- Email: [email protected]
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histologically proven lymph node metastases in comparison with negative lymph nodes patients. The lung cancer patients with the presence of CTCs in the peripheral blood or the bone marrow using c-met had significantly shorter overall survival and higher hazard ratio (p < 0.024; HR = 4.39 [95% CI: 1.59–12.22], resp. p < 0.008; HR = 5.08 [95% CI: 1.79–14.43]). In addition, patients with the presence of CTCs detected in bone marrow using CEA and/or c-met had significantly shorter overall survival (p < 0.025; HR = 3.08 [95% CI: 1.26−7.5]). Conclusion: The present study demonstrates that the presence of CTCs is a negative prognostic factor in lung cancer patients. CTCs detection using PCR based methods can identify patients with advance disease for whom radical surgery is of small benefit. Prospective studies should confirm usefulness of this method for the selection of patients for radical surgery. Acknowledgement: This work was financially supported by CZ1.05/2.1.00/ 01.0030 and IGA UP LF_2012_017. 788 The Influence of the Combined Treatment with 13-cis Retinoic Acid and Thalidomide on the Growth of U251 Glioblastoma Xenografts D. Milanovic1 , A.L. Grosu1 , G. Niedermann1 . 1 Klinik fur ¨ Strahlenheilkunde, Freiburg, Germany Introduction: 13-cis retinoic acid (RA) and Thalidomide (THAL) show some clinical effects in Glioblastoma (GBM) patients as sole agents or in combination with other chemotherapeuticals. RA is a differentiation agent acting on cell cycle regulation and on EGFR-mediated growth stimulation. However, RA may induce expression of homeobox (HOX) genes, i.e. developmental regulators during embryogenesis that are normally inactive in adults but may become active during carcinogenesis. RA-induced HOXB7, may in turn induce bFGF which is a potent angiogenic and mitogenic factor, and this might limit the usefulness of RA in the treatment of GBM. THAL has immunomodulatory and anti-angiogenic effects and can downregulate bFGF. In our previous work we shoved that in in vitro condition THAL inhibits RA gene expression of homeobox B7 in human GBM cells. The purpose of the present study was to test the influence of RA and THAL, as sole agents and in combination on the growth of U251 glioblastoma xenografts. Material and Methods: 1.5×106 U251 cells were inoculated s.c. into the right hind limb of NMRI-Foxn1nu athymic female nude mice. Animals were randomly assigned in 4 groups (7−11 animals/group) for treatment: control, RA, THAL and RA + THAL. The animals were treated daily (Monday-Friday) via intragastric tube with RA (30 mg/kg), THAL (30 mg/kg), or RA (30 mg/kg) plus THAL (30 mg/kg). Xenografts from sacrifed animals were used for hematoxylin and eosin staining. Results: The treatment was excellent tolerated; no side effect was observed. RA and THAL as sole agents did not affect the tumor growth in comparison to untreated controls. However, combined treatment caused a significant decrease in tumor volume. The final tumor volume were: control = 1.37±0.2 cm3 , RA = 1.38±0.23 cm3 , THAL = 1.43±0.35 cm3 , RA + THAL = 0.69±0.075 cm3 . Hematoxylin and eosin staining of xenografts showed marked hypocellularity in case of combined treatment. Conclusions: Additive growth inhibition by RA and THAL may be achieved in U251 glioblastoma xenografts. These results support our prevoious findings from in vitro experiments. Because RA and THAL are well tolerated in patients, these data encourage further clinical studies on combinations of these compounds. Molecular-biological analysis of xenografts is underway and it will be presented on the meeting. 789 GAGE5, MAP3K2 and TCEA1 Are Potential Predictive Markers for Chemoradioimmunotherapy With Interferon-alpha and 5-fluorouracil of Pancreatic Cancer Patients A. Bazhin1 , T. Rusch1 , S. Bulashevska2 , J. Werner1 . 1 University Heidelberg, Department of General Surgery, Heidelberg, Germany, 2 German Cancer Research Centre (DKFZ), Division of Theoretical Bioinformatics, Heidelberg, Germany Background: Nowadays the importance of individualised therapy finds its way more and more into the awareness of scientists and medical doctors. Pancreatic carcinoma has a particularly poor prognosis with a median survival of 6 months. The standard treatment today is surgical resection and subsequent adjuvant chemotherapy. This is possible in about 20% of all patients, and results in a median survival of over 20 months. A recent multicenter trial on intensified aduvant chemoradioimmuntherapy with interferon-alfa, the CapRi trial, also could not increase survival compared to standard chemotherapy, 5-fluorouracil alone. Nevertheless the outcome of over 30 months median survival represented the best ever reported outcome for patients with resected pancreatic cancer in a randomized trial. The main aim of the work was to identify predictive molecular markers for patients’ selection for chemoradioimmunotherapy with interferon-alfa. Methods: RNA from the frozen tumor tissues of patients with higher and lower overall survival (OS) was isolated and used for gene expression profiling with Illumina technology. Bioinformatics was applied to select differentially
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expressed genes. The selected gene candidates were further validated by Real-Time PCR. Survival analysis with gene candidates as predictors was applied to find predictive markers with respect to OS and disease-free survival (DFS). Results: 12 gene candidates were selected from the Illumine array data as potential candidates for their subsequent evaluation as predictive markers. Expression level of these genes was measured with Real-Time PCR. Afterwards we applied the Cox Proportional Hazard model on the 12 selected genes to identify the significant predictors of the OS and disease-free survival (DFS). Highly significant predictors for the OS of the patients appeared to be GAGE5 (p-value = 0.0099), as well as MAP3K2 (p-value = 0.055) and RTEL1 (p-value = 0.06). 3 genes were detected to be highly significant predictors for the DFS: GAGE5 (p-value = 0.0088), MAP3K2 (p-value = 0.02444) and TCEA1 (p-value = 0.0304). Conclusion: Expression level of GAGE5, MAP3K2 and RTEL1 has been found to be predictive for the OS, and GAGE5, MAP3K2 and TCEA1 − for the DFS of patients undergoing chemoradioimmunotherapy. These markers should be prospectively evaluated in a future clinical trial. 791 Results of Immunohistochemical Staining of Cell Cycle Regulators Predict a Progression-free Survival of Pituitary Adenoma Y.Z. Kim1 , K.H. Kim1 , E.H. Lee2 . 1 Sungkyunkwan University Samsung Changwon Hospital, Neurosurgery and Neurooncology, Changwon, South Korea, 2 Sungkyunkwan University Samsung Changwon Hospital, Pathology, Changwon, South Korea Background: In spite of advancement of surgical techniques for pituitary adenoma, recurrences develop over time, and in several series, 1.3% to 40% of pituitary adenoma patients are reported to experience recurrence. The studies of different proliferation and molecular biology could not confirm their prognostic value relative to a potential recurrence. In order to detect the role of cell cycle regulation in recurrence of pituitary adenoma, we examined the expression of regulatory proteins in cell cycle of pituitary adenoma. Material and Methods: From January 2000 to December 2009, we performed immunohistochemial (IHC) staining for secreting cells in adenophysis of pituitary gland and cell cycle regulatory proteins (p16, p15, p57, CDK4, CDK6, Rb protein, and Cyclin D1) on the formalin-fixed paraffin-embedded (FFPE) samples which were surgically resected in 156 newly diagnosed pituitary adenoma patients. We categorized the pituitary adenomas according to the clinical manifestations of elevated pituitary hormone, the results of biochemical study of serum hormone, and IHC staining of secreting cells in adenophysis of pituitary gland; functional adenoma, clinical silent adenoma, true silent adenoma, and null cell adenoma. Mean follow-up duration was 76.5 months (range from 24.5 months to 139.2 months). Results: Mean age was 48.9 years (range from 24 years to 85 years). Considering categorization of pituitary adenoma, 59 patients (38.1%) were categorized as functional adenoma, 19 patients (12.3%) as clinical silent adenoma, 57 patients (36.8%) as true silent adenoma, and 21 patients (13.5%) as null cell adenoma. In the result of IHC staining for secreting cell of adenophysis of pituitary gland, positive staining for ACTH secreting cell was found in 37 samples (23.9%), prolactin secreting cell in 88 samples (56.8%), GH secreting cell in 41 samples (26.5%), TSH in 42 samples (27.1%), luteinizing hormone (LH) secreting cell in 49 samples (31.6%), and follicular stimulating hormone (FSH) secreting cell in 52 samples (33.5%). In the results of IHC staining for cell cycle regulatory protein, positive staining for p16 in 79 samples (51.0%), p15 in 11 samples (7.1%), p21 in 14 samples (9.0%), CDK4 in 53 samples (34.2%), CDK6 in 5 samples (3.2%), Rb protein in 57 samples (36.8%), and Cyclin D1 in 74 samples (47.7%). Among 156 patients, 55 patients (35.3%) experienced radiological recurrences. Median progression free survival is 21.3 months (range from 3.4 months to 80.6 months). Multivariate analysis using Cox regression model for progression-free survival showed that adenoma with extension outside of sellar trucica (p = 0.047), positive staining for p16 (p = 0.045), positive staining for Rb protein (p = 0.007), functional adenoma (p = 0.029), and null cell adenoma (p = 0.008) were associated with longer progression-free survival. Conclusions: The IHC staining of FFPE samples of pituitary adenoma for pituitary hormone secreting cell and cell cycle regulatory protein is suggested to be a helpful method predicting a recurrence of pituitary adenomas. 793 Activity of Lenalidomide in Vitro and in Vivo Models of Bortezomib-resistant Mantle Cell Lymphoma Involving the Modulation of C-myc/p27 Axis A. Moros1 , I. Saborit-Villarroya1 , P. Perez-Gal ´ an ´ 1 , A. Mart´ınez2 , E. Campo2 , D. Colomer1 , G. Roue´ 1 . 1 IDIBAPS, Hemato-oncology Department, Barcelona (Barcelona), Spain, 2 Hospital Cl´ınic, Hematopathology Unit Department of Pathology, Barcelona (Barcelona), Spain Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy
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and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the clinical practice, not all the patients respond and relapsed frequently occurres after initial response. When comparing the behavior of both bortezomib-resistant and bortezomib-sensitive cell lines in a xenotransplant mouse model, we observed an increased tumorigenecity of bortezomibresistant cells in vivo, suggesting a major capacity of these tumors to interact with lymphoid microenvironment. As the immunomoduladory drug lenalidomide has been shown to modulate tumor-stroma interaction in several B cell malignancies, we assessed the activity in vitro and in vivo of this agent either alone or combined with the proteasome inhibitor in both bortezomibresistant and bortezomib-sensitive samples. Lenalidomide single agent was found to exert modest antitumoral activity in 2/10 MCL cell lines, corresponding to those cells with either primary or acquired resistance to the proteasome inhibitor. Conversely, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a 10−50 mg/kg/day regimen of lenalidomide, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated group (p < 0.05). The corresponding biopsies harbored several hallmarks of lenalidomide activity in malignant B cells such as CD80 and CD40L upregulation, together with a remarkable decrease in mitotic index, c-myc down-regulation, p27 cytosolic accumulation and caspase-3 processing. Similarly, bortezomib-resistant MCL cell lines treated for 72h with 1 mM lenalidomide showed lower c-myc levels, as well as p27 accumulation, caspase-3/7 activity and apparition of hypodiploid cells. When combined to bortezomib therapy (0.15 mg/kg, twice a week), lenalidomide induced a 37% and a 66% inhibition of tumor growth when compared to lenalidomide and vehicle groups, respectively (p = 0.02). In accordance, lenalidomide showed synergistic effect in vitro with bortezomib in co-culture system associating the MCL cell line Jeko-1 to the dendriticlike cells BDCM, by modifying the secretion pattern of these latest. Altogether, these results suggest that single agent lenalidomide is preferentially effective in MCL cases resistant to bortezomib, by targeting c-myc-driven tumorigenesis. Additionally, lenalidomide may overcome the protection offered by lymphoid tumor microenvironment toward bortezomib treatment, thus warranting a promising clinical activity of lenalidomide-bortezomib combination in MCL cases refractory to bortezomib. 794 Mutant P53 Functions in Vivo − Dominant-negative Effect is Cell-type Specific, Dose-dependent and Regulates Acute Response Whereas Gain-of-function Property is Mutation-type Specific K. Sabapathy1 , M.K. Lee1 , W.W. Teoh1 , W.M. Tong2 , Z.Q. Wang3 . 1 National Cancer Centre Singapore, Singapore, Singapore, 2 Chinese Acad of Medical Science, Beijing, China, 3 Leibniz Institute for Age Research, Jena, Germany Background: Mutations in p53 occur during the course of tumorigenesis, and are found in over 50% of all human cancers. The mutant allele therefore coexists with the wild-type allele in the cell for a time period till the latter is lost due to loss-of-heterozygozity (LOH), leaving behind the mutant allele in some cancers. Functionally, mutant p53 has been shown in several instances to lead to inhibition of wild-type p53 activity when both co-exist, in a phenomenon termed as the dominant-negative (DN) effect, or exert gain-of-function (GOF) properties upon loss of the other wild-type allele. However, the specific roles of mutant p53’s DN or GOF properties in regulating acute response and longterm tumorgenesis are unclear. Material and Methods: We have generated ‘knock-in’ mouse strains expressing varying levels of the R246S mutant p53 (equivalent to the human R249S mutant), to study mutant p53 properties during acute p53 activation and long-term tumorigenesis, using both cellular and several in vivo tumor models. Results and Discussion: We show that DN effect of mutant p53 on p53dependent-transactivation is universally observed in all cell types after acute p53 activation, either by DNA damage or by nutlin treatment, whereas the effect on cellular outcome (e.g. apoptosis) is cell-type specific, only apparent in some cell types, regardless of their differentiation state. Threshold levels of mutant p53 are required for the manifestation of the DN effect, as reducing mutant p53 levels abrogated the effect. Importantly, while mutant p53’s DN effect protected against radiation-induced acute cell-death, it did not accentuate tumorigenesis, highlighting that the initial p53 response after damage is not relevant for tumor suppression. Furthermore, the R246S mutant did not promote tumorigenesis compared to p53−/− mice in IR or oncogene-mediated tumor models, even in the absence of MDM2, unlike the R172H mutant, highlighting specificity of mutant p53 in promoting GOF. Conclusion: Together, these data demonstrate that mutant p53’s DN property only affects acute responses, whereas GOF is not universal, being mutationtype specific.
