bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4029: Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702)
Authors: Furuta Megumi , Sakakibara-Konishi Jun , Kikuchi Hajime , Yokouchi Hiroshi , Nishihara Hiroshi , Minemura Hiroyuki , Harada Masao , Yamazaki Shigeo , Akie Kenji , Fujita Yuka , Takamura Kei , Kojima Tetsuya , Harada Toshiyuki , Minami Yoshinori , Watanabe Naomi , Oizumi Satoshi , Suzuki Hiroyuki , Nishimura Masaharu , Dosaka-Akita Hirotoshi , Isobe Hiroshi
Published: August 2019

Background:Delta-like protein 3 (DLL3) is a Notch ligand that has an important role in the tumorigenesis of small cell lung cancer (SCLC). Recently, rovalpituzumab tesirine (Rova-T), a DLL3-targeted antibody-drug conjugate, has been developed for treating SCLC and has shown improved clinical response in patients with high DLL3 expression. DLL3 is a transcriptional target of the achaete-scute homolog-1(ASCL1) transcription factor, which is involved in pulmonary neuroendocrine cell development. However, the relationship between DLL3 and/or ASCL1 expression and the clinical features of SCLC remains unknown, especially for early-stage resected SCLC. This study aimed to investigate the expression of DLL3 and ASCL1 in resected SCLC samples using immunohistochemical analysis.

Materials and Methods:We collected 95 formalin-fixed and paraffin-embedded SCLC samples, which were surgically resected at 11 institutions participating in either the Hokkaido Lung Cancer Clinical Study Group Trial (HOT) or the Fukushima Investigative Group for Healing Thoracic Malignancy (FIGHT) between 2003 and 2013. Immunohistochemistrystaining was performed to investigate the correlation between the expression of either DLL3 or ASCL1 and clinicopathological features of study patients.

Results:Seventy-seven (83%) of 93 immunohistochemically evaluable samples were positive for DLL3 (expression in ≥ 1% of tumor cells), and DLL3-high expression (≥ 75%) was observed in 44 samples (47%). Sixty-one (64%) of 95 samples were positive for ASCL1 (expression in ≥ 5% of tumor cells). A positive correlation was observed between DLL3 and ASCL1 expression. DLL3 and ASCL1 expression were not associated with survival in SCLC patients. DLL3 and ASCL1 were more prevalent in patients with advanced clinical disease and pure SCLC, respectively.

Conclusion:DLL3 and ASCL1 were highly expressed in surgically resected SCLC patients.

Citation Format: Megumi Furuta, Jun Sakakibara-Konishi, Hajime Kikuchi, Hiroshi Yokouchi, Hiroshi Nishihara, Hiroyuki Minemura, Masao Harada, Shigeo Yamazaki, Kenji Akie, Yuka Fujita, Kei Takamura, Tetsuya Kojima, Toshiyuki Harada, Yoshinori Minami, Naomi Watanabe, Satoshi Oizumi, Hiroyuki Suzuki, Masaharu Nishimura, Hirotoshi Dosaka-Akita, Hiroshi Isobe. Analysis of DLL3 and ASCL1 in surgically resected small cell lung cancer (HOT1702) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4029.
Abstract 4039: Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy
Authors: Krupa Rachel , Richardson Robin , Ontiveros Priscilla , Schonhoft Joseph , Byun Jiyun , Lu David , Nisperos Sean , Wang Yipeng , Landers Mark , Dittamore Ryan
Published: August 2019

Purpose: Activated immune cell subpopulations and circulating tumor cells in patients receiving checkpoint inhibitor therapy were characterized with the aim of developing more sensitive diagnostic tools for prediction of immunotherapy response or resistance.

Methods: Blood samples from bladder, kidney, and prostate cancer patients undergoing checkpoint inhibitor therapy were collected at baseline and on-therapy at convenience and sent to Epic Sciences for processing. Red blood cells were lysed and nucleated cells were plated onto glass slides and stained with CTC (pan-CK, CD45, PD-L1 and DAPI) and immune panels (CD4, CD8, Ki-67, and DAPI). 2-3 million cells per patient were imaged using high throughput digital pathology pipelines and changes in immune cell sub-populations and changes in circulating tumor cell (CTC) burden with PD1/PD-L1 immunotherapy were assessed.

Results: A method to profile activated (CD8+Ki-67+ and CD4+Ki-67+) leukocyte subpopulations and PD-L1+ CTCs in a single blood sample was developed and feasibility was demonstrated in cell lines, healthy donor (HD), and patient blood. CTCs were detected in 73% (24/33) of patients tested. Of the 33 baseline samples tested, 12% (4/33) had PD-L1+ CTCs detected. No PD-L1+ CTCs were detected in on-therapy samples. Of 14 patients with matched samples, 57% (8/14) patients had an increase in activated CD4+ leukocytes and 36% (5/14) patients had an increase in activated CD8+ leukocytes in on-therapy samples compared to baseline. A decrease in activated CD4+ leukocytes was observed in 21% (3/14) patients and a decrease in activated CD8+ leukocytes was also observed in 7% (1/14) patients with therapy.

