Clinical and microscopic features of generalized discoid lupus erythematosus in dogs (10 cases)

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1 Vet Dermatol 2016; 27: 488 e131 Clinical and microscopic features of generalized discoid lupus erythematosus in dogs (10 cases) Frane Banovic*, Keith E. Linder, Maarja Uri, Michael A. Rossi** and Thierry Olivry DOI: /vde *Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, 2200 College Station Road, Athens, GA 30602, USA Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27607, USA Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA Small Animal Clinic, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia **Veterinary Skin and Allergy Specialists, Veterinary Referral Center of Colorado, 3550 South Jason Street, Englewood, CO 80110, USA Correspondence: Thierry Olivry, NC State University, College of Veterinary Medicine, 1060 William Moore Drive, Raleigh, NC 27607, USA. Background Generalized discoid lupus erythematosus (GDLE) is a newly recognized canine variant of chronic cutaneous lupus erythematosus (CLE) that is not well characterized. Hypothesis/Objectives We report herein the signalment, clinical signs, treatment outcome, histopathology and immunological findings of 10 dogs with GDLE. Methods Inclusion criteria were: (i) a >3 month history of generalized skin lesions indicating a chronic or recurrent nature; (ii) skin lesions resembling those of human GDLE; (iii) histopathology of CLE (lymphocyte-rich interface dermatitis). Direct immunofluorescence (IF) and antinuclear antibody serology were investigated whenever possible. Results Various breeds were affected in their mid- to late adulthood. Selection criteria of generalized multifocal, annular ( discoid ) to polycyclic plaques with pigment changes, erythematous margin, adherent scaling, follicular plugging and central alopecia were shown in all dogs. In nine dogs, plaques contained mild to moderate central scarring with depigmentation and/or hyperpigmentation. There were no dogs in which the disease progressed to systemic lupus erythematosus within a median follow-up of 2.5 years. Per inclusion criteria, interface dermatitis occurred with basement membrane zone (BMZ) thickening, suprabasal apoptosis and/or dermal fibrosis in some dogs. Infundibular interface folliculitis was common; it sometimes transitioned to mural folliculitis in lower follicle segments, and occurred with follicular and sebaceous gland atrophy. The direct IF revealed patchy deposition of immunoglobulin IgG and IgM at the BMZ. Lesions responded to a variety of treatments, including ciclosporin, hydroxychloroquine, topical tacrolimus and tetracycline/niacinamide. Relapses were common after medications were tapered. Conclusions and clinical importance These observations support the existence of a canine homologue of human GDLE. Introduction In humans, the Sontheimer Gilliam classification divides the skin lesions that can be encountered in lupus erythematosus (LE) into those with characteristic microscopic changes of lymphocyte-rich interface dermatitis with basal keratinocyte damage (i.e. lupus-specific skin diseases = cutaneous LE or CLE) and those without such microscopic pattern (i.e. LE-nonspecific skin diseases). 1,2 Furthermore, LE-specific skin diseases are subdivided into acute, subacute and chronic types (i.e. ACLE, SCLE and CCLE, respectively); 1 these designations refer to both the lesional morphology and the average duration Accepted 24 August 2016 Sources of Funding: This study was self-funded. Conflict of Interest: No conflicts of interest have been declared. 488 of individual skin lesions. 1 A purely histological classification is impossible, because a significant overlap exists between the various clinical subtypes of CLE. 3 Among the several variants of human CCLE [e.g. discoid LE (DLE), verrucous (hyperkeratotic) LE, chilblain LE, lupus tumidus and lupus profundus], DLE represents the most common form: it is divided into a localized variant where skin lesions are confined to the head and neck, and the generalized form, in which skin lesions also occur below the neck. 4 The classic skin lesions of human DLE usually consist of early erythematous and variably scaly macules or papules that slowly evolve into coin-shaped (i.e. discoid) plaques with adherent scales, follicular plugging (i.e. comedones) and peripheral hyperpigmentation presumed to occur secondarily to inflammation. These discoid plaques can coalesce and develop central scarring and depigmentation. 4 Atypical presentations of generalized DLE have been reported in patients of differing 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e131.

2 Canine generalized discoid lupus ethnic groups, such as Indian people from the Asian subcontinent. 5 8 In these patients, the morphological appearance of lesions can vary from hyperpigmented macules 5,6 to hyperkeratotic, hyperpigmented plaques with erythematous border. 7,8 At this time, well characterized manifestations of canine CLE that fall under the category of CCLE include the long recognized, localized, facial-predominant DLE, 9 11 exfoliative CLE (ECLE) of German shorthaired pointers 12 and mucocutaneous lupus erythematosus (MCLE). 13 Several single case reports have suggested the existence of other generalized variants of CCLE in dogs The purpose of this paper is to describe the clinical features, histopathology, immunopathology and treatment outcome of 10 dogs with skin lesions resembling those of generalized DLE (GDLE) of humans. Herein, we propose canine GDLE to be a second variant to be added to the classic facial (i.e. localized) DLE first reported in dogs in Materials and methods Case selection Electronic medical records of canine CCLE cases prospectively collected at North Carolina State University s Veterinary Hospital Dermatology Service between 2009 and 2015 were reviewed. One other case was added after an ad hoc questionnaire was sent to the Vet- Derm list (vetderm@lists.ncsu.edu). Dogs were included if they fulfilled all of the following three criteria: 1 a >3 month history of generalized skin lesions indicating their chronic or recurrent nature; 2 the presence of skin lesions resembling those of human GDLE; that is the presence of annular (discoid) to polycyclic plaques with dyspigmentation, adherent scaling, follicular plugging and central alopecia below the neck); 3 the presence of microscopic lesions typical of CLE (i.e. a lymphocyte-rich interface dermatitis with basal cell damage). Dogs with macular, vesiculobullous, typical and atypical target skin lesions, and histopathological findings of cytotoxic lymphocytic interface dermatitis with keratinocyte apoptosis occurring throughout multiple epidermal layers suggestive of erythema multiforme variants [minor (EMm) and major (EMM)] 18 were excluded. We also did not include German shorthaired pointers with lesions typical of ECLE 12 or dogs with skin lesions characteristic of MCLE. 13 Histopathology Histopathological changes were identified and scored for severity in haematoxylin and eosin-stained skin biopsy sections. Changes were scored subjectively as absent, mild, moderate or marked and the results provided normally reflected the most developed lesional area identified in a set of biopsies. Because a cell-rich lymphocytic interface dermatitis was an inclusion criterion, histological sections were evaluated for morphological variation, distribution and severity thereof. The severity of keratinocyte apoptosis and lymphocytic satellitosis of apoptotic cells in the basal layer of the epidermis were scored separately from that in suprabasal layers. Dispersal of epidermal pigment to dermal macrophages (pigmentary incontinence), epidermal hyperpigmentation, epidermal ulcers, atrophy, hyperplasia and hyperkeratosis were also scored, and the presence of orthokeratosis and parakeratosis was noted. Basement membrane zone (BMZ) thickening, dermal fibrosis and the severity of lichenoid inflammatory infiltrates (subepidermal band-like infiltrate of inflammatory cells) were graded and the predominant inflammatory cell types were recorded. Hair follicles were evaluated for inflammatory infiltrates at all levels (folliculitis patterns), keratinocyte apoptosis, follicular atrophy, infundibular hyperkeratosis and perifollicular fibrosis. Sebaceous gland inflammation and atrophy were recorded. Direct immunofluorescence (IF) In order to detect the presence of immunoglobulin (IgG, IgA, IgM) and activated complement C3 deposits along the BMZ, direct IF was performed as described previously. 12 The frequency, distribution (including extension along the basement membrane of hair follicles) and characteristics of deposits were described subjectively. The detection of a thin-to-thick linear deposit at the BMZ, either continuous or interrupted (i.e. patchy), was considered a positive lupus band test (LBT). 19 Evaluation for systemic lupus erythematosus In order to rule out concurrent systemic lupus erythematosus (SLE), further investigations included combinations of serum antinuclear antibodies (ANA) titre, serum chemistry profile, complete blood count and urinalysis. The detection of serum ANA was performed using a variety of techniques depending upon the laboratory used for such testing. Clinical management and prognosis Data on signalment, history, clinical signs and treatment outcome for each case were collected and analysed. Details on previous, initial and maintenance immunosuppressive therapy (dose, frequency and length of treatment for each drug or combination of drugs) were reviewed for each case. Additionally, any adverse effects related to drug therapy, concurrent diseases and frequencies of initial disease relapses were reported. Results Clinical summary Ten dogs met the inclusion criteria. Three of the cases included herein have been previously published as single case reports. 14,16,17 Selected dogs included seven pure bred and two cross bred dogs: two were Chinese crested dogs and Labrador retrievers; there was one each of the following pure breeds: miniature pinscher, Leonberger, shih tzu and toy poodle. The age of onset of GDLE skin lesions varied between five and 12 years of age (median 9 years), whereas the female-to-male ratio was one; all dogs were castrated. Three dogs had a previous history of a skin disease: one dog each suffered from either cutaneous melanocytoma, atopic dermatitis or liver associated necrolytic migratory erythema (NME). Before the initial visit, skin lesions had been present between 3 and 24 months (median: 9 months). The initial lesions most commonly reported by the owners were erythematous macules, papules and plaques with erosions and scaling; these were first noticed usually on the neck and trunk. Upon presentation all dogs had lesions on the neck, dorsum and lateral thorax. Lesions were also present on the head in seven of 10 cases (70%), whereas abdomen and medial and lateral aspects of proximal limbs were affected in eight dogs (80%). According to the inclusion criteria, skin lesions included generalized multifocal, annular (discoid) to polycyclic plaques with dyspigmentation, an erythematous margin, adherent scaling, follicular plugging and central alopecia (Figure 1). In nine dogs (90%), the plaques evolved with mild to moderate ulcerations, central atrophic or hypertrophic scarring and 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e

3 Banovic et al. a b c d Figure 1. Typical skin lesions of generalized discoid lupus erythematosus (GDLE) in dogs at initial presentation (a) Two annular hyperpigmented plaques with focal central depigmentation and scarring (Case 2). (b) An annular to polycyclic plaque consisting of central alopecia, erythema, depigmentation and atrophic scarring with peripheral hyperpigmented rim and scaling (Case 3). (c, d) Annular coin-shaped plaques with characteristic depigmentation, atrophic (c) and hypertrophic (d) scarring at inactive centre and hyperpigmentation with erosions and adherent scaling at peripheral active border (Case 5). pigmentation changes (depigmentation and hyperpigmentation; Figure 1). Generalized hyperpigmented to depigmented macules/patches with occasional thick adherent scaling, central alopecia and erythematous margin were seen in nine dogs (90%; Figure 2). In one apricot miniature poodle, reddish brown patches and plaques with large scales were present throughout the body, and there was extensive spontaneous alopecia that occurred even outside of the more focal inflammatory lesions (Figure 3). Four dogs (40%) had mucocutaneous regions involved with plaques appearing most commonly on or around the genitalia (Figure 4). One Chinese crested dog concurrently had depigmentation, deep erosions and loss of architecture of the nasal planum (Figure 4). Hyperpigmented macules/plaques with adherent scaling and follicular plugging were observed on the concave pinnae and 490 caudal (lateral) ear margins of three dogs (Figure 5a,b). An unusual pattern of reticulated (net-like) hyperpigmentation was visible on the ventral abdomen and lateral thorax in two cases (Figure 5c,d). There were no systemic signs observed in any dog, apart from the pruritus and pain at the site of lesions in four (40%) and three dogs (30%), respectively. A complete blood count revealed mild regenerative anaemia, serum chemistry results included mild hypoalbuminemia and moderate elevations of liver enzymes in two of 10 dogs (20%), whereas urinalysis results were unremarkable in all dogs. Histopathology A total of 25 biopsy specimens were evaluated (2 4 per dog). The cell rich lymphocytic interface dermatitis was present in all cases (this was an inclusion criterion, 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e131.

4 Canine generalized discoid lupus a b c d Figure 2. Close up of the macular/patch type skin lesions present on the thorax of dogs with GDLE (a) Well demarcated annular to polycyclic hyperpigmented patches with severe adherent scaling, partial alopecia and mild scarring in the centre (Case 10). (b) Well demarcated annular hyperpigmented macules to patches with prominent follicular plugging, scaling, peripheral erythematous rim and central ulceration (Case 3). (c, d) Hyperpigmented annular macules and patches with central loss of tissue architecture and prominent silver adherent scales surrounded by a mild peripheral erythematous margin (d) (Case 7). however). Moderate to marked basal cell degeneration, also an inclusion criterion, consisted of keratinocyte vacuolation, apoptosis and/or disappearance (Figure 6). The distribution of these lesions varied from diffuse to focal or peri-ulcerative; in two cases the severity of basal cell injury led to microscopic intrabasal cleft formation. Apoptosis in suprabasal epidermal layers (Figure 7) was frequently observed (eight of 10; 80%): it was mild (1 10 cells per biopsy section) in five cases (50%) and moderate (10 25 cells per section) in three others (30%). Overall, the suprabasal apoptosis was less severe than the interface change observed for each case. Lymphocytic satellitosis of apoptotic basal cells (Figure 6) was robust and present in all cases, whereas the lymphocyte targeting of apoptotic suprabasal cells occurred in fewer dogs (four of eight; 50%). Similarly, lymphocytic exocytosis was prominent in the deep epidermis and was absent or mild in superficial epidermal layers. Foci of epidermal atrophy were uncommon (three of 10; 30%), and most cases (seven of 10; 70%) exhibited mild to moderate, diffuse, epidermal hyperplasia. Interface dermatitis was associated with foci of partial epidermal collapse, with flattening and elongation of keratinocytes occurring in an otherwise hyperplastic epidermis (Figure 6). A mild and occasionally marked orthokeratotic hyperkeratosis occurred in most cases with occasional interruption by foci of parakeratosis. Moderate to marked pigmentary incontinence colocalized with foci of epidermal depigmentation and/or areas of epidermal hyperpigmentation in all cases. Moderate to marked BMZ thickening was detected in focal areas in several cases (six of 10; 60%). A focal to diffuse and mild to marked lichenoid, dermal inflammatory infiltrate composed of lymphocytes mixed with a similar amount or fewer plasma cells was seen in eight cases (80%; Figure 6). In two dogs that lacked the lichenoid infiltrate, as well as in cases where lichenoid infiltrates showed limited distribution in comparison to the interface dermatitis, the lymphocytes were still numerous in the basal epidermal layer. A mild perivascular inflammatory pattern was observed in the superficial 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e

