bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 5351: A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA
Authors: Doh Jung H. 
Published: July 2017

Abstract
Extensive archives of formalin-fixed, paraffin-embedded (FFPE) cancer tissues and next generation sequencing (NGS) capabilities provide rich resources for identifying and validating new cancer biomarkers. However, the ability to conduct FFPE-NGS studies has been limited by poor nucleic acid purification methods that are generally difficult and cumbersome to perform, particularly with RNA. FormaPure DNA was recently launched to provide a simplified workflow to extract DNA from FFPE curls. Its nucleic acid binding chemistry employs Beckman Coulter's proprietary SPRI (solid phase reversible immobilization) technology, which purifies nucleic acids without centrifugation or vacuum manifold. The SPRI technology ensures stronger binding and higher quality purification than other silica-coated bead methods that typically lead to lower quality results due to instances of nucleic acid shearing. Here, we present a modified FormaPure DNA workflow to purify RNA from FFPE samples, offering the flexibility to examine both genomic and transcriptomic roles in cancer biomarkers studies. We show that our extractions yield significantly more RNA (up to 2.5 µg per FFPE curl) and take a fraction of the time to perform compared to other benchmarks. Furthermore, we demonstrate that purified RNA can be used to generate high quality NGS data. Lastly, our extraction method can be automated on a variety of Beckman Coulter’s Biomek liquid handling workstations to offer an integrated solution for high-throughput FFPE-RNA-NGS experiments: RNA from 96 FFPE samples can be purified for NGS in less than 5 hours with minimal hands-on time.
Citation Format: Jung H. Doh. A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA [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 5351. doi:10.1158/1538-7445.AM2017-5351
Abstract 536: Novel identification of STAT1 as a crucial mediator of ETV6-NTRK3-induced tumorigenesis
Authors: Park Jinah , Kim Junil , Sorensen Poul H. , Kim Seong-Jin 
Published: July 2017

Abstract
Chromosomal rearrangements that facilitate tumor formation and progression through activation of oncogenic tyrosine kinase are frequently observed in cancer. The ETV6-NTRK3 (EN) fusion has been implicated in various cancers, including infantile fibrosarcoma, secretory breast carcinoma and acute myeloblastic leukemia, and has exhibited in vivo and in vitro transforming ability. In the present study, we analyzed transcriptome alterations using RNA-Seq in EN-transduced NIH3T3 fibroblasts to identify the mechanisms that are involved in EN-mediated tumorigenesis. Through functional profile assessment of EN-regulated transcriptome alterations, we found that EN up-regulated genes mainly associated with cell motion, membrane invagination, and cell proliferation, while down-regulated genes are involved in cell adhesion, which described the transforming potential and increased proliferation in EN-transduced cells. Notably, KEGG pathway analysis identified the JAK-STAT signaling pathway with the highest statistical significance. Moreover, Ingenuity Pathway Analysis and gene regulatory network analysis identified the Stat1 transcription factor and its target genes as top EN-regulated molecules. We further demonstrated that EN enhanced STAT1 phosphorylation but attenuated STAT1 acetylation, thereby inhibiting the interaction between NF-κB p65 and acetylated STAT1. Consequently, nuclear translocation of NF-κB p65 and subsequent anti-apoptotic NF-κB activity were increased by EN. Taken together, here we report, for the first time, STAT1 as a significantly EN-regulated transcription factor and a crucial mediator of EN-induced tumorigenesis. [This work was supported by a National Research Foundation grant of Korea (NRF-2014M3A9B5073918) funded by the Korea government.]
Citation Format: Jinah Park, Junil Kim, Poul H. Sorensen, Seong-Jin Kim. Novel identification of STAT1 as a crucial mediator of ETV6-NTRK3-induced tumorigenesis [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 536. doi:10.1158/1538-7445.AM2017-536
Abstract 5361: Predesigned gene content for rapid deployment of custom oncology panels
Authors: Barry Andrew J. , Emerman Amy , Bowman Sarah , Patel Kruti , Dimalanta Eileen , Adams Scott , Henig Noa , Stewart Fiona , Hendrickson Cynthia , Davis Theodore , Elfe Charles 
Published: July 2017