Conclusions: We developed a liquid biopsy-based platform that can simultaneously measure immune related biomarkers in CTCs and leukocytes at single cell resolution from ~1 mL of blood. The platform has extreme sensitivity over conventional methodologies and immune cell populations existing in less than 1% of the total are readily quantified. Efforts to identify morphological sub-populations within each canonical leukocyte category and comparison of leukocyte and CTC biomarker panels to patient outcomes are ongoing.

Citation Format: Rachel Krupa, Robin Richardson, Priscilla Ontiveros, Joseph Schonhoft, Jiyun Byun, David Lu, Sean Nisperos, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4039.
Abstract 4071: A novel combination therapy of high intensity focused ultrasound and PDL1 blockades against advanced breast cancer
Authors: Abe Shinya , Osada Takuya , Kaneko Kensuke , Zhong Pei , Lyerly Herbert K.
Published: August 2019

Background: Previous studies have reported that tumor debris and inflammation made by high-intensity focused ultrasound (HIFU) therapy induced the antitumor immune response, however, HIFU as a monotherapy is still not potent enough to eradicate tumors and treat distant metastasis. We have reported that mechanical HIFU (M-HIFU), that will mechanically destroy tumor cells/tissues through acoustic cavitation, induced stronger antitumor immune response compared to conventional thermal HIFU (T-HIFU) that will cause thermal ablation of tumors. In the present study, we established an immune based combination therapy of M-HIFU and immune checkpoint blockade to enhance systemic antitumor immune response and treated distant/metastatic tumors in murine breast cancer models.

Methods and Results: HER2 oncogene-dependent murine breast cancer cell line, MM3MG-HER2, was established in our lab. In mice with bilateral implantation of MM3MG-HER2 tumors, M-HIFU monotherapy induced stronger HER2-specific cellular response and inhibited the growth of HIFU-untreated distant tumors as well as HIFU treated-tumors, more potently than T-HIFU. Flow cytometry and immunohistochemical analysis of tumor microenvironment revealed significantly stronger accumulation of activated T cell and NK cells in M-HIFU-treated tumors. On the other hand, M-HIFU induced stronger expression of programmed death-ligand 1 (PD-L1) on various immune cells in both sides of tumors than T-HIFU therapy or no treatment control. Based on these findings, we investigated the combination therapy of M-HIFU and PD-1/PD-L1 axis blockades, and found significantly enhanced tumor-specific cellular immune response compared to each monotherapy, which resulted in improved therapeutic effect against distant tumors as well as HIFU treated-tumors. Furthermore, immune cell depletion studies demonstrated that both CD8+ T cells and NK cells played an essential role for the antitumor efficacy in this combinatory therapy against both HIFU-treated tumors and untreated distant tumors.

Conclusion: this study provides strong rational evidence that M-HIFU combined with PD-1/PD-L1 axis blockades could be a promising treatment strategy against advanced breast cancer with metastatic lesion.

Citation Format: Shinya Abe, Takuya Osada, Kensuke Kaneko, Pei Zhong, Herbert K. Lyerly. A novel combination therapy of high intensity focused ultrasound and PDL1 blockades against advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4071.
Abstract 4086: Preclinical evaluation of pharmacokinetics, pharmacodynamics and efficacy of the dual CD73-A2AR Inhibitors
Authors: Chikkanna Dinesh , Narayanan Kishore , Panigrahi Sunil , Priyadarshini Garima , Goyal Megha , Mani Jiju , Samiulla DS , dadhania Sagar , Aravind AB , Daginakatte Girish , Anthony Thomas , Nellore Kavitha , Samajdar Susanta , Ramachandra Murali
Published: August 2019

As a potent immunosuppressor adenosine is essential for maintaining tissue homeostasis and preventing an overzealous immune response during inflammation and infection. However, adenosine generated within the tumor microenvironment by the action of ecto-nucleotidases including CD73 hinders the immune reaction towards cancer cells by signaling through adenosine receptors such as high affinity A2AR expressed on immune cells. Inhibitions of either adenosine generation or signaling by inhibiting CD73 or A2AR have been shown to be effective therapeutic approaches. Interestingly, the co-blockade of CD73 and A2AR results in a more pronounced anti-tumor activity than blockade of either, likely due to increased CD73 expression upon A2AR inhibition and compensatory activity of other adenosine receptors such as A2BR. In view of this, we sought to discover and develop small molecule inhibitors that dually target CD73 and A2AR with oral bioavailability for ease of administration and use in combination with other anti-cancer therapies. Aurigene’s dual inhibitors exhibit potent inhibition of both A2AR and CD73 in respective biochemical and cellular assays. High potency translated into rescue of NECA or AMP induced repression of IFNΥ and IL2 in human PBMCs. Lead compounds exhibited desirable drug-like properties including solubility, permeability, lack of CYP inhibition, and pharmacokinetic exposure. In Syngeneic tumour model, treatment with lead compounds resulted in significant tumour growth inhibition while correlating well with tumour drug levels and modulation of pharmacodynamic markers. In summary, we have identified first-in-class dual inhibitors with good drug like properties, which showed significant antitumor efficacy. Evaluation of these lead compounds in additional tumour models and in vivo toxicity studies is currently under way.

