bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4874: Interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immunotherapy
Authors: Yang Xi , Tietje Ashlee , Yu Xianzhong , Wei Yanzhang
Published: July 2016

Whereas cancer immunotherapy with cytokines demonstrates effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system and block the Fas-mediated apoptosis by down-regulating its expression. In the present study, we report the design and preliminary evaluation of the antitumor activity of a novel fusion protein:mIL-12/FasTI, consisting of mIL-12 and the transmembrane and intracellular domains of Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-ã production and cytotoxicity. Enhanced caspase 3 activity of the clones when co-cultured with NK cells demonstrated that apoptosis was induced through Fas/FasL signaling pathway. The bifunctional protein is clearly more effective than IL-12 alone. It, therefore, represents a promising new therapeutic agent for cancer treatment when combined with tumor cell-specific gene delivery.

Citation Format: Xi Yang, Ashlee Tietje, Xianzhong Yu, Yanzhang Wei. Interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immunotherapy. [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 4874.
Abstract 4878: Quantitative cell-based bioassays for therapeutic development targeting immune checkpoint and co-stimulatory receptors
Authors: Grailer Jamison , Stecha Pete , Wang Jun , Hartnett Jim , Fan Frank , Cong Mei , Cheng Zhi-jie Jey
Published: July 2016

Immunotherapy harnesses the immune system to fight cancer and has proven to be a very promising therapeutic strategy. Drug targets in cancer immunotherapy include both inhibitory and co-stimulatory immune receptors on T cells or NK cells, in particular. Current approaches to assay immunotherapy biologics rely on primary cells, are highly variable, and are not suitable for a quality control environment during drug development. We have developed a panel of cell-based assays using a bioluminescent reporter platform that can quantitatively determine the potencies of antibodies and ligand proteins targeting immune checkpoint receptors and co-stimulatory receptors including PD-1, CTLA-4, LAG-3, GITR, 4-1BB, OX40 and CD40. For each target, a stable cell line was generated in an immune cell background to stably express an immune checkpoint or co-stimulatory receptor and a luciferase reporter driven by a response element specifically responding to signaling induced by TCR or directly from the immune receptor. These bioassays reflect biological mode-of-action for each class of drug candidate and are able to determine the potencies for on-market biologic drugs including PD-1 antibodies pembrolizumab and nivolumab, and CTLA-4 antibody ipilimumab. The assay signals are robust, specific, and have good repeatability and linearity. Therefore they can serve as valuable tools for drug screening, QC lot release and stability studies in immunotherapy drug development.

Citation Format: Jamison Grailer, Pete Stecha, Jun Wang, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based bioassays for therapeutic development targeting immune checkpoint and co-stimulatory receptors. [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 4878.
Abstract 4894: Relationship of characterization of the immunological microenvironment and pathological response in advanced rectal cancer after oxaliplatin-based neoadjuvant chemotherapy
Authors: Sekizawa Kentaro , Ichikawa Yasushi , Ishibe Atsushi , Suwa Hirokazu , Momiyama Masashi , Kato Ikuma , Ota Mitsuyoshi , Endo Itaru
Published: July 2016

Background:

Characterization of the immunological microenvironment has been demonstrated to be associated with prognosis and the effect of chemotherapy of various cancers. Then it has been shown by their role several studies in 5-FU-based neoadjuvant chemoradiotherapy for rectal cancer. However, their role in neoadjuvant chemotherapy (NAC) for rectal cancer remains unknown. The objective of this study was to examine the relationship between immune reactions and the pathological response to induction oxaliplatin-based NAC in patients with advanced rectal cancer.

Methods:

A total of 65 patients with advanced rectal cancer who underwent induction oxaliplatin-based NAC followed by surgical resection were enrolled. The resected tumor specimens were counted positive cell of tumor infiltrating lymphocytes (TILs) and macrophages by using immunohistochemical staining for CD3, CD8, Foxp3, CD86, CD206. We analyzed for the relationship between the numbers of TILs or macrophages at the stroma in resected specimens and the pathological response to induction oxaliplatin-based NAC.

Results:

The numbers of Foxp3+ TILs in resected specimens were strongly correlated with pathological response. Patients with higher number of Foxp3+ TILs showed poor pathological response (p<0.001). There were no statistically significant differences between the number of CD3+, CD8+ TILs and pathological response, but patients with higher ratio of CD8+/FOXP3+ TILs was associated with good pathological response (p = 0.023). No associations were observed between the numbers of CD86+, CD206+ macrophages and pathological response.

Conclusions:

In rectal cancer patients, T lymphocyte-mediated immune reactions significantly correlated with pathological response of oxaliplatin-based NAC for advanced rectal cancer. These were similar to the T lymphocyte-mediated immune responses in the neoadjuvant radiation therapy for rectal cancer.

Citation Format: Kentaro Sekizawa, Yasushi Ichikawa, Atsushi Ishibe, Hirokazu Suwa, Masashi Momiyama, Ikuma Kato, Mitsuyoshi Ota, Itaru Endo. Relationship of characterization of the immunological microenvironment and pathological response in advanced rectal cancer after oxaliplatin-based neoadjuvant chemotherapy. [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 4894.
Abstract 4898: Effects of chemotherapeutic agents on the tumor immune microenvironment
Authors: Belvin Marcia P. , Luoh Shiuh-Ming , Cheung Jeanne , McNamara Erin , Cubas Rafael , Kim Jeong
Published: July 2016

Immunotherapeutic agents have shown dramatic success in oncology in recent years and several have been approved in melanoma and lung cancer. One strategy for extending the benefit of immunotherapy is to evaluate these agents in combination with standard of care chemotherapy, with the goal of bringing the benefit of immunotherapy into earlier lines of treatment.

We have carried out a systematic evaluation of the effects of different classes of chemotherapeutic agents (including alkylating agents, platinum based agents, and taxanes) on the tumor immune microenvironment of syngeneic murine tumor models (CT26 and MC38) in two different strains of mice (BALB/c and C57BL/6). We found that different chemotherapeutic agents caused markedly different changes in the composition and cell phenotype of the tumor immune environment. The majority of chemotherapeutic agents resulted in a mild to moderate increase in CD8+ T cells in the tumor. Some agents also altered the activation state of these CD8+ cells as measured by the level of PD-1 and IFN-gamma. A subset of the chemotherapeutic agents resulted in profound changes in the number of CD4+ cells, including regulatory T cells, as well as changes in myeloid subpopulations including monocytes, neutrophils, and macrophages. In addition to single agents studies, we carried out combination studies of selected chemo agents with simultaneous addition of anti-PD-L1. Several of these combinations caused pronounced increases in efficacy above single agent treatment. We will present the combination pharmacodynamic marker changes and efficacy of these treatments.

Our findings demonstrate that chemotherapeutic agents can stimulate the tumor immune environment, at least transiently, through multiple mechanisms. Different subsets of chemotherapeutic agents display unique changes in the tumor immune infiltrate, and may require different immunotherapeutic approaches. These findings provide a rationale for testing immunotherapy-chemotherapy combinations in the clinic.

Citation Format: Marcia P. Belvin, Shiuh-Ming Luoh, Jeanne Cheung, Erin McNamara, Rafael Cubas, Jeong Kim. Effects of chemotherapeutic agents on the tumor immune microenvironment. [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 4898.
Abstract 4902: A mouse GITRL fusion protein drives T-cell activation and antitumor activity in preclinical mouse models of cancer
Authors: Leyland Rebecca , Watkins Amanda , Mulgrew Kathy A. , Bamber Lisa , Tigue Natalie J. , Dick Emily , Holoweckyj Nick , Young Lesley , Morrow Michelle , Hammond Scott A. , Leow Ching Ching , Wilkinson Robert W. , Stewart Ross
Published: July 2016

GITR is a member of the TNFR superfamily of proteins and is expressed on resting regulatory T-cells and on other T-cells following activation. Signals through GITR have been shown to drive increased T-cell activity and reduced regulatory T-cell function. In order to explore the potential of therapeutically targeting GITR in a cancer setting, we generated a mouse GITRL fusion protein (mGITRL FP) consisting of the extracellular domain of mGITRL linked to a structural domain and an IgG Fc domain. The antitumor activity and pharmacodynamic effects of this molecule were then explored in the colorectal syngeneic model of cancer (CT26). Treatment of mice with mGITRL FP mediated anti-tumor activity that was dependent on isotype and exposure. The anti-tumor activity could be attributed at least in part to the increased activation and proliferation status of CD8+ and CD4+ T-cells, as evidenced by increases in Ki67 expression and ICOS upregulation. Intratumourally we observed a significant decrease in the frequency of CD4+ T-cells (including T regs), but a corresponding increase in cytotoxic CD8+ T-cells. OX40 is another member of the TNFR superfamily, that has similar expression and functions to GITR. In order to better understand the potential differences between targeting of these two pathways, the activity and pharmacodynamic effects of the mGITRL FP were additionally compared and contrasted to those of a mOX40L FP and the observed differences will be discussed.

Citation Format: Rebecca Leyland, Amanda Watkins, Kathy A. Mulgrew, Lisa Bamber, Natalie J. Tigue, Emily Dick, Nick Holoweckyj, Lesley Young, Michelle Morrow, Scott A. Hammond, Ching Ching Leow, Robert W. Wilkinson, Ross Stewart. A mouse GITRL fusion protein drives T-cell activation and antitumor activity in preclinical mouse models of 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 4902.
Abstract 4928: MiR expression profiles can predict response to systemic treatment in patients with advanced colorectal cancer
Authors: Poel Dennis , Neerincx Maarten , Sie Daoud L.S. , van Grieken Nicole C.T. , Shankaraiah R , van der Wolf F. S.W. , van Waesberghe J. H.T.M , Burggraaf J. D. , Eijk Paul P. , Ylstra Bauke , Verhoef Cees , van de Wiel Mark A. , Verheul Henk M.W. , Buffart Tineke E.
Published: July 2016

Background and aim: Patients with advanced colorectal cancer (mCRC) are commonly treated with systemic treatment consisting of fluoropyrimidine-based regimens being ineffective in 20-25% of the patients. Currently, selection criteria for patients to predict who will respond to this treatment is lacking. The aim of this study is to identify which patients will respond to first line fluoropyrimidine-based treatment based using microRNA (miR) expression profiles in order to avoid ineffective treatment.

