bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 547: Radio- and chemotherapy causes up-regulation of immunoinhibitory ligands pd-l1 and galectin-9 in gastric cancer
Authors: Petersen Sven H.
Published: July 2016

Apart from the cytotoxic effect of radiation- or chemotherapy it has become evident that these treatments are able to induce an immune response which can play an

additional and pivotal role in clearing tumours. This treatment-induced immunogenic tumour cell death (ICD) enables the host's immune system to recruit to the tumour and recognise and kill tumour cells which can even extend to yet untreated metastases, a phenomenon called “abscopal effect”. For the first time, we found evidence that radiation and/or chemo-treatment stimulates a pronounced up-regulation of both, PD-L1 and Galectin-9 on pre-apoptotic and apoptotic gastric tumour cells. Both these proteins exert immuno-modulatory signals which in turn may dampen the aimed immunogenicity of the treatment. PD-L1, which is the ligand to programmed cell death protein 1 (PD1) on T cells, and its upregulation is commonly used by cancer cells to evade immunity. PD-L1 causes T cell apoptosis and hence an inhibition of T cell driven anti-tumour immunity. Galectin-9 exerts immunoinhibitory functions and is, together with the immunostimulatory molecule HMGB1, a competitive binding partner for the receptor T-cell immunoglobulin domain and mucin domain 3 (Tim-3). Once engaged by its ligand, Tim-3 either exerts stimulatory or inhibitory effects, depending on ligand, cell type, maturation state and Tim-3 expression rate. Together with PD-L1, Galectin-9 enables a complex interplay between cancer cells and cells of the adaptive immune response expressing PD1 and Tim-3. Based on these results, we are investigating the mechanism responsible for PD-L1 and Galectin-9 upregulation and the effect on the efficacy of radio- as well as chemotherapy on tumour eradication. Additionally, due to their potentially enhancing effect on anti-tumour immunity, we are studying the therapeutic value of blocking Abs specific for human PD1 and/or Tim-3 in cancer immunotherapy.

Citation Format: Sven H. Petersen. Radio- and chemotherapy causes up-regulation of immunoinhibitory ligands pd-l1 and galectin-9 in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 547.
Abstract 548: Characterization of myeloid cells in bladder cancer
Authors: Mukherjee Neelam , Zhao Xiang Ru , Ji Niannian , Svatek Robert S.
Published: July 2016

Both cancer-associated inflammation and tumor-mediated immune suppression have been linked with expansion of myeloid cells in multiple tumor types, including bladder [1]. We previously observed a predominance of M2-like macrophages in mouse bladder tumors [2]. However, little is known about the characteristics of these myeloid cells in human bladder cancer. In this study, we analyzed myeloid cells from tumor tissues collected from patients with muscle-invasive bladder cancer. We used flow cytometry to characterize the different subsets of these myeloid cells.Myeloid cells were identified in the bladder tissue. Our results showed that bladder cancer patients have two major CD11b+CD33+ myeloid derived suppressor cell subsets: granulocyte-type HLADR−CD15+ cells and monocyte-type HLADR+CD14+ cells. Analysis of CD45+ CD11b+cells demonstrated that bladder tumors are dominated by phenotypical M2-like (CD163+ CD80lo) CD163+ M2 macrophages.

The primary treatment modality for bladder cancer is Bacillus Calmete-Guerin (BCG), a live attenuated mycobacterium that is instilled into the bladder via a urethral catheter [3]. This agent remains one of the most effective immune therapies to date. However, though it is effective in prevention of tumor recurrence, response is heterogeneous and tumor recurrence is observed in up to 50% of patients over time. The predominance of immunosuppressive myeloid cells subsets among TILs in human bladder cancer suggests myeloid cells could be potential targets for therapeutic intervention in bladder cancer. We investigated the role of macrophages in BCG therapy in the MB49 bladder cancer model. Depletion of macrophages by liposomal clodronate (LC) during MB49 tumor challenge resulted in decreased tumor growth compared to BCG monotherapy suggesting that macrophages have a critical role in BCG immunotherapy. This study characterizes these myeloid populations in bladder cancer patients and also investigates their potential role in bladder cancer development and therapeutic response.

References:

1.

Eruslanov, E., et al., Circulating and tumor-infiltrating myeloid cell subsets in patients with bladder cancer. International journal of cancer. Journal international du cancer, 2012. 130(5): p. 1109-1119.

2.

Svatek, R.S. et al., Sequential intravesical mitomycin plus Bacillus Calmette-Guérin for non-muscle-invasive urothelial bladder carcinoma: translational and phase I clinical trial. Clinical Cancer Research, 2015. 21(2): p. 303-11

3.

Askeland, E.J., et al., Bladder Cancer Immunotherapy: BCG and Beyond. Advances in urology, 2012. 2012: p. 181987.