Citation Format: Dinesh Chikkanna, Kishore Narayanan, Sunil Panigrahi, Garima Priyadarshini, Megha Goyal, Jiju Mani, DS Samiulla, Sagar dadhania, AB Aravind, Girish Daginakatte, Thomas Anthony, Kavitha Nellore, Susanta Samajdar, Murali Ramachandra. Preclinical evaluation of pharmacokinetics, pharmacodynamics and efficacy of the dual CD73-A2AR Inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4086.
Abstract 4089: Antitumor activity of eribulin in combination with anti-PD1 antibody in a mouse syngeneic breast cancer model
Authors: Semba Taro , Tabata Kimiyo , Ozawa Yoichi , Miyano Saori Watanabe , Matsui Junji , Funahashi Yasuhiro
Published: August 2019

Eribulin is a synthetic analog of halichondrin B, which is isolated from the marine sponge natural product, as the first halichondrin class of microtubule dynamics inhibitors. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. Eribulin shows antitumor activity through cell death induction by an antimitotic effect, and modulation of tumor microenvironment that include vascular remodeling, and reversal of EMT in some of preclinical subcutaneous xenograft models. Effects of eribulin on tumor microenvironment led us to test combination of eribulin with anti-PD1 blockades, and Phase2 clinical study of eribulin in combination with pembrolizumab for metastatic BC patients is in progress. In this study, we investigated antitumor activity of eribulin in combination with anti-PD1 mAb in preclinical mouse BC model using 4T1 cells. Because parent 4T1 cells express P-glycoprotein (Pgp) and eribulin is a substrate of Pgp, we established Pgp-knockout (Pgp-KO) mouse 4T1 BC cells to examine combination antitumor activity of eribulin with anti-PD1 antibody. We used a clone 31 of Pgp-KO 4T1 (#31) cells, which maintains similar tumor phenotypes to parent 4T1 orthotropic transplantation (OT) models, such as growth rate and formation of metastasis. Eribulin inhibited in vitro proliferation of Pgp-KO 4T1 (#31) cells with a lower concentration compared to parent 4T1 cells. Eribulin were intravenous administered at 1 mg/kg with either Q7Dx2 or Q4Dx3 in the combination with anti-PD1 mAb, 200 µg/head, Q3Dx10 in the Pgp-KO 4T1 (#31) OT model (6 mice per a treatment group). Anti-PD1 mAb did not clearly show in vivo antitumor activity. Treatments of eribulin by Q4Dx3 showed enhanced antitumor activity in the combination compared to each single treatment, and all tumors became non-palpable in the combination group. On the other hand, weekly treatments of eribulin did not enhance antitumor activity in the combination with anti-PD1 mAb, although eribulin single treatment significantly inhibited in vivo tumor growth of Pgp-KO 4T1 (#31) tumors. These preclinical results provide preclinical rationale for clinical testing of eribulin in combination with anti-PD1 blockades in patients with BC, and further preclinical analysis of MOA of combination antitumor activity of eribulin with anti-PD1 antibody is warranted.

Citation Format: Taro Semba, Kimiyo Tabata, Yoichi Ozawa, Saori Watanabe Miyano, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of eribulin in combination with anti-PD1 antibody in a mouse syngeneic breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4089.
Abstract 4091: Evaluating the efficacy of a STING agonist in a murine model of prostate cancer
Authors: Gurkan Jonathan , Choi Jae Eun , Tien Jean , Cieślik Marcin , Franchi Luigi , Chinnaiyan Arul M.
Published: August 2019

Despite the promise of checkpoint inhibitor therapy, prostate cancer has remained resistant to this treatment. Innate immune agonists, however, have been shown to have anti-tumor effects in various cancer types, such as melanoma and colon cancer, and have been nominated to be used in combination with approved anti-PD1/PD-L1 drugs. For instance, STING agonist + anti-PD-1 combination therapy has been demonstrated in a syngeneic mouse model of melanoma. Other innate immune agonists, such as TLR7/8 and TLR9 have also been shown to have potential anti-tumor effects. We hypothesized that innate immune agonists may be effective in the MycCaP syngeneic mouse model of prostate cancer. To compare the efficacy of three innate agonists targeting the STING (3’3’ cGAMP), TLR7/8 (CL097), and TLR9 (ODN2395) pathways, FVB mice were first injected bilaterally with MycCaP subcutaneous tumors. Upon establishment of tumors, mice were administered 3 doses of a single-sided intratumoral injection of each drug. The injected tumors had responses of 50% (6/12), 23% (3/13), and 8% (1/13) for the STING, TLR7/8 and, TLR8 agonists, respectively. Contralateral tumors showed no significant regression.

Published data have reported inherent resistance of MycCaP tumors to anti-PD-1 treatment in vivo. Given our results, we hypothesize that the addition of a STING agonist could enhance efficacy of anti-PD-1 therapy in this model. Mice with MycCaP tumors were administered anti-PD1 alone (n=10) or in combination with bilateral injections of the STING agonist (n=14). Combined therapy resulted in significant reduction in tumor size (71%) compared to anti-PD1 alone (9%). Additionally, there was a significant increase in the Ifnb1 (p=.04), Ifng (p=.008), Tnf-α (p=.006), IRF3 (p=.003) and II6 (p<.0001) gene expression in the combination group showing enhancement of the STING pathway genes. Combined, these findings suggest that targeting the STING pathway may have a modest local anti-tumor effect when compared to other innate immune pathways in prostate cancer. The efficacy of anti-PD-1 therapy was significantly enhanced when combined with a STING agonist, possibly through enhancement STING pathway genes. Our data suggest that combination therapy with a STING agonist and anti-PD1 therapy may have potential anti-tumor efficacy in prostate cancer. Future studies will examine these effects on both local and abscopal tumors.

