There is an increasing demand for accurate endotyping of patients according to their pathogenesis to allow more targeted treatment. We explore a combination of blood-based joint tissue metabolites ...(neoepitopes) to enable patient clustering through distinct disease profiles. We analysed data from two RA studies (LITHE (N = 574, follow-up 24 and 52 weeks), OSKIRA-1 (N = 131, follow-up 24 weeks)). Two osteoarthritis (OA) studies (SMC01 (N = 447), SMC02 (N = 81)) were included as non-RA comparators. Specific tissue-derived neoepitopes measured at baseline, included: C2M (cartilage degradation); CTX-I and PINP (bone turnover); C1M and C3M (interstitial matrix degradation); CRPM (CRP metabolite) and VICM (macrophage activity). Clustering was performed to identify putative endotypes. We identified five clusters (A-E). Clusters A and B were characterized by generally higher levels of biomarkers than other clusters, except VICM which was significantly higher in cluster B than in cluster A (p<0.001). Biomarker levels in Cluster C were all close to the median, whilst Cluster D was characterised by low levels of all biomarkers. Cluster E also had low levels of most biomarkers, but with significantly higher levels of CTX-I compared to cluster D. There was a significant difference in ΔSHP score observed at 52 weeks (p<0.05). We describe putative RA endotypes based on biomarkers reflecting joint tissue metabolism. These endotypes differ in their underlining pathogenesis, and may in the future have utility for patient treatment selection.
Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in ...collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders.
A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls.
The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively.
Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.
Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as ...osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies.
There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a ...misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope – formation or degradation of tissues, and even signaling potential of proteins.
PubMed was searched for collagen, neo-epitope, biomarkers. Results: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident.
We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.
BackgroundAccurate patient stratification is critical if medical professionals are to adopt a precision medicine approach when planning clinical trials or prescribing medication. This approach ...results in a superior level of drug response in the target group, a reduction in adverse effects and reduced costs for payers.Best practice treatment recommendations and disease activity markers such as DAS28, as opposed to a treat to target approach, guide current treatment of arthritic disease. There is currently a lack of tools to enable patient stratification, in part due to traditional biomarkers reflecting systemic inflammation rather than the target tissue.ObjectivesIn this paper, we explore the use of a combination of novel tissue specific biomarkers for patient clustering with the objective of identifying different disease profiles.MethodsFour biomarker substudy cohorts were pooled for this study, including two RA studies; LITHE (n=574) and OSKIRA-1 (n=131) and two OA studies; SMC1 (n=447) and SMC2 (n=81) all of which have been described in detail before. Whilst the principle focus was to examine RA patient profiles, OA studies were included to enrich the cohort with a non-RA population. OSKIRA and LITHE both had measurements at 24 weeks with additional measurements at 52 week s in LITHE.Several serological biomarkers were measured in each cohort, selected due to the specific tissue metabolite they represent. These included: C2M (cartilage degradation); CTX-I and PINP (bone resorption and formation); C1M and C3M (interstitial matrix degradation); CRPM (CRP metabolite) and VICM (macrophage activity).Each biomarker was log transformed and min-max normalised in order to allow for direct comparison of each of the variables. Patient clustering was performed using Ward hierarchical clustering and the number of clusters determined using the GAP statistic. ANOVA test was used to identify differences in delta change in radiographic scores at 24 and 52 weeks in the RA placebo groups (n=271) only.ResultsClustering analysis resulted in five different clusters (A-E). Cluster A and B were both comprised of >98% RA patients. Cluster D was comprised mainly of OA patients whilst clusters C and E were a mix of OA and RA patients.Clusters A and B were characterised by high levels of all biomarkers compared to other clusters except for VICM, which is significantly lower in cluster A than in cluster B (Tukey test p<0.001). Biomarker levels in Cluster C were all close to the median. Cluster D was characterised by low levels of all biomarkers compared to other clusters with significantly lower C2M levels, whilst cluster E also had low levels of markers, yet with significantly higher levels of CTX-1 compared to cluster D.When looking at the RA placebo groups there were no difference in change in SHP score at 24 weeks between the groups, (n=271, LITHE, OSKIRA), but a significant difference in SHP change 52 weeks (n=83, p<0.05, LITHE).Abstract AB0250 – Figure 1ConclusionsWe have identified putative RA profiles based on novel serological biomarker status. Whether patients in particular clusters may benefit from specific targeted treatments, according to their tissue turnover profile, will be investigated further.Disclosure of InterestNone declared
Rheumatoid arthritis (RA) is a chronic inflammatory disease with fluctuating course of progression. Despite substantial improvement in treatments in recent years, treatment response is still not ...guaranteed. The aim of this study was to identify variation in Disease Activity Score 28 (DAS28) of RA patients in response to Tocilizumab, and to investigate both molecular and clinical factors influencing response. Clinical and biochemical data for 485 RA patients receiving Tocilizumab in combination with methotrexate were extracted from the LITHE phase III clinical study (NCT00106535), and post-hoc analysis conducted. Latent class mixed models were used to identify statistically distinct trajectories of DAS28 after the initiation of treatment. Biomarker measurements were then analysed cross-sectionally and temporally, to characterise patients by serological biomarkers and clinical factors. We identified three distinct trajectories of drug response: class 1 (n = 85, 17.5%), class 2 (n = 338, 69.7%) and class 3 (n = 62, 12.8%). All groups started with high DAS28 on average (DAS28 > 5.1). Class 1 showed the least reduction in DAS28, with significantly more patients seeking escape therapy (p < 0.001). Class 3 showed significantly higher rates of improvement in DAS28, with 58.1% achieving ACR response levels compared to 2.4% in class 1 (p < 0.0001). Biomarkers of inflammation, MMP-3, CRP, C1M, showed greater reduction in class 3 compared to the other classes. Identification of more homogenous patient sub-populations of drug response may allow for more targeted therapeutic treatment regimens and a better understanding of disease aetiology.
During cancer the otherwise tightly controlled homeostasis of the extracellular matrix (ECM) is disturbed. The protein composition changes, the ECM stiffens and increased levels of proteases are ...secreted. The combination of these processes result in release of specific protein fragments (e.g. collagens) to the circulation, which when measured may reflect disease pathogenesis.
To investigate if biomarkers of protease-degraded collagen could differentiate ovarian and breast cancer patients from healthy controls when measured in serum.
The levels of markers reflecting MMP-degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12) collagen were assessed in serum from ovarian cancer patients (n= 10), breast cancer patients (n= 14) and healthy controls (n= 49) using validated ELISAs. The markers were compared using one way ANOVA and AUC was calculated.
All markers were significantly elevated in serum from ovarian cancer patients (p< 0.0001) and breast cancer patients (p< 0.04-0.0001) compared to healthy controls. Furthermore, diagnostically the markers were able to differentiate ovarian (AUROC 90%-93%) and breast cancer patients (AUROC 76%-93%) from healthy controls, with C1M being the strongest differentiator of disease vs.
Four serum biomarkers reflecting altered MMP-mediated collagen turnover were able to differentiate ovarian and breast cancer patients from healthy controls.
Disease modifying treatments for dementia are only just surfacing, and their development is still significantly hindered by the lack of validated tools for identification of subjects with subclinical ...disease. Much interest has been taken in developing accessible non-invasive serum biomarkers of neurodegeneration. Recent studies have identified caspase-3-cleaved tau (Tau-C), and ADAM-10 cleaved tau (Tau-A) as possible markers of preclinical neurodegenerative disease.
To explore if serum levels of Tau-A and Tau-C change as a consequence of neurodegeneration.
Cohort study with measurement of biomarkers and genome sequencing at baseline with follow-up after an average of 14 years.
Postmenopausal Danish women from the Prospective Epidemiological Risk Factor (PERF) cohort (n=4968) Methods: Genotyping data was used to perform a Mendelian randomization analysis of serum levels of Tau-A and Tau-C in relation to a diagnosis of dementia at follow-up. A dementia diagnosis was defined as a composite of an all-cause dementia diagnosis derived from the Danish National Health registries, a self-reported diagnosis of dementia and/or cognitive test scores suggestive of dementia. Serum levels of Tau-A and Tau-C were measured blinded in samples from baseline in a CAP certified lab. The association with dementia was assessed using bi-directional one- and two- sample Mendelian randomization.
A lead single nucleotide polymorphism (SNP) was identified for Tau-A (rs10414043) and Tau-C (rs429358), respectively were identified. Both were located in the APOE/C1 cluster on chromosome 19. APOE and EPOC1 variants were associated with lower levels of Tau-A and Tau-C levels - effect size -0.13, 95%CI -0.17 - -0.09 log2 (ng/mL), p=7.05e-11 for rs10414043 association with Tau-A and effect size -0.12, 95%CI -0.15 - -0.08 log2 (ng/mL), p=2e-11 for rs429358 association with Tau-C. When incorporating genetic data from a larger genetic study we found that Alzheimer's disease was marginally associated with a decreased Tau-A and Tau-C levels (Odds Ratio 0.97, 95%CI 0.93 - 1.00. No association was found in the forward Mendelian randomization analysis.
By combining genotype data with serum measurements of the novel biomarkers Tau-A and Tau-C, we conclude that Tau-A and Tau-C levels change because of neurodegeneration. We also conclude that lower serum-values of the biomarkers are associated with the presence of genetic variants commonly found in individuals suffering from late-onset Alzheimer's Dementia. These findings add to the growing data pointing towards Tau-A and Tau-C as valuable biomarkers for neurodegeneration.
BackgroundOsteoarthritis (OA) is a heterogeneous disease described by a combination of joint pain, physical disability and radiographic alterations leading to joint failure and total joint ...replacement (TJR). Commonly used endpoints in OA trials are worsening of pain and joint space narrowing. TJR is normally not considered an endpoint. Age and female gender are considered as major risk factors for developing OA.ObjectivesWe hypothesise that TJR can be used as an endpoint in OA outcome studies within reasonable time frame. To investigate the basis for this hypothesis, we explored the prevalence and incidence of TJR as a reflection of joint failure in the Prospective Epidemiologic Risk Factor (PERF I) study.MethodsA total of 5,855 Danish postmenopausal women aged 49–88 enrolled in the Prospective Epidemiologic Risk Factor (PERF I) study during 1999–2001 (baseline). Three, six and twelve year follow-up data from the Danish National registry was collected in end of 2014, including occurrence of TJR, OA and other relevant diagnosis. Also, women where at baseline and in 2014 asked whether they had a TJR or OA. The biomarker C1M was measured in baseline serum samples. The PERF I study was carried out in accordance with ICH-GCP and the study protocol was approved by the local ethics committees.ResultsThere were 798 women that had their first TJR between baseline and 12 year follow-up; giving an incidence proportion of 13.6%. The TJR women were on average 1 year older (p=0.010) and heavier (1.7 kg/cm2, p<0.0001), compared to women with no TJR in the follow-up period. The incidence after three and six years, of first ever TJRs, were 171 and 362 corresponding to an incidence proportions of 2.9% and 6.2%. Next we investigated the TJR incidence rates at 3, 6 and 12 years in different subgroup of women: 1) All, PERF I women that experience their first ever TJR (5855); 2) Prior TJR, had a TJR before baseline (266); 3) OA diagnose at baseline but no prior ;TJR (1757) and 4) OA diagnose at baseline and high C1M (>40 ng/mL, median) that after baseline underwent there first ever TJR (841). Group 1 is as described above. The 3, 6 and 12 incidence rates were 9.0, 17.3% and 29.7% for the prior TJR group, 6.2, 12.1% and 23.3% for the OA group, and 7.6, 13.6% and 25.1% for the OA +C1M group. The age-dependent prevalence and incidence for the first TJR. The prevalence was insignificant for the age group younger than 60 years old (<0.1%). The prevalence increased steadily from the age group 60 to age group 85; from 0.1% to 13.2%.ConclusionsWithin a timeframe of 3, 6 or 12 years TJR incidence for women with an OA diagnosis reached 6, 12% and 23%, which was a doubling compared to the All population. The incidence increased by adding a single diagnostic measure. This reflects that TJRs are frequent amongst elderly women and that if designed minutiously, such as including specific diagnostic criteria (e.g. biomarker, OA diagnose), it may be feasible to conduct clinical studies with TJR as an endpoint. However, special attention much be directed to the objectiveness of the criteria for TJR. This may build a case for design of outcome studies (joint failure) for developing drugs in OA.Disclosure of InterestA. Bay-Jensen Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience, C. Bager Employee of: Proscion, A. Bihlet Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, C. Thudium Employee of: Nordic Bioscience, I. Byrjalsen Employee of: Nordic Bioscience, H. Nielsen Employee of: Nordic Bioscience, J. Andersen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, B. Riis Shareholder of: Nordic Bioscience, C. Christiansen Shareholder of: Nordic Bioscience, M. Karsdal Shareholder of: Nordic Bioscience, Grant/research support from: ApproacH (IMI support), Employee of: Nordic Bioscience
Lysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 ...neo-epitope generated through the release of the signal peptide during LOXL2 maturation.
An ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16).
A technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001-0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49-84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001).
A specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.
PDF