5 Banovic et al. a b Immunopathology Antinuclear antibody serology was determined for eight of 10 dogs (80%) and low ANA serum titres (1:20 1:40) were detected in seven of these eight cases (88%). Although ANA serology was positive at low dilutions in most dogs, the lack of systemic signs and laboratory results to suggest renal or haematological involvement excluded a diagnosis of coexisting SLE. 20 Direct IF revealed fine to thick, patchy deposition of IgG and IgM along the dermo-epidermal junction of lesional paraffinc d Figure 3. Skin lesions of generalized discoid lupus erythematosus in an apricot poodle. Generalized brown coloured patches and plaques with large scales and extensive spontaneous alopecia throughout the body (a, b, d) and involving the dorsum of head and ears (c) (Case 9). dermis, whereas the middle and deep dermis usually contained minimal or no inflammation (Figure 6). A mild to marked superficial, laminar dermal fibrosis (five of 10; 50%) extended from the BMZ, and was sometimes paucicellular. Occasional ulcers (four of 10; 40%) were associated with mild neutrophilic dermatitis and crusts. Lesions indicative of vasculitis or panniculitis were not observed in any section. Histological changes in adnexa were evaluated for eight of 10 dogs (80%), excluding the two hairless Chinese crested dogs. Mild to moderate perifollicular lymphoplasmacytic inflammation was observed in all eight cases; the inflammation surrounded the infundibulum and tapered inwards through the level of the isthmus (Figure 8). A moderate to marked lymphocytic interface folliculitis involved the hair follicle infundibula of these eight cases, and the changes were similar to the basal cell degeneration, suprabasal apoptosis and lymphocytic exocytosis with satellitosis observed in the epidermis (Figure 8). The interface folliculitis that extended to the isthmus was usually mild and occasionally moderate; however, this distinction was difficult to make in cases of advanced follicular atrophy. Lymphocytic mural folliculitis (Figure 8) was mild to moderate in the infundibulum of seven cases (88%) and usually milder in the lower follicular segments of all cases. Scattered individual and loose clusters of lymphocytes infiltrated the external root sheath of anagen 492 stage, telogen stage and atrophic follicles. Very rarely, occasional individual lymphocytes infiltrated an anagen hair bulb. Hair follicle atrophy was observed in all cases and (Figure 8) ranged from diffuse and advanced to focal and mild, with atrophic follicles sometimes bordering anagen follicles. Sebaceous gland atrophy was also seen in all dogs and was more commonly associated with atrophic hair follicles; the severity ranged from mild and partial in at least one biopsy. The inflammation within the glands was limited to the walls of sebaceous ducts that sometimes contained a few lymphocytes; infiltration of sebaceous gland lobules was not observed. Follicular hyperkeratosis and infundibular dilation were mild or absent in most cases. Perifollicular fibrosis was uncommon (one of eight cases; 13%), mild and restricted to the infundibulum ESVD and ACVD, Veterinary Dermatology, 27, 488 e131.

6 Canine generalized discoid lupus a b c d Figure 4. Skin lesions affecting mucocutaneous junctions in dogs with GDLE (a) Well-demarcated bilaterally symmetrical diffuse hyperpigmentation with adherent scaling and partial alopecia on the muzzle, including periocular areas. Central depigmentation, scarring and ulcerations involve the nasal planum and bilaterally the medial canthus of the eyes (Case 5). (b) Multifocal hyperpigmented macules with mild erythematous rim on prepuce (Case 5). (c) Anal/perianal diffuse hyperpigmentation with multifocal depigmentation, scarring and adherent scaling. Note the linear ulcerations in friction areas; this case suffered from liver associated necrolytic migratory erythema. The development of GDLE type skin lesions in this area is suggestive of the Koebner phenomenon (Case 5). (d) Diffuse hyperpigmented alopecic patch/plaque with multifocal depigmentation, scarring and ulceration at the external vulva and perivulvar skin (case 79). embedded skin sections in nine of 10 dogs (90%) (Figure 9). An additional positive LBT for IgA and C3 was found in three (30%) and two (20%) cases, respectively. Positive LBTs were detected for four and two immunoreagents in three (30%) and seven (70%) of 10 dogs, respectively. Clinical management and prognosis Information on treatment outcome was available for all dogs. Prior to the initial visit, the skin lesions were present between three and 24 months, with a median of approximately 9 months. Drugs prescribed prior to the diagnosis of GDLE was made included short courses of antimicrobial drugs (e.g. cefalexin, clindamycin, enrofloxacin; eight of 10 cases, 80%) and a single glucocorticoid course (four of 10 cases, 40%); these regimens resulted in only transient improvement of skin lesions in two dogs (20%). A spontaneous resolution of cutaneous lesions was not seen in any case. In one dog, an initial four month combination of doxycycline and niacinamide, thrice daily at appropriate dosages ( mg per dog three times daily), 21 did not result in clinical improvement. Once the coexisting SLE was ruled out and the diagnosis of GDLE diagnosis had been made, a recommendation to avoid excessive sun exposure was given to the owners for all dogs, and three different therapeutic modalities were initiated: Group 1 (six of 10 dogs; 60%) was commenced with oral glucocorticoids (1 2 mg/kg/ day, progressively tapered over a month) given with oral ciclosporin (range mg/kg, mean 4.8 mg/kg once daily, Atopica; Elanco Animal Health; Greenfield, IN, USA), in conjunction with ketoconazole in three dogs 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e

