Tumor Biol. (2010) 31 (Suppl 1):S39–S65 DOI 10.1007/s13277-010-0092-y

Oral Presentations

# International Society of Oncology and BioMarkers (ISOBM) 2010

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of stem-like cells which are susceptible to oncogenic factors.

DOES hCG OR hCG-BETA PLAY A ROLE IN BREAST CANCER CELL BIOLOGY? OP-2 R Iles (1), S Butler (1) (1) Centre for Investigative and Diagnostic Oncology, Middlesex University, UK Aim: hCG’s involvement in breast cancer has been antithetical: the detection of ectopically expressed hCGbeta by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. We review the current literature to reconcile these differences. Method: Literature review and analysis. Results: Recent work has hCGbeta is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression—are phenomena which clearly apply to breast cancer. Conversely, hCg has been shown to alter gene expression and malignant behaviour of breast cancer cells and a clinical trial is underway to see if the incidence of breast cancer is reduced in women who received exogenous hCG. Conclusion: We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population

COMPARATIVE ELISA STUDY OF NF-KAPPAB P65 AND P50, ITS INHIBITOR IKAPPAB, AND UPSTREAM EFFECTOR PROTEIN KINASE AKT1 EXPRESSION AND ACTIVITY IN THE TUMORS OF BREAST CANCER PATIENTS Elena Gershtein (1), A Platova (1), A Scherbakov (1), G Tchemeris (1), N Kushlinsky (1) (1) Russian N.N. Blokhin Cancer Research Center, Moscow, Russia Aims: NF-kappaB signaling activation plays a critical role in conferring hormone and broad-spectrum drug resistance on breast cancer cells, but clinical data supporting these experimental findings are equivocal. Aims: Analysis of NFkB p65 and p50, its IkB alpha inhibitor, and upstream effector protein kinase Akt1 expression and/or activation state in human breast cancer in relation to receptor status and clinico-pathologic factors. Methods: 119 breast cancer patients were enclosed in the study. The following ELISA kits were used: «NF-kBP65 (Total)» (Invitrogen), «TransAM NFkB p65» and «TransAM NFkB p50» (Active Motif), «PathScan Total Akt1 Sandwich ELISA Kit», «PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit», «PathScan Total IkBalpha Sandwich ELISA Kit» and «PathScan Phospho- IkBalpha (Ser32) Sandwich ELISA Kit» (Cell Signaling Technology). Results: A coordinate increase of NF-kB p65 and p50 DNA binding activities was demonstrated in 95% of

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breast cancer samples as compared to adjacent histologically unchanged tissue. NF-kBp65 activity positively correlated with its protein content. The activities of both subunits were positively associated with the contents of IkB alpha and Akt1. NF-kBp50 activity was several times higher than that of p65 and correlated with the level of phosphorilated Akt1. No significant associations were found between the parameters studied and disease stage, tumor size and histology, lymph node status, but HER2+ tumors were characterized by an increase of total NF-kBp65 content independent on steroid receptor status and not accompanied by an enhancement of its DNA binding activity. Conclusion: Increased NF-kB p65 and p50 DNA binding activity, as well as p65, IkB alpha and Akt1 protein content appear to be a widespread feature of human breast cancer. There are no significant associations between these markers (except NF-kB p65 content) and main breast cancer clinicopathologic features including steroid receptors and HER2 status.

BRCA2 genes underwent the BRCA-RA Assay in which 20 genes, previously found to be differently expressed between normal and mutated BRCA, were tested for their expression level. For each gene, an ROC curve was built and a cut-off point was extracted. Results: Of the 57 participants, 26 had BRCA1, 13 had BRCA2, 1 had in both and 17 had no mutation. 17 of the 20 genes tested in the BRCA-RA assay were found to be statistically significant. Under-expression of 6 genes was determined as the cut-off threshold. i.e., a total score of ≥6 implies that the subject is suspected to be with a mutated BRCA (1,2 or both) gene. The sensitivity and specificity of the BRCA-RA assay were determined as 95% and 88.2%, respectively. Conclusions: The study demonstrates the applicability of the BRCA-RA Assay as a tool for assessing carriers of deleterious/null mutations in BRCA1 and/or BRCA2 genes.

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THE PROGNOSTIC RELEVANCE OF SERUM CA27.29 LEVEL IN PRIMARY BREAST CANCER PATIENTS BEFORE ADJUVANT CHEMOTHERAPY—RESULTS OF THE GERMAN SUCCESS TRIAL

A NOVEL RT-PCR ASSAY FOR THE ASSESSMENT OF BRCA MUTATIONS Tamar Peretz(1, 2), A Salmon(1, 2) (1) Micromedic Technologies, Ramat Gan, Israel (2) Sharett Institute of Oncology, Hadassah University Hospital, Jerusalem, Israel Aim: BRCA1/BRCA2 mutation carriers are at a higher risk for developing HBOC and other types of cancer. Mutations in BRCA1/BRCA2 predispose cells to an increased risk of mutagenesis and transformation after exposure to radiation. Normal human lymphoblastoid cells with heterozygous BRCA1 and BRCA2 mutations show increased radiosensitivity. Heterozygous mutation carriers show a different response to DNA damage compared with non-carriers. It was suggested to identify genes which are differentially expressed in lymphocytes from BRCA1/BRCA2 deleterious/null mutation carriers following radiation stress, and to validate whether changes in these genes may be indicative of deleterious/null mutations in BRCA1/BRCA2. Micromedic’s BRCA-RA is an RT-PCR based kit for the identification of carriers of null/deleterious BRCA mutations. The study’s aim was to determine the sensitivity and specificity of the BRCA-RA assay in identifying women with deleterious/null mutations in BRCA1 and/or BRCA2 genes. Methods: 57 subjects who underwent a genetic test identifying deleterious/null mutations in BRCA1 and/or

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J Neugebauer (1), U Andergassen (1), C Schindlbeck (2), A Schneeweiss (3), T Zwingers (4), W Lichtenegger (5), MW Beckmann (6), K Friese (1), W Janni (7), B Rack (1) for the SUCCESS study group (1) Department of Gynecology and Obstetrics, Klinikum der Ludwig-Maximilians-Universitaet, Munich, Germany; (2) Klinikum Traunstein, Traunstein, Germany; (3) Department of Gynecology and Obstetrics in the National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany; (4) Estimate, Augsburg, Germany; (5) Department of Gynecology and Obstetrics, Charité Medical University, Berlin, Germany; (6) Frauenklinik der Universitaet Erlangen, Erlangen, Germany; (7) Department of Gynecology and Obstetrics, Heinrich-Heine-Universtitaet, Duesseldorf, Germany Aims: While tumor markers are frequently used to assess treatment efficacy in metastatic breast cancer, there is lack of evidence regarding the role of MUC-1 markers in primary disease. The value of CA27.29 in the adjuvant setting was prospectively evaluated in the German multicenter SUCCESS study. Methods: The German SUCCESS trial is a multicenter phase III study comparing FEC-Docetaxel (Doc) vs. FEC-Docetaxel-Gemcitabine (Doc-G) and 5 versus 2 years of Zoledronate as adjuvant treatment in patients

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with node positive or high risk node negative primary breast cancer. In this trial serum CA27.29 level has been prospectively evaluated in 3202 patients before and immediately after adjuvant chemotherapy as well as 2 and 5 years thereafter. CA27.29 was measured with the ST AIA-PACK CA27.29 reagent using MUC-1 for AIA600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for positivity was >31 U/ml. Results: Mean CA27.29 serum level before adjuvant chemotherapy was 19,3 U/ml (SD +/- 15,5) in both arms. 8,0% (n=127) of patients in the FEC-Doc-G arm and 7,4% (n=120) in the FEC-Doc arm had a marker of more than 31 U/ ml. Mean CA27.29 serum levels were significantly higher in patients with lobular carcinoma (p=0.001), with positive lymph nodes (p=0.02) and post-menopausal patients (p< 0.001). After a median follow-up period of 34 months 233 patients relapsed and 108 patients died. CA27.29 before chemotherapy was a significant prognostic marker for disease-free survival (DFS) (p<0.0001) and overall survival (OAS) (p<0.0001) in univariate and multivariate analysis. Conclusion: These findings indicate the independent prognostic relevance of serum CA27.29 levels in primary breast cancer patients before adjuvant treatment. Further follow-up within the SUCCESS trial will show whether initial CA27.29 level could serve as a tool for adjuvant treatment monitoring. OP-5 THYROID HORMONE RECEPTOR (TR) EXPRESSION IN MALIGNANT BREAST TUMORS N Ditsch (1), B Toth (2), Miriam Lenhard (1), K Friese (1), D Mayr (3), U Jeschke (1) (1) Department of Gynaecology and Obstetrics, University of Munich, Munich, Germany; (2) Department of Gynaecological Endocrinology and Fertility, University of Heidelberg, Heidelberg, Germany; (3) Department of Pathology, University of Munich, Munich, Germany Aims: Until now the relationship between thyroid status and breast cancer is unknown. Hormone dependency of the mammary gland and the similarity of TR with estrogen and progesterone receptors lead to remark of TR as marker in breast cancer patients. The aim of this study was an analysis of TR expression in patients with sporadic breast cancer. Methods: Immunohistochemical staining was performed in 181 samples of malignant breast tumours. The expression of TRalpha1/2 and TRbeta1/2 was examined. Staining reaction was performed with the ABC method and intensities were analysed using the IRS-score.

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Results: TRalpha1/2 as TRbeta1/2 were expressed in the nuclei of breast cancer cells. Expression of TRalpha1/2 was present in about 57% of breast cancer patients. TRbeta1/2 expression was found in almost 60% of breast cancer patients. Clinical parameters as tumour growth, nodal status and metastasis correlated significantly with the expression of TRalpha or TRbeta. Conclusion: The expression of TR—based on immunoreactive exploration—seems to be an important point in the pathophysiology of malignant breast tumours and might influence prognosis. OP-6 PROGNOSTIC SIGNIFICANCE OF CYTOKERATIN MARKERS IN BREAST CANCER—A META ANALYSIS Vivian Barak (1), AC Aronsson (2), I Novikov (3), T Peretz (1), R Einarsson (2) (1) Immunology Laboratory for Tumor Diagnosis,Oncology Dep; (3) Hadassah- Hebrew University Medical Center, Jerusalem, Biostatistical Unit,Gertner Institute for Epidemiology and Health Policy Research; (2) Israel, and IDL Biotech AB, Stockholm, Sweeden Aims: A meta-analysis evaluating the potential of Cytokeratin Markers TPS and TPA, compared to established Tumor Markers CA15-3, CEA for evaluating the clinical status of advanced Breast Cancer patients (pts), their response to therapy and Lead time to metastatic disease. This Meta-analysis should improve statistical power by combining information from many small studies during the last 25 years, to estimate an overall ‘effect’ by summarizing knowledge via a single quantity, to determine ability to draw overall conclusions and random vs. systemic variations. Methods and Patients: A Meta-analysis of 45 studies, dating from 1985 up to now,summarizing 3090 advanced Breast Cancer pts. It included Descriptive/graphical methods ( L’Abbe plot, Forest plot, Funnel plot), estimation of a common effect and between-studies heterogeneity, mainly using a random effect model. Results: Sensitivity (%) of markers from all studies was recalculated (95% CI ) and found; CA15-3- 67.9 % (67.8 / 68.0), CEA - 53.0 % (52.8/53.1), TPS- 71.1% (71.0/71.2), TPA- 70.6 % (70.3/70.9) and the combinations CA15-3+ TPS- 81.7 % (81.5/81.9) or CA15-3 +CEA+TPA -89.0 % (88.7/89.3). Clinical correlations to marker levels, overall estimates (95% CI ) were: TPS0.795 (0.594, 0.903), TPA- 0.638 (0.047, 0.898), CA153- 0.554 (0.340, 0.713), CEA - 0.614 (0.405, 0.761). Lead time for local recurrences or Metastases formation

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(months, 95% CI) were: TPS - 4.9 (3.11, 6.82), TPA 2.5 ( 1.76, 3.13), CA15-3- 4.4 (2.93,5.93), or the pannel CEA+TPA+CA15-3 - 11.9 (2.93, 20.89). Conclusion: We conclude that cytokeratin markers TPS/ TPA demonstrated the best response to therapy and the most important information for prognosis in advanced Breast Cancer pts.Using the combination of CEA, CA15-3 and TPA revealed the highest sensitivity followed by TPS and CA15-3. OP-7 SOLUBLE UROKINASE PLASMINOGEN ACIVATOR RECEPTOR SERUM LEVELS IN BREAST CANCER PATIENTS J Tarapacz (1), Urszula Rychlik (1), Z Stasik (1), E Wójcik (1), JK Kulpa (1) (1) Center of Oncology – Maria Sklodowska-Curie Memorial Institute, Cracow Division, Poland Aims: The urokinase plasminogen activator receptor (uPAR) is an essential component of the urokinase plasminogen activator (uPA) system which plays an important role in many processes involved in cancer invasion and metastasis. In the opinion of many investigators, elevated levels of uPAR as well as uPA and PAI-1 correlate with poor patient prognosis. The aim of the presented study was the evaluation of soluble uPAR, CA 15-3, CEA and CRP levels in breast cancer patients, in respect to selected clinical parameters. Methods: Studies of suPAR, CA 15.3, CEA and CRP, were performed in 85 breast cancer patients qualified to surgery. Results: Levels of suPAR>3.8 ng/mL, CA 15.3>25 U/mL, CEA>5.0 ng/mL and CRP>3.0 mg/L in breast cancer group under study were found in: 12.9 %, 44.7 %, 10.6 %, and 30.6 % of patients, respectively. There were no significant correlations between the analyzed parameters. Significantly higher concentrations of CA 15.3 and CRP were presented by patients in more advanced stages of disease ([T1+T2] vs. [T3+T4]), at weak, insignificant tendency to higher levels of suPAR and CEA. No significant differences in levels of the four analyzed parameters were found between groups selected in respect to: age ([<50] vs. [>50]), lymph nodes ([N0] vs. [N1]), HER2 status ([−] vs. [3+]). Those with ER(−)/PgR (−) had a tendency to higher, however insignificant, levels of suPAR in comparison to the remaining patients. Significantly higher concentrations of suPAR and CA 15.3 were observed in patients with CRP levels higher than 3.0 mg/L.

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Conclusions: In breast cancer patients, suPAR seems to be associated with chronic inflammation. OP-8 SPECIFICITY OF AN INDIVIDUAL REFERENCE LIMIT (IRL) COMPARED WITH CONVENTIONAL CUTOFF DURING INTENSIVE POST-OPERATIVE MONITORING OF DISEASE-FREE BREAST CANCER PATIENTS WITH SERUM CEA-TPA-CA15.3 TUMOR MARKER (TM) PANEL A Nicolini (1), P Ferrari (1), S Fancelli (1), M Collareta (1), M Conte (2), L Anselmi (1), P Berti (2), P Miccoli (2) (1) Department of Internal Medicine, University of Pisa, Italy; (2) Department of Surgery, University of Pisa, Italy Aim: 124 disease-free breast cancer patients after mastectomy were intensively monitored with a serum CEA-TPA-CA15.3 tumor marker (TM) panel. To improve cost-effectiveness ratio of the panel, specificity has been evaluated using as the cut-off value an individual reference limit (IRL). Methods: Recently we used the commercial kit cut-off value, and constant elevation (CE) or progressive increase (PI) in one or more TM were considered as significant increases to suspect a relapse. The high tumor marker value was considered to be PI when it was 30% or more higher than in the sample taken 2-3 weeks following the initial elevated value. Otherwise, 2 equally high values were regarded to be a CE. In this study, an IRL has been applied that was calculated with 5 consecutive serum values regularly obtained during 7.1+1.5 (m + sd; 5-9 range) months at the beginning of the follow-up. When a TM value was higher than IRL, the following sample was taken 1–2 weeks following the elevated value. Three consecutive values higher than IRL were considered a significant increase to suspect a relapse. In the 124 patients, tumor marker determinations were performed every 3 months. Results: Using the commercial kit cut-off value, we have previously reported about 2.3%, 25%, 4% and 31% CE and/or PI for CEA, TPA, CA15.3 and a CEA-TPA-CA15.3 association, respectively, with CEA-TPA-CA15.3 panel. In this study, total determinations were 266 and high values of CEA, TPA, CA15.3 and CEA-TPA-CA15.3 association were 2, 52, 4 and 57 in 1 (0.8%), 35 (28%), 3 (2.4%) and 38 (31%) patients. However, only one (0.8%) patient was falsely suspected of relapse with TPA and CEA-TPACA15.3 panel. Conclusions: IRL as cut-off value strongly increased specificity of serum and CEA-TPA-CA15.3 panel compared to using a conventional cut-off. These preliminary

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data suggest that an appropriate use of IRL permits to carry out in these patients an intensive monitoring with a favorable cost-effectiveness ratio.

OP-9 IDENTIFICATION OF RECURRING TUMOR-SPECIFIC SOMATIC MUTATIONS IN ACUTE MYELOID LEUKAEMIA BY TRANSCRIPTOME SEQUENCING

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which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in a cohort of 95 CNAML patients was 2%. Conclusion: Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation. OP-10

PA Greif (1,2), SH Eck (3), A Benet-Pagès (3), NKon standin (1,2), A Dufour (2), A Vetter (1), H Popp (2), B Lorenz-Depiereux (3), T Meitinger (3,4), TM Strom (3,4), SK Bohlander (1,2)

EMBRYONIC AND FETAL HEMOGLOBINS ARE NOT ONLY INDICATORS OF HYPOXIA, BUT ALSO POSSIBLY ADDITIONAL MARKERS IN MONITORING PATIENTS WITH HEMOBLASTOSIS

(1) Clinical Cooperative Group Leukemia, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany; (2) Department of Medicine III, Universität München, Munich, Germany; (3) Institute of Human Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany; (4) Institute of Human Genetics, Technische Universität München, Munich, Germany

Dina Nikulina (1), Y. Kriventcev (1), R. Bisalieva (1), L. Zakljakova (1), N. Borisova (1)

Aims: Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing using next generation technology was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer as well as cytognetically normal acute myeloid leukaemia (CN-AML). Despite its technical feasibility, whole genome sequencing is still time consuming and cost intensive. As an alternative approach, here we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Methods: Mutations were detected by comparing the transcriptome sequence of a CN-AML with the corresponding remission sample. In a single Genome Analyzer II run, we generated 2.9 Gb of CN-AML and 1.25 Gb of remission transcriptome sequence from the same patient. 66% of AML reads and 78% of remission reads mapped to exon regions. 10,896 and 6,077 Genes were sequenced with an average coverage of seven or higher for AML and remission sample, respectively. By comparing the 63,159 Single Nucleotide Variants (SNVs) discovered in the CN-AML sample with the remission sample, we identified 3 non-synonymous mutations specific to the tumor sample. Results: We found 3 tumor-specific somatic mutations. Among them is a nonsense mutation affecting the RUNX1 gene, which is a frequent mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. A second missense mutation was identified in SHKBP1,

(1) Medical Academy, Astrakhan, Russia Aims: To evaluate immunochemical tests to embryonic and fetal hemoglobin (HbE and HbF) in the diagnosis of conditions accompanied by tissue hypoxia in newborns and myeloproliferative marrow diseases. While the study of HbF and HbE in hypoxia is absolutely sound, the aim to study them in diseases associated to the pathological condition of the myeloid population of pluripotent cells especially in erythremia is based on modern conceptions ascribing erythremia to the marrow myelo-proliferative disease group. Methods: HbE and HbF were studied with the use of in-house designed immuno-chemical monovalent test systems in 196 samples of newborns’ umbilical blood, 56 samples of blood donors and 125 samples of hemoblastosis patients (nosologic groups from 18 to 38 patients). Results: The HbF level in newborns with severe intrauterine hypoxia was 1.3–1.7 times as high as in normal newborns. The HbF level in normal newborns was 1077.1±49.2 9 mg/l (boys) and 1211.4±75.0 mg/ l (girls). In intrauterine hypoxia it was 1422.6±51.3 (boys) and 2306.9±92.3 (girls). For the first time HbE was registered in the blood of newborns with severe intrauterine hypoxia (30.23%). In girls in this case the primitive hemoglobin was detected twice as often as in boys (39.13% versus 20.0%, respectively). Also for the first time HbE is detected in patients with erythremia (66.67%) and myeloleukemia (in 49.41% of the subacute form, in 24.44% of the acute form, in 20.53% of the chronic form; from 3.71±0.20 to 4.07±0.23 mg/l); in the acute and chronic lymphoid leukemia HbE was not revealed.

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Conclusions: The findings indicate a possible diagnostic role of early antenatal types of haemoglobin for the estimation of the hypoxic severity and the differential diagnosis of hemoblastosis.

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mesenchymal phenotype. Although hCGβ is secreted in to the culture media the effects maybe due to other factors produced by SCaBER cells. OP-12

OP-11 ECTOPIC HCGBETA MAY INDUCE EPITHELIALMENSENCHYMAL TRANSITION ON HUMAN KERA TINOCYTES IN VITRO AND THIS COULD PROMOTE TUMOUR PROGRESSION AND INVASION

IMMUNE DEPRESSION EFFECT OF OLIGOPEPTIDES FROM PREGNANCY SPECIFIC GLOBULIN AND CARCINOEMBRYONIC ANTIGEN A Kazimirsky (1), J Salmasi (1), T Makarov (1), G Porjadin (1), A Terentiev (1)

Xu Song Wen (1), D Li (1), L Ghali (1), R Iles (1) (1) Russian State Medical University, Moscow, Russia (1) Centre for Investigative & Diagnostic Oncology, Middlesex University, UK Aims: Our more recent work on cervical cancer showed that there was a positive correlation between hCGβ expression and tumour progression, invasion and metastasis. However, the mechanism by which hCGβ participates in these malignant aspect of oncogenesis is still unknown. Our most recent study has suggested that the hCGβ expressed by tumors is structurally different from that derived during pregnancy. This study aims to examine the effect of tumour cell derived hCGβ on cellular transformation. Methods: Conditioned medium (CM) from ScaBER cell line (HTB-3, ATCC), a stable bladder cancer cell line that is known to over express hCGβ, was used to incubate human keratinocytes (HK, ScienCell) in culture. HK cells were also incubated with either conditioned media from TCL-1 (trophoblast cell line) or 3T3 cells (mouse fibroblast cell line) or by adding recombinant hCGβ to control medium. HK cells were cultured for 72 hours and cellular morphological changes observed. The expression of Cytokeratin, Vimentin and E-cadherin proteins was studied by immunocytochemistry. The percentage and intensity of positive stained cells were evaluated by averaging of six individual fields under light microscope. Results: Exposure to SCaBER CM resulted in a significant spindled shape change in HK morphology while those treated with TCL-1, 3T3 or recombinant hCGβ showed little change. Immunohistochemistry staining analysis showed decreased expression of cytokeratin (80% weak to 100%strong), increased expression of Vimentin (75% strong to none staining) and down regulation of E-cadherin (50% medium to 100% strong) in HK that were incubated with SCaBER conditioned media. Conclusion: The data shows that factors present in the SCaBER cell CM transform HK cells from an epithelial to

Aims: to study immune depression effect of oligopeptides from Pregnancy Specific Globulin (PSG) and Carcinoembryonic Antigen (CEA) that consisted by definition of influencing synthetic peptides LDSYQCT (AFP14-20), YVCQ (CEA-peptide) and three peptides of PSG: YECE (E-peptide), YQCE (Q-peptide), YVCG (V-peptide) on lymphocytes populations of peripheral blood of healthy donors. Methods: all researches are executed on peripheral blood lymphocytes of 20 healthy donors. The lymphocytes were cultivated under standard conditions during 16 hours. The final concentration of peptides in culture was 10–7 M, then the lymphocytes were washed from peptides and were undergoing immunophenotyping. Results: all investigated peptides have shown a strong immune depression effect. They reduced the quantity of Tlymphocytes (CD3+-cells) and their subpopulations Thelper lymphocytes (CD4+-cells) and cytotoxic T lymphocytes (CD8+-cells) from 32% to 48 %. At the highest degree these peptides influence the quantity of T- helper lymphocytes. Among all investigated peptides with the highest immune depressive activity concerning Tlymphocytes appeared YVCQ (CEA-peptide). This peptide also in the highest degree reduced the maintenance of NKcells (CD16+-lymphocytes) practically two-fold. However all investigated peptides did not change the maintenance of subpopulation of NK-cells (CD56+-lymphocytes). LDSYQCT, YECE, YQCE, YVCG peptides did not influence the maintenance of B-lymphocytes (CD20, CD72, mIgM, mIgG). YVCQ (CEA-peptide) considerably reduced of the maintenance of B-lymphocytes 2–3-fold. Conclusion: the received results demonstrate that we have revealed a peptide site responsible for an immune depression effect both of PSG–pregnancy specific globulin and CEA cancer specific globulin. Research is supported by the grant of the Russian Humanitarian Scientific Fund (RHSF) No.: 09-06-00241A

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OP-13 STROMAL DERIVED FACTOR 1A (SDF-1A), A HOM ING FACTOR FOR MESENCHYMAL PROGENITOR CELLS, IS ELEVATED IN TUMOR TISSUE AND PLASMA OF GLIOMA PATIENTS M Timmer (1), H Theiss (2), K Juerchott (3), C Ries (4), I Paron (1), W Franz (2), Virginia Egea (4), Ke-Tai Guo (1), J Selbig (3), JC Tonn (1), C Schichor (1) (1) Department of Neurosurgery, Ludwig-MaximiliansUniversity, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany; (2) Medical Department I, Ludwig-Maximilians-University, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany; (3) Bioinformatics Group, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany; (4) Division of Clinical Chemistry and Clinical Biochemistry, Surgical Department, Ludwig-Maximilians-University, Campus Innenstadt, Nussbaumstr. 20, 80336 Munich, Germany Aims: Malignant gliomas are a fatal disease without sufficient possibilities for early diagnosis and a lack of chemical markers to detect remission or relapse. The recruitment of progenitor cells like mesenchymal stem cells (MSC) is a main feature of gliomas. SDF-1, a chemokine produced in glioma cell lines, enhances migration in MSC and was associated with cell survival and apoptosis in gliomas. Therefore, this study was performed as a first step to evaluate SDF-1 as chemical marker for gliomas and the interaction of MSC and SDF-1. Methods: SDF1 blood levels of 44 patients and 10 healthy volunteers were analyzed before and after surgery and before and after corticosteroid intake. Tumor tissue was gained from resections of malignant gliomas. In addition, glioma cell lines and human MSC from bone marrow and gliomas were analyzed using rt-PCR, microarray data, cytokine array, cell invasion assay and ELISA. Results: In glioblastoma tissue, immunohistochemistry revealed that SDF-1 and its receptor CXCR4 are expressed in regions of angiogenesis and necrosis, qPCR showed that SDF-1 is elevated and public expression data indicates that CXCR4 is up-regulated. The latter data also illustrates that SDF-1 could be up- or downregulated in glioma compared to normal brain in a transcript specific manner. In plasma, SDF-1 is elevated in glioma patients. The level is reduced by both, dexamethasone intake and surgery. Dexamethasone also decreased SDF-1 production in MSC in vitro.

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SDF-1 stimulates invasion of hMSC in a dose dependent manner. Conclusion: Taken together, we show that SDF-1 is a potent chemoattractant of progenitor cells like hMSCs and its expression is elevated in glioma tissue, resulting in elevated SDF-1-levels in the patient’s plasma samples. For the first time, we describe an influence of corticosteroid intake on biological variables measured in malignant glioma patients. The fact that elevated SDF-1 plasma levels are significantly decreased after tumor resection could be a hint that SDF-1 could act as tumor marker for gliomas. OP-14 METHODOLOGICAL AND CLINICAL PROGRESS IN THYROID TUMOR MARKERS A Carpi (1), A Nicolini (2) (1) Department of Reproduction and Ageing, University of Pisa, Pisa, Italy; (2) Department of Internal Medicine, University of Pisa, Pisa, Italy Aims: New techniques of improving diagnostic reliability of thyroid nodules are needed to asses whether progress in thyroid tumour markers can improve preoperative diagnosis of thyroid nodules. Methods: Analysis of medical literature in PubMed. Results: Two systems of biology concepts, genomics and proteomics, have recently been considered as thyroid tumor markers. Tissue microarray studies can produce genetic maps, and proteomics uncover patterns of protein expression of TTM derived from preoperative biopsies and specimens. Proteomic analysis from various tissue sources can provide useful information regarding the overall state of a thyroid cancer cell. However, despite the realization that these emerging technologies hold great promise, there are still significant obstacles to the routine use of TTM. These include equivocal thyroid nodule tissue morphologic interpretations, inadequate standardization of methods, and monetary costs. Interpretative shortcomings are frequently due to the relative scarcity of cellular material from fine-needle aspiration biopsy (FNAB) specimens. This can be rectified with the large needle aspiration biopsy (LNAB) technique and is exemplified by the favorable performance of galectin-3 determinations on LNAB specimens. LNAB and galectin-3 immunodetection on LNAB reduce the number of inadequate and indeterminate follicular nodules at FNAB cytology. Galectin-3 detection on LNAB shows a specificity for preoperative diagnosis of thyroid nodules significantly higher than FNAB cytology.

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Conclusion: Thyroid tissue tumor marker research includes important methodologic and clinical progress. The detection of galectin-3 on preoperative LNAB improves preoperative selection and diagnosis of thyroid nodules.

have on well characterised antibodies commonly used to recognise hCG.

