631: Impact of TP53 accumulation on ovarian cancer prognosis in BRCA1 mutation carriers

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628 MED15 in head and neck squamous cell carcinoma: Clinical and molecular implications

630 Receptor tyrosine kinases as novel therapeutic targets in head and neck squamous cell carcinoma

A. Offermann1 , Z. Shaikhibrahim1 , R. Halbach1 , M. Braun1 , G. Kristiansen2 , ¨ 3 , S. Perner1 . 1 Institute of F. Bootz3 , R. Mikut4 , M. Reischl4 , A. Schrock Pathology University Hospital of Bonn, Department of Prostate Cancer Research, Bonn, Germany, 2 University Hospital of Bonn, Institute of Pathology, Bonn, Germany, 3 University Hospital of Bonn, Department of Otorhinolaryngology, Bonn, Germany, 4 Karlsruhe Institute of Technology, Applied Computer Science, Karlsruhe, Germany

1 A. Von Massenhausen ¨ , M. Deng1 , W. Vogel1 , A. Queisser1 , S. Perner1 . 1 Institute of Pathology, Department of Prostate Cancer Research, Bonn, Germany

Introduction: Head and Neck Squamous Cell Carcinoma (HNSCC) development and progression depends on various dysregulated pathways. Regulation of diverse pathways is mediated by the Mediator complex. The Mediator subunit MED15 is essential for TGFb signaling and involved in breast and prostate cancers. We investigated whether MED15 is implicated in HNSCC. Material and Method: We performed immunohistochemistry (IHC) for MED15 on 324 tissue samples from 195 HNSCC patients and assessed Ki67 and pSMAD3 as marker of TGFb. MED15 knockdown followed by proliferation and migration assays were performed in HNSCC cells. Cells were treated with TGFb1, followed by analysis of pSMAD3 and MED15. Results and Discussion: We found MED15 to be overexpressed in 35% of primary tumors, 30% of lymph node metastases, and 70% of recurrences in contrast to no/low expression in benign tissues. MED15 overexpression in primaries from patients who developed recurrences was associated with a higher mortality rate and occurred at much higher frequency in oral cavity/oropharynx tumors, compared to hypopharyngeal/laryngeal tumors. Furthermore, MED15 expression correlates significantly between primaries and corresponding lymph node metastases. MED15 expression correlates with proliferative activity in tissues, and MED15 knockdown reduces proliferation and migration in cells. We observed an association between MED15 and TGFb signaling in tissues, along with TGFb leading to increased MED15 expression in cells. Conclusion: Taken together, our results implicate MED15 for the first time in HNSCC, and suggest that MED15 overexpression is a clonal event during HNSCC progression. Our findings further suggest that MED15 may serve as a prognostic marker for recurrence, and a therapeutic target in HNSCC patients. No conflict of interest. 