7 Banovic et al. a b c d Figure 5. Canine GDLE (a) Hyperpigmented macules and plaques with erythema, adherent scaling, crusting and follicular plugging of the concave pinnae and caudal (lateral) pinna margin. Note that this this pattern of clinical involvement is highly specific for human discoid lupus erythematosus. (Case 3). (b) Diffuse hyperpigmentation with adherent scaling on the pinna (Case 5). (c) An unusual pattern of reticulated (net-like) hyperpigmentation with alopecia on the ventral abdomen. Inset: Higher magnification shows classic coin-shaped plaques with central depigmentation, scarring and peripheral hyperpigmentation (Case 3). (d) Reticulated hyperpigmentation pattern developing from coalescing GDLE skin lesions (Case 2). (range mg/kg, mean 2.3 mg/kg once daily; Teva Pharmaceuticals; Sellersville, PA, USA), Group 2 (three of 10 dogs; 30%) received topical tacrolimus 0.1% ointment (twice daily, Protopic; Astellas Pharma; Deerfield, IL, USA) and oral hydroxychloroquine (HCQ) (5 mg/kg once daily, Gallipot; St Paul, MN, USA); and a single dog (10%) was started solely on the tetracycline/niacinamide combination (45 mg/kg of each drug three times a day). A complete remission of clinical signs (scarring alopecia remained in some lesions) following treatment occurred after 3 6 months (median 4.5 months) in five dogs (50%; two dogs each from Group 1 and 2; the dog from Group 3), whereas an approximately 75% partial remission was seen after 1 6 months (median 5.5 months) in the other five dogs (50%). An attempt to discontinue or taper the treatment resulted in the rapid recurrence of typical skin lesions in six dogs (60%); re-introducing the treatment modality at lower frequency of administration (e.g. ciclosporin every other day; topical tacrolimus twice weekly without HCQ) maintained remission of clinical signs in 494 five dogs. In one dog that had several disease flares while receiving HCQ, oral ciclosporin (5 mg/kg once daily) was introduced and induced complete clinical remission over the following four months. Discussion Although there are two large case series of dogs with classic, facial (nasal) planum-predominant localized DLE, 10,11 there are three other case reports of dogs with an apparent generalized variant of this disease. 14,16,17 Herein, we describe the signalment, clinical signs and treatment outcomes of 10 dogs with generalized skin lesions with clinical, microscopic and immunopathological features of human GDLE. Generalized DLE affected various breeds of dogs and their crosses with an equal representation of male and female dogs. In humans with GDLE, women are affected more often than men (female-to-male ratio of 1.5). 22 Interestingly, and surprisingly, German shepherd dogs, a 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e131.

8 Canine generalized discoid lupus Figure 6. Histopathology of canine GDLE. A marked, subepidermal, band-like (lichenoid) infiltrate of lymphocytes with fewer plasma cells is associated with a lymphocytic interface dermatitis reaction. Basal keratinocyte vacuolation and apoptosis (inset, arrows) are moderate to marked and are associated with pigmentary incontinence. The hyperplastic epidermis is multifocally attenuated. Basement membrane zone thickening is present (inset, arrowhead). The middle and deep dermis have minimal perivascular inflammation. Case 2; haematoxylin and eosin, magnification 1009, inset magnification Figure 7. Histopathology of canine GDLE. Mild lymphocytic interface dermatitis is associated with multifocal suprabasal keratinocyte apoptosis (arrows). Case 7; Haematoxylin and eosin, magnification breed predisposed to develop several forms of LE, such as SLE, localized facial DLE 11 and MCLE, 13 were not represented in this population. This discrepancy may be explained by the German shepherd dog breed not being predisposed to GDLE, by the small size of our study group or by a possible clinical misdiagnosis of this disease as idiopathic lichenoid dermatoses based on histopathological identification of a lichenoid tissue reaction in dogs. 26 Indeed, one dog from our series had been initially diagnosed and referred with such a diagnosis. The age of onset of GDLE was variable, with most dogs exhibiting their first skin lesions in mid to late adulthood. A comparable age of onset is reported for humans with DLE, as the disease most commonly develops in the fourth to fifth decade. 