Myriam Gharbi

Fibulin 3 peptides Fib3-1 and Fib3-2 are potential biomarkers of osteoarthritis.

OBJECTIVE This study was undertaken to identify new biomarkers of osteoarthritis (OA) by proteomics analysis and to develop specific immunoassays to detect and quantify them. METHODS Proteomics analysis was performed in urine samples from 10 women (mean±SD age 76.0±5.0 years) undergoing knee replacement surgery due to severe OA and 5 healthy women (mean±SD age 25.6±2.6 years). Protein content was analyzed by 2-dimensional differential gel electrophoresis. Protein spots that exhibited an OA:control abundance ratio of ≥1.5 were identified by mass spectrometry. Specific enzyme-linked immunosorbent assays were developed and validated in serum obtained from 236 healthy subjects ages 20-64 years and from 76 patients with severe radiologic knee OA (mean±SD age 68.8±11.9 years). Immunohistochemical analysis was performed on articular cartilage from tibial plateaus. RESULTS Thirteen proteins within spots that were significantly modified between groups were identified. Two peptides of fibulin 3, named Fib3-1 and Fib3-2, were of particular interest. Two antisera directed against these peptides were used to develop immunoassays. Compared with age-matched healthy subjects, median levels of serum Fib3-1 and Fib3-2 were elevated in OA patients (54.6 pM versus 85.1 pM [P<0.0001] and 144.4 pM versus 191.4 pM [P<0.0001], respectively). Using area under the receiver operating characteristic curve analysis, we demonstrated that Fib3-1 and Fib3-2 levels discriminate between OA and normal populations. Immunostaining revealed the presence of Fib3-1 and Fib3-2 in chondrocytes and in the extracellular matrix of the superficial layer of the fibrillated cartilage. CONCLUSION Our findings indicate that Fib3-1 and Fib3-2 are potential biochemical markers for the diagnosis of OA.

Application for proteomic techniques in studying osteoarthritis: a review.

After the genomic era, proteomic corresponds to a wide variety of techniques that study the protein content of cells, tissue, or organism and that allow the isolation of protein of interest. It offers the choice between gel-based and gel-free methods or shotgun proteomics. Applications of proteomic technology may concern three principal objectives in several biomedical or clinical domains of research as in osteoarthritis: (i) to understand the physiopathology or underlying mechanisms leading to a disease or associated with a particular model, (ii), to find disease-specific biomarker, and (iii) to identify new therapeutic targets. This review aimed at gathering most of the data regarding the proteomic techniques and their applications to osteoarthritis research. It also reported technical limitations and solutions, as for example for sample preparation. Proteomics open wide perspectives in biochemical research but many technical matters still remain to be solved.

Identification of differential pattern of protein expression in canine osteoarthritis serum after anterior cruciate ligament transection: a proteomic analysis.

Osteoarthritis (OA) management remains a great challenge and there is considerable effort to understand its pathophysiology and to identify new therapeutic targets and biomarkers. Canine OA surgically induced by the transection of the anterior cruciate ligament (ACLT) is a widely used and relevant model. This study reports a proteome mapping of dog serum and an analysis of the differentially expressed proteins between before and after ACLT. In the first part of the study, 261 picked protein spots were identified from preparative 2D gels and 71 different proteins were identified among the 261 spots present on the reference map. Canine serum proteome mapping reveals the presence of proteins of interest, such as fetuin B, complement C3 and C1s and pregnancy zone protein. The comparison between serum from dogs before and after ACLT reveals the differential expression of several proteins that could play a key role in the pathogenesis of OA. A number of proteins, such as fetuin B and complement C3, were increased in dog OA serum whereas others, such as hyaluronan binding protein 2, inter-alpha-trypsin inhibitor H4 (ITIH4), complement C1s and C4 and haptoglobin were decreased. Some of these proteins could be candidate biomarkers for diagnosis, prognosis and treatment evaluation. The results of the study also reinforced the similarities between dog experimental OA and human cases of OA.

Soluble biomarkers development in osteoarthritis: from discovery to personalized medicine

Abstract Context: Specific soluble biomarkers could be a precious tool for diagnosis, prognosis and personalized management of osteoarthritic (OA) patients. Objective: To describe the path of soluble biomarker development from discovery to clinical qualification and regulatory adoption toward OA-related biomarker qualification. Methods and results: This review summarizes current guidance on the use of biomarkers in OA in clinical trials and their utility at five stages, including preclinical development and phase 1 to phase 4 trials. It also presents all the available regulatory requirements. Conclusions: The path through the adoption of a specific soluble biomarker for OA is steep but is worth the challenge due to the benefit that it can provide.

148 FIBULIN-3 FRAGMENTS (FIB3-1 AND FIB3-2) ARE POTENTIAL NEW BIOMARKERS FOR THE DIAGNOSIS OF OSTEOARTHRITIS

By comparison with healthy subjects, Fib3-1 and FiB3-2 serum levels are found elevated in patients with knee OA. Furthermore, the immunohistochemical findings indicate that fibrillated cartilage is a major source of fibulin-3 fragments. Specific immunoassays were developed using two rabbit antisera with high specificity for Fib3-1 and Fib3-2 respectively, ELISAs were validated in serum population collected from healthy subjects and patients with knee OA. Antisera did not recognize native fibulin-3 and did not cross-react with each fragment. Immunohistochemistry analysis was performed on articular cartilage from tibial plateaus 20-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 40 60 80 100 **

AB0767 Effect of Chondroitin Sulfate on Soluble Biomarkers of Osteoarthritis: How To Analyze and Interpret The Results from An Open-Label Trial in Unilateral Knee Osteoarthritis Patients

Background Changes in the level of biomarkers specific of osteoarthritis (OA) could help not only for the diagnosis but also for the monitoring of the disease progression and efficacy of a therapeutic intervention. Objectives The aim of this study was to investigate the effects of chondroitin sulfate (CS) on the serum levels of biomarkers in patients with knee OA. Methods Seventy two patients with unilateral symptomatic knee OA were involved in a post-authorization open-label study. Patients treated with CS (800 mg/day) were evaluated 5 times from D-30 to 6-month. The primary outcome was the % relative change in serum biomarkers (Coll2–1, Coll2–1NO2, Fib3–2). Secondary outcomes were the evaluation of pain (VAS) and function (Lequesnes Index). Responders and non-responders were classified according to OMERACT-OARSI recommendations. Finally, an original cut-off method was applied to categorize patients and interpret variations in serum levels of Coll2–1. Results Patients from either ITT or PP populations showed no difference in the serum biomarkers levels at baseline. Most of the biomarkers levels decreased after 1 month of treatment but no significant differences were reported. However when considering responders and non-responders from the ITT population, a significant difference was found for Coll2–1 at 3 months (p=0.030) and 6 months (p=0.038) (ACA approach). A decrease in pain (VAS) and an improvement in function (LI) were recorded throughout the visits (p<0.01). The decrease in pain was significant at 1, 3 and 6 months in ITT and PP populations when compared to baseline. The improvement of function was shown to be significant after 6 months. Finally, an intra-batch cut-off of 22% was determined for Coll2–1 assay based on the variation of Coll2–1 between two blood collections in non-arthritic adult subjects. This value allowed the definition of metabolic responders as patients with variation of Coll2–1 by more than 22%.This means that a variation of 22% or less in the serum level of Coll2–1 consisted in a variation related to homeostasis. In contrast, a variation over this limit revealed either an increase or a decrease in cartilage catabolism. Considering these categories, CS decreased Coll2–1 serum levels between baseline and 1-month visit compared to the value of Coll2–1 before treatment (screening visit) which can be interpreted as a drastic reduction of the proportion of patients with an increase of Coll2–1 over 22% (reduction from 13% to 3). It also consisted in a more important proportion of patients with a decrease in Coll2–1. Conclusions CS was effective modulating the metabolic status of KOA patients trough the reduction of Coll2–1 levels in responders and the diseases symptoms. Biomarkers are important tools for the monitoring of OA disease and of treatment. The main goal in biomarker research is to qualify them as surrogate endpoints in clinical trials. This study proposes a new approach for the analysis and the interpretation of the variation in biomarker levels and introduces the notion of metabolic responders. Disclosure of Interest None declared

124 IDENTIFICATION OF NOVEL BIOLOGICAL MARKERS OF OSTEOARTHRITIS BY A PROTEOMIC APPROACH

the disease in a rural Japanese population. We have performed the survey in Matsudai town Niigara city, Japan since 1979 and the survey was performed every 7 years. In this survey, checkup included an interview, physical examination, anteroposterior standing radiographs of both knees. In this survey, knee OA was defined as being present in a knee if radiographic grade of 2 or higher with Kellegren and Lawrence (KL) scale were detected. In current study, subjects were female ages were between 60 and 80 years who attended the both 4th (2000) and 5th (2007) project of the Matsudai knee OA survey. In 5th survey, we measured urinary N-terminal crosslinking telopeptide of type I collagen (NTx) and Cterminal crosslinking telopeptide of collagen type II (CTX-II) of 564 female aged 60 and older. Occurrence of knee OA was defined if a grade 0 or 1 at the 4th survey advanced to grade 2 or higher at 5th servay. Progression of the disease was defined if a grade 2 at the fourth survey advanced to grade 3 or higher at 5th survey. We compared the data of NTx and CTX-II between occurrence and non-occurrence groups as well as between progression and non-progression groups in age ranged 60-70 year-old and 7180 year-old. Statistical significance of a difference between two continuous variables tested by Mann-Whitney U test and a p-value of less than 0.05 was considered statistically significant. Results: In younger participants, ages between 60 to 70 years, showed the mean value of CTX-II in non-occurrence and occurrence group were 231±212 (ng/mmolCr) and 315±255 (ng/mmolCr), respectively. The mean value of CTX-II in nonprogressive OA and progressive OA were 276±246 (ng/mmolCr) and 416±314 (ng/mmolCr). We found occurrence and progression group showed significantly higher value of CTX-II in comparison non-incident or non-progression groups (47% and 50% increase, respectively) in aged between 60 to 70-year-old. In younger participants, incident group and progression group were higher amount of NTX than nonoccurrence and progression group, but they are not statistically significant. People older than 71, there is no statistically significant difference in the value of CTX-II as well as NTx between incident and non-incident as well as between progression and non-progression groups. Conclusions: Currently, there are no available biomarkers which predict the disease progression of knee OA. Current study suggested that CTX-II might be a candidate as an OA incident as well as progression marker especially relatively younger female (60-70 years old) in this retrospective study of Matsudai knee OA survey.