Background
B cells play an important role in the pathophysiological process of rheumatoid arthritis (RA), presumably through B-T-cell interaction and auto-antibody production. Targeted depletion of B cells with a monoclonal antibody (mAb) such as rituximab (anti-CD20) appears to be efficient and cost-effective in patients with RA that is refractory to disease-modifying anti-rheumatic drugs (DMARDS) and anti-tumor necrosis factor-alpha therapy (anti-TNF) [
1‐
3]. Nevertheless, 30–50% of patients with RA do not respond to rituximab [
4,
5]. Treatment could be more efficient if potential responders to rituximab could be selected before treatment or early during treatment.
Molecular imaging with positron emission tomography (PET) might be a predictive tool for therapeutic outcome in RA: PET allows non-invasive 3D visualization and quantification of pathophysiological processes at the picomolecular level, by binding of radiolabeled agents to any affected tissue in the whole body [
6]. Apart from prediction of disease outcome in RA [
7,
8], our group has also previously demonstrated that PET predicts infliximab outcome as early as two weeks after initiation of treatment [
9]. This predictive value for therapeutic outcome was later confirmed by Roivainen et al. for early DMARD combination treatment [
10].
In our laboratory we have experience with good manufacturing practice (GMP)-labeling of mAbs with the PET isotope Zirconium-89 (
89Zr) [
11,
12]. Zirconium-89 has a physical half-life of about 78.4 hours and can be stably coupled to mAbs.
89Zr-labeled rituximab has been successfully applied for imaging and radioimmunotherapy of CD20-positive B-cell lymphomas [
13]. In fact, this “immuno-PET” technique showed more tumor-positive lymph nodes than the standard fluorodeoxyglucose (
18F-FDG; glucose metabolism) PET scans [
13].
89Zr-rituximab PET imaging may not only be interesting for visualization of B cells in B-cell lymphoma but also for other B-cell-related immune activity in the body. In RA, apart from the joint synovium, studies have demonstrated that B cells also play an important role in lymph nodes in patients with RA [
14‐
16].
Methods
In this study, we investigated whether in vivo biodistribution of 89Zr-rituximab in RA, with special focus on hand joints and lymph nodes, was associated with clinical response to rituximab. We also collected lymph node biopsies for analysis of B cells prior to rituximab treatment, and after treatment with rituximab for 4 weeks, in order to investigate the potential association between histological findings and imaging results.
Patients
Twenty rituximab-naïve patients with RA were included between October 2010 and November 2014. Inclusion criteria were: patients (>18 years of age) with at least two clinically inflamed joints in the hands/wrists and a clinical indication for rituximab treatment, and stable treatment with DMARDs for at least 2 weeks and previous failure or intolerance to at least one anti-TNF drug. Anti-TNF had to be discontinued at least 4 weeks before initiation of rituximab treatment. Patients were not eligible if being treated with >10 mg daily dose of prednisolone at the time of inclusion, if they had been treated with investigational drugs within the previous 3 months or if they were pregnant or breast-feeding. The study protocol was approved by the VUmc Medical Ethics Committee. All patients gave written informed consent prior to participation in the study.
Discussion
Our study is the first that applied novel, non-invasive PET imaging of B cells by 89Zr-rituximab to investigate whether biodistribution of rituximab at baseline is related to clinical response to rituximab treatment. 89Zr-rituximab PET demonstrated clinically active joints, even partly in clinically silent joints, and there was a significant association between quantitative 89Zr-rituximab uptake in the hand joints and clinical response to rituximab treatment at 24 weeks. Moreover, by using a T/B cutoff value of 4.0 in PET-positive hand joints we found a potential positive predictive value of 90% for clinical response after 24 weeks rituximab treatment, while clinical and serological data were not distinctive. Apart from quantitative differences in joint uptake, there were no 89Zr-rituximab biodistribution differences in the lymph nodes or internal organs between responders and non-responders.
Specific targeting of B cells by
89Zr-rituximab was supported by positive associations between quantitative lymph node PET data and baseline CD22
+ cell count (as a surrogate marker for B cells) in histological lymph node analysis, and by positive associations between post-treatment CD22
+ B cells in lymph nodes and quantitative
89Zr-rituximab uptake on PET in the hand joint. These findings are in line with results from our colleagues, Jauw et al
. who found that tumor uptake of
89Zr-rituximab correlated positively with CD20 expression in tumor biopsies in patients with diffuse, large Bcell lymphoma [
22]. Other observations in this study also underlined the specificity of targeting B cells. Besides retention of
89Zr-rituximab in arthritic joints over time (while cleared from blood), specificity of uptake of
89Zr-rituximab in arthritic joints was also supported by our finding of significantly higher
89Zr-rituximab uptake in the joints of responders vs. non-responders, despite identical levels of disease activity at baseline. The lack of association between lymph node uptake on PET and clinical response may have been caused by the limited spatial resolution of PET of 4 mm, thus, positive lymph nodes may have been missed by PET.
Potentially,
89Zr-rituximab administration following the therapeutic dose of rituximab could result in competition of CD20 binding in the target, although it was administered within one hour. Nevertheless we chose this design to show the biodistribution of rituximab as used in the therapeutic setting in daily clinical practice. The kinetics of antibody influx in inflammatory targets are rather slow (from hours up to several days) [
11], but partial blockade of CD20 binding sites by unlabeled rituximab at the time of the labeled rituximab infusion cannot be excluded. If therapeutic doses negatively influenced binding of the tracer then we may even have underestimated
89Zr-rituximab uptake in the synovium and lymph nodes. On the other hand, targeting of
89Zr-rituximab in the joints and lymph nodes may also be influenced positively by infusion of the therapeutic dose of rituximab just prior to injection of labeled
89Zr-rituximab, as the spleen has been recognized as the “sink” for rituximab binding during the first passage in circulation [
11,
13]. Therefore, after saturation of the spleen, more
89Zr-rituximab may have become available for other CD20 targets such as the arthritic joints. For future clinical mAb studies in RA, dose escalation studies could answer this question and help define the optimal study design.
The finding that PET imaging with a
89Zr-labeled therapeutic antibody is able to predict therapeutic response of the antibody is in line with recent findings by Gebhart et al
. They showed that pre-treatment
89Zr-trastuzumab imaging in combination with early
18F-FDG PET response assessment after one cycle of trastuzumab was promising for the identification of non-responders after three cycles (PPV100% and NPV92%) in patients with breast cancer [
23].
There were 2 out of 20 patients in our study who did not show any 89Zr-rituximab uptake in the joints despite having a clinical response. The explanations for this may be relatively low Bcell counts (these patients had approximately 1500 CD22+ cells/mm2 in the lymph nodes vs. approximately 3000 cells/mm2 in PET-positive patients) and/or general low inflammatory activity in the joints in these patients, even though they did not differ clinically from other (PET-positive) responders. Finally, PET scans may have been false negative in these two patients.
Apart from
89Zr-rituximab PET, the serological status (RF and/or anti-CCP) has previously also been indicated as a potential predictive biomarker of therapeutic response. A meta-analysis, analyzing four placebo-controlled, phase II or phase III clinical trials, indicates that seropositive patients respond better to rituximab than seronegative patients [
24]. We could not confirm this in our cohort and this discrepancy can be due to the relatively small sample size of this study. Nevertheless, in this relatively small number of patients, the PET approach did reveal the predictive potential of
89Zr-rituximab PET imaging with discriminative value between responders and non-responders by demonstrating and quantifying the radiolabeled drug in arthritic joints. Actually, the level of
89Zr-rituximab uptake in the hand joints did not correlate with any clinical or laboratory parameter at baseline. None of the clinical or laboratory data collected at baseline in our study differed between responders and non-responders.
Conclusions
In conclusion, Bcell imaging in joints by 89Zr-rituximab PET-CT showed a clear association with clinical response at 24 weeks in patients with RA. This technique has potential value to select potential responders before initiation of rituximab treatment. This finding should be validated in larger cohorts, also in relation to other potential predictive biomarkers, in particular the serological status. Potentially, non-invasive, whole body 89Zr-rituximab PET-CT also holds promise for stratification and monitoring of anti-Bcell therapies in other Bcell-driven autoimmune diseases, such as systemic lupus erythematosus and Sjögren’s disease.
Acknowledgements
We would like to thank Hoffmann-La Roche, the Netherlands, for financial support of this investigator-initiated study. In addition, we would like to thank Ewa Platek (research nurse) for excellent patient care, research support and assessment of clinical parameters.