27 As defined by our inclusion criteria (i.e. lesions resembling those of human GDLE), erythematous disc-like plaques with adherent scaling that extended into hair follicles and dyspigmentation were seen in all of our cases. The central loss of normal skin texture and scarring that occurred as the result of long term persistent DLE activity was present in the centre of skin lesions of all but one dog. Follicular plugging (i.e. comedones) was a prominent feature of canine GDLE, as it is in the human disease. However, the characteristic carpet tack sign seen in human DLE, 6 which are follicular-sized keratotic spikes similar in appearance to carpet tacks at the under surface of removed adherent scales, unfortunately was not assessed in these cases. However, papular, corneocyte-plugged follicles within areas of hyperpigmentation in the concave pinnae and caudal (lateral) ear margin, a pattern highly specific for human DLE, 6 was also seen in several of the canine cases. Although any ethnic group can be affected with human DLE, collective data indicate that skin lesions with typical inflammatory hypo- and hyperpigmentation are more frequent in darker-skinned individuals, whereas in patients with light skin, plaques usually appear grey or have minimal pigment alteration. 6 Interestingly, a diffuse reticulated hyperpigmentation on the ventral abdomen and lateral thorax was present in two dogs. Such an unique pigmentation pattern has been reported in a dog with an alopecic variant of CCLE, 15 dogs with MCLE 13 and recently in a dog with a presumptive idiopathic EM. 18 In humans with DLE, discrete plaques can develop on the nasal, conjunctival and genital mucosa. 27,28 Although four dogs included in the present study exhibited typical DLE lesions in mucocutaneous regions, most commonly in the genital/perigenital area, there are important differences between their character and that of lesions seen in dogs with MCLE. 13 In the latter, characteristic extensive erosive skin lesions, sometimes with peripheral hyperpigmentation, that are restricted to mucocutaneous regions predominate, but the depigmentation and scarring of GDLE are typically not seen in these dogs. 13 Remarkably, one dog with GDLE and liver-associated NME developed classical DLE lesions of depigmentation, erythema, scarring and crusting at perioral, periocular and perianal areas, long after the development and apparent healing of the initial NME-associated oedematous and erosive lesions. The occurrence of DLE lesions in this dog at previous NME-associated mucocutaneous locations could be evidence suggesting that GDLE lesions, similarly to those of human DLE, can follow any form of cutaneous trauma, a response known as the Koebner phenomenon. 29 In humans affected with the generalized variant of DLE, a positive ANA titre is frequently found, and it represents a risk factor for development of SLE within 5 years after the initial diagnosis of skin lesions. 30 Seven dogs in this report had a low positive ANA serum titre, but a progression to the exhibition of additional criteria for SLE was not seen in any dog within a median follow-up of 2.5 years (range from 0.5 to 6 years). To the best of the authors knowledge, the progression from a CCLE variant to SLE has been reported only in one dog. 15 The histology of DLE in humans is characterized by a lichenoid interface dermatitis reaction pattern, which was an inclusion criterion in this study (i.e. cell-rich, lymphocytic interface dermatitis). 1,2,31,32 Like typical, fully developed DLE in humans, interface dermatitis occurred in a hyperplastic epidermis with focal areas of epidermal atrophy, epidermal hyperpigmentation or depigmentation, and pigmentary incontinence. 3,32,33 Similarly to human GDLE, the interface reaction (vacuolar degeneration, apoptosis and loss of basal cells) was well developed and 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e

9 Banovic et al. a b c Figure 8. Histopathology of canine GDLE (a) The deep infundibulum and isthmus of a hair follicle with lymphocytic interface folliculitis and mural folliculitis associated with perifollicular lymphoplasmacytic inflammation, pigmentary incontinence and mild follicular hyperkeratosis (Case 9). (b) In this inferior portion of an anagen stage hair follicle, mild lymphocytic mural folliculitis is present in the external root sheath. A few lymphocytes (arrows) are present in the follicle wall and inflammation in the perifollicular dermis is absent (case 9). (c) A hair follicle unit has marked hair follicle atrophy (arrows) and a few small islands of external root sheath epithelium contain a few lymphocytes (Case 4). Magnification Haematoxylin and eosin. more severe than is typical of localized DLE in dogs. 31,32 As a consequence of this severity, microscopic intrabasal clefts were observed in two cases. Apoptosis of suprabasal keratinocytes occurs in canine 13,34 and human CLE 32,35 and was common in our dogs with GDLE. Interface dermatitis and basement membrane thickening were more prominent than suprabasal apoptosis and satellitosis; the latter two findings typically are also seen in the context of EM and toxic epidermal necrolysis. 8 Interestingly, occasional foci of grouped suprabasal apoptosis, in conjunction with lymphocytic satellitosis, were observed and mimicked an EM-type reaction pattern 18 in a few skin sections. Similar observations of increased 496 suprabasal apoptosis were identified in association with skin chronicity in ECLE of German shorthaired pointers, 34 and they have been reported for canine localized DLE as well. 31 With the addition of these cases, suprabasal keratinocyte apoptosis is now recognized in several different forms of CCLE in dogs, including localized DLE, 31 GDLE, MCLE 13 and ECLE. 34 It should be noted that solar injury is an additional factor that both exacerbates LE lesions in dogs and humans, 1,2 and sun induced damage could contribute to the development of apoptosis of suprabasal keratinocytes. 1,2,31,32 Lichenoid inflammation, a subepidermal band of lymphocytic infiltration, predominated in our cases with 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e131.

