University of Groningen. Hidradenitis suppurativa Dickinson-Blok, Janine Louise

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1 University of Groningen Hidradenitis suppurativa Dickinson-Blok, Janine Louise IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Dickinson-Blok, J. L. (2015). Hidradenitis suppurativa: From pathogenesis to emerging treatment options [Groningen]: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 HIDRADENITIS SUPPURATIVA FROM PATHOGENESIS TO EMERGING TREATMENT STRATEGIES Janine Louise Dickinson-Blok

3 ISBN: ISBN: (e-version) 2015 by J.L. Dickinson-Blok, Groningen, The Netherlands Financial support for the publication of this thesis was provided by: AbbVie BV, ALK-Abelló BV, Almirall, Celgene BV, Fagron BV, Flen Pharma, Galderma Benelux BV, Janssen-Cilag BV, La Roche-Posay, Leo Pharma BV, Molnlycke Health Care, Rijksuniversiteit Groningen, Studiefonds Dermatologie, Universitair Medisch Centrum Groningen, Will Pharma. Design and layout: Printing: Arjan Veening, Id Graficus, Assen, The Netherlands GVO drukkers & vormgevers B.V., Ede, The Netherland

4 HIDRADENITIS SUPPURATIVA FROM PATHOGENESIS TO EMERGING TREATMENT STRATEGIES Proefschrift ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 30 september 2015 om uur door Janine Louise Blok geboren op 11 juli 1984 te Deventer

5 Promotor Prof. dr. M.F. Jonkman Copromotor Dr. B. Horváth Beoordelingscommissie Prof. dr. B. van der Lei Prof. dr. E.P. Prens Prof. dr. G.B.E. Jemec

6 Paranimfen Katarzyna Gostyńska Ineke Janse

7 Contents List of abbreviations 7 Chapter 1. Introduction 9 Chapter 2. Increased expression of integrin α6β4 at the basement membrane 23 zone lining the sebaceous glands in hidradenitis suppurativa Chapter 3. The possible association of hidradenitis suppurativa and Down 37 syndrome: are impaired Notch signaling and immunological abnormalities the missing links? Chapter 4. Gene expression profiling in skin and blood in hidradenitis 45 suppurativa Chapter 5. Systemic therapy with immunosuppressive agents and retinoids 51 in hidradenitis suppurativa: a systematic review Chapter 6. Ustekinumab in hidradenitis suppurativa: a clinical open label 79 study with analyses of the protein expression profile in serum Chapter 7. Skin-tissue-sparing excision with electrosurgical peeling (STEEP): 97 a surgical treatment option for severe hidradenitis suppurativa Hurley stage II/III Chapter 8. Surgery under general anesthesia in severe hidradenitis 107 suppurativa: a study of 363 primary operations in 113 patients Chapter 9. General discussion and future perspectives 125 Chapter 10. Dutch summary / Nederlandse samenvatting 137 Appendices List of publications 145 Dankwoord Curriculum vitae

8 List of abbreviations AE BMZ DLQI FPSU HiSCR HS IF IFE IL m-hslasi mss PAS PGA SFJ STEEP Th-cells TNF-α VAS adverse event basement membrane zone daily life quality index folliculopilosebaceous unit hidradenitis suppurativa clinical response hidadenitis suppurativa immunofluorescence interfollicular epidermis interleukine modified hidradenitis suppurativa-lesion area and severity index modified Sartorius scale periodic acid schiff physician s global assessment sebofollicular junction skin tissue sparing excision with electrosurgical peeling T-helper cells tumor-necrosis-factor-α visual analogue scale

9

10 1Introduction J.L. Dickinson-Blok 9

11 Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease affecting approximately 1-4% of the general population. 1 The disease is characterized by painful deep-seated nodules and abscesses. In a later stage, epitheliazed sinus tracts are formed in the dermis and subcutaneous fat. The lesions seen in HS are mainly restricted to the body folds, like the axillary, inguinal and anogenital regions. 2 These locations have several characteristics in common: 1) the skin contains apocrine glands, 2) a predisposition to mechanical friction and 3) humid conditions. Lesions commonly heal with hypertrophic fibrous scarring resulting in complete architectural loss, cosmetic disfigurement and in some cases even movement impairment. 3 Not surprisingly, patient s quality of life is impaired to a great extent. 4 In fact, it has been found that quality of life scores are worse in HS compared to other distressing chronic dermatoses like atopic dermatitis, psoriasis, Darier s disease and Hailey-Hailey disease. 5 In addition to their professional career, patient s intimate, sexual and social relationships are adversely affected by the disease. HS has also an impact on society, as it is associated with frequent and/or long-term sick leaves. 6 Due to embarrassment, ignorance and neglect of the patient, as well as a lack of knowledge of regarding HS under certain medical specialists, diagnostic delays of several years are not uncommon. 7,8 Pathogenesis The pathogenesis of HS is still largely unknown. The term hidradenitis suppurativa dates back to a time where it was assumed to be primarily a disease of the apocrine sweat glands. 3 Although, it is now generally accepted that the hair follicle is primarily involved while the associated apocrine gland is affected in only the minority of patients as a secondary event (figure 1). 9 The role of bacteria in this inflammatory process remains elusive. Fulminant discharge suggests bacterial involvement but cultures in microbiological studies have been shown to be negative or mainly revealed commensal bacteria of the skin or intestine, dependent on the investigated body location Psoriasiform hyperplasia, follicular hyperkeratosis and occlusion are early events in the disease process It has been suggested that these histopathological changes result from subclinical inflammation initiated by keratinocytes reacting to commensal skin bacteria. 16 Additionally, a recent study suggested that fragility of the sebofollicular junction (SFJ) as part of the folliculopilosebaceous unit (FPSU) could contribute to the inflammatory activity by a defect in the follicular basement membrane zone (BMZ) that allows the release of follicular content into the surrounding dermis. 17 Massive inflammation as a result of immune system activation occurs upon complete rupturing of the occluded hair follicle. 10

12 Epidermis FPSU Sebaceous gland Apocrine gland Hair follicle Dermis Subcutaneous fat Figure 1. The folliculopilosebaceous unit (FPSU) in the skin. * sebofollicular junction (SFJ) In addition, overproduction of interleukin (IL)-1b and tumor necrosis factor (TNF)-α from the innate immune system as well as IL-10, IL-12, IL-17 and IL-23 from the adaptive immune system have been demonstrated in HS skin. 18,19 Epithelialized sinus tracts may be formed from epithelial strands in the dermis in response to these cytokines. This may facilitate access for (commensal) bacteria, leading to repetitive inflammation, further extension of the disease, and subsequently a vicious circle is made with ever increasing architectural destruction. Unraveling what cytokines are predominant in the inflammatory cascade is an important step for a better understanding of the HS pathogenesis and for identifying therapeutic targets. Epidemiology The prevalence of HS varies between studies and is estimated to be 1-4% in Europe, these numbers are mostly derived from population based questionnaires. 20,21 Substantial lower prevalence rates were found in the United States, varying from to 0.078%. 22,23 Females are three times more often affected than men. 1,20,24 First symptoms typically occur in the second or third decades of life but disease onset during childhood is not exceptional

13 About 80% of HS patients has a history of smoking, making it a well-known risk factor for HS. 20,24,28,29 The exact pathogenic mechanism remains unclear, however, tobacco smoking may induce HS by promoting follicular occlusion, augmenting the innate immune system and triggering pro-inflammatory cytokine release. 30 The association between obesity and HS has also widely been recognized and may result from increased mechanical friction of the skin and inducing a pro-inflammatory state. 20,29,31 Both smoking and obesity are associated with higher disease severity. 31 The role of hormones in HS remains controversial, especially regarding androgens. Studies have shown that HS improves in women on anti-androgen therapy. 32,33 Also, HS rarely develops in postmenopausal women, a phase in life that is characterized by relative hypo-androgenism. 34 However, it has been demonstrated that free androgen levels are not consistently elevated in women with HS. 35 Multiple studies have suggested that HS is associated with several co-morbidities, including morbus Crohn, metabolic syndrome, hypertension, diabetes mellitus and polycystic ovarian syndrome (PCOS). 23,36-38 Finally, it has been recognized that HS runs in families, indicating that genetic factors are also important. (24) In fact, loss-of-function mutations in genes encoding for the g-secretase protein complex have been identified in familial HS. 39,40 Inactivation of g-secretase may result in altered Notch signaling which may promote the formation of epidermal cysts and contribute to the continuing inflammatory activity in HS by dysfunction of the innate immune system. 30 Classification and monitoring disease severity There is wide diversity in the clinical appearance of HS regarding severity, disease location and whether there is predomination of inflammatory nodules or sinus tracts and fistulas. The Hurley classification (grade I through III) is a well-known and commonly used system to express disease severity by determination of the character and the extensiveness of the lesions (figure 2)

14 Hurley I Localized disease. Single or multiple abscesses. No sinus tracts or scarring. Hurley II Recurrent abscesses. Single or multiple sinus tracts and scarring. Lesions separated by healthy skin. Hurley III Multiple interconnected abscesses and sinus tracts. Involvement of the entire affected area. Figure 2. The Hurley stages of lesions in HS. Although the Hurley classification is convenient to use in daily practice, its major disadvantage is that it is a static rather than a dynamic scoring system and therefore inappropriate for monitoring therapeutic effects over time. In recent years, several dynamic scoring systems have been developed, including the modified Sartorius score (mss) 29 and the Hidradenitis Suppurativa Clinical Response (HiSCR). 42 The recently proposed HiSCR is actually the first 13

15 score defining a validated practical clinical endpoint. 42 Unfortunately, in previous studies no uniformly applied clinical endpoint was applied to assess treatment effectiveness, making it difficult to compare these trials with each other. The identification of a specific biomarker for HS could support disease monitoring. Recent evidence suggests that the soluble IL-2 receptor (sil2r) and S100A8/A9 may be putative candidates for distinguishing HS patients from healthy controls. 43,44 However, more studies are needed to establish their usefulness in monitoring treatment efficacy and to identify other potential biomarkers. Treatment of HS Treatment of HS is a challenge as many patients are resistant to therapy. Recently Zouboulis et al. 45 developed a treatment guideline for HS. Although this guideline is of great help in ordering the currently available therapeutic options, the evidence for individual therapies remains relatively sparse. Three primary goals should be pursued in the treatment of HS: 1) to treat acute painful lesions, 2) to heal chronic lesions in the maintenance phase and 3) to prevent the development of new lesions. The main general therapeutic options are topical agents, systemic medication and surgical interventions. The strategy for achieving the treatment goals is dependent on the severity of HS and the expertise of the center of treatment. The Hurley classification is a practical tool to give direction to the choice of therapy. Topical therapies The only topical treatments that have been studied in HS are resorcin 15% cream and topical clindamycin. 46 These agents can be applied as monotherapy in Hurley stage I disease. In Hurley stage II or III disease it is mainly used as adjuvant or as maintenance therapy. Acute painful lesions may be treated with intralesional triamcinolon 0.1% acetonide 10 mg/ml. Systemic therapies Systemic agents comprise anti-inflammatory, immunosuppressive medication and retinoids. Systemic antibiotics are used for both their anti-inflammatory and anti-bacterial effect. These agents are indicated for Hurley II and III disease as well as in widely spread Hurley I disease. The choice for a specific systemic antibiotic is mainly dependent on clinical experience, as studies are still limited. Most evidence exists for oral tetracyclin and combinational therapy with clindamycin and rifampicin The systemic retinoids acitretin and isotretinoin were introduced to the therapeutic arsenal of HS based on their immunomodulatory effects and 14

16 their ability to normalize epithelial cell differentiation. Immunosuppressive therapy is indicated in severe inflammatory disease (Hurley stage II or III) and a wide variety of agents has been studied, including dapsone, methotrexate, ciclosporin and biologicals, like the TNF-α inhibitors infliximab and adalimumab. Unfortunately, the quality of performed studies is frequently poor and the number of randomized controlled trials is only limited. Therefore, consensus on what systemic agent is most effective in HS is still not achieved. Surgical treatment Surgery is required for Hurley stage II and III disease, as epithelialized cysts and sinus tracts will still remain present once inflammation has been treated. In the acute phase simple incision and drainage is appropriate for relieving pressure of acute painful abscesses. However, this is a symptomatic rather than a definite treatment, as lesions will recur. Therefore, surgical removal of all lesional tissue is the preferred approach in HS. Sparing healthy tissue to a maximum while lesional tissue is completely removed could be an appropriate surgical aim in HS. This aim may be achieved with the deroofing technique. 51,52 The so-called deroofing is a suitable technique for Hurley stage I or limited stage II disease as lesions are superficially removed. However, in severe HS deroofing does not suffice since lesions may extend into the subcutaneous fat. Furthermore, severe HS is frequently dominated by fibrotic tissue, which cannot be removed during deroofing. Removal of this tissue is of importance as it may contain skin appendages that serve as a source of recurrence, and prevent adequate wound contraction and subsequent healing. Therefore in moderate to severe HS, wide excision of the entire affected area is frequently used, especially by surgeons. 53 A disadvantage of this approach is that it causes large defects with a serious risk on contracture formation and long healing times. Surgery may be performed with cold steel, electrosurgery or a CO 2 laser. 52,54,55 Finally, several types of wound healing techniques have been proposed for HS, including healing by secondary intension or primary closure by sutures, skin grafts or flaps. 9 Exploring current and new surgical approaches in severe HS is needed to identify what techniques are superior regarding surgical outcomes in terms of radical lesional tissue removal, healing time and complications. In conclusion, treatment of HS is still difficult despite the numerous options, leading to frustration in both patients and in doctors. Studies are needed to investigate currently available treatments and to explore new systemic and surgical treatments for the development of general treatment guidelines. 15

17 AIMS AND OUTLINE OF THIS THESIS HS has a severe impact on quality of life and treatment is, despite the numerous options, in many cases still unsatisfactory. To develop new and improved treatment strategies, the fundamentals of the pathogenesis of HS need to be further unraveled. Furthermore, clinical trials are needed to investigate the effectiveness of (new) systemic and surgical treatments as well as to determine their therapeutic value in HS. The aims of this thesis are: To investigate the principles of the HS pathogenesis by focusing on histopathological changes of the hair follicle and to study the role of specific protein upregulation in the inflammatory cascade. To study the effectiveness of established and new systemic agents for the treatment of HS. To explore new surgical techniques to provide tools for clinicians dealing with HS. Chapter 2 describes the expression of the main glycoproteins at the basement membrane zone in pilosebaceous units of HS patients by performing immunofluorescence stainings on perilesional skin. In Chapter 3 an association between Down s syndrome and HS is hypothesized based on defective Notch signaling as a result of functional g-secretase deficiency. Chapter 4 describes the gene expression profile of hidradenitis suppurativa in skin and blood. In Chapter 5 we systematically review the current literature to explore the effectiveness of systemic treatment with immunosuppressive agents and retinoids in HS. In Chapter 6 the effectiveness and safety of the IL-12/IL-23 inhibitor ustekinumab is prospectively studied in HS patients. Chapter 7 and 8 focus on the skin tissue sparing excision with electrosurgical peeling (STEEP) technique as a surgical method for moderate to severe HS and describes its results over a time span of 14 years. Chapter 9 summarizes the main findings of this thesis and provides a general discussion for future studies. 16

18 REFERENCES 1. Jemec GB. Clinical practice. Hidradenitis suppurativa. N Engl J Med 2012; 366: Revuz J. Hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2009; 23: Jemec G, Revuz J, Leyden J. Hidradenitis Suppurativa. Berlin: Springer-Verlag, Wolkenstein P, Loundou A, Barrau K, et al. Quality of life impairment in hidradenitis suppurativa: a study of 61 cases. J Am Acad Dermatol 2007; 56: Matusiak L, Bieniek A, Szepietowski JC. Psychophysical aspects of hidradenitis suppurativa. Acta Derm Venereol 2010; 90: Esmann S, Jemec GB. Psychosocial impact of hidradenitis suppurativa: a qualitative study. Acta Derm Venereol 2011; 91: Vazquez BG, Alikhan A, Weaver AL, et al. Incidence of Hidradenitis Suppurativa and Associated Factors: A Population-Based Study of Olmsted County, Minnesota. J Invest Dermatol 2013; 133: Mebazaa A, Ben Hadid R, Cheikh Rouhou R, et al. Hidradenitis suppurativa: a disease with male predominance in Tunisia. Acta Dermatovenerol Alp Panonica Adriat 2009; 18: Alikhan A, Lynch PJ, Eisen DB. Hidradenitis suppurativa: a comprehensive review. J Am Acad Dermatol 2009; 60:539-61; quiz Lapins J, Jarstrand C, Emtestam L. Coagulase-negative staphylococci are the most common bacteria found in cultures from the deep portions of hidradenitis suppurativa lesions, as obtained by carbon dioxide laser surgery. Br J Dermatol 1999; 140: Sartorius K, Killasli H, Oprica C, et al. Bacteriology of hidradenitis suppurativa exacerbations and deep tissue cultures obtained during carbon dioxide laser treatment. Br J Dermatol 2012; 166: Matusiak L, Bieniek A, Szepietowski JC. Bacteriology of Hidradenitis Suppurativa - Which Antibiotics are the Treatment of Choice? Acta Derm Venereol 2014;. 13. Jemec GB, Hansen U. Histology of hidradenitis suppurativa. J Am Acad Dermatol 1996; 34:

19 14. von Laffert M, Stadie V, Wohlrab J, Marsch WC. Hidradenitis suppurativa/acne inversa: bilocated epithelial hyperplasia with very different sequelae. Br J Dermatol 2011; 164: van der Zee HH, de Ruiter L, Boer J, et al. Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions. Br J Dermatol 2012; 166: van der Zee HH, Laman JD, Boer J, Prens EP. Hidradenitis suppurativa: viewpoint on clinical phenotyping, pathogenesis and novel treatments. Exp Dermatol 2012; 21: Danby FW, Jemec GB, Marsch WC, von Laffert M. Preliminary findings suggest hidradenitis suppurativa may be due to defective follicular support. Br J Dermatol 2013; 168: van der Zee HH, de Ruiter L, van den Broecke DG, et al. Elevated levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-alpha and IL-1beta. Br J Dermatol 2011; 164: Schlapbach C, Hanni T, Yawalkar N, Hunger RE. Expression of the IL-23/Th17 pathway in lesions of hidradenitis suppurativa. J Am Acad Dermatol 2011; 65: Revuz JE, Canoui-Poitrine F, Wolkenstein P, et al. Prevalence and factors associated with hidradenitis suppurativa: results from two case-control studies. J Am Acad Dermatol 2008; 59: Jemec GB, Heidenheim M, Nielsen NH. The prevalence of hidradenitis suppurativa and its potential precursor lesions. J Am Acad Dermatol 1996; 35: Cosmatos I, Matcho A, Weinstein R, et al. Analysis of patient claims data to determine the prevalence of hidradenitis suppurativa in the United States. J Am Acad Dermatol 2013; 69: Shlyankevich J, Chen AJ, Kim GE, Kimball AB. Hidradenitis suppurativa is a systemic disease with substantial comorbidity burden: a chart-verified case-control analysis. J Am Acad Dermatol 2014; 71: Canoui-Poitrine F, Revuz JE, Wolkenstein P, et al. Clinical characteristics of a series of 302 French patients with hidradenitis suppurativa, with an analysis of factors associated with disease severity. J Am Acad Dermatol 2009; 61: Palmer RA, Keefe M. Early-onset hidradenitis suppurativa. Clin Exp Dermatol 2001; 26:

20 26. Lewis F, Messenger AG, Wales JK. Hidradenitis suppurativa as a presenting feature of premature adrenarche. Br J Dermatol 1993; 129: Mengesha YM, Holcombe TC, Hansen RC. Prepubertal hidradenitis suppurativa: two case reports and review of the literature. Pediatr Dermatol 1999; 16: Schrader AM, Deckers IE, van der Zee HH, et al. Hidradenitis suppurativa: a retrospective study of 846 Dutch patients to identify factors associated with disease severity. J Am Acad Dermatol 2014; 71: Sartorius K, Emtestam L, Jemec GB, Lapins J. Objective scoring of hidradenitis suppurativa reflecting the role of tobacco smoking and obesity. Br J Dermatol 2009; 161: Melnik BC, Plewig G. Impaired Notch-MKP-1 signalling in hidradenitis suppurativa: an approach to pathogenesis by evidence from translational biology. Exp Dermatol 2013; 22: Kromann CB, Ibler KS, Kristiansen VB, Jemec GB. The influence of body weight on the prevalence and severity of hidradenitis suppurativa. Acta Derm Venereol 2014; 94: Mortimer PS, Dawber RP, Gales MA, Moore RA. A double-blind controlled cross-over trial of cyproterone acetate in females with hidradenitis suppurativa. Br J Dermatol 1986; 115: Kraft JN, Searles GE. Hidradenitis suppurativa in 64 female patients: retrospective study comparing oral antibiotics and antiandrogen therapy. J Cutan Med Surg 2007; 11: Zumoff B, Strain GW, Miller LK, Rosner W. Twenty-four-hour mean plasma testosterone concentration declines with age in normal premenopausal women. J Clin Endocrinol Metab 1995; 80: Barth JH, Layton AM, Cunliffe WJ. Endocrine factors in pre- and postmenopausal women with hidradenitis suppurativa. Br J Dermatol 1996; 134: van der Zee HH, van der Woude CJ, Florencia EF, Prens EP. Hidradenitis suppurativa and inflammatory bowel disease: are they associated? Results of a pilot study. Br J Dermatol 2010; 162: Sabat R, Chanwangpong A, Schneider-Burrus S, et al. Increased prevalence of metabolic syndrome in patients with acne inversa. PLoS One 2012; 7:e Gold DA, Reeder VJ, Mahan MG, Hamzavi IH. The prevalence of metabolic syndrome in patients with hidradenitis suppurativa. J Am Acad Dermatol 2014; 70:

21 39. Pink AE, Simpson MA, Desai N, et al. Mutations in the gamma-secretase genes NCSTN, PSENEN, and PSEN1 underlie rare forms of hidradenitis suppurativa (acne inversa). J Invest Dermatol 2012; 132: Wang B, Yang W, Wen W, et al. Gamma-secretase gene mutations in familial acne inversa. Science 2010; 330: Hurley HJ. Axiillairy hyperhidrosis, apocrine bromhidrosis, hidradenitis suppurativa, and familial benign pemphigus: surgical approach. In: Dermatologic Surgery(In: Roenigh, R.K. Roenigh, HH, ed): Marcel Dekker, New York, 1989; Kimball AB, Jemec GB, Yang M, et al. Assessing the validity, responsiveness and meaningfulness of the Hidradenitis Suppurativa Clinical Response (HiSCR) as the clinical endpoint for hidradenitis suppurativa treatment. Br J Dermatol 2014; 171: Matusiak L, Bieniek A, Szepietowski JC. Soluble interleukin-2 receptor serum level is a useful marker of hidradenitis suppurativa clinical staging. Biomarkers 2009; 14: Wieland CW, Vogl T, Ordelman A, et al. Myeloid marker S100A8/A9 and lymphocyte marker, soluble interleukin 2 receptor: biomarkers of hidradenitis suppurativa disease activity? Br J Dermatol 2013; 168: Zouboulis CC, Desai N, Emtestam L, et al. European S1 guideline for the treatment of hidradenitis suppurativa/acne inversa. J Eur Acad Dermatol Venereol 2015; 29: Clemmensen OJ. Topical treatment of hidradenitis suppurativa with clindamycin. Int J Dermatol 1983; 22: Jemec GB, Wendelboe P. Topical clindamycin versus systemic tetracycline in the treatment of hidradenitis suppurativa. J Am Acad Dermatol 1998; 39: Gener G, Canoui-Poitrine F, Revuz JE, et al. Combination therapy with clindamycin and rifampicin for hidradenitis suppurativa: a series of 116 consecutive patients. Dermatology 2009; 219: van der Zee HH, Boer J, Prens EP, Jemec GB. The effect of combined treatment with oral clindamycin and oral rifampicin in patients with hidradenitis suppurativa. Dermatology 2009; 219: Bettoli V, Zauli S, Borghi A, et al. Oral clindamycin and rifampicin in the treatment of hidradenitis suppurativa-acne inversa: a prospective study on 23 patients. J Eur Acad Dermatol Venereol 2014; 28:

22 51. van der Zee HH, Prens EP, Boer J. Deroofing: a tissue-saving surgical technique for the treatment of mild to moderate hidradenitis suppurativa lesions. J Am Acad Dermatol 2010; 63: van Hattem S, Spoo JR, Horvath B, et al. Surgical treatment of sinuses by deroofing in hidradenitis suppurativa. Dermatol Surg 2012; 38: Rompel R, Petres J. Long-term results of wide surgical excision in 106 patients with hidradenitis suppurativa. Dermatol Surg 2000; 26: Alharbi Z, Kauczok J, Pallua N. A review of wide surgical excision of hidradenitis suppurativa. BMC Dermatol 2012; 12: Madan V, Hindle E, Hussain W, August PJ. Outcomes of treatment of nine cases of recalcitrant severe hidradenitis suppurativa with carbon dioxide laser. Br J Dermatol 2008; 159:

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24 2Increased expression of integrin α6β4 at the basement membrane zone lining the sebaceous glands in hidradenitis suppurativa J.L. Blok, I.C. Janse, B. Horváth, M.F. Jonkman Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands Acta Derm Venereol Jun 30. doi: / [Epub ahead of print] 23

25 ABSTRACT The pathogenesis of hidradenitis suppurativa (HS) is still elusive. A recent study of the folliculopilosebaceous unit (FPSU) in HS patients showed that the basement membrane zone (BMZ) of the sebofollicular junction was almost devoid of periodic acid schiff (PAS) positive material, a staining method for glycoproteins. By performing PAS and immunofluorescence stainings for type XVII collagen, type VII collagen, laminin-332 and integrin α6β4 on skin biopsies from body folds of patients and controls, we aimed to identify whether these glycoproteins are differently expressed in HS. We found normal PAS staining along the sebofollicular junction in HS, and could therefore not confirm any weakening of the BMZ. Conversely, we demonstrated increased expression of integrin α6β4 lining the sebaceous glands in HS patients. The other BMZ components were normally expressed. Enhanced expression of integrin α6β4 along sebaceous glands in HS could result from alterations in the skin microbioma and enhance inflammation. 24

26 INTRODUCTION Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent painful nodules, abscesses, sinus tracts and scarring. The disease is primarily located at the apocrine gland bearing skin, including the armpits and groins. 1 Quality of life in HS patients is severely affected due to the appearance of the skin, associated pain and the filthy odor that is often secreted from the inflammatory lesions. 2 The pathogenesis of HS has not been clarified yet. Several histological studies have shown that the hair follicle is involved at an early stage of the disease process. 3-6 These studies demonstrated that follicular occlusion is present in the majority of patients, an event that eventually results in rupturing of the hair follicle with subsequent inflammation and sinus tract formation. However, the question remains what the driving mechanism behind this follicular occlusion is. In a recent study, diminished periodic acid schiff (PAS) staining was found at the basement membrane zone (BMZ) of the sebofollicular junction (SFJ) at the folliculopilosebaceous units (FPSU) of HS patients. 3 Since this deficiency was also found in perilesional skin, the authors suggest that the PAS-negative gaps represent primary defects in the BMZ leading to fragility of the hair follicle. Consequently there is release of follicular contents into the dermis, triggering an inflammatory reaction. Diminished expression in one of the glycoproteins in the BMZ of the SFJ might explain the PAS-gaps. However, there was not stained for specific glycoproteins. The aim of this study was to document the expression of the most important BMZ components, including type XVII collagen, type VII collagen, laminin-332, and integrin α6β4, of the follicular epidermis relative to the interfollicular epidermis in HS, and to compare the expression ratio with healthy controls. 25

27 MATERIAL AND METHODS Design and setting This study was performed at the department of Dermatology at the University Medical Center Groningen. The local institutional review board approved the study. Patients Patients who were scheduled for surgical treatment of HS with the deroofing technique or the STEEP procedure 4,5 were included after written informed consent was obtained. Healthy volunteers were considered eligible for participation when they had no skin disease at the armpits and had given written informed consent. The age of all individuals was 18 years or older. Collection procedure Up to 17 perilesional samples were obtained from axillary or inguinal HS skin by four mm punch biopsy, immediately frozen in liquid nitrogen, and subsequently stored at -80 degrees Celsius. Additionally, after injection with local anaesthesia consisting of 1cc 1% lidocaine/ adrenaline (1: ), four mm punch biopsies were obtained from axillary skin of eight healthy volunteers that served as controls. Skin samples lacking an associated sebaceous gland with the hair follicle were excluded. Staining procedure PAS staining of the skin samples was performed. Briefly, after periodic acid solution oxidation, tissue sections were immerged in Schiff s reagent and counterstained with hematoxylin. Immunofluorescence (IF) staining for type XVII collagen, type VII collagen, laminin 332, integrin α6 and β4 was performed. The procedures for IF staining and image collection have been described in detail previously. 6 The following monoclonal antibodies were used: VK1 against type XVII collagen (dr. H. H. Pas, Groningen, The Netherlands), LH7:2 against collagen type VII (gift from Dr I. Leigh, London, UK), K140 against laminin β3 (gift from dr. M. Marinkovich, Stanford, U.S.A.), 58xβ4 against integrin β4 (gift from dr. Sonnenberg, Amsterdam, Netherlands) and GOH3 against integrin α6 (gift from dr. Sonnenberg, Amsterdam, Netherlands). Fluorescein-conjugated goat anti rat IgG (Southern Biotechnology Associates, Birmingham, U.S.A) and Alexa 488-conjugated goat anti mouse IgG (Molecular Probes, Eugene, U.S.A) were used as secondary steps. 26

28 Assessments and statistical analysis The intensity of the stainings was measured at the 6 segments of the FPSU: 1) the interfolliclar epidermis (IFE), 2) the superior segment (SS) of the hair follicle, 3) the inferior segment (IS) of the hair follicle, 4) the sebofollicular junction (SFJ) and 5) the sebaceous gland (SG). The SS was defined as the part of the hair follicle extending from the IFE to the SFJ. The IS was defined as the part extending from the SFJ to the bulb. The SFJ was defined as the transition zone from the hair follicle to the sebaceous gland. The SFJ of skin samples stained with IF was identified by the presence of fat globules characteristic of the sebaceous gland and by comparison of the skin sample with the PAS staining of that same biopsy. For each skin sample the intensity of all performed PAS and IF stainings at the aforementioned individual segments were analyzed using Image J software. Subsequently, the ratio of individual segments to the IFE was calculated for both the PAS and IF stainings, with the IFE serving as an internal control for each skin sample. The Mann-Whitney U test was performed to compare the differences in these ratios between patients and controls. 27

29 RESULTS Skin biopsies were obtained from a total of 17 HS patients and eight controls. Biopsies of ten patients and two controls were excluded due to the lack of an associated sebaceous gland. Eventually, biopsies of seven HS patients (two women, five men) and six controls (three women, three men) were studied. PAS staining As previously described by Danby et al. 3 the IFE showed a continuous and regular PAS staining pattern in all biopsies with a mean intensity of pixels (SD 11.6) in controls and pixels (SD 10.5) in patients. Therefore, the IFE served as a suitable internal control. There were no statistically significant differences between the ratios of the mean intensity of the PAS staining at the individual segments of the FPSU to the IFE in patients and controls (figures. 1-2). Type VII collagen, type XVII collagen, laminin 332 For these IF stainings no statistically significant differences were found between patients and controls in the ratios of the mean staining intensity at the individual FPSU segments to the IFE. Integrin β4 and α6 At the sebaceous gland, the mean intensity of integrin β4 declined compared to the IFE in both patients and controls (figures. 1-2). The ratio of the mean intensity of integrin β4 at the sebaceous gland to the IFE was significantly higher in patients (0.41) compared to controls (0.14) (p=0.004). This implicates that integrin β4 expression at the sebaceous gland was relatively higher in patients. IF-staining for integrin α6 also revealed a significantly (p=0.011) higher level of expression at the sebaceous gland in HS compared to controls (figures. 1-2). It is plausible that the expression patterns of integrin α6 en β4 followed the same pattern, since they are dimerized in the integrin α6β4-complex. The relative expression of integrin α6β4 was not altered at the remaining FPSU segments in HS compared to controls. 28

30 Control skin IFE SS IS SFJ SG PAS Integrin β4 Integrin a6 IFE Patient skin SS SFJ SG IS PAS Integrin β4 Integrin a6 Figure 1. PAS and IF staining of the IFE and SFJ/SG in control and patient skin. There were no differences in the intensity of the PAS staining between the IFE and the SFJ/SG in both controls and patients. The ratio of the staining intensity of integrin α6 and β4 of the SG to the IFE is higher in patients than in controls. Scale bar = 20 µm. 29

31 Ratio segment to IFE 1,1 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 PAS FPSU segment Patient Controls Ratio segment to IFE 1,1 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Integrin b4 FPSU segment Ratio segment to IFE 1,1 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Integrin a4 FPSU segment Figure 2. Ratio of PAS and integrin α6β4 expression compared to the IFE. The bars represent the ratio of the mean PAS and integrin α6β4 staining intensities of the individually assessed folliculopilosebaceous segments to the IFE. IFE: interfollicular epidermis; SS: superior segment; SFJ: sebofollicular junction; SG: sebaceous gland; IS: inferior segment. *statistically significant difference 30

32 DISCUSSION This study demonstrates that integrin α6β4 was relatively upregulated along the BMZ of the sebaceous glands in HS patients. In contrast to Danby et al, 3 reduced PAS positivity at the SFJ in HS was not observed, neither did we find any differences in PAS positivity within the remaining hair follicle segments. Therefore, we cannot confirm that BMZ fragility at the SFJ is responsible for leakage of follicular content into the surrounding dermis. One can speculate about the cause and consequences of integrin α6β4 upregulation in HS. Integrins are a family of heterodimeric glycosylated transmembrane receptors. They come primarily to expression in organs lined with stratified epithelium, like the skin and lungs. In human epidermis, integrin expression is restricted to the basal layer. 7 The β4 integrins are primarily found at the basement membrane zone where they are located transmembranous in hemidesmosomes. In addition to an adhesive function, integrins are important signaling molecules that have bidirectional actions. Integrins show affinity to several extracellular proteins and are therefore involved in a variety of pathological processes, including oncogenesis, immune responses and inflammatory reactions. 8,9 In HS integrines possibly have a cell signaling function. For instance, previous studies on pulmonary tissue have demonstrated that integrin β4 expression increased upon binding with pathogenic microorganisms. 8,10 It has been hypothesized that integrines, just as toll-like receptors, function as pattern recognition receptors (PRRs) that upon interaction with bacteria induce cellular responses that acitivate the innate immune system and inflammation. 8 The role of bacteria in the pathogenesis of HS is still elusive, as previous microbiological studies found a wide range of bacteria associated with HS However, new molecular techniques using a genomic approach revealed that there is greater bacterial diversity in the skin than previously assumed based on results of culturing methods. 14 Van der Zee et al, 15 hypothesized that alterations in the microbioma of HS skin stimulate keratinocytes to produce antimicrobial peptides and pro-inflammatory cytokines, resulting in the typical histological changes observed early on in the disease process. Additionally, alterations in the microbioma could explain why HS lesions are mainly localized at the body folds, as the bacterial community is different in these moist areas compared to the relatively dry areas of the body. 14,16 Similar to lung tissue, changes in the bacterial community are possibly responsible for the integrin α6β4 upregulation we found in HS patients and may contribute to activation of the immune system. Finally, increased expression of α6β4 may also contribute to the development of squamous cell carcinoma (SCC), a well-known complication of HS. 17,18 In fact, mice with aberrant α6β4 expression showed a greater infiltration of immunosuppressive cells during tumor promotion, a phenomenon that may contribute to the susceptibility of SCC formation

33 In conclusion, in this study we demonstrate that integrin α6β4 is upregulated in sebaceous glands of HS patients. This upregulation could result from alterations in the skin microbioma and may contribute to the inflammatory reaction seen in HS as well as to the increased risk of SCC development in HS. Integrin α6β4 could therefore be a putative treatment target for HS in the future. Characterization of the skin microbioma in more detail could provide further insight in the role of bacteria in HS and may provide a rationale for specific antibiotic treatments. 32

