Analysis of The Immunoserological Profile of Autoimmune ...

Analysis of The Immunoserological Profile of Autoimmune Skin Diseases (Reference ? 2013.03.003)

Notice of Assessment

April 2014

DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en sant? et en services sociaux (INESSS), and that version can be consulted at e_maladies_cutanees_auto-immune.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS's permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation. While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.

1 GENERAL INFORMATION

1.1 Requestor: CHUM

1.2 Application for Review Submitted to MSSS: April 11, 2013

1.3 Application Received by INESSS: November 1, 2013

1.4 Notice Issued: February 28, 2014

Note: This notice is based on the scientific and commercial information submitted by the requestor and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.

2 TECHNOLOGY, COMPANY, AND LICENCE(S)

2.1 Name of the Technology

Analysis of the immunoserological profile of autoimmune skin diseases using indirect immunofluorescence on specific substrates and ELISA (enzyme-linked immunosorbent assay) microplates.

2.2 Brief Description of the Technology, and Clinical and Technical Specifications

The immunoserological analysis of autoimmune skin diseases consists of detecting serum autoantibodies that target protein components of the skin. Two methods are included in the requestor's application: Indirect Immunofluorescence on a Mosaic of Six Substrates (BIOCHIP MosaicsTM by EUROIMMUN)11: monkey esophagus, salt-split human skin, cells expressing desmoglein 1 and 3, BP23012 (C-terminal globular domain), and BP180 (Figure 1). Each substrate is in a microarray13 available for simultaneous analysis.

The technique consists of bringing into contact the serum dilution (1:10) and the substrates. The serum autoantibodies attach themselves to the antigenic determinant of the target protein. The antigenantibody complex is detected by an (IgG) antibody tagged with a fluorochrome, fluorescein isothiocyanate, and examined using a conventional fluorescence microscope.

The anti-intercellular substance antibodies against desmoglein 1 and desmoglein 3 react with the surface antigens of keratinocytes. The analysis of the esophagus tissue section displays a granular fluorescence. As it is difficult to differentiate between desmoglein 1 and desmoglein 3, specific transfected cells are used to establish an accurate diagnosis. When the autoantibodies against BP180 or BP230 are present, a fluorescence is observed on the epidermis of the skin sample separated at the dermal-epidermal junction by a saline solution. The epidermal basal membrane of the esophagus is visible as a fine linear colouring between the stratum basale and the connective tissue. These

11 EUROIMMUN. Autoantibodies against antigens of the skin [website]. Available at: (viewed November 21, 2013). 12 BP: bullous pemphigoid. 13 A microarray is an analytical and diagnostic tool measuring approximately 1 cm2. It consists of a glass or silicon support on which proteins are fixed. (Universcience glossary [website]. Available at: ).

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antibodies can be differentiated by means of BP180-NC16A-4X coated BIOCHIPs and cells specifically transfected with BP230 (C-terminal globular domain) [EUROIMMUN, 2014]. Figure 1: BIOCHIP mosaic

Source: Zarian et al., 2012. Figure reproduced with permission of the author.

ELISA Microplate Method for the specific detection of autoantibodies: various ELISA kits using 5 recombinant antigens are available for a quantitative analysis of the target proteins, with the exception of envoplakin, for which the analysis is semiquantitative (characterization of the autoantibodies or identification of the target epitopes). These antigens are extracellular domain of desmoglein 1 or desmoglein 3, envoplakin, tetramer of the NC16A domain of BP180 (BP180-NC16A4X), and the C-terminal segment of BP230 (BP230-CF). The ELISA method comprises a microplate with 8 individual reagent wells. The detection of monospecific antibodies is carried out in several steps: 1) serum dilution 1:100; 2) incubation with the recombinant antigen on solid support; and 3) incubation with a conjugate (monoclonal anti-human IgG antibody) coupled with peroxidase. If the samples are positive, the specific IgG, IgA, and IgM antibodies will attach to the corresponding antigenic site. The quantitative results, expressed in U/mL, are obtained by spectrophotometric measurement of the intensity of the colorimetric reaction [EUROIMMUN, 2014]. Other kits from the Japanese company Medical & Biological Laboratories (MBL) are available. 2.3 Company or Developer EUROIMMUN (Medizinische Labordiagnostika AG) (Germany). 2.4 Licence(s): Not applicable. 2.5 Patent, If Any: Not applicable. 2.6 Approval Status (Health Canada, FDA) The ELISA (IgG) kits from EUROIMMUN have been approved by Health Canada: anti-desmoglein 1 (No. 82882); anti-desmoglein 3 (No. 82881); anti-envoplakin (No. 91661); anti-BP180-NC16A-4X (No. 74292); anti-BP230-CF (No. 91721). IIFT: Dermatology Mosaic 7 (EUROIMMUN) was approved by Health Canada in July 2013 (No. 91721). The FDA has approved the ELISA (IgG) semi-quantitative test kits for anti-BP180 and anti-BP230, MBL International Corporation (k07196114); the qualitative or semi-quantitative kits for anti-BP180-4X ELISA (IgG), EUROIMMUN US Inc. (k08361515); and the semi-quantitative and qualitative kits for the

