interest in understanding the pathogenesis of RA increased the importance of studying the biomarkers involved in different stages of the disease and diagnosis

1. BIOLOGICAL MARKERS IN
RHEUMATOLOGY
PRESENTER :DR.MADHAVAN

2. LESSON PLAN
Introduction:1 min
History:1min
Pathogenesis: 2mins
Clinical feature:3 mins
Diagnostic modalities :5 mins
Diagnostic biomarkers:3mins
Prognostic biomarkers: 3 mins
Biomarkers for the monitoring the disease activity:3 mins
Biomarkers for the response to biologic therapy:3mins
Summary:2 mins
Questions:5 mins

3. OBJECTIVES
At the end of the class all the
students should be able to know the
1.Diagnostic biomarkers
2.Prognostic biomarkers
3.Biomarkers for the monitoring the
disease activity
4.Biomarkers for the response in
biologic therapy

4. INTRODUCTION
Rheumatoid arthritis (RA) is a chronic inflammatory
disease with autoimmune disease, joint involvement that leads to
deforming and destructive arthritis of synovial joints along with multiple
systemic manifestations
The aetiology of RA remains unknown, multiple mechanisms
being involved in the physio pathogenic chain
So, an interest in understanding the pathogenesis of RA
increased the importance of studying the biomarkers involved in
different stages of the disease and diagnosis as well
Ref: Balanescu A. Poliartrita reumatoida si boli inrudite. Ionescu R. Esentialul in Reumatologie. Ed. Medicala Amaltea; 2006

5. • By the term biological markers (biomarkers ) mean an objective
molecular indicator or substitute of pathological processes that possess
1. Predictive utility
2. Diagnostic value
3. Prognostic value
• Biological markersare widely used that can provide an objective
measure of
1. Normal range
2. Pathogenic processes
3. Pharmacological responses to a therapeutic intervention

6. HISTORY
• Guillaume de Baillou (1538–1616) was a
French physician born in Paris
• He gave the first modern descriptions
of Rheumatism and arthritis in the book
Liber de Rheumatismo

7. EPIDEMIOLOGY
It is more common in women then men
The ratio is 2-3:1
Peak incidence is 35-50 years of age

8. PREVALENCE
The worldwide prevalence of RA has been estimated as 0.24 percent
based upon the Global Burden of Disease 2010 Study
Most epidemiologic studies of RA have been conducted in Caucasians
As a result, the risk factors commonly high from these populations
The incidence and prevalence of RA is 10 times greater in Native
Americans than those of most population groups
Del Puente A, Knowler WC, Pettitt DJ, Bennett PH. High incidence and prevalence of rheumatoid arthritis in Pima Indians. Am J Epidemiol 1989;
129:1170.

9. ETIOLOGY
• Monozygotic twins :12-15%
• Major histocompatibility complex class II loci is the prime culprit
that holds the DR4BETA chain (DRB*0401 &DRB*0404)
• Class III gene TNF alpha ,heat shock protein 70 (HSP70 )
• CD4 T cells are key mediators of tissue damage, both in the joint
and in extra-articular lesions infections : mycoplasm ,epstein barr
virus , cytomegalo virus, parvo virus, rubella virus

10. Risk of RA Protective Against RA

11. • Cigarette smoking and coffee consummation considered to be strong
association with RA factor and Anti CCP positive RA
• Alcohol consumption, use of olive oil, fish oil and statins may lower
the risk up to 40 % of developing RA and Anti CCP positive RA
• Nullyiparity with early menopause have high risk of RA
• Whereas breast feeding have a protective effect against RA

12. PATHOGENESIS

14. NEED FOR BIOMARKERS

15. • The major role of biomarkers can be objectified by comparing the
diagnostic criteria's
• The first diagnostic criteria given by American College of
Rheumatology (ACR) in the year 1987 for the diagnosis of RA,
which includes Rheumatoid Factor (RF) as a biomarker along with
other criteria's

17. • Although RF analysis is one of the serological tests in the American
College of Rheumatology (ACR) criteria, RF is nonspecific and may
be present in healthy individuals or in those with other autoimmune
diseases
Ref:Dorner T, Egerer K, Feist E, Burmester GR (2004) Rheumatoid factor revisited. Curr Opin Rheumatol 16(3):246–
253

18. •The most specific RA autoantibody system known is
directed against citrullinated antigens
Ref: Schellekens GA, Jong BA de, Hoogen FH van den et al (1998) Citrulline is an essential constituent of
antigenic determinants recognized by rheuma- toid arthritis-specific autoantibodies. J Clin Invest 101(1):273–
281

19. • Later the new American College of Rheumatology (ACR)/ European
League Against Rheumatism(Eular) 2010 criteria for the early diagnosis
of RA was proposed
• It uses four serological tests, namely
1. Rheumatoid factor (RF)
2. Antibodies against cyclic citrullinated proteins(anti –CCP )/Anti-
citrullinated protein/peptide antibody(ACPA)
3. Erythrocyte sedimentation rate (ESR)
4. C- reactive protein (CRP)

22. OTHER BIOMARKERS
• Currently, the ACR/ EULAR 2010 criteria
uses the rheumatoid factor (RF) and
antibodies against cyclic citrullinated
proteins (anti-CCP)
• Besides them, other diagnostic biomarkers
that can help the early diagnosis of RA
were identified`

23. Rheumatoid
factor
Anti- CCP
Anti –MCV
Anti Carp
14-3-3 eta
protein

24. RHEUMATOID
FACTOR
•Rheumatoid factors
(RFs) are polyreactive
IgM antibodies
produced by a subset
of B lymphocytes and
bind to the Fc portion
of the IgG molecule

25. The occurrence of IgA, IgG as well as IgM-rheumatoid
factors is widely established by Kunkel et al in
1966,Dunne et al in 1979
In juvenile rheumatoid arthritis (JRA), the sensitivity is
much lower than in RA i.e (5%), suggesting that RF is of
little diagnostic aid in JRA

26. RF above 20 IU/ml is not considered enough to diagnose RA
Other autoimmune diseases conditions, chronic infections,
diabetes, bacterial endocarditis, cancer, normal aging may
express RF
Sensitivity :60 to 80% for RA
Specificity :80 to 95%.
High titres of IgM RF correlate with RA disease activity and
extra-articular involvement (vasculitis in particular)

27. The normal range of RF is from 0-
20 IU/ml.
But RF above 20 IU/ml is not
considered enough to diagnose
RA, as their other reasons for
the rise

28. • Few data have been reported that
use of disease-modifying anti-
rheumatic drugs (DMARDs), and
significant decrease in RF titre
after treatment with TNF-blocking
agents and with anti CD20
antibodies Rituximab

29. Anti- CCP
• Anticyclic citrullinated peptide (anti-CCP) The
normal level of anti-CCP is less than 20 Units by
different measurement notation, that is, as less than
20 EU/ml
• This test is 97% specific for RA if it is present.
Once a patient develops a positive anti-CCP, it will
usually remain positive, despite remission.

30. • About 20% of RA patients are seronegative, meaning that
their RF and anti-CCP lab results both continue to come
back negative. In these cases, the the diagnosis based on
physical examination and radiological findings
• RF and anti-CCP are not used to monitor disease activity,
because they both tend to remain positive despite remission.

31. SEDIMENTATION RATE
The ESR is not specific for RA, such as bad processing, infection, and
aging in patients over the age of 50
Normal levels for men: 0-15 mm/hr to 0-20mm/hr :women :0-20
mm/hr/ to 0-30mm/hr
It is calculated by measuring the rate at which red blood cells sediment
in a test tube in one hour
Erythrocyte sedimentation rate or ESR is a simple measure of
inflammation

32. MUTATED
CITRULLINATED
VIMENTIN
ANTIBODIES
(Anti –MCV)
Vimentin is a protein and a
major constituent of the intermediate
filament family of proteins, expressed
in normal mesenchymal cells and is
known to maintain cellular integrity
and provide resistance against
stress

33. • Vimentin can be citrullinated (deamination) the conversion of
amino acid arginine in a protein into the amino acid citrulline a
reaction mediated by peptidyl arginine deiminase with the
formation of anti-vimentin antibodies
• To improve the quality of the test, a mutation was performed in
which arginine residues are replaced with glycine causing the
formation of MUTATED CITRULLINATED VIMENTIN
ANTIBODIES (Anti-MCV)

34. • Enhanced antibody reactivity against the mutated citrullinated vimentin
(MCV) was found in the serum of Results in anti-MCV showed
increased diagnostic accuracy
Ref: Bang H, Egerer K, Gauliard A et al (2007) Mutation and citrullination modifies vimentin to a novel au- to antigen for
rheumatoid arthritis. Arthritis Rheum 56(8):2503–2511

35. • A meta-analysis from 2010 that included 14
studies, in which the mutated citrullinated
vimentin antibodies (anti-MCV) and anti-
CCP for the RA diagnosis were tested,
concluded that there is no difference
between the two tests. Thus, the anti-
MCV may be a 2rd line diagnostics if ,
suspected RA, but with anti-CCP and RF
negative
Ref: Luime JJ, Colin EM, Hazes JM, Lubberts E. Does anti-mutated citrullinated vimentin have additional value as a serological marker in the diagnostic and
prognostic investigation of patients with rheumatoid arthritis?. A systematic review. Ann Rheum Dis. 2010 Feb;69(2):337–344.

36. • 14-3-3 eta protein belongs to the family of 14-
3-3 proteins that consists of 7 isoforms, it is
located intracellularly, being externalized in
the inflammatory process where it can be
citrullinated
• The 14-3-3 (η) proteins act like chaperonins
they prevent misfolding of proteins during
stressful situations such as high heat
14-3-3 ETA (η) PROTEIN
Ref: Maksymowych WP, Marotta A. 14-3-3η: a novel biomarker platform for
rheumatoid arthritis. Clin Exp Rheumatol. 2014;32(Suppl. 85):35–39.

37. In a study conducted on 619 subjects, 14-3-3 eta
protein sensitivity and specificity for RA was 77% and 93%
respectively. In the early stages of the disease, the determination
of protein 14-3-3 eta along with RF and anti-CCP increases the
diagnostic rate from 72% (RF + anti-CCP) to 78% (RF + anti-
CCP + 14-3-3eta )
Ref: Maksymowych WP, Naides SJ, Bykerk V, Siminovitch KA, van Schaardenburg D, Boers M, et. Serum 14-3-3η is a novel
marker that complements current serological measurements to enhance detection of patients with rheumatoid arthritis. J
Rheumatol. 2014 Nov;41(11):2104–2113

38. • This is the newest blood test to be used in the diagnosis of
rheumatoid arthritis. People with RA seem to have elevated levels of
the 14-3-3η protein in their blood, the 14-3-3η test may be used with
other lab tests in the early diagnosis of RA
The value of 14-3-3η more then ≥0.19 ng/ml

39. ANTIBODIES AGAINST
CARBAMYLATED PROTEINS
(ANTI-CARP)
• Anti-carbamylated protein antibodies (anti-
CarP) are reported to be associated with
increased disease activity and with more
severe joint damage in rheumatoid arthritis
(RA) patients

40. • In 2011, In a study performed on
2086 patients with early RA, anti-
CarP, anti-CCP and RF (Ig M) were
tested. Results showed,
1. Anti-CCP sensitivity and
specificity of 54% and 96%
2. RF had 59%, respectively 91%
3. Anti-CarP 44% and a
specificity of 89% for the

41. PROGNOSTIC
BIOMARKERS
• RF presence and high titers were correlated
with an increased risk of developing RA
• The risk may increase even 26 times if the
initial titers of RF are > 100 IU/ ml
Nielsen SF, Bojesen SE, Schnohr P, Nordestgaard BG. Elevated
rheumatoid factor and long term risk of rheumatoid arthritis: a
prospective cohort study. BMJ. 2012;345:e5244

42. •Patients with RF positive ,
usually develop more
aggressive forms of the
disease, with a more severe
functional impairment
Rheumatoid nodules
formation
Paimela L, Palosuo T, Leirisalo-Repo M, Helve T, Aho K. Prognostic value of quantitative measurement of rheumatoid factor in
early rheumatoid arthritis. Br J Rheumatol. 1995 Dec;34(12):1146–1150

43. • In a study that included 279 patients with early forms of RA, who were followed
for five years by clinical examination, serological and imaging tests, the presence
of anti-CCP at diagnosis was associated with a more important radiological
progression and severe forms of disease
Ref: Ronnelid J, Wick MC, Lampa J, et al. Longitudinal analysis of citrullinated protein/ peptide antibodies (anti-CP) during 5
year follow up in early rheumatoid arthritis: anti-CP status predicts worse disease activity and greater radiological
progression. Ann Rheum Dis. 2005;64:1744–1749

44. • The presence of other biomarkers was associated with more severe
forms of RA, such as anti-MCV, 14-3-3 eta protein, but these tests are
not commonly used and need to be implemented in guidelines

