bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3313: iPSC-derived natural killer cells expressing EGFR-CAR against glioblastoma
Authors: Yu Melissa , Mansour Anthony G. , Teng Kun-Yu , Sun Guoqiang , Shi Yanhong , Caligiuri Michael A.
Published: September 2020

Background: Natural Killer (NK) cells are a type of immune cells that belong to the innate immune system and executes a rapid immune reaction against cancer cells and viruses without prior activation. The huge success of chimeric antigen receptor (CAR)-T cells has motivated scientists to genetically modify human NK cells with CARs to further improve their tumor killing capacity. To engineer NK cells with a CAR, NK cells can be isolated from peripheral blood and umbilical cord blood. NK cells can also be derived from induced pluripotent stem cells (iPSCs) to produce a homogenous and standardized group of NK cells for the treatment of cancer. Here, we test the feasibility to engineer our EGFR-CAR construct into iPSC-derived hematopoietic stem cells (iPSC-HSC) to generate EGFR-CAR NK cells that will eradicate glioblastoma (GBM).

Material and Methods: iPSCs-derived HSCs were differentiated into NK cells using co-culture with a feeder stroma of OP9-DL1 cells in the presence of human cytokines and NK cells were assessed by flow cytometry. Expanded NK cells (with K562 feeder cells expressing mb-IL-21 and 4-1BB) and HSCs from peripheral blood were efficiently transduced with a retroviral vector, carrying a CAR targeting EGFR. CAR expression was assessed by flow cytometry. After transduction, the CAR-NK cells were collected for cytotoxicity assay to test their efficacy. CAR-HSCs were differentiated into CAR-NK cells utilizing OP9-DL1 stroma cells.

Results: Our results demonstrated that iPSCs can be fully differentiated into NK cells expressing typical NK cell surface markers including CD56, NKp46, CD16 and NKG2A, etc. In addition, HSCs can be engineered with EGFR-CAR without affecting their differentiation into CAR-NK cells. Our engineered EGFR-CAR NK cells showed an enhanced antitumor activity against GBM compared to NK cells without the EGFR-CAR.

Conclusion: Collectively, our study suggests the feasibility of engineering CAR-EGFR NK cells from iPSCs for effective “off the shelf” immunotherapy. Our approach can be utilized to generate an unlimited number of CAR-NK cells to treat patients with cancer such as GBM in the clinical setting.

Citation Format: Melissa Yu, Anthony G. Mansour, Kun-Yu Teng, Guoqiang Sun, Yanhong Shi, Michael A. Caligiuri. iPSC-derived natural killer cells expressing EGFR-CAR against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3313.
Abstract 3364: Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer
Authors: Bailis Julie M. , Lutterbuese Petra , Thomas Oliver , Locher Kathrin , Harrold John , Boyle Michael , Wahl Joachim , Li Shyun , Sternjak Alexander , Henn Anja , Dahlhoff Christoph , Naegele Virginie , Rattel Benno , Raum Tobias , Coxon Angela
Published: September 2020

Advanced gastric cancer remains a disease of high unmet medical need. In the United States, most patients present with symptomatic, incurable disease and prognosis is poor, with a 5-year survival rate of less than 10%. BiTE® (bispecific T cell engager) immune therapy activates a patient's own T cells to kill tumor cells and has the potential to overcome common mechanisms of therapy resistance. We generated fully human, half-life extended (HLE) BiTE® molecules against the tumor antigens MUC17 and CLDN18.2 for the treatment of gastric cancer. The mucin MUC17 is a protein normally found in the mucosal layer of intestinal epithelial cells that is delocalized and expressed in 45% of gastric tumors. The claudin CLDN18.2 is a protein normally found in the cellular tight junctions of gastric mucosa and intestinal epithelium that is delocalized and expressed in >60% of gastric tumors. AMG 199 (MUC17 HLE BiTE®) and AMG 910 (CLDN18.2 HLE BiTE®) show potent cytotoxic activity against gastric cancer cell lines that express MUC17 or CLDN18.2, respectively, in vitro, and promote significant tumor growth inhibition against established gastric tumor xenograft models in vivo. In preclinical non-human primate (NHP) toxicology studies, both molecules show evidence for BiTE® target engagement, including T cell activation and proliferation, but demonstrate different effects on target-expressing tissue. Weekly administration of AMG 199 is well tolerated in NHP with minimal findings in MUC17-expressing normal tissues. In contrast, treatment with AMG 910 led to direct cell killing of CLDN18.2-expressing gastric mucosal cells in NHP, a finding which was fully reversible once treatment was stopped. AMG 199 and AMG 910 may offer the potential to improve outcomes in advanced gastric patients worldwide.

