1. RESULTS INTRODUCTION METHODS CONCLUSIONS ACKNOWLEDGMENTS.
Effect of Disease Modifying Anti-Rheumatic Drugs (DMARDs) on Anti-CCP2 and Anti-Citrullinated Protein Antibody (ACPA) Levels During Longitudinal Assessments in Rheumatoid Arthritis Patients
Makoto Soejima1, Aarat M. Patel1, Danielle Goudeau1, Donald M. Jones1, Christine L. Amity1, Lynne M. Frydrych1, Derek Sippel1,
Dawn McBride1, Heather F. Eng2, David Kyle2, Melissa Saul3, Stephen R. Wisniewski2, Larry W. Moreland1 and Marc C. Levesque1 in collaboration with the RACER physicians working group*
University of Pittsburgh, 1Department of Medicine, Division of Rheumatology and Clinical Immunology 2Graduate School of Public Health, Department of Epidemiology 3Department of Biomedical Informatics, Clinical Research Informatics Services
Table 1: Clinical and demographic characteristics of RACER subjects at baseline
Oral DMARDs
n = 70
Biologics only
n = 10
Oral DMARDs plus Biologics
n = 35 p
Female, n of patients (%)
55 (79)
9 (90)
29 (83)
0.6482
Age, mean (SD) yrs
61.1 (14.0)
60.3 (7.2)
57.3 (14.2)
0.4097
Race, Caucasian: African American
58: 12
8: 2
32: 3
0.4499
Disease duration, yrs
12.0 ( )
32.0 ( )
8.0 ( )
0.0011*
CRP, mg/dl
0.9 ( )
0.5 ( )
0.5 ( )
0.3580
RF, IU/ml
77.0 ( )
222.0 ( )
87.0 ( )
0.3840
Baseline DAS, mean (SD)
4.1 (1.6)
4.2 (1.9)
4.2 (1.5)
0.9725
Baseline CDAI, mean (SD)
20.9 (16.5)
23.6 (17.8)
25.3 (16.9)
0.4476
Figure 1: Percentage change from baseline of serum CCP2 and ACPA.
RESULTS
INTRODUCTION
CCP2
P = for CCP2
Age, sex, race, RF levels, and disease activity scores (DAS28) were similar among RA subjects in different treatment groups within each cohort (Tables 1 and 2).
Among anti-CCP2 positive RA subjects, individual ACPAs bound distinct subsets of citrullinated peptides (Table 3).
Anti-CCP2, anti-P6c, and anti-P20c levels decreased during oral DMARD therapy. In contrast, etanercept treatment was associated with no change in anti-CCP2, anti-P6c, and anti-P20c levels (Figure 1).
N-memory and memory subsets of B cells were increased in RA patients treated with oral DMARDs compared to normal subjects and RA patients treated with biologics (p < 0.03; Figure 2). N-memory subsets of B cells tended to decease after treatment with a TNF antagonist (P = 0.07; Figure 4).
Pre-switch memory B cells (IgD+, CD27+) in patients with RA on anti-TNF therapy were significantly lower than in control subjects (p = ; Figure 3).
The percentage of naïve and memory B cells did not change between after treatment with a TNF antagonist. IgD− CD27 − memory B cells significantly decreased after treatment with a TNF antagonist (P = 0.01).
Anti-citrullinated protein antibodies (ACPA) are specific makers for rheumatoid arthritis (RA) and may have a pathogenic role.
A commercial ELISA assay (anti-CCP2) is widely used to detect ACPA and forms the basis for an important diagnostic test for RA. The peptide sequence of CCP2 is proprietary and has not been published.
Several studies have addressed whether anti-CCP2 levels decrease during treatment but the results vary between studies and these other studies have not controlled for differences in disease activity. No studies have examined the effects of therapy on levels of individual ACPA that bind known citrullinated antigens.
Aim: To determine the effects of anti-rheumatic therapies on levels of individual ACPA and anti-CCP2 and to correlate these changes with changes in B cell subsets associated with ACPA production.
Percentage changes (%)
Table 2: Clinical and demographic characteristics of TEAR RA subjects at baseline AE
n = 83 AT
n = 38 SE
n = 63 ST
n = 22
MTX
n = 20 p
Female, n of patients (%)
59 (71)
27 (71)
44 (70)
17 (77)
13 (65)
0.9380
Age, mean (SD) yrs
50.2 (12.0)
48.1 (13.0)
50.3 (11.5)
51.4 (10.0)
48.2 (12.8)
0.7818
Disease duration, months
1.0 ( )
1.0 ( )
1.1 ( )
1.1 ( )
1.1 ( )
0.8537
ESR, mm/h
35.0 ( )
35.5 ( )
36.0 ( )
32.0 ( )
26.5 ( )
0.2520
CRP, mg/l
8.8 ( )
5.2 ( )
5.9 ( )
8.2 ( )
2.5 ( )
0.0656
RF positive, n of patients (%)
77 (92.8)
37 (97.4)
59 (93.7)
21 (95.5)
19 (95)
0.8882
Baseline DAS, mean (SD)
6.1 (1.0)
6.1 (0.9)
5.8 (1.0)
5.6 (1.0)
0.2159
ESR: erythrocyte sedimentation rate, CRP: C-reactive protein, RF: Rheumatoid factor, DAS: Disease activity score, CDAI: Clinical disease activity index, AE: aggressive etanercept, AT: aggressive triple DMARDs, SE: Step up etanercept, ST: Step up triple DMARDs, P values: chi square for trend and ANOVA
P6C
P = for anti-P6c
Percentage changes (%)
Table 3: Percentage of serum ACPA in RACER subjects versus healthy control subjects
Name
Peptides Sequences
Description
RA CCP2+
(n = 102)
Control
(n = 58) P4
EGGGVXGPXVVEXHQSACKDS
Fibrinogen a Cit
10% 0% P6
THSTKXGHAKSXPVXGIHTS
Fibrinogen a Cit
46%
P6C
THCTKXGHAKSXPVXGIHTC
Fibrinogen a Cit cyclic
66% P8
SAVRAXSSVPGVXLLQDSVDF
Vimentin Cit 4%
P10
STXSVSSSSYXXMFGG
Vimentin 2-17-Cit
19%
P12
VYATXSSAVXLXSSVP
Vimentin Cit
13%
P14
NEEGFFSAXGHRPLDKK
Fibrinogen b 6-22-Cit
50%
P18
KIHAXEIFDSXGNPTV
a-Enolase 5-20-Cit
44%
P20
SHQESTXGRSRGRSGRSGS
Filaggrin Cit
40%
P20C
HQCHQESTXGRSRGRCGRSGS
Filaggrin Cit cyclic
78% 3%
METHODS
P20C
P = for anti-P20c
CONCLUSIONS
Study design of TEAR trial and description of RACER registry subjects: One hundred fifteen RA patients with 3 longitudinal samples in the University of Pittsburgh Medical Center (UPMC) Rheumatoid Arthritis Comparative Effectiveness Research (RACER) registry and 226 RA patients with 4 longitudinal samples in the Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) registry were clinically assessed at each visit and samples were analyzed.
Statistical analysis: Mixed effects regression model, ANOVA, Mann-Whitney U test and unpaired t-test.
Percentage changes (%)
This is the first study to demonstrate a differential effect of DMARD therapy versus anti-TNF therapy on ACPA levels in patients with RA.
Anti-TNF therapy was associated with an initial decrease in ACPA levels and a later increase in ACPA levels. The mechanism for the later increase in ACPA levels during anti-TNF therapy is not understood.
Consistent with other published studies (Anolik et al), anti-TNF therapy was associated with a lower percentage of pre-switch IgD+ CD27+ and post-switch IgD− CD27+ memory B cells. There were no differences in plasmablast numbers in RA subjects with treatment and compared to healthy control subjects.
Consistent with other published studies (Souto-Carneiro et al), there were few changes in B cell subsets after treatment with TNF antagonists (Figure 4).
In the future, we will correlate changes in ACPA levels with B cell subset numbers and determine whether changes in B cell subset numbers are specific to anti-TNF therapies versus other biologic therapies.
Figure 3: Data from flow cytometry analysis of naïve and memory B cell subsets from RACER subjects treated with oral DMARDs or oral DMARDs plus biologics.
Group 1; Oral DMARDs only (MTX ,SSZ, HCQ and LEF)
Group 2; Oral DMARDs plus Biologics (TNF antagonists, Non-TNF antagonists)
Group 3; Biologics only
Baseline
Follow up 1 (12 weeks)
Follow up 2 (24 weeks )
Groups
Rheumatoid Arthritis Comparative Effectiveness Research (RACER)
Figure 2: Data from flow cytometry analysis of B cell subsets from RACER subjects treated with oral DMARDs or oral DMARDs plus biologics. 4 70 4 3 75 7 5 60 5 60 16 53 11 73 6
IgD
IgD 9 8 21 2 3 11 1 6 16 1 15 9 28 8 13
Healthy Control
MTX-RA
MTX plus TNF antagonist-RA
Healthy Control
MTX-RA
MTX plus TNF antagonist-RA
SCREENING
BASELINE
MTX + Etanercept (Arm#1)
MTX + SSZ/HCQ (Arm#2)
MTX + Etanercept (Arm#3)
MTX + SSZ/HCQ (Arm#4)
24 weeks
78 weeks
MTX
Treatment of Early Aggressive Rheumatoid Arthritis (TEAR)
102 weeks
TEAR Subjects in Arms 1 and 2, respectively received aggressive early therapy with MTX plus etanercept (AE; Arm 1) or MTX plus SSZ plus HCQ (AT; Arm 2). Subjects in Arms 3 and 4 received MTX monotherapy through Visit 2 (Week 24). If DAS28 at that visit was <3.2, MTX monotherapy was continued.; if DAS28 was >= 3.2, subjects were stepped up to either MTX plus etanercept (SE; Arm 3) or MTX plus SSZ plus HCQ (ST; Arm 4).
