bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 502: In vitro characterization of antibodies targeting the SIRPα-CD47 phagocytosis checkpoint using tumor-associated macrophages from HuGEMM™ mice
Authors: Yan Xuefei , Zhang Hongjuan , Liu Yi , Zhang Yahong , Zheng Lei , Zang Mingfa , An Annie Xiaoyu , Xu kevin Qijin , Ouyang Davy , Li Henry , Huang Yujun 
Published: July 2021

Abstract
Introduction: The signal-regulatory protein SIRPα-CD47 pathway is a phagocytosis checkpoint for macrophages and other innate immune cells. Tumor cells ubiquitously express CD47, and blocking SIRPα-CD47 interactions has been shown to promote cancer cell elimination by macrophage phagocytosis. The impact of SIRPα-CD47 blockade may be further improved by targeting prophagocytic receptors (Fc receptors) to enhance phagocytosis for anti-CD47 antibodies. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in late-stage tumors. Currently, drug development targeting CD47-SIRPα is rapidly growing, requiring robust in vitro macrophage/tumor cell co-culture assays to evaluate new agents targeting this pathway. Here, we report on the use of macrophages from an engineered mouse model expressing human SIRPα, which may serve as a good tool to test drug candidates.
Methods: We have developed and characterized the human CD47 and SIRPα double knock-in mouse model (CD47/SIRPα HuGEMM) via CRISPR/Cas9 targeting. To characterize the effects of anti-human CD47 antibodies on phagocytosis and tumor cell killing, TAMs were isolated from tumor-bearing HuGEMM mice for in vitro macrophage/tumor cell co-culture.
Results: Human CD47 expressing tumor cells, such as murine MC38-hCD47 and human Jurkat cells, were labeled with CFSE and used as target cells for antibody-dependent cellular phagocytosis (ADCP) and tumor cell death. The percentage of CFSE-positive among macrophages, and the percentage of dead cells among CFSE-positive tumor cell death, were measured by flow cytometry. We observed significant ADCP effects by TAM and killing of tumor cells after in vitro anti-CD47 antibody treatment. The contribution to ADCP by SIRPα-CD47 blockade and/or Fc effector function of antibodies was further investigated. We also evaluated the effect of SIRPα-CD47 blockade on the modulation of TAM phenotypes.
Conclusion: Our data show that the CD47/SIRPα HuGEMM system provides an excellent tool for in vitro functional assays for drug candidates targeting the SIRPα-CD47 pathway, to evaluate the effect on ADCP by macrophages, activation of macrophages, and overall tumor cell death.
Citation Format: Xuefei Yan, Hongjuan Zhang, Yi Liu, Yahong Zhang, Lei Zheng, Mingfa Zang, Annie Xiaoyu An, kevin Qijin Xu, Davy Ouyang, Henry Li, Yujun Huang. In vitro characterization of antibodies targeting the SIRPα-CD47 phagocytosis checkpoint using tumor-associated macrophages from HuGEMM™ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 502.
Abstract 504: NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells
Authors: Barham Whitney J. , Wen Ti , Li Ying , Zhang Henan , Liu Cindy , Hirdler Jacob , Gicobi Joanina , Lucien-Matteoni Fabrice , Yan Yiyi , Dong Haidong 
Published: July 2021

Abstract
Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy.
Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.
Abstract 532: Association of pCR and the 8-gene signature: NRG Oncology/NSABP B-41
Authors: Song Nan , Tan Xiaoqing E. , Wang Ying , Kim Rim S. , Bandos Hanna , Tang Gong , Mamounas Eleftherios , Geyer Charles E. , Rastogi Priya , Jacobs Samuel A. , Srinivasan Ashok , Lucas Peter C. , Paik Soonmyung , Wolmark Norman , Swain Sandra M. , Pogue-Geile Katherine L. 
Published: July 2021

