Laboratory Diagnosis and Clinical Profile of Anti-p200 Pemphigoid

Importance  Anti-p200 pemphigoid is a rare subepidermal autoimmune blistering disease characterized by autoantibodies against a 200-kDa protein in the basement membrane zone. Anti-p200 pemphigoid is probably often misdiagnosed because of low availability of diagnostic assays and expertise and classified as bullous pemphigoid or epidermolysis bullosa acquisita.

Objective  To clinically characterize patients with anti-p200 pemphigoid, identified by using indirect immunofluorescence microscopy on skin substrates deficient in type VII collagen and laminin-332 (knockout analysis), to validate this technique by immunoblot with dermal extract, and to incorporate direct immunofluorescence serration pattern analysis in the diagnostic algorithm.

Design, Setting, and Participants  This was a retrospective study performed from January 2014 to June 2015 with biobank patient materials and clinical data for the period 1998 to 2015 from the single national referral center on autoimmune bullous diseases. Patients were selected based on a dermal side binding on 1-mol/L salt (sodium chloride)-split human skin substrate by indirect immunofluorescence microscopy, not diagnosed epidermolysis bullosa acquisita or anti–laminin-332 mucous membrane pemphigoid.

Main Outcomes and Measures  Indirect immunofluorescence microscopy knockout analysis was performed and diagnosis of anti-p200 confirmed by immunoblot with dermal extract. Clinical, histological, and immunological findings were registered. Autoantibodies against laminin γ1 were determined by immunoblot.

Results  Twelve patients with anti-p200 pemphigoid (7 male and 5 female; mean age, 66.6 years) were identified using the indirect immunofluorescence microscopy knockout analysis. Direct immunofluorescence microscopy showed a linear n-serrated IgG deposition pattern along the basement membrane zone in 9 of 11 patients. The diagnosis was confirmed by immunoblot showing autoantibodies against 200-kDa protein in dermal extract in 12 of 12 patients. Autoantibodies against recombinant laminin γ1 were detected by immunoblot in 8 of 12 patients. Remarkable similarities were seen in clinical features with predominantly tense blisters on hands and feet, resembling dyshidrosiform pemphigoid. Mucosal involvement was seen in 6 (50%) of the patients.

Conclusions and Relevance  Predominance of blisters on hands and feet may be a clinical clue to the diagnosis of anti-p200 pemphigoid. Direct immunofluorescence microscopy serration pattern analysis and indirect immunofluorescence microscopy knockout analysis are valuable additional techniques to facilitate the diagnosis of anti-p200 pemphigoid.

Anti-p200 pemphigoid is a rare subepidermal autoimmune bullous disease (sAIBD) originally described in 1996 and characterized by autoantibodies against a 200-kDa protein, localized within the lower lamina lucida of the basement membrane zone (BMZ).^1-3 Anti-p200 pemphigoid is probably often misdiagnosed and classified as bullous pemphigoid (BP) or epidermolysis bullosa acquisita (EBA) because of low availability of diagnostic assays and expertise.⁴ Serum samples of most patients with anti-p200 pemphigoid seemed to recognize laminin γ1, and therefore the term anti–laminin γ1 pemphigoid was proposed for the disease.⁵ However, until now, ex vivo and in vivo studies showed no direct pathogenic role of anti–laminin γ1 antibodies, and the molecular identity of the pathogenic 200-kDa autoantigen still remains unclear.^6,7

Approximately 100 cases have been published so far, and the incidence is likely higher; the clinical characteristics and disease course are not well known.⁴ The heterogeneous clinical presentation of anti-p200 pemphigoid may mimic BP, linear IgA dermatosis and inflammatory EBA.^4,8 Moreover, the clinical course of anti-p200 pemphigoid is reported to be more benign compared with EBA and even BP.⁴

Diagnosis of anti-p200 pemphigoid is based on a combination of histopathologic analysis, direct immunofluorescence (DIF) microscopy, and immunoserologic testing. Histopathologic analysis alone does not allow differentiating anti-p200 pemphigoid from other sAIBDs, and a linear deposition of IgG and C3c along the BMZ by DIF is found in all IgG-mediated subtypes of sAIBD.⁹ Dermal side binding on indirect immunofluorescence microscopy on 1-mol/L salt (sodium chloride)-split skin (IIF SSS) enables to differentiate anti-p200 pemphigoid from other sAIBD but not from EBA and anti–laminin-332 mucous membrane pemphigoid (anti–LN-332 MMP). The differentiation is of importance because in anti–LN-332 MMP oncological screening may be indicated and in EBA often a refractory response to treatment is seen.

The laboratory diagnosis of anti-p200 pemphigoid can be made by immunoblot with human dermal extract but is established in only a few specialized laboratories.^1,4 In addition, IIF microscopy knockout analysis on skin sections from patients with hereditary epidermolysis bullosa completely lacking expression of type VII collagen and laminin-332 has been used prior in experimental studies to provide the evidence that the 200-kDa autoantigen is different from type VII collagen and laminin-332.^10,11 To further facilitate the laboratory diagnosis of anti-p200 pemphigoid, we incorporated IIF microscopy knockout analysis and DIF serration pattern analysis to the diagnostic algorithm. Furthermore, identification of a series of patients with anti-p200 pemphigoid allowed profiling clinical characteristics, disease course, and immunopathological features.

Box Section Ref ID

Patients were selected from our biobank of the single national referral center on autoimmune bullous diseases in the Netherlands for the period of 1998 to 2015. These patients could be newly diagnosed, referred to our hospital, or referred for laboratory diagnostics. Inclusion criteria were (1) dermal side binding of serum IgG on 1-mol/L SSS human skin substrate by IIF microscopy of patients not diagnosed as having EBA or anti–LN-332 MMP, and (2) linear IgG deposition along the BMZ by DIF microscopy. To distinguish patients with anti-p200 pemphigoid from patients with anti–LN-332 MMP and EBA, DIF serration pattern analysis was implemented and IIF microscopy knockout analysis was performed with IgG and/or IgG4 on skin sections from patients with hereditary epidermolysis bullosa completely lacking expression of type VII collagen (recessive dystrophic epidermolysis bullosa [RDEB], severe generalized) and laminin-332 (junctional epidermolysis bullosa [JEB], severe generalized), as described previously.^10,12 IgG4 was being used for IIF microscopy knockout analysis in patients with low serum titers of autoantibodies, to reduce background staining on the skin substrate. This retrospective study was performed in line with the guidelines of the University Medical Center Groningen ethics committee; according to the national regulations in the Netherlands, no approval by the ethics committee is needed.

Lesional skin biopsy specimens were examined by light microscopy of hematoxylin-eosin–stained paraffin sections and the blister level mapped by immunohistochemical analysis using a human type IV collagen monoclonal antibody (Ventana Medical Systems).

The DIF and IIF microscopy studies were performed using the methods previously described.¹³ Cryosections of 4 µm from perilesional skin biopsy specimens were studied by DIF microscopy for anti-BMZ deposits of IgG, IgA, IgM, and C3c. Routine DIF serration pattern analysis was performed using a Leica DMRA microscope with a standard 40 × dry objective and 10 × ocular.

Immunoblot analyses were performed as previously described on 2 types of extracts: (1) human dermal extract,¹ labeling a 200-kDa protein, and (2) recombinant monomeric C-terminus fragment of human laminin γ1 (hLAMC1-cterm).¹⁴

