Tumor Biol. (2012) 33 (Suppl 1):S81–S116 DOI 10.1007/s13277-012-0540-y

ABSTRACTS

Poster Presentations

P1 CANCER STEM CELLS, CIRCULATING TUMOR CELLS P1.1 Circulating tumor cells as a biomarker for the patients with castration resistant prostate cancer M. Jančíková1, V. Mikulová1, O. Čapoun2, V, Soukup2, H. Honová3, T. Zima1 1

Institute of Medical Biochemistry and Laboratory Diagnostics; 2Department of Urology; 3Department of Oncology, General University Hospital in Prague, First Faculty of Medicine, Charles University in Prague Background: Castration resistant prostate cancer (CRPC) is an advanced stage of the prostate cancer. Patients suffering from the CRPC exhibit castration levels of testosterone, increasing levels of prostate specific antigen (PSA) and presence of metastases. Circulating tumor cells (CTCs) are rare cells detached from the primary tumor into the blood stream. They are an important part of the metastatic process. The presence of CTCs is thus highly probable in the blood of the patients with CRPC and it may allow us to study the role of CTCs in the metastatic process. Aims: Our aim is to monitor the presence of CTCs in the blood of the patients with CRPC in the different stages of the treatment. Ultimately, by comparing our data with the clinical information we shall evaluate the potential of CTCs as a prognostic marker for CRPC. Methods: We enriched the fraction of CTCs from the peripheral blood of the patients by the immunomagnetic beads coated with antibodies against EpCAM and HER2 (AdnaGen, Germany). We isolated mRNA from this fraction (AdnaGen, Germany). Using the PCR methods we determined the expression of tumor-associated genes: PSMA, PSA and EGFR. Blood samples were drawn at the time of CRPC diagnosis, after the 4th cycle of systemic therapy and after the complete treatment.

Results: Ten patients with CRPC have been tested for the presence of CTCs from December 2011. Initially, all of them were identified as CTCs positive with high levels of expression of PSA. PSMA and EGFR have been expressed in very different levels. This shows that the CTCs are heterogeneous and could be used as a marker for personalised therapy. After the 4th cycle of systemic therapy, we observed a decrease in the levels of the studied genes. One patient was even CTC negative. Conclusion: Our first results indicate that the CTCs can possibly serve as a powerful prognostic marker and as a marker for the therapy efficiency for the CRPC patients. We believe that further results will reveal the possibility of their use in personalised medicine.

P1.2 The PI3K/Akt/β-catenin/CBP pathway regulates plasticity between the cancer stem-like and non cancer stem-like states Kaijie He, Tong Xu, Yucheng Xu, Michael Kahn and Amir Goldkorn University of Southern California Background: Cancer stem-like cells (CSC) – a subpopulation of self-renewing, tumorigenic, drug resistant tumor cells – are thought to promote cancer formation, therapy resistance and disease progression. Recently, we and others reported that CSC can arise through direct conversion of non-CSC to CSC. Aims: We investigated the role of PI3K/Akt/β-catenin/CBP signaling in mediating the phenotypic plasticity between the non-CSC and CSC states. Methods: Fluorescence activated cell sorting (FACS) and Hoechst dye exclusion were applied to breast and bladder cancer cell lines to isolate side populations (SP) of cells enriched for CSC properties (high tumorigenicity and drug resistance) versus non-side-populations (NSP) of cells

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lacking these properties. GFP(+) NSP were re-combined with GFP(–) SP to enable tracking of the two phenotypes in culture over time. The cells were treated with pharmacological and siRNA inhibitors targeting members of the PI3K/Akt/β-catenin/CBP pathway, and their effects on SP size and NSP-to-SP conversion were measured. Results: Inhibition of PI3K/Akt signaling with pharmacologic agents (LY294002, Akt inhibitor IV) or with anti-AKT siRNA significantly reduced NSP-to-SP conversion. PI3K/Akt inhibition exerted these effects in part by inducing an active (de-phosphorylated) state of GSK3β, a downstream protein that potentiates β-catenin degradation. Conversely, inhibition of GSK-3β with BIO (6bromoindirubin-3’-oxime) induced β-catenin de-

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phosphorylation, activation and nuclear translocalization, resulting in significantly increased SP size, whereas siRNA knockdown of β-catenin signficantly decreased SP size. Further downstream, blocking β-catenin’s interaction with its co-factor CBP using the specific inhibitor ICG001 significantly decreased SP size and sphere formation, as did siRNA knockdown of CBP. Conclusion: Our results demonstrate that PI3K/Akt/β-catenin/CBP signaling plays a key role in mediating phenotypic plasticity between non-CSC and CSC. Therapeutic targeting of this pathway may therefore disrupt the replenishing of CSC from the non-CSC population, thereby overcoming the therapy resistance and disease progression attributed to the CSC phenotype.

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P2 EPIGENOMICS / PROTEOMICS P2.1 Evaluation of primary brain tumors utilizing the ion ampliseqTM cancer panel for the detection of 739 cosmic mutations in 46 cancer genes Kenneth L. Muldrew and Jennie L. Lovett University of Toledo Medical Center, Toledo, Ohio, USA Background: Many malignant primary central nervous system tumors have a poor prognosis, and therapy remains limited. Gliomas exhibit point mutations in isocitrate dehydrogenase, 1p and/or 19q loss, and show perturbations in the RTK, p53, and Rb pathways. Nextgeneration sequencing has allowed for rapid discovery of these types of mutations in different tumor types and may become more useful in the clinical laboratory setting for prognosis and therapy. Aims: We describe utilization of the Ion AmpliseqTM Cancer Panel assay and the IonTorrent Personal Genome machine (PGM) to identify mutations in 46 tumor suppressor and oncogenes from brain tumor samples. Methods: DNA was extracted from unstained paraffinembedded tissue slides of sixteen primary brain tumors

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including 7 glioblastoma multiforme, 4 oligodendrogliomas, 2 astrocytomas, 1 mixed oligoastrocytoma and 2 ependymomas. Single tube multiplex (190 amplicons) PCR amplification was performed and libraries were prepared using the Ion AmpliseqTM Cancer Panel (Applied Biosystems, USA). The libraries were sequenced using the Ion PGM and data was analyzed using the Ion Torrent browser, Variant Caller plugin, and Integrative Genomics Viewer (IGV). Variants calls were considered true variants if the region of interest had 100 or greater fold coverage. Results: One of 16 samples yielded low quality sequencing data and was excluded. Thirty-one mutation variants in 17 genes were detected by the assay. 29 of 31 variants were point mutations (19 non-synonymous), and 2 were deletions. As expected for the gliomas, mutations were detected in the EGFR, TP53, and PDGFRA genes. Compared to Sanger sequencing for IDH1 mutations, the assay correctly classified all 15 samples. 14 samples had a single point mutation in the RET gene associated with Hirschsprung’s disease and Medullary Carcinoma of the thyroid. Thirteen specimens had a single point mutation in the APC gene. Conclusion: The Ion AmpliseqTM cancer panel is robust, and correctly identified the IDH1 mutation status in all fifteen brain tumor samples, and detected mutations in genes previously known to be involved in glioma oncogenesis.

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P3 BREAST CANCER

P3.2 Targeting Breast Cancer Stem Cell by Glycomic Approach

P3.1 Discordance between molecular markers (ER, HER2 and TOP2A) before and after neoadjuvant chemotherapy (NAC) in locally advanced breast cancer (LABC)

Yu, AL; Chen, IJ; Lin, YC; Lin, JJ; Yeo, HL; Chang, CH; Lin, RJ; Liao, KH; Lee, CH; Hsu, CW; Yu, JC

M.K.Tuxen1, E.Balslev2, S.Cold3, U.B.Tange4, D.Carlsen1, D.L.Nielsen1 Department of Oncology, Herlev University Hospital, Herlev1, Department of Pathology, Herlev University Hospital, Herlev2, Department of Oncology, Odense University Hospital, Odense3, Department of Oncology, Rigshospitalet, Copenhagen4, Denmark. Background: Studies comparing samples from primary tumor with corresponding relapsed tumor have demonstrated discordances in ER, PgR and HER2 status. The knowledge about influence of NAC in LABC on expression of molecular markers is limited. Aims: Aim of the present analysis was to investigate and compare ER, HER2 and TOP2A status between core needle biopsies before NAC and surgical specimens after NAC. Methods: Patients (pts) with LABC were enrolled in Danish neoadjuvant trial PO050903. Pts received 4 cycles of pegylated liposomal doxorubicin (Caelyx®) 35 mg/m2 and cyclophosphamide 600 mg/m2 on q21d followed by 4 cycles of docetaxel 100 mg/m2 q21d. Pts with HER2-positive tumors were concurrently treated with trastuzumab 8→6 mg/kg q21d for 8 cycles. ER was determined by immunohistochemistry, TOP2A by FISH and HER2 by both analyses. Results: 47 pts, out of 49 enrolled pts, were assessable for response evaluation. 10 pts (21 %) had HER2-positive tumor and 7 (15 %) had triple negative tumor. Median tumor size was 7 cm (range 2-15 cm). Clinical response rate was 83 %, 3 pts (6 %) had a clinical complete response and 36 (77 %) had a partial response. Pathological complete response (pCR) rate was 19 % (9 pts). ER and HER2 were evaluated in 47 patients. ER decreased in one pt (2 %). HER2 expression changed in 6 pts (13 %), from negative to positive in 5 pts (11 %) and from positive to negative in 1 pt (2 %). We were able to evaluate TOP2A on 44 paired specimens before and after NAC. TOP2A data will be presented at the conference. Conclusion: Our study demonstrated that there is indeed a change in molecular markers before and after NAC in pts with LABC. Discordance of HER2 expression tended to be relatively high in this study (13 %). HER2 status and ER status should be re-evaluated on surgical tissue specimens after NAC in order to select optimal adjuvant treatment.

Genomics Research Center, Academia Sinica, Taiwan; University of California in San Diego, USA; General Surgery, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan Background: Aberrant glycosylation is a feature of cancer cells. GD2 is highly expressed in neuroectodermal tumors. I have pursued immunotherapy of neuroblastoma with antiGD2 from preclinical to international phase III randomized trial leading to a dramatic 20 % increase in the cure rate in high risk neuroblastoma. This is the first mAb targeting a glycan shown to be effective for cancer immunotherapy Another potential glycan target is Globo H, a hexasaccharide overexpressed on many epithelial cancers such as colon, ovarian, gastric, pancreatic, lung, prostate and breast cancers, etc.. Aims: To examine whether Globo H is expressed in breast cancer stem cells (BCSCs), and examine the role of its synthetic enzymes. Methods: FACS analysis of glycan expression in clinical breast cancer specimens and studies of the effects of silencing/overexpression of genes involved in Globo H synthesis on breast cancer survival. Results: We examined 53 human breast cancer specimens and found Globo H to be present in BCSCs, although to a much lesser extent than non-BCSCs. We also showed for the first time the expression of Gb5, the precursor of Globo H, in BCSCs in >60 % of tumors. Relapse-free survival was significantly higher in Globo H negative patients (13/44) than Globo H positive patients (p<0.05).Immunization of mice with Globo H-KLH induced antibody reactive with not only Globo H but also Gb5, suggesting that Globo H-based vaccine will target BCSCs. These findings pave the way for our development of a phase II/III clinical trial of Globo H vaccine for metastatic breast cancer, which is ongoing in Taiwan, Hongkong and USA. In addition, we delineated the involvement of both fucosyl transferases 1 and 2 (FUT1/ FUT2) which catalyze α1,2-linked fucosylation, in Globo H synthesis. Silencing and overexpression of FUT1/FUT2 revealed that both FUT1 and FUT2 play an important role in the growth, adhesion, migration and mammosphere forming capacity of breast cancer, and may serve as a therapeutic target for breast cancer. Conclusion: Immunotherapy targeting Globo H or inhibition of its synthetic enzymes, FUT1/FUT2 may provide effective therapy directed against breast cancer stem cells.

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P3.3 Paclitaxel blood management: analytical validation of a rapid automated immunoassay P. Kenny1, D. J. Cline1, G. D. Lundell1, H. Zhang1, R. L. Harney1, H. K. Riaz1, Y. Li1, J. B. Courtney1, I. Baburina1, C. Delaney2, R. A. Hilgar3, M. Miyazaki4, S. J. Salamone1. Saladax Biomedical, Inc., Bethlehem, USA, 2Quest Diagnostics, Valencia, USA, 3University Hospital Essen, Germany, 4FALCO biosystems, Ltd., Kyoto, Japan

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P3.4 Relationship between plasma homocysteine and CK19 and c-erb-B2 expression in breast cancer patients receiving chemotherapy F.L.A. Fonseca 1,2 , L.A. Azzalis 2 , B.T. Marinelli 1 , R. Yoshihara1, R.K. Kunyioshi1, F. Gerhke1, B. Alves1, V.A. Vilas Boas1, A. Del Giglio1

1

Background: Paclitaxel (Taxol®, Abraxane®) is used in many regimens for breast, non-small-cell-lung and ovarian cancer. Paclitaxel (PTX) is a candidate for dose management: multiple studies have demonstrated a greater than 10fold interpatient variability of clearance rates with BSAbased dosing, a pharmacokinetic (PK) pharmacodynamic relationship has been demonstrated, and high exposure has been shown to be a good predictor of severe & febrile neutropenia which is experienced by > 90 % of all patients treated with PTX. The requirements to run physical methods limit their use in the clinical laboratory for PTX dose management. Aims: This study was undertaken to validate a PTX immunoassay assay as a tool to identify and manage patients at high risk of suffering from extreme toxicity on regimens containing PTX. Methods: A PTX immunoassay (MyPaclitaxel™) developed at Saladax was used on the Beckman AU400/640 and Roche c111. Precision, accuracy and linearity were evaluated by CLSI protocols at four laboratories. Method comparison was done versus a validated HPLC-MS/ MS method using samples (n 0113) collected from patients on PTX therapy in accordance with an IRB approved protocol. Results: The following results were generated in our laboratory and verified at three external laboratories. Three controls and four patient pools were used in precision studies. Reproducibility CV was 2.2 % - 5.5 %, and within-laboratory CV 3.0 - 9.9 %. Linearity was demonstrated from 30 to 300 ng/mL. Lower limits of detection and quantitation were 11 and 19 ng/mL, respectively. The method comparison produced R200.9956 for the linear regression, with slope 0 1.002 and intercept 0 -3.029. Cross-reactivity in the assay was ≤5 % for co-administered drugs, <1 % for unrelated drugs, and ≤3.4 % for major metabolites. Conclusion: The analytical performance of an automated immunoassay for PTX has been validated, providing a tool for the individualized dosing using PK-guided dose management.

Oncology Department – Faculdade de Medicina do ABC, Santo André, SP, Brazil 2Instituto de Ciências Químicas, Farmacêuticas e Ambientais – Unifesp, Diadema, SP, Brazil 1

Background: Breast cancer is the most common cancer among women and the second most common in the world. In Brazil, mortality rates remain high for breast cancer, probably because of late diagnosis. In solid tumors, latent cells may spread from the primary tumor and remain in the body after the removal of the tumor and they are not easily detectable by conventional methods. These cells correspond to minimal residual disease (MRD). CK-19 and c-erb-B2 seem to be markers of MRD and may be modified in breast cancer patients undergoing chemotherapy. Their expression in the mononuclear fraction of peripheral blood and their correlation with plasma homocysteine could clarify whether tumor cells circulating in peripheral blood contribute to the thromboembolic event frequently observed in patients receiving chemotherapy. Aims: To evaluate CK-19 and c-erb-B2 expression at diagnosis of breast cancer, 3 and 6 months after the beginning of the treatment and correlate them with both plasma homocysteine and clinical data. Methods: RNA samples obtained from peripheral blood of 35 breast cancer patients and from 23 healthy women were analyzed. Results were correlated to clinical data. Results: Differences were observed in the expression of CK-19 and c-erb-B2 between healthy women and women with breast cancer at diagnosis. Changes in the expression of those markers at 3 and 6 months were also found. On the other hand, no statistically correlation between homocysteine and the expression of genes CK-19 and c-erb-B2 were found. Concerning clinical data, there was statistically significant correlation (p0 0.009) only between homocysteine and menopausal status. Conclusion: Results have shown that plasma homocysteine was increased in breast cancer patients undergoing chemotherapy and it was associated to the treatment. Moreover, other markers could be related to homocysteine, but they were not assessed in this study.

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P3.5 Proliferative background in healthy women carriers of BRCA1/2 mutation

P3.6 Usefulness of new biomarkers in breast tumors diagnostics in comparison with traditional tumor markers

B. Nisman1, L. Kadouri1, T. Allweis1, T. Hamburger1, S. Gronowitz2, T. Peretz1

R. Kucera1, S. Svobodova1,3, A. Narsanska2, O. Topolcan1, M. Cerna2, J. Vrzalova1, R. Fuchsova1, Zedníková I., I. Treskova2, V. Treska2

Department of 1Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel and 2Group of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden. Background: BRCA1 and BRCA2 are important tumorsuppressor genes involved in the control of cell growth. We hypothesized that mutations in BRCA1/2 genes may lead to the changes in serum expression of proteins implicated in cell proliferation. Aims: We investigated the association of BRCA1/2 mutation carriage with serum activity of thymidine kinase 1 (TK1) and serum levels of extracellular domain of Epidermal Growth Factor Receptor 1 (sEGFR). Methods: TK1 and sEGFR were determined with DiviTum (Biovica) and Quantikine (R&D systems) ELISA assays respectively using two groups of women namely, control i.e. healthy blood donors (Group 1, n 070) matched for age with healthy carriers of BRCA1/2 mutation (Group 2, n070). Results: The analysis of the 140 serum samples from the two groups did neither reveal any correlation between age and sEGFR or TK1 (r00.01 and r00.02), nor between the two biomarkers (r00.09). The TK1 activity in women from Groups 2 was found to be significantly higher than in the control Group 1 (p<0.001). Both BRCA1 and BRCA2 mutation carriers from Group 2 demonstrated higher serum TK1 activity than control women (for all p<0.001). In Group 2 significantly higher sEGFR was found in serum of women with BRCA1 compared to those with BRCA2 mutation (p00.006) and control women from Group 1 (p 00.013). The continuous relationship between two markers level and risk of BRCA1/2 mutation carriage as logarithmic function was found (TK1 for BRCA1/2 carriage: 2.1, p00.001 and sEGFR for BRCA1 carriage: 1.07, p00.005). Elevated combined proliferative index of two markers adjusted for age was found in 39 % of women from Group 2, and 14 % of them during followup developed breast cancer (BC) with median time to diagnosis of 19 (range 13-22) months. Elevated proliferative index was significantly associated with development of BC (p00.02). Conclusion: Increased proliferative background is an important property of healthy women carrieying BRCA1/2 mutation indicating an increased risk for development of BC.

Laboratory of Immunoanalysis, 2Department of Surgery Faculty Hospital in Pilsen and Medical Faculty in Pilsen and 3 IIIrd Internal Medicine Clinic, 1st Medical Faculty, Charles University in Prague, Czech Republic. 1

Background: Based on WHO data the breast cancer is one of the most frequent cancer types among females worldwide. Mortality rates tend to increase continuously. Aims: Study Aim is to evaluate if it would be possible to use the new biomarkers for a brief evaluation of the clinical status of patients and distinguishing between benign and malignant tumors prior to biopsy and histological examination. We compared these new possibilities with the traditional tumor markers for breast tumors. Methods: Group of patients consisted of 115 females, 89 with breast cancer and 26 with benign breast tumors. IGF1 serum levels were measured using an IRMA (Immunotech, France). IGFBP3 serum levels using an IRMA (DiaSource, Belgium). Leptin, HGF, EGF, TGF and VEGF were measured using an xMAP Luminex multiplex panel (Merck,USA). Serum levels of CEA and CA 15-3 were measured using a DxI instrument (Beckman Coulter, USA). TPA-M and MonoTotal were measured using IRMA (IDL, Sweden). Serum samples were collected prior to surgery. The SAS 9.2 was used for all statistical analysis. Results: HGF and Leptin were found to show a statistically significant difference between the two groups. The mean of HGF in malign diseases prior to surgery was 3 370 pg/mL compared to 1 799 pg/mL in benign tumors with ρ00.0016. The mean of Leptin in malign diseases prior to surgery was 25 729 pg/ml compared to 13 522 pg/mL in benign tumors with ρ00.0458. Other biomarkers were not significant. Conclusion: Only HGF and Leptin seem to be promising new potential markers for distinguishing between benign and malign tumors. Other tested biomarkers are not useful to distinguish between malignant and benign disease. Traditional tumor markers are currently used for therapy effect monitoring of cancer diseases. Our findings fully correspond to this practice. Acknowledgements: Supported by the project of MOH of the Czech Rep. for conceptual development of research organization 00669806 - Faculty Hospital in Pilsen.

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P3.7 Dachshund homologue 2 is a marker of good prognosis in breast cancer

P3.8 Level changes of steroid-linked proteins as potential diagnostic and prognostic criteria in breast cancer

Marie Fridberg 1 , Björn Nodin 1 , Mathias Uhlén 2,3 , Karin Jirström1

N. Scvortsova, D. Nikulina, A. Kokhanov, T. Vorobyeva, O. Dolina

1

Department of Oncology and Department of Biochemistry, of State Medical Academy, Astrakhan, Russia

Department of Clinical Sciences, Pathology, Lund University, Skåne University Hospital, 221 85 Lund, Sweden, 2 Department of Proteomics, AlbaNova University Center, Royal Institute of Technology, 106 91 Stockholm, Sweden, 3 Science for Life Laboratory, Roy Background: Breast cancer is the most common form of cancer in women and 1 of 9 will be diagnosed with this disease during their life time. There are several clinically distinct subgroups of breast cancer, differing in prognosis, and the diagnostic criteria of today are not sufficient to clearly distinguish between these groups. Hence, there is an urgent need to identify new prognostic and treatment predictive biomarkers for this cancer form. While Drosophila has a single DACH gene, two DACH genes have been identified in humans: DACH1 and DACH2. We recently reported the association between a high DACH2 expression and poor prognosis in epithelial ovarian cancer, a novel finding indicating an important role for this protein in tumorigenesis. Here, we report for the first time a connection between a high protein expression of DACH2 and good prognosis in breast cancer. These findings are in line with previous reports for the more extensively studied DACH1 protein, which has been coupled to poor prognosis in ovarian cancer and to good prognosis in breast cancer. Aims: The present study aimed at investigating the prognostic role of DACH2 in breast cancer. Methods: The protein expression of DACH2 was analysed by immunohistochemistry in tissue micro arrays (TMA) with tumours from 498 breast cancer patients. Nine breast cancer cell lines was analysed by Western blot for the expression of DACH2 and DACH1. Results: A significant correlation with DACH2 to ER (p<0.001), PR (p00.047) and HER2 (p00.008) status was observed in the tumours analysed. DACH2 expression was also found to be an independent prognostic marker for a prolonged disease free survival (p00.003) in the cohort. Nine breast cancer cell lines were analyzed for DACH2 expression by western blot and 6 of them showed a strong expression of the protein. All cell lines showed DACH1 expression. Conclusion: Our results demonstrate that a high expression of DACH2 in breast cancer tumors is an independent prognostic marker for good prognosis and correlates to ER, PR and HER2 expression.

Background: In the previous study, we obtained data on level changes of some steroid-linked proteins in biological fluids of cancer patients. Among these proteins were pregnancy associated alfa2-glycoprotein (α2-PAG) (in different malignant tumors), sex hormone binding globulin (SHBG) and transcortin (in hormone-dependent tumors). Aims: Investigate the dependence of the level of steroidrelated protein on the condition of patients with breast cancer before and after treatment. Methods: Proteins were determined by immunochemical methods (ELISA, IDA) in samples of blood serum of 150 patients with breast cancer. Monitoring the level of SHBG, α2-PAG and transcortin in the blood of women was made from diagnosis until the end of combined treatment. After 6 years was analyzed the dynamics of the disease in most cases (estimated duration and the state of remission, one-year mortality and 5-year survival rate of patients). Results: Patients were divided into groups according to the level detected in beginning of disease steroid-related proteins. SHBG level in the blood of breast cancer patients aged under 59 increased in 2 times as high as normal level, but transcortin decreased by half its normal level. Lack of SHBG level increase in patients aged 60 and upwards evidences that in this woman group of breast cancer patients a concurrent action of estrogen and glucocorticoid excess favorable to tumor process development in mammary gland. α2-PAG increased 1.7-fold higher than in the healthy group. At the level of α2-PAG below 3 mg/l average life of 83.3 % of patients was 2-4 years, at more 5 mg/l 93.2 % women have the long-term remission of five years or more. At a high level of SHBG are 75 % of patients in remission over 6 years. A similar correlation was not found for transcortin. A higher α2-PAG level in patients with positive dynamics can be associated with its participation in the formation of the active state of the immune system. The level of SHBG shows quantitative changes in the profile of steroid hormones. Conclusion: Level detection of these proteins may help to assess the hormonal status of patients with breast cancer, the effectiveness of the treatment, and possibly make a prognosis about the dynamics of disease progression.

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P4 OVARIAN CANCER

P4.2 Expression of TGF-β and its receptor variants in ovarian carcinoma

P4.1 The comparison of ca 125 and he4 dynamics in ovarian cancer (oc) patients monitoring

N. Gutgold1, B. Davidson2,3, R. Reich1 1

N.S. Sergeeva, N.V. Marshutina, I.I. Alentov, M.P. Solokhina, I.A.Korneeva, E.G. Novikova FSBI “Moscow P.A. Hertzen Research Oncological Institute” Ministry Health RF, Moscow, Russia Background: СА 125 is the serological tumor marker traditinally used for ovarian cancer (OC) patients monitoring. However, some patients do not exhibit increasing levels of СА 125 in clinical relapse. A marker that may complement CA 125 in this setting is HE4 Aims: The aim of this investigation was therefore to compare CA 125 and HE4 dynamics in monitoring of OC patients. Methods: Levels of CA 125 and HE4 were measured in serum of 31 OC patients on ARCHITECT (ABBOTT Diagnostics, USA). The mean monitoring duration was 25,6 month (18-64 month). In total, 514 СА 125 tests and 478 НЕ4 tests were performed. For all measured points the radio of absolute marker level to its discriminative level (DL) was calculated. As DL for СА 125 35 U/ml and for НЕ4 - 70/140 pmol/l (in accordance with menopausal status before treatment) were used. Additionally, the cofficients of rank correlation (CRC) between CA125 and HE4 levels were calculated for all steps of treatment and monitoring. The scheme of OC patients treatment: neoadjuvant chemotherapy (NCh), surgery, adjuvant chemotherapy (ACh), chemotherapy (Ch) of proved relapse. Results: Before treatment, the levels as CA 125 and HE4 were increased in serum of all patients. Before treatment, the multiplicity excess of DL for CA 125 was equal to 83,1 and for HE4 equal to 8,0. The average decreasing dynamic of CA125 and HE4 during NCh was similar. After surgery in 46 % of cases, only one of these two markers stay increased. After 6 courses of Ach, both CA125 and HE4 levels became “normal”. In 36 % cases of proved relapse one of two markers stay in normal limits (in 22,5 % cases - СА 125 and in 13,5 % cases – НЕ4). In all other cased both CA 125 and HE4 levels were elevated. CRC between markers levels before treatment was 0,6, in remission -0,05, in proved relapse - 0,36. The multiplicity DL excess in point of proved relapse was equal 11,1 for CA125 and 2,5 - for HE4. The alterations of CA125 and HE4 during chemotherapy of relapses in some cases significantly different. Conclusion: HE4 levels in some cases gave the additional information to CA 125 in monitoring therapy in OC patients.

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, 2Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway 3 The Medical Faculty, Unive Background: Transforming growth factor-β (TGF-β) initiates a wide range of cellular responses, which may be opposing ones. These include proliferation, differentiation, adhesion, invasion, progression and apoptosis, depending on the target cells. Alternatively-spliced transcript variants encoding different isoforms have been identified for one of the TGF-β ligands (TGF-β2) and for all three TGF-β receptors. Ovarian cancer (OC) is the leading cause of death from gynecological cancers in Western countries. The disease is usually diagnosed at an advance stage (FIGO stages III–IV). Primary solid tumors, solid metastases, and effusions in the peritoneal (ascites) and pleural cavities characterize the tumor as it progresses. The role of TGF-β in ovarian cancer progression has not been studied in depth. Aims: To investigate the expression and clinical role of TGF-β and its receptors in OC. Methods: Hundred human ovarian carcinoma biopsies of primary, solid metastases and pleural and peritoneal effusions were analyzed for splice variant ligands and receptors of TGF-β using real-time PCR. Results: All receptors were expressed more dominantly in effusions when compared to primary tumors and solid metastases. (TGF-β-R1a p00.01, TGF-β-R1b p00.001, TGFβ-R2a,b p<0.0001, TGF-β-R3 p<0.004). TGF-β1 and TGF-β2a were expressed more dominantly in effusions when compared to primary tumors and solid metastases (p00.002), while TGF-β3 was expressed more dominantly in metastases when compared to primary tumors and effusions (p00.007). Significant correlation was also observed with clinical parameters: stage and grade regarding TGF-β2b and TGFβ-R1a expression, resistance to chemotherapy regarding TGF-β-R2a expression. Conclusion: It is concluded that TGF-β-R2a might dominate progression from solid tumors to effusions. To our best knowledge, this is the first study to show association between anatomic site, tumor progression and treatment outcome and expression of TGF-β and its receptors in OC.

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P4.3 Expression of the global regulator SATB1 is an independent factor of poor prognosis in high grade epithelial ovarian cancer 1§

Björn Nodin , Charlotta Hedner Marie Fridberg1, Karin Jirström1

1,2

, Mathias Uhlén

3,4

,

P4.4 Comparison of HE4 with other tumor markers in ovarian cancer diagnostics S. Svobodova1,3, R. Kucera1, O. Topolcan1, J. Presl2, J. R. Fuchsova1,Vrzalova1, Z. Rokyta2, L. Betincova2, Z. Novotny2 Laboratory of Immunoanalysis, 2Department of Gynecology and Obstetrics, Faculty Hospital in Pilsen and Faculty of Medicine, Pilsen, 3IIIrd internal Medicine Clinic, 1st Medical Faculty, Charles University in Prague, Czech Republic

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Background: The global gene regulator Special AT-rich sequence-binding protein 1 (SATB1) has been reported to reprogramme tumour cells into a more malignant phenotype and associate with poor clinical outcome in several cancer forms. Aims: In this study, the expression and prognostic impact of SATB1 was examined in epithelial ovarian cancer (EOC). Methods: Immunohistochemical expression of SATB1 was examined in tissue microarrays with 151 incident EOC cases from two prospective, population-based cohorts. Kaplan Meier analysis and Cox proportional hazards modelling were used to assess the impact of positive vs negative SATB1expression on ovarian cancer specific survival (OCSS) and 5-year overall survival (OS). The effects of siRNA-mediated silencing of SATB1 on the expression of hormone receptors, Ki67 and checkpoint proteins in A2780 ovarian cancer cells were examined. Results: Positive SATB1 was denoted in 35/151 (23.2 %) cases.There was an inverse correlation between SATB1 expression and histological grade (R00.23, p00.005) and a positive correlation with DACH2 (R00.0.28, p00.001), pChek1 (R00.26, p00.002) and minichromosome maintenance protein 3(MCM3) (R00.17, p00.042). Univariable Cox regression analysis revealed that SATB1 expression, while not prognostic in the full cohort was a negative prognostic factor for both OCSS and 5-year OS in high grade tumours (n 0105) (HR02.20, 95 % CI 1.29-3.75 and HR 0 HR02.00, 95 % CI 1.14-3.50, respectively). This association remained significant in multivariable analysis, adjusted for age and clinical stage (HR02.23, 95 % CI 1.23-4.07 for OCSS and HR02.06, 95 % CI 1.11-3.03, respectively). Conclusion: These results demonstrate that SATB1 expression is an independent factor of poor prognosis in high grade EOC and correlates with cellular processes involved in the maintenance of DNA integrity. Further investigations into the underlying mechanisms and potential clinical utility of these observations are warranted.

Background: Ovarian cancer is the leading cause of gynecological cancer death, representing 5 % of all cancers in women. It has a poor prognosis, mainly because of the late detection. Mortality is still high. Aims: Study Aim is to evaluate the benefits of the determination HE4 and to evaluate if it is possible to use it to distinguish between benign and malignant tumors. We compared the HE4 marker with the following tumor markers (CA 125, CA 19-9, CEA, TK, TPS and MonoTotal). Methods: 176 females were divided into two groups. The first consisted of 19 females with ovarian cancer and the second of 157 females with benign ovarian tumors. Serum levels of HE4 were measured using an EIA kit (Fujirebio Diagnostics, Sweden). Serum levels of CA 15-3, CEA and CA 19-9 were measured using a DxI instrument (Beckman Coulter, USA). TK was measured using RIA (Immunotech, Czech Republic). TPS and MonoTotal were measured using IRMA (IDL, Sweden). The SAS 9.2 was used for all statistical analysis. The Wilcoxon test was used to compare distributions of values between benign and malignant tumors. Results: When HE4 was evaluated at 95 % specificity, the HE4 cut-off was 86.1 pmol/L at a sensitivity of 83.33 %, PV + of 68.18 %, PV- of 98.01 % and AUC of 0.9534. Comparing the parameters of serum levels between the benign and malignant groups of patients HE4 a statistically significant difference were found (p<0.0001). CA 125, CEA, TPS and MonoTotal show a statistically significant difference, too. CA 19-9 and TK were not significant. Conclusion: Determination of HE4 levels is an appropriate tool for improving primary detection of ovarian cancer. HE4 together with CA125 and CEA broadens the range of differential diagnosis. TPS and MonoTotal confirmed their status of markers of proliferation. These two markers can be used to monitor the activity and aggressiveness of the tumor. Acknowledgements: Supported by the project of MOH of the Czech Rep. for conceptual development of research organization 00669806 - Faculty Hospital in Pilsen.

Department of Clinical Sciences, Division of Pathology, Lund University, 2University and Regional Laboratories Region Skåne, SE-221 85 Lund, Sweden, 3Department of Proteomics, AlbaNova University Center, Royal Institute of Technology, 106 91 Stockholm, S

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P4.5 Serum Human Epididymis Protein 4 (HE4) levels are less frequently elevated than CA125 in women with different types of endometriosis JM. Escudero*, M. Rius+, MA. Martínez-Zamora+, P. Fusté+, JM Augé*, X. Filella*, J. Balasch+, F. Carmona+, R. Molina* +Institut Clínic of Gynecology, Obstetrics and Neonatology. *Laboratory of Clinical Biochemistry. Hospital Clínic of Barcelona Background: Elevations of CA125 suggesting malignancy are a problem in premenopausal women with endometriosis. There are two types of endometriosis: ovarian endometrioma (OE) or deep infiltrating endometriosis (DIE). Human epididymis protein 4 (HE4) is a new biomarker for ovarian cancer but its serum levels in endometriosis patients are not well known. Aims: We evaluated serum concentrations of HE4 and CA125 in women with various types of endometriosis. Methods: CA 125 and HE4 serum concentrations were determined in 114 women with endometriosis before laparoscopic surgery. Benign endometriosis was confirmed in all cases during surgery and they were classified as having ovarian endometrioma (OE) alone (37 patients), deep infiltrating endometriosis (DIE) alone (30 patients) or both (47 patients). Results: 43 patients presented with serum CA125 levels higher than 40 U/mL (17 OE alone, 7 DIE alone and 19 OE + DIE) and 13 patients had serum HE4 levels higher than 150 pmol/L (7 OE alone, 3 DIE alone and 3 OE + DIE) (p<0.0001). The comparison of serum concentrations of HE4 between the three groups showed higher levels of HE4 in OE alone patients compared to DIE alone patients (p00.04) (OE alone:49.7+/-18.2; DIE alone:40.2+/-15.4; DIE + OE:42.9+/-13.5). The comparison of serum concentrations of CA125 between the three groups showed no statistical differences (p00.2) (OE alone:48.1+/-47.7; DIE alone:39.9+/-36.2; DIE + OE:29.7+/- 31.8). Conclusion: HE4 is elevated less frequently than CA125 in endometriosis patients. Patients with OE alone have more frequently increased HE4 levels than patients with DIE alone.

P4.6 Kallikreins 6 and 10 are prognostic markers of ovarian cancers V. Barak, Y. Sherman, V. Doviner, D.Edelman, T.Peretz and E.P. Diamandis

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Immunology Lab. for Tumor Diagnosis, Oncology Dep., Pathology Dep., Hadassah-Hebrew University Medical Center, Jerusalem, Israel and Pathology Lab., Medicine Dep, Mount Sinai Hospital, Toronto, ON, Canada Background: Previous studies indicated that 2 members of the human kallikrein (KLK) family, KLK6 and KLK10, are highly expressed in epithelial Ovarian Cancers and may serve as independent adverse prognostic markers. Their expression in Ovarian Cancer cytosolic extracts correlated well with unfavorable prognosis in patients with otherwise apparently good prognosis (KLK6) and in the late-stage ovarian cancer group (KLK10). Aims: we examined the immunostaining of the various Ovarian Neoplasms and evaluated correlations between nits intensity and the histological sub-typing and grading of tumors, defining the potential use of them as a morphologicprognostic marker. Methods: We analyzed specimens from28 normal ovarian tissues, 29 ovarian tissues with benign epithelial neoplasms, 20 with various mesenchymal tumors and 49 carcinomas, various histological sub-types. We employed KLK6 andKLK10-specific polyclonal rabbit antibody and avidin-biotin to localizeKLK6 and KLK10 respectively, by IHC. Results: Both KLK s were markedly expressed in the cytoplasm of carcinoma cells of various histological types and grades. They were absent in normal stromal cells ,in tumor cells of mesenchymal origin, and negligibly expressed in cells of the epithelial component of benign ovarian neoplasms. The staining intensity in the positive carcinoma cells was found to differentiate well between the main two histological subtypes, so that the serous papillary carcinoma portrayed, a higher staining intensity than the mucinous carcinoma. Comparison of staining characteristics in the subgroups (histological grading), revealed a tendency towards incereasing intensity of expression the higher the tumor grade, particularly accentuated in foci of Anaplastic Ca. Correlations between high KLKs expression and high CA 125 serum levels, were demonstrated (p = 0.04). Conclusion: Our results indicate that IHC expression of both KLK6, KLK10 in Ovarian cancers follows the path of the classical morphological criteria for tumor aggressiveness, and may be a useful diagnostic tool for more accurately defining therapeutic response of the tumors.

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P5 LUNG CANCERS

P5.2 Evaluation of thymidine kinase 1 activity in the serum of lung cancer (LC) patients

P5.1 Computed tomography findings predicting invasiveness of thymoma

B. Nisman1, H. Nechushtan1, H. Biran2, S. Gronowitz3, T. Peretz1

Edith M. Marom, Cesar A. Moran, Ping Liu, Arlene M. Correa, Edward S. Kim, Ritsuko Komaki, Jeremy J. Erasmus, Wayne L. Hofstetter, David C. Rice, Stephen G. Swisher

Department of 1Oncology, Hadassah and Hebrew University Medical Centre, Jerusalem, Israel. 2Lung Cancer Unit, Division of Oncology, Sheba Medical Center, Tel Hashomer, Israel, 3Group of Clinical Virology, Department of Medical Sciences, Uppsala Universit

The University of Texas MD Anderson Cancer Center Background: Thymoma is the most common primary neoplasm of the anterior mediastinum. The Masaoka staging system, which is based on the gross and microscopic invasive properties of the tumor at surgery, is the most widely used system in clinical practice and correlates with prognosis. Patients with stage III and IV disease should receive neoadjuvant therapy prior to surgery as it provides a survival advantage compared to adjuvant therapy and facilitates surgical resection. Aims: To identify preoperative computed tomography (CT) findings associated with thymoma invasiveness prior to surgical resection and with clinical outcome. Methods: We retrospectively reviewed CT scans of 99 thymoma patients surgically treated at our institution between September 1999 and April 2010. Chest CT findings documented were size, volume and heterogeneity of primary tumor; abutment of mediastinal vessels; and presence of calcifications, lobulation, infiltration of fat surrounding tumor, adjacent pulmonary changes, adenopathy, and pleural nodularity. Results: Our study group consisted of 53 (54 %) men and 46 (46 %) women, age 18-79 (mean: 53.2) years. Masaoka pathologic stages were stage I for 10 (10 %), stage II for 48 (48 %), stage III for 21 (21 %), and stage IV for 20 (20 %). The median radiologic tumor size was 7 cm (range: 2.521 cm). A multivariable logistic regression model showed that primary tumors with pre-chemotherapy radiologic tumor size ≥7 cm (odds ratio [OR]: 3.18, 95 % confidence interval [CI]: 1.16-8.67, p00.02), a lobulated tumor contour (OR: 8.20, 95 % CI: 1.63-41.35, p00.01), and infiltration of surrounding fat (OR: 3.76, 95 % CI: 1.45-9.78, p00.007) were more likely to have stage III or IV disease. Cox’s proportional hazard model showed that the presence of pulmonary nodules on staging CT was the only imaging parameter associated with shorter progression-free survival (hazard ratio [HR]: 4.93, 95 % CI: 1.60-15.17, p00.005) and overall survival (p00.03). Conclusion: The primary tumor CT imaging features can differentiate between stage I/II and stage III/IV disease and thus help identify patients more likely to benefit from neoadjuvant therapy.

Background: Tumor cell proliferation is an important parameter in evaluation prognosis in LC. Measuring cell proliferation is possible with a specific assay of the metabolic enzyme thymidine kinase (TK1) involved in DNA synthesis. Aims: To measure pretreatment TK1 activity in serum of LC patients and correlate the results with clinicopathological parameters. Methods: The TK1 activity was measured with DiviTum (Biovica) assay in serum of 68 patients with benign lung disease (BLD), 209 patients with Non-Small Cell Lung Cancer (NSCLC) and 74 patients with Small Cell Lung Cancer (SCLC). Results: Significantly higher activity of TK1 was found in patients with NSCLC and SCLC compared to those with BLD (p00.01 and p00.0007). TK1 activity was higher in SCLC than NSCLC (p00.007). In the NSCLC group TK1 activity was significantly associated with age, disease stage and performance status-PS (p00.003, p00.0003 and P00.01, respectively), while in SCLC only with disease stage (p00.027). At cut-off 580 Du/L corresponding to 100 % specificity, TK1 assay supplied sensitivity of 27 % in the detection of metastatic disease. A univariate analysis showed a significant association of TK1 with survival in SCLC (p00.01) and NSCLC (p00.001). In multivariate analysis, PS (RR02.5, p00.0001), TK1 activity (RR01.7, p00.01) and stage (RR0 1.5, p00.04) in NSCLC and PS (RR02.1, p00.02), and TK1 activity (RR01.2, p00.03) in SCLC were found as significant variables with an independent impact on survival. Conclusion: Pretreatment serum TK1 activity measured with DiviTum assay is a significant parameter demonstrating association with survival in two major entities of LC.

P5.3 Plasma Pro-gastrin-releasing peptide in lung cancer: prospective study B. Nisman1, H. Nechustan1, H. Biran2, N. Ramu1 and T. Peretz1 1

Department of Oncology, Hadassah and Hebrew University Medical Centre, Jerusalem

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Background: Pro-gastrin-releasing peptide (ProGRP) is a precursor of the neuropeptide hormone produced frequently by small cell lung cancer (SCLC) and considered as potential marker for this disease. Aims: In this prospective study we estimated the diagnostic value of ProGRP. Methods: Plasma levels of were measured with the ARCHITECT (Abbott) assay in 319 individuals, namely 70 patients with benign pulmonary disease (BPD), 110 patients with NSCLC, 40 patients with SCLC and 100 apparently healthy blood donors (control). Results: In control group the median and 95 percentile for ProGRP were 40 pg/ml and 70 pg/ml. There was no correlation of ProGRP with age (r00.12) or association with gender (p00.39). Significantly high levels of ProGRP were found in SCLC (median and 95 percentile: 703 pg/ml and 27286 pg/ml) compared to BPD (40 pg/ml and 74 pg/ml) or NSCLC (38 pg/ml and 121 pg/ml) (for all, P<0.0001). The cut-off value 100 pg/ml discriminating SCLC from NSCLC and BBD at 95 % specificity supplied sensitivity of 92 %. Although the levels of ProGRP in limited stage of SCLC were significantly lower than in extensive stage (p<0.045), the rate of positive results was about the same. Conclusion: Plasma ProGRP is a potential circulating biomarker for discriminating SCLC from NSCLC and BPD.

P5.4 A nanomaterial-based breath test for short-term follow-up after lung tumor resection YY. Broza 1 , R. Kremer 2 , U. Tisch 1 , A. Gevorkyan 1 , A. Shiban1, L. Anson Best,2 and H. Haick1 1

The Department of Chemical Engineering and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel. 2Thoracic Surgery Division, Rambam Health Care Campus, Haifa 31096, Israel Background: Early detection and treatment follow-up are of prime interest for lung cancer (LC) management. Breath analysis is currently attracting much research interest as a potential future non-invasive and costeffective complementary method for early LC detection. Several studies have used spectrometric methods and/or chemical sensors arrays to show that the profiles of volatile organic compounds (VOCs) in the breath of LC patients differ from those of healthy persons without lung nodules Aims: Test the feasibility of nanomaterial-based sensors for identifying the breath-print of early-stage LC, and evaluate breath analysis biomarkers as a short-term follow-up method after LC resection.

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Methods: Breath samples were collected from a small patient cohort prior and after lung resection. 17 patients, aged 50-80 years that were all scheduled for lung resection after being diagnosed with small, potentially malignant lung nodules by conventional methods. Samples were analyzed, using two independent, complimentary approaches. This first approach was the chemical analysis of breath VOCs between pre-surgery and postsurgery LC, using GC-MS combined with SPME. The second approach was the analysis with an array of nanomaterial-based sensors, combined with multivariate discriminant factor analysis as statistical pattern recognition algorithm. Results: A nanomaterial-based sensor-array distinguished between pre-surgery and post-surgery LC states, as well as between pre-surgery LC and benign states. In contrast, the same sensor-array distinguished neither between presurgery and post-surgery benign states, nor between LC and benign states after surgery. Conclusion: Results suggest that the observed pattern was associated with the presence of malignant lung tumors. The proof-of-concept presented here has initiated a large-scale clinical study that could lead to a future diagnostic breath test for early, curable LC, and could aide frequent, non-invasive post-surgery follow-up of LC patients

P5.5 NSE and ProGRP in assessment of long-term survival of patients with limited disease small cell lung cancer E. Wojcik, B. Sas-Korczynska, U. Rychlik, JK. Kulpa Center of Oncology – M. Skłodowska-Curie Memorial Institute, Krakow Division, Poland Background: Stage, sex, weight loss, LDH, and NSE have been reported to be prognostic factors in SCLC patients. Value of ProGRP in this respect is rarely evaluated and opinions about its prognostic utility are vary considerably. Aims: The aim of this study was to check the prognostic values of NSE and ProGRP for assessment of SCLC-LD patients long-term survival. Methods: Between 2001 and 2010, 88 patients with LDSCLC were treated with combined chemo- and chest radiotherapy. Patients who responded to systemic therapy were given additional prophylactic cranial irradiation. NSE and ProGRP were determined before each course of chemotherapy and then at 3 and 6 months after end of treatment. Results: During treatment, ProGRP concentration was observed to decrease more slowly than that of NSE. The present

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study revealed prognostic values for both tumor markers directly prior to four consecutive courses of chemotherapy. The discriminant values for disease free survival (DFS) were as follows: 300, 110, 45, 20 pg/ml for ProGRP and 39, 15, 10 and also 10 ng/ml for NSE, respectively before 1st, 2nd, 3th and 4th courses. Multivariate analysis showed that ProGRP were independent prognostic factors of DFS before 1st and 2nd course, NSE before 3rd course, NSE and ProGRP before 4th course, while before 5th course – only the performed prophylactic cranial radiation (PCI). Conclusion: NSE and ProGRP levels, not only before but also during treatment, are related with disease free survival of SCLC-LD patients. ProGRP is more important at start of treatment, and the next course, whereas NSE before the 3rd course. Both tumor markers are independent prognostic factors before 4th course of chemotherapy at the time of PCI initiation.

DFI and OS were found only in specific subgroups according to tumor type and stage. We found longer OS in patients with adenocarcinoma with higher expression of RRM1 mRNA (p0 0.002). We found longer OS in patients with SCC with higher expression of BRCA1 mRNA (p00.041). In NSCLC patients of stage III, we found longer DFI in patients with higher expression of RRM1 (p00.004) and ERCC1 (p00.038). Conclusion: Patients who had been treated with adjuvant chemotherapy and had shown lower expression of repair genes had adverse prognosis. We observed that the assessment of DNA repair gene level in primary tumor treated by surgical resection had prognostic significance and did not predict response to adjuvant chemotherapy. Study was supported by SVV project of LF UK Plzen no. SVV-2012-264806 and for conceptual development of research organization 00669806 - Faculty Hospital in Pilsen, Czech Republic.

P5.6 Relevance of ERCC1, RRM1 and BRCA1 in surgically treated non-small cell lung cancer patients

P5.7 Identification of unique HLA peptides presented on SCLC cells

Pesta Martin, Kulda Vlastimil, Fiala Ondrej, Safranek Jarmil, Topolcan Ondrej, Krakorova Gabriela, Cerny Radim, Pesek Milos

Shelly Kalaora, Arie Admon

Faculty of Medicine and University hospital in Pilsen, Charles University in Prague, Czech Republic

Background: Lung cancer accounts for 12 % of all new cases of cancers worldwide with small cell lung carcinoma (SCLC) representing 13 % of the newly diagnosed lung cancers. Treatment of SCLC remains challenging because of its rapid growth, early dissemination and development of drug resistance during the course of the disease. Many current studies aim at developing more specific ways of treating cancer. These include, immunotherapy, which utilizes the specificity of the immune system in order to attack the transformed cells. Aims: The research focuses on discovery of peptides presented by the HLA complexes of SCLC cells with potential to serve as targets for immunotherapy. Since the expression of class I HLA molecules in SCLC cells is lower than in normal lung cells, our goal is also to find treatments, including cytokines (such as interferon gamma, INFγ) and other chemotherapy agents, to increase the expression of HLA molecules on the cells’ surface and facilitate recovery of specific peptides. Methods: We used the human SCLC cell line NCI-H82 and NCI-H69. The HLA molecules were collected from cell lysate which were immunoaffinity purified with the mAb W6/32 bound to Amino Link beads. The acid eluted peptides were concentrated by disposable C18 columns and analyzed by capillary chromatography and tandem massspectrometer analysis on a OrbitrapXL mass spectrometer. Peptides were identified using multiple search engines: Pep-

Background: Recent studies have shown that assessment of predictive molecular markers could be helpful for estimation of response rate to chemotherapy. Aims: The aim of our study was to assess relation of mRNA levels of DNA repair genes ERCC1, RRM1 and BRCA1 in surgically resected tumor tissue in patients who received adjuvant chemotherapy to disease free interval (DFI) and overall survival (OS). We investigated if potential residual tumor cells after resection reflect properties of a primary tumor and response to chemotherapy according to level of predictive markers with the respect to current knowledge. Methods: We studied group of 90 patients with NSCLC, who had undergone curative lung resection, 59 of them were subsequently treated with adjuvant chemotherapy, DFI and OS were evaluated only in this subgroup. Quantitative estimation of mRNA of selected genes in paired (tumor and control) lung tissue samples was performed by RT real-time PCR. Results: We found lower mRNA expression of ERCC1 (p&<0.001) and RRM1 (p00.023) in NSCLC tumor tissue compared to normal lung tissue. Comparing expression in histological subtypes we recorded higher mRNA expression of ERCC1 (p0 0.021), RRM1 (p00.011) and BRCA1 (p00.011) in adenocarcinoma than in squamous cell carcinoma (SCC). The differences in

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Miner, MaxQuant and Proteome Discoverer (including Sequest and Mascot). Results: We identified about 800 different HLA class I peptide, from 8 different purifications of untreated NCIH82 and NCI-H69 cell lines. After treatment with 500U/ ml of interferon gamma for 48 hours, we identified above 2500 HLA class I peptides, from just two purifications of the same cell lines. Similar results, indicating a significant increase in HLA class I expression, were also noticed when

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Western blot, flow cytometry and confocal microscopy analyses were perfromed. Conclusion: A small percentage of the identified HLA peptides were derived from putative tumor associated antigens. These genes are known to be expressed especially in cancer cells or in immune privileged tissues. Such peptides and their source proteins are potentially advantageous as candidates for cancer vaccines or tumor markers.

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P6 GASTROINTESTINAL CANCERS P6.1 Polymyositis as a paraneoplastic process in colon cancer Daniel M. Broman Mid-Cheshire NHS Foundation Trust Background: Colorectal cancer most often presents with a change in bowel habit, weight loss or with bleeding per rectum. Much less commonly, colorectal cancer may present as part of a paraneoplastic syndrome. Polymyositis is a rare disease most often considered a complement-mediated idiopathic inflammatory myopathy manifested by proximal muscle weakness. However, polymyositis may also be part of a paraneoplastic syndrome associated with an underlying malignancy. The relationship between polymyositis and malignancy is well known, but it has been suggested that tumours of the large bowel are rarely complicated by myositis. Aims: Our aim was to describe our case report of an 82-yearold gentleman with a presumed musculoskeletal or neurological deficit who was subsequently diagnosed with metastatic colon cancer in the setting of progressive fatigue and muscle weakness. Concurrently, we review the current literature looking at the relationship between cancer and polymyositis. Methods: Our method was a descriptive report of our case including a review of current literature on this topic. Results: Colorectal cancer rarely presents with symptoms such as muscle weakness, however it is important to be aware of the possibility of an underlying malignancy when seeing patients with symptoms which are suggestive of polymyositis. Conclusion: The diagnosis of polymyositis in the elderly population should raise suspicion of an underlying malignancy. This case demonstrates the value of knowledge about associated paraneoplastic syndromes, as this may help solve uncommon but serious diagnostic conundrums. Early diagnosis of malignancy is critical in cases of polymyositis. We suggest that patients over the age of 45, with a newly diagnosed myositis, should be screened for occult cancer, including a thorough history and extensive examination, and investigations including blood tests for haematological and biochemical screening and assessment of tumour markers (CEA, CA-125, PSA, etc.).

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Cancer Genetics and Stem Cell Group, Division of PreClinical Oncology, School of Clinical Sciences; Medical School, University of Nottingham, UK Background: Colorectal cancer(CRC) is the 3rd most common cancer worldwide accounting for 10 % of cancer morbidity.FBXW7 tumor suppressor forms the substrate recognition component of the SCF(SKP1-CULL-F-box)E3 type ligase complex involved in the Ubiquitin Proteasome System and is heavily implicated in CRC.P53 tumor suppressor,the 'guardian angel' gene,is a transcriptional regulator of cell cycle progression.Upon genotoxic stress it undergoes nuclear stabilization to prevent propagation of defective DNA.Phosphorylation of p53 at Serine15(S15) is one of the earliest events mediating such stabilization.FBXW7 is a transcriptional target of p53 and a “p53 dependent” link between the 2 has been reported Aims: Novel findings in our lab demonstrate the aberrant induction of phosphorylated p53 at S15[phosphop53(S15)] in FBXW7-deficient CRC cells,correlating phospho-p53(S15) induction with FBXW7 mutations.Validation of such phenomenon formed the main purpose of this project Methods: 2 inter-supporting in vitro biological assays,immunofluorescence and Western blotting,were performed in the wild-type and FBXW7-deficient human CRC cell line HCT116.In vivo validation was performed using immunohistochemical staining in wild-type(n06) and FBXW7-mutated (n05) human CRC tissues Results: Phospho-p53(S15) induction was evident in the FBXW7-mutated CRC cells and the results were highly statistically significant (p<0.0005).Herein, we report for the first time an “FBXW7 dependent” relationship between the 2 tumor suppressors. Furthermore,the results point towards the suitability of phospho-p53(S15) as an independent indicative marker of CRC with an FBXW7-mutated locus Conclusion: Validation of increased phopsho-p53(S15) protein induction in FBXW7 human CRCs suggests that the expression of the former may be regulated through FBXW7 mediated degradation,implicating the 2 tumor suppressors (FBXW7&P53) in an autoregulatory loop.Also,the results point towards the suitability of phospho-p53(S15) as an independent indicative marker of CRC with an FBXW7-mutated locus and could be exploited for the potential use of phospho-p53(S15) in the molecular characterization of FBXW7-associated carcinogenesis and the clinical prospect this entails

P6.2 Validation of phospho-proteomically identified human phospho-p53(S15) protein induction in colorectal cancer with FBXW7 mutation

P6.3 Comparison of four tumour marker assays for determination of AFP, CEA, PSA, and fPSA in serum

Kalakouti E., Li N., Nateri AS.

G. Hintereder and H. Welle-Scharmann

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Department of Laboratory Medicine, J.W.Goethe University Hospital, Frankfurt, Germany and DiaSorin Deutschland GmbH, Dietzenbach, Germany Background: Serial determinations of tumour markers are well established in the management of cancer patients for monitoring of disease progression and effectiveness of treatment. The main clinical use of tumour markers is in the post-operative follow-up of cancer patients. Increased marker levels may detect disease progression prior to the appearance of clinical symptoms. Aims: To compare serum measurements of the tumour marker assay for AFP, CEA, PSA, and fPSA on different analyzers, the MODULAR® E170, IMMULITE® 2000, and LIAISON® XL Analyzer with respect to correlation and throughput. Methods: A minimum of 37 patient samples spanning the assay range were tested for each tumour marker and dose correlation was calculated by regression analysis. We recorded the time needed to run the samples on IMMULITE® 2000 and LIAISON® XL Analyzer to assess productivity of the analyzers. Results: Method comparison showed clear correlation between methods with correlation coefficients of 0,94 or higher and widely agreement between methods. Time recorded for sample processing revealed higher throughput for LIAISON® XL Analyzer in comparison to IMMULITE® 2000. Conclusion: The tumour marker assays showed satisfactory compatibility on all tested analyzers. Throughput assessment confirmed suitability of the LIAISON® XL Analyzer for medium-high volume laboratories

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of miR-187 were measured by a miRNA quantitative RT-PCR (qRT-PCR) analysis system. The correlation between miR187 level and multiple clinical variables were then checked by Mann–Whitney test and Wilcoxon matched pairs test. For exogenous miR-187 expression, pre-mir-187 sequence was cloned into a lentivirus vector and cotransfected with helper plasmids into 293T cells to produce lentiviruses. The miR-187 stable clones of AGS-GFPM2, Kato III and SCM-1 cell lines were established by lentiviral infection. These stable clones were applied to analyze the tumorigenic phenotypes including migration assay, anchorage-independent growth and tumor xenografts. The miR-187 blocker was transfected into stable clones to suppress the expression of miR-187. Results: Significant higher level of miR-187 in the plasma of GC patients than in the controls was identified. Such increase was also present in stage I patients subset and earlier tumors without nodal involvement. In vitro assays indicated that exogenous miR-187 expression induced the migration and anchorage-independent growth of GC cells. Such induction was reversed with the treatment of miR-187 blocker, suggesting an oncogenic role of miR-187 expression in GC. Exogenous miR-187 expression also drastically enhanced the tumorigenecity of SCM-1 cells on nude mice, while the xenografic tumor growth was attenuated by miR-187 blocker. Conclusion: This study concludes that miR-187 drives the oncogenesis of GC. Plasma level of miR-187 could be potential biomarker of GC. The blockage of miR-187 may be validated as a new therapeutic trial against GC.

P6.5 Results of an international proficiency study with Alpha-1-Fetoprotein (AFP) P6.4 Up-regulation of miR-187 as potential biomarker of gastric carcinoma Su-Shun Lo, Pei-Shih Hung, Kuo-Wei Chang National Yang Ming University Background: MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the translation or degradation of target gene mRNAs by RNA interference. They are involved in diverse disease processes including oncogenesis. Our previous study identified a panel of miRNAs aberrantly expressed in gastric carcinoma (GC) tissues (Oncogene, 2012;31(2):226-37). Among those, miR-187 was found markedly up-regulated in GC tissues. Aims: To evaluate the role of oncological function of miR187 in gastric cancer Methods: Blood samples of GC patients were obtained before operation. Resected tissues from 33 GC patients along with paired non-cancerous mucosa were obtained from the patients underwent surgery. The expression levels

G.M. Oremek1, N. Bewarder2, M. Zwirner3 1

Zentrum Innere Medizin, Fachbereich Laboratoriumsmedizin, Universitätsklinikum Frankfurt, 60590 Frankfurt, Germany, 2 BIOREF GmbH, 63776 Mömbris, Germany 3Dept Obst & Gynecol, University Tuebingen 71157 Tuebingen, Germany Background: The between test comparability of immunological test systems is still present. This can lead to misinterpration of tumor marker time courses. Aims: The aim of the present study was to evaluate the long term quality of AFP determinations regarding between test comparability and precision. Methods: The 25 participating laboratories applied a liquid human BIOREF-control serum consisting of 3 different AFP concentration levels in their routine measurement procedures. During a 12 months study period control samples were measured once per week and reported monthly for statistical evaluation summarizing in 792 results which were performed on 8 different commercially available test systems.

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Results: The mean values of the individual laboratories ranged between 4.4 – 6.3 ng/ml, 48.3 – 65.7 ng/ml and 155.6 – 212.4 ng/ml, for the low, medium and high control, respectively. These deviations seem to be partly due to the different test systems applied since the relative deviation between the laboratory mean values and the method specific mean values was only 0.3 to 8.5 %. The coefficients of variation (CV) of the single laboratories ranged from 1.3 to 16.1 % depending on the AFP concentration. For the low control, 7 of the 25 laboratories exhibited a CV of more than 10 %. Several test systems and in consequence the liquid AFP control which was stored at 8°C exhibited excellent long term stability. Conclusion: The accuracy as well as the precision of most of the tests included was acceptable.The between test comparability should further improved. The participation at external quality assessment schemes combined with a longterm internal quality control with a test-independent native control serum is advisable for every laboratory in order to detect changes over time.

Expression changes were statistically validated using the REST 2009 software. Results: For the pilot study we have used paired tumor and healthy tissue samples from 56 patients. Out of the twelve genes, ten had significantly different expression levels, six of them were downregulated in tumor (CLDN23, SLC26A2, MIER3, MAPK1, DSTN and ACSL5) and four upregulated (CTHRC1, VSNL1, VCAN, LGR5). At the moment we are correlating expression data with the clinical informations with the goal to identify possible prognostic or predictive markers. Conclusion: In the study we have identified ten genes with the different expression level between the tumor and healthy tissue. Based on the preliminary statistical data, some of these changes correlate with the clinical data and the final results are going to be presented during the conference. Project is supported by grants IGA MZ CR NR 12025 and SVV 264-808.

P6.6 Change in the expression of selected genes in the colorectal cancer development

J. Van de Put1, C.Mombrial2, T.Bossman3

P6.7 CISBIO Chromogranine A ELISA kit evaluation on Stratec Gemini

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DIAsource Immunoassays, Louvain-la-Neuve, Belgium Cisbio Bioassays, Codolet, France - 3Asbach Medical Products (AMP), Obrigheim, Germany

P.Pitule , M.Cedikova , V.Liska , I.Hlavata , M.Kralickova , P. Soucek3, V.Treska2

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Background: More and more clinical biology laboratories are equipped with automated systems which provide many advantages. There are many companies in the market developing and manufacturing such instruments. CISBIO which develops and manufactures immunoassays to be run manually wants to give this chance to their customers. Aims: Chromogranine A, or CGA, is a protein acting as a pro-hormone in neuroendocrine cells. It's especially well known as a tumorous marker. Cisbio manufactures CGA ELISA kit on microplate support. Looking for automated solution they found Gemini, a fully automated ELISA analyser system manufactured by Stratec (German company which has 33 years' experience developing automates for OEM partners in biomedical field). Gemini is an open system meaning that a large range of ELISA assays on microplate support can be adapted successfully to it. DIAsource has been working with Gemini for more than 3 years and has obtained experience in adapting and validating assays to it. Cisbio asked DIAsource to carry out CGA ELISA kit validation on Stratec Gemini. Methods: Validation on Gemini is a process divided in two parts. Firstly, it consists in adaptations for running the kit components properly in the machine. Secondly, analytical performances are analysed. New adaptations

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Department Histology and embryology, Medical faculty in Pilsen, Charles University in Prague, 2Department of Surgery, Teaching Hospital in Pilsen, 3National Institute of Public Health Background: Colorectal cancer is one of the most common malignant disease in the Czech republic and prediction of the disease progression or selection of the best treatment for the particular patient is very complicated. We are trying to find new markers of the disease progression or treatment response to help to select appropriate care for the patients and to improove quality of their lives. Aims: We would like to identify genes with the different expression levels between the tumor and healthy tissue in the well-defined group of patients. Our ultimate goal is to use differently expressed genes during the diagnosis as the prognostic or predictive factor. Methods: We performed literature and database search and selected set of 12 genes for our pilot study (ACSL5, VSNL1, DSTN, SAMD3, CTHRC1, MAPK1, CLDN23, SLC26A2, MIER3, CAPN10, VCAN a LGR5). Some of these genes are already connected to the malignant process in other types of cancer. After the identification of the best reference genes and optimalization of qPCR reactions, we have measured the expression levels of our candidate genes.

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or assay programming changes are realised in order to get best performances. For the validation samples have been collected in the same conditions among biomedical labs in Germany with collaboration of AMP company. These samples have been stored frozen (-20°C) before testing. As a first step an assay programming has been made up on Gemini. Once a protocol reached our expectations a correlation was established by comparing manual and Gemini methods on the same day and in the same environmental conditions. 39 patient samples have been assayed. Results: Some adaptations have been realised successfully so as to use CGA kit material properly. Standard OD’s on Gemini are slightly lower than those obtained manually. Slight OD difference is inherent in this system. Correlation curve gives a r2 and a slope close to 1 (y00.98x - 2.08;r200.98). Conclusion: Analytical results from both methods match well. Cisbio CGA is validated and now available on Stratec Gemini.

P6.8 Kiss-1 methylation and protein expression patterns contribute to diagnostic and prognostic assessments in colon cancer Noemi Pompas, 1Patricia Moya, 1Sergio Esteban, 2Antonio Fernandez-Suarez, 3Marisa Maestro, 4Manuel Morente, 1 Marta Sánchez-Carbayo*.

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grade (p00.011), predicted recurrence (p00.009), metastasis (p00.004), disease-free (p00.034), and overall survival (p00.015). In the second validation cohort, KiSS-1 methylation predicted disease-specific survival (p00.030). In the training set, cytoplasmic KiSS-1 expression was significantly higher in non-neoplastic biopsies as compared to colon tumors (p<0.0005). In the validation set, loss of cytoplasmic expression correlated with tumor stage (p00.007), grade (p00.035), recurrence (p00.017), and disease-specific survival (p00.022). Conclusion: KiSS-1 was revealed epigenetically modified in colon cancer. The diagnostic and prognostic utility of KiSS-1 methylation and expression patterns suggests their assessment for the clinical management of colon cancer patients.

P6.9 Does neo-adjuvant radiotherapy interferes with the interpretation of the Immunohistochemistry stain (IHC) for Mismatch Repair Proteins (MMR) in patients with Rectal Cancer Alex Vilkin1, Marisa Halperin2, Sara Morgenstein3, Shlomo Birkenfeld4, Niv Yaron1 and Zohar Levi1

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Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain; 2Biochemistry Department, Hospital Alto Guadalquivir, Andujar, Jaen, Spain; 3Biochemistry Department, Hospital Clinico Universitario, Madrid, Spain; 4Tumor

Background: KISS1 is a metastasis suppressor lost in several solid malignancies Aims: We evaluated the clinical relevance of KiSS-1 methylation and its protein expression in colon cancer. Methods: The epigenetic silencing of KiSS-1 by hypermethylation was tested in colon cancer cells (n05) before and after azacytidine treatment. KiSS-1 methylation was evaluated by MS-PCR in colon cancer cells and tissues (n 0352) grouped in a training set (n062), and two independent validation cohorts (n0100, and n0190). KiSS-1 protein expression was analyzed by immunohistochemistry on tissue arrays. Results: KiSS-1 hypermethylation correlated with transcript and protein expression loss, being increased in vitro by azacytidine. Methylation rates were 53.1 %, 70.0 % and 80.0 % in the training and validation sets, respectively. In the training set, KiSS-1 methylation rendered a diagnostic accuracy of 72.7 % (p00.002). Combination of KiSS-1 methylation and serum CEACAM (p00.001) increased the prognostic utility of CEACAM alone (p00.022). In the first validation set, KiSS-1 methylation correlated with tumor

The Gastroenterology Dept. Rabin Medical Center, Petach Tikva, Israel. Background: The use of IHC for MMR from paraffin embedded material for the detection of Lynch Syndrome in colorectal cancer patients has been widely accepted in clinical practice. Aims: We aimed to assess whether the neo-adjuvant radiotherapy (Rx) interferes with the quality of stain for the MMR proteins, as compared to the material before the operation. Methods: Included patients with stage II-III rectal cancer treated at Beilinson Campus with neo-adjuvant radiotherapy and had paraffin embedded material from both the colonoscopy before Rx and the operation post Rx. IHC stain for MLH1, MSH2,MSH6 and PMS2 was performed. Then, we scored the intensity of the stain using a semi quantified method [Score: percent of stained cells (0-4 scale)*intensity (0-3 scale)] by a dedicated pathologist blinded to the data. Results: 20 patients were included, mean age 61.2±11.2 y. The mean score of the stain was significantly higher in the pre Rx material as compared to post Rx material for MLH1, MSH2,MSH6 and PMS2 [ 11.2±2.5 vs. 5.3±3.9; 11.0±2.0 vs. 6.0 ± 3.7;10.8± 2.3 vs. 4.3 ± 2.4;8.4± 4.4 vs. 3.5 ± 3.5; p<.001 for all]. Null or weak stain was observes in 4/80 (5.0 % slides of the pre Rx as compared to 19/80 slides (23.8 %) of the post Rx material (p00.001). Conclusion: Our data suggest that IHC for MMR from paraffin embedded material after Rx might be misleading. The material obtained before Rx should be prioritized.

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P7 UROLOGICAL CANCERS (PROSTATE, BLADDER) P7.1 Objective responses and survival in eighteen patients with advanced urothelial urinary bladder cancer scheduled to undergo T-cell based adoptive immunotherapy A.M.Sherif1,E.Radecka2 ,M.N.Hasan1, S.Shabo3, P.Marits4, M.Karlsson4, M.C.Schumacher1, O.Winqvist4 Karolinska University Hospital, Dept.of Urology, 2Karolinska University Hospital, Dept. of Diagnostic Radiology, Stockholm,Sweden, 3Mälarsjukhuset County Hospital, Dept. of Surgery and Urology,Eskilstuna,Sweden, 4Karolinska University Hospital 1

Background: Expected 2 year survival in urothelial urinary bladder cancer (UBC) with metastatic lymph node involvement is low, regardless of standard oncological therapy. Tumor reactive lymphocytes are present in sentinel nodes draining UBC and display immunologic function upon restimulation in vitro. Metinel nodes (MNs) drain secondarily from metastatic tumor and also possess tumor reactive lymphocytes. Aims: To determine by computerized tomography (CT) if treatment with expanded autologous T-helper cells in patients with metastatic UBC, could result in objective responses. To evaluate overall survival in thse patients. Methods: All 18 patients were staged T2-T4bN1-2 and/or M0-M1 or MX.MNs and tumor draining nodes were excised in conjunction with intended cystectomy. T-lymphocytes were extracted with subsequent enhancement and expansion of tumor specific T-helper cells, followed by reinfusion.Regular CT-investigations were performed and overall survival calculated. Results: In 9/18 patients the treatment was feasible.In 2/9 treated patients,objective responses were detected by CT defined as significantly diminished nodal metastatic lesions.These two patients displayed increased overall survival. Conclusion: An adoptive immunotherapy based on expanded autologous T-cells from tumor draining lymph in advanced UBC was evaluated. Objective/CT-verified responses according to RECIST-criteria were detected in 2/ 9 treated patients. The described therapy might be a future treatment option in metastasized and/or advanced unmetastasized high risk UBC.

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Oncology Department – Faculdade de Medicina do ABC, Santo André, SP, Brazil 2Instituto de Ciências Químicas, Farmacêuticas e Ambientais – Unifesp, Diadema, SP, Brazil

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Background: Although prostate cancer (PC) is very prevalent among men, relatively little is known about the molecular mechanisms involved in the development and progression of the disease. In other words, it is important to find markers that could predict which patients have tumors with aggressive invasive potential to spread outside the prostate. A number of studies have identified E-cadherin, a cell adhesion protein, as a marker of early neoplastic change in PC. Moreover, studies on prostate cancer have shown that matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) are produced by the interstitial monocytesmacrophages and fibroblasts localized in the tumor focus, as well as by neoplastic cells and that their expression is generally correlated with tumor differentiation. Aims: To evaluate plasmatic E-cadherin and MMP-13 at diagnosis of prostate cancer, 3 and 6 months after the beginning of the treatment and correlate those values with PSA and clinical data, such as Gleason score and perineural invasion. Methods: This study included 29 patients with prostate adenocarcinoma and 10 healthy men. Blood samples were collected for E-cadherin and MMP-13 measurement and their relationship with total, free PSA and clinical data were analysed using SPSS 15.0. Results: Our results have shown that E-cadherin (p < 0.001) and the ratio E-cadherin/MMP-13 (p < 0.001) could be markers for PC. Moreover, E-cadherin/MMP-13 ratio correlated with Gleason score (p00.047) and age (p00.0291). On the other hand, no differences were observed in plasmatic levels of E-cadherin and MMP-13 at diagnosis of prostate cancer, 3 and 6 months after the beginning of the treatment. Conclusion: Other different biomarkers remain to be identified that potentially could improve the evaluation of prognostic of the PC patient.

P7.3 Relationship between glasgow prognostic score (gps) and cyfra 21-1 and cea in muscle-invasive bladder cancer patients (pilot study) U. Rychlik, JK. Kulpa, J. Nowak-Sadzikowska, E. Wójcik, J. Jakubowicz Center of Oncology,Krakow Division, Poland

P7.2 Plasma levels of E-Cadherin and MMP-13 in prostate cancer patients C.M. Bonaldi1, L.A. Azzalis2, V.A.Vilas Boas1, C.G.B. de Oliveira1 F.L.A. Fonseca1,2

Background: The relationship between cancer and inflammation are subject of many investigations. Systemic inflammatory response is widely regarded as a bad prognostic factor. Aims: The aim of presented study was the evaluation of relationship between CYFRA 21-1 and CEA levels in serum

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and urine and selected factors of inflammation in muscleinvasive bladder cancer patients. Methods: The determinations of serum CYFRA 21-1, CEA, C-reactive protein (CRP) and albumin (ALB) levels and urine concentrations of both markers and creatinine were performed before treatment in 86 muscle-invasive bladder cancer patients and in reference group of 37 healthy persons. For each person under study Glasgow Prognostic Scored (GPS). Concentrations of CYFRA 21-1 and CEA in the urine are given per mg creatinine. Results: In bladder cancer patients, in comparison to the reference group, significantly higher CYFRA 21-1 and CEA serum and urine concentrations were found. In the group of bladder cancer patients in more advanced stages T3-4 significantly higher CYFRA 21- in serum and both markers in urine levels there were observed than in those in stage T2. Patients with Karnofsky PS ≤ 60 in comparison to those with PS ≥ 80 had significantly higher serum CYFRA 21-1 and urine concentration of both markers. Patients with complete response

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(CR) after therapy had pretreatment CYFRA 21-1 serum and urine levels lower than remain. Moreover, in group of patients without complete response there were observed significant tendency to higher frequency of pretreatment GPS 1 and 2. When the group of bladder cancer patients were divided in respect to GPS values, significantly higher serum and urine CYFRA 21-1 levels were observed in group with GPS 2 in comparison with those with GPS 0 or 1. Patients deceased before 12 months in comparison with those surviving longer, had only significantly higher pretreatment CYFRA 21-1 serum levels. Moreover in the group of patients who died before 12 months, more frequently were found those with pretreatment GPS 1 and 2 than the others. Conclusion: 1. Muscle-invasive bladder cancer patients with pretreatment GPS 2 represents higher probability of shorter survival 2. An elevated serum and urine level of CYFRA 21-1 seems to be associated with poorer prognosis.

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P8 NEW ANTI - CANCER THERAPIES P8.1 Induction of anti-tumor immune responses by ablation of the primary tumor with pulsed electric currents Hochman I.1,2, Confino H.2, Efrati M.2, Korenstein R.1 & Keisari Y.2 1 Departments of Pharmacology and 2 Immunology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel Background: Ablation is a local treatment, which is used for in situ destruction of solid tumors. in situ tumor ablation can result in propagation of antigenic molecules, which can activate immunological reactions against the tumor that may subsequently destroy residual malignant cells. Thus, in order to better fight malignant antigenic cancer and eliminate metastases we take the immunotherapeutic approach which claims that the tumor can serve as its own antigenic vaccine provided that the tumor cells are killed effectively inside the body. We performed electrochemical ablation of tumors using a treatment in which a train of unipolar-pulsed electric currents was delivered (PECTA). Aims: Defined the parameters required for maximal ablation of tumors by PECTA, and the following manifestation of antitumor immunity. Methods: Balb/c mice bearing subcutaneous tumors (7-9 mm in diameter) of murine mammary adenocarcinoma (DA3) or colon carcinoma (CT26) cells were treated with 25-100 coulombs (C) per cm3 of tumor tissue. Intratumoral electrodes delivering electric currents of 30 mA were used. PECTA mediated elimination of the primary tumors was measured and compared to surgical removal of the tumors. To test anti-tumor responses, mice cured by PECTA or surgery were reinjected with tumor cells (challenge assay), or splenocytes from such treated mice were mixed with tumor cells and implanted subcutaneously in naive mice (Winn assay). Results: Primary tumor ablation: A direct correlation was found between the amount of charge delivered and the rate of primary tumor ablation. Treatment with 100 C/cm3 cured 100 % of the mice (ablation defined as no recurrence for 3-4 months). Anti-tumor immunity: Tumor cells injected into PECTA cured mice grew slower than in surgery cured mice (challenge assay). After the injection of splenocytes/tumor cell mixture to naïve mice, splenocytes from PECTA treated mice were more efficient in inhibiting tumor growth and prolonged survival compared to splenocytes from surgery treated mice or normal mice (Winn assay). Conclusion: The results indicate that PECTA successfully ablated DA3 and CT26 tumors and stimulated the immune response against the tumor and metastatic lesions.

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P8.2 Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody act synergistically to suppress human hepatic cancer cells in vitro and vivo Y. Kato1, H. Hirata2, M. Tsujisaki3, T. Matsune4, S. Sasaki5, Y. Shinomura5, K. Imai6 Sapporo Shirakabadai Hospital, Sapporo, 2Hirata Hiromi Clinic, Hakodate, 3Tenshi Hospital, Sapporo, 4Shirakaba Pharmacy, Sapporo, 5Medical University, Sapporo, 6University of Tokyo, Tokyo, Japan 1

Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and ranks as the fifth most frequent cancer, overall, and the third leading cause of cancer death in the world. At present, effective therapeutic options available for HCC are limited. Consequently, the prognotsis for these patients is poor. New approaches to the HCC therapy must be still studied. Aims: To identify a novel target for antibody therapy agaist HCC we investigated the fibroblast gwowth factor receptor 1 (FGFR1) expression in HCC, and studied its clinical application. Methods: Cytometric and immunocytochemical analyses as well as Western blot examination were made to investigate the regulation of the FGFR1 expression by interferon-α/β in several human HCC cell lines. In addition, we tested the efficacy of combined treatment with an anti-FGFR1 monoclonal antibody (mAb) and interferon-α/β in a murine xenograft model of human HCC. Results: Interferon-α/β induced the expression of FGFR1 in human HCC cell lines. An anti-FGFR1 mAb, whose target is the induced FGFR1, can effectively inhibit the growth and survival of HCC cells in vitro and in vivo. The combination of interferon-α, anti FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro on HCC. Conclusion: The combination therapy with an anti-FGFR1 antibody and Interferon-α/β is a promising approach to the treatment of HCC.

P8.3 The effect of the p30 helper epitope from the tetanus toxin on DNA immunization with a gene gun Šmahel Michal, Poláková Ingrid, Dušková Martina, Ludvíková Vierka Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic Background: CD4+ T helper cells (Th) play important role in priming and expansion of CD8+ cytotoxic T lymphocytes

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(CTL). In DNA vaccination, CD4+ T-cell help can be enhanced by a fusion of a gene encoding an immunization protein with a foreign gene or its part providing Th epitopes. Tetanus toxin (TT) is often used as a source of universal helper epitopes. Residues flanking helper epitopes in a protein substantially influence the immunogenicity of the epitopes. The key factor that affects proteolytic processing of antigens and the generation of peptides carrying helper epitopes is probably conformational flexibility which correlates with the presence of protease-sensitive sites. Aims: Analysis of the effect of helper epitope localization in a protein molecule and the effect of fusion protein cellular localization on immune responses induced by DNA vaccines delivered by a gene gun. Methods: The TT helper epitope p30 (aa 947-967) was fused with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16 and cellular localization of the fusion constructs was altered with signal sequences targeting proteins into endoplasmic reticulum (ER) and endosomal/lysosomal compartment. Moreover, based on the published X-ray crystallographic analysis, enlarged version of the epitope containing potential proteasesensitive sites, was also utilized. After DNA immunization of mice with a gene gun, activation of TT-specific CD4+ and E7specific CD8+ T cells was examined. Results: The p30 epitope enhanced E7-specific CTL response, but only in constructs without the added signal sequences. After localization of the fusion proteins into ER, N-glycosylation of the p30 epitope and preferential induction of the TT-specific type 2 Th response reduced the induction of the E7-specific response. The enlargement of the p30 epitope with the flanking residues did not improve the effect of immunization. Conclusion: The efficacy of DNA vaccination against the E7 antigen was increased with the p30 epitope, but this beneficial effect was inhibited after localization of the fusion proteins into ER. This project was supported by grants NT/11541-4 from the Czech Ministry of Health and P501/12/1761 from the Czech Science Foundation.

P8.4 COMBAT in vitro: Enhancement of the retinoidinduced cell differentiation by LOX/COX inhibitors in cell lines derived from pediatric solid tumors P. Chlapek1,3, M. Redova1, J. Neradil1,2, K. Zitterbart2, M. Hermanova3, J. Sterba2, R. Veselska1,2 1

Dept. of Experimental Biology, School of Science, Masaryk University, Brno; 2Dept. of Pediatric Oncology, University Hospital Brno; 3Institute of Pathologic Anatomy, St. Anne’s University Hospital, Brno (Czech Republic)

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Background: Special regimen called COMBAT (Combined Oral Maintenance Biodifferentiating and Antiangiogenic Therapy) was developed at our university and was reported as effective in treatment of children with relapsed and/or high-risk pediatric solid tumors. In this protocol, the chemotherapy agents are combined with administration of celecoxib and isotretinoin. Aims: This study was aimed at explaining possible synergistic effects of these compounds using cell lines derived from neuroblastomas and medulloblastomas. Based on our previous results, we focused especially on the mechanisms of enhancement of the retinoid-induced differentiation by its combined treatment with inhibitors of lipoxygenases (LOX) or cyclooxygenases (COX). Methods: Two neuroblastoma (SK-N-BE(2) and SH-SY5Y), and two medulloblastoma (Daoy and D283 Med) cell lines were chosen for this study. Due to intracellular isomerisation of retinoids, all-trans retinoic acid (ATRA) was applied in combinations with caffeic acid (CA, inhibitor of 5 lipoxygenase) and celecoxib (CX, inhibitor of cyclooxygenase-2). The antineoplastic effects of these compounds and their combinations were evaluated using proliferation assays, cell morphology assessment, detection of differentiation markers, and analyses of cell cycle and gene expression. Results: Enhanced cell differentiation was found after combined treatment with ATRA and LOX/COX inhibitors, especially in neuroblastoma cell lines. Expression profiling showed a concentration-dependent increase in expression of genes associated with control of cell differentiation, genes participating in regulation of cytoskeleton rearrangements, and genes involved in mitochondrial metabolism. Conclusion: Both on a cellular and molecular levels, the results obtained proved that ATRA-induced differentiation was enhanced by its combined application with LOX/COX inhibitors. Supported by grants IGA MZCR NR/9341-3, CZ.1.07/ 2.3.00/20.0183, and GACR 204/08/H054.

P8.5 Preoperative use of biological therapy does not influence liver regeneration after large resection - porcine experimental model V. Liska, V. Treska, H. Mirka, J. Benes, O. Vycital, J. Bruha, P. Pitule, T. Skalicky, A. Sutnar, A. Chlumska, J. Racek, L. Trefil, J. Finek and L. Holubec Medical School and Teaching Hospital Pilsen, Charles University Prague, Czech Republic Background: Targeted biological therapy is becoming a standard in personalized medicine for patients with advanced stages of cancer. Aims: The aim of this work was to study the influence of isolated biological therapy administered immediately before

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extended liver resection on liver function and regenerative capacity of future liver remnant (FLR) in a large-animal experiment. Methods: Nineteen piglets were included in this study (10 in the control group and 9 in the experimental group). A port-acath was introduced into the superior caval vein. On days 11 and 4 before liver resection, cetuximab was administered via this port at 400 mg/m2 of piglet body surface. Physiologic solution was applied to the control group. The resection of the left lateral, left medial and right medial hepatic lobes was performed (reduction of 50-60 % of liver parenchyma).Blood samples were collected at different times before the operation and after liver resection. Serum levels of bilirubin, urea, creatinine, alkaline phosphatase, gammaglutamyltransferase, cholinesterase, aspartate aminotransferase, alanine aminotransferase, albumin, C-reactive protein and transforming growth factor-β1 were assessed. The ultrasonographic examinations at different times were performed preoperatively and after liver resection to assess the liver volume. The biopsies from the liver parenchyma were examined for proliferative activity, binocluated hepatocytes, size of hepatocytes, and the length of the lobuli. Results: There were no important complications of administration of biologic therapy during the operation or throughout the perioperative period. There was no statistically significant difference in regeneration of FLR nor were any differences in biochemical, immunoanalytical and histological parameters detected. Conclusion: The achieved results of comparable liver regeneration in both the experimental and control groups confirms the use of biological treatment with cetuximab in the preoperative period for minimizing the recovery period. This study was supported by the grant IGA MZ CR 12025.

P8.6 The efficacy of IGF-IR blockade for human gastrointestinal carcinomas is independent for mutation status of k-ras Y. Adachi1,2, Y. Matsunaga1, H. Yamamoto1, H. Ohashi1, K. Nosho1, H. Suzuki1, Y. Arimura1, T. Endo2, Y. Kato2, K. Imai3, and. Y. Shinomura1

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First Department of Internal Med, Sapporo Medical Univ, Sapporo (Japan), 2Sapporo Shirakaba-dai Hospital, Sapporo (Japan), and 3Inst.of Med Sci, Univ of Tokyo, Tokyo (Japan). Background: Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of many tumors, including gastrointestinal carcinomas. We have previously shown successful therapy for colorectal, pancreatic, gastric, and esophageal cancers using recombinant adenoviruses expressing dominant negative IGF-IR (IGF-IR/dn). Mutation in k-ras is a critical genetic change for gastrointestinal tumors. Aims: In this study, we sought to evaluate the effect of IGFIR blockade, using a monoclonal antibody (mAb) for IGFIR or IGF-IR/dn, on the progression of human gastrointestinal cancers with/without k-ras mutation. Methods: We assessed the effect of IGF-IR blockade on signal transduction, proliferation, and survival in six gastrointestinal cancer cell lines with or without k-ras mutation, colon and pancreatic adenocarcinoma, esophageal squamous cancer; and hepatoma. Combination effects of IGF-IR inhibition and chemotherapy were studied. Then IGF-IR blockade was evaluated in the treatment for human gastrointestinal. cancer xenografts in nude mice. Results: IGF-IR blockade inhibited autophosphorylation of IGF-IR and the downstream signals. IGF-IR inhibition suppressed proliferation and up-regulated chemotherapy induced apoptosis in all cell lines. Moreover, the combination of the mAb and chemotherapy was effective against tumors on mice. The effect of IGFIR blockade is not influenced by the mutation stasis of k-ras. The mAb reduced expression of IGF-IR but not insulin receptor in tumors on mice. The drug did not affect on neither murine body weight nor blood concentrations of glucose, insulin, IGFBP-3, and growth hormone. Conclusion: IGF-IR might be a good molecular therapeutic target in human gastrointestinal carcinomas even if k-ras mutated types.

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P9 NEW EMERGING THERAPY TARGETS P9.1 Functions of CTCF and its testis specific paralog BORIS revealed by analysis of knockout mice and ChIP-seq data D. Loukinov, E. Pugacheva, S. Robinson, S. Rivero-Hinojosa, V. Lobanenkov Molecular Pathology Section, Laboratory of Immunogenetics, NIAID/NIH Background: CTCF recently earned the title of “master weaver of the genome“ acknowledging its remarkable versatility and multifunctionality. CTCF protein contains an 11 zinc fingers DNA recognition domain that is flanked with N- and C-termini. This gene is exceptionally conserved throughout evolution from Drosophila to humans. Depending on the genomic context, CTCF acts as a transcriptional repressor or activator as part of hormone-dependent composite silencers. It plays an important role at least in some enhancers and is capable of forming intra- and inter-chromosomal loops via its intrinsic property to form di- and multi-mers. In contrast, BORIS appeared relatively late in evolution by duplication of the CTCF gene and became strictly germ cell specific only in mammals. BORIS evolved rapidly and conservation even

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between mice and men is high in the DNA recognition domain, significant in the N-termini but is insignificant in the C-termini. In humans BORIS is expressed in over a dozen of alternatively spliced isoforms. BORIS is expressed in embryonic stem cells and is frequently detected in various cancers. Aims: Deciphering CTCF and BORIS functions in germ cell development and cancers Methods: Knockout mice, ChIP-seq, bioinformatics Results: Ctcf knockout leads to early embryonic lethality. Conditional Ctcf knockout also leads to death of cells or tissues affected that make it difficult to decipher Ctcf functions.The Boris knockout phenotype is restricted to spermatogenesis defects although the mice are fertile. Compound knockout (Ctcf+/-,Boris-/-) present synergistic phenotype and are largely infertile on male transmission and display partial embryonic lethality (about 25 %) on maternal transmission. Ctcf heterozygous mice are prone to tumor development, especially when challenged with radiation or ENU. All tumors analyzed from those animals are Boris positive. ChIP –seq for CTCF and BORIS had been performed in several cancer cell lines. Many interesting cancerrelated loci, including c-MYC, hTERT, INK-ARF are occupied by BORIS. Implications of abberrant BORIS occupancy will be discussed. Conclusion: CTCF ensures coordinated gene expression patterns in somatic cells while aberrant BORIS expression alters this pattern leading to malignant phenotypes.

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P10 NEW BIOMARKERS and APPLICATIONS

Department of Pathology University of Calgary and Calgary Laboratory services

P10.1 Aberrant l1cam affects tumor behavior and chemosensitivity in anaplastic thyroid carcinoma

Background: IGF-1 is an oncogenic protein that promotes growth, proliferation and differentiation in many neoplasms through the mTor (Rapamycin) pathway. IGF-1R expression in different types of cancers has been linked to tumor proliferation and progression. IGF-1R has been shown to be expressed in many neoplasms including breast and gastric cancer. There are promising studies that show tumor response to anti-IGF-1R agents such as somatostatins and metphormins in the treatment of these neoplasms. Treatments, other than surgical excision, of liposarcoma have for the most part, proven ineffective. To our knowledge, no studies have been undertaken to evaluate the level of expression of IGF-1R in liposarcomas inspite of studies showing IGF-1R gene mutation this neoplasm. Aims: In this pilot study we examined the expression of IGF1R in cases of liposarcoma using immunohistochemistry Methods: All cases were confirmed histologically. Immunohistochemical identification of IGF-1R was performed using the G11 antibody (Ventana MicrosystemsTM) with a dilution 1:8 and the use epitope exposure (antigen retrieval). Results: There were 38 cases of liposarcoma; including well differentiated liposarcoma (n015), myxoid liposarcoma (n0 10), pleomorphic liposarcoma (n05), and dedifferentiated liposarcoma (n04) and round cell liposarcoma (n-2). Genetic confirmation was obtained in 21 cases with the DDIT3 gene rearrangement seen in 13 cases and ring chromosomes in 5. Three cases were negative. IGF-1R expression was seen in 8 cases (21 %). This included one case of well differentiated liposarcoma, one case of round cell liposarcoma and 6 cases of myxoid liposarcoma. The staining was mainly membranous, heterogeneous and patchy. Conclusion: IGF-1R is expressed in liposarcoma; especially myxoid liposarcoma. Larger studies are needed to examine IFG-1R expression in liposarcoma. Correlation with genetic mutation in the tumors and IGF-1 gene rearrangement is also warranted. Confirmation of such expression may help identify liposarcoma that would potentially benefit from anti IGF-1R therapies.

Koon Soon. Kim, Minho. Shong, Jin Man. Kim Department of Pathology, Cancer Research Institute, Regional Cancer Center, and Infection Signaling Network Research Center, Chungnam National University School of Medicine, Daejeon, Korea Background: Anaplastic thyroid carcinoma (ATC) is one of the most invasive human cancers and has a poor prognosis. Molecular target of ATC that determine its highly aggressive nature of ATC remain unidentified. Aims: This study investigated L1CAM expression and its role in tumorigenesis of ATC. Methods: Expression of L1CAM was evaluated by immunohistochemical analyses of tissue samples from 9 ATC, 10 nodular hyperplasia, and 200 papillary thyroid carcinoma. We investigated the role of L1CAM in proliferation, migration, invasion, and chemoresistance using shRNA knockdown experiments in human ATC cell lines. Finally, we evaluated the role of L1CAM on tumorigenesis using ATC xenograft assay in a nude mouse model. Results: L1CAM expression was not detectable in normal follicular epithelial cells of the thyroid, nodular hyperplasia or in differentiated papillary thyroid carcinoma. In contrast, all 9 cases of ATC samples showed specifically higher expression of L1CAM in the invasive area of the tumor. Specific knockdown of L1CAM in the ATC cell lines, FRO and 8505C, caused a significant decrease in the proliferative, migratory, and invasive capabilities of the cells. Suppression of L1CAM expression in ATC cell lines increased chemosensitivity to gemcitabine or paclitaxel. Finally, in an ATC xenograft model, depletion of L1CAM markedly reduced tumor growth and increased the survival of tumor-bearing mice. Conclusion: We report that L1CAM is highly expressed in the samples taken from ATC patients. L1CAM plays an important role in determining tumor behavior and chemosensitivity in cell lines derived from ATCs. Therefore, we suggest that L1CAM may be an important therapeutic target in ATC patients.

P10.3 MicroRNA expression in primary and metastatic uterine Leiomyosarcoma Y. Ravid1, M. Formansky, Y. Smith4, V.M. Abeler2,3, R. Reich1, B. Davidson2,3

P10.2 Insulin-like growth factor-1 receptor (IGF-1R) expression in liposarcoma as a potential target for therapy. A pilot study W. A. Mourad, MD, FCAP, FRCPC, L. DiFrancesco, MD, FRCPC

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Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, 2Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway 3 The Medical Faculty, Unive

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Background: Leiomyosarcoma (LMS) is a malignant neoplasm of smooth muscle cells that arise in the uterus or in soft tissue throughout the body. LMS comprise 1 % of the uterine malignancies and 45 % of uterine sarcomas. Patients usually present with abnormal uterine bleeding and often are para-menopausal. In general, uterine LMSs are very aggressive tumors with a high recurrence rate. Currently, only limited therapeutic options exist for patients diagnosed with LMS and the lack of prognostic markers and limited understanding of the biological mechanism underlying LMS complicate the clinical management of these tumors. Aims: Our aim is to examine the differentially expressed miRNA profile of primary and metastatic human uterine LMS. Methods: Eight primary and eight metastatic LMS were analyzed using TaqMan low density miRNA array cards and bioinformatics tools were used to identify their predicted targets and the molecular networks they may affect. Results: 352 miRNAs that putatively regulate the expression of 3751 genes were identified to be differentially expressed between the two sites. Consistent with the miRNA-predicted targets, using Onto-Tools suite, 5 significant potential pathways were predicted. Analysis of gene expression array of the same samples revealed the activation of two common pathways, TGF-β and Wnt. Conclusion: To our knowledge, this is the first study to evaluate the differential expression of miRNAs in LMS progression and propose a potential target for future therapy of the disease.

P10.4 Exosome-derived miRNAs in breast and ovarian malignant fluids N. Federman 1 , O. Vaksman 1 , Y. Smith 4 , R. Reich 1 B. Davidson2,3 1

Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, 2Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway 3 The Medical Faculty, Unive Background: Effusions might accumulate in serosal cavities as a consequence of benign and malignant processes. They usually contains cells of various origins and fluid rich in proteins and cell-derived small particles. Exosomes, naturally occurring biological nanovesicles have been shown to contain lipids, proteins, and nucleic acid sequences including miRNAs. It has been postulated that this content might be considered as Wa messenger systemW that exhibits paracrine bioactivities and facilitates tumor communication within the local tumor microenvironment and distantly through transfer of regulatory messages to other cells.

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Aims: Our aim is to examine the differentially expressed acellular miRNA profile in breast and ovarian carcinomaderived serous fluids. Methods: Exosomal microRNA from effusion fluids of breast and ovarian carcinoma patients were extracted and analyzed using TaqMan, low density miRNA array cards and bioinformatics tools to identify their predicted targets and the molecular networks they may affect. Results: 222 miRNAs were detected, of which 12 were ovarian effusion-specific and 9 were breast effusion-specific. Consistent with the miRNA-predicted targets, using OntoTools suite, MAPK, Focal Adhesion, mTOR and WNT pathways were prominent in ovarian carcinoma while the PI-PLC and Ras-Erk pathways were involved in breast carcinoma. Conclusion: To our knowledge, this is the first study to evaluate the differential expression of miRNAs in effusion fluidderived exosomes in these two malignancies. The potential activation of the various pathways indicates on significant difference between the exosomal signaling in these two diseases.

P10.5 Blood-circulated proteins and microRNAs as potential biomarkers for monitoring of lung cancer I.Zborovskaya, V.Aushev, V.Shevchenko, A.Komelkov, O.Kovaleva, S. Kovalev, M.Akselrod, K.Lactionov, K.Archipova, A.Shneiderman, V.Krutovskikh* Oncogenes Regulation Department, N.N.Blokhin Russian Cancer Research Center, Moscow. * IARC, WHO, Lyon, France Background: Circulated miRNAs and proteins are the most promising as potential biomarkers for monitoring of cancer progression, particularly for such initially asymptomatic and aggressive tumors as lung cancer. However, results of previous attempts to identify specific circulating miRNAs and proteins were inconsistent, due to their individual variability in blood. To overcome this, we compared circulating miRNA profiles and quota of proteins in patients with lung squamous cell carcinomas (LSCC) before and after tumor removal, assuming that postsurgical levels of cancer specific biomarkers would decrease. Aims: Investigate blood-circulated proteins and miRNAs as potential biomarkers for monitoring of lung cancer. Methods: 30 patients with LSCC of T1-T3 stages were examined. Blood samples were taken at 2 points: 3 days before surgery, and 7-10 days after. For miRNA profiling “microRNA Ready-to-Use PCR panels” from Exiqon were used. Extracted RNA was reverse transcribed using Universal cDNA Synthesis kit and cDNA was processed on an ABI 7900HT. For validation, qPCR was performed in triplicate and Ct means were used. Peptides were analyzed by LC-

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ESI-MS/MS and LC-MALDI-MS/MS. MS spectra were acquired in a fully automatic mode on an Ultraflex II mass spectrometer. ESI-MS/MS spectra were acquired on a microTOF-Q mass spectrometer with an UltiMate Nanop-LC. Results: We compiled a list of 94 miRNAs, expression of which at least once was reported as deregulated in lung and other invasive SCC. Ct for the total set of 94 microRNAs were analysed. 94,5 % miRNAs from the designed list were successfully detected.The miRNAs that significantly decreased in the blood after LSCC surgery were: miR-205, -19a, -19b, -451, -30b, and some others. Previously, it has been shown that 809 proteins were identified in the plasma samples of the same patients taken before surgery. The expression of 55 proteins was increased at transition from the control to LSCC I-II and III stages. We haven’t detect most of them in postsurgical plasma samples. Conclusion: The methodology involving profiling the plasma miRNA and proteome is developed for the search of tumor markers of lung cancer monitoring.

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incubation at 37oC simplifies the assay and makes it easier to automatize. This combined with use of soluble standards increased the reproducibility of the assay. Comparison between the original and the modified DiviTum assay showed similar values for cancer patients as compared to healthy individuals. A better resolution of cancer patients (including breast, lung, prostate, pancreas and blood malignancies) from healthy individuals was found using DiviTum, as compared to REA-TK or nonradioactive Liaison TK. Further, in healthy carrying BRCA mutation or benign breast lumps increased levels of cell division was detected with DiviTum assay but not Liaison TK assay. Pretreatment s-TK efficiently predicted recurrence and survival for different types of cancer. The effect therapy on cell division in patients with spread cancer illustrates the capacity of the improved assay. Conclusion: S-TK by DiviTum detects 10 times less tumor cell division than other TK analyses due to low signal from healthy. This makes total body cell division analyses a useful parameter for solid cancers. For the implementation in daily practice more clinical research addressing all types of cancer is required.

P10.6 Total body cell division the ultimate biomarker for personalized medicine in cancer? J. S. Gronowitz1, B. Nisman2, Tamar Peretz2 1

P10.7 Improving cancer classification and interpretation using advanced gene expression analysis

Dept of Medical Sciences, Uppsala University and Biovica International, Uppsala, Sweden. 2 Dept of Oncology, Hadassah and Hebrew University Medical Centre, Jerusalem, Israel

David Amar, Ofer Lavi, Hershel Safer, Gideon Dror and Ron Shamir

Background: The hallmark of cancer is cell division. In clinical chemistry a simple automatable assay for monitoring and prognoses of patients with solid tumors using serum is needed. For this purpose thymidine kinase activity (s-TK) is an excellent biomarker as it is only expressed in G1-S and in G2 and leaves the cell by mitotic exit. Aims: To compare an updated s-TK assay to other TK assays, and show results addressing, other markers, and clinical parameters in various types of cancers. Methods: S-TK was determined using gen 1 and gen 2 DiviTum assay. These are based on BrdU phosphorylation with concomitant processing to BrdUTP and its immobilization into a growing DNA-strand anchored in a 96-well microtitre plate, followed by ELISA quantification of DNA amount produced. Serum material analyzed comprised both retrospective cohorts and recently sampled sera. Results: Working time of DiviTum assay was reduced from 24 h to <7 h by use of 2 μl sample and modified reaction conditions. Technically less sample dilution and

Background: Gene expression profiles, measured by microarrays or next generation sequencing of mRNA, allow systematic comparison of cancer patients and healthy individuals. Standard approaches for analysis of such data include building a classifier that can automatically predict the phenotype of a new patient, and differential expression analysis to detect genes that are altered in the cancer patients as compared to the healthy controls. Aims: We wish to develop computational methods for two analysis goals: (1) to improve the classification power and (2) to detect differential co-expression between and within gene groups in order to find disease-specific behavior of pathways and to detect disease-specific microRNAs. Methods: We present two computational tools that facilitate classification, biomarker selection and interpretation when applied to gene expression profiles of cases and controls. The first tool, called NICK, uses protein-protein interaction networks as auxiliary information in constructing a support vector machine (SVM) classifier. The second tool, called DICER, looks for gene modules that manifest different correlation patterns in the disease class compared to the control class.

Tel-Aviv University, The Academic College of Tel-Aviv-Yaffo

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Results: In tests on six breast cancer and three lung cancer datasets, NICK had better discriminative power than SVM and outperformed other classifiers that use network information. When applied to cancer, neurodegenerative disorders, and inflammatory disease profiles, DICER's modules manifested high functional enrichment in signaling and metabolic pathways. Moreover, DICER dissected known pathways into homogenous subunits such that the inter-unit correlation changed significantly between the disease class and the control class, thereby revealing inner structures in disease regulatory networks. In addition, DICER modules pointed out disease-specific microRNA families. Conclusion: Our methods can improve cancer gene expression classification and functional analysis and provide new insights on the disease.

P10.8 New biomarkers in diagnostics and treatment of Glioblastoma multiforme Jiri Polivka, 2,3Jiri Polivka jr., 4Ondrej Topolcan, 1Vladimir Rohan, 5Vladimir Priban

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Department of Neurology, Faculty Hospital in Pilsen and Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen. 2Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen. 3 Department Background: Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults with limited effectiveness of standard therapy (surgery, radiotherapy and chemotherapy with temozolomide). The progress in genomics in GBM, in the detection of new milestones of oncogenesis‚ abnormalities in signalling pathways, tumor microenvironment and pathological angiogenesis over the past decade are of great importance for management of GBM. Aims: The research project “Interdisciplinary complex care of patients with GBM in the context of personalised medicine“, supported by Ministery of Health of Czech Republic is presented. The aim is to estimate the role of novel prognostic biomarkers (Isocitrate dehydrogenases 1 and 2, CpG island methylator phenotype, promoter methylation status of the MGMT gene, biomarkers of tumor angiogenesis) and to include them into optimal management of GBM patients in Western and Eastern Bohemia and to create multivariate model for patients outcome. Methods: Systematic literature review of the role of biomarkers, assessment of biomarkers to clinical practice and multivariate modelling for optimal clinical management of GBM. Results: Preliminary results of 24 patients with GBM are presented.

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Conclusion: The overview of GBM biomarkers and their importace in the management of patients with GBM is presented and discussed. The research project concerning GBM with preliminary results will be presented in this topic.

P10.9 Chromogranin A as a useful neuroendocrine marker in patients with pheochromocytoma R. Bilek, L. Safarik, T. Zelinka Institute of Endocrinology, General Teaching Hospital, Prague, Czech Republic Background: Chromogranin A (CGA) is a member of granins (chromogranins A-C and secretogranins III-VI), which are a family of acid proteins present in the secretory granules of a wide variety of endocrine and neuro-endocrine cells. CGA (439 amino acid protein preceded by an 18residue signal peptide and encoded on chromosome 14) belongs to the granin family contained in secretory vesicles of chromaffin adrenal cells. Serum CGA increases in patients affected by pheochromocytoma and other diseases of the chromaffin system. Aims: The aim of the study was to characterize chromogranin A in plasma of pheochromocytoma patients and control individuals, in order to further evaluate the usefulness of CGA as a marker in the circulation for the management of the pheochromocytoma tumors. Methods: Blood samples for measurement of plasma CGA were collected in 38 pheochromocytoma patients 48±16 (mean ± standard deviation) years old immediately before surgery. Controls consisted of 12 patients without pheochromocytoma aged 53±11 years who underwent surgery of adrenal gland because of non-functioning adrenal masses. A solid-phase two-site immunoradiometric assay was used with primary immobilized monoclonal antibodies and secondary radioiodinated monoclonal antibodies, both directed against the central domain of the molecule (145-245), which is less sensitive to proteolysis (CIS bio international, France; CGA-RIACT). Results: In controls, plasma CGA levels were 50±27 (mean ± standard deviation) ng/ml. In patients with pheochromocytoma, plasma levels of CGA were 15-fold higher than those of controls (725±511 ng/ml), and returned to normal values after removal of the tumor. The relationship between CGA and metanephrines will be presented. Conclusion: Plasma CGA concentration is an effective marker of pheochromocytoma with association to malignity and tumour mass. CGA should be considered together with metanephrines in the diagnosis of pheochromocytoma and in monitoring response and relapse.

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P10.10 Early identification of hepatocellular carcinoma C H Middleton1, W Irving2, J F R Robertson1, A Murray3, J McElveen3, J Allen3, B J Thomson2, S Holdenrieder4, C J Chapman1. 1

C.E.A.C, University of Nottingham, Nottingham City Hospital, UK. 2Division of Microbiology, Queen's Medical Centre, Nottingham, UK. 3Oncimmune Ltd, Nottingham City Hospital, UK. 4University Hospital Bonn, Germany Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer and third most common cause of cancer-related death worldwide. Major risk factors include alcoholism and HBV/HCV infection. Current surveillance methods are sub-optimal; early detection and diagnosis of HCC is paramount to improve prognosis by enabling early treatment prior to the onset of metastatic disease. The literature widely documents the presence of an immune response, in the form of IgG antibodies raised to tumour-associated antigens (TAAs) in cancer patient serum. Autoantibodies (Aabs) provide an in vivo amplification of carcinogenesis, months to years before the tumour bulk becomes otherwise clinically detectable and their measurement may be used as an aid to early diagnosis in a number of solid tumours. Aims: To characterise the autoimmune response in the sera of patients with HCC and liver disease. Methods: Recombinant TAAs identified from the literature as being over-/aberrantly expressed or mutated in liver disease and HCC, were produced in E.coli using high-throughput methods and purified according to manufacturer’s protocols. An abridged version of the commercial EarlyCDT semi-automated ELISA was used to analyse the aab response to 41 TAAs, in the serum of a) HCC patients, b) HBV, HCV and non-viral cirrhosis patients, and c) age- and sex-matched healthy volunteers. Results: A detectable aab response to TAAs was confirmed in the serum of patients with HCC. A 21- antigen panel gave an overall specificity of 92 % and sensitivity of 45 %. The best single antigen gave a specificity 98 % and sensitivity of 9 %. A panel of 8 compared with 4 TAAs resulted in increased panel sensitivity; 17 % to 30 %, whilst maintaining overall panel specificity at 96 %. Conclusion: Results illustrate the value of panel-antigen vs single-antigen mediated aab detection and suggest that an optimised TAA panel would improve on current goldstandards serving as a cost-effective means to improve prognosis worldwide.

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Dept. of Chem. Eng. & Russell Berrie Nanotechnology Inst., Technion, Israel Inst. of Technology, Haifa, Israel. 2 Dept. of Oncology, First Affiliated Hospital of Anhui Medical Univ., Hefei China. 3Liver Cancer Inst.& Zhon Background: While early detection of lung cancer might improve the 5 years survival by 3-4 folds, there is no approved screening protocol for high risk patients. In contrast, breath testing, which links specific volatile organic compounds (VOCs) in exhaled breath to lung cancer conditions, has been showing growing evidence during the last decade. Aims: The overreaching goal of this study is to tailor a novel Nano Artificial Olfactory System (NaNose®) in order to compare the signatures and compositions of exhaled VOCs in people with pulmonary nodules (PNs) as a part of a fast, non-invasive diagnostic method. Methods: Breath samples were collected from 74 volunteers with PNs in a cross-sectional comparative survey conducted in University of Colorado Cancer Center and Denver Veterans Affairs Medical Center (Denver, CO, USA). The exhaled breath was analyzed by the NaNose® and, for comparsion, by GC-MS for determining the chemical nature of the VOCs. Pattern recognition methods were used then to analyze the results obtained from GC-MS and NaNose® and to correlate the results with the clincal data. Results: Discriminant function analysis of the signals of the sensor array containing 5-nm gold nanoparticle sensors and HBC-functionalized random network carbon nanotube sensors discriminated benign from malignant PNs in a high-risk cohort based on lung cancer related VOCs profiles (p<0.0001; accuracy 88±2 %). Further, it discriminated NSCLC from SCLC (p00.0015; accuracy 94±1 %) and between early vs. advanced disease (p<0.0001; accuracy 88±2 %). Conclusion: The results provide a launching pad towards obtaining an inexpensive, compact tool that is amenable to widespread screening and that has a potential for direct and real-time.

P10.12 RECAF serum test discriminates with high sensitivity and specificity pancreatic cancer samples from normal samples Osredkar J1, Suhadolc K1, Štabuc B2, Janša R2, Moro R3, Tcherkassova Janneta3, Moro-Vidal R3 1

P10.11 Non-invasive breath analysis of pulmonary nodules M. Hakim, N. Peled, P. Bunn, Y. Miller, T. Kennedy, J. Mattei, J. Mitchell, F. Hirsch and H. Haick

Institute of Clinical Chemistry and Biochemistry, University Medical Centre Ljubljana, Slovenia 2Department of Gastroenterology, Division of Internal Medicine, University Medical Centre Ljubljana, Slovenia 3Pacific Biosciences Research Centre (PBRC), Ric

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Background: Pancreatic cancer is the 4th leading cause of cancer-related death in the US and has the highest mortality rate of all cancers. Tumors are diagnosed at late stages, when less than 20 % can to be removed by surgery. At that stage chemo and radiotherapy are mostly palliative in nature. There are no early diagnostic tools at the moment to detect pancreatic cancer. RECAF is an oncofetal cancer marker that is present during fetal life in different tissues. Its levels decrease after the fetus is born, to be elevated again when cancer cells are present. RECAF has been shown to be elevated in the serum of patients with a variety of malignancies. Aims: To ascertain if serum RECAF is increased in pancreatic cancer. Methods: The samples (21 pancreatic cancer and 18 normal control serum samples) were blinded and sent to PBRC for testing. 24 additional normal control serum samples from Promedex were also tested blind.

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Results: Using a cutoff value of 4,516 RECAF units, 18/21 pancreatic cancer samples were true positives for RECAF (sensitivity 0 85.7 %) while 2/42 normal controls were false positives (specificity 0 95.2 %). The area under the curve of a receiver operating characteristic test (ROC) was 0.88. The mean RECAF unit for pancreatic cancer was 8,964 and for normal controls it was 2,946. The difference was significant using a median test (p0<0.0001) and a two-tailed t-test (p03.6E-06). Conclusion: Even though pancreatic cancer is not one of the most common types of cancer, its mortality rate is the highest of all cancers and there are no effective diagnostic tools. In this study we show that the RECAF serum test can discriminate normal and pancreatic serum samples with high sensitivity and specificity. More samples and from early stages in particular, need to be tested to establish the usefulness of RECAF in detecting pancreatic cancer.

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P11 LEUKEMIA & LYMPHOMA

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Conclusion: Our results indicate that GAS5 and lincRNAp21 are differentially expressed in MM and that GAS5 can be a candidate biomarker for MM diagnosis.

P11.1 Expression levels of circulating gas5 and lincrna-p21 in multiple myeloma M. Isin1, E. Ozgur1, N. Erten2, U. Gezer1, N. Dalay1 1

I.U. Oncology Institute, Department of Basic Oncology, I.U. Istanbul Medical Faculty, Department of Internal Medicine

P11.2 TK-Thymidine kinase- A new biomarker for lymphoma and leukemia Barak V., Kalickman I., Fridman M, and Gatt M.

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Background: Multiple Myeloma (MM) is a very common hematological malignancy with various clinical consequences and is characterized by the expansion of a plasma cell clone in the bone marrow. New therapeutic approaches give better cure opportunities to the patients but unfortunately, overall survival in MM patients is limited to a median of five years. Nearly 70 % of the human genome is transcribed to RNA and a considerable portion of these transcripts are long non-coding RNAs (lncRNA) which are involved in various cellular processes. One of these molecules is GAS5 which renders cells sensitive to apoptosis by regulating glucocorticoid activity and lincRNA-p21 acts as a scaffold for a protein complex involved in gene silencing and its expression is stimulated by p53 protein. Aims: In our study, we aimed to investigate the relation of growth arrest and cell survival related lncRNAs in MM patients. For this purpose we enrolled GAS5 and lincRNAp21 and we aimed to investigate the circulating levels of lincRNA-p21 and GAS5 lncRNA levels and their correlation with the stage of the disease. Methods: 63 MM patients and 40 healthy controls were enrolled for the study. Total RNA was isolated from 200 μl of plasma and cDNA synthesized. Real-time PCR was carried out using SyberGreen Chemistry (Roche-Germany) to investigate GAS5 and lincRNA-p21 lncRNAs. Analysis of the lncRNA expression levels were performed using basic relative quantification. Results: We found a significant difference in the expression levels of GAS5 (p00.004), no difference was observed for lincRNA-p21 expression (p00.70) in MM patients compared with the healthy group. Interestingly, both lncRNA expression levels were related to the disease state and a correlation between two candidate lncRNAs was also observed.

Immunology Lab. for Tumor Diagnosis, Oncology and Hematology Depts, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Background: Thymidine Kinase (TK) is an enzyme in the salvage pathway for pyrimidines, involved in the nucleic acid synthesis and considered to be a proliferation biomarker. TK, in its old version, was shown previously to be a significant prognostic marker in a number of malignant diseases, both in solid and hematological diseases. Today it is not used routinely in clinical settings. Aims: Our study aimed to assess the usefulness of a new kit that measures TK serum level in a variety of Hematological Malignancies. Methods: We studied correlations between TK levels (Diasorin) and: The routine marker β2-Microglobulin, patients survival and a longitudinal follow-up of TK levels in Multiple Myeloma.194 patients with Hematological Malignancies and 40 Controls were included in this study. The upper limit of the normal values range was determined to be 7.7 U/l, based on average+2SD. Results: No significant correlation was found between TK and β2-microglobulin serum levels. However, in some pts, there was a similar pattern of changes in levels, correlated to clinical parameters. In a long term follow-up of patients with Multiple Myeloma, increases in TK levels were accompanied by recurrence or increase in severity of disease. A statistically significant decreasing trend was found in the TK levels in Multiple Myeloma pts., during the first 3 m of treatment (P<0.005) which correlated to survival. Conclusion: This preliminary study of the new Diasorin TK Biomarker in Hematological malignancies shows prognostic significance of increasing levels and correlation to survival of those patients.

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P12 MELANOMA P12.1 Detecting the V600 BRAF mutation by allele-specific PCR

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P12.2 Melanoma and biomarkers R. Kucera1, I. Treskova2, O. Topolcan1, S. Svobodova1,3, J. Vrzalova1, R. Fuchsova1 Laboratory of Immunoanalysis, 2Department of Surgery, Faculty Hospital in Pilsen and Faculty of Medicine, Pilsen, 3 IIIrd internal Medicine Clinic, 1st Medical Faculty, Charles University in Prague, Czech Republic. 1

Regine Dahse and Hartwig Kosmehl Institute of Pathology, HELIOS Clinical Center Erfurt, Germany Background: The BRAF gene encodes a serinethreonine kinase belonging to the MAPK kinase pathway. BRAF mutations result in proliferation and growth advantage of tumor cells. The most common BRAF mutation is the V600E transversion which has been found in about 50 % of melanoma, 44 % of papillary thyroid carcinoma and in about 10 % of colorectal cancer, ovarian, breast and lung cancer. The frequency of BRAF V600 and the dependence of tumor cell growth and survival on BRAF V600 activity have pointed to this mutation as a promising therapeutic target. Clinical trials with small-molecule BRAF kinase inhibitors for the treatment of cancers harboring the activating V600 mutation show promising preliminary results. Aims: The aim of this study was to design a BRAF mutation screening assay based on PCR and to evaluate the methodology in clinical diagnostics. Methods: Our assay includes one common forward (BF) and two separate reverse primers (BR and BMu). BF: 5’CTCTTCATAATGCTTGCTCTGATAGG-3’; BR: 5’AGTTGAGACCTTCAATGACTTTCTAGT-3’; BMu: 5’CCCACTCCATCGAGATTTCT-3’. The forward primer BF and the reverse primer BR amplify a 273 bp fragment of both mutant and wildtype alleles and thus serve as amplification control. The second reverse primer (BMu) is specific for the mutated allele at the 3’- end. This primer together with BF generates an 143 bp product only in the presence of the V600E (GTG>GAG) and the V600K (GTG>AAG) mutation. PCR cycling is performed with a touch-down PCR. The discrimination of the allele-specific PCR fragments is done by agarose gel electrophoresis. Results: The specificity of the allele-specific PCR was tested in 20 previously sequenced DNA samples of patients with melanoma harbouring the V600 mutation or the BRAF wildtype. It demonstrates 100 % specificity (i. e. detection of only the normal or only the mutant allele), 100 % sensitivity (i. e. no spurious PCR fragments), and acceptable yield. Conclusion: Our protocol provides a rapid, sensitive, and cost-effective BRAF screening method for clinical routine diagnostics.