14 Food and Drug Administration (FDA). 510(k) Substantial equivalence determination decision summary. 510(k) Number: k071961. Available at: . 15 510(k) Number: k083615. Available at: .

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measurement of anti-desmoglein 1 and anti-desmoglein 3 (IgG), EUROIMMUN US Inc. (k09196916). No approval was found for the Dermatology Mosaic 7 (EUROIMMUN) kit.

2.7 Weighted Value: 148

3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES

3.1 Targeted Patient Group

Patients with autoimmune bullous diseases who are being treated in an oncodermatology service.

3.2 Targeted Disease(s)

Acquired autoimmune bullous diseases are a heterogeneous group of rare disorders of the skin and external mucous membranes characterized by the production of autoantibodies against the surface antigens of keratinocytes and the components of the desmosomes17 (anti-intercellular substance) and other antigens that are not part of the junctional complexes [Grando, 2012]. These autoantibodies are responsible for the loss of intercellular structure by acantholysis (or loss of adhesion between skin cells18) and the formation of intraepidermal bullae (pemphigus). The autoantibodies may be directed against the dermal-epidermal junction (anti-basement membrane), causing a change in one of the components (hemidesmosomes, anchoring filaments) and the formation of subepidermal bullae (bullous pemphigoid). Different antigents (proteins) are associated with various clinical forms [HAS, 2011b; Humbel, 2003]:

pemphigus foliaceus: desmoglein 1; pemphigus vulgaris: desmogleins 1 and 3; paraneoplastic pemphigus: plakins (desmoplakins 1 and 2, envoplakin, and periplakin), desmogleins

1 and 3, BPAG119, plectin (HD1); bullous pemphigoid: BPAG1 and BPAG 2; cicatricial pemphigoid: BPAG1 and BPAG2, LABD20 Antigen 1, integrin, laminin 5, type VII collagen; Linear IgA bullous dermatosis: BPAG1 or BPAG2, LABD, type VII collagen; IgA pemphigus: desmocollins 1 and 2; Pemphigoid gestationis or herpes gestationis: BPAG2, BP180-NC16A; Acquired epidermolysis bullosa: type XVII collagen.

Bullous pemphigoid is the most common clinical form,21 with an incidence of 43 cases per million inhabitants per year in the United Kingdom and 7 to 13 cases per million inhabitants per year in other parts of Europe [Venning et al., 2012]. It affects individuals aged 70 and over and manifests in its classical form as pruritus, erythema, and the formation of bullae, with no mucosal involvement or Nikolsky's sign.22 Atypical forms are accompanied by mucosal involvement, particularly of the buccal mucosa [HAS, 2011a]. These forms are associated with significant morbidity (e.g., severe itching and

16 510(k) Number: k091969. Available at: . 17 Desmosome: protein ensuring intercellular adhesion and interkeratinocyte junctions. 18 Two main isoforms are targeted: desmoglein 1, expressed primarily in the superficial epithelium, and, to a lesser extent, in the oral mucosa; desmoglein 3 expressed primarily in the epithelium of the oral musoca and the suprabasal epidermal layer. 19 BPAG1: bullous pemphigoid Antigen 1. 20 LABD: linear IgA bullous dermatosis. 21 Bernard P. Bullous pemphigoid [website]. Available at: (viewed January 24, 2014). 22 Nikolsky's sign is a skin finding in which the top layers of the skin slip away from the lower healthy layers when rubbed. It indicates intraepidermal cleavage (acantholysis) during pemphigus. (Coll?ge National des Enseignants de Dermatologie. Item 116: Dermatoses bulleuses auto-immunes. Available at: ).

