e17032 Background: In cancer, systematic analysis of mRNA expression levels can contribute to define a molecular network of lung carcinogenesis and establish predictive and prognostic molecular markers. Altered mRNA expression in certain angiogenic and anti-angiogenic genes such as vascular endothelial growth factor family of ligands and their receptors together with other molecules implicated in angiogenesis could predict disease outcome and add prognostic information at diagnosis. Methods: We performed RT-qPCR in frozen head and neck cancer specimens from untreated patients who had undergone surgical resection (n = 23). Samples were processed for mRNA extraction following standard procedures and quantification of gene expression was expressed as relative concentration normalized by an endogenous gene. The following angiogenic genes were quantified: PDGF-A, PlGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, COX-2, bFGF, HGF, IL-8; and anti-angiogenic genes: Angiomotin and endostatin. Results: Median age was 56.5 years, range (31–79), 17 patients presented squamous carcinoma of the larynx; 8 patients were stage III and the other 15 stage IV. None of the patients received pre-surgery chemo or radiotherapy and 10 patients reveived post-surgery treatment. Our results show that tumor samples had significant higher expression of PDGF, PlGF, COX-2, and IL-8 and a deep down-regulation of angiomotin. However, those data show no correlation between the levels of expression and stage of the disease. Conclusions: Our results reveal that there is an increase in the expression of the pro-angiogenic mediators PDGF, PlGF, COX-2, and IL-8 and a downregulation of angiomotin that may mediate the inhibitory effect of angiostatin on tube formation and the migration of endothelial cells toward growth factors during the formation of new blood vessels in the tumoral area. This data suggest that more research are needed in other angiogenic mediators that VEGF, that PlGF and angiomotin have some role in cancer progression and could be a promising new biomarker in head and neck cancer. No significant financial relationships to disclose.

Tyrosine kinases are promising targets for personalized medicine, and new drugs are currently in phase 2 and phase 3 clinical trials. However, expression analysis of tyrosine kinases as predictive biomarkers is still not a standard approach. Furthermore, only limited studies have investigated the expression of tyrosine kinase receptors on the protein level. In this study, we analysed a well-characterised group of soft tissue sarcomas for different tyrosine kinase receptors and correlated the results with clinicopathological parameters, including survival.275 soft tissue sarcomas of our Sarcoma center at the Ludwig-Maximilians-University (LMU) were reinvestigated and categorized according to the current WHO classification system. The tumor collective included undifferentiated pleomorphic sarcomas (n = 81), leiomyosarcomas (n = 50), synovial sarcomas (n = 27), liposarcomas (n = 51), angiosarcomas (n = 43) and other soft tissue sarcomas (n = 23).On protein levels, high expression of VEGFR1 was detected immunohistochemically in 61%, VEGFR2 (KDR) in 11%, VEGFR3 in 64%, PDGFRA in 42% and PDGFRB in 73%. High expression of VEGFR1-3 and PDGFRB was significantly correlated with higher grading (G2 vs G3, p < 0.05), and high VEGFR2 was significantly correlated with decreased patients’ survival (p < 0.001).Tyrosine kinase receptors showed a distinct expression pattern in soft tissue sarcomas. High expression of VEGFR2 (KDR) is significantly associated with decreased patients’ survival. High VEGFR 1-3 and PDGFRB are significantly correlated with higher tumor grading. Protein signatures might be evaluated before targeted therapy to give a rationale for an eligible personalized therapy.

e22070 Background: HLA-G is a human non-classical MHC molecule, mainly expressed in the trophoblast whose main function is to suppress immunologic activity that allows maternal tolerance to phoetus. On the other hand, infiltrating regulatory T lymphocytes (Treg) could promote peripheral inmunotolerance fo oncogenic transformation. Methods: We performed real-time PCR in frozen oral cancer specimens from untreated patients who had undergone surgical resection (n=22) and in normal oral mucosa from healthy subjects (n=10). Samples were processed for mRNA extraction and quantification of gene expression was expressed as relative concentration by an endogenous gene. To asses the presence of infiltrating Treg we analysed CTLA-4, Foxp-3, IL-10, TGF-beta, CD4, CD8, CXCR4, CD127 and CD25 and also we determined HLA-G levels. We correlate the expression of immunologic mediators with clinical variables. Results: Patients presented squamous carcinomas of the tongue (n=12) or gum (n=10) and stages ranged from I to IV (stage I=4; II=9; III=4; IV=5). 10 patients presented well-differentiated lesions and the other 12 moderately-differentiated cells. Eight patients received post- surgery chemo and radiotherapy. Our results show that tumor samples had significant higher expression of the CTLA-4, Foxp-3, TGF-beta and CD127 genes than normal tissue. However, those data showed no correlation between the levels of expression and clinico-pathologic variables. When patients were grouped according to tumor size, there was a trend in the way that bigger tumoral lesions expressed relative higher amounts of Treg. By contrast we could not observe an increase of the expression of HLA-G in patients. Conclusions: Our results reveal that there is an increase in the expression of Treg in oral cancer patients. These Treg in tumoral tissues might contribute to the impairment of immunological rejection of the neoplasic transformation. Conversely we have not been able to demonstrate tumoral expression of HLA-G as a strategy to escape from immunosurveyance. Further analysis of this cells and their function is important in order to develop new therapeutic strategies. No significant financial relationships to disclose.

e22207 Background: NSCLC is a major cause of cancer-related death worldwide. The prognosis for lung cancer patients is poor with 5-years survival rates being less than 15%. It is known that angiogenesis is an essential event for solid tumour growth. Vascular endothelial growth factor (VEGF) family of ligand and receptors (VEGFR) are described as powerful angiogenic factors. VEGF belongs to a protein family, within which Placental growth factor (PlGF) is a member, they bound to their receptors at the membrane levels, gathering a cascade of intracellular events. In this study, we examine the expression of angiogenic genes in NSCLC samples correlating the expression of these genes between them and with clinicopathological variables. Methods: We performed real-time quantitative polymerase chain reaction (RT-qPCR) to assess the expression of VEGF, PlGF, VEGFR1 and VEGFR2 in frozen lung cancer specimens from untreated NSCLC patients who had undergone surgical resection (n=21). For this purpose, RNA was extracted and RTqPCR was performed using TaqMan® probes. Relative quantification was calculated by Pfaffl formulae, using an endogenous gene for normalization. We correlate the expression of the angiogenic genes between them and with other biologic variables. Statistical analysis were done using the SPSS 13.0 software. Results: Our results show that tumor samples have higher expression of PlGF than normal tissue. The expression of PlGF and VEGF correlates with the expression of their receptors in the group of samples analyzed. The expression of VEGFR1 and VEGFR2 was also significant correlated. We found a significant correlation between the levels of expression of PlGF and the tumor size (p= 0.023, Spearman's test), whereas no relation was found between the expression of the genes and the histology or stage of disease. Conclusions: Our results reveal that, in NSCLC, PlGF mRNA is higher in tumor than in normal tissue and is positively correlated with the tumor size and with the expression of angiogenic receptors. Theses finding could indicate that PlGF have some role in lung cancer progression and may be a promising new biomarker in NSCLC, but still more investigations are necessary with a larger number of samples. Supported by Instituto de Salud Carlos III (Fondo de Investigación Sanitario Grant). No significant financial relationships to disclose.

