Discovery of Innovative Therapies for Rare Immune-Mediated Inflammatory Diseases via Off-Label Prescription of Biologics: The Case of IL-6 Receptor Blockade in Castleman’s Disease

Biologics have revolutionized the field of clinical immunology and proven to be both effective and safe in common immune-mediated inflammatory diseases (IMIDs) such as rheumatoid arthritis, inflammatory bowel diseases, and various hematological disorders. However, in patients with rare, severe IMIDs failing on standard therapies, it is virtually impossible to conduct randomized controlled trials. Therefore, biologics are usually prescribed off-label in these often severely ill patients. Unfortunately, off-label prescription is sometimes hampered in these diseases due to a lack of reimbursement that is often based on a presumed lack of evidence for effectiveness. In the present article, we will discuss that off-label prescription of biologics can be a good way to discover new treatments for rare diseases. This will be illustrated using a case of multicentric Castleman's disease, an immune-mediated lymphoproliferative disorder, in which off-label tocilizumab (humanized anti-IL-6 receptor blocking antibody) treatment resulted in remarkable clinical improvement. Furthermore, we will give recommendations for monitoring efficacy and safety of biologic treatment in rare IMIDs, including the use of registries. In conclusion, we put forward that innovative treatments for rare IMIDs can be discovered via off-label prescription of biologicals, provided that this is based on rational arguments including knowledge of the pathophysiology of the diseas

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3(+ )T cells, CD38(+ )plasma cells and CD68(+ )macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials

Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy - a single centre, open-label study

With the development of increasing numbers of potential therapeutic agents in inflammatory disease comes the need for effective biomarkers to help screen for drug efficacy and optimal dosing regimens early in the clinical trial process. This need has been recognized by the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group, which has established guidelines for biomarker validation. To seek a candidate synovial biomarker of treatment response in psoriatic arthritis (PsA), we determined whether changes in immunohistochemical markers of synovial inflammation correlate with changes in disease activity scores assessing 28 joints (ΔDAS28) or magnetic resonance imaging synovitis scores (ΔMRI) in patients with PsA treated with a biologic agent. Twenty-five consecutive patients with PsA underwent arthroscopic synovial biopsies and MRI scans of an inflamed knee joint at baseline and 12 weeks after starting treatment with either anakinra (first 10 patients) or etanercept (subsequent 15 patients) in two sequential studies of identical design. DAS28 scores were measured at both time points. Immunohistochemical staining for CD3, CD68 and Factor VIII (FVIII) was performed on synovial samples and scored by digital image analysis (DIA). MRI scans performed at baseline and at 12 weeks were scored for synovitis semi-quantitatively. The ΔDAS28 of the European League Against Rheumatism good response definition (>1.2) was chosen to divide patients into responder and non-responder groups. Differences between groups (Mann Whitney U test) and correlations between ΔDAS28 with change in immunohistochemical and MRI synovitis scores (Spearman's rho test) were calculated. Paired synovial samples and MRI scans were available for 21 patients (8 anakinra, 13 etanercept) and 23 patients (8 anakinra, 15 etanercept) respectively. Change in CD3 (ΔCD3) and CD68 expression in the synovial sublining layer (ΔCD68sl) was significantly greater in the disease responders compared to non-responders following treatment (P = 0.005 and 0.013 respectively). ΔCD3, but not ΔCD68 or ΔFVIII, correlated with both ΔDAS28 (r = 0.49, P = 0.025) and ΔMRI (r = 0.58, P = 0.009). The correlation of ΔCD3 with ΔDAS28 and ΔMRI following biologic treatment in this cohort contributes to the validation of ΔCD3 as a synovial biomarker of disease response in PsA, and supports the further evaluation of ΔCD3 for predictive properties of future clinical outcome

Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets

Objectives To investigate the expression and activation of mitogen-activated protein kinases in patients with early arthritis who are disease-modifying antirheumatic drug (DMARD) naive. Methods A total of 50 patients with early arthritis who were DMARD naive (disease duration <1 year) were prospectively followed and diagnosed at baseline and after 2 years for undifferentiated arthritis (UA), rheumatoid arthritis (RA) (1987 American College of Rheumatology (ACR) and 2010 ACR/European League Against Rheumatism (EULAR) criteria), or spondyloarthritis (SpA). Synovial biopsies obtained at baseline were examined for expression and phosphorylation of p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunohistochemistry and digital analysis. Synovial tissue mRNA expression was measured by quantitative PCR (qPCR). Results ERK and JNK activation was enhanced at inclusion in patients meeting RA criteria compared to other diagnoses. JNK activation was enhanced in patients diagnosed as having UA at baseline who eventually fulfilled 1987 ACR RA criteria compared to those who remained UA, and in patients with RA fulfilling 2010 ACR/EULAR criteria at baseline. ERK and JNK activation was enhanced in patients with RA developing progressive joint destruction. JNK activation in UA predicted 1987 ACR RA classification criteria fulfilment (R-2=0.59, p=0.02) after follow-up, and disease progression in early arthritis (R-2=0.16, p <0.05). Enhanced JNK activation in patients with persistent disease was associated with altered synovial expression of extracellular matrix components and CD44. Conclusions JNK activation is elevated in RA before 1987 ACR RA classification criteria are met and predicts development of erosive disease in early arthritis, suggesting JNK may represent an attractive target in treating RA early in the disease proces

