The Diagnostic Role of Biomarkers in. Rheumatoid Arthritis: The Old and New!

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1 Review Article 9 Copyright University of Medicine, Tirana AJMHS 2015; Vol. 46 (1): The Diagnostic Role of Biomarkers in Rheumatoid Arthritis: The Old and New! Arbi Pecani 1, Zamira Ylli 2, Margarita Kurti-Prifti 2, Elizana Petrela-Zaimi 3, Genc Sulçebe 2 1 Rheumatology Unit, Department of Internal Medicine and Medical Specialties. La Sapienza University of Rome, Rome, Italy. 2 Service of Immunology and Histocompatibility, Department of Laboratory Medicine, Faculty of Medicine, University of Medicine, Tirana and University Hospital Centre Mother Teresa, Tirana, Albania. 3 Department of Health and Environment, Faculty of Public Health, University of Medicine, Tirana and Statistics Unit, University Hospital Center Mother Teresa, Tirana, Albania. Abstract Rheumatoid Arthritis (RA) is a systemic autoimmune inflammatory disease that affects mainly the joints in about 0.5 to 1% of the general population. Early detection and treatment of this disease prevents its progression and the subsequent irreversible damage of the joints. In addition to the important role of anticitrullinated peptide antibodies (ACPA) and rheumatoid factor (RF) in the clinical diagnosis of RA, new biomarkers are required to achieve an early detection of this progressive disease. In the last decade, an important role in the early RA diagnosis has been attributed to the autoantibodies directed to mutated citrullinated vimentin (MCV). Also, polymorphic genetic biomarkers such as HLA-DRB1 locus with its RA predisposing shared epitope (SE) alleles, have shown interesting results in this area. Recently, a new group of autoantibodies directed against carbamylated proteins have been discovered. These autoantibodies, together with some autoantigens described by different research groups represent a new group of biomarkers with an interesting potential toward the diagnosis of RA. This review will provide an updated view of the new biomarkers in RA and discuss their potential role toward the wind of change in RA diagnosis. Keywords: parathyroid gland (PG), primary hyperparathyroidism (PHPT), adenoma of PG, hyperplasia of PG, carcinoma of PG. Address for correspondence: Arbi Pecani, Rheumatology Unit, Department of Internal Medicine and Medical Specialties. La Sapienza University of Rome, Rome, Italy. arbipecani@gmail.com

2 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis 10 INTRODUCTION Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology that affects about 0.5% to 1% of the general population [1]. The disease is characterized by chronic joint inflammation often leading to bone and cartilage destruction, as well as the presence of autoantibodies: the rheumatoid factor (RF) in particular and more recently the anticitrullinated peptide antibodies (ACPAs) (1). An early and aggressive treatment can alter the course of the disease, preventing irreversible damage of the joints as well as improving their function and the quality of life in patients affected by this disease (2). New diagnostic criteria for RA are available since 2010, as a joint initiative between the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) (3) (Table 1). These criteria have included the presence of anti-citrullinated peptide antibodies for the diagnosis of RA, assisting the rheumatologist toward a better and earlier classification of the disease. Yet, a significant number of patients with early arthritis do not fulfill these criteria and are wrongly labeled as undifferentiated arthritis (4). Thus, there is still a need for new diagnostic biomarkers aiming toward an earlier diagnosis of the disease. Rheumatoid Factor (RF) and Anti-citrullinated peptide antibodies (ACPA) The rheumatoid factor (RF) is an autoantibody, which may be of IgM, IgG or IgA isotype and was first mentioned in 1922 (5). RF recognizes the CH2 and CH3 domains in the Fc segment of human IgG (6). For over fifty years, the laboratory diagnosis of RA has relied on the detection of rheumatoid factor, which was one of the seven criteria established in 1987 by the American College of Rheumatology for the diagnosis of the disease (7). Only in 2010, ACPA were added as one of the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) disease classification criteria for RA (3). Historically, the anti-perinuclear factor (APF) was the first citrulline-binding autoantibody discovered in the sera of rheumatoid arthritis (RA) patients (8). Later on, it was reported that RA sera contain anti-keratine antibodies (AKA) and for a long time the combination of these biomarkers has been used to aid the diagnosis of RA (9). Only in 1998 it was demonstrated that APF and AKA recognize similar epitopes and for this purpose the conversion of arginine to citrulline is essential (10, 11). Therefore, APF and AKA were broadly categorized as anticitrullinated peptide antibodies, leading to the development of a novel assay for the diagnosis of RA.

