Conny J. van der Laken

Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients

OBJECTIVE To determine the expression of folate receptor beta (FRbeta) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FRbeta than methotrexate (MTX). METHODS Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FRbeta protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FRbeta messenger RNA (mRNA) levels were determined by reverse transcription-polymerase chain reaction analysis. Binding affinities of FRbeta for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FRbeta-transfected cells. Efficacy of FRbeta-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FRbeta-transfected cells. RESULTS Immunohistochemical staining of RA synovial tissue showed high expression of FRbeta on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FRbeta mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FRbeta had a high binding affinity (20-77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FRbeta-transfected cells. CONCLUSION Abundant FRbeta expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity-binding folate antagonists, of which BCG 945 may be a prototypical representative.

The value of ultrasonography in predicting arthritis in auto-antibody positive arthralgia patients: a prospective cohort study

IntroductionUltrasonography (US) has better sensitivity than clinical evaluation for the detection of synovitis in early rheumatoid arthritis (RA). Patients presenting with arthralgia and a positive anti-citrullinated protein antibodies (ACPA) and/or Rheumatoid Factor (IgM-RF) status are at risk for developing RA. In the present study, US utility and predictive properties in arthralgia patients at risk for the development of arthritis were studied.Methods192 arthralgia patients with ACPA and/or IgM-RF were included. Absence of clinical arthritis was confirmed by two physicians. US was performed by one of two trained radiologists of any painful joint, and of adjacent and contralateral joints. Joint effusion, synovitis and power Doppler (PD) signal in the synovial membrane of the joints and tenosynovitis adjacent to the joint were evaluated and classified on a 4-grade semi-quantitative scale. Grade 2-3 joint effusion, synovitis, tenosynovitis and grade 1-3 Power Doppler signal were classified as abnormal.ResultsForty-five patients (23%) developed arthritis after a mean of 11 months. Inter-observer reliability for synovitis and PD was moderate (kappa 0.46, and 0.56, respectively) and for joint effusion low (kappa 0.23). The prevalence of tenosynovitis was too low to calculate representative kappa values. At joint level, a significant association was found between US abnormalities and arthritis development in that joint for joint effusion, synovitis and PD. At patient level, a trend was seen towards more arthritis development in patients who had US abnormalities for joint effusion, synovitis, PD and tenosynovitis.ConclusionsUS abnormalities were associated with arthritis development at joint level, although this association did not reach statistical significance at patient level. US could potentially be used as a diagnostic tool for subclinical arthritis in seropositive arthralgia patients. However, further research is necessary to improve test characteristics.

Macrophage positron emission tomography imaging as a biomarker for preclinical rheumatoid arthritis: Findings of a prospective pilot study

OBJECTIVE To conduct a prospective pilot study to determine whether macrophage targeting by 11C-(R)-PK11195 positron emission tomography (PET) can visualize subclinical synovitis in arthralgia patients who have anti-citrullinated protein antibodies (ACPAs). METHODS Twenty-nine arthralgia patients who were positive for ACPAs but did not have clinical arthritis were studied. High (spatial)-resolution 11C-(R)-PK11195 PET scans of the hands and wrists were performed. For all metacarpophalangeal, proximal interphalangeal, and wrist joints (i.e., 22 joints per patient), tracer uptake was scored semiquantitatively (0-3 scale) by 2 observers who were blinded with regard to the clinical data. Patients were followed up prospectively for 24 months to investigate the development of clinical arthritis. RESULTS Overall agreement and kappa values for the readings of the 2 observers were, respectively, 97% and 0.91 (95% confidence interval [95% CI] 0.74-1) at the patient level and 99% and 0.81 (95% CI 0.65-0.96) at the joint level. In 4 patients, at least 1 and as many as 5 PET-positive joints (score≥1) were found at baseline. Within 2 years of followup, 9 patients had developed clinical arthritis. This included all 4 patients with positive findings on the 11C-(R)-PK11195 scan, who developed clinical arthritis in the hand/wrist region, as identified on PET scans. Of the 5 remaining arthritis patients with negative findings on PET scans, 2 developed arthritis in the hand joints and 3 developed arthritis at locations outside the field of view of the PET scanner. CONCLUSION Subclinical arthritis in ACPA-positive arthralgia patients could be visualized by 11C-(R)-PK11195 PET scanning and was associated with development of arthritis within 2 years of followup. This indicates that 11C-(R)-PK11195 PET may be useful in determining arthritis activity in the preclinical phase of RA.