Abstract
Efficient utilization of targeted gene panels for oncology research is challenged by the wide variation in gene constituents specific to a given study. While focused gene panels efficiently provide the necessary depth of coverage for low frequency variant detection, the high costs and design challenges associated with de novo panel design present challenges.
The NEBNext DirectTM technology utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. The approach rapidly hybridizes both strands of genomic DNA with biotinyated probes prior to strepdavidin bead capture, enzymatic removal of off-target sequence, and conversion of captured molecules into sequencer-ready libraries. This results in a unique read coverage profile that results in uniform coverage across a given target. Unlike alternative hybridization methods, the approach does not necessitate upfront library preparation, and instead converts the captured molecules into dual-indexed illumina sequencer compatible libraries containing an 8 basepair sample ID and a 12bp Unique Molecule Index (UMI). The UMI individually tags each molecule prior to the final PCR amplification of the library, enabling identification of PCR duplicate molecules. The result is a 1-day protocol that enables the preparation of sequence-ready libraries from purified genomic DNA specific to the gene content included in the panel.
We have designed and developed baits specific to the full exonic content of 450 genes associated with cancer. These are designed, balanced, and pooled on a per gene basis, and can be combined into customized panels, allowing rapid turnaround of specific custom gene subsets. Here, we present the ability to rapidly deploy custom gene panels across a variety of panel sizes and content, while maintaining high specificity, uniformity of coverage across target content, and sensitivity to detect nucleic acid variants from tumor samples.
Citation Format: Andrew J. Barry, Amy Emerman, Sarah Bowman, Kruti Patel, Eileen Dimalanta, Scott Adams, Noa Henig, Fiona Stewart, Cynthia Hendrickson, Theodore Davis, Charles Elfe. Predesigned gene content for rapid deployment of custom oncology panels [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 5361. doi:10.1158/1538-7445.AM2017-5361
Abstract 5362: Targeting BRCA1 and BRCA2 with NEBNext Direct™
Authors: Adams Scott M. , Patel Kruti M. , Emerman Amy B. , Bowman Sarah K. , Elfe Charles D. , Henig Noa , Russello Salvatore , Barry Andrew , Davis Theodore , Hendrickson Cynthia L. 
Published: July 2017

Abstract
The screening and detection of germline BRCA1 and BRCA2 mutations are critical for the effective management of patients with breast or ovarian cancer and for the identification of individuals with a high risk of developing these cancers. In addition, somatic detection of pathogenic variants in BRCA1/2 can influence treatment decisions due to the susceptibility of tumors with BRCA mutations to PARP inhibitors.
Here we introduce the NEBNext DirectTM BRCA enrichment panel for the interrogation of BRCA1 and BRCA2 by Illumina sequencing. NEBNext DirectTM is a novel, hybridization-based method to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes. This approach includes features such as the incorporation of unique molecule identifiers (UMIs) and the ability to capture degraded DNA, enabling accurate detection of low-frequency variants from formalin-compromised DNA and other challenging sample types.
We applied the NEBNext Direct BRCA1/2 panel to frozen tissue and achieved a high specificity for the BRCA targets (96% of the sequenced reads mapped to BRCA1 and BRCA2 and 80% of the sequenced bases were within the targeted regions). In addition, the resulting libraries were highly uniform in coverage, with 91% of the targeted bases covered at a value of at least 50% of the mean depth of coverage and 100% of the bases covered at 20% of the mean or greater. Some variability in the specificity was observed with formalin-fixed, paraffin embedded (FFPE) samples, and this effect was dependent on the quality of the FFPE DNA. The UMIs were used to identify PCR duplicates prior to variant calling, as well as for error correction, enabling the accurate detection of low-frequency variants. The library preparation for all samples was completed in one day, and the entire process of library preparation, sequencing on the Illumina Miseq, and data analysis was completed in a total of two days.
In summary, we demonstrate that application of the NEBNext DirectTM method to the enrichment of BRCA1 and BRCA2 provides a tractable approach for the rapid and highly sensitive analysis of these cancer-associated genes.
Citation Format: Scott M. Adams, Kruti M. Patel, Amy B. Emerman, Sarah K. Bowman, Charles D. Elfe, Noa Henig, Salvatore Russello, Andrew Barry, Theodore Davis, Cynthia L. Hendrickson. Targeting BRCA1 and BRCA2 with NEBNext Direct™ [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 5362. doi:10.1158/1538-7445.AM2017-5362
Abstract 5363: Measuring gene expression at the tumor microenvironment: A comparison between nCounter PanCancer Immune Profiling Panel and Oncomine Immune Response Research Assay
Authors: Mongan Ann , Tom Warren , Au-Young Janice , Pankov Aleksandr , Ganpule Gauri , Hyland Fiona 
Published: July 2017

