Helena Idborg

Systems biology of SLE : biochemical characterisation of subgroups within SLE for improved diagnosis and treatment

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with symptoms ranging from skin related problems to more severe cardiovascular effects. The heterogenous representation of the disease might be the reason for the lack of efficient treatment. The authors hypothesise that subgroups of SLE can be characterised by different biochemical pathways and that specific biomarkers along these pathways can be identified. In this study, the authors have utilised the Karolinska lupus cohort that consists of 320 SLE patients and 320 age-matched controls. Two main subgroups were defined: One group was defined as having SSA and SSB antibodies and a negative lupus anticoagulant test (LAC), that is, a ‘Sjögren-like’ group. The other group was defined as being negative for SSA and SSB antibodies but positive in the LAC test. According to previous studies these patients are at increased risk for cardiovascular events as compared to the ‘Sjörgren-like’ group. A pilot study was designed and EDTA-plasma from selected patients in these two groups and controls were analysed using a proteomic and metabolomic approach. Pathway analysis was then performed on the obtained data. The pilot study showed that it was possible to differentiate the two subgroups of SLE based on the proteomic profile. From the proteins found to be significantly different between the groups, several proteins known to be involved in SLE were detected, for example, Apolipoprotein A1 and complement factor 3. In addition, proteins that to our knowledge have not been reported earlier to correlate with SLE, for example, Apolipoprotein M, were detected and are subject for further investigations. Apolipoprotein E was one of the proteins that was found to be significantly different between the two subgroups of SLE and will be investigated in the entire cohort. Preliminary data from metabolomics demonstrate that it is possible to separate patients from controls and the authors found for example that tryptophan levels were lower in SLE patients. Pathway analyses of proteomics and metabolomics data strongly predict that the changes in SLE patients compared to controls are associated with inflammation and immunity related pathways. This project will provide new knowledge about SLE taking several complex systems into account simultaneously. Using selected biomarkers it will be possible to identify more homogenous patient populations for clinical trials and thereby increase the efficacy. The systems biology approach is likely to identify pathways that may lead to better understanding of the disease, identification of novel drug targets and biomarkers supporting improved diagnosis of SLE.

05.01 Protein profiling in plasma reveals molecular subgroups in systemic lupus erythematosus

Objective Systemic Lupus Erythematosus (SLE) is a heterogeneous systemic autoimmune disease that is currently lacking specific diagnostic biomarkers. The diversity within the patients might obstruct clinical trials and could reflect differences in underlying pathogenesis. Our objective was to identify protein profiles that could be used for diagnosis and to identify molecular subgroups within SLE for patient stratification subjected to different treatment. Method In a cross-sectional study we performed protein profiling of 695 plasma samples from SLE patients and matched controls. This was achieved by utilising an antibody suspension bead array targeting 367 proteins. T-test and ROC analysis was performed to identify differences in the protein profiles between SLE and controls. Unsupervised K-means clustering was performed to identify data-driven SLE subgroups. Results We report that the novel proteins MMP1, SELE, and S100A12 can distinguish between SLE patients and controls with an area under curve of 0.80 in a ROC analysis. In addition, 28 proteins were found to show differences (corrected p-value<0.05) between SLE patients and controls. By unsupervised clustering we identified an IRF5, NOS3 and CLDN8-driven subgroup, an ARID2 and SELE-driven subgroup and one subgroup characterised by low SLC22A2 levels. Conclusion We have identified potential biomarkers of SLE that may be used to improve the diagnosis of SLE patients. Our suggested panel of biomarkers needs to be validated in an additional SLE cohort and also in relation to other systemic autoimmune diseases before it can be used as a diagnostic test. We have also identified subgroups characterised by different molecular patterns, indicating underlying pathogenic differences. These patient groups might benefit from different treatment strategies. Our work adds new information to today’s view of classifying the heterogeneous subgroups within SLE and the importance of personalised medicine.

Abstract 3986: Characterization of prostaglandin signaling in primary neuroblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Neuroblastoma (NB) is an embryonic childhood tumor of the sympathetic nervous system. Despite intensive treatment patients in the high-risk group of NB shows a poor prognosis. We have shown the importance of inflammation in NB development and progression and that the pro-inflammatory lipid mediator prostaglandin E2 (PGE2) acts as a paracrine survival factor in NB. We have also shown that mPGES-1, essential for PGE2 synthesis, promotes growth and survival of several malignancies. In this study we focus on the abundance of prostaglandins and their specific enzymes in primary NB of different biological and clinical subsets in the aim of finding a more specific and targeted NB treatment. Levels of prostaglandins were assayed in 40 primary human NB tumors by mass spectrometry (MS) showing elevated levels of PGE2 in high risk 11q-deleted NB compared to low-risk NB whereas elevated levels of prostaglandin D2 (PGD2) could be coupled to NB with favorable clinical outcome. The expression of COX-1, COX-2, mPGES-1, L-PGDS, H-PGDS and 15-PGDH were analyzed in tumors by immunohistochemistry (IHC). Expression of mPGES-1 and L-PGDS was found in all analyzed tumors. Double stainings of mPGES-1 and the specific NB cell marker GD2 were performed on tumors with different biology using immunofluorescence. In low-risk tumors mPGES-1 is not expressed by the tumor cells whereas co-localization were found in 11q deleted high-risk tumors. To consider the level of inflammation and PGE2 signaling in these tumors we analyzed cell markers of both the innate and adaptive immune system together with mPGES-1. In all tumors mPGES-1 expressing cells are found in clusters closely together with T-cells, B-cells, dendritic cells and M1/M2 macrophages. Together this suggests an involvement of inflammatory prostaglandins in neuroblastoma development and tumor progression opening up new strategies for targeted therapies. Citation Format: Anna Kock, Karin Larsson, Linda Ljungblad, Helena Idborg, Marina Korotkova, Lotta Elfman, John-Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Characterization of prostaglandin signaling in primary neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2014-3986

Abstract 2746: Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Neuroblastoma (NB) cells are enriched in the omega-6 fatty acid arachidonic acid, the substrate for the cyclooxygenase (COX) enzymes and prostaglandin biosynthesis. The inducible isoform, COX-2 is overexpressed in NB and NB cells produce prostaglandin E2 (PGE2) that acts as an autocrine and/or paracrine survival and proliferation factor. Downstream of the COX enzymes, specific synthases are responsible for the production of the respective prostaglandins. Microsomal prostaglandin E2 synthase-1 (mPGES-1) specifically converts PGH2 to PGE2 and is thought to primarily couple to COX-2. The aim of this study was to investigate if inhibition of mPGES-1 could represent an alternative therapeutic approach to COX- inhibition in NB. Methods: Western blot and immunohistochemistry were used for protein detection. Cell viability of seven NB cell-lines treated with the mPGES-1 inhibitor CAY1052 was determined by MTT-assay. Stable mPGES-1 knockdown SK-N-BE2 clones were established using shRNA and the clonogenic capacity was analysed by clonogenic assay. To study the in vivo effect of COX inhibition, four week old homozygous TH-MYCN mice were treated with 10mg/L diclofenac for two weeks. Ex vivo analyses of COX-metabolites in tumors were performed by LC-MS/MS. Results: Expression of mPGES-1 in human NB tumors and in NB cell lines could be detected. Inhibition of mPGES-1 reduced NB cell growth in vitro and knockdown of mPGES-1 significantly reduced the clonogenic capacity. Expression of COX-1, COX-2 and mPGES-1 in TH-MYCN tumors could be detected and treatment with the dual COX-inhibitor diclofenac significantly reduced tumour weight, compared to untreated animals. Ex vivo analysis of tumor tissues from treated animals revealed a significantly decreased level of COX metabolites compared to controls. The expression of mPGES-1was not affected by the treatment. Furthermore, cells staining positive for cleaved caspase-3 were more prevalent in treated tumors indicating apoptosis Induction. Conclusion: We found that mPGES-1 is expressed in NB, with a potential role for PGE2 synthesis and tumor growth. mPGES-1 represents an alternative therapeutic target for inhibiting NB growth through specific PGE2 inhibition and the TH-MYCN model is well suited for in vivo studies with this purpose. Citation Format: Anna Kock, Agnes Rasmuson, Marina Korotkova, Helena Idborg, John Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Microsomal prostaglandin E2 synthase-1 may provide a novel specific therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2013-2746