Citation Format: Julie M. Bailis, Petra Lutterbuese, Oliver Thomas, Kathrin Locher, John Harrold, Michael Boyle, Joachim Wahl, Shyun Li, Alexander Sternjak, Anja Henn, Christoph Dahlhoff, Virginie Naegele, Benno Rattel, Tobias Raum, Angela Coxon. Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3364.
Abstract 3415: Mutant BRAF small molecule inhibition enhances oncolytic herpes virus immunotherapy through increased immune cell recruitment and activation in melanoma
Authors: Bozhanova Galabina , Jennings Victoria , Pedersen Malin , Kyula Joan , Patin Emmanuel , Hassan Jehanne , Ono Masahiro , Harrington Kevin , Melcher Alan
Published: September 2020

Small molecule inhibitors of the MAPK pathway have demonstrated rapid responses in melanoma patients, but their use is hindered by the frequent development of acquired resistance. Oncolytic viruses are a novel class of immunotherapeutics which selectively replicate inside cancer cells, causing lysis and subsequent activation of anti-tumor immunity. BRAF and MEK inhibitors have previously been shown to synergise with oncolytic viruses to kill melanoma cells, but the consequences of these interactions on immune responses have not been assessed. Here, we show that the combination of PLX4720 (BRAFV600E inhibitor) and intratumoral oncolytic herpes virus HSV1716 led to a significant tumor reduction and improved survival over single-agent treatment in a murine BRAFV600E melanoma model. The combination led to a significant increase of PD-L1+ neutrophils and monocytes at an early time point following therapy administration, suggesting an inflammatory switch in the tumor microenvironment. Importantly, the combination led to a significant increase in cytotoxic CD8+ cells at a later time point, which was accompanied by an increase in CD25+ Foxp3+ T-regulatory (Treg) cells. Addition of an anti-CD25 antibody to the PLX4720/HSV1716 doublet led to complete eradication of melanoma tumors in vivo. Current efforts are focused on deciphering T-cell dynamics during this therapy using a novel technology, Timer of cell kinetics and activity (Tocky). Tocky uses a fluorescent Timer protein which spontaneously changes its emission spectrum from blue to red, reporting real-time activation of the T-cell receptor and subsequent activation of immune cells in vivo. Using Tocky, we are identifying specific immune subsets that are stimulated following therapy and the real-time dynamics of antigen recognition and subsequent cell activation. These findings form the basis of future testing of drug-virus combinations in an effort to identify potent cytotoxic, as well as immuno-modulatory, strategies and their detailed mechanism of action.

Citation Format: Galabina Bozhanova, Victoria Jennings, Malin Pedersen, Joan Kyula, Emmanuel Patin, Jehanne Hassan, Masahiro Ono, Kevin Harrington, Alan Melcher. Mutant BRAF small molecule inhibition enhances oncolytic herpes virus immunotherapy through increased immune cell recruitment and activation in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3415.
Abstract 3460: Effects of feeding soy protein isolate diet on hepatic methylation and oxidative stress in DMBA-induced mammary tumor in obese rat model
Authors: Hakkak Reza , Melnyk Stepan , Korourian Soheila
Published: September 2020

The obesity epidemic is in the United States and world for past two decades. There is a link between obesity and chronic diseases development such as diabetes, cardiovascular disease and certain types of cancers including breast cancer. Obesity has a significant impact on the metabolic profiles for a variety of cellular-tissue-organ levels in animals and humans. The objective of this study was to determine the effects of obesity and a soy protein isolate (SPI) diet on liver methylation and oxidative stress using 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor model. We hypothesize that SPI diet is capable to reduce the liver damage by reducing oxidative stress and modifying methylation status. After one week of acclimation, five weeks old female lean and obese Zucker rats were randomly fed AIN-93-G diet with either casein (CAS as control) or SPI as source of protein. All rats were orally gavaged at age 50 days with 65 mg/kg DMBA. Rats were weighed and palpated twice weekly and sacrificed 22 weeks post-DMBA treatment. Liver sample metabolites concentrations were measured using HPLC with Electrochemical Detection and LC-MS. Our results shows that: 1) Obesity increased significantly (P<0.001) body weight for both CAS and SPI diets; 2) 69% of the Lean casein rats developed mammary tumors compared to 50% in lean soy group (p= 0.176) and in the obese group, 76% of the soy-fed rats developed mammary tumors compared to 15% (P<0.001) of obese casein- fed rats; 4) Obesity in both diets significantly modify liver methylation and oxidative stress status; 5) lean SPI-fed rats significantly (P<0.025) increased SAM/SAH “methylation ratio” compare to lean CAS-fed rats; 6) obese SPI-fed rats significantly (P<0.001) decrease level of Homocysteine in liver and increase significantly (P<0.001) Methionine/Homocysteine ratio compared CAS obese rats; 7) obesity caused a significant increase in oxidative stress (GSH/GSSG ratio decrease) in liver for both CAS (P<0.004) and SPI (P<0.01) diets and 8) obese SPI fed rats significantly (P<0.02) decreased oxidative in liver (GSH/GSSG ratio increase) compared to obese CAS fed rats. In summary, we found that SPI diet capable make significant impact on methylation status by increasing a “methylation ratio”, decrease level of toxic intracellular compound homocysteine, and improving redox environment in liver triggered by obesity in mammary tumor model.

Citation Format: Reza Hakkak, Stepan Melnyk, Soheila Korourian. Effects of feeding soy protein isolate diet on hepatic methylation and oxidative stress in DMBA-induced mammary tumor in obese rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3460.
Abstract 3464: Vitamin D supplementation to improve doxorubicin-induced cardiotoxicity in triple negative breast cancers
Authors: Lee Kevin J. , Wright Griffin , Wiggins Leigh Ann , Schuler Michele , Gassman Natalie R.
Published: September 2020

Doxorubicin (Dox) is a first-line treatment for triple negative breast cancer (TNBC), which lacks the molecular targets for hormone or growth factor therapy. Although highly effective as a chemotherapeutic agent, Dox induces severe side effects including cardiotoxicity that can cause patients to stop treatment early and impact the long-term health of patients in remission. Reactive oxygen species (ROS) are the predominant contributing factor to Dox-induced cardiotoxicity. Here, we sought to use the antioxidant vitamin D to provide a cardioprotective environment to prevent or reduce cardiotoxicity in a mouse model of TNBC. Supplementation with vitamin D (10,000 IU/kg) in chow for two weeks significantly increased the active vitamin D metabolite (25(OH)VD3) in plasma from 61.2 ± 4.5 nM (Mean ± SEM) to 85.0 ± 6.3 nM (P < 0.05). Supplemented and normal chow mice were then inoculated with the syngeneic 4T1 mouse model of TNBC, and subjected to 10 mg/kg Dox once per week for two weeks. Cardiotoxicity was monitored through non-invasive echocardiography weekly. After two weeks, mice on standard chow with moderate levels of vitamin D exposed to 10 mg/kg Dox showed clear signs of cardiotoxicity represented by a decrease in cardiac output from 9.7 ± 0.5 mL/min in control mice to 4.8 ± 0.9 mL/min in mice treated with Dox (P < 0.001); a decrease in ejection fraction from 49.2% ± 1.9 in control mice to 35.7% ± 5.9 in Dox mice (P < 0.05); and a decrease in fractional shortening from 24.4% ± 1.2 in control mice to 16.9% ± 3.1 in Dox mice (P < 0.05). Mice treated with Dox also showed detectable levels of cardiac troponin (0.04 ± 0.01 ng/mL) in circulating plasma indicative of cardiac damage, while troponin levels in control mice were undetectable. Vitamin D supplementation during exposure to Dox prevented cardiotoxicity in all parameters tested with levels of cardiac output of 7.4 ± 0.9 mL/min compared to 4.8 ± 0.9 mL/min in Dox mice; ejection fraction of 55.3% ± 3.4 compared to 35.7% ± 5.9 in Dox mice (P < 0.01); and fractional shortening of 28.1% ± 2.1 compared to 16.9% ± 3.1 in Dox mice (P < 0.01). Cardiac troponin levels were also undetectable similar to control mice. Vitamin D supplementation during Dox exposure resulted in cardiac function not significantly different than control, untreated mice. These data support further examination of vitamin D supplementation during chemotherapy to prevent cardiac toxicity and to enhance long-term outcome of TNBC patients.

Citation Format: Kevin J. Lee, Griffin Wright, Leigh Ann Wiggins, Michele Schuler, Natalie R. Gassman. Vitamin D supplementation to improve doxorubicin-induced cardiotoxicity in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3464.
Abstract 3510: The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS)
Authors: Buko Alexander , Cheng Leo , Gustev Andrew , Feldman Adam
Published: September 2020

Background: Prostate cancer (PCa) pathogenesis is influenced by alterations in cellular metabolism. Metabolomics measures these biochemical changes to create global tissue metabolite profiles. Urinary studies are noninvasive and can potentially identify biomarkers for PCa.

Objective: We used Capillary Electrophoresis Mass Spectrometry (CE-MS) to analyze urine from men undergoing prostate biopsy to investigate their metabolomic profiles.

Methods: We retrospectively analyzed 40 urine samples (20 from bx demonstrating PCa, 20 from benign) that were prospectively collected from men undergoing prostate biopsy. Analysis of charged metabolites done with Agilent CE-MS system (J Proteome Res. 2:488; 2003). Screening of potential biomarkers was performed with statistical protocols and pathway analyses. Metabolites with levels below the detection limit in all samples were excluded. Relative abundances of metabolites were normalized to levels of creatinine. Pathway analysis with MetaboAnalyst (Metabolites 9:57; 2019).

Conclusions: CE-MS analysis identified several metabolic pathways that were altered in urine of men with PCa. These metabolites are involved in steroid, aromatic, microorganism and SCFA processes and warrant targeted studies which are underway in our lab. If validated, they have potential to serve as non-invasive biomarkers for PCa diagnosis and therapeutics

Citation Format: Alexander Buko, Leo Cheng, Andrew Gustev, Adam Feldman. The metabolomic profile of urine from prostate cancer patients using capillary electrophoresis mass spectrometry (CEMS) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3510.
Abstract 3533: Germline POLE and POLD1 proofreading domain mutations in endometrial carcinoma from Middle Eastern region
Authors: Siraj Abdul K. , Parvathareddy Sandeep K. , Bu Rong , Iqbal Kaleem , Siraj Sarah , Masoodi Tariq , Ghazwani Laila , Kong Yan , Sabido Maria A. , Albalawy Wafaa , Victoria Ingrid F. , Galvez Mary J. , Al-Kuraya Khawla S.
Published: September 2020

Endometrial carcinoma (EC) accounts for 5.8% of all cancers in Saudi females. Although most ECs are sporadic, 2%-5% tend to be familial, being associated with Lynch syndrome and Cowden syndrome. In this study, we attempted to uncover the frequency, spectrum and phenotype of germline mutations in the proofreading domain of POLE and POLD1 genes in a large cohort of ECs from Middle Eastern region. We performed Capture sequencing and Sanger sequencing to screen for proofreading domains of POLE and POLD1 genes in 432 EC cases, followed by evaluation of protein expression using immunohistochemistry. Variant interpretation was performed using PolyPhen-2, MutationAssessor, SIFT, CADD and Mutation Taster. In our cohort, four mutations (0.93%) were identified in 432 EC cases, two each in POLE and POLD1 proofreading domains. Furthermore, low expression of POLE and POLD1 was noted in 41.1% (170/1414) and 59.9% (251/419) of cases, respectively. Both the cases harboring POLE mutation showed high nuclear expression of POLE protein, whereas, of the two POLD1 mutant cases, one case showed high expression and another case showed low expression of POLD1 protein. Our study shows that germline mutations in POLE and POLD1 proofreading region are a rare cause of EC in Middle Eastern population. However, it is still feasible to screen multiple cancer related genes in EC patients from Middle Eastern region using multigene panels including POLE and POLD1.

Keywords: POLE; POLD1; Endometrial carcinoma; Proof-reading domain mutations; Germline.

Citation Format: Abdul K. Siraj, Sandeep K. Parvathareddy, Rong Bu, Kaleem Iqbal, Sarah Siraj, Tariq Masoodi, Laila Ghazwani, Yan Kong, Maria A. Sabido, Wafaa Albalawy, Ingrid F. Victoria, Mary J. Galvez, Khawla S. Al-Kuraya. Germline POLE and POLD1 proofreading domain mutations in endometrial carcinoma from Middle Eastern region [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3533.
Abstract 3549: Low frequency of germline mutations in POLE and POLD1 proofreading domains in Middle Eastern colorectal cancers
Authors: Bu Rong , Siraj Abdul K. , Iqbal Kaleem , Parvathareddy Sandeep K. , Masoodi Tariq , Siraj Nabil , Siraj Nabil , Al-Rasheed Maha , Siraj Sarah , Kong Yan , Ahmed Saeeda O. , Al-Obaisi Khadija A.S. , Victoria Ingrid G. , Al-Kuraya Khawla S.
Published: September 2020

Colorectal cancer (CRC) is one of the main causes of cancer related morbidity and mortality. Only ~ 5% of all CRCs occur as a result of mutations in well-defined CRC predisposing genes. The underlying causes in genetically unexplained CRC cases from Middle Eastern region are still unknown. We performed Capture sequencing and Sanger sequencing to screen the proofreading domains of DNA Polymerase Epsilon (POLE) and DNA Polymerase Delta 1 (POLD1) genes to find out the genetic causes in over 1100 CRC cases from Middle Eastern region. In our cohort, five mutations (0.44%) were identified in 1135 CRC cases, four in POLE gene (0.35%, 4/1135) and one (0.1%, 1/1135) in POLD1 gene. These mutations have been infrequently reported in Western populations. Furthermore, low expression of POLE was noted in 38.9% (417/1071) of cases and showed significant association with lymph node involvement (p=0.0184) and grade 3 tumors (p=0.0139). Whereas, low expression of POLD1 was observed in 51.9% (555/1069) of cases and was significantly associated with adenocarcinoma histology (p=0.0164), larger tumor size (T3 and T4 tumors; p=0.0012) and stage III tumors (p=0.0341). Our findings revealed that there was a low frequency of

mutations in POLE and POLD1 proofreading region in CRC cases and established that the mutations in the proofreading domain of these two genes were a rare cause of CRC in Middle Eastern region. It is feasible to screen multiple cancer related genes in CRC patients from Middle Eastern region using multigene panels including POLE and POLD1.

Citation Format: Rong Bu, Abdul K. Siraj, Kaleem Iqbal, Sandeep K. Parvathareddy, Tariq Masoodi, Nabil Siraj, Nabil Siraj, Maha Al-Rasheed, Sarah Siraj, Yan Kong, Saeeda O. Ahmed, Khadija A.S. Al-Obaisi, Ingrid G. Victoria, Khawla S. Al-Kuraya. Low frequency of germline mutations in POLE and POLD1 proofreading domains in Middle Eastern colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3549.
Abstract 3553: Three-dimensional (3D) telomere signatures of sporadic pediatric papillary thyroid carcinoma (PTC)
Authors: Sisdelli Luiza , Cordioli Maria Isabel , Vaisman Fernanda , Monte Osmar , Longui Carlos , Cury Adriano N. , Freitas Monique O. , Rangel-Pozzo Aline , Mai Sabine , Cerutti Janete
Published: September 2020

The incidence of thyroid carcinoma has increased worldwide, including in pediatric patients. Papillary thyroid carcinoma (PTC) is the most common subtype. Previous studies have suggested that clinical presentation and outcome diverge between pediatric and adult PTC and that this difference is likely due to distinct genetic alterations. BRAF V600E point mutation is highly prevalent in adults, while genetic fusions (RET/PTC, AGK-BRAF, ETV6-NTRK3) are the most prevalent events in the pediatric population. As telomere loss or dysfunction results in aneuploidy and chromosomal rearrangements, which can promote carcinogenesis, we investigated the three-dimensional (3D) structure of the telomeres in the nuclei from 21 pediatric patients (≤18 y.o), representing 16 PTC cases (with paired normal and tumor tissues for five of them) and five normal thyroid tissues from patients undergoing thyroidectomy for reasons other than cancer. We performed quantitative fluorescence in situ hybridization (FISH), 3D imaging, and 3D telomere analysis using TeloView® software to examine telomere dysfunction. The parameters examined included total number of signals (numbers of telomeres), total number of telomere aggregates, a/c ratio (indicating the phase of the cell cycle), total intensity (telomere length) and nuclear volume. Thyroid cancer cells showed lower average intensity of signals (p<0.0001) and lower intensity signals (p<0.0001), as well as higher nuclear volume (p<0.0001), compared to normal thyroid cells. In addition, tumour cells showed a higher a/c ratio (p<0.0001), indicating that they are more actively dividing than normal cells, which correlates with the high proliferation rate of cancer cells. We did not observe any alterations related to the total number of telomere signals and total number of aggregates, which suggests no significant aneuploidies occurring with these samples. The 3D nuclear telomere signature was able to detect differences in telomere architecture in tumours, compared to normal. Further analysis is necessary to define whether nuclear telomere architecture and specific genetic alterations observed in pediatric thyroid cancer are associated.

Citation Format: Luiza Sisdelli, Maria Isabel Cordioli, Fernanda Vaisman, Osmar Monte, Carlos Longui, Adriano N. Cury, Monique O. Freitas, Aline Rangel-Pozzo, Sabine Mai, Janete Cerutti. Three-dimensional (3D) telomere signatures of sporadic pediatric papillary thyroid carcinoma (PTC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3553.
Abstract 3566: Lysyl oxidase is associated with the epithelial–mesenchymal transition of gastric cancer cells in hypoxia
Authors: Yashiro Masakazu , Kasashima Hiroaki , Yamamoto Yurie , Kuroda Kenji , Sera Tomohiro , Sugimoto Atsushi , Kushiyama Shuhei , Togano Shingo , Okuno Tomohisa , Miki Yuichiro , Ohira Masaichi
Published: September 2020

Purpose: Hypoxic environments are randomly scattered throughout carcinomas. Although hypoxia is cytotoxic to both cancer cells and normal cells, some types of cancer cells acquire characteristics that allow them to survive and progress in a hypoxic environment. Hypoxia is considered to be associated with aggressive tumor phenotypes of gastric carcinomas, including the ability to metastasize. Hypoxia is clinically associated with metastasis and poor patient outcome, however the underlying processes remain unclear. It has been reported that lysyl oxidase (LOX) is a hypoxia-responsive factor and is associated with the malignant progression of carcinoma. The aim of this study was to clarify the relationship between the epithelial–mesenchymal transition (EMT) and LOX in gastric cancer cells under hypoxia.

Methods: Two gastric cancer cell lines, OCUM-2MD3 and OCUM-12, were used in an in vitro study. The effect of siLOX on the EMT and motility of gastric cancer cells under hypoxic condition was analyzed by RT-PCR, western blot, wound-healing assay, and invasion assay. Correlations between LOX expression and the clinicopathological features of 544 patients with gastric carcinoma were examined immunohistochemically.

Results: Hypoxic conditions increased the number of polygonal or spindle-shaped cells of EMT in gastric cancer cells. The EMT of cancer cells induced by hypoxia was inhibited by treatment with LOX siRNA. Taken together, the number of migrating and invading gastric cancer cells in hypoxia was significantly decreased by LOX knockdown. LOX siRNA significantly increased the E-cadherin level and decreased the vimentin level of gastric cancer cells. LOX expression was significantly associated with invasion depth, tumor differentiation, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal metastasis. Multivariable analysis revealed that LOX was an independent parameter for overall survival.

Conclusion: HIF-1α might increase the expression of LOX, which affects the EMT of gastric cancer cells in hypoxic conditions via down-regulation of E-cadherin level and up-regulation of vimentin level. LOX expression may be a useful prognostic factor for patients with gastric cancer.

Citation Format: Masakazu Yashiro, Hiroaki Kasashima, Yurie Yamamoto, Kenji Kuroda, Tomohiro Sera, Atsushi Sugimoto, Shuhei Kushiyama, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Masaichi Ohira. Lysyl oxidase is associated with the epithelial–mesenchymal transition of gastric cancer cells in hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3566.
Abstract 358: Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer
Authors: Nielsen Boye Schnack , Holmstrom Kim
Published: September 2020

Tumor necrosis factor alpha (TNF) is a potent cytokine reported to have several functions, including stimulation of inflammation, directing cell death and cell proliferation. The cellular origin of TNF has generally believed to be the inflammatory cells especially because macrophages are prone to induction of TNF. Using in situ hybridization (ISH) based on TNF mRNA probes (RNAscope) we recently reported that TNF mRNA is also seen in cytokeratin positive cancer cells (CC) and is associated with morphologic appearance interpreted as CC branching (Møller et al, IJMS 2019, doi: 10.3390/ijms20081907). To determine the fraction of CC-derived TNF mRNA signal at different areas in colon cancers we developed a cell and color segmentation-based image analysis tool to process fluorescence signals in digital slides. The approach allowed quantification of the TNF mRNA-positive CC and TNF mRNA-positive stromal cells in the digital slides previously reported, that were also stained for cytokeratin, microRNA-21 (miR-21) and DAPI nuclear stain. In the two cases with most abundant TNF mRNA ISH signal, both showing moderate-to-high budding index, the fraction of TNF-mRNA positive CC in the outer periphery (approximately 500 µm) was 8% and 20%, respectively. The frequency of the TNF-positive CC increased towards the invasive front from 7% (33/500 CC per 0.25 mm2) to 35% (48/137 CC), and from 8% (127/1536 CC per 0.50 mm2) to 28% (108/389 CC), respectively. The upregulation is in agreement with initial qualitative evaluation. In the same areas, the fraction of TNF mRNA positive stromal cells among all TNF mRNA-positive cells was only 8% in both cases, indicating that the majority of the TNF mRNA originates from CC. The fraction of the TNF mRNA-positive CC that were also miR-21 positive was 85% and 43%, respectively, which was higher than the prevalence of the overall miR-21 positive CC, 55% and 30%, suggesting frequent co-expression with miR-21. In conclusion, we have developed a method to obtain quantitative estimates from digital multiplex-stained slides with RNAscope and LNA fluorescence ISH and immunofluorescence. More samples will need to be processed to better understand the importance of TNF expression in cancer cell invasion.

Citation Format: Boye Schnack Nielsen, Kim Holmstrom. Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 358.
Abstract 3632: Genomic profiling in circulating tumor DNA from Chinese advanced gastric adenocarcinomas patients
Authors: ZHANG ZHE , Chen Hui , Zhou Yifan , Zhang Bei , Bai Yuezong , Guo Lieping
Published: September 2020

Background and Objective: Circulating tumor DNA (ctDNA) is considered to be an ideal sample for advanced unresectable cancer patients to perform gene sequencing to guide clinical therapy, which might help to avoid tumor heterogeneous, especially in gastric adenocarcinomas (GACs). However, the ctDNA genomic profiling of Chinese advanced GACs is still missing.

Methods: Blood samples were obtained from 122 advanced GACs patients. Target sequencing were performed on ctDNA using a validated commercial 150-gene-panel NCC-GP150, which includes 100 microsatellite loci. Blood tumor mutation burden (bTMB) and blood microsatellite instability (bMSI) were calculated according to the NGS results. Statistical analysis was performed by the Mann-Whitney U test and Fisher exact test using GraphPad Prism.

Results: In 122 patients, 47 (38.5%) were female and 75 (61.5%) were male, with the median age of 61 (range 24 to 84). A total of 660 somatic variations were identified in 118 genes as indicated by a maximum somatic allele frequency (MSAF) of >0.3%. One hundred and two (83.6%) patients carried at least one somatic variation and 84 (68.9%) patients had at least on pathogenic or likely pathogenic variations. The median pathogenic or likely pathogenic variations was 2.5 per patient in our cohort. The most frequent altered genes were TP53 (36.9%), LRP1B (18%), MYC (14.8%), RBM10 (13.9%), ERBB2 (13.9%), KRAS (12.3%), AR (11.5%) and FAT1 (10.7%). From the perspective of signaling pathway, the variations occurred frequently in RTK-Ras-MAPK pathway (53.3%), cell cycle pathway (52.5%), DNA damage repair (DDR) pathway (22.1%), Wnt pathway (18.9%), PI3K/AKT pathway (16.4%), and TGF-β pathway (2.5%). The median bTMB was 3 (mean 5.27, range 0 to 52); and 8 (7.8%) patients had high bMSI. The bMSI-H patients had significantly higher bTMB than bMSS patients (22 vs 3, p<0.0001) and patients harboring DDR pathway gene variations had significantly higher bTMB than wildtype subgroups (6 vs 3, p=0.0004).

Conclusions: Our study revealed a comprehensive genomic profiling of advanced Chinese GACs patients. And the relationship between the genomic profiling and target-therapy as well as immunotherapy will be studied in an enlarged group of GACs patients.

Citation Format: ZHE ZHANG, Hui Chen, Yifan Zhou, Bei Zhang, Yuezong Bai, Lieping Guo. Genomic profiling in circulating tumor DNA from Chinese advanced gastric adenocarcinomas patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3632.
Abstract 3661: Association of mammographic density with blood DNA methylation
Authors: Lucia Rachel McFarland , Huang Wei-Lin , Alvarez Andrea , Masunaka Irene , Ziogas Argyrios , Goodman Deborah , Odegaard Andrew O. , Norden-Krichmar Trina M. , Park Hannah Lui
Published: September 2020

Background: Mammographic density is a strong risk factor for breast cancer. While numerous genetic variations have been shown to be associated with mammographic density, an epigenetic signature of mammographic density has not yet been identified. The objective of this study was to identify a DNA methylation signature associated with mammographic density.

Methods: Blood samples were collected from 197 postmenopausal women 45-65 years old with no personal history of breast cancer. For each participant, mammographic density according to the BI-RADs density classification was obtained from the most recent mammogram report. White blood cell DNA methylation at more than 850,000 CpG sites was measured using the Illumina HumanMethylationEPIC BeadChip. Differential methylation was assessed using genome-wide, probe-level, and regional approaches after filtering and normalization using the recommended steps for DNA methylation microarray data. All analyses were adjusted for appropriate confounding variables.

Results: On average, genome-wide DNA methylation was higher in women with elevated mammographic density. Additionally, DNA methylation at 343 individual sites and in 34 regions was significantly associated with mammographic density after false discovery rate correction. Most density-associated sites were hypermethylated. The top differentially methylated sites were located near cell cycle regulatory genes (ICMT, DESI2, TCF21/TARID, ROBO3), as well as those encoding cytoskeletal or plasma membrane proteins (PALLD, ANO7, ATP8B3, MTUS2). We observed hypermethylation near the promoters of genes including RUFY1 and SLFN12 and within the bodies of genes including LCN6, SNED1, and LRFN1. Functional analysis of the differentially methylated sites revealed enrichment of gene ontology categories including angiogenesis and regulation of cell adhesion, as well as apoptotic signaling in response to DNA damage.

Conclusions: White blood cell DNA methylation differed according to mammographic density in a sample of postmenopausal women. Probe-based and regional analysis identified differentially methylated regions located near genes implicated in cancer. These differentially methylated regions may serve as a signature of mammographic density, and future studies of the genes implicated may provide a deeper understanding of the connection between mammographic density and breast cancer risk.

Citation Format: Rachel McFarland Lucia, Wei-Lin Huang, Andrea Alvarez, Irene Masunaka, Argyrios Ziogas, Deborah Goodman, Andrew O. Odegaard, Trina M. Norden-Krichmar, Hannah Lui Park. Association of mammographic density with blood DNA methylation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3661.
Abstract 3681: Selective vulnerability of GBM PDX to a panel of EGFR tyrosine kinase inhibitors
Authors: Tang Nanyun , Reid George , Miller C Ryan , Berens Michael E.
Published: September 2020

Glioblastoma (GBM), a particularly aggressive form of primary brain tumor, has poor survival due to a lack of effective treatments and high recurrence. Epidermal growth factor receptor (EGFR) (mapped to 7p 11.2), is frequently amplified and mutated (>50%) in GBM, and is suggested as a potential therapeutic target. To correlate chemo-vulnerability with genomic aberrations and further determine biomarkers for targeted therapies, a panel of seven EGFR tyrosine kinase inhibitors were tested in a selected nine human GBM patient-derived xenograft (PDX) panel; these preclinical models harbor different mutation combinations of CDKN2A deletion (C), PTEN deletion (P), wild-type EGFR (E) and/or EGFRvIII (Ev3) overexpression. A single agent drug dose response assay (DDR) was performed on GBM PDX neurospheres using drug concentrations ranging from 100uM to 0.5nM with a 12-point serial 3-fold dilution scheme. The cytotoxic efficacy of EGFR inhibition was dependent on both the PDX line and the EFGR inhibitor applied. GBM59 and GBM76 – both triple CEv3P mutants – were the most responsive to EGFR inhibition relative to the other GBM models tested. For GBM59, the IC50 values of afatinib, canertinib and neratinib, were sub-micromolar, and low micromolar (<3uM) for erlotinib, gefitinib and lapatinib. GBM76 had IC50 values of 1-8uM with all seven EGFR inhibitors including AZD3759. However, GBM155 and GBM126 with CEv3 but wildtype PTEN only responded to afatinib, canertinib and neratinib at low micromolar IC50 values (<3uM) but not to erlotinib, gefitinib and lapatinib. A deletion in PTEN may sensitize GBM cells with CEv3 mutations to erlotinib, gefitinib and lapatinib in vitro. The other group of GBM PDX lines (C only) represented by GBM122, GBM150 and GBM182 responded well to afatinib, canertinib and neratinib with IC50 values of 2- 9uM. GBM156 with CE had a similar profile to the group of C only or CEv3. GBM56 with CP showed the most resistance with mild responses to afatinib, canertinib and neratinib, as observed in its high micromolar IC50 values (6-9uM). Overall, neratinib is the most effective compound among the tested EGFR inhibitors based on IC50 values. Canertinib, the triple tyrosine kinase inhibitor against EGFR/HER2/ErbB-4, is the second most effective agent. Afatinib has similar drug efficacy to canertinib. All three compounds moderately inhibit all GBM PDX lines. The drug response mechanism and its correlation with the mutation status of CDKN2A, EGFR, and PTEN will be further investigated cross a broad array of GBM PDX models. Supported by NIH NCI R01 CA204136

Citation Format: Nanyun Tang, George Reid, C Ryan Miller, Michael E. Berens. Selective vulnerability of GBM PDX to a panel of EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3681.
Abstract 3710: Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer
Authors: Hossian A K M Nawshad
Published: September 2020

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Among the 18.1 million of total cancer patients last year, 2.1 million of the new cases were of LC worldwide. The poor prognosis, higher incidence, and mortality rate make this disease a devastating type of cancer. Cell cycle inhibition is one of the prominent choices among researchers in cancer treatment. Different cell cycle inhibitors are developed by scientists where the cyclin-dependent kinase (CDK) inhibitors are among the most frequently researched. Just recently, 3 CDK inhibitors received FDA-approval for treatment of specific breast cancer subtypes, but no CDK inhibitors currently exist for LC patient treatment. Thus, there is a need for molecules that regulate cell cycle progression and CDK expression. Micro RNAs (miRNAs) are gene-expression regulatory molecules. Among several miRs, we selected miR-143 and miR-506 to regulate CDK1 and CDK4/6, respectively. qPCR and RNA-sequencing indicated that this combinatorial miR therapy caused downregulation of the respective CDK1, 4, and 6 genes in H358 and H1975 lung cancer cell lines. This RNA inhibition corresponded to protein levels downregulation, as detected by western blot analysis. Further investigation found that this treatment caused significant halt of the cell cycle progression, which is markedly stronger than the commercially available CDK inhibitors, Ribociclib and Flavopiridol. We also identified the combined miR-143 and miR-506 caused significant increase of the apoptotic cell population at both 24 and 48 h post-treatment, as detected by an Annexin V/PI analysis. Interestingly, low or no apoptotic impact was observed in HFL1 normal lung fibroblast cells by the combinatorial miR treatment, indicating a cancer-cell specific activity. Upstream pathway analysis through RNA-sequencing using Ingenuity Pathway Analysis software (IPA; Qiagen) indicated a potential ERK-mediation on the activity of the combinatorial miR treatment. Our analysis of the ERK pathway using MAPK (ERK) inhibitors indicated an ERK-independent cell cycle inhibitory activity of the two miRs. Through further analysis of the RNA-Seq data, we observed a potential inhibition of the cellular migration and invasion activity of the cells, which was confirmed by a cell migration assay. In summary, combinatorial treatment of miR-143 and miR-506 has a significant impact on the cell cycle, overall survival, and migration of LC cells, which still merits further study.

Citation Format: A K M Nawshad Hossian. Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3710.
Abstract 3724: BMAL1 rewires mitochondrial metabolism and promotes tumor progress of triple negative breast cancer during insulin resistance
Authors: Ramos Cassandra A. , Ouyang Ching , Qi Yue , Chung Yiyin , Cheng Chun-Ting , LaBarge Mark A. , Seewasldt Victoria L. , Ann David K.
Published: September 2020

Insulin resistance is a hallmark of metabolic diseases and negatively associated with breast cancer outcomes. While much work has dedicated to circadian dysregulation, metabolic diseases in breast cancer progression, the molecular mechanism connecting all these perspectives together has not yet established. To understand this underlying mechanism, we use in vitro and in vivo insulin-resistant triple-negative breast cancer (TNBC) models, illustrating that insulin resistance in TNBC reciprocally alters mitochondrial respiration and the expression of genes in the circadian rhythm pathway. Furthermore, genetic knockdown of BMAL1, an important circadian transcription factor, ablates key adaptations to insulin resistance, such as increased pyruvate flux, oxygen consumption, and lipid storage. Our in vivo study demonstrates that orthotopically transplanted E0771 mouse breast cancer cells with BMAL1 knocked down grow significantly faster than the parental counterpart in mice fed with the high-fat diet. This new understanding of BMAL1 expands our insights into circadian control of tumor metabolism and may lead to novel treatments for breast cancer.

Citation Format: Cassandra A. Ramos, Ching Ouyang, Yue Qi, Yiyin Chung, Chun-Ting Cheng, Mark A. LaBarge, Victoria L. Seewasldt, David K. Ann. BMAL1 rewires mitochondrial metabolism and promotes tumor progress of triple negative breast cancer during insulin resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3724.
Abstract 3757: Examination of the epidermal growth factor 3 (HER3) dysregulations in hepatocellular carcinoma
Authors: Ming Sheung In , Chan Lo Kong , Ho Daniel W.H. , Ng Irene O.L.
Published: September 2020

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a particularly high mortality rate. Treatment options for HCC are limited. Curative treatments including surgical resection and liver transplantation are only applicable to early diagnosed patients. For advanced HCC patients, the currently available first-line molecularly targeted medications are suffering from low rates of response and high inter-patients variability providing limited survival benefits. Receptor tyrosine kinases (RTKs) are plasma membrane-associated protein kinases which control the activation of intracellular signal transductions in response to the extracellular stimuli. Activation of specific RTKs has been implicated in various kinds of human cancers, and pharmacological inhibition of cancer associated RTKs is one of the key strategies of modern targeted therapy. However, a comprehensive and systematic assessment of the RTKs dysregulations in terms of expression and mutation in HCC is still much awaited.

In this study, RNA-sequencing analysis was performed in paired HCC tissues to examine the expression profiles of 58 RTKs. Also, the mutational status of these RTKs in HCC tissues was examined by targeted DNA-sequencing analysis. Our systemic analyses have revealed that Epidermal Growth Factor Receptor 3 (HER3) was significantly upregulated and recurrently mutated in HCC. Independent qPCR analysis for HER3 in 101 paired HBV-associated HCC tumor and non-tumorous samples validated that HER3 was upregulated in HCC. More importantly, upregulation of HER3 was found to be significantly correlated with the presence of metastatic features of HCC including tumor microsatellite formation (p=0.015) and venous invasion (p=0.001). Neuregulin-1 (NRG-1) is the best-known ligand for HER3. Stimulation of HER3 activation by the ligand NRG-1 could induce HER3 phosphorylation, and activate the downstream PI3K/AKT and MAPK signaling pathways, and signficantly promoted the proliferation and migration of HCC cells. Conversely, CRISPR/Cas9-mediated HER3 knockout in Huh7 and PLC cells significantly reduced the NRG-1 induced functional changes. Consistently, our preliminary results demonstrated that HER3 knockout Huh7 cells forms relatively smaller tumors in a subcutaneous tumor xenograft model in vivo. Together, our results demonstrated that HER3 dysregulation plays a supporting role in HCC development. Targeting HER3 may potentially derive new therapeutic strategy for treatment of HCC.

Citation Format: Sheung In Ming, Lo Kong Chan, Daniel W.H. Ho, Irene O.L. Ng. Examination of the epidermal growth factor 3 (HER3) dysregulations in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3757.