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795 Protein Pathway Activation Mapping of KRAS Mutated NSCLC Clinical Samples Reveals Systemic Pathway Alterations and Point to New Therapeutic Targets for EGFR Resistant Tumors R. Pellicani1 , A. Zupa2 , A. Silvestri1 , J. Deng3 , M. Aieta4 , P. Musto5 , D. Nitti6 , C. Belluco7 , J. Wulfkuhle3 , E. Petricoin3 . 1 CRO-National Cancer Institute, Experimental Oncology 2, Aviano, Italy, 2 I.R.C.C.S.CROB-National Cancer Institute, Laboratory of Molecular Oncology, Rionero in Vulture, Italy, 3 George Mason University, Centre for Applied Proteomics and Molecular Medicine, Manassas, USA, 4 I.R.C.C.S.CROB-National Cancer Institute, Division of Medical Oncology, Rionero in Vulture, Italy, 5 I.R.C.C.S.CROB-National Cancer Institute, Onco-Hematology Department, Rionero in Vulture, Italy, 6 University of Padua, Clinica Chirurgica 2nd, Padova, Italy, 7 CRO-National Cancer Institute, Division of Surgical Oncology, Aviano, Italy Introduction: Lung cancer is the leading cause of cancer-related death for solid tumors world-wide. Since current therapeutic approaches offer a limited survival benefit, it is of critical importance to discover new biomarkers for patients selection for treatment as well as new drug targets. It is known that mutations in KRAS, BRAF, and EGFR are drivers of non-small cell lung cancer (NSCLC) growth and response to targeted agents, however little is known about protein network alterations that arise as a consequence of the underpinning mutation. In order to understand these linked events we determine the correlation between mutational status and phosphoproteomic profile of NSCLC samples, and focus on the effects of KRAS mutation, the most prevalent mutation in these tumors. Material and Methods: NSCLC adenocarcinoma samples were collected from chemo-na¨ıve patients and snap frozen. For mutational analysis each sample was macrodissected to obtain at least 80% of tumor epithelium and KRAS mutational status was analyzed by Sanger sequencing. For phosphoproteomic profiling, laser capture microdissected cancer epithelial cells were subjected to protein pathway activation mapping using Reverse Phase Protein Microarrays to determine the level of expression/activation of 128 key signaling proteins known to be involved in tumor development and progression. Results and Discussion: 23 NSCLC adenocarcinoma specimens were carefully selected for analysis based on mutational status of KRAS, EGFR, BRAF, PTEN and PIK3CA. 52% of adenocarcinoma tumors revealed KRAS mutations in codons 12 or 13 and WT for all other genes evaluated, whereas 48% of the samples contained no mutations in KRAS or any gene analyzed. Protein pathway activation mapping revealed, amongst others, increased phosphorylation/expression of MAPK signaling network (BRAF and ELK), RTK activation (HER3 and HER4, c-KIT), PKC signaling increases (PKC alpha, PKC C, and PKC zeta/lambda) as well as TGF signaling activation (SMAD 1,2,5) in KRAS mutated tumors. Conclusions: Our results demonstrated that a combined approach of DNA mutational profiling along with functional protein pathway mapping may provide new insights into the functional effects of key genomic mutational event. Moreover, the identification of specific activation profiles of signaling proteins that correlate with the presence of a mutation point to their potential role as new drug targets for the treatment of anti-EGFR therapy resistant patients. 796 Establishment of Primary Endometrial Carcinoma Cell Cultures as a Preclinical Tool for Drug Screening − Methods and Characterization S. Schrauwen1 , L. Coenegrachts1 , D. Garcia-Dios1 , C. Luyten1 , G. Verbist1 , I. Vergote2 , D. Lambrechts3 , F. Amant2 . 1 KU Leuven, Department of Oncology − Division Gynecological Oncology, Leuven, Belgium, 2 KU Leuven − University Hospitals Leuven, Department of Oncology − Division Gynecological Oncology, Leuven, Belgium, 3 VIB − KU Leuven, Vesalius Research Center − Department of Oncology, Leuven, Belgium Introduction: Endometrial cancer (EC) is the most common gynecological cancer. Treatment options for primary advanced and recurrent disease are limited, making development of new treatment strategies essential. Our objective was to establish primary EC cell cultures to provide a cellular model for both in vitro and in vivo studies, including screening of new drugs. Material and Method: After informed consent, fresh endometrial tumor biopsies were collected. Primary cell cultures were established after collagenase digestion and fibroblast depletion using CD90 for negative selection. They were validated by (i) measurement of telomerase activity, (ii) dynamic monitoring of cell proliferation (xCELLigence), and (iii) immunocytochemical stainings (CK, VIM, a-SMA, CD90). In vivo tumorigenicity was determined in a s.c. xenograft model. Mutation analysis (Sequenom) was performed to detect hot spot mutations in genetic pathways that are known to be affected in EC (e.g. PI3K/PTEN/AKT and EGFR/KRAS pathway). The mutation profile of the primary tumor, cell culture and xenograft tumor was compared. Results and Discussion: Establishment of grade 1 endometrial tumors was not feasible, whereas the success rate for grade 2 and 3 tumors was ~30% (10 out of 32). Cell cultures can be cultured for >25 passages. Telomerase activity of all primary cell cultures was similar to the activity of the HEC1A commercial cell line. Doubling time of the cell cultures ranged between
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histologically proven lymph node metastases in comparison with negative lymph nodes patients. The lung cancer patients with the presence of CTCs in the peripheral blood or the bone marrow using c-met had significantly shorter overall survival and higher hazard ratio (p < 0.024; HR = 4.39 [95% CI: 1.59–12.22], resp. p < 0.008; HR = 5.08 [95% CI: 1.79–14.43]). In addition, patients with the presence of CTCs detected in bone marrow using CEA and/or c-met had significantly shorter overall survival (p < 0.025; HR = 3.08 [95% CI: 1.26−7.5]). Conclusion: The present study demonstrates that the presence of CTCs is a negative prognostic factor in lung cancer patients. CTCs detection using PCR based methods can identify patients with advance disease for whom radical surgery is of small benefit. Prospective studies should confirm usefulness of this method for the selection of patients for radical surgery. Acknowledgement: This work was financially supported by CZ1.05/2.1.00/ 01.0030 and IGA UP LF_2012_017. 788 The Influence of the Combined Treatment with 13-cis Retinoic Acid and Thalidomide on the Growth of U251 Glioblastoma Xenografts D. Milanovic1 , A.L. Grosu1 , G. Niedermann1 . 1 Klinik fur ¨ Strahlenheilkunde, Freiburg, Germany Introduction: 13-cis retinoic acid (RA) and Thalidomide (THAL) show some clinical effects in Glioblastoma (GBM) patients as sole agents or in combination with other chemotherapeuticals. RA is a differentiation agent acting on cell cycle regulation and on EGFR-mediated growth stimulation. However, RA may induce expression of homeobox (HOX) genes, i.e. developmental regulators during embryogenesis that are normally inactive in adults but may become active during carcinogenesis. RA-induced HOXB7, may in turn induce bFGF which is a potent angiogenic and mitogenic factor, and this might limit the usefulness of RA in the treatment of GBM. THAL has immunomodulatory and anti-angiogenic effects and can downregulate bFGF. In our previous work we shoved that in in vitro condition THAL inhibits RA gene expression of homeobox B7 in human GBM cells. The purpose of the present study was to test the influence of RA and THAL, as sole agents and in combination on the growth of U251 glioblastoma xenografts. Material and Methods: 1.5×106 U251 cells were inoculated s.c. into the right hind limb of NMRI-Foxn1nu athymic female nude mice. Animals were randomly assigned in 4 groups (7−11 animals/group) for treatment: control, RA, THAL and RA + THAL. The animals were treated daily (Monday-Friday) via intragastric tube with RA (30 mg/kg), THAL (30 mg/kg), or RA (30 mg/kg) plus THAL (30 mg/kg). Xenografts from sacrifed animals were used for hematoxylin and eosin staining. Results: The treatment was excellent tolerated; no side effect was observed. RA and THAL as sole agents did not affect the tumor growth in comparison to untreated controls. However, combined treatment caused a significant decrease in tumor volume. The final tumor volume were: control = 1.37±0.2 cm3 , RA = 1.38±0.23 cm3 , THAL = 1.43±0.35 cm3 , RA + THAL = 0.69±0.075 cm3 . Hematoxylin and eosin staining of xenografts showed marked hypocellularity in case of combined treatment. Conclusions: Additive growth inhibition by RA and THAL may be achieved in U251 glioblastoma xenografts. These results support our prevoious findings from in vitro experiments. Because RA and THAL are well tolerated in patients, these data encourage further clinical studies on combinations of these compounds. Molecular-biological analysis of xenografts is underway and it will be presented on the meeting. 789 GAGE5, MAP3K2 and TCEA1 Are Potential Predictive Markers for Chemoradioimmunotherapy With Interferon-alpha and 5-fluorouracil of Pancreatic Cancer Patients A. Bazhin1 , T. Rusch1 , S. Bulashevska2 , J. Werner1 . 1 University Heidelberg, Department of General Surgery, Heidelberg, Germany, 2 German Cancer Research Centre (DKFZ), Division of Theoretical Bioinformatics, Heidelberg, Germany Background: Nowadays the importance of individualised therapy finds its way more and more into the awareness of scientists and medical doctors. Pancreatic carcinoma has a particularly poor prognosis with a median survival of 6 months. The standard treatment today is surgical resection and subsequent adjuvant chemotherapy. This is possible in about 20% of all patients, and results in a median survival of over 20 months. A recent multicenter trial on intensified aduvant chemoradioimmuntherapy with interferon-alfa, the CapRi trial, also could not increase survival compared to standard chemotherapy, 5-fluorouracil alone. Nevertheless the outcome of over 30 months median survival represented the best ever reported outcome for patients with resected pancreatic cancer in a randomized trial. The main aim of the work was to identify predictive molecular markers for patients’ selection for chemoradioimmunotherapy with interferon-alfa. Methods: RNA from the frozen tumor tissues of patients with higher and lower overall survival (OS) was isolated and used for gene expression profiling with Illumina technology. Bioinformatics was applied to select differentially
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expressed genes. The selected gene candidates were further validated by Real-Time PCR. Survival analysis with gene candidates as predictors was applied to find predictive markers with respect to OS and disease-free survival (DFS). Results: 12 gene candidates were selected from the Illumine array data as potential candidates for their subsequent evaluation as predictive markers. Expression level of these genes was measured with Real-Time PCR. Afterwards we applied the Cox Proportional Hazard model on the 12 selected genes to identify the significant predictors of the OS and disease-free survival (DFS). Highly significant predictors for the OS of the patients appeared to be GAGE5 (p-value = 0.0099), as well as MAP3K2 (p-value = 0.055) and RTEL1 (p-value = 0.06). 3 genes were detected to be highly significant predictors for the DFS: GAGE5 (p-value = 0.0088), MAP3K2 (p-value = 0.02444) and TCEA1 (p-value = 0.0304). Conclusion: Expression level of GAGE5, MAP3K2 and RTEL1 has been found to be predictive for the OS, and GAGE5, MAP3K2 and TCEA1 − for the DFS of patients undergoing chemoradioimmunotherapy. These markers should be prospectively evaluated in a future clinical trial. 791 Results of Immunohistochemical Staining of Cell Cycle Regulators Predict a Progression-free Survival of Pituitary Adenoma Y.Z. Kim1 , K.H. Kim1 , E.H. Lee2 . 1 Sungkyunkwan University Samsung Changwon Hospital, Neurosurgery and Neurooncology, Changwon, South Korea, 2 Sungkyunkwan University Samsung Changwon Hospital, Pathology, Changwon, South Korea Background: In spite of advancement of surgical techniques for pituitary adenoma, recurrences develop over time, and in several series, 1.3% to 40% of pituitary adenoma patients are reported to experience recurrence. The studies of different proliferation and molecular biology could not confirm their prognostic value relative to a potential recurrence. In order to detect the role of cell cycle regulation in recurrence of pituitary adenoma, we examined the expression of regulatory proteins in cell cycle of pituitary adenoma. Material and Methods: From January 2000 to December 2009, we performed immunohistochemial (IHC) staining for secreting cells in adenophysis of pituitary gland and cell cycle regulatory proteins (p16, p15, p57, CDK4, CDK6, Rb protein, and Cyclin D1) on the formalin-fixed paraffin-embedded (FFPE) samples which were surgically resected in 156 newly diagnosed pituitary adenoma patients. We categorized the pituitary adenomas according to the clinical manifestations of elevated pituitary hormone, the results of biochemical study of serum hormone, and IHC staining of secreting cells in adenophysis of pituitary gland; functional adenoma, clinical silent adenoma, true silent adenoma, and null cell adenoma. Mean follow-up duration was 76.5 months (range from 24.5 months to 139.2 months). Results: Mean age was 48.9 years (range from 24 years to 85 years). Considering categorization of pituitary adenoma, 59 patients (38.1%) were categorized as functional adenoma, 19 patients (12.3%) as clinical silent adenoma, 57 patients (36.8%) as true silent adenoma, and 21 patients (13.5%) as null cell adenoma. In the result of IHC staining for secreting cell of adenophysis of pituitary gland, positive staining for ACTH secreting cell was found in 37 samples (23.9%), prolactin secreting cell in 88 samples (56.8%), GH secreting cell in 41 samples (26.5%), TSH in 42 samples (27.1%), luteinizing hormone (LH) secreting cell in 49 samples (31.6%), and follicular stimulating hormone (FSH) secreting cell in 52 samples (33.5%). In the results of IHC staining for cell cycle regulatory protein, positive staining for p16 in 79 samples (51.0%), p15 in 11 samples (7.1%), p21 in 14 samples (9.0%), CDK4 in 53 samples (34.2%), CDK6 in 5 samples (3.2%), Rb protein in 57 samples (36.8%), and Cyclin D1 in 74 samples (47.7%). Among 156 patients, 55 patients (35.3%) experienced radiological recurrences. Median progression free survival is 21.3 months (range from 3.4 months to 80.6 months). Multivariate analysis using Cox regression model for progression-free survival showed that adenoma with extension outside of sellar trucica (p = 0.047), positive staining for p16 (p = 0.045), positive staining for Rb protein (p = 0.007), functional adenoma (p = 0.029), and null cell adenoma (p = 0.008) were associated with longer progression-free survival. Conclusions: The IHC staining of FFPE samples of pituitary adenoma for pituitary hormone secreting cell and cell cycle regulatory protein is suggested to be a helpful method predicting a recurrence of pituitary adenomas. 793 Activity of Lenalidomide in Vitro and in Vivo Models of Bortezomib-resistant Mantle Cell Lymphoma Involving the Modulation of C-myc/p27 Axis A. Moros1 , I. Saborit-Villarroya1 , P. Perez-Gal ´ an ´ 1 , A. Mart´ınez2 , E. Campo2 , D. Colomer1 , G. Roue´ 1 . 1 IDIBAPS, Hemato-oncology Department, Barcelona (Barcelona), Spain, 2 Hospital Cl´ınic, Hematopathology Unit Department of Pathology, Barcelona (Barcelona), Spain Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy
Poster Sessions
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and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the clinical practice, not all the patients respond and relapsed frequently occurres after initial response. When comparing the behavior of both bortezomib-resistant and bortezomib-sensitive cell lines in a xenotransplant mouse model, we observed an increased tumorigenecity of bortezomibresistant cells in vivo, suggesting a major capacity of these tumors to interact with lymphoid microenvironment. As the immunomoduladory drug lenalidomide has been shown to modulate tumor-stroma interaction in several B cell malignancies, we assessed the activity in vitro and in vivo of this agent either alone or combined with the proteasome inhibitor in both bortezomibresistant and bortezomib-sensitive samples. Lenalidomide single agent was found to exert modest antitumoral activity in 2/10 MCL cell lines, corresponding to those cells with either primary or acquired resistance to the proteasome inhibitor. Conversely, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a 10−50 mg/kg/day regimen of lenalidomide, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated group (p < 0.05). The corresponding biopsies harbored several hallmarks of lenalidomide activity in malignant B cells such as CD80 and CD40L upregulation, together with a remarkable decrease in mitotic index, c-myc down-regulation, p27 cytosolic accumulation and caspase-3 processing. Similarly, bortezomib-resistant MCL cell lines treated for 72h with 1 mM lenalidomide showed lower c-myc levels, as well as p27 accumulation, caspase-3/7 activity and apparition of hypodiploid cells. When combined to bortezomib therapy (0.15 mg/kg, twice a week), lenalidomide induced a 37% and a 66% inhibition of tumor growth when compared to lenalidomide and vehicle groups, respectively (p = 0.02). In accordance, lenalidomide showed synergistic effect in vitro with bortezomib in co-culture system associating the MCL cell line Jeko-1 to the dendriticlike cells BDCM, by modifying the secretion pattern of these latest. Altogether, these results suggest that single agent lenalidomide is preferentially effective in MCL cases resistant to bortezomib, by targeting c-myc-driven tumorigenesis. Additionally, lenalidomide may overcome the protection offered by lymphoid tumor microenvironment toward bortezomib treatment, thus warranting a promising clinical activity of lenalidomide-bortezomib combination in MCL cases refractory to bortezomib. 794 Mutant P53 Functions in Vivo − Dominant-negative Effect is Cell-type Specific, Dose-dependent and Regulates Acute Response Whereas Gain-of-function Property is Mutation-type Specific K. Sabapathy1 , M.K. Lee1 , W.W. Teoh1 , W.M. Tong2 , Z.Q. Wang3 . 1 National Cancer Centre Singapore, Singapore, Singapore, 2 Chinese Acad of Medical Science, Beijing, China, 3 Leibniz Institute for Age Research, Jena, Germany Background: Mutations in p53 occur during the course of tumorigenesis, and are found in over 50% of all human cancers. The mutant allele therefore coexists with the wild-type allele in the cell for a time period till the latter is lost due to loss-of-heterozygozity (LOH), leaving behind the mutant allele in some cancers. Functionally, mutant p53 has been shown in several instances to lead to inhibition of wild-type p53 activity when both co-exist, in a phenomenon termed as the dominant-negative (DN) effect, or exert gain-of-function (GOF) properties upon loss of the other wild-type allele. However, the specific roles of mutant p53’s DN or GOF properties in regulating acute response and longterm tumorgenesis are unclear. Material and Methods: We have generated ‘knock-in’ mouse strains expressing varying levels of the R246S mutant p53 (equivalent to the human R249S mutant), to study mutant p53 properties during acute p53 activation and long-term tumorigenesis, using both cellular and several in vivo tumor models. Results and Discussion: We show that DN effect of mutant p53 on p53dependent-transactivation is universally observed in all cell types after acute p53 activation, either by DNA damage or by nutlin treatment, whereas the effect on cellular outcome (e.g. apoptosis) is cell-type specific, only apparent in some cell types, regardless of their differentiation state. Threshold levels of mutant p53 are required for the manifestation of the DN effect, as reducing mutant p53 levels abrogated the effect. Importantly, while mutant p53’s DN effect protected against radiation-induced acute cell-death, it did not accentuate tumorigenesis, highlighting that the initial p53 response after damage is not relevant for tumor suppression. Furthermore, the R246S mutant did not promote tumorigenesis compared to p53−/− mice in IR or oncogene-mediated tumor models, even in the absence of MDM2, unlike the R172H mutant, highlighting specificity of mutant p53 in promoting GOF. Conclusion: Together, these data demonstrate that mutant p53’s DN property only affects acute responses, whereas GOF is not universal, being mutationtype specific.