Citation Format: Jonathan Gurkan, Jae Eun Choi, Jean Tien, Marcin Cieślik, Luigi Franchi, Arul M. Chinnaiyan. Evaluating the efficacy of a STING agonist in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4091.
Abstract 4094: The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo
Authors: Lu Hailing , Betancur Alec , Meulen Jan ter
Published: August 2019

Background: G100, which contains the synthetic TLR4 agonist Glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water emulsion (SE) for intratumoral therapy, is currently in clinical development in combination with pembrolizumab for the treatment of B-cell follicular lymphoma (FL). The current study was undertaken to investigate the interaction of G100 with rituximab, the anti-CD20 mAb that is standard-of-care treatment for FL.

Methods: In vitro studies were performed using peripheral blood mononuclear cells (PBMC) from healthy human donors to investigate the effect of GLA-AF (an aqueous formulation of GLA) on natural killer (NK) cell activation. IFNγ production in NK cells after activation with plate-bound anti-CD16 or target K562 leukemia cells was measured by intracellular cytokine staining (ICS). Rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) was measured using pretreated or untreated PBMC and rituximab-coated Raji Burkitt’s lymphoma cells as target cells. In vivo studies were carried out in Balb/c mice with bilateral A20 lymphoma. Mice received G100 alone (10μg, intratumoral, 3 injections/week), anti-CD20 alone (clone 5D2, provided by Genentech, 200μg, intraperitoneal, 1 injection/week), or G100+anti-CD20 for a total of 3 weeks.

Results: Incubation of PBMC from 3 different donors with GLA-AF (1 μg/mL) resulted in NK cell activation as demonstrated by increased IFNγ production, as well as enhanced response to the K562 target cells and anti-CD16. When PBMC from 5 normal donors were used in rituximab-mediated ADCC assay, pretreated PBMCs had significantly enhanced lysis of target Raji cells compared to untreated PBMC (p<0.05). In mice with bilateral A20 tumors, the combination of G100 with anti-CD20 resulted in significantly better tumor control and prolonged survival (n=5 per group, p<0.05).

Conclusions: G100 enhances NK cell activity and rituximab-mediated ADCC in human PBMC. Combination of G100 and anti-CD20 mAb has synergistic anti-tumor effects in a mouse lymphoma model. These data support clinical testing of G100 with rituximab in patients with FL.

Citation Format: Hailing Lu, Alec Betancur, Jan ter Meulen. The TLR4 agonist G100 enhances rituximab-mediated ADCC in vitro and has synergistic anti-tumor effects with anti-CD20 mAb in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4094.
Abstract 4100: Receptor occupancy and neo-epitope specific T-cell repertoires in patients with solid tissue tumors following anti-PD-1 therapy
Authors: Ashok Devika , Peng Songming , Yuen Benjamin , Walters Matt , Young Stephen W. , Seitz Lisa
Published: August 2019

Background:

In patients undergoing chemotherapy and/or immune therapy, endogenous T cells within the tumor micro-environment (TME) and in the periphery are subject to dynamic changes. Particularly, exhausted T cells in the TME and peripheral blood mononuclear cells (PBMCs) isolated from whole blood express the inhibitory receptor, Programmed Cell Death Receptor 1 (PD-1). A small but significant number of these T cells can recognize neo-epitopes (neoE) arising from patient specific mutations. We determined receptor occupancy (RO) in patients treated with monoclonal anti-PD-1 antibody AB122, which is currently in clinical development.

Methods: Multi-color flow cytometry was used to determine RO post-AB122 administration using a directly-conjugated competitive antibody to AB122. In addition, we determined RO values using the established method for nivolumab using a biotinylated anti-human IgG4 to detect bound anti-PD-1. The assays were also optimized to determine RO in whole blood from patients with solid tumors treated with AB122. Receptor occupancy using both methods were compared. Further, whole exome sequencing from archival formalin-fixed, paraffin-embedded (FFPE) tissue was performed and T cells that recognized patient-specific neoE resulting from tumor-specific mutations were identified in patient blood.

Results:

In the dose-escalation study for AB122 monotherapy arm, a variety of dosing regimens have been evaluated, including Q2W, Q3W and Q4W. The number of doses received ranged from 1 to 20. Data from patients in the 80 and 240 mg Q2W cohorts showed that AB122 serum concentrations ≥1.5 μg/mL (equivalent to 10 nM) are associated with full receptor occupancy. The RO associated with AB122 dosing is maintained when dosed in combination with the potent A2aR/A2bR dual adenosine receptor antagonist AB928 in an ongoing clinical trial.

Conclusions:

Full RO is observed with both cohorts of AB122 monotherapy (80 and 240 mg Q2W) and in the 240 mg Q2W combination with AB928. Study of peripheral target engagement and expansion of neoE-specific T cell populations in a longitudinal study of solid tumor patients treated with AB122 could provide insight towards designing successful combination immune therapy regimens in future clinical trials with AB122.

Citation Format: Devika Ashok, Songming Peng, Benjamin Yuen, Matt Walters, Stephen W. Young, Lisa Seitz. Receptor occupancy and neo-epitope specific T-cell repertoires in patients with solid tissue tumors following anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4100.
Abstract 4117: Novel bead-based multiplex assay for the quantification of human immune checkpoint biomarkers
Authors: Lehmann Jason , Rughwani Priyanka , Zhao Amy , Jiang Weiping , Ji Shaoquan , Sun Binggang
Published: August 2019

Immune checkpoint molecules are important regulators of immune activation. This regulation is critical to maintaining immune homeostasis, and is driven by a complex interplay of both co-stimulatory and inhibitory signals. These checkpoints are key to preventing autoimmunity in healthy individuals; however, cancer cells often exploit this system by modulating the expression of these molecules in order to suppress anti-tumor responses during host immuno-editing. The ability to accurately quantify expression levels of checkpoint molecules is critical to ongoing biomedical research efforts exploring potential therapeutic interventions to overcome tumor-mediated immune-evasion.

To further aid these ongoing research efforts, we have developed a new bead-based multiplex assay panel specifically designed to quantify several human oncology-related immune checkpoint biomarkers including sCD25 (IL-2Ra), GITR, 4-1BB, sCD27, B7.2 (CD86), free active TGF-β1, CTLA-4, PD-L1, PD-L2, PD-1, TIM-3, LAG-3, Galectin-9. The panel utilizes fluorescence-encoded microsphere technology and is designed for use on commonly available flow cytometers. The panel has been extensively validated for specificity, dilution linearity, cross-reactivity, and inter- and intra-assay precision.

This multiplex panel is a robust tool for measuring the concentrations of several immune checkpoint mediators in human serum, plasma, and cell culture supernatant samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA-based quantification methods.

Citation Format: Jason Lehmann, Priyanka Rughwani, Amy Zhao, Weiping Jiang, Shaoquan Ji, Binggang Sun. Novel bead-based multiplex assay for the quantification of human immune checkpoint biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4117.
Abstract 4118: Molecular cytometry for immunotherapy trials
Authors: Lomas Woodrow E. , Winters Aidan F. , Alexandre Jason , Martin Jody , Bansal Nidhanjali , Nakamoto Margaret , Gaylord Brent , Saksena Suraj , Chattopadhyay Pratip K.
Published: August 2019

The ability to simultaneously query both protein and mRNA expression on tens of thousands of single cells has emerged only recently, with the development of CITE-seq, REAP-seq, and Ab-seq platforms. Each technology relies on antibodies conjugated to oligonucleotide tags, followed by capture of antibody-stained cells for single cell RNA-sequencing. The technologies have important advantages over flow cytometry, chiefly in that they allow study of a limitless number of parameters, and single cell RNA sequencing, which cannot identify cell populations with as much verity as protein/antibody-based analysis. We characterized the newest molecular cytometry technique (Ab-seq, BD Biosciences) by titrating nearly 100 antibodies (in a single staining experiment and sequencing run), and demonstrated that oligonucleotide antibodies exhibit the same staining characteristics and patterns as flow cytometry antibodies. We also comprehensively examined cells discordant for protein and mRNA expression, in order to discriminate true features of immune cell biology from artifacts caused by drop-out in sequencing assays. Importantly, we also found that mRNA expression could guide the discrimination of cells expressing a protein versus those lacking expression, and define a remarkably low limit of detection for the technology: 1-5 molecules. This remarkable sensitivity will allow detection of cells expressing even the lowest levels of immune exhaustion and checkpoint molecules in cancer immunotherapy trials, and could enable better biomarker discovery than existing technologies. Moreover, this approach allows more detailed characterization of the tumor microenvironment and periphery than ever before. Indeed, in a lymphoma patient, we show that cells expressing both PD1 and CTLA4 carry a unique and specific signature that includes markers never revealed by flow cytometry or other technologies, which might represent new targets for immunotherapy. In sum, this presentation will characterize this new technology and demonstrate the power and promise of new molecular cytometry tools.

Citation Format: Woodrow E. Lomas, Aidan F. Winters, Jason Alexandre, Jody Martin, Nidhanjali Bansal, Margaret Nakamoto, Brent Gaylord, Suraj Saksena, Pratip K. Chattopadhyay. Molecular cytometry for immunotherapy trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4118.
Abstract 4126: Inferring target occupancy from fitting nonlinear-PK data with mechanistic PKRO model for Pembrolizumab
Authors: Hua Fei , Lin Lin , Gruenbaum Lore , Millard Bjorn L. , Burke John M. , Apgar Joshua F.
Published: August 2019

Purpose: Pembrolizumab (Pembro), an anti-PD-1 antibody, has been approved for several cancer indications at 200 mg or 2 mg/kg IV every 3 weeks (Q3W). Understanding the percent target receptor occupancy (RO) for PD-1 at this approved dose can help with dose selection for other anti-PD-1 drugs in development. However, RO can be challenging to determine through direct experimental measurements. A mechanistic PKRO model was developed to fit to Pembro phase 1 PK data and was then used to predict RO at 2 mg/kg IV dosing Q3W.

Methods: A mechanistic PKRO model describes linear clearance of Pembro, tissue distribution of Pembro, binding to PD-1, binding to soluble PD-1 (sPD-1) and clearance of drug-PD-1 complex, clearance of drug-sPD-1 complex as well as drug distribution to tumor and binding to PD-1 in the tumor compartment. Binding between Pembro and PD-1 was fixed to 29 pM (Pembro BLA, Pharmacology Review) and half-life of internalization of drug-PD-1 complex was set to 2 hours.

Results: Since a wide range of sPD-1 concentration has been reported, different sPD-1 concentration was used to fit to the PK data. The model predicted PD-1 expression level in circulation to be approximately 10.8 pM with no soluble PD-1. In the presence of soluble PD-1, PD-1 expression is predicted to be higher to capture the non-linear PK. The model was then used to predict RO at 2 mg/kg IV dosing Q3W and > 98% RO was predicted in the tumor compartment with a range of sPD-1 concentrations from 0 to 3000 pg/mL tested. The impact of Kd and drug-PD-1 turnover rate on predicted RO was explored. When either of the parameters was varied by 10-fold higher or lower from its nominal value, the model still predicted greater than 90% RO at 2 mg/kg dose.

Conclusion: A mechanistic PKRO model was built to capture Pembro phase I PK data and then predicted near complete PD-1 target receptor occupancy at the approved dose. This approach with expanded model scope has the potential to be used to inform dose selection for other anti-PD-1, or I/O therapeutics, as a single therapy or in combination.

Citation Format: Fei Hua, Lin Lin, Lore Gruenbaum, Bjorn L. Millard, John M. Burke, Joshua F. Apgar. Inferring target occupancy from fitting nonlinear-PK data with mechanistic PKRO model for Pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4126.
Abstract 4139: Q702, selective Axl, Mer and CSF1R triple kinase inhibitor with dual potentials leading to tumor regression: Immuno-oncology therapy and targeted cancer therapy
Authors: Yang Yeong-In , Kang Hwankyu , Park Dongsik , Jeon Yeejin , Kim Jeongjun , Choi Baejung , Kim Jaeseung , Rho Jinkyung , Lee Jaecheol , Nam Kiyean
Published: August 2019

Functional composition of tumor micro-environment (TME) has been regarded as one of the most important factors in tumor progression. Cancer cells induce immune suppressive condition in TME in order to evade immune system as well as to promote metastasis. Axl, Mer and CSF1R play important roles in making tumor friendly TME by increasing regulatory T cells (Tregs), M2 macrophages, myeloid-derived suppression cells (MDSCs) and suppressing antigen presentation. On the other hand, in cancer cells, overexpression of Axl is reported to have correlation with poor prognosis of patients through promoting epithelial-to-mesenchymal transition (EMT) and inducing drug resistance to chemo or targeted therapy. Q702 is an orally available selective Axl, Mer and CSF1R triple kinase inhibitor under IND enabling studies. In this poster, we present dual potentials of Q702 leading to tumor regression through: 1) immune stimulating activities of Q702 through decreasing Tregs, M2 macrophages, MDSCs population and promoting antigen presentation, 2) direct cytotoxic activity in tumor models including acute myeloid leukemia (AML) and EGFR TKI resistant non-small cell lung cancer (NSCLC).

Citation Format: Yeong-In Yang, Hwankyu Kang, Dongsik Park, Yeejin Jeon, Jeongjun Kim, Baejung Choi, Jaeseung Kim, Jinkyung Rho, Jaecheol Lee, Kiyean Nam. Q702, selective Axl, Mer and CSF1R triple kinase inhibitor with dual potentials leading to tumor regression: Immuno-oncology therapy and targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4139.
Abstract 4140: Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy
Authors: Lee Kyungik , Jun Seungah , Byun EunYoung , Lee Hosun , Lee Yongtaek , Moon MiJin , Kim Yu-Yon , Kang Hyun Jeong , Ahn YoungGil , Kim YoungHoon , Suh Kwee Hyun
Published: August 2019

Introduction: Extracellular adenosine produced at high concentrations within the tumor micro-environment (TME) and suppresses immune function via inhibition of immune cell activation. Targeting adenosine receptors has emerged as a novel method to activate anti-tumor immunity. In particular, A2A adenosine receptor (A2AR), one of the G-protein-coupled-receptors, exhibits immunosuppressive functions. Herein, we suggest novel A2AR antagonist lead compounds as a highly potent antagonist of A2AR for immunotherapy.

Materials and Methods: Novel A2AR antagonist lead compounds were designed using CADD and synthesized as the active biologic inhibitory compound. The protein preparation and molecular docking were performed using Glide (Schrödinger). A radioligand binding assay was performed to evaluate the affinities of A2AR Antagonists for the human adenosine A2AR. The potencie of A2AR antagonists were determined by cAMP assay in HEK293-hA2AR cells and cAMP-mediated pCREB assay in human CD8+ T cells from whole blood. In vivo CT26 and MC38 syngeneic tumor models were used to assess the therapeutic effect of A2AR antagonists.

Results: A2AR antagonist lead compounds showed strong binding affinities toward human A2AR. They also potently inhibited the NECA-mediated production of intracellular cAMP in HEK293 cells expressing human A2AR. Elevated intracellular cAMP following A2AR activation results in the phosphorylation of CREB (cAMP response element-binding protein). A2AR antagonist lead compounds treatment inhibited pCREB in NECA-stimulated HEK293-hA2AR and human CD8+ T cells. A2AR antagonist lead compounds inhibited tumor growth of mouse syngeneic tumor models as a single agent and combination with anti-PD-L1. In combination with anti-PD-L1, A2AR antagonist lead compounds had remarkable antitumor activities in multiple mouse tumor models, including restoration of immune responses in models that incompletely responded to anti-PD-L1 monotherapy.

Conclusion: These results showed the potencies of A2AR antagonist lead compounds with high capability of A2AR inhibition. Blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-PD-L1 therapy. A2AR antagonist lead compounds demonstrate a novel approach to anti-cancer immunotherapy.

Citation Format: Kyungik Lee, Seungah Jun, EunYoung Byun, Hosun Lee, Yongtaek Lee, MiJin Moon, Yu-Yon Kim, Hyun Jeong Kang, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4140.
Abstract 4181: Awareness of human papillomavirus (HPV) infection among men and women with oral cancer
Authors: Miller Assia , Rollins Latrice , Banerjee Ananya
Published: August 2019

Background There is a high level of public awareness of association human papillomavirus (HPV) infection and genital cancer. Though recent research showed that 26 million Americans have oral HPV infection, the information on awareness of association HPV infection with oral cancer among men and women is limited.

Methods The objective of our study was to assess population awareness of and knowledge about the association between HPV and oral cancer among men and women. We obtained cross-sectional data from the National Cancer Institute's Health Information National Trends Surveys (HINTS 2007-2015, N=25,410). We applied a Wald chi-square test to examine the association of HPV awareness in participants with oral cancer compared to participants with other forms of cancer stratified by gender.

Results: Among men with oral cancer (N=10) 16 % were aware of HPV while among men with other forms of cancer (N=3557) 44% were aware of HPV (p=0.0924). Among women with oral cancer (N=5) 56 % were aware of HPV while among women with other forms of cancer (N=5631) 64% were aware of HPV (p=7971).

Conclusion: There is decreased awareness and knowledge of HPV in men with oral cancer promoting opportunities to target communication about a vaccine.

Citation Format: Assia Miller, Latrice Rollins, Ananya Banerjee. Awareness of human papillomavirus (HPV) infection among men and women with oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4181.
Abstract 4187: Racial disparities in kidney cancer incidence across South Texas, Texas, and United States using TCR, SEER and NPCR registries (2004-2014)
Authors: Perez Casey , Shah Pankil K. , Mathews Aaron , Munoz Edgar , Bokov Alex , Kaushik Dharam , Michalek Joel , Ramirez Amelie , Rodriguez Ronald , Shah Dimpy P.
Published: August 2019

Introduction: Despite overall decrease in incidence of cancers in U.S., kidney cancer is one out of the only seven cancers with increasing incidence. Limited research has been conducted using national, state-wide, and regional data, especially comparing racial and ethnic disparities in incidence and outcomes for kidney cancer.

Methods: Kidney cancer incidence and outcomes from 2004 to 2014 were compared using Texas Cancer Registry, National Program for Cancer Registries, and Surveillance, Epidemiology and End Results. Age-adjusted incidence rates (per 100,000 population) and temporal trends, reporting sources, diagnosis, patient and tumor characteristics between Hispanics and Non-Hispanic Whites (NHWs) were compared for each state. The Kaplan-Meier method was used for relative survival analyses, which were limited to Texas due to data availability.

Results: A total of 566,716 patients were identified. The temporal trend shows a sustained increase in kidney cancers in U.S. and a consistently higher incidence in TX and South Texas (STX) over the past decade, with Hispanics in particular having the highest annual percent change (APC) among the 20-29 year old age group (5.3 in U.S. and 7.7 in TX, respectively). Age-adjusted kidney cancer incidence was 26.2 in STX, 24.5 in TX and 21.8 in U.S. Both males and females had a higher incidence in STX and TX compared to U.S. (35.1, 32.5 vs. 29.6 for males and 18.8, 17.9 vs.15.2 for females). Overall, Hispanic males in STX had the highest kidney cancer incidence (37.7) throughout U.S. The difference in incidence rate in Hispanics compared to NHWs was significant in STX (4.9) and TX (3.4), compared to U.S. (-0.9). Tumor grade was only available in 60% of cancers in TCR compared to 70% in U.S. Despite localized tumor stage being the most common presentation for all regions, the 5-year survival was lower in Hispanics compared to NHWs in STX (68% vs. 73%) and TX (69% vs. 71%).

Conclusions: Kidney cancer rates were higher in STX and TX when compared to U.S., especially among Hispanic males. Relative 5-year survival between Hispanics and NHWs were similar in TX and STX. Improvement in cancer reporting and detailed studies examining causes for racial/ethnic disparities in incidence in the top 15 states with highest disparities, especially TX, are needed.

Citation Format: Casey Perez, Pankil K. Shah, Aaron Mathews, Edgar Munoz, Alex Bokov, Dharam Kaushik, Joel Michalek, Amelie Ramirez, Ronald Rodriguez, Dimpy P. Shah. Racial disparities in kidney cancer incidence across South Texas, Texas, and United States using TCR, SEER and NPCR registries (2004-2014) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4187.
Abstract 419: Single cell-driven secreted proteins as a surrogate biomarker for Cetuximab therapy in colorectal cancer
Authors: Park Ye-Lim , Kim Hwang-Phill , Kang Jun Kyu , Lim Yoo Joo , Song Sang-Hyun , Han Sae-Won , Kim Tae-You
Published: August 2019

Epidermal growth factor receptor (EGFR) blockade is efficacious in KRAS wild-type of colorectal cancer, but acquired resistance is inevitably generated. Here, we established the 5 acquired resistant single cell models (CR cell lines) to cetuximab and investigated the mechanism of acquired resistance using single cell-based whole-exome sequencing and transcriptional profiling. Of these, 4 cell lines have KRAS Q61L mutation and the other has BRAF V600E mutation. From gene enrichment analysis (GSEA), we found that epithelial to mesenchymal transition (EMT) occurs and CXCL chemokine family, which are known as secreted proteins, are overexpressed in the CR cell lines compared with parental cells. Interestingly, we found that CXCL chemokines were strongly secreted/expressed in KRAS/BRAF mutant cell lines and plasma/serum samples from colorectal cancer patients who acquired KRAS/BRAF mutation during cetuximab therapy. CXCL chemokines are regulated coordinately by Slug and NF-B transcription factors and trans-activate EGFR through CXCR2/MMP1/sHB-EGF axis. This process activates MAPK pathway via autocrine. To overcome the resistance to cetuximab, combination treatment of cetuximab and MEK/BRAF inhibitor had a larger antitumor effect compared to the single agents, decreasing the expression of MAPK pathway and secretion of CXCL chemokines. This finding highlights that CXCL chemokines, as a secreted proteins circulating in the bloodstream, show correlation with KRAS/BRAF mutation and could be clinical biomarkers for predicting resistance to anti-EGFR therapy in CRC.

Citation Format: Ye-Lim Park, Hwang-Phill Kim, Jun Kyu Kang, Yoo Joo Lim, Sang-Hyun Song, Sae-Won Han, Tae-You Kim. Single cell-driven secreted proteins as a surrogate biomarker for Cetuximab therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 419.
Abstract 4197: Long-term follow-up of a racially and ethnically diverse population of patients with localized prostate cancer who did not undergo initial active treatment
Authors: Slezak Jeff , Eeden Stephen Van Den , Cannavale Kimberly L. , Chein Gary W. , Chao Chun R.
Published: August 2019

Background: Long-term studies of men who were not initially treated for prostate cancer have been limited, especially among ethnic minorities. We sought to provide detailed long-term outcomes of a large, diverse cohort of men diagnosed with prostate cancer between 1997 and 2007 in a large managed care organization.

Methods: Incident prostate cancer cases diagnosed at localized stage were identified using a SEER cancer registry. Treatments were identified by the cancer registry and patients’ medical records. Men were excluded if an initial course of treatment was started within 6 months of diagnosis. All patients were followed to identify later initiation of treatment, suspected prostate cancer metastasis or death. Suspected metastases were manually reviewed and confirmed. Death due to prostate cancer (PCD) was identified using the underlying cause of death on the death certificate. Competing risks methods were used to estimate the cumulative incidence of treatment initiation or metastasis (naive of treatment), and the incidence of death due to prostate cancer or other causes.

Results: A total of 6207 men were included: 60.4% white, 18.9% Black, 15.2% Hispanic and 5.5% Asian or Pacific Islander (API). At 19 years after diagnosis, the estimated incidence of treatment initiation, metastasis, PCD, and death due to other causes was 39.1%, 13.4%, 12.0% and 52.9%, respectively. Rates of treatment initiation at 19 years were highest in Hispanic (44.5%) and Black (44.7%) men, compared to 35.4% in white and 38.6% in API men. Rates of treatment-naïve metastasis as well as PCD were highest in Blacks (19.3% and 14.1%, respectively) and lowest among Hispanics (7.7% and 9.9%, respectively). API men had low rates of PCD through the first 10 years after diagnosis (2.4% compared to 5.6-6.9% for other groups), after which rates increased and at 19 years their estimated rates of PCD (12.0%) approximated those of whites (11.7%).

Conclusions: Significant risks of metastasis and prostate cancer death remains even 15 years after diagnosis among men who were initially untreated. Notably, there is an apparent increasing risk of PCD in API more than 10 years after diagnosis.

Citation Format: Jeff Slezak, Stephen Van Den Eeden, Kimberly L. Cannavale, Gary W. Chein, Chun R. Chao. Long-term follow-up of a racially and ethnically diverse population of patients with localized prostate cancer who did not undergo initial active treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4197.