629 Expression and role of the embryonic protein SOX2 in head and neck squamous cell carcinoma A. Schrock ¨ 1 , M. Bode2 , A. Franzen2 , L. Heasley3 , C. Lengerke4 , S. Perner2 , A. Queisser2 . 1 University Hospital of Bonn, Department of Otorhinolaryngology/Head and Neck Surgery, Bonn, Germany, 2 Institute of Pathology University Hospital of Bonn, Department of Prostate Cancer Research, Bonn, Germany, 3 University of Colorado Anschutz Medical, Department of Craniofacial Biology, Aurora, USA, 4 University Hospital of Tuebingen, Medical Center II, Tuebingen, Germany Introduction: Recently, SOX2 has been identified as a potential lineage specific oncogene in lung squamous cell carcinomas (LSCC). Since head and neck squamous cell carcinomas (HNSCC) are morphologically and clinically highly related to LSCC, we hypothesized that SOX2 also plays an oncogenic role in this tumor entity. Material and Method: We assembled a cohort of 496 patients with HNSCC, including 253 metastases and 135 recurrences. SOX2 amplification (FISH) and SOX2 protein expression (IHC) were correlated with molecular and clinico-pathological parameters. In order to investigate the functional role of SOX2 in human HNSCC, SOX2 knockdown and overexpression in SCC25 cells were generated by lentiviral constructs and subjected to cell cycle analysis, proliferation and apoptosis assays. Furthermore, SOX2 expression was correlated with the expression of proliferation and apoptosis related proteins in primary HNSCC samples. Results and Discussion: SOX2 amplification was detected in 21% of primary HNSCC and mostly observed in a concordant manner between primary tumors and corresponding metastatic tissues. Overall, SOX2 amplification resulted in protein overexpression and was mutually exclusive with HPV infection. SOX2 protein overexpression was associated with clinico-pathological parameters of worse outcome. Functionally, SOX2 induced the expression of the antiapoptotic protein BCL-2 and enhanced resistance to apoptosis-inducing agents including cisplatin, indicating SOX2 as a mediator of therapy resistance in human HNSCC. Conclusion: Targeting SOX2 and related molecular downstream pathways such as BCL-2 may enhance therapy efficacy in SOX2 expressing HNSCC. No conflict of interest.

Introduction: Malignant tumors of the head and neck region are the 6th most common malignancy world wide in which squamous cell carcinoma of the head and neck (HNSCC) are the most prevalent. However, next to surgery and radio and/or chemotherapy therapy options remain limited. Recently, the concept of personalised medicine through targeted tumor therapies has become an area of high interest. An example are receptor tyrosine kinases (RTKs) that are surface proteins and offer suitable targets. They bind growth factors, cytokines and hormones and regulate normal cellular processes as well as tumorigenesis. Small molecule inhibitors or antibodies for several RTKs are available. Since some RTKs are known to play a role in HNSCC we aimed to identify more RTKs that could be novel therapeutic targets for this tumor entity. Materials and Methods: In-silico analysis of available data from The Cancer Genome Atlas (TCGA) was performed to identify RTKs that are frequently mutated in HNSCC (n = 306). Subsequently immunohistochemistry (IHC) of these RTKs was done on a HNSCC cohort (n = 336). SCC25 cells were transfected with FGFR3 (fibroblast growth factor receptor 3) using Screenfect A and positive clones were selected under G418 treatment. Results: We identified 24 RTKs that are mutated in at least 2% of patients with HNSCC. Seven are already well described in head and neck cancer. Out of the remaining we performed IHC on our HNSCC cohort for RTKs for which small molecule inhibitors are available. ROS1 (c-ros oncogene 1, receptor tyrosine kinase) that is mutated in 5.6% and ALK (anaplastic lymphoma receptor tyrosine kinase) that is mutated in 2.6% of patients showed no expression in tumor tissue whereas FGFR3 (fibroblast growth factor receptor 3) and DDR2 (discoidin domain receptor tyrosine kinase 2) that are both mutated in 2% of cases showed positive IHC in 23.8% and 21.4% of primary tumors, respectively. Normal tissue showed positive staining in 8% of cases for both RTKs. We will also do IHC staining for RET (ret proto-oncogene) (mutation frequency 2.6%) as well as AXL (AXL receptor tyrosine kinase) (mutation frequency 2%) and MERTK (c-mer proto-oncogene tyrosine kinase) (mutation frequency 2%). Promising RTKs will be stably overexpressed in HNSCC cells. Subsequently, proliferation, migration/invasion as well as colony formation will be analysed upon treatment with and without small molecule inhibitors. As proof of principle, FGFR3 was already successfully stably overexpressed in SCC25 cells. Conclusions: Our data suggest that DDR2 and FGFR3 could be novel therapeutic targets in HNSCC. In-vitro analysis will underline our findings. Further studies will show if more RTKs can be included as novel therapeutic targets in HNSCC. No conflict of interest. 631 Impact of TP53 accumulation on ovarian cancer prognosis in BRCA1 mutation carriers I.K. Rzepecka1 , L. Szafron1 , A. Felisiak-Golabek1 , A. Podgorska1 , J. Kupryjanczyk1 . 1 Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Department of Pathology, Warsaw, Poland Introduction: BRCA1 and TP53 are involved in the cellular response to chemotherapeutic drugs and their dysfunction may have an additive effect on patients outcomes. We aimed to evaluate the clinical effect of BRCA1 mutations in relation to TP53 accumulation status on patient survival in highgrade ovarian tumors. Material and Method: The study group consisted of 159 high-grade ovarian carcinomas previously analyzed for BRCA1 germline mutations and TP53 accumulation. The patients were treated with standard taxane-platinum or cisplatin-cyclophosphamide regimens (118 and 41 patients, respectively). The Kaplan–Meier method, log-rank test and the Cox’s regression model were used for survival analyses. The statistical analysis was conducted for all patients and separately for the subgroups of patients with or without tumor TP53 accumulation. Results and Discussion: BRCA1 was mutated in 40 (25.2%) of 159 ovarian cancers. In a subgroup of 57 patients without TP53 accumulation [TP53(−)], median overall survival for BRCA1 carriers was significantly longer than for sporadic cases (43.3 vs. 27.6 months, respectively; P = 0.041). In multivariate analysis in this subgroup, the presence of BRCA1 mutation independently decreased death rates by 59% (HR 0.41, P = 0.046) compared with BRCA1 mutation-negative patients. In a subgroup of 102 patients with TP53 accumulation [TP53(+)], as well as in the entire patients group, BRCA1 mutation did not significantly influence median OS compared with noncarriers. In the TP53(−) subgroup, median disease-free survival was 14.5 months for BRCA1-positive patients and 11.1 months for non-carriers (P = 0.15). In multivariate analysis, there was a strong trend toward decreased recurrence rate in carriers compared with sporadic cases (HR 0.31, P = 0.070). When

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 DFS was evaluated in the TP53(+) subgroup, the observed median DFS was significantly shorter for BRCA1 mutation carriers than for those with sporadic disease (7.7 vs. 18.4 months, respectively; P = 0.010). The fact of being a BRCA1 mutation carrier increased the recurrence rate (HR 2.50, P = 0.004) compared with non-carriers in multivariate analysis. Conclusion: We observed the advantage in survival for BRCA1 carriers over non-carriers but only in a subgroup of patients without TP53 accumulation in their tumors. Interestingly, in a subgroup of patients with dysfunctional TP53 protein, BRCA1 carriers had worse disease-free survival than patients with sporadic disease. No conflict of interest. 632 Tamoxifen resistance can be overcome by salinomycin treatment 2 A. Sommer1 , F. Mickler2 , A. Herrmann3 , A. Hermawan3 , C. Brauchle ¨ , E. Wagner3 , P. Knyazev1 , A. Ullrich1 , A. Roidl3 . 1 Max-Planck-Institute of Biochemistry, Department of Molecular Biology, Martinsried, Germany, 2 LMU Munich, Physical Chemistry, Munchen, ¨ Germany, 3 LMU Munich, Department of Pharmacy, Munchen, ¨ Germany

Introduction: Luminal A and Luminal B breast cancer have the highest prevalence among women and are usually well treatable with endocrine therapy. Nevertheless resistance to tamoxifen treatment often occurs and thus worsens the prognosis for breast cancer patients. Our goal was to circumvent all three major resistance mechanisms in ERa (estrogen receptor alpha)positive breast cancer types, i.e. cancer stem cells, overexpression of MDR-1 and the ligand independent activation of ERa, by a combined treatment of tamoxifen and salinomycin. Material and Method: We utilized cytotoxicity assays and Western blotting to analyze the effect of the combined treatment on cell viability and ERasignaling. To analyze endocytosis of EGFR and Her2 we performed spinning disk confocal microscopy in living cells. Additionally, the mammosphere forming potential was measured and a molecular evolution assay was utilized to demonstrate the efficacy of the combinational treatment. Results and Discussion: Our study demonstrates that tamoxifen treatment induces the ligand independent activation of ERa by activating members of the EGFR-family. Salinomycin on the other hand inhibits the phosphorylation of Her2 and Her3 in Luminal A and B breast cancer cell lines and is therefore a putative and novel additive to tamoxifen therapy. We observed an increased fusion of endosomes with lysosomes hampering the recycling of EGFR and Her2 and finally leading to an inhibition of receptor tyrosine kinase signaling. Hence, the combination of both drugs shows an increased cytotoxic effect compared to single treatment and prevented the activation of ERa via Her2 and Her3. The combination of tamoxifen and salinomycin additionally inhibited mammosphere forming potential, a marker for cancer stem cells. Finally, the combination does not create resistant cell clones as demonstrated by the molecular evolution assay and eradicates both cell lines after 6 treatment cycles. Conclusion: We postulate a novel treatment strategy for ERa-positive breast cancer to circumvent all three major resistance mechanisms: cancer stem cells, MDR-1 overexpression and the ligand independent activation of ERa via Her2 and Her3. This combinational therapy of tamoxifen and salinomycin might improve clinical outcome of breast cancer patients. No conflict of interest. 634 Real-time RT-PCR analysis of galectin-3 expression in fine-needle aspirates of thyroid papillary carcinoma and thyroid non-neoplastic lesions I. Samija1 , N. Matesa1 , Z. Kusic1 . 1 University Hospital Center Sestre Milosrdnice, Oncology and Nuclear Medicine, Zagreb, Croatia Background: The aim of this study was to evaluate galectin-3 as a potential marker for preoperative diagnosis of malignant thyroid lesions. For that purpose galectin-3 expression in fine-needle aspiration (FNA) samples was analyzed by real-time RT-PCR and compared between thyroid papillary carcinoma and thyroid non-neoplastic lesions. Material and Methods: Real-time RT-PCR analysis of galectin-3 expression was performed on RNA isolated from aspirates obtained by ultrasound guided FNA from 105 patients with papillary carcinoma, Hashimoto thyroiditis and nodular goiter. Galectin-3 expression was quantified by DDCt method using pooled FNA samples from normal thyroid tissue as calibrator. Results of realtime RT-PCR analysis were evaluated against the definitive FNA diagnosis. Results: With a cut-off value of 0.97 determined by ROC curve analysis 26 out of 27 (96%) papillary carcinoma, 9 out of 31 (29%) Hashimoto thyroiditis and 32 out of 47 (68%) nodular goiter samples were positive for galectin-3 expression. Galectin-3 expression has shown accuracy of 60.0%, sensitivity of 96.3% and specificity of 47.4% in discriminating between samples with papillary carcinoma and non-neoplastic thyroid lesions. Proportion of galectin-3 positive samples was significantly higher in samples with papillary carcinoma compared to samples with non-neoplastic lesions (p < 0.0001, Fisher’s exact test).

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Conclusions: Real-time RT-PCR analysis of galectin-3 expression in FNA samples has shown high sensitivity, but relatively low specificity in discriminating between papillary thyroid carcinoma and non-neoplastic lesions. Therefore, its potential value as a marker for preoperative diagnosis of malignant thyroid lesions is limited. No conflict of interest. 635 CA IX inhibitor 4-(3 -(3 ,5 -dimethylphenyl)ureido)phenyl sulfamate (S4) as an adjuvant to doxorubicin chemotherapy reduces metastatic dissemination R.G. Gieling1 , M. Babur1 , B.A. Telfer1 , K.J. Williams1 . 1 University of Manchester, Hypoxia and Therapeutics Group Manchester Pharmacy School, Manchester, United Kingdom Background: Carbonic anhydrase (CA, EC 4.2.1.1) IX (CA IX) is strongly overexpressed in most solid tumours and the expression negatively correlates with the prognosis of cancer patients. CA IX activity is important in regulation of pH in tumours (H2 O + CO2 ↔ HCO−3 + H+ ), allowing tumour cells to survive in an otherwise hostile microenvironment. The uptake of the weak-base doxorubicin (Dox), a drug clinically used in cancer chemotherapy, is very dependent on the pH in tumours. The extracellular acidic pH in tumours reduces the uptake of Dox and hence the therapeutic efficiency. Our aim was to determine whether Dox cancer chemotherapy would benefit from increasing of the extracellular pH in tumours by inhibition of CA IX. We tested the recently developed sulfamate CA inhibitor S4 specific for the human tumour-associated IX/XII isoforms with Ki = 7 nM and 2 nM, respectively. Material and Method: We tested the effects of S4 and/or Dox in cultures of three-dimensional (3D) spheroids from FaDu (hypopharyngeal) carcinoma cells grown to a diameter such that hypoxia became evident. Human tumour xenografts were established by growing FaDu and MDA-MB-231 (breast) subcutaneously on the lower back of female nu/nu CBA mice. S4 at 10 mg kg−1 (daily, 5× per week) and Dox at 5 mg kg−1 (weekly) were injected intraperitoneally for the duration of the study, starting when tumour volumes reached 125 and 225–300 mm3 respectively (n = 5 per group). Tumours and lungs were excised when the primary tumour volumes reached 1000 mm3 . Small pieces of fresh lung tissue were used for ex vivo clonogenic assays to evaluate possible metastasis formation. Results and Discussion: In spheroid cultures of FaDu cells the results show that S4 enhances the therapeutic effect of Dox as indicated by a 59% lower number of colonies in the clonogenic survival assay (Dox only vs Dox + S4). In vivo the chronic use of S4 on its own did not affect the primary tumour growth of both cell lines. S4 as an adjuvant to Dox chemotherapy gives a moderate enhanced growth delay of ~2 days in the FaDu model with a total cumulative volume ten days after the start of the treatment of 83% (TCVd10 ) (Dox only is 100%). S4 as an adjuvant to Dox did not exacerbate Dox toxicity (no substantial additional weight loss). Metastatic lung burden was 63% (FaDu) and 40% (MDA-MB-231) lower with combined treatment versus the Dox only group. Conclusion: We conclude that the CA IX inhibitor S4 enhances the anticancer activity of doxorubicin in human tumour xenografts of FaDu and MDAMB-231. No conflict of interest. 637 The PATH biobank: German breast cancer patients approve of genome wide association studies C. Mayer1 , U. Ohlms1 , C. Waldner1 , D.C. Schmitt1 , H. Bodenmuller ¨ 1, T. Anzeneder1 . 1 Stiftung PATH, Biobank, Muenchen, Germany Background: PATH is a biobank providing high-quality fresh-frozen breast cancer specimens to research groups. Being a patient-driven foundation, PATH operates according to high ethical standards. The need for a re-consent for genomic wide association studies (GWAS) on biobank material is a subject of controversial discussion. Here, we present an overview about the PATH biobank and PATH’s approach for obtaining consent to GWAS. Material and Methods: The PATH biobank consists of a centralized database and a decentralized bio repository. The samples are collected and stored in seven institutes for pathology at certified German breast cancer centers. Tumor tissue, along with normal adjacent tissue and blood serum aliquots are processed, labeled and stored according to unique SOPs. Informed consent for the storage of their specimen and its use for research is obtained from patients before storage in the biobank. However, GWAS have not been explicitly mentioned in these consent forms. A letter explaining risks and benefits associated with GWAS and a re-consent form for GWAS were sent to 31 patients in a pilot trial. Patients not responding to the first letter received two reminders. Results: Since 2004, more than 7,200 breast cancer patients have given their informed consent to the storage and analysis of their tissue and blood serum for research purposes. Tumor stages T1 or T2 were present in 93% of donors. The distribution of molecular subtypes among PATH patients according to the St. Gallen International Expert Consensus was: 68% Luminal A, 11% Luminal