Abstract
The tumor microenvironment, especially infiltrating T lymphocytes and inflammatory molecules, is believed to be highly relevant to the tumor’s sensitivity to cancer checkpoint blockade therapy. At the same time, the exact markers that are predictive of response for each therapeutic agent are still the subject of active investigations. To address the need for better understanding of the effect of different T cell subsets, antigen presentation, and tumor killing, gene expression profiling presents an attractive means to simultaneously evaluate the tumor microenvironment and cancer cells. In this study we compare the results and performance of the nCounter PanCancer Immune Profiling Panel and the Oncomine Immune Response Research Assay, both of which are designed to measure the expression of genes indicative of an immune response and potential immune-editing activities by tumor cells. The nCounter panel detects gene expression by counting unique probes that hybridize target mRNA, while the Oncomine panel employs NGS to sequence and count reads derived from the targets. While both panels are designed to work with FFPE samples, The nCounter panel expects 100 ng of unamplified RNA, while the Oncomine panel requires only 10 ng total RNA with its AmpliSeq technology. The two panels share 254 common genes, which constitute the basis for this comparison. Across 12 cancer samples (breast, H&N, melanoma, NSCLC, and RCC), results show that the Oncomine panel offers 20% higher dynamic range, thereby providing more robust readouts about the differences among samples. More importantly, with virtually no background noise, the underlying NGS technology provides an absolute zero readout for non-expressing genes which significantly improves the sensitivity for detecting low expressing genes, as their presence can be confirmed by as few as two reads. The two technologies show moderate correlation (R ~ 0.7), with the Oncomine panel more strongly correlating with qPCR (R ~ 0.9). Finally, when clustered using all genes on the panel, only the Oncomine panel provides clear stratification of cancer types, thus allowing the panel to be used for tissue type confirmation in addition to evaluating the immune response.
Citation Format: Ann Mongan, Warren Tom, Janice Au-Young, Aleksandr Pankov, Gauri Ganpule, Fiona Hyland. Measuring gene expression at the tumor microenvironment: A comparison between nCounter PanCancer Immune Profiling Panel and Oncomine Immune Response Research Assay [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 5363. doi:10.1158/1538-7445.AM2017-5363
Abstract 5366: Detection of low-frequency somatic variants using single-molecule, real-time sequencing
Authors: Baybayan Primo , Nolden Laura 
Published: July 2017

Abstract
Detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a low level, is challenging. Single Molecule, Real-Time (SMRT®) Sequencing is ideal for minor variant detection because of its ability to sequence single molecules with very high accuracy (&gt;QV40) using the circular consensus sequencing (CCS) approach.
Here, we characterize the Sequel System for the detection of low-frequency somatic variants using constructs containing mutations in coding regions in EGFR, NPM1, AKT1 and JAK2 representing deletion, insertion, substitution and homopolymer variants. Wild type and mutant amplicons, provided by SeraCare, were mixed and serially diluted from 10% down to 0.1% allelic frequency. Independent SMRTbell libraries were constructed for each dilution point, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants. We will demonstrate sensitivity of the PacBio Systems to detect mutations down to 0.1%.
Citation Format: Primo Baybayan, Laura Nolden. Detection of low-frequency somatic variants using single-molecule, real-time sequencing [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 5366. doi:10.1158/1538-7445.AM2017-5366
Abstract 5369: Epigenetic, transciptomic and ubiquitomic changes associated with BAP1 loss in uveal melanoma
Authors: Kurtenbach Stefan , Kuznetsov Jeffim N. , Field Matthew G. , Reddy Rohit , Sanchez Margaret I. , Decatur Christina L. , Harbour J William 
Published: July 2017

Abstract
Uveal melanoma (UM) is the most common primary and aggressive ocular cancer. Up to 50% of the patients develop metastasis, which are notoriously resistant to all forms of therapy and despite medical treatment leads to death within a mean time of 5-7 months, with a mortality rate of over 90%. We previously described that gene expression profiling can be used to classy UM tumors into two basic categories, class 1 (low metastatic risk) and class 2 (high metastatic risk). We further described that inactivating mutations in the ubiquitin hydrolase BAP1 are found in over 85% of class 2 tumors. BAP1 is involved in removing ubiquitin from specific proteins to regulate their function, like histone H2A, thereby regulating gene expression.
Building on these findings, we have generated uveal melanoma and melanocyte cell lines that allow for the inducible knockdown of BAP1. First, we performed RNAseq on multiple cell lines before and after BAP1 knockdown and compare the results to gene expression of class 1 and class 2 tumors. We identified significant overlap in key genes linked to metastasis between the primary tumors, uveal melanoma and melanocyte cell lines. Using ChIP-seq to interrogate uveal melanoma cells depleted of BAP1, we identified changes in genome wide histone marks and RNA polymerase localization associated with BAP1. To gain further insight into non-histone deubiquitination targets of BAP1, we performed ubiquitination proteomic profiling using the UbiScan® technology and identified a list of high probability BAP1 substrates.
Taken together, these complementary genome-wide investigations provide a global picture of the cellular functions of BAP1 and they provide novel insights into the metastasis-promoting effect of BAP1 loss in UM.
Citation Format: Stefan Kurtenbach, Jeffim N. Kuznetsov, Matthew G. Field, Rohit Reddy, Margaret I. Sanchez, Christina L. Decatur, J William Harbour. Epigenetic, transciptomic and ubiquitomic changes associated with BAP1 loss in uveal melanoma [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 5369. doi:10.1158/1538-7445.AM2017-5369
Abstract 5373: 3D genome architecture changes during cancer cell migration and metastasis
Authors: McCord Rachel Patton , Golloshi Rosela 
Published: July 2017

Abstract
Metastasis, the most deadly part of cancer, results from cancer cell migration and invasion from a tumor into surrounding tissues. During this migration, the cell often has to squeeze and deform its nucleus through tight spaces. This nuclear squeezing is a rate-limiting step in migration, and the deformations can even rupture the nucleus. The organization of the genome inside the nucleus into loops, domains, and territories is important for proper gene regulation, DNA replication, and genome repair. However, little is known about the characteristics of nuclear organization that might allow for the nuclear deformation of metastatic cancer cells. We are using Chromatin Conformation Capture (Hi-C) and microscopy techniques to study genome organization during cancer cell migration. Using photoconvertible fluorophore Dendra2-H4 we draw and monitor patterns on nuclei of cells migrating through dense collagen matrices, Transwell filters, or microfluidic channels. Our data show changes in these patterns, indicating spatial reorganization of the genome during migration. Previous work has suggested that cancer cell migration requires global condensation of the genome. Some cancer chemotherapeutics act on epigenetic marks to decondense chromatin. We are testing whether such chromatin decondensation affects the physical deformability of the nucleus and thus effective migration. We have used Transwell and wound healing migration assays to monitor migration of cells treated with histone deacetylase inhibitors and histone methyltransferase inhibitors. Our results show that the drugs inhibit migration in a Transwell assay but not in the wound healing assay. This suggests that genome decondensation prevents the passage of the nucleus through narrow 3D spaces. But, a condensed genome organization is not necessary for 2D cell motility, where nuclei are unconfined. Hi-C experiments on migrating cells with deformed nuclei and drug-treated cells will characterize the specific rearrangements in genome structure that occur during cancer cell migration and whether these specific changes are counteracted by epigenetically active drugs.
Citation Format: Rachel Patton McCord, Rosela Golloshi. 3D genome architecture changes during cancer cell migration and metastasis [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 5373. doi:10.1158/1538-7445.AM2017-5373
Abstract 5376: Requirement of TET2 in MLL-AF9 mediated leukemogenesis
Authors: Nath Dipmoy , Mohi Golam 
Published: July 2017

Abstract
TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine resulting in DNA demethylation. Loss-of-function mutations or deletions of TET2 have been found in several myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). Moreover, deletion of TET2 leads to myeloid malignancies in mice. Therefore, TET2 has been suggested to play a tumor suppressor role in myeloid malignancies. In this study, unexpectedly, we have uncovered a tumor promoter function for TET2 in MLL rearranged leukemia.
Chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene have been found in ~10% of acute leukemia and are associated with poor prognosