bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 759: Highly-sensitive detection of EGFR mutation in non-small cell lung cancer using mutant-enrichment method
Authors: Chien Hung-Chi
Published: July 2017

Detection of mutations in EGFR in non-small cell lung cancer is important for the clinical decision-making process of molecularly targeted therapies, including Iressa®, Tarceva®, and Tagrisso®. In this study we developed an assay allowing the detection of EGFR gene mutations being conducted in the routine diagnostic procedure and its analytic and clinical performance were evaluated using different types of specimens. The EGFR mutation assay in this study allows for detecting EGFR exon 19 deletion (Del), exon 20 insertion (Ins), and mutation in T790M, L858R, S768I and L861Q of rare amounts in a normal genetic background. Enhancement of detection sensitivity was achieved with the developed mutant enrichment method which combines mutation-specific primers and 3'-phosphorylation oligonucleotides as blockers for inhibiting wild-type gene from DNA amplification. Analytic performance for each mutant was assessed using the control plasmids containing the mutant genes of known amounts as well as the genomic DNA extracted from mutant cell lines and Horizon FFPE samples. The assay performance were also evaluated using clinical samples in parallel with the FDA-approval companion diagnostic product therascreen® EGFR RGQ PCR Kit . The sensitivity of the assay evaluated using control plasmids for Del, T790M , L858R, S768I, L861Q mutation and Ins was 5%, 0.5%, 1%, 0.5%, 0.1% and 0.5% respectively, and was 1%, 0.5%, 0.5%, 0.5%, 0.1% and 0.5% respectively when applying on HDx reference standards. Diagnostic results for the 116 clinical samples for identifying mutations of del, T790M, L858R, S768I, L861Q and Ins was 92.2%, 100%, 99.1%, 100%, 97.4% and 100% respectively concordance with the therascreen® EGFR RGQ PCR Kit, including 17 cases of multiple mutations. The overall diagnostic results for all mutations evaluated with PPA (positive percent agreement), NPA (negative percentage agreement) and OPA (overall percentage agreement) were 83.3%, 98.2% and 90.5%, respectively. The three samples firstly diagnostic as false positive were retested and confirmed to be Exon 19 del by DNA sequencing. The assay demonstrated good sensitivity when compared with commercial product. More clinical studies are being performed to establish relationship between drug efficacy and different mutation combinations.

Citation Format: Hung-Chi Chien. Highly-sensitive detection of EGFR mutation in non-small cell lung cancer using mutant-enrichment method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 759. doi:10.1158/1538-7445.AM2017-759
Abstract 763: Analytical validation of Ki67/CD8 duplex IHC assay using computational tissue analysis (cTATM)
Authors: Kearney Staci J. , Black Joshua C. , Landis Benjamin J. , Koegler Sally , Hirsch Brooke , Gianani Roberto
Published: July 2017

Chromogenic multiplex immunohistochemistry (IHC) assays enable investigation of the spatial relationships between tumor and immune cells, which is thought to be important for understanding and predicting therapeutic response. Development and analytical validation of multiplex IHC assays enables the use of such assays to simultaneously investigate multiple biomarkers as predictors of clinical response. In this study, we analytically validated a chromogenic duplex IHC assay that quantifies Ki67 and CD8 in formalin-fixed, paraffin-embedded non-small cell lung cancer tissues. Five performance criteria were selected and evaluated based on Clinical Laboratory Standards Institute guidelines: reportable range, analytical sensitivity, analytical specificity, accuracy, and precision. Similar to analytical validation studies for monoplex IHC assays, this study utilized a reference method and multiple days of staining. The percentage of cells positive for Ki67 nuclear staining and/or CD8 membrane staining were quantified using our computational Tissue Analysis (cTATM) platform. Performance of the Ki67/CD8 chromogenic duplex IHC assay was considered acceptable for the five criteria evaluated. Once the performance of the assay was established, additional exploratory cTA-based endpoints were examined, including the quantification of each biomarker in the tumor compartment and the tumor microenvironment, and analysis of the spatial arrangement of immune cells relative to tumor cells. In conclusion, Flagship’s cTA platform allows for more consistent quantification of individual analytes on dual-stained tissue sections, enabling investigation of complex biological questions that cannot be achieved with traditional tissue-based manual endpoints.

Citation Format: Staci J. Kearney, Joshua C. Black, Benjamin J. Landis, Sally Koegler, Brooke Hirsch, Roberto Gianani. Analytical validation of Ki67/CD8 duplex IHC assay using computational tissue analysis (cTATM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2017-763
Abstract 765: Prognostic impact of Philadelphia chromosome in elderly acute lymphoblastic leukemia patients ineligible for hematopoietic stem cell transplantation
Authors: Byun Ja Min
Published: July 2017

Elderly acute lymphoblastic leukemia (ALL) patients are often unfit for neither dose-intensive chemotherapy nor stem cell transplantation, thus their clinical outcomes have been dismal. The increased incidence of BCR-ABL translocation with increasing age also contributed to poor prognosis but, it was our conjecture that the introduction of potent tyrosine kinase inhibitors (TKI) targeting ABL, BCR-ABL translocation might not deliver negative prognostic impact in elderly ALL. Considering that many elderly Ph- ALL patients do not tolerate cytotoxic chemotherapy required for effective tumor control, we supposed that efficacy of ABL TKIs and their combination strategy with low dose cytotoxic chemotherapy would result in better survival for elderly Ph+ ALL compared to their Ph- counterparts.

This study was a multicenter longitudinal cohort study of elderly de novo ALL (≥ 60 years) recruited from 11 hospitals in Korea from January 2005 to December 2015. We considered Ph+ if it was positive in at least one of three techniques: conventional cytogenetics, fluorescence in situ hybridization, or quantitative real-time polymerase chain reaction (qRT-PCR).

Among the 96 patients enrolled, there were 50 Ph- ALL patients (52.1%) and 46 Ph+ ALL patients (47.9%). There were no differences between the 2 groups regarding age, sex distribution, performance status, and induction regimens. For treatment outcomes evaluation, the 5 Ph+ ALL patients not receiving TKI were eliminated and 41 Ph+ ALL patients treated with TKI were compared with 50 Ph- ALL patients (Table). Complete remission (CR) was attained in 27 (54.0%) of patients in the Ph- group versus 35 (85.4%) in the Ph+ group (P=0.001). The median RFS was significantly longer in the Ph+ group (3.1 months for Ph- vs. 8.9 months for Ph+, P=0.017). Induction death and induction failure occurred significantly more often in the Ph- group. The median OS was 6.3 months for Ph- group versus 10.3 months for Ph+ group (P=0.033).

In conclusion, using real world data, this study reports the possible favorable impact of Ph+ in TKI-era for elderly patients.

Citation Format: Ja Min Byun. Prognostic impact of Philadelphia chromosome in elderly acute lymphoblastic leukemia patients ineligible for hematopoietic stem cell transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2017-765
Abstract 766: Comorbidities limiting recruitment of non-small cell lung cancer (NSCLC) patients in early phase trials
Authors: Duma Narjust , Wang Yucai , Mansfield Aaron
Published: July 2017

Background: Early phase trials (EPCT) provide significant value by evaluating the possible benefits of new agents. The eligibility criteria in EPCT are usually rigorous and may exclude many patients (pts) commonly seen in clinical practice. Our objective was to identify the most common comorbidities excluded in EPCT for NSCLC.

Methods: ClinicalTrials.gov was queried on August 1st of 2016. 299 interventional drug trials were extracted from 2010 to 2016. We studied the trial characteristics including: experimental therapy, location, funding and eligibility criteria. Exclusion of CNS metastasis was treated as a binary variable (strict exclusion vs. allowed). Logistic regressions were used for statistical analysis.

Results: Of the 299 trials, 164 (55%) were phase II, 81 (27%) phase I and 54 (18%) phase Ib/II. 164 (55%) were conducted in the United States (US), 45 (15%) in Asia, 39 (13%) in Europe and 51 (17%) were international. 171 (57%) trials were funded by the industry, 54 (18%) by government agencies and 74 (25%) were investigator initiated. Survival was an end-point in 97 (32%) trials. 70 (23%) trials required a repeat lung biopsy. 54 (18%) trials excluded pts >75 years of age. 99 (33%) trials required creatinine levels <1.5 mg/dl, 132 (44%) liver enzymes <2.5 and total bilirubin <1.5 of the upper limit of normal. 88 (29%) trials required Hgb>9 g/dl and 141 (47%) excluded pts with platelets <100,000/dl. In terms of comorbidities, 84 (28%) trials excluded pts with atrial fibrillation, 94 (31%) with any anticoagulation therapy and 120 (40%) HIV positive pts. CNS involvement was a significant exclusion factor, 123 (41%) trials excluded pts with CNS metastasis. On univariate analysis, the odds of CNS metastasis exclusion were significantly higher in immunotherapy trials (OR 1.72, 95%CI 1.2-2.4, p<0.01) and lower in European trials (OR 0.76, 95%CI 0.6-0.9, p<0.005). In multivariate analysis, US based trials had higher odds of exclusion of CNS metastasis (OR: 3.2, 95%CI 1.4-7.5, p<0.05).

Conclusions: Many of the EPCTs excluded pts with advanced age, cytopenias and other common comorbidities, eliminating the possibility of many of our aging pts from participation. Regarding CNS disease, our analysis suggests the type of agent and location may influence the trial design about CNS metastasis inclusion. Investigators should review whether sufficient justification exists for every exclusion criterion before their incorporation in future trial protocols.

Citation Format: Narjust Duma, Yucai Wang, Aaron Mansfield. Comorbidities limiting recruitment of non-small cell lung cancer (NSCLC) patients in early phase trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2017-766
Abstract 768: The utility of a breast cancer registry for improved cancer management in developing countries
Authors: McGarity DeAnna , Epps Brandon , Maynor Alexis , Anderson Tania , Alema-Mensah Ernest , Fray Delroy , Aung Maung , Holden Kisha , Lillard James , Rivers Brian , Beech Derrick
Published: July 2017

Background

Breast cancer is one of the leading causes of cancer related deaths in the island nation of Jamaica, West Indies. Cancer and other non-communicable diseases have increased in many developing nations in the Caribbean as well as Central and South America. The health systems of these developing countries continue to provide care to patients with limited resources. The use of a breast cancer registry can assist in care coordination of these patients and allow targeted screening and treatment programs in under-resourced environments.

Methods

In collaboration with the Western Regional Health Authority, a breast cancer registry database was developed in RedCap to provide a comprehensive analysis of breast cancer in under resourced environment. 53 patients from Atlanta were compared to 78 patients from Jamaica These patients were entered into the breast cancer registry from June 1, 2014 to March 31, 2015 by the attending physician and de-identified. Demographics, tumor related factors, treatment, survival and health behavior information was collected by the attending physician. The findings were then analyzed using Statistical Analysis System (SAS) and Data Analysis and Statistical Analysis (STATA): Fisher Exact test, chi-square and multiple regressions.

Results

The median of patient ages were 61 years old (range 38 - 81 years) for the U.S. patients and 53 years old (range 26 - 84 years old) for Jamaican women. For the U.S. patients, 84% were Black, 4% White, and 12.24% were Latin or Asian. The majority of the patients were diagnosed with stage 1 invasive ductal breast cancer. In contrast, the majority of the Jamaican patients were diagnosed with late-stage breast cancer. Utilizing the data from the breast cancer registry, we were able to identify parishes (counties) with disproportionately high rates of late-staged cancer at the time of diagnosis.

Conclusion

The use of a disease specific registry can improve care coordination and identify actionable target areas for enhanced screening and treatment. It is our hope to frequent review the dashboards associated with the registry data by providers, medical centers and health systems. This will optimize care coordination and allow timely improvements in cancer diagnosis and management in developing countries.

Citation Format: DeAnna McGarity, Brandon Epps, Alexis Maynor, Tania Anderson, Ernest Alema-Mensah, Delroy Fray, Maung Aung, Kisha Holden, James Lillard, Brian Rivers, Derrick Beech. The utility of a breast cancer registry for improved cancer management in developing countries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2017-768
Abstract 775: Identification of activated signal transduction pathways and molecules in the HMGB-1 induced angiogenesis
Authors: Kwon Yujin , Shin Sujin , Kim Won Kyu , Kim Hoguen
Published: July 2017

High-mobility group box-1 (HMGB-1) is a highly conserved protein playing various roles both in the nucleus and cytosol and known to be expressed in almost all types of mammalian cells. Secreted HMGB-1 has been most intensively studied and reported as an immune cell derived cytokine regulating systemic inflammation, signal transduction, and activation of endothelial cells (EC). Especially, HMGB-1 over-expression is now considered as a hallmark in cancers based on its involvement in cell proliferation, inflammatory microenvironment, invasion, metastasis, and sustained angiogenesis. Numerous studies have shown that secreted HMGB-1 directly or indirectly promotes angiogenesis by activation of ECs. However, mechanisms of HMGB-1 mediated angiogenesis have not yet been fully understood, especially, in terms of genomic changes caused by HMGB-1 treatment. Moreover, differences between vascular endothelial growth factor (VEGF) mediated and HMGB-1 mediated angiogenesis have not been studied.

To explore the roles of HMGB-1 in angiogenesis, we first confirmed whether HMGB-1 treatment renders ECs angiogenic by evaluating proliferation, migration, wound-healing, and tubule formation of ECs after HMGB-1 treatment. In addition, we performed gene expression microarray to clarify how HMGB-1 treatment induced EC activation at various time points and subsequent angiogenic features according to time-dependent gene expression changes.

We show that HMGB-1 treatment induces proangiogenic features to ECs such as stimulation of tubule formation, promotion of proliferation, and accelerated wound healing. By analysis of microarray data, we found that VEGF and HMGB-1 commonly promoted the activation of MAPK, cell proliferation, and wound healing pathways which directly regulate angiogenesis. On the other hand, axon guidance, gap junction, P53 signaling, cell cycle, and cell differentiation pathways were specifically up-regulated by HMGB-1. Taken together, HMGB-1 leads to angiogenesis via upregulation of both angiogenic and nonangiogenic genes.

Citation Format: Yujin Kwon, Sujin Shin, Won Kyu Kim, Hoguen Kim. Identification of activated signal transduction pathways and molecules in the HMGB-1 induced angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2017-775
Abstract 776: Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry
Authors: Helali Aya El , McCabe Nuala , Dickson Naomi , Perez Lara Dura , Harkin Denis P , Wilson Richard , Kennedy Richard
Published: July 2017

Introduction:

Platinum resistant High Grade Serous Ovarian Cancer (HGSOC) has a poor outcome with limited treatment options. Angiogenesis is a key pathological feature of ovarian cancer and anti-angiogenics have dominated the field of drug development in EOC, particularly in the second-line setting (Marachini et al 2013). In this study we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents.

Method:

A review of phase III anti-angiogenic clinical trials was used to investigate the association between platinum resistance and response to anti-angiogenic agents. To investigate the effect of chemotherapy on predefined ovarian cancer molecular subgroups (Gourley, et al. J Clin Oncol 32:5s, 2014), we analysed 35 matched pre- and post-chemotherapy samples by gene expression. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the mechanisms of cisplatin section pressure and shift to an angiogenic phenotype. This was further validated in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy.

Results:

Critical review of 22 phase III anti-angiogenic trials suggested that there was a better response to anti-angiogenics following previous platinum-based chemotherapy. Our analysis demonstrated that 67% of treatment naïve tumours that were initially classified as non-angiogenic shifted to an angiogenic biology, which was associated with platinum resistance. Additionally we found that cisplatin resistant cancer cell lines demonstrated hallmarks of vascular mimicry and an associated increase in vessel density using an angiogenesis Matrigel plug assay in Athymic nude mice (p-value=<0.0001). In addition, cell lines established from platinum-resistant patients as well as cell lines made platinum resistant in-vitro, demonstrated overexpression of VEGFa which would be expected to stimulate angiogenesis.

Conclusion:

We have demonstrated that platinum-resistance in HGSOC is associated with angiogenic biology supporting the use of anti-angiogenic agents in this setting.

Citation Format: Aya El Helali, Nuala McCabe, Naomi Dickson, Lara Dura Perez, Denis P Harkin, Richard Wilson, Richard Kennedy. Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 776. doi:10.1158/1538-7445.AM2017-776
Abstract 78: Highly efficient antibody drug conjugation with a novel photoactivation strategy
Authors: Gardiner Elisabeth , Forster Karin , McKenzie Robert
Published: July 2017

Meditope SnAP (site-specific novel antibody platform) technology is a novel way to target molecules to antibodies in a site specific, non-destructive fashion. A “meditope” is a proprietary peptide that can bind a meditope-enabled antibody in a unique site and in such a way that antigen binding is not compromised. This specific peptide antibody interaction can be readily employed for site-specific covalent conjugation of cytotoxic payloads to any meditope-enabled antibody. To facilitate covalent conjugation, the meditope peptide has been rationally designed with a photoactivatable side chain and the generation of the antibody-drug conjugate (ADC) occurs in a highly efficient manner following the specific binding of the meditope to the enabled antibody. To test the hypothesis that photoactivation would be an efficacious and homogenous way to make an ADC, meditope-enabled ADCs were designed and tested in vitro and in vivo. Toxin conjugation was tested, as was efficiency of covalent interaction following short-term exposure of the antibody peptide conjugates to a nondestructive wavelength of 340 nM. Photoactivation proved to be a highly efficient and scalable way to make an ADC. Payload stability was confirmed in vivo with multiple toxins and in vivo potency of two unique ADCs were tested in an EGFR positive gastric cancer xenograft as well as a HER2 positive ovarian cancer xenograft. The meditope-enabled photogenerated anti-HER2 ADC was benchmarked against conventionally conjugated T-DM1 and showed equivalent efficacy, but with the site-directed conjugated meditope ADC using less toxin. Meditope-directed photoactivated-controlled conjugation can be readily extended to any meditope-enabled antibody, resulting in a novel way to develop ADCs. This methodology, with its high efficiency, speed and homogeneity has evident advantages for the development of antibody-drug conjugates.

Citation Format: Elisabeth Gardiner, Karin Forster, Robert McKenzie. Highly efficient antibody drug conjugation with a novel photoactivation strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 78. doi:10.1158/1538-7445.AM2017-78
Abstract 781: Functional consequence of the p53 codon 72 polymorphism in colorectal cancer
Authors: Katkoori Venkat R. , Manne Upender , Bumpers Harvey
Published: July 2017

Background: The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) and CRC health disparities. In this study, we determine the functional consequence of this polymorphism in CRC following both in-vitro cell based and in-vivo mouse model studies.

Experimental Design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. We selected a CRC cell line Caco2, which does not express p53 for in vitro studies. Severe combined immunodeficient mice were inoculated with CRC cells (HT29, SW480, and LS174) to establish tumor xenografts (tumors). Tumor angiogenesis was assessed in tumors by immunostaing for CD31. Sequencing analysis for codon 72 polymorphism of p53 was performed using genomic DNA purified from tumors. Western blot (WB) or immunostaining analyses for proteins of signaling mechanisms were performed to assess the functional consequence of P72 phenotype of p53.

Results: Our study demonstrated that P72 tumors had well established vascularity, while R72 tumors had very poor vascularization. Indeed, the mean micro vessel density was higher in P72 tumors than in R72 tumors. WB analyses revealed that P72wt or mutant phenotypes effectively induced the activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Up-regulation of phosphorylated SEK1/MKK4, an upstream kinase of p38 MAPK was associated with P72wt or mutant phenotypes. This activation was accompanied by up-regulation of phosphorylated-MAPKAPK-2, -Hsp27, and -CREB, downstream targets of p38 MAPK. Increased activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. Furthermore, suppression of RAF/MEK/ERK activation was significantly higher in cells that express R72wt phenotype compared to cells that express P72wt or mutant phenotypes. The expression of P72wt or mutant phenotypes displayed decreased expression of E cadherin and/or an increased expression of vimentin, fibrinectin, CD44, thereby promoting CRC metastasis.

Conclusions: These findings offer significant novel insights into the mechanism by which P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, these studies suggest a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251.

Citation Format: Venkat R. Katkoori, Upender Manne, Harvey Bumpers. Functional consequence of the p53 codon 72 polymorphism in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 781. doi:10.1158/1538-7445.AM2017-781
Abstract 783: Cancer cell-derived extracellular vesicles stimulate tumor angiogenesis by delivering VEGF to endothelial cells
Authors: Ko Song Yi , Naora Honami
Published: July 2017

Angiogenesis is essential for tumor growth and metastasis, and is orchestrated by a repertoire of growth factor signaling pathways that stimulate endothelial cell growth, migration and vessel formation. Recent studies have shown that angiogenesis can also be regulated by cell-derived extracellular vesicles. Exosomes are small endosomal-derived membrane vesicles that contain various biomolecules such as RNA and proteins, and are increasingly thought to play important roles in transferring informational cargo between cancer cells and stromal cells. In the majority of studies to date, the effects of cancer cell-derived exosomes have been attributed to their RNA cargo. In this study, we identified that exosomes derived from ovarian, colon and renal cancer cells contain abundant vascular endothelial growth factor (VEGF). Cancer cell-derived exosomes were found to activate the VEGF signaling pathway in endothelial cells and to stimulate endothelial cell migration and tube formation. Furthermore, our studies using inhibitors of VEGF signaling demonstrated that the stimulatory effects of cancer cell-derived exosomes on endothelial cells depend on the presence of VEGF in exosomes and are mediated via the VEGF signaling pathway. These findings indicate that cancer cell-derived exosomes promote tumor angiogenesis by delivering VEGF to endothelial cells.

Citation Format: Song Yi Ko, Honami Naora. Cancer cell-derived extracellular vesicles stimulate tumor angiogenesis by delivering VEGF to endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 783. doi:10.1158/1538-7445.AM2017-783
Abstract 789: Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells
Authors: Valdivia Andres , Racordon Dusan , Aravena Raul , Mingo Gabriel , Sandoval Alejandra , Bravo Maria Loreto , Cuello Mauricio A. , Kato Sumie , Erices Rafaela , Ramirez Carolina , Gonzalez Pamela , Sanchez Beatriz , Corvalan Alejandro H. , Owen Gareth I.
Published: July 2017

Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process.

Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT).

Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry.

Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers.

Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001

Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2017-789
Abstract 79: Trastuzumab and C6.5 diabody armed with deBouganin overcome drug resistance to ADCs comprised of anti-microtubule agents
Authors: Chooniedass Shilpa , Dillon Rachelle L. , Premsukh Arjune , Adams Gregory P. , MacDonald Glen C. , Cizeau Jeannick
Published: July 2017

DeBouganin (deB) is a de-immunized form of bouganin, a Ribosome Inactivating Protein (RIP) that when internalized blocks protein synthesis thereby leading to cell death. When conjugated to trastuzumab (T-deB) or genetically attached to the C6.5 diabody, deBouganin was more potent than DM1 and unaffected by mechanisms of resistance to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment in vitro were re-exposed to T-DM1, T-MMAE, or treated with T-deB or an anti-HER2 C6.5 diabody-deBouganin fusion protein. C6.5 diabody-deBouganin and T-deB were potent against HCC1419 and BT-474 cells surviving T-DM1 or T-MMAE treatment. However, the surviving cell populations were resistant to T-DM1, T-MMAE, DM1, MMAE and taxol treatment. In addition, cross-resistance was seen against trastuzumab-duocarmycin which contains a payload with a cell cycle independent mechanism of action. The contribution of multi-drug resistance, Bcl-2 family members and other survival pathways accounting for the resistant phenotype will be discussed. Overall, the data suggest that treatment with chemotherapeutics or ADCs comprised of small molecule compounds such as anti-microtubule agents, can lead to the outgrowth of tumor cells resistant to similar agents. In contrast, antibodies and antibody fragments armed with deBouganin can overcome these mechanisms of resistance and may therefore represent a more effective treatment option.

Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Gregory P. Adams, Glen C. MacDonald, Jeannick Cizeau. Trastuzumab and C6.5 diabody armed with deBouganin overcome drug resistance to ADCs comprised of anti-microtubule agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2017-79
Abstract 791: Inhibitory effects of theaflavin-3, 3′-digallate (TF3) on ovarian cancer cells
Authors: Chen Yi C. , Rankin Gary O.
Published: July 2017

Theaflavin-3, 3'-digallate (TF3) is a black tea polyphenol produced from polymerization and oxidization of green tea ployphenols epicatechin gallate and (−)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anti-cancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the current research, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cells induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by down-regulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/ mTOR/ p70S6K/ 4E-BP1 pathway and Akt/ c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally impaired cancer cells induced angiogenesis. Nevertheless, TF3 didn’t have any influence on MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

Citation Format: Yi C. Chen, Gary O. Rankin. Inhibitory effects of theaflavin-3, 3′-digallate (TF3) on ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2017-791
Abstract 793: Human endothelial progenitor cells: A new target for anti-vascular therapy
Authors: Rahoma Ghada Ben , Tuli Neha , Maniyar Rachana , Chakraborty Sanjukta , Singh Sarnath , Mittelman Abraham , Tiwari Raj K.
Published: July 2017

Breast cancer affects one in eight women in the USA. Early diagnosis and newer treatment modalities have rendered breast cancer manageable. However, triple negative breast cancer is still difficult to treat and warrantes a search for newer targets. One strategy that has emerged in cancer research involves targeting of tumor associated blood vessels which provide growing tumors with oxygenated blood and growth factors necessary for maintenance and metastasis. Antiangiogenic drug therapy is transient and has not been able to gain mainstream therapeutic modality. We discovered that endothelial progenitor cells (EPCs) are mobilized from the bone marrow to the tumor site and contribute to the development of breast tumor vessel formation in an estrogen dependent manner. Therefore, characterization of tumor associated endothelial progenitor cells in breast cancer may provide a more specific antivascular therapy. Using the highly proliferative human umbilical cord blood derived EPCs, having the phenotype (CD133+, CD34+, VEGFR-2+), the effect of growth factor and chemokine rich EPCs conditioned medium (CM) was assessed in luminal (MCF-7), and post-EMT (MDA-MB-231) breast carcinoma cell lines. We observed an initial halt in cellular proliferation in MCF-7 followed by a significant increase in proliferation after forty eight hours of treatment. On the other hand, MDA-MB-231 showed decreased proliferation even after forty eight hours of treatment. Treating the EPCs with breast cancer conditioned medium resulted in morphological and cellular growth changes in the EPCs. MDA-MB-231 CM resulted in an increase of the EPCs proliferation and differentiation by increasing the number of spindle shaped attaching cells, and MCF-7 CM resulted only in an increase in the differentiation rate by increasing the number of cell clusters. This increase in EPCs proliferation and differentiation associated with MDA-MB-231 CM treatment might explain the invasiveness of this breast cancer cells through the increase in the tumor associated neovascularization. The analysis of the paracrine interaction between breast cancer cells and EPCs along with the associated cellular changes will facilitate identification of the interactive mediators and subsequent development of effective antivascular therapy.

Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Raj K. Tiwari. Human endothelial progenitor cells: A new target for anti-vascular therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 793. doi:10.1158/1538-7445.AM2017-793
Abstract 794: Potential role of DLL4 in uveal melanoma vascular mimicry
Authors: Escandon Julia , Field Matthew G. , Kurtenbach Stefan , Kuznetzov Jeffim , Decatur Christina L. , Harbour J William
Published: July 2017

Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma.

Citation Format: Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. Potential role of DLL4 in uveal melanoma vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2017-794
Abstract 800: Enhancing tumor killing abilities of NK cells by targeting CD16 shedding, and ADAM17
Authors: Mishra Hemant K. , Wu Jianming , Kaufman Dan S. , Walcheck Bruce
Published: July 2017

CD16 consists of two isoforms (CD16a and CD16b) encoded by two highly homologous genes that differ by only 6 amino acids in their extracellular regions. CD16b is expressed by human neutrophils and CD16a by human natural killer (NK) cells. The ectodomain regions of both CD16 isoforms are cleaved proximal to the cell membrane by a proteolytic process referred to as ectodomain shedding. We demonstrate that the membrane metalloprotease ADAM17 cleaves CD16 in isolated leukocytes and in human patients. By mass spectrometry analysis, we determined 3 adjacent cleavage sites in neutrophil CD16b and one cleavage site in NK cell CD16a that occurred at the same location as the predominant cleavage site in CD16b, which is interesting considering CD16b is linked to the plasma membrane via a GPI anchor and CD16a is a transmembrane protein. Antibody-dependent cell cytotoxicity (ADCC) by NK cells is a key mechanism in the anti-cancer effects of therapeutic antibodies, and CD16a exclusively recognizes tumor-bound antibodies. Surface levels of CD16a are rapidly down-regulated upon NK cell activation by cytokines, target cell interaction, and tumor infiltration, which is associated with impaired ADCC. Thus blocking this process has important clinical significance. We contend that maintaining high surface levels of CD16a during NK cell-based immunotherapy will enhance their killing of antibody-bound tumor cells. In ongoing studies, we are examining pharmaceutical and gene-targeting means of preventing CD16a cleavage as a novel therapeutic strategy to enhance the anti-cancer effects of NK cells.

Note: This abstract was not presented at the meeting.

Citation Format: Hemant K. Mishra, Jianming Wu, Dan S. Kaufman, Bruce Walcheck. Enhancing tumor killing abilities of NK cells by targeting CD16 shedding, and ADAM17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2017-800
Abstract 801: A zebrafish model of NF1-mutant melanomas that lack activating mutations of BRAF or NRAS
Authors: He Shuning , Mansour Marc R. , Layden Hillary M. , Rodig Scott J. , Patton E. Elizabeth , Look A. Thomas
Published: July 2017

Cutaneous melanoma is the most lethal type of skin cancer, with ~76,380 newly diagnosed melanoma and ~10,130 melanoma-associated deaths per year in the US. Thus, there is a need for improved understanding of the molecular pathogenesis and more effective targeted therapies for this devastating disease. The recent work of The Cancer Genome Atlas Network has defined melanoma as an RTK/RAS-driven solid tumor that can be classified into four genomic subtypes: BRAF-mutant, RAS-mutant, NF1-mutant, and triple-wild-type. This landmark study highlighted the important role of the previously understudied NF1 tumor suppressor in melanoma pathogenesis, especially for the 9% of melanoma patients who have acquired inactivating NF1-mutations, but lack hotspot mutations that activate BRAF or RAS. To date, animal models have not been developed for the NF1-mutant subtype of melanoma, which has significantly impaired the development of novel therapeutic strategies for this subtype. Here we report the first zebrafish model for NF1-mutant melanoma, which we generated by combining the loss of nf1 with loss of both pten and p53. The resultant compound mutant zebrafish develop aggressive melanomas from the age of 7 weeks and the tumor penetrance is 80% by the age of 18 weeks. We demonstrate further that these high-risk zebrafish melanomas were exclusive of hotspot mutations of braf and nras. Sustained inhibition of both MEK and PI3K suppressed tumor progression in vivo, whereas inhibition of MEK or PI3K alone was insufficient to suppress the growth of these tumors. Surprisingly, single agent therapy with rapamycin, an MTOR inhibitor, proved even better for short- and long-term suppression of tumor cell growth in nf1/pten-mutant melanomas. Thus our model appears ideal for the testing of drugs that will prove uniquely active for the significant subset of NF1-mutant, BRAF/NRAS-wildtype human melanomas.

Citation Format: Shuning He, Marc R. Mansour, Hillary M. Layden, Scott J. Rodig, E. Elizabeth Patton, A. Thomas Look. A zebrafish model of NF1-mutant melanomas that lack activating mutations of BRAF or NRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2017-801
Abstract 802: Loss of function of Arid1a synergizes with MYCN in neuroblastoma pathogenesis
Authors: Shi Hui , Tao Ting , Kadoch Cigall , Look Thomas
Published: July 2017

ARID1A is a key component of the SWI/SNF-A (BAF) chromatin-remodeling complex that is known to modulate chromatin structure and gene transcription. Recent exome and whole-genome resequencing has identified SWI/SNF as the most frequently mutated chromatin remodeler in human cancers, and ARID1A as the most highly mutated component among the SWI/SNF subunits, primarily in solid tumors. In high-risk neuroblastoma, ARID1A is mutationally inactivated in one allele in ~1% of patient tumors. In addition, ARID1A is deleted in one allele in at least 85% of cases with chromosome 1p deletions, which is the most common deletion found in neuroblastoma. Thus, ARID1A appears to function as a haploinsufficient tumor suppressor in a substantial fraction of high-risk neuroblastomas. The arid1a gene is duplicated in zebrafish, and the two genes are hence named arid1aa and arid1ab. We targeted each allele of the two genes using CRISPR/Cas genome editing system and recovered stable lines with loss-of-function mutations in each arid1a gene. arid1ab but not arid1aa homozygous mutation showed embryonic lethality. We bred these lines with a transgenic zebrafish line expressing high levels of MYCN in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. Loss of one copy of either arid1aa or arid1ab dramatically accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma. Loss of additional copies of arid1aa or arid1ab accelerated the tumor onset even futher, with the most rapid onset in compound heterosygotes of both genes. The compound heterosygotes showed increased fraction of sympathoadrenal cells in active cell cycle compared to the MYCN transgenic fish with wild type arid1a allales. Our long-term goal is to use the zebrafish model to elucidate the mechanisms underlying the highly dose-dependent tumor suppressor role of this component of the BAF chromatin remodeling complex in neuroblastoma, as a first step toward designing targeted therapies synthetic lethal with reduced expression levels of ARID1A.

Citation Format: Hui Shi, Ting Tao, Cigall Kadoch, Thomas Look. Loss of function of Arid1a synergizes with MYCN in neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2017-802