bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1292: Validation study on quantitative analysis method of interrelationships between immune cells and prostate epithelial and cancer cells
Authors: Kramer Gero , Kenner Lukas , Szekeres Thomas , Shariat Shahrokh , Schwarz Sarah
Published: August 2015

Context based image analysis of immunohistochemically stained sections has become a topic after several studies have demonstrated that T-cell infiltration is important for survival and of prognostic value. Software algorithms for CD3 detection, epithelial gland detection and location analysis were validated using CD3 stained prostate biopsies.

Prostate biopsies (n = 96, 38 with and 58 without tumor areas) were stained with anti-CD3-antibody (DAKO) and hematoxylin and were scanned. For validation, epithelial cell (EC) structures were manually annotated in StrataQuest 5.0 (TissueGnostics) using virtual biopsies. Automated enumeration of CD3 positive cells was controlled visually. For image processing, StrataQuests Easy Gland detection algorithm (SQ-GD) was used as provided. Briefly, hematoxylin shade was used for EC nuclei detection. EC structures were segmented using nuclei compactness and area. EC density was used to detect glands. To determine the relationships, distance transform from EC was added as virtual channel. EC proximity for CD3 distribution analysis were grouped into (i) within EC, (ii) 0-20 μm, (iii) 20-40 μm, (iv) 40-60 μm or (v) more from the EC border.

Comparison of automated EC detection by SQ-GD with manually annotated EC showed a significant correlation of both methods (Pearson r = 0.74) although SQ-GD detects significantly more EC. The reason for that was that SQ-GD had to be adjusted so that missing EC areas were below 5%.

CD3+/mm2 In ECCD3+/mm2 in 0 to 20μm EC proximityCD3+/mm2 in 20 to 40μm EC proximityCD3+/mm2 in 40 to 60μm EC proximityCD3+/mm2 in more than 60μm EC proximitymanualautomanualautomanualAutomanualautomanualautoTumor300±230390±213244±131148±126231±131191±138202±134168±151159±128128±184Non tumor416±586430±240270±209147±127243±168180±143240±143171±140172±128124±277Correlation0.8230.7380.7100.8020.815

In conclusion, SQ-GD algorithm detects EC in more than 95% of EC-glands and tumor areas. EC, tumor EC and CD3+ cells detection was highly sensitive to staining artifacts at biopsy borders and to tissue folding, both of which had to be manually excluded. Interestingly, we observed no differences between the biopsies negative for prostate cancer and the positive ones. It is noteworthy that the T-cell numbers gradually decrease in correlation with the distance to the epithelium suggesting a certain immunogenicity of EC.

Note: This abstract was not presented at the meeting.

Citation Format: Gero Kramer, Lukas Kenner, Thomas Szekeres, Shahrokh Shariat, Sarah Schwarz. Validation study on quantitative analysis method of interrelationships between immune cells and prostate epithelial and cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1292. doi:10.1158/1538-7445.AM2015-1292
Abstract 130: PF-06463922, a novel next generation ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models
Authors: Friboulet Luc , Zou Helen , Kodack David P. , Engstrom Lars D. , Li Qiuhua , West Melissa , Tang Ruth W. , Wang Hui , Tsaparikos Konstantinos , Wang Jinwei , Timofeevski Sergei , Dinh Dac M. , Lam Hieu , Lam Justine L. , Yamazaki Shinji , Hu Wenyue , Patel Bhushankumar , Bezwada Divya , Mahmood Sidra , Lifshits Eugene , Affolter Timothy , Lappin Patrick B. , Gukasyan Hovhannes , Lee Nathan , Deng Shibing , Jain Rakesh K. , Johnson Ted W. , Shaw Alice T. , Fantin Valeria R. , Smeal Tod
Published: August 2015

Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology.

For 1st and 2nd generation ALK inhibitors acquired resistance due to ALK kinase domain mutations and/or pharmacological drug resistance are major causes for disease relapse. Here, we report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor with sub to low nanomolar cell potency against ALK fusions and all known clinically-acquired resistant mutations. PF-06463922 exhibited marked cytoreductive activity in tumor xenografts driven by various ALK mutants. Furthermore, PF-06463922 led to significant regression of EML4-ALK driven lung cancer brain metastasis and prolonged mouse survival. Compared to other clinically available ALK inhibitors, PF-06463922 is unique in its superior potency against a broad spectrum of acquired ALK mutations, including the highly resistant G1202R mutant and its robust antitumor activity in the brain. Furthermore, PF-06463922 demonstrated remarkable selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 may be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to ALK secondary mutations and/or brain metastases.

Citation Format: Luc Friboulet, Helen Zou, David P. Kodack, Lars D. Engstrom, Qiuhua Li, Melissa West, Ruth W. Tang, Hui Wang, Konstantinos Tsaparikos, Jinwei Wang, Sergei Timofeevski, Dac M. Dinh, Hieu Lam, Justine L. Lam, Shinji Yamazaki, Wenyue Hu, Bhushankumar Patel, Divya Bezwada, Sidra Mahmood, Eugene Lifshits, Timothy Affolter, Patrick B. Lappin, Hovhannes Gukasyan, Nathan Lee, Shibing Deng, Rakesh K. Jain, Ted W. Johnson, Alice T. Shaw, Valeria R. Fantin, Tod Smeal. PF-06463922, a novel next generation ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2015-130
Abstract 1302: High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials
Authors: Sarikonda Ghanashyam , Ashok Devika , Pahuja Anil , Lameh Jelveh , Tangri Shabnam , Dakappagari Naveen
Published: August 2015

The renaissance of cancer immunotherapies and the positive clinical responses observed with chimeric antigen receptor modified T cells in the hematomalignancy setting has stimulated substantial interest in monitoring immune cell activation and suppression to determine efficacy, prognosis and safety in new agent investigational trials. However, immunophenotyping capacity has been limited and required multi-test tube panels. To address the growing needs of clinical trials, we have developed three fit-for-purpose high complexity (10 or more markers) T-cell phenotyping flow cytometry panels on a qualified LSR FortessaTM platform that enables detection of up to 15 markers in a single test tube. The first panel allows identification of multiple phenotypes along the T-cell differentiation pathway, namely, T-naive (TN), T-stem cell memory (TSCM), T-effector memory (TEM) and T-central memory (TCM) and T-effector memory RA+ (TEMRA). The second panel identifies the most common helper T-cell phenotypes such as Th1, Th2, Treg, and Th17. A third panel determines the functional status of T-cells (e.g., activation vs. suppression) but also enables quantitation of important checkpoint inhibitor expression (e.g., PD-1) on T-cells of interest. These high complexity flow cytometry panels can serve as powerful tools for comprehensive examination of T-cells in a small volume of patient specimen. We believe these new flow cytometry panels will have a substantial impact on the determination of efficacy and safety correlates of immunomodulating agents administered alone or in combination to patients with leukemia.

Citation Format: Ghanashyam Sarikonda, Devika Ashok, Anil Pahuja, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. High complexity flow cytometry panels to monitor target expression, T-cell activation and suppresssion by novel immunotherapies in hematomalignancy clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1302. doi:10.1158/1538-7445.AM2015-1302
Abstract 1305: In vitro analysis of T cells effector vs regulatory function in patients with metastatic melanoma and its correlation to patient outcome
Authors: Hegde Upendra P. , Jha Sidharth S. , Chakraborty Nitya G.
Published: August 2015

Back ground: Metastatic melanoma is an aggressive tumor of the epidermal melanocytes with high mortality rates in patients. The anti- melanoma immune response has been extensively studied while immune based treatment strategies are showing promising activity in melanoma patients. A number of immune characteristics are evaluated as predictive and prognostic biomarker.

Methods: With approval from Institution's regulatory board, we analyzed T cells from peripheral blood of 12 metastatic melanoma patients for baseline frequency of T cells reacting against melanoma related Mart-1 27-35 epitope. We expanded CD 8+ T lymphocytes by exposing them to the Mart-127-35 epitope pulsed on autologous mononuclear cell derived antigen presenting cells ex vivo. Using magnetic bead separation techniques, we also isolated from the peripheral blood CD4+CD25+ (naturally occurring Treg) and CD4+CD25- cells and analyzed in vitro proliferation of CD4+CD25- T cells in response to anti CD3 antibody and PHA and analyzed the effects of CD4+CD25+ cells on their proliferation. Finally, we induced CD4+ T cells into Th1 or Th2 type responses and analyzed their function in vitro.

Results: There was variable degrees of CD8+effector T lymphocytes responses to the antigens in in vitro cultures from all the patients studied and their cytokine profile noted. The percentage of nTreg activity in the PBL averaged about 5% that demonstrated suppression of the CD4+CD25- T cells proliferation in response to anti CD3 antibody in about 50% of patients, but lacked suppression activity in about 50% of the patients. It was possible to generate Th1 type cells from all the patients but generation of the Th2 type cells was limited to 50%. Favorable immune characteristics were observed in majority of the patients who are responding to the treatment.

Conclusion: Our findings suggest that in vitro functional T cell immune characteristics could be examined in patients diagnosed with metastatic melanoma. Examination of these characteristics in larger number of patients diagnosed with melanoma might provide predictive marker for immune intervention.

Citation Format: Upendra P. Hegde, Sidharth S. Jha, Nitya G. Chakraborty. In vitro analysis of T cells effector vs regulatory function in patients with metastatic melanoma and its correlation to patient outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2015-1305
Abstract 1309: Fitness of T cells for use in cellular therapy of pediatric malignancies
Authors: Singh Nathan , Hulitt Jessica , Grupp Stephan A. , Barrett David M.
Published: August 2015

Despite promising clinical trial results, many patients are ineligible to receive cellular therapy, either due to an inability to harvest sufficient lymphocytes, or that the harvested cells fail to yield a clinically viable product. The ideal time to harvest these cells remains unclear, and is of significant clinical relevance.

We collected blood from pediatric patients with acute lymphoblastic leukemia (ALL) or non-Hodgkin lymphomas (NHL) at time of diagnosis and after each cycle of chemotherapy. We plated these samples overnight to deplete adherent cells, quantified the CD3+ T cell population by flow cytometry, combined these T cells with CD3/CD28 stimulatory beads at a ratio of 3 beads/cell, and measured expansion over time. These studies (Table 1) revealed that ALL samples harvested earlier in therapeutic course demonstrated enhanced ex vivo expansion. We stratified our samples into standard (SR) and high-risk (HR+VHR) disease and found that SR patients have even more robust early expansion and that this expansion ability is maintained, while high-risk samples lose ex vivo expansion ability after consolidation. Broadening this analysis to lymphoma revealed that only a fraction of NHL diagnosis samples expand ex vivo, and nearly no expansion is observed after initiation of chemotherapy.

We also examined T cell phenotype from our ALL samples, and found a correlation between expansion capability and presence of naïve T cells, and that larger terminal effector pools correlate with poor expansion.

These data represent the most detailed analysis of T cell number, function and phenotype during chemotherapy for pediatric B cell malignancies to date. They suggest that T cell fitness is considerably affected by type of chemotherapy as well as underlying malignancy, and that fitness may correlate with phenotype. These findings have direct clinical relevance, suggesting that T cells should be harvested as early as possible after diagnosis. This practice has the potential to significantly expand the pool of patients eligible for cellular therapy.

Table 1. Percentage of samples with >5-fold ex vivo expansion

ALL (total)ALL (SR)ALL (HR+VHR)NHLDiagnosis80907025*Post induction82768412*Post consolidation668052*0*Post interim maintenance647846*0*Post delayed intensification618534*0*Maintenance37500*0**p<0.01 compared to ALL (SR)

Citation Format: Nathan Singh, Jessica Hulitt, Stephan A. Grupp, David M. Barrett. Fitness of T cells for use in cellular therapy of pediatric malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1309. doi:10.1158/1538-7445.AM2015-1309
Abstract 1318: A protein microarray signature of autoantibody biomarkers for the detection of HER2+ breast cancers
Authors: Katchman Benjamin A. , Alam Rizwan , Field Matthew S. , Viloria Jennifer , Wallstrom Garrick , LaBaer Joshua , Engstrom Paul , Anderson Karen S.
Published: August 2015

Introduction:

Breast cancer is a heterogeneous disease, clinically defined by expression of the biomarkers ER, PR, and HER2, with 15-30% of breast cancers overexpressing the HER2 protein. Antibodies to tumor antigens that are present in patient sera are potential biomarkers for early detection and prognosis of breast cancer. The purpose of this study is to identify a panel of novel serum biomarkers for the early diagnosis of HER2+ breast cancer.

Methods:

We screened sera from breast cancer patients for autoantibodies using custom nucleic acid programmable protein microarrays (NAPPA). Plasmid cDNAs encoding 10,000 full-length human proteins with c-terminal GST or FLAG tags were printed on arrays with anti-GST or anti-FLAG antibodies, and transcribed and translated in situ using HeLa expression lysate. Serum samples were obtained at diagnosis from patients with stage I-III HER2+ breast cancer classified by IHC and/or FISH testing (HER2+/ER-/PR-, 47%; HER2+/ER+/PR+, 48%; HER2+/ER-/PR+, 5%). Control sera were age-matched and obtained from women undergoing screening mammography or diagnostic mammography for benign breast disease. The microarrays were screened with sera from 29 cases and 29 controls. 850 antigens were selected, displayed in microarray format, and screened with an independent set of sera 27 cases, 29 benign breast disease controls, and 25 healthy controls. Bound IgG was detected by fluorescence.

Results:

From the initial 10,000 proteins, we identified 850 proteins using cutoff values of AUC (0.68), sensitivity (0.29), and pAUC (0.0075) at 94% specificity for further analysis. From these, 50 candidate autoantibody biomarkers for the detection of HER2+ breast cancer were identified based on visual analysis and statistical cutoff values of 15% sensitivity at 98% specificity and K-statistic (≥ 0.9).

Conclusion:

We identified a 50-autoantibody serum signature for the detection of HER2+ breast cancer. Blinded validation studies are ongoing.

Citation Format: Benjamin A. Katchman, Rizwan Alam, Matthew S. Field, Jennifer Viloria, Garrick Wallstrom, Joshua LaBaer, Paul Engstrom, Karen S. Anderson. A protein microarray signature of autoantibody biomarkers for the detection of HER2+ breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2015-1318
Abstract 1325: Evaluation of immuno-oncology related treatment in syngenic and human reconstituted immune systems
Authors: Hillairet de Boisferon Marc , Bichat Francis , Mignard Caroline , Tizon Xavier , France Damien , Mirjolet Jean-François
Published: August 2015

Immunotherapy based on monoclonal antibodies targeting cancer cells is now developed as a valid approach to treat cancer. Modulation of novel immune checkpoints and other targets are highly promising approaches against cancer and many other diseases. They have the potential to activate the immune system and to establish an active defense against pathological conditions. In addition, antibodies, antibody fragments, and other biologics can also have a strong impact on the immune system which needs to be evaluated early on. In order to develop and accurately evaluate these immunology linked approaches, appropriate preclinical models with relevant immunological readouts are needed at different stages of therapy development. Ideally, methods should be available that allow predictive readouts in vivo and ex vivo.

A comprehensive panel of tools was constructed and validated aiming at evaluating the modulation of the immune system by new therapies.

In immunocompetent mice, immune cells were studied for the detection of their cell surface markers, induction of proliferative phenotype, antigen-specific T lymphocyte detection, secretion of soluble mediators using FACS phenotyping, cytometric bead assay (CBA) or Luminex multiplex technologies and ELISPOT. We report on a panel of syngenic tumor models (4T1, B16-F10, CT26, EMT6, AY27, LLC1, MBT-2 and Renca) our capacity to correlate subpopulation of immune infiltrating cells (as T cells, macrophages, NK cells, granulocytic and monocytic cells) and the therapeutic effects of critical antibodies directed against Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death 1 membrane protein (PD-1).

In immunodeficient mice, the reconstitution of the mature human immune system with PBMC or naïve human immune system using hematopoietic stem cells was used to evaluate the modulation of those immune cells by therapies through human cytokine release and onset of graft versus host disease.

All of these tools were used in the context either of rodent syngenic models or humanized mouse models. In addition to their use in immune system modulating anti-cancer therapies, these humanized or syngenic models also have a potential application in many other therapeutic areas such as autoimmune diseases, inflammation, anti-infectives.

Citation Format: Marc Hillairet de Boisferon, Francis Bichat, Caroline Mignard, Xavier Tizon, Damien France, Jean-François Mirjolet. Evaluation of immuno-oncology related treatment in syngenic and human reconstituted immune systems. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1325. doi:10.1158/1538-7445.AM2015-1325
Abstract 1329: Identification of neoantigens recognized by autoantibodies in chemotherapy-treated pancreatic cancer patients
Authors: Mandili Giorgia , Mazza Emanuela , Capello Michela , Giordano Daniele , Cappello Paola , Novelli Francesco
Published: August 2015

Pancreatic ductal adenocarcinoma (PDA) 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, 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 neo-antigens can be induced by chemotherapy and targeted by passive or active immunotherapy. We have previously identified a number of Tumor Associated Antigens (TAA) that are recognized by autoantibodies in PDAC patients. One of these, alpha-enolase, was found to have good antigen capability and immunization with DNA coding for alpha-enolase effectively enhanced the survival of genetically engineered mice that develop spontaneously PDAC. To discover neo-antigens that might be selected for immunotherapy, we have analysed antibody response in PDAC patients’ sera before and after chemotherapy. The reactivity of PDAC patients’ sera obtained before and after two rounds of chemotherapy was analysed on 2-dimensional gel electrophoresis proteome map of CF-PAC pancreatic cancer cell line and the antigens recognized by autoantibodies were identified by mass spectrometry. A number of antigens, mainly metabolic enzymes, proteins involved in transcription and structural proteins, were recognized by autoantibody only after one or two rounds of chemotherapy. The identification of common and individual antigens from chemotherapy treated-patients formed the platform for ongoing immunological studies aimed to assess the ability of these neo-antigens, compared to that identified before chemotherapy treatment, to induce specific helper and cytotoxic response to PDAC. A stronger and sustained immune response correlated to survival is expected to be induced by neo-antigens. Validation of these neo-antigens will allow to develop specific immunotherapy combined with chemotherapy.

Citation Format: Giorgia Mandili, Emanuela Mazza, Michela Capello, Daniele Giordano, Paola Cappello, Francesco Novelli. Identification of neoantigens recognized by autoantibodies in chemotherapy-treated pancreatic cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1329. doi:10.1158/1538-7445.AM2015-1329
Abstract 1331: Targeting CD47 triggers macrophage-dependent brain tumor destruction in a clinical relevant model
Authors: Zhu Huaiyang , Yang Ning , Leiss Lina , Rygh Cecilie B , Mitra Siddhartha S , Cheshier Samuel H , Huang Bin , Bjerkvig Rolf , Enger Per Ø , Weissman Irving L , Li Xingang , Wang Jian
Published: August 2015

Glioblastoma multiform (GMB), as a major unsolved clinical challenge, remains among the most aggressive malignancies in human brain with poor prognosis around 10-14.6 months survival. Due to their infiltrative character in the limited space, it is very difficult to be completely resected surgically. Although radiotherapy and chemotherapy that were given to patients after surgery may improve overall survival, the treatment outcomes in general are poor. The tumors eventually recur. Thus, new therapeutic strategy for GBM is urgently needed. It has been shown that CD47, a tumor cell surface marker, through binding its receptor SIRPα on macrophages plays as “don't eat me signal” and thus, by blocking the interaction of CD47 with SIRPα could lead to tumor destruction. So, we developed a standardized protocol for a novel xenograft surgical resection model which mimicked the process of operation in clinic. We hypothesized that the mechanical surgical procedure of tumor resection may create an inflammatory tumor environment, leading to recruitment of immune cells including macrophages. We believed the presence of these macrophages was needed to mediate the anti-tumor effects of the CD47 antibody which we administered postoperatively. The aims of the project are: 1) Translating brain tumour immunotherapy from the lab bench via preclinical studies to the clinic; 2) Establishing a novel xenograft surgical resection model to mimic the clinical setting of tumour recurrence in patients. Specifically, we will: 1) In vitro quantify and functionally characterize tumour-associated macrophages from treated vs. untreated tumours; 2) Using our clinical relevant animal models we want to determine the efficacy of targeting CD47 therapy.

In this study, we have developed a standardized protocol for a novel xenograft surgical resection model to parallel the treatment seen in glioma patients. The model accounts for the therapeutic benefit of surgical resection in pre-clinical models and demonstrates that recurrent tumors show higher proliferation and angiogenesis comparing to non-resected tumors. Moreover, the resection site is highly infiltrated by both CD68 and pSTAT1 positive macrophages. CD47 antibody treatment group has longer survival time than other groups.

Citation Format: Huaiyang Zhu, Ning Yang, Lina Leiss, Cecilie B Rygh, Siddhartha S Mitra, Samuel H Cheshier, Bin Huang, Rolf Bjerkvig, Per Ø Enger, Irving L Weissman, Xingang Li, Jian Wang. Targeting CD47 triggers macrophage-dependent brain tumor destruction in a clinical relevant model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1331. doi:10.1158/1538-7445.AM2015-1331
Abstract 1340: Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2
Authors: Xia Yang , Tao Huimin , Hu Yangyang , Zhou Li , Bao Yangyi , Lundy Steven K , Huang Shiang , Li Qiao , Chang Alfred E
Published: August 2015

In cancer immunotherapy, effector B cells can inhibit spontaneous tumor metastases as well as local tumor growth in animal models. We have previously reported that antitumor effector B cells generated from tumor-draining lymph node (TDLN) could directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. The number of TDLN B cells increased following transfer. As a result, adoptive transfer of TDLN B cells conferred tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer, 4T1. Removal of IL-10, either by using IL-10-/- TDLN B cells or by systemic neutralization of IL-10 using anti-IL-10 antibody, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. In recent studies, we found that tumor cell death mediated by effector B cells involves the CXCR4/CXCL12 as well as Fas/FasL interactions. Blockade of CXCR4 and FasL using a CXCR4-specific inhibitor, AMD3100, and anti-FasL antibody, respectively, reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Of note, blockade of CXCR4 and FasL concurrently inhibited B cell-mediated direct killing of tumor cells in an additive fashion, suggesting that both CXCL12/CXCR4 and Fas/FasL pathways are involved in the direct killing of 4T1 tumor cells by 4T1 TDLN B cells. In other experiments, we found that TDLN B cells express IL-2 receptors (IL-2R) and the ability of the TDLN B cells to inhibit metastasis was significantly enhanced with exogenous IL-2 administration. Besides our previously reported Fas/FasL pathway and IL-10-mediated suppression, our recently identified CXCL12/CXCR4 pathway and IL-2-mediated augmentation of the biological function of effector B cells suggests these B cells may represent an important component of the host immune response against tumor.

Citation Format: Yang Xia, Huimin Tao, Yangyang Hu, Li Zhou, Yangyi Bao, Steven K Lundy, Shiang Huang, Qiao Li, Alfred E Chang. Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1340. doi:10.1158/1538-7445.AM2015-1340
Abstract 1350: The combination of a murine iNKT cell-activating antibody and IL-12 exhibits antitumor activity in a mouse melanoma model
Authors: Yuan Weiming , Wen Xiangshu
Published: August 2015

Invariant natural killer T (iNKT) cells are innate-like T cells with an invariant iTCR with potent anti-tumor activity. Current anti-tumor clinical trials using the iNKT cell ligand, a-galactosylceramide (a-GalCer) have had limited success. One major limitation of a-GalCer-based therapies is the bioavailability of lipid drugs and current therapies require isolation and a-GalCer loading of dendritic cells prior to administration (Richter et al Blood 121:423, 2013). We have engineered a murine iNKT TCR-targeting monoclonal antibody (NKT14m) that activates iNKT cells directly without dendritic cells or glycolipids. We have evaluated the in vivo anti-tumor efficacy with the established B16 mouse melanoma model. Based on preliminary results that the potency of NKT14m is less than that of a-GalCer, we reasoned that the addition of IL-12 stimulation would be beneficial for iNKT cell activation (Yue et al PNAS 102:11811, 2005). Currently, the toxicity of IL-12 has restricted its use in clinical trials. We evaluated a non-toxic dose of IL-12 alone and in combination with NKT14m. C57BL/6 mice were injected with 400K B16F10 melanoma cells on Day 0. On Day 2 they were divided into untreated, IL-12 treated (0.1 ug/mouse/day for 12 days), NKT14m treated (100 ug/mouse once on Day 2) and the combination of NKT14m+IL-12 using the same dose regimens. On Day 14 mice were sacrificed and lung tumors counted. We detected a synergistic effect in the combination of NKT14m+IL-12 (Table 1). These findings represent a new paradigm of iNKT cell activation for their anti-tumor function.

Table 1 - Lung Tumors in Treatment GroupsExperimental GroupsControlIL-12NKT14mIL-12+NKT14mNumbers of metastatic tumors150+/-67117+/-2555+/-2923+/-10

Citation Format: Weiming Yuan, Xiangshu Wen. The combination of a murine iNKT cell-activating antibody and IL-12 exhibits antitumor activity in a mouse melanoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1350. doi:10.1158/1538-7445.AM2015-1350
Abstract 1366: Mdm2 antagonist RG7388 does not interfere with tumor angiogenesis in vitro and in vivo
Authors: Lehmann Christian , Friess Thomas , Lorenzon Erica , Herting Frank , Dangl Markus
Published: August 2015

MDM2 interacts with the transcription factor p53 via its N-terminal domain and is the most prominent and best described natural inhibitor of the p53 tumor suppressor pathway. Apart from this very well investigated function, MDM2 has been reported to be involved in angiogenesis via various mechanisms. Published data indicate that MDM2 interacts directly with and activates HIF1a but can also display HIF1a independent effects such as VEGF mRNA stabilization or regulation of ERK1/2 signaling. Taken together, these reports suggest that MDM2 antagonists might have the potential to inhibit tumor-angiogenesis or age-related macular degeneration.

In order to test the potential anti-angiogenic activity of interfering with MDM2-p53 binding, we utilized the highly selective, clinical stage MDM2 antagonist RG7388. First, it was tested if RG7388 influences the induction of response to hypoxic conditions using a HRE-reporter cell line, followed by analyzing the interference of RG7388 with human endothelial cell tubule formation. The in-vitro results showed no statistically significant inhibition of HIF1a facilitated GFP reporter activation nor any effect on HUVEC tubule formation, viability or proliferation. In a mouse cornea pocket assay in-vivo, systemic administration of a MDM2 antagonist did not prevent VEGF-A or FGF-2 induced vessel formation. Finally, RG7388 and vehicle treated SJSA xenograft tumors were explanted and analyzed in detail for changes in vessel architecture. No effects on microvascular density or vessel structure were observed. Taken together, these data indicate that RG7388, a highly selective MDM2 antagonist that binds to the p53-binding pocket of MDM2 and prevents interaction of MDM2 with p53, is not suitable for anti-angiogenic treatment. One possible explanation for the lack of anti-angiogenic activity is the increased potency/specificity of RG7388 resulting in higher selectivity and fewer off target effects compared to Nutlin-3a.

Citation Format: Christian Lehmann, Thomas Friess, Erica Lorenzon, Frank Herting, Markus Dangl. Mdm2 antagonist RG7388 does not interfere with tumor angiogenesis in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1366. doi:10.1158/1538-7445.AM2015-1366
Abstract 1370: Activin receptor-like kinase 1 ligand trap reduces microvascular density and improves chemotherapy efficiency to various solid tumors
Authors: Hawinkels Lukas JAC , Garcia de Vinuesa Amaya , Paauwe Madelon , Kruithof-de Julio Marianna , Heijkants Renier , Goumans Marie-Jose , ten Hagen Timo , ten Dijke Peter
Published: August 2015

Anti-angiogenic therapy, mostly targeting vascular endothelial growth factor (VEGF), has been clinical applied in cancer patients for the last decade. However, often resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new anti-angiogenic strategies are needed. In this study we analyzed the potential anti-angiogenic effects of activin-receptor-like kinase (ALK)1-Fc protein. ALK1-Fc sequesters bone morphogenetic protein (BMP)-9 and -10 and prevents binding of these ligands to endothelial ALK1, which controls angiogenesis. Treatment of mice with breast cancer, head and neck cancer, or melanomas strongly decreased the tumors’ microvascular density, but this effect was not accompanied by a reduction in tumor volume. In contrast to anti-VEGF therapy, we observed decreased hypoxia, and increased staining for pericytes, consistent with the notion that ALK1-Fc may have induced a normalization of the remaining tumor vessels. Next we found that combined regimen of ALK1-Fc with chemotherapy inhibited tumor growth in the breast- and head and neck cancer models more efficiently than chemotherapy alone. Our results provide strong rational to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase-II clinical trials with ALK1-Fc.

Citation Format: Lukas JAC Hawinkels, Amaya Garcia de Vinuesa, Madelon Paauwe, Marianna Kruithof-de Julio, Renier Heijkants, Marie-Jose Goumans, Timo ten Hagen, Peter ten Dijke. Activin receptor-like kinase 1 ligand trap reduces microvascular density and improves chemotherapy efficiency to various solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1370. doi:10.1158/1538-7445.AM2015-1370
Abstract 1372: Acquired resistance to sunitinib is not associated with functional re-vascularization in 786-O renal cell carcinoma xenografts
Authors: Robinson Simon P.
Published: August 2015

Anti-angiogenic therapy has shown considerable efficacy in metastatic renal cell carcinoma (mRCC). However, the ability of drugs such as sunitinib to delay tumour progression and extend survival is limited, due to both innate and evasive drug resistance, thought to arise through alternative, VEGF-independent mechanisms of tumour re-vascularization. Furthermore, there are no validated biomarkers that predict which mRCC patients will benefit from anti-angiogenic therapy. In investigating the response of 786-O RCC xenografts to sunitinib, a model of acquired resistance has been established with chronic treatment. The aims were to evaluate fractional blood volume (fBV) derived from susceptibility contrast MRI with ultrasmall superparamagnetic iron oxide (USPIO) particles as a non-invasive imaging biomarker of response, and interrogate the vascular phenotype of 786-O xenografts exhibiting acquired resistance to sunitinib.

Mice bearing established xenografts underwent MRI prior to and two weeks after daily treatment with 40mg/kg sunitinib. USPIO particle uptake was reduced in all treated tumours, resulting in a highly significant reduction in fBV in the absence of any change in tumour volume (Table 1). This response was associated with a significant reduction in the uptake of the perfusion marker Hoechst 33342. The average pre-treatment tumour fBV was significantly positively correlated (R2 = 0.92, p<0.0001) with sunitinib-induced changes in tumour fBV across the cohort.

Additional tumour-bearing mice exhibiting acquired resistance to sunitinib (designated 786-O-R) were imaged when their tumours reached at least 4x their volume at commencement of treatment. Resistant 786-O- R xenografts revealed a suppressed fBV whilst still being treated with sunitinib.

Pre-treatment tumour fBV is a predictive biomarker of subsequent response to sunitinib in RCC. Acquired resistance to sunitinib is not associated with functional re-vascularization in 786-O RCC xenografts.

Table 1

786-O (n = 9)786-O-R (n = 4)PrePostPostTumour volume (mm3)153 ± 17148 ± 24471 ± 63fBV (%)8.3 ± 22.4 ± 0.5**2.1 ± 0.5fBV / Tumour volume6.8 ± 22.5 ± 1*0.5 ± 0.1Hoechst 33342 uptake (%)13.5 ± 56.4 ± 1*n.a.

Citation Format: Simon P. Robinson. Acquired resistance to sunitinib is not associated with functional re-vascularization in 786-O renal cell carcinoma xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1372. doi:10.1158/1538-7445.AM2015-1372
Abstract 1383: Phenotype-based development of novel therapies for the treatment of colorectal cancer
Authors: Butler Clare T. , Murphy Adrian , Gallagher William M. , O'Sullivan Jacintha , Kennedy Breandán
Published: August 2015

Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Treatment of late stage CRC using Bevacizumab®, a targeted anti-VEGF, produces insufficient patient response rates and acquired tumour resistance. There is a clinical need for development of improved treatment options.

Materials and Methods: Previously, anti-angiogenic screens in Tgfli1:EGFP zebrafish identified Quininib, a small molecule drug as effective as Bevacizumab®at reducing tumour volume in vivo and which reduces tumour CD31 expression in xenografted mice. Here, structural analogues of Quininib were synthesised to identify novel chemical entities with higher anti-angiogenic and anti-inflammatory efficacy. Analogues were examined for anti-angiogenic effects in the zebrafish intersegmental-vessel assay. Hits were progressed for safety analysis in cytotoxic assays, anti-angiogenic efficacy in aortic explants, and effects on cell proliferation and tubule formation using HT29_Luc2 and HMEC cells. Leads were then tested in ex vivo human CRC tumour explants to determine effects on angiogenic and inflammatory factor secretion. A CRC-specific murine xeongraft model was created by subcutaneous injection of HT29_Luc2 cells for anti-tumorigenic analysis of the Quininib analogues.

Results and Discussion: Five out of 12 drug candidates (CTB_1, CTB_2, WP_1, WP_2 and WP_11) reduced developmental angiogenesis in fli1:EGFP zebrafish significantly more than Quininib (98% vs 67%, ***P<0.01). These analogues were well-tolerated in human endothelial and CRC cell cytotoxic assays and were more effective than Quininib at reducing tubule formation and angiogenic factor secretion in HMEC cells. Two analogues reduced aortic ring vessel sprouting equally or more effectively than Quininib. Preliminary data suggests that certain analogues can reduce inflammatory and angiogenic factor secretion from ex vivo human CRC explants more so than Quininib. We successfully established a CRC-specific murine tumour xenograft model. Using a ranking efficacy table, we chose analogues to analyse their anti-tumorigenic and anti-metastatic effects, by i/p injection in an optimised model of HT29_Luc2-CRC tumourigenesis in Balb/c nude mice.

Conclusion: We identified 5 analogues which are significantly more effective than Quininib within in vitro and ex vivo models of inflammation and angiogenesis. Our highest ranking analogue will be tested in murine xenografts to determine effects on tumour burden and metastasis.

Citation Format: Clare T. Butler, Adrian Murphy, William M. Gallagher, Jacintha O'Sullivan, Breandán Kennedy. Phenotype-based development of novel therapies for the treatment of colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1383. doi:10.1158/1538-7445.AM2015-1383
Abstract 1388A: Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system
Authors: Kokatam Srinivasan , Tiwari Kanchan , Schroeder Jenny , Toell Andrea , Hussain Lubna , Kapoor Preeti
Published: August 2015

Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning's Matrigel product.

The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology.

In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™-2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ vector, and it was shown that the Nucleofection™ process had no deleterious effect on the tube formation potential of HUVEC on Corning's Matrigel product.

Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning's Growth Factor Reduced Matrigel product in comparison to control samples.

Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment.

Citation Format: Srinivasan Kokatam, Kanchan Tiwari, Jenny Schroeder, Andrea Toell, Lubna Hussain, Preeti Kapoor. Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388A. doi:10.1158/1538-7445.AM2015-1388A
Abstract 139: Activating ERBB4 mutations in non-small cell lung cancer
Authors: Kurppa Kari J. , Denessiouk Konstantin , Johnson Mark S. , Elenius Klaus
Published: August 2015

Genes encoding the ErbB receptor tyrosine kinases (EGFR/ERBB1, ERBB2, ERBB3, and ERBB4) are key regulators of cellular proliferation, survival, and differentiation, and thus represent potent proto-oncogenes. In particular, mutations or copy number variations of EGFR or ERBB2 are present in human malignancies and serve as predictive markers for targeted therapies. Recent efforts to comprehensively characterize the mutational landscape of human cancers have identified frequent somatic mutations in ERBB4 in various cancer types, such as non-small cell lung cancer (NSCLC), melanoma, and colorectal cancer. However, the significance of mutated ERBB4 in cancer remains elusive.

Here, we have functionally characterized nine ERBB4 mutations previously identified in lung adenocarcinoma. Four out of the nine mutations, Y285C, D595V, D931Y and K935I, were found to be activating, increasing both basal and ligand-induced ErbB4 phosphorylation. According to structural analysis, the four activating mutations were located at critical positions at the dimerization interfaces of the ErbB4 extracellular (Y285C, D595V) and kinase (D931Y and K935I) domains. Consistently, the mutations enhanced ErbB4 dimerization and increased the trans activation in ErbB4 homodimers and ErbB4/ErbB2 heterodimers. The expression of the activating ERBB4 mutants promoted survival of NIH 3T3 cells in the absence of serum. Interestingly, serum starvation of NIH 3T3 cells expressing the ERBB4 mutants only moderately increased the phosphorylation of canonical ErbB signaling pathway effectors Erk1/2 and Akt as compared to wild-type ERBB4. In contrast, the mutations clearly enhanced the proteolytic release of signaling-competent ErbB4 intracellular domain.

These results suggest the presence of activating, oncogenic mutations of ERBB4 in non-small cell lung cancer.

Citation Format: Kari J. Kurppa, Konstantin Denessiouk, Mark S. Johnson, Klaus Elenius. Activating ERBB4 mutations in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 139. doi:10.1158/1538-7445.AM2015-139