EliA Mi-2

1. EliA Mi-2 Launch Presentation 2011-12-19 EM

2. Content • Indications for testing • Clinical background • The antigen • Methods to detect anti-Mi-2 • Product Details – Technical Performance • Clinical Performance • Competition • Sales argumentation

3. Indications for testing • Differential diagnosis of myopathy/myositis1 • Suspicion of idiopathic inflammatory myopathies (IIM)2 • Differential diagnosis of poly-, dermato- and inclusion body myositis2 • Follow-up of a positive EliA CTD Screen, if common antibodies are negative 1 Conrad K et al (2002) 2 Ghirardello A et al (2005)

4. Clinical background • Idiopathic inflammatory myopathies (IIM) are a group of diseases which differ from the much more common myopathies caused by non-inflammatory processes. • IIM are characterized by an inflammatory infiltrate of the skeletal muscle. • IIM are classified into three major forms 3: • Polymyositis (PM) • Dermatomyositis (DM) • Inclusion body myositis (IBM, not associated with specific antibodies). Rare in jounger subjects, more frequent in those >50 years. • PM or DM can be associated with cancer or with other connective tissue diseases or overlap syndromes (e.g. PM/DM, PM/Scl) • No prevalence data on IIM are available. However, IIM is rare, with an incidence of an estimated 4-10 cases per 1.000.000/year 4 3 Ghirardello A et al (2011) 4 Prieto S and Grau JM (2010)

5. Polymyositis • Definition: subacute myopathy that evolves over weeks to months • Least common IIM in all age groups • Affects adults but rarely children, onset usually above 18 years • Sex Ratio: male:female = 1:2-3 • Symptoms: • Muscle weakness in shoulders, pelvis or thighs (first symptoms) • Symmetric pain • Difficulties with speech and swallowing • Mimics many other myopathies and remains a diagnosis of exclusion • Prognosis depends on severity, but often curable with steroids and immunosuppression

6. Dermatomyositis • Definition: acute or chronic inflammatory disease of muscle and skin • Most common IIM in all age groups, most frequent IIM in children • Affects subjects of all ages, also children • Sex Ratio: male:female = 1:2-3 • Symptoms: • Muscle weakness in shoulders, pelvis or thighs (first symptoms) • Symmetric pain • Typical rash (face, neck, shoulders, chest, elbows, knees) usually preceding muscle weakness • Joint contractures • Difficulties with speech and swallowing • Prognosis depends on severity, but often curable with steroids and immunosuppression

7. Diagnosis of PM and DM Three examinations are usually done: • Serum muscle enzyme measurement (creatine kinase most sensitive,sometimes also aspartate kinase, alanine aminotransferases, lactate dehydrogenase, aldolase) • Electromyography • Muscle biopsy • Autoantibodies are useful clinical markers

8. Autoantibodies in IIM • Can be grouped in: • Myositis-specific antibodies (e.g. Jo-1, PL-7, PL-12, Mi-2, SRP) • Myositis-associated antibodies, not specific for myositis (e.g. U1RNP, Ro52) • Tissue-specific antibodies, not specific for myositis • App. 70% of PM and DM patients show positive autoantibodies 4 4 Prieto S and Grau JM (2010)

9. Mi-2 antibodies • Mi-2 antibodies are detectable in 15-31% of adult dermatomyositis patients but are rare (<1%) in polymyositis patients. 5 • Thus, Mi-2 antibodies are highly specific for DM. • Due to the low sensitivity a negative anti-Mi-2 antibody result does not rule out the possibility of dermatomyositis.1 • More than 90% of anti-Mi-2 positive patients have dermatomyositis.2 • The antibody is named after a 60-year-old female dermatomyositis patient called Mi.2 1 Conrad C et al (2010) 2 Ghirardello A et al (2005) 5 Targoff IN (2007)

10. Anti-Mi-2 is a prognostic marker • In contrast to synthetase antibodies (Jo-1, PL-7, PL-12) positive patients those positive for anti-Mi-2 antibodies show • a relative mild disease course 1 • rare synovitis exhibitions, lung manifestations or Raynaud’s phenomenon 1 • Mi-2 antibodies are associated with an increased risk for cancer. 1 • Mi-2 antibodies are detectable in the early stage of myositis development. 1 • A positive Mi-2 antibodies result is useful for the exclusion of paraneoplastic myositis. 2 1 Conrad K et al (2002) 2 Ghirardello A et al (2005)

11. The antigen • Two forms of Mi-2 proteins with high similarity (almost 70%) were identified:Mi-2α (208 kDa) and Mi-2β (218 kDa).5,6 Only Mi-2β seems to be the specific target for autoantibodies. 4 • Mi-2 seems to play a role in regulation of transcription. • Mi-2β is a part of a nuclear complex named “nucleosome remodelling deacetylase (NuRD)”. 5,6 • EliA Mi-2 uses human recombinant Mi-2β protein produced in the baculovirus/insect cell system. 4 Prieto S and Grau JM (2010) 5 Targoff IN (2007) 6 Targoff IN (2000)