e22108 Background: An increase in VEGF expression in tumour or some blood compartments (i.e. serum or plasma) has been found in solid tumours of various origins. Several studies have suggested that ligands and receptors of the VEGFs/VEGFR system play an important role in tumour growth and is associated with metastasis and poor prognosis. The aim of our study was to investigate the usefulness of plasmatic VEGFR2 quantification as a new biomarker in advanced NSCLC. Methods: We studied 106 healthy controls (c) and 467 advanced NSCLC patients (p) (stage IIIB and IV) treated with cisplatin and docetaxel. Blood samples were collected before chemotherapy and the plasmatic levels of the VEGFR2 were determined by ELISA. Results: In the NSCLC group, the median age was 59.9, range (31–80); 82% were males. The histological subtypes were: 31.4% squamous, 49.8% adenocarcinoma, 15.3% large cell and undifferentiated and 3.5% other. There was a significant difference in the plasmatic levels of VEGFR2 between c and p (mean± SEM): 6318±152 ng/ml and 8141± 119 ng/ml, respectively (p<0.0001). On the other hand, we found no statistical differences according to sex, histology, or stage. The area under the ROC curve was 0.743 indicating that VEGFR2 is an adequate biomarker for the discrimination between c and p. Dividing the cohort in two subgroups according to VEGFR2 levels: high (>9473,9 ng/ml) and low (≤ 9473,9 ng/ml), we found significant difference in terms of Time to Progression (TTP). Patients with higher levels of VEGFR2 had a median TTP of 204 days whereas in the group with lower expression the median was 164 days, (p= 0.039). Conclusions: In advanced NSCLC, we found higher levels of soluble VEGFR2 in p than in c. There was a correlation between higher expressions of soluble VEGFR2 with better prognosis, in terms of TTP, therefore a more thorough understanding in the role of the plasmatic quantification of this angiogenic receptor in advanced NSCLC p seems to be an important task. No significant financial relationships to disclose.

The sentinel lymph node (SLN) concept is that lymphatic flux from a primary tumor initially flows into a SLN. The mechanism mediating tumor metastasis within SLNs remains largely unknown; however, primary tumors overexpressing vascular endothelial growth factor (VEGF)-A appear to induce SLN lymphangiogenesis prior to metastasis in animal model. Our aim was to further investigate the capacity of VEGFs to induce lymphangiogenesis within SLNs and to assess their role in SLN metastasis in non-small cell lung carcinoma (NSCLC).Real-time quantitative RT-PCR was used to assess expression of mRNAs encoding several VEGFs (VEGF121, VEGF165, VEGFR1, VEGFR2, VEGFR3, VEGF-C and VEGF-D) in resected lymph node specimens from 35 NSCLC patients, after which we compared their expression SLNs and non-SLNs. In addition, expression of the lymphatic endothelium-specific hyaluronan receptor (LYVE)-1 was used to assess lymphangiogenesis in SLNs and non-SLNs.Immunohistochemical staining revealed substantial expression of LYVE-1 in SLNs. Moreover, levels LYVE-1 mRNA were significantly higher in SLNs than non-SLNs (P < 0.05), as were levels of VEGF121 and VEGFR2 mRNA (P < 0.01 and P = 0.02, respectively). In addition metastasis-positive SLNs showed significantly higher levels of VEGF121, VEGF-C and VEGF-D mRNA than metastasis-negative SLNs (P < 0.001, P = 0.01 and P = 0.01, respectively), and VEGF121 induced the proliferation of lymphatic endothelial cells (P < 0.01).Our findings suggest that active lymphangiogenesis is ongoing within SLNs from NSCLC patients, even before metastasis. This lymphangiogenesis may be promoted by upregulation of VEGF121, which may in turn act in part via induction of VEGF-C.

20618 Background: Tumour cachexia is a complex syndrome characterized by a progressive weight loss. Ubiquitin and leptin are important mediators of protein and lipid turnover in muscle and adipose tissue. The objective of this study was to investigate the usefulness of plasma measurements of leptin and ubiquitin as prognostic marker of cachexia in advanced non-small cell lung cancer (NSCLC) patients. Methods: The cohort consisted in 50 patients with advanced NSCLC that received first-line therapy with platinum combinations. Patients received a median of 6 chemotherapy cycles and none nutritional supplementations. Concentration levels of leptin and ubiquitin were determined in plasma by a sandwich quantitative ELISA in the baseline, before therapy. In parallel were analysed 70 age-matched controls. Results: Median age was 61, range [20–78], 74% males, 37% stage IIIB and 63% IV and 86% PS 0–1. The histological subtypes were: 34% squamous cell carcinoma, 34% adenocarcinoma, 26% undifferentiated. Median leptin and ubiquitin levels differ significantly between controls and patients: leptin 14.479 vs 3.735 pg/ml (p=0.035) and ubiquitin 0.0686 vs 0.0294 AUs (p=0.018). Median leptin levels were higher in women 15.334 vs 3.160 pg/ml (p=0.002). Leptin and ubiquitin levels increased with the number of chemotherapy cycles but were only significant for leptin (p=0.044). There were not differences in basal or sequential levels of leptin and ubiquitin if we divided the cohort in patients with or without anorexia. Splitting the cohort in two groups attending to median levels of leptin and ubiquitin TTP and OS curves were not significantly different. Leptin and ubiquitin levels are not correlated with other clinical or anthropometrical variables. Conclusions: Leptin and ubiquitin levels are significabtly different between controls and patients but there is not a correlation between patient’s clinical characteristics and plasma levels of these cachexia mediators. No significant financial relationships to disclose.

