Identification of synovial biomarkers of response to experimental treatment in early‐phase clinical trials in spondylarthritis

Objective

To identify biomarkers for effective treatment in early‐phase clinical trials of spondylarthritis (SpA), by analyzing which synovial features can be reliably identified in patients with SpA.

Methods

Synovial biopsies were performed at weeks 0 and 12 in 20 SpA patients treated with infliximab, 20 treated with etanercept, and 12 who were not treated. Primary clinical outcome measures were patient and physician global assessment of disease activity. Extensive histologic evaluation included assessment of lining layer hyperplasia, vascularity, markers of cellular infiltration, and metalloproteinases (MMPs) in the lining and sublining layers.

Results

Changes in levels of CD163 (resident tissue macrophages) in the lining, and CD163, MMP‐3, and myeloid‐related protein 14 (MRP‐14; infiltrating myeloid cells) in the sublining correlated significantly with changes in the primary clinical outcomes. Comparison between responders (n = 35) and nonresponders (n = 17) showed differences in the degree of change in the levels of CD163 in the lining and CD163, MMP‐3, and CD3 in the sublining, whereas trends in change in the levels of MRP‐8 and MRP‐14 in the lining and sublining were similar in the 2 groups. Accordingly, the highest differences in standardized response means (SRMs) between the 2 groups were found for CD163 in the lining, MMP‐3, CD163, CD3, and MRP‐8 in the sublining, and the level of polymorphonuclear cells (PMNs). When comparing treated and untreated patients, high differences in SRMs were again found for CD163 in the lining, MMP‐3, CD163, and MRP‐8 in the sublining, and PMNs. These parameters performed prognostically as well as the erythrocyte sedimentation rate and better than the C‐reactive protein level. Class prediction analysis yielded a 90% correct prediction using 8 synovial parameters, as follows: lining and sublining CD163, MRP‐8, and MRP‐14, sublining MMP‐3, and PMNs. In validation analyses with independent samples, effective treatment was correctly predicted in 24 of 30 SpA patients and in 2 of 2 placebo‐treated patients.

Conclusion

Changes in synovial macrophage subsets, PMN levels, and MMP‐3 expression reflect response to treatment in SpA. The ability of these parameters to correctly identify effective therapy makes them interesting biomarkers for use in early‐phase clinical trials in SpA.

     

Novel approaches for the treatment of rheumatoid arthritis: lessons from the evaluation of synovial biomarkers in clinical trials

The analysis of synovial biomarkers is increasingly used in the context of innovative trial design in rheumatoid arthritis (RA). This approach, which is generally well tolerated by patients, has been used to provide insight into the pathogenesis of RA and the mechanism of action of therapy, as well as for screening purposes during early drug development.     

Detection of cystic fibrosis transmembrane conductance regulator activity in early-phase clinical trials

Advances in our understanding of cystic fibrosis pathogenesis have led to strategies directed toward treatment of underlying causes of the disease rather than treatments of disease-related symptoms. To expedite evaluation of these emerging therapies, early-phase clinical trials require extension of in vivo cystic fibrosis transmembrane conductance regulator (CFTR)-detecting assays to multicenter trial formats, including nasal potential difference and sweat chloride measurements. Both of these techniques can be used to fulfill diagnostic criteria for the disease, and can discriminate various levels of CFTR function. Full realization of these assays in multicenter clinical trials requires identification of sources of nonbiological intra- and intersite variability, and careful attention to study design and statistical analysis of study-generated data. In this review, we discuss several issues important to the performance of these assays, including efforts to identify and address aspects that can contribute to inconsistent and/or potentially erroneous results. Adjunctive means of detecting CFTR including mRNA expression, immunocytochemical localization, and other methods are also discussed. Recommendations are presented to advance our understanding of these biomarkers and to improve their capacity to predict cystic fibrosis outcomes.     

Measuring treatment response in Alzheimer's disease clinical trials

Alzheimer's disease (AD) is a progressive disorder that negatively impacts cognitive, behavioral, and functional abilities. Because of the complex nature of this disease, it is crucial to design assessment procedures that accurately track disease progression across a wide variety of symptom domains. One important use of such techniques is to assess the effectiveness of therapeutic compounds in AD clinical trials. A number of outcome measures that assess cognitive, behavioral, functional, or global ability have been developed for this purpose. This paper describes the assessment measures that are most commonly used in AD trials. The inherent strengths and limitations of each evaluative technique are summarized, as well as how these outcome measures are useful to the practicing clinician.     

