1. Presented by Jill Buyon, M.D. at the September 29, 2003 meeting of the Arthritis Advisory Committee

2. Low High

3. The Enduring Role of anti-dsDNA and Complement Proteins in Diagnostic Testing (Back to Basics) anti-dsDNA abs specific to SLE anti-dsDNA abs can deposit in the glomerulus (high avidity, IgG, cationic, fix complement ) Evidence of complement consumption indicates immune complex-driven inflammation genetic alterations in early complement proteins of classical pathway) are associated with SLE Association between genetic polymorphisms in Fc  R alleles (IIa) and renal disease

4. --> C1 esterase activity C4 --C1--> C4a + C4b C2 --C1--> C2a + C2b C4b,2b C3 C3b C4b,2b,3b C5 C5b C3a C5a MAC Classical Pathway C3b C3b,Bb,3b C5b C3a C5a MAC C3b,BbC3b,Bb,P P C3bB D + Ba C3b + B C3 --> C3a + C3b (spontaneous) (stable) Alternative Pathway + C6 C7 C8 C9 + C6 C7 C8 C9 chemotactic factor anaphylotoxin chemotactic factor anaphylotoxin DNA-IgG anti-dsDNA + C1 Glomerulonephritis Fetal loss

5. Neutrophil activation Endothelial cell activation (priming) Leukothrombosis Resting EC.................................... ICAM-1 CR3 C3a C5a Resting PMN IL-1ß TNF  C1q C3a C5a C5b-9 aEC aPL E-selectin...................................................................... Vaso-occlusive plug.......

7. Playing Rules for Evaluation of the Biomarker f Define Assay for Measurement AssayBindingIsotypeDNASens/Spec Crithidiahigh + low affinity absIgM or IgGdsDNAspec>sens Farrhigh affinity abs IgM and IgGss and dsDNA ELISAhigh + low affinity absIgM or IgGss or dsDNAsens>spec anti-DNA abs Complement Assay ComponentSpecimenMeasurement Immunochemical Native C3, C4serumNephelometry Functional integrity CH 50 EDTA plasmaRBC lysis Catabolic state Activation C3aEDTA plasmaELISA Define parameters of change for these candidate biomarkers

8. Does the candidate biomarker: predict flare? associate with flare? respond to therapy in parallel with favorable clinical outcome? An association between a factor and the risk of a disease does not guarantee that drug-induced changes in that factor will produce a corresponding change in the risk.

9. Percent of Visits with Flares, Categorized by Prior and Concurrent Changes in Levels of Anti-dsDNA (Total 574 visits, overall flare rate = 19%) Ho et al, AR, 2001 Flare P (SLEDAI > 3) Prior  DNAabs ELISA >10% (70 visits) 30% 0.007 Prior  DNAabs ELISA >25% (45 visits) 29% N.S. Prior  DNAabs Critidia >2 dilutions (72 visits) 39%0.001 Concurrent  DNAabs ELISA (89 visits) 30%0.002 Concurrent  DNAabs Crithidia (112 visits) 29%0.0002 Prior: between visits 2 months and 1 month before visit with flare Concurrent: between previous visit and current visit

10. Reanalysis of Ho and Petri Data: Likelihood Ratio Kavanaugh et al, Arth Rheum, 2001 LR for a positive test: Extent to which a positive test increases pretest likelihood of disease (10 is high) sensitivity 1-specificity LR for association of flare and  dsDNA abs by Crithidia = 2.7 LR for a negative test: To determine the post test probability of disease after a negative result (0.10 is low) 1-sensitivity specificity LR for association of flare and  dsDNA abs by Crithidia = -.081 Conclusion: these tests had limited utility in predicting or excluding lupus flares

11. Clinically Active Serologically Quiescent (CASQ) SLE 1 (514 patients followed at the Toronto Lupus Clinic 1991-1995) 62 patients had CASQ : 43 with CNS, renal and/or vasculitis 58 patients had followup after last CASQ defining visit 9 remained CASQ for 3 yrs 23 became inactive 5 became serologically active but clinically stable (SACS) 21 became clincially and serologcially active Gladman et al, J Rheum, 2003

12. Evaluation of the Sensitivity and Specificity of C3, C4, CH50, anti- dsDNA and C3a for Detection of Lupus Flares within 3 months (Tseng et al, Arth Rheum suppl, 2001) Cohort:Patients enrolled in Safety of Estrogen in Lupus Erythematosus National Assessment (SELENA) randomized double-blind placebo controlled trial 496 female patients enrolled from 9/96 – 3/02 SLE patients given either HRT/placebo or OCP/placebo for 1 year Analytes measured:C3, C4, CH50, C3a and anti-dsDNA baseline, q monthly x 3, and then q 3 months over a 12 month period Disease activity: SELENA SLEDAI and PGA Outcomes: Severe flares, Mild/moderate flares

13. MeasurementsDefinition of Positive Tests for Detection of Lupus Flares C3a  50% increase from previous visit * and absolute level  500 ng/ml CH50  25% decrease from previous visit C3  25% decrease from previous visit C4  25% decrease from previous visit Anti-dsDNA Antibodies  25% increase from previous visit Approach * Previous visit must have occurred within 3 months from date of measurement