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. METHOD: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). RESULTS: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. CONCLUSION: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance

The Features of the Synovium in Early Rheumatoid Arthritis According to the 2010 ACR/EULAR Classification Criteria

OBJECTIVES: It has been shown in early arthritis cohorts that the 2010 ACR/EULAR criteria for rheumatoid arthritis (RA) enable an earlier diagnosis, perhaps at the cost of a somewhat more heterogeneous patient population. We describe the features of synovial inflammation in RA patients classified according to these new criteria. METHODS: At baseline, synovial tissue biopsy samples were obtained from disease-modifying antirheumatic drug (DMARD)-naïve early RA patients (clinical signs and symptoms <1 year). Synovial tissue was analyzed for cell infiltration, vascularity, and expression of adhesion molecules. Stained sections were evaluated by digital image analysis. Patients were classified according to the two different sets of classification criteria, autoantibody status, and outcome. FINDINGS: Synovial tissue of 69 RA patients according to 2010 ACR/EULAR criteria was analyzed: 56 patients who fulfilled the criteria for RA at baseline and 13 who were initially diagnosed as undifferentiated arthritis but fulfilled criteria for RA upon follow up. The synovium at baseline was infiltrated by plasma cells, macrophages, and T cells as well as other cells, and findings were comparable to those when patients were selected based on the 1987 ACR criteria for RA. There was no clear cut difference in the characteristics of the synovium between RA patients initially diagnosed as undifferentiated arthritis and those who already fulfilled classification criteria at baseline. CONCLUSION: The features of synovial inflammation are similar when the 2010 ACR/EULAR classification criteria are used compared to the 1987 ACR criteria

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

Background: Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man. Methods/Principal Findings: We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (99m-Tc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4x10(-3) (0.95-5.1x10(-3)) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion. Conclusions/Significance: The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention

EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis

The Study Group for Risk Factors for Rheumatoid Arthritis was established by the EULAR Standing Committee on Investigative Rheumatology to facilitate research into the preclinical and earliest clinically apparent phases of rheumatoid arthritis (RA). This report describes the recommendation for terminology to be used to define specific subgroups during different phases of disease, and defines the priorities for research in this area. Terminology was discussed by way of a three-stage structured process: A provisional list of descriptors for each of the possible phases preceding the diagnosis of RA were circulated to members of the study group for review and feedback. Anonymised comments from the members on this list were fed back to participants before a 2-day meeting. 18 participants met to discuss these data, agree terminologies and prioritise important research questions. The study group recommended that, in prospective studies, individuals without RA are described as having: genetic risk factors for RA; environmental risk factors for RA; systemic autoimmunity associated with RA; symptoms without clinical arthritis; unclassified arthritis; which may be used in a combinatorial manner. It was recommended that the prefix ‘pre-RA with:’ could be used before any/any combination of the five points above but only to describe retrospectively a phase that an individual had progressed through once it was known that they have developed RA. An approach to dating disease onset was recommended. In addition, important areas for research were proposed, including research of other tissues in which an adaptive immune response may be initiated, and the identification of additional risk factors and biomarkers for the development of RA, its progression and the development of extra-articular features. These recommendations provide guidance on approaches to describe phases before the development of RA that will facilitate communication between researchers and comparisons between studies. A number of research questions have been defined, requiring new cohorts to be established and new techniques to be developed to image and collect material from different sites

Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis.

BACKGROUND: Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development. METHODS: Twenty-four patients with RA, 23 individuals positive for autoantibodies but without clinical disease (RA risk group) and 14 seronegative healthy control subjects underwent ultrasound-guided inguinal lymph node (LN) biopsy. Human LNSCs were isolated and expanded in vitro for functional analyses. In analogous co-cultures consisting of LNSCs and peripheral blood mononuclear cells, αCD3/αCD28-induced T-cell proliferation was measured using carboxyfluorescein diacetate succinimidyl ester dilution. RESULTS: Fibroblast-like cells expanded from the LN biopsy comprised of fibroblastic reticular cells (gp38+CD31-) and double-negative (gp38-CD31-) cells. Cultured LNSCs stably expressed characteristic adhesion molecules and cytokines. Basal expression of C-X-C motif chemokine ligand 12 (CXCL12) was lower in LNSCs from RA risk individuals than in those from healthy control subjects. Key LN chemokines C-C motif chemokine ligand (CCL19), CCL21 and CXCL13 were induced in LNSCs upon stimulation with tumour necrosis factor-α and lymphotoxin α1β2, but to a lesser extent in LNSCs from patients with RA. The effect of human LNSCs on T-cell proliferation was ratio-dependent and altered in RA LNSCs. CONCLUSIONS: Overall, we developed an experimental model to facilitate research on the role of LNSCs during the earliest phases of RA. Using this innovative model, we show, for the first time to our knowledge, that the LN stromal environment is changed during the earliest phases of RA, probably contributing to deregulated immune responses early in disease pathogenesis

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted