bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2563: A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency
Authors: Lewis Samantha R. , Peterson Shaun , Oostdik Kathryn , Tomlinson Heather 
Published: August 2019

Abstract
Use of biomarkers for patient treatment stratification is an increasingly important topic with the recent FDA approval of Pembrolizumab and Nivolumab for microsatellite instability (MSI) high/mismatch repair (MMR) deficient cancers. This ground-breaking approval allows physicians to make patient treatment decisions based on molecular biomarker status. Thus, performance of detection methods for these biomarkers is of great importance. The gold standard for microsatellite instability (MSI) analysis is PCR followed by fragment analysis using capillary electrophoresis to resolve DNA fragment size. Recently, PCR followed by melt curve analysis and Next Generation Sequencing (NGS) have been presented as alternative approaches. In this set of experiments, these methods will be compared using a subset of human cancer samples.
A cohort of matched cancer and adjacent normal FFPE samples were used for this comparison. From this cohort, individuals were selected that contained subtle MSI shifts, low tumor volume, or heterozygosity at microsatellite loci. Samples from these individuals were tested for MSI by either fragment or melt curve analysis or by sequencing. Samples were then classified as MSI-High, MSI-Low or microsatellite stable (MSS) according to manufacturer specifications for melt curve analysis or by an interpretive software for fragment analysis and NGS. Preliminary results indicate that there may be differences in sensitivity when using challenging samples with melt curve analysis compared to the gold standard method, fragment analysis.
The standard method for determining MSI status utilizes PCR and fragment analysis. Recently, melt curve analysis and NGS have been proposed as alternative methods. We present preliminary findings of decreased sensitivity with melt curve compared to fragment analysis. Additional studies with a larger, more diverse sample set will be required to define this relationship further.
Citation Format: Samantha R. Lewis, Shaun Peterson, Kathryn Oostdik, Heather Tomlinson. A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2563.
Abstract 2564: Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR
Authors: Yi Eunhee , Verhaak Roel 
Published: August 2019

Abstract
Glioblastoma (GBM) is the most lethal brain tumor; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. Recurrence after therapy for GBM is unavoidable. There are substantial differences among the cells of GBM tumors in the abundance and types of genetic material. This heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. A recent study has suggested that the majority of GBM activate oncogenes through amplification extrachromosomal DNA (ecDNA). Despite the speculation that ecDNA is a key factor of tumor heterogeneity, how ecDNA is propagated and distributed among—and how it behaves within—cancer cells is completely unknown. To address this gap in knowledge, this study focused on developing a novel cytogenetic tool that enables visualization and tracking ecDNA behavior in live GBM cells. We found breakpoint sequences resulting from genome rearrangements during ecDNA formation by performing computational analysis whole genome sequencing data from a pair of primary/recurrent GBM neurospheres. Each breakpoint represents a unique target sequence for ecDNA-specific labeling. Breakpoint were validated by breakpoint-PCR, focusing on breakpoints shared by the primary and recurrent neurosphere. We determined location and quantity of all ecDNA breakpoints through breakpoint-FISH (BP-FISH) analysis, which showed that all signal was found outside of the conventional chromosomes in the metaphase spread, validating the extrachromosomal nature of the ecDNA breakpoints. The breakpoint quantities, reflecting the number of ecDNA elements per cell, varied between primary and recurrent neurosphere suggesting that ecDNAs quantitatively changed during GBM evolution. These results emphasize the necessity of tracking ecDNA dynamics to answer the question of how ecDNA is distributed into the daughter cells. Our findings will be strong evidence to make ecDNA-specific CRISPR system in further research. Tracing ecDNA dynamics will provide new insight into the impact of ecDNA on cancer evolution.
Citation Format: Eunhee Yi, Roel Verhaak. Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2564.
Abstract 2567: TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability
Authors: Lu Wei-ting , Zalmas Lykourgos-Panagiotis , Webber Thomas , Kanu Nnennaya , Swanton Charles 
Published: August 2019

Abstract
TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in “trunk” clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing.
Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution.
Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed.
Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Thomas Webber, Nnennaya Kanu, Charles Swanton. TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2567.
Abstract 2569: Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair
Authors: Selemenakis Platon , Maranon David , Wiese Claudia 
Published: August 2019

Abstract
Genomic instability is one of the enabling hallmarks of cancer and can arise by exposure to several factors: endogenous challenges such as stress to DNA replication forks or exogenous compounds such as exposure to ionizing radiation (IR) and other environmental mutagens. These insults can lead to the induction and propagation of mutations through the generation of multiple DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different repair pathways. One of these pathways is homologous recombination (HR), a relatively faithful DSB repair pathway that relies on the availability of a homologous template for DNA repair synthesis.
DNA strand exchange in HR is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. In human cells, RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While RAD54 translocates on double-stranded DNA and promotes DNA strand separation, RAD51AP1 enhances the assembly of the synaptic complex. However, if RAD54 and RAD51AP1 work independently or together in the HR reaction is unclear. Since functional loss of either RAD54 or RAD51AP1 each leads to a moderate defect in HR, we speculate that RAD54 and RAD51AP1 may act in parallel in the promotion of RAD51 activity. We hypothesize that simultaneous inactivation of both RAD54 and RAD51AP1 may lead to a synthetic phenotype in HR impairment.
The main focus of this study is to investigate the potential synergism between RAD51AP1 and RAD54 during late stages of the HR reaction to improve targeted cancer therapy. We are investigating if loss of RAD54 in human cells exacerbates the phenotype of RAD51AP1-deficiency and renders cells more sensitive to DNA damaging agents. We have generated RAD51AP1 knockout HeLa cells using CRISPR/Cas9 technology. In these cells, we have also inactivated the RAD54 gene using a very similar approach. Moreover, we have generated RAD54 knockout (KO) cells in a wild type RAD51AP1 background. All cell lines are tested for their response to chemotherapeutic agents in cell survival and DNA replication assays. Our first results show that the double KO cells are more sensitive to mitomycin C exposure than single KO cells, suggesting that RAD51AP1 and RAD54 function largely independently of each other in the HR reaction.
The results from our investigation are expected to lead to the potential development of new cancer drugs targeted at ancillary factors of RAD51 in late stages of the HR reaction.
Citation Format: Platon Selemenakis, David Maranon, Claudia Wiese. Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2569.
Abstract 2571: Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations
Authors: Ghosh Anindita , Richardson Christine A. 
Published: August 2019

Abstract
The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia.
Citation Format: Anindita Ghosh, Christine A. Richardson. Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2571.
Abstract 2572: Integrated viral sequences in esophageal cancer tissue
Authors: Patten Victoria A. , Mwaphaga Lamech , Mathew Christopher G. , Hendricks Denver T. , Parker M. Iqbal 
Published: August 2019

Abstract
The human genome exhibits a significant degree of invasion by viral genomes. During the course of evolution, several RNA and DNA viruses have become integrated into the vertebrate genome and there is no reason to believe that the process has ended. Integrated viral DNA may account for as much as 10% of the human genome and it is very likely that they may act as carcinogens by altering normal cellular gene expression.
In this study we have compared the viral DNA sequences present in normal and tumor tissues from esophageal squamous cell carcinoma (ESCC) samples. DNA was extracted from paired normal and tumor biopsies and subjected to whole genome sequencing. Raw data run folders were converted to FASTQ files and progressed through a QC pipeline. Normal and tumor data sets were then blasted and aligned against an extensive viral sequence database using specialized DRAGEN software, so that all integrated viral DNA fragment sequences were identified. The identified viral sequences were then compared between the paired normal and tumor samples.
Using this approach, a large number of viral DNA sequences were identified; these included human papilloma viruses, Herpes simplex viruses, adenoviruses and Hepatitis C viruses. Furthermore, some unexpected viruses of interest were also identified. These include the Autographa Californica Nucleopolyhedrovirus, the Emiliana Huxleyi virus 86, the Gryllus bimaculatus nudivirus and the Trichodysplasia spinulosa-associated polyomavirus, present in different tumour DNA samples.
Amplification and rearrangements of endogenous retroviruses such as the Human Endogenous Retrovirus K113 were also observed. Endogenous retroviruses (ERV) can serve as long-lasting viral reservoirs and provide evidence for the coexistence between retroviruses and their hosts over many generations. Defective ERVs can also recombine to affect the expression of adjacent genes.
It has been proposed that integrated viral sequences may play a possible role in the etiology and promotion of diseases such as cancer, thus targeted studies of specific viral sequences may elucidate the mechanisms of gene disruption and may ultimately lead to the development of more effective therapeutic interventions.
Note: This abstract was not presented at the meeting.
Citation Format: Victoria A. Patten, Lamech Mwaphaga, Christopher G. Mathew, Denver T. Hendricks, M. Iqbal Parker. Integrated viral sequences in esophageal cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2572.
Abstract 2582: Investigating the origin and function of circulating miRNA in lung cancer
Authors: Colangelo Tommaso , Mazzarelli Francesco , Cuttano Roberto , Dama Elisa , Melocchi Valentina , Graziano Paolo , Fiordelisi Fabiola , Bianchi Fabrizio 
Published: August 2019

Abstract
MicroRNAs (miRNAs) are small, highly conserved noncoding RNA molecules involved in the regulation of gene expression. MicroRNAs are resistant to harsh conditions and stably exist in body fluids (e.g. saliva, urine, breast milk, blood). Circulating cell-free miRNAs (cf-miRNA) were shown to be effective biomarkers for the early diagnosis of cancer. Furthermore, cf-miRNA were also found in exosomes that are nano-sized extracellular vesicles which exchange molecular information among cells. However, the understanding of origin of cf-miRNAs and of biological function still remains elusive. We recently identified a signature of cf-miRNAs diagnostic for lung cancer which we now hypothesize being released by lung cancer cells (EP-cf-miRNA) and by tumor microenvironment (TME-cf-miRNA). In particular, two cf-miRNAs, miR-29a and 223-3p, were selected as prototypes of EP- and TME-cf-miRNAs for further investigations.We found that the expression of miR-223-3p was higher in TME than in cancer cells of a cohort of 19 lung adenocarcinoma. In contrast, miR-29a-3p was frequently overexpressed (≥2 fold) in cancer cells. Interestingly, the serum concentration of cf-miR-29a was lower than miR-223-3p and this also in exosomes.We next investigated the expression profile of miR-29a/223-3p in a panel of lung normal and adenocarcinoma cell lines (N=14). miR-29a/223-3p were both expressed intracellularly and in exosomes. Of note, the miR-29a expression level resembles that observed in lung cancer cells from FFPE samples. Conversely, miR-223-3p expression was lower in the adenocarcinoma cell lines which further confirm a more TME origin. We are now expanding our analysis by screening the whole set of cf-miRNAs by high-throughput qPCR profile (OpenArray technology) and by applying laser capture microdissection to investigate single cancer epithelial cells and TME. Our work provides proof of principle demonstration of an effective approach to investigate cf-miRNA origin to unravel the role of cf-miRNA in lung adenocarcinoma.
Citation Format: Tommaso Colangelo, Francesco Mazzarelli, Roberto Cuttano, Elisa Dama, Valentina Melocchi, Paolo Graziano, Fabiola Fiordelisi, Fabrizio Bianchi. Investigating the origin and function of circulating miRNA in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2582.
Abstract 2612: Roles of USF1 in breast tumorigenesis and disease progression
Authors: Lewis Jessica , Mahmud Iqbal , Tian Guimei , Liao Daiqing 
Published: August 2019

Abstract
Breast cancer accounts for nearly one-quarter of all cancer diagnoses and is the principal cause of cancer-related mortality in women worldwide. Triple negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with ER+, PR+ and HER2+ breast cancer subtypes. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. USF1 (upstream stimulatory factor 1), a gene linked to drive lipogenesis and cellular proliferation, is overexpressed in human malignancies, yet its contribution to cancer remains unclear. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), we found that USF1 expression is significantly higher in TNBC patient samples. Also, USF1 gene expression positively correlates with key lipogenic enzymes. Significantly, we found that high expression of USF1 in breast cancer correlates with decreased patient survival. We therefore hypothesize that USF1 promotes breast tumorigenesis and progression by activating lipogenic gene expression. We conducted pilot in vitro studies to determine the influence of USF1 expression and cell proliferation. It was demonstrated that knockdown of USF1 decreased cellular proliferation in 2D cell culture of the TNBC cell line MDA-MB-231. We also assessed the effects on USF1 expression levels on in vivo tumor growth. We found that USF1 overexpression and knockdown enhanced and reduced tumor growth in vivo, respectively. Further studies are underway to determine the mechanisms by which USF1 promotes TNBC tumorigenesis and metastatic progression. Our studies will shed lights on roles of USF1 in breast cancer tumor biology. (Supported by McKnight Foundation, Bankhead-Coley Cancer Research Program and James and Esther King Biomedical Research Program, Florida Department of Health.)
Citation Format: Jessica Lewis, Iqbal Mahmud, Guimei Tian, Daiqing Liao. Roles of USF1 in breast tumorigenesis and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2612.