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Quelhas da Costa R, Aguirre-Alastuey ME, Isenberg DA, Saracino AM. Assessment of Response to B-Cell Depletion Using Rituximab in Cutaneous Lupus Erythematosus. JAMA Dermatol. 2018;154(12):1432–1440. doi:10.1001/jamadermatol.2018.3793
What is the response to B-cell depletion therapy with rituximab in severe active cutaneous lupus erythematosus among patients with systemic disease?
This cohort study of 50 patients with cutaneous lupus erythematosus reports a complete clinical response of the cutaneous manifestations of systemic disease to rituximab of 20 patients (40%) at 6 months and 24 (52%) at 12 months. Acute cutaneous lupus erythematosus and nonspecific lupus erythematosus showed better clinical responses, whereas only 2 of 6 (33%) with subacute disease at both time points and 5 of 12 (42%) and 5 of 11 (46%) with chronic disease demonstrated a complete clinical response at 6 and 12 months, respectively.
Study findings suggest that B-cell depletion therapy using rituximab may be effective in treating severe active cutaneous lupus erythematosus in some patients with systemic disease, especially those with acute and nonspecific types.
Cutaneous lupus erythematosus (CLE) can be severe and treatment resistant. B-cell depletion therapy (BCDT) with rituximab is well recognized in organ involvement in systemic lupus erythematosus (SLE), but its efficacy in cutaneous manifestations is less well established.
To evaluate the outcomes of BCDT in CLE and its clinical subtypes in the setting of associated SLE.
Design, Setting, and Participants
This single-center, retrospective, cohort study was performed at the adult tertiary referral Rheumatology Department of University College London Hospital, London, United Kingdom, from January 1, 2000, through March 31, 2016, with 12-month follow-up completed on March 31, 2017. Adult patients with carefully classified CLE and mucocutaneous British Isles Lupus Assessment Group (BILAG) grade A or B who were treated with rituximab BCDT were selected from a prospective database of 709 patients with SLE. Data were analyzed from April through December 2017.
Main Outcomes and Measures
Clinical response was examined at 6 and 12 months after treatment for CLE and its subtypes acute CLE (ACLE), subacute CLE (SCLE), chronic CLE (CCLE), and nonspecific LE (NSLE). A complete response was defined as achieving BILAG grade D; partial response, BILAG grade C; stable disease, no change; and disease flare, change from BILAG grade C or D to grade A or B.
A total of 50 patients with SLE were eligible for inclusion; mean (SD) age at diagnosis was 26.9 (12.1) years, and 49 (98%) were women. Twenty-one patients had ACLE; 6, SCLE; 10, CCLE; and 11, NSLE (including 2 with concurrent ACLE and CCLE). Overall, at 6 months, 38 patients (76%) improved their mucocutaneous BILAG grade A or B status, including 20 (40%) with a complete response. At 12 months, 28 of 46 patients (61%) maintained this response, including 24 (52%) with a complete response. Two of 6 patients (33%) with SCLE showed a complete response at 6 and 12 months. Five of 12 patients (42%) with CCLE showed a complete response at 6 months, and 5 of 11 (45%), at 12 months. Fifteen patients (30%) required further rituximab therapy within 12 months for cutaneous involvement.
Conclusions and Relevance
B-cell depletion therapy using rituximab appears effective in patients with SLE and severe active CLE; however, outcomes are variable in those with SCLE and CCLE subtypes.
Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune rheumatic disease. Cutaneous manifestations are frequent, occurring in as many as 80% of cases.1,2 The clinical heterogeneity of cutaneous lupus erythematosus (CLE) is well recognized. Four major subtypes are defined according to the modified Gilliam grouping system.3 Many clinical phenotypes can present within each of these major subtypes. Discoid LE (DLE) is the most common form of chronic CLE (CCLE), and as many as 25% of those with SLE have discoid lesions.4 Lupus erythematosus tumidus (dermal lupus) is now often separately subcategorized as intermittent CLE, rather than CCLE, owing to its fluctuating clinical course. Nevertheless, CLE can be severe, widespread, and treatment resistant, with potential scarring and a negative effect on quality of life.
Of relevance, possible immunopathogenic variability between clinical CLE subtypes is increasingly highlighted. B-cell–related mechanisms are likely implicated in at least some skin lesions, given their role in SLE; however, non–B-cell pathways are also recognized.5-13 For example, keratinocyte apoptosis and associated production of cytokines and chemokines is well recognized, predominant T-cell infiltrates are described, and plasmacytoid dendritic cells also appear important in some CLE subtypes.6-11 Clinically, less autoantibody production (20% in DLE) and a weaker association of CCLE with SLE (6%-28%) also support the notion of variable underlying pathogenic pathways in different clinical scenarios.3,14,15
Treatment of CLE commonly focuses on strict photoprotection, topical therapies, systemic corticosteroids, antimalarials, or broadly immunosuppressive corticosteroid-sparing agents. More recently, treatment with biologic agents targeting B cells, such as rituximab, have shown efficacy in many inflammatory conditions, including SLE. Although the 2 major clinical trials of rituximab in SLE (Exploratory Phase II/III SLE Evaluation of Rituximab [EXPLORER]16 and Lupus Nephritis Assessment With Rituximab [LUNAR]17 studies) failed to prove its efficacy, this failure was postulated as likely owing to poor trial design. In contrast, numerous observational studies have indicated its efficacy,18,19 particularly in renal,20,21 musculoskeletal, and hematologic manifestations.22 However, the value of B-cell depletion therapy (BCDT) in treatment of the cutaneous manifestations of LE remains poorly described and unclear.23,24 The literature is limited to case reports and observational studies, usually with only short-term follow-up. Furthermore, some published treatment protocols have included cyclophosphamide (concurrent with rituximab) and others have not. Some authors have indicated positive outcomes in ACLE and SCLE,25 perhaps supporting the concept of shared B-cell immunopathogenesis with SLE. However, responses are highly variable (Table 1),18,25-34 and some cases of possible rituximab-induced flares of SCLE and CCLE are described.33 Efficacy in the case of CCLE appears especially unpredictable, ranging from no response in some observational studies33,34 to 37.5% responders in another.25 Such inconsistent reported responses and documented disease flares may provide further indication of unique immunogenic variability within and between subtypes of CLE, even among SLE cohorts.
To help address these issues, we report, to our knowledge, one of the largest cohorts of patients with SLE and severe mucocutaneous involvement treated with rituximab BCDT. We assess clinical response overall and according to CLE subtype (with a unique focus on the less well-studied SCLE and CCLE forms) at 6 and 12 months after treatment.
This retrospective observational study included patients with SLE treated with rituximab at the adult tertiary referral Rheumatology Department of the University College London Hospital (UCLH), London, United Kingdom, from January 1, 2000, through March 31, 2016. Patients were enrolled through the UCLH Rheumatology database, which contains a prospective record of clinical, serologic, and treatment variables. Patients were included in the analysis if they were older than 18 years, had a diagnosis of SLE according to the American College of Rheumatology revised criteria,35 and had severe manifestations of cutaneous involvement (classified according to the modified Gilliam grouping system3) with a mucocutaneous British Isles Lupus Assessment Group (BILAG) grade A or B (see the paragraph that follows for definition of these grades), requiring treatment with BCDT using rituximab. No institutional review board approval was requested because this study was considered an audit and UCLH does not require formal research study approval or informed consent for audits.
Cutaneous involvement was initially assessed in a combined dermatology-rheumatology clinic and then retrospectively reviewed by one of our dermatologists (A.M.S.) according to clinical and/or histologic findings recorded immediately before the administration of rituximab. The BILAG 2004 system was used to classify the severity of skin involvement,5 where grade A denotes severe disease, requiring more than 20 mg of prednisolone or equivalent and/or commencing or increasing immunosuppressive treatments; grade B, moderate disease, requiring lower doses of immunosuppression (eg, <20 mg of prednisolone); grade C, mild persistent activity, requiring symptomatic therapy only; grade D, past disease, with no current activity; and grade E, the organ system has never been affected.
B-cell depletion therapy was administered according to the protocol at the time of treatment; until 2007, this protocol consisted of a 2-week spaced intravenous administration of two 1-g doses of rituximab, both followed the day after by an infusion of 750 mg of cyclophosphamide; from 2007 onwards, 1 infusion of 750 mg of cyclophosphamide was given on the day after the first rituximab infusion only.19 Each patient was followed up every 2 to 3 months for at least 12 months after rituximab administration. Follow-up was completed on March 31, 2017.