Using the laboratory tests (vide infra), 12 patients were identified as having anti-p200 pemphigoid. Their mean age was 66.6 years (range, 28-91 years), with a male to female ratio of 1.4:1 (7 [58%] were male, 5 [42%] were female). Patient characteristics are shown in Table 1. The initial clinical presentation of anti-p200 pemphigoid was exclusively cutaneous in 6 of 12 patients (50%), and concomitant mucosal and cutaneous in the 6 other patients (50%). None of the patients had the mucosa exclusively or predominantly affected. All 12 patients showed tense skin blisters, while pruritus was present in 11 of 12 patients. The distribution of skin lesions showed remarkable similarities in 9 of 12 of the patients with tense blisters localized on the hands and feet, resembling dyshidrosiform pemphigoid (Figure 1A and B). Circumscript monomorphic blisters were observed in patient 3 (Figure 1D). The bullous eruption was generalized on the skin and mucosa in patients 2 and 6 (Table 1). Neither scarring nor milia formation was observed in any patient. Mucosal lesions consisted of gingivitis, blistering on the tongue, and on buccal and/or anogenital mucosa (Figure 1C). The eyes or larynx were never involved. Psoriasis was not observed in any patient.

All 12 patients received (super)potent topical corticosteroids during the disease course (Table 1). One patient (8%) with localized disease (Figure 1D) achieved clinical remission with daily lesional application of clobetasol propionate, 0.05% cream; likewise, 2 of 12 patients (17%) achieved clinical remission with additional doxycycline, 200 mg daily. In 2 of 12 patients, topical whole-body clobetasol propionate, 0.05% cream, application was being used at onset of disease. Nine patients (75%) received systemic oral corticosteroids, mostly prednisolone, 0.5 mg/kg/d, and, in case of severe disease (in patients 2 and 11), up to 1.0 mg/kg/d. Systemic oral corticosteroids were often combined with dapsone (4 of 12 patients) or azathioprine (3 of 12), the adjunctive treatment was continued after achieving clinical remission to prevent relapses. Clinical remission on therapy was achieved in 7 of 12 patients within a mean (SD) duration of 7.2 (5.3) months. Clinical remission off therapy was seen in 4 of 12 patients, with a mean time to remission of 18.5 (16.7) months (Table 1). Five patients (42%) experienced relapse during follow-up, and 3 patients (25%) died during follow-up (patients 1, 9, and 10). Detailed information on follow-up of patient 1 was not available; she was in her 90s and died of natural causes. Patient 9 had achieved disease control after 2 months, until she died of cardiac failure during surgery not related to the disease. Patient 10 died from cardiac arrest in his 80s, 18 months after diagnosis.

In 11 of 12 patients with anti-p200 pemphigoid, lesional skin biopsy specimens were available (Table 2). In all biopsy specimens, subepidermal blistering was observed along with mild to dense inflammatory infiltrates in the upper dermis. The inflammatory infiltrate was exclusively composed of neutrophils in 5 patients (42%), whereas a mixed infiltrate of neutrophils and eosinophils was seen in 5 other patients (Table 2). In 2 patients, papillary microabscesses were observed combined with an exclusively neutrophilic infiltrate (patient 7) and neutrophilic spongiosis (patient 9), respectively. In patient 11, only eosinophilic spongiosis was seen. Staining of type IV collagen was performed in 10 of 11 lesional biopsies. In all patients, type IV collagen was mapped to the dermal side (the floor) of the blister (Table 2).

Direct immunofluorescence microscopy of perilesional skin biopsy specimens showed linear depositions of IgG and C3c along the BMZ in all 12 patients (Table 3). In addition, depositions of IgA and/or IgM along the BMZ were present in 7 patients (58%) and 2 patients (17%), respectively. An n-serrated immunodeposition pattern along the BMZ was identified in 9 of 11 available biopsy specimens (82%). In the remaining 2 patients, the serration pattern of the linear deposition was considered undetermined by 2 observers (J.M.M. and G.F.H.D.). The sera of all anti-p200 patients showed binding of IgG to the dermal side of the split in IIF SSS, and additional binding of IgA was found in 4 of 12 patients (33%). Knockout analysis by IIF microscopy of all 12 sera showed a positive reactivity on skin substrates deficient in type VII collagen and laminin-332, and on control healthy human skin (Table 3, Figure 2). Immunoblot analysis with human dermal extract demonstrated reactivity to the 200-kDa protein in all 12 patients. Reactivity to the recombinant C-terminus of laminin γ1 was seen in 8 of 12 patients (67%) (Table 3).