10 Canine generalized discoid lupus a b Figure 9. Direct immunofluorescence of canine GDLE (a) Thick and patchy IgG deposition along the epidermal basement membrane zone. (i.e. positive lupus band test; arrowheads). Inset: Magnification of IgG deposition with fluorescent round cells in the superficial dermis that represent IgG-positive plasma cells, a common finding at inflamed mucocutaneous junctions [Case 2; anti-igg fluorescein with diamidino phenylindole (DAPI), 109 magnification]. (b) Thick and patchy IgM deposition along the epidermal basement membrane zone. (Case 5; anti-igm fluorescein with DAPI, 109 magnification). GDLE, and similarly to late stage DLE lesions in humans, 3 this band was sometimes greatly diminished in areas of superficial dermal fibrosis. Additionally, a lichenoid band was absent in two cases, which is a feature of early DLE lesions in humans. 3 In our dogs, a perivascular pattern of inflammation was mild superficially and usually absent in the middle and deep dermis, which is similar to dogs with localized DLE. 31 This is in contrast to humans with localized DLE or GDLE where perivascular inflammation is often well developed throughout the dermis. 3,32 Thickening of the BMZ occurred in 60% of our cases and this is a characteristic, but not specific, feature of CCLE in dogs and humans ,31,32 Clinical scarring was attributed to the superficial dermal fibrosis observed histologically. Increased epidermal and dermal mucin deposits have been proposed as an additional diagnostic factor for localized DLE in the dog; 36 however, the detection of skin mucin deposits alone has limited specificity in humans and is common in other dermatoses such as EM, lichen planus and fixed drug reactions. 37 Detailed studies characterizing skin mucin distribution and deposition in canine CLE variants (localized DLE, GDLE, ECLE, VCLE, MCLE) and other diseases (EM variants, ischemic dermatopathy) are necessary to elucidate whether mucin deposition in the skin of dogs with CLE is a specific finding and is able to distinguish between different disease entities. Similarly to human CLE forms, in which histopathological findings can overlap and may not clearly differentiate between subtypes, 3,38 our study suggest that biopsy findings alone do not differentiate GDLE from other forms of canine CCLE (localized DLE, MCLE and ECLE) or SLE. Scarring alopecia occurs on the head in localized DLE in dogs but histological descriptions of follicular changes are lacking. 9 11,31 In humans, when localized DLE involves the scalp, scarring alopecia is relatively common, occurring in 34% of DLE skin sections in one study; it is associated with lymphocytic interface folliculitis and follicular atrophy. 39 Human DLE is classified as a primary scarring (cicatricial) alopecia because permanent alopecia occurs clinically and direct lymphocytic targeting of the follicle (interface folliculitis) is associated with follicular atrophy and, ultimately, loss of the follicular epithelium the follicular scarring event Concentric laminar fibrosis occurs around the infundibulum and mid-follicle but it is a mild, late stage event and is not the main reason for classification of DLE as a scarring alopecia In our study of canine GDLE, alopecia occurred in nearly all cases and lymphocytic interface folliculitis involved the infundibulum and extended into the isthmus as for human DLE. 43,44 Lymphocytic mural folliculitis was also common, usually milder and involved the infundibulum, isthmus and inferior hair follicle segments, typically sparing the bulb. This mural pattern mirrors that of human DLE, where it is also called a panfollicular pattern, but it is considered to be minimal and poorly described. 39,43 Follicular atrophy was present in all cases and ranged from partial to complete. The combination of lymphocytic interface folliculitis, advanced follicular atrophy and lack of hair regrowth in a subset of dogs supports the classification of canine GDLE as a primary scarring alopecia. 41,42 Perifollicular fibrosis was not a feature in our study, however. Follicular hyperkeratosis, plugging and dilation occurred in our cases clinically, as is reported in human DLE, but related histological changes in follicles were very limited, a clinicopathological discrepancy that some authors have noted for human DLE. 32,39 Appropriate biopsy site selection for GDLE lesions is at the periphery of the lesion, which presumably has less advanced follicular injury than older central areas, and this could explain the limited infundibular changes and perifollicular fibrosis histologically in the present study. Sebaceous gland atrophy in our cases was sometimes complete and often mild and partial, which is similar to descriptions of human DLE, but lacked the mild glandular lymphocytic infiltrate that can occur. 32 In dogs with ECLE, partial to complete sebaceous gland atrophy occurs in synchrony with follicular atrophy; it is associated with a mild lymphocytic periglandular infiltrate, which we did not see in our cases. 12 The LBT initially was considered to be quite specific for human CLE but controversies regarding terminology (number of different deposits at dermo-epidermal junction) and standardization (lesional versus nonlesional skin, sun exposed versus sun protected skin) have clouded its diagnostic value. 6 A positive LBT in sun protected 2016 ESVD and ACVD, Veterinary Dermatology, 27, 488 e

