### A1 Intratumoral immunotherapy. B16-F10 murine melanoma model #### J. Ženka1, V. Caisová1, O. Uher1, P. Nedbalová1, K. Kvardová1, K. Masáková1, G. Krejčová1, L. Paďouková1, I. Jochmanová2, K. I. Wolf3, J. Chmelař1, J. Kopecký1 ##### 1Department of Medical Biology, Faculty of

Epithelial–mesenchymal transition (EMT) is a key event that is involved in the invasion and dissemination of cancer cells. Although typically considered as having tumour-suppressive properties, transforming growth factor (TGF)-β signalling is altered during cancer and has been associated with the invasion of cancer cells and metastasis. In this study, we report a previously unknown role for the cytoplasmic promyelocytic leukaemia (cPML) tumour suppressor in TGF-β signalling-induced regulation of prostate cancer-associated EMT and invasion. We demonstrate that cPML promotes a mesenchymal phenotype and increases the invasiveness of prostate cancer cells. This event is associated with activation of TGF-β canonical signalling pathway through the induction of Sma and Mad related family 2 and 3 (SMAD2 and SMAD3) phosphorylation. Furthermore, the cytoplasmic localization of promyelocytic leukaemia (PML) is mediated by its nuclear export in a chromosomal maintenance 1 (CRM1)-dependent manner. This was clinically tested in prostate cancer tissue and shown that cytoplasmic PML and CRM1 co-expression correlates with reduced disease-specific survival. In summary, we provide evidence of dysfunctional TGF-β signalling occurring at an early stage in prostate cancer. We show that this disease pathway is mediated by cPML and CRM1 and results in a more aggressive cancer cell phenotype. We propose that the targeting of this pathway could be therapeutically exploited for clinical benefit.

Background: HAGE protein is a known immunogenic cancer-specific antigen.Methods: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n = 2147): early primary (EP-BC; n = 1676); primary oestrogen receptor-negative (PER-BC; n = 275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n = 196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated.Results: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGE +) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGE + expression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE + did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE + and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P = 0.000001), and progression-free survival was worse in those patients who had HAGE + residual disease (P = 0.0003).Conclusions: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC.

1098 Background: Recently TOP2A alteration was found to be a predictor for ATC-CT and our neural network analysis of BC gene expression array (GEA) data has revealed SPAG5 gene as a major hubs in both TOP2A and proliferation pathways. In this study the molecular and clinicopathological functions of SPAG5 was investigated in BC and OVC. Methods: (1) A series of 171 BC was evaluated for SPAG5 gene copy number (using aCGH) and mRNA expression (using GEA) which were validated in 5 independent databases. (2) The expression of SPAG5 protein was evaluated pre-clinically in BC and OVC cell lines and in both 40 normal breast tissues and a series of 1650 primary BC and was correlated to clinicopathological and other biomarkers. (3) The association between SPAG5 and response to CT was investigated in a) 350 ER negative BC treated with adjuvant ATC-CT, b) 250 BC treated with neoadjuvant (NEO-A)-ATC-CT, and c) 200 primary OVC treated with cisplatinum based adjuvant CT. Results: (1) 5% and 15% of the 171 BC showed amplification and gain of SPAG5 locus, respectively, at 17q11.2. SPAG5 mRNA expression displayed a significant correlation with its copy number (p< 0.0001). (2) 30% and 20% of ovarian and BC respectively, showed overexpression of SPAG5 protein (+). In BC, SPAG5+ at both mRNA and protein levels showed a significant association with aggressive phenotypes, high mitosis, ER-, high grade, p53 mutation and epithelial mesenchymal transition phenotypes (ps <0.0001). SPAG5 mRNA (+) was statistically associated with poor survivals (p<0.0001). (3) In ER- BC treated with adjuvant ATC-CT, SPAG5 negative (-)had 7-times higher risk of progression compared with SPRAG+ BC (p<0.0001). SPAG5+ BC received NEO-A-ATC based CT achieved 38% pathological complete response (pCR) vs. 6% of SPAG5- (p<0.0001). After controlling to other predictors for pCR, SPAG5 was an independent predictor (HR; 2.4; p=0.001). Similarly, SPAG5- OVCs were resistant to platinum (p<0.001) and independently associated with poor survival (p<0.001). Conclusions: SPAG5 is an important novel gene implicated in the survival of BC and OVC cells and its protein expression is an independent predictor for anthracycline/ cisplatinum CT.

1013 Background: Recently, we have confirmed that HAGE is involved in promoting proliferation as assessed by increased thymidine incorporation and our preliminary results using shRNA to permanently knockdown HAGE expression also suggests the involvement of HAGE in tumor motility and metastasis. In this study we aimed to analyze the expression of HAGE in large well-characterized BC cohorts to determine its relationship with other clinico-pathological parameters and to investigate its prognostic value. Methods: HAGE protein expression was assessed in: a) 40 normal breast tissue (NBT), b) 60 invasive BCs and their matching NBT, c) BC cell lines, d) A series of 1650 consecutive cases of primary BC who treated with adjuvant CMF and/or endocrine therapies. Further validation was performed in 2 independent series of high risk ER- BC: a) 300 ER –BC who did not received any CT and b) 396 ER- BC treated with adjuvant anthracycline (ATC) based CT. Results: The NBT showed negative HAGE expression (HAGE-) throughout. HAGE overexpression (HAGE+) was observed in 10% of BC and was significantly associated with aggressive clinico-pathological features including: ER-, high grade and triple negative phenotypes. Moreover, HAGE+ expression showed an adverse outcome with a 2-4 fold increase in the risk of death, recurrence and metastases (ps<0.00001) compared to HAGE-; ps<0.0001. Using a multivariate Cox regression model including ER status, grade, size and tumour stage, HAGE expression was confirmed as a powerful independent prognostic factor (p<0.0001). The poor clinical outcome of HAGE+ was further confirmed in high risk (NPI>3.4) ER- patients who did not received any CT (p<0.0001). While, adjuvant CT either CMF or ATC had a positive impact on HAGE+/high risk ER- BC as HAGE+ had a similar risk of death, recurrence and distant metastases to HAGE- expression. Conclusions: This is the first report which shows HAGE to be a potential predictor for poor prognosis in BC patients, and may be an attractive novel target for molecular and vaccine therapy for those patients. A prospective trial of adjuvant chemotherapy/vaccine to confirm this finding is warranted.