15075 Background: Serum hTERT levels have been demonstrated as prognostic markers in several malignancies. VEGF is the most important angiogenic factor and EGFR is an oncogen with prognostic implications in cancer. Our objective was to analyze the prognostic role of serum concentrations of hTERT, VEGF and EGFR in metastatic colorectal patients and their association with other clinical variables. Methods: Retrospective analysis with 55 patients diagnosed of colorrectal carcinoma stages II-IV diagnosed between 2001 and 2005. Blood samples were collected before any clinical intervention. Concentration levels of VEGF and EGFR were determined by a sandwich quantitative ELISA. Serum free DNA was isolated by using Qiagen affinity columns and hTERT levels were assessed by RT-PCR using a Taqman assay. Results: Median age was 70 years; 60% males; 8% stage II, 56% stage IIIB and 36% IV; 62% curative and 38% palliative surgery; 58% not presented metastasis. Median hTERT levels increased significantly with stage: II, 298 ng/ml; III, 545; III, 1460; p=0.048. hTERT values differ significantly between those patients that received curative surgery versus those with palliative: 492 ng/ml vs 1224, p=0.042. EGFR, VEGFR and hTERT serum levels were not correlated with pre-operative CEA levels, presence of anaemia or metastasis or relapse after surgery. Splitting the cohort in two groups attending to median levels of serum EGFR, VEGFR and hTERT, time to progression and overall survival curves were not significantly different. Conclusions: hTERT levels, but not EGFR and VEGF increase significantly with disease severity or stage. We have not found any other correlation between these molecular mediators and any patient's clinical characteristics. No significant financial relationships to disclose.

The interaction of cadmium with genes involved in oxidative stress, cadmium metabolism and transport pathways on albuminuria can provide biological insight on the relationship between cadmium and albuminuria at low exposure levels.We tested the hypothesis that specific genotypes in candidate genes may confer increased susceptibility to cadmium exposure.Cadmium exposure was estimated by inductively coupled plasma mass spectrometry (ICPMS) in urine from 1397 men and women aged 18–85 years participating in the Hortega Study, a representative sample of a general population from Spain. Urine albumin was measured by automated nephelometric immunochemistry. Abnormal albuminuria was defined as urine albumin greater than or equal to 30 mg/g.The weighted prevalence of abnormal albuminuria was 6.3%. The median level of urine cadmium was 0.39 (IQR, 0.23–0.65) μg/g creatinine. Multivariable-adjusted geometric mean ratios of albuminuria comparing the two highest to the lowest tertile of urine cadmium were 1.62 (95% CI, 1.43–1.84) and 2.94 (95% CI, 2.58–3.35), respectively. The corresponding odds ratios of abnormal albuminuria were 1.58 (0.83, 3.02) and 4.54 (2.58, 8.00). The association between urine cadmium and albuminuria was observed across all participant subgroups evaluated including participants without hypertension, diabetes or chronic kidney disease. We observed Bonferroni-corrected statistically significant interactions between urine cadmium levels and polymorphisms in gene SLC30A7 and RAC1.Increasing urine cadmium concentrations were cross-sectionally associated with increased albuminuria in a representative sample of a general population from Spain. Genetic variation in oxidative stress and cadmium metabolism and transport genes may confer differential susceptibility to potential cadmium effects.