Material and methods: Total RNA was isolated from 88 fresh frozen colorectal cancer tissue samples consisting of ≥ 70% tumor cells, collected from patients with mCRC. MiR expression profiles were generated by next generation sequencing using the Illumina High Seq 2000 platform. Of all patients clinical and pathological data, including treatment response based on RECIST criteria, were collected. Class prediction and miR selection were performed using the GRidge package in R. Penalized selection and internal cross validation were used to select miRs predictive for treatment response.

RESULTS: Next generation sequencing resulted in a mean of 10.087.107 (range 6.114.932 to 74.313.067) reads per sample corresponding to 2567 unique mature miR sequences, including 457 novel candidate and 2110 known miRs sequences (miRbase version 19). Penalized regression analysis on tumor specific miRs identified an expression profile which was predictive for clinical benefit (defined as response and stable disease) from first line treatment.

Conclusion: With miR profiling of CRC tissue samples response prediction to first line fluoropyrimidine-based treatment in patients with mCRC is possible. We foresee that selection of treatment using miR expression profiling will avoid unnecessary treatment related toxicity and improve outcome for patients with mCRC

Citation Format: Dennis Poel, Maarten Neerincx, Daoud L.S. Sie, Nicole C.T. van Grieken, R Shankaraiah, F. S.W. van der Wolf, J. H.T.M van Waesberghe, J. D. Burggraaf, Paul P. Eijk, Bauke Ylstra, Cees Verhoef, Mark A. van de Wiel, Henk M.W. Verheul, Tineke E. Buffart. MiR expression profiles can predict response to systemic treatment in patients with advanced colorectal 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 4928.
Abstract 4940: Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas
Authors: Jacobsen Max , Sandusky George , Shah Kavita
Published: July 2016

Pancreatic cancer is the 4th most common cause of cancer deaths in this country. Close to 50,000 deaths occur every year. This is due to the fact that most patients are not diagnosed until presentation at a stage IV. In a recent study, it is estimated that more than 10 years go by between the first genetic mutation and the formation of invasive cancer. More mutations occur in metastatic lesions after an additional 6 years. Both SNAIL and TWIST have gained more attention as biomarkers because that contribute to E-cadherin dysregulation that allows the tumor cells to invade. In this study we evaluated 17 cases of pancreatic ductal adenocarcinoma using twist-1 antibody to evaluate tumor cell expression in these cases. All paraffin blocks were retrieved from the IU pathology department files via an IRB approved protocol. The unstained slides were stained with primary antibody Twist-1 using the DAKO Flex system. Control sections were treated with an isotype control using the same concentration as primary antibody to verify the staining specificity. The Aperio whole slide system was used to image the slides. Computer-assisted morphometric analysis of digital images was carried out using the Aperio Image scope software using the positive pixel algorithm. The algorithm was used to quantify the Twist-1 positivity of normal pancreas, and pancreatic ductal adenocarcinoma. This algorithm is the only FDA approved algorithm for human clinical trials. The immunostaining of Twist-1 was found only in the ductal epithelial carcinoma cells in the pancreas. There was no staining in the stroma in any of the normal pancreas and ductal pancreatic carcinoma cases. We analyzed 5 normal pancreata, and 12 pancreatic ductal adenocarcinomas. The data from the image analysis was 3.38% expression in normal pancreata and 11.44% expression in the pancreatic ductal adenocarcinoma. In summary, the image analysis data demonstrates the overexpression of Twist-1 in stage IV pancreatic adenocarcinomas compared to the normal pancreas. This suggests that this marker may be a predictor of tumor invasiveness and metastasis.

Citation Format: Max Jacobsen, George Sandusky, Kavita Shah. Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas. [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 4940.
Abstract 4941: Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia
Authors: Li Qing , Wang Hao , Zogopoulos George , Shao Qin , Liu Jun-Li , Gao Zu-Hua
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types, with a 5-year survival rate <5%. Tumor specific biomarkers that can aid in early detection and treatment monitoring are urgently needed in order to improve the outcomes. Human Reg family proteins (Reg1A, Reg1B, Reg3A, Reg3G and Reg4) are a group of calcium-dependent (C-type) lectins that promote cell growth and respond to inflammatory stimulants. We examined their associations with pancreatic ductal adenocarcinoma (PDAC). In patients with PDAC, the serum levels of Reg1A and 1B were significantly elevated (p = 0.0003 and 0.0018), which correlated with TNM stages of the patients. By using immunohistochemistry (IHC), the expression of Reg1A, Reg1B and Reg4 was observed in the malignant glandular epithelium of pancreatic intraepithelial neoplasia (PanIN) and invasive PDAC. The intensity of Reg1A and Reg1B staining positively correlated with the histological grades of PanIN. The serum levels and tissue expression of Reg1A and Reg1B were negatively correlated with the differentiation grades of invasive PDAC. Additionally, patchy expression of Reg1A andReg3A/G was also observed in areas with acinar-to-ductal metaplasia (ADM) adjacent to cancer cells. Our data suggests that the changes in Reg proteins were closely associated with the carcinogenesis of PDAC. Reg1A and 1B proteins showed specificity for pancreatic ductal differentiation and could be used as clinical markers for early detection of PDAC, monitoring therapeutic response and predicting tumor recurrence after therapy and tumor metastasis. The expression of Reg family proteins in both ADM and PanIN cells supports the notion that ADM could be an alternative mechanism of PDAC carcinogenesis.

