bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 405: The role of GALNT14 in chemoresistant and metastatic osteosarcoma
Authors: Prudowsky Zachary D. , Patel Tajhal , Rajapakshe Kimal , Coarfa Christian , Shuck Ryan , Rainusso Nino , Yustein Jason
Published: July 2021

Introduction: Osteosarcoma is the most common bone tumor in children and adolescents. Many patients have disease progression through standard chemotherapy regimens of methotrexate, cisplatin, and doxorubicin. Presently, there are no salvage therapies that have added significant survival benefits, leaving minimal options for relapsed and resistant disease. Additionally, there are no clinically significant biomarkers for this disease that would predict treatment failures. N-acetylgalactosaminyltransferase 14 (GALNT14) is an enzyme that initiates O-linked glycosylation to outer membrane-bound and extracellular proteins. Using transcriptomic analyses, we identified GALNT14 overexpression correlated with poor tumor necrosis and survival outcomes. Therefore, we hypothesize that GALNT14 contributes to metastatic and chemoresistant phenotypes in pediatric osteosarcoma.

Methods: Transcriptomic data from the Therapeutically Applicable Research for Generating Effective Treatments (TARGET) database and RNA-sequencing of institutional patient-derived xenografts (PDXs) revealed that overexpression of GALNT14 correlated to poor necrosis rates. Gene Set Enrichment Analysis (GSEA) also identified upregulation of glycosylation pathways. Aberrant expression of GALNT14 was confirmed using quantitative polymerase chain reaction (qPCR) and Western blot analyses in various osteosarcoma cell lines. Transient GALNT14 knockdown and overexpression were then analyzed for effects on proliferation, invasion, migration, and chemotherapy response in vitro. Kaplan-Meier curves demonstrating overall survival (OS) and event-free survival (EFS) were established using data from the TARGET database.

Results: GALNT14 expression was higher in both commercial and PDX cell lines that were known to be more prone for chemoresistance and invasiveness. The highest quartile of patients based on GALNT14 overexpression had 40% OS and 25% EFS, compared to 80% (p=0.002) and 75% (p<0.001) for the lowest quartile of expression, respectively.

Summary: GALNT14 shows promise as a predictive marker of chemoresistance and metastasis for pediatric osteosarcoma. Future functional genomic studies through gain and loss-of-function assays will evaluate the role of GALNT14 in osteosarcoma chemoresistance and metastatic potential.

Citation Format: Zachary D. Prudowsky, Tajhal Patel, Kimal Rajapakshe, Christian Coarfa, Ryan Shuck, Nino Rainusso, Jason Yustein. The role of GALNT14 in chemoresistant and metastatic osteosarcoma [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 405.
Abstract 407: Belantamab Mafodotin detection by protein electrophoresis: Assessing interference for defining clinical response
Authors: McClure Fiona J.
Published: July 2021

Multiple Myeloma (MM) is a life threatening incurable cancer of the bone marrow resulting in plasma cell expansion and over production of plasma proteins or ‘M' proteins. Belantamab Mafodotin (Belamaf) is an antibody drug conjugate (ADC) for the treatment of multiple myeloma. The antibody component is an afucosylated IgG1 directed against B-cell maturation antigen (a protein expressed on normal B lymphocytes and multiple myeloma cells) conjugated to the microtubule inhibitor monomethylauristatin F (MMAF).Current criteria for assessing clinical response include changes in serum or urine M-protein levels by serum protein electrophoresis and immunofixation electrophoresis (IFE). Therapeutic use of ADC's has the potential to interfere with M-protein assessment by electrophoresis thus confounding clinical trial results. We aimed to modify an existing electrophoresis (EPS) platform (SAS-1 plus) to identify Belamaf in both saline and patients sera using a specific Anti-mcMMAF antibody.Objectives:1)To determine if Belamaf can be detected by serum protein electrophoresis and/or immunofixation electrophoresis methods that are used to measure ‘M' protein in MM patients. 2)If Belamaf can be detected by this assay, then determine if the detectable levels could interfere with defining clinical outcomes such as complete response*.By modifying an existing platform and using an mcMMAF specific antibody we were able to detect Belamaf in patients sera with no ‘M' protein at concentrations down to 0.5g/L by IFE. In multiple myeloma patients whose total protein concentrations were in excess of 90g/L Belamaf could be detected at 1.5g/L. Belamaf in patients sera can only be identified by anti-mcMMAF as anti-IgG and anti-IgKappa will detected those proteins present in sera in both MM and healthy patients.Belamaf could not be detected below 0.5g/L either by EPS or IFE using Anti-mcMMAF. As therapeutic Cmax levels of Belamaf are expected to be 0.04g/L, it will not be identified by EPS or IFE using the SAS-1 plus system. Further refinement of the SAS-1 plus system may achieve greater sensitive down to 0.2g/L (0.2g/L was detected using anti- IgG) however increased sensitivity and optimisation is highly unlikely to detected therapeutic concentrations of Belamaf.* Complete Response: Patients with CR must show that no abnormal monoclonal proteins (also called M-proteins) are present in the blood serum or urine.

