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Meta-analyses have demonstrated that low-dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the antiplatelet action of low-dose aspirin likely contributes to its antimetastatic effect. Cycloxooxygenase-2 (COX-2)–derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low-dose aspirin also inhibits PGE2 biosynthesis. We show that low-dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (P < 0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells, its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells upregulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet–tumor cell aggregates. In conclusion, low-dose aspirin has a significant effect on extraplatelet cyclooxygenase and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low-dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells. Cancer Prev Res; 9(11); 855–65. ©2016 AACR.

PDF file - 16K, SLC1A5 protein is overexpressed in NSCLC tissues

PDF file - 28K, SLC1A5 mediates response to Gln-dependent growth

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PDF file - 20K, Inhibition of SLC1A5 activity reduces Gln uptake and cell growth of NSCLC cells

PDF file - 15K, Silencing SLC1A5 in SCC cells by siRNA attenuates cell growth and reduces glutamine uptake

Purpose: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non–small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival.Experimental Design: To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines.Results: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na+-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling.Conclusions: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival. Clin Cancer Res; 19(3); 560–70. ©2012 AACR.

PDF file - 39K, Expression of SLC1A5 in lung cancer cell lines

Somatostatin receptor 2 (SSTR2) is overexpressed in a majority of neuroendocrine neoplasms, including small-cell lung carcinomas (SCLCs). SSTR2 was previously considered an inhibitory receptor on cell growth, but its agonists had poor clinical responses in multiple clinical trials. The role of this receptor as a potential therapeutic target in lung cancer merits further investigation. We evaluated the expression of SSTR2 in a cohort of 96 primary tumors from patients with SCLC and found 48% expressed SSTR2. Correlation analysis in both CCLE and an SCLC RNAseq cohort confirmed high-level expression and identified an association between NEUROD1 and SSTR2. There was a significant association with SSTR2 expression profile and poor clinical outcome. We tested whether SSTR2 expression might contribute to tumor progression through activation of downstream signaling pathways, using in vitro and in vivo systems and downregulated SSTR2 expression in lung cancer cells by shRNA. SSTR2 downregulation led to increased apoptosis and dramatically decreased tumor growth in vitro and in vivo in multiple cell lines with decreased AMPKα phosphorylation and increased oxidative metabolism. These results demonstrate a role for SSTR2 signaling in SCLC and suggest that SSTR2 is a poor prognostic biomarker in SCLC and potential future therapeutic signaling target.

Meta-analyses have demonstrated that low-dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the antiplatelet action of low-dose aspirin likely contributes to its antimetastatic effect. Cycloxooxygenase-2 (COX-2)–derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low-dose aspirin also inhibits PGE2 biosynthesis. We show that low-dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (P < 0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells, its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells upregulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet–tumor cell aggregates. In conclusion, low-dose aspirin has a significant effect on extraplatelet cyclooxygenase and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low-dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells. Cancer Prev Res; 9(11); 855–65. ©2016 AACR.

New therapeutic strategies are desperately needed in lung cancer. Targeting cancer-specific biochemical phenotypes, including those determined metabolically, represents an alternative approach to treating patients with lung cancer, the leading cause of cancer deaths in the USA and worldwide.1 L-Glutamine (Gln) is an essential amino acid for non-small cell lung cancer (NSCLC) growth in vitro and in vivo.2–4 Gln has been shown to sustain tumor growth under hypoxia,5 to mediate K-RAS-driven lung cancer growth6 and to support the autophagy-mediated prosurvival pathway in B-RAFV600-driven lung tumors.7 Nonetheless, the specific contribution of Gln transporters to lung cancer remains largely unknown. What’s new? New strategies to overcome lung cancer mortality depend heavily on the discovery of novel therapeutic targets. In non-small cell lung cancer (NSCLC), a possible target is solute linked carrier family 1A, member 5 (SLC1A5), a major glutamine transporter in NSCLC. This study furthers the promise of SLC1A5 by showing that its expression levels in lung cancer cells can predict cell sensitivity to the inhibitor gamma-L-glutamyl-p-nitroanilide (GPNA). In NSCLC cell lines, SLC1A5 inactivation led to glutamine starvation and oxidative stress-mediated autophagy and apoptosis. In NSCLC patients, SLC1A5 expression was associated with poor overall survival. We recently identified by in-depth shotgun proteomic analysis of stage I NSCLCs several therapeutic targets including the solute linked carrier family 1A, member 5 (SLC1A5).8 We were the first to report that SLC1A5 functions as the primary transporter of Gln in a sodium-dependent manner in NSCLC.9 Inhibition of SLC1A5 attenuates cell growth and mTOR signaling.9,10 Although pharmacological strategies to inhibit Gln metabolism using amino acid analogs such as acivicin and 6-diazo-5-oxo-L-norleucine (DON) have been investigated,3,11 the lack of selectivity of these agents has shifted the efforts to developing agents directed at specific nodes of glutamine metabolism instead.12 Given the emerging role of Gln metabolism in cancer13 and the differential expression of SLC1A5 in NSCLC,9 we tested the hypothesis that elevated SLC1A5 expression is a key prosurvival mechanism that promotes NSCLC progression by increasing tumor cells to transport and utilize Gln available in the microenvironment. We investigated the prognostic value of SLC1A5 expression in NSCLC and examined the therapeutic potential of targeting its Gln transport activity in cell and xenografts by inhibiting SLC1A5-dependent Gln transport.

The molecular determinants of lung cancer risk remain largely unknown. Airway epithelial cells are prone to assault by risk factors and are considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into the molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushing specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified 2,869 proteins, of which 312 proteins demonstrated a trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for 7 months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results provide a rationale for the development of potentially new chemopreventive strategies and selection of patients for surveillance programs.

Aberrant expression of RNA-binding proteins has profound implications for cellular physiology and the pathogenesis of human diseases such as cancer. We previously identified the Fragile X-Related 1 gene (FXR1) as one amplified candidate driver gene at 3q26-29 in lung squamous cell carcinoma (SCC). FXR1 is an autosomal paralog of Fragile X mental retardation 1 and has not been directly linked to human cancers. Here we demonstrate that FXR1 is a key regulator of tumor progression and its overexpression is critical for nonsmall cell lung cancer (NSCLC) cell growth in vitro and in vivo. We identified the mechanisms by which FXR1 executes its regulatory function by forming a novel complex with two other oncogenes, protein kinase C, iota and epithelial cell transforming 2, located in the same amplicon via distinct binding mechanisms. FXR1 expression is a candidate biomarker predictive of poor survival in multiple solid tumors including NSCLCs. Because FXR1 is overexpressed and associated with poor clinical outcomes in multiple cancers, these results have implications for other solid malignancies.

Introduction: Small cell lung cancer (SCLC) is a high grade neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths. Treatment in SCLC has not changed significantly in the last 20 years. SCLC initially responds well to chemotherapy, but inevitably recurs. Characterization of tumor heterogeneity and changes in SCLC cell signaling and phenotypes after chemotherapy could yield new insights and therapeutic options. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of more than 40 proteins in single cells and offers the opportunity to identify new subpopulations including potential cancer stem cell populations as well as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with one cycle of carboplatin/etoposide or saline injection. Tumors were harvested at ~2000mm3, disaggregated, and cryopreserved. PDX samples were stained with a 22 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. ViSNE analysis and biaxial gating were used to identify major subpopulations of interest. Results: PDX tumors released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. ACK buffer and enzymatic dissociation yielded the best quality cells by depleting red blood cells. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and iridium intercalator was used to identify nucleated cells. Single cell protein expression and phosphorylation was analyzed using viSNE and yielded at least 9 distinct subpopulations based on density islands with neuroendocrine (CD56+) and non-neuroendocrine (CD56-) populations. Chemotherapy treated cells had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included 2-3 fold expansion of SOX2+, CD117+, and pSTAT3+ populations with chemotherapy treatment. A small CD44+ tumor subpopulation identified in the chemotherapy treated cells was not present in the matched mock treated tumor suggesting a potential chemotherapy resistant/ stem- like subpopulation. Kinase activity showed stable p-AKT overall, but increased p-S6 in the chemotherapy treated cells. Conclusions: Mass cytometry was able to identify multiple neuroendocrine and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of multiple stem-like signaling factors. This work demonstrates the utility of mass cytometry and viSNE as novel techniques to identify subpopulations associated with chemotherapy resistance for future targeting and demonstrates the feasibility of this technique for characterizing signaling heterogeneity in human SCLC tumors. Citation Format: Jonathan M. Lehman, Nalin Leelatian, Bradford Harris, Megan Hoeksema, Zou Yong, Deon B. Doxie, Jonathan M. Irish, Pierre P. Massion. Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2017-3935