Abstract
Background: Lapatinib (L), a HER2 signaling, tyrosine kinase inhibitor, demonstrated numerically higher pCR in NSABP B-41 when added to paclitaxel (AC→P) and trastuzumab (T) following doxorubicin + cyclophosphamide (62% v 52.5%). We previously validated an 8-gene signature that predicted the degree of T benefit in NSABP B-31 and NCCTG9831. The purpose of this study is to determine the association of pCR with the 8- gene T-benefit groups, enabling the possibility of stratifying patients (pts) who do or do not receive benefit from L.
Methods: Normalized B-41 nCounter® Breast Cancer 360 gene expression data was used to define the three T-benefit groups: large-, moderate-, and no-. The 8-gene signature was modified to use only 7 genes because one of the 8 genes was not included in the nCounter code set. The ability of the 7-gene signature to predict T benefit was equivalent to the 8-gene signature when tested in B-31. Comparisons within each treatment arm were made with Fisher's exact test.
Results: The pCR rates were significantly different among the three groups in the AC→P+T arm: Large: 24/28 (86%); Moderate: 13/32 (41%); and No: 1/9 (11%); p&lt;0.001, but were not significantly different in the other two arms; a trend for significance was seen in the AC→P+T+L arm (Table 1). We also tested if L would improve pCR when added to AC→P+T in any of the 7-gene benefit groups compared to T. There was a non-significant, numerical increase in pCR in the no-benefit group in the AC→P+T+L arm (44%) v the AC→P+T arm (11%) (p=0.29).
Conclusions: The significant association of the modified 8-gene benefit groups with pCR in the AC→P+T arm suggests this signature could identify pts who may benefit from dual HER2-targeted neoadjuvant therapy. This could be tested in a meta-analysis including other neoadjuvant trials.
Support: Lombardi CCC; BCRF; GSK; P30CA051008; Genentech; NSABP
Table 1.pCR rates in trastuzumab benefit groups in NSABP B-41pCR rates in Breast and NodesTreatmentNo BenefitIntermediateLarge BenefitEntire Cohortp valueAC→P+T1/9 (11.1%)13/32(40.6%)24/28 (85.7%)38/69 (55.1%)&lt;0.001AC→P+L2/7 (28.6%)11/26 (42.3%)14/31 (45.2%)27/64 (42.2%)0.82AC→P+T+L4/9 (44.4%)11/25 (44.0%)20/27 (74.1%)35/61 (57.4%)0.051
Citation Format: Nan Song, Xiaoqing E. Tan, Ying Wang, Rim S. Kim, Hanna Bandos, Gong Tang, Eleftherios Mamounas, Charles E. Geyer, Priya Rastogi, Samuel A. Jacobs, Ashok Srinivasan, Peter C. Lucas, Soonmyung Paik, Norman Wolmark, Sandra M. Swain, Katherine L. Pogue-Geile. Association of pCR and the 8-gene signature: NRG Oncology/NSABP B-41 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 532.
Abstract 551: Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer
Authors: Dong Xiaoxi , Zhang Minghua , Zheng Tiantian , Tan Lu , Xie Feng , Wang Amy Xiaohong , Du Pan , Jia Shidong , Lu Chris , Yu Jianjun 
Published: July 2021

Abstract
Loss of expression of the PTEN gene is associated with poorer prognosis in many solid tumors. While cfDNA-based liquid biopsy testing are gradually adopted as the routine testing method in the clinical setting, most of them are unable to reliably detect copy number loss and/or are limited reporting copy number amplification events only.
Method: We have developed a capture-based CLIA-certified NGS assay, PredicineCARE, to robustly identify tumor-derived aberrations including SNVs, Indels, fusions, copy number amplifications and deletions from blood samples. Analytical validation of the assay was performed with commercial standard reference materials and contrived cell lines. Copy number loss of PTEN was also confirmed by Immunohistochemistry (IHC) assay in 15 clinical samples. Clinical utility of PTEN copy number loss was further established by its significant association with poor outcome in clinical patients with metastatic castration-resistant prostate cancer (mCRPC).
Results: Analytical validation of the PredicineCARE assay demonstrated its super sensitivity and robustness of detecting homozygous copy number loss at approximately 10% tumor fraction. Both the sensitivity and Positive Prediction Value reached 100% when overall gene copy number is below 1.75 copies. When comparing to tissue IHC results reviewed by two independent pathologists, PredicineCARE liquid biopsy assay revealed 86.7% concordance with IHC in detecting PTEN loss (80% sensitivity, 100% specificity, and 100% PPV). The copy number loss was further confirmed by low-pass whole-genome sequencing of cfDNA samples. When the assay was applied to profile mCRPC patients, copy number loss of PTEN was detected at the prevalence rate of 19.2% (10/52) and 35.9% (23/64) respectively. Notably, patients harboring PTEN copy number loss showed significantly worse overall survival in the two cohorts independently (log-rank P = 0.0001 and 0.0004 respectively).
Conclusion: We have developed PredicineCARE for clinical use, a highly sensitive cfDNA-based assay to detect gene copy number variants in addition to gene mutations and fusions. Its ability to detect gene copy number loss in low fraction of tumor contents as a valuable molecular biomarker holds great potential for clinical applications and patient selections in personalized precision oncology, particularly for investigational drugs targeting the AKT-PI3K-PTEN pathway.
Citation Format: Xiaoxi Dong, Minghua Zhang, Tiantian Zheng, Lu Tan, Feng Xie, Amy Xiaohong Wang, Pan Du, Shidong Jia, Chris Lu, Jianjun Yu. Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 551.
Abstract 579: Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis
Authors: Tanaka Junko , Nakagawa Tatsuo , Ishida Takeshi , Kamura Yoshio , Shiratori Akiko , Harada Kunio , Uematsu Chihiro 
Published: July 2021

