percent change in Bcl-2 expression (p=0.012), with
significantly higher Bcl-2 count at EOS in the doxycycline group (p=0.031) (Table 3).
Adverse events
Adverse events were mild and did not necessitate discontinuation of therapy in either
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group. Adverse effects, mainly gastric upset, were significantly more encountered in the
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doxycycline group (p=0.001) (Table 3).
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Further analysis within the doxycycline group
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Within the doxycycline group, mSWAT, CAILS, pruritus score, DLQI score and H&E
score of MF were reduced significantly from baseline (p=0.013, p=0.001, p=0.001, p=0.01, and
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p=0.007 respectively) (Table 4).
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The percent reductions in mSWAT and CAILS showed no significant correlation with the
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percent changes in the H&E score of MF (p=0.948; r=-0.017, p=0.581; r=0.139 respectively),
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CD3 count (p=0.906; r=-0.03, p=0.839; r=0.052) or Bcl-2 count (p=0.072; r=-0.447, p=0.194;
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r=-0.331).
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Two patients with partial response at EOS continued the same dose of doxycycline for
another 12 weeks. No further improvement was achieved and one developed new lesions. Both
were shifted to PUVA. Fourteen patients with stable disease at EOS received doxycycline (400
mg daily) for another 12 weeks. Three didn’t show any further improvement and 2 developed
new lesions. All 5 patients were shifted to PUVA. The 9 remaining patients were lost to follow-
up.
Discussion
With partial response rate of 11.1% compared to 50% in the PUVA group, it is obvious
that doxycycline can't substitute PUVA in treating early stages of MF. Although TTR showed no
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significant difference between both groups, the percent reduction in mSWAT and CAILS, was
significantly less in the doxycycline group (-11% and -20%) compared to PUVA (-45% and -
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47%).
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Although doxycycline has caused significant reduction in both mSWAT and CAILS
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scores from baseline, the percent reduction in CAILS score (-20%) was higher than the percent
reduction in mSWAT (-11%) indicating more influence on index lesion skin score than on the
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actual disease extent. The CAILS score includes assessment of erythema, scaling and
pigmentation in addition to lesion elevation and size. It appears that the mild improvement
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achieved by doxycycline reflects mainly some effects on various parameters of this score.
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Angiogenesis has been found to be increased in lesional skin of early MF compared to normal
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skin reflecting the degree of erythema perceived in MF lesions [41]. Doxycycline has a well-
known anti-angiogenic effect through its inhibitory effect on vascular endothelial growth factor
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Moreover, the inhibitory effects of doxycycline on inflammatory cytokines and MMPs may also
skin where it mediates degradation of the extracellular matrix and basement membrane
(-47% and -45% respectively) reflecting its potent multi-target effect in MF thus it retains its
position as the cornerstone in the treatment of early disease stages [46]. It has achieved complete
remission rates of 90%, 76% and 78% in stages IA, IB and IIA respectively [47].
clinical trials of MF [31], we assessed, as secondary outcomes, changes in the H&E score of MF,
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CD3 and Bcl-2 counts in an attempt to study the effect of doxycycline on apoptosis of T cells in
comparison to PUVA. With 3% and 8% reductions in the H&E score and CD3 count in the
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doxycycline group compared to 88% and 65% in the PUVA group respectively, one can clearly
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conclude that doxycycline has a very weak T cell apoptotic effect in early stage MF patients.
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Within the doxycycline group, the H&E score was reduced significantly from baseline to EOS,
however the CD3 count showed no significant difference. The H&E score used in this study
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assesses several parameters, one of them includes the density of the dermal infiltrate. Reactive
inflammatory cells are known to be present in the dermal infiltrate of MF especially in early
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stages and may include B cells and other non-T cells [48,49]. Accordingly, doxycycline via its
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anti-inflammatory effect might have minimally affected the density of the dermal infiltrate in
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general rather than significantly inducing T cell apoptosis. The superior effect of PUVA in
reducing the H&E score and CD3 count can be explained by the multiple mechanisms by which
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In the current RCT, a significant difference existed between the doxycycline group and
the PUVA group as regards the percent change in Bcl-2 count with Bcl-2 increasing in the
doxycycline group and decreasing in the PUVA group. The decreased expression by PUVA is
consistent with the results of other studies which explained that this Bcl-2 reduction is an
important pathway of induction of cell death through loss of the anti-apoptotic proteins [52]. The
increased expression of Bcl-2 in the doxycycline group, although not significant, may explain the
resistance to apoptosis of T cells which was manifested by the lack of significant reduction in the
CD3 count within the doxycycline group and the significantly less percent reduction compared to
PUVA.
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apoptotic agent in malignancy. The only previous study assessing the apoptotic effect of
doxycycline in CTCLs was an in vitro study conducted on multiple different cell lines from
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patients with MF and Sézary syndrome [26]. They found that doxycycline achieved a significant
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dose dependent apoptosis of T cells through inhibiting TNF induced NF-κB activation and
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through reactive oxygen species. It appears that what is achieved in an in vitro setting cannot be
expected to be the same inside the human body where many players interact together creating a
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completely different milieu. Also, the stage of the disease is a crucial factor, where the blood
resident T cell lymphocyte subtypes of late stage MF might be more vulnerable to the
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doxycycline apoptotic actions than those of the early stage skin disease.
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apoptotic injury [53]. Previous work on neu2ral tissue suggested anti-apoptotic actions of
tetracyclines attributed to increased Bcl-2 in certain conditions of tissue injury [54]. These
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studies might advocate that doxycycline can activate both apoptotic and anti-apoptotic pathways
We found no significant correlation within the doxycycline group between the percent
change in the clinical scores (mSWAT and CAILS) and the percent change in the H&E score,
CD3 or Bcl-2 counts, further supporting the idea that the minimal doxycycline achieved effects
in early stage MF in the current study are mainly mediated through its anti-inflammatory effect
From patients' symptoms and psychological perspective, the current study showed that
the overall patient satisfaction (pruritus and DLQI scores) with doxycycline was comparable to
that of PUVA. Doxycycline was a more feasible mode of treatment to most patients compared to
PUVA therapy which necessitated 3 weekly visits to the phototherapy unit. On the other hand,
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the doxycycline associated side effects in the form of GIT upset and nausea were significantly
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The choice of the 200 mg daily dose in the current study was based on the standard
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guidelines for acne [55]. According to data available from a registered clinical trial
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NCT02341209,53677 where a 400mg daily dose of doxycycline is given for 5months-1year for
relapsed cases of CTCL (results not available yet), patients with partial response in the current
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study were given doxycycline 200mg daily for another 3 months and patients with stable disease
were given doxycycline 400mg daily for another 3 months. However, neither dosage
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The lack of satisfactory therapeutic efficacy in the current study compared to the
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cell lymphoma, after 6 weeks of 200 mg doxycycline daily therapy can be explained by the
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current understanding of this type of T cell proliferation which was downgraded in the current
actual lymphoma based on limited clinical risk, localized disease, and similarity to clonal drug
reactions [28,56]. And so, doxycycline seems to have acted mainly in this case as an anti-
inflammatory agent.
Moreover, the satisfactory effect of doxycycline in treating cases of ocular adnexal
lymphoma is explained by the fact that most of these cases are marginal zone lymphomas
associated with Chlamydophila psittaci infection for which doxycycline has been suggested as a
In conclusion, doxycycline can't be used as a sole agent in the treatment of early stages of
MF since it has achieved significantly less therapeutic efficacy compared to PUVA in a 3 months
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treatment duration. Changes achieved are mainly attributed to its anti-inflammatory effects. The
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small number of patients and short treatment duration were limitations in this study. Moreover,
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the potential risk of disease progression while on an experimental treatment prevented us from
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trying doxycycline for periods longer than 6 months. The value of using doxycycline as an
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Fig. 1: Patient's flow diagram at EOS according to CONSORT guidelines for reporting RCTs.39
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Fig. 2: A patient in the doxycycline group achieving partial response at EOS
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New tumors (T3) in patients with T1, T2 or T4
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only skin disease or Loss of response: in those
Progressive disease
with complete or partial response, increase of
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skin score of greater than the sum of nadir plus
50% baseline score
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Any disease recurrence in those with complete
Relapse
response
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Table 2: Baseline characteristics in doxycycline and PUVA groups before therapy§
Range 1 - 26 2 - 25
Duration (years) 0.051
Median 5 9.50
Disease Ia 3 (16.7%) 2 (11.1%)
stage
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Ib 13 (72.2%) 14 (77.8%) 1
Number
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(%) IIa 2 (11.1%) 2 (11.1%)
Range 5 - 92 7 - 78
mSWAT 0.563
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Median 36.5 46
CAILS Mean±SD 54.83 ± 16.35 56.5 ± 17.2 0.913
Range 0-8 0-5
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Pruritus score 0.265
Median 2.5 2
Range 0 - 22 2 - 19
DLQI score 0.791
Median 6an 7
H&E score of MF Mean±SD 5.97 ± 0.88 6.61 ± 1.4 0.203
Range 228 - 924 46 - 803
CD3 count/5HPF ˂0.001*
Median 713 356.5
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Range 0-273 2-217
Bcl-2 count /5HPF 0.433
Median 67 54
§
Data is presented as mean±SD if normally distributed and as range and median if non-normally
distributed; *p<0.05 is significant M: Males, F: Females; mSWAT: Modified Severity Weighted
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Assessment Tool; CAILS: Composite Assessment of Index Lesion Severity; DLQI: Dermatology
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Life Quality Index; H&E: Hematoxylin and Eosin; HPF: High Power Field.
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Table 3: Summary of the results of the RCT at EOS§
Doxycycline PUVA group
P value
group (n=18) (n=18)
Complete response 0 (0%) 0 (0%)
Partial response 2 (11.1%) 9 (50.0%)
ORR Stable disease 14 (77.8%) 9 (50.0%) 0.016*
Number (%) Progressive disease 2 (11.1%) 0 (0%)
Relapse 0 (0%) 0 (0%)
Percent change Range -51.1 - 61.3 -82.2 - (-10.2)
0.001*
in mSWAT score at EOS Median -11.05 -44.95
mSWAT
Range 6.5 - 83 5 - 70
mSWAT at EOS 0.389
t
Median 29 23
ip
TRR (weeks) Mean±SD 12 ± 0 10.22 ± 2.11 0.436
Percent change Range -67.1 - 16.1 -81 - (-11.1)
0.001*
cr
CAILS in CAILS score at EOS Median -20.3 -46.85
CAILS at EOS Mean±SD 42.28 ± 10.94 32.22 ±15.78 0.017*
us
Percent change Range -100 - 0 -100 - 50
0.719
Pruritus in pruritus score at EOS Median -48.55 -80
score Range 0-4 0-3
Pruritus score at EOS 0.481
Medianan 0 0
Percent change Range -83.3 - 66.7 -100 - 0
0.265
in DLQI score at EOS Median -26.1 -38.1
DLQI score
Range 0 - 12 0 - 14
M
DLQI score at EOS 0.355
Median 5 4
Percent change Range -85.7 - 0 -100 - 0
˂0.001*
H&E score in H&E score at EOS Median -3.85 -88.25
d
§
Data is presented as mean±SD if normally distributed and as range and median if non-normally
distributed, *p<0.05 is significant ; ORR: Objective Response Rate; mSWAT: Modified Severity
Weighted Assessment Tool; CAILS: Composite Assessment of Index Lesion Severity; DLQI:
Dermatology Life Quality Index; H&E: Hematoxylin and Eosin; HPF: High Power Field
§
Table 4: Comparison between baseline and EOS values within the doxycycline group
Doxycycline (baseline) Doxycycline (at EOS) P value
(n=18) (n=18)
Range 5 - 92 6.5 – 83
mSWAT 0.013*
Median 36.5 29
CAILS Mean±SD 54.83 ± 16.35 42.28 ± 10.94 0.001*
Range 0-8 0-4
Pruritus score 0.001*
Median 2.5 0
Range 0 - 22 0 - 12
DLQI score 0.01*
Median 6 5
H&E score of
Mean±SD 5.97 ± 0.88 4.67 ± 1.75 0.007*
MF
t
CD3 Range 228 - 924 123 - 1171
ip
0.372
count/5HPF Median 713 548.5
Bcl-2 Range 0-273 0 - 411
0.478
cr
count/5HPF Median 67 81.5
§
Data is presented as mean±SD if normally distributed and as range and median if non-
normally distributed; *p<0.05 is significant ; mSWAT: Modified Severity Weighted Assessment
us
Tool; CAILS: Composite Assessment of Index Lesion Severity; DLQI: Dermatology Life
Quality Index; H&E: Hematoxylin and Eosin; HPF: High Power Field
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