Identification and Evaluation of Novel Synovial Tissue Biomarkers in Rheumatoid Arthritis by Laser Scanning Cytometry

Christiane Fueldner; Anja Mittag; Jens Knauer; Maria Biskop; Pierre Hepp; Roger Scholz; Ulf Wagner; Ulrich Sack; Frank Emmrich; Attila Tárnok; Joerg Lehmann

Disclosures

Arthritis Res Ther. 2012;14(1) 

In This Article

Abstract and Introduction

Abstract

Introduction Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA.
Methods Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis.
Results CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis.
Conclusions In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by infiltration of cells into the synovial tissue and progressive destruction of cartilage and bone. Cell types known to be involved in RA pathogenesis in the joint are, among others, mononuclear immune cells and fibroblasts.[1]

For successful therapeutic intervention for RA with the focus on individualized medicine, it is useful to have procedures for specific and sensitive diagnosis as well as exact disease staging. It is important to identify patients with destructive disease prognosis in need of intensive treatment and to spare others from potential side effects. Therefore, tools for early and reliable diagnosis, monitoring inflammatory progress and controlling therapeutic success, are of utmost importance.

Early disease staging in RA according to American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria is in addition to the enumeration of involved small and large joints based mostly on blood tests measuring the erythrocyte sedimentation rate (ESR) and levels of C-reactive protein (CRP), rheumatoid factor (RF), and anti-citrullinated protein antibodies (ACPAs).[2] Such serologic parameters do not necessarily reflect biologic actions in the target tissue of the patient and, thus, provide only imprecise information on disease activity. Despite the great need for confirmed diagnosis in RA, no specific laboratory test is available (excellently reviewed by Nakamura[3]). However, in the last decade, monoclonal antibodies (mAbs) leading to immune-modulation of the underlying pathogenic process in RA, started a therapeutic revolution. These mAbs can be radiolabeled and applied for specific diagnostic tests. The scintigraphic detection of these radiolabeled mAbs allows direct visualization of the synovitis of RA. The combination of the assessment of disease-specific cellular biomarkers directly in the joint and noninvasive high-resolution in vivo imaging techniques, such as immunoscintigraphy or immuno-positron-emission tomography (PET), are suitable approaches to determine alterations in the joints and hence offer valuable tools for sensitive and specific diagnosis in RA.[4–7]

This study aimed to identify appropriate biomarkers for RA intended to be further validated and envisioned to be used in immunoscintigraphy or immuno-PET. To find RA-specific biomarkers, we used synovial tissue samples from patients with a long-term course of RA and from patients with acute non-RA arthritis as a control group. Several mAbs directed against cellular-surface molecules on cells, associated with the pathogenesis of RA, including adhesion molecules, activation markers, and receptors,[8–12] were tested for their potential to identify appropriate RA biomarkers by discriminating RA and acute arthritis. This was realized by quantitative tissue analysis using LSC combined with advanced statistical analysis (see Additional file 1, Figure S1 for an overall scheme of the test procedure). As a high-content screening technique, LSC is the method of choice. Although microscope based, it is unbiased and highly reproducible. Furthermore, LSC is not restricted to a few fields of view, but the whole specimen is measured,[13] making this method an appropriate technique for drawing conclusions about marker expression in the tissue of origin.

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