Evidence for Updating the Core Domain Set of Outcome Measures for Juvenile Idiopathic Arthritis: Report from a Special Interest Group at OMERACT 2016

Objective. The current Juvenile Idiopathic Arthritis (JIA) Core Set was developed in 1997 to identify the outcome measures to be used in JIA clinical trials using statistical and consensus-based techniques, but without patient involvement. The importance of patient/parent input into the research process has increasingly been recognized over the years. An Outcome Measures in Rheumatology (OMERACT) JIA Core Set Working Group was formed to determine whether the outcome domains of the current core set are relevant to those involved or whether the core set domains should be revised.Methods. Twenty-four people from the United States, Canada, Australia, and Europe, including patient partners, formed the working group. Guided by the OMERACT Filter 2.0 process, we performed (1) a systematic literature review of outcome domains, (2) a Web-based survey (142 patients, 343 parents), (3) an idea-generation study (120 parents), (4) 4 online discussion boards (24 patients, 20 parents), and (5) a Special Interest Group (SIG) activity at the OMERACT 13 (2016) meeting.Results. A MEDLINE search of outcome domains used in studies of JIA yielded 5956 citations, of which 729 citations underwent full-text review, and identified additional domains to those included in the current JIA Core Set. Qualitative studies on the effect of JIA identified multiple additional domains, including pain and participation. Twenty-one participants in the SIG achieved consensus on the need to revise the entire JIA Core Set.Conclusion. The results of qualitative studies and literature review support the need to expand the JIA Core Set, considering, among other things, additional patient/parent-centered outcomes, clinical data, and imaging data

Longitudinal micro-CT as an outcome measure of interstitial lung disease in TNF-transgenic mice

<div><p>Introduction</p><p>Rheumatoid arthritis associated interstitial lung disease (RA-ILD) is a debilitating condition with poor survival prognosis. High resolution computed tomography (CT) is a common clinical tool to diagnose RA-ILD, and is increasingly being adopted in pre-clinical studies. However, murine models recapitulating RA-ILD are lacking, and CT outcomes for inflammatory lung disease have yet to be formally validated. To address this, we validate μCT outcomes for ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA.</p><p>Methods</p><p>Cross sectional μCT was performed on cohorts of male TNF-Tg mice and their WT littermates at 3, 4, 5.5 and 12 months of age (n = 4–6). Lung μCT outcomes measures were determined by segmentation of the μCT datasets to generate Aerated and Tissue volumes. After each scan, lungs were obtained for histopathology and 3 sections stained with hematoxylin and eosin. Automated histomorphometry was performed to quantify the tissue area (nuclei, cytoplasm, and extracellular matrix) and aerated area (white space) within the tissue sections. Spearman’s correlation coefficients were used to evaluate the extent of association between μCT imaging and histopathology endpoints.</p><p>Results</p><p>TNF-Tg mice had significantly greater tissue volume, total lung volume and mean intensity at all timepoints compared to age matched WT littermates. Histomorphometry also demonstrated a significant increase in tissue area at 3, 4, and 5.5 months of age in TNF-Tg mice. Lung tissue volume was correlated with lung tissue area (ρ = 0.81, p<0.0001), and normalize lung aerated volume was correlated with normalized lung air area (ρ = 0.73, p<0.0001).</p><p>Conclusions</p><p>We have validated in vivo μCT as a quantitative biomarker of ILD in mice. Further, development of longitudinal measures is critical for dissecting pathologic progression of ILD, and μCT is a useful non-invasive method to study lung inflammation in the TNF-Tg mouse model.</p></div

Histomorphometry method for identifying cell nuclei, cytoplasm, extracellular matrix, red blood cells and alveolar space with Visiopharm.

<p>To quantify histomorphometric areas, lungs were stained with Hematoxylin and Eosin, whole slides were scanned, imported into Visiopharm and a ROI was drawn around the margin of the lung (<b>A,</b> ROI = blue dotted line). A custom application was developed to classify the assorted colors within the ROI of the stained slide into 3 categories (Blue, Pink, and White) by training Visiopharm on the Red/Green/Blue pixel intensity ranges. This occurs through manual selection of a training data set of RGB values for each category and application of a Bayesian discrimination algorithm to determine a set of RGB ranges for each category. An example training data set with >15 pixels for each category is presented in <b>B</b> with the intensity of the Red, Blue and Green pixels plotted against each other with each category circled together in 3D space. Note how each category has a distinct range of intensities allowing discrimination of cell nuclei (Blue category) from cytoplasm, ECM and RBCs (Pink category) from alveolar space (White category). Once defined, this classification was applied to every slide in the data set. Representative H and E (<b>A</b> and <b>D</b>) and classified slides (<b>C</b> and <b>E</b>) are shown to demonstrate the specificity of our classification method (bronchiole = *; artery = #). For arteries and arterioles that were not filled with RBCs, manual classification was performed to change the space from White to Pink after inspection of the epithelial layer (#, <b>E</b>).</p

In vivo micro-CT quantification of aerated and tissue lung volume.

<p><b>A)</b> A 2D representative μCT transverse, inferior cross section of a WT lung at cervical vertebra 8 shows the margin of the lung inside of the abdominal cavity. <b>B)</b> This margin is then used with semi-automated tools in Amira to segment the lung (Blue overlay) and a 3D reconstruction can be generated (<b>C</b>). The lung is further segmented into the conducting airway (Grey, <b>C</b>) and into the left (*) and right (#) lobes for more specific analysis (<b>A-C</b>). The raw intensity values from either the whole lung segment or a smaller portion are extracted to generate intensity histograms (<b>D</b>). To delineate aerated lung volume from tissue lung volume a thresholding operation must be developed. The most abundant volume in a healthy lung is the interface between air and the alveolar epithelium. As measured by μCT, the most abundant density in 3–12 month old WT mice is -256 HU (<b>E</b>). Using this threshold, aerated and tissue lung volume can be quantified as shown in a representative 2D overlay (<b>F</b>, Green = Aerated Volume, Orange-Red = Tissue Volume, Blue = Conducting Airway) and 3D reconstructions (<b>G</b>, Aerated Volume; <b>H</b>, Tissue Volume, Blue = Conducting Airway). Note the distinct bronchiole and arteriole structures in <b>G</b> and <b>H</b>.</p

TNF-Tg male mice have greater tissue area compared to WT litter mates measured via histomorphometry.

<p>Blue, Pink and White areas were measured on 3 sections >200 μm apart for 3, 4, 5.5, and 12-month-old male TNF-Tg and WT littermates. Representative H and E images from a 12-month WT (<b>A</b>), and 3 (<b>B</b>), 4 (<b>C</b>), 5 (<b>D</b>) and 12 (<b>E</b>) month TNF-Tg mice show a clear increase in cells in the TNF-Tg mice at all timepoints. Quantifying each histomorphometric category demonstrates a significant increase in Total area at 3 and 5.5 months (<b>F</b>), Blue area at all timepoints (<b>G</b>) and Blue + Pink at 3, 4, and 5.5 months (<b>H</b>) for TNF-Tg mice compared to their WT littermates. Due to the known volumetric changes that occur in the histologic processing solutions (i.e. formalin, ethanols) and the nature of lung tissue being sensitive to changes due to its composition, we normalized all the measurements to the total area (<b>I</b>, <b>J</b>, <b>K</b> and <b>L</b>). As expected, percent Blue area (<b>J</b>) at all timepoints and percent Blue + Pink (<b>L</b>) at 3, 4 and 5.5 months were significantly increased in TNF-Tg mice compared to WT littermates. Interestingly, when normalized to total area, there was a significant decrease in the White area at 3, 4, and 5.5 months in TNF-Tg mice compared to WT littermates (<b>K;</b> *p<0.0125, **p<0.01, ***p<0.001, M±SD, n = 4–6).</p

Power Doppler ultrasound phenotyping of expanding versus collapsed popliteal lymph nodes in murine inflammatory arthritis.

Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic "expanding" phase of the disease, and then suddenly "collapse" during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553 ± 0.007 vs. 0.008 ± 0.003; p0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare

Relationship between Lymph Node Volume and Pain following Certolizumab Therapy for Rheumatoid Arthritis Flare: A Pilot Study

Objectives The mechanisms that trigger flare in rheumatoid arthritis (RA) are unknown. In murine arthritis models, dysfunctional lymph node (LN) drainage is associated with joint flare. To examine if LN alterations are associated with RA flare, we analyzed the change in LN volume via contrast-enhanced magnetic resonance imaging (CE-MRI) in patients with active joint flare at baseline and 16 weeks after certolizumab pegol (CZP) therapy. We also assessed the changes in popliteal or epitrochlear LN volumes versus the Rheumatoid and Arthritis Outcome Score (RAOS) (knee), or the Michigan Hand Questionnaire (MHQ; wrist/hand), and Disease Activity Score 28 (DAS28), at baseline and 16 weeks. Results Total LN volume in 7 of 10 patients with measurable LN on CE-MRI significantly decreased 16 weeks after CZP therapy (mean decrease 37%; P = 0.0019). Improvement in knee pain measured by the RAOS ( P = 0.03) inversely correlated with a decrease in total popliteal LN volume ( R 2 = 0.94). All patients demonstrated significant improvement in DAS28 (mean decrease 1.48; P = 0.0002). For flare in the hand, significant improvement in activities of daily living (ADL) as measured by the MHQ was observed (left hand mean improvement 20%; P = 0.02; right hand mean improvement 37%; P = 0.03). Conclusion RA patients with the smallest change in LN volume during anti-tumor necrosis factor (anti-TNF) therapy experienced the greatest pain relief in symptomatic knee joints. Moreover, the remarkably linear inverse correlation between LN volume and joint pain observed in this small clinical pilot provides initial evidence to support the concept that dynamic changes in draining LN volume are a biomarker of clinical response to therapy in RA