bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1650: Targeting CD47- SIRPα interaction by novel peptide-based antagonists
Authors: Sasikumar Pottayil G. , Gundala Chennakrishnareddy , Gowda Nagaraj M. , Naremaddepalli Sudarshan S. , Bhumireddy Archana , Nair Rashmi , Balasubramanian Wesley Roy , Lakshminarasimhan Anirudha , Dodheri Samiulla S. , Aithal Kiran , Ramachandra Raghuveer K. , Daginakatte Girish , Ramachandra Murali
Published: July 2017

Background: Cluster of differentiation (CD47) is a trans-membrane glycosylated protein which is upregulated in several cancers. Increased expression of CD47 on tumor cells is associated with immune evasion and cancer progression. CD47 through its interaction with signal regulatory protein alpha (SIRPα), a cell-surface molecule on macrophages inhibits phagocytosis of tumor cells. Disrupting CD47-SIRPα interactions by monoclonal antibodies targeting CD47 and recombinant SIRPα proteins have been used as therapeutic strategies for treating cancer. Our objective was to discover and develop peptide/peptidomimetic based CD47 antagonists for disrupting CD47-SIRPα interactions.

Methods: Through rational design based on crystal structure of CD47/SIRPα interacting interface, we designed peptides having potential to disrupt CD47-SIRPα interactions. FACS based cellular binding assay was developed to assess the binding affinity of CD47 antagonists. SIRPα protein labelled with fluorescent dye was incubated with Jurkat T cells expressing high levels of CD47 in the presence/absence of peptides. Binding affinity was measured by decrease in fluorescence. Functional activity of the peptides was evaluated in a FACS-based phagocytosis assays, in which tumor cells were incubated with human/mouse macrophages in the presence/absence of CD47 antagonists.

Results: We identified CD47 antagonists demonstrating disruption of CD47-SIRPα interaction in a cellular binding assay. These peptides significantly inhibited phagocytosis of different tumor cells by macrophages. The lead CD47 antagonist displaying good ADME properties including moderate oral bioavailability was evaluated in a B16F10 syngeneic mouse tumor model. The lead CD47 antagonist inhibited primary tumor growth as well tumor metastasis to lungs. Biomarker characterization and efficacy studies in additional tumor models are ongoing.

Citation Format: Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Nagaraj M. Gowda, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Rashmi Nair, Wesley Roy Balasubramanian, Anirudha Lakshminarasimhan, Samiulla S. Dodheri, Kiran Aithal, Raghuveer K. Ramachandra, Girish Daginakatte, Murali Ramachandra. Targeting CD47- SIRPα interaction by novel peptide-based antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1650. doi:10.1158/1538-7445.AM2017-1650
Abstract 1653: Modeling tumor immuno-dynamics to predict patient survival and immunotherapy efficacy
Authors: Akers Nicholas K. , Schadt Eric E. , Losic Bojan
Published: July 2017

State of the art models of cancer survival are increasingly utilizing molecular data that feature the number of non-synonymous mutations, or a network based stratification of mutations, as significant predictors. It is widely hypothesized that this association between mutations and survival is a result of neo-epitopes that induce a robust anti-tumor immune response. Naively this implies that patient neo-epitope burden should better predict survival than that of mutations alone, however this has generally been difficult to prove. Indeed, mutations and predicted neo-epitopes explain roughly the same survival variance, likely due to the intertwined difficulties of neo-epitope prioritization and the often immuno-suppressive tumor microenvironment.

Reasoning that integrating leading order immune response with novel neo-epitope prioritization will lead to superior performance, we built a statistical model to quantify the influence of tumor immune-dynamics on patient survival. Neoepitopes (MHC-I/II) were predicted from mutations and filtered on a self-ligandome. The clonal structure of these mutations and the distribution of resulting epitopes were assessed as predictive features. Additionally, the existing response to epitopes was assayed by incorporating TCR and BCR sequence counts and entropy as a proxy for clonal tumor infiltrating lymphocyte expansion.

Leveraging the cancer genome atlas (TCGA) for 9 major cancer types including bladder (BLCA), breast (BRCA), colorectal (COAD/READ), glioblastoma (GBM), liver (LIHC), lung (NSCLC), melanoma (SKCM), pancreatic (PAAD), and uterine (UCEC) cancer, we obtained omics data for a total of 2,866 patients. Random forest based recursive feature elimination was used to determine which features were most likely to be predictive while overfitting was controlled with k-fold cross validation. A cox proportional hazard model of survival as a function of mutation burden and functions of neoepitope and immune response predictors was constructed using the selected features. Our results confirm that neoepitope and immune response based predictors for survival often significantly outperform mutation burden alone and simultaneously suggest a quantitative classification of immunotherapy efficacy across cancer subtypes.

