Sandra Hermann

Multigene analysis of Rb pathway and apoptosis control in esophageal squamous cell carcinoma identifies patients with good prognosis

Deregulation of cell‐cycle G1‐restriction point control by disruption of Rb‐pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin‐dependent kinase inhibitor p16INK4a and the proapoptotic Bcl‐2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G1 restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16INK4a, Rb, p21CIP/WAF‐1, p53, Bax and Bcl‐2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP‐PCR of exons 5–8. Loss of p16INK4a, Rb, p21CIP/WAF‐1 or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl‐2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21CIP/WAF‐1, p16INK4a and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.

Bax expression in benign and malignant thyroid tumours: dysregulation of wild-type P53 is associated with a high Bax and P21 expression in thyroid carcinoma.

The purpose of our study was to determine the expression of the pro‐apoptotic BAX protein in relation to the mutational status of BAX and p53 (as transcriptional activator of the BAX gene) in benign and malignant thyroid tissue. In 47 patients with thyroid tumours (14 follicular and 3 papillary carcinomas, 14 adenomas and 16 goitres), the DNA was screened for mutations of BAX (exon 1–6) and p53 (exon 5–8) by single‐strand conformation polymorphism polymerase chain reaction (SSCP‐PCR). Furthermore, the protein expression of BAX, p53 and p21 (which is also increased transcriptionally by p53) was investigated by immunohistochemistry. Surprisingly, we observed elevated BAX levels in patients with thyroid carcinomas compared with patients with adenomas (unpaired t‐test: p<0.05) or with goitres (p<0.02). This is in clear contrast to other carcinomas where BAX is frequently inactivated which correlates to a poor prognosis (Sturm et al. J. Clin. Oncol. 1999;17:1364–74.). There were no significant differences of the BAX levels between goitres or the adenomas. In the SSCP‐PCR analysis, no BAX mutations were detectable. P53 mutation analysis by SSCP‐PCR did not reveal any functional p53 mutations in the patients with carcinomas, adenomas or goitres. Nevertheless, patients with carcinomas showed an overexpression (preferentially cytoplasmic) of p53 protein compared with patients with benign tumours (p<0.05). The absence of p53 mutations suggests that the overexpressed p53 is wild type. This is in line with the expression profile of BAX and p21, which showed a higher protein expression in these p53 positive tumours (p<0.05 in the carcinomas compared with the non‐malignant lesions). Consequently, the overexpressed p53 might be a correlate for dysregulation without loss of function. This, in turn, might be a reason for the good outcome of some patients with thyroid cancer.

Monocyte alterations in rheumatoid arthritis are dominated by preterm release from bone marrow and prominent triggering in the joint

Objective Rheumatoid arthritis (RA) accompanies infiltration and activation of monocytes in inflamed joints. We investigated dominant alterations of RA monocytes in bone marrow (BM), blood and inflamed joints. Methods CD14+ cells from BM and peripheral blood (PB) of patients with RA and osteoarthritis (OA) were profiled with GeneChip microarrays. Detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and mobilisation. Cytometric profiling determined monocyte subsets of CD14++CD16−, CD14++CD16+ and CD14+CD16+ cells in BM, PB and synovial fluid (SF) and ELISAs quantified the release of activation markers into SF and serum. Results Investigation of genes differentially expressed between RA and OA monocytes with reference transcriptomes revealed gene patterns of early myeloid precursors in RA-BM and late myeloid precursors along with reduced terminal differentiation to CD14+CD16+monocytes in RA-PB. Patterns associated with tumor necrosis factor/lipopolysaccharide (TNF/LPS) stimulation were weak and more pronounced in RA-PB than RA-BM. Cytometric phenotyping of cells in BM, blood and SF disclosed differences related to monocyte subsets and confirmed the reduced frequency of terminally differentiated CD14+CD16+monocytes in RA-PB. Monocyte activation in SF was characterised by the predominance of CD14++CD16++CD163+HLA-DR+ cells and elevated concentrations of sCD14, sCD163 and S100P. Conclusion Patterns of less mature and less differentiated RA-BM and RA-PB monocytes suggest increased turnover with accelerated monocytopoiesis, BM egress and migration into inflamed joints. Predominant activation in the joint indicates the action of local and primary stimuli, which may also promote adaptive immune triggering through monocytes, potentially leading to new diagnostic and therapeutic strategies.

Single source dual-energy computed tomography in the diagnosis of gout: Diagnostic reliability in comparison to digital radiography and conventional computed tomography of the feet

OBJECTIVES To investigate the diagnostic value of single-source dual-energy computed tomography (SDECT) in gouty arthritis and to compare its capability to detect urate depositions with digital radiography (DR) and conventional computed tomography (CT). METHODS Forty-four patients who underwent SDECT volume scans of the feet for suspected gouty arthritis were retrospectively analyzed. SDECT, CT (both n=44) and DR (n=36) were scored by three blinded readers for presence of osteoarthritis, erosions, and tophi. A diagnosis was made for each imaging modality. Results were compared to the clinical diagnosis using the American College of Rheumatology (ACR) classification criteria. RESULTS The patient population was divided into a gout (n=21) and control (n=23) group based on final clinical diagnosis. Osteoarthritis was evident in 15 joints using CT and 30 joints using DR (p=0.165). There were 134 erosions detected by CT compared to 38 erosions detected by DR (p<0.001). In total 119 tophi were detected by SDECT, compared to 85 tophi by CT (p=0.182) and 25 tophi by DR (p<0.001). SDECT had best diagnostic value for diagnosis of gout compared to DR and conventional CT (sensitivity and specificity for SDECT: 71.4% and 95.7%, CT: 71.4% and 91.3% and DR: 44.4% and 83.3%, respectively). For all three readers, Cohens kappa for DR and conventional CT were substantial for all scoring items and ranged from 0.75 to 0.77 and 0.72-0.76, respectively. For SDECT Cohens kappa was good to almost perfect with 0.77-0.84. CONCLUSIONS SDECT is capable to detect uric acid depositions with good sensitivity and high specificity in feet, therefore diagnostic confidence is improved. Using SDECT, inter-reader variance can be markedly reduced for the detection of gouty tophi.

Ultra-low-dose CT detects synovitis in patients with suspected rheumatoid arthritis

Purpose To prove the feasibility and measure the diagnostic accuracy of contrast-enhanced ultra-low-dose CT (ULD-CT) for the depiction of inflammatory soft-tissue changes (synovitis, tenosynovitis and peritendonitis) in patients with arthritis of the hand. Materials and methods In this institutional review board–approved study, 36 consecutive patients over the age of 50 with suspected rheumatoid arthritis underwent ULD-CT (estimated radiation exposure <0.01  mSv) and MRI of the hand with weight-adapted intravenous contrast administration. ULD-CT subtraction and MR images were assessed for synovitis, tenosynovitis and peritendonitis by three readers using a modified Rheumatoid Arthritis MRI Score (RAMRIS). Patients were asked which modality they would prefer for future examinations. Sensitivity and specificity of ULD-CT for detection of inflammatory changes were calculated using MRI as standard of reference. The sum scores were correlated using Pearson’s r. Results All 36 patients showed synovitis in MRI. ULD-CT had 69% sensitivity on the patient level and 65% on the joint level with 87% specificity. Sensitivity was higher in patients with more severe inflammation (80% for MRI RAMRIS >1). There was almost perfect correlation between the modified RAMRIS sum scores of ULD-CT and MRI (Pearson’s r=0.94). Regarding preferences for future examinations, 85% preferred ULD-CT over MRI. ULD-CT detected more differential diagnoses than MRI (8 vs 2/12). Conclusion Contrast-enhanced ULD-CT of the hand allows for depiction of soft-tissue inflammation at the hand and can be achieved using very low radiation exposure (<0.01 mSv). ULD-CT may evolve to a fast and comfortable alternative to MRI, although it is not as sensitive as MRI for detecting mild disease.

05.08 Increased turnover of monocytes in patients with rheumatoid arthritis identified by transcriptome and cytometric profiling

Background Targeting molecules involved in monocyte activation is an important treatment strategy for RA. In this study we aimed to determine monocyte maturation and activation from bone marrow (BM) via blood into synovial fluid (SF) by investigating monocytes transcriptomes and by cytometric profiling of classical (CD14++CD16-), intermediated (CD14++CD16+) and non-classical (CD14+CD16+) monocytes. Materials and methods CD14+ cells from BM and blood of RA and osteoarthritis (OA) patients were profiled with Affymetrix microarrays. A detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and egress from BM induced by G-CSF (granulocyte colony-stimulating factor). Cytometric profiling of CD14, CD16, HLA-DR and CD163 expression were used to determine monocyte subsets and to follow their activation and differentiation in BM, blood and SF. Results Transcriptomes of RA-BM monocytes exhibited i) pronounce gene pattern of early myeloid precursors from BM and ii) weak gene pattern of late myeloid precursors from BM. Transcriptomes of RA blood monocytes demonstrated i) pattern of late myeloid precursors from BM and ii) reduced pattern of terminally differentiated CD14+CD16+ monocytes from blood. Cytometric profiling of BM, blood and SF monocytes in RA and OA showed that all three body compartments have their own distribution of monocyte subsets. BM was characterised with classical and intermediate subsets and both subsets showed decreased CD16 expression in RA when compared to OA. As expected, blood was characterised with three subsets, and RA blood showed decreased CD14 and HLA-DR expression on classical monocytes and reduced frequency of non-classical subset. In RA-SF, classical monocytes were absent, intermediate were most dominant and cell-phenotype with low CD16 expression but similar to non-classical monocytes was related to macrophages. Cell frequency of intermediate subset in SF positively correlated with inflammation (ESR; R>0.85) and showed the highest expression of HLA-DR, CD14, CD163. Conclusions Monocyte turnover is increased in RA and characterised with accelerated monocytopoiesis, faster BM egress and migration into inflamed joints. Permanent monocyte activation in the joint and their role in linking innate and adaptive immunity, which is targeted by biologics, emphasises their high diagnostic value and relevance for therapeutic stratification.

A6.11 Immunoclust based analysis of cytometric profiles reveals immunophenotypic changes in synovial fluid compared to peripheral blood cells in rheumatoid arthritis

Background and objectives Flow cytometry offers quantification of multidimensional characteristics at single cell level for millions of cells. Multiplex flow cytometry or mass cytometry enable to screen for dozens of antigens on a single cell. Conventional analysis of such data requires user defined gating and is time consuming. Using the new bioinformatics tools immunoClust for automated and user-independent analysis, we investigated the complexity of phenotypic changes of immune cells upon migration from peripheral blood (PB) to synovial fluid (SF) in rheumatoid arthritis (RA). Materials and methods Seven paired samples of PB and SF from RA patients were stained in 10 different antibody cocktails and data investigated by the newly developed immunoClust pipeline for sample specific populations and differences between PB and SF. Population clustering and comparative meta-clustering assume finite mixture models and use Expectation Maximisation (EM)-iterations with integrated classification likelihood (ICL) criterion to stabilise the number of reasonable clusters. For meta-clustering, a probability measure on Gaussian distributions was invented, which is based on the Bhattacharyya Coefficients. Meta-clusters were manually annotated and classified. The clustering tools of immunoClust are available as open source R-package in Bioconductor. Results Automated clustering with 46 different surface markers detected all major leukocyte subsets and several activation markers in PB and SF samples including neutrophils, eosinophils, T-cells and sub-populations, monocytes, B-cells, NK-cells and dendritic cells. The comparison revealed about 10 highly significant changes per staining cocktail. For example the percentage of monocytes/macrophages was doubled in SF and dominated by CD16+ cells, the frequencies of effector/memory subpopulations of lymphocytes were increased and naïve T-cells and B-cells were almost completely absent. In addition several unexpected populations like CCR7+ monocytes were found in SF only. Conclusion In conclusion, the results give a reasonable starting point to face the next field of research for marker detection and prediction analysis. The data will be further exploited for changes in cell activation and differentiation in SF in order to screen for these populations also in PB. This approach is not only applicable to fluorescence-based flow data but could be also used for multi-parametric data sets generated by mass spectrometry-based cytometry (CyTOF).

AB0014 Nanoparticles as MRI Contrast Agent for Early Diagnosis of RA: Effects of Amino-PVA-Coated SPIONS on CD4+ T Cell Activity

Background In medical applications nanotechnology provides new opportunities for diagnostic and therapeutic interventions in a variety of human diseases. Superparamagnetic iron oxide nanoparticles (SPION) are used as high-sensitive enhancer for magnetic resonance imaging, where they represent a promising tool for early diagnosis of destructive diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). However, safety aspects still represent crucial problems for the further development of nanotechnology based products. Therefore, the focus of our work here was to identify more in detail putative unwanted effects of amino-polyvinyl alcohol-coated (a-PVA) SPION on human immune cell functions. Objectives Since we could demonstrate in former studies that professional phagocytes are activated by a-PVA-SPION, we here focused on the influence of these nanoparticles on human T helper cell activity. Methods PBMCs were isolated from blood samples obtained from healthy donors (HD, n≥9) or patients suffering from RA (n≥11). Primary human CD4 positive T cells were separated via MACS-Sort and incubated with the mitogen PHA-L (5μg/ml) and/or varying doses of a-PVA-SPION (1 μg/ml, 10 μg/ml, and 100 μg/ml) or left untreated for 20 h (analysis of caspase-3/7-activity, intracellular ATP content and CD25 expression) or 72 h (analysis of proliferation and CD25 expression). Cells were incubated under either normoxia (app. 18%O2) or hypoxia (1%O2) in order to mimic conditions found in the circulation and in the inflamed joint, respectively. Results We observed for PHA-L/a-PVA-SPION co-stimulated T cells from HD a decrease in cell count (all p<0.05) whereas the caspase-3/7-activity is increased (all p<0.05) in these samples, as expected. Although, we observed that T cells from RA patients are more susceptible to low-dose a-PVA-SPION-induced apoptosis than T cells from HD (p<0.05), in both groups a-PVA-SPION do not activate CD4+ T cells per se and do not influence mitogen-mediated T cells activation with regard to CD25 expression and cell proliferation. Nevertheless, we were able to demonstrate that CD4+ T cells obtained from RA patients and healthy subjects differ in their response to mitogen stimulation and oxygen availability. Conclusions PVA-SPION at concentrations up to 100μg/ml do neither activate nor significantly influence mitogen-stimulated CD4+ T cells activation and have negligible influence on T cells apoptosis. Disclosure of Interest None declared

FRI0040 A Novel Approach to Quantify Morning Stiffness in Patients with Rheumatoid Arthritis

Background In addition to joint swelling and tenderness, patients with rheumatoid arthritis (RA) commonly experience morning symptoms of joint stiffness associated with pain that result in impaired function causing morbidity and productivity loss with early disease as well as those with low disease activity or remission1. However, the degree of morning stiffness has never been described. Objectives Although RA patients recognize morning stiffness as being one of the four most significant symptoms to manage, validated morning stiffness patient-reported outcome (PRO) measures are lacking. Therefore, we have developed an approach to objectively quantify finger joint stiffness. The device has been approved by the ethical committee for the use in patients, and has also already obtained a national technical certification. We here present the first results of an ongoing study with this device. Methods So far, nine female postmenopausal RA patients affected by morning stiffness of at least one hour agreed to participate in this study and underwent repetitive measurements of evening and on following morning cycle. These measurements quantified the passive resistance of an affected MCP joint against an externally applied torque while the finger was fixed in the device and passively moved from a completely extended position (referred to as 0°) to a flexed position of 60°. Measurement related sensors and advanced analysis algorithms for the investigation of resulting hysteresis curves enabled both the stiffness and dissipated energy to be evaluated at definite flexion-extension angles. A one-way ANOVA was then applied to test for differences in both parameters between repetitive evening and morning measurements. Results The stiffness of the MCP joint (given in Nm/°) showed highest absolute values at extreme positions, i.e. at 0° and 60°, while lower values were detected at angles in between (Figure 1). At all flexion angles except 0°, stiffness was – as expected – always more pronounced in the morning (red bars) compared with evening measurements (blue bars). The difference found at 40° was statistically significant (p=0.038). Similar results (not shown) were obtained for dissipated energy with a significantly higher value in the morning than in the evening at a flexion angle of 50° (p=0.047). Conclusions Even though the number of patients examined was low and the individual variation of absolute values between subjects is (known to be) considerable, our findings indicate that our approach is capable to differentiate stiffness and dissipated energy in the morning from that measured in the evening. The quantification of these parameters offers for the first time an option for objective evaluation the possibility to objectively evaluate and quantify this important patient-reported outcome in RA. Furthermore, such biomechanical assessments are also very likely able to quantify treatment effects on morning stiffness in the future. References Da Silva, JAP et al.: Impact of impaired morning function on the lives and well-being of patients with rheumatoid arthritis. Scand J Rheumatol. Vol. 125 (2011), pp. 6-11. Acknowledgements This study was funded by Horizon Pharma, USA. Disclosure of Interest H. Boeth: None declared, G. Duda: None declared, D. Hinzmann: None declared, S. Hermann: None declared, W. Taylor: None declared, R. Ehrig: None declared, T. Witaschek: None declared, F. Buttgereit Grant/research support from: Dr. Buttgereit received consultancy fees, honoraria and travel expenses from Merck Serono, Horizon Pharma (formerly Nitec Pharma) and Mundipharma International Ltd, and grant support from Merck Serono and Horizon Pharma