11 Banovic et al. nonlesional skin appears to have the highest diagnostic specificity for clinical association with SLE. 19 Furthermore, most authors agree that the LBT specificity and predictive value increases with the number of immunoreactants detected at the dermal epidermal junction. 45,46 In our series, a linear deposition of IgG and IgM at the dermo-epidermal basement membrane zone (i.e. a positive LBT) of lesional skin was found in 90% of cases, resembling the findings seen in human DLE lesions. 45,47 Interestingly, the most commonly detected immunoreactant deposited in facial-predominant canine localized DLE was C3 (90 100%), whereas IgG and IgM were revealed in 40 70% of cases, respectively. 10,11 These variable results between canine localized and generalized DLE could be related to differences in tissue fixation techniques (frozen versus formalin), antigen retrieval methods and/or immunofluorescence staining protocols. To investigate the value of performing DIF in canine CLE diagnostic investigation, future studies regarding the sensitivity and specificity of a positive LBT for the diagnosis of CLE variants are warranted. Currently, there are no medications approved specifically for the treatment of human CLE. 48 Besides photoprotection, glucocorticoids and/or calcineurin inhibitors remain the classic topical approach for localized DLE, whereas the systemic drug of choice is the antimalarial drug HCQ. 48 In this report, various treatment regimens were successfully used to treat dogs with GDLE. Oral HCQ in conjunction with topical tacrolimus application helped to induce and maintain remission of skin lesions in two dogs with this disease. Although retinal toxicity limits the use of HCQ in humans, it was not observed in any dog in this case series or in dogs with ECLE. 34,49 In our study, a remarkable improvement or a complete remission in GDLE skin lesions followed treatment with ciclosporin along with a short course of glucocorticoids at its onset. Given that ciclosporin and tacrolimus share a similar mechanism of action on T-lymphocytes, and that tacrolimus ointment has been used successfully for topical treatment of human 47 and canine localized DLE, 50,51 we had inferred that ciclosporin might be beneficial for treatment of canine GDLE. Interestingly, ciclosporin is no longer recommended for treatment of human CLE due to a relatively unfavourable risk benefit profile. 48 Since 1992, antibiotics of the tetracycline family along with niacinamide have been suggested to be beneficial for treatment of canine immune-mediated skin diseases, including the CLE variants; localized DLE, 20,52 ECLE 52 and MCLE. 13,52 In our case series, one dog did not respond to a 4 month combination of doxycycline and niacinamide, whereas another dog had a complete remission of signs with tetracycline and niacinamide. In humans, evidence exists on the equipotency of different tetracycline for therapy of bullous pemphigoid, 53 as some authors successfully used doxycycline or minocycline as an alternative to tetracycline. Although tetracycline and doxycycline were shown to be relatively similar in effectiveness to treat canine lupoid onychodystrophy (idiopathic onychitis/ onychomadesis), 54 there are no data on which of the tetracyclines is more effective, and whether niacinamide is of synergistic value for treatment of CLE variants in dogs; this needs to be studied further. 498 In conclusion, we report herein 10 dogs with an unique clinical phenotype as well as histological and immunofluorescence findings that resemble the features of generalized human DLE patients. Skin lesions of canine GDLE appear to respond to a wide range of treatments, but half of the cases experienced relapses upon the tapering of drug dosages. Our limited outcome data suggest that ciclosporin should be considered as a potentially effective therapeutic option for canine GDLE, especially for dogs refractory to HCQ, niacinamide and tetracycline therapy. 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A retrospective study regarding the treatment of lupoid onychodystrophy in 30 dogs and literature review. J Am Anim Hosp Assoc 2003; 39: Resume Contexte Le lupus erythemateux disco ıde generalise (GDLE) est un variant canin nouvellement reconnu du lupus cutane chronique (CLE) qui n est pas bien defini. Hypotheses/Objectifs Nous rapportons ici le signalement, les signes cliniques, l efficacite des traitements, l histopathologie et les donnees immunologiques de 10 chiens atteints de GDLE. Methodes Les criteres d inclusion etaient (i) a>3 mois de commemoratifs de lesions cutanees generalisees correspondant a une atteinte chronique ou recidivante; (ii) des lesions cutanees ressemblant a celles de la GDLE de l homme; (iii) l histopathologie de CLE (dermatite d interface riche en lymphocytes). L immunofluorescence directe (IF) et la serologie anticorps antinucleaires ont ete etudiees si possible ESVD and ACVD, Veterinary Dermatology, 27, 488 e