Abstract
Background
Digital PCR (dPCR) is a promising method for liquid biopsies and is able to quantify nucleic acids more sensitively than real-time PCR. However, dPCR has a large fluctuation in the fluorescence intensity of the droplets or wells due to insufficient PCR in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping.
Methods
A sample is digitized into a silicon chip with up to ~104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that are complimentary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, dye color of the probe, and melting temperature (Tm). Because the Tm of the PCR products does not considerably depend on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping.
Results
The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF and, eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. G12D, G12V and G12R-mutant KRAS alleles were detected with FAM-labeled probes, G13D-mutant KRAS allele and wildtype KRAS allele were detected with HEX-labeled probes, G12S and G12C-mutant KRAS alleles were detected with Cy5-labeled probes, and V600E-mutant BRAF allele and wildtype BRAF allele were detected with CAL Fluor Red 610-labeled probes. The results showed that the peaks of the Tm distributions of the DNA groups were separated by 3.5 °C on average, while the standard deviation of the Tm distributions was 0.23 °C on average. The difference in Tm was 15 times larger than the standard deviation, enabling more accurate genotyping than when using the fluorescence intensity.
Conclusion
We have proposed a measurement method that combines dPCR with melting curve analysis, and successfully performed the multiplexed genotyping assay. We plan to evaluate the effect of this method on measurement using clinical specimens of pancreatic cancer patients in the future.
Citation Format: Junko Tanaka, Tatsuo Nakagawa, Takeshi Ishida, Yoshio Kamura, Akiko Shiratori, Kunio Harada, Chihiro Uematsu. Multiplex digital PCR combined with melting curve analysis for detecting KRAS mutations towards pancreatic cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 579.
Abstract 596: Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer
Authors: Barbirou Mouadh , Manjunath Yariswamy , Miller Amanda , Ramirez Arturo , Ericson Nolan , Staveley-O'Carroll Kevin F. Kevin F. , Warren Wes , Li Guangfu , Tonellato Peter J. , Kaifi Jussuf T. 
Published: July 2021

Abstract
Purpose: Circulating tumor cells (CTCs) may be novel biomarkers to predict cancer prognosis and monitor therapeutic efficacy. In the current study, we investigated the use of the time-dependent enumeration and targeted DNA sequensing of single CTCs as liquid biopsy based biomarkers of non-small cell lung cancer (NSCLC) patients for diagnosis, prognosis, therapeutic choice and outcome monitoring.
Experimental Design: In a prospective study, we tested the AccuCyte® system (RareCyte®, Inc. Seattle, WA) for CTC enumeration in NSCLC using FDA criteria (CK/EpCAM+/CD45- with a nucleus). Whole blood (7.5 ml) from healthy donors was spiked with human lung adenocarcinoma (A549 cell line) cells at specific numbers (N=0, 100, 200, 1000). CTC detection and single-CTC picking was performed. NSCLC subjects' (N=20) and chronic smokers without cancer on screening low-dose CT subjects' (N=10) blood were collected (amount, method, biotechnology). For all blood samples, CTC detection and single CTC picking was performed. DNA was isolated from CTCs (N=3) and white blood cells (N=2) per sample. DNA libraries were prepared for targeted oncogene characterization using the CleanPlex® OncoZoom® panel. Somatic variant analysis and comparisons were performed.
Results: AccuCyte enumeration of A549 cells spiked at four levels in healthy subjects' whole blood showed (Spiked #, Detected #): (0, 0); (100, 76); (200, 208); (1000, 1223). NSCLC subjects' (N=20; age: 64.85±6.64; BMI: 28.00±8.15sex ratio: 0.42) blood had a mean CTC count of 13.4 (SD=52, median=0, range [0, 237]). Ten screening controls without cancer (64.2±5.81, 1, 28.50±6.13, 0.66) had a mean CTC count of 0.20 (0.42, 0, [0, 1]). Significant association was noted regarding a higher number of CTCs found in NSCLC compared to screening subjects without cancer according to the (p= 0.020, Wilcoxon signed rank). CTC numbers increased with NSCLC AJCC stage. Somatic variant analysis on patient-matched single CTCs and WBCs is ongoing.
Conclusions: The molecular investigation of patient matched single CTCs and white blood cells will fundamentally advance understanding on the NSCCL carcinogenic signature of these blood born cells and their potential value as cancer detection and therapy management liquid biopsy-based biomarkers.
Citation Format: Mouadh Barbirou, Yariswamy Manjunath, Amanda Miller, Arturo Ramirez, Nolan Ericson, Kevin F. Kevin F. Staveley-O'Carroll, Wes Warren, Guangfu Li, Peter J. Tonellato, Jussuf T. Kaifi. Single circulating tumor cell enumeration and targeted sequencing in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 596.
Abstract 60: Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment
Authors: Good Charly R. , Kuramitsu Shunichiro , Samareh Parisa , Donahue Greg , Ishiyama Kenichi , Ma Yujie , Wellhausen Nils , Tian Lifeng , Agarwal Sangya , Guedan Sonia , Aznar M. Angela , Alexander Katherine A. , Zhang Zhen , Singh Nathan , Richardson Max W. , Watanabe Keisuke , Tanyi Janos L. , O'Hara Mark H. , Ruella Marco , Lacey Simon F. , Moon Edmund K. , Schuster Stephen J. , Albelda Steven M. , Lanier Lewis L. , Young Regina M. , Berger Shelley L. , June Carl H. 
Published: July 2021

Abstract
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains largely ineffective in solid tumors. A major factor leading to the reduced efficacy of CAR T cell therapy is T cell dysfunction, and the mechanisms mediating this dysfunction are under investigation. Here we establish a robust model to study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer. Continuous antigen exposure results in hallmark features of exhaustion including reduced proliferation capacity and cytotoxicity, and severe defects in cytokine production. Here we identified a transcriptional signature at both population and single-cell levels in CAR T cells after continuous antigen exposure. In addition, TCR lineage tracing revealed a CD8+ T-to-NK-like T cell plasticity that results in reduced tumor cell killing. The transcription factors SOX4 and ID3 are specifically expressed in the dysfunctional CAR NK-like T cells and are predicted to be master regulators of the dysfunction gene expression signature and the post-thymic acquisition of an NK-like T cell fate. Finally, we identified the emergence of NK-like CAR T cells in a subset of patients after infusion of CAR T cells. The findings gleaned from this study shed light on the plasticity of human CAR T cells and suggest new approaches to improve the efficacy of CAR T cell therapy in solid tumors by preventing or revitalizing CAR T cell dysfunction.
Citation Format: Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Greg Donahue, Kenichi Ishiyama, Yujie Ma, Nils Wellhausen, Lifeng Tian, Sangya Agarwal, Sonia Guedan, M. Angela Aznar, Katherine A. Alexander, Zhen Zhang, Nathan Singh, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O'Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 60.
Abstract 600: Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin
Authors: Chow Jennifer , Tevis A Anders , Lo Edward , Clein Alisa , Sabath Daniel E. , Ramirez Arturo B. , Kaldjian Eric P. , George Tad 
Published: July 2021

Abstract
Protein expression of synaptophysin (SYP) is characteristic of neuroendocrine subtype tumors. In prostate cancer, neuroendocrine differentiation is correlated with disease progression, poor prognosis, and treatment resistance. Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and patient stratification. Here we describe the validation of a biomarker IF assay for enumeration of CTCs and characterization of their SYP expression. The 0920-VB SYP CTC assay workflow includes processing blood samples to slides (AccuCyte® Sample Preparation System), staining slides with a panel of fluorescent markers (RarePlex® Staining Kit and Ventana® DISCOVERY® ULTRA immunostaining system), and multiparameter imaging and analysis (CyteFinder® Instrument). The panel consists of a nuclear dye, and antibodies against cytokeratins and EpCAM (to identify epithelial CTCs), CD45 (to exclude white blood cells) and SYP. Both clinical and spike-in samples were used for assay validation. The model CTCs used to generate spike-in samples included 22Rv1 (prostate carcinoma, SYP positive) and BT-474 (breast carcinoma, SYP negative). Performance metrics for the assay included accuracy, sensitivity, specificity, repeatability, and intermediate precision of SYP detection and CTC enumeration. Single-cell SYP mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. An MFI threshold for SYP positivity was established by maximizing the classification accuracy of the positive and negative cell lines. Using this threshold, 98.7% of 22Rv1 cells were correctly classified as SYP-positive, and 99.7% of BT474 cells were correctly classified as SYP-negative with an inter-run coefficient of variation of 13.9% for the 22Rv1 cell line. In clinical prostate, breast and colon cancer patient samples, subsets of CTCs were found to be SYP positive with the expected cytoplasmic localization of the marker. In summary, this liquid biopsy assay provides an analytically sensitive and specific method for CTC enumeration and SYP biomarker expression analysis, allowing non-invasive detection of neuroendocrine differentiation.
Citation Format: Jennifer Chow, A Anders Tevis, Edward Lo, Alisa Clein, Daniel E. Sabath, Arturo B. Ramirez, Eric P. Kaldjian, Tad George. Liquid biopsy for neuroendocrine differentiation: Validation of a circulating tumor cell assay for synaptophysin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 600.