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795 Protein Pathway Activation Mapping of KRAS Mutated NSCLC Clinical Samples Reveals Systemic Pathway Alterations and Point to New Therapeutic Targets for EGFR Resistant Tumors R. Pellicani1 , A. Zupa2 , A. Silvestri1 , J. Deng3 , M. Aieta4 , P. Musto5 , D. Nitti6 , C. Belluco7 , J. Wulfkuhle3 , E. Petricoin3 . 1 CRO-National Cancer Institute, Experimental Oncology 2, Aviano, Italy, 2 I.R.C.C.S.CROB-National Cancer Institute, Laboratory of Molecular Oncology, Rionero in Vulture, Italy, 3 George Mason University, Centre for Applied Proteomics and Molecular Medicine, Manassas, USA, 4 I.R.C.C.S.CROB-National Cancer Institute, Division of Medical Oncology, Rionero in Vulture, Italy, 5 I.R.C.C.S.CROB-National Cancer Institute, Onco-Hematology Department, Rionero in Vulture, Italy, 6 University of Padua, Clinica Chirurgica 2nd, Padova, Italy, 7 CRO-National Cancer Institute, Division of Surgical Oncology, Aviano, Italy Introduction: Lung cancer is the leading cause of cancer-related death for solid tumors world-wide. Since current therapeutic approaches offer a limited survival benefit, it is of critical importance to discover new biomarkers for patients selection for treatment as well as new drug targets. It is known that mutations in KRAS, BRAF, and EGFR are drivers of non-small cell lung cancer (NSCLC) growth and response to targeted agents, however little is known about protein network alterations that arise as a consequence of the underpinning mutation. In order to understand these linked events we determine the correlation between mutational status and phosphoproteomic profile of NSCLC samples, and focus on the effects of KRAS mutation, the most prevalent mutation in these tumors. Material and Methods: NSCLC adenocarcinoma samples were collected from chemo-na¨ıve patients and snap frozen. For mutational analysis each sample was macrodissected to obtain at least 80% of tumor epithelium and KRAS mutational status was analyzed by Sanger sequencing. For phosphoproteomic profiling, laser capture microdissected cancer epithelial cells were subjected to protein pathway activation mapping using Reverse Phase Protein Microarrays to determine the level of expression/activation of 128 key signaling proteins known to be involved in tumor development and progression. Results and Discussion: 23 NSCLC adenocarcinoma specimens were carefully selected for analysis based on mutational status of KRAS, EGFR, BRAF, PTEN and PIK3CA. 52% of adenocarcinoma tumors revealed KRAS mutations in codons 12 or 13 and WT for all other genes evaluated, whereas 48% of the samples contained no mutations in KRAS or any gene analyzed. Protein pathway activation mapping revealed, amongst others, increased phosphorylation/expression of MAPK signaling network (BRAF and ELK), RTK activation (HER3 and HER4, c-KIT), PKC signaling increases (PKC alpha, PKC C, and PKC zeta/lambda) as well as TGF signaling activation (SMAD 1,2,5) in KRAS mutated tumors. Conclusions: Our results demonstrated that a combined approach of DNA mutational profiling along with functional protein pathway mapping may provide new insights into the functional effects of key genomic mutational event. Moreover, the identification of specific activation profiles of signaling proteins that correlate with the presence of a mutation point to their potential role as new drug targets for the treatment of anti-EGFR therapy resistant patients. 796 Establishment of Primary Endometrial Carcinoma Cell Cultures as a Preclinical Tool for Drug Screening − Methods and Characterization S. Schrauwen1 , L. Coenegrachts1 , D. Garcia-Dios1 , C. Luyten1 , G. Verbist1 , I. Vergote2 , D. Lambrechts3 , F. Amant2 . 1 KU Leuven, Department of Oncology − Division Gynecological Oncology, Leuven, Belgium, 2 KU Leuven − University Hospitals Leuven, Department of Oncology − Division Gynecological Oncology, Leuven, Belgium, 3 VIB − KU Leuven, Vesalius Research Center − Department of Oncology, Leuven, Belgium Introduction: Endometrial cancer (EC) is the most common gynecological cancer. Treatment options for primary advanced and recurrent disease are limited, making development of new treatment strategies essential. Our objective was to establish primary EC cell cultures to provide a cellular model for both in vitro and in vivo studies, including screening of new drugs. Material and Method: After informed consent, fresh endometrial tumor biopsies were collected. Primary cell cultures were established after collagenase digestion and fibroblast depletion using CD90 for negative selection. They were validated by (i) measurement of telomerase activity, (ii) dynamic monitoring of cell proliferation (xCELLigence), and (iii) immunocytochemical stainings (CK, VIM, a-SMA, CD90). In vivo tumorigenicity was determined in a s.c. xenograft model. Mutation analysis (Sequenom) was performed to detect hot spot mutations in genetic pathways that are known to be affected in EC (e.g. PI3K/PTEN/AKT and EGFR/KRAS pathway). The mutation profile of the primary tumor, cell culture and xenograft tumor was compared. Results and Discussion: Establishment of grade 1 endometrial tumors was not feasible, whereas the success rate for grade 2 and 3 tumors was ~30% (10 out of 32). Cell cultures can be cultured for >25 passages. Telomerase activity of all primary cell cultures was similar to the activity of the HEC1A commercial cell line. Doubling time of the cell cultures ranged between