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BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer.METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F).RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.

Background Plasma YKL-40 is an inflammatory biomarker. No useful biomarker exists in patients with psoriasis or psoriatic arthritis. Objective To measure YKL-40 and high-sensitivity C-reactive protein (hs-CRP) in patients with psoriasis or psoriatic arthritis before and during treatment. Methods In 48 patients with psoriasis, we measured YKL-40, hs-CRP and Psoriasis Area and Severity Index (PASI) at inclusion and in a subgroup of 14 patients, we repeated the measurements after four to six weeks of methotrexate treatment. In 42 patients with psoriatic arthritis, we measured YKL-40 and hs-CRP at inclusion and during 48 weeks of adalimumab treatment. The patients with psoriatic arthritis were divided into responders and non-responders. Results In patients with psoriasis, the baseline median PASI score was 10.8 and baseline YKL-40 was 45 μg/L. Seventeen per cent had elevated plasma YKL-40 compared with healthy subjects. Baseline PASI and YKL-40 were not correlated (rho = 0.14, P = 0.347) and YKL-40 and hs-CRP remained unchanged after treatment. In patients with psoriatic arthritis, the median pretreatment YKL-40 was 112 μg/L and 43% had elevated YKL-40. YKL-40 decreased in 33 patients who responded to adalimumab (from 112 μg/L to 68 at 48 weeks, P = 0.007). Hs-CRP decreased (from 4.65 mg/L to 0.91, P = 0.013) in the responders. In the non-responders (n = 9), YKL-40 and hs-CRP remained unchanged. Conclusions YKL-40 is elevated in many patients with psoriatic arthritis, but not in patients with psoriasis. YKL-40 decreased in patients with psoriatic arthritis who responded to treatment. YKL-40 may be a useful biomarker to monitor the effect of treatment with tumour necrosis factor-α inhibitors in patients with psoriatic arthritis.

At least 30% of patients with rheumatoid arthritis (RA) do not respond to biologic agents, which emphasizes the need of predictive biomarkers. We aimed to identify microRNAs (miRNAs) predictive of response to adalimumab in 180 treatment-naïve RA patients enrolled in the OPtimized treatment algorithm for patients with early RA (OPERA) Study, an investigator-initiated, prospective, double-blind placebo-controlled study. Patients were randomized to adalimumab 40 mg (n=89) or placebo-adalimumab (n=91) subcutaneously in combination with methotrexate. Expressions of 377 miRNAs were determined using TaqMan Human MicroRNA LDA, A Card v2.0 (Applied Biosystems). Associations between miRNAs and treatment response were tested using interaction analyses. MiRNAs with a P-value

Background The medical treatment of inflammatory rheumatic diseases has improved during the last decades. However, many patients experience treatment failure due to lack of effectiveness or serious adverse events. Biomarkers are promising future tools to predict treatment outcome on the patient level (personalized treatment), but these biomarkers still remain to be identified. The newly started nationwide Danish Rheumatologic Biobank (DRB) provides the infrastructure needed to promote research within this field. The protocol “Identification of biomarkers that improve diagnostics, predict treatment responses, adverse events or prognosis among patients with inflammatory rheumatic disease followed in the Danish nationwide DANBIO registry” (approvals: H-2–2014–086 and GLO-2015–6) is the first national study within DRB. DRB is built on the infrastructure of the Danish CancerBiobank [1] and is funded by The Danish Rheumatism Association and the hospital owners (Danish Regions). Objectives The aim of DRB is to promote the identification of new biomarkers for personalised treatment of patients with inflammatory rheumatic diseases. We here present the first protocol in DRB. Methods DRB is established through the collaboration between Depts. of Rheumatology and Depts. Clinical Biochemistry at hospitals in all regions of Denmark. Currently, 9 hospitals participate. Patients are included after having signed an informed consent. Blood samples are collected 1) cross-sectionally (i.e. patients provide one blood sample); 2) longitudinally (i.e. patients are enrolled when they start a new disease-modifying anti-rheumatic drug (DMARD, biologic or conventional synthetic) and provide blood samples at 0, 3, 6, 12, 24, 36, 48 and 60 months and at time of treatment withdrawal. Synovial fluid, tissue and urine may also be collected. Samples are collected, handled and stored (at minus 800C) according to nationally approved Standard Operating Procedures (SOPs) and all data regarding the samples are registered in the nationwide database, the Biobank of Danish Regions. Sample-associated high-quality clinical data are registered prospectively in the nationwide quality and research registry, DANBIO, e.g. patient demographics and patient-reported outcomes, previous and current treatment (DMARDs) including reasons for withdrawal, and physician-reported outcomes (clinical characteristics, treatment response, imaging) [2]. Results From May 2015 to January 2016, 840 patients with rheumatoid arthritis (67%), axial spondyloarthritis (22%), psoriatic arthritis (9%) and other diagnoses (2%) had been included in the study, and had contributed 1,239 blood sample packages stored at minus 800C, each including whole blood (1x1.5 ml), buffy coat (1x1.5 ml), EDTA plasma (2x2 ml), serum (4x2 ml) and whole blood in PAXgene RNA tubes (2x2.5 ml). Conclusions The establishing of DRB has been successful with >1,200 samples collected. Sampling from patients included in the first biomarker protocol continues and the first scientific results are expected in 2016. DRB is unique due to the nationwide and parallel collection of clinical data and biological material from patients with inflammatory rheumatic diseases. References www.cancerbiobank.dk Rheumatology (Oxford). 2011 Jan;50(1):69–77 Disclosure of Interest None declared

Background According to national guidelines issued in 2015, a non-medical switch from originator infliximab (IFX) to biosimilar IFX was conducted in all Danish patients with inflammatory rheumatic diseases treated in routine care. Objectives To investigate effects of the switch on serum (s) IFX and anti-drug antibodies (ADA) in patients with rheumatoid arthritis (RA), axial spondyloarthrits (SpA) and psoriatic arthritis (PsA). Methods We included originator infliximab treated patients, who switched to and were treated with biosimiilar infliximab for >2 months with 2 available trough level serum samples (baseline immediately before switching/follow-up 2–4 months after switching time of 2nd–4th infusion). Patients stopping treatment earlier were not included. Trough sIFX 30AU/l median-high. sIFX and ADA were analyzed using automated in-house assays at OUS-Radiumhospitalet. Characteristics and outcomes were registered in the DANBIO registry. Remission was defined by disease activity indices (DAS28 Results 96 pts (192 samples) from 4 departments (49 RA, 27 SpA, 10 PsA, 10 other) were included (age 52 (43–62) years, 47% women). Previous IFX treatment duration was 7.5 (5.1–10.3) years. Follow-up was after 81 (71–90) days. 58 patients (60%) received concomitant methotrexate (15 (10–20) mg/wk). Baseline IFX dose was 3.1 (3.0–4.8) mg/kg every 7 (6–9) weeks. At baseline, 60% of pts had low sIFX and 29% very low and at follow-up it was 57% and 29%, respectively. At baseline, 14 pts (15%) had medium-high ADA, at follow up 16%. Median sIFX was lower at baseline vs. follow-up (1.8 (0.8–5.8) vs 2.4 (0.8–6.2) mg/l, p=0.006) whereas ADA were similar (p=0.7). Six of 58 pts with low baseline sIFX had high sIFX at follow-up, and 3 of 38 pts with high baseline sIFX had low sIFX at follow-up. Similar numbers for low vs. medium–high ADA at baseline and follow-up were 2/81 and 3/15. For the rest, sIFX and ADA remained stable between baseline and follow-up in 87/96 (91%) and 91/96 (95%), respectively (Figure). MTX use was not associated with sIFX or ADA (both p>0.05). Patients with low sIFX received lower IFX doses than pts with sIFX ≥3mg/l (255 (200–320) mg vs. 300 (250–400) mg, p=0.02) and with longer intervals (8 (6–10) weeks vs. 6 (6–6)weeks, p 0.05). Conclusions In this highly selected group of patients treated with originator infliximab for >5 years, non-medical switch to biosimilar IFX had no negative impact on sIFX or ADA 2–4 months following switch. At baseline, 60% of patients had low sIFX but few patients had ADA, perhaps indicating low immunogenicity in these patients. Disclosure of Interest None declared

