1. 15.06.2011 / EM EliA Mi-2 Launch Presentation 2011-12-19 EM

2. 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. 3 Indications for testing Differential diagnosis of myopathy/myositis 1 Suspicion of idiopathic inflammatory myopathies (IIM) 2 Differential diagnosis of poly-, dermato- and inclusion body myositis 2 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. 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. 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. 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. 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. 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) AutoantibodyAntigen identity/functionFrequency in PM/DM Jo-1Histidyl-aminoacyl-tRNA synthetase20-30 % PL-7Threonyl-aminoacyl-tRNA synthetase2-5 % PL-12Alanyl-aminoacyl-tRNA synthetase2-5 % SRPSignal recognition particle4-6 % Mi-2Helicase transcription regulator10-15%

9. 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. 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. 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. 12 Methods to detect anti-Mi-2 IFA on HEp-2 cells: undefined homogenous/fine speckled pattern, which generally spares nucleoli and metaphase chromosomes 2. 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. 13 Product Details Product name EliA Mi-2 Well Package size 24 Article number 14-5604-01 negative < 7 U/ml Normal range equivocal 7 – 10 U/mlpositive > 10 U/ml Measuring range 0.4 U/ml to ≥ 302 U/ml

14. 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. 15 EliA Mi-2 – Precision Excellent precision on all Phadia instruments Sample Mean Value U/ml Intra-Run CV % Inter-Run CV % Phadia 10019.53.12.5 239.86.34.5 3248.84.95.5 Phadia 25018.46.22.3 231.37.41.8 3238.08.33.7 Phadia 250019.73.35.0 234.85.16.6 3238.26.23.8

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

17. 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. Most blood donors far below cut-off, good discrimination Equivocal TestUnitnMean value 95-% percentile 99-% percentile EliA Mi-2EliA U/ml4000.7 U/ml1.1 U/ml2.3 U/ml

18. 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. 19 EliA Mi-2 Sensitivity myositis (PM+DM)4.0 % Sensitivity dermatomyositis (DM) 10.0 % Specificity 100 % Positive predictive value (PPV) (for DM) 100 % Negative predictive value (NPV) (for DM) 65.2 % Positive likelihood ratio (LR+) (for DM) ∞ Negative likelihood ratio (LR-) (for DM) 0.9 Sensitivity in the range reported in the literature, but depending on the sample panel. Optimal specificity gives this test an excellent clinical value. Clinical Performance Cut-off Equivocal range

20. 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. 21 Comparison to Euroimmun Line Blot (Euroline Myositis Profile) EliA Mi-2Euroimmun Line Blot Sensitivity myositis (PM+DM)4.0 %2.0 % Sensitivity dermatomyositis (DM) 10.0 % Specificity all controls 100 %97.8 % Specificity CTDs + RA as controls 100 %95.6 % Specificity infections + tumors as controls 100 % EliA equally sensitive but more specific than Euroimmun line blot.

22. 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. 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. 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. 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 EliA Mi-2 Sensitivity myositis (PM+DM)4.0 % Sensitivity dermatomyositis (DM) 10.0 % Specificity 100 % Positive predictive value (PPV) for DM 100 % Negative predictive value (NPV) for DM 65.2 % Positive likelihood ratio (LR+) for DM ∞ Negative likelihood ratio (LR-) for DM 0.9

26. 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. 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. 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. Autoimmunity 38: 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. 29 EliA Mi-2 Pinpoints PM/DM patients