Citation Format: Fiona J. McClure. Belantamab Mafodotin detection by protein electrophoresis: Assessing interference for defining clinical response [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 407.
Abstract 411: New estimators for mediation effects in complex sampling schemes: A comparison with applications
Authors: Mai Yujiao , Srivastava Deo Kumar
Published: July 2021

Mediation analysis examines the indirect effects via a third variable on the pathway between exposures and outcomes. For the finite-population surveys, that often use stratified cluster sampling, the conventional methods assuming simple random sampling are not appropriate. The point estimation of the effect can be corrected using sampling weights, but the standard error needs further improvement. Recent studies have integrated mediation analysis with balanced repeated replication for standard error estimation. Alternative estimators include Jackknife repeated replication (JRR), bootstrap, and Taylor series linear approximation (TSE). This study proposes new standard error estimators by incorporating the classic linear mediation analysis with JRR, bootstrap, and TSE for complex surveys and compares them using Monte Carlo simulations. The preliminary results show that replication methods have lower Type-I error rates and higher confidence-interval coverage rates than conventional methods. The proposed methods are applied to National Health Interview Surveys to explore the mediation effects on the pathway between cancer survivors' health status and employment status.

Citation Format: Yujiao Mai, Deo Kumar Srivastava. New estimators for mediation effects in complex sampling schemes: A comparison with applications [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 411.
Abstract 412: Blockade of beta-catenin pathway combined with Aurora kinase activity inhibition enhances antitumor effects in adrenocortical cancer cells
Authors: Maria Andrea G. , Borges Kleiton S. , Lira Regia C. , Thome Carolina H. , Berthon Annabel , Drougat Ludivine , Faucz Fabio R. , Faca Vitor M. , Tone Luiz G. , Stratakis Constantine A.
Published: July 2021

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is very low and current treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity and side effects. Therefore, identification of new biological targets to improve ACC treatment is crucial. Beta-catenin alterations have been found in approximately 30% of ACCs and blockade of Wnt-beta-catenin showed to decrease adrenal steroidogenesis and increase apoptosis of ACC cells, NCI-H295, in vitro and in a mouse model. Aurora kinases are known to play an important role in cell division during G1-M phase and their aberrant expression is correlated with poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinase activity combined with beta-catenin pathway blockade could promote better antitumor effects in NCI-H295 cells. We studied the in vitro effects of AMG 900, an aurora kinase activity inhibitor and PNU-74654, a beta-catenin pathway blocker, in proliferation, migration, colony establishment and invasion of NCI-H295 and SW-13 ACC cell lines. Our results show that the combination of AMG 900 with PNU-74654, decreases cell proliferation and viability, compared to either one of the single treatments. Regarding cell invasion and clonogenesis inhibition, AMG 900 was more efficient than PNU-74654, and its combination with the latter did not improve these effects. On the other hand, PNU-74654 was more effective in decreasing cortisol secretion. Taken together, our data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway could bring new approaches for designing new drugs to treat certain ACCs.

Citation Format: Andrea G. Maria, Kleiton S. Borges, Regia C. Lira, Carolina H. Thome, Annabel Berthon, Ludivine Drougat, Fabio R. Faucz, Vitor M. Faca, Luiz G. Tone, Constantine A. Stratakis. Blockade of beta-catenin pathway combined with Aurora kinase activity inhibition enhances antitumor effects in adrenocortical cancer 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 412.
Abstract 458: Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma
Authors: Diaz Jose Jonathan , Scoggin Tatiana , Ramos Jason
Published: July 2021

Introduction: Alpha thalassemia/mental retardation syndrome X-linked (ATRX) plays a role in chromatin regulation and maintenance of telomeres. It regulates incorporation of histone H3.3 into telomeric chromatin. ATRX is also a major component of various essential cellular pathways such as DNA replication and repair, chromatin higher-order structure regulation, and gene transcriptional regulation. ATRX loss was observed in grades II/III astrocytomas, oligoastrocytomas, oligodendrogliomas, and glioblastomas. In grades II/III gliomas, most ATRX-loss cases had IDH1/2 mutations. Assessment of ATRX loss by IHC staining captures the majority of mutations, giving a reasonable sensitivity in neuropathology diagnostics. In this study, the specificity and sensitivity of ATRX were analyzed on multiple tissues focusing on Astrocytoma and Glioblastoma tissues.

Materials and Methods: IHC staining has been performed by using formalin-fixed paraffin-embedded tissue with standard deparaffinization steps and a heat-induced retrieval method using a citrate buffer solution in a pressure cooker (Decloaking chamber). Antibody was incubated for 30 minutes, followed by HRP detection and DAB solution for visualization. Slides were stained using semi-automated staining system (intelliPATH). ATRX polyclonal antibody [Biocare Medical, California, USA] diluted in a carrier at an optimal concentration with standard protocol.

Results: ATRX stained 44% of diffuse and anaplastic astrocytoma and 57% of glioblastoma. Also, cross-reactivity testing determined ATRX polyclonal may stain normal brain in some cases. A more comprehensive data evaluation shown below:

Conclusions: ATRX polyclonal shows a potential advantage when diagnosing brain tumors (especially astrocytoma and glioblastomas) even if some cross reactivity can be observed. ATRX in conjunction with other brain marker antibodies can offer a better tool in diagnosing brain tumors.

Diseased Tissue Specificity and SensitivityTissuePositive CasesTotal Cases% +Astrocytoma163644Glioblastoma4757Breast Cancer132748Colon Cancer84020Lung Cancer245246Prostate Cancer164238Normal Tissue Cross-ReactivityCerebrum51145Cerebellum2367

Citation Format: Jose Jonathan Diaz, Tatiana Scoggin, Jason Ramos. Characterization of ATRX polyclonal, application of immunohistochemical analysis on astrocytoma and glioblastoma [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 458.
Abstract 461: Pan-liver disease single cell-based deconvolution reveals γδ2 T cells as a marker in hepatocellular carcinoma development
Authors: Shankar Rama , Haskins Jeremy , Paithankar Shreya , Chen Bin
Published: July 2021

Hepatocellular carcinoma (HCC) is the most common form of primary adult liver cancer and the fourth leading cause of cancer-related death worldwide. HCC incidence is highest most often in people with chronic liver diseases, yet the effect of cell composition on the progression of chronic liver diseases to HCC remains unknown. Using gene biomarkers of twenty cell-types identified from healthy liver scRNA-seq data, we estimated cell compositions of six chronic liver diseases and HCC based on their bulk RNA-seq samples. We observed a decrease in the enrichment of various health cells as the progression of liver diseases. Compared to a healthy state, liver fibrosis and HCC present higher enrichment of γδ2 T cells and lower enrichment of central venous liver sinusoidal endothelial cells (LSECs). High enrichment of γδ2 T cells was specifically observed in HCV and/or HBV positive HCC and advanced HCC. γδ2 T cell is one of the nine cell types that are associated with HCC prognosis. The analysis of multiple independent datasets confirmed that γδ2 T cells enrichment is more associated with poor prognosis in patients with lower Alpha-fetoprotein (AFP) level and HCV/HBV positive patients. Following pan-cancer analysis suggested enrichment of γδ2 T cells is associated with poor prognosis in kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, and low grade gliomas. In addition, this type of cells is also enriched in the blood samples of various chronic liver diseases and HCC, indicating the potential of being diagnostic markers. In summary, the integrative bioinformatics analysis identified γδ2 T cells as a marker for HCC progression.

Citation Format: Rama Shankar, Jeremy Haskins, Shreya Paithankar, Bin Chen. Pan-liver disease single cell-based deconvolution reveals γδ2 T cells as a marker in hepatocellular carcinoma development [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 461.
Abstract 463: Features consistent with a small-cell neuroendocrine like (SC/NE-L) transformation can be readily detected and quantified in circulating tumor cells (CTCs)
Authors: Scher Howard I. , Jendrisak Adam , Gilbertson Cole , Gill Audrey , Barnett Ethan , Gopalan Anuradha , Keegan Niamh , Zaidi Samir , Benoliel Hannah , Emily Carbone , Wang Eva , Lu James , Tubbs Alisa , Anderson Amanda , Jones Joshua , Schonhoft Joseph D. , Wenstrup Rick
Published: July 2021

Background: The increasing availability and earlier use of life prolonging drugs targeting the androgen receptor signaling axis (ARSI) has resulted in an increase in the frequency of late state tumors with “small cell/neuroendocrine like (SC/NE-L) phenotypes” similar to small-cell lung cancer (SCLC). These tumors are typically heterogenous wherein the small-cell component represents only a portion of CTC's present. Definitive pathologic criteria to diagnose small-cell neuroendocrine transformation are lacking, and the eligibility criteria across trials in this space inconsistent, limiting the ability to relate outcomes between studies. We hypothesize that an analytically validated assay for a rigorously defined “small-cell CTC” phenotype would clarify the identification and diagnosis of SC/NE-L tumors.

Methods: Using the WHO guidelines for small-cell diagnosis in tissue as reference, we defined an equivalent set of single-cell criteria for defining a CTC with small cell or neuroendocrine like (SC/NE) histology that include: a circulating CD45- cell with a high nuclear to cytoplasm area ratio, small size, high circularity, lack of detectable nucleoli, salt and pepper chromatin, SC/NE or tumor-related protein expression (DLL3, CD56, CK etc.), and other features associated with lineage plasticity (i.e., CK speckling). Clinical feasibility of feature extraction was explored on CTCs detected using the Epic Sciences platform in the blood of patients with small-cell lung and late state prostate cancers.

Results: Using the Epic Sciences platform, morphologic features associated with small-cell or neuroendocrine transformation can be readily identified and quantified automatically in CTCs in a liquid biopsy from patients with small-cell lung and prostate cancers.

Conclusions: Identifying small-cell to neuroendocrine like (SC/NE-L) CTC subtypes may aid in establishing that lineage transformation to a SC/NE-L phenotype has occurred where invasive single site tissue biopsies may not be informative or feasible.

Citation Format: Howard I. Scher, Adam Jendrisak, Cole Gilbertson, Audrey Gill, Ethan Barnett, Anuradha Gopalan, Niamh Keegan, Samir Zaidi, Hannah Benoliel, Carbone Emily, Eva Wang, James Lu, Alisa Tubbs, Amanda Anderson, Joshua Jones, Joseph D. Schonhoft, Rick Wenstrup. Features consistent with a small-cell neuroendocrine like (SC/NE-L) transformation can be readily detected and quantified in circulating tumor cells (CTCs) [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 463.
Abstract 477: Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps
Authors: Yu Ming , Wang Ting , Carter Kelly T. , Baker Kelsey , Redman Mary W. , Vickers Kathy , Dzubinski Lynda Ann , Schoen Robert E. , Grady William M.
Published: July 2021

The purpose of this study is to evaluate the use of molecular markers for more accurately predicting the individual risk of metachronous polyps or cancer during colonoscopic surveillance. Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the United States. The risk of CRC varies among people; individuals who develop adenomas are at heightened risk of forming metachronous adenomas and interval cancer, compared to those without a personal history of adenoma or CRC. Yet, even in these high risk individuals, only 25-30% will develop subsequent adenomas. To date, few studies have evaluated the use of molecular markers for predicting the risk of metachronous lesions. We have demonstrated that aberrantly methylated mir342/EVL is present at a higher frequency in the normal colon of people with CRC compared to people with no colon cancer or polyps, suggesting it may indicate a field cancerization process. Further, we developed an ultra-sensitive quantitative Methylation-specific ddPCR (MS-ddPCR) assay to accurately determine the level of rare EVL methylation in the normal colon mucosa. To investigate the correlation of EVL methylation in normal mucosa and the risk of developing polyps during the follow-up colonoscopy, we conducted a retrospective cohort study (N=136 subjects) and EVL methylation status was determined in normal colon mucosa samples from these subjects. Among them, 71 subjects had polyps detected within 12 years of the index colonoscopy, and 11 subjects were polyp-free at all subsequent colonoscopic exams done within 8-16 years of the index colonoscopy (54 subjects did not meet the criteria for follow up, so were excluded from the following analysis). Applying the standard Inverse Probability Weighted Logistic Regression (IPWLR) model, we found that higher level of EVL methylation in the normal colon mucosa at the time of the index colonoscopy (which was when the tissue sample was collected) indicated an increased risk of having polyps detected within 12 years, compared to subjects with no polyps detected by colonoscopy done within 8-16 years (Odds Ratio (95% CI) = 4.35 (1.14, 16.60); p-value = 0.0317). Our results suggest that EVL methylation level in the normal colon mucosa can be used as a risk biomarker for metachronous adenomas.

Citation Format: Ming Yu, Ting Wang, Kelly T. Carter, Kelsey Baker, Mary W. Redman, Kathy Vickers, Lynda Ann Dzubinski, Robert E. Schoen, William M. Grady. Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps [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 477.
Abstract 494: Novel human B7-H3 knock-in mice demonstrate efficacy of anti-B7-H3 immunotherapy in vivo
Authors: Zhao Lei , Li Huilin , Liu Yujie , Yu Zhaoxue , Lin Eugene
Published: July 2021

In recent years, immune checkpoint inhibitors (ICIs), particularly against PD-1/PD-L1, have revolutionized cancer treatment. However, ICI monotherapy only benefits a portion of cancer patients, while co-blockade of PD-1/PD-L1 and CTLA-4 is associated with increased IrAEs. Combined targeting of other immune checkpoints with PD-1/PD-L1 rather than CTLA-4 may be safer. B7-H3, also known as CD276, is a newer member of the same B7/CD28 superfamily as PD-1 and PD-L1. Its transcripts are ubiquitously expressed in normal tissues and solid tumors, but the protein is expressed in tumor tissues and by antigen-presenting cells, such as DC or macrophages, where it inhibits T cells and contributes to tumor immune evasion. Importantly, B7-H3 is overexpressed on a wide range of human solid cancers and correlates with negative prognosis and poor clinical outcome in patients. Recent studies have shown that B7-H3 also drives tumor growth, invasion, migration, angiogenesis, and metastasis beyond its immune regulatory roles. Thus, B7-H3 is an interesting new target for immunotherapy. To investigate B7-H3 function in vitro and evaluate in vivo efficacy of B7-H3 antibodies, Biocytogen generated a B7-H3 humanized mouse (B-hB7-H3) and B7-H3-expressing MC38 cell line (MC38-hB7-H3). In the mice, exons 3-4 of the mouse B7-H3 gene, which encode the extracellular domain, are replaced by human B7-H3 exons 3-6. Human B7-H3 mRNA was detectable in spleen and bone marrow of only B-hB7-H3 mice, and B7-H3 antibodies bound well to hB7-H3+ MC38 cells. Furthermore, using the MC38-hB7-H3 and EL4 tumor models, we showed robust efficacy of anti-human B7-H3 antibodies in inhibiting tumor growth in vivo, which was amplified in mice receiving combined human B7-H3 and mouse PD-1 blockade. Taken together, B7-H3 humanized mice are a useful tool for in vivo efficacy evaluation and selection of candidate human B7-H3 antibodies for advancement to clinical trials. In addition, Biocytogen has generated B7-H3/PD-1 and B7-H3/PD-1/PD-L1 multi-gene humanized mice to evaluate B7-H3/PD-1/PD-L1 combination therapies in vivo.

Citation Format: Lei Zhao, Huilin Li, Yujie Liu, Zhaoxue Yu, Eugene Lin. Novel human B7-H3 knock-in mice demonstrate efficacy of anti-B7-H3 immunotherapy in vivo [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 494.
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

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

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

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<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%)<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

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

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

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

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

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.