bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 4127: Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM.
Authors: Gu Jian 
Published: August 2014

Abstract
Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. We have developed methods for small RNA and whole transcriptome analysis of FFPE samples on the Ion Torrent PGM™ System using Ion total RNA-Seq Kit v2 and RiboMinus Eukaryote System v2.
Paired FFPE and fresh-frozen RNA samples from lung adenocarcinoma tissue were used in this study. Small RNA libraries were prepared using the Ion Total RNA-Seq kit v2 with modified protocol, followed by sequencing on PGM™ system. Results show that small RNA extracted from FFPE sample was successfully converted to small RNA library. Although percentage of reads mapped to miRBase was slightly lower when compared to fresh-frozen sample, the overall miRBase coverage and detection sensitivity were comparable. For whole transcriptome analysis, RiboMinus Eukaryote System v2 kit was used to deplete rRNA from FFPE samples, followed by library construction using the Ion Total RNA-Seq kit v2 with a modified protocol. Significant reduction in rRNA reads was observed after sequencing. High RefSeq correlations were observed between fresh-frozen and FFPE samples.
In conclusion, a full spectrum of gene expression data, from both small RNA and whole transcriptome, were generated from FFPE samples using Ion Total RNA-Seq kit v2 with modified protocols. High correlations were observed between paired fresh-frozen and FFPE samples, demonstrating the reliability of the modified workflow for gene expression profiling on FFPE samples. This study provides feasibility for systematic gene expression analysis on FFPE samples from cancer and other diseases.
Citation Format: Jian Gu. Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2013-4127
Abstract 4132: Rapid microfluidic-based isolation of tumor cells from pleural effusions for cytodiagnostics.
Authors: Che James , Mach Albert , Go Derek , Talati Ish , Ying Yong , Rao Jianyu , Di Carlo Dino , Kulkarni Rajan 
Published: August 2014

Abstract
Pleural effusions accumulate in patients with cancer or other illnesses and are often drained to provide symptomatic relief. Cells within this fluid are concentrated and analyzed to determine the cause of effusion. In patients with cancer, tumor cells may be present in the effusions and provide a source of cells for further diagnostics and mutational analysis. However, blood cell components and other non-cellular materials can create a significant background and can obscure the search for tumor cells. Furthermore, blood cell impurities can adversely impact the search for genomic or proteomic changes in the tumor cells by creating a large wild type background. A purity of at least 20% of target cells is necessary to ensure that background signal is suppressed. We have developed a microfluidic based “Centrifuge Chip” for the rapid isolation and purification of cancer cells. The Centrifuge Chip utilizes microvortices to trap cells based on size without the need for affinity-based capture. Isolated cells can subsequently be released on-demand and are viable to grow or can be analyzed for molecular changes. We utilized this chip for the isolation of tumor cells and mesothelial cells at high purity from pleural effusions and similar complex fluids such as blood. We then stained the cells with CK/DAPI for tumor cells and CD45 for white blood cells. Samples with high numbers of CK positive cells were deemed suspicious for malignancy; all our results were cross-checked to the standard Cytospin results. We routinely achieved improved purities in all samples, with greater than 70-fold increase in purity for samples positive for malignancy, and are developing a quantitative PCR (QPCR) based system to confirm the presence of activating EGFR or KRAS mutations in lung cancer cells isolated from patients with malignant pleural effusions secondary to lung cancer. The Centrifuge Chip can achieve rapid isolation of tumor cells from complex biofluids and allows for easier access to such cells for molecular analysis that will provide clinicians with critical information required for targeted cancer therapies.
Citation Format: James Che, Albert Mach, Derek Go, Ish Talati, Yong Ying, Jianyu Rao, Dino Di Carlo, Rajan Kulkarni. Rapid microfluidic-based isolation of tumor cells from pleural effusions for cytodiagnostics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4132. doi:10.1158/1538-7445.AM2013-4132
Abstract 4137: An improved assay system for cell migration and invasion.
Authors: Partridge Jeff , Rimsa Stephen , Flaherty Paula 
Published: August 2014

Abstract
In vitro cell migration and invasion assays are frequently used as model systems for quantifying the directed movement of cells towards a chemoattractant stimulus, or to measure how a particular drug, antibody or extra cellular matrix (ECM) coating affects that movement. Analysis of this movement is often accomplished through the use of visual methods that are either time-consuming, labor-intensive, or subjective. To address these issues, an improved fluorescence blocking version of the Boyden chamber was developed. As cells migrate or invade through a unique fluorescence blocking microporous membrane, they can be detected using a bottom-reading spectrofluorometer or fluorescence microscope. Cells remaining in the upper chamber of the insert are shielded from this detection, allowing for quantitation of cell numbers in a homogeneous assay system.
The technology blocks ≥ 98% transmission of light from 360 - 690 nm as measured by transmission spectrophotometry, and improvements have been made over previous embodiments such that &gt;50% more light in the range of 360 - 490 nm is blocked. These enhancements now permit the use of the blue fluorescent Hoechst dyes and DAPI for direct cell enumeration and multiplexing. Functional applications of this technology were demonstrated in several well-established assays: endpoint staining in a model tumor invasion system yielded a Z’-factor &gt;0.5; real-time chemotaxis of monocytes demonstrated a peak signal between 25-35 minutes; and migration of human endothelial cells to VEGF was demonstrated with high reproducibility (CV &lt; 15%). Additionally, the measured IC50 values of chemotherapeutic compounds including paclitaxel and doxorubicin remained consistent with previously reported results. FluoroBlok™ cell culture inserts can efficiently be used for high-throughput cell migration studies, are automation friendly and yield highly reproducible results.
Citation Format: Jeff Partridge, Stephen Rimsa, Paula Flaherty. An improved assay system for cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4137. doi:10.1158/1538-7445.AM2013-4137
Abstract 4141: Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme.
Authors: Mueller Claudius , deCarvalho Ana C. , Mikkelsen Tom , Calvert Valerie , Espina Virginia , Liotta Lance A. , Petricoin Emanuel F. 
Published: August 2014

Abstract
Introduction & Methods: Glioblastoma tumor cells are generally assumed to be mixed with few distinct non-tumorigenic cell types and few cells of the microenvironment in general, owing to their location in the brain and the high tumor cell density compared to normal brain parenchyma. However, even small cell populations can have a significant impact on the abundance of phosphorylated proteins. We have analyzed the level and/or phosphorylation of 133 proteins in laser capture microdissected glioblastoma primary tumors (n=39) and compared the results to non-tumor cell enriched, whole tissue lysates.
Results: A large portion (44 percent) of endpoints were significantly different between matched tumor cell enriched and non-enriched samples - even when limiting the investigation to tumor samples with at least 90 percent tumor content. EGFR protein levels, which is highly expressed in tumor cells compared to normal brain parenchyma, increased in enriched and non-enriched samples following EGFR mutation, while EGFR phosphorylation was only significantly increased in tumor cell enriched samples. In contrast, PTEN, which has a higher expression in normal brain compared to tumor cells, was only found to be reduced in tumor cell enriched samples from patients with either PTEN mutation or loss in PTEN copy number, with no difference seen in non-enriched samples. Patient stratification by either phosphorylated EGFR, Akt, mTOR, Stat1, VEGFR2 or Bcl-2 based on non-tumor cell enriched samples was dramatically different from tumor cell enriched samples, showing significant misclassification for up to 40 percent of patients even in high tumor content samples.
Conclusions: This study demonstrates the critical necessity for selected tumor cell population procurement when investigating protein levels and phosphorylation in glioblastoma. Only tumor cell enriched samples correctly reflected expected tumor biology in the face of EGFR and PTEN mutations. Moreover, tumor cell enrichment had a dramatic effect on the stratification of clinical patient samples that would form the basis for targeted therapy selection.
Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Valerie Calvert, Virginia Espina, Lance A. Liotta, Emanuel F. Petricoin III. Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2013-4141
Abstract 4151: A novel, fast, and efficient technology for the preparation of high quality NGS libraries.
Authors: Kurihara Laurie , Beaubien Ron , Perry Jeff , Kelchner Vanessa , McUsic Drew , Mishra Anjali , Laliberte Julie , Chupreta Sergey , Makarov Vladimir 
Published: August 2014

Abstract
Today's next-generation sequencing (NGS) investigators and clinicians must extract and analyze complex sequence data from a wide range of sample types. Preparing fragment libraries from these samples for whole genome, exome or targeted gene panel sequencing by NGS is a laborious process, characterized by long prep times and sample compatibility constraints due to DNA input quantity, genomic complexity, and molecular integrity. The workflows for most commercially-available kits are variations on traditional modifications for the repair, adaptation, and selection of DNA fragments, requiring 2-4 hours of preparation time before sequencing. As a consequence, NGS users cite sample preparation as the second most time-consuming step and bottleneck in their NGS workflows (following data analysis and interpretation; Bioinformatics, Oct. 2011). We present a novel technology that enables faster and more efficient NGS fragment library preparation, which increases yield and is compatible with many sample types, including damaged, cross-linked samples derived from FFPE or ChIP or samples with limiting DNA quantity. This method conveys these advantages because it does not require intact, double-stranded DNA for adapter ligation, and the streamlined workflow results in significant time savings relative to the workflows of commercial products. Side-by-side comparisons of sequencing data from libraries prepared using this new technology versus those from existing commercial products demonstrates equivalent or improved genome coverage, and suitability for damaged or cross-linked samples.
Citation Format: Laurie Kurihara, Ron Beaubien, Jeff Perry, Vanessa Kelchner, Drew McUsic, Anjali Mishra, Julie Laliberte, Sergey Chupreta, Vladimir Makarov. A novel, fast, and efficient technology for the preparation of high quality NGS libraries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2013-4151
Abstract 4152: New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids.
Authors: Gutierrez Sara , Strug Ivona , Cappione Amedeo , Smith Janet L. , Mabuchi Masaharu , Nadler Timothy 
Published: August 2014

Abstract
Microvesicles, such as exosomes, are small particles secreted by many types of cells under both normal and pathological conditions such as cancer. These microparticles contain RNA and protein (both membrane-bound and cytosolic) from their cells of origin and thus represent a burgeoning resource for biomarker identification. While plasma and urine offer convenient, non-invasive access to sufficient sample volumes for screening purposes, major obstacles to this effort include the problematic issues of exosome isolation methods, the presence of over-abundant proteins or interfering cell debris, and the wide dynamic range of protein expression. It is clearly recognized in many fields that the quality of sample preparation ultimately impacts their performance in downstream analyses; this is particularly true where the target in question is a low abundant protein. Here we present an ultrafiltration-based method for the isolation of microvesicles from cell culture media as well as body fluids. To assist in process optimization, we used a novel infrared (IR) based biomolecule detection system to evaluate lysis and extraction conditions and analyze protein and lipid content. This IR system is less influenced by reducing agents and detergents than either BCA or Bradford assays. Following isolation, samples were analyzed using a rapid immunodetection technique, permitting detection of biomarkers more quickly and efficiently than ever before.
Citation Format: Sara Gutierrez, Ivona Strug, Amedeo Cappione, Janet L. Smith, Masaharu Mabuchi, Timothy Nadler. New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2013-4152
Abstract 4159: Expression of miRNAs associated with DNA repair capacity in women with breast cancer.
Authors: Matta Jaime L. , Morales Luisa , Ortiz Carmen , Zayas Celestino , Suarez Erick 
Published: August 2014

Abstract
MicroRNAs (miRNA) are endogenous, short (19-24 nucleotides) non-protein-coding RNAs that regulate gene expression at the post-transcriptional level via binding to 3′-untranslated regions of protein-coding transcripts. A low DNA repair capacity (DRC) is an important risk factor for breast cancer (BC). We are currently investigating whether miRNAs expression is associated with dysregulation of DRC in women with BC. To that effect, we obtained plasma from 30 BC cases and 30 controls selected based on their DRC levels. DRC was measured in lymphocytes using a host cell reactivation assay. Samples were analyzed for miRNA expression utilizing protocols from Applied Biosystems (Life Technologies). After QA/QC, 27 BC cases and 29 controls whose DRC data was available from our cohort study were selected and miRNAs isolated from plasma. The miRNA expression profiling was performed utilizing the RT-PCR TaqMan Array Human MicroRNA A Cards v 2.0 (Applied Biosystems). Single-stranded cDNA was synthesized from 200 ng of total RNA in 8 Multiplex RT primer pool reactions containing stem-looped RT primers that were specific to mature miRNAs. U6 snRNA-001973 was selected for normalization based on our own experimental validations. To quantify the association of the miRNA expression the fold change () was estimated for every detector with the p-values based on the t-test. Significant candidate miRNAs were determined with the false discovery rate of 4%. The results showed that twelve miRNAs are underexpressed in patients with BC relative to controls. These candidate miRNAs were associated with the expression of twenty seven DNA repair genes. Two of these genes (RPA3, MMS19L) are part of the NER pathway which has recently been linked to breast carcinogenesis. Our pilot study data suggests that differential expression of some miRNAs might be associated with dysregulation of DRC in women with BC.
Supported by grants S06 GM008239-20, MBRS-RISE GM082406 [NIH-NIGMS] and 1SCA157250-2 from the NCI Center to Reduce Health Disparities, and NIH-MBRS Program (NIGMS).
Citation Format: Jaime L. Matta, Luisa Morales, Carmen Ortiz, Celestino Zayas, Erick Suarez. Expression of miRNAs associated with DNA repair capacity in women with breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4159. doi:10.1158/1538-7445.AM2013-4159
Abstract 4160: miR-34a as a radio sensitizer of triple negative breast cancer.
Authors: Bhardwaj Anjana , Ganesan Nivetha , Molkentine David , Raju Uma , Bedrosian Isabelle 
Published: August 2014

Abstract
Introduction: Triple negative breast cancer (TNBC) is known to be relatively resistant to radiation therapy resulting in a comparatively higher rates of locoregional recurrence compared with non-TNBC patients. The mechanisms behind this relative radio resistance remain unknown. The goal of this study is to investigate the role of three miRNAs namely, miR-34a, miR-21, and miR-210, which are implicated in DNA damage-repair process, in the response of TNBC cell lines to radiation therapy. Methods: Seven TNBC breast cancer cell lines were used for the study. The levels of miR-34a, miR-21 and miR-210 were evaluated by taqman- based quantitative PCR assay. These cells were exposed to 2 Gy radiation dose and colonogenic survival was assessed after about 2 weeks. Survival fraction after 2 Gy (SF2), which is the ratio of number of cell colonies formed after 2 Gy radiation vs. the number of cell colonies with out radiation, was determined. The SF2 factor is a reliable index to measure radio sensitivity, greater the SF2 factor more radio resistant the cells are. Results: The SF2 value of the TNBC cells varied from 0.31 to 0.74 with MDA-MB-468, HCC-70 being the most radio sensitive cell lines and MDA-MB-436, BT-20 being the most radio resistant cell lines. MDA-MB- 231, BT-549 and Hs 578T were found to be moderately radio resistant breast cancer lines. The oncomiR -21 expression levels in these 7 TNBC cell lines varied from 0.85 to 4.18 fold with a very poor correlation with SF2 (r2=0.08). The oncomiR 210 levels varied from 0.49 to 9.19 fold and also did not exhibit any correlation with radio resistance in TNBC cells (r2= 0.141). Whereas the tumor suppressor miR, miR-34a expression showed an inverse correlation with radio resistance (r2=0.8) in TNBC cells. The radio resistant cell line (e.g., MDA-MB-436, SF2 = 0.74) expressed significantly low levels of miR-34a (0.016 fold) relative to miR -34a levels (set as 1) in HCC 70, which was found to be a radiosensitive cell line with SF2 of 0.309. Conclusions: Our preliminary data indicates that miR 34a may play a role in the response of TNBC cells to radiotherapy.
Citation Format: Anjana Bhardwaj, Nivetha Ganesan, David Molkentine, Uma Raju, Isabelle Bedrosian. miR-34a as a radio sensitizer of triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4160. doi:10.1158/1538-7445.AM2013-4160
Abstract 4166: MicroRNA-449a enhances radiosensitivity in lung adenocarcinoma cells.
Authors: Liu Yi-Jyun , Lin Yu-Fen , Chen Yi-Fan , Luo En-Ching , Sher Yuh-Ping , Tsai Mong-Hsun , Chuang Eric Y. , Lai Liang-Chuan 
Published: August 2014

Abstract
Lung cancer is the leading cause of cancer-related mortality worldwide. Radiotherapy is often applied for treating lung cancer, but it often fails because of the relative non-susceptibility of lung cancer cells to radiation. MicroRNAs (miRNAs) have been reported to modulate the radiosensitivity of lung cancer cells and have the potential to improve the efficacy of radiotherapy. The purpose of this study was to identify a miRNA that can adjust radiosensitivity in lung adenocarcinoma cells. Two lung adenocarcinoma cell lines (CL1-0 and CL1-5) with different metastatic ability and radiosensitivity were used. In order to understand the regulatory mechanisms of differential radiosensitivity in these isogenic tumor cells, both CL1-0 and CL1-5 were treated with 10 Gy radiation, and were harvested respectively at 0, 1, 4, and 24 h after radiation exposure. The changes in expression of miRNA upon irradiation were examined using Illumina Human microRNA BeadChips. Twenty-five miRNAs were identified as having differential expression post-irradiation in CL1-0 or CL1-5 cells. Among these miRNAs, miR-449a, which was down-regulated in CL1-0 cells at 24 h after irradiation, was chosen for further investigation. Overexpression of miR-449a in CL1-0 cells effectively increased irradiation-induced DNA damage and apoptosis, altered the cell cycle distribution and eventually led to sensitization of CL1-0 to irradiation.
Citation Format: Yi-Jyun Liu, Yu-Fen Lin, Yi-Fan Chen, En-Ching Luo, Yuh-Ping Sher, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. MicroRNA-449a enhances radiosensitivity in lung adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4166. doi:10.1158/1538-7445.AM2013-4166