Laboratory diagnosis of anti-p200 pemphigoid was feasible by using IIF microscopy knockout analysis in our series of 12 patients. The clinical profile of this subtype of pemphigoid had a remarkable acral distribution of blisters on hands and feet, resembling dyshidrosiform pemphigoid. Moreover, mucosal involvement was seen in half of the patients. The disease course of these 12 patients with anti-p200 pemphigoid was more severe than so far reported in the literature; treatment with systemic corticosteroids was needed in three-quarters of patients.

The clinical presentation of anti-p200 pemphigoid has been described as highly variable mimicking BP, linear IgA dermatosis, and the inflammatory variant of EBA.^8,15 The findings in these 12 patients showed a common denominator in initial clinical presentation with acral distribution of tense blisters on hands and feet. Although not exclusive to anti-p200 pemphigoid, the acral blisters may give a clinical clue to diagnosis. In contrast to mechanobullous EBA, no scarring or milia formation was observed. In most patients, skin blistering was limited to the extremities; only 2 patients presented with a generalized bullous eruption. Involvement of oral and/or anogenital mucosa was observed in 50% of patients, which was much higher than previously estimated in literature in about 20% of reported cases.⁸ However, the mucosal lesions were not a dominant feature in the patients and less severe (no ocular or laryngeal involvement) than in patients with anti–LN-332 MMP. Although a rare disease, owing to a relatively low number of patients (12), these clinical findings do not allow us to draw conclusions on general characteristics of anti-p200 pemphigoid. Patients with anti-p200 pemphigoid tend to be slightly younger compared with those with BP, which mainly affects patients older than 70 years.^8,16 Based on routinely performed IIF SSS in the diagnostic algorithm for sAIBD, we estimate anti-p200 pemphigoid to constitute approximately 1.3% of seropositive patients with sAIBD in our clinic (unpublished data). For the differential diagnosis of sAIBD with dermal side binding by IIF SSS, the ratio of anti-p200 pemphigoid, anti–LN-332 MMP and EBA was 23.5% (total sAIBD, 1.3%), 21.6% (total sAIBD, 1.2%), and 54.9% (total sAIBD, 3.0%), respectively. Diagnosis of anti-p200 pemphigoid and anti–LN-332 MMP can be confirmed only based on serologic test results. Consequently, seronegative patients diagnosed as having EBA based on the linear u-serrated anti-BMZ immunodeposition by DIF are not included in this calculation. Summarizing, 1 in 4 patients with dermal side binding by IIF SSS was diagnosed as having anti-p200 pemphigoid.

No treatment recommendations are known for anti-p200 pemphigoid; therapy usually corresponds with standard treatment for BP. The clinical course of anti-p200 pemphigoid has previously been described as variable, but usually more benign compared with that of EBA, and even BP.⁴ In contrast, in our study population, most (75%) received systemic corticosteroids and often adjuvant immunosuppressive drugs. Nevertheless, 5 patients (42%) experienced relapse during follow-up. Although they had a more severe disease course than was expected, a number of patients with localized disease showed a prompt response to immunosuppressive treatment.

Currently the diagnosis of anti-p200 pemphigoid is based on DIF microscopy showing linear depositions of IgG and C3c along the BMZ and dermal side binding by IIF on 1 mol/L SSS.^4,9 Using the latter technique, anti-p200 pemphigoid can be differentiated from BP but not from EBA and anti–LN-332 MMP. For the first time, to our knowledge, routine DIF serration pattern analysis was included in the diagnostic algorithm of anti-p200 pemphigoid (Figure 3). To perform DIF serration pattern analysis, it is sufficient to use a conventional IF microscope with 40 × dry objective and 10 × ocular.¹³ The linear n-serrated pattern is found in all sAIBD with immunodeposits located in hemidesmosomes, lamina lucida, or lamina densa. In contrast, in EBA and in bullous systemic lupus erythematosus, a linear u-serrated pattern is seen corresponding with the ultralocalization of type VII collagen in the sublamina densa zone.¹³ Described previously by Vodegel et al¹³ in 2 patients, our findings confirm that anti-p200 pemphigoid is characterized by a linear n-serrated pattern by DIF microscopy. This finding corresponds with the observed ultrastructural localization of anti-p200 immunodeposits along the lower lamina lucida and upper lamina densa of the BMZ.³

The histopathological features do not allow to distinguish anti-p200 from other sAIBD.^3,9 Nevertheless, the mainly neutrophilic inflammatory dermal infiltrate is in contrast to a typically eosinophilic infiltrate in BP and may prompt a histopathologist to consider the diagnosis of anti-p200 pemphigoid and perform DIF microscopy and serological studies to define or exclude the diagnosis. Type IV collagen in anti-p200 pemphigoid locates to the blister floor and may be helpful to differentiate it from EBA, wherein type IV collagen is found on the blister roof.¹⁷

Previously, Dainichi et al⁵ described the presence of antibodies against laminin γ1 in 90% of anti-p200 pemphigoid sera, confirmed by enzyme-linked immunosorbent assay (ELISA) using the recombinant C-terminus of laminin γ1. In our study, 8 of 12 patients with anti-p200 pemphigoid (66.7%) showed reactivity against hLAMC1-cterm by immunoblot. Differences in patient population may play a role; for example, an association with psoriasis has been reported in up to 30% of cases, mostly in Asian patients.^2,8,18 In contrast, coexistence of psoriasis was not observed in any of our cases. Moreover, this difference might be explained by reactivity of autoantibodies to epitopes of laminin γ1 outside of hLAMC1-cterm or with another 200-kDa dermal protein. Although relapses occurred only in patients with reactivity to laminin γ1, no correlations were seen with disease area and severity. Reactivity to laminin γ1 by immunoblot or ELISA may be useful as a diagnostic marker for anti-p200 pemphigoid.

Laboratory techniques, such as immunoblot with dermal extract or recombinant laminin γ1 and IIF microscopy knockout analysis, may not be available in most medical centers. In contrast, DIF serration pattern analysis can be performed in routine laboratories and allows identification of patients with EBA. In patients with dermal side binding of autoantibodies on IIF SSS suspected for anti-p200 pemphigoid, it is recommended to specify the diagnosis of anti-p200 pemphigoid in referral centers for autoimmune blistering disease.

Laboratory diagnosis of anti-p200 pemphigoid is possible not only by immunoblot with human dermal extract, but also by IIF microscopy knockout analysis on skin substrates deficient in type VII collagen and laminin-332. We add DIF serration pattern analysis to the diagnostic algorithm to differentiate anti-p200 pemphigoid (n-serrated) from EBA (u-serrated). Clinical clues of anti-p200 pemphigoid in our series of 12 patients with anti-p200 pemphigoid include acral distribution of blisters without scarring or milia, mild mucosal involvement in half of them, and a slightly younger age of onset of disease compared with patients with BP.

Corresponding Author: Marcel F. Jonkman, MD, PhD, Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands (m.f.jonkman@umcg.nl).

Accepted for Publication: March 24, 2016.

Published Online: May 11, 2016. doi:10.1001/jamadermatol.2016.1099.

Author Contributions: Drs Meijer and Jonkman had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Meijer, Pas, Jonkman.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Meijer.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Meijer.

Obtained funding: Jonkman.

Administrative, technical, or material support: Meijer, Diercks, Schmidt, Jonkman.

Study supervision: Pas, Jonkman.

Conflict of Interest Disclosures: None reported.

Previous Presentation: This study was presented as an oral e-poster at the 24th European Academy of Dermatology and Venerology Congress; October 7-11, 2015; Copenhagen, Denmark.

Additional Contributions: We thank the patients for their participation. Janny Zuiderveen, Gonnie Meijer, and Miranda Nijenhuis BSc (Immunodermatology Laboratory, Groningen Center for Blistering Diseases) provided technical assistance. Piet Toonder (Department of Dermatology, University Medical Center Groningen) provided excellent photography. There was no compensation for their contributions.