BACKGROUND: Elderly patients with primary colorectal cancer (CRC) are less frequently treated with adjuvant chemotherapy than younger patients due to concerns regarding toxicity and efficiency. We investigated how age, performance status (PS) and comorbidity influence treatment outcomes.PATIENTS AND METHODS: A retrospective single-centre study of 529 patients with stages II-III CRC treated with adjuvant chemotherapy (5-fluorouracil/capecitabine+/÷oxaliplatin) from 2001 to 2011 at Herlev Hospital, Denmark. Baseline characteristics, chemotherapy and outcome were analysed with respect to age after adjusting for PS and comorbidity.RESULTS: Elderly patients (>70 years) had significantly more comorbidity (pCONCLUSIONS: Choice of regimen, primary dose reduction and given dose intensity in patients treated with adjuvant chemotherapy for CRC were highly dependent on age. However, age had no impact on DFS and CRC mortality. Comorbidity in younger patients and PS in all patients were associated with shorter DFS and higher CRC mortality.

Objective Osteoarthritis (OA) is a complex heterogeneous joint disease affecting more than 35 million people worldwide. The current gold standard diagnostic investigation is the plain radiograph, which lacks sensitivity. Biochemical markers have the potential to act as adjunct markers for imaging in the assessment of knee OA. We undertook this study to determine the association between individual biochemical markers and radiographic features, and to establish whether the association is strengthened when selected biochemical markers are combined into a single factor (a theoretical marker). Methods Twenty serum and urinary biochemical markers were analyzed in 119 patients with predominantly tibiofemoral knee OA. Pearson's correlation was performed, and corresponding coefficients of determination (R2) were calculated to determine the association between biochemical markers and a range of imaging features from radiographs and dual x-ray absorptiometry of the knee. Biochemical markers demonstrating a significant association (P < 0.05) with a specific imaging feature were combined by principal components analysis (PCA). Pearson's correlation was repeated to establish whether the combined panel of biochemical markers showed a stronger association with imaging than the best single marker. Results Fourteen biochemical markers showed significant associations with one or more imaging features. By combining specific panels of biochemical markers to form factors, the association of markers with imaging features (R2) increased from 11.9% to 22.7% for the Kellgren/Lawrence (K/L) score, from 5.9% to 9.2% for joint space width (JSW), from 6.6% to 10.8% for sclerosis, from 13.5% to 22.6% for osteophytes, and from 12.0% to 14.2% for bone mineral density (BMD). Biochemical markers identifying patients with osteophytes overlapped with those correlated with a high K/L score, while markers of subchondral BMD formed a completely separate group. Biochemical markers of JSW included markers associated with both osteophytes and BMD. Conclusion The PCA results suggest that biochemical marker combinations may be more sensitive than individual biochemical markers for reflecting structural damage in patients with knee OA. The differences in biochemical marker profiles associated with osteophytes compared with those associated with subchondral BMD raise the possibility that these 2 processes, commonly seen in bone in knee OA, have underlying biologic differences.