45. BIOMARKERS FOR THE MONITORIZATION OF
THE DISEASE ACTIVITY
• In the “treat to target” recommendations, 3 composite scores
for the monitorization of the disease
• Disease activity score (DAS 28)
• Simple disease activity index (SDAI)
• Clinical disease activity index (CDAI)

46. • The disadvantage of these scores is the degree of subjectivity of some
of the criteria
• Moreover, a significant proportion of the patients with negative
inflammatory tests, still have active disease
• So, For a better monitorization of the disease activity “multi-
biomarkers disease activity test (MBDA)” has been developed
MULTI-BIOMARKERS DISEASE ACTIVITY
TEST (MBDA)

48. INTERPRETATION
• Depending on the values of this score, the
disease activity can be classified into
• Mild (1-28)
• Moderate (29-43)
• Severe (>44)
• The usefulness of this score determined the
commercialization of Vectra DA test, which
is now available for clinicians

50. Vectra DA test
• Vectra is the currently validated multi-biomarker blood test to measure
rheumatoid arthritis (RA) disease activity.
• It is validated for use in adults diagnosed with RA and has been studied
in more than 1,700 patients from multiple cohorts
• Vectra integrates 12 key protein biomarkers into a single, objective
score between 1 and 100 to classify RA disease activity
Ref: Curtis JR, et al. Validation of a Novel Multi-Biomarker Test to Assess Rheumatoid Arthritis Disease Activity, Arthritis Care Res (Hoboken).
2012; 64(12):1794-1803.

51. HOW IS VECTRA DIFFERENT FROM OTHER LAB TESTS?
That’s a major benefit by this test
The usual lab tests may measure inflammation indirectly ESR or
measure only one biomarker of inflammation like C-reactive protein
(CRP) whereas Vectra measures 12 key proteins that represent multiple
RA biological pathways at a time

52. Additionally, the
utility of ESR and
CRP is limited
because they are
normal in a large
percentage of
patients with RA
In contrast, Vectra
can frequently
detect elevated
disease activity even
when CRP is low
Ref: Hambardzumyan, K, et al. Annals of the Rheumatic Diseases 2014. doi:10.1136/annrheumdis-2013-204986

53. PREDICTIVE BIOMARKERS OF THE
RESPONSE TO BIOLOGIC THERAPY
• The introduction of biological therapies in the management of RA
represented a major step forward for a better control of the disease
• A better understanding of the pathophysiological mechanism
underlying AR led to this development

54. Therapies currently available are
Anti TNF α it includes
• Infliximab (IFX)
• Adalimumab (ADA)
• Etanercept (ETA)
• Certolizumab (CER)
• Golimumab pegol (GOL)

55. Anti-lymphocyte B - rituximab (RTX)
Interleukin 1 ant-agonists receptor
anakinra (ANA)
Interleukin 6 receptor ant-agonists -
tocilizumab (TCZ)
Blocking co-stimulation of T lymphocyte -
abatacept (ABA)

56. RESPONDERS VS NON-RESPONDERS
• Despite this progress, studies reported that 20-40% of the
patients were declared nonresponders after starting a biologic
therapy
• High costs and patient exposure to severe adverse reactions
(ex. infections) determined the need to identify biomarkers
that can distinguish pretreatment responders vs. nonresponder
patients
Ref:Chaves Chaparro LM, Salvatierra Ossorio J, Raya Álvarez E. Predictors of response to biologic therapies in rheumatoid
arthritis. Reumatol Clin. 2011;7:141–144.

57. OTHER BIOMARKERS
Cartilage oligomeric matrix protein (COMP) is a specific
serological marker, evaluates the articular cartilage degradation and
its turnover, its detectable in the blood and synovial fluid
Serum calprotectin is a protein that has the ability to bind
calcium, belonging especially to leukocytes, but can also be found
in monocytes or macrophages. High levels of calprotectin can
appear in various inflammatory conditions
Survivin, a tumoral biomarker, which belongs to the family of
inhibitors of apoptosis, has been reported in patients with RA

58. BIOMARKERS FOR THE PREDICTION OF THE
RESPONSE TO BIOLOGIC THERAPY

59. IMMUNOGENICITY
The is one possible cause of non-responsiveness to a biological treatment
Due to appearance of anti-drug antibodies (ADAs)
One of the strategies proposed to decrease the immunogenicity of biological
agents by Methotrexate. It reduces the production of ADAs, perhaps by
inhibiting lymphocytes
Due to the unique structure between the anti-TNF agents is less immunogenic.
There are rare cases for the appearances of ADAs against anti-TNF agents

60. THANK YOU