OP-15

GENE EXPRESSION ANALYSIS FOLLOWING RADI ATION INDUCED MITOTIC CATASTROPHS IN HELA HEP2 CELLS

STRUCTURAL MODELLING OF HUMAN CHORION IC GONADOTROPIN: THE EFFECT OF NICKING AND GLYCOSYLATION ON THE MOLECULAR FOLDING AND LIKELY EPITOPE RECOGNITION BY HCG ANTIBODIES S Butler (1), N Gibbons (1), SU Rehman (1), R Iles (1) (1) Centre for Investigative and Diagnostic Oncology, Middlesex University, Middlesex, UK Aim: Human chorionic gonadotropin is one of four members of the glycoprotein hormone family, so named because between 20–30% of the molecular mass is accounted for by glycosylation. In 1994 the crystal structure of hCG was elucidated and since then the model generated has become the accepted structure of hCG and its molecular variants. With the variation in the extent of glycosylation and nicking effectively giving rise to charge fluctuations across the entire molecule we propose that hCG undergoes topological shifts. Thus, the original model does not fully represent the molecule in its native state due to an additional carboxyl terminal peptide (CTP) and glycosylation which is tremendously variable. In addition, “nicking” of the Keutman loop is also likely to have a pronounced effects on molecular folding. We propose that these shifts may give rise to epitiope changes effecting antibody recognition. Methods: Using bioinformatic tools we have examined the effect of variable CTP, glycosylation and nicking and we have characterized a panel of variant hCG molecules. Results: We have built a “random coil” model of the CTP using sequence editor in hyperchem which when attached to the main peptide shows a limited number of thermodynamically stable conformations and provide evidence for the most likely formation. We have also demonstrated the effect of the normal and hyper-glycosylated O-linked sugars on the CTP and reveal not only three dimensional shifts but also potential secondary structural modifications. N-Linked oligosaccaraides (GGG GGM GGGF) and nicking at 4 sites in the Keutman loop also bring about conformational shifts. Conclusion: We highlight significant changes to the folding in hCG as a result of introducing these molecular changes and suggest the effect that these alterations may

OP-16

T Stigbrand (1), T Lindgren (1), D Eriksson (1), L Johansson (2), K Riklund (3) (1) Department of Immunology, University of Umeå, Umeå, Sweden; (2) Department of Radiation Physics, University of Umeå, Umeå, Sweden; (3) Department of Diagnostic Radiology, University of Umeå, Umeå, Sweden Aims: To explore changes in the gene expression profile during radiation induced mitotic catastrophes. Methods: We measured global gene expression in HeLa Hep2 tumor cells of 24.500 human genes following exposure to 5 Gy of ionizing radiation (60Co) on a bead chip array(Illumina). The raw data were normalized and statistically analysed using BeadStudio. Genes with less than a 2-fold change in expression and a p-value >0.05 were discarded. Signalling pathways and processes significantly altered following irradiation were explored using Metacore. Results: At 6 h following irradiation the expression was changed only for a few genes including histone H2 and H4, essential for activation of a DNA-damage checkpoint. Striking changes appeared at later time-points. From 1296 hours post irradiation a significant fraction of the genes with altered expression were found to be involved in cell cycle progression and its regulation. The significant changes were seen for genes in several mitotic processes, and those involved in the G2/M and the spindle assembly checkpoints. Also centrosome associated genes(amplification, separation, maturation) displayed an increased expression. The gene expression alterations were confirmed for a number of genes, including Aurora kinase A and B, cyclin B, Plk1, HSET and Survivin, by Quantitative Real-Time RT-PCR. Conclusions: This study indicates specific characteristics in the altered gene expression pattern induced by irradiation, which can be linked to the sequential steps observed in HeLa Hep2 cells during a mitotic catastrophe. There are currently a number of inhibitors in clinical trials that can specifically target and potentially inhibit some of these genes including Aurora kinase inhibitors. Therapeutic strategies employing these alterations might potentiate future therapy and enhance tumor cell killing.

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OP-17 SHOX2 DNA METHYLATION—A VALIDATED BIO MARKER FOR DETECTING LUNG CANCER IN BRONCHIAL ASPIRATES JK Field (1), D Dietrich (2), C Kneip (2), O Raji (1), T Liloglou (1), A Seegebarth (2), T Schlegel (2), N Flemming (2), S Rausch (2), J Distler (2), M Fleischhacker (3), V Liebenberg (4), T Giles (5), M Walshaw (6), C Wabbourton (7), B Schmidt (8) (1) Roy Castle Lung Cancer Research Programme Department of Surgery and Oncology, University of Liverpool Cancer Research Centre, Liverpool L3 9TA, UK; (2) Epigenomics AG, Berlin, Germany; (3) Medizinische Klinik m.S. Hämatologie Onkologie, Charité-Universitätsmedizin, Berlin, Germany; (4) Metanomics Health GmbH, Berlin Germany; (5) Department of Pathology, Royal Liverpool Broadgreen University Hospital Trust, Prescot St. Liverpool, L7 8XP; (6) Respiratory Medicine, Royal Liverpool & Broadgreen University Hospital Trust, Thomas Drive, Liverpool, L14 3LB, UK; (7) Thoracic Medicine Aintree Chest Centre Aintree University Hospitals NHS Foundation Trust, Aintree House, University Hospital Aintree, Longmoor Lane, Liverpool, L9 7AL, UK; (8) Universitätsklinik und Poliklinik für Innere Medizin I, Universitätsklinikum Halle (Saale), Halle, Germany Aims: DNA methylation plays an important role in biological processes such as development and cellular differentiation. DNA methylation has been shown to play a major role in carcinogenesis and cancer progression, suggesting that it may be a valuable source of biomarkers. Hypermethylation of SHOX2 in bronchial lavage has previously been shown in a case control study with more than 500 patients to be a clinically useful tumor marker for identifying subjects with lung cancer, especially if histological and cytological findings after bronchoscopy are ambiguous. To facilitate the use of SHOX2 DNA methylation in a diagnostic setting, the “Epi proLung BL Reflex Assay” was developed and the biomarker was validated in an independent patient dataset. Methods: The assay is comprised of the three individual kits “DNA Preparation Kit” for the preparation of bisulfite converted DNA, “Real-time PCR Kit” for the determination of SHOX2 DNA methylation, and “Work Flow Control Kit” for controlling the entire detection process. This study describes the analytical performance of the test and the validation of the biomarker in a blinded and randomized case control study comprised of 250 patients (125 cases, 125 controls). Results: The assay was shown to be a robust and reliable diagnostic tool for identifying patients with lung cancer

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using Saccomanno-fixed bronchial lavage specimens. The assay detected patients with lung cancer for a sensitivity of 78% [CI=69–86]. Specificity of the test was 96% [CI=90– 99] resulting in an accuracy (area under the ROC curve, AUC [95% CI]) of 0.95 [CI=0.91–0.98]. Conclusions: The new CE-marked IVD, Epi proLung BL Reflex Assay, allows the accurate analysis of SHOX2 DNA methylation in Saccomanno-fixed bronchial lavage specimens from patients undergoing first-time bronchoscopy for suspected lung cancer. The test result may be used by physicians to aid in the diagnosis of lung cancer as an adjunct to existing clinical and pathological information. OP-18 DIAGNOSTIC VALUE OF BIOMARKER DICKKOPF-1 (DKK1) IN LUNG CANCER PATIENTS Dagmar Michel (1), F Spelsberg (2), K Krocker (1), D Nagel (1), S Chan (3), K Hofmann (1), S Schuster (1), B Dowell (3), R Hatz (2), P Stieber (1) (1) Institute of Clinical Chemistry, (2) Surgical Department, University Hospital of Munich – Campus Grosshadern, Germany (3) Cancer Discovery Research, Abbott Laboratories, Abbott Park, IL, USA Aim: To investigate the diagnostic capacity of DKK1 as compared to established biomarkers in lung cancer (LC). Methods: DKK1 levels were measured retrospectively by ELISA (Quantikine, R&D; USA) in sera of 218 untreated LC patients (28 small cell lung cancer (SCLC), 102 non small cell lung cancer (NSCLC: 44 squamous, 36 adeno, 22 large cell). Sera of 50 patients with benign lung diseases (BLD) and 27 healthy individuals (HI) served as reference groups. CYFRA 21-1, NSE, CEA, and ProGRP were analyzed in parallel. Results: DKK1 levels were significantly higher in patients with lung cancer than in HI and patients with BLD. At 95% specificity for HI (resp. BLD) the sensitivities were: DKK1 36.9% (15.4%), CYFRA 21-1 58.8% (30%), CEA 34.6% (27.7%), NSE 28.5% (20.8%), ProGRP 26.2% (16.9%). There was no significant correlation of DKK1 and tumor histology. In SCLC (NSCLC) the sensitivities at 95% specificity (vs. HI) were: DKK1 42.9% (35.3%), CYFRA 21-1 46.4% (55.9%), CEA 46.4% (31.4%), NSE 75.0% (15.7%), ProGRP 64.3% (16.9%). The combination of DKK1 with CEA or CYFRA 21-1 significantly improves the discriminative power (vs. HI) for NSCLC (AUC 84.8% resp. 86.5%) and with NSE or ProGRP for SCLC (AUC 90.9% resp. 86.8%). Referring to BLD as reference group none of the various combinations between DKK1 and the established markers led to a

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significant increase of AUC. In NSCLC (stage I) the AUC (vs HI) were: DKK1 76.4%, CYFRA 21-1 81.2% and CEA 74.6%. DKK1 combined with CYFRA 21-1 or CEA significantly improves the discriminative power for NSCLC (stage I) as compared to HI with an AUC of 87.9% resp. 82.8%. Conclusions: DKK1 is not superior to CYFRA 21-1 as best biomarker in lung cancer, in combination with established biomarkers it significantly increases the discriminative power for lung cancer diagnosis, especially in stage I of NSCLC in combination with CYFRA 21-1. Investigations on large patient numbers including smokers and COPD patients as reference group must be performed.

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Conclusion: MonoTotal represents a new tumor marker for NSCLC with clinical significance for progression diagnosis and prognosis. This pilot study should be confirmed in a multi-center clinical trial. It seems that its measurement prior surgery and its regular monitoring after the surgery will result in quality life improvement in patients with NSCLC. This study was supported research project VZ MSM 0021620819. OP-20

OP-19

THE COMBINATION OF MESOTHELIN AND CEA SIGNIFICANTLY IMPROVES THE DIFFERENTIATION BETWEEN MESOTHELIOMA, BENIGN ASBESTOSIS AND LUNG CANCER

MONOTOTAL—MARKER FOR NON-SMALL CELL LUNG CARCINOMA (NSCLC)

T Muley (1), C Stolp (1), M Meister (1), H Hoffmann (2), H Dienemann (2), FJF Herth (3), J Schneider (4)

M Prazakova (1), O Topolcan (1), S Svobodova (1), J Vrzalova (1), L Pecen (1), G Krakorova (1), V Treska (1), M Spisakova (1), L Holubec jr. (1), M Pesek (1)

(1) Translational Research Unit, (2) Dept. of Surgery, (3) Dept. of Pneumology and Respiratory Medicine, Thoraxklinik, University of Heidelberg,Germany (4) Institute and Policlinic for Occupational and Social Medicine, JustusLiebig University, Giessen, Germany

(1) Charles University Prague - Medical faculty Plzen and Faculty hospital Plzen, Czech Republic Aim: To determine clinical significance of tumor marker MonoTotal in patients with NSCLC and to compare it with so far routinely tested tumor markers. Methods and Patients: Cancer group of 93 patients with NSCLC stage I–III, surgery performed at Surgery clinic Faculty hospital Pilsen (2005–2007) according to histological structure (34 patients with adenocarcinoma, 59 patients with epidermoid cancer and control group of 20 patients with benign lung disease and 20 healthy subjects). The following parameters were measured prior surgery and during the 3-year follow-up period every three months using routine immunoanalytical methods: MonoTotal (MT), TPS, TPA, CYFRA 21-1, SCCA, CEA, TK and Chromogranin A. Results: When evaluating pre-operative values we have found a significant serum level increase of all cytokeratines in epidermoid cancer. Elevated MT levels were the most significant both in absolute serum levels and in statistical significance. Diagnostic sensitivity of MT was only 43% prior surgery, but this sensitivity was the highest one. Adenocarcinoma showed only CEA to be significantly elevated. MonoTotal was significantly elevated in NCSLC progression in both histology types (compared to remission values, its sensitivity was 72 %). Pre-operative MT serum levels correlated in both histology types with DFI and OS. TPA correlated only with DFI in epidermoid cancer and CYFRA and CEA only with OS in adenocarcinoma.

Aim: Soluble mesothelin related proteins (SMRP) have been reported as potential markers for the diagnosis of malignant pleural mesothelioma (MPM). We wondered, whether a combination with a CEA-test might improve the relatively low diagnostic yield of the SMRP-test. Methods: 100 newly diagnosed MPM patients and 29 MPM patients at tumour relapse/progression were compared to 75 patients with benign asbestosis and 139 patients with lung cancer. SMRP serum concentrations were measured with MesomarkTM-Kit (Fujirebio Diagnostics, Malvern, USA). CEA was measured with Advia Centaur immunoassay (Siemens, USA). Statistical analyses were done with SPSS 17.0 (Chicago, Illinois, USA). Results: The results of the SMRP-test alone have been recently published (Schneider et al., 2008). The combination of Mesothelin and CEA increased the power to differentiate between MPM, lung cancer, and benign asbestosis. Whereas CEA was found to be low expressed in MPM and therefore nonspecific for the detection of MPM, most lung cancer patients had elevated CEA serum levels. The area under curve (AUC) of the receiver operator characteristics (ROC) curve for Mesothelin alone was found to be only 0.72. When Mesothelin was combined with CEA the AUC of the resulting ROCcurve increased to 0.978. The sensitivity rate was 92% at 95.7% specificity (68% at 99.3%; 67% at 100%) for the differentiation between MPM and lung cancer. The AUC

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of the ROC-curve for the differentiation between MPM and benign asbestosis was 0.878 for the combination of Mesothelin and CEA. The sensitivity rate was 55% at 95% specificity (49% at 99%; 47% at 100%). In contrast, the AUC for the Mesothelin-test alone was only 0.73, and for the CEA-test alone 0.16. Conclusion: The diagnostic yield of the Mesomark-test can be improved when combined with a CEA test in regard to the differential diagnosis between MPM and lung cancer and between MPM and benign asbestosis. For diagnosis the use of both tests should be recommended. OP-21 A PROGNOSTIC SCORE BASED ON CLINICAL FAC TORS AND BIOMARKERS FOR ADVANCED NONSMALL CELL LUNG CANCER J Trapé (1), J Montesinos (2), S Catot (2), J Buxó (2), J Franquesa (1), M Sala (1), M Domenech (2), F Sant (3), JM Badal (3) (1) Service of Clinical Chemistry. Althaia Xarxa Assistencial de Manresa; (2) Service of Oncology, Althaia Xarxa Assistencial de Manresa; (3) Service of Pathology, Althaia Xarxa Assistencial de Manresa, Spain Aims: Survival of patients diagnosed of advanced stage non-small cell lung cancer (NSCLC) is poor. Several studies have demonstrated that the performance status (PS), tumour stage and treatment are independent prognostic factors for survival. Other studied variables include age, histology, CNS metastases, serum tumour markers, LDH, albumin Il-6 and CRP. The objective of the present study is to determine the prognostic impact of clinical factors and biomarkers in patients with advanced stages of NSCLC and establish a prognostic classification of these patients. Methods: A prospective study of 130 patients with nonoperable NSCLC stages IIIA-IV with follow-up until death of all patients. CEA, CA125, CYFRA21-1, albumin, LDH, VSG and leucocytes levels were determined. Results: Multivariate analysis showed PS(ECOG) > 1 HR=2.4 (95%CI, 1.5–3.9), stage IV HR=2.23 (95%CI, 1.4–3.4), no treatment HR = 2.5 (95%CI, 1.5–4.1), CA125>35U/mL HR=2.4 (95% CI, 1.5–3.7), CYFRA 21-1 > 3.6 ng/mL HR = 1.9 (95% CI, 1.2–2.9) and leucocytes >10000/μL HR=1.8 (95%CI, 1.1–2.7), as independent prognostic factors for survival. One point was assigned for each adverse prognostic factor (APF) except for treatment. Patients were classified into three groups according to the number of APF: “low risk” 0–1

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APF; medium risk 2–3 APF (HR=2.8; 95%CI, 1.6–4.9) and high risk 4–5 APF (HR=9.2; 95%CI, 4.8–17.9). Mean survival of patients was 15 months (95%CI, 6.2– 23.8) in the low risk group, 6 months (95%CI, 5.5–6.5) in the medium risk group and 2 months (95%CI, 1.1– 2.9) in the high risk group. When classifying patients according to whether or not they had received chemotherapy, both groups presented similar results to the total group as higher numbers of APF correlated with shorter survival. Conclusion: The application of a SCORE which includes clinical data and biomarkers may improve the prognostic classification of these patients. Further prospective validation studies are needed to confirm these results. OP-22 PROGNOSTIC VALUES OF ProGRP AND IL-6 IN SMALL CELL LUNG CANCER PATIENTS? E Wojcik (1), B Sas-Korczynska (1), Z Stasik (1), JK Kulpa (1) (1) Center of Oncology – M. Sklodowska-Curie Memorial Institute, Cracow Division, Poland Aims: Small cell lung cancer is a rapidly growing neoplasm that represents a strong tendency to dissemination. Although the diagnostic sensitivity and specificity of ProGRP was found to be higher than NSE, only limited and inconsistent data are available on the prognostic value of this marker in SCLC. Apart from the classical prognostic factors, unfavorable impact on lung cancer patients’ survival involved the presence of systematic inflammatory response. Interleukin-6 is regarded as one of main regulators of inflammatory responses and also a factor which plays a significant role in the pathophysiology of cancer. Assessment of influence of relationship between systemic inflammation and tumor markers on prognosis of small cell cancer patients was analyzed. Methods: Studies of ProGRP, NSE, CYFRA 21-1, CEA, SCC-Ag, and IL-6 were performed in 122 small cell lung cancer patients (LD – 74, ED - 48) and in 65 healthy persons. Results: Patients with small cell lung cancer presented significantly higher levels of all assessed tumor markers as well as IL-6, in comparison with reference group. The frequency of elevated levels of analyzed tumor markers were as follows: ProGRP – 69.6 %, NSE – 71.3 %, CYFRA 21-1 – 40.2 %, CEA – 25.0 %, SCC-Ag – 2.5%, LDH – 49.2 % and IL-6 – 58.2 %. In SCLC patients under study, IL-6 was correlated only with NSE and LDH.

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Univariate analysis revealed significant relations between patients’ overall survival and pretreatment levels of all, apart from SCC-Ag, analyzed tumor markers. Multivariate analysis showed that independent, unfavourable prognostic factors were more advanced disease and IL-6 level higher than 6.0 ng/mL. Conclusions: & &

IL-6, besides extensiveness of disease, seems to be an independent prognostic factor in SCLC patients SCLC patients with simultaneously ProGRP levels lower than 230 pg/mL and IL-6 lower than 6.0 ng/ml represent the group with relatively better prognosis.

Results: We identified 13 lung cancer specific (p<0.05) monoclonal antibodies capable to detect non small cell lung cancer patients at stage I. Majority of the cognate antigens are detectable in lung cancer cells via immunohistology, suggesting that these cancer markers originate from the cancer cells. An antibody panel has been validated on multiple cohorts and shows over 80% accuracy. Conclusion: The results are sufficiently convincing to start the development of a new diagnostic tool (i) for the early detection of lung cancer in patient populations at risk (COPD), and (ii) to contribute to more accurate diagnosis of patients who present with small (<1 cm) nodules detected by spiral CT.

OP-23 OP-24 DISCOVERY OF LUNG CANCER BIOMARKERS BY PROFILING THE PLASMA PROTEOME WITH MONO CLONAL ANTIBODY PROTEOMICS TECHNOLOGY M Guergova-Kuras (1), I Kurucz (2), W Hempel (1), N Tardieu (1), J Kádas (2), C Malderez-Bloes (1), A Jullien (1), Y Kieffer (1), M Hincapie (3), A Guttman (3), E Csánky (4,5), B L Karger (3), L Takács (1,2) (1) Biosystems International SAS, Evry, France; (2) Biosystems International Kft., Debrecen, Hungary; (3) Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA; (4) Department of Pulmonology, Medical and Healthcare Center of University Debrecen, Debrecen, Hungary; (5) Department of Pulmonology, Semmelweis Hospital and Healthcare Center, Miskolc, Hungary Aims: Currently, lung cancer 5 year survival probability is between 15 and 20%. This is due to the fact that the majority of the patients are diagnosed at late stages (stage III–IV). Our aim was to develope a widely applicable plasma proteome profiling approach in which we generate large mAB libraries directed against the natural plasma proteome that are screened for disease specific antigens to discover early cancer markers from clinical samples. Methods: Here we show the results of a lung cancer study encompassing screening of plasma samples from control and lung cancer cohorts with >3000 mABs generated against the plasma proteome of lung cancer patients. Four clinical cohorts, totalling 305 patients with lung cancer, 247 healthy controls and 141 patients with other inflammatory lung diseases and non-related cancers were tested via high throughput ELISA screening.

THE ROLE OF NITRIC OXIDE IN HUMAN LUNG CANCER: A MODEL SYSTEM BASED ON A CELL LINE EXPOSED TO LONG-TERM NITRIC OXIDE JA Radosevich (1,2), BJ Vesper (1), A Onul (1), WA Paradise (1), KM Elseth (1), J Xue (1), GK Haines III (3), G Tarjan (4), A Goel (5) (1) Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois-Chicago, Chicago, IL, USA; (2) Jesse Brown VAMC, Chicago, IL, USA; (3) Department of Pathology, Yale University School of Medicine, New Haven, CT, USA; (4) Department of Pathology, John H. Stroger, Jr. Hospital of Cook County, Chicago, IL, USA; (5) Internal Medicine, Baylor Research Institute, Dallas, TX, USA Aims: There are currently only a handful of biomarkers that can be used to predict the response to therapy and/or the development of resistance to various therapies. This is in part because we do not have a complete understanding of what drives these processes. Advances in the field have been hampered by the lack of model systems to study. Our laboratory has developed a new model system in which we adapt cell populations to Nitric Oxide. This adaptation mimics what is seen in the clinical setting. Methods: By gradually adapting a human lung cancer cell line, A549, to high Nitric Oxide (HNO) levels by increasing the concentration of an NO donor over time, we have been able to produce a new cell line, A549-HNO. Results: A549-HNO cells are enriched for an aggressive phenotype. The A549-HNO cells have an identical morphology to the untreated “parent” cell line (A549) from which it originated. Compared to the A549 parent

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cells, the A549-HNO cells grew more aggressively in both standard and low-nutrient media, were more resistant to X-ray radiation/UV/high temperatures, expressed various CD antigen markers, and were more resistant to taxol. Fluorescence activated cell sorting analysis revealed that the A549-HNO cells have: 1) a greater percentage of cells in S-phase of the cell cycle, 2) a significantly greater population of cells that exclude Hoechst 33342 than the parent A549 cells, and 3) no change in ploidy when compared to the parent cell line. Gene chip array studies show that aldehyde dehydrogenase-1 (ALDH1) is up-regulated in the A549-HNO cell line, relative to the parent cell line. Conclusion: Collectively these results support the idea that the HNO adaptation process has enriched the cell population with an aggressive phenotype. This cell line model system will be useful to probe the cellular and gene expression properties of these adapted cells and provides a model system to look for biomarkers that predict response/ resistance to various therapies. OP-25 VERIFICATION OF BIOMARKER GENEEXPRESSION ASSAYS FOR PREDICTIVE DIAGNOSTICS WITHIN THE HER2/EGFR PATHWAY Stefanie Froehner (1), A Roesler (1), R Büttner (2) (1) Roche Diagnostics GmbH, Roche Applied Science Penzberg, Germany; (2) Institute of Pathology, University Hospital of Bonn, Germany Aims: The HER2/EGFR signaling pathway plays an important role in cancer development e.g. in the development of mammary carcinomas. Different genes being involved in this pathway show genetic defects, influencing the tumor development. Our aim was to establish and verify a toolbox of expression assays for ten genes being involved in the HER2/EGFR signaling pathway. We verified these assays on a breast cancer collective comprising 106 FFPET samples and correlated these data to different clinicopathologic patient parameters. Methods: RNA was isolated from tumor tissue slides and adjacent tissue slides of one patient. Gene expression was analysed using a quantitative one-step RT-PCR based on the use of hydrolysis probes. Relative quantification was performed using two reference genes: MRPL19 and ALAS1. Results: Gene overexpression in a considerable amount of patient samples has been observed for AKT1, HER2, HER3 and E-Cadherin. Gene underexpression in a

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considerable amount of patient samples has been observed for EGFR, PIK3CA, PTEN, BRAF and ECadherin. We identified, as expected, a correlation between DAKO score and the HER2 expression status. We investigated furthermore the relationship between ER/PR status, HER2 status and the clinicopathologic parameters T and G and showed that HER2 positive, ER/ PR negative patients have higher T and G values by trend compared to the HER2 negative, ER/ PR positive patients showing lower T and G values by trend. Finally we recognized correlations for HER2/HER3, HER2/ PTEN, AKT1/AKT2. HER2 and HER3 are both overexpressed in 34% of the samples. HER2 and PTEN show an inverse correlation, 32% of the samples showed HER2 overexpression and PTEN underexpression. AKT1 and AKT2 expression in the samples showed a similar expression level pattern. Conclusion: In summary we verified ten gene expression assays within the HER2/ EGFR pathway using a mamma collective comprising 106 breast cancer (BC) patient samples and showed that some genes may serve as biomarkers for BC. OP-26 IS AN ELEVATED PRO-GASTRIN-RELEASING PEP TIDE LEVEL IN WELL AND MODERATELY DIFFER ENTIATED PULMONARY NEUROENDOCRINE TUMORS AN INDICATION FOR A MORE AGGRES SIVE TREATMENT? Catharina M. Korse (1), JMG Bonfrer (1), A Vincent (2), BG Taal (3) (1) Department of Clinical Chemistry, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands; (2) Department of Biometrics, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam; (3) Departement of Medical Oncology, The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Aims: Pro-gastrin-releasing peptide (proGRP) is a recently identified biomarker of small cell lung cancer, a disorder of neuroendocrine tissue differentiation.The aim of this study was to investigate the diagnostic and prognostic value of proGRP in comparison with the customary tumor marker Chromogranin A(CgA), in well and moderately differentiated NETs. Methods: Serum samples were obtained in 285 patients, diagnosed with NET (247 well and 38 moderately

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differentiated), and 295 healthy subjects. The ROC technique was used to assess specificity and sensitivity in the identification of tumor location (lung vs. other). Unadjusted and adjusted Cox proportional hazards models were constructed to determine the association between patients characteristics and tumor markers with overall survival. Results: The upper limit of normal for CgA was established as 88 μg/l and for proGRP as 53 ng/l. High proGRP-levels were only found in pulmonary tumors and unknown sites. ROC-curve indicated a cut-off level of 90 ng/l, with a specificity of 99% and a sensitivity of 43% in distinguishing primary pulmonary tumor from other tumors. In the adjusted Cox model CgA and proGRP were strongly associated with survival (HR=1.72, p<0.0001 and HR= 2.87, p=0.0004 respectively). Conclusion: Elevated proGRP in well and moderately differentiated NET is a strong indication for a primary tumor in the lung. Clinicians should consider an earlier use of a more aggressive treatment in patients with pulmonary neuroendocrine tumors who present with both an elevated proGRP and an elevated CgA. After CgA, proGRP could be a complementary tumor marker for treatment monitoring in patients with pulmonary NET.

Results: We demonstrate through site-directed mutagenesis and multiple biochemical techniques that C48 alkylates Cys468 in Stat3, a residue at the DNA interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines and that C48 inhibits proliferation and survival of tumor cell lines harbouring constitutive Stat3 DNA-binding activity. In addition, C48 shows anti-tumor efficacy in a xenograft and a syngeneic mouse model using human and mouse breast cancer cells, respectively Conclusions: We have identified a small molecule, C48, as a selective Stat3 inhibitor. Collectively, we provide evidence that selective alkylation of Stat3 at Cys468 constitutes a viable site for further development of selective Stat3 inhibitors for cancer treatment.

OP-27

(1) Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain; (2) Urology Department, Hospital Central de Asturias, Oviedo, Spain; (3) Pathology Department, Hospital Central de Asturias, Oviedo, Spain

ALKYLATION OF CYSTEINE 468 IN STAT3 DEFINES A NOVEL SITE FOR THERAPEUTIC DEVELOPMENT R Buettner (1), R Rashid (1), R Corzano (1), M Senthil (1), J Lin (1), M Hedvat (1), A Schroeder (1), A Mao (1), A Herrmann (1), J Yim (1), H Li (1), Y-C Yuan (1), K Yakushijin (1), F Yakushijin (1), N Vaidehi (1), G Gugiu (1), T-D Lee (1), R Yip (1), Y Chen (1), R Jove (1), D Horne (1), J-C Williams (1) (1) Beckman Research Institute, City of Hope Comprehensive Cancer Center, Los Angeles, California, USA Aims: To develop small molecule inhibitors of Stat3. Stat3 is a latent transcription factor that promotes cell survival and proliferation and is frequently found constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Methods: A combination of chemical, biophysical, and biological techniques are used to identify and determine the mechanism of C48 as a selective inhibitor of Stat3.