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impetiginization23), which affects quality of life. The mortality rates at 1 year, as reported in four studies reviewed by Schmidt et al. [2012], range between 19% and 29%.

Gestational pemphigoid is a rare form that occurs in the second or third trimester of pregnancy and manifests as severe pruritus, urticaria, and blisters that first appear on the abdomen [Ingen-Housz-Oro et al., 2011].

Pemphigus is an even rarer disease. According to the literature, its incidence ranges from 1 to 16 new cases per million inhabitants per year [Joly and Sin, 2011]. The form known as pemphigus vulgaris presents as delayed mucosal (especially buccal erosions) and cutaneous (bullous eruptions) involvement, with a positive Nikolsky's sign when involvement is severe. The sequelae, mainly due to cutaneous, ophthalmologic, and buccal involvement, and the side effects of immunosuppressive therapy, are irreversible. When there is no mucosal involvement, it is referred to as superficial pemphigus or pemphigus foliaceus [HAS, 2011b].

Paraneoplastic pemphigus is the most severe form, associated with other neoplastic, especially lymphoproliferative, diseases [Humbel, 2003]. It is extremely rare, and its prevalence is unknown.24

3.3 Number of Patients Targeted

The requestor estimates the provincial volume for the next three years to be approximately 100 patients per year.

3.4 Medical Specialties and Other Professions Involved

Oncodermatology and hematology-immunology.

3.5 Testing Procedure

Blood tests are performed in a test centre using venipuncture.

4 TECHNOLOGY BACKGROUND

4.1 Nature of the Diagnostic Technology: Single test.

4.2 Brief Description of the Current Technological Context

Two tests are currently available to confirm the diagnosis of pemphigus or bullous pemphigoid: the histological examination of a skin sample from an intact blister, and direct immunofluorescence of a skin biopsy. The histological examination is used to specify the epidermal location of the bullae [Joly et al., 2011; Humbel, 2003]. Direct immunofluorescence of peribullous skin shows linear deposits of IgG and complement C3 along the dermal-epidermal junction with a linear fluorescence pattern (bullous pemphigoid) or on the surface of keratinocytes with a honeycomb fluorescence pattern of the epithelium (pemphigus) [Ingen-Housz-Oro et al., 2011; Joly et al., 2011].

Indirect immunofluorescence has long served as a reference method to detect anti-skin autoantibodies in serum, using monkey esophagus or human salt-split skin as a substrate [Tampoia et al., 2012a]. This technique allows the detection of anti-basement membrane zone (anti-BMZ) antibodies in the malpighian epithelium with a linear fluorescence (bullous pemphigoid) and of anti-intercellular substance antibodies (anti-ICS) in the malpighian epithelium with a honeycomb-patterned fluorescence of the epithelium (pemphigus) [Humbel, 2003]. According to certain authors, these procedures are difficult to standardize and depend on the skills of the individuals using them [Charneux et al., 2011].

23 Microbial secondary infection resembling impetigo on affected skin that was not initially infected, such as eczema. 24 Dubertret L. Paraneoplastic pemphigus [website]. Available at: (viewed January 24, 2014).

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4.3 Brief Description of the Advantages Cited for the New Technology

BIOCHIP Mosaics offers a full antigen spectrum in a single analysis and can be used to differentiate between pemphigus vulgaris and pemphigus foliaceus.

ELISA is a simple technology that yields results in one day, which allows a large number of samples to be tested in a relatively short time [Lee et al., 2012]. It provides quantitative results and does not require experienced personnel [Atzori et al., 2008].

Based on the information provided in the application submitted by the requestor, BIOCHIP Mosaics and ELISA technology provide a specific approach that allows an accurate diagnosis to be established in 90% of patients.

4.4 Cost of Technology and Options: Not assessed.

5 EVIDENCE

5.1 Clinical Relevance

5.1.1 Other Tests Replaced This test could replace the detection of cutaneous anti-basement membrane antibodies (pemphigoid) (code 20700) and anti-intercellular substance antibodies (pemphigus) (code 20715) by indirect immunofluorescence.

5.1.2 Diagnostic or Prognostic Value Analysis of the immunoserological profile confirms the clinical diagnosis of autoimmune skin diseases by identifying the antigen targeted by the autoantibodies. Analysis also distinguishes various clinical forms, particularly pemphigus vulgaris or pemphigus foliaceus and atypical forms of bullous pemphigoid.

In the case of pemphigus, a correlation was reported between the immunological profile of the autoantibodies and the clinical phenotype. The presence of anti-desmoglein 3 is specific to mucosal involvement, and the presence of anti-desmoglein 1 and 3 is specific to mucocutaneous involvement [Joly et Sin, 2011]. Moreover, the higher the levels of autoantibodies, the more severe the clinical condition [HAS, 2011b; Joly and Sin, 2011].

In the early stages or in atypical forms, bullous pemphigoid may be mistaken for various types of dermatoses and localized drug reactions. Clinical and histopathological criteria, and particularly the development of immunological techniques involving ELISA, allow a serological diagnosis to be made for most patients [Schmidt et al., 2012].

Correlation Between Antidesmoglein 1 and 3 Titres (Using ELISA) and the Severity of Pemphigus The degree to which the disease has spread is clinically assessed according to severity, the number and extent of mucocutaneous lesions, the number of erosions (mainly oral), the number of new blisters since diagnosis, and the intensity of the pruritus.

Pemphigus Vulgaris In the case of pemphigus vulgaris, most of the studies show a significant correlation between levels of anti-desmoglein 1 and 3 and the extension or activity of the disease when the diagnosis is established (Table 1).

Belloni-Fortina et al. [2009] did not observe a correlation between anti-desmoglein 1 and 3 titres and mucocutaneous spread as they monitored the progression of the disease. By contrast, HerreroGonz?lez et al. [2010] reported a significant correlation between levels of anti-desmoglein 3 and an

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unfavourable disease prognosis (several refractory episodes, chronic disease) (p < 0.001), but they did not observe any association with levels of anti-desmoglein 1 antibodies (p value not available).

Another study [Abasq et al., 2009] reported a reduction in levels of anti-desmoglein 1 (from 113.8 U/mL to 22.3 U/mL; p < 0.001) in patients with pemphigus vulgaris who had mucocutaneous lesions and showed complete remission of their skin lesions. Levels of anti-desmoglein 3 decreased from 148.4 U/mL to 99.4 U/mL per day (p < 0.001) in patients who showed complete remission of their mucosal lesions. The decrease in levels of anti-desmoglein 3 was not as significant as that of antidesmoglein 1 in patients with mucocutaneous pemphigus or pemphigus foliaceus (cutaneous). For 11 patients with pemphigus foliaceus or pemphigus vulgaris who had recurrent mucocutaneous lesions, anti-desmoglein 1 levels were significantly higher (p = 0.03) during periods of relapse than during periods of remission. However, no significant difference was noted in levels of anti-desmoglein 3 between periods of complete remission and periods of relapse (p = 0.13) of pemphigus vulgaris mucosal lesions.

Table 1: Relationship between anti-desmogleins 1 and 3 titres by ELISA and the severity of pemphigus vulgaris

STUDY

ASSESSMENT OF THE SEVERITY OR EXTENT

OF THE DISEASE

AUTOANTIBODIES

Herrero-Gonz?lez et al., Extent of

Anti-Dsg1

2010

mucocutaneous lesions Anti-Dsg3

Schmidt et al., 2010

Number of mucocutaneous lesions (PDAI25)

Anti-Dsg1 Anti-Dsg3

Belloni-Fortina et al., 2009

Number of mucosal lesions

Anti-Dsg1 Anti-Dsg3

Extent of cutaneous lesions

Anti-Dsg1 Anti-Dsg3

Daneshpazhooh et al., 2007

Severity of cutaneous involvement

Anti-Dsg1 Anti-Dsg3

Number of oral erosions

Anti-Dsg1 Anti-Dsg3

Sharma et al., 2006

Cutaneous surface affected by vesicles, bullae, or erosions

Anti-Dsg1 Anti-Dsg3

Number of oral erosions

Anti-Dsg3

Abbreviations: Dsg = desmoglein; n/a = not applicable; NR = not reported.

