bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3108: Exploring base excision repair variants as potential defining events in cancer patient populations and characterization of models for pre-clinical studies
Authors: Riches Lucy C. , Brown Helen , Dalgliesh Gillian , Fletcher Lynn , Sherwood James , Shea Kerry , Lau Alan , O'Connor Mark J. 
Published: August 2012

Abstract
Background Deficiencies in base excision repair (BER) may increase the cell's dependence on alternative repair pathways and so define a patient population for novel inhibitors of DNA damage repair. Notably, cancer associated molecular events in DNA polymerase beta (POLα) have been described in various disease backgrounds including colorectal cancer (CRC), gastric, breast and prostate (1-5). Although functional consequence has been linked to certain POLα mutations and splice variants, their prevalence in defined disease segments remains largely undefined. Aim We set out to ascertain the prevalence of POLα deficiencies in CRC, and subsequently investigated protein and gene expression profiling as alternative methods for defining BER defects in cancer. Methods/Results A comprehensive study of clinical (CRC; N=64), tumour explant (CRC; N=36) and cell line (mixed origin; N=280) samples revealed that cancer-associated mutations in the polymerase encoding domain of POLα (1-3) are not common events in CRC. Additionally, a previously described splice variant of POLα lacking part of the polymerase domain (3-4), was assayed and found to be expressed at similar levels in CRC and normal tissue. This would suggest that expression of the POLα splice variant is not a defining element of CRC in western populations. Expression profiling using Fluidigm Taqman revealed a subset of BER genes more highly expressed in CRC clinical samples and mixed cell lines compared with normal tissue. While protein expression profiles did not reflect relative gene expression in selected cell line models, we observed an additional low molecular weight band in a subset of cells profiled for POLB expression. Furthermore, we report variations in the expression of the scaffold protein XRCC1 in colorectal and breast cell lines and propose that this could also provide a mechanism for variation in DNA repair capacity. Summary Although from our studies sequencing and transcript analysis of POLα do not provide suitable markers of BER deficiency in CRC, we report aberrant banding patterns in POLα by Western blotting and variation in XRCC1 protein expression in a subset of cell lines analyzed. We hypothesize that these variants may confer a BER deficient phenotype. 1. Lang, T. et al. (2004Proc. Natl Acad. Sci. USA, 101, 6074-6079. 2. Tan, Xiao-Hui, et al Cancer Letters: 2005: 220: 101-114 3. Wang, L et al Cancer Research, 1992; 52:4824-4827 4. Bhattacharyya, N. et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10324-10329
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3108. doi:1538-7445.AM2012-3108
Abstract 3113: Effects of an up-regulated ATR-CHEK1 pathway in head and neck squamous cell carcinomas
Authors: Parikh Rahul A. , Lewis Dale W. , Gollin Susanne M. 
Published: August 2012

Abstract
Background: The ATR-CHEK1 pathway is upregulated in a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with loss of the G1 cell cycle checkpoint. We hypothesized that the upregulated ATR-CHEK1 pathway protects HNSCC from mitotic catastrophe (cell death) by enhancing the G2M phase checkpoint and that inhibition of this pathway may sensitize HNSCC to DNA damaging agents. Methods and Results: In HNSCC, we observed gain, amplification or translocation of the ATR gene in 8 of 20 cell lines studied; whereas the CHEK1 gene is partially lost in 12 of 20 cell lines. ATR and CHEK1 FISH in paraffin sections of primary head and neck squamous cell carcinomas confirmed our findings in HNSCC cell lines. We observed that both ATR and CHEK1 are overexpressed by quantitative PCR in five of eight HNSCC cell lines with loss of the G1 cell cycle checkpoint. Inhibition of ATR and CHEK1 with caffeine or specific inhibition with the respective siRNAs results in increased susceptibility of HNSCC to ionizing radiation by clonogenic cell survival assay. siRNA-mediated ATR or CHEK1 knockdown leads to loss of G2M checkpoint accumulation and increased sub-G0 apoptotic population by flow cytometric analysis. The mechanism of cell death after ATR and CHEK1 knockdown is by mitotic catastrophe, as evidenced by increased premature chromatin condensation. Conclusions: The ATR-CHEK1 pathway is up-regulated in a subset of HNSCC with loss of G1 phase cell cycle checkpoint. The up-regulated ATR-CHEK1 pathway protects these HNSCC from mitotic catastrophe by enhancing the G2M phase cell cycle checkpoints and inhibition of ATR and/or CHEK1 increase the sensitivity of the HNSCC to ionizing radiation. We conclude that inhibition of the up-regulated ATR-CHEK1 pathway in HNSCC may potentiate or increase the efficacy of the current therapeutic modalities for HNSCC.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3113. doi:1538-7445.AM2012-3113
Abstract 3178: Can DNA from archived formalin-fixed paraffin embedded (FFPE) cancer tissues be used for somatic mutation analysis in next generation sequencing
Authors: Palescandolo Emanuele , Jones Robert , Raza Alina , Sunkavalli Ashwini , Brastianos Priscilla K. , Ducar Matthew , Go Christina , Roden Christine , Hatton Charles , Hanna Megan , Mills Adri , Adusumilli Ravali , Kumar Prateek , Meyerson Matthew L. , William Hahn C. , MacConaill Laura , Hummelen Paul Van 
Published: August 2012

Abstract
Formalin-fixed paraffin embedded (FFPE) tissues are far more abundant in most tissue banks and pathology departments than fresh or fresh frozen (FF) samples, but typically yield varying degrees of degraded DNA as a result of the fixation process. The quality of FFPE tissues may vary based on a number of factors such as: age of the block, fixation time, storage and handling conditions. Being able to harness the power of next generation sequencing technologies to genomically characterize these abundant and diverse achieved samples would be tremendously valuable to the cancer research community and would enable the use of this material for clinical purposes. The goal of this study was to assess the performance of FFPE samples in next generation sequencing applications. We monitored close to 100 samples from tumor and normal tissues of FFPE, FF and blood origin. These samples underwent several processes for whole-exome or targeted sequencing, including DNA fragmentation, size selection, library preparation, and hybrid-capture enrichment. We observed variable performance across these samples at several of the above steps, which correlated predominantly with FFPE tissues and the age of block. Other quality metrics that showed relative lower performance of FFPE DNA were the cluster density, duplication rate or library complexity. Most FFPE samples still generated good quality sequence, however, older FFPE blocks, over 10 years, may need more input DNA or higher sequence depth to reach minimum coverage if somatic mutation analysis is the goal. There was no difference in performance between DNA from tumor and normal tissues.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3178. doi:1538-7445.AM2012-3178
Abstract 3180: A fast solution to NGS library preparation with low nanogram DNA input
Authors: Liu Pingfang , Lohman Greg , Cantor Eric , Langhorst Brad , Apone Lynne , Munafo Daniela , Sumner Christine , Stewart Fiona , Evans Thomas C. , Dimalanta Eileen T. , Davis Theodore 
Published: August 2012

Abstract
Next generation sequencing (NGS) has significantly impacted cancer genetics, enabling a comprehensive characterization of genomic abnormalities in the cancer genome at very high resolution. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized genome-based diagnosis and targeted cancer therapy a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. To overcome this challenge, we have developed a fast library preparation method using novel NEBNext reagents and adaptors. This method enables library construction from a minimal quantity of DNA (&lt; 5 ng), and can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3180. doi:1538-7445.AM2012-3180
Abstract 3185: Rapid and efficient methods for preparing rRNA-depleted and directional RNA-Seq libraries from low-input and FFPE RNA samples
Authors: Sooknanan Roy , Hitchen John 
Published: August 2012

Abstract
Massively parallel sequencing of cDNA libraries (RNA-Seq) is rapidly becoming the preferred method for transcript profiling, and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. Preparing NGS RNA-Seq libraries typically require first isolating rRNA-depleted RNA or enriching for poly(A)+ mRNA. However, total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) patient tissue samples is normally fragmented, making it not suitable for rRNA-depletion using some commercially available kits or poly(A)+ enrichment, which results in a 3′ sequence bias. Here, we present an efficient “single-pass” rRNA-depletion method (Ribo-Zero™ technology) for use with as little as 100 ng total RNA input from either intact or fragmented (e.g., FFPE) RNA samples. Additionally, an improved, more user-friendly version of the ScriptSeq™ RNA-Seq method (ScriptSeq™ v2) is used to rapidly prepare directional (∼99% strandedness) RNA-Seq libraries in about 2.5 hours, in a single-tube workflow, from either the intact or fragmented Ribo-Zero™ treated RNA samples. The ScriptSeq™ method does not require end-polishing, adaptor-ligation, cDNA fragmentation, or gel-size selection. The combined Ribo-Zero™ and ScriptSeq™ workflow is completed in about 5 hours, generating cluster-ready NGS libraries that contain &lt;2 % of reads that map to rRNA sequences (nuclear- and mitochondrial-encoded) and ∼98% of reads that map to the genome, while maintaining the representation of coding and non-coding transcripts, independent of polyadenylation. This reduction in rRNA sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads required for transcriptome analysis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3185. doi:1538-7445.AM2012-3185
Abstract 3186: Comparative analysis of different total RNA sequencing approaches
Authors: Sumner Christine J. , Munafo Daniela , McReynolds Larry , Langhorst Brad , Liu Ping , Apone Lynne , Stewart Fiona , Dimalanta Eileen , Davis Theodore 
Published: August 2012

Abstract
Initial transcriptomic studies largely relied on hybridization-based microarray technologies and offered a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over wide ranges of levels. Massively parallel cDNA sequencing, or Total RNA-Seq, has allowed many advances in the characterization and quantification of transcriptomes; an offer a new appreciation for the complexity of the Transcriptome, encompassing a multitude of previously unknown coding and non-coding RNA species, particularly small RNAs, including micro RNAs. This work investigates the performance of different strategies for Total RNA-Seq using multiple next generation sequencing platforms. Standard RNA-sequencing approaches generally require double-stranded cDNA Synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Here, we examine the performance of strand-specific RNA libraries made by direct ligation of adaptor on to the RNA. We analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation) and we provide a comparative data analysis (library complexity, continuity of gene coverage, strand specificity and 3′and 5′-end bias analysis). Identification and analysis of small RNA by deep sequencing requires preparation of a di-tagged cDNA library, which leads to adaptor-dimer formation that strongly contaminates the library. We have developed a novel method to generate di-tagged small RNA libraries free of adapter-dimer contamination without introducing any additional enzymatic steps or gel purifications. This method has optimized the 3′adaptor-ligation reaction to recover and to increase representation of the 2′-O-modified RNAs present in a biological sample. To reduce cost and increase sample throughput we have developed a barcode strategy to tag samples during library construction. The multiplexed libraries can then be pooled together before size selection, reducing the number of steps in the workflow. This technique reduces bias by ligation, increases representation of modified small RNAs and simplifies workflow during library construction for small RNA analysis and discovery.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3186. doi:1538-7445.AM2012-3186
Abstract 3196: In vivo drug sensitivity testing of CTCs isolated using the FMSA device
Authors: Gallant Jean-Nicolas , Cheng Hairong , Harouaka Ramdane , Zheng Siyang , El-Deiry Wafik S. 
Published: August 2012

Abstract
BACKGROUND: Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response; indeed, the enumeration of fixed CTCs with the Johnson and Johnson Veridex CellSearch System is FDA-approved for assessment of patient prognosis in metastatic breast, prostate, and colorectal cancer. The opportunity to isolate viable CTCs using the Flexible Micro Spring Array (FMSA) device – a novel microfluidic device that enriches CTCs by two physical parameters: size and flexibility – offers the possibility of testing cancer treatments in real time and rapidly providing information back to clinicians regarding the best course of treatment. OBJECTIVES: In this study, we evaluated the possibility of isolating viable CTCs from human blood using the FMSA device, growing these cells in a mouse xenograft model, and determining successful treatments for metastatic disease. METHODS: GFP-labeled HCT-116 cells were spiked into samples of blood from healthy donors to mimic the blood of patients with metastatic disease. This spiked blood was filtered using the FMSA device, and the recovered cells were implanted into the flanks of immunodeficient mice for study. A series of in vivo experiments were carried out to investigate the effect of chemotherapy on the resulting tumors. These tumors were molecularly characterized, compared to tumors derived from non-filtered cells, and analyzed for potential differences resulting from FMSA enrichment. Studies are ongoing to further investigate the use of this model and approach to culture CTCs from patients. RESULTS & CONCLUSIONS: Cancer cells isolated from blood using the FMSA can proliferate when implanted subcutaneously into the flanks of immunodeficient mice – developing into tumors within two weeks. Although they grow at rates that depend on the number of cancer cells found in each mL of blood, these tumors provide a means for testing the effects of chemotherapy on CTCs in vivo. Using this model, treatments targeted towards CTCs could be assessed and provide real-time information to clinicians regarding treatment interventions for patients with metastatic cancer and detectable CTCs. It may also be possible to use this model as the primary mode for CTC culture as the in vivo environment may provide soluble factors, cell-to-cell interactions, and physical parameters that are not easily mimicked by in vitro culture. The approach has potential to help with efforts to develop personalized therapy options for patients with advanced cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3196. doi:1538-7445.AM2012-3196
Abstract 3198: The CEE-Selector Assay: A tool for the identification of rare allele variants
Authors: Alexiadis Vassilios , Watanaskul Tim , Kosco Karena , Arnold Lyle 
Published: August 2012

Abstract
Molecular assays for the identification of rare allele occurrences are important tools for proper cancer classification and treatment. A prime example is the T790M mutation in EGFR which leads to resistance to the tyrosine kinase inhibitors gefitinib (Iressa®) and erlotinib (Tarceva®) used in the treatment of non-small cell lung cancer (NSCLC). Identification of the T790 mutation in cancer-shed particles in blood (either as whole cells or subcellular vesicles) calls out the need for an alternative cancer treatment. We have developed a highly sensitive PCR-based assay which allows the identification of the T790M mutation in blood plasma (either when present in mRNA or genomic DNA). The assay combines Real-Time PCR as well as melt curve analysis of the mutant PCR product and is followed by sequencing to verify the presence of the mutation. The Selector Assay is based on a wild-type specific PCR blocker and allows the mutant template to be amplified in a high background of wild-type template. A few copies of T790M mutant can be detected in greater than a 1000-fold excess of wild-type. Data using the Selector Assay with clinical lung cancer samples showing the identification of T790M in genomic DNA as well as mRNA in blood plasma and in CTCs will be presented. The Selector Assay can be applied to other mutations relevant to cancer and is a valuable tool for clinical diagnostics.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3198. doi:1538-7445.AM2012-3198
Abstract 3199: Miniaturized, multiplexed, highly sensitive protein NanoArray assay for measuring biomarkers in dried blood spot (DBS) samples
Authors: Dixon Douglas B. , Ivanov Ivan D. , Gubbins Earl J. , Harris Richard R. , Coen Michael L. , Helfrich Rosalind , Jamil Haris 
Published: August 2012

Abstract
As the utility of new biomarkers for diagnosis and treatment of disease become better understood, physicians and researchers are faced with increasingly critical decisions regarding how to best distribute their valuable patient samples. While simply taking more blood would seem a reasonable solution, a recent publication in the Archives of Internal Medicine (Salisbury, et.al., Arch Intern Med. 2011;171(18):1646-1653) addresses the very real problem of hospital-acquired anemia (HAA) brought on by blood loss resulting from blood drawn for clinical assays. Also in many resource constrained countries, the lack of adequate infrastructure such as hospitals, laboratories, transportation systems as well as qualified personnel pose major obstacles for patient access to clinical testing. This has revived interest in collecting dried blood spots (DBS) for clinical testing. DBS samples provide a number of benefits when compared with drawing venous blood: they require minimal blood volume (usually 50µl per spot), they can easily be collected from infants and children, and they can be used to collect blood samples where collection and storage of serum or plasma is not feasible. However for DBS samples to be a viable solution for clinical assay, the assay method must be sensitive and yet require very small sample volumes. NanoInk, Inc. has developed a highly sensitive assay based on dip-pen nanolithography (DPN™) using the NanoArrayer3000™. Utilizing a 48 subarray slide platform printed with a nano-scale antibody array we have created a miniaturized multiplexed sandwich ELISA with sensitivities as low as the 100's of femtograms/ml while using sample volumes that range from 2-4µl/well. Using our proprietary elution protocol, we are able to elute biomarkers from a standard 3.2mm DBS card punch sufficient to provide triplicate assay values while still keeping the samples concentrated enough to measure lower concentration biomarkers. Our NanoInk DBS cytokine assay has been validated using eluted blood standards and QC samples and shows good well-to-well and slide-to-slide variability. NanoInk's multiplexed assay format can also be customized to include a wide array of biomarkers. This multiplex capability, in combination with our proprietary sample and standard elution protocol, now allows clinicians to accurately measure the concentration of multiple biomarkers using convenient small-volume DBS samples. The results of an ongoing study for measuring biomarker levels in patient samples using our 5-cytokine DBS assay will be presented.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3199. doi:1538-7445.AM2012-3199