Citation Format: Neelam Mukherjee, Xiang Ru Zhao, Niannian Ji, Robert S. Svatek. Characterization of myeloid cells in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 548.
Abstract 550: Evaluating changes in T cell activation by measuring expression of IL-2, CTLA-4, NFAT, and NF-kB in Jurkat cells
Authors: Bement Jacquelyn M. , Miyamoto Yuko J.
Published: July 2016

Insufficient stimulation of T cells due to activated checkpoint pathways can lead to ineffective clearance of tumor cells and thus, tumor growth and cancer. Developing methods to measure changes in the activation of such pathways are needed to evaluate the efficacy of immunotherapy approaches to treating cancer by altering T cell activation. Current methods that use measurments of IL-2 expression are limited by the fact that IL-2 is not specific to T cell activation and that it provides little insight into the mechanisms by which activation is affected. This project aims to address such limitations by determining whether changes in T cell activation can be effectively evaluated by measuring expression of CTLA-4, NFAT, and NF-kB, in addition to IL-2. Jurkat cells were cultured and activated with PMA (100 ng/mL) and ionomycin (500 ng/mL) for five hours. Staining for IL-2 and CTLA-4 with antibodies and transfection of the cells with genetic constructs that link NFAT and NF-kB to fluorescent proteins provided the means to link expression of fluorescence to expression of the molecules of interest. FACS analysis was then used to measure changes in the expression of the four signaling molecules in response to the induced changes in activation. Activation of the cells resulted in increased expression of all four molecules. Following activation, fluorescence due to CTLA-4, NFAT, and NF-kB expression changed by average fold increases of 2.58, 1.10 and 1.13, respectively. By measuring these key elements of T cell signaling pathways in addition to IL-2, these methods provide a potentially more effective alternative to current methods for evaluating the efficacy of immunotherapeutic treatments for cancer.

Citation Format: Jacquelyn M. Bement, Yuko J. Miyamoto. Evaluating changes in T cell activation by measuring expression of IL-2, CTLA-4, NFAT, and NF-kB in Jurkat cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 550.
Abstract 571: Pregnancy associated plasma protein A (PAPP-A) is a potential novel therapeutic target in Ewing sarcoma
Authors: Heitzeneder Sabine , Shern John F. , Khan Javed , Mackall Crystal L.
Published: July 2016

Background: Ewing sarcoma (EWS) is the second most common bone malignancy in the pediatric population. Despite intensive treatment regimens, 5-year overall survival for patients with metastatic or relapsed disease is less than 20%, indicating a clear need for novel targeted therapies. The importance of IGF signaling in EWS is illustrated by activity of antagonistic targeting of IGF-1R in clinical trials, although this approach is plagued by rapid development of resistance. Pregnancy Associated Plasma Protein A (PAPP-A) anchors to cell surface proteoglycans and mediates metalloproteinase activity, that cleaves IGFs from IGFBP-4, IGFBP-2 and IGFBP-5, thus increasing local bioavailability of bioactive, free IGF. PAPP-A is expressed at high levels by placental trophoblasts, where it induces high levels of IGFs, which are required for fetal growth.

Methods: In an attempt to identify novel targetable cell surface antigens in EWS, we performed ribosomal depleted RNA sequencing in 122 EWS samples (49 cell lines and 73 tumor samples) and 96 normal samples of variable tissues.

Results: PAPP-A was identified as one of the top 5 membrane associated proteins overexpressed in EWS (> than 4-fold) compared to normal tissue (p = 3.89E-30). PAPP-A showed substantial expression in tumors (median log2FPKM = 3.933, n = 122) and minimal expression in normal tissue (median log2FPKM = -1.564, n = 96). We confirmed cell surface expression of PAPP-A by flow cytometry on 10 EWS cell lines tested, and identified PAPP-A in the supernatant of 12 EWS cell lines, measured by ELISA. Targeting the PAPP-A locus in the EWS cell line EW8 utilizing CRISPR/Cas9 completely abrogated PAPP-A secretion in single cell clones and significantly diminished bioactive IGF-1 levels in EWS cultures, while increasing IGF-1 bound to IGFBP-4, results which confirm absent PAPP-A metalloproteinase activity. Gene knockout of PAPP-A in EW8 resulted in decreased cell growth in vitro.

Conclusions: We have identified PAPP-A as a novel potential cell surface target in Ewing sarcoma, characterized by high level tumor expression, limited normal tissue expression and biological significance in this tumor. Ongoing work is focused on studying growth of PAPP-A KO cell lines, and assessing whether PAPP-A neutralizing antibodies may mediate antitumor effects in EWS.

Citation Format: Sabine Heitzeneder, John F. Shern, Javed Khan, Crystal L. Mackall. Pregnancy associated plasma protein A (PAPP-A) is a potential novel therapeutic target in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 571.
Abstract 572: A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity
Authors: Lu Dan , Polonskaya Zhanna , Zhang Haifan , Luna Xenia , Martomo Stella , Zhang Zhikai , Zhong Zhaojing , Wu Yan , Patel Jeegar , Tonra James , Witte Larry , Waksal Sam , Zhu Zhenping
Published: July 2016

The importance of VEGF/VEGFR2 signaling in cancer growth and metastasis has been clearly highlighted by the therapeutic benefits of bevacizumab (Avastin), a humanized antibody to VEGF, and Cyramza, a fully human antibody to VEGFR2, in multiple cancer treatment modalities. Recently, immune checkpoint antagonistic antibodies to PD1 and PDL1 have shown some success in a variety of clinical settings across multiple cancer types. The overall clinical efficacy of these individual antibody therapies has been, however, rather limited and, only evident in a fraction of patients. To this end, combinations of antibodies against both VEGF/VEGFR2 and PD1/PDL1 may represent promising approaches to further enhance the antitumor efficacy of individual antibody therapies. In this study, we engineered a bispecific anti-PDL1 x anti-VEGFR2 antibody using two fully human antibodies derived from antibody phage display libraries. In this bispecific format, a high affinity single chain antibody to PDL1 was genetically fused to the C-terminus of the heavy chain of a conventional IgG antibody against VEGFR2. The bispecific antibody was efficiently expressed in mammalian cells and could be purified to homogeneity via single step affinity chromatography. The bispecific antibody retained the binding activity of its parental antibodies to PDL1 and VEGFR2, and strongly blocked both VEGF/VEGFR2 and PDL1/PD1 interaction. Further, the bispecific antibody inhibited VEGF-stimulated VEGFR2 phosphorylation and proliferation of endothelial cells, and promoted proliferation of human T cells and secretion of cytokine such as IL2 and INFγ. The bispecific antibody is currently being evaluated in vivo in relevant animal models.

Citation Format: Dan Lu, Zhanna Polonskaya, Haifan Zhang, Xenia Luna, Stella Martomo, Zhikai Zhang, Zhaojing Zhong, Yan Wu, Jeegar Patel, James Tonra, Larry Witte, Sam Waksal, Zhenping Zhu. A novel anti-PDL1 x anti-VEGFR2 bispecific antibody for enhanced antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 572.
Abstract 582: Novel insights into the antitumor activity of an antibody specific for transferrin receptor 1 (ch128.1) in an in vivo model of human multiple myeloma
Authors: Leoh Lai Sum , Kim Yoon Kyung , Martinez-Maza Otoniel , Daniels-Wells Tracy R. , Penichet Manuel L.
Published: July 2016

The transferrin receptor 1 (TfR1), also known as CD71, is a type II transmembrane homodimeric protein involved in cellular iron uptake and regulation of cell growth. The high levels of TfR1 expression on cancer cells, its extracellular accessibility, and its central role in cancer pathology make it an attractive target for antibody-mediated cancer therapy. We have previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which exhibits direct cytotoxic activity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. In addition, we showed that ch128.1 is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) against malignant B cells in vitro. Importantly, ch128.1 shows exceptional antitumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige) in an early disease setting. Intriguingly, this activity is observed even in malignant cells that show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. In order to study the mechanism of the in vivo antitumor activity, we generated a ch128.1 mutant with abolished binding to FcγR and the complement component C1q (L234A/L235A/P331S). Interestingly, this antibody mutant exhibited a total lack of in vivo protection against MM. This lack of antitumor activity is not due to increased clearance as determined by bioavailability studies. If fact, the two antibodies showed comparable affinity for the mouse FcRn (the neonatal Fc receptor, also known as the Brambell receptor) as determined via surface plasmon resonance analysis. This result suggests a critical role for the antibody Fc fragment in ch128.1-mediated antitumor activity. However, preliminary in vivo studies showed that depletion of complement using cobra venom factor (CVF) does not decrease the antitumor activity of ch128.1, suggesting that CDC is not a relevant mechanism of action, at least in the model used. Furthermore, we recently found that treatment with ch128.1 also significantly increased survival in late MM disease. Further studies aiming to provide a better understanding of the in vivo activity conferred by this antibody are in progress. Our results suggest that ch128.1 can be effective in the therapy of incurable human B-cell malignancies such as MM.

Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Otoniel Martinez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Novel insights into the antitumor activity of an antibody specific for transferrin receptor 1 (ch128.1) in an in vivo model of human multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 582.
Abstract 598: Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization
Authors: Dumitru Calin D. , Gardiner Elisabeth , Williams John , Zer Cindy
Published: July 2016

Therapeutic antibodies can bind to cell-surface receptors, prompting receptor internalization and effectively blocking further stimulation by the cognate receptor ligands. Receptor internalization is also exploited by antibody-drug conjugates (ADC) to promote effective delivery of cytotoxic payload to the inside of the cell. Meditope Biosciences has developed a way to use its SnAP (Site-specfi novel Antibody Platform) technology to promote enhanced internalization of antibody receptor complexes. The Meditope's SnAP technology functionally enables antibodies to bind to specific peptides, termed “meditopes’, and this property can be used to directly facilitate receptor crosslinking when meditope enabled antibodies are bound to cell surface receptors. Here we show that the affinity of the meditope enabled antibodies (trastuzumab anti-HER2 and gemtuzumab anti-CD33) for cell surface receptors results in the formation of an antibody receptor complex that can be detected by native gel electrophoresis and gel filtration. Binding of the meditope peptide to the enabled antibody does not interfere with normal antigen-antibody interaction and the formation of the peptide bound antibody:receptor complexes results in accelerated internalization of the complex, as shown by decreased surface fluorescence of Alexa 488-labeled SnAP peptides as well as by increased pHrodo-Red increased fluorescence of internalized antibodies when they reach the endosomal compartment. Finally, as a result this increased internalization, the downregulation of specific antibody mediated signaling events are much more effectively downregulated in the presence of the meditope peptide complex as compared to the antibody alone.