TEST

TYPE

RESUL

T

Kruskal-

n/a

Wallis

Spearman NR

Spearman NR

Spearman Pearson

0.746 0.382

?0.245 0.295

0.429 0.217

ANOVA

n/a

test

P VALUE

< 0.001 < 0.001 < 0.001 < 0.001

0.001 0.006 0.102 0.009 < 0.001 < 0.001 < 0.036 < 0.011 0.026 0.278

0.039

Avgerinou et al. [2013] reported the mean difference in anti-desmoglein 1 and 3 titres between the beginning of treatment and 12 months after treatment in 40 patients with pemphigus vulgaris. They observed a significant decrease in anti-desmoglein 1 (from 140.82 to 31.56; p = 0.003) and antidesmoglein 3 (from 178.08 to 126.43; p = 0.009) titres in 25 patients who showed clinical improvement. In the 7 patients who showed clinical deterioration, anti-desmoglein 1 titres did not change significantly, whereas anti-desmoglein 3 increased from 176.70 to 245.92; p = 0.028. No changes were observed in the 8 patients in stable clinical condition. The authors conclude that anti-

25 PDAI (pemphigus disease area index): score 3: > 3 lesions, score 2: 2-3 lesions; score 1: 1 lesion; score 0: no lesions.

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desmoglein 1 and 3 can be useful for the clinical follow-up of pemphigus vulgaris and to direct therapeutic treatment.

Pemphigus Foliaceus

Two studies showed divergent results (Table 2). Although a correlation was established between levels of anti-desmoglein 1 and the extent of cutaneous involvement for a small number of patients (n = 5) in one study [Schmidt et al., 2010], no correlation was observed by Herrero-Gonz?lez et al. [2010], who noted that this might be a result of the small sample size (n = 7).

Table 2: Relationship between anti-desmoglein1 titres by ELISA and the severity of pemphigus foliaceus

STUDY

ASSSESSMENT OF THE SEVERITY OR EXTENT OF THE

DISEASE

AUTOANTIBODIE S

Herrero-Gonz?lez et al., 2010

Extent of

Anti-Dsg1

cutaneous lesions

Schmidt et al., 2010

Number of

Anti-Dsg1

cutaneous lesions

Abbreviations: Dsg = desmoglein; n/a = not applicable; NR = not reported.

TEST

KruskalWallis Spearma n

COEFFICIEN T

n/a NR

P VALUE

0.091 < 0.001

Abasq et al. [2009] reported a significant decrease in the level of anti-desmoglein 1, from 123.8 U/mL to 76.8 U/mL (p = 0.03), in patients with pemphigus foliaceus who had a complete remission of their skin lesions, after initial treatment and after a 90-day follow-up.

Correlation Between Anti-BP180 and Anti-BP230 Titres by ELISA and the Severity of Bullous Pemphigoid

As anti-BP180 is strongly associated with bullous pemphigoid activity and severity (Table 9), and is recommended for monitoring of the progression of the disease.

An anti-BP180 titre of 27 UI/mL is a good indicator of clinical recurrence during the year following cessation of treatment, with a positive predictive value of 90.9% and a negative predictive value of 51.2% [Bernard et al., 2009].

A study reported significantly lower values of anti-BP180 during follow-up of patients without recurrences (p = 0.03) [Le Sach?-de Peufeilhoux et al., 2012]. The significance of anti-BP230 has not been demonstrated in typical cases of bullous pemphigoid [Le Sach?-de Peufeilhoux et al., 2012].

Correlation Between BIOCHIP Mosaic-based Indirect Immunofluorescence and Anti-desmoglein 1 and 3 Titres by ELISA

For 9 patients (3 cases of bullous pemphigoid, 3 cases of pemphigus foliaceus, and 3 cases of pemphigus vulgaris), Van Beek et al. [2012] reported a correlation between anti-BP180 and anti-desmoglein 1 and 3 titres--measured using the two methods (BIOCHIP Mosaic-based indirect immunofluorescence and ELISA)--and the activity of the disease26 during follow-up. The authors concluded that ELISA is the most appropriate test to monitor autoantibody titres.

26 Score 4: > 10 lesions; score 3: 4 to 10 lesions; score 2: 1 to 3 lesions; score 1: clinical remission with immunosuppressive therapy; score 0: clinical remission without immunosuppressive therapy.

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