Background: The incidence of melanomas has been increasing over the past decades. According to WHO statistics 132 000 melanoma skin cancers occur currently each year globally, as an ozone levels have been depleting, the next incidence increase of melanomas is estimated. Aims: Study aim is to evaluate the usefulness of new biomarkers for melanoma detection. Methods: Our group of patients with melanomas consisted of 103 persons. Our healthy control group consisted of 59 persons. We evaluated the serum levels of the following biomarkers. IGF1 serum levels were measured using an IRMA radioisotope IGF1 assay kit (Immunotech, France). IGFBP3, OPG, OPN, EGF and VEGF were measured using an xMAP Luminex multiplex panel (Merck ,USA). Serum samples were collected prior to surgery or other form of treatment. The SAS 9.2 was used for all statistical analysis. A summary of statistical findings for age and serum levels of each of the analytes was presented. The Wilcoxon test was used to compare distributions of values between melanoma group and the group with the healthy persons. Results: We observed statistically significant differences in four biomarkers. The median of IGF1 in melanoma group was 154.1 ng/mL compared to 111.2 ng/mL in group of healthy persons with ρ00.0038. The median of IGFBP3 in melanoma group was 1153 ng/mL compared to 532.2 ng/mL in group of healthy persons with ρ0 0.0001. The median of OPG in melanoma group was 320.7 pg/mL compared to 233.4 pg/mL in group of healthy persons with ρ00.0001. The median of OPN in melanoma group was 16038 pg/ml compared to 8535 pg/mL in group of healthy persons with ρ00.0001. EGF and VEGF were not significant. Conclusion: Our findings regarding statistically significant differences in four biomarkers are optimistic. Increasing of OPN was observed in many studies recently. Our results confirm this fact. EGF and VEGF are not possible to use for the detection of melanomas. Acknowledgements: Supported by the project of MOH of the Czech Rep. for conceptual development of research organization 00669806 - Faculty Hospital in Pilsen and IGA grant project NT11017-5/2010.

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P13 TUMOR CELL BIOLOGY

Department of Biochemistry and Clinical Laboratory Diagnostic of State Medical Academy, Astrakhan, Russia

P13.1 CRABP1 stimulates tumorigenicity and metastasis of rsv-transformed fibroblasts

Background: Earlier we have established that level of pregnancy associated alpha2-glycoprotein (α2-PAG) raises not only at pregnancy but at an inflammation and tumors at studying large clinical material (14536 samples of blood serum and other biological liquids). Its maximum level had been noted at various forms of inflammation with the autoimmune component. Aims: To define probable general points of pathogenesis of an inflammation and tumors were studied immunomodulatory properties of α2-PAG, its possible communication with interleukins and apoptosis factors, dependence of its production from level of hormones. Methods: Serum proteins were determined by immunochemical methods. We used 118 samples of breast cancer patient blood; 102 - of children with kidney diseases, 262 of patients with inflammatory diseases; 139 - of uterine lavage of women with endometrial pathology. Results: Had been revealed communication between rising of α2-PAG level and decreasing of IgA, IgG and lymphocytes in a blood of healthy persons and patients with inflammatory diseases 2.Immunosuppressive role of α2-PAG was revealed by reducing of AFC quantity and of lien mass to 11,51 % and 13,25 %. 3. α2-PAG, TNFα and IL4 had authentically higher levels at autoimmune diseases, than at simple inflammation and identical dynamics of their level was in disease course. Apoptosis inductor DR5 had higher level also, but it had no such precise correlation in dynamics. 4. Maximum α2-PAG level (28 mg/l) had been revealed at the endometrial cancer among hormone-dependent tumours. Authentic change of α2PAG level was revealed from a dose of estrogen up to 47 mg/l. Similar dependence was founded at a breast cancer. These facts correspond to data on affinity of α2-PAG to estrogen. Conclusion: Inflammatory, autoimmune and tumoral diseases have the general pathogenetic parts sold through uniform morphological structures and molecular mechanisms, connected with change of the organism immunoreactivity. Therefore rising of α2-PAG occurs not due to a possible inflammation around of a tumor as considered earlier, and due to activation of the general mechanisms for an inflammation and tumors or due to other additional protein functions, important for development of a tumor.

Y. Kainov, I. Favorskaya, L. Trukhanova, G. Chemeriz, A. Komelkov, E. Tchevkina Oncogenes Regulation Department, Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Russia, Moscow Background: CRABP1 (Cellular Retinoic Acid Binding Protein) is a small cellular protein, which localizes predominantly in the cytoplasm and plays a substantial role in retinoic acid transport and metabolism. To date there is no experimental evidence of CRABP1 participation in metastasis stimulation and tumor progression. Aims: In this study we examined the role of CRABP1 in the regulation of transformed fibroblasts tumorigenicity and metastatic activity in vivo. Methods: A panel of Rous Sarcoma Virus-transformed Syrian hamster primary fibroblasts with different level of metastatic activity was used as an appropriate model to study CRABP1-dependent modulation of metastasis in immunocompetent animals. Methods: Transfection, molecular cloning, retroviral/lentiviral infections, shRNA-mediated gene knockdown, western-blot hybridization, spontaneous metastatic activity assay (subcutaneous injection of cells), experimental metastatic activity assay (intravenous injection of cells), tumorigenicity assay. Results: In the present study we showed a correlation between CRABP1 protein expression and the level of transformed cells with spontaneous metastatic activity. We also demonstrated that CRABP1 overexpression increased the tumorigenecity of cells with low metastatic potential. Moreover, CRABP1 knockdown in a highmetastatic cell line resulted in a significant reduction of both tumorigenecity and spontaneous metastatic activity of the studied cells. In addition, the reduction of CRABP1 expression in a high-metastatic cell line decreased the size of lung metastases formed after intravenous injection of cells. Conclusion: The presented data suggests that CRABP1 strongly contributes to the highly malignant phenotype of transformed mesenchymal cells.

P13.3 CpG methylation and transcription factor c-Myb regulate human Vav1 expression in hematopoietic and cancer cell lines

P13.2 Biological role of inflammation markers defines rising of their level at tumours

Lena Ilan and Shulamit Katzav

D. Nikulina, T. Vorobyeva, P. Ivanov, Ju. Vorobyeva, N. Scvortsova, T. Oganesyan

Developmental Biology and Cancer Research, IMRIC, Hadassah Medical School - Hebrew University, Jerusalem, Israel

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Background: Vav1 is a signal transducer protein exclusively expressed in the hematopoietic system. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma and lung cancer. Immunohistochemical analysis of primary human lung cancer tissue revealed that stronger Vav1 staining was associated with larger tumor size. Knockdown of Vav1 in lung cancer cell line reduced proliferation in agar and tumor growth in nude mice. Aims: to elucidate the mechanisms regulating vav1 gene transcription in hematopoietic and lung cancer cell lines. Methods: site-directed mutagenesis, luciferase reporter assay, bisulfite sequencing, electrophoretic mobility shift assay and western blotting. Results: We demonstrate that mutations in transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Depletion of cMyb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines, led to a dramatic reduction in vav1 expression in these cells. Consistent with this, over expression of the factor activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. Bisulfite sequencing revealed that vav1 promoter was completely unmethylated in human lymphocytes, but shows various degree of methylation in tissues that do not normally express vav1. The vav1 promoter does not contain CpG island upstream to the transcription start site; however, we demonstrated that methylation of a single CpG dinucleotide at a consensus Sp1 binding site interferes with protein binding in vitro. Conclusion: Our data reveal two regulatory mechanisms for vav1 expression: activation by c-Myb and repression by CpG methylation in 5’ regulatory sequences that interrupts with binding of transcription factors. Mutation of other sites able to bind transcription factors suggests that additional factors regulate vav1 expression as well. Taken together, vav1 promoter methylation status and expression of Vav1 and cMyb may be used as markers for tumor aggressiveness.

P13.4 Tumor associated neutrophil (tan) develop pro-tumorigenic properties during tumor progression I. Mishalian, R. Bayuch, L. Zolotriob, L. Levy, Z.G. Fridlender

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Institute of Pulmonary Medicine, Hadassah-Hebrew University Medical Center Background: It is becoming increasingly well recognized that tumor develops a complex immunosuppressive network that can paralyze the effector arm of the immune system. We and others have shown that key players in this immunosuppressive network are the tumor associated neutrophils (TAN). TAN can have antitumorigenic ('N1') or protumorigenic ('N2') function. However, there is limited work on the roles and characteristic of TAN during tumor progression. Aims: We hypothesized that the phenotype of TAN is dependent on the stage of tumor development, with neutral or antitumorigenic, N1-like phenotype in early tumor development, and tumor-promoting, N2-like phenotype in established tumors. We aimed to further characterize TAN's phenotype at different time points during tumor development. Methods: Neutrophils phenotype was evaluated at early (7 day) and late (14 day) stages after tumor injection, by comparing their properties to each other and to naïve BM neutrophils. Results: We found that TAN were more capable of killing tumor cells ex-vivo at early stages of tumor development than TAN from late stages. Our data suggests some possible mechanisms for this ability - production of H2O2, NO and possibly expression of TNF-α by TAN from early stages. Gene expression analysis revealed that TAN possesses a WmixedW phenotype, expressing both N2-associated genes (CCl17, ARG, IL-10) and N1-associated genes (CXCL9, ICAM-1, iNOS). However it seems that TAN from established tumor express high levels of N2- associated genes which support tumorigenesis. Systemic neutrophil depletion after tumor establishment resulted in significantly reduced tumor growth, whereas depletion at early stage of tumor development did not show such an effect Conclusion: Our results show that TAN at early stages of tumor growth have more of an N1 phenotype. They are more cytotoxic to tumor cells both directly and indirectly. Later in tumor-growth they acquired a more WN2-likeW phenotype, hence supporting tumor growth. Understanding the effect of tumor on neutrophils, as well as the way these cells support or fight cancer will help to develop strategies to direct the immune system against the tumor.

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P14 VEGF- ROLE IN CANCER & ANTI VEGF THERAPIES

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P14.2 PLD is a downstream partner of Arf6 and RalB in stimulation of transformed hamster fibroblasts proliferation and metastasis

P14.1 The study of angiogenesis, microvessel characteristics and microRNAs in Glioblastoma multiforme

E. Tchevkina, O. Kovaleva, A. Knizhnik, I. Zborovskaya, V.Rybko

1,3

Jiri Polivka Jr., 2Jiri Polivka, 4Martin Pesta, 5Ondrej Hes, 4Ondrej Topolcan, 1Zbynek Tonar

Oncogenes Regulation Department, N.N.Blokhin Russian Cancer Research Center, Moscow

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Background: Previously we have showed that Ral proteins mediate SMA (spontaneous metastatic activity)stimulative effects of Ha-ras in HET-SR cells, and that RalB is more potent in SMA stimulation than its homolog RalA. Ral proteins cooperate with Arf6 to activate PLD, well-known second-messenger producer that regulates membrane traffic, cytoskeletal reorganization and cell survival. Aims: Investigate the role of RalB- and Arf6-dependent PLD activation in stimulation of SMA and proliferation rate. Methods: HET-SR, a well characterized cell line of RSVtransformed hamster fibroblasts was used as a convenient model for tumor progression and SMA study on immunocompetent animals. Using the “gain-of-function” approach we compared the SMA-stimulative effect of three effector loop mutants of activated RalB each of which was unable to interact with certain RalB effector: with PLD (RalBΔN11), RalBP1 (RalBD49N) or exocyst complex (RalBD49E). Studied Arf6 variants included wild type (Arf6WT), constitutively active form (Arf6Q67L) or mutant unable to interact with PLD (Arf6N48I). Methods: molecular cloning, retroviral infection, western blotting, SMA test, PLD activity assay, proliferation assay. Results: RalB ΔN11 mutant was the only one incapable of stimulating SMA evidencing that interaction with PLD is crucial for RalB-dependent metastasis rise. Expression of Arf6 (WT or Q67L) had no effect on cell motility, invasion or SMA. Moreover, simultaneous expression of RalA/RalB and Arf6 gave no additional stimulation of SMA compared to the effect of RalA/RalB alone. Arf6 (WT or Q67L, but not N48I) effected both proliferation rate and PLD activity, pointing to the significance of Arf6-PLD interaction in proliferation dynamics. PLD mediated an Arf6 effect on mTORC1 downstream targets (S6K1, S6 and 4E-BP1 proteins) involved in translation initiation. Arf6 upregulated Erk1/2 and p38 MAP kinases activity in a PLD-dependent manner. Arf6PLD-mTOR and p38, but not Erk1/2, contributed to Arf6dependent proliferation stimulation. Conclusion: PLD plays the key role in RalB-dependent increase of SMA and Arf6-dependent proliferation rise mediating Arf6-PLD-mTORC1-S6K1-S6/mTORC1-4E-BP1 signaling.

Department of Histology and Embryology, 5Department of Pathology, Faculty of Medicine in Pilsen, Charles University in Prague, Pilsen, 2Department of Neurology, Faculty Hospital in Pilsen and Faculty of Medicine in Pilsen, Charles University in Prague Background: Glioblastoma multiforme (GBM) is the most malignant, extremely invasive and hard to treat primary brain tumor in adults. The angiogenesis is widely examined in GBM. The incorporation of bevacizumab in the standard treatment of relapsed glioblastoma and an increasing number of new antiangiogenic agents in development support the importance of further research of GBM angiogenesis. Another extensively examined area in GBM cancerogenesis is the role of novel small non-coding RNAs (MicroRNAs, miRNAs). Aims: The aim of this study was to examine the parameters of angiogenesis together with selected microRNAs as prognostic biomarkers for 24 GBM patients in relation to clinicopathological characteristics. The study is supported by the project Ministry of Health, Czech Republic for conceptual development of research organization 00669806 – Faculty Hospital in Pilsen, Czech Republic. Methods: The expression of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor-1α (HIF-1α) were measured by immunohistochemistry. The immunostaining of CD31, CD34 and CD105 endothelial markers were used for the determination of microvessel characteristics. A set of miRNAs (miR-21, miR-20a, miR15a, miR-214) in formalin fixed paraffin embedded tissue was analyzed by RT-PCR. Results: Preliminary results of 24 GBM patients are presented together with their angiogenesis expression profiles, microvessel parameters and miRNAs levels. The correlations with clinicopathological characteristics are also involved. Conclusion: The study of GBM biomarkers of angiogenesis and neovascularisation together with the analysis of selected miRNA is presented. The role of these parameters as the prognostic and predictive biomarkers for patients with GBM is discussed in the context of preventive, predictive and personalized medicine.

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P15 TUMOR BIOLOGY AND METASTASIS P15.1 Monocyte activation model to study cytokine involvement in angiogenesis and inflammation promoting metastasis Raphael Nir; Eliezer Zomer SBH Sciences, Natick, USA Background: Monocyte subset populations have been reported to differentiate into pro-inflammatory and anti-inflammatory macrophages. We investigated the use of the THP-1 cell line as an in-vitro model to evaluate this differentiation and to develop potential therapeutics for inflammation. PBMCs (Peripheral Blood Mononuclear Cells) have traditionally been used for in-vitro studies of anti-inflammatory agents. We find that use of these primary cells does not yield consistent results: PBMCs are inherently complex due to their heterogeneous sources, and variation between batches is common. Furthermore, it is well documented that PBMCs can be stimulated by extremely low levels of LPS; concentrations of less than 0.1 ng/ml LPS result in the secretion of various cytokines including TNFa, IFNg, IL-8, and IL-6. Aims: A powerful substitute for PBMCs is the human THP1 cell line. Derived from the peripheral blood of a young

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male with acute monocytic leukemia, this cell line (ATCC; TIB-202) is advantageous in its macrophage function, reproducibility, the fact that there is no donor variability, and the relative ease and reasonable cost of growing large numbers of cells. Methods: We will report the development of an in-vitro THP-1 cell-based model for lead drug optimization with the intent of paving the way for the discovery of a new anti-inflammation or anti-cancer drug. Heretofore we have concluded the following: that 1 ng/ml LPS can induce THP1 to secrete TNFa; that treating cells with known macrophage activators PMA in serum-free media will result in the secretion of IL-8 and of Gal-3, a protein known to be involved in the pathology of cancer metastasis and inflammation; that Glatiramer Acetate (GA), an immunomodulatory agent used in the treatment of multiple sclerosis, can modulate the secretion of TNFa by LPS; and that exposure to GA in the presence of IFNg results in a significant increase in the secretion of IP-10 and MIG. Results: We have initiated an in-silico model based on the interaction of Gal-3 with the nuclear export receptor CRM1 and are currently investigating the biological activity of more than 100 compounds on THP-1, a panel of human cancer cell lines, and on normal human cells. Conclusion: We are excited to collect data to support services for the investigation of drugs for cancer, inflammation, and fibrotic disease.