Citation Format: Calin D. Dumitru, Elisabeth Gardiner, John Williams, Cindy Zer. Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 598.
Abstract 60: PTEN expression, cholesterol metabolism, and lethal prostate cancer
Authors: Stopsack Konrad H. , Gerke Travis A. , Mucci Lorelei A. , Rider Jennifer R.
Published: July 2016

BACKGROUND: We previously showed that mRNA expression of squalene monooxygenase (SQLE), part of the cholesterol synthesis pathway, is associated with lethal prostate cancer. In-vitro studies suggest that loss of PTEN expression and resulting PI3K pathway activation drive cholesterol ester accumulation in aggressive prostate cancers through sterol regulatory element-binding protein (SREBP) and acyl coenzyme A-cholesterol acyltransferase (ACAT1) activity. In two prospective cohorts, we studied whether lower PTEN expression is associated with cholesterol metabolism and how this relates to lethal prostate cancer.

METHODS: We analyzed men with prostate cancer from the prospective prostatectomy Health Professionals Follow-up Study and Physicians’ Health Study. 105 men had lethal cancer and 284 men non-lethal disease without metastases at 8 years of follow-up. Whole-transcriptome mRNA expression profiling data was available from diagnostic prostate tumor specimens. Linear regression models were used to assess continuous and categorical associations, and Pearson correlation coefficients were calculated. Logistic regression was used to obtain odds ratios (ORs) and 95% confidence intervals (CIs) of lethal prostate cancer.

RESULTS: Both lower PTEN and higher SQLE expression were associated with higher Gleason grade (p trend < 0.001 each). Correlations of PTEN with SREBF1 (r = -0.08, p = 0.1), SREBF2 (r = -0.09, p = 0.07), and ACAT1 (r = 0.04, p = 0.4) were weak. SREBF2 was correlated with SQLE (r = 0.33, p < 0.001) but not with ACAT1 (r = -0.01, p = 0.8). Low PTEN was associated with lethal cancer (OR, 1.50 per 1 standard deviation [SD] decrease; 95% CI, 1.20 to 1.87; p = 0.001). High SQLE was associated with lethal cancer (OR 2.13 per 1 SD increase, 95% CI, 1.63 to 2.79; p < 0.001). SREBF1, SREBF2, and ACAT1 were not associated with lethal cancer (all p > 0.05). Adjusting for PTEN mildly attenuated the association of SQLE with lethal cancer (OR, 2.07; 95% CI, 1.57 to 2.73), as did additional adjustment for age, Gleason grade, and stage (SQLE: OR, 1.75; 95% CI, 1.29 to 2.40). In the lowest quartile of PTEN expression, SQLE was less strongly associated with lethal cancer (OR 1.74; 95% CI, 1.20 to 2.52) compared to the upper three quartiles (OR 2.47; 95% CI, 1.69 to 3.60; p interaction = 0.2).

CONCLUSION: Low PTEN mRNA expression and high SQLE are features of aggressive prostate cancers. However, at the time of prostatectomy, SREBF1/2 expression and resulting ACAT1 expression do not appear to be major characteristics of prostate cancers with lethal outcomes.

Citation Format: Konrad H. Stopsack, Travis A. Gerke, Lorelei A. Mucci, Jennifer R. Rider. PTEN expression, cholesterol metabolism, and lethal prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 60.
Abstract 624: Humanized mouse models for evaluation of cancer therapies
Authors: Duchamp Olivier , Bichat Francis , Mignard Caroline , Ghiringhelli François , Mirjolet Jean-François ,   IMODI Consortium
Published: July 2016

Mice with a humanized immune system, so called “humanized” mouse models, have been established to study the complex interaction of the human immune system during human disorders. In case of cancer research, the in vivo model ideally should recapitulate the biological characteristics of the human tumor and of the related tumor microenvironment in patient such as immune system.

Human immune system is reconstituted in immunodeficient mice using either human PBMCs or hematopoietic stem cells (HSCs). Humanized mice bearing human target tumor cells constitute relevant models for evaluation of cancer therapeutics such as bispecific antibodies, immune cell targeting antibodies.

In vivo proof of concept studies were performed with humanized mice xenografted with either human disseminated lymphoma or with human solid tumors (tumor cell line and Patient Derived Xenograft model, Her2 positive breast, colon, ovarian and lung cancers) and treated with therapeutic antibodies.

Biomarkers of response to immune cell targeting therapies were evaluated by flow cytometry as well as immunohistochemistry analyses. In case of disseminated B-cell lymphoma grafted into mice reconstituted with human PBMC, efficacy of antibody was evidenced by an increased survival and a high decrease of human B cells in mouse bone marrow. Lung and ovarian PDX developed in HSC reconstituted BRGS mice induced an increase in Tregs and a change in T/B ratio in blood and spleen samples. Furthermore, we evidenced the presence of Tregs within human PDX tumor by immunohistochemistry.

Citation Format: Olivier Duchamp, Francis Bichat, Caroline Mignard, François Ghiringhelli, Jean-François Mirjolet, IMODI Consortium. Humanized mouse models for evaluation of cancer therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 624.
Abstract 632: Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma
Authors: Lin WT , Xia L , Wen Dan Dan , Tsang CM , Lo KW , Lung ML , Tsao George Sai-Wah
Published: July 2016

Nasopharyngeal carcinoma (NPC) is a common cancer among ethnic Cantonese living in Hong Kong and southern China. It is closely associated with Epstein-Barr virus (EBV) infection. Unfortunately, there are very few representative xenografts and cell lines established from NPC available for investigation. Most of the NPC xenografts established have been passaged in immune deficient animals for over 20 years and may not be representative of the original NPC in patients. For in vitro NPC cell lines, there is only one cell line which retains the EBV genomes. Other NPC cell lines have all lost their EBV episomes. Furthermore, many of these NPC cell lines are found to be contaminated with genetic components from HeLa cells (HPV16 genome) which raised issues on their origins and limited their uses as NPC cells. There is great urgency in establishment new NPC cell lines both in vivo and in vitro for various research investigations and for the study of pathogenic role of EBV infection in NPC. We have carried out continuous efforts since 2010 to attempt establishment of new NPC xenografts and cell lines from surgical and biopsies NPC tissues. NPC tissues from resected recurrent NPC and biopsies from primary NPC were explanted to subrenal capsular sites and maintained for four months to one year to observe for growth of new NPC xenografts. Attempts to establish new NPC celles were also carried out. At present, we have successfully established 3 NPC xenografts (Xeno 23, Xeno 32, Xeno 47) which could be passages at subcutaneously sites and one in vitro NPC cells (NPC43) which harbors EBV genomes. The growth properties and genetic alterations of these newly established NPC cell lines have been characterized. Novel genetic alterations and growth signaling pathways were observed in these newly established NPC cell lines and may represent driver mutations and signaling pathways essential for progression of NPC. Profiling of EBV gene expression of these newly established NPC cell lines revealed predominant latent EBV infection and expression of EBV-encoded mRNA from the BART transcripts which are characteristic of EBV infection in NPC. The establishment of new NPC cells will contribute to investigate the pathogenesis of NPC and its intimate relationship with EBV infection.

Funding acknowledgement: University Grant Council (HK) grants (AoE NPC (AoE/M-06/08); TBRS (T12-401/13R); GRF); University of Hong Kong (CRCG and SRT cancer)

Citation Format: WT Lin, L Xia, Dan Dan Wen, CM Tsang, KW Lo, ML Lung, George Sai-Wah Tsao. Establishment and characterization of xenografts and cell lines trom nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 632.
Abstract 637: Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis
Authors: Konkol Yvonne , Valta Maija , Bernoulli Jenni , Taimen Pekka , Boström Peter , Härkönen Pirkko , Halleen Jussi , Tuomela Johanna M.
Published: July 2016

Bone metastases are frequent and fatal outcome of advanced prostate cancer. Many of the currently used preclinical models lack typical characteristics of the heterogenic human disease. We improved existing methodology by using fresh patient-derived material in a xenograft model of bone metastasis.

Clinical prostate tumor specimens were collected from robotic-assisted laparoscopic radical prostatectomy operations in Turku University Hospital (Turku, Finland). Patient-derived tumor tissue of Gleason grade 8-10 tumors (n = 5) were cut into small pieces, digested overnight, and then cell suspension was intratibially inoculated into the bone marrow cavity of nude mice (n = 8-10/patient). X-ray pictures were taken and blood samples were collected once a month. Mice were sacrificed 6 months after tumor cell inoculation, and hind limbs and lungs were collected for histological and immunohistochemical analysis. PSA was measured from serum using commercial kit.

X-rays demonstrated osteosclerotic bone lesions. Immunohistochemical analysis showed that 80% of tumors in bone expressed similar characteristics compared with original tumor (androgen receptor AR, prostate specific antigen PSA, proliferation marker Ki67). However, no changes were seen in serum PSA. Lung metastases were detected in 50% of tumor-bearing mice. Interestingly, the most of lung metastases were negative for AR and PSA, which indicates that the cell population that forms metastases may be undifferentiated clone of heterogenic tumor and therefore the most aggressive.

Our platform provides new tools for prostate cancer research.

Citation Format: Yvonne Konkol, Maija Valta, Jenni Bernoulli, Pekka Taimen, Peter Boström, Pirkko Härkönen, Jussi Halleen, Johanna M. Tuomela. Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 637.
Abstract 645: The effect of rhBMP-2 on in vitro osteosarcoma tumorigenesis and on pulmonary growth and development
Authors: Geller David S. , Chung So Hak , Zhang Wendong , Piperdi Sajida , Freeman Carrie , Hoang Bang , Yang Rui , Roth Michael , Gill Jonathan , Gorlick Richard
Published: July 2016

Introduction: Complete surgical resection is a critical component of care for patients with osteosarcoma (OS). Limb-salvage surgery is performed in over 90% of cases. Non-union is a well-recognized complication of allograft reconstructions, frequently performed following tumor extirpation. Bone Morphogenetic Protein-2 (BMP-2) has been shown to be osteoinductive and could theoretically improve allograft-host bone union. The purpose of this investigation is to evaluate the effect of exogenous recombinant human BMP-2 (rhBMP-2) on osteosarcoma.

Methods: Functional assays were performed using five standard osteosarcoma cell lines and three patient derived xenograft cell lines. Random migration motility was measured via wound healing scratch assay. Migration (haptotaxis) was quantitatively measured using Boyden Chamber assay. Invasion was assessed using the Biocoat Matrigel Invasion Kit. Cell proliferation rate was measured by using MTT reagent. Metastatic disease was assessed using a xenograft murine model.

Results: Findings suggest that exogenous rhBMP-2 does not meaningfully affect osteosarcoma migration, invasion or proliferation across all tested lines. Average corrected lung weight in the single-dose rhBMP-2 experiment was 1.56 g in the experimental group versus 1.67 g in the control group (p = 0.69). Average corrected lung weight in the triple-dose rhBMP-2 experiment was 1.23 g in the experimental group versus 1.06 g in the control group (p = 0.07).

Conclusions and Future Directions: Results support the contention that exposure of osteosarcoma to exogenous rhBMP-2 does not increase either in vitro tumorigenesis or pulmonary disease growth and development within this experimental model. While results are compelling, additional characterization and confirmation of these findings are necessary safety prerequisites prior to proposing clinical application.

Citation Format: David S. Geller, So Hak Chung, Wendong Zhang, Sajida Piperdi, Carrie Freeman, Bang Hoang, Rui Yang, Michael Roth, Jonathan Gill, Richard Gorlick. The effect of rhBMP-2 on in vitro osteosarcoma tumorigenesis and on pulmonary growth and development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 645.
Abstract 649: Biobank collaboration enables cancer modeling with high quality biospecimens and data
Authors: Shopland Lindsay S. , Emery Ivette , Hoffert Todd , Aalberg Rise , Helbig Petra , LaPierre Susan E. , Sanders Vicky , Mills Kevin D. , Hill Tara , Sheikh Aysha , Jones Michael , Breggia Anne , Rueter Jens
Published: July 2016

According to the National Cancer Institute, the lack of standardized, high-quality biospecimens is one of the most significant roadblocks to progress in cancer research. This shortage especially affects the generation of murine xenograft models of human cancers. Maine, with the third largest age-adjusted cancer incidence rate in the nation, has abundant cancer specimens potentially available for research. To meet the need for quality biospecimens, the biobanks at Eastern Maine Medical Center and Maine Medical Center have established a collaboration to jointly procure cancer specimens from Maine patients. Through this collaboration, the cooperating biobanks provide researchers with tissue, blood, bone marrow and other body fluid specimens from a broad spectrum of solid and hematologic cancers. The specimens from hematologic cancer patients have > 90% viability and demonstrate anticipated biomarker composition by flow cytometry. The comparatively high frequency of engraftment of solid tumors in immunodeficient mice is also indicative of the quality of the samples procured by both repositories. Currently, the biobanks are providing fresh ovarian tumors to develop new xenograft models for poorly engrafting tumors in collaboration with the Jackson Laboratory's Patient Derived Xenograft Program. Both biobanks are integrated with clinical and pathology practices and, therefore, the specimens are annotated with in-depth, de-identified patient datasets. With support from the Maine Cancer Foundation, the biobanks have created the Maine Cancer Biospecimen Portal (MBCP), a website that provides one-stop access to annotated cancer biospecimens and to consultation services for translational research study design. The collaboration of the EMMC and MMC biobanks provides a resource of high quality biospecimens for advancement of both in vivo and in vitro translational research such as mouse models of human cancers.

Citation Format: Lindsay S. Shopland, Ivette Emery, Todd Hoffert, Rise Aalberg, Petra Helbig, Susan E. LaPierre, Vicky Sanders, Kevin D. Mills, Tara Hill, Aysha Sheikh, Michael Jones, Anne Breggia, Jens Rueter. Biobank collaboration enables cancer modeling with high quality biospecimens and data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 649.
Abstract 65: In vivo pooled shRNA library screens identify cancer genes driving epithelial ovarian cancer
Authors: Kodama Michiko , Kodama Takahiro , Newberg Justin , Copeland Neal G. , Jenkins Nancy A.
Published: July 2016

Background & Aims: Epithelial ovarian cancer (EOC) is the 5th leading cause of cancer-related deaths in the USA. Most cases of EOC are diagnosed at an advanced stage with peritoneal carcinomatosis (PC), which seriously affects mortality. To identify therapeutic targets for EOC, we performed in vivo loss of function screens using pooled shRNA libraries. Methods: To discover tumor suppressor genes (TSGs) involved in PC formation, the human EOC cell line, SKOV3, was transduced with a pooled shRNA library containing 42,450 shRNAs targeting 7490 druggable genes. Transduced cells that were highly evasive as measured in Boyden chamber assays were then injected intraperitonealy into female nude mice. Eighteen PC tumors that developed were then sequenced and enriched shRNAs identified. SKOV3 cells transduced with the druggable shRNA library were also injected into nude mice without further in vitro manipulation and 12 PC tumors sequenced in order to identify potential oncogenes as measured by shRNA depletion. Results: Enrichment screening of invasive cells identified 34 potential TSGs targeted by two or more shRNAs. Knockdown of the two most highly ranked genes, RAD51C and TXN, by individual shRNAs, was subsequently shown to accelerate PC formation following intraperitoneal injection of transduced SKOV3 cells. Individual knockdown of these two genes was subsequently shown to increase cell invasion in Boyden chamber assays and adhesion to the extracellular matrix in vitro. These results suggest that RAD51C and TXN are suppressors of human EOC metastasis through their negative regulation of cell adhesion and cell invasion. Finally, shRNA depletion screening identified 17 potential oncogenes. These genes included three known oncogenes, ERBB2, RAF1 and FOS, in addition to eight genes that showed a significant positive correlation between their mRNA levels and poor patient survival in human EOCs. Knockdown of the second ranked oncogene, KPNB1, significantly decreased in vitro cell proliferation in multiple human EOC cell lines, suggesting that KPNB1 may be a new potential therapeutic target of human EOC. KPNB1 knockdown in SKOV3 cells downregulated AURKA, MYBL2, CCNE1 and CDK2 mRNA levels, suggesting that KPNB1 may function by positively regulating the cell cycle. Conclusion: In vivo pooled shRNA library screens are a powerful tool for identifying novel cancer genes and new potential drug targets in human epithelial ovarian cancer.

Citation Format: Michiko Kodama, Takahiro Kodama, Justin Newberg, Neal G. Copeland, Nancy A. Jenkins. In vivo pooled shRNA library screens identify cancer genes driving epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 65.
Abstract 651: Male mouse model of canine and human inflammatory breast cancer (IBC)
Authors: Caceres Sara , Peña Laura , Illera Maria J. , Silvan Gema , de andres Paloma J. , Gao Hui , Woodward Wendy A. , Reuben James M. , Illera Juan C.
Published: July 2016

Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with poor survival in women. Canine IBC has been proposed as a good surrogate model for the study of the human disease. Male breast cancer is a rare disease, accounting for less than 1% of all malignancies in men. Although rarely, IBC has been also found in men. The aim was to evaluate the capacity of male SCID mice to develop IBC tumors after the injection of canine and human IBC cell lines (IPC-366 and SUM149, respectively). Eighty SCID mice: 40 male and 40 females, 6-8 weeks old were used. Suspensions of 106 IPC-366 and SUM149 cells were implanted subcutaneously into the fourth inguinal mammary gland in 20 male and 20 female mice (of each cell line), respectively. Mice were inspected twice a week for the development of tumors. Tumors were monitored by palpation and measured by calipers. Mice were sacrificed when tumors reached 1.5 cm3 and tumors and organs were removed for immunohistochemical (IHC) characterization. A portion of tumor was used for hormonal analysis (Progesterone, Estradiol, Estrone sulphate, Testosterone, Androstenedione). Results revealed that IPC-366 reproduced tumors in 90% (male) vs 100% (female) after 2 weeks p.i. SUM149 reproduced tumors in 40% (male) vs 80% (female) at 4 weeks p.i. Both cell lines produced metastasis in lungs. Higher metastatic rates found in females than in males (IPC-366: 90% vs 20%; SUM149: 80% vs 50%). EIA hormonal analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors (p< 0.05). IHC analysis revealed that male and female tumors shared immunophenotipic characteristics. All xenografts were highly proliferative (high Ki-67 indexes), triple negative (ER-, PR-, HER-2-), vimentin positive and contained less than 10% of cells positive to AE1-AE3 and CK-14. In conclusion, IPC-366 and SUM149 cell lines can reproduce tumors in male mice capable of metastasize. Male and female xenograft models shared immunophenotypic characteristics. Male mice model would be useful for future IBC research using a different endocrine environment that could help to better understand the androgens function in canine and human IBC.

Citation Format: Sara Caceres, Laura Peña, Maria J. Illera, Gema Silvan, Paloma J. de andres, Hui Gao, Wendy A. Woodward, James M. Reuben, Juan C. Illera. Male mouse model of canine and human inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 651.
Abstract 653: Identification of metastasis promoting genes using the PiggyBac insertional mutagenesis system
Authors: Tinghu Qiu N. , Vera Ramirez Laura , Rad Roland , Green Jeffrey E.
Published: July 2016

The Myc oncogene is overexpressed in approximately 30% of human breast cancers. Mice carrying the mammary tumor virus LTR/c-myc (MMTV-c-Myc) develop mammary tumors, but display a relatively low rate of metastases to the lung. However, not all cells that express the transgene develop neoplasms indicating that Myc-induced tumorigenesis requires additional cooperating events for full transformation. To identify genes that cooperate with Myc-induced tumorigenesis leading to the acceleration of tumor progression or an increase in metastases in MMTV-Myc transgenic mice, we have employed the PiggyBac (PB) transposon system.

The PiggyBac system used in our studies was (developed by Rad et al, Science, 2010) is composed of two elements, the ATP-1 PiggyBac transposon and transposase. ATP-1 transposons are mobile DNA elements that can randomly integrate throughout the genome in the presence of transposase resulting in the activation or loss of gene function. The insertion sites can be identified through sequencing. It has been used successfully for genomic modification of cells in vivo leading to cancer gene discovery. In order to better adapt the PB system for studing mammary gland biology, we have designed a system to express a hyperactive transposase and luciferase specifically in the mammary gland using the MMTV LTR (MMTV-hyTPase-luc). The efficiency of MMTV-hyTPase-luc expression was first examined in vitro by transfection with an ATP-1 containing plasmid using the HC11 mouse mammary epithelial cell line. Selection for integration of ATP1 in the cells using puromycin selection and crystal violet staining, RT-QPCR and luciferase assays confirmed that MMTV-hyTPase-luc led to efficient integration of ATP-1 into the HC11 cells. HC11 cell proliferation further increased in the presence of dexamethasone, a hormone that stimulates MMTV promotor function. The MMTV-HyPB-Luc construct was subsequently used to generate transgenic mice and multiple founder lines are being analyzed for mammary specific transposase expression. The best expressing line will be crossed with MMTV-cMyc mice and mice carrying multiple copies of the ATP1 transposon. Transposon integration sites will be determined from tumors or metastases in mice with accelerated tumor development and/or increased metastases compared to controls. Further molecular characterization and validation of candidate genes will identify novel genes that cooperate with Myc to augment tumorigenesis and metastases.

Citation Format: Qiu N. Tinghu, Laura Vera Ramirez, Roland Rad, Jeffrey E. Green. Identification of metastasis promoting genes using the PiggyBac insertional mutagenesis system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 653.
Abstract 66: High-throughput screening using patient derived tumor cell to conduct personalized medicine
Authors: Kim Seung T. , Park Charny , Kim Sun , Lee Won , Park Joon , Kim Kyoung-Mee , Park Woongyang , Lee Jeeyun , park young , Jang Jiryeon
Published: July 2016

Background:

Patient-derived cell (PDC) model is one of prospective technologies to investigate drug response for precision medicine. Previously drug sensitivity derived PDC model has been studied in colorectal cancer organoid culture or lung cancer model. They revealed that PDC model recapitulates genomic characteristic and drug high-throughput screening is amenable. We established PDC models from metastatic cancer cohort of multiple cancer types, and revealed genomic characteristics and matched drug response.

Method:

Target sequencing was performed using OncoPanel. We called SNV, and CNV from target sequencing using GATK, then clinically unassociated SNPs were eliminated. Somatic submodules were identified from integrative variant profiles using HotNet referring HPRD. Drug high-throughput screening was performed SEMCO platform. In order to identify sensitive drug and variant gene pairs, drug sensitivity was tested by ANOVA using IC50 values.

Result:

Target sequencing of exome were generated from our 165 PDC models, and drug high-throughput screening was performed from 30 PDCs and ant-cancer 15 drugs. We tried integrative analysis approaches from genomic profile including mutation and copy number alteration, and identified genomic modules related with Fanconi, Hedgehog, FGFR receptor, Cytokine-cytokine receptor pathway. Variants of known oncogenes were successfully identified in MET, BRAF, FGFR and PIK4CA. Especially novel and recurrent mutations were identified in FGFR2, and corresponding samples indicated AZD4547 sensitivity as well as known drug response case such as BRAF V600E targeting Vemurafenib.

Conclusion:

Our study explains that PDC cohort sustains cancer related pathways, and its drug response follows genomic features. It is expected that our research would contribute to facilitate precision medicine from PDC model to utilize genomics and drug response.

Citation Format: Seung T. Kim, Charny Park, Sun Kim, Won Lee, Joon Park, Kyoung-Mee Kim, Woongyang Park, Jeeyun Lee, young park, Jiryeon Jang. High-throughput screening using patient derived tumor cell to conduct personalized medicine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 66.