Noninvasive imaging of macrophages in rheumatoid synovitis using 11C-(R)-PK11195 and positron emission tomography.

OBJECTIVE Noninvasive imaging by positron emission tomography (PET) of macrophages in inflamed joints of patients with rheumatoid arthritis (RA) may allow early detection of disease activity. We undertook this study to investigate whether rheumatoid synovitis can be visualized by PET using the tracer 11C-(R)-PK11195, which binds to peripheral benzodiazepine receptors (PBRs) on macrophages. METHODS Knee joints of 11 RA patients with active arthritis of at least 1 knee joint were imaged with 11C-(R)-PK11195 PET. Tissue uptake of 11C-(R)-PK11195 was quantified. PET was followed by arthroscopy of the most inflamed knee joint of each RA patient. Synovial tissue samples were subjected to immunohistochemical staining. RESULTS 11C-(R)-PK11195 uptake on the PET scans was significantly higher in severely inflamed joints than in joints with moderate or mild signs of inflammation. In addition, tracer uptake in contralateral uninflamed knee joints of RA patients was significantly higher than in uninflamed joints of control patients without inflammatory joint disease, suggesting the presence of subclinical disease activity. PET tracer uptake in joints correlated significantly with PBR staining in the sublining of synovial tissue. PBR staining correlated significantly with CD68 staining of macrophages. CONCLUSION 11C-(R)-PK11195 PET imaging allows noninvasive in vivo imaging of macrophages in rheumatoid synovitis and possibly even in subclinical synovitis. Noninvasive visualization of macrophages may be useful both for detecting early synovitis and for monitoring synovitis activity during treatment.

Involvement of breast cancer resistance protein expression on rheumatoid arthritis synovial tissue macrophages in resistance to methotrexate and leflunomide

OBJECTIVE To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF). METHODS Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed. RESULTS BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients. CONCLUSION These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

Evaluation of the Novel Folate Receptor Ligand (18F) Fluoro-PEG-Folate for Macrophage Targeting in a Rat Model of Arthritis.

IntroductionDetection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[11C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [18F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.Methods[18F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [18F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[11C]PK11195.Results[18F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF . In the rat model, [18F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [18F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [18F]fluoro-PEG-folate were increased compared with those of (R)-[11C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [18F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.ConclusionsThe novel PET tracer [18F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[11C]PK11195. These results warrant further exploration of [18F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.

Promising potential of new generation translocator protein tracers providing enhanced contrast of arthritis imaging by positron emission tomography in a rat model of arthritis

IntroductionEarly diagnosis of and subsequent monitoring of therapy for rheumatoid arthritis (RA) could benefit from detection of (sub)clinical synovitis. Imaging of (sub)clinical arthritis by targeting the translocator protein (TSPO) on activated macrophages is feasible using (R)-[11C] PK11195-based positron emission tomography (PET), but clinical applications are limited by background uptake in peri-articular bone/bone marrow. The purpose of the present study was to evaluate two other TSPO ligands with potentially lower background uptake in neurological studies, [11C]DPA-713 and [18F]DPA-714, in a rat model of arthritis.MethodsTSPO binding of DPA-713, DPA-714 and PK11195 were assessed by in vitro competition studies with [3H]DPA-713 using human macrophage THP-1 cells and CD14+ monocytes from healthy volunteers. In vivo studies were performed in rats with methylated bovine serum albumin-induced knee arthritis. Immunohistochemistry with anti-TSPO antibody was performed on paraffin-embedded sections. Rats were imaged with [11C]DPA-713 or [18F]DPA-714 PET, followed by ex vivo tissue distribution studies. Results were compared with those obtained with the tracer (R)-[11C]PK11195, the established ligand for TSPO.ResultsIn THP-1 cells, relative TSPO binding of DPA-713 and DPA-714 were 7-fold and 25-fold higher, respectively, than in PK11195. Comparable results were observed in CD14+ monocytes from healthy volunteers. In the arthritis rat model, immunohistochemistry confirmed the presence of TSPO-positive inflammatory cells in the arthritic knee. PET images showed that uptake of [11C]DPA-713 and [18F]DPA-714 in arthritic knees was significantly increased compared with contralateral knees and knees of normal rats. Uptake in arthritic knees could be largely blocked by an excess of PK11195. [11C]DPA-713 and [18F]DPA-714 provided improved contrast compared with (R)-[11C]PK11195, as was shown by significantly higher arthritic knee-to-bone ratios of [11C]DPA-713 (1.60 ± 0.31) and [18F]DPA-714 (1.55 ± 0.10) compared with (R)-[11C]PK11195 (1.14 ± 0.19).Conclusions[11C]DPA-713 and [18F]DPA-714 clearly visualized arthritis and exhibited lower (peri-articular) bone/bone marrow uptake than (R)-[11C]PK11195. These features merit further investigation of these tracers for early diagnosis and therapy monitoring of RA in a clinical setting.

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

IntroductionPositron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [18F]FDG and [11C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [18F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.MethodsIn a stepwise approach different PET tracers were investigated. First, whole body [18F]FDG and [11C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [18F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.ResultsNo increased [18F]FDG and [11C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [18F]Fluoride PET-CT, [18F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [18F]FDG depicted only three lesions, with an uptake of five times lower compared to [18F]Fluoride, and again no [11C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [18F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [18F]Fluoride PET positive lesions.ConclusionsOur PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [18F]Fluoride. In contrast to active RA, inflammation tracers [18F]FDG and [11C](R)PK11195 appeared to be less useful for AS imaging.

Three-Year Clinical Outcome Following Baseline Magnetic Resonance Imaging in Anti–Citrullinated Protein Antibody–Positive Arthralgia Patients: An Exploratory Study

Advanced imaging may be useful in the detection of subclinical synovitis (i.e., synovitis that cannot be detected by clinical examination) in anti–citrullinated protein antibody (ACPA)–positive arthralgia patients, and it may contribute to timely assessment of which individuals will eventually develop rheumatoid arthritis (RA) (1,2). Therefore, in this pilot study we investigated whether magnetic resonance imaging (MRI) can visualize subclinical inflammation in the hands and/or wrists of ACPA-positive arthralgia patients, and we determined the relationship between baseline MRI and development of clinical arthritis during 3 years of followup. MRI scans in healthy volunteers were included for comparison. The study was embedded in a cohort study that recruited seropositive arthralgia patients at the rheumatology outpatient clinics of the VU University Medical Center and Jan van Breemen Research Institute Reade (3). During 26 months, all arthralgia patients with positive ACPA status (3) (independent of IgM–rheumatoid factor status) were consecutively asked to participate in the present MRI substudy. Inclusion and exclusion criteria have been reported previously (2,3). Baseline MRI was performed on 28 included patients and 4 healthy volunteers without a history of joint disorders or clinical arthritis. Development of clinical arthritis was monitored according to the schedule of the cohort study during at least 3 consecutive years. MRI sequences (Siemens Sonata 1.5T MR scanner) were chosen according to Outcome Measures in Rheumatology (OMERACT) guidelines (4). STIR and 3-dimensional T1-weighted magnetization-prepared rapid gradient-echo images were obtained before and after intravenous gadolinium administration. Synovitis and bone marrow edema were scored by 2 independent observers (NA, CD) according to the OMERACT RA MRI Scoring (RAMRIS) system (4). The MRI protocol included scanning of all proximal interphalangeal (PIP) joints (PIP joints 1–5), metacarpophalangeal (MCP) joints (MCP joints 1–5), and wrist joints of both hands. At the patient level, MRI positivity was defined as the presence of synovitis and/or bone marrow edema in at least 1 joint/bone. Individual cumulative MRI scores (range 0–288) were calculated by summing synovitis and bone marrow edema scores of each hand/wrist joint. At baseline, the median age of the 28 patients was 44 years (interquartile range [IQR] 37–53 years). Twenty-three patients (82%) were women. The median duration of arthralgia was 15 months (IQR 11–35 months). The median age of the 4 healthy controls (1 man, 3 women) was 31 years (IQR 26–56 years). At baseline, MRI abnormalities were frequently found. In 26 of 28 patients (93%), MRI synovitis was present in 1 joint of both hands/wrists (Figure 1A). Ten of 26 patients had a synovitis score of 2 in 1 joint. A synovitis score of 3 was not observed. Bone marrow edema was present in only 3 of 28 patients (11%). MRIs in all healthy controls showed signs of mild synovitis (score of 1) in 1 joint (range 3–23). A score of 2 for synovitis was found in 1 joint of 1 healthy control. Bone marrow edema was not observed on MRIs in healthy controls. The presence of MRI abnormalities at baseline was not associated with dichotomous outcome of development of clinical arthritis (yes/no). Twelve of 28 patients (43%) developed clinical arthritis and were subsequently diagnosed as having RA according to the American College of Rheumatology/European League Against Rheumatism 2010 classification criteria (5). In 10 of 12 patients, arthritis was observed in hand and/or wrist joints. At the patient level, 11 of 12 patients who developed clinical arthritis and all 16 patients who did not develop clinical arthritis had a positive baseline Figure 1. A, Baseline T1-weighted contrast-enhanced magnetic resonance imaging (MRI) scan of the hand/wrist joints of an anti– citrullinated protein antibody (ACPA)–positive arthralgia patient who developed arthritis in hand/wrist joints during 3-year followup. Arrows indicate MRI signs of synovitis (score of 2). B, Cumulative MRI scores (including score of 1 for synovitis and bone marrow edema) of patients with and those without development of arthritis during 3-year followup. Symbols represent individual patients; horizontal bars show the median. C, Survival curves for all included patients, comparing the group with an MRI synovitis score of 2 in at least 1 joint (solid line) and the group with an MRI synovitis score of 1 in at least 1 joint (dashed line). Patients with a synovitis score of 2 developed arthritis faster than those with a synovitis score of 1. Plus sign indicates a censored patient. D, Cumulative MRI scores (including score of 1 for synovitis and bone marrow edema) in relation to age, in individual ACPA-positive arthralgia patients and individual healthy controls.

18F-Fluorodeoxyglucose Positron Emission Tomography in Elderly Patients with an Elevated Erythrocyte Sedimentation Rate of Unknown Origin

Patients with an elevated erythrocyte sedimentation rate (ESR) and non-specific symptoms often pose a diagnostic dilemma. PET/CT visualises infection, inflammation and malignancy, all of which may cause elevated ESR. The objective of this study was to determine the contribution of 18F-fluorodeoxglucose positron emission tomography (PET/CT) in the diagnostic work-up of referred patients with an elevated ESR, in whom initial routine evaluation did not reveal a diagnosis. We conducted a combined retrospective (A) and prospective (B) study in elderly (>50 years) patients with a significantly elevated ESR of≥50 mm/h and non-specific complaints. In study A, 30 patients were included. Malignancy (8 patients), auto-inflammatory disease (8 patients, including 5 with large-vessel vasculitis) and infection (3 patients) were suggested by PET/CT. Two scans showed non-specific abnormalities and 9 scans were normal. Of the 21 abnormal PET/CT results, 12 diagnoses were independently confirmed and two alternative diagnosis were made. Two diagnoses were established in patients with a normal scan. In study B, 58 patients in whom a prior protocolised work-up was non-diagnostic, were included. Of these, 25 PET/CT-scans showed suspected auto-inflammatory disease, particularly large-vessel vasculitis (14 cases). Infection and malignancy was suspected in 5 and 3 cases, respectively. Seven scans demonstrated non-specific abnormalities, 20 were normal. Of the 40 abnormal PET/CT results, 22 diagnoses were confirmed, 3 alternative diagnoses were established. Only one diagnosis was established in the 20 patients with a normal scan. In both studies, the final diagnosis was based on histology, clinical follow-up, response to therapy or additional imaging. In conclusion, PET/CT may be of potential value in the diagnostic work-up of patients with elevated ESR if routine evaluation reveals no diagnosis. In particular, large-vessel vasculitis appears to be a common finding. A normal PET/CT scan in these patients suggests that it is safe to follow a wait-and-see policy.