14. Definition of flares Severe Flare Change in SLEDAI to > 12 New/worse: CNS SLE Vasculitis Nephritis Myositis Plt 0.5mg/kg/d Hospitalization New Cytoxan, Azathioprine or Methotrexate Increase in PGA to >2.5 Mild or Moderate Flare Change of SLEDAI >3 New/worse: Lupus rash Nasopharyngeal ulcers Pleuritis Pericarditis Arthritis Fever (SLE) Any  in Prednisone to < 0.5mg/kg/d Added NSAIDS or Plaquenil for disease activity Physician Global Assessment (PGA) increase >1.0, and < 2.5

15. 1.496 Total Patients (328 HRT patients + 168 OCP patients) : 428 patients had C3a and/or CH50 available 496 patients had C3, C4, anti-dsDNA available 2.Flares (including multiple flares in patients): 491 mild/moderate flares 39 severe flares Patients Available for Evaluation and Outcomes

16. Mild / Flares ModerateSevereFlares MeasurementsSensitivitySpecificitySensitivitySpecificity C3 (  by 25%) 11%92%21%92% C4 (  by 25%) 18%88%21%88% CH50 (  by 25%) 16%88%29%87% Anti-dsDNA (  by 25%) 32%77%39%77% C3a (  by 50%) 18%90%11%89% C3a  500 ng/ml 38%76%54%73% Sensitivity and Specificity of Analytes to Predict Flares Limitations/ Implications U tility of analytes improved if definition of positive tests less stringent. Analytes q 3 months insufficient, monthly may improve prediction of flares. Absence of abnormal analytes does not equate with clinical stability\ but presence may be predictive of flares. A priori treatment of patients with abnormal analytes may be appropriate since few patients would be unnecessarily exposed.

17. Inclusion Criteria  Anti-DNA abs present within 2 years  Prednisone <15 mg  No active infection  Stability of disease and medications for 2 months Serologically Active, Clinically Stable SLE Objective: To evaluate steroid treatment in averting flares when elevations of plasma C3a are accompanied by rising anti-dsDNA titers in stable or inactive patients Principal Investigator: Steve Abramson Collaborators: Chung-E Tseng Jill P. Buyon Michael Belmont Betty Diamond Meggan Mackay

18. Study Design  Patients followed monthly for 12-18 months  History and physical, analytes, and SLEDAI Randomization Criteria Rise of C3a (> 50% and absolute level  500 ng/ml) Rise of anti-DNA (>25%) from visit within 1-2 months Absence of clinical activity PlaceboPrednisone : 30 mg X 2 wks 20 mg X 1 wk 10 mg X 1wk

19. 180 RANDOMIZED (Serological flare, clinically stable) 41 NON-RANDOMIZED 139 Completed no serological or clinical flare 92 Stopping point 47 Clinical flare 11 No clinical flare 30 Clinical flare with or without serologic flare 21 Voluntary drop out 11 Exclusion criteria 9 Protocol Violation 7 Mild to Moderate 5 Severe 6 Flow Chart of Patients Followed in the Observational Study (up to 18 months) Asian 17% African-American 22% Hispanic 46% Caucasian 15%

20. Analysis of Severe Flares  90 Days Severe Flare No Flare Prednisone Placebo 021 6 14 Fisher’s exact test = 0.009

21. Placebo 3 renal 1 CNS C3a 1 pyoderma gangrenosum, pancytopenia 1 pleural effusion 1 month 2 month 3 month Randomization:Timing and Clinical Features of the 6 Severe Flares Anti-DNA Prednisone (no severe flares) 30mg X 1wk 20mg X 1wk 10mg X 1wk Randomization

22. Summary of Results of Outcome Variables by Treatment Groups VariablePrednisonePlaceboP-Value SLEDAI after 1 month-0.571.600.016 SLEDAI after 2 months-0.500.65NS SLEDAI after 3 months0.110.10NS C3a after 1 month-310-185NS C3a after 2 months-241-203NS C3a after 3 months-235-179NS dsDNA after 1 month-207-940.036 dsDNA after 2 months-161292NS dsDNA after 3 months-58-14NS C3 after 1 month1.52-6.3NS C3 after 2 months0.85-0.8NS C3 after 3 months-0.8NS C4 after 1 month2.30.10.037 C4 after 2 months0.450NS C4 after 3 months0.215.0NS

23. Serial Measurements of Analytes in Representative Patients From Placebo and Prednisone Groups -2-10123 Prednisone C3a -2-10123 500 250 0 Prednisone DNA 1500 1000 500 0 -2-10123 Placebo C3a -2-10123 2000 1000 0 2072 Placebo DNA 984 618 434 378 128 1500 1000 500 0 Clinical Flare Rand

24. Clinical laboratory correlation in SLE is a heterogeneous relationship Unanswered Questions 1. Are these serologic parameters useful as predictors of flare and/or in assessment of flare and response to therapy? 2. Which tests are best and are combinations superior? 3. What is the optimal time interval in which to study a patient? 4. What is the outcome being measured i.e. definitions of flare, and in what organ, renal could be most relevant? Anti-DNA abs and C as Candidate Biomarkers for Clinical Trials in SLE

25. " One easily believes what one earnestly hopes for " The Roman dramatist Terrence Ability of Immune Tests to Predict Clinical Exacerbations in SLE C3Anti-DNAClinical Evidence  none necessarily  active nephritis  active extrarenal  active nephritis and extrarenal TABLE 13.4, p252... Dubois Textbook, Chapter: Complement and SLE, Schur and Glickstein