RNA-binding proteins (RBPs) are the master regulators of mRNA processing and translation and are often aberrantly expressed in cancer. We have recently identified Fragile X Mental Retardation-Related 1 (FXR1) as a novel cancer gene in non-small cell lung cancer (NSCLC) and its expression is correlated with poor prognosis in multiple human cancers. FXR1 encodes an RNA binding protein (RBP) that belongs to the family of fragile X-related proteins including the fragile X mental retardation protein (FMR1) and FXR2. Inactivation of FMR1 expression is the cause of the Fragile X syndrome in humans. Little is known for the function of FXR1 in human cancers. In this study, endogenous FXR1 or a Flag-tagged FXR1 was immunoprecipitated from H520, a lung squamous carcinoma cell line, and analyzed by shotgun proteomics. The Flag-tagged FXR1 was also transfected into human HEK293 cells and followed by co-immunoprecipitation and shotgun proteomic analysis. In total we found 206 proteins enriched in H520 with more than two-fold change spectral counts over the IgG control. Of the 206 proteins, 49 were detected in HEK293 cells as well and 157 were only detected in H520. KEGG pathway analysis indicated enrichment of proteins involved in ribosomal function, RNA transport and proteasome. To identify lung cancer related proteins, we interrogated the mRNA expression of 206 proteins in The Cancer Genome Atlas (TCGA) lung cancer dataset (1013 tumors,109 normal), in a combined NSCLC dataset (1392 tumors, 240 normal) from the Gene Expression Omnibus (GEO) and in a previously reported dataset of putative FXR1 target mRNAs and found 75 altered genes in common. Among these, we found that 24 genes were not only unregulated in all NSCLC samples (n = 2405, FDR Citation Format: Jun Qian, Yong Zou, Megan Hoeksema, Bradford Harris, Heidi Chen, Pierre Massion. Identification of FXR1-associated protein complexes in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2873.

Purpose: We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non–small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival. Experimental Design: To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines. Results: Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na+-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling. Conclusions: These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival. Clin Cancer Res; 19(3); 560–70. ©2012 AACR.

Advances in proteomic analysis of human samples are driving critical aspects of biomarker discovery and the identification of molecular pathways involved in disease etiology. Toward that end, in this report we are the first to use a standardized shotgun proteomic analysis method for in-depth tissue protein profiling of the two major subtypes of nonsmall cell lung cancer and normal lung tissues. We identified 3621 proteins from the analysis of pooled human samples of squamous cell carcinoma, adenocarcinoma, and control specimens. In addition to proteins previously shown to be implicated in lung cancer, we have identified new pathways and multiple new differentially expressed proteins of potential interest as therapeutic targets or diagnostic biomarkers, including some that were not identified by transcriptome profiling. Up-regulation of these proteins was confirmed by multiple reaction monitoring mass spectrometry. A subset of these proteins was found to be detectable and differentially present in the peripheral blood of cases and matched controls. Label-free shotgun proteomic analysis allows definition of lung tumor proteomes, identification of biomarker candidates, and potential targets for therapy.

Background: Meta-analysis of the trials evaluating the effect of aspirin on cardiovascular outcomes demonstrated that aspirin prevented mortality from adenocarcinomas by 47% and reduced metastasis by 46%. Equivalent benefit was seen with both high and low doses of aspirin. We hypothesize that low dose aspirin blocks metastasis by inhibiting platelet COX-1 and also by inhibiting the biosynthesis of the pro-metastatic PGE2. Methods and Results: The effect of a range of doses of aspirin on PGE2 biosynthesis in three lung adenocarcinoma cell lines was compared with that in washed platelets. PGE2 was measured by GC/MS. In the cancer cell lines, aspirin inhibits COX-2-dependent PGE2 production with IC50s equivalent to or less than that for platelet COX-1. The IC50s were: Platelet = 19.8 ± 1.5 μM; H2122 = 19.5 ± 5 μM; HCC827 = 3.9 ± 2 μM; A549 = 1.6 ± 0.4 μM. To explore the extra-platelet effects of low dose aspirin in vivo, we examined the effect of aspirin 81 mg daily for 2 weeks on the biosynthesis of PGE2 and prostacyclin as reflected by their respective urinary metabolites in 54 healthy humans. The PGE2 metabolite was measured by LC/MS/MS and the prostacyclin metabolite by GC/MS. This dose of aspirin inhibited PGE2 production by 45% (p < 0.0001) and reduced prostacyclin by 37% (p < 0.0001). Adherence of platelets to tumor cells facilitates metastasis. We determined the effect of adherence of ADP-activated washed platelets to lung adenocarcinoma cells (A549) on the PGE2 biosynthetic pathway. COX-2 expression was determined by Western blot, and cytosolic calcium by Fura-2 AM. Activated platelets increased cytosolic calcium in A549 cells by 22% (p < 0.001), increased expression of COX-2 by 8.6 fold (p Conclusion: These findings form the basis for a hypothesis that the remarkable effect of low dose aspirin on adenocarcinoma prevention and reduction in metastatic behavior could result from inhibition of platelet activation in concert with inhibition of platelet-induced PGE2 biosynthesis and direct inhibition of the catalytic activity COX-2 in circulating tumor cells. Work funded by the Thoracic Program of the Vanderbilt Ingram Cancer Center Citation Format: John Oates, Pierre Massion, Bjorn Knollmann, James Smith, Elias Haddad, Philip Lammers, Denise Oram, Taneem Amin, Bradford Harris, Megan Hoeksema, Hyun Hwang. Low dose aspirin that reduces mortality from lung adenocarcinoma inhibits both platelet COX-1 and the biosynthesis of PGE2. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR05.

Introduction: We recently reported that solute carrier family A1 member 5 (SLC1A5) controls glutamine (GLN) uptake and modulates cell growth, oxidative stress and mTOR signalling pathway. SLC1A5 is a transmembrane, high affinity glutamine transporter that is overexpressed and located at the plasma membrane in non-small cell lung cancer (NSCLC). GLN deprivation has been shown to induce cell death in several types of cancers, however the exact mechanism by which SLC1A5 targeting affects NSCLC survival remains unknown. We hypothesize that inhibition of SLC1A5 deprives cells from GLN which induces autophagy which eventually leads to cell starvation and ultimately apoptotic cell death. Methods: To test our hypothesis we targeted SLC1A5 by siRNA or by its specific inhibitor, GPNA, in a panel of 6 NSCLC lung cancer cell lines (express high level of SLC1A5). For comparison 2 human bronchial epithelial cell lines (HBEs) (express low level of SLC1A5) were used. The effects of targeting SLC1A5 on growth, GLN uptake, ATP level, autophagy, and cell death were examined. Markers of autophagy and cell death were analysed using western blot, nuclear staining and cell cycle analysis. Results: SLC1A5 inactivation by GPNA or by siRNA resulted in a significant decrease in cellular GLN uptake in NSCLC cell lines, while HBE cell lines were unaffected. Decrease in GLN uptake was accompanied by an increase in autophagy as evident by decrease in cell size, increase of LC3-II and decrease if LC3-I markers of autophagy. In addition, we observed a significant drop in ATP levels by 2 folds with increasing doses of GPNA. Markers of internal apoptotic pathway that include decrease of mitochondrial potential and cleaved caspases-3 and 9 were detected in response to increasing doses of GPNA. No change was observed in external apoptotic markers, caspases 8 or 1. In addition a 2-10 fold increase (based on the cell line) of sub-G1 was observed in NSCLC cell lines but not in HBE cell lines. Conclusion: Our results show that targeting SLC1A5 in NSCLC cells induces apoptotic cell death. Cells starve due to GLN deprivation and undergo autophagy which leads to a decrease in ATP level resulting in apoptotic cell death. These findings suggest that targeting SLC1A5 may have therapeutic implications in NSCLC. Grant funding: Lung Cancer Research Foundation Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B161. Citation Format: Mohamed Hassanein, Jun Qian, Megan Hoeksema, Marie Jacobovitz, Fredrick T. Harris, Bradford Harris, Pierre P. Massion. SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B161.