Cartilage biomarkers in ankylosing spondylitis: relationship to clinical variables and treatment response

OBJECTIVE: Ankylosing spondylitis (AS) is a progressive disease in which chronic inflammation can lead to extensive new bone formation throughout the spine. At present, few measures of the activity or extent of the disease are available. In this study, we sought to determine whether markers of cartilage synthesis and degradation could provide such quantitative measures. METHODS: Serum samples from 23 patients receiving infliximab treatment for AS were obtained at baseline and at weeks 2, 6, 14, and 22. Patients were stratified with respect to joint involvement and baseline levels of inflammatory markers, and responders were defined according to the Assessments in Ankylosing Spondylitis 20% criteria. Serial measurements of interferon-gamma, tumor necrosis factor alpha, transforming growth factor beta (TGFbeta), interleukin-10 (IL-10), and IL-1 were done at each time point. The following biomarkers were measured by enzyme-linked immunosorbent assay: the proteoglycan aggrecan 846 epitope, a marker of cartilage turnover; C-propeptide of type II collagen (CPII), a biosynthesis marker; and the Col2-3/4(long mono) (C2C) and Col2-3/4(short) (C1-2C) neoepitopes, reflecting collagen cleavage of type II collagen and type I/type II collagen, respectively. RESULTS: At baseline, patients with AS demonstrated significant elevations in serum levels of CPII, the 846 epitope, and the CPII-to-C2C (CPII:C2C) ratio (but not C2C or C1-2C) compared with normal controls. Of the biomarkers examined, only CPII:C2C showed a correlation with the C-reactive protein (CRP) level. Among the biomarker-cytokine relationships, TGFbeta demonstrated a trend toward a positive correlation with the 846 epitope. CONCLUSION: In AS, elevated serum levels of CPII and the 846 epitope may be related to biosynthetic turnover of hyaline cartilage and the intervertebral discs but may also reflect progressive bone formation as a result of endochondral ossification. The correlation of the CPII:C2C ratio with CRP suggests that the CPII:C2C ratio might prove to be a useful marker of disease activity in AS.     

Biomarkers of treatment response in clinical trials of novel antituberculosis agents

Global initiatives have been launched to develop improved tuberculosis chemotherapy. New drugs and potential treatment-shortening regimens require careful assessment in clinical trials, but existing markers of treatment outcome-clinical cure and relapse-require prolonged follow-up of patients. There is, therefore, a need to find alternative biomarkers or surrogate endpoints predictive of response. Effective treatment requires drugs with sterilising activity to produce clinical cure without relapse, and thus a useful biomarker for a drug under trial must predict the likelihood of relapse. We explore the strengths and weaknesses of existing biomarkers, which assess either host response or mycobacterial load. Change in mycobacterial burden is likely to be the best indicator of treatment outcome, but the optimum study techniques remain undefined. Finally, we propose methods to assess candidate markers, and how these candidate markers could be implemented in future clinical trials.     

Recommendations on use of biomarkers in alcoholism treatment trials

BACKGROUND: Biochemical markers of heavy drinking are playing increasingly prominent roles in alcohol treatment efficacy studies, especially in those designed to evaluate medications. Among these roles are serving as inclusion or exclusion criteria for research participants, corroboration of self-report of drinking status, assessment of the safety of the agent being evaluated, and determination of treatment outcome. METHODS: Recent alcohol medication development trials that included biomarker information were reviewed and critiqued from the perspectives of how biomarker measures were used and how findings on them were reported. RESULTS: Although generally the application of biomarkers as inclusion criteria is not recommended, they may aid in exclusion of potential subjects (e.g., elevated liver function measures in trials of agents that could result in liver damage). Biomarkers are most commonly used as indicators of outcome, usually serving as secondary outcome variables. The relationship of outcome findings on biomarker and self-report measures is positive, but only moderate. As used to date, biomarkers of drinking tend to be less sensitive than well-standardized and properly administered self-report measures. Nevertheless, they do provide a useful, unique source of information on drinking status. CONCLUSIONS: The contribution of biomarkers to alcoholism clinical research would be enhanced if certain design strategies were incorporated into their application and if critical information were included in the research publication. This article offers a series of recommendations to improve on their use in a research context.     

Identification of biomarkers that predict response to treatment of lupus nephritis with mycophenolate mofetil or pulse cyclophosphamide

Objective

There is a need to identify clinical characteristics and/or biomarkers that can predict treatment outcome in lupus nephritis. To this end, we utilized data from the Aspreva Lupus Management Study to identify possible baseline and early predictors of renal response to mycophenolate mofetil (MMF) or intravenous (IV) cyclophosphamide (CYC).

Methods

Patients with class III–V lupus nephritis were randomized to MMF or IV CYC. We assessed predictors of renal response, including baseline demographic, clinical, laboratory, and histologic characteristics, as well as early clinical and laboratory data, obtained within the first 2 months of therapy. Odds ratios (ORs) and 95% confidence intervals for renal response were calculated for each putative predictor.

Results

Normalization of C3, C4, or both by week 8 was strongly predictive of renal response at week 24 (ORs 2.5, 2.6, and 2.9, respectively; P < 0.05). Reduction in proteinuria by ≥25% by week 8 was predictive of renal response at week 24 (OR 3.2, P < 0.05). Reduction in anti–double‐stranded DNA (anti‐dsDNA) by week 8 was not predictive of renal response. Only 3 baseline characteristics (C4 level, time since diagnosis of lupus nephritis, and estimated glomerular filtration rate [GFR]) were predictive of renal response; the remaining characteristics (age, age at lupus nephritis onset, time since diagnosis of systemic lupus erythematosus, sex, histopathologic class, anti‐dsDNA antibody level, C3 level, level of proteinuria, and use of angiotensin‐converting enzyme inhibitors, statins, or hydroxychloroquine) were not.

Conclusion

This study demonstrates that baseline C4 level, time since diagnosis of lupus nephritis, baseline estimated GFR, early normalization of complement, and reduction in proteinuria independently predict renal response to therapy at 6 months.     

Interleukin‐17–positive mast cells contribute to synovial inflammation in spondylarthritis

Objective

Studies comparing spondylarthritis (SpA) to rheumatoid arthritis (RA) synovitis suggest that innate immune cells may play a predominant role in the pathogenesis of SpA. Recent observations have indicated a marked synovial mast cell infiltration in psoriatic SpA. We therefore undertook the present study to investigate the potential contribution of mast cells to synovial inflammation in SpA.

Methods

Synovial tissue and fluid were obtained from patients with either nonpsoriatic or psoriatic SpA (n = 82) and patients with RA (n = 50). Synovial biopsy tissue was analyzed by immunostaining and used in ex vivo cultures. Synovial fluid was analyzed by enzyme‐linked immunosorbent assay.

Results

We observed a strong and specific increase of c‐Kit–positive mast cells in the synovium from patients with SpA compared to the synovium from patients with RA synovitis, which was independent of disease subtype (nonpsoriatic versus psoriatic), disease duration, and treatment. Staining of mast cell granules, analysis of synovial fluid, and results in ex vivo tissue culture did not indicate increased degranulation in SpA synovitis. However, mast cells expressed significantly more interleukin‐17 (IL‐17) in SpA than in RA synovitis, and mast cells constituted the major IL‐17–expressing cell population in the SpA synovium. Ex vivo targeting of synovial mast cells with the c‐Kit inhibitor imatinib mesylate significantly decreased the production of IL‐17 as well as other proinflammatory cytokines in synovial tissue cultures. Analysis of paired pre‐ and posttreatment synovial tissue samples indicated that the mast cell/IL‐17 axis in SpA was not modulated by effective tumor necrosis factor (TNF) blockade.

Conclusion

The specific and TNF‐independent increase in IL‐17–expressing mast cells may contribute to the progression of synovial inflammation in peripheral SpA.

