bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5446: Genetic bases of testicular seminoma and non-seminoma tumors
Authors: Martin-Gimeno Paloma , Paumard-Hernandez Beatriz , Calvete Oriol , Benitez Javier
Published: September 2020

Testicular Germ Cell Tumor (TGCT) is the most common cancer type in men between 15-45 years old (1 per 250 men). Around 1-2% of all cases are familial (with at least two affected individuals within the same family), between 3-5% are bilateral (they develop two tumors, one in each testicle), and the rest are sporadic patients. There are two main types of histologies, seminomas and non-seminomas, and there are also tumors that contain both seminoma and non-seminoma cells, and are called mixed tumors. No great genetic differences have been found so far between them.

In order to identify susceptibility genes that could explain these histological differences, we performed Whole Exome Sequencing (WES) in peripheral blood of 61 members from 17 families with at least two affected members with TGCT. Twenty one members had seminoma, seven members had non-seminomas, and nine had mixed tumor. We did not find significant genetic differences between the two histological types at the germline level. Therefore, an evaluation at a somatic level was carried out to determine if the differences between the tumor types are in the tumor.

To this end, WES of blood and tumor tissue was performed in three bilateral cases with different histologies (a seminoma in one testicle and a non-seminoma in the other). The comparison between pairs of tumors from each of the patients showed that they shared in average a 30% of the somatic mutations between both the histologies. Even more, 27 genes were shared among the two tumors in the three patients. We next performed independency tests, comparing the somatic similarities and differences between the two tumors from each patient (intrapatient) and the two histologies (interpatients), which suggest a common genetic background that could predispose to testicular cancer development. We are currently performing gene ontology analysis with the genes that were shared among the pairs of tumors from each patient.

Partially supported by CIBERER and FIS 16/00440

Citation Format: Paloma Martin-Gimeno, Beatriz Paumard-Hernandez, Oriol Calvete, Javier Benitez. Genetic bases of testicular seminoma and non-seminoma tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5446.
Abstract 5448: Integrative analysis of microRNA expression identifies two biologically distinct and clinically relevant subtypes of head and neck squamous cell carcinoma
Authors: Holt Jeremiah R. , Jo Heejoon , Walter Vonn , Zhao Xiaobei , Hayes David Neil , Ko Yoon Ho
Published: September 2020

Purpose: The objective of this study is to investigate the potential role of microRNAs (miRNA) as molecular markers of tumor heterogeneity in head and neck squamous cell carcinoma (HNSCC).

Experimental Design: Here, we analyzed 562 HNSCC samples, 88 from a novel cohort and 474 from The Cancer Genome Atlas (TCGA), using miRNA-microarray and miRNA-seq, respectively. Using an integrative correlations method followed by miRNA expression-based hierarchical clustering, we validated miRNA clusters across cohorts, and evaluation of clusters by logistic regression and gene ontology approaches revealed subtype-based clinical and biological characteristics.

Results: We identified two statistically significant tumor subtypes and named them ‘epithelial' and ‘stromal' based on correlations with functional target gene ontology in relation to differing stages of epithelial cell differentiation. Each subtype demonstrated significant differences in terms of mRNA signature (p < 0.001), primary tumor sites (p < 0.001) and HPV status (p < 0.001), and multivariate analysis associated the stromal subtype with a worse prognosis (HR = 1.5646, p = 0.006). We also observed several dysregulated miRNA families and clusters that function as regulators of subtype-specific gene expression networks in HNSCC.

Conclusion: Our findings suggest miRNAs determine HNSCC subclassifications through coordinating growth and differentiation programs in epithelial cells. These results delineate developmental miRNA signatures characterizing the phenotypic diversity between HNSCC subtypes, providing an expanded framework for the pathogenesis and personalized treatment of HNSCC.

Citation Format: Jeremiah R. Holt, Heejoon Jo, Vonn Walter, Xiaobei Zhao, David Neil Hayes, Yoon Ho Ko. Integrative analysis of microRNA expression identifies two biologically distinct and clinically relevant subtypes of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5448.
Abstract 5454: A data driven pipeline to detect somatic mutations in oncology
Authors: Shortt Katherine , Johnson Christopher , Ramesh Archana , Neuwerth Tom , Giauque Chris , Raghunath Sharanya
Published: September 2020