The primary outcome of the analysis was overall cutaneous response to BCDT at 6 and 12 months. The secondary outcome was the cutaneous response according to different CLE subtypes (ACLE, SCLE, CCLE, and NSLE) at 6 and 12 months. We also assessed those patients requiring additional cycles of rituximab during the 12-month follow-up.
Cutaneous treatment responses were defined as a complete response when the BILAG grade A or B reduced to D, partial response when the BILAG grade A or B improved to C, a stable disease when no improvement in BILAG grade A or B was seen, or a flared disease when cutaneous involvement worsened (BILAG grade of C or D worsened to A or B) at 6 or 12 months. Those patients requiring a further BCDT cycle within the 12-month follow-up were considered separately. Indications justifying an additional BCDT cycle were maintaining stable disease at 3 to 6 months; having a cutaneous disease flare (worsening of the BILAG grade C or D to A or B); or having a new systemic disease flare without worsening cutaneous involvement. The effect of BCDT on organ systems other than mucocutaneous was not investigated in this study.
Data were analyzed from April through December 2017. Statistical analysis was performed using SPSS software (version 25; IBM Corporation). Continuous variables with normal distribution were described as mean (SD). Dichotomous variables were described as the number of positive events (percentage of available data). Nondichotomous categorical variables were described by the number of events (percentage of available data) on each category. Confidence intervals were calculated using the Poisson based method for counts, and 2-sided P < .05 was deemed significant.
From an overall cohort of 709 patients with SLE, 150 were treated with rituximab, of whom 57 had a mucocutaneous BILAG grade of A or B. Six patients were excluded owing to unclassifiable dermatological manifestations and 1 owing to previous inclusion in a randomized clinical trial for anti–B-cell activating factor monoclonal antibody. Fifty patients remained for inclusion (49 women [98%] and 1 man [2%]; mean [SD] age at diagnosis, 26.9 [12.1] years). Table 2 describes further demographic and clinicopathological characteristics.
Regarding CLE subtypes, ACLE was most prevalent (21 [46%]). Six patients had SCLE features (12%), 10 had CCLE (24%), and 11 had NSLE (22%). Of note, 2 patients satisfied ACLE and CCLE criteria (one with generalized DLE and the other with localized DLE) and were included in both subtypes in the treatment response evaluation; there were no differences in responses between their 2 CLE subtypes. Of the 10 patients with CCLE alone, 7 had generalized DLE (affecting the head, neck and trunk, and/or limbs), 1 had localized DLE (affecting the head and neck only), and 2 had lupus panniculitis. Disease severity before rituximab treatment was described as a mucocutaneous BILAG grade A in 15 patients (30%) and as grade B in 35 patients (70%).
Forty patients were rituximab naive. Ten patients had received previous treatment with rituximab (7 [14%] with 1 cycle, 2 [4%] with 2 cycles, and 1 [2%] with 3 cycles); of these, 9 were treated for noncutaneous involvement and 1 was treated for cutaneous involvement as a child. Only 2 patients had been treated within the preceding year (11 months), and both had recorded mucocutaneous BILAG grade B. All others received their preceding BCDT more than 12 months earlier.
Forty-six patients were treated with 2 infusions of 1 g of rituximab for their severe cutaneous involvement. Of the 4 patients who only received a single infusion of 1 g of rituximab, 1 had a decompensation of chronic liver disease after the first infusion, 1 developed a chest infection after the first infusion and received the second infusion after 4 months, 1 developed symptoms compatible with a possible allergic reaction, and 1 was unable to attend the hospital owing to social circumstances.
In accordance with protocol, different cumulative doses of cyclophosphamide were administered at different times during the study period. Ten patients received 1500 mg; 33, 750 mg; and 7, no cyclophosphamide (owing to contraindications or patient refusal). No statistically significant differences or trends toward a variable treatment response were noted between these treatment groups.
At 6 months after rituximab treatment, 38 patients (76%) demonstrated a clinical response (Table 3). Twenty patients (40%) demonstrated a complete response overall, with those with the NSLE subtype having the highest proportion of patients with a BILAG grade reduction to D (5 of 11 [45%]). Of the remaining patients, 18 (36%) had a partial response and 8 (16%) maintained stable disease.
At 12 months after rituximab infusion, 28 of the 46 patients (61%) with clinical follow-up available demonstrated a clinical response (Table 3). Twenty-four patients (52%) demonstrated a complete response, with the ACLE and NSLE subtypes having similarly high response rates (12 of 21 [57%] and 6 of 10 [60%], respectively). Of note, those with NSLE included those with alopecia, vasculitis, and/or mouth ulcers. These cutaneous manifestations occurred concurrently in many patients, and hence this study was not powered to report differences in clinical responses within the clinically variable NSLE group.