     

Improved early-phase insulin response after candesartan treatment in hypertensive patients with impaired glucose tolerance

As the effect of renin-angiotensin system (RAS) blockade on beta-cells in clinical situations remains unclear, new evidence has been presented that angiotensin-converting enzyme (ACE) inhibitors and angiotensin vertical line vertical line receptor blockers (ARBs) may delay or prevent the development of insulin resistance and diabetes through novel mechanisms. This study aimed to determine the effects of ARBs on insulin excretion by beta-cells. Hypertensive patients with impaired glucose tolerance were randomly divided into two groups: group A (n = 6), which received 8 mg/day of oral candesartan for three months, and controls (n = 6). Before and after administration, a 75 g oral glucose tolerance test was conducted to compare various parameters. No significant differences in age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting glucose, or fasting immunoreactive insulin (IRI) were identified between the groups before administration. After three months, there were no significant changes in BMI, SBP, and DBP for the controls and in BMI and DBP for group A. However, SBP was significantly decreased from 144 +/- 2.6 mmHg to 125 +/- 4.6 mmHg in group A. Insulinogenic index tended to be slightly decreased for controls, but was significantly increased from 0.32 +/- 0.0 to 0.47 +/- 0.1 for group A. No significant changes in HOMA-R were identified in either group. To the best of our knowledge, no previous studies have documented a RAS inhibitor improving early-phase insulin response; thus, the present study may be the first of its kind.     

Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis

OBJECTIVE: To create greater understanding of the changes in synovial tissue parameters that occur in conjunction with clinical response by using an effective therapy, in order to facilitate the planning of future studies with therapeutic agents for rheumatoid arthritis (RA). METHODS: Twenty-one patients with active RA were randomized to receive either oral prednisolone (n = 10) or placebo (n = 11) for 2 weeks. In all patients, synovial tissue biopsy specimens were obtained by arthroscopy directly before treatment and after 14 days of treatment. Immunohistochemical analysis was performed to characterize the cell infiltrate and vascularity. Stained tissue sections were analyzed by digital imaging. Statistical analysis was performed using an analysis of covariance model. RESULTS: After treatment, the mean Disease Activity Score in 28 joints (DAS28) was 2.0 units lower (95% confidence interval [95% CI] 1.0-3.0) in patients who received prednisolone than in those who received placebo. In the prednisolone group, the mean (+/-SD) DAS28 decreased from 6.27 +/- 0.95 to 4.11 +/- 1.43 after therapy; minimal change was observed in the placebo group. For macrophages, the estimated effect of prednisolone was large. Patients receiving active treatment had fewer (mean 628 cells/mm(2) [95% CI 328-927]) macrophages after therapy compared with those receiving placebo. A reduction in the total number of CD68+ macrophages, from 1,038 +/- 283 cells/mm(2) before treatment to 533 +/- 248 cells/mm(2) after treatment, was observed in the prednisolone group. There were clear trends toward decreased infiltration by T cells, plasma cells, and fibroblast-like synoviocytes after active treatment. We observed a trend toward a reduction in alphavbeta3+ newly formed blood vessels and expression of vascular growth factors after prednisolone therapy. CONCLUSION: Prednisolone therapy in RA is associated with a marked reduction in macrophage infiltration in synovial tissue, suggesting that synovial macrophage numbers could be used as a biomarker for clinical efficacy.     

Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials

Inflammation of synovium is one of the hallmarks of rheumatoid arthritis (RA). Analysis of synovial tissue has increased our understanding of RA pathogenesis, aided in identifying potential therapeutic targets and has been used in the response and mechanistic evaluation of antirheumatic treatments. In addition, studies are ongoing, aimed at the identification of diagnostic and prognostic biomarkers in the synovium. This paper outlines the currently used procedures for sampling and processing of synovial tissue, and presents a standardised recommendation to support multicentre translational research.     

Design of clinical trials with biological response modifiers

Biological response modifiers (BRMs) include biological and chemical agents which can increase host resistance against tumor growth and also biological agents which can have direct effects on tumor cells, by inducing cytolysis, growth inhibition, and/or differentiation. It is becoming increasingly clear that initial phase I clinical trials with BRMs need to be designed considerably differently from those for chemotherapeutic agents. In addition to determining the toxicity of each agent and its maximal tolerated dose, it is important to evaluate its effects on relevant immunologic and other host responses and to determine the optimal biological response modifying dose (OBRMD). Also, since most BRMs are likely to be effective mainly for treatment of cancer patients with low tumor burdens, and the biological response modifying effects of an agent may vary with the extent of disease, it seems necessary to first perform a phase IA trial in patients with advanced cancer, with an emphasis on determining toxicity and possibly the maximal tolerated dose. Then a phase IB trial will be performed, with patients with minimal or even undetectable tumor burden, to determine the OBRMD. These phase I trials will then allow planning for phase II trials for evaluation of antitumor effects, at doses and in cancer patients which might be expected to be favorable for detection of efficacy.