12. Methods to detect anti-Mi-2 • IFA on HEp-2 cells: undefined homogenous/fine speckled pattern, which generally spares nucleoli and metaphase chromosomes2. This staining pattern is typical but not specific for Mi-2 antibodies.1 • Immunoprecipitation (IP) of radiolabeled cell culture extracts or recombinant proteins (gold standard).2 • Immunodiffusion on calf thymus extract (less sensitive than IP) 3 • Western Blot (WB) using native extracts or recombinant Mi-2β. Highly specific, but low sensitivity. 2 • Line Blot (LB). Less accurate than IP. 7 • ELISA tests not commercially available. • Available methods either laborious or not quantitative or not available as single analyte tests. 1 Conrad K et al (2002) 2 Ghirardello A et al (2005) 7 Ghirardello A et al (2009)

13. Product Details

14. EliA Controls • Mi-2 activity is not included in the EliA ANA Positive Control. • Due to expected low volume and difficult serum supply we are not able to offer EliA Controls for EliA Mi-2. • This means that both the EliA ANA Positive Control and the EliA IgG/IgM/IgA Negative Control must not be used with EliA Mi-2.

15. EliA Mi-2 – Precision Excellent precision on all Phadia instruments

16. EliA Mi-2 – Serum/Plasma Correlation Both serum and all types of plasma can be used

17. EliA Mi-2 – Blood Donors and Normal Range 400 blood donors were measured on Phadia 250. A substudy using 70 blood donors on Phadia 100 and Phadia 2500 showed good comparison. Equivocal Most blood donors far below cut-off, good discrimination

18. Clinical Performance • Sample panel used in the evaluation:280 clinically defined samples Patients: • 100 PM or PM/DM Controls: • 50 CTD (SLE, Sjögren, MCTD, PM/DM) • 40 RA • 70 infections (HCV, HBV, EBV, bacterial) • 20 tumors

19. Clinical Performance Cut-off Equivocal range Sensitivity in the range reported in the literature, but depending on the sample panel. Optimal specificity gives this test an excellent clinical value.

20. Competition • No ELISA based tests available • Some Euroimmun Line Blots include Mi-2, but no single Mi-2 test available • IIF not conclusive for Mi-2, as pattern is not specific for Mi-2 • Immunoprecipitation

21. Comparison to Euroimmun Line Blot(Euroline Myositis Profile) EliA equally sensitive but more specific than Euroimmun line blot.

22. Sales Arguments • First single anti-Mi-2 test for routine use, saving costs for additional tests • First automated anti-Mi-2 test, reducing the workload for your lab personnel • Clear differentiation between idiopathic inflammatory myopathies and other connective tissue diseases • Reliable follow-up of a positive EliA CTD Screen • Reliable follow-up of a positive IIF result

23. Sales Arguments • First single anti-Mi-2 test for routine use, saving costs for additional tests • To date no single tests are available for anti-Mi-2, but only Line Blots including Mi-2 among many other antigens, making anti-Mi-2 testing expensive. • EliA Mi-2 brings single anti-Mi-2 testing to the routine lab, making it flexible and cost-efficient.

24. Sales Arguments • First automated anti-Mi-2 test, reducing the workload for your lab personnel • The possibility of single testing combined with automation makes anti-Mi-2 testing cost-efficient, saving both labour time and costs for other tests which are not needed. Phadia 100 Phadia 250 Phadia 2500 Phadia 5000

25. Sales Arguments • Clear differentiation between idiopathic inflammatory myopathies and other connective tissue diseases • EliA Mi-2 pinpoints PM/DM without generating false positives. This results in an optimal positive predictive value and provides reliable early guidance for the physicians. Cut-off Equivocal range

26. Sales Arguments • Reliable follow-up of a positive EliA CTD Screen • If EliA CTD Screen is positive but the most frequent antibodies are negative, this may be due to anti-Mi-2. Both tests are perfectly aligned. • If PM/DM is suspected and EliA CTD Screen is positive, EliA Mi-2 is the follow-up test of choice, providing clear, quantitative results of high clinical value

27. Sales Arguments • Reliable follow-up of a positive IIF result • If IIF shows a pattern (undefined homogenous/fine speckled pattern, which generally spares nucleoli and metaphase chromosomes) which is indicative of, but not specific for anti-Mi-2, EliA Mi-2 can unequivocally confirm or rule out the presence of anti-Mi-2 due to its optimal specificity

28. References 1 Conrad K et al (2002). Mi-2 Antibodies. In: Autoantigens, Autoantibodies, Autoimmunity, Vol 2, Conrad, Sack (eds), Pabst, Lengerich: 107-109 2 Ghirardello A et al (2005): Anti-Mi-2 antibodies. Autoimmunity38: 79-83 3 Ghirardello A et al (2011). Cutting edge issues in polymyositis. Clin Rev Allerg Immunol 41: 197-189 4 Prieto S and Grau JM (2010). The geoepidemiology of autoimmune muscle disease.Autoimmun Rev 9: A330-A334 5 Targoff IN (2007): Myositis Autoantibodies: Aminoacyl-tRNA synthetase, signal recognition particle, Mi-2, and PM-Scl Autoantibodies. In: Autoantibodies, Vol 2, Shoenfeld Y, Gershwin ME, Meroni PL (eds), Elsevier, Amsterdam: 577-589 6 Targoff IN (2000): Update on myositis-specific and myositis associated autoantibodies. Curr Opin Rheumatol 12: 475–481 7 Ghirardello A et al (2009). Commercial blot assays in the diagnosis of systemic rheumatic diseases. Autoimmun Rev 8: 645-649

29. EliA Mi-2 Pinpoints PM/DM patients