At 6 months after rituximab infusion, 5 of the 6 patients with SCLE (88%) and 10 of 12 with CCLE (83%) demonstrated a clinical response by improving their BILAG grade A or B status. This included a complete response in 2 of 6 patients (33%) with SCLE and 5 of 12 (42%) with CCLE (Table 3).
At 12 months after rituximab infusion, 3 of 6 patients (50%) with SCLE and 7 of 11 (64%) with CCLE demonstrated a clinical response. This included a complete response in 2 (33%) with SCLE and 5 (45%) with CCLE.
All patients with CCLE with a complete response at 6 months maintained their complete response at 12 months. However, 3 of 7 patients (43%) demonstrating a partial response or stable disease at 6 months required an additional rituximab infusion before 12 months for severe stable or worsening CCLE.
Among the 4 patients who received a single 1-g rituximab infusion only, 1 with ACLE maintained stable disease at 6 and 12 months (receiving 750 mg of cyclophosphamide), 1 with SCLE obtained a partial response at 6 and 12 months (receiving 750 mg of cyclophosphamide), and 2 with ACLE achieved a complete response at 6 and 12 months (one receiving 750 mg of cyclophosphamide and the other rituximab only) (Table 3).
Of the 50 patients who received BCDT with rituximab for their severe cutaneous involvement, 32 (64%) required an additional treatment cycle (15 patients within the 12-month follow-up period and 17 after the 12-month follow-up period). These patients included 21 (66%) with cutaneous involvement and 10 (31%) with noncutaneous involvement. One patient did not have this follow-up information available.
Of the 21 patients who underwent a second cycle of rituximab for severe skin disease, 2 (1 with ACLE and 1 with SCLE) had received their first BCDT cycle within the previous 6 months. Both patients had stable severe cutaneous disease, which had been unresponsive to the first treatment cycle. All others received their additional rituximab infusion at 6 to 12 months of follow-up.
Regarding the administered drugs, 18 patients were treated with 2 rituximab infusions of 1 g. Of these, 1 patient received an additional 1500 mg of cyclophosphamide; 14, an additional 750 mg of cyclophosphamide; and 3, no additional cyclophosphamide. The second infusion of cycle 2 was withheld in 1 patient because of a probable allergic reaction. The remaining 2 patients received a single 1-g infusion of rituximab (one was completing a delayed first cycle and the other refused a second infusion), both without cyclophosphamide.
At 6 months after the second rituximab cycle, 12 of 20 patients (60%) showed a clinical response (Table 3). Of these, 7 patients (35%) demonstrated a complete response; 5 (25%), a partial response; and 8 (40%), stable disease. At 12 months after the second rituximab cycle (when BILAG grades were available for 18 of 20 patients), 9 (50%) overall maintained a clinical response, including 5 (28%) with a complete response and 4 (22%) with a partial response.
Four patients received a third rituximab cycle from 6 to 12 months after the second cycle. Two of these patients had ongoing severe but stable cutaneous disease (1 with NSLE and 1 with CCLE), and 2 had a cutaneous flare (1 with ACLE and 1 with CCLE) (Table 3).
Of the 49 patients whose CD19 counts were available, 48 achieved B-cell depletion after rituximab therapy. Notably, of the patients whose CLE flared, all but 2 had repleted B cells at the time of flare and/or additional BCDT.
Both patients who had a skin flare despite ongoing B-cell depletion had CCLE at baseline and at the time of flare. The first patient had a partial clinical response to the initial rituximab cycle, but the disease flared with worsening CCLE and the patient received a second rituximab cycle at 11 months, despite maintained B-cell depletion at that time. The second patient had a complete clinical response to the initial rituximab cycle; however, at 8 months after treatment a CCLE flare occurred despite ongoing B-cell depletion. A second cycle was given, with a favorable complete clinical response (with complete B-cell depletion). However, the CCLE flared 8 months later despite maintained B-cell depletion. A third cycle of BCDT was given.
To our knowledge, this is one of the largest studies to date to assess the response to BCDT of CLE and its subtypes, occurring in the setting of SLE. Our study also has unique longer-term follow-up beyond 6 months.