The analysis of Next Generation Sequencing (NGS) data relies on workflows that integrate different bioinformatics tools. Designing workflows that are assay agnostic and have a robust computational framework for result generation in a timely manner is particularly important for clinical applications of NGS. These technologies play an important role in classifying a patient's tumor at a molecular level however, there is still a need to develop workflows that generate consistent mutation profiles for oncology. We developed a bioinformatics tool to analyze DNA and RNA sequencing data in a single pipeline and to specifically detect somatic mutations in oncology clinical workflows. Our automated pipeline report contains extensive quality metrics for each sample and generates a robust mutation annotation and filtration module vital for clinical use. We use data driven filters and algorithms that predict the effectiveness of mutations to filter out mutations of poor quality or mutations without clinical significance. We applied our pipeline to standard cell lines and various cancer FFPE samples acquired from the Intermountain Healthcare Biorepository. Our pipeline successfully processes different mutation classes including Single Nucleotide Variants (SNVs), small (<20 bp) and large (>21 bp) insertions and deletions (InDels), gene fusions, and exon skipping mutations. In addition, we established a reference set that allows us to call Copy Number Variants (CNVs) in a gene panel designed to detect clinically relevant mutations in lung cancers. Comparison of this pipeline to a validated external workflow yield high concordance of clinically significant mutations. We conclude that our pipeline offers a sensitive and specific methodology to analyze DNA and RNA sequencing data to clinically relevant results from low input amplicon-based assays for various solid tumor cancer types.

Citation Format: Katherine Shortt, Christopher Johnson, Archana Ramesh, Tom Neuwerth, Chris Giauque, Sharanya Raghunath. A data driven pipeline to detect somatic mutations in oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5454.
Abstract 5460: Detecting somatic DNA copy number differences with cyclic shift testing
Authors: Walter Vonn , Choi Hyo Young , Zhao Xiaobei , Zevallos Jose , Hayes D. Neil
Published: September 2020

The most basic differential expression analysis involves the identification of genes that exhibit distinct expression patterns in two cohorts. Surprisingly, even though there are numerous computational methods for detecting and assessing the statistical significance of somatic DNA copy number (CN) gains and losses in a single cohort, there are no tools for detecting and assessing the statistical significance of CN differences between two cohorts. Instead, typically each cohort is analyzed separately and distinct findings are presented as evidence of CN differences.

Cyclic shift testing was originally introduced as a method to identify and assess the statistical significance of recurrent somatic CN gains and losses. Subsequent work explored theoretical statistical underpinnings of cyclic shift testing. Here we extend the previous results by showing that cyclic shift testing can be applied to detect and assess the statistical significance of CN differences between two cohorts.

Human papilloma virus (HPV) infection is a risk factor for head and neck squamous cell carcinoma (HNSC), and it is known that HPV+ and HPV- HNSC have distinct mutational and gene expression profiles. We apply cyclic shift testing to detect and assess the statistical significance of CN differences between HPV+ and HPV- HNSC using data from The Cancer Genome Atlas HNSC cohort. Our approach detects statistically significant CN differences in regions that are known to contain HNSC drivers (7p, 9p21, 11q13, 11q14-qter), as well as regions of chr14, chr16, and others that are less well characterized.

Citation Format: Vonn Walter, Hyo Young Choi, Xiaobei Zhao, Jose Zevallos, D. Neil Hayes. Detecting somatic DNA copy number differences with cyclic shift testing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5460.
Abstract 5493: Mathematical modeling of antibody nanoconjugates transport quantifies the impact of the tumor microenvironment on drug penetration
Authors: Vaghi Cristina , Rodallec Anne , Sicard Guillaume , Correard Florian , Fanciullino Raphaelle , Ciccolini Joseph , Poignard Clair , Benzekry Sebastien
Published: September 2020

One of the major challenges in breast cancer treatment is the high toxicity associated with cytotoxic agents. To overcome this issue, we developed nanoparticles conjugated with cancer cell specific antibodies (trastuzumab) to improve drug delivery to the tumor, while sparing healthy tissues. These antibody nano-conjugates (ANCs) consist in docetaxel-encapsulated liposomes engrafted with trastuzumab on the surface. Intra-tumor penetration properties of these ANCs are not fully understood and could be improved. To this end, mathematical modeling can provide quantitative tools to obtain a description of the ANCs transport inside a tumor. Few models in literature characterize the nanoparticle penetration in tumors. We derived a multi-scale model to obtain a description of the fluid flow and nanoparticle transport in the tumor interstitium and vessels. Moreover, there is a lack of studies that integrate spatial data to theoretical models. Here, we aim at filling this gap.

Experimental data - carried on human MDA-MB-231 xenografts - included ex-vivo microscopy images of tumor sections obtained from different regions of the tumor (central and peripheral) using fluorescence imaging. This data allowed to recover the tumor microstucture. Our multi-scale model consisted of two Darcy equations for the fluid flow and two convection-diffusion equations governing the ANCs penetration, including the porosity of the medium. Eventually, our model was calibrated with the ex-vivo imaging data.

Simulations confirmed that the tumor microsctructure influences the macroscale flow dynamics and ANC delivery in the tumor interstitium. The model also provides quantitative predictions of the impact of the tumor microenvironment (e.g., heterogeneity, vessel leakage and interstitium permeability) on the accumulation and penetration of ANCs. This methodology could be further applied to personalize the dose of injected nanoparticles according to a patient's specific tumor histology.