34 REFERENCES 1. Jemec GB. Clinical practice. Hidradenitis suppurativa. N Engl J Med 2012; 366: Matusiak L, Bieniek A, Szepietowski JC. Psychophysical aspects of hidradenitis suppurativa. Acta Derm Venereol 2010; 90: Danby FW, Jemec GB, Marsch WC, von Laffert M. Preliminary findings suggest hidradenitis suppurativa may be due to defective follicular support. Br J Dermatol 2013;168: Blok JL, Spoo JR, Leeman FW, Jonkman MF, Horvath B. Skin-Tissue-sparing Excision with Electrosurgical Peeling (STEEP): a surgical treatment option for severe hidradenitis suppurativa Hurley stage II/III. J Eur Acad Dermatol Venereol 2015; 29: van Hattem S, Spoo JR, Horvath B, Jonkman MF, Leeman FW. Surgical treatment of sinuses by deroofing in hidradenitis suppurativa. Dermatol Surg 2012; 38: Vodegel RM, Jonkman MF, Pas HH, de Jong MC. U-serrated immunodeposition pattern differentiates type VII collagen targeting bullous diseases from other subepidermal bullous autoimmune diseases. Br J Dermatol 2004;151: Oldak M, Smola-Hess S, Maksym R. Integrin beta4, keratinocytes and papillomavirus infection. Int J Mol Med 2006;17: Ulanova M, Gravelle S, Barnes R. The role of epithelial integrin receptors in recognition of pulmonary pathogens. J Innate Immun 2009; 1: Berman AE, Kozlova NI. Integrins: structure and functions. Membr Cell Biol 2000;13: Gravelle S, Barnes R, Hawdon N, Shewchuk L, Eibl J, Lam JS, et al. Up-regulation of integrin expression in lung adenocarcinoma cells caused by bacterial infection: in vitro study. Innate Immun 2010;16: Lapins J, Jarstrand C, Emtestam L. Coagulase-negative staphylococci are the most common bacteria found in cultures from the deep portions of hidradenitis suppurativa lesions, as obtained by carbon dioxide laser surgery. Br J Dermatol 1999; 140: Sartorius K, Killasli H, Oprica C, Sullivan A, Lapins J. Bacteriology of hidradenitis suppurativa exacerbations and deep tissue cultures obtained during carbon dioxide laser treatment. Br J Dermatol 2012; 166:

35 13. Matusiak L, Bieniek A, Szepietowski JC. Bacteriology of Hidradenitis Suppurativa - Which Antibiotics are the Treatment of Choice? Acta Derm Venereol 2014; 94: Grice EA, Segre JA. The skin microbiome. Nat Rev Microbiol 2011; 9: van der Zee HH, Laman JD, Boer J, Prens EP. Hidradenitis suppurativa: viewpoint on clinical phenotyping, pathogenesis and novel treatments. Exp Dermatol 2012; 21: SanMiguel A, Grice EA. Interactions between host factors and the skin microbiome. Cell Mol Life Sci 2015; 72: Lavogiez C, Delaporte E, Darras-Vercambre S, et al. Clinicopathological study of 13 cases of squamous cell carcinoma complicating hidradenitis suppurativa. Dermatology 2010; 220: Maalouf SW, Theivakumar S, Owens DM. Epidermal alpha6beta4 integrin stimulates the influx of immunosuppressive cells during skin tumor promotion. J Dermatol Sci 2012; 66:

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38 3The possible association of hidradenitis suppurativa and Down syndrome: are impaired Notch signaling and immunological abnormalities the missing links? J.L. Blok 1, M.F. Jonkman 1, B.Horváth 1 1 Department of Dermatology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands Published in the British Journal of Dermatology, 2014; 170:

39 ABSTRACT Hidradenitis suppurativa (HS) is an inflammatory skin disease of unknown origin. Recently, it has been demonstrated that mutations in several genes encoding for the protease g-secretase (GS), including presenilin-1 (PSEN1), probably play a major role in the pathogenesis through impairment of the Notch signaling pathway. Mutations in PSEN1 are also associated with Alzheimer s disease (AD), a condition that is strongly related to Down syndrome (DS). HS occuring in patients with DS has been described in only five patients so far. Here we describe five new cases. An association between HS and DS is reasonable since trisomy of chromosome 21 leads to overexpression of the amyloid precursor protein (APP) resulting in a change of the substrate pool for GS processing at the expense of the Notch receptors. Consequently, Notch signaling is impaired in DS predisposing these individuals to HS. APP itself may also directly influence keratinocytes resulting in the typical histopathological features seen in HS. Finally, the relatively high prevalence of obesity amongst DS patients as well as alterations in their immune system could underlie the possible association between both conditions. To confirm our hypothesis, further studies are needed investigating the expression of Notch receptors and APP in the epidermis of DS patients. 38

40 Hidradenitis suppurativa (HS) is an inflammatory skin disease that usually arises after puberty and severely impairs quality of life. Smoking, obesity, genetics and abnormalities of the immune system are important risk factors for the development of HS. (1) The latter makes that the disease is generally responsive to immunosuppressive agents, including to tumor-necrosis-factor (TNF)-α inhibitors. 1 Our knowledge regarding the pathogenesis, risk factors and treatment may be further enhanced by studying conditions that tend to co-occur with HS. Here, we describe five cases where HS occurred in Down syndrome (DS). The cases are summarized in table 1 and figure 1. The disease characteristics and course of patients 1-3 have several similarities: they have therapy resistant disease located at the (ano)genital area and a pre-pubertal onset. Patients 4 and 5 are monozygotic twins whose other family members were not affected by HS. DS is caused by trisomy of chromosome 21 and occurs in approximately 1 of 1000 newborns. The co-occurrence of HS and DS has previously been described in three men and two women. 2-4 The phenotype of DS is complex and includes a broad range of cognitive and neurological deficits. Two hypotheses have been proposed regarding the cause of the DS phenotype: the developmental instability hypothesis states that developmental pathways are disrupted through a general genetic imbalance whereas the gene-dosage theory implies that increased expression of certain genes on chromosome 21 is responsible. We think that the genetic abnormalities of DS might also predispose these individuals to the development of HS. Previously, loss-of-function mutations in the genes encoding for the g-secretase (GS) complex have been identified in familial HS, including nicastrin (NCSTN), presenilin-1 (PSEN1) and presenilin enhancer 2 (PSENEN). 5 GS is a transmembranous enzyme complex that enhances intracellular Notch signaling by cleavage of the Notch receptor (figure. 2). Humans have four Notch receptors of which Notch-1 and Notch-2 are predominantly expressed in the epidermis. 6 GS-deficiency and inhibition of Notch-1 and -2 in mice causes replacement of hair follicles by epidermal cysts and diminished sebaceous gland differentiation, which are typical features of HS. 5 Impaired Notch signaling also inhibits the generation of natural killer cells and causes an insufficient suppression of the innate immune system once it is activated, resulting in a compromised defense mechanism and continuing inflammatory activity, respectively. 5 GS is also a key player in the development of Alzheimer s disease (AD). By the fourth decade of life characteristic β-amyloid (Aβ) brain plaques start to develop in DS that eventually give rise to AD. Aβ is a product resulting from GS-cleavage of the amyloid precursor protein (APP). APP is also strongly expressed in the human epidermis. 7 The gene encoding for APP has shown to be located on chromosome 21 and its expression is therefore, in accordance with the gene dosage hypothesis, probably increased in DS. APP and one of its other cleavage products sappα (secretory N-terminal ectodomain of APP) stimulate keratinocyte adhesion, migration 39

41 and proliferation. 7 This makes DS patients prone to keratinocyte hyperproliferation and follicular plugging, which are major histopathological features of HS. Furthermore, Berezovska et al. 8 demonstrated that APP and the Notch receptor are competitive substrates for GS and that Notch-1 signaling was diminished in primary neurons overexpressing APP. The increased amounts of APP that need to be processed by GS in DS might therefore occur at the expense of Notch processing. The genes encoding for the Notch receptors and GS are in contrast to APP not located on chromosome 21. Thus, increased APP expression might represent the missing link between HS and DS by enhancing keratinocyte activity as well as by being a competitive substrate for Notch receptors, leading to impairment of Notch-1 and -2 signaling (figure. 2). Finally, obesity and a dysregulated immune system might also contribute to an association between DS and HS. The majority of the patients in our case series were overweight or obese (four out of five patients). Indeed, the prevalence of obesity is higher in children with DS compared to healthy children, making them more prone for the development of HS as well as to a more severe course of the disease. Additionally, DS patients are more susceptible to the development of infections and autoimmune disorders, like celiac disease, due to intrinsic immunological defects. 9 With five new cases we strengthen the thought that DS and HS are associated. We hypothesize that this results from increased APP expression, an altered immune system and increased prevalence of obesity in DS. Further studies comparing the expression of Notch receptors and APP in the epidermis of DS patients and controls are needed to confirm our hypothesis. Patient 1 Patient 2 Patient 3 Patient 4 a Patient 5 a Seks Female Female Male Female Female Age (years) Age at disease onset (years) BMI (kg m -2 ) Family history Negative Negative Negative Negative b Negative b Location affected by HS Genital area Anogenital area Genital area and upper Armpits and groins Armpits and groins legs Treatment history SA, anti-androgens, SA, i.l. Corticosteroids, TA, SA SA, surgery SA, surgery prednisone, surgery etanercept, infliximab, surgery Current treatment infliximab 5 mg kg -2 every 4 weeks i.l. Corticosteroids Doxycycline 100 mg daily None None a Monozygotic twin sisters; b except for the monozygotic twin sister no other family members were affected. BMI, body mass index; HS, hidradenitis suppurativa; i.l., intralesional; SA, systemic antibiotics; TA, topical antibiotics. Table 1. Characteristics of the patients with Down syndrome 40

42 (a) (b) Figure 1. (a) Patient 1 with erythematous nodules, fistulae and scarring located on the genital area, before treatment with infliximab. (b) Four months later after four infusions of infliximab; the hidradenitis suppurativa has improves to some extent but there are still multiple active nodules and fistulae present. Aβ Aβ Notch receptor Aβ Notch receptor extracellular GS extracellular GS intracellular intracellular NICD NICD NICD NICD Nucleus Nucleus (a) NICD (b) NICD Figure 2. (a) Normal situation: amyloid precursor protein (APP) and the Notch receptor are both processed by g-secretase (GS). APP is cleaved by GS whereupon β-amyloid (Aβ) is released. The Notch receptor is also cleaved by GS leading to the release of the nuclear intracellular domain (NICD). NICD enters the nucleus where it activates the transcription factor for the Notch target genes that enhance epidermal differentiation and immune regulation. (b) patients with Down syndrome: increased expression of APP changes the substrate pool of GS. Increased amounts of GS are required for APP processing occurring at the expense of the Notch receptor. This leads to a functional GS deficiency for Notch processing whereupon the release of the NICD is prevented resulting in impaired intracellular Notch signaling. 41

43 REFERENCES Alikhan A, Lynch PJ, Eisen DB. Hidradenitis suppurativa: a comprehensive review. J Am Acad Dermatol 2009; 60:539-61; quiz Borbujo Martinez J, Bastos Amigo J, Olmos Carrasco O, et al. Suppurative hidradenitis in Down s syndrome. Apropos of three cases. An Esp Pediatr 1992; 36: Mebazaa A, Ben Hadid R, Cheikh Rouhou R, et al. Hidradenitis suppurativa: a disease with male predominance in Tunisia. Acta Dermatovenerol Alp Panonica Adriat 2009; 18: Mengesha YM, Holcombe TC, Hansen RC. Prepubertal hidradenitis suppurativa: two case reports and review of the literature. Pediatr Dermatol 1999; 16: Melnik BC, Plewig G. Impaired Notch-MKP-1 signalling in hidradenitis suppurativa: an approach to pathogenesis by evidence from translational biology. Exp Dermatol 2013; 22: Massi D, Panelos J. Notch signaling and the developing skin epidermis. Adv Exp Med Biol 2012; 727: Herzog V, Kirfel G, Siemes C, Schmitz A. Biological roles of APP in the epidermis. Eur J Cell Biol 2004; 83: Berezovska O, Jack C, Deng A, et al. Notch1 and amyloid precursor protein are competitive substrates for presenilin1-dependent gamma-secretase cleavage. J Biol Chem 2001; 276: Kusters MA, Verstegen RH, Gemen EF, de Vries E. Intrinsic defect of the immune system in children with Down syndrome: a review. Clin Exp Immunol 2009; 156:

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46 4GENE EXPRESSION PROFILING IN SKIN AND BLOOD IN HIDRADENITIS SUPPURATIVA J.L. Blok 1, K. Li 2 ; C. Brodmerkel 2 ; B. Horváth 1, M.F. Jonkman 1 1. Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands 2. Janssen Research & Development, LLC, Spring House, PA, U.S.A. Submitted 45

47 Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by recurrent abscesses, nodules and sinus tract formation that is mainly localized in the body folds. The pathogenesis of HS is poorly understood. Mutations in genes encoding for essential compounds of the transmembrane protease g-secretase, including NCSTN, PSEN1 and PSENEN, have been identified in familial HS. 1,2 These mutations may result in impaired Notch signaling promoting cyst formation and continuing inflammatory activity. 3 Additionally, certain single nucleotide polymorphisms (SNPs) at the promoter region of the tumour necrosis factor - alpha (TNF-α) gene were found to be associated with a greater susceptibility for the development of HS as well as to influence the natural course of the disease. 4 Furthermore, elevated levels of IL-1, TNF-α and IL-10 were demonstrated in both lesional and perilesional skin of HS patients, 5 and increased expression of the IL-23/Th-17 pathway was found in lesional skin. 6 However, the mechanism behind these pro-inflammatory changes in HS is still largely unknown. To acquire a better understanding of the molecular pathogenesis of HS, we performed mrna microarray studies to compare gene expression in lesional skin to healthy skin of HS patients. Also, the gene expression profile in whole blood of patients and unaffected individuals was determined. Seventeen HS patients were included. Whole blood was collected from all subjects and ten healthy controls. Skin biopsies of 3 mm were obtained from inflammatory lesions in all 17 patients. Additionally, an extra biopsy from clinically healthy skin of the upper arm or leg was obtained from 13 patients. The skin samples were immediately frozen in liquid nitrogen and subsequently stored at -80 degrees Celsius. mrna was extracted from skin samples using the Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen Inc., Valencia, CA) and from whole blood with the Qiagen PAXgene blood RNA kit according to the manufacturer s instructions. RNA was hybridized to the GeneChip HT HG-U133+ PM Array (Affymetrix, Santa Clara, CA). Pathway analyses were conducted with QIAGEN s Ingenuity Pathway Analysis (IPA, ingenuity). Array Studio software version 8.0 (OmicSoft Corp., St. Morrisville, NC) was used to analyze microarray data. Dysregulated genes were identified using a general linear model. Expression modulation with a fold change >2 or <-2 and an FDR-adjusted p-value < 0.05 were considered significant. A significant difference was observed in mrna expression between lesional and clinically healthy skin of HS patients, with over 1145 probe sets representing over 800 genes having at least a two-fold change (p <0.05). Patients with the most dysregulated gene profile were more likely to have longstanding disease (>15 years) (figure 1a). Pathway analyses of the modulated genes were mostly related to inflammation, including cell adhesion, diapedesis and 46

48 extravasation as well as immune cell signaling and communication pathways (figure 1b). An interesting finding is involvement of the atherosclerosis signaling pathway in lesional HS skin as it supports the current thought that the inflammatory response in HS is associated with metabolic syndrome. 7 Expression of NCSTN, PSEN1 and PSENEN was not modulated in lesional compared to clinically healthy skin of patients. No significant differences were identified in whole blood mrna expression between patients (N=17) and healthy controls (N=10) (data not shown). In summary, we demonstrated significant altered gene expression in lesional HS skin compared to clinically healthy skin of patients. This, in addition to the finding that whole blood RNA expression did not differ between HS patients and healthy subjects, implicates that activated cells in HS reside in affected tissue. This may be due to migration of leucocytes from the circulation into skin tissue in response to released inflammatory chemokines. Our results implicate that the inflammatory reaction is restricted to the skin of specific anatomical areas and HS may therefore be considered as a localized rather than a generalized skin disease. However, our results must be interpreted with caution as the sample size was relatively small. Regardless whether local dysregulation of the atherosclerotic pathway is caused by the inflammatory process or a secondary event due to an unhealthy lifestyle, the clinician should be aware of metabolic syndrome in HS patients as early detection and treatment may prevent cardiovascular complications. As perilesional skin of HS patients already shows histological abnormalities, 8 one may hypothesize that the skin type of HS patients in general is genetically different from normal human skin, making patients prone to the development of HS lesions under certain mechanical and lifestyle triggers. Therefore, investigating differences in gene expression between clinically healthy skin from predilection sites of HS patients and skin of unaffected individuals from the same sites would be an interesting additional study. 47

49 (a) Skin type Disease duration Non lesional Lesional 5 15 (b) Granulocyte Adhesion and Diapedesis Agranulocyte Adhesion and Diapedesis Atherosclerosis Signaling Hepatic Fibrosis / Hepatic Stellate Cell Activation Primary Immunodeficiency Signaling Communication between Innate and Adaptive Immune Cells Dendritic Cell Maturation Complement System Systemic Lupus Erythematosus Signaling Leukocyte Extravasation Signaling - log (p-value) Figure 1. (a) The heat-map of differentially expressed genes in HS skin based on mrna microarray analysis. A significant difference was observed in mrna expression between lesional (grey boxes) and non-lesional patient skin (red boxes). Patients with the most dysregulated gene profile were more likely to have longstanding disease. (b) The top ten canonical pathways involved in the disease profile of patient skin. 48

50 REFERENCES 1. Wang B, Yang W, Wen W, et al. Gamma-secretase gene mutations in familial acne inversa. Science 2010; 330: Pink AE, Simpson MA, Brice GW, et al. PSENEN and NCSTN mutations in familial hidradenitis suppurativa (Acne Inversa). J Invest Dermatol 2011; 131: Melnik BC, Plewig G. Impaired Notch-MKP-1 signalling in hidradenitis suppurativa: an approach to pathogenesis by evidence from translational biology. Exp Dermatol 2013; 22: Savva A, Kanni T, Damoraki G, et al. Impact of Toll-like receptor-4 and tumour necrosis factor gene polymorphisms in patients with hidradenitis suppurativa. Br J Dermatol 2013; 168: van der Zee HH, de Ruiter L, van den Broecke DG, et al. Elevated levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-alpha and IL-1beta. Br J Dermatol 2011; 164: Schlapbach C, Hanni T, Yawalkar N, Hunger RE. Expression of the IL-23/Th17 pathway in lesions of hidradenitis suppurativa. J Am Acad Dermatol 2011; 65: Gold DA, Reeder VJ, Mahan MG, Hamzavi IH. The prevalence of metabolic syndrome in patients with hidradenitis suppurativa. J Am Acad Dermatol 2014; 70: van der Zee HH, de Ruiter L, Boer J, et al. Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions. Br J Dermatol 2012; 166:

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52 5Systemic therapy with immunosuppressive agents and retinoids in hidradenitis suppurativa: a systematic review J.L. Blok 1, S. van Hattem 1, M.F. Jonkman 1, B.Horváth 1 Department of Dermatology 1, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands Published in the British Journal of Dermatology, 2013;168:

53 ABSTRACT Hidradenitis suppurativa (HS) is a difficult disease to treat. Although the pathogenesis of this inflammatory skin disease is largely unknown, the important role of the immune system has been demonstrated in both experimental and clinical studies. Clinicians are therefore increasingly prescribing systemic treatments with immunosuppressive agents, but the more traditional systemic retinoids, especially isotretinoin, also remain relatively common therapies. In order to provide an overview of all currently available systemic immunosuppressive agents and retinoids for the treatment of HS, a systematic search was performed using MEDLINE and EMBASE databases. All published papers concerning systemic retinoids or immunosuppressive treatment for HS in adults were included. The primary endpoint was the percentage of significant responders, moderate responders and non-responders. Other endpoints were the relapse rate and adverse events. In total 87 papers were included, comprising 518 patients with HS who were treated with systemic retinoids, biologic agents or other immunosuppressive agents including colchicine, ciclosporin, dapsone or methotrexate. The highest response rates were observed with infliximab, adalimumab and acitretin. Overall, the quality of evidence was low and differed between the agents, making direct comparisons difficult. However, based on the amount of evidence, infliximab and adalimumab were the most effective agents. Acitretin was also effective in HS, although the quality of the evidence was low. The therapeutic effect of isotretinoin is questionable. Randomized controlled trials are needed to confirm the effectiveness of acitretin as well as to identify the most effective immunosuppressive agent in HS. 52

54 INTRODUCTION Hidradenitis suppurativa (HS), also known as acne inversa, is a chronic inflammatory skin disease characterized by recurrent, painful, deep-seated nodules and abscesses. In an advanced stadium sinus tracts are formed, eventually leading to fibrotic scars, dermal contractures and induration of the skin. 1,2 Lesions typically occur on inverse, apocrine gland-bearing skin, like the axillary, inguinal and anogenital regions. Quality of life is greatly impaired in HS. 3,4 In addition to lifestyle changes, therapeutic options include topical and systemic antibiotics, antiandrogens, systemic retinoids, immunosuppressive agents, laser treatment, and surgery. 5-7 Since an effective monotherapy is lacking, it is often required to combine different treatment modalities to achieve some improvement. Although the pathogenesis of HS is largely unknown, follicular hyperkeratinisation followed by follicular occlusion is a primary feature of HS Several factors probably contribute to these histological changes, including smoking, sweating, obesity and hormonal changes. 12 The important role of the immune system in HS has been underlined in recent studies, where several observations have been observed, including involvement of the interleukin (IL)-12/ Th1 IL-23/Th17 pathways, and increased TNF-α in the skin and serum In addition, there is a deficiency of IL-22 and IL-20 in lesional HS skin leading to decreased antimicrobial protein (AMP) levels, making the skin prone to bacterial infection. 16 In conclusion, both clinical and experimental studies support the use of anti-inflammatory drugs and retinoids in the treatment of HS and several different types of these agents are currently available. However, there is no consensus on which agent is most effective for HS. Therefore, this review aims (i) to evaluate all existing evidence to date for the use of systemic immunosuppressive agents and systemic retinoids in HS and (ii) to assess their current position in the treatment of HS. 53

55 PATIENTS AND METHODS Inclusion and exclusion criteria Included in the study were all fully published papers that reported on the clinical effects of any systemic immunosuppressive agents or systemic retinoids in HS localized at the typical inverse. Patients had to be 18 years or older. Studies not exclusively dealing with HS were excluded, unless data for HS could be extracted separately. Studies were excluded if insufficient details were given on treatment regime in respect of dosing, treatment duration and concomitant immunosuppressive medication. There were no language restrictions. Types of outcome measures The efficacy of treatment was classified for each patient as significant response, moderate response or nonresponse. A significant response was defined as a reduction of the Sartorius score with 50%, improvement in quality of life of >50% or if stated so by the authors. A moderate response comprised score reductions <50% or if stated so by the authors. The primary endpoint comprised the percentage of significant responders, moderate responders and nonresponders. If a study did not report individual results, all patients of that study were categorized corresponding to the reported mean results. Dropouts were considered to be nonresponders. The secondary endpoint was the percentage of responders that relapsed during or after discontinuation of treatment, and the tertiary endpoint comprised adverse events (AEs). Identification of studies Databases were systematically searched by two independent authors (SvH and JLB) for studies dated up to May A search was conducted using EMBASE (search terms: hidradenitis suppurativa /exp OR hidradenitis suppurativa OR (hidraden* AND suppurativ*) OR acne inversa OR inverse acne ) and Medline (search terms: Hidradenitis Suppurativa [MeSH] OR (hidraden* AND suppurativ*) OR acne inversa OR inverse acne ). Reference lists of included papers and relevant reviews were manually searched to identify additional papers. Data extraction Two authors (JLB and SvH) independently conducted data extraction by using standardized forms. Discrepancies between the researchers were resolved through discussion. Authors were not contacted for missing data. Data were analysed by means of descriptive statistics. 54

56 Quality assessment The quality of evidence was assessed by grading as follows: A, systematic review or metaanalysis, randomized controlled trial with consistent findings, or all-or-none observational study; B, lower quality clinical trial or study with limitations and inconsistent findings, cohort study or case-control study; C, consensus guidelines, usual practice, expert opinion, or case series

57 RESULTS Figure 1 shows the process of study selection, at the end of which 87 papers were included, comprising a total of 518 patients. The immunosuppressive therapies evaluated in these papers were biologics, colchicine, ciclosporin, methotrexate and dapsone. Treatment with systematic retinoids included the use of acitretin and isotretinoin. Two papers dealt with two immunosuppressive agents and these studies are therefore discussed in subheadings of the Results section. 18,19 The level of evidence of included papers is described for each immunosuppressive agent in Table 1. A summary of the results is described in Figure 2. 56

58 Figure 1. Study selection 57

59 Biologics Adalimumab Studies: We identified 15 papers studying a total of 68 patients One study had a randomized double-blind placebo-controlled design (evidence level A). 31 In one retrospective cohort study, the effectiveness of adalimumab was compared to infliximab (evidence level B). 19 Four other studies had an evidence level of B, 20,21,23,32 and the remaining 9 studies had level C. 18,22,24-30 Dosing regimes varied from mg in a frequency ranging from weekly to every other week. The treatment duration was 1 year in three studies, 21,24,26 6 months in six studies 18,20,22,27,31,32 and unclear in six studies. 19,23,25,28-30 One patient was simultaneously to adalimumab treated with methotrexate for the first 2 months. 26 The follow-up time varied between studies, ranging from 13 weeks-29 months. Primary endpoints: in total, 30/68 patients (44%) showed a significant response to adalimumab, 24 patients (35%) had a moderate response and 14 patients did not respond (21%). (Figure 2) Secondary endpoints: one paper reported that the majority of the seven responding patients showed recurrence of HS after 1 year of follow-up; however, individual numbers could not be extracted. 19 Occurrence of relapse was described for 35 of the remaining 42 responders: 22/35 (66%) relapsed within 3-10 months after discontinuation of treatment. 21,23,25,26,28,31 Seven of the 35 responders (20%) relapsed during treatment, but symptoms improved in all when the dose of adalimumab was increased. 23,26,28 Tertiary endpoints: Adverse events (AEs) are described in Table 2. Six papers did not report on AEs. 22,24,27-30,32 58

60 Immunosuppressive agent (total nr of papers) Nr of level (% within Nr of level A evidence group) B evidence (%within Nr of level group) C evidence Biologicals a b 71 (% within % of % of non group) responders responders Adalimumab Etanercept Infliximab Ustekinumab Retinoids Acitretin/etretinate Isotretinoin Other Ciclosporin Dapsone Colchicine Methotrexate a one paper compared adalimumab with infliximab, and is included as level B for both adalimumab and infliximab. 19 b One paper describes the efficacy of adalimumab and etanercept; therefore it has been included as level C for both adalimumab and etanercept. 18 Table 1. Level of evidence for all included studies Etanercept Studies: nine papers comprising 54 patients evaluated the effect of etanercept on HS. 18,33-40 One study had a randomized double-blind placebo controlled design (evidence level A); however, after 12 weeks all patients received open-label etanercept. 33 We included only those 10 patients who were initially allocated to etanercept group. Five papers had evidence level B 34,35,37,39,40 and 3 papers level C. (18,36,38) Dosing schedules varied from 25 mg to 50 mg once or twice weekly to 100 mg weekly. Treatment duration was 3 months in two papers, 34,35 6 months in two 33,39 and around 1 year or longer in four papers. 18,36-38 The follow up time was weeks. Long term results of the patients described by Giarmellos et al. 35 were reported in a separate paper. 41 Primary endpoints: a significant response to etanercept was observed in 21/54 patients (39%), whereas nine patients (17%) had moderate improvement and 24 patients (44%) did not respond to the treatment. (Figure 2) Secondary endpoints: in total 18/30 responders (60%) relapsed after treatment was discontinued. The time to relapse ranged from immediately after stopping of treatment until 8 months thereafter, but the majority had recurrence of HS lesions within 2 months. Tertiary endpoint: Table 2 describes the tertiary endpoints. One study did not report on AEs

61 Infliximab Studies: the efficacy of infliximab was evaluated in 42 papers, comprising 147 patients. One study had a randomized double-blind placebo controlled design (evidence level A) but after 8 weeks all patients received infliximab. 42 Only those 15 patients who were initially allocated to infliximab were included. Evidence levels B and C were found in seven 19,43-48 and 34 studies respectively. One study compared the effect of infliximab with another treatment, namely adalimumab. 19 Almost all 147 patients received intravenous infliximab 5 mg/kg at weeks 0, 2 and 6. In 10 studies treatment was discontinued after these three administrations. 19,46,57,61,63,65-67,70,82 However, the majority of patients received maintenance therapy every 6-8 weeks. Dosing schedules were not clear in five papers. 50,69,71,74,75 The duration of treatment was >1 year in nine studies. 45,48,49,53,56,60,64,79,80 In four papers patients, in addition to infliximab, patients received methotrexate, which might have prevent the formation of autoantibodies. 45,49,60,64 Simultaneously to infliximab, patients were treated with azathioprine in two studies, 70,72 prednisolone in one study, 77 prednisolone and ciclosporin in one study, 68 and with oral azathioprine and methylprednisolone in one study. 71 Primary endpoints: a significant improvement was seen in 74/147 patients (50%); 57 patients (39%) showed moderate improvement and 16 patients (11%) had no response (Figure 2) Secondary endpoints: Only 10/131 responders (8%) experienced recurrence of HS during treatment, 48,49,55,60,68,79 and 26 responders (20%) relapsed within 2 weeks to 3 years after discontinuation of therapy. 42,46,52-54,57,62,63,67,73,75 One paper reported that the majority of patients had recurrence of HS one year after discontinuation of treatment, however, individual numbers could not be extracted. 19 Tertiary endpoints: Fourteen studies did not report on AEs. 50,52,55,59-61,65,66,69-71,74,78,82 AEs were observed in 19 studies (Table 2). 19,42-48,57,63,64,68,72,73,75,76,79-81 Ustekinumab Studies: Two papers comprising a total of four patients, evaluated the effect of ustekinumab (both evidence level C). 83,84 The patients received 45 mg ustekinumab at weeks 0, 4 and 12. Subsequently, one patient received injections every 3 months, 84 and three patients every 2 months. 83 Three patients were treated for at least 6 months and two of them were probably still on treatment at the time the paper was written. 83 Primary endpoints: two of the four patients (50%) showed a significant response, one patient had a moderate response (25%) and one patient (25%) did not respond. (Figure. 2) Secondary endpoints: one responding patient had temporary relapses every 2 weeks prior to 60

62 his next injection, but after administration. 84 In another responding patient lesions recurred after 6 months. 83 The dose ustekinumab was therefore increased to 90 mg and his disease has improved ever since. The remaining one responding patient did not relapse during treatment. 83 Tertiary endpoint: AEs were reported in one paper (Table 2). 83 Retinoids Isotretinoin Studies: seven papers evaluated the effect of oral isotretinoin, and comprised a total of 174 patients. Level B evidence was found in four papers and level C in three The daily dosages of isotretinoin were mg/kg and treatment duration was 4-12 months. One patient was pretreated with prednisone and erythromycin, followed by the gradual introduction of isotretinoin. 89 Primary endpoints: significant improvement was observed in 32/174 patients (18%), moderate improvement in 30/174 patients (17%) and no response was observed in 112 patients (64%) (Figure 2). Secondary endpoints: one study comprising 14 responders did not mention whether recurrences occurred after cessation of therapy. 85 Of the remaining 48 responders, six patients (13%) relapsed within a couple of months after discontinuation of treatment. Tertiary endpoint: Two studies did not report on AEs. 85,89 All remaining 18 patients experienced AEs (Table 2). Acitretin and etretinate Studies: Acitretin is a metabolite of etretinate and has replaced treatment with etretinate in a variety of disorders, as it has a much shorter elimination half-life and is equally effective. Six papers reported on the treatment of HS with these retinoids, and comprised 22 patients The level of evidence was B in two studies; 92,96 the remaining papers were level C. Patients treated with etretinate received daily doses of mg/kg and the daily doses for acitretin ranged from mg/kg. The duration of treatment was 3-39 months. Primary endpoints: significant improvement was seen in 16 of 22 patients (73%), five patients (23%) improved moderately and one patient (5%) did not respond to the therapy (Fig. 2). Secondary endpoints: No relapses during therapy were described. Acitretin or etretinate treatment was eventually discontinued in 17 patients. Within six months after cessation of therapy, six of 17 patients (35%) had recurrence. Eight patients (47%) relapsed >1 year after discontinuation of treatment. 61

63 Tertiary endpoint: The AEs that were reported are shown in table 2. Two studies did not report on AEs. 93,97 For one study, data on AEs could not be extracted separately for HS. 96 Other therapies Dapsone Studies: the effect of dapsone was described in three papers all with evidence level C In total 34 patients were treated with doses of mg daily during months. The majority of patients was still on treatment at the time of study closure. Primary endpoints: A significant improvement was seen in 12/34 patients (35%) showed a significant response, seven patients (21%) had a moderate response and 15 patients (44%) did not respond (Figure 2). Secondary endpoints: Two studies reported that discontinuation of therapy led to a rapid recurrence of HS lesions in all patients, and that dapsone treatment could therefore not be terminated. 99,100 Two out of nine responders in the study of Yazdanyar et al. 98 also rapidly relapsed after cessation of treatment; however, re-introduction of dapsone led to rapid improvement. Tertiary endpoint: Adverse events are shown in table 2. Colchicin Studies: we identified one paper (evidence level B) describing eight patients who were treated with colchicine 0.5 mg twice daily during 4 months. 101 Primary endpoints: Two out of eight patients (25%) had a moderate respons and six out of eight patients (75%) did not respond to colchicines (Figure 2). Secondary endpoints: these were not stated. Tertiairy endpoint: The observed AEs are shown in Table 2. Ciclosporin Studies: we identified three papers (evidence level C) on ciclosporin Four patients were treated with ciclosporin 2-6 mg/kg daily for 4-15 months. Two patients were concomitantly treated with prednisolone and oral antibiotics. 102,103 Primary endpoints: a significant response was observed in two of four patients (50%) and the remaining two patients had a moderate response (Figure 2). 62

64 Secondary endpoints: in one patient ciclosporin was discontinued after 4 months, leading to a recurrence 4 months later. 102 Two patients were still on treatment at the time the paper was published and did not experience any relapses. It was not reported whether the last patient experienced relapse. 104 Tertiary endpoint: These were not reported in any of the studies. Methotrexate Studies: we identified one paper that reported on the effectiveness of methotrexate in HS. 105 It concerned an open study in which two patients received a weekly dose of 12.5 mg and one patient received 15 mg. Treatment duration was 6 weeks, 4 months or 6 months. Primary endpoints: none of the three patients responded to the treatment with methotrexate (Figure 2). Secondary endpoints: since none of the patients showed a response to the treatment, time to relapse was not applicable. Tertiary endpoint: Adverse events were not reported. 63

65 SI = significant responders. MI = moderate responders. NR = non-responders. N = number of patients. Figure 2. Overview of total number of papers and treated patients for each agent, including response rates. 64

66 Immunosuppressive agent (number of treated patients) Adalimumab (68) Etanercept (54) Observed adverse events (frequency) Painful injection site,a mild infections (10), non-specific gastrointestinal symptoms (3), non-specific rash (3), fatigue (3), elevated liver enzymes (2), reactivation of Epstein-Barr virus (1), severe infusion reaction urticaria (1), facial cellulitis (1), irritation ears (1), hoarseness (1), headache (1), dry eyes (1), muscle chest pain (1), dry skin (1), hay fever (1), Injection site reaction,a upper respiratory tract infection (4), nausea (3), paresthesias (2), chest pain (2), cellulitis (2), muscle cramps (1), flu-like symptoms (1), hypertension (1), elevated cholesterol (1) Infliximab (147) Non-specific side effects (14), headache (7), acute arthritis/myalgia (8), hypersensitivity reactions (5), influenza-like illness (4), dizziness (3), asthenia (3), numbness in legs/neuropathy (3), skin rash (3), anaphylactic shock (1), pneumococcal sepdis (1), localized tuberculosis infection (1), pustular lesions lower limbs (1), fever (1), hypertension (1), herpes zoster (1), colon cancer (1), cervical abscess (1), dyspnoea (1), lupus like reaction (1) Nr of patients that discontinued treatment due to adverse events 1 (23) 3 (34) 31 (43-46,48,57,63,68,72,73,75,76,78-80) Ustkeinumab (4) Cystitis (1), psoriasiform dermatitis (1), HS lesion infected by staphylococcus aureus None (1) Isotretinoin (174) Cheilitis/xeorsis (15), usual side efectsb (3), arthralgia (1), headache (1) 10 (86) Acitretin/etretinate (22) Cheilitis/xerosis (13), alterations in lipids (4), altered triglyceride levels (3), hypertrichosis/photosensitivity (2), alopecia (2), depression/fatigue (1), headache (1), loss of concentration (1), elevated cholesterol (2), buzzing ears (1), joint pain (1) Dapsone (34) Anemia/hemolysis (4), nausea (3), dizziness (2), tiredness (2), headache (2), elevated None bilirubine (1), rash (1), gloomy mood (1), malaise (1) Colchicine (4) Nausea (3), diarrhea (3) 1 (101) Methotrexate (3) Adverse events not stated none Ciclosporin (4) Adverse events not stated none a some studies reported that this event occurred in 'several patients', without mentioning exact numbers. b Probably xerosis/cheilitis 2 (92,95) 65

67 DISCUSSION To the best of our best knowledge, this is the first systematic review specifically aimed to analyze all currently available evidence of immunosuppressive agents and systemic retinoids for the treatment of HS. In total 518 patients were analysed, divided over 87 papers. The majority of patients (n = 273) was treated with a biological agent. Overall, the quality of the included papers was low; only three randomized controlled trials were identified, all on biologics. 31,33,42 The majority of papers were case reports or series, bringing along a risk of publication bias. Two papers were not identified by our search strategy due to the fact that they were not incorporated in Medline or EMBASE. 96,97 Based on our results, the most effective agents for HS were infliximab, adalimumab and acitretin with respectively 89%, 79% and 96% of patients, respectively, responding to treatment. However, as the results of acitretin were based on far fewer patients and were of a lower level of evidence than the results for infliximab and adalimumab, caution must be taken when directly comparing the efficacy of these agents. The positive results of infliximab and adalimumab are in accordance with the findings of Van Rappard et al. 106 Acitretin for HS is barely mentioned in the literature, however, its positive effect is pharmacologically reasonable, as the primary event in HS is follicular occlusion and acitretin induces normalization of the epithelial cell proliferation and differentiation. 107,108 Not surprisingly, isotretinoin is ineffective for HS as this agent primarily works on sebaceous glands, which are not involved in the pathogenesis of HS. 109,110 The observation that 35% of treated patients still responded to isotretinoin, is more likely to be due to the immunomodulatory effects of this retinoid. 111 The highest quality of evidence was identified for etanercept, which enables us to conclude that the efficacy (56% responders) was relatively low. Only a few patients have been treated with ustekinumab, ciclosporin, dapsone, methotrexate and colchicine. It has been shown that the IL-12/IL-23 pathway is upregulated in HS, therefore there is a rationale for the efficacy of ustekinumab (an inhibitor of this pathway), and the first results of this agent are indeed promising. 83,84 However, clinical trials are needed to confirm its effect. The same applies for ciclosporin; although all patients responded to treatment, this agent has been studied in only four patients, making it impossible to draw any definite conclusions. The efficacy of dapsone is doubtful, methotrexate as a monotherapy seems of little value and colchicine is not effective in HS. Although long-term results and relapse rates were not available for many papers on biologics, recurrence of HS occurred frequently during therapy or within a couple of months after cessation of biologic therapy. In contrast, Boer and Nazary 92 achieved long-term remission (i.e. >1 year) in a majority of patients treated with acitretin, indicating that this is probably also 66

68 effective on the long term. However, this observation needs to be confirmed in bigger trials since only 12 patients were included. Adverse events were observed with all agents, except for ciclosporin and methotrexate, where it was not stated. The highest number of withdrawals due to AEs occurred with infliximab and isotretinoin. Other reviews also showed that the risk of withdrawal is higher during infliximab therapy compared with adalimumab and etanercept therapy. 106,112 The most common AE during acitretin therapy is cheilitis which can be very disturbing for patients. Moreover, the most important disadvantage of acitretin is that it has extremely teratogenic side effects. 113 Therefore, this agent should mainly be reserved for men and sterilized or postmenopausal women. A limitation of this review, and any other review on HS treatment, is heterogeneity between the studies in respect of study design, number of included participants, the severity of HS and the timing and methods for outcome assessments. Therefore, caution must be taken in directly comparing the different treatment options of HS. In conclusion, this review indicates that, based on the evidence today, infliximab and adalimumab are the most effective immunosuppressive agents for HS. Additionally, acitretin is a promising agent and definitely worth considering in men and sterilized or postmenopausal women, although high quality evidence is lacking for its administration in HS. Also, these data strongly indicate that there is a need for randomized controlled clinical trials in order to identify the most effective treatment targets and the most effective therapy for HS. 67

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71 28. Scheinfeld N. Treatment of coincident seronegative arthritis and hidradentis supprativa with adalimumab. J Am Acad Dermatol 2006; 55: Alzaga Fernandez AG, Demirci H, Darnley-Fisch D, Steen DW. Interstitial keratitis secondary to severe hidradenitis suppurativa: A case report and literature review. Cornea 2010; 29: Koilakou S, Karapiperis D, Tzathas C. A case of hidradenitis suppurativa refractory to anti-tnfalpha therapy in a patient with Crohn s disease. Am J Gastroenterol 2010; 105: Miller I, Lynggaard CD, Lophaven S, et al. A double-blind placebo-controlled randomized trial of adalimumab in the treatment of hidradenitis suppurativa. Br J Dermatol 2011; 165: Van der Zee, Laman JD, De Ruiter L, et al. Adalimumab (antitumour necrosis factor-(alpha)) treatment of hidradenitis suppurativa ameliorates skin inflammation: An in situ and ex vivo study. Br J Dermatol 2012; 166: Adams DR, Yankura JA, Fogelberg AC, Anderson BE. Treatment of hidradenitis suppurativa with etanercept injection. Arch Dermatol 2010; 146: Lee RA, Dommasch E, Treat J, et al. A prospective clinical trial of open-label etanercept for the treatment of hidradenitis suppurativa. J Am Acad Dermatol 2009; 60: Giamarellos-Bourboulis EJ, Pelekanou E, Antonopoulou A, et al. An open-label phase II study of the safety and efficacy of etanercept for the therapy of hidradenitis suppurativa. Br J Dermatol 2008; 158: Zangrilli A, Esposito M, Mio G, et al. Long-term efficacy of etanercept in hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2008; 22: Cusack C, Buckley C. Etanercept: effective in the management of hidradenitis suppurativa. Br J Dermatol 2006; 154: Henderson RL,Jr. Case reports: treatment of atypical hidradenitis suppurativa with the tumor necrosis factor receptor-fc fusion protein etanercept. J Drugs Dermatol 2006; 5: Sotiriou E, Apalla Z, Ioannidos D. Etanercept for the treatment of hidradenitis suppurativa. Acta Derm Venereol 2009; 89: Lopez-Martin C, Tortajada Goitia B, Faus Felipe V, et al. Partial response to etanercept in the treatment of hidradenitis suppurativa. Farm Hosp 2011; 35:189.e1-189.e4. 70

72 41. Pelekanou A, Kanni T, Savva A, et al. Long-term efficacy of etanercept in hidradenitis suppurativa: results from an open-label phase II prospective trial. Exp Dermatol 2010; 19: Grant A, Gonzalez T, Montgomery MO, et al. Infliximab therapy for patients with moderate to severe hidradenitis suppurativa: a randomized, double-blind, placebo-controlled crossover trial. J Am Acad Dermatol 2010; 62: van Rappard DC, Mekkes JR. Treatment of severe hidradenitis suppurativa with infliximab in combination with surgical interventions. Br J Dermatol 2012; 167: Delage M, Samimi M, Atlan M, et al. Efficacy of infliximab for hidradenitis suppurativa: assessment of clinical and biological inflammatory markers. Acta Derm Venereol 2011; 91: Brunasso AM, Delfino C, Massone C. Hidradenitis suppurativa: are tumour necrosis factor-alpha blockers the ultimate alternative? Br J Dermatol 2008; 159: Mekkes JR, Bos JD. Long-term efficacy of a single course of infliximab in hidradenitis suppurativa. Br J Dermatol 2008; 158: Fernandez-Vozmediano JM, Armario-Hita JC. Infliximab for the treatment of hidradenitis suppurativa. Dermatology 2007; 215: Usmani N, Clayton TH, Everett S, Goodfield MD. Variable response of hidradenitis suppurativa to infliximab in four patients. Clin Exp Dermatol 2007; 32: De Souza A, Solomon GE, Strober BE. SAPHO syndrome associated with hidradenitis suppurativa successfully treated with infliximab and methotrexate. Bull NYU Hosp Jt Dis 2011; 69: Dos Santos CH, Netto PO, Kawaguchi KY, et al. Association and management of Crohn s disease plus hidradenitis suppurativa. Inflamm Bowel Dis 2011; 18:E801-E von Preussen AC, Flux K, Hartschuh W, Hartmann M. Acne inversa successfully treated with infliximab. Int J Dermatol 2011; 58: Husein-ElAhmed H, Fernandez-Pugnaire MA, Ruiz-Carrascosa JC. Severe hidradenitis suppurative in an HIV-positive male: use of multiple treatment modalities, including tumor necrosis factor blockade. AIDS Patient Care STDS 2011; 25: Alecsandru D, Padilla B, Izquierdo JA, et al. Severe refractory hidradenitis suppurativa in an HIV-positive patient successfully treated with infliximab. Arch Dermatol 2010; 146:

73 54. Lasocki A, Sinclair R, Foley P, Saunders H. Hidradenitis suppurativa responding to treatment with infliximab. Australas J Dermatol 2010; 51: Poulin Y. Successful treatment of hidradenitis suppurativa with infliximab in a patient who failed to respond to etanercept. J Cutan Med Surg 2009; 13: Antonucci A, Negosanti M, Negosanti L, et al. Acne inversa treated with infliximab: different outcomes in 2 patients. Acta Derm Venereol 2008; 88: Elkjaer M, Dinesen L, Benazzato L, et al. Efficacy of Infliximab treatment in patients with severe Fistulizing Hidradenitis Suppurativa. J Crohns Colitis 2008; 2: Montes-Romero JA, Callejas-Rubio JL, Sanchez-Cano D, et al. Amyloidosis secondary to hidradenitis suppurativa. Exceptional response to infliximab. Eur J Intern Med 2008; 19:e Pedraz J, Penas PF, Garcia-Diez A. Pachyonychia congenita and hidradenitis suppurativa: no response to infliximab therapy. J Eur Acad Dermatol Venereol 2008; 22: Goertz RS, Konturek PC, Naegel A, et al. Experiences with a long-term treatment of a massive gluteal acne inversa with infliximab in Crohn s disease. Med Sci Monit 2009; 15:CS Kwan C, Chong L. A chinese man with chronic recalcitrant hidradenitis suppurativa successfully treated with infliximab. Hong Kong J Dermatol Venereol 2008; 16: Moschella SL. Is there a role for infliximab in the current therapy of hidradenitis suppurativa? A report of three treated cases. Int J Dermatol 2007; 46: Fardet L, Dupuy A, Kerob D, et al. Infliximab for severe hidradenitis suppurativa: transient clinical efficacy in 7 consecutive patients. J Am Acad Dermatol 2007; 56: Thielen AM, Barde C, Saurat JH. Long-term infliximab for severe hidradenitis suppurativa. Br J Dermatol 2006; 155: Rosi YL, Lowe L, Kang S. Treatment of hidradenitis suppurativa with infliximab in a patient with Crohn s disease. J Dermatolog Treat 2005; 16: Mekkes J, Hommes D. Treatment of suppurative hidradenitis with surgical deroofing and infliximab. Nederlands Tijdschrift voor Dermatologie en Venereologie 2004; 14:

74 67. Adams DR, Gordon KB, Devenyi AG, Ioffreda MD. Severe hidradenitis suppurativa treated with infliximab infusion. Arch Dermatol 2003; 139: Sullivan TP, Welsh E, Kerdel FA, et al. Infliximab for hidradenitis suppurativa. Br J Dermatol 2003; 149: Lebwohl B, Sapadin AN. Infliximab for the treatment of hidradenitis suppurativa. J Am Acad Dermatol 2003; 49:S275-S Roussomoustakaki M, Dimoulios P, Chatzicostas C, et al. Hidradenitis suppurativa associated with Crohn s disease and spondyloarthropathy: response to anti-tnf therapy. J Gastroenterol 2003; 38: Katsanos KH, Christodoulou DK, Tsianos EV. Axillary hidradenitis suppurativa successfully treated with infliximab in a Crohn s disease patient. Am J Gastroenterol 2002; 97: Martinez F, Nos P, Benlloch S, Ponce J. Hidradenitis suppurativa and Crohn s disease: response to treatment with infliximab. Inflamm Bowel Dis 2001; 7: van Rappard DC, Mooij JE, Baeten DL, Mekkes JR. New-onset polyarthritis during successful treatment of hidradenitis suppurativa with infliximab. Br J Dermatol 2011; 165: Torres T, Selores M. Treatment of hidradenitis suppurativa with infliximab. An Bras Dermatol 2010; 85: Garcia-Rabasco AE, Esteve-Martinez A, Zaragoza-Ninet V, et al. Pyoderma gangrenosum associated with hidradenitis suppurativa: a case report and review of the literature. Actas Dermosifiliogr 2010; 101: Lozeron P, Denier C, Lacroix C, Adams D. Long-term course of demyelinating neuropathies occurring during tumor necrosis factor-alpha-blocker therapy. Arch Neurol 2009; 66: Obadia DL, Daxbacher EL, Jeunon T, Gripp AC. Hidradenitis suppurativa treated with infliximab. An Bras Dermatol 2009; 84: Deschamps M-, Payet S, Tournadre A, et al. Efficacy of infliximab in the treatment of follicular occlusion triad. Ann Dermatol Venereol 2010; 137: Pedraz J, Dauden E, Perez-Gala S, et al. Hidradenitis suppurativa. Response to treatment with infliximab. Actas Dermosifiliogr 2007; 98:

75 80. Benitez-Macias J, Garcia-Gil D, Brun-Romero F. Fatal pneumococcal sepsis in a patient with hidradenitis suppurativa treated with infliximab. Med Clin 2008; 131: Maalouf E, Faye O, Poli F, et al. Fatal epidermoid carcinoma in hidradenitis suppurativa following treatment with infliximab [2]. Ann Dermatol Venereol 2006; 133: Suys E, D Heygere F. Infliximab for acne inversa (alias hidradenitis suppurativa)? Ned Tijdschr Dermatol Venereol 2005; 15: Gulliver WP, Jemec GB, Baker KA. Experience with ustekinumab for the treatment of moderate to severe Hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2012; 26: Sharon VR, Garcia MS, Bagheri S, et al. Management of Recalcitrant Hidradenitis Suppurativa with Ustekinumab. Acta Derm Venereol 2012; 8: Soria A, Canoui-Poitrine F, Wolkenstein P, et al. Absence of efficacy of oral isotretinoin in hidradenitis suppurativa: a retrospective study based on patients outcome assessment. Dermatology 2009; 218: Boer J, van Gemert MJ. Long-term results of isotretinoin in the treatment of 68 patients with hidradenitis suppurativa. J Am Acad Dermatol 1999; 40: Dicken CH, Powell ST, Spear KL. Evaluation of isotretinoin treatment of hidradenitis suppurativa. J Am Acad Dermatol 1984; 11: Norris JF, Cunliffe WJ. Failure of treatment of familial widespread hidradenitis suppurativa with isotretinoin. Clin Exp Dermatol 1986; 11: Fearfield LA, Staughton RC. Severe vulval apocrine acne successfully treated with prednisolone and isotretinoin. Clin Exp Dermatol 1999; 24: Jones DH, Cunliffe WJ, King K. Hidradenitis suppurativa-lack of success with 13-cis-retinoic acid. Br J Dermatol 1982; 107: Brown CF, Gallup DG, Brown VM. Hidradenitis suppurativa of the anogenital region: response to isotretinoin. Am J Obstet Gynecol 1988; 158: Boer J, Nazary M. Long-term results of acitretin therapy for hidradenitis suppurativa. Is acne inversa also a misnomer? Br J Dermatol 2011; 164:

76 93. Chow ET, Mortimer PS. Successful treatment of hidradenitis suppurativa and retroauricular acne with etretinate. Br J Dermatol 1992; 126: Hogan DJ, Light MJ. Successful treatment of hidradenitis suppurativa with acitretin. J Am Acad Dermatol 1988; 19: Scheman AJ. Nodulocystic acne and hidradenitis suppurativa treated with acitretin: a case report. Cutis 2002; 69: Stewart W. Etretinate in other diseases of keratinization. In: Medicines Publishing Foundation Symposium Series(Anonymous ) Oxford, 1984; Vahlquist A, Griffiths W. Retinoid therapy in hidradenitis suppurativa - a report of a case. Retinoids Today Tom 1990; 18: Yazdanyar S, Boer J, Ingvarsson G, et al. Dapsone therapy for hidradenitis suppurativa: a series of 24 patients. Dermatology 2011; 222: Kaur MR, Lewis HM. Hidradenitis suppurativa treated with dapsone: A case series of five patients. J Dermatolog Treat 2006; 17: Hofer T, Itin PH. Acne inversa: a dapsone-sensitive dermatosis. Hautarzt 2001; 52: Van Der Zee HH, Prens EP. The anti-inflammatory drug colchicine lacks efficacy in hidradenitis suppurativa. Dermatology 2011; 223: Rose RF, Goodfield MJ, Clark SM. Treatment of recalcitrant hidradenitis suppurativa with oral ciclosporin. Clin Exp Dermatol 2006; 31: Buckley DA, Rogers S. Cyclosporin-responsive hidradenitis suppurativa. J R Soc Med 1995; 88:289P-290P Gupta AK, Ellis CN, Nickoloff BJ, et al. Oral cyclosporine in the treatment of inflammatory and noninflammatory dermatoses. A clinical and immunopathologic analysis. Arch Dermatol 1990; 126: Jemec GB. Methotrexate is of limited value in the treatment of hidradenitis suppurativa. Clin Exp Dermatol 2002; 27:

77 106. van Rappard DC, Limpens J, Mekkes JR. The off-label treatment of severe hidradenitis suppurativa with TNF-alpha inhibitors: a systematic review. J Dermatolog Treat 2013; 24: von Laffert M, Stadie V, Wohlrab J, Marsch WC. Hidradenitis suppurativa/acne inversa: bilocated epithelial hyperplasia with very different sequelae. Br J Dermatol 2011; 164: Laffert MV, Helmbold P, Wohlrab J, et al. Hidradenitis suppurativa (acne inversa): Early inflammatory events at terminal follicles and at interfollicular epidermis. Exp Dermatol 2010; 19: Jemec GBE, Gniadecka M. Sebum excretion in Hidradenitis suppurativa. Dermatology 1997; 194: Rigopoulos D, Larios G, Katsambas AD. The role of isotretinoin in acne therapy: why not as first-line therapy? facts and controversies. Clin Dermatol 2010; 28: Dispenza MC, Wolpert EB, Gilland KL. Systemic isotretinoin therapy normalizes exaggerated TLR-2-mediated innate immune responses in acne patients. JID 2012; 132: Singh JA, Wells GA, Christensen R, et al. Adverse effects of biologics: a network meta-analysis and Cochrane overview. Cochrane Database Syst Rev 2011; 2:CD Katz HI, Waalen J, Leach EE. Acitretin in psoriasis: an overview of adverse effects. J Am Acad Dermatol 1999; 41:S7-S12. 76

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80 6Ustekinumab in hidradenitis suppurativa: A clinical open label study with analyses of the protein expression profile in serum Janine L. Blok, MD 1 ; Katherine Li, MS 2 ; Carrie Brodmerkel, PhD 2 ; Péter Horvátovich PhD 3, Marcel F. Jonkman, MD PhD 1 ; Barbara Horváth, MD PhD 1 1 Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands 2 Janssen Research & Development, LLC, Spring House, PA, U.S.A. 3 Department of Pharmacy, Analytical Biochemistry, Faculty of Mathematics and Natural Sciences, University of Groningen, Groningen, The Netherlands Submitted 79

81 ABSTRACT Introduction Treatment of hidradenitis suppurativa (HS) is difficult and the search for effective therapies continues. Objectives To evaluate the efficacy of ustekinumab in HS. To discover a potential biomarker. Material and methods Seventeen patients were included in this open label study and treated with 45 to 90 mg ustekinumab at weeks 0, 4, 16 and 28. Proteomic technology and enzyme-linked assay analysis (ELISA) was applied on serum. Results Twelve patients completed the protocol. Moderate to marked improvement of the modified Sartorius Score was achieved in 82% of patients at week 40 and the hidradenitis suppurativa clinical response (HiSCR-50) in 47%. There was significant modulation in the expression of 54 serum proteins in patients at baseline compared to normal controls. Involved pathways were related to inflammation, immune cell signaling, and tissue morphology and development. Four of these (FSH, LH, HCG and LTA4H) were significant modulated at the end of treatment. Good responders had milder disease and lower expression of leukotriene A4-hydrolase (LTA4H). IL- 2R, TNF-α, IL17A and IL-17F were not elevated in patient serum and did not change during treatment. Conclusion The majority of patients showed improvement with ustekinumab. Although no biomarker was discovered, low LTA4H concentrations with mild disease severity may be predictive for the effectiveness of ustekinumab. 80

82 Introduction Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by recurrent abscesses and sinus tract formation. 1 The clinical severity of HS is commonly classified according to the Hurley stages. 2 HS is painful and disfiguring, impairing quality of life to a great extent. 3,4 Genetic susceptibility, smoking and obesity are important risk factors for the development of HS. 5 A dual approach is usually adopted in the treatment of HS. First, the inflammatory reaction is inhibited with systemic anti-inflammatory or immunosuppressive agents. Lesions resistant to systemic therapy, like sinus tracts, are subsequently surgically removed. 6 Commonly used immunosuppressive agents are tumor-necrosis-factor-α (TNF-α) inhibitors, like infliximab and adalimumab. 7 However, in a substantial number of patients TNF-α inhibitors are ineffective or cause adverse events requiring discontinuation of therapy. 7,8 Therefore, there is still a need for new effective immunosuppressive agents in HS. The pathogenesis of HS has not been clarified yet. It has been suggested that follicular plugging followed by the release of follicular material into the dermis are primary events that activate the immune system. 9 Unraveling what cytokines are involved has been the main objective of several studies. Recently, it has been shown that the interleukin-23/ T-helper 17 cells (IL-23/Th17) and IL-12/Th1 pathways come to expression in HS skin. 10,11 Ustekinumab is a human IgG1k monoclonal antibody that binds with high affinity to the p40 subunit of IL-12 and IL-23. Thereby, these cytokines are prevented from interacting with their IL-12Rβ1 receptor protein that is expressed on the surface of T-cells and natural killer cells. 12 Certain genetic variations within the gene encoding for the common IL-12βR1 subunit of the IL-12/IL23 receptor have shown to be associated with a more severe course of HS. 13 Ustekinumab has been approved for the treatment of psoriasis The effectiveness of ustekinumab in HS has been retrospectively studied in a total of seven patients with conflicting results With this open label prospective study we investigated the efficacy and safety of ustekinumab in a group of 20 patients with moderate to severe HS. To identify candidate biomarkers for HS we performed proteomics and immunoassays on serum of patients and healthy volunteers. 81

83 Material and Methods The manuscript was approved by the institutional review board. Study patients Subjects were recruited from May 2012 until March Patients with moderate to severe HS (Hurley stage II-III) with a treatment history of at least one systemic anti-inflammatory/ immunosuppressive agent or surgery were eligible for participation. The diagnosis of HS was made by a dermatologist. Design and intervention The trial had a prospective uncontrolled open-label design. The washout period for systemic immunosuppressive medication was at least 3 months. All patients received ustekinumab according to the psoriasis dosing regimen. (14) Subcutaneous injections were administered at weeks 0 and 4 (induction phase) and week 16 and 28 (maintenance phase). Each injection contained 45 mg ustekinumab, with subjects weighing >100 kg receiving 90 mg per injection. The intervention period was set to 40 weeks, consisting of the treatment phase (weeks 0-28) followed by a post-treatment phase of 12 weeks. Topical resorcinol 15% cream or incision and drainage of acutely painful were the only allowed escape treatments. Assessments were performed by the same investigator at baseline (week 0) and weeks 4, 10, 16, 22, 28, 34 and 40. The study was approved by the local institutional review board and registered with ClinicalTrials.gov (NCT ). Assessments of disease severity At every visit, the modified Sartorius scale (mss) and modified hidradenitis suppurativa-lesion area and severity index (mhslasi) were assessed. The mss is a dynamic scoring system for HS and includes the number of involved anatomical regions, the type and number of lesions, the extent of involvement and the Hurley stage. 21 The mhslasi reflects the degree of inflammatory activity by assessing the level of experienced pain, redness, edema and lesional discharge. 22 A 50% reduction in these scores was considered as a marked response; 25-<50% reduction as moderate; >0-<25% reduction as mild and 0 as non-response. A visual analogue scale (VAS) to determine the degree of experienced pain ranging from 0 mm (no pain) to 100 mm (maximum pain), the Dermatology Life Quality Index (DLQI) and Skindex-29 questionnaires were used to investigate patient-reported outcomes. 82

84 Outcomes and follow-up The primary endpoint was the proportion of patients with marked improvement ( 50% reduction) of the mss and mhslasi scores at week 40. Secondary outcomes included the mean change in patient s reported pain, Skindex-29 and DLQI. Regarding the Skindex-29, the cut-off scores as proposed by Prinsen et al. 23 were applied. Improvement of the Skindex-29 was considered as clinically meaningful if the score of a specific domain went at least one scale down compared to week 0. A DLQI of 0-1 corresponds to no effect on patient s quality of life, an index of 2-5 to a small effect, 6-10 to a moderate effect, to a very large effect and to an extremely large effect on patient s quality of life. 24 A reduction of 5 points on the DLQI was considered as clinically meaningful improvement. 25 We performed a post-hoc analysis using the Hidradenitis Suppurativa Clinical Response (HiSCR), which is a recently validated endpoint for assessing HS treatment effectiveness. 26 Responders (HiSCR achievers) are defined as patients with at least 50% reduction in the number abscesses or inflammatory nodules, without an increase in draining fistulas (HiSCR-50). This endpoint was added to support our clinical scores as the mss and mhslasi may not be optimal in assessing inflammatory manifestations. 27 Sample collection and analyses Protein analyses from serum: A total of 1129 proteins were measured in serum by somalogics (high content proteomics) using SOMAscan (SomaLogic Inc. Boulder, CO) at baseline and at week 40. Pathway analyses: Pathway analyses were conducted with QIAGEN s Ingenuity Pathway Analysis (IPA, www. qiagen.com/ingenuity). Immunoassays: Enzyme-linked Immunosorbent Assay (ELISA) was performed on serum at baseline, week 4, 16, 28 and 40 for TNF-α (MesoScale Discovery, Rockville, MD) and the soluble IL-2 receptor (sil2r) (Alpco, Salem, NH) according to the manufacturer s instructions. Serum IL17A and IL17F were measured by the Erenna Immunoassay system according to the manufacturer s 83

85 instructions (Singulex, Alameda, CA). Based on the protein Somalogics data measured at week 0 and 40, additional ELISA analyses were performed for leucotriene A4-hydrolase (LTA4H), follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (HCG) at weeks 0, 4, and 40. Safety assessments Serious adverse events were reported to the competent Health Authorities and the ethics committee within 24 hours in addition to completion of a Suspected Unexpected Serious Adverse Reaction form. A Data Safety Monitoring Board gathered every six months. Statistical analysis Descriptive statistics (mean, percentages, minimum and maximum) were performed for the outcome variables. Efficacy analyses were conducted on the intention-to-treat (ITT) population that included all patients who received at least one dose of study medication. If a patient dropped out it was attempted to obtain further patient assessments for the primary outcomes, if not the missing data were handled by carrying the last observation forward. The Wilcoxon rank test was used to analyze the efficacy of treatment. The criterion for statistical significance was p<0.05. Analyses were performed with SPSS Statistics 22. Array Studio software version 8.0 (OmicSoft Corp., St. Morrisville, NC) was used to analyze proteomics data. Blood samples from healthy volunteers were used as controls for serum proteomics. Expression modulation was analyzed using a general linear model. A >1.5 fold change and an FDR-adjusted p-value < 0.05 were considered significant. Scatter plots were made with Matlab version (R2014a). 84

86 Results Patients and drop-outs A total of 26 patients were screened for entering the study. Seventeen (four men, 13 women) instead of the initially planned 20 patients were included, as the trial medication expired due to a slower recruitment rate than expected. Patient s characteristics are described in table 1. Five subjects dropped out prematurely. The mean age was 35 years (range 20-53), mean BMI 28.3 kg/m 2 and mean disease duration was 18 years. Primary endpoints (mss and mhslasi) At week 40 improvement of the mss was marked in six of 17 patients (35%), moderate in eight patients (47%), mild in one patient (6%) and in two patients (12%) there was no change or worsening. The mean mss of the ITT population significantly reduced from at baseline to at week 40 (46.33% improvement; p=0.001) (fig. 1a). Improvement of the mhslasi at week 40 was marked in three of 17 patients (18%), moderate in six patients (35%), mild in three patients (18%) and in five patients (29%) there was no change or worsening. The mean mhslasi of the ITT population significantly reduced from at baseline to at week 40 (25.5% improvement; p=0.011) (fig. 1a). Secondary endpoints (Skindex-29, VAS, DLQI) At week 40 clinically meaningful improvement on the Skindex-29 overall domain was observed in six of 17 subjects (35%), on the functioning domain in eight subjects (47%), on the emotions domain in four subjects (24%) and on the symptoms domain in three subjects (18%) (fig. 1c). At baseline, the DLQI indicated that HS had a very large or extremely large effect on daily life in 71% of subjects, at week 40 this was 59% (fig. 1d). At week 40 clinically meaningful improvement of the DLQI had occurred in seven patients (41%). The mean reported pain on a VAS was 5.8 out of 10 at the start of treatment and 4.6 at week 40. Post-hoc analysis with HiSCR The number of HiSCR-50 achievers increased from the induction phase through to week 22. At week 40, eight out of 17 patients (47%) were HiSCR50 achievers (fig. 1b). 85

87 Escape treatments Resorcinol 15% cream was used for troublesome inflammatory lesions by four patients and incision and drainage in one patient. The protocol was violated in two patients who received intralesional corticosteroids for painful lesions that were not suitable for incision and drainage. Safety assessments The most common adverse events were headache, fatigue and upper respiratory tract infections. All events were mild and temporary. Protein expression in the serum Fifty-four proteins were significantly differentially expressed in serum of patients (n=17) at baseline compared to healthy controls (n=10) (fig. 2a). The top involved dysregulated pathways were related to inflammation, immune cell signaling, as well as tissue morphology and development (fig. 2b). Significant modulation of the leucotriene A4-hydrolase (LTA4H), follicle stimulating hormone (FSH), luteizing hormone (LH) and human chorionic gonadotropin (HCG) were identified at week 40 in the four best responders (subjects 6, 12, 14, 15). Serum ELISA for LTA4H, FSH, LH and HCG As LTA4H, FSH, LH and HCG were identified as potential biomarkers with protein microarray analysis, these were further analyzed with ELISA. No linear correlation between clinical disease severity and serum concentrations was identified, making these proteins not suitable as biomarkers for treatment effectiveness. However, good responders showed a trend towards having a relatively less severe clinical phenotype as well as lower concentrations of LTA4H (fig. 3). Serum ELISA for sil-2r, TNF-α, IL17A and IL-17F There were no significant differences in serum concentrations of sil-2r, TNF-α, IL17A and IL-17F between patients and controls, nor did we identify a significant decrease in any of these cytokines during ustekinumab treatment (data not shown). 86

88 Subject number Gender Age History BMI (kg/m2) Smoking status Disease duration** mss Treatment history 1 F 37 Eczema, hypothyreodism, hypertension 32.0 Former T. and i.l. corticosteroids, antibiotics, deroofing 2 F 48 Asthma, depression 37.0 Former Resorcine15%, antibiotics, isotretinoin, deroofing 3 M 23 Conglobate acne 23.0 Active 7 91 Prednisolone, isotretinoin, antibiotics, infliximab 4 F 51 Diabetes 34.4 Active Resorcine15%, antibiotics, prednisolone, infliximab, deroofing, excision 5 F 32 Depression 38.0 Active Resorcine15%, antibiotics, incision/drainage, deroofing 6 F Active Corticosteroids i.l., antibiotics, IPL-EPI, etanercept, deroofing 7 F 42 Depression 24.0 Active Resorcine15%, antibiotics, deroofing, excision 8 M 53 Benign fasciculation syndrome 22.0 Former Resorcine15%, antibiotics, incision/drainage 9 M 21 Depression, conglobate acne 25.3 Active T. and i.l. corticosteroids, resorcine 15%, isotretinoin, antibiotics, incision/ drainage 10 F 22 Conglobate acne, eczema 22.0 Active T. corticosteroids, resorcine15%, antibiotics, isotretinoin, incision/drainage, excision 11 F 23 Depression 26.0 Active 5 63 I.l. corticosteroids, resorcine 15%, antibiotics, incision and drainage, deroofing, IPL-EPI*** 12 F 44 Kidney stones, hypertension 32.0 Active Resorcine15%, topical and intralesional corticosteroids, antibiotics, deroofing, excision 13 M 33 Depression 23.1 Active Antibiotics, isotretinoin, infliximab, excision 14 F 40 Asthma, depression, hyperlipidemia 23.9 Active Resorcine15%, antibiotics 15 F 43 Hypercholesterolemia 24.9 Active 5 99 Antibiotics, resorcine15% incision and drainage, deroofing 16 F 20 Crohn s disease, depression 32.7 Active 9 52 Topical resorcine15%, deroofing 17 F 33 Eczema, heterozygous sickle cell anemia 40.6 Former I.l. corticosteroids, antibiotics, incision/drainage, deroofing *at the start of the study in years;**in years IPL-EPI = Intense pulse light epiliation; mss = modified Sartorius Score; T. = topical; i.l.=intralesional Table 1. Patient characteristics 87

89 (a) (c) (b) (d) Figure 1. Clinical scores during ustekinumab treatment (a) Percentual reduction of the mss and mhslasi during ustekinumab treatment. (b) The number of patients achieving >50% improvement of HiSCR during ustekinumab treatment. (c) Improvement in Skindex-29 occurred for each domain with worsening of the scores during the post-treatment phase. (d) The impact of HS on daily life as measured by the DLQI. The number of patients is presented within the bars. Arrows represent ustekinumab administration points 88

90 Disease Controls HS patients Natural Killer Cell Signaling Fc Epsilon RI Signaling Insulin Receptor Ephrin Receptor T Cell Receptor Signaling HGF Signaling ncreatic Adenocarcinoma Signaling Role of Tissue Factor in Cancer Ovarian Cancer Signaling PI3K Signaling in B Lymphocytes - log (p-value) Figure 2. Disease profile in serum (A) The heat-map of the HS disease profile in serum based on somalogics. The expression of 54 proteins was significantly different between HS (red boxes) and healthy controls (grey boxes) at baseline. (B) The top 10 involved canonical pathways in serum of HS patients. 89

91 Figure 3. Scatter plot for the correlation between LTA4H and the absolute HiSCR. There is no linear correlation between the concentration of LTA4H and the HiSCR. Note that good responders (green circle) have lower HiSCR combined with lower LTA4H concentrations compared to non-responders (red circle). 90

92 Discussion This is the first prospective study investigating the efficacy of ustekinumab in HS. Both primary outcomes (mss and mhslasi) showed a significant reduction of the mean at week 40. Additionally, moderate to marked improvement of the mss and mhslasi was seen in the majority (82% and 53% respectively) of patients. This suggests that ustekinumab may be applied for the treatment of HS as the reduction in mss is comparable to studies on adalimumab and infliximab. 8,28 The mss reduced >50% in 35% of patients. Other studies considered reductions of 30% to 40% as therapeutic responses. 29,30 The HiSCR-50 was achieved in 47%. Considering 50% improvement of the mss as clinically meaningful in HS is therefore probably too strict. The psoriasis dosing regimen may not have been sufficient for HS, further explaining why only a minority met the primary endpoint. Indeed, inflammatory marker levels are higher in HS than in psoriasis. 10 Increasing the administration frequency, as has been shown to improve the results of adalimumab in HS, as well as increasing the dosage, which has been safely applied in Crohn s disease, may therefore improve the effectiveness of ustekinumab. 8,31 Almost half of the patients showed clinically meaningful improvement on the functioning domain of the Skindex-29 at week 40. Quality of life scores worsened at the post-treatment phase, suggesting that longer treatment duration may lead to prolonged effectiveness. 12 Upper respiratory tract infections were one of the most common adverse events. The increased infection risk during ustekinumab treatment is caused by suppression of Th-17 cell differentiation, as IL-17 provides immunity against microbes like staphylococcus and candida. We could not confirm the potential of sil2r as a serum biomarker in HS. 32,33 Neither could we put IL-17A, IL-17F and TNF-α forward as potential biomarkers. However, HS patients had a significant different serum protein profile compared to controls, indicating that several proteins are secreted towards the circulation by cells present in inflammatory skin. The significant modulation in LH, FSH and HCG after treatment in the best responders, who were all female, is difficult to interpret as these hormones naturally fluctuate during the menstrual cycle. In psoriasis no correlation was found between disease severity and concentrations of FSH and LH in male patients. 34 However, estradiol concentrations were associated with milder disease severity, confirming the potential protective role of estrogen in inflammatory diseases. Therefore, the role of sex hormones in HS also remains an interesting topic for future studies. We show significant modulation in the expression of LTA4H in the best ustekinumab responders. LTA4H is an intracellular enzyme released by epithelial cells and macrophages. It converts leukotriene A4 (LTA4) into leukotriene B4 (LTB4), a pro-inflammatory mediator capable of recruiting and activating a wide range of immune cells, including neutrophils. 91

93 The subsequent LTA4H release from neutrophils counteracts the inflammatory reaction by degradation of neutrophilic attractant peptides. 35 This dual effect may be responsible for the lack of a clear linear correlation between LTA4H serum concentration and clinical disease severity. However, the LTA4H concentration in combination with disease severity (HiSCR) may be used to predict the effect of ustekinumab: low concentrations with a relatively mild clinical disease severity may be associated with a good response. Limitations of this study include the small number of patients and the lack of a control group. Furthermore, there was a relatively high dropout rate, increasing the risk of bias. This was restricted to a minimum by analyzing the ITT population. In summary, this study shows that ustekinumab is well tolerated and that the standard psoriasis dosing schedule improved HS in the majority of patients. The dosing schedule may need to be intensified for HS to achieve sufficient immune suppression. Modulated pathways in serum of HS patients were related to inflammation, immune cell signaling, as well as tissue morphology and development. A biomarker for measuring treatment effects in HS was not identified. However, low LTA4H serum concentrations in combination with relatively mild disease severity may be predictive for the efficacy of ustekinumab. 92

94 REFERENCES 1. Revuz J. Hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2009; 23: Hurley HJ. Axiillairy hyperhidrosis, apocrine bromhidrosis, hidradenitis suppurativa, and familial benign pemphigus: surgical approach. In: Dermatologic Surgery(In: Roenigh, R.K. Roenigh, HH, ed): Marcel Dekker, New York, 1989; Matusiak L, Bieniek A, Szepietowski JC. Psychophysical aspects of hidradenitis suppurativa. Acta Derm Venereol 2010; 90: Wolkenstein P, Loundou A, Barrau K, et al. Quality of life impairment in hidradenitis suppurativa: a study of 61 cases. J Am Acad Dermatol 2007; 56: Alikhan A, Lynch PJ, Eisen DB. Hidradenitis suppurativa: a comprehensive review. J Am Acad Dermatol 2009; 60:539-61; quiz Blok JL, Spoo JR, Leeman FW, et al. Skin-Tissue-sparing Excision with Electrosurgical Peeling (STEEP): a surgical treatment option for severe hidradenitis suppurativa Hurley stage II/III. J Eur Acad Dermatol Venereol 201; 29: Blok JL, van Hattem S, Jonkman MF, Horvath B. Systemic therapy with immunosuppressive agents and retinoids in hidradenitis suppurativa: a systematic review. Br J Dermatol 2013; 168: Kimball AB, Kerdel F, Adams D, et al. Adalimumab for the treatment of moderate to severe Hidradenitis suppurativa: a parallel randomized trial. Ann Intern Med 2012; 157: Danby FW, Jemec GB, Marsch WC, von Laffert M. Preliminary findings suggest hidradenitis suppurativa may be due to defective follicular support. Br J Dermatol 2013; 168: van der Zee HH, de Ruiter L, van den Broecke DG, et al. Elevated levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10 in hidradenitis suppurativa skin: a rationale for targeting TNF-alpha and IL-1beta. Br J Dermatol 2011; 164: Schlapbach C, Hanni T, Yawalkar N, Hunger RE. Expression of the IL-23/Th17 pathway in lesions of hidradenitis suppurativa. J Am Acad Dermatol 2011; 65: Benson JM, Peritt D, Scallon BJ, et al. Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders. MAbs 2011; 3:

95 13. Giatrakos S, Huse K, Kanni T, et al. Haplotypes of IL-12Rbeta1 impact on the clinical phenotype of hidradenitis suppurativa. Cytokine 2013; 62: Leonardi CL, Kimball AB, Papp KA, et al. Efficacy and safety of ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with psoriasis: 76-week results from a randomised, double-blind, placebo-controlled trial (PHOENIX 1). Lancet 2008; 371: Papp KA, Langley RG, Lebwohl M, et al. Efficacy and safety of ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with psoriasis: 52-week results from a randomised, double-blind, placebo-controlled trial (PHOENIX 2). Lancet 2008; 371: Griffiths CE, Strober BE, van de Kerkhof P, et al. Comparison of ustekinumab and etanercept for moderate-to-severe psoriasis. N Engl J Med 2010; 362: Sharon VR, Garcia MS, Bagheri S, et al. Management of recalcitrant hidradenitis suppurativa with ustekinumab. Acta Derm Venereol 2012; 92: Gulliver WP, Jemec GB, Baker KA. Experience with ustekinumab for the treatment of moderate to severe hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2012; 26: Baerveldt EM, Kappen JH, Thio HB, et al. Successful long-term triple disease control by ustekinumab in a patient with Behcet s disease, psoriasis and hidradenitis suppurativa. Ann Rheum Dis 2013; 72: Martin-Ezquerra G, Masferrer E, Masferrer-Niubo M, et al. Use of biological treatments in patients with hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2015; 29: Sartorius K, Emtestam L, Jemec GB, Lapins J. Objective scoring of hidradenitis suppurativa reflecting the role of tobacco smoking and obesity. Br J Dermatol 2009; 161: Xu LY, Wright DR, Mahmoud BH, et al. Histopathologic study of hidradenitis suppurativa following long-pulsed 1064-nm Nd:YAG laser treatment. Arch Dermatol 2011; 147: Prinsen CA, Lindeboom R, de Korte J. Interpretation of Skindex-29 scores: cutoffs for mild, moderate, and severe impairment of health-related quality of life. J Invest Dermatol 2011; 131: Hongbo Y, Thomas CL, Harrison MA, et al. Translating the science of quality of life into practice: What do dermatology life quality index scores mean? J Invest Dermatol 2005; 125:

96 25. Basra MK, Fenech R, Gatt RM, et al. The Dermatology Life Quality Index : a comprehensive review of validation data and clinical results. Br J Dermatol 2008; 159: Kimball AB, Jemec GB, Yang M, et al. Assessing the validity, responsiveness and meaningfulness of the Hidradenitis Suppurativa Clinical Response (HiSCR) as the clinical endpoint for hidradenitis suppurativa treatment. Br J Dermatol 2014; 171: van der Zee HH, Laman JD, Boer J, Prens EP. Hidradenitis suppurativa: viewpoint on clinical phenotyping, pathogenesis and novel treatments. Exp Dermatol 2012; 21: van Rappard DC, Leenarts MF, Meijerink-van t Oost L, Mekkes JR. Comparing treatment outcome of infliximab and adalimumab in patients with severe hidradenitis suppurativa. J Dermatolog Treat 2012; 23: Savva A, Kanni T, Damoraki G, et al. Impact of Toll-like receptor-4 and tumour necrosis factor gene polymorphisms in patients with hidradenitis suppurativa. Br J Dermatol 2013; 168: Fardet L, Dupuy A, Kerob D, et al. Infliximab for severe hidradenitis suppurativa: transient clinical efficacy in 7 consecutive patients. J Am Acad Dermatol 2007; 56: Sandborn WJ, Gasink C, Gao LL, et al. Ustekinumab induction and maintenance therapy in refractory Crohn s disease. N Engl J Med 2012; 367: Matusiak L, Bieniek A, Szepietowski JC. Soluble interleukin-2 receptor serum level is a useful marker of hidradenitis suppurativa clinical staging. Biomarkers 2009; 14: Wieland CW, Vogl T, Ordelman A, et al. Myeloid marker S100A8/A9 and lymphocyte marker, soluble interleukin 2 receptor: biomarkers of hidradenitis suppurativa disease activity? Br J Dermatol 2013; 168: Cemil BC, Cengiz FP, Atas H, et al. Sex hormones in male psoriasis patients and their correlation with the Psoriasis Area and Severity Index. J Dermatol 2015; 42: Snelgrove RJ. Leukotriene A4 hydrolase: an anti-inflammatory role for a proinflammatory enzyme. Thorax 2011; 66:

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98 7SKIN-TISSUE-SPARING EXCISION WITH ELECTROSURGICAL PEELING (STEEP): A SURGICAL TREATMENT OPTION FOR SEVERE HIDRADENITIS SUPPURATIVA HURLEY STAGE II/III J.L. Blok 1, MD; J.R. Spoo 1, MD PhD; F.W.J. Leeman 2, MD; M.F. Jonkman 1, MD PhD; B. Horváth 1, MD PhD 1 Department of Dermatology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 2 Department of Dermatology, Antonius Hospital, 8601 ZK Sneek, The Netherlands Published in the Journal European of the Academy of Dermatology and Venereology, 2015;29:

99 ABSTRACT Background: Surgery is the only curative treatment for removal of the persistent sinus tracts in the skin that are characteristic of severe hidradenitis suppurativa (HS). Complete resection of the affected tissue by wide excision is currently regarded as the preferred surgical technique in these cases. However, relatively large amounts of healthy tissue are removed with this method and suitable skin-tissue saving techniques aiming at creating less extensive surgical defects are therefore needed in severe HS. Method: We describe a skin-tissue saving surgical technique for HS Hurley stage II-III disease: the Skin-tissue-sparing Excision with Electrosurgical Peeling (STEEP) procedure. Discussion: In contrast to wide excisions that generally reach into the deep subcutaneous fat, the fat is maximally spared with the STEEP procedure by performing successive tangential excisions of lesional tissue until the epitheliazed bottom of the sinus tracts has been reached. From here secondary intention healing can occur. In addition, fibrotic tissue is completely removed in the same manner since this also serves as a source of recurrence. This tissue sparing technique results in low recurrence rates, high patient satisfaction with relatively short healing times and favorable cosmetic outcomes without contractures. 98

100 INTRODUCTION Hidradenitis suppurativa (HS) is an inflammatory skin disease characterized by painful deepseated nodules and abscesses that mainly occur on apocrine gland bearing skin. 1 In a later stage, sinus tracts surrounded by extensive fibrosis are formed in the dermis that in severe cases even extend into the deep subcutaneous fat. These sinus tracts serve as a source for the characteristic chronically recurring inflammation in HS. The Hurley classification is frequently used to reflect disease severity by determination of: (i) the character of the lesions (solitary nodules or abscesses correspond to stage I disease while stage II/III disease is characterized by sinus tract formation); and (ii) the extensiveness of the lesions (differentiation of stage II from stage III disease is made by determining whether or not there is healthy skin between the lesions). 2 Despite the numerous therapeutic options it is still difficult to treat HS, especially in severe cases (Hurley stage II or III disease). Severe HS is characterized by both inflammation and a permanent destruction of the normal skin architecture by the epithelialized sinus tracts and fibrotic scars. Therefore, treatment requires a combined approach in order to be successful. First, inflammatory activity needs to be improved. This can be achieved by systemic antiinflammatory or immunosuppressive treatment. However, (non-inflammatory) sinus tracts and fibrotic scars will remain present after systemic therapy. The only way to permanently remove these sinus tracts and fibrotic tissue is by means of surgery. Currently, the classic deroofing technique and wide excision are regarded as the preferred surgical methods for treating HS Hurley stage I/II and stage II/III respectively. 3,4 However, Hurley stage II/III disease remains a surgical challenge and the surgical treatment has dual aspects. On the one hand, the goal of surgery is to achieve complete removal of lesional tissue. On the other hand, it is important to spare as much healthy tissue as possible to prevent the formation of serious contractures and to promote wound healing. The latter is not achieved with wide excision. To meet both goals to a maximum we have developed a surgical technique for severe HS where the advantages of both wide excision and the deroofing technique are combined: the Skin-Tissue-sparing Excision with Electrosurgical Peeling (STEEP) procedure. 99

101 Technique The STEEP procedure is performed under general anesthesia. The procedure starts with palpation of the affected area to localize inflammatory nodules and fibrosis. Visible sinus tracts are then sondaged with a probe to investigate their extensiveness. The sinus roof is subsequently electrosurgically incised with a wire loop tip coupled to an Erbotoom (Erbe USA Inc. Surgical Systems). This method is similar to the deroofing technique as described previously for Hurley stage I/II disease. (3,4) Subsequently all lesional tissue, including fibrosis that is identified by palpation, is removed from the incision on to the deeper skin layers by successive tangential electrosurgical transsections (figure 1). The epithelialized sinus floors and subcutaneous fat are left intact where possible. This procedure of incising sinuses and tangential peeling off affected tissue is continued until the whole area is clear of lesional tissue and fibrosis. Finally, the wound margins are meticulously checked with a probe for the presence and removal of any residual sinus tracts. Hemostasis is achieved by using the coagulation mode of the Erbotoom. The wound margins (i.e. healthy tissue) are injected with triamcinolonacetonide 10-20mg and bupivacaine 0.5%(10ml) to prevent hypergranulation. Wounds are left open to heal by secondary intention. Post-operative wound care consists of twice daily irrigation followed by alginate and silicone dressings. Pain is managed with acetaminophen, non-steroidal anti-inflammatory drugs or, in severe cases, opiates. Generally, patients can leave the hospital on the day of surgery. Figure 2 illustrates an example of the satisfying cosmetic results we achieve with the STEEP procedure. This 46-year old woman with severe therapy resistant HS in the genital area was initially treated with intravenous infliximab for six months. After five infusions, inflammatory activity had improved and the STEEP procedure was then successfully performed in two separate sessions. Currently (i.e. three years later), the disease is still in remission. Additionally, her quality of life has significantly improved, as reflected in a reduction of her Dermatology Life Quality Index (DLQI) from 28 points (maximum: 30) at the beginning of infliximab treatment to 3 points at eight months after the final STEEP procedure. 100

102 Epidermis Dermis Sinus tract (non-inflammatory) Inflammatory nodule Subcutaneous fat (a) (b) Figure 1. Wide excision (a): red dotted line represents the cutting surface. Healthy tissue is removed and resection reaches into the subcutaneous fat. STEEP-procedure (b): inflammatory nodules, sinus tracts and scar tissue are localized by a probe or with palpation. Affected tissue is then peeled off layer by layer by means of multiple tangential transsections (red dashed lines). The epithelial lining is spared in non-inflammatory sinus tracts but is totally excised in inflammatory sinus tracts (blue dashed lines). The procedure is repeated until a plane has been obtained that is free of lesional tissue. The result is that the final defect is smaller compared to wide excision. (a) (b) Figure 2. Right groin before treatment with the STEEP procedure. Sondage with a tissue forceps demonstrates the depth of the sinus tract (a). The right groin 24 weeks after the STEEP procedure (b). 101

103 DISCUSSION Treatment of HS is a challenge for both physicians and patients due to its complex and largely unknown pathogenesis. It is generally accepted that in order to achieve satisfying results in severe cases (Hurley stage II/III disease), a dual therapeutic approach is required. 5 Usually, the first step is inhibition of inflammatory activity with anti-inflammatory or immunosuppressive agents, including infliximab. 5, 6 However, systemic treatment will not restore the skin s original architecture, meaning that epithelialized cysts and sinus tracts will remain present in the affected skin once the inflammation has been treated. This may facilitate access for (commensal) bacteria, leading to repetitive inflammation and further extension of the disease, with ever-increasing architectural destruction. 7 This vicious circle can only be interrupted by surgical removal of these residual lesions. The STEEP procedure is a promising tissue-saving surgical technique for HS Hurley stage II/III. Tissue-saving surgical techniques are importance to HS for two main reasons: (i), HS especially occurs in the body folds, which are areas prone to the formation of contractures after surgery and (ii) large interconnected skin areas are frequently involved, making it even more important to reduce the size of the already large surgical defects to a minimum. A suitable tissue-saving surgical technique for Hurley stage I or limited stage II disease is the deroofing technique. 3,4 However, in case of extensive Hurley stage II/III disease dominated by fibrotic tissue, the deroofing technique is ineffective since fibrosis is not removed. Removal of fibrotic tissue is important because it may contain skin appendages that serve as a source for recurrence and also prevent adequate wound contraction and subsequent healing. In addition, the deroofing technique is too time-consuming in severe HS. Surgical intervention by means of wide excision is therefore often considered as the most effective treatment in these cases. 8 However, the STEEP procedure has several advantages over both wide excision and the deroofing technique in severe HS. First, wide excisions always reach into the healthy deep subcutaneous fat, while the STEEP procedure with its successive tangential transsections leaves the epithelialized bottoms of the sinus tracts and a large extent of the subcutaneous fat intact, leading to more superficial and smaller defects (fig 2). This results in relatively shorter healing times and fewer complications, such as contracture formation. Furthermore, recurrence rates around the operated area are reduced to a minimum since at the end of the STEEP procedure residual affected tissue is identified and removed by sondaging the final wound margins with a probe and extensive palpation. Tissue-sparing surgery in HS can also be accomplished with the use of a CO 2 laser, as previously demonstrated The main advantage of CO 2 laser incision is that proper hemostasis is achieved allowing adequate visualization of remaining lesional tissue. During the STEEP procedure prompt cauterization of bleeding vessels can be easily achieved 102

104 as well by switching between the surgery and electrocautery mode of the electrosurgical unit using a foot pedal. For the performance of multiple transversal sections as we describe for our surgical technique, electrosurgery has some important advantages over CO 2 laser, namely: (i) the depth of vertical incisions with transversal electrosection is more easily controlled and adjusted by the surgeon. This leaves the epithelialized sinus bottom intact and warrants deep excision of fibrotic and inflammatory tissue at the same time while a CO 2 laser removes a continuous horizontal plane of one depth, making it less precise and less tissue-sparing; (ii) laser treatment is more expensive in terms of purchasing the device, the need for a special room to perform the treatment and safety notices; and (iii) a basically trained dermatosurgeon can perform electrosurgery, while laser treatment requires more experience and skills. In our clinic, we have performed the STEEP procedure under general anesthesia in 156 patients with Hurley stage II/III disease between 2004 and The feedback from the patients is generally very positive. Patients with extensive disease at multiple locations were usually operated in several sessions. The wounds are allowed to heal by secondary intention since in our experience, and that of others, it is time-efficient and leads to good cosmetic and functional results. 14,15 Furthermore, it allows prolonged wound drainage, diminishing the risk of wound infection. 14 In conclusion, we consider the STEEP procedure with electrosurgery superior over wide resections and deroofing in Hurley stage II/III disease for several reasons: (i) recurrence rates are low as it specifically aims at complete removal of lesional and fibrotic tissue; (ii) it saves healthy tissue to a maximum which leads to rapid healing, satisfying cosmetic results and prevention of contractures; (iii) healing time is further improved by allowing re-epithelization of the defects from the intact epitheliazed sinus floors and dermal tissue where possible rather than from subcutaneous fat; and (iv) in contrast to laser surgery the procedure can be performed by basically trained dermatologic surgeons. 103

105 REFERENCES 1. Revuz J. Hidradenitis suppurativa. J Eur Acad Dermatol Venereol 2009; 23: Hurley HJ. Axiillairy hyperhidrosis, apocrine bromhidrosis, hidradenitis suppurativa, and familial benign pemphigus: surgical approach. In: Dermatologic Surgery (Roenigh R.K, Roenigh HH,eds). New York: Marcel Dekker, 1989; Van der Zee HH, Prens EP, Boer J. Deroofing: a tissue-saving surgical technique for the treatment of mild to moderate hidradenitis suppurativa lesions. J Am Acad Dermatol 2010; 63: van Hattem S, Spoo JR, Horvath B, Jonkman MF, Leeman FW. Surgical treatment of sinuses by deroofing in hidradenitis suppurativa. Dermatol Surg 2012; 38: Van Rappard DC, Mekkes JR. Treatment of severe hidradenitis suppurativa with infliximab in combination with surgical interventions. Br J Dermatol 2012; 167: Blok JL, van Hattem S, Jonkman MF, Horvath B. Systemic therapy with immunosuppressive agents and retinoids in hidradenitis suppurativa: a systematic review. Br J Dermatol 2013; 168: van der Zee HH, Laman JD, Boer J, Prens EP. Hidradenitis suppurativa: viewpoint on clinical phenotyping, pathogenesis and novel treatments. Exp Dermatol 2012; 21: Alikhan A, Lynch PJ, Eisen DB. Hidradenitis suppurativa: a comprehensive review. J Am Acad Dermatol 2009; 60: Jain V, Jain A. Use of lasers for the management of refractory cases of hidradenitis suppurativa and pilonidal sinus. J Cutan Aesthet Surg 2012; 5: Lapins J, Marcusson JA, Emtestam L. Surgical treatment of chronic hidradenitis suppurativa: CO2 laser stripping-secondary intention technique. Br J Dermatol 1994; 131: Madan V, Hindle E, Hussain W, August PJ. Outcomes of treatment of nine cases of recalcitrant severe hidradenitis suppurativa with carbon dioxide laser. Br J Dermatol 2008; 159: Hazen PG, Hazen BP. Hidradenitis suppurativa: successful treatment using carbon dioxide laser excision and marsupialization. Dermatol Surg 2010; 36: Bratschi HU, Altermatt HJ, Dreher E. Therapy of suppurative hidradenitis using the CO2-laser. Case report and literature review. Schweiz Rundsch Med Prax 1993; 82:

106 14. Bieniek A, Matusiak L, Chlebicka I, Szepietowski JC. Secondary intention healing in skin surgery: our own experience and expanded indications in hidradenitis suppurativa, rhinophyma and non-melanoma skin cancers. J Eur Acad Dermatol Venereol 2013; 27: Wollina U, Tilp M, Meseg A, Schonlebe J, Heinig B, Nowak A. Management of severe anogenital acne inversa (hidradenitis suppurativa). Dermatol Surg 2012; 38:

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108 8SURGERY UNDER GENERAL ANESTHESIA IN SEVERE HIDRADENITIS SUPPURATIVA: A STUDY OF 363 PRIMARY OPERATIONS IN 113 PATIENTS J.L. Blok, MD 1 ; M. Boersma, MD 1 ; J.B. Terra, MD PhD 1 ; J.R. Spoo, MD PhD 1 ; F.W.J. Leeman, MD 2 ; E.R. van den Heuvel, MD PhD 3 ; J. Huizinga, MSc, RN 1 ; M.F. Jonkman, MD PhD 1 ; B. Horváth, MD PhD 1 1 Department of Dermatology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 2 Department of Dermatology, Antonius Hospital, 8601 ZK Sneek, The Netherlands 3 Department of Epidemiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands Published in the Journal of the European Acadamy of Dermatology and Venereology, 2015 doi: /jdv

109 ABSTRACT Background: Treatment of hidradenitis suppurativa (HS) is a difficult undertaking, especially since there is no consensus on what surgical technique is preferred. At our center severe HS (Hurley II/III) is operated under general anesthesia, mostly with the STEEP-procedure. Objectives: To investigate characteristics, surgical outcomes and patient satisfaction of HS patients who underwent deroofing or STEEP under general anaesthesia. Methods: A clinical records-based retrospective analysis was conducted of all patients who had surgery under general anesthesia between 1999 and Patient satisfaction was retrospectively investigated with questionnaires. Results: A total of 482 operations (363 primary operations and 119 re-operations) was performed during the study period. The proportion of women in the included population was 68%. The median diagnostic delay (patient s and doctor s delay) was 6.5 years. Relapses occurred after 29.2% of primary operations. Women had higher relapse rates than men (odds ratio 2.85 [1.07;7.61]). Hypergranulation of the wound was the most common complication and occurred in 7% of all operations. The median score patients attributed to the medical effect of surgery was 8 out of 10 (zero corresponding to very dissatisfied and 10 to very satisfied). Conclusion: The diagnostic delay in HS is long due to a lack of knowledge in both patients and health care professionals, indicating that there is a need for education. Deroofing and the STEEP are effective surgical procedures in severe cases of HS and lead to a relatively high patient satisfaction. The post-operative relapse risk is higher in women. Prospective studies are required for the development of clear guidelines on the appropriate choice of surgery. 108

110 INTRODUCTION Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with varying phenotypes. The clinical severity is often classified according to the Hurley stages. 1 Stage I is characterized by abscesses and nodules while in stage II and III sinus tracts have been formed. The presence of healthy skin between the lesions differentiates Hurley stage II from III disease. Several treatment modalities are available for HS and usually a combination of different treatment modalities is required. An effective topical treatment is resorcinol 15%. 2 However, severe cases require systemic and surgical treatment. Systemic treatment options include acitretin, antibiotics and tumour-necrosis-factor-α (TNF-α)-inhibitors. 3-6 Though these systemic agents may be able to improve the inflammatory component, the architectural skin destructions that may have been formed as a consequence of HS remain. Therefore, surgery is mandatory in the treatment of severe HS. There is no consensus on the most suitable surgical technique for HS. Optional methods are (i) treatment of each individual lesion with deroofing, 7,8 carbon dioxide laser treatment or excision; 9 (ii) radical excision of the entire affected area with wide margins; 10 and (iii) excision of the entire area encompassing all lesions with the skin-tissue-saving excision with electrosurgical peeling (STEEP) technique; 11 A detailed description of the STEEP technique was recently published by our group. 11 In short, the STEEP aims at removing all lesional tissue by performing subsequent tangential transections (fig. 1, chapter 7). In contrast to wide excision, which serves the same goal, the STEEP technique saves healthy tissue to a maximum. Regardless of the applied surgical technique, wound closure for HS may be achieved with sutures, skin grafts or flaps, or by secondary intention. 12 Surgery is performed under local or general anesthesia, a choice usually depending on the extensiveness of HS. At our specialized day care center, we are able to perform surgery in HS patients under general anesthesia. The use of general anesthesia enables us to treat large surface areas without being limited by the maximum allowed amount of local anesthetics. There is an indication for the STEEP procedure when patients have extensive stage II or III disease. In these cases the deroofing technique is too superficial and therefore not sufficient as fibrotic scar tissue that may serve as a source of relapsing disease, is not being removed. When patients with Hurley stage I or limited stage II disease refuse to be operated with the deroofing technique under local anesthesia, general anesthesia is also applied. In both, the STEEP and the deroofing technique the wounds heal by secondary intention. In this retrospective study we describe the characteristics of HS patients who underwent deroofing or the STEEP-procedure under general anesthesia. Furthermore, we evaluate the characteristics, outcomes and patient satisfaction regarding these treatments. 109

111 MATERIAL AND METHODS Study design and subjects The medical charts of all patients who underwent surgical treatment under general anesthesia between May 1999 and January 2013 at our day care center were retrospectively analyzed by two authors (J.L.B and M.B). Our day care center applies strict criteria regarding body mass index (BMI): patients with a BMI 35 kg/m 2 are refused for operation. The data were stored in a database (SPSS Statistics 20 IBM, Armonk, New York, USA). The authors randomly checked each other s data extraction. Primary parameters: Characteristics of patients and surgeries Patients characteristics included gender, age, smoking status, BMI and family history. Disease severity was defined as the Hurley stage at the first visit to our department. Diagnostic history included the diagnostic delay (sum of patient s and doctor s delay) and the number of visited doctors before referral to our specialized center was achieved. The use of co-medication for HS was only recorded if it had been used 2 months post-operatively. Secondary parameters: Surgical outcomes These included the percentage of remissions, post-operative general disease activity, relapses due to irradical surgery, natural disease progression and complications. Surgeries were divided into primary operations and re-operations. Primary operations were surgeries performed for the first time in a specific anatomical area. Any subsequent surgical procedures within that anatomical area were defined as re-operations. General disease activity was defined as postoperative inflammatory activity (abscesses, nodules or fistulas) occurring within the operated anatomical area after a primary operation and was further subdivided into relapses due to irradical surgery and natural progression of the disease. Relapses due to irradical surgery were defined as inflammatory activity occurring within or at the border of the surgical scar and considered unknown when the post-operative follow-up period was <8 weeks. Inflammation in the operated anatomical area occurring outside the surgical scar was considered as natural disease progression. Complications were recorded for all surgeries, including re-operations. 110

112 Factors influencing the risk of relapse It was investigated whether disease location, gender, age, smoking status, BMI, and the use of co-medication were associated with the occurrence of relapses. Tertiary parameters: Patient reported outcomes Patients opinion about their surgical treatment was assessed with questionnaires. Patients who were <18 years, mentally retarded or deceased were excluded. Patients were asked to name the best treatment they ever had for HS and to rate their satisfaction regarding the cosmetic and medical outcomes of the surgery on a scale from 0 (very dissatisfied) to 10 (very satisfied). Medical ethical committee In the Netherlands, retrospective studies of patient records and short questionnaires do not need to be reviewed by a medical ethical committee. Statistical analysis Patients and surgical characteristics and outcomes were analyzed with descriptive statistics. The chi-square test was used to compare differences between groups. A p-value <0.05 was considered significant. A generalized linear mixed model (GLMM) was selected to investigate whether the aforementioned variables influenced the relapse risk. A logit link function and a random intercept were applied to address for repeated measurements in subjects (in multiple subjects >1 anatomical area was operated). First, the variables were individually put into the model. If the P-value of the fixed effect or the interaction effect with disease location was below 0.25, the variable was selected for a multivariable model. In the second part of the analysis a multivariable model was created containing all variables including their interaction with location. Backward elimination was applied at the significance level of The least significant terms were eliminated one by one, keeping the model hierarchical. The analyses were conducted with the procedure GLIMMIX of SAS institute. 111

113 RESULTS Primary outcomes Characteristics of patients and surgeries We included 113 HS patients in this study (male: female ratio = 1:2.1) (Table 1). Hurley stage I disease was present in 11.5% of patients, stage II in 77.9% and stage III in 10.6%. In 23 patients the first symptoms appeared at the age of 16 years or younger and in five patients before the age of 11. The median diagnostic delay was 6.5 years. On average, patients had seen 2.35 doctors before they were referred to our specialized center and in 27.4% an accurate diagnosis of HS had not been made until then. The median time between disease onset and surgery under general anesthesia was 12.0 years (IQR seven-18 years). The groins and armpits were affected in 85.8% and 62.8% of patients respectively (table 2). The buttocks were significantly more often involved in men compared to women (69.4% vs. 41.6% respectively, P=0.008). The main indication for general anesthesia was the extensiveness of disease (64.6%), followed by request of the patient (30.1%), mental retardation since consciousness during surgery may be traumatic for these patients and might hinder the procedure (2.7%) and location of the disease (2.7%). Secondary parameters Surgical outcomes A total of 482 regions were operated under general anesthesia in 113 patients (363 primary operations and 119 re-operations). The mean number of locations treated within a single surgical procedure was 2.4. Most primary operations were performed in the groins (40.5%), followed by the armpits (22.9%), buttocks (19.8%) and genital area (10.2%) (table 3). Complications developed in 16% of the 482 operations with the highest percentage occurring in the armpits (33.7%). Hypergranulation of the wound was the most common complication and occurred in 7.2% of all operations, followed by wound infections and post-operative bleeding which both occurred in 1.8% of operations. Remission at the operated anatomical area was achieved in 132 of 363 primary operations (Fig. 2). Disease activity at the operated anatomical area eventually developed after 230 of 363 primary operations of which 124 were considered as natural disease progression and 106 as true relapses due to irradical surgery. 112

114 Factors influencing the risk of relapse For the first screening the variables disease location (P = 0.113), gender (P = 0.037) and smoking (P = 0.141) were selected for the multivariable model (GLMM). The second screening for variables investigating a possible interaction effect with location, indicated that both gender (P = 0.111) and smoking (P = 0.061) should be selected. After backward elimination the final model only contained the variable gender (P = ). The odds ratio [95% CI] for gender was 2.85 [1.07; 7.61], indicating that women have a significant higher risk of relapses after surgery. Tertiary outcomes Patient satisfaction The questionnaire was sent to 105 of 113 patients. Home addresses were missing in three patients, three patients had been deceased and two patients were mentally retarded. A total of 66 of 105 patients responded. There were no significant differences between responders and non-responders with respect to gender (P = 0.93) and relapses due to irradical surgery (p=0.42). Surgery under general anesthesia was the best treatment for HS according to 76% of responders. The median satisfaction scores for the medical and cosmetic effects of surgery were, respectively, 8.0 and 6.0 out of

115 n % Sex Men Women Smoking status Never smoked Missing: 3 Current/former smoker BMI (kg/m2) Normal weight ( 24) Missing: 3 Overweight (>24-29) Obesity ( 30) Hurely stage I II III Age at onset of disease Mean (SD) 23.1 (8.2) Missing: 6 16 years >16-39 years years Previously visited doctors Mean number of visited doctors (range)* 2.35 (0-7) Missing: 50 Specialist who made diagnosis: -General practitioner Dermatologist Surgeon Gastroenterologist Other Median diagnostic delay in years (IQR**) 6.5 ( ) Missing: 59 Median time between disease onset and surgery under general anesthesia in years (IQR***) Missing: ( ) Previous treatments Incision/drainage Wide excision Deroofing under local anesthesia Systemic antibiotics Isotretinoin Biologicals *Before referral to our centre; **Standard deviation; ***Interquartile range Table 1. Patient characteristics 114

116 Location Total, n=113 Men, n=36 Women, n=77 P-value1 n % n % n % Armpits Groins Genital area Buttocks * Submammary area Abdominal fold Retroauricular/neck Other areas * 1Chi-square test; 2mons pubis, scrotum or labia; 3cheeks (n=1), upper legs (n=14), chest (n=5), underneath stoma pouch (n=1), popliteal space (n=3), back (n=2) rump bone (n=5) *Significant difference Table 2. Affected locations 115

117 Armpits Groins Genital area Buttocks Other areas* Total Number of operations Primary operations Re-operations Total Hurley stage, n (% of primary operations) I 12 (14.5) 19 (12.9) ) 25 (34.7) 11 (45.8) 78 (21.4) II/III 71 (85.5) 126 (85.7) 22 (9.4) 44 (61.1) 13 (54.2) 276 (76.0) Unknown - 2 (1.4) 4 (10.8) 3 (4.2) - 9 (2.5) Co-medication, n Tetracyclines Clindamycine/rifampicin Erythromycin Other antibiotics Steroids Biologicals Complications, n (% of total operations) None 65 (66.3) 183 (89.3) 48 (94.1) 88 (86.3) 21 (80.8) 406 (84.0) Wound infection 2 (2.0) 4 (2.0) 1 (2.0) 1 (1.0) 1 (3.8) 9 (1.9) Nervce irritation 4 (4.1) (3.8) 5 (1.0) Bleeding 1 (1.0) 3 (1.5) 1 (2.0) 3 (2.9) 1 (3.8) 9 (1.9) Stricture 3 (3.0) (0.6) Pain >4 weeks 1 (1.0) 1 (0.5) 1 (2.0) 2 (2.0) - 5 (1.0) Other** 22 (22.4) 14 (6.8) - 6 (5.9) 2 (7.7) 44 (9.1) Follow-up in months Median (IQR) (9-89) (14-93) (11-67) (17-91) (21-79) ( ) *Mammary region (n = 5), chest (n = 2), upper legs (n = 4), abdomen (n = 7), neck (n = 1), retroauricular area (n = 1), popliteal stump (n = 1), sacrum (n = 3). **Hypergranulation (n = 35), hypertrophic scar (n = 4), hyperpigmentation (n = 2), delayed wound healing (i.e. >3 months; n = 3). Table 3. Characteristics of surgery in each location 116

118 Figure 2. Outcomes of primary operations for each anatomical area. 117