- Email: [email protected]
Identifying changes in mutational dynamics in patients with early breast cancer undergoing neoadjuvant chemotherapy
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 BAF180. GCN5 is a histone acetyltransferase (HAT) responsible for promoting transcriptional activation. GCN5 contains a HAT domain and a bromodomain, both of which were predicted as druggable by assessment with Fpocket 2.0. Here focus is on the bromodomain. In this work it was shown that the bromodomain represents a promising target for tool inhibitor development. A structure guided approach was used to design chemical libraries of compounds which might offer improved selectivity and potency compared to micromolar fragments from unpublished work (Structural Genomics Consortium) for the closely related homolog PCAF (P300/CBPassociated factor). These libraries were synthesised and screened in house using a thermal shift assay and hits were tested by Isothermal titration calorimetry to confirm any actives. No conflict of interest. 855 Evaluating the efficiency of hyaluronic acid for specific tumour targeting A. Spadea1 , J.M. Rios de la Rosa1 , M. Mehibel1 , A. Marianne2 , N. Tirelli3 , I.J. Stratford1 . 1 University of Manchester, School of Pharmacy, Manchester, United Kingdom, 2 Astrazeneca, Drug Targeting, Manchester, United Kingdom, 3 University of Manchester, Inflammation and Repair and School of Materials, Manchester, United Kingdom Background: The success of cancer treatment has primarily been hindered by the non-specific effects of conventional anticancer therapies. The development of a delivery system capable of specifically targeting the tumours could be a promising strategy to overcome this issue. One of the currently most investigated cancer targets is the hyaluronic acid (HA) membrane receptor CD44, which is overexpressed in most cancers. CD44 is also considered a reliable marker of cancer-initiating cells and its expression levels are associated with tumour relapse after therapy and poor prognosis. The aim of this project was to evaluate the efficiency of HA to specifically bind CD44 and thereby, selectively target tumour cells. In order to achieve this, a panel of cancer cell lines and normal fibroblasts were screened for CD44 expression and for their ability to binding and internalise HA. Material and Methods: Human dermal fibroblasts (HDF) and a wide panel of cancer cell lines selected from breast, prostatic, head and neck, pancreatic, ovarian, colorectal, thyroid and endometrial cancer were evaluated for their CD44 expression (total and different variants) using western blotting, flow cytometry and immunofluorescence techniques. The uptake of HA was evaluated with flow cytometry and microscopy techniques. Results: The analysis of CD44 expression showed distinct levels and isoform profiles in the different cancer cell lines. Overall, the level of cellular uptake of HA correlated, in most cell lines, with the level of CD44 expression. Further, all the cells expressing CD44 were found able to bind and internalise HA. The lowest HA internalisation was observed in Ishikawa endometrial cancer cells, which have a low and non-homogeneous expression of the receptor. Interestingly, HDF did not internalise large quantities of HA despite their high CD44 expression, and this could be due to receptor inactivation. Surprisingly, LNCaP, CD44 negative prostate cancer cells, showed unspecific uptake of HA. Thus, part of our future work will be to investigate the specificity of this CD44 targeting, analyzing the HA uptake into cells where the receptor has been blocked with anti-CD44 antibodies or transiently knocked down with anti-CD44 siRNA. Conclusions: In order to effectively use hyaluronic acid as a selective targeting agent for tumours, we need to understand the interplay between CD44 expression and functionality, and the underlying mechanism(s) for unspecific uptake. The experiments described here are a first step to help us achieve this understanding. No conflict of interest. 856 High HMGN5 expression confers resistance to tamoxifen and its expression is predictive of response to tamoxifen in ER+ breast cancer patients D. Elias1 , L. Suntharalingam1 , H. Vever1 , A. Lykkesfeldt2 , M. Bak3 , H. Ditzel1,4 . 1 University of Southern Denmark, Cancer and Inflammation Research, Odense C, Denmark, 2 The Danish Cancer Society, Breast Cancer Group- Cell Death and Metabolism, Copenhagen, Denmark, 3 Odense University Hospital, Department of Pathology, Odense, Denmark, 4 Odense University Hospital, Department of Oncology, Odense, Denmark Introduction: A significant proportion of ER+ breast cancers do not respond to first line anti-estrogen treatments leading to patients suffering from the unnecessary side effects of treatments they do not benefit from. This underlines the urgent need for biomarkers capable of accurate separation of patients into treatment categories for optimal patient management. This project is aimed at identifying biomarkers capable of classifying ER+ breast cancer patients that are likely to respond to anti-estrogen therapy. Materials and Methods: Gene array was conducted on tamoxifen-resistant breast cancer cell lines using Affymetrix gene chip. The expression levels of selected genes at mRNA level were further evaluated in two cohorts
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of breast cancer patients. Pathway analysis was conducted using Ingenuity Pathway Analysis software. Gene knockdown was performed using siRNA transfection on MCF7-derived tamoxifen-resistant and -sensitive breast cancer cell lines. Cell number and proliferation was measured using crystal violetbased colorimetric and BrdU incorporation methods, while apoptosis was measured using a cell death detection kit. Protein expression was measured in tissue microarrays of 312 ER+ breast cancer patients treated with tamoxifen as adjuvant therapy by immunohistochemistry and correlated with clinical outcome. Results: Approximately 1100 genes showed marked alteration in expression in tamoxifen-resistant breast cancer cell lines versus the parental breast cancer cell lines. Among these genes, 400 also exhibited similar alteration in 2 cohorts of 316 and 201 ER+ primary breast tumors when patients who experienced recurrence within 5 years of surgery compared to those who were disease free for 7 years following surgery. Among these genes, High Mobility Group Nucleosome Binding Domain 5 (HMGN5), a chromatin binding protein affecting histone modifications, was found to be a central molecule in pathways associated with cell proliferation and death. Knockdown of HMGN5 in tamoxifen-resistant cell lines resulted in increased sensitivity to tamoxifen (p = 0.011) as determined by crystal violet-based colorimetric assay, enhanced tamoxifen induced cell death (p = 0.041), while no significant effect on proliferation (p = 0.071) was observed. Knockdown of HMGN5 did not significantly affect the proliferation (p = 0.12) or apoptotic death (p = 0.065) of the parental tamoxifen-sensitive cell line. Evaluation of the expression of HMGN5 using immunohistochemistry in a cohort of ER+ breast cancer patients (n = 312) treated with tamoxifen showed that increased expression of HMGN5 correlated significantly (p < 0.04) with shorter disease free survival. Conclusion: HMGN5 may play a significant role in tamoxifen induced cell death and its expression may serve as a prognostic marker for tamoxifen response in ER+ breast cancer. No conflict of interest. 857 Identifying changes in mutational dynamics in patients with early breast cancer undergoing neoadjuvant chemotherapy S.J. Sammut1 , S.F. Chin1 , O.M. Rueda1 , S.J. Dawson2 , M. Callari1 , E. Provenzano3 , J. Abraham4 , L. Hughes-Davies4 , H. Earl4 , C. Caldas1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom, 2 Peter MacCallum Cancer Centre, Molecular Biomarkers and Translational Genomics Laboratory, Victoria, Australia, 3 Cambridge University Hospitals NHS Foundation Trust, Department of Histopathology, Cambridge, United Kingdom, 4 Cambridge University Hospitals NHS Foundation Trust, Department of Oncology, Cambridge, United Kingdom Background: Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer. Not only does it improve rates of breast conservation surgery, but also enables prediction of local recurrence and survival by using tumour response to treatment as a surrogate. In addition, the neoadjuvant setting provides a unique opportunity to study in-vivo the impact of cytotoxic systemic therapy on the biology of treatment na¨ıve breast cancer. Our aim was to identify the change in clonal dynamics and mutational profile of treatment na¨ıve early breast cancer while subjected to the selection pressures of chemotherapy and targeted therapies. Materials and Methods: 74 patients with Stage I-III breast cancer receiving neoadjuvant chemotherapy were recruited to the Trans-NEO translational study. Sequential tumour biopsies were taken, when possible, prior to commencing chemotherapy, midway through treatment and on completion of chemotherapy. Somatic variants and copy number alterations were characterised by whole exome (minimum 80x coverage) and shallow whole genome sequencing of tumour and peripheral blood leucocytes at all available time points to generate a comprehensive landscape of changes in genomic alterations during chemotherapy. Using Bayesian clustering methods, the change in clonal composition of tumours during the duration of chemotherapy was characterised, allowing the genomic identification of tumour clones that were susceptible to chemotherapy, as well as others that had not been eliminated by treatment. Results: Of all patients analysed, 22 (30%) had complete pathological response, 19 (26%) had an excellent response (<10% tumour cellularity on biopsy), 27 (36%) had a good response and 6 (8%) had no or very modest response on imaging and histopathological assessment. Resistance to treatment was associated with maintained copy number alterations and minimal change in the prevalence of the dominant breast cancer driver genes. We show that the chemotherapy agent switch classically done midway during treatment to improve response is efficacious and helps eliminate resistant clones generated or maintained in the first half of the treatment regimen. Additionally, we were able to identify residual mutational changes that might belong to remaining tumour cells. Conclusions: The neoadjuvant setting offers an important opportunity to research molecular predictors of response to therapy and prognosis. The detailed analysis of the somatic mutational landscape change during
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chemotherapy offers clinically important insight by characterising the emergence of adapting tumour cells that are resistant to cytotoxic agents delivered. No conflict of interest. 858 Plasma miRNAs as prognostic markers in colorectal cancer M. Maierthaler1 , A. Benner2 , L. Jansen3 , P. Knebel4 , J. Chang-Claude5 , M. Hoffmeister3 , H. Brenner3 , B. Burwinkel1 . 1 German Cancer Research Center DKFZ, Clinical Epidemiology, Heidelberg, Germany, 2 German Cancer Research Center DKFZ, Biostatistics, Heidelberg, Germany, 3 German Cancer Research Center DKFZ, Clinical Epidemiology and Aging Research, Heidelberg, Germany, 4 University of Heidelberg, Department of GeneralVisceral and Transplantation Surgery, Heidelberg, Germany, 5 German Cancer Research Center DKFZ, Cancer Epidemiology, Heidelberg, Germany Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide with 1.36 million new cases annually. Approximately 20% of diagnoses are made in the metastatic stage with a median survival time of less than two years. The lack of effective treatment options for the late CRC stages necessitates to better understand the processes of distant metastasis formation and to find efficient markers for the patients’ prognosis. Circulating microRNAs (miRNAs) have been proposed as minimally invasive prognostic markers for various types of cancers, including CRC. Before, we had identified the diagnostic plasma miRNA set “PROMPT” for the early detection and prognosis of breast cancer metastasis which included miRNAs especially important in the epithelial-to-mesenchymal transition and the reverse process, the mesenchymal-to-epithelial transition. Here, we aimed to evaluate the levels of circulating miRNAs that might serve as prognostic markers for CRC with a specific focus on miRNAs known to be involved in these metastasis formation processes. Material and Method: After comprehensive literature study, 95 miRNAs, including quality control miRNAs, were selected for the generation of Custom TaqMan® Array MicroRNA Cards. First, plasma samples of primary and metastatic colon cancer patients, each group consisting of 20 samples with good and 20 samples with bad prognosis, were screened using the array cards. Good prognosis was defined as no event (relapse, metastasis or cancerrelated death), occurring for more than 5 years in primary and 2.5 years in metastatic cases and bad prognosis as an event occurring within one year. Identified and selected candidate miRNAs were further validated by RT-qPCR in a plasma sample set of 543 (309 primary and 234 metastatic) CRC patients. The association of the miRNA levels with patients’ survival and the prognostic subtypes was analyzed with uni- and multivariate logistic regression and Cox proportional hazards regression models. Results and Discussion: We found miRNAs, including members of the miR-200 family, to be associated with relapse free survival, overall survival and different clinicopathological characteristics. These miRNAs as well as the influence of the time-point of blood collection will be discussed. Conclusion: Identified miRNA markers could be of potential use in the development of a molecular multi-marker blood test for CRC prognosis. No conflict of interest. 859 SEARCHBreast; making surplus material from in vivo models of breast cancer available for research B. Morrissey1 , K. Blyth2 , P. Carter3 , C. Chelala3 , L. Jones3 , I. Holen4 , V. Speirs1 . 1 University of Leeds, Leeds Institute of Cancer and Pathology, Leeds, United Kingdom, 2 Cancer Research UK Beatson Institute, Cancer Research UK Beatson Institute, Glasgow, United Kingdom, 3 Queen Mary University London, Barts Cancer Institute, London, United Kingdom, 4 University of Sheffield, Medical School, Sheffield, United Kingdom Introduction: Significant advances in unravelling the mechanistic details associated with breast cancer development and therapy have been made possible by pre-clinical studies in mice. Due to the nature of in vivo studies, a substantial number of animals are sacrificed, generating surplus tissue and other material that, is archived at the time of study conclusion and therefore not of immediate use to the researcher but which could be used by other research groups to answer additional scientific questions. There is then a pressing need to make such material transparent. Material: SEARCHBreast (Sharing Experimental Animal Resources: Coordinating Holdings − Breast) was developed to facilitate the sharing of archival tissue between researchers by making material derived from mouse models of breast cancer more visible and accessible to the research community. The project has a secure database to upload or request animal material relevant to breast cancer which is available to share in collaboration. Results and Discussion: The SEARCHBreast database contains information on thousands of tissue samples available for immediate use following a simple online request system. Samples are available from over 25 different mouse models including transgenic and xenograft material. There are many different types of tissue available including tumours, mammary glands, mammary epithelial cells, lung metastasis, liver, brain, and bone. Information including
GEM alleles, background strain, cell lines, metastatic sites & penetrance, transplantation sites, & hormone receptor status is also available Importantly these samples are ready to use with most available as paraffin embedded blocks or histological slides. Conclusion: SEARCHBreast enables researchers to access well-characterised, material to enhance their research, without the need to carry out new in vivo studies. The immediate availability of these tissues will save the researcher time and money, and via, connecting members, SEARCHBreast enables scientists with little experience with in vivo work, the opportunity to perform experiments which could increase the quality of their outputs, enhancing their work. No conflict of interest. 860 Phosphorylated YB-1 (Y-box binding protein 1) as a biomarker of poor prognosis in patients with breast cancer S. Casimiro1 , M. Bettencourt1 , A. Fernandes1 , T.R. Pacheco1 , M. MaiaMatos1 , A.L. Costa2 , C. Abreu2 , A. Alves3 , L. Correia3 , L. Costa2 . 1 Instituto de Medicina Molecular, Translational Oncobiology- LCosta Lab, Lisboa, Portugal, 2 Hospital de Santa Maria − Centro Hospitalar Lisboa Norte, Oncology Division, Lisboa, Portugal, 3 Hospital de Santa Maria − Centro Hospitalar Lisboa Norte, Pathology Division, Lisboa, Portugal Background: YB-1 (Y-box binding protein 1) is a multifunctional cold-shock protein constitutively expressed in tissues. YB-1 plays a major role in most cellular functions and has been implicated in all hallmarks of cancer. Elevated YB-1 expression has been correlated with cancer progression and poor prognosis in several types of cancers, including breast cancer (BC), where high YB-1 expression seems to be related to lower overall survival (OS) and distant metastasis free-survival (DMSF) across all subtypes. The phosphorylated form of YB-1 (p-YB-1) seems to be involved in the transcriptional regulation of important genes like EGFR and ERBB2, but its role as a biomarker is unknown. In this study we assessed the prognostic value of p-YB-1 at diagnosis or relapse, in patients with advanced BC. Materials and Methods: In this retrospective study we included 65 patients diagnosed with BC, either metastatic at diagnosis or that relapsed at distant sites on the course of the disease. Expression of p-YB-1 was evaluated by immunohistochemistry in 65 primary tumors and 32 paired metastases, using an anti-human p-YB-1 (Ser102) antibody (Cell Signaling), and the EnVision Detection System (Dako). Negative control was obtained by pretreatment with Calf alkaline phosphatase. Staining intensity was classified from 0 to 3, and samples were scored according to the percentage of cells with a cytoplasmatic staining intensity 2, and the positivity or percentage of cells with nuclear staining for p-YB-1. Cytoplasmatic p-YB-1 expression was considered high at a cut-off of 35% or 20%, for primary tumors or metastases, respectively, as determined using Cutoff Finder 2.1 (Institute of Pathology, Charite´ − Universitatsmedizin ¨ Berlin) and OS as endpoint. Differences in clinicopathological characteristics were tested using Fisher exact test. Time to event outcomes were analysed using Cox models. Results: High cytoplasmatic p-YB-1 expression was found in 23% of primary tumors and 32.3% of metastases. In primary tumors, high p-YB-1 was associated with ER/PR negativity (P = 0.006 and P = 0.037, respectively), and with lower DMFS (P = 0.0108, HR 0.3058, 95% CI 0.1230–0.7606), but not significantly with OS (P = 0.0687, HR 0.4789, 95% CI 0.2168–1.058). Among metastases, high p-YB-1 was correlated with PR negativity (P = 0.030). In this setting, high cytoplasmatic p-YB-1 was prognostic of decreased OS (P = 0.009, HR 0.2394, 95% CI 0.08186–0.7002). Nuclear expression of p-YB-1 was not indicative of prognosis. Conclusions: Our study shows that p-YB-1 may be important an important prognostic biomarker of relapse in patients with BC, and of decreased OS in patients with metastatic disease, especially in patients with ER−/PR− tumors. No conflict of interest. 861 A gemcitabine based peptide conjugate with improved metabolic properties, dual mode of action and efficacy in animal models T. Karampelas1 , E. Skavatsou1 , O. Argyros1 , C. Tamvakopoulos1 . Biomedical Research Foundation- Academy of Athens, Pharmacology and Pharmacotechnology, Athens, Greece 1
Background: Although gemcitabine (gem) has shown efficacy in various types of cancer, it is characterized by weaknesses that limit its anticancer potential. The most important limitation of gem’s use is associated with its rapid inactivation upon administration. We have previously shown that conjugation of gem to a GnRH-R ligand peptide (molecule GSG) enhanced its metabolic stability systemically and locally (in the cancer cell), resulting in improved efficacy in a castration resistant prostate cancer (CRPC) animal model. In order to enrich our previous findings, we developed methodologies that would allow us to further elucidate the mode of action of GSG regarding its potential role in the activation of gem, with respect to its capacity to activate the GnRH-R in the pituitary and with respect to toxicity potential particularly in comparison to gem.
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of breast cancer patients. Pathway analysis was conducted using Ingenuity Pathway Analysis software. Gene knockdown was performed using siRNA transfection on MCF7-derived tamoxifen-resistant and -sensitive breast cancer cell lines. Cell number and proliferation was measured using crystal violetbased colorimetric and BrdU incorporation methods, while apoptosis was measured using a cell death detection kit. Protein expression was measured in tissue microarrays of 312 ER+ breast cancer patients treated with tamoxifen as adjuvant therapy by immunohistochemistry and correlated with clinical outcome. Results: Approximately 1100 genes showed marked alteration in expression in tamoxifen-resistant breast cancer cell lines versus the parental breast cancer cell lines. Among these genes, 400 also exhibited similar alteration in 2 cohorts of 316 and 201 ER+ primary breast tumors when patients who experienced recurrence within 5 years of surgery compared to those who were disease free for 7 years following surgery. Among these genes, High Mobility Group Nucleosome Binding Domain 5 (HMGN5), a chromatin binding protein affecting histone modifications, was found to be a central molecule in pathways associated with cell proliferation and death. Knockdown of HMGN5 in tamoxifen-resistant cell lines resulted in increased sensitivity to tamoxifen (p = 0.011) as determined by crystal violet-based colorimetric assay, enhanced tamoxifen induced cell death (p = 0.041), while no significant effect on proliferation (p = 0.071) was observed. Knockdown of HMGN5 did not significantly affect the proliferation (p = 0.12) or apoptotic death (p = 0.065) of the parental tamoxifen-sensitive cell line. Evaluation of the expression of HMGN5 using immunohistochemistry in a cohort of ER+ breast cancer patients (n = 312) treated with tamoxifen showed that increased expression of HMGN5 correlated significantly (p < 0.04) with shorter disease free survival. Conclusion: HMGN5 may play a significant role in tamoxifen induced cell death and its expression may serve as a prognostic marker for tamoxifen response in ER+ breast cancer. No conflict of interest. 857 Identifying changes in mutational dynamics in patients with early breast cancer undergoing neoadjuvant chemotherapy S.J. Sammut1 , S.F. Chin1 , O.M. Rueda1 , S.J. Dawson2 , M. Callari1 , E. Provenzano3 , J. Abraham4 , L. Hughes-Davies4 , H. Earl4 , C. Caldas1 . 1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom, 2 Peter MacCallum Cancer Centre, Molecular Biomarkers and Translational Genomics Laboratory, Victoria, Australia, 3 Cambridge University Hospitals NHS Foundation Trust, Department of Histopathology, Cambridge, United Kingdom, 4 Cambridge University Hospitals NHS Foundation Trust, Department of Oncology, Cambridge, United Kingdom Background: Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer. Not only does it improve rates of breast conservation surgery, but also enables prediction of local recurrence and survival by using tumour response to treatment as a surrogate. In addition, the neoadjuvant setting provides a unique opportunity to study in-vivo the impact of cytotoxic systemic therapy on the biology of treatment na¨ıve breast cancer. Our aim was to identify the change in clonal dynamics and mutational profile of treatment na¨ıve early breast cancer while subjected to the selection pressures of chemotherapy and targeted therapies. Materials and Methods: 74 patients with Stage I-III breast cancer receiving neoadjuvant chemotherapy were recruited to the Trans-NEO translational study. Sequential tumour biopsies were taken, when possible, prior to commencing chemotherapy, midway through treatment and on completion of chemotherapy. Somatic variants and copy number alterations were characterised by whole exome (minimum 80x coverage) and shallow whole genome sequencing of tumour and peripheral blood leucocytes at all available time points to generate a comprehensive landscape of changes in genomic alterations during chemotherapy. Using Bayesian clustering methods, the change in clonal composition of tumours during the duration of chemotherapy was characterised, allowing the genomic identification of tumour clones that were susceptible to chemotherapy, as well as others that had not been eliminated by treatment. Results: Of all patients analysed, 22 (30%) had complete pathological response, 19 (26%) had an excellent response (<10% tumour cellularity on biopsy), 27 (36%) had a good response and 6 (8%) had no or very modest response on imaging and histopathological assessment. Resistance to treatment was associated with maintained copy number alterations and minimal change in the prevalence of the dominant breast cancer driver genes. We show that the chemotherapy agent switch classically done midway during treatment to improve response is efficacious and helps eliminate resistant clones generated or maintained in the first half of the treatment regimen. Additionally, we were able to identify residual mutational changes that might belong to remaining tumour cells. Conclusions: The neoadjuvant setting offers an important opportunity to research molecular predictors of response to therapy and prognosis. The detailed analysis of the somatic mutational landscape change during
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EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
chemotherapy offers clinically important insight by characterising the emergence of adapting tumour cells that are resistant to cytotoxic agents delivered. No conflict of interest. 858 Plasma miRNAs as prognostic markers in colorectal cancer M. Maierthaler1 , A. Benner2 , L. Jansen3 , P. Knebel4 , J. Chang-Claude5 , M. Hoffmeister3 , H. Brenner3 , B. Burwinkel1 . 1 German Cancer Research Center DKFZ, Clinical Epidemiology, Heidelberg, Germany, 2 German Cancer Research Center DKFZ, Biostatistics, Heidelberg, Germany, 3 German Cancer Research Center DKFZ, Clinical Epidemiology and Aging Research, Heidelberg, Germany, 4 University of Heidelberg, Department of GeneralVisceral and Transplantation Surgery, Heidelberg, Germany, 5 German Cancer Research Center DKFZ, Cancer Epidemiology, Heidelberg, Germany Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide with 1.36 million new cases annually. Approximately 20% of diagnoses are made in the metastatic stage with a median survival time of less than two years. The lack of effective treatment options for the late CRC stages necessitates to better understand the processes of distant metastasis formation and to find efficient markers for the patients’ prognosis. Circulating microRNAs (miRNAs) have been proposed as minimally invasive prognostic markers for various types of cancers, including CRC. Before, we had identified the diagnostic plasma miRNA set “PROMPT” for the early detection and prognosis of breast cancer metastasis which included miRNAs especially important in the epithelial-to-mesenchymal transition and the reverse process, the mesenchymal-to-epithelial transition. Here, we aimed to evaluate the levels of circulating miRNAs that might serve as prognostic markers for CRC with a specific focus on miRNAs known to be involved in these metastasis formation processes. Material and Method: After comprehensive literature study, 95 miRNAs, including quality control miRNAs, were selected for the generation of Custom TaqMan® Array MicroRNA Cards. First, plasma samples of primary and metastatic colon cancer patients, each group consisting of 20 samples with good and 20 samples with bad prognosis, were screened using the array cards. Good prognosis was defined as no event (relapse, metastasis or cancerrelated death), occurring for more than 5 years in primary and 2.5 years in metastatic cases and bad prognosis as an event occurring within one year. Identified and selected candidate miRNAs were further validated by RT-qPCR in a plasma sample set of 543 (309 primary and 234 metastatic) CRC patients. The association of the miRNA levels with patients’ survival and the prognostic subtypes was analyzed with uni- and multivariate logistic regression and Cox proportional hazards regression models. Results and Discussion: We found miRNAs, including members of the miR-200 family, to be associated with relapse free survival, overall survival and different clinicopathological characteristics. These miRNAs as well as the influence of the time-point of blood collection will be discussed. Conclusion: Identified miRNA markers could be of potential use in the development of a molecular multi-marker blood test for CRC prognosis. No conflict of interest. 859 SEARCHBreast; making surplus material from in vivo models of breast cancer available for research B. Morrissey1 , K. Blyth2 , P. Carter3 , C. Chelala3 , L. Jones3 , I. Holen4 , V. Speirs1 . 1 University of Leeds, Leeds Institute of Cancer and Pathology, Leeds, United Kingdom, 2 Cancer Research UK Beatson Institute, Cancer Research UK Beatson Institute, Glasgow, United Kingdom, 3 Queen Mary University London, Barts Cancer Institute, London, United Kingdom, 4 University of Sheffield, Medical School, Sheffield, United Kingdom Introduction: Significant advances in unravelling the mechanistic details associated with breast cancer development and therapy have been made possible by pre-clinical studies in mice. Due to the nature of in vivo studies, a substantial number of animals are sacrificed, generating surplus tissue and other material that, is archived at the time of study conclusion and therefore not of immediate use to the researcher but which could be used by other research groups to answer additional scientific questions. There is then a pressing need to make such material transparent. Material: SEARCHBreast (Sharing Experimental Animal Resources: Coordinating Holdings − Breast) was developed to facilitate the sharing of archival tissue between researchers by making material derived from mouse models of breast cancer more visible and accessible to the research community. The project has a secure database to upload or request animal material relevant to breast cancer which is available to share in collaboration. Results and Discussion: The SEARCHBreast database contains information on thousands of tissue samples available for immediate use following a simple online request system. Samples are available from over 25 different mouse models including transgenic and xenograft material. There are many different types of tissue available including tumours, mammary glands, mammary epithelial cells, lung metastasis, liver, brain, and bone. Information including
GEM alleles, background strain, cell lines, metastatic sites & penetrance, transplantation sites, & hormone receptor status is also available Importantly these samples are ready to use with most available as paraffin embedded blocks or histological slides. Conclusion: SEARCHBreast enables researchers to access well-characterised, material to enhance their research, without the need to carry out new in vivo studies. The immediate availability of these tissues will save the researcher time and money, and via, connecting members, SEARCHBreast enables scientists with little experience with in vivo work, the opportunity to perform experiments which could increase the quality of their outputs, enhancing their work. No conflict of interest. 860 Phosphorylated YB-1 (Y-box binding protein 1) as a biomarker of poor prognosis in patients with breast cancer S. Casimiro1 , M. Bettencourt1 , A. Fernandes1 , T.R. Pacheco1 , M. MaiaMatos1 , A.L. Costa2 , C. Abreu2 , A. Alves3 , L. Correia3 , L. Costa2 . 1 Instituto de Medicina Molecular, Translational Oncobiology- LCosta Lab, Lisboa, Portugal, 2 Hospital de Santa Maria − Centro Hospitalar Lisboa Norte, Oncology Division, Lisboa, Portugal, 3 Hospital de Santa Maria − Centro Hospitalar Lisboa Norte, Pathology Division, Lisboa, Portugal Background: YB-1 (Y-box binding protein 1) is a multifunctional cold-shock protein constitutively expressed in tissues. YB-1 plays a major role in most cellular functions and has been implicated in all hallmarks of cancer. Elevated YB-1 expression has been correlated with cancer progression and poor prognosis in several types of cancers, including breast cancer (BC), where high YB-1 expression seems to be related to lower overall survival (OS) and distant metastasis free-survival (DMSF) across all subtypes. The phosphorylated form of YB-1 (p-YB-1) seems to be involved in the transcriptional regulation of important genes like EGFR and ERBB2, but its role as a biomarker is unknown. In this study we assessed the prognostic value of p-YB-1 at diagnosis or relapse, in patients with advanced BC. Materials and Methods: In this retrospective study we included 65 patients diagnosed with BC, either metastatic at diagnosis or that relapsed at distant sites on the course of the disease. Expression of p-YB-1 was evaluated by immunohistochemistry in 65 primary tumors and 32 paired metastases, using an anti-human p-YB-1 (Ser102) antibody (Cell Signaling), and the EnVision Detection System (Dako). Negative control was obtained by pretreatment with Calf alkaline phosphatase. Staining intensity was classified from 0 to 3, and samples were scored according to the percentage of cells with a cytoplasmatic staining intensity 2, and the positivity or percentage of cells with nuclear staining for p-YB-1. Cytoplasmatic p-YB-1 expression was considered high at a cut-off of 35% or 20%, for primary tumors or metastases, respectively, as determined using Cutoff Finder 2.1 (Institute of Pathology, Charite´ − Universitatsmedizin ¨ Berlin) and OS as endpoint. Differences in clinicopathological characteristics were tested using Fisher exact test. Time to event outcomes were analysed using Cox models. Results: High cytoplasmatic p-YB-1 expression was found in 23% of primary tumors and 32.3% of metastases. In primary tumors, high p-YB-1 was associated with ER/PR negativity (P = 0.006 and P = 0.037, respectively), and with lower DMFS (P = 0.0108, HR 0.3058, 95% CI 0.1230–0.7606), but not significantly with OS (P = 0.0687, HR 0.4789, 95% CI 0.2168–1.058). Among metastases, high p-YB-1 was correlated with PR negativity (P = 0.030). In this setting, high cytoplasmatic p-YB-1 was prognostic of decreased OS (P = 0.009, HR 0.2394, 95% CI 0.08186–0.7002). Nuclear expression of p-YB-1 was not indicative of prognosis. Conclusions: Our study shows that p-YB-1 may be important an important prognostic biomarker of relapse in patients with BC, and of decreased OS in patients with metastatic disease, especially in patients with ER−/PR− tumors. No conflict of interest. 861 A gemcitabine based peptide conjugate with improved metabolic properties, dual mode of action and efficacy in animal models T. Karampelas1 , E. Skavatsou1 , O. Argyros1 , C. Tamvakopoulos1 . Biomedical Research Foundation- Academy of Athens, Pharmacology and Pharmacotechnology, Athens, Greece 1
Background: Although gemcitabine (gem) has shown efficacy in various types of cancer, it is characterized by weaknesses that limit its anticancer potential. The most important limitation of gem’s use is associated with its rapid inactivation upon administration. We have previously shown that conjugation of gem to a GnRH-R ligand peptide (molecule GSG) enhanced its metabolic stability systemically and locally (in the cancer cell), resulting in improved efficacy in a castration resistant prostate cancer (CRPC) animal model. In order to enrich our previous findings, we developed methodologies that would allow us to further elucidate the mode of action of GSG regarding its potential role in the activation of gem, with respect to its capacity to activate the GnRH-R in the pituitary and with respect to toxicity potential particularly in comparison to gem.