CD38
CD27
P =
P =
P =
P =
P =
P <
Number of subjects
Normal n = 17
No DMARD n = 4
MTX n = 22
MTX + TNF n = 19
Non-TNF n = 10 % % % % %
ACKNOWLEDGMENTS
P =
P =
P =
P =
P =
P =
P =
P =
P = % % % % %
Funding sources:
Figure 4: Data from flow cytometry analysis of B cell subsets from RACER subjects before and 6 months after treatment with MTX plus a TNF antagonist.
Number of subjects n = 13
P value
Naïve B cell P = Median: 57.8 (Before) vs 58.4 (After)
N-Memory P = Median: 10.4 (Before ) vs 7.6 (After)
Transitional P = Median: 4.29 (Before) vs 4.66 (After)
Memory P = Median: 11.6 (Before) vs 8.6 (After)
Early Memory P = Median: 11.9 (Before) vs 12.2 (After)
Plasmablast P = Median: 1.13 (Before) vs 1.19 (After)
IgD+CD27- Naïve P = Median: 58.0 (Before) vs 58.4 (After)
IgD+CD27+ DP P = Median: 3.91 (Before) vs 4.22 (After)
IgD-CD27-DN P = Median: 16.9 (Before) vs 13.5 (After)
IgD-CD27+ P = Median: 19.4 (Before) vs 20.8 (After)
REFERENCES
Natalia Wegner, et al: Autoimmunity to specific citrullinated proteins gives the first clues to etiology of rheumatoid arthritis. Immunology Journal reviews 2010; 233:
Marijn Vis, et al. IgM-rheumatoid factor, anti-citrullinated peptides, and anti-citrullinated human fibrinogen antibodies decrease during treatment with tumor necrosis factor blocker infliximab in patients with rheumatoid arthritis. J Rheumatol 2010; 35:
Wouter H. Bos, et al. Differential response of the rheumatoid factor and anti-citrullinated protein antibodies during adalimumab treatment in patients with rheumatoid arthritis. J Rheumatol 2008; 35:
Benjamin A Fisher, et al. Antibodies to citrullinated a-enolase peptide 1 and clinical and radiological outcomes in rheumatoid arthritis. Ann Rheum Dis 2011.
Raimon Sanmart, et al. Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis. Arthritis Research & Therapy 2009; 11: R135
Margarida Souto-Carneiro, et al. Alterations in peripheral blood memory B cells in patients with active rheumatoid arthritis are adependent on the action of tumor necrosis factor. Arthritis Research & Therapy 2009; 11: R84.
Rita A. Moura, et al. Alterations on peripheral blood B-cell subpopulations in very early arthritis patients. Rheumatology 2010; 49:
Annett M. Jacobi, et al. Activated memory B cell subsets correlate with disease activity in systemic lupus erythematous. Arthritis & Rheumatism 2008; 58:
Jennifer H. Anolik, et al. Cutting Edge: Anti-tumor necrosis factor therapy in rheumatoid arthritis inhibits memory B lymphocytes via effects on lymphoid germinal centers and follicular dendric cell networks. Journal of Immunology 2008; 180:
Transitional B cell 5 10 15 20
IgD+CD27+ DP 10 20 30 40
IgD+CD27- Naive 60 80
100
Before
After
IgD-CD27- DN 50
IgD-CD27+ memory
N-memory 5 10 15 20
Naive 20 40 60 80
100
RF, CRP, ESR
CCP2 and Individual ACPA levels (ELISA)
Disease activity score (DAS28, CDAI)
Clinical and Lab index
B cell subsets (Flow cytometry) % % % % %
Memory 10 20 30
Early Memory 20 40 60
Plasmablast 5 10 15 * % % % % %
Before
After
Before
After
Before
After
*RACER PHYSICIANS WORKING GROUP
Antonio Achkar MD, Rohit Aggarwal MD, Noah Bass MD, Alan Berg MD, Sherwood Chetlin MD,
Daniel DeLo MD, Robyn Domsic MD, Kenneth Gold MD, Kimberly Liang MD, Douglas Lienesch MD, Kathleen McKinnon DO, Thomas Medsger MD, Niveditha Mohan MD, Chester Oddis MD, Thaddeus Osial Jr MD, Terence Starz MD, Bertrand Stolzer MD, Elizabeth Young MD