We report an overall clinical response of 76% at 6 months, which included 40% complete responses and 36% partial responses. At 12 months, the overall clinical response decreased to 61%, including 52% complete responses. Interestingly, a higher proportion of complete responses was observed at 12 months (24 patients [52%]) and, as could perhaps be expected, a higher proportion of patients with stable or flared disease (18 patients [39%]), some of whom received further BCDT. Acute CLE and NSLE responded most consistently, whereas SCLE and CCLE show complete response rates of less than 50%.
One of the first retrospective studies performed in our UCLH cohort27 presented an overall clinical response to rituximab of 87.4% at 6 months, but only 16 patients had mucocutaneous involvement. A recent study by Md Yusof et al34 described one of the largest populations of patients with SLE receiving rituximab and used a treatment protocol that did not include concurrent cyclophosphamide; the investigators reported a similar proportion of patients with CLE and clinically significant responses at 6 months, but no longer-term assessment. In contrast, Vital et al33 reported a clinical response in 9 (34.6%) of 26 patients with SLE mucocutaneous involvement. However, this group also included patients with BILAG grade C, and hence results are not directly comparable to ours. This difference may also imply a lower response in less severe baseline disease. Of note, the treatment protocol used by Vital et al33 was rituximab only, without cyclophosphamide, and this may also be a confounding factor contributing to our relatively higher response rates. Other authors28,29,32 have reported mixed results of the efficacy of rituximab for cutaneous involvement, but the samples were small, with variable treatment protocols (without or without cyclophosphamide), and the tools used to assess disease activity included the Mexican Systemic Lupus Erythematosus Disease Activity (SLEDAI), Safety of Estrogens in Systemic Lupus Erythematosus National Assessment SLEDAI, and European Consensus Lupus Activity Measurement, which are global scoring systems with very minimal description of cutaneous involvement and hence limited ability to capture any significant effect of BCDT on specific mucocutaneous manifestations. Further, definitions of treatment response, disease relapse, and flare are difficult to ascertain clearly in other studies.33,34
Regarding the effect of BCDT according to different CLE subtypes, the evidence is relatively scarce and involves small numbers overall. In light of a strong association with SLE, its activity, and hence assumed shared B-cell–dependent immunopathologic features, the high rate of positive clinical responses achieved by patients with ACLE and NSLE in our study was expected and in keeping with other reports.25,34 Notably also, complete response rates improved from 6 to 12 months after the first rituximab cycle for patients with these CLE subtypes.
In contrast, SCLE and CCLE have illustrated more variable outcomes. A previous report by Hoffman et al25 evaluated 17 patients from our larger population and described a trend of no response to rituximab in patients with SLE and CCLE. Similarly, no responders were found among those with CCLE in the cohort reported by Vital et al.33 Using a larger sample from the same Leeds cohort, Md Yusof et al34 described, once again, a significant absence of response in the CCLE group. Authors have postulated that this lack of response may be a consequence of potentially different and B-cell–independent pathogenesis in CCLE overall or in CCLE subtypes, which could be in keeping with the less strong association of CCLE with SLE, as well as autoantibody positivity. In addition, alternative non–B-cell pathways may be triggered and used after BCDT in this CLE subtype. These theories could be in keeping with our finding that 2 patients with CCLE experienced a flare (in 1 patient twice), despite maintained B-cell depletion after rituximab infusion.
Overall, in our large cohort with longer-term follow-up, no statistically significant differences occurred in response rates between CLE groups, and CCLE had a greater than 60% response at 6 and 12 months. In fact, the CCLE group included the best responses at 6 months, with 84% showing a clinically significant response. In particular, those with a complete response had good results and maintained their complete response at 12 months. However, patients with CCLE who responded partially or maintained stable disease at 6 months had worse results at 12 months. A plausible explanation for this variable response within our cohort is elusive but could again imply the possibility of different immunopathogenic patient subsets, whereby some patients with CCLE have more prominent B-cell–independent involved pathways (before and/or after BCDT) and hence respond differently. For example, localized DLE is less strongly associated with SLE compared with generalized DLE, suggesting immunopathologic findings less associated with B cells or SLE in the former. Importantly however, all patients in our and other reported cohorts18,25-32 had SLE (not isolated CLE) diagnosed according to the revised criteria of the American College of Rheumatology.