3 11 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis Table 1. The 2010 ACR/EULAR classification criteria for RA (3) Classification criteria for RA A score > 6 is considered as the cut-off point for RA diagnosis. Categories Joint involvement 1 large joint 2 10 large joints 1 3 small joints 4 10 small joints >10 joints (at least 1 small joint) Serology (at least 1 test result is needed for classification) Negative RF and negative ACPA Low-positive RF or low-positive ACPA High-positive RF or high-positive ACPA Acute-phase reactants (at least 1 test result is needed for classification) Normal CRP and normal ESR Abnormal CRP or normal ESR Duration of symptoms <6 weeks 6 weeks Score (0-5) (0-3) (0-1) 0 1 (0-1) 0 1 ACPA = anti-cyclic citrullinated peptide antibodies; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; RA = rheumatoid arthritis; RF = rheumatoid factor. ACPAs are mostly detected using the anti-cyclic citrullinated peptide (CCP) antibody assay. The first commercially available ACPA assay (1 st generation or cyclic citrullinated peptide or CCP-1) relied on a peptide derived from the filaggrin protein (12). The second generation of cyclic citrullinated peptide tests (CCP-2), has been identified by screening peptide libraries of high complexity with sera of RA patients (13). In contrast, the third generation was designed by combinatorial peptide engineering and contains multiple citrullinated epitopes displayed in a conformational structure to increase epitope exposure and thus the immunoreactivity (14). This is meant especially for early RF-negative RA patients, since these patients have antibodies to only one or very few epitopes (15). The antigen compositions of these latest assays are

4 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis 12 not yet publicly available as patents are pending, but apart from the main differences in substrates, all the currently available commercial ACPA assays use the ELISA method and similar dilutions (1:100), diluents, controls, conjugates, and interpretation. The combination of both RF and ACPA predict RA with an improved sensitivity of 60% to 80% and a specificity of 95% to 99% (15). Details of the studies concerning the sensitivity and 48%, when compared to the Northern and Western European populations (16). Interestingly, the ACPA positivity rate in the Albanian RA patients is similar to the rate of these autoantibodies reported in an Italian group of RA patients, where a positivity rate of 44 % was reported (16, 17). A number of studies have documented the appearance of ACPA prior to the onset of the disease. specificity of ACPA and RF used alone and combined for the diagnosis of RA are summarized in Table 2. Table 2. Sensitivity and specificity of ACPA and FR in the diagnosis of RA Autoantibody Sensitivity (%) Specificity (%) Ref. ACPA [15, 18] ACPA 1 st generation [15] ACPA 2 nd generation [19] ACPA 3 rd generation [20] RF [21] ACPA 2 nd + RF [22] ACPA = anti-cyclic citrullinated peptide antibodies; RF = rheumatoid factor Data regarding the positivity of these antibodies differ from country to country, apparently in function of the genetic background of the population. For example, ACPA and RF positivity are reported to be in lower rate in In this regard, ACPA were identified with a median of 4.8 years before RA diagnosis (23), with a maximum time distance of 10 years (24), demonstrating its significant predictive value for the eventual early diagnosis of the disease. Albanian RA patients, respectively 46% and

5 13 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis On the other hand, ACPA have been found to be positively correlated with higher erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) serum level, number of swollen joints and worse physician global assessment rating, while the presence of RF is positively correlated with increased ESR and CRP but there is no association with other disease activity markers (25). Furthermore, ACPA are useful in identifying RA patients who are more likely to develop bone damage and who may not be identified by RF testingalone (26). In a 6 year follow up study, ACPA-positive RA patients had significantly more radiological damage than ACPA-negative patients (p < 0.05). RF was also associated with increased radiological damage at 6 years (p < ) (27). Of note, current or previous cigarette smoking has been associated with a positive ACPA test. This is really interesting because there is an association between tobacco use and the development of RA, as well as an increased RA disease activity (28). The fact that ACPA can be found early in the course of the disease (29), their association with more joint destruction and greater disease activity and the striking gene-environment interaction between the Human Leukocyte Antigen (HLA) system (such as HLA-DRB1 shared epitope) (30) and smoking, described only in ACPA positive RA patients, suggest that these autoantibodies carry a pathological role in the development of the disease (23, 25). ACPA are able to activate both FcR-positive cells and the complement system, providing further support for their possible role in this disease pathogenesis (31, 32). All these evidences support the idea that ACPA define a separate patho-physiological entity within RA (33). Some reports describe a decrease in the ACPA titer following successful treatment of RA (34). However, the decrease is usually modest and should not drive treatment decisions. ACPA positive patients usually remain positive despite treatment. Antibodies to citrullinated vimentin and mutated citrullinated vimentin (MCV) Vimentin is a dynamic intermediary filament, very important for the cell structure as it is involved in the regulation of mechanical stress between chondrocytes and matrix tissue (35). Citrullination of vimentin results in the production of an autoantigen expressed in synovial tissue, formerly known as antigen Sa, which stands for Savoie, the name of the patient in whom it was first identified (36). Anti-Sa antibodies have been found to have a high specificity of >98%, but a limited sensitivity of 22% to 40% in patient with RA. Moreover, they have a high predictive value of about 84% to 99% for RA and are closely associated with extra-articular manifestation and severe joint involvement (36, 37). Citrullination and mutation can influence the antigenicity of vimentin. Bang et al. found an antigenic mutated

6 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis 14 isoform of vimentin in a human fibroblast cell, resulting from the replacement of several glycine residues by arginine residues (38). This finding brought to the production of a in vitro assay named the anti-mutated citrullinated vimentin (anti-mcv) ELISA test. Studies on the sensitivity and specificity of anti-mcv ELISA are rather contradictory. Most of the studies have shown a higher sensitivity with a lower specificity of anti-mcv compared to ACPA, but it should be noted that the anti-mcv sensitivity varied with the disease duration and it was found to be 81 % in established RA (>2 years duration) and 92% in early RA (<2 years duration) (39). The overall sensitivity and specificity of anti- MCV was 84% and 87% (40). Like ACPA, anti-mcv antibodies are suitable for the early diagnosis of RA, with comparable sensitivity (55.3% versus 59.3% respectively), specificity (92.1% vs. 92.3% resp.) and positive predictive value (95.8% vs. 96.1% resp.) (41). Moreover, a significant correlation has been found between anti-mcv antibody titers and both the severity and disease activity of RA (42). In patients with active RA, the anti-mcv titer was higher compared to patients with milder RA (43). Experiments of cross-reactivity between anti- MCV and ACPA have shown that only a part of the anti-mcv antibodies react with ACPA and vice versa, indicating that these antibodies target different epitopes (37). Anti-MCV antibodies were associated with the shared epitope (SE) and protein tyrosine phosphatase non-receptor type 22 (PTPN22), while ACPA were only associated with SE (44).These findings highlight the important role of anti-mcv antibodies in the pathogenesis of RA, demonstrating that this test has almost an equal value as ACPA for the diagnosis of RA (42). Genetic biomarkers Multiple genetic studies have been carried out to identify and validate other possible RA biomarkers. Several genetic polymorphisms have been identified in this context, but none of them has been conclusive as the primary predisposing genetic determinant of the disease. A well-known genetic biomarker in RA, is the HLA-DRB1 locus with the predisposing alleles DRB1*01:01, 01:02, 04:01, 04:04, 04:05, 04:08, 04:10, 10:01, 14:02, sharing a similar amino acid sequence, known as the shared epitope (SE) (45). These predisposing alleles have been linked to an increased presence of ACPA, RF and a more severe disease progression (46). These findings have been confirmed even in an Albanian RA study population reporting a significant increase of the HLA-DRB1*04 and at a lesser degree of HLA-DRB1*01 allele frequencies among ACPA and RF positive patients (16). Contrarily, HLA DRB1*11:02, 11:03, 13:01, 13:02,01:03 and 04:02 have been associated with a absence of circulating ACPA and are called as protective alleles in RA (47). In the Albanian study, it has been reported that the predisposing HLA-DRB1*01 and HLA-

7 15 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis DRB1*04 alleles if calculated together, are found in 29.0% of the RA patients, while the corresponding result for the HLA-DRB1*11 and HLA-DRB1*13 alleles is 56.0%. The high frequency of these protective alleles, combined with the low frequency of the predisposing alleles in this population could probably explain the rather low positivity rates of ACPA and RF antibodies among Albanian RA patients and perhaps also the relatively lower prevalence and gravity of RA in Southern Europe and Mediterranean countries (16). Other single nucleotide polymorphism (SNP) and MicroRNA (mirna) based studies clearly confirm that different genetic susceptibility loci contribute to the risk for the development of RA (48). Table 3 provides a summary of all these studies. However, the value of the genetic biomarkers in the RA diagnosis and prognosis remains a topic for further research (49). Antibodies against carbamylated proteins (Anti-CarP antibodies) Carbamylation is a non-enzymatic post translational modification that changes the binding properties of many proteins to their receptors (52). Carbamylation is a particular type of post-translational modification, in which isocyanic acid binds to the amine group of an amino-acid, mostly resulting in the conversion of lysine into homocitrulline. In contrast to citrullination, which is enzimatically mediated (by PAD), carbamylation is a chemical reaction that uses cyanate present in the human organism and in equilibrium with urea (52). Inflammation can enhance carbamylation through a mechanism that depends on myeloperoxidase released from the neutrofilic cells during inflammation (53). One recent study showed that carbamylation induces an autoantibody response. Shi et al identified anticarbamylated antibodies (anti-carp) in ACPApositive and ACPA-negative RA patients, using carbamylated foetal calf serum (FCS) as the antigen and non-modified FCS as control. In their cohort study, 16% of ACPA negative patients were positive for anti-carp IgG and 30% for anti-carp IgA, while from the ACPA positive patients, 73% resulted positive for anti- CarP IgG and 51% for anti-carp IgA (54). Other studies have demonstrated that carbamylated human fibrinogen is specifically recognised by autoantibodies in patients with RA (55) and the presence of anti-carp antibodies in ACPA negative RA patients was strongly associated with a more severe joint damage (54). Furthermore, the presence of anti-carp antibodies was associated with a higher risk of developing RA in arthralgia patients, indipendently of ACPA (56). These autoantibodies are not only present in adult patients, but can also be found in juvenile idopathic arthritis (JIA), the most common rheumatic disease in children (57). Therefore, anti-carp antibodies can be a useful biomarker

8 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis 16 to detect both ACPA negative and ACPA positive pre-ra patients and newly diagnosed RA patients of all ages, who should undergo into an early and agressive clinical intervention. (58). In order to achieve this goal, it is important to identify new biomarkers that can improve the early diagnosis and detect seronegative RA patients. Table 3. Other genetic biomarkers in Rheumatoid Arthritis: Single Nucleotide Polymorphysm (SNP) and micro RNA (mirna) (50,51) Markers having a strong risk association in a genome-wide functional SNP screen in RA - Central portion of the ancestral 8,1HLa haplotype, identified as A1-B8-DRB1*03. - HLA class II region of certain HLA-DRB1*0404 haplotypes not associated to SE. - Centromeric region of HLA-DRB1 locus in SNP mapping of MHC region in ACPA positive RA patients showed:hla-dob (beta-chain), antigen peptide transporter 2(TAP2), HLA- DPB1, collagen typex1 alpha2 genes. - missense SNP (R620W) in protein tyrosine phosphatase non-receptor type 22 gene (PTPN22). Markers having a moderate risk association in a genome-wide functional SNP screen in RA - A new 100kb region on chromosome 9 identified as TRAF1-C5 and a TNF receptor associated gene. - Two independent alleles at chromosome 6q23. - A variant allele of STAT4 (signal transducer and activator transcription 4). - PADI4 (peptidylarginine deiminase type 4) polymorphisms, have been identified as a RA susceptibility locus and associated with anti-ccp antibodies. This polymorphism was identified in Japanese, North American and Swedish RA population but couldn t be replicated in other European RA population except the German population. Dysregulated mirna - Aberrant expression of mirna plays an important role in the immunopathogenesis of RA, especially in identifying those cases that would benefit from early initiation of biologic therapies. Other discoveries of biomarkers in early RA Recent findings support the evidence that early treatment of RA can lead to drug-free remission In this context, some autoantigens have been described by two research groups:

9 17 Pecani A. Diagnostic Role of Biomarkers in Rheumatoid Arthritis a- Using a phage display technology with pooled sera from early and seronegative RA patients, Somers et al, identified 14 different antigens. The sensitivity of these new biomarkers varied from 2% - 29% with a specificity of 95% - 100%. In ACPA negative RA patients, these autoantibodies were found in 44% - 67% of patients. Despite the importance of these findings for an early diagnosis and more adequate treatment of RA, the multiplex testing required for the identification of this autoantigens is not yet a common approach in the routine diagnostics (59). b- Auger et al, using a proteomic approach, identified PAD4 (peptydil arginine deiminase type 4) and BRAF (v raf murine sarcoma viral oncogene homologue B1) as new autoantigens in RA. Furthermore, they described the epitopes on both these antigens opening the road to alternative ways toward an early RA detection (60). CONCLUSION The biomarkers described above show promising results toward an early diagnosis and better treatment of RA patients. They rise the hope that individuals who are at risk to develop the disease can be identified in a pre-clinical phase. Besides the established clinical utility of ACPA and RF, new biomarkers like anti-carp seem to have an important role in the identification of early pre-ra and especially in the identification of ACPA negative RA patients, suggesting that the combination of different biomarkers carries the potential to identify much more cases with RA. Still, further studies are needed to identify the exact role of these new biomarkers in the diagnostic algorithm of RA. This wide range of autoantibodies can be used in near future in the detection and classification of RA subtypes. Acknowledgements: Not available Conflict of interest disclosure: Not available REFERENCES 1. Scott DL, Wolfe F, Huzinga TW. Rheumatoid arthritis. Lancet 2010; 25;376: Resman-Targoff BH, Cicero MP. Aggressive treatment of early rheumatoid arthritis: recognizing the window of opportunity and treating to target goals. Am J Manag Care. 2010;16 (9 Suppl):S Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, et al. Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 2010;62:(9): Krabben A, Abhishek A, Britsemmer K, Filer A, Huizinga TW, et al. Risk of rheumatoid arthritis development in patients with unclassified arthritis according to the 2010 ACR/EULAR criteria for rheumatoid

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