Presented byJill Buyon, M.D.

at the September 29, 2003

meeting of theArthritis Advisory Committee

Low

High

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 FcR alleles (IIa) and renal disease

--> C1 esterase activity

C4 --C1--> C4a + C4bC2 --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,PP

C3bB D

+ Ba

C3b + B

C3 --> C3a + C3b (spontaneous)

(stable)

Alternative Pathway

+ C6C7C8C9

+ C6C7C8C9

chemotactic factoranaphylotoxin

chemotactic factoranaphylotoxin

DNA-IgG anti-dsDNA + C1

GlomerulonephritisFetal loss

Neutrophil activation

Endothelial cell activation (priming)

Leukothrombosis

RestingEC

.. ..

.. .. . ..

.. ..

. ...

. ... ...

. .. . ...

. ..ICAM-1CR3

C3aC5a

Resting PMN

IL-1ßTNFC1qC3aC5aC5b-9aECaPL

E-selectin. ..

.. .. . ..

.. .. .. .. ..

.

. ...

. .. ..

..

..

. . ...

. .. . ...

. ... ...

. ... ..

.. .. .. .

... .Vaso-occlusive plug

. ...

. ..

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>sensFarr 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 lysisCatabolic state Activation C3a EDTA plasma ELISA

Define parameters of change for these candidate biomarkers

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.

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 flareConcurrent: between previous visit and current visit

Reanalysis of Ho and Petri Data: Likelihood RatioKavanaugh et al, Arth Rheum, 2001

LR for a positive test: Extent to which a positive test increases pretest likelihood of disease (10 is high)

sensitivity1-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

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

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

Measurements Definition 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

Definition of flares

Severe Flare

Change in SLEDAI to > 12

New/worse: CNS SLEVasculitis

Nephritis MyositisPlt<60,000

Hemolytic anemia Requiring:

Doubling of PrednisonePrednisone >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 rashNasopharyngeal ulcers

PleuritisPericarditis

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

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

Mild / Flares

Moderate Severe Flares

Measurements Sensitivity Specificity Sensitivity Specificity

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 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.

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. BuyonMichael Belmont

Betty DiamondMeggan Mackay

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

180

RANDOMIZED(Serological flare, clinically stable)

41

NON-RANDOMIZED

139

Completedno serological or clinical flare

92

Stopping point

47Clinical

flare

11

No clinicalflare

30Clinical flare

with or without serologic flare

21

Voluntarydrop out

11

Exclusioncriteria

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%

Analysis of Severe Flares 90 Days

Severe Flare No Flare

Prednisone

Placebo

0 21

6 14

Fisher’s exact test = 0.009

Placebo3 renal1 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 1wk30mg X 1wk20mg X 1wk10mg X 1wk

Randomization

Summary of Results of Outcome Variables by Treatment Groups

Variable Prednisone Placebo P-Value

SLEDAI after 1 month -0.57 1.60 0.016

SLEDAI after 2 months -0.50 0.65 NS

SLEDAI after 3 months 0.11 0.10 NS

C3a after 1 month -310 -185 NS

C3a after 2 months -241 -203 NS

C3a after 3 months -235 -179 NS

dsDNA after 1 month -207 -94 0.036

dsDNA after 2 months -161 292 NS

dsDNA after 3 months -58 -14 NS

C3 after 1 month 1.52 -6.3 NS

C3 after 2 months 0.85 -0.8 NS

C3 after 3 months -1.0 -0.8 NS

C4 after 1 month 2.3 0.1 0.037

C4 after 2 months 0.45 0 NS

C4 after 3 months 0.21 5.0 NS

Serial Measurements of Analytes in Representative PatientsFrom Placebo and Prednisone Groups

116153 167

7897

136

0

250

500

-2 -1 0 1 2 3

380 356

618

191

426

267

0

500

1000

1500

-2 -1 RAND 1 2 3-2 -1 0 1 2 3

PrednisoneC3a

-2 -1 0 1 2 3

500

250

0

PrednisoneDNA

1500

1000

500

0

499608

835

693

9181028

0

500

1000

1500

-2 -1 RAND 1 2 3-2 -1 0 1 2 3

PlaceboC3a

0

1000

2000

-2 -1 0 1 2 3-2 -1 0 1 2 3

2000

1000

0

2072

•PlaceboDNA

984

618

434378

128

1500

1000

500

0

Clinical Flare

Clinical Flare

Rand

Rand

Rand Rand

Clinical laboratory correlation in SLE is a heterogeneous relationship

Unanswered Questions1. 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

" One easily believes what one earnestly hopes for " The Roman dramatist Terrence

Ability of Immune Tests to Predict Clinical Exacerbations in SLE

C3 Anti-DNA Clinical Evidence

none necessarily

active nephritis

active extrarenal

active nephritis and extrarenal

TABLE 13.4, p252... Dubois Textbook, Chapter: Complement and SLE, Schur and Glickstein