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Presented by Jill Buyon, M.D. at the September 29, 2003 meeting of the

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

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Presented by Jill Buyon, M.D. at the September 29, 2003 meeting of the

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

  2. High Low

  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 FcgR alleles (IIa) and renal disease

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

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

  6. Playing Rules for Evaluation of the Biomarker f Define Assay for Measurement Assay Binding Isotype DNA Sens/Spec Crithidia high + low affinity abs IgM or IgG dsDNA spec>sens Farr high affinity abs IgM and IgG ss and dsDNA ELISA high + low affinity abs IgM or IgG ss or dsDNA sens>spec anti-DNA abs Complement Assay Component Specimen Measurement Immunochemical Native C3, C4 serum Nephelometry Functional integrity CH50 EDTA plasma RBC lysis Catabolic state Activation C3a EDTA plasma ELISA Define parameters of change for these candidate biomarkers

  7. 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.

  8. 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 Prior: between visits 2 months and 1 month before visit with flare Concurrent: between previous visit and current visit Flare P (SLEDAI > 3) Prior h DNAabs ELISA >10% (70 visits) 30%0.007 Prior h DNAabs ELISA >25% (45 visits) 29%N.S. Prior h DNAabs Critidia >2 dilutions (72 visits) 39% 0.001 Concurrent i DNAabs ELISA (89 visits) 30% 0.002 Concurrent i DNAabs Crithidia (112 visits) 29% 0.0002

  9. 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 hdsDNA 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 hdsDNA abs by Crithidia = -.081 Conclusion: these tests had limited utility in predicting or excluding lupus flares

  10. 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

  11. Evaluation of the Sensitivity and Specificity of C3, C4, CH50, anti-dsDNA and C3a for Detection of Lupus Flareswithin 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

  12. Approach * Previous visit must have occurred within 3 months from date of measurement

  13. Definitionof flares Mild or Moderate Flare Change of SLEDAI >3 New/worse: Lupus rash Nasopharyngeal ulcers Pleuritis Pericarditis Arthritis Fever (SLE) Any h in Prednisone to < 0.5mg/kg/d Added NSAIDS or Plaquenil for disease activity Physician Global Assessment (PGA) increase >1.0, and < 2.5 Severe Flare Change in SLEDAI to > 12 New/worse: CNS SLE Vasculitis Nephritis Myositis Plt<60,000 Hemolytic anemia Requiring: Doubling of Prednisone Prednisone >0.5mg/kg/d Hospitalization New Cytoxan, Azathioprine or Methotrexate Increase in PGA to >2.5

  14. Patients Available for Evaluation and Outcomes • 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

  15. Sensitivity and Specificity of Analytes to Predict Flares Limitations/ Implications Utility 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.

  16. 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 Inclusion Criteria • Anti-DNA abs present within 2 years • Prednisone <15 mg  • No active infection • Stability of disease and medications for 2 months

  17. 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 Prednisone : 30 mg X 2 wks 20 mg X 1 wk 10 mg X 1wk Placebo

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

  19. Analysis of Severe Flares  90 Days Severe Flare No Flare Prednisone 0 21 6 14 Placebo Fisher’s exact test = 0.009

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

  21. Summary of Results of Outcome Variables by Treatment Groups

  22. Placebo C3a 2000 1000 0 -2 -1 0 1 2 3 2072 • Placebo DNA 984 618 434 378 128 -2 -1 0 1 2 3 Serial Measurements of Analytes in Representative Patients From Placebo and Prednisone Groups 1500 1000 500 0 1500 1000 500 0 Prednisone C3a Rand Clinical Flare Rand 500 250 0 -2 -1 0 1 2 3 Prednisone DNA Rand Rand Clinical Flare -2 -1 0 1 2 3

  23. Anti-DNA abs and C as Candidate Biomarkers for Clinical Trials in SLE 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?

  24. TABLE 13.4, p252... Dubois Textbook, Chapter: Complement and SLE, Schur and Glickstein Ability of Immune Tests to Predict Clinical Exacerbations in SLE C3 Anti-DNA Clinical Evidence i hh none necessarily ii hhh active nephritis i hhh active extrarenal iii h active nephritis and extrarenal " One easily believes what one earnestly hopes for " The Roman dramatist Terrence

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