Citation Format: Qing Li, Hao Wang, George Zogopoulos, Qin Shao, Jun-Li Liu, Zu-Hua Gao. Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia. [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 4941.
Abstract 4955: Isolation and characterization of circulating tumor cells (CTCs) in breast and prostate cancer: Comparison of Harpoon CTC assay performance with the CellSearch CTC Test
Authors: Connelly Mark , Chianese David , Morano Carrie , Bui Thai , Powell Shemeeakah , Ngoubilly Noel , Sanders Renouard , Barber Tom , Kapur Ravi , Maheswaran Shyamala , Toner Mehmet , Haber Daniel
Published: July 2016

Circulating tumor cells (CTCs) can provide important information about a patient's prognosis. Currently, the CELLSEARCH® CTC Test (hereafter referred to as CS) is the only US Food and Drug Administration-cleared assay for isolating and enumerating CTCs. This technology uses a CTC antigen-dependent method to isolate CTCs that may limit its ability to detect CTCs with low or no epithelial cell adhesion molecule (EpCAM) expression. We evaluated the Harpoon CTC Isolator and Chip, which uses an antigen-independent, negative depletion method to enrich CTCs from patient blood samples. As a result of the negative depletion method, the final Harpoon Product (ie, CTC-enriched cell suspension) contains CTCs that express cytokeratin (CK) and EpCAM (canonical CTCs) as well as those with low or no expression of these antigens (non-canonical CTCs). After isolation, CTCs were spun onto microscope slides and imaged using a Vectra Multispectral Imaging System. We obtained performance data on an initial prototype Harpoon assay (Harpoon + Vectra [Hrp+Vec]) and compared CTC enumeration between Hrp+Vec and CS using blood samples from 36 breast and prostate cancer patients. From 7.5 mLs of blood, the Hrp+Vec assay recovered 79.0% to 93.5% of canonical EpCAM+/CK+ CTCs recovered by CS. The Hrp+Vec assay also successfully detected non-canonical CTCs (EpCAM-/CK+ or EpCAM+/CK-) in these samples. At the CS clinical cutoff for prognostic value, where ≥5 CTCs is associated with poor prognosis, the Hrp+Vec assay was 97% concordant with CS. Hrp+Vec also detected CTCs in 12.5% (2/16) more patient samples than CS. Finally, a consensus analysis of all Vectra scan results (including both canonical and non-canonical CTCs) demonstrated that the Hrp+Vec assay detected between 90.5% and 108.9% of the number of CTCs as identified by CS. These results indicate that, when compared with CS, the prototype Hrp+Vec assay detected CTCs with similar efficiency and identified more samples as CTC-positive in these breast and prostate cancer patients. Importantly, the Hrp+Vec assay did enrich non-canonical CTCs thought to be missed by CS, and ongoing efforts are aimed to further characterize these cell populations.

Citation Format: Mark Connelly, David Chianese, Carrie Morano, Thai Bui, Shemeeakah Powell, Noel Ngoubilly, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation and characterization of circulating tumor cells (CTCs) in breast and prostate cancer: Comparison of Harpoon CTC assay performance with the CellSearch CTC Test. [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 4955.
Abstract 4972: Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin
Authors: Vaidya Bhavesh , Vulfson Peter , Sanders Renouard , Klein Donna , Goldberg Shalom , Jacobs Steve , Rao Chandra
Published: July 2016

Prostate specific membrane antigen (PSMA), a transmembrane glycoprotein, is a clinically validated marker of prostate cancer. PSMA is overexpressed in primary and metastatic prostate cancer when compared to normal prostate tissue which makes it an attractive target for immunotherapy. Several antibodies are being considered to target PSMA for the development of an anticancer drug. The expression levels of PSMA on tumor cells may play a role in response to treatment with anti PSMA drugs. Therefore, it is important to monitor PSMA expression on tumor cells before and during treatment. We used CellSearch® CTC assay to determine the presence of PSMA on circulating tumor cells (CTCs) using anti PSMA Centyrin as a biomarker. Centyrins are a class of alternative scaffold molecules which are proteins of small size (∼ 10 kDa). They are engineered to bind to target molecules with high specificity and sensitivity. The anti PSMA Centyrin was tested for specificity and sensitivity using tissue cultured tumor cell lines spiked into normal healthy blood and then compared to anti PSMA antibody. The anti PSMA Centyrin showed 100% positivity with the high PSMA expressing cell line (LNCap), 25% positivity with the low PSMA expressing cell line (22Rv1) and 0% positivity with the PSMA negative cell line (PC3-9). The anti PSMA Centyrin was further tested with prostate patient blood samples to determine the expression levels of PSMA on CTCs. The patient samples which contain CTCs are positive for anti PSMA Centyrin and the percentage of CTCs positive for PSMA ranged from 25-100%. Thus, PSMA expression on CTCs can be determined using CellSearch® system and anti PSMA Centyrin as a biomarker in a clinical trial.

Citation Format: Bhavesh Vaidya, Peter Vulfson, Renouard Sanders, Donna Klein, Shalom Goldberg, Steve Jacobs, Chandra Rao. Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin. [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 4972.
Abstract 4974: Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker
Authors: li jitian , Dai Liping , Lei Ningjing , Xing Mengtao , Casiano Carlos A. , Zhang Jianying
Published: July 2016

Prostate specific antigen (PSA) level test in blood has been widely implemented for the early detection and prediction of prostate cancer (PCa). However, lack of specificity led to overdiagnosis, following by unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers are urgently needed to complement PSA by enhancing its sensitivity and specificity in the diagnosis of PCa. Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with TAAs. In this study, we identified and characterized nucleophosmin 1 (NPM1) with 33 kDa molecular weight as an autoantigen by using serological proteomics analysis (SERPA) approach. In the further validation study, it was observed that the level of anti-NPM1 in sera from patients with PCa was significantly higher than that from benign prostatic hyperplasia (BPH) and healthy individuals and receiver operating characteristic (ROC) curve analysis showed high diagnostic value for PCa (area under the curve (AUC):0.805). Interestingly, when considering anti-NPM1 combined with PSA level in sera from PCa patients at the early stage as well as BPH, 100% PCa patients could be identified correctly, while 69.2% patients with BPH who has elevated PSA level were found anti-NPM1 negative. The data suggested that anti-NPM1 can elicit humoral immune response in PCa and might be a potential biomarker for the immunodiagnosis of PCa, and it would be a supplementary of PSA examination to distinguish PCa from BPH.

Key words: prostate cancer; nucleophosmin 1 (NPM1); tumor-associated antigens (TAAs); autoantibodies; serological proteomics analysis (SERPA)

Citation Format: jitian li, Liping Dai, Ningjing Lei, Mengtao Xing, Carlos A. Casiano, Jianying Zhang. Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker. [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 4974.
Abstract 4975: Detection and characterization of circulating and disseminated tumor cells in patients with bladder and prostate cancer
Authors: Verdone James E. , van der Toom Emma E. , Pienta Kenneth J. , Gorin Michael A.
Published: July 2016

Introduction: Increasing evidence suggests that cancer cells display dynamic molecular changes in response to systemic therapy. Circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in bone marrow represent a readily available source of cancer cells with which to measure this dynamic process. To date, many strategies to isolate and characterize these cells have been described. These techniques, however, have been limited by the requirement for positive or negative selection thus resulting in cell losses. Herein we report a method to detect and characterize CTCs and DTCs in matched peripheral blood and bone marrow samples without need for a selection step.

Methods: Following blood or bone marrow aspirate collection, nucleated cells were extracted using a modified density-media centrifugation technique. Nucleated cells were spread on positively charged slides and stained with epithelial markers (Pan-cytokeratin and EpCAM) and a leukocyte marker CD45. High throughput scanning microscopy was performed followed by image processing and subsequent ranking for probability of being a cancer cell. Candidate cancer cells were defined as events which were positive for PanCK and/or EpCAM but negative for CD45. Candidate cells were independently confirmed with high resolution microscopy.

Results: Cytokeratin-positive, EpCAM-positive DTCs and CTCs were readily detectable in patients with clinically-localized bladder and prostate cancer without need for positive or negative selection.

Conclusions: Future work aims to incorporate disease specific makers to confirm that the observed cells truly represent cells of either bladder or prostate origin. This method offers the flexibility to preserve samples for downstream molecular assays including single cell isolation and sequencing. All told, this assay provides a novel means to detect and characterize CTCs and DTCs without positive or negative selection methods.

Citation Format: James E. Verdone, Emma E. van der Toom, Kenneth J. Pienta, Michael A. Gorin. Detection and characterization of circulating and disseminated tumor cells in patients with bladder and 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 4975.
Abstract 4982: BNC105 induces tumor micro-environment changes which enhance the efficacy of checkpoint inhibitor therapy in preclinical models
Authors: Inglis Daniel J. , Beaumont Donna M. , Leske Annabell F. , Scherer Michaela A. , Lavranos Tina C.
Published: July 2016

BNC105 is a Phase II stage drug that specifically targets tubulin causing rapid depolymerisation. The therapeutic window of BNC105 has been shown to be superior compared to similar compounds in its class with highly specific action on tumor vasculature and minimal off target activity. BNC105 shows evidence of strong anti-cancer efficacy in vitro and in animal models not only through destruction of tumour microvasculature but also through strong suppression of cancer cell proliferation. Complementary studies in models of chronic lymphocytic leukaemia have shown that BNC105 also activates pro-apoptotic proteins, which mediate cancer cell death. Recent studies have been conducted to assess changes in the tumour microenvironment that make BNC105 amenable to combination with immune checkpoint inhibitors.

Strong synergy with BNC105 has been seen with anti-PD-1 (97%TGI in combination compared to 40% with BNC105 and 74% with anti-PD-1 as monotherapies) and anti-CTLA-4 (70%TGI in combination compared to 27% with BNC105 and 14% with anti-CTLA-4 as monotherapies) using syngeneic MC38 and CT26 colorectal murine tumor models respectively. We hypothesised that the rapid BNC105 induced tumor microvasculature destruction and onset of tumor hypoxia and necrosis leads to an efflux of tumor antigens being presented to the immune system. This has the potential to elicit an immune response against tumors with a lower mutational load than those that would otherwise activate the immune system using checkpoint inhibitors alone. An increase in tumoral IFNgamma can be associated with the presence of antigen-stimulated lymphocytes within the tumor. IFNgamma levels, CD3+ and CD8+ cells were assessed in tumors treated with BNC105 to elucidate the changes in the recruitment of T cell populations within the tumor microenvironment. A more rapid rate of maturation of dendritic cells upon presentation of antigen is also a potential mechanism associated with changes in the tubulin content of the dendritic cell.

The elucidation of the mechanism whereby the disruption of micro-vasculature caused by BNC105 further validates the complementary functional role of BNC105 in immune activation with checkpoint inhibitors. These findings support the investigation of combining BNC105 with immune checkpoint inhibitors with the view of progressing such combinations to clinical evaluation.

Citation Format: Daniel J. Inglis, Donna M. Beaumont, Annabell F. Leske, Michaela A. Scherer, Tina C. Lavranos. BNC105 induces tumor micro-environment changes which enhance the efficacy of checkpoint inhibitor therapy in preclinical models. [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 4982.
Abstract 4984: Chemotherapy induces a coordinate autoantibody and T-cell response against tumor-associated antigens in pancreatic cancer patients
Authors: Novelli Francesco , Mandili Giorgia , Mazza Emanuela , Bulfamante Sonia Bulfamante , Giordano Daniele , Follia Laura , Cappello Paola
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult cancer to treat, both for lack of effective screening method and for resistance phenomenon. At present surgical resection is the only potentially curative option, but the absence of early symptoms or clinical-pathological markers results in diagnosis at a late, inoperable stage in mostly of cases. Once diagnosed, a number of chemotherapy (CTX), radiation and combination therapy regimens are usually used to treat patients, but responses remain poor. However, chemotherapy is able to enhance tumor immunogenicity. Thus more immunogenic tumor associated antigens (TAA) can be induced by CTX and targeted by passive or active immunotherapy. We have previously identified a number of TAAs that are recognized by autoantibodies in PDAC patients. One of these, alpha-enolase (ENO1), was found to have good antigenic capability and immunization with DNA coding for ENO1 effectively enhanced the survival of genetically engineered mice that develop spontaneously PDAC. To discover TAAs that might be selected for immunotherapy, we have analyzed autoantibody response in 19 PDAC patients’ sera before and after CTX. The reactivity of PDAC patients’ sera obtained was analyzed on 2-dimensional gel electrophoresis proteome map of CF-PAC1 pancreatic cancer cell line and TAAs recognized by IgG autoantibodies were identified by mass spectrometry. The antibody response against 25 proteins was induced or up-regulated after CTX. Among CTX-induced TAA identified, only four, namely ENO1, FUBP1, CK8 and G6PDH were found able to ex novo induce or up-regulate IgG response in more than seven patients were selected and their ability to activate proliferation of autologous PBMC was evaluated following in vitro stimulation with the corresponding recombinant proteins. Notably, mostly of autologous T cells obtained after CXT displayed an increased proliferation in response to at least one or more of the four selected TAA after in vitro stimulation compared to autologous T cells obtained before CTX. Experiments are in progress to validate these CTX- induced TAA in a mouse model of PDAC and to evaluate the therapeutic value of the combination of CTX with the immunotherapy against these TAAs.

Citation Format: Francesco Novelli, Giorgia Mandili, Emanuela Mazza, Sonia Bulfamante Bulfamante, Daniele Giordano, Laura Follia, Paola Cappello. Chemotherapy induces a coordinate autoantibody and T-cell response against tumor-associated antigens in pancreatic cancer patients. [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 4984.
Abstract 4988: Reprogramming the immunologic microenvironment through radiation and Axl targeting
Authors: Aguilera Todd A. , Rafat Marjan R. , Castellini Laura , Kariolis Mihalis S. , vonEbyen Rie , Graves Edward E. , Giaccia Amato J.
Published: July 2016

There is increasing evidence that hypofractionated high dose ionizing radiation (RT) can enhance antitumor immune responses in many cancers. In some cases the combination of RT and checkpoint immunotherapy can enhance antitumor immune responses. Here, we developed a model to study the genetic, microenvironmental, and immunologic factors of immune mediated antitumor responses after radiation. Two different tumor clones from the same parental transgenic PyMT mammary carcinoma model reveal similar growth characteristics but different responses to RT primarily due to an antitumor immune response. The responsive Py117 tumors increase expression of PD-L1 after RT and the antitumor response is enhanced with immune checkpoint antibodies (Ab) targeting PD-1 and CTLA-4. However the unresponsive Py8119 tumors do not respond to radiation or combination immunotherapy. Key differences in the microenvironment include greater infiltration of suppressive macrophages, lower MHCI on tumor cells, and low T cell infiltrates. Upon profiling differences in the tumors we found that Axl a receptor tyrosine kinase of the TAM family associated with invasion and metastasis in a broad range of tumors was overexpressed on the surface of Py8119 cells but not Py117. We genetically knocked out Axl in Py8119 cells using CRISPR/Cas technology and found greater tumor latency and enhanced radiosensitivity in the mouse but not in culture. The effects are largely immune mediated as the RT response was diminished in nude mice. On analysis of the tumor cells we found that MHCI is significantly increased and secretion of a number of chemokines is diminished after Axl targeting. In the tumor microenvironment there was significant decrease in macrophages, increase in myeloid dendritic cells, and resulting influx of T cells by 10 days after RT. These data suggest that Axl may not only mediate invasion and metastasis but can influence immunoserveilance and response to therapy through suppression of antigen presentation and supporting a tumor promoting immune microenvironment through chemokine signaling.

Citation Format: Todd A. Aguilera, Marjan R. Rafat, Laura Castellini, Mihalis S. Kariolis, Rie vonEbyen, Edward E. Graves, Amato J. Giaccia. Reprogramming the immunologic microenvironment through radiation and Axl targeting. [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 4988.
Abstract 4999: Cellular mechanism of action of bispecific T-cell engager (BiTE®) antibody constructs targeting Folate Receptor 1
Authors: Bailis Julie M. , Orf Jessica N. , Sternjak Alexander , Belmontes Brian , Case Ryan , Grinberg Natalia , Chang Wesley , Hinkle Beth , Friedrich Matthias
Published: July 2016

Bispecific T-cell engaging (BiTE®) antibody constructs connect T cells to target-positive cells to induce cytotoxicity. Clinical proof of concept was achieved in B cell malignancies with the anti-CD19 BiTE® Blincyto®, and several BiTE® antibody constructs are currently in development for solid tumor indications. With the goal of developing a BiTE® for ovarian cancer, we focused on targeting the Folate Receptor (FOLR1). FOLR1 mRNA and protein are highly expressed in >80% of ovarian tumor samples. The FOLR1 protein localizes to the cell membrane of ovarian tumor epithelial cells, whereas in normal tissues such as kidney tubules expression is restricted to the apical cell layer. We generated human FOLR1-BITE® antibody constructs that cross react to cynomolgus monkey and here present the preclinical characterization of these molecules. FOLR1-BiTE® antibody constructs had low nanomolar binding affinity for FOLR1 but did not bind to other FOLR family members. The FOLR1-BiTE® antibody constructs recognized diverse epitopes of the FOLR1 protein. In vitro, binding of FOLR1-BiTE® to CD3 on T cells and FOLR1 on tumor cells led to upregulation of CD25, CD69 and GITR on T cells, as well as release of cytokines including TNF, IFNγ, IL-6 and MCP-1. T cell activation by FOLR1-BiTE® was strictly target cell-dependent. Using time lapse imaging, we demonstrated that T cells were recruited to cancer cells expressing FOLR1 to form a cytolytic synapse and induce target cell lysis. Cytotoxicity mediated by FOLR1-BiTE® antibody constructs occurred even at very low target expression levels (1000 receptors per cell), and was not impacted by the presence of soluble receptor at levels found in ovarian cancer patients. FOLR1-BiTE® antibody constructs also mediated redirected lysis of cell lines that were resistant to standard of care chemotherapy. The in vivo activity of the FOLR1-BiTE® was explored in mouse xenograft studies where immunocompromised mice were implanted with mixtures of human T cells and human cancer cell lines. Daily intraperitoneal administration of the BiTE® inhibited tumor growth, even though the serum half-life of the BiTE® was just a few hours.

Citation Format: Julie M. Bailis, Jessica N. Orf, Alexander Sternjak, Brian Belmontes, Ryan Case, Natalia Grinberg, Wesley Chang, Beth Hinkle, Matthias Friedrich. Cellular mechanism of action of bispecific T-cell engager (BiTE®) antibody constructs targeting Folate Receptor 1. [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 4999.
Abstract 50: 2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic cancer
Authors: Zielinski Rafal , Fokt Izabela , Rusin Aleksandra , Skora Stanislaw , Priebe Waldemar
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is one of the most hypoxic and subsequently glycolytic tumors. Thus the inhibition of glycolysis appears to be an attractive therapeutic approach that should be explored.

Methods: A series of 2-deoxy analogs of D-glucose, namely, 2-deoxy-D-glucose (2-DG), 2-fluoro-D-glucose (2-FG), 2-chloro-D-glucose (2-CG), 2-bromo-D-glucose (2-BG), and 2,2-difluoro-D-glucose (2,2-diFG) were synthesized in our laboratory. The ability of 2-deoxy-2-halo-D-glucose derivatives to inhibit glycolysis in pancreatic cancer cell lines was tested using Seahorse 96 analyzer and XF Glycolysis Stress Test kit. The cellular ATP level was measured using CellTiter-Glo assay. Viability of cells cultured in normoxic and hypoxic conditions were assessed using MTS assay.

Results: 2,2-diFG and 2-FG were significantly more potent inhibitors than 2-DG of the Extracellular Acidification Rate (ECAR) of medium in oligomycin-stimulated PDAC cells (complete inhibition at <12.5 mM, whereas, the 100 mM is a commonly used concentration for 2-DG in Seahorse 96 experiments). Interestingly, 2-bromo (2-BG) and 2-chloro (2-CG) analogs have a limited or no effect on glycolysis at tested concentration. Consistently, both, 2,2-diFG and 2-FG, inhibited proliferation of pancreatic cancer cell lines more potently than 2-DG in either normoxic and hypoxic conditions, with the differential effects being significantly more pronounced under hypoxia.

Conclusion: Both, 2,2-diFG and 2-FG have a superior to 2-DG ability to inhibit glycolysis in vitro and should be further evaluated in vivo in the PDAC models as a potential anticancer agents.

Citation Format: Rafal Zielinski, Izabela Fokt, Aleksandra Rusin, Stanislaw Skora, Waldemar Priebe. 2-Deoxy-2-halo-D-glucose derivatives inhibition of glycolysis in pancreatic 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 50.
Abstract 5006: Docetaxel-platinum-fluorouracil (TPF) induction chemotherapy increases PD-L1 positivity and CD8+ / FOXP3+ immune infiltrates in advanced head and neck squamous cell carcinoma (HNSCC)
Authors: Leduc Charlotte , Adam Julien , Louvet Emilie , Bernard Marine , Maingot Elodie , Even Caroline , Olaussen Ken A. , Temam Stéphane , Soria Jean-Charles , Postel-Vinay Sophie
Published: July 2016

Introduction: Checkpoint inhibitors targeting programmed cell death-1/-ligand1 (PD-1/-L1) have demonstrated promising clinical activity in locally advanced head and neck squamous cell carcinoma (LA-HNSCC), with overall response rates (ORR) of ≈20% in unselected patients (pts). Moreover, significant improvements have been observed when selecting pts on PD-L1 positivity, with ORR reaching 50-78%. It is therefore crucial to optimize therapeutic sequence between induction chemotherapy (Docetaxel, Platinum, Fluorouracile,TPF) and anti-PD-1/-L1 in order to maximize the number of pts that benefit from these drugs. Methods: All patients with LA-HNSCC treated at Gustave Roussy between 2006 and 2013 by induction TPF followed by surgery were considered. Only patients for who paired samples (pre and post-TPF) were available were kept for further analysis. PD-L1 expression was quantified by immunochemistry (IHC) (clone E1L3N, Cell Signaling) on a Ventana Discovery Ultra autostainer by a senior pathologist according to a validated protocol. PD-L1 IHC staining on tumor cells (TC) and tumor-infiltrating immune cells (IC) was quantified with a positivity threshold of ≥5%. The density of CD8+ and FOXP3+ lymphocytes was also assessed. Results: Out of 313 pts receiving induction TPF, 86 underwent surgery; paired samples were available for 21. Baseline PD-L1 expression was positive in only 2 and 5 samples for TC and IC respectively. An increased PD-L1 expression was observed post-TPF in 3 (14%) and 15 (71%; p = 0.0003, T-test) samples for TC and IC respectively. Tumor-infiltrating CD8+ and FOXP3+ populations also significantly increased post-TPF. Conclusion: TPF induction in LA-HNSCC increases PD-L1 positivity on tumor-infiltrating immune cells as well as CD8+ and FOXP3+ populations. This suggests that anti-PD-1/-L1 therapy might be more effective as a second-line than first-line strategy, but warrants clinical evaluation. IHC staining resultspre-TPFpost-TPFpaired samplesmedianCImedianCIT-testPD-L1 positive IC (%)2110-10100-300.0003***PD-L1 positive IC (%)2100-2010-200.08 NSCD8+ cell density (n/mm3)171194-77942653-11900.016*FoxP3 cell density (n/mm3)179126-1801774-4670.018*

Citation Format: Charlotte Leduc, Julien Adam, Emilie Louvet, Marine Bernard, Elodie Maingot, Caroline Even, Ken A. Olaussen, Stéphane Temam, Jean-Charles Soria, Sophie Postel-Vinay. Docetaxel-platinum-fluorouracil (TPF) induction chemotherapy increases PD-L1 positivity and CD8+ / FOXP3+ immune infiltrates in advanced head and neck squamous cell carcinoma (HNSCC). [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 5006.