OP-28 MYOPODIN METHYLATION IS A PROGNOSTIC AND BCG PREDECTIVE BIOMARKER IN PATIENTS WITH T1G3 BLADDER CANCER M Alvarez-Múgica (1,2), V Cebrian (1), JM FernándezGómez (2), F Fresno (3), S Escaf (2), M SánchezCarbayo (1)

Aims: The Bacillus of Calmette-Guérin (BCG) is a standard treatment to reduce tumor recurrence and delay progression of high-risk non-muscle invasive bladder tumors. However, it is not clear yet which T1G3 patients are more prone to display a more aggressive clinical behavior or be susceptible to respond to BCG. The aim of this study was to evaluate the role of myopodin methylation as a clinical outcome prognosticator and predictive biomarker for BCG response in patients with T1G3 bladder tumors. Methods: The methylation status of myopodin was analyzed on tumor specimens belonging to 170 patients with T1G3 bladder cancer, being a subset of them undergoing BCG treatment (n=108). Myopodin methylation was assessed by methylation-specific polymerase chain reactions (MS-PCR). Recurrence, progression into muscle invasive tumors and disease-specific overall survival rates were analyzed using competing risks regression analysis. Results: Among the 170 cases analyzed, 72 of them recurred (42.4%), 36 progressed (21.2%), and 24 died of

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the disease (14.1%). Univariate and multivariate survival analyses revealed that myopodin methylation was significantly associated with an increased recurrence rate (p= 0.004), progression (p=0.002), and shorter disease-specific overall survival (p=0.020). Interestingly, in a subset of cases treated with BCG, myopodin methylation was also related to an increased recurrence rate (p=0.011), progression (p=0.030), and a shorter disease-specific overall survival (p=0.028). Conclusion: Epigenetic analyses revealed that myopodin methylation was associated with the tumor aggressiveness and clinical outcome of patients with T1G3 disease. Myopodin methylation distinguished patients responding to BCG from those who may require a more aggressive therapeutic approach.

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C4-2 cells were incubated with CD4+ and CD8+ T cells alone or if PSMA-negative DU 145 cells were incubated with T cells and the PSMAxCD3 bsc diabody. In an in vivo mouse model, treatment with the PSMAxCD3 bsc diabody and human T cells significantly inhibited tumor growth compared to control groups. Conclusion: The PSMAxCD3 bsc diabody induces highly efficient T cell-mediated lysis of prostate cancer cells in vitro and in vivo. It thus bears a high potential for immunotherapy of prostate cancer. OP-30

OP-29

NEW PREDICTIVE AND PROGNOSTIC BIOMARKERS IN COLORECTAL CANCER PATIENTS WITH UNRE SECTABLE LIVER METASTASES UNDERGOING SE LECTIVE INTERNAL RADIATION THERAPY

TARGETED THERAPY OF PROSTATE CANCER WITH A PSMAxCD3 BISPECIFIC SINGLE-CHAIN DIABODY

Yvonne Fehr (1), S Holdenrieder (1), RT Hoffmann (2), K Tatsch (3), T Jakobs (2), D Nagel (1), P Stieber (1)

Kerstin Fortmüller (1), K Alt (1), P Wolf (1), P Bühler (1), U Elsässer-Beile (1)

(1) Institute of Clinical Chemistry, University-Hospital Munich-Grosshadern, Munich, Germany; (2) Institute of Clinical Radiology, University-Hospital MunichGrosshadern, Munich, Germany; (3) Clinic of Nuclear Medicine, University-Hospital Munich-Grosshadern, Munich, Germany

(1) Department of Urology, Experimental Urology, University Hospital Freiburg, Freiburg, Germany Aims: The prostate specific membrane antigen (PSMA) is an excellent target for immunotherapy because it is selectively expressed on the surface of prostate epithelial cells and upregulated in all stages of prostate cancer. We constructed a bispecific single-chain (bsc) diabody directed against PSMA and the CD3 subunit of the T cell receptor complex to induce lysis of PSMA-expressing prostate cancer cells by redirecting human cytotoxic T lymphocytes. Methods: Binding properties of the PSMAxCD3 bsc diabody and expression of T cell activation markers were analyzed by flow cytometry. The WST-1 cell viability assay was used to determine the lysis of prostate cancer cells in vitro. For testing the therapeutic potential of the diabody in vivo, SCID mice bearing C4-2 prostate cancer xenograft tumors were treated with diabody and human peripheral blood lymphocytes. The control groups received human T cells alone or a PBS vehicle. Results: The PSMAxCD3 bsc diabody showed specific binding to the PSMA-expressing prostate cancer cell line C4-2 and the CD3-expressing Jurkat T cell line. Incubation of C4-2 prostate cancer cells with freshly isolated human CD4+ and CD8+ T cells and diabody in vitro induced a dose-dependent cell lysis of C4-2 cells and upregulation of the T cell activation markers CD25 and CD69. Neither cell lysis nor upregulation of activation markers was observed if

Aims: Selective internal radiation therapy (SIRT) is a new and effective locoregional anticancer therapy for colorectal cancer patients with liver metastases. Markers for prediction of therapy response and prognosis are needed for the individual management of those patients undergoing SIRT. Methods and Patients: Blood samples were prospectively and consecutively taken from 49 colorectal cancer patients with extensive hepatic metastases before, three, six, 24 and 48 hours after SIRT to analyze the concentrations of nucleosomes and further laboratory parameters, and to compare them with the response to therapy regularly determined three months after therapy and with overall survival. Results: Circulating nucleosomes, cytokeratin-19 fragments (CYFRA 21-1), carcinoembryonic antigen (CEA), C-reactive protein (CRP) and some liver markers increased already 24 hours after SIRT. Pretherapeutical levels of CYFRA 21-1, CEA, cancer antigen 19-9 (CA 19-9), asparate-aminotransferase (AST) and lactate dehydrogenase (LDH) as well as 24 hours values of nucleosomes were significantly higher in patients suffering from disease progression (N=36) than in non-progressive patients (N=13). Concerning overall sur-

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vival, CEA, CA 19-9, CYFRA 21-1, CRP, LDH, AST, choline esterase (CHE), gamma-glutamyl-transferase, alkaline phosphatase, and amylase (all 0 h, 24 h) and nucleosomes (24 h) were found to be prognostic relevant markers in univariate analyses. In multivariate CoxRegression analysis, the best prognostic model was obtained for the combination of CRP and AST. When 24 hours values were additionally included, nucleosomes (24 h) further improved the existing model. Conclusion: Panels of biochemical markers are helpful to stratify pretherapeutically colorectal cancer patients for SIRT therapy and to early estimate the response to SIRT therapy. OP-31 TREATMENT UTILITY AND EARLY DETECTION OF METASTASIS IN UVEAL MELANOMA BY BIOLOGI CAL MARKERS Vivian Barak (1), S. Frenkel (2), K. Hendler (2), I Kalickman (1), J. Pe’er (2) (1) Immunol Lab for Tumor Diagnosis, Oncology and Ophthalmology; (2) Departments, Hadassah -Hebrew Uni versity Medical Center, Jerusalem, Israel Aims: We have previously shown that serum biomarkers as Osteopontin (OPN), S-100, Melanoma-inhibitory activity (MIA), and Tissue Polypeptide-Specific Antigen (TPS) are significantly elevated in patients (pts) with metastatic Uveal Melanoma, as compared to disease-free patients. The aim of this study was to examine the impact of treating the primary Uveal Melanoma by enucleation or brachytherapy on the levels of serum biomarkers and study the prediction of liver metastasis by increases in those biomarkers. Methods and patients: Levels of serum biomarkers were analyzed for 75 Uveal Melanoma pts. Biomarker levels were measured using the ELISA method. A matched-pairs analysis was used to compare baseline (pre-treatment) marker levels with those measured at 1, 4, and 10 months after treatment. Differences in biomarker levels were analyzed for the entire group or for each treatment group separately, and correlated to metastasis status vs disease free status of pts. Results: Of the 75 pts, 62 underwent brachytherapy and 13 were enucleated. One month following treatment S-100, MIA, and TPS levels did not change, while OPN increased (11.51 to 13.11 ng/ml, p=0.0430). Four months after treatment there were no changes in marker levels from baseline, except for a significant decrease in S-100 levels in the enucleated pts (0.09 to 0.06 μg/l, p=0.0492). S-100 levels decreased in the brachytherapy patient group (0.11 to

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0.08 μg/l, p=0.0251) only after 10 months. OPN, MIA, TPS, and S -100 significantly (p=0.002) increasing levels were predictive of metastases development. Conclusions: Local treatment (enucleation) of primary Uveal Melanoma resulted in a significant decrease in S100 levels after 1 m and in OPN levels after 4 m. Significant increases in all 4 markers detected earlier than CT and liver enzyme the development of metastases in the liver from Uveal Melanoma. OP-32 ACTIVATED MACROPHAGES CONTAINING TUMOR MARKER IN COLON CARCINOMA: A PROOF OF CONCEPT? Tjitske JE Faber (1), D Japink (1), MPG. Leers (2), MN Sosef (1), MF von Meyenfeldt (4), M Nap (3) Departments of (1) General Surgery, (2) Clinical Chemistry and Hematology (3) Clinical Pathology, Atrium Medical Center Parkstad, 6401 CX Heerlen, the Netherlands; (4) Department of Surgery, Maastricht University Medical Center, 6202 AZ Maastricht, the Netherlands Aims: The presence of CEA-containing activated macrophages in peripheral blood has been demonstrated in patients with colorectal carcinoma using flow cytometry. Macrophages in non-tumoral environment migrate towards sites of injury, phagocytose debris and return to the bloodstream. It seems likely that tumor marker containing activated macrophages are present in the stroma of colorectal carcinoma. Next, they could follow either a haematogenic or lymphogenic route to peripheral blood. The Aim is to assess the presence of tumormarker containing activated macrophages in the stroma of colon carcinoma and in regional, metastasis-free lymph nodes using immunohistochemical stains on serial sections. Methods: from 10 cases of colon carcinoma, 10 samples of tumor tissue and 10x2 regional metastasis-free lymph nodes were cut in serial sections and stained for CD68 to identify macrophages. Serial sections were stained for CEA-, cytokeratin- or M30-presence. Slides were digitalised and then visually inspected using two monitors, comparing the CD68 stain to the tumor marker stain to evaluate the presence of tumor marker positive macrophages. Results: three stage I tumors, one stage II tumor and six stage III tumors were analysed. Macrophages containing CEA, cytokeratin and M30 could be identified in tumor stroma but also in metastasis-free regional lymph nodes using this method. The distribution varied for the different markers.

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Conclusions: the presence of macrophages containing CEA, cytokeratin or M30 in tumor stroma and lymph nodes from patients with colon carcinoma could be confirmed in this series using serial immunohistochemistry. This finding supports the concept of activated macrophages after phagocytosing cell debris being transported through the lymphatic system. These results further support the potential of these activated and tumor marker containing macrophages to serve as an additional tool for diagnosis and follow-up.

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focus on non-invasive detection of pre-malignant colorectal adenomas (EPITEK). Initial biomarker identification will be followed by clinical studies using plasma from 2000 average risk screening colonoscopies to identify the best 2–3 biomarkers for colorectal adenoma. OP-34

OP-33

METHYLATION OF NEUROG1 IN SERUM IS A SENSITIVE MARKER FOR THE DETECTION OF EARLY COLORECTAL CANCER

DETECTION OF COLORECTAL ADENOMAS IN BLOOD USING DNA METHYLATION BIOMARKERS

A Herbst (1), K Rahmig (1), P Stieber (2), A Ofner (1), A Crispin (3), J Neumann (4), R Lamerz (1), FT Kolligs (1)

Antje Kretzschmar (1), R Tetzner (2), M Ebert (1)

(1) Department of Medicine II, (2) Institute of Clinical Chemistry, (3) Institute of Medical Informatics, Biometry and Epidemiology, and (4) Institute of Pathology, University of Munich, Munich, Germany

(1) Klinikum rechts der Isar, Technische Universität München, Germany; (2) Epigenomics AG, Berlin, Germany Aims: Colorectal cancer (CRC) is the second most common cause of cancer death in Europe although highly curable when detected early or at premalignant stage. A blood-based test capable to detect both cancer and advanced adenomas could increase the compliance with a CRC screening test. Recent results from a prospectively enrolled CRC screening cohort with of >7900 patient samples demonstrated that methylated SEPT9 DNA (mSEPT9) in plasma from average risk screening patients predicts CRC with sensitivity of 67% (95% CI: 47–78%) and specificity of 88% (CI: 86–90%) (Church. DDW. 2010). A CE-marked blood test based on mSEPT9 (Epi proColon) has been developed and is now available. Preliminary research revealed that a combination of methylated ALX4 DNA and mSEPT9 detected 71% of late stage adenomas at a specificity of 82% (Tänzer. PLoSOne. 2010). Building on these preliminary results, the EPITEK Study was designed to identify biomarkers of colorectal adenoma. Methods: DNA methylation marker candidates were analyzed on 18 adenoma and 7 normal tissues and confirmed to be differentially methylated in polyps. Additional selection was done based on methylation status in normal peripheral blood. Results: 5 DNA methylation markers were identified based on adenoma tissue. Real time PCR assays have been developed for marker validation with plasma samples from individuals with confirmed polyp status. Conclusion: In summary, the molecular detection of epigenetic biomarkers in blood can be used for CRC diagnosis and the first validation of an epigenetic CRC test in a prospective clinical study has been successfully completed with mSEPT9. Ongoing clinical studies will

Aims: Colorectal cancer is the third most common cancer and a major cause of cancer-related deaths. Early detection of colonic lesions can reduce the incidence and mortality of colorectal cancer. Colonoscopy is the screening test for colorectal cancer with the highest sensitivity, but its acceptance in the general public is rather low. To identify suitable tumor-derived markers that could detect colorectal cancer at early stages in blood samples, we analyzed the methylation status of a panel of genes in sera of affected patients. Methods: Using methylation-specific quantitative PCR, we analyzed the methylation of ten marker genes in sera of 32 healthy individuals and 15 patients with metastasized colorectal carcinomas. Methylated marker genes that were detected in at least 50% of these samples were further analyzed in a tumor stage set consisting of 95 patients with colorectal cancers (stages UICC I to IV). Methylation of the marker NEUROG1 was studied in 45 healthy individuals and 97 colorectal cancers (UICC I and II). Results: HLTF, HPP1/TPEF and NEUROG1 DNA methylation was detectable in 60%, 67%, and 93% of patients with advanced colorectal cancers, respectively. Whereas HLTF and HPP1/TPEF preferentially detected advanced and metastasized colorectal cancers, NEUROG1 methylation was detectable in UICC stages I to IV at a similar rate (73%, 55%, 73%, and 67%, respectively). At a specificity of 91% NEUROG1 reached a sensitivity of 61% (CI 50.4–70.6%) for the detection of early stage colorectal cancers. Furthermore, detection of NEUROG1 methylation was independent of age and gender. Conclusion: Methylation of the NEUROG1 gene is frequently found in sera of patients with colorectal cancers independent of tumor stage. The quantitative detection of NEUROG1 DNA methylation in serum of patients with

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colorectal cancers might be a suitable approach for the noninvasive screening for early stage colorectal cancers. OP-35 CD24: A BLOOD-BASED ASSAY FOR THE EARLY DETECTION OF COLORECTAL CANCER Nadir Arber (1, 2) (1) Micromedic Technologies, Ramat Gan, Israel, (2) Integrated Cancer Prevention Center, Tel Aviv Medical Center and Tel Aviv University, Israel Aim: CRC is a major health concern worldwide, typically developing over many years from its precursor lesion the adenomatous polyp, and providing ample opportunity for early intervention. Early diagnosis of CRC has been shown to improve prognosis and in turn to decrease disease-associated morbidity and mortality. A number of screening modalities are recommended for adenoma and CRC detection, each with related advantages and disadvantages that impact patient acceptance and compliance. A simple, noninvasive test that could reliably identify colorectal adenomas or early carcinomas, will have great utility for CRC early detection as well as high compliance by the general population. The aim of the study was to determine the sensitivity and specificity of a blood-based CD24 assay in detecting colorectal adenomas. Methods: Blood samples from 277 subjects (77 with CRC, 67 with colorectal adenomas and 133 healthy), >40y old which were referred for colonoscopy, were analyzed for CD24 levels in peripheral blood leukocytes using a Western blot assay. Results: Overall sensitivity and specificity for detecting colorectal adenoma (inclusive of optically detectable early adenoma, late adenoma, and cancer) was 72.2% & 79.7%, respectively. Sensitivity of adenoma detection (excluding CRC) was 74.6% and sensitivity of CRC detection was 70.1%. Conclusion: The CD24 assay demonstrated good specificity and sensitivity and superior performance over FOBT, thus positioning it as a useful and reliable tool for the early detection of colorectal cancer. OP-36 TWO SIGNATURES FOR A NEW BLOOD-BASED TEST FOR COLORECTAL CANCER SCREENING S Monnier-Benoit(1), Laura Ciarloni (1), S Hosseinian (1), N Imaizumi (1), S Therianos (1), L Wieczorek (1), C Nichita (2), G Dorta (2), C Rüegg (3) (1) DIAGNOPLEX SA, Lausanne, Switzerland; (2) Centre Hospitalier Universitaire Vaudois (CHUV) - Gastro-Enterology

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and Hepatology Department, Lausanne, Switzerland; (3) Division of Pathology, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland Aims: Adenoma and early colorectal cancer (CRC) screening tools are unsatisfactory. There is a large unmet screening need calling for an accurate, non-invasive and cost-effective test to screen early neoplastic and pre-neoplastic lesions. Our goal is to identify biomarker combinations to develop a CRC screening test, based on a multi-gene assay performed on peripheral blood mononuclear cells (PBMC). Methods: A pilot study was conducted on 92 subjects. Colonoscopy revealed 21 CRC, 30 adenomas larger than 1 cm and 41 healthy controls. 103 biomarkers were selected by 2 approaches: a candidate gene approach based on literature review and whole transcriptome analysis by Illumina® TAG profiling. Blood samples were taken from each patient and PBMC purified. Total RNA was extracted and the 103 biomarkers were tested by multiplex RT-qPCR on the cohort. Different univariate and multivariate statistical methods were applied on the PCR data and 60 biomarkers, with significant p-value (<0.01) for most of the methods, were selected. Results: These biomarkers are involved in several different biological functions, such as cell adhesion, cell motility, cell signaling, cell proliferation, development and cancer. Two distinct molecular signatures derived from the biomarker combinations were established based on penalized logistic regression to separate patients without lesion from those with CRC or adenoma. Signatures were validated using bootstrapping method, leading to a separation of patients without lesion from those with CRC (Se 67%, Sp 93%, AUC 0.87) and from those with adenoma larger than 1 cm (Se 63%, Sp 83%, AUC 0.77). In addition, the organ and disease specificity of these signatures was confirmed by means of patients with other cancer types and inflammatory bowel diseases. Conclusions: The 2 defined biomarker combinations effectively detect the presence of CRC and adenomas with high sensitivity and specificity. A prospective and multicentric study is underway to validate these results. OP-37 PATIENTS WITH METASTASIZED COLORECTAL CARCINOMAS HAVE AN UNFAVORABLE PROGNO SIS WHEN METHYLATED HLTF AND HPP1 TUMOR DNA IS DETECTED IN SERUM AB Philipp (1), P Stieber (2), D Nagel (2), F Spelsberg (3), A Herbst (1), FT Kolligs (1) (1) Department of Medicine II, University of Munich, Germany; (2) Institute of Clinical Chemistry, University of Munich, Germany; (3) Department of Surgery, University of Munich, Germany

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Aims: Hypermethylation of CpG islands is a frequent epigenetic alteration in colorectal carcinomas. We identified helicase-like transcription factor (HLTF) and hyperplastic polyposis 1 (HPP1) as methylation markers in serum of patients with CRC. So far no data is available on the prognostic relevance of these genes when detected in serum of patients with metastasized tumors. The aim of this project was to elucidate the prognostic relevance of methylated HLTF and HPP1 DNA compared to the carcinoembryonic antigen (CEA) in serum of patients with metastasized CRC. Methods: We examined pretherapeutic serum samples of 311 patients with CRC (208 UICC I–III, 103 UICC IV). CEA was determined using the AXsym system (Abbott Diagnostics). Free DNA was extracted from the serum samples. After bisulfite treatment of the DNA, quantitative realtime PCR was performed to determine the amount of methylated HTLF and HPP1 DNA. Results: Serum methylation of HLTF and HPP1 was found in 11% (23/208) and 5% (11/208) of UICC I–III and 24% (25/103) and 51% (53/103) of UICC IV patients. Methylation of the genes HLTF (p=0.0024) and HPP1 (p<0.0001) occurred significantly more frequently in serum samples of patients with metastasized CRC than in those of patients without distant metastases. In this patient group overall survival was significantly shortened when methylation of HLTF (p=0.0005) or HPP1 (p=0.0003) or CEA values> 27 ng/ml (median of UICC IV group, p=0.0020), respectively, were found. Multivariate analysis showed that CEA (HR 1.6; p=0.039) as well as methylation of HLTF and/or HPP1 (HR 2.0; p=0.004) are independent prognostic factors in UICC IV patients. Conclusion: Detection of methylated HLTF or HPP1 DNA and elevated CEA in serum of patients with metastasized CRC is correlated with a worse prognosis. Therefore, HLTF and HPP1 are potentially additive to CEA in identifying patients with an elevated risk. Prospective studies must examine whether these patients need a more intense therapy. OP-38 IDENTIFYING DEREGULATED MICRORNAS AS POTENTIAL BIOMARKERS FOR MALIGNANT TRANSFORMATION OF HUMAN HEPATOCELLUAR ADENOMA ASSOCIATED WITH TYPE IA GLYCOGEN STORAGE DISEASE

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Storage Disease Program, University of Florida College of Medicine, Gainesville, Florida, USA; (4) Charles Dent Metabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK; (5) Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Aims: Hepatocellular adenoma (HCA) is a frequent longterm complication of type I glycogen storage disease (GSD I) and malignant transformation to hepatocellular carcinoma (HCC) is known to occur in some cases. This study was conducted to identify miRNAs differentially expressed in HCA associated with GSD Ia that could serve as biomarkers for risk assessment of malignant transformation. Methods: Massively parallel sequencing analysis was performed in paired HCA and normal liver from seven GSD Ia cases. Statistical analysis of sequencing reads mapped to known miRNA genes was done with Partek Genome Suite. Differential expression of interesting miRNAs was validated by quantitative RT-PCR. Results: Eighty-seven differentially expressed miRNAs were identified by using paired sample t-test analysis. Among them, 43 showed a fold change of at least two. Over-expression of six miRNAs and under-expression of two miRNAs that were involved in tumorigenesis in various tumor types were validated by quantitative RTPCR in paired HCA and normal liver from 10 GSD Ia cases. Conclusion: Among the significantly over-expressed miRNAs, miR-21, miR-34a, and miR-224 have been reported to be up-regulated in human HCC tissues and HCC cell lines, suggesting these miRNAs may be involved in HCA as well as HCC development. Therefore, they could serve as biomarkers for malignant transformation of GSD Ia HCA and are worthy for further investigation. OP-39 PROGNOSIS OF PATIENTS WITH HEPATOCELLULAR CARCINOMA: A RETROSPECTIVE ANALYSIS OF 330 PATIENTS AND CORRELATION WITH THE ESTABLISHED HCC-STAGING SYSTEMS M op den Winkel (1), D Nagel (2), J Sappl (1), G Straub (1), C Zech (3), P Stieber (2), FT Kolligs (1)

P S. Kishnani (1), LY Chiu (2), D Bali (1), D Koeberl (1), S Austin (1), D Weinstein (3), E Murphy (4), K Boyette (1), YT Chen (1, 5), Ling-Hui Li (2, 5)

(1) Medical Department 2, University of Munich, Germany; (2) Institute of Clinical Chemistry, University of Munich, Germany; (3) Department of Clinical Radiology, University of Munich, Germany

(1) Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA; (2) National Genotyping Center, Academia Sinica, Taipei, Taiwan (3) Glycogen

Aims: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, with the highest incidence in Asian countries and the third world. Primarily due to viral

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hepatitis and alcoholic induced liver cirrhosis, it has shown an increasing incidence in the western world, as well. At time of diagnosis, treatment options for HCCpatients are often limited: Only a minor portion of patients is amenable for potentially curative treatment options, i.e. liver transplantation, surgical resection or radiofrequency ablation (RFA). The major fraction of patients is amenable for palliative treatment strategies only, including transarterial chemoembolisation (TACE) or systemic therapy with sorafenib. Several “prognostic models” have been developed to stage HCC. The aim of this study was to analyze survival rates for HCC-patients in our institution and to perform a correlation with the three major staging systems: Barcelona Clinic Liver Cancer (BCLC), Cancer of the Liver Italian Program (CLIP) and Okuda. Methods: We performed a retrospective analysis of medical records at our institution dating from 2001 to 2009 and identified 405 patients with HCC. We analyzed clinical and laboratory parameters, survival curves and treatment modalities and included only those patients with sufficient data for utilization in all staging systems. Results: Overall, 330 eligible patients were identified and were staged by using the three staging systems. Within each staging system, the different levels (ranging from minimal to advanced disease) showed a significantly different course (p< 0.0001), e.g. Okuda I vs. III (median survival 28.6 vs 2.5 months). Overall median survival was 16 months. Furthermore, we identified the following laboratory parameters as being independent negative prognostic factors for survival: Albumin, AFP, Bilirubin, GOT and CRP (each p<0.0001). Conclusion: Survival analysis of HCC-patients in our institution showed a good correlation with the major HCC-staging.

Aims: Approximately 22,000 new ovarian cancer cases are diagnosed each year with approximately 14,000 deaths as a result of the disease. 75% of these new cases are diagnosed with late stage disease (Stage III and IV), which has a five year survival rate of approximately 20%. The aim of this study is to develop ELISA assays to complement CA125 in order to shift diagnosis of ovarian cancer towards Stage I and II, where the five year survival rate is approximately 85%. Methods: Recognizing CA125 as a monitoring tool for ovarian cancer patients, we have spent the last fifteen years selecting marker candidates to complement CA125 to improve monitoring and for a panel of markers to efficiently diagnose early stage disease in post menopausal women. These markers were selected from approximately 25,000 genes because of their over-expression in ovarian tumors, being secreted or released from the surface of the ovarian tumors, and having either low or no expression in normally functioning adult tissues. The selected candidates are TADG14 (KLK8), TADG-15 (Matriptase), SCCE (KLK7), MMP-7 (Pump-1), ALP (Antileukoprotease/SLPI), TADG-12/12D (TMPRSS3 + variant), and Hepsin (TMPRSS1). Results: Thus far we have cloned the TADG series and expressed all the recombinant products using baculovirus technology in insect cells. In addition to the five ELISA assays we have developed, we are in the final stages of developing ELISA assays for TADG-12/12D and Hepsin. Antibodies to both markers are currently in production. Conclusion: The current five assays have been validated for their usefulness on an archived tumor serum bank with IRB approval. We will present the current status of these five assays in advancing monitoring of patients with cancers that do not elaborate CA125 and the usefulness of these markers in recognizing early stage (I&II) disease.

OP-40

OP-41

NEW BIOMARKERS HELP MONITOR PATIENTS DEFICIENT IN CA125 AND MOVE DIAGNOSIS FROM LATE STAGE TOWARD STAGE I AND II

THE UTILITY OF HE4, CA125 AND ROMA IN DIFFER ENTIAL DIAGNOSIS OF BENIGN AND MALIGNANT GYNEACOLOGICAL DISEASES

WC Hitt (1), PJ Stone (1), RA Dennis (2), MJ Cannon (3), K O’Brien (1), D Rose (4), K Shigemasa (5), JB Beard (1), TJ O’Brien (1)

JM Escudero (1), JM Auge (1), X Filella (1), L Foj (1), R Molina (1)

(1) University of Arkansas for Medical Sciences, Department of Obstetrics and Gynecology; (2) Central Arkansas Veterans Healthcare System; (3) University of Arkansas for Medical Sciences, Department of Microbiology and Immunology; (4) University of Arkansas for Medical Sciences, Department of Neurobiology; (5) National Hospital Organization Fukuyama, Department OB/GYN

(1) Biochemistry Department, Unit of Cancer Research, Hospital Clinic, Barcelona Aims: A pelvic mass will be diagnosed in approximately 20% of all women, yet only a small percentage of them represent ovarian cancer. Use of CA125 in diagnosis of pelvic masses is limited because CA 125 lacks expression in the early stages of ovarian cancer and has poor

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specificity in benign gynaecological diseases. The aim of this study was to assess the diagnostic utility of HE4, CA125 and ROMA in patients with different forms of benign and malign pelvic masses. Methods: Serum levels of CA125 and HE4 were determined using a CMIA on ARCHITECT® in 299 benign gynaecological diseases, 39 patients with endometrial cancer, 19 patients with cervical squamous malignancy and 123 ovarian cancer. ROMA (Risk of Ovarian Malignancy Algorithm) selects a high risk group for ovarian cancer according to HE4 and CA125 levels, and menopausal. Results: Excluding patients with renal failure, HE4 and CA125 levels correlated to the histological type (mainly serous-papilar) and stage of the tumor. HE4 was more sensitive in early stage ovarian cancer and CA125 in advanced stages. Specificity was 98.4%, 67.6% and 85.2% for HE4, CA125 and ROMA, respectively. Sensitivity was 76.1%, 84.6%, and 90.8% for HE4, CA125 and ROMA, respectively. AUC-ROC curves were 0.939, 0912 and 0.849 for HE4, CA125 and ROMA, respectively, in the differential diagnosis of benign gynaecological diseases and ovarian cancer. Conclusion: HE4 had a significantly higher specificity than CA125 and similar sensitivity for discriminating benign versus malignant ovarian tumors, obtaining the best results in combination. ROMA improves the specificity compared to CA125 alone and increases the sensitivity for the diagnosis of ovarian cancer. OP-42 MULTIPLEX PANEL FOR OVARIAN CARCINOMA Jindra Vrzalova (1), O Topolcan (1), Z Novotny (2), J Presl (2), M Pesta (3) (1) Immunoanalytic Laboratory, Dep.of Nuclear Medicine, Faculty Hospital in Pilsen, Czech Republic; (2) Dep. of Gyneacology, Faculty Hospital in Pilsen, Czech Republic; (3) Central Isotopic laboratory, Fac. of Medicine in Pilsen, Charles University in Prague Aim: In our study the commercially available xMAP multiplex panel specifically designed for ovarian cancer was tested for the measurement of serum levels of tumour biological activity markers: Macrophage Migration Inhibitory Factor (MIF), prolactin (PRL), CA-125, leptin, osteopontin (OPN) and IGF-II and compare to other tumour markers. Methods: Cancer groups: ◦ 50 patients with ovarian carcinoma ◦ 7 p. with border line tumour of the ovary (BTO)

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Benign groups: ◦ 30 p. with benign ovarian cysts (BOC) ◦ 30 p. with endometriosis ◦ 11 p. with cardiac insufficiency Serum levels of multiplex markers were measured by Beadlyte® Human Cancer Biomarker Panel from Millipore-Upstate (USA) and Luminex 100 instrument (Luminex, USA). Simultaneously, levels of CA125, TK, TPS, HE4 and Monototal were measured by routine immunoanalytic methods. Results: From the multiplex markers, the best ROC characteristics were observed for CA125, IGFII and OPN. HE4 marker had the best ROC characteristic from all measured markers for differentiation of ovarian carcinoma and BOC or endo-metriosis. In the multiplex panel, significant differences in marker levels between ovarian carcinoma and BCO were found for CA125, OPN and IGFII (lower in carcinoma) and between ovarian carcinoma and endometriosis for IGFII, OPN, PRL (for both higher in carcinoma). Levels of all multiplex markers and of CA125, HE4 and TK were altered in patients with cardiac failure. For OPN, MIF and HE4 the levels were different in BTO compared to BCO. CA125 either measured by multiplex or routine method was higher in endometriosis compared to benign cysts. Conclusion and prospective: Multiplex enables an easy simultaneous measurement of multiple markers and could enable the use of a scoring system in the future for clinical use in distinguishing among clinical diagnoses to overstep the imperfection of nowadays used tumour markers. The study was supported by the research grant project NS10258-3. OP-43 WHICH BIOMARKERS ADD TO CA 125 IN THE DIAGNOSIS OF OVARIAN CANCER? A Burges (1), M Lenhard (1), D Mayr (2), D Nagel (3), P Stieber (3) (1)Department of Obstetrics and Gynecology, (2) Department of Pathology, (3) Department of Clinical Chemistry, University of Munich - Campus Grosshadern, Munich, Germany Aims: Ovarian cancer leads to an increased release of several non organ or tumor specific biomarkers. Depending on the profile of release of each marker in benign and malignant diseases the sum of the different markers might lead to an increase of their diagnostic or differential diagnostic capacities.

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Methods: We investigated retrospectively the sera (stored at −80°C) of 109 healthy women (81 premenpausal (prem), 28 postmenopausal (postm)), 396 patients with various benign gynecological disorders (endometriosis, uterus myomatosus, ovarian cysts, benign adnex tumors, bleeding disorders, cervical dysplasia (226 prem, 170 postm)), and 147 patients suffering from ovarian cancer (low malignant potential (LMP): 16; ovarian cancer (OC) stage I: 23, stage II: 15, stage III: 83, stage IV: 10 (32 prem, 115 postm)) at time of diagnosis before first treatment using the following parameters: CA 125 (Abbott, ARCHITECT, USA), CYFRA 21- 1 and CA 72-4 (Elecsys, Roche, Germany), as well as HE4 (Elisa, Fujirebio, USA). Results: All 4 biomarkers investigated showed higher concentrations in the benign diseases as compared to healthy individuals and again a higher release as compared to both control groups in borderline tumors (LMP). In ovarian cancer patients the strongest release could be observed for CA 125 (median 393 U/ml, 95th percentile: 8321 U/ml), the median being >20 fold higher than in the benign disease group. CA 72-4 was released to the highest extent in mucinous epithelial carcinomas, CA 125 and HE4 in serous epithelial carcinomas. A correlation with tumor size and stage could be observed for all 4 markers. Conclusion: CA 125 is the best single marker in the diagnosis of ovarian cancer. The combined analysis with one or several of the other biomarkers leads to a significantly superior profile of diagnostic efficacy of ovarian tumors. Especially for screening purposes it is important to investigate if other malignant or benign conditions might lead to the same pattern of release of biomarkers. OP-44 THE UTILITY OF SERUM HUMAN EPIDIDYMIS PROTEIN 4 (HE4) IN PATIENTS WITH MALIGNANT AND NON-MALIGNANT DISEASES: COMPARISON WITH CA 125 JM Escudero (1), JM Auge (1), X Filella (1), L Foj (1), R Molina (1) (1) Biochemistry, Unit of Cancer Research, Hospital Clinic, Barcelona Aims: The aim of this study was to assess HE4 levels in healthy subjects and in patients with benign and malignant diseases of various origins, and to compare them with CA125.

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Methods: CA125 and HE4 serum levels were determined by CMIA on ARCHITECT® in 111 healthy people, 479 patients with benign pathologies (206 benign gynaecological diseases) and 437 malignant diseases (123 ovarian cancer). 35 U/mL and 150 pmol/L were used as cut-off, respectively. Results: HE4 and CA125 were abnormal in 2% and 4% of healthy people. Abnormal serum levels of HE4 and CA125 were found in 12.3% and 36.9% of patients with benign diseases, respectively. Renal failure showed significantly higher HE4 (p=0.001) than other benign diseases, with a wide range of results. HE4 specificity was significantly higher than CA125 in benign diseases, excluding renal failure. Both HE4 and CA125 had significantly higher concentrations in ovarian cancer than in other malignancies (p<0.001). However, HE4 was also elevated in approximately 30% of patients with NSCLC and patients with endometrial cancer. By contrast abnormal CA125 was found in several malignancies (NSCLC, breast, primary liver cance, lymphomas). When the diagnostic utility of both tumor markers was compared (excluding patients with renal failure), a significantly higher area under the curve was obtained with HE4 than with CA125 for the differential diagnosis of benign versus malignant diseases, or in the diagnosis of gynaecological diseases. Conclusions: Renal failure and serous effusions are the main sources of HE4 false positive results. HE4 specificity is higher than CA125 in benign diseases and in relation to gynaecological malignancies. OP-45 IS RENAL FAILURE A RELEVANT INFLUENCING FACTOR ON HE4 VALUES? Sophie Fürst (1), A Burges (1), M Lenhard (1), A Kirschenhofer (1), L Hertlein (1), D Mayr (3), D Nagel (2), K Hofmann (2), K Krocker (2), A Burges (1), P Stieber (2) (1) Department of Obstetrics and Gynecology, University of Munich, Germany; (2) Department of Clinical Chemistry, University of Munich, Germany; (3) Department of Pathology, University of Munich, Germany Aims: During the last years HE4 has been described as a relevant biomarker in addition to CA 125 in differential diagnosis of ovarian tumors. Single case reports revealed that there might be an influence of renal function on the serum concentration of HE4. Methods: We investigated retrospectively HE4 and CA 125 (ARCHITECT, Abbott, US) in sera of 1236 patients, 570 with various benign diseases, 666 with different malignant diseases and correlated the results with the corresponding

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creatinine level. As cut off (co) Creatinine values (Crea) ≤1 mg/dl in women and ≤1.2 mg/dl in men were regarded as normal. Results: In the various groups of benign and malignant diseases the frequency of increased creatinine levels was similar with 5–30%, in benign urological disorders 58%. The HE4 medians for Crea ≤ co versus > co differed among the benign diseases only for benign urological diseases including BPH, in the other groups only single increased HE4 values in the group Crea > co influenced 95th percentile or range. At 289 pM HE4 reached 100% specificity for benign diseases (if Crea ≤ co). On the basis of this cut off HE4 had a sensitivity of 42% for ovarian cancer if Crea ≤ co and 53% if Crea > co. Only in few patients with other cancer diseases HE4 reached these high levels. Concerning the profile of specificity and sensitivity of HE4 for benign gynaecologic disorders (255xCrea ≤ co; 70xCrea > co) vs. Ovarian Cancer/borderline tumors (100xCrea ≤ co; 30xCrea > co) the AUC was the same for Crea ≤ co and Crea > co (88,5% vs. 88.4%). Conclusions: In patients with a reduced renal function HE4 levels as well as combinations of HE4 with other biomarkers must be interpreted with caution and should not be the single indicator for invasive diagnostic or treatment procedures. OP-46 WHY ARE PLASMA CONCENTRATIONS OF AFAMIN REDUCED IN OVARIAN CANCER? L Fineder (1), B Dieplinger (2), G Wietzorrek (1), I Braicu (3), J Sehouli (3), I Cadron (4), I Vergote (4), S Mahner (5), Pl Speiser (6), R Zeillinger (6), H Dieplinger (1,7) (1) Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria; (2) Department of Laboratory Medicine, Konventhospital Barmherzige Brüder, Linz, Austria; (3) Department of Gynecology, Campus Virchow, Charité University Hospital, Berlin, Germany; (4) Division of Gynecological Oncology, Department of Obstetrics and Gynecology, University Hospitals Leuven, Katholieke Universiteit Leuven, Belgium; (5) Department of Gynaecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; (6) Department of Obstetrics and Gynaecology, Medical University of Vienna, Vienna, Austria; (7) Vitateq Biotechnology GmbH, Innsbruck, Austria Aims: Comparative proteomics identified the vitamin E binding plasma protein afamin as potential novel tumor marker for ovarian cancer (OC). For further validation,

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afamin plasma concentrations were measured in a pilot and a large case/control study. Afamin was significantly decreased in preoperative OC patients compared to controls and increased to control values after successful tumor removal. The aim of this study was to evaluate and specify the diagnostic utility of afamin in OC patients recruited within the OVCAD study and to elucidate possible mechanisms for decreased afamin levels in OC. Methods: We measured afamin by specific ELISA in plasma and ascites from 230 OC patients. 107 OC patients were analysed preoperatively and 6 months after tumor removal and chemotherapy. Patients were subdivided in responding and non-responding to chemotherapy. To investigate a possibly increased consumption of afamin by OC tissue that could explain the diminished plasma levels in OC patients, we studied afamin uptake in HTP77 cells, an established OC cell line, by immuno-blotting and immunohistochemistry. Results: Afamin plasma concentrations were significantly reduced in OC patients and correlated significantly with those in the respective ascites fluids (n=230, rs=0.203; p= 0.004). An increase to values of healthy controls was only observed in patients responding to chemotherapy (p< 0.001), whereas afamin values in non-responders did not change (p=0.202). OC tissue showed massive immunohistochemical reactions with antibodies against human afamin. Studies in HTP77 cells with exogenously added recombinant human afamin showed substantial uptake of afamin as demonstrated by immunoblotting and immunohistochemistry. Conclusions: These data clearly indicate an additional role of afamin in predicting response to thearapy. They furthermore point towards an increased consumption of afamin as response-to-disease mechanism to explain the decreased afamin levels in OC. OP-47 EXTERNAL VALIDATION OF AN ARTIFICIAL NEU TRAL NETWORK (ANN) AND TWO NOMOGRAMS FOR PROSTATE CANCER DETECTION T Ecke (1), P Bartel (1), S Hallmann (1), S Koch (2), J Ruttloff (1), H Cammann (3), M Lein (4,5), K Miller (4), C Stephan (4). (1) Department of Urology, HELIOS Hospital, Bad Saarow, Germany; (2) Institute of Pathology, HELIOS Hospital, Bad Saarow, Germany; (3) Institute of Medical Informatics, Charité - Universitätsmedizin Berlin, Germany; (4) Department of Urology, Charité - Universitätsmedizin Berlin, Germany; (5) Berlin Institute for Urological Research, Germany

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Aims: We performed an external validation of the artificial neural network (ANN) program “ProstataClass” with data in daily routine to increase prostate cancer (PCa) detection rate and to reduce unnecessary biopsies. The diagnostic accuracy of this ANN for prostate cancer detection rate was compared with total PSA (tPSA), percent free PSA (% fPSA), another ANN built on own data (ANN-Saarow), and two nomogram models. Methods: A total of 282 patients were included in the study. The Beckman Access PSA assay was used for 195 patients, the Roche Elecsys 2010 for 87 patients. Pretreatment PSA was measured prior to digital rectal examination (DRE) and twelve core systematic transrectal ultrasound (TRUS) guided biopsies. The individual ANN predictions were generated with the use of the ANN application for the Beckman Access and for the Roche Elecsys 2010 PSA and free PSA assays, which rely on age, tPSA, %fPSA, prostate volume, and DRE. The new ANN-Saarow built on the 282 patients was further compared with the probabilities of two tested nomograms. Diagnostic validity of tPSA, %fPSA, and the ANN was evaluated by ROC curve analysis and comparisons of observed versus predicted probabilities. Results: tPSA ranged from 4.01 to 9.99 ng/ml, %fPSA from 4% to 48%, respectively. Overall, 101 (35.8%) PCa were detected. The areas under the ROC curve (AUCs) were 0.501 for tPSA, 0.669 for %fPSA, 0.694 for ANNCharité, 0.713 for nomogram I, 0.742 for nomogram II, and 0.749 for the ANN-Saarow showing a significant advantage for the ANN-Saarow (p=0.008) and nomogram II (p= 0.009) compared with %fPSA while the other model did not differ from %fPSA (p=0.15 and p=0.41). Conclusions: The ANN-Saarow showed the largest AUC, but nomogram I the best intraclass correlation coefficient in our cohort. Our results showed limitations of multivariate models when external validations were independently performed.

The major disadvantage of current nucleic acid-based highthroughput technologies is the disability to detect posttranslational protein modifications. Neither can these methods match the dynamic range nor kinetic time scales of cancerrelated protein changes occuring on the level of oxidation, phosphorylation or proteolytic cleavage of proteins. Next to spatial and temporal resolution, the bioinformatic integration of consecutive molecular snapshots of fast changes will be considered. Methods: We will discuss analytical and statistical requirements of quantitative differential proteomics using isotopic labeling and mass spectrometry. In terms of pathways and mechanisms cancer biology can be strongly overlapping with inflammatory, immune and ageing processes. Thus, cancer biomarkers have to be profiled against a background of complex and stochastic protein signatures. Results: Annexin A3 is an example of successful discovery, validation and development of a protein biomarker for prostate cancer. We started from a well characterized and controlled panel of clinical samples (biopsies), were proceeding with a differential protein analysis, the development of specific antibodies and a test for detection of the marker in urine of patients. Finally we eventually could validate the marker employing diagnostic clinical studies and tissue arrays. Conclusion: Annexin A3 now offers a novel tool for an urgent clinical need, with a strong correlation to prostate cancer progression.

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(1) Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Germany; (2) Institute of Pathology, Dresden University of Technology, Germany; (3) Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, The Netherlands; (4) Department of Urology, Dresden University of Technology, Germany

THE CHALLENGE OF PROTEIN BIOMARKERS: FINDING THE NEEDLE IN THE HAY STICK AND VALIDATING IT; ANNEXIN A3 IN PROSTATE CANCER

OP-49 KALLIKREIN-RELATED PEPTIDASE 4 (KLK4)— A POTENTIAL BIOMARKER IN PROSTATE CANCER Lina Seiz (1), M Kotzsch (2), F Sweep (3), S Fuessel (4), A Lossnitzer (2), M Schmitt (1), V Magdolen (1)

A Schrattenholz (1), GP Schwall (1), K Groebe(1) (1) ProteoSys AG, Carl-Zeiss-Str.51, 55129 Mainz, Germany Aims: The search for proteomic cancer biomarkers is essentially an epigenetic exercise, because the condensed information of relatively small genomes unfolds on the level of a dazzling number of proteinaceous species.

Aims: To date, PSA (prostate-specific antigen, KLK3) is the best-established clinical marker routinely used for prostate cancer screening and monitoring of disease recurrence in follow-up care. However, limitations such as false positive results in patients with benign prostatic hyperplasia and false negative results in the event of residual disease restrict the use of PSA as an exclusive diagnostic and prognostic marker. To analyze whether

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KLK4, another member of the human kallikrein-related peptidase family, may represent a clinically relevant factor, we aimed to evaluate the expression profile of KLK4 in prostate cancer patients. Methods: A cohort of prostate cancer patients (n=44) was investigated for KLK4 expression. Tissue microarrays representing tumor tissue and the corresponding tumorfree areas of each patient were immunohistochemically stained using two polyclonal antibodies directed against KLK4 (pAbs 617A + 617C)[1]. Semiquantitative score values for tumor and tumor-free areas based on staining intensity and the percentage of positively stained cells were related to clinicopathological parameters including Gleason score, tumor stage, grading, and PSA serum levels. Results: Distinct KLK4 immunostaining was observed in malignant glandular epithelial cells with both antibodies, whereas glandular cells in tumor-free areas were stained only weakly. For pAb 617A a highly significant association (P=0.005) was found between the tumor score values and tumor stage, which has strong impact on a patient’s prognosis. KLK4 expression was significantly lower in stage pT3+4, i.e. non-organ-confined disease, as compared to organ-confined pT1+2 tumors. Conclusions: Our results indicate that KLK4 may represent an applicable marker in prostate cancer with stage-related prognostic value. 1 Seiz L, Kotzsch M, Grebenchtchikov NI, Geurts-Moespot AJ, Fuessel S, Goettig P, Gkazepis A, Wirth MP, Schmitt M, Lossnitzer A, Sweep F, Magdolen V. Biol. Chem. 2010; 391: 391–401. OP-50 PET IMAGING OF PROSTATE CANCER XENOGRAFTS USING DIFFERENT ANTI-PSMA ANTIBODIES AND FRAGMENTS Karen Alt (1), S Wiehr (2), W Ehrlichmann (3), U ElsässerBeile (1) (1) Department of Urology,University Hospital Freiburg, Germany; (2) Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation, University of Tübingen, Germany; (3) Radiopharmacy, Department of Radiology, University of Tübingen, Germany Aims: Prostate cancer continues to represent a major health problem, and there is an urgent need for the development of more effective diagnostic and therapeutic strategies. The prostate specific membrane antigen (PSMA) is an excellent target for prostate cancer because it is selectively expressed in the prostate and upregulated in all stages of the disease. The present work reports on the in vivo behaviour and

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tumor uptake of three anti-PSMA monoclonal antibodies (mAbs) in a SCID mouse Xenograft model. Methods: The three monoclonal antibodies 3/F11, 3/A12, 3/E7 and Fab/F(ab′)2 fragments of 3/A12 were conjugated with DOTA and 64Cu. SCID mice bearing PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts of about 30–50 mm3 volume were used for the imaging experiments. Each animal received an i.v. injection of 20–30 μg radiolabeled mAb corresonding to 7.6– 11.5 MBq. PET scans were performed 3, 24 and 48 h after tracer application. After the last scan the in vivo biodistribution was determined by gamma-counting. For PSMA blocking experiments the mice were injected with 250 μg or 700 μg non-radioactive antibody 3 hrs prior to the injection of the corresponding radiolabeled mAb. Results: PET images revealed a high uptake of the three mAbs in PSMA positive tumors and only a minimal distribution in the PSMA-negative Du 145 tumors and other organs. The tumor-to-background ratio in the C4-2 tumors was 32.03±14.24 (3/A12), 34.67±8.18 (3/F11) and 26.8±8.64 (3/E7) at 48 h. In contrast, the Fab and F(ab′)2 fragments of 3/A12 were highly spread in the kidney but not in the C4-2 tumors. Conclusion: Due to the high and specific uptake of the 64Cu-labeled mAbs in PSMA positive tumors, these mAbs represent excellent tools for prostate cancer imaging. OP-51 CIRCULATING PLASMA BMP6 mRNA, CELL-FREE DNA, NUCLEOSOMES AND H3K27 METHYLATION LEVELS IN PROSTATE CANCER U Deligezer (1), F Yaman (2), E Darendeliler (2), Y Dizdar (2), S Holdenrieder (3), M Kovancilar (1), N Dalay (1) (1) Istanbul University Oncology Institute, Department of Basic Oncology, Istanbul, Turkey; (2) Istanbul University Oncology Institute, Department of Radiation Oncology, Istanbul, Turkey; (3) Institute of Clinical Chemistry, University of Munich, Munich, Germany Aims: We evaluated the utility of post-treatment plasma levels of the circulating bone-morphogenetic protein-6specific mRNA (cBMP6 mRNA), cell-free DNA (cfDNA), apoptotic nucleosomes and Histone H3 lysine 27 trimethylation (H3K27me3), in discriminating metastatic prostate cancer (PCa) from organ confined, locally controlled disease. Methods: Peripheral blood was taken from the patients at the end of therapy, and quantitative PCR was performed to amplify cBMP6 mRNA or cf-DNA from plasma while apoptotic nucleosomes and H3K27me3 were determined by

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ELISA-based approaches. Following blinded measurements, the markers were compared between the patients with local (n=22), local advanced (n=11) or metastatic disease (n=28). Results: Of the four markers investigated, the cBMP6 mRNA and H3K27me3 levels revealed significant differences between the three subgroups. We found higher levels of cBMP6 mRNA in the patients with metastases than in those with localized (p=0.001) or local advanced disease (p=0.05). When compared to cBMP6, H3K27me3 displayed an inverse distribution and was significantly lower in the patients with metastatic disease than in those with localized (p=0.05) or local advanced disease (p=0.024). There was no correlation between the different markers and total PSA levels or Gleason score at diagnosis. Conclusion: Our study provides evidence that posttreatment analysis of cBMP6 mRNA and H3K27me3 may be used to distinguish metastatic PCa from organ confined, locally controlled disease.

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The values of [-2]proPSA and the phi index do not differentiate the clinical stage defined with DRE (T1c vs. T2a). The serum levels of [-2]proPSA and the parameters % [-2]proPSA and the phi index were significantly different for patients with PCa, Gleason <7 (n=41) compared to patients PCa with Gleason > 7 (n=61). For a clinical decision point of phi fixed at 23 (sensitivity 94%), the majority of PCa not detected (88%) below the cut off were Gleason 6 (n=7), and one of PCa Gleason 7 was not detected. Conclusion: These preliminary results indicate a relationship between the aggressiveness of PCa and [-2]proPSA, % [-2]proPSA and the phi index. These parameters preferably detect PCa with high Gleason and majority of non-detected PCa are Gleason 6 OP-53 SERUM THYMIDINE KINASE AND VASCULAR EN DOTHELIAL GROWTH FACTOR (VEGF) IN RENAL CELL CARCINOMA

OP-52 DETECTION OF AGGRESSIVE PROSTATE CANCER USING [-2]PROPSA AND THE PROSTATE HEALTH INDEX JS Blanchet (1), S Vincendeau (4). X Durand (2), JN Ramirez (3), K Bensalah (4), B Guille (4), A Houlgatte (2) (1) Beckman Coulter, Inc., Nyon, Switzerland; (2) Val de Grace Hospital, Department of Urology, Paris, France; (3) Val de Grace Hospital, Department of Biochemistry, Paris, France; (4) Hôpital Pontchaillou, Department of Urology, Rennes, France Aims: Previous studies have described the relationship between the aggressiveness of prostate cancer (PCa) (Gleason > 7) and serum levels of [-2]proPSA, an isoform of PSA. Recently, Beckman Coulter has developed the Prostate Health Index (phi) which combines the results of total PSA, free PSA and [-2]proPSA, measured with the Hybritech p2PSA assay. Data from a multicenter clinical evaluation of [-2]proPSA and phi were analyzed to identify a possible relationship with the aggressiveness of PCa detected. Methods: After six months of recruitment, 250 men (107 with and 143 without PCa) with a total PSA level between 1.8–8.0 ng/mL and digital rectal examination(DRE) nonsuspicious were included in the study. All PCa cases were confirmed by biopsies with 10 cores or more. The serum total PSA, free PSA and [-2]proPSA were measured on a Beckman Coulter DxI800 automated immunoassay analyzer. Results: The analysis was performed on 102 patients with PCa for which Gleason score information was available.

B Nisman (1), H Nechushtan (1), V Yutkin (2), T Peretz (1), S Gronowitz (3), D Pode (2) Departments of (1) Oncology and (2) Urology, Hadassah and Hebrew University Medical Center, Jerusalem, Israel; (3) Group of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden Aims: Thymidine kinase 1 (TK1) is the key enzyme involved in DNA synthesis and important proliferation marker. VEGF is a well-known inducer of angiogenesis. We investigated the factors’ association with disease recurrence in patients (pts) with renal cell carcinoma (RCC). Methods: We measured preoperatively the serum TK1 activity and VEGF levels in patients with RCC and benign kidney tumors (BKT) using a quantitative ELISA and correlated the results to clinicopathological parameters. Results: Significantly higher levels of TK1 and VEGF were found in 131 RCC pts compared to 44 healthy participants (p<0.001 and p=0.001), but not compared to 29 patients with BKT (p=0.82 and p=0.2). There was a significant association between the TK1 activity and T stage (P=0.03), but not grade (P=0.47). No associations between VEGF levels and stage (p=0.06) or grade (p= 0.83) could be observed. Only TK1 showed a prognostic significance in terms of recurrence-free survival. In multivariate Cox analysis TK1, adjusted for stage, grade and tumor necrosis was shown as an independent predictor of disease recurrence (HR=3.9, P=0.03). During the first 24 hours after surgery TK1 activity showed a significant decline (P= 0.002), followed by an increase, with a

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maximum activity at 7–14 days (P<0.001). Afterwards, the enzyme activity gradually decreased and achieved plateau at 6–8 weeks. A similar pattern was observed for VEGF excluding the initial decline. A significant correlation was found between TK1 and VEGF (R=0.30; p=0.002) during follow-up. The changes in these two factors reflected postoperative wound healing. In patients with metastatic RCC the significant rise in serum TK1 and VEGF levels indicated disease progression. Conclusions: The measurement of TK1 in pts with RCC before nephrectomy can be useful for predicting recurrence and stratifying the patients into risk groups for possible adjuvant treatment. The combined use of TK1 and VEGF may be helpful in the follow-up of RCC. OP-54 MULTIPLEXED METHYLATION PROFILES OF TUMOR SUPPRESSOR GENES IN BLADDER CANCER Laura Grau (1), M José Cabello (1), N Franco (1), E Orenes (1), M Alvarez (2), A Blanca (3), O Heredero (4), A Palacios (4), M Urrutia (4), JM Fernández (2), A LópezBeltrán (3), M Sánchez-Carbayo (1) (1) Tumor Markers Group, Molecular Pathology Program, Spanish National Cancer Center, Madrid, Spain; (2) Urology Department, Hospital Central de Asturias, Oviedo, Spain; (3) Pathology Department, Hospital Reina Sofía, Córdoba, Spain; (4) Urology Department, Hospital Universitario de Salamanca, Salamanca, Spain

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Aims: Changes in DNA methylation of tumor suppressors can occur early in carcinogenesis, being potentially important early indicators of cancer. The objective of this study was to assess the methylation of 25 tumor suppressor genes in bladder cancer using a methylationspecific multiplex ligation-dependent probe amplification assay (MS-MLPA). Methods: Initial analyses in bladder cancer cell lines (n= 14), and fresh-frozen primary bladder tumor specimens (n= 31) supported the panel of genes selected being altered in bladder cancer. MS-MLPA was optimized for its application in body fluids using two independent training and validation sets of urinary specimens (n=146) including bladder cancer patients (n=96) and controls (n=50). Results: BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%) were the most frequently methylated genes in bladder tumors, being WT1 methylation significantly associated with tumor stage (p=0.011). WT1 and PAX5A were identified as methylated tumor suppressors. BRCA1, WT1 and RARB were also the most frequently methylated genes in urinary specimens. ROC curves analyses revealed significant diagnostic accuracies in both urinary sets for BRCA1, RARB and WT1. Conclusion: The novelty of this report relates to applying MS-MLPA, a multiplexed methylation technique for tumor suppressors in bladder cancer, and also in body fluids. Methylation profiles of tumor suppressor genes were clinically relevant for histopathologic stratification of bladder tumors, and offered a non-invasive diagnostic strategy for the clinical management of patients affected with uroepithelial neoplasias.