Regarding flares in cutaneous disease, none occurred within 6 months of the first or second cycle of rituximab. However, from 6 to 12 months, flares evolved in 7 cases, including 2 patients each with ACLE, SCLE, and CCLE and 1 with NSLE. Most of these patients received further rituximab (Table 3). No patients switched CLE subtype on flaring after rituximab treatment, and B cells (CD19 counts) had recovered in all but 2 patients with CCLE. Other investigators33,36,37 have also reported flares of all CLE subtypes after rituximab treatment, as well as new cases of CCLE after rituximab treatment. This finding again raises the possibility of differing immunopathologic findings and/or the triggering of alternative pathways in certain CLE subtypes, CCLE in particular. Notably, in our entire cohort we only report 2 cases of new-onset CLE after rituximab infusion. Both patients developed SCLE, but 20 and 51 months after rituximab treatment, so the relevance of a posttreatment effect is dubious. Only 6 patients in our cohort had SCLE overall, making it difficult to draw any significant conclusions regarding this group. The 2 patients, who initially had complete responses at 6 months, maintained this response. However, 2 patients with partial responses experienced a flare by 12 months, and this pattern of response was similar in other groups (Table 3).
Although the subsample was small, we found an overall trend toward a lower clinical response among those receiving a second cycle of rituximab within 12 months of the first, especially by 12-month follow-up (Table 3). Interestingly, Md Yusof et al34 found that 1 in 8 patients with SLE do not achieve clinical benefit or B-cell depletion on additional cycles of rituximab, regardless of prior response. Further larger and prospective studies are needed to explain this finding in the context of mucocutaneous involvement, and the usefulness or otherwise of additional treatment cycles.
This study has several limitations, most related to its retrospective nature and small numbers within each CLE subgroup. The latter means the study is not powered to detect differences in responses between groups. However, data were extracted from a prospective database. These data were only available prospectively at 6 and 12 months, which precludes information on time to response; rituximab is known to deplete pathogenic antibodies at 2 to 3 months. To reduce misclassification bias, we excluded patients in whom available records were unreliable, inaccurate, or insufficient to accurately describe their CLE subtype. All patients in our cohort have SLE meeting the American College of Rheumatology revised criteria for the classification of SLE35; hence, we have not studied the effect of rituximab on CLE occurring in isolation. Most of the patients in our study received concurrent cyclophosphamide, and although no significant difference in treatment response was seen between these groups, numbers were small and no definite conclusion can be drawn on the possible additional clinical effect of combined therapy. This also remains an issue with interstudy comparisons throughout the literature, with variable treatment protocols used in different centers. Finally, although validated for use in mucocutaneous involvement in SLE (based on the physicians’ intention to treat), the BILAG system is a gross measure of extent and severity of dermatological involvement. A more detailed and dermatological-specific system is the Cutaneous Lupus Disease Area and Severity Index (CLASI).
Overall, our results show good clinical response to BCDT using rituximab in all forms of mucocutaneous involvement, especially in patients with ACLE and NSLE. Overall, more than 50% of patients in each subtype improved their BILAG grade A or B status at 6 (40% complete responders) and 12 (52% complete responders) months after treatment. Although patients with CCLE responded well, numbers were small and further study of this new result in our study is warranted. Almost one-third of patients required further rituximab treatment within 12 months for severe skin disease and showed a trend toward lower clinical responses overall.
Accepted for Publication: September 2, 2018.
Corresponding Author: Amanda M. Saracino, FACD, Centre for Rheumatology and Connective Tissue Diseases, Division of Medicine, Department of Inflammation, 2nd Floor, University College London Medical School Building, Royal Free Campus, Rowland Hill Street, London NW3 2PF, United Kingdom (firstname.lastname@example.org).
Published Online: October 31, 2018. doi:10.1001/jamadermatol.2018.3793
Author Contributions: Drs Quelhas da Costa and Aguirre-Alastuey are co–first authors; Drs Isenberg and Saracino are co–last authors. Drs Quelhas da Costa and Aguirre-Alastuey had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Concept and design: Aguirre-Alastuey, Isenberg, Saracino.
Acquisition, analysis, or interpretation of data: Quelhas da Costa, Aguirre-Alastuey.
Drafting of the manuscript: All authors.
Critical revision of the manuscript for important intellectual content: Saracino.
Statistical analysis: Quelhas da Costa.
Administrative, technical, or material support: Aguirre-Alastuey, Saracino.
Supervision: Isenberg, Saracino.
Conflict of Interest Disclosures: None reported.
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