# Presentation: 733494

## Presented by

- Jill Buyon, M.D.
- at the
- September 29, 2003
- meeting of the
- Arthritis 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 FcR alleles (IIa) and renal disease

## Classical Pathway

**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,Bb**

**C3b,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

## .

**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**

## Slide 6

## anti-DNA abs

- 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

## 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 flare
- Concurrent:        between previous visit and current visit

## 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

## 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) 

- <date/time>

- 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

## Approach

- <date/time>

| 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 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**

**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**

## 496 Total Patients (328 HRT patients +  168 OCP patients) : 

- <date/time>

**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**

## Sensitivity and Specificity of Analytes to Predict Flares

- <date/time>

|  | 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****   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.  **
**  **
** **

## Inclusion Criteria

**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

## Study Design

**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

- Placebo

- Prednisone :  30 mg X 2 wks
- 	         20 mg X 1 wk
- 	         10 mg X 1wk

## Flow Chart of Patients Followed in the Observational Study (up to 18 months)

- <date/time>

**180**

- RANDOMIZED(Serological flare, clinically stable)

**41**

- NON-RANDOMIZED

**139**

- _Completed_
- no serological or clinical flare

**92**

- Stopping point

**47**

- Clinicalflare

**11**

- No clinicalflare

**30**

- _Clinical 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*

## 	      1 month	   2 month	  3 month

- 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
- 30mg X 1wk
- 20mg X 1wk
- 10mg X 1wk

- Randomization

##  Summary of Results of Outcome Variables by Treatment Groups

- <date/time>

-  **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 Patients

**From Placebo and Prednisone Groups**

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

***Prednisone***
**C3a**

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

**500**
**250**
**0**

***Prednisone***
**DNA**

**1500**
**1000**
**500**
**0**

**1500**
**1000**
**500**
**0**

- Clinical   Flare

- Clinical Flare

- Rand

- Rand

- Rand

- Rand

- 				      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**

## " 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