Citation Format: Nicholas K. Akers, Eric E. Schadt, Bojan Losic. Modeling tumor immuno-dynamics to predict patient survival and immunotherapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1653. doi:10.1158/1538-7445.AM2017-1653
Abstract 1658: Establishment of CD137 humanized mouse model for efficacy assessment of agonistic anti-CD137 therapeutic antibodies
Authors: Ouyang Davy Xuesong , Chen Gang , Wang Zhensheng , Zheng Lei , An Annie Xiaoyu , Wery Jean-Pierre , Liu Jay , Dong Xin , Li Henry Q. X.
Published: July 2017

CD137 belongs to the TNF receptor super family, and its activation is essential to the function of T cells and NK cells. Multiple agonistic CD137 antibodies are currently being tested in humans with advanced cancer, which expected to become an important new immuno-oncology therapeutics in clinic. It is important to establish preclinical models for efficacy assessment of investigational CD137 antibodies before entering clinic. Currently available animal models cannot meet this need. Syngeneic mouse tumor models provide a useful platform for testing surrogate immuno-oncology therapies, but cannot be used for testing human therapeutic antibodies due to the species specificity; human immunity reconstituted models, by human PBMC or hematopoietic stem cell inoculation into immune deficient mice, suffering from highly variable responses, are not robust enough to provide a reliable system for efficacy studies. Establishing target humanized models by replacing mouse therapeutic target with human counterpart while maintaining normal mouse immunity is a practical approach to evaluate human therapeutic antibodies in vivo. Using CRISPR-Cas9 gene editing, we have engineered human CD137 knock-in model expressing chimeric human/mouse CD137 composed of human extracellular and transmembrane domains (Exon 4-7), with intact mouse signal peptide and intracellular domain. FACS analysis of splenocytes derived from homozygous knock-in mice showed that CD3/CD28 stimulated T cells only express chimeric CD137 reactive to human CD137 antibodies, but not mouse CD137; Yet the expression levels of the chimeric CD137 in the knock-in mice are generally lower than endogenous mouse protein in wild-type C57BL/6 mice. At present, we are testing the efficacy of reference human CD137 agonistic antibodies by treating these homozygous knock-in mice grafted with syngeneic MC38 tumor cells. Data will be presented at the conference.

Citation Format: Davy Xuesong Ouyang, Gang Chen, Zhensheng Wang, Lei Zheng, Annie Xiaoyu An, Jean-Pierre Wery, Jay Liu, Xin Dong, Henry Q. X. Li. Establishment of CD137 humanized mouse model for efficacy assessment of agonistic anti-CD137 therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2017-1658
Abstract 1661: Humanized mouse models for immune-oncology biotherapeutics: Development, validation and applications
Authors: Chen Gang , Cai Bin , Zhou Feng , Zhou Jerry , Liu Jie , Dong Xin
Published: July 2017

With clinical success and FDA approval of Ipilimumab, Nivolumab, Pembrolizumab and Atezolizumab, developing biotherapeutics against immune checkpoint targets, like CTLA-4, PD-1/PDL-1 and others, becomes hot pursuits for big pharma and biotech. However, traditional syngeneic models with surrogate murine reagents or human immune cell re-constitution in immune deficient mice are useful but not reliable and robust due to the species specificity and highly variable responses. It is crucial to establish additional preclinical models for efficacy assessment of investigational immune-oncology antibodies before entering clinic. Establishing human therapeutic target engineered mouse models by replacing mouse gene with human counterpart while maintaining intact mouse immunity is a practical approach for in vivo studies.

Applying CRISPR-Cas9 and other gene editing techniques, we have engineered genetically a series of single and double human immune-checkpoint genes, like PD-1/PDL-1, CTLA-4, TIM3, CD137 and others, knock-in model expressing chimeric human/mouse targets composed of human extracellular domains, with intact mouse signal peptide and intracellular domain. FACS analysis of immune cells derived from homozygous knock-in mice showed that activated cells only express chimeric targets reactive to anti human target antibodies, but not mouse gene specific antibodies. We have been testing the efficacy of reference antibodies by treating these homozygous knock-in mice inoculated with syngeneic murine tumor cells. Other applications, like target validation, combination therapy and biomarker discovery, are also explored.

Note: This abstract was not presented at the meeting.

Citation Format: Gang Chen, Bin Cai, Feng Zhou, Jerry Zhou, Jie Liu, Xin Dong. Humanized mouse models for immune-oncology biotherapeutics: Development, validation and applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1661. doi:10.1158/1538-7445.AM2017-1661
Abstract 1664: Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants
Authors: Head Jonathan F. , Phillips Jeffrey T. , Jiang Xianpeng , Elliott Robert L.
Published: July 2017

Developing animal tumor models for human cancer vaccines creates a tool to investigate the mechanism of action, variations in formulation, dosing schedules, and combinations with other forms of cancer therapy. In this study we developed a mouse whole cell mammary cancer vaccine model with both subcutaneous and intraperitoneal injection of the vaccine. The vaccine, containing 4T1 mouse mammary cancer cells (1,000,000 cells), IL-2 (0.2 µg) and GM-CSF (0.1 µg) in a total volume of 150 µl, was injected either subcutaneously into the backs of BALB/c mice or intraperitoneally. There were six injections of the vaccine (weeks 1, 2, 3, 7, 11, 15) and one week after the last injection 100,000 4T1 cells from cell culture in a volume of 100 µl were injected into the scapular region of the mice. At 22 days post 4T1 tumor transplantation there was a 37% inhibition of the growth of the 4T1 tumor in the mice receiving the subcutaneous injection of the vaccine and a 42% inhibition of the growth of the 4T1 tumor in the mice receiving the intraperitoneal injection of the vaccine. The inhibition of 4T1 mammary tumor growth in this mouse model with subcutaneous injection of a whole cell vaccine gives further support for the previously reported efficacy in a Phase 1/2 clinical trial of our therapeutic breast cancer vaccine containing autologous and allogeneic breast cancer cells in the adjuvant setting. The inhibition of 4T1 tumor growth with intraperitoneal injection suggests a delivery method that may be applicable to ovarian cancer and cancer patients with malignant ascites.

Citation Format: Jonathan F. Head, Jeffrey T. Phillips, Xianpeng Jiang, Robert L. Elliott. Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2017-1664
Abstract 1691: USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors
Authors: Kumar Suresh , Wu Jian , Wang Liqing , Wang Feng , Sokirniy Ivan , Wang Hui , Sterner David , Grove Charles , Weinstock Joseph , Mattern Michael , Hancock Wayne
Published: July 2017

Tumor microenvironment (TME) plays critical role in suppressing the immune system’s ability to recognize and destroy cancer cells. Although T cell checkpoint blockers such as anti-PD1 antibodies unleash anti-tumor immune response against a variety of tumors, the complex immunosuppressive tumor milieu necessitates development of additional therapeutic agents to potentiate active drugs and expand the realm of the revolutionary cancer immunotherapy. The presence of highly immunosuppressive Foxp3+ T-regulatory cells (Tregs) in the TME has been correlated with poor prognosis. Thus, depletion of Tregs or impairment of Treg function is considered an attractive therapeutic approach. USP7, a deubiquitylase (DUB) implicated as a critical node in several cancer signaling pathways has emerged as an essential factor in maintaining Treg functions. Treg specific deletion of USP7 impairs Treg function leading to lethal autoimmunity. Using the UbiProTM discovery platform, Progenra has identified selective covalent USP7 inhibitors that were subjected to lead optimization and preclinical evaluation. Potent and selective candidate USP7 inhibitors have been shown to impair Treg functions ex vivo and in vivo as well as exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. Most importantly, Progenra’s USP7 inhibitors enhances the efficacy of anti-PD1 antibody and cancer vaccines. Along with the already established direct anti-tumor activities of USP7 inhibitors, these studies provide a strong rationale for combining USP7 inhibitors to suppress Treg functions and improve the efficacy of currently approved cancer immunotherapy agents.

Citation Format: Suresh Kumar, Jian Wu, Liqing Wang, Feng Wang, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Joseph Weinstock, Michael Mattern, Wayne Hancock. USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2017-1691
Abstract 17: Anti-metastatic platinum through glycan targeting in breast cancer
Authors: Katner Samantha J. , Peterson Erica Peterson J. , Katsuta Eriko , DeMasi Stephanie C. , Koblinski Jennifer , Takabe Kazuaki , Farrell Nicholas P.
Published: July 2017

The high affinity of polynuclear platinum compounds (PPCs) for heparan sulfate (HS) is mediated through a “sulfate clamp” and PPC interactions can be conceptualized as “polyarginine” mimics. Strong HS-PPC binding protects the oligosaccharide against sulfate loss through metalloshielding.1 Metalloshielding will in principle affect HS interactions with relevant enzymes and proteins such as heparanase and growth factors, similar in concept to inhibition of DNA-protein binding through modification of DNA structure and conformation.2 PPCs inhibit cleavage of a model pentasaccharide by heparanase and further modulate bFGF binding to HS, and bFGF-induced migration and signaling in colon cancer cells.3 The end-point of functional modulation of HS interactions is inhibition of angiogenesis and metastasis.3 Following proof-of-principle of strong in vivo anti-metastatic activity of PPCs in clinically relevant breast cancer models,4 this contribution examines the underlying mechanism of platinums as anti-metastatic agents. We report on the cellular effects of PPC-HS metalloshielding on multiple growth factor signaling pathways (bFGF, HB-EGF) and specific syndecan (Sdc)-dependent signaling in breast cancer. Coupled with previously demonstrated DNA binding, PPCs represent intrinsically dual- function agents combining platinum cytotoxicity with anti-angiogenic effects derived from first principles through glycan targeting.

[1] Mangrum, J.B., Engelmann, B.J., Peterson, E.J., Ryan, J.J., Berners-Price, S.J., Farrell, N.P. Chemical Communications 2014, 50, 4056-4058.

[2] Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. Molecules 2015, 20, 6342-6388.

[3] Peterson, E.J., Daniel, A.G., Katner, S.J., Bohlmann, L., Chang,C., Bezos, A., Parish, C.R., von Itzstein, M., Berners-Price, S.J., Farrell, N.P. Chem. Sci. 2016, DOI: 10.1039/C6SC02515C.

[4] E. Katsuta, S. Demasi, S. Katner, H. Aoki, E. Peterson, E., N.P. Farrell and K. Takabe. (2016). Proc. AACR New Orleans, LA. Abstract #3064.

DC 1-5 April 2017

Citation Format: Samantha J. Katner, Erica Peterson J. Peterson, Eriko Katsuta, Stephanie C. DeMasi, Jennifer Koblinski, Kazuaki Takabe, Nicholas P. Farrell. Anti-metastatic platinum through glycan targeting in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2017-17
Abstract 1705: Slide-based multi-parametric immunophenotyping of human blood samples by cyclic immunofluorescence using the Accucyte-CyteFinder system
Authors: Lin Jia-Ren , U'Ren Lance , Baker Gregory J. , Nordberg Joshua J. , Maliga Zoltan , Stilwell Jackie L. , Kaldjian Eric P. , Sorger Peter K.
Published: July 2017

Immunophenotyping is an approach to measuring the abundance and functional state of immune cells as a means to understand mechanisms of homeostasis, identify biomarkers of disease, and measure therapeutic and adverse responses to drugs such as immune checkpoint inhibitors. Such studies are conventionally performed using flow cytometry together with combinations of immunomarkers that allow for identification of cell type, maturation state, and activation status. Here we describe a method for profiling circulating human leukocytes with highly-multiplexed immunofluorescence microscopy. The method has distinct advantages over flow cytometry in that it has greater sensitivity for detecting rare cell populations, allows for repeat analysis and long-term storage of precious biological samples, and obviates the requirement for spectral deconvolution. Using an open-source multiplexed immunofluorescence protocol referred to as cyclic immunofluorescence (CycIF), and commercially available reagents and instruments (AccuCyte and CyteFinder; RareCyte Inc.), we show that imaging can provide 16-plex, single-cell intensity data in addition to information on cellular morphology. Analytic technologies borrowed from the field of mass cytometry, such as ViSNE and Phenograph, allow for downstream phenotypic analysis and high-dimensional biomarker discovery. Current efforts now aim to expand the number of validated immune targets and combine imaging with single-cell picking and sequencing technologies.

Citation Format: Jia-Ren Lin, Lance U'Ren, Gregory J. Baker, Joshua J. Nordberg, Zoltan Maliga, Jackie L. Stilwell, Eric P. Kaldjian, Peter K. Sorger. Slide-based multi-parametric immunophenotyping of human blood samples by cyclic immunofluorescence using the Accucyte-CyteFinder system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2017-1705
Abstract 1738: Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients
Authors: Marx Sascha , Splittstöhser Maximilian , Paland Heiko , Seifert Carolin , Juettner Madlen , Boehm Andreas , Ritter Christoph A. , Bien-Moeller Sandra , Schroeder Henry W. , Rauch Bernhard
Published: July 2017

Objective

Glioblastoma patients (GBM) suffer from an increased incidence of cardiovascular events. Platelets are well known as main player of the primary hemostasis, but have a broad range of additional functions. The formation of heterotypic conjugates between platelets and leukocytes (PLC) represents a pro-inflammatory surrogate marker and is usually increased after platelet activation. The aim of the present study was to evaluate the platelet activation status and the rate of circulating PLC in GBM.

Methods

Blood samples were drawn of consecutive patients before surgery for a suspected glioblastoma. The formation of PLC and several parameters of platelet activation were determined by flow cytometry before and after stimulation with either ADP or the thrombin receptor-activating peptide (TRAP) in vitro: expression of P-Selectin, CD63, CD40L and fibrinogen-binding to the activated GPIIb/ IIIa. Blood samples from age and gender matched healthy volunteers were used as controls. Statistical analysis was done with the Mann-Whitney-Test.

Results

Final analysis included 22 patients with histopathological proven virgin glioblastoma (9f, 13m, mean age 67.5 years, range from 55 to 86 years) and their respective controls. Basal platelet activation and in vitro platelet reactivity was increased in GBM. The difference got significant in the basal expression of CD63 (2.8% versus 1.9%, p=0.008), the Fibrinogen-binding after ADP-stimulation (110.3 MFI versus 63.1 MFI, p=0.04) and the CD63 expression after TRAP-stimulation (38.4% versus 33.3%, p=0.04). Furthermore, a reduced number of circulating PLC and in vitro PLC formation was seen in GBM without getting statistically significant.

Conclusions

In this preliminary report, we show for the first time an increased level of platelet activation and agonist-induced platelet reactivity in GBM. Both could be a reflection of the pro-thrombotic status in these patients. Interestingly, the formation of PLC was not increased, but in tendency decreased. Whether this observation potentially mirrors the intratumoral, anti-inflammatory microenviroment in GBM remains unclear.

Citation Format: Sascha Marx, Maximilian Splittstöhser, Heiko Paland, Carolin Seifert, Madlen Juettner, Andreas Boehm, Christoph A. Ritter, Sandra Bien-Moeller, Henry W. Schroeder, Bernhard Rauch. Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2017-1738
Abstract 1744: RNAseq identified immune signatures associated with adverse outcome in high-risk neuroblastoma
Authors: Wei Jun S. , Zhang Shile , Kuznetsov Igor , Song Young K. , Asgharzadeh Shahab , Sindiri Sivasish , Wen Xinyu , Patidar Rajesh , Auvil Jaime M. Guidry , Gerhard Daniela S. , Seeger Robert , Maris John M. , Khan Javed
Published: July 2017

Despite the success of multimodal therapies, the mortality and morbidity remains substantial for patients with high-risk neuroblastoma (NBL). Sequencing of paired tumor/normal DNA of NBL has revealed a low somatic mutation burden and few recurrent somatically-mutated genes. Here we hypothesize that whole transcriptome sequencing (WTS) in patients with high-risk NBL tumor will yield valuable insights into the biology of this disease. We performed deep WTS of 150 NBLs (129 high-risk stage 3 and 4; and 21 stage 4S tumors) of which majority of them had whole genome sequencing or whole exome sequencing of case-matched tumor/normal pairs through the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. We identified correlations between gene expression and clinical parameters of patients such as MYCN-amplification and survival to provide understandings of high-risk NBL biology.

Consensus clustering using gene expression profiles of tumors revealed 4 subgroups with distinct survival probability (p<0.01). Among them, the group with the best outcome consisted of all stage 4S and 4S-like young patients; whereas the worst survival group was made up mostly of high-risk patients with MYCN-amplification. In order to identify the gene expression patterns correlating with outcome in the high-risk patients without MYCN-amplification, we further examined if any other known gene expression signatures can further stratify these patients using Gene Set Enrichment Analysis (GSEA). Among several molecular signatures, a previously reported 157-gene MYCN activation signature was significantly correlated with the poor outcome in high-risk patients without MYCN-amplification. Of note, we identified CD8, cytolytic, and activated nature killer (NK) cell signatures that were significantly associated with outcome in these patients. These findings were validated in an independent dataset. Our data has an important clinical implication for a subset of high-risk neuroblastoma patients without MYCN-amplification that have an activated MYCN signature may benefit from immune-modulating agents such as immune check point inhibitors which should be tested in context of clinical trials.

Citation Format: Jun S. Wei, Shile Zhang, Igor Kuznetsov, Young K. Song, Shahab Asgharzadeh, Sivasish Sindiri, Xinyu Wen, Rajesh Patidar, Jaime M. Guidry Auvil, Daniela S. Gerhard, Robert Seeger, John M. Maris, Javed Khan. RNAseq identified immune signatures associated with adverse outcome in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1744. doi:10.1158/1538-7445.AM2017-1744
Abstract 1745: Multifocal prostate cancer has high degree of genomic heterogeneity
Authors: Løvf Marthe , Zhao Sen , Axcrona Ulrika , Johannesen Bjarne , Hoff Andreas M. , Bakken Anne Cathrine , Carm Kristina Totland , Myklebost Ola , Meza-Zepeda Leonardo A. , Lie Agnes K. , Axcrona Karol , Lothe Ragnhild A. , Skotheim Rolf I.
Published: July 2017

Prostate cancer is the most common cancer type among men in the Western world. Most prostate cancers are multifocal with individual tumors harboring different aggressiveness. In recent genomics studies, the multifocal nature of prostate cancer has been investigated only in small sample cohorts. Here we have investigated the intra-organ mutational spectra of multiple tumors from a large cohort of prostate cancer patients. From radical prostatectomies performed between 2010 and 2012 at Oslo University Hospital, 43 prostatectomy specimens with multiple and clearly separated tumors were identified based on histology. From each of these prostatectomy specimens, DNA from frozen samples from 2-3 different tumor foci and corresponding normal tissue samples were analyzed by high-coverage whole-exome sequencing, adding up to a total of 159 samples. We identified 3093 somatic substitutions, insertions and deletions, with an average of 27 alterations per tumor sample. Both known and novel significantly mutated genes were identified and their distribution among different tumor foci from the same prostate was examined. Mutations in genes such as SPOP, MED12, and FOXA1 have previously been identified in prostate cancer and were also found to be frequently mutated in this cohort. However, the same mutations were rarely found in multiple tumor foci within the same prostate. In fact, for 13 out of the 43 examined patients there were no common mutations among tumors from the same prostate. For 12 patients, we found only one overlapping mutation among tumor foci. Whereas the overall list of mutated genes (n = 2101) overlap significantly with the 594 genes of the Cancer Gene Census (100/594; p=9.6e-06), this was not the case for 142 genes with overlapping mutations among different tumor foci (2/594; p=0.63). This indicates that the overlapping mutations are not typical cancer driving genes. To conclude, results from exome sequencing of multifocal prostate cancer show a large degree of heterogeneity in genomic alterations between different tumor foci within the same prostate. With very few common inter-foci mutations, an implementation of genome-based personalized prostate cancer medicine will require sampling of all tumor foci to tailor optimal treatment.

Citation Format: Marthe Løvf, Sen Zhao, Ulrika Axcrona, Bjarne Johannesen, Andreas M. Hoff, Anne Cathrine Bakken, Kristina Totland Carm, Ola Myklebost, Leonardo A. Meza-Zepeda, Agnes K. Lie, Karol Axcrona, Ragnhild A. Lothe, Rolf I. Skotheim. Multifocal prostate cancer has high degree of genomic heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1745. doi:10.1158/1538-7445.AM2017-1745
Abstract 1753: Refining breast cancer characterization through single-cell analysis of ex vivo reprogrammed tumor and adjacent normal cells
Authors: Nakshatri Harikrishna , Anjanappa Manjushree , Cardoso Angelo , Cheng Lijun , Mohamad Safa , Gunawan Andrea , Rice Susan , Dong Yan , Li Lang , Srour Edward
Published: July 2017

Discovery of inter-individual functional variations in gene regulatory elements and the observation that tumor and normal tissues of the same organ are in different differentiation states necessitate rethinking of gene expression based subclassification/characterization of tumors. To address this issue, we performed single cell genomics of breast tumors and adjacent-normal cells propagated using epithelial reprogramming growth conditions for a short duration. Epithelial cells analyzed were either unselected for specific subpopulation or phenotypically defined undifferentiated ALDH+/CD49f+/EpCAM+ luminal progenitors present in the normal breast, which express both basal and luminal-enriched genes. Expression of 93 genes that included PAM50 intrinsic subtype classifier and stemness-related genes was analyzed in 420 tumor and 284 adjacent-normal cells. ALDH+/CD49f+/EpCAM+ tumor and normal cells clustered differently compared to unselected tumor and normal cells. PAM50 genes-set analyses of ALDH+/CD49f+/EpCAM+ populations efficiently identified major and minor clones of tumor cells with the major clone resembling clinical parameters of the tumor. Similarly, stemness-associated gene set identified clones with divergent stemness pathway activation within the same patient sample. This refined technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, assays presented here may enable precise identification of genes deregulated in cancer, early identification of therapeutically-targetable minor population of tumor cells, and eventually to refinement of precision therapeutics.

Citation Format: Harikrishna Nakshatri, Manjushree Anjanappa, Angelo Cardoso, Lijun Cheng, Safa Mohamad, Andrea Gunawan, Susan Rice, Yan Dong, Lang Li, Edward Srour. Refining breast cancer characterization through single-cell analysis of ex vivo reprogrammed tumor and adjacent normal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2017-1753
Abstract 1783: Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging
Authors: Chen Xiao , Yang Yizeng , Katz Sharyn
Published: July 2017

Background: Inhibition of thymidylate synthase (TS) results in a transient compensatory “flare” in thymidine salvage pathway activity which is measureable with 18F-thymidine (FLT) positron emission tomography (PET) at 2 hrs of exposure to therapy. Here we examine the predictive value of the FLT-PET measured “flare” for NSCLC sensitivity to pemetrexed, a commonly used TS inhibitor, in a preclinical model.

Experimental Design: Resistance to pemetrexed therapy was induced in two sensitive human NSCLC cell lines, H460 and H1299, by overexpressing TS. TS overexpression was confirmed with RT-PCR and Western blotting and pemetrexed-resistance confirmed with IC50 assays. The presence of a pemetrexed-induced DNA salvage pathway “flare” was then measured using 3H-thymidine in both pemetrexed-sensitive (H460 and H1299) and resistant (H460R and H1299R) lines in vitro, as well as inherently resistant NSCLC cell lines CALU6, H522, H650, H661, H820, H1838, and validated with FLT-PET in vivo using H460 and H460R xenografts.

Results: Overexpression of TS induced resistance to pemetrexed in the H460 and H1299 with IC50 for H460, H1299, H460R and H1299R measured as 0.141 μM, 0.656 μM, 22.842 μM, 213.120 μM, respectively. Significant increases in DNA salvage pathway activity (“flare”) was observed at 2hrs of therapy in the pemetrexed-sensitive H460 and H1299 lines but not the resistant H460R and H1299R, CALU6, H522, H650, H661, H820, H1838 cell lines in vitro using 3H-thymidine assays. Similarly, FLT “flare” was observed in the pemetrexed-sensitive H460 xenograft tumors but not the pemetrexed-resistant H460R xenograft tumors with post-therapy increases in measured FLTmax of 58% (STD 12.1) and 10.8% (STD 7.3) respectively.

Conclusions: FLT-PET imaging of TS inhibition may provide an early indicator of NSCLC sensitivity to pemetrexed.

Citation Format: Xiao Chen, Yizeng Yang, Sharyn Katz. Early detection of thymidylate synthase resistance in non-small cell lung cancer with FLT-PET imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1783. doi:10.1158/1538-7445.AM2017-1783
Abstract 1784: Early feasibility and development of multiplexed, single-reaction assays for ALK, ROS1 and RET novel ddPCR RNA fusions
Authors: Mellert Hestia , Alexander Kristin , Jackson Leisa , Roda Galen , Cooper Samantha , Marr Dianna , Jones Stephen J. , Charrington Nia , Pestano Gary
Published: July 2017

We have previously described a targeted genomic Laboratory Developed Test (LDT) that includes variant specific Droplet Digital™ PCR (ddPCR) testing for EGFR, KRAS and BRAF in plasma. This test supports the rapid delivery of molecular diagnostic test results, with >95% of tests results delivered in 72 hours of receipt in our Laboratory. This test then meets the key clinical need for the delivery of results that can result in faster treatment decisions. Additionally, the test may be of utility for those patients who need mutation results quickly or for whom tissue may be unavailable or insufficient for molecular testing. This is especially true for patients diagnosed with non-small cell lung cancer (NSCLC). In this report we will update on new test concepts created using the recently available design software engine for ddPCR assays. Specifically, we will describe studies on the development of single-reaction, multiplexed assays for the respective detection of ROS1 (up to 11 variants), RET (up to 8 variants) and EML4-ALK (v1 - v3). Design considerations, specificity and sensitivity, as well as reproducibility and robustness studies for these complex assays will be reviewed. Similar studies were conducted for the development of the commercially available test for the EML4-ALK fusion variants. EML4-ALK concordance studies compared the fusions found in blood with known positives and negatives found using FISH and PCR based methods (n=24 evaluable matched pair samples). Clinical sensitivity, specificity and concordance were 85%, 100% and 92% respectively. In this study we also report on test performance of the ALK RNA fusion test over 3 consecutive months of testing. Of note, we have delivered greater than 95% of tests (n = 272 samples) with an observed positive sample frequency of 2%. The robust detection of rare variant, RNA fusions from plasma within 72 hours represents a molecular testing option of value to patients with NSCLC and their physicians.

Citation Format: Hestia Mellert, Kristin Alexander, Leisa Jackson, Galen Roda, Samantha Cooper, Dianna Marr, Stephen J. Jones, Nia Charrington, Gary Pestano. Early feasibility and development of multiplexed, single-reaction assays for ALK, ROS1 and RET novel ddPCR RNA fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2017-1784
Abstract 1786: Predictive value of LRP8, KPNA2 and GDF15 expression to anthracycline/taxane based chemotherapy response in patients with locally advanced breast cancer
Authors: Maldonado-Mtz Hector , Oscar Alonso-Luna , Flores-Perez Ali , Abelardo Meneses-Garcia , Ruiz-Garcia Erika B. , Lopez-Camarillo Cesar , Vega Horacio Astudillo-de la
Published: July 2017

Background: Chemotherapy plays a major role in its treatment, though overtreatment is frequent since its choice is based on clinicopathological prognostic data. The identification and validation of new predictive profiles or markers continues to be relevant. New findings may contribute to achieve better results in this area, as well as to provide new targets of the pathways involved in chemoresistance. Karyopherin α2 (KPNA2), low density lipoprotein receptor-related protein 8 (LRP8) and growth and differentiation factor 15 (GDF15), are three candidate predictive biomarkers in breast cancer, previously identified on DNA microarrays reports.

Methods: Using tissue microarrays technology and immunohistochemistry, we compared the expression of these three markers between cases of breast cancer with response to chemotherapy (absence of recurrence), and those with failure (with recurrence). All patients received chemotherapy based on anthracyclines and taxanes. Potential confounding factors, as known recurrence and progression risk factors, were controlled for during the comparison.

Results: 20 out of 63 patients had tumor recurrence. Potential confounding factors were similar among cases with response and failure to chemotherapy. For KPNA2 no differences were found between cases with response or failure. LRP8 showed higher expression in cases with failure to chemotherapy (mean 7.22 vs 14.01%, p =0.025). In Cox multivariable regression analysis, which included potential confounding factors as covariates, only LRP8 remained as a significant predictive factor (HR 1.28, p=0.016). GDF15 showed no expression in our cases, nor in an external set of cases.

Conclusions: We demonstrated increased expression of LRP8 in breast cancer cases recurring after chemotherapy. KPNA2 and GDF 15 showed no predictive value for chemotherapy response in our study. LRP8 is a potential predictive marker worth of further investigation for its role in chemoresistance as well as for its potential validation.

Note: This abstract was not presented at the meeting.

Citation Format: Hector Maldonado-Mtz, Alonso-Luna Oscar, Ali Flores-Perez, Meneses-Garcia Abelardo, Erika B. Ruiz-Garcia, Cesar Lopez-Camarillo, Horacio Astudillo-de la Vega. Predictive value of LRP8, KPNA2 and GDF15 expression to anthracycline/taxane based chemotherapy response in patients with locally advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1786. doi:10.1158/1538-7445.AM2017-1786
Abstract 1787: miR-150 as a predictor of hematopoietic reconstitution after myeloablation
Authors: Yadav Marshleen , Chakravarti Arnab , Jacob Naduparambil K.
Published: July 2017

The identification of biomarkers that allow evaluation of myeloablation and hematopoietic reconstitution time frame is important for tailoring the radiation or chemo therapy in cancer patients. The fate of hematopoietic cells is regulated at multiple stages by various genes and regulatory microRNAs. In this context, miR-150 has been demonstrated as a major regulator of lymphoid lineage differentiation and development. Our mouse model studies shows a novel role of miR-150 in imparting radiation resistance. We observed a prolonged survival benefit in mice where both the copies of miR-150 genes are deleted in comparison to that of wildtype mice in the same C57BL/6 background (p<0.05). The survival benefit after total body irradiation was found to be associated with lower apoptosis and early reconstitution in hematopoietic stem cells (lin-ckit+sca1+/-) in bone marrow. Furthermore, the recovery kinetics at sub lethal dose (6Gy) showed that HSCs in miR-150-/- mice recovers faster. Myeloid (Mac1+Gr1+) and lymphoid (CD45+CD19+) lineage cells reconstitutes within 14 days after radiation in KO mice compared to 21 days or more in WT mice. We observed a statistically significant upregulation in myeloid cells in KO mice which implicates an early recovery from radiation induced neutropenia and hence more survivability. In addition, the enhanced bone marrow reconstitution was reflected in peripheral blood counts where a noticeable difference (p<0.05) in blood RBCs lymphocytes, monocytes, neutrophils, and basophiles were noted when compared sub lethally irradiated KO with WT mice. Altogether we demonstrate that the level of miR-150 regulates the bone marrow cells distribution and hence could be targeted for achieving accelerated hematopoietic recovery after myeloablation. These findings may be translated to human cancer patients for therapy guiding based on miR-150 expression level. Besides its use in prediction of reconstitution, miR-150 in the circulating system could provide a direct readout of functional marrow and hence hematological acute radiation syndrome (H-ARS).

Citation Format: Marshleen Yadav, Arnab Chakravarti, Naduparambil K. Jacob. miR-150 as a predictor of hematopoietic reconstitution after myeloablation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1787. doi:10.1158/1538-7445.AM2017-1787
Abstract 1793: Telomere length and mitochondrial DNA copy number variations as predictors for survival in hepatocellular carcinoma patients treated with sorafenib
Authors: Yan Dong
Published: July 2017

Background: The study aims to evaluate the potential predictive value of relative telomere length (RTL) and mitochondrial DNA (mtDNA) copy number variations for survival in hepatocellular carcinoma (HCC) patients treated with sorafenib.

Methods: The study was conducted in 120 patients with pathologically or radiologically confirmed HCC who were treated with sorafenib as their first-line or second-line monotherapy. RTL and mtDNA copy number in peripheral lymphocytes were measured using quantitative real-time polymerase chain reaction (PCR)-based method. ANOVA and non-parametric tests were applied to compare the RTL and mtDNA copy number between groups. Progression-free survival (PFS) and overall survival (OS) time was analyzed using Kaplan Meier plot, log-rank test, and Cox proportional hazards regression model.

Results: A longer telomere was observed in patients with early stage disease or normal body weight and in Hispanics compared to their counterparts. Patients with longer RTL compared to those with shorter RTL had significantly better PFS (6.90 vs 4.40 months, P=0.048) and OS (16.53 vs 7.72 months, P=0.006). Cox regression analysis demonstrated that longer RTL was an independent predictor for OS (hazard ratio [HR]=0.47, 95% confidence interval [CI]=0.29-0.76, P<0.01) in all patients and for PFS in patients received sorafenib (HR=0.37, 95% CI=0.15-0.94, P=0.01) after adjusting for other clinical predictors. RTL was positively correlated to mtDNA copy numbers (r=0.312, P=0.001), but no significant association of mtDNA copy numbers and PFS or OS was observed.

Conclusion: RTL, but not mtDNA copy number, might be a potential predictor for OS and for response to sorafenib treatment in HCC.

Note: This abstract was not presented at the meeting.

Citation Format: Dong Yan. Telomere length and mitochondrial DNA copy number variations as predictors for survival in hepatocellular carcinoma patients treated with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1793. doi:10.1158/1538-7445.AM2017-1793
Abstract 1801: Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts
Authors: Gaustad Jon-Vidar , Simonsen Trude G. , Andersen Lise Mari K. , Rofstad Einar K.
Published: July 2017

Background: Studies comparing the effect of antiangiogenic agents targeting different angiogenic pathways are sparse. The purpose of this study was to compare the effect of properdistatin and sunitinib treatment in a preclinical model of malignant melanoma. Properdistatin is a small peptide derived from the thrombospondin-1 domain of the plasma protein properdin, and sunitinib is a tyrosine kinase inhibitor targeting several receptors including the vascular endothelial growth factor receptors.

Methods: R-18 human melanoma xenografts growing in dorsal window chambers were treated with properdistatin, sunitinib, or vehicle. Parameters describing the morphology of tumor vasculature were assessed from high-resolution transillumination images, and BST (blood supply time; the time needed for arterial blood to flow from the main supplying artery to downstream microvessels) was assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by using pimonidazole as a hypoxia marker.

Results: Properdistatin treatment selectively removed small-diameter vessels and reduced BST, whereas sunitinib treatment reduced the density of small- and large-diameter vessel similarly and did not change BST. These observations imply that properdistatin treatment reduced geometric resistance to blood flow and improved vascular function, whereas sunitinib treatment did not affect vascular function. Accordingly, sunitinib-treated tumors showed higher hypoxic fractions than properdistatin-treated tumors.

Conclusions: Properdistatin and sunitinib both inhibited angiogenesis, but had distinctly different effects on vascular morphology, vascular function, and extent of hypoxia in R-18 human melanoma xenografts.

Citation Format: Jon-Vidar Gaustad, Trude G. Simonsen, Lise Mari K. Andersen, Einar K. Rofstad. Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2017-1801