Objective To examine if the determination of the levels of serological tumor markers at time of relapse had any predictive value for chemoresistance in the second-line treatment of ovarian cancer patients. Methods From a registry of consecutive single-institution patients with epithelial ovarian carcinoma pretreated with paclitaxel plus platinum, we selected 82 patients with (a) solid tumor recurrence, and (b) second-line chemotherapy consisting of topotecan (platinum-resistant disease) or paclitaxel plus carboplatin (platinum-sensitive disease). Stored serum samples were analyzed for the biochemical tumor markers tetranectin, YKL-40, CASA (cancer-associated serum antigen), and CA 125. The serum tumor marker levels at time of relapse were correlated with response status at landmark time after 4 cycles of second-line chemotherapy. Univariate and multivariate logistic regression analyses (chemoresistant vs non-chemoresistant disease) were performed. Results At landmark time, 26% of patients had progression according to the GCIG (Gynecologic Cancer Intergroup) progression criteria. In univariate logistic regression analysis, the tumor markers tetranectin (OR 0.4; 95% CI: 0.2–0.8; p=0.008), YKL-40 (OR 1.8; 95% CI: 1.0–3.3; p=0.045), and CASA (OR 1.8; 95% CI: 1.2–2.7; p=0.007) had predictive value for second-line chemoresistance, whereas serum CA 125 had no predictive value. In a multivariate logistic regression analysis, serum tetranectin and CASA both had independent predictive value for chemoresistance. The combined determination of tetranectin and CASA had a specificity of 90% with 33% sensitivity for the prediction of chemoresistance (area under the receiver operating characteristic curve = 0.78; 95% CI: 0.66–0.91; p=0.001). Conclusion Low serum levels of tetranectin, or high serum levels of CASA or YKL-40, are associated with increased risk of second-line chemoresistance in patients with ovarian cancer.

Background Single nucleotide polymorphisms (SNPs) in the C-reactive protein ( CRP) gene are implicated in the regulation of the constitutional CRP expression and its response to pro-inflammatory stimuli. Previous reports suggest that these effects may influence instruments for clinical decision-making based on CRP, e.g. DAS28. Objectives To investigate the associations between 7 CRP SNPs, their haplotypes, the serum level of CRP and DAS28 in recent onset, treatment naive RA (rheumatoid arthritis) patients. Methods 315 DMARD and steroid naive RA patients with disease duration Results The minor allele of rs1205 C>T was associated with decreased CRP levels at baseline (p=0.03). The TT genotype showed a 50% reduction in CRP from 16.7 to 8.4 mg/l (p=0.005) compared to the CC genotype. This association was not observed after one year of active treatment (p=0.38) (Table 1). The common H2 haplotype, characterized by the T allele of rs1205, was associated with a 26% reduction in CRP at baseline (p=0.04), but no effect was observed at year 1 (p=0.47) (Table 2). No other SNP or haplotype were associated with CRP at baseline or year 1 (p≥0.09). We observed no associations between SNPs or haplotypes and DAS28 scores at baseline or year 1 (p≥0.10) Conclusions This study shows that DAS28 can be used for clinical decision-making without adjustment for CRP gene variants, as CRP genotypes and haplotypes were only marginally associated with CRP levels and without any association to the DAS28 score. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1357

Background Recently we reported, that DAS28CRP rd month triple therapy with MTX, sulphasalazine (SZS) and hydroxychloroquine (HCQ) 1 . Addition of adalimumab (ADA) at baseline yielded even better clinical responses and remission rates after treatment for one year. Objectives The purpose of the present investigation was to study whether the improved clinical response obtained by adding ADA to MTX + i.a. TRIAM injections can be maintained after withdrawal of ADA. Methods Patients with ERA were randomized to receive i.a. TRIAM (40 mg/ml) in any swollen joint in combination with MTX (20 mg/wk) for two years and placebo-ADA (MTX+PLA) or MTX+ADA (40 mg eow) during the first year. Peroral glucocorticoid was not allowed. After 1 yr, ADA/PLA was withdrawn. If patients had swollen joints and DAS28(CRP)>3.2 during yr 2, SZS and HCQ were added, and if active disease persisted, ADA (40 mg eow) replaced SZS and HCQ in both treatment arms. Clinical response was assessed by DAS28(CRP), CDAI, SDAI and ACR/EULAR remission criteria. Analysis was by ITT with last observation carried forward. Medians (5%/95% percentiles) or percentage. Mann-Whitney or Pearson’s chi-square tests. Results Baseline characteristics were similar in the MTX+PLA and MTX+ADA groups (DAS28(CRP) 5.6 (3.8-7.3) vs. 5.5 (3.8-7.8), NS). Table shows 1 and 2 years results. Serious adverse events were seen in 10 MTX+PLA patients (malignancy: 2, infections: 5, other: 2) and in 16 MTX+ADA patients (malignancy: 3, infections: 5, other: 8). Conclusions Combined MTX and i.a. TRIAM provided excellent disease control at 2 years’ follow-up of ERA patients. The improved clinical responses observed during additional ADA administration could not be maintained after withdrawal of ADA. Radiographic data are needed to evaluate the overall effect of 1 yr of ADA as induction therapy in combination with MTX and i.a. TRIAM in ERA References Horslev-Petersen K et al. Ann Rheum Dis 2012: Disclosure of Interest None Declared

Background Response to anti-TNF therapy is highly variable in patients with rheumatoid arthritis (RA). Predictors of treatment response have been identified, but their ability to predict response in individual patients is poor (1). MicroRNAs (miRNAs) have been suggested to influence susceptibility to RA and disease severity (2). Objectives To identify predictive miRNAs associated with treatment response in 180 patients with RA (disease duration Methods Patients received methotrexate 7.5 mg weekly (increased to 20 mg/week within two months) in combination with adalimumab (n=89)/placebo-adalimumab (n=91) 40 mg subcutaneously every other week. If swollen joints were present, they were injected with triamcinolone. Treatment response after 1 year was assessed according to EULAR response (good vs. moderate/poor), DAS28(CRP) remission, and ACR/EULAR Boolean remission criteria. MiRNAs were purified from pretreatment whole blood. Expressions of miRNAs were determined using TaqMan® Human MicroRNA LDA, A Card v2.0 (Applied Biosystems). Raw Cycle threshold (Ct) values of each miRNA were pre-processed using quantile normalization. Interaction between miRNAs and treatment was tested for each miRNA, and potential predictive miRNAs (p-values Results In the adalimumab-group/placebo-group 82%/74% achieved EULAR good response, 74%/49% achieved DAS28(CRP) remission, and 48%/30% EULAR/ACR Boolean remission. After backwards elimination a low expression of miR-22 in the adalimumab group was associated with response using EULAR response (OR: 12.18, 95% CI: 2.30-140.45, p=0.01) and DAS28(CRP) remission (OR: 44.74, 95% CI: 5.99-736.18, p=0.001) as outcome parameters. Similarly, high expressions of miR-23b (OR: 0.48, 95% CI: 0.24-0.86, p=0.03) and miR-758 (OR: 0.20, 95% CI: 0.03-0.86, p=0.05) in the adalimumab group were associated with response using DAS28(CRP) remission as outcome parameter. A high expression of miR-629 in the adalimumab group (OR: 0.45, 95% CI: 0.23-0.82, p=0.01) was associated with response using ACR/EULAR remission. Conclusions We identified several miRNAs in whole blood from patients with early RA associated with treatment response to adalimumab. Validation studies are needed to assess the utility of these miRNAs as predictive biomarkers in patients with RA. References Prajapati R et al. Pharmacogenomics 2011;12:1571-85. Trenkmann M et al. Clin Rev Allergy Immunol 2010;39:10-9. Horslev-Petersen K et al. Arthritis Rheum 2011; 63 (Supplement 10): 394. Disclosure of Interest S. B. Krintel: None Declared, C. Dehlendorff: None Declared, M. Hetland: None Declared, K. Horslev-Petersen: None Declared, K. Andersen: None Declared, P. Junker: None Declared, J. Podenphant: None Declared, T. Ellingsen: None Declared, P. Ahlqvist: None Declared, H. Lindegaard Consultant for: MSD, Merck, Roche, A. Linauskas: None Declared, A. Schlemmer: None Declared, M. Dam: None Declared, I. Hansen: None Declared, H. C. Horn: None Declared, A. Jorgensen: None Declared, J. Raun: None Declared, C. Ammitzboll: None Declared, M. Ostergaard: None Declared, K. Stengaard-Pedersen: None Declared, J. Johansen: None Declared