Citation Format: Cristina Vaghi, Anne Rodallec, Guillaume Sicard, Florian Correard, Raphaelle Fanciullino, Joseph Ciccolini, Clair Poignard, Sebastien Benzekry. Mathematical modeling of antibody nanoconjugates transport quantifies the impact of the tumor microenvironment on drug penetration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5493.
Abstract 5544: Newcastle Disease Virus in human derived tumors: Understanding immune recruitment and activation via virally delivered IL-12
Authors: Durham Nicholas M. , McGlinchey Kelly , Burke Shannon , Creasy Todd , Rath Nicola , Monks Noel , Tammali Ravinder , Schifferli Kevin , Holoweckyj Nick , Hayes Susie , Jones Emma , Kelly Elizabeth J. , Carroll Danielle , Harper James , Streicher Katie
Published: September 2020

Oncolytic viruses (OV) are defined by their intrinsic or engineered tropism for tumor cells. Understanding the drivers of susceptibility to infection as well as the downstream intracellular signaling and immune activation profile is critical in identifying the patients that will respond to OV therapy. Newcastle Disease Virus (NDV) is a negative stranded RNA avian paramyxovirus, here modified to encode human GM-CSF or IL-12. Patient derived xenografts (PDX) can more accurately reflect the genetic, epigenetic, and architectural heterogeneity of human tumors and were therefore selected to model the in vivo activity of NDV:GM-CSF. Eighty-nine independent models derived from patients with Bladder, Colorectal, Head and Neck, Kidney, Liver, Lung, Ovarian, Pancreatic, and Breast tumors were studied. Mice were dosed intravenously with NDV:GM-CSF, monitored for pharmacodynamic and pharmacokinetic changes 48 hours after the first dose, and followed for tumor growth inhibition. Objective response was observed in 68% of treated mice, with responses observed in all indications. Using total stranded RNAseq, we were able to assess changes in human tumor, murine immune infiltrate, and viral RNA. Results showed that NDV:GM-CSF treatment leads to increases in transcripts of inflammatory chemokines and cytokines such as RANTES and CXCL9, with induction directly correlated to level of viral replication. Additionally, we saw increases in NKp46 and IL-12R, markers of NK cells. To assess if IL-12 would augment immune activation, we treated surgically resected human tumors with NDV:IL-12. NDV:IL-12 was capable of infecting tumor cells, generating IL-12 protein, and driving a strong IFNγ response. Here we show that NDV can infect a wide variety of human tumors across multiple indications and drive a potent immune activation which can be modulated by transgenes. This data provides insight into what types of tumors could be treated with NDV:GM-CSF or NDV:IL-12.

Citation Format: Nicholas M. Durham, Kelly McGlinchey, Shannon Burke, Todd Creasy, Nicola Rath, Noel Monks, Ravinder Tammali, Kevin Schifferli, Nick Holoweckyj, Susie Hayes, Emma Jones, Elizabeth J. Kelly, Danielle Carroll, James Harper, Katie Streicher. Newcastle Disease Virus in human derived tumors: Understanding immune recruitment and activation via virally delivered IL-12 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5544.
Abstract 5545: Neutralization of interleukin-10 by genetically engineered macrophages increase cancer cell death in metastatic colorectal cancer
Authors: Labadie Kevin P. , Kreuser Shannon A. , Brempelis Katherine J. , Jiang Xiuyun , Daniel Sara K. , Crane Courtney A. , Pillarisetty Venu G.
Published: September 2020

Background

Interleukin-10 (IL-10) is an immunosuppressive cytokine that inhibits innate and adaptive antitumor immune responses in the tumor microenvironment (TME). Antibody neutralization of IL-10 in organotypic tumor slice cultures (TSC) in colorectal cancer liver metastases (CRCLM) leads to cancer cell death through. To target IL-10 blockade to the TME, we genetically engineered macrophages (GEM) to produce an IL-10 neutralizing antibody which we hypothesized would reactivate tumor infiltrating immune cells and increase cancer cell death in TSC.

Methods

We created a lentiviral construct to transduce GEMs with a publicly available neutralizing IL-10 antibody sequence (BT-063). CD14+ monocytes were isolated from healthy donor peripheral blood mononuclear cells and differentiated into macrophages, which were transduced with lentivirus to create control (cGEMs) and anti-IL-10 GEMs (αIL10 GEMs). To test the efficacy of αIL10 GEMs, punch biopsies (6 mm) were obtained from resected CRCLM, sliced to 250 um thickness with a vibratome and placed in individual culture wells. Slices were treated in triplicates with either 20ug/mL of human monoclonal αIL10 (αIL10), 1e5 cGEMs or αIL10 GEMs + 20ug/mL αIL10. GEMs and tumor cells were visualized with a Leica SP8X confocal microscope and apoptosis was measured with SR-FLICA fluorescent poly-caspase assay kit. Supernatant interferon-gamma (IFN-γ) levels were measured with Bioplex immunoassay and IL-10 neutralizing antibody was quantified with an enzyme-linked immunosorbent assay.

Results

Allogenic GEMs were transduced with BT-063-T2A-CD19t at 31% efficiency. cGEMs and αIL10 GEMs persisted in tumor slice culture for at least 6 days. αIL10 GEM-treated slice cultures contained 6.6ng/mL and 6.5ng/mL of αIL10 antibody after 3 and 6 days, respectively. At day 6 in culture, cancer cell apoptosis was increased in the αIL10 treated groups with 7%, 15%, 25%, 31% and 39% of cells were apoptotic in the no treatment control, cGEM, αIL10, cGEM + αIL10 and αIL10 GEM groups, respectively (One-way ANOVA, p = 0.0004). Elevation in IFN-γ was observed on day 6 in slices treated cGEMs (7.2ng/mL+4.3) and αIL10 GEMs (9.2ng/mL+5) compared to isotype control.

Conclusion

GEMs transduced with lentivirus encoding IL-10 neutralizing antibody maintain its production and are associated with increased cancer cell apoptosis.

Citation Format: Kevin P. Labadie, Shannon A. Kreuser, Katherine J. Brempelis, Xiuyun Jiang, Sara K. Daniel, Courtney A. Crane, Venu G. Pillarisetty. Neutralization of interleukin-10 by genetically engineered macrophages increase cancer cell death in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5545.
Abstract 5565: Skin-targeted combinatorial DNA immunization overcomes tumor resistance to PD1 therapy
Authors: Falo Louis D.
Published: September 2020

Despite the remarkable treatment benefits associated with interventional checkpoint blockade for solid tumors, many patients ultimately progress on therapy. The development of novel strategies that can overcome resistance to checkpoint therapy remains a considerable unmet clinical need. In this context, we developed a novel tumor immunization strategy that is multifunctional and combines co-delivery of plasmid DNA encoding antigen and molecules driving immunostimulatory function and antigen targeting. Specifically, we targeted expression of plasmid DNA encoding the stress response transcription factor XBP1 and the melanoma-associated antigen TRP2 fused to heat shock protein HSP70. Transient skin-localized expression promotes a pro-immunogenic skin microenvironment and the induction, recruitment, and maintenance of Type-1 cytotoxic CD8 T cells (Tc1), including Tc1 T-cell infiltrates within the tumor microenvironment (TME), and tumor regression in a PD1-resistant mouse melanoma model. These data suggest a novel therapeutic strategy in which combined effects of localized XBP1 and HSP70 antigen conjugation establishes a PD1-responsive TME for effective and durable treatment of PD1-resistant cancers.

Citation Format: Louis D. Falo. Skin-targeted combinatorial DNA immunization overcomes tumor resistance to PD1 therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5565.
Abstract 5591: Differential profiles of pro-inflammatory chemokines identified in patients with prostate cancer and benign prostatic hyperplasia
Authors: Reyes Niradiz , Tiwari Raj , Geliebter Jan
Published: September 2020

Background: Prostate cancer (PCa) is the most frequently diagnosed malignant disease and the second leading cause of cancer-related deaths of men in the United States. The development of PCa has been linked to inflammation; thus, systemic inflammatory biomarkers may be useful to differentiate PCa from other prostatic disorders, such as benign prostatic hyperplasia (BPH). Proinflammatory chemokines are among the factors that regulate inflammatory processes through the recruitment of leukocytes to the sites of infection or injury. The aim of the present study was to compare the serum profiles of pro-inflammatory chemokines of patients newly diagnosed either with PCa or BPH.

Methods: Serum was prepared from blood samples collected at the time of diagnosis from a cohort of patients newly diagnosed with PCa or BPH. Protein concentrations of chemokines were measured in each group of samples with a multiplex ELISA kit specific for the quantification of 12 proinflammatory chemokines: CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL22, CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11. Results were further validated for selected chemokines using single analyte ELISA kits. This work was approved by the Institutional Review Board of the University of Cartagena.

Results: Our study showed that PCa patients had a significantly different proinflammatory chemokine profile compared to BPH patients. Serum samples in the PCa group had significant higher levels of CXCL8, CCL2, CCL3, CCL4, CXCL11 and CXCL1 compared with the BPH group.

Conclusion: Our study showed that PCa patients had a high proinflammatory chemokine profile. Additional studies are required to determine whether proinflammatory chemokine profiles can serve as a diagnostic signature to differentiate between PCa and BPH, and whether proinflammatory chemokine profiles are correlated with clinical outcomes of PCa patients.

Keywords: Prostate cancer, chemokine, inflammation, Benign Prostatic Hyperplasia

Citation Format: Niradiz Reyes, Raj Tiwari, Jan Geliebter. Differential profiles of pro-inflammatory chemokines identified in patients with prostate cancer and benign prostatic hyperplasia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5591.
Abstract 5592: MOSPD2 a novel target for bi-specific antibody mediated solid tumor cells immune death
Authors: Salem Yaniv , Yacov Niva , Propheta-Meiran Oshrat , Kafri Pinhas , Mendel Itzhak , Breitbart Eyal
Published: September 2020

Introduction: We have previously identified motile sperm domain-containing protein 2 (MOSPD2) as key regulator of monocyte motility. While MOSPD2 is normally expressed by myeloid cells with low/undetectable levels in healthy tissues, high expression of MOSPD2 is detected in multiple solid tumors and cancer cell lines. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer but not in in-situ tumors, and our data indicate that it is essential for breast cancer cell metastasis in vitro and in-vivo. Based on its abundant-and specific solid tumor expression profile, MOSPD2 may serve as a target for immune-mediated targeting of tumor cells.

Experimental procedure: We have developed CD3xMOSPD2 bi-specific IgG antibodies (BsAb) for killing of MOSPD2+ cancer cells. Binding of the BsAbs to cancer cells and T-cells was validated by flow cytometry. For efficacy, cancer cell lines were co-cultured with effector CD8 T-cells in the presence of CD3xMOSPD2 BsAb or a control IgG. Cancer cell survival and T-cell activation was determined by flow-cytometry and ELISA for IFN-gamma.

Results: Flow-cytometry confirmed surface binding of BsAbs to cancer cell lines and CD8 T-cells. Employing CD3xMOSPD2 IgG BsAb on cancer cell lines induced T-cell activation and yielded profound death of target tumor cells.

Conclusions: CD3xMOSPD2 bi-specific antibodies potentially offer a new immuno-oncology therapeutic tool for treatment of various MOSPD2-positive solid tumors.

Citation Format: Yaniv Salem, Niva Yacov, Oshrat Propheta-Meiran, Pinhas Kafri, Itzhak Mendel, Eyal Breitbart. MOSPD2 a novel target for bi-specific antibody mediated solid tumor cells immune death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5592.
Abstract 5596: Dynamic monitoring of plasma DAMP proteins during multimodal anticancer treatment in advanced lung cancer patients
Authors: Inoue Hiroyuki , Tsutsumi Hirono , Shiraishi Yoshimasa , Okamura Koji , Nakanishi Yoichi , Okamoto Isamu
Published: September 2020

Immunogenic cell death characterized by the release of damage-associated molecular patterns (DAMPs) from dying cancer cells is a potential contributor to the synergistic antitumor effects of the combination of cytotoxic chemotherapy with immune checkpoint blockade. However, little information on the kinetics of circulating DAMP levels in cancer patients has been available. We evaluated the possible effects of various anticancer treatment modalities on the kinetics of plasma DAMP proteins in lung cancer patients. In a prospective observational study, plasma concentrations of three representative DAMP proteins–high-mobility group box 1 (HMGB1), calreticulin (CRT), and histone H3–in 50 patients with advanced lung cancer were measured at five time points during the first cycle of anticancer therapy by ELISA assay. The plasma levels of HMGB1 and CRT increased during the first treatment cycle in 42 (84%) and 35 (70%) patients, respectively, with these increases being detectable as early as day 3. The maximum fold changes in HMGB1 and CRT concentrations was associated with clinical response. Among the various treatment modalities, cytotoxic chemotherapy induced the greatest increases in the concentrations of these DAMPs. There was no correlation between the maximum fold changes in the plasma levels of HMGB1 and CRT. Meanwhile, histone H3 was essentially undetectable in most patients. In conclusion, serial monitoring of plasma revealed that anticancer treatment modalities increased the circulating levels of DAMPs with their association with clinical response, suggesting that agents capable of increasing the release of DAMPs into plasma might induce immunogenic cell death in advanced lung cancer patients.

Citation Format: Hiroyuki Inoue, Hirono Tsutsumi, Yoshimasa Shiraishi, Koji Okamura, Yoichi Nakanishi, Isamu Okamoto. Dynamic monitoring of plasma DAMP proteins during multimodal anticancer treatment in advanced lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5596.
Abstract 5618: Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice
Authors: Guo Yanan , Zhou Xiaofei , Su Youhong , Ng Gary , Guo Chaoshe
Published: September 2020

Interleukin-2 receptor alpha chain (also called as CD25) is a 55 kD glycoprotein, also known as IL-2Ra, Ly-43, p55, or Tac. It is expressed on activated T and B cells, thymocyte subset, pre-B cells, and T regulatory cells. IL-2Ra and IL-2Rβ (CD122), together with the common gamma chain (IL2Rg)(CD132), form the high affinity signaling IL-2 receptor. Its affinity for IL2 and cellular function are tightly regulated and vary in different cell types. The high frequency of IL-2Ra on the surface of many different haematological tumor cells is now well established and, apart from its prognostic significance, IL-2Ra may be present on leukemic stem cells and enable oncogenic signaling pathways in leukemic cells. Additionally, high IL-2Ra expression in activated circulating immune cells and Tregs is a factor that has already been exploited by IL2 immunotherapies for treatment of tumors and autoimmune disease. The relative clinical safety and efficacy of administering anti- IL-2Ra radioimmunoconjugates and immunotoxins in various haematological tumor indications has been established and clinical trials of a novel IL-2Ra-directed antibody drug conjugate are underway.

Biocytogen has generated IL-2Ra humanized mouse for both in vitro functional validation of signaling pathway and in vivo efficacy evaluation of IL-2Ra antibodies. The targeting strategy is that the genomic DNA covering exons 2~6 of mouse IL-2Ra gene which encode the extracellular domain are replaced by the counterpart of human IL-2Ra genomic DNA covering exons 2~6. Human IL-2Ra is mainly detectable on the T cells in spleen of the IL-2Ra humanized mice. The humanized mice are highly similar to the wild-type mice in basal leukocyte subpopulations, including T/B cells, NK cells, DC, granulocytes and monocytes/macrophages. STAT-5 phosphorylation induced by mouse IL2 protein is blocked by anti-human IL-2Ra antibodies, suggesting that the functional pathways in IL-2Ra humanized mice work well. The colon cancer model established in IL-2Ra humanized mice is undergoing, anti-human IL-2Ra antibodies showed good efficacy in inhibiting tumor growth.

Taken together, IL-2Ra humanized mice are a useful tool for in vivo efficacy evaluation of human IL-2Ra antibodies that can be advanced to human clinical trials.

Citation Format: Yanan Guo, Xiaofei Zhou, Youhong Su, Gary Ng, Chaoshe Guo. Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5618.
Abstract 5627: BALB/c-hCD3E transgenic mice to evaluate humanCD3-bispecific antibodies
Authors: Ju Cunxiang , Zhang Mingkun , Jia Dongjing , Mao Rui , Tang Jing , Li Shuai , Zhao Jing , Gao Xiang
Published: September 2020

T cell-mediated immunotherapy is an attractive strategy for treatment of various tumors. T-cell bispecific antibody (TCB) can binds tumor associated antigen (TAA) and CD3 molecule (especially epsilon chain, CD3ϵ), and directs target specific killing of cells carrying the TAA. However, there is a challenge to evaluate the therapeutic efficacy of TCB due to the lack of immune competent animal that have both human immune system and TAA. Considering that CD3ϵ plays a critical role in formation and function of the TCR-CD3 complex while human CD3ϵ (hCD3ϵ) shares only 47% homology with mouse CD3ϵ (mCD3ϵ) in the extracellular domain, we established a CD3ϵ BAC transgenic model (BALB/c-hCD3E) carrying hCD3E gene and its entire regulatory sequence to meet this need. These transgenic mice co-express human and mouse CD3ϵ in over 90% of T-cells. Besides, the expression pattern of hCD3ϵ in spleen and thymus of BALB/c-hCD3E mice are similar with mCD3ϵ in wild type mice. With a normal immune system, splenic T cells from this strain could be activated by either anti-hCD3ϵ or anti-mCD3ϵ antibodies to produce IFNγ, IL2 and TNFα in vitro. We also tested the T cell-dependment cellular cytotoxicity on BALB/c-hCD3E mice use blinatumomab (bscCD19xCD3), the results shown significant anti-tumor effect. It's worth noting that further knockout of mCd3e in BALB/c-hCD3E mice cause remarkable reduce of splenic T cells, strong tumor inhibition of anti-mCTLA4 was observed in BALB/c-hCD3E but not BALB/c-hCD3E/mCd3e-KO mice, demonstrate that mCD3ϵ is indispensable for T-cell development and function in BALB/c-hCD3E transgenic mice. Taken together, these findings indicate that BALB/c-hCD3E mice with intact mCd3e is a novel and valuable model to assessing the therapeutic efficacy of TCBs.

Citation Format: Cunxiang Ju, Mingkun Zhang, Dongjing Jia, Rui Mao, Jing Tang, Shuai Li, Jing Zhao, Xiang Gao. BALB/c-hCD3E transgenic mice to evaluate humanCD3-bispecific antibodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5627.
Abstract 5637: Investigating tumor intrinsic PD-1/PD-L1 signaling in canine osteosarcoma cell lines as a spontaneous model for human disease
Authors: Katarzyna Dziubek , Kocikowski Mikolaj , Vojtesek Borek , Argyle David , Lisowska Malgorzata , Hupp Ted , Parys Maciej
Published: September 2020

There is limited advancement in osteosarcoma therapy in humans. Interestingly, dogs spontaneously develop very similar disease at higher frequency, making canines a perfect model to study OS. Recent introduction of immune checkpoint blockade has revolutionized cancer therapy. Despite huge success, immunotherapy is still not effective in some group of patients or cancer types, and largely ineffective in sarcomas. Until recently, immune-checkpoint therapy was considered to block the interaction between PD-L1 expressed by cancer cells and PD-1 receptor localized on T-cells. Surprisingly, latest studies showed that cancer cells not only express PD-L1 but also PD-1 what is associated with increased growth, proliferation and survival via mTOR pathway signalling. Our group use canine osteosarcoma cell lines to investigate the role of PD-1/PD-L1 intrinsic signalling in disease pathogenesis. Flow cytometry confirmed PD-1 spontaneous expression on many OS cell lines, which ranged between 0.26% and 33.3%. Phenotypic changes were observed in PD-1/PD-L1 transfected over-expressing cells (OE). Strikingly, when grown in 3D culture, over-expressing cells shown accelerated growth and formation of spheres. Upregulation of pS6RP in PD-1 and PD-L1 OE cell lines indicated increased mTOR pathway signaling as demonstrated by Western Blot analysis. To confirm the impact of PD-1 antibody on canine cells, cell viability assay was performed after PD-1 blocking antibody treatment. Interestingly although in many of the cell lines, the addition of the antibody did not alter the growth, in one cell line the growth accelerated, while in another the proliferation decreased. Our data implies that the PD-1/PD-L1 intrinsic signalling may be important in canine osteosarcoma and targeting it could help enhance the therapeutic effect in both dogs and humans.

Citation Format: Dziubek Katarzyna, Mikolaj Kocikowski, Borek Vojtesek, David Argyle, Malgorzata Lisowska, Ted Hupp, Maciej Parys. Investigating tumor intrinsic PD-1/PD-L1 signaling in canine osteosarcoma cell lines as a spontaneous model for human disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5637.
Abstract 5660: Characterisation of novel CD73 antibodies as a therapeutic method of adenosine regulation
Authors: Gernon Grainne , Grooby Suzanne , Tonkin Louise , Bitterwolf Anja-Christina , Stewart Lorna , Deken Marcel , Shah Pritom , Ewings Katherine , Johnson Zoe
Published: September 2020

CD73 is a membrane-bound nucleotidase receptor which is frequently overexpressed in the tumour microenvironment and can be found on both tumour and immune cell types. Its function is to catalyse the conversion of adenosine monophosphate (AMP) to adenosine and phosphate and it has been proposed as a therapeutic target in cancer due to adenosines role in tumour immune suppression.

Using multiple approaches, a series of novel CD73 antibodies have been characterised for their therapeutic potential and benchmarked against agents currently undergoing clinical evaluation Inhibition of CD73 activity was evaluated using an Amplex Red-based coupled adenosine assay against both human and mouse CD73, and kinetics of antibody binding were determined using BioLayer Interferometry. Cellular assays were then utilised to further evaluate the antibodies in vitro. The ability of the CD73 antibodies to internalise was evaluated using two different methods, a Fab-ZAP killing assay and the IncuCyteTM FabFluor internalisation assay. The antibodies have also undergone functional studies, investigating the ability of the CD73 antibodies to disrupt the production of adenosine in tumour cells. We demonstrate that the novel CD73 antibodies inhibit CD73 function by two different mechanisms; direct inhibition of enzyme activity and modulation of cell surface expression, both of a which have therapeutic potential to disrupt CD73-mediated adenosine production and therefore reduce antitumor immune responses.

Ongoing studies are investigating the in vitro and in vivo efficacy of the clone selected for further development as a monotherapy and in combination with a novel small molecule inhibitor in murine models of cancer.

Citation Format: Grainne Gernon, Suzanne Grooby, Louise Tonkin, Anja-Christina Bitterwolf, Lorna Stewart, Marcel Deken, Pritom Shah, Katherine Ewings, Zoe Johnson. Characterisation of novel CD73 antibodies as a therapeutic method of adenosine regulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5660.
Abstract 5663: Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models
Authors: Nisthal Alex , Dragovich Matthew , Pong Erik W. , Zeng Veronica , Hedvat Michael , Bonzon Christine , Avery Kendra , Rashid Rumana , Ardila Connie , Muchhal Umesh S. , Moore Gregory L. , Chu Seung Y. , Desjarlais John R.
Published: September 2020

Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells.

Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5663.
Abstract 5706: Identifying regulators of cancer heterogeneity with phenotypic sorting and single cell sequencing
Authors: Chen Kevin , Fraley Stephanie Irene
Published: September 2020

Intratumoral heterogeneity is a major challenge to the effective treatment of cancer. The genomic, transcriptomic, and epigenetic differences among tumor cells give rise to diverse phenotypes, some of which can persist in dysregulated environmental conditions and survive therapeutic attempts. Single cell sequencing (scSeq) technologies have improved our understanding of the transcriptional variation among tumor cells. However, it remains unclear how cell behavior is related to these alterations. Here, we link genotype to phenotype by employing a method of fluorescently labeling and isolating tumor cell subpopulations based on user-defined phenotypic characteristics from within a biologically relevant three-dimensional (3D) environment for subsequent scSeq and molecular analyses (Pheno-scSeq). We compare this Pheno-scSeq technique to traditional agnostic clustering of scSeq data and find significant differences. We then use Pheno-scSeq to guide our dissection of the mechanistic basis of heterogeneity in the invasive capacity of triple negative breast cancer cells. After seeding cells in 3D collagen and photoconverting based on invasion phenotype, we find that cells undergoing collective invasion are defined by a transcriptional program of migration, anabolism, and proliferation, suggesting a “go and grow” state contrasting with the “go or grow” hypothesis. Cells that do not invade form acini and are defined by a transcriptional program enriched for catabolism, senescence, and immune regulation. Functional assessment of the gene sets validates the relevance of the transcriptional program and reveals differential Human Leukocyte Antigen localization associated with differential migration phenotypes. Treatment with doxorubicin and paclitaxel reveals that acini cells are resistant to drug-induced death, while collectively invasive cells are more susceptible. Modulation of cell-matrix adhesion is explored to determine the potential for reprogramming between the two phenotypes and to identify couplings between migration, immune, and metabolic phenotypes. This study lends deeper insight into the mechanisms underlying phenotypic heterogeneity and may inform the success of precision oncology initiatives.

Citation Format: Kevin Chen, Stephanie Irene Fraley. Identifying regulators of cancer heterogeneity with phenotypic sorting and single cell sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5706.
Abstract 5714: Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma
Authors: Issaq Sameer , Mendoza Arnulfo , Rosales Tracy , Heske Christine , Thomas Craig , DeBerardinis Ralph , Helman Lee
Published: September 2020

Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma (ES), translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact ES cellular metabolism, raising the possibility of targeting metabolic dependencies as a novel therapeutic strategy. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis, is highly expressed in human ES cells and that EWS-FLI1 regulates PHGDH expression and de novo serine synthesis. PHGDH knockdown resulted in decreased ES cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of ES. Additionally, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish ES cellular proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited ES cell proliferation and tumorigenesis, indicating that ES cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for ES tumorigenesis, and that targeting serine metabolism should be further investigated as a potential therapeutic strategy for ES.

Citation Format: Sameer Issaq, Arnulfo Mendoza, Tracy Rosales, Christine Heske, Craig Thomas, Ralph DeBerardinis, Lee Helman. Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5714.
Abstract 5726: A-to-I RNA deaminase deregulation in pre-leukemia stem cell evolution
Authors: Jiang Qingfei , Holm Frida , Isquith Jane , Mark Adam , Mason Cayla , Reynoso Eduardo , Morris Isabella , Ma Wenxue , Diep Raymond , Pham Jessica , Nasamran Chanond , Xu Guorong , Sasik Roman , Rosenthal Sara Brin , Birmingham Amanda , Crews Leslie , Pineda Gabriel , Whisenant Thomas , Fisch Kathleen , Jamieson Catriona
Published: September 2020

While inflammation induced ADAR1 RNA deaminases protect the human genome from retroviral integration, deregulation promotes therapeutic resistance in many malignancies. However, the combinatorial role of these deaminases in pre-leukemia stem cell (pre-LSC) evolution to therapy resistant LSC had not been elucidated. Thus, we performed whole genome sequencing (WGS) analysis of 43 CD34+pre-leukemic myeloproliferative neoplasm (MPN) samples compared with matched saliva and non-MPN controls andwhole transcriptome sequencing (RNA-seq) analysis of 113 FACS-purified hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) from MPN, acute myeloid leukemia (AML) and healthy young and aged samples. During MPN progression, inflammation-driven ADAR1 isoform p150 upregulation corresponded with increased Adenosine-to-Inosine (A-to-I) transitions in both MPN stem and progenitor populations. We identified unique A-to-I editing events in coding regions that are associated with either normal stem progenitors or with MPN progenitors. In addition, STAT3 transcript hyper-editing leads to STAT3beta splice isoform expression. Moreover, lentiviral ADAR1p150 overexpression enhanced replating as well as beta-catenin activation, which was reversed by lentiviral shRNA ADAR1 knockdown. In summary, innate immune deaminase deregulation fuels pre-LSC evolution to LSC and may represent a vital LSC therapeutic vulnerability.

Citation Format: Qingfei Jiang, Frida Holm, Jane Isquith, Adam Mark, Cayla Mason, Eduardo Reynoso, Isabella Morris, Wenxue Ma, Raymond Diep, Jessica Pham, Chanond Nasamran, Guorong Xu, Roman Sasik, Sara Brin Rosenthal, Amanda Birmingham, Leslie Crews, Gabriel Pineda, Thomas Whisenant, Kathleen Fisch, Catriona Jamieson. A-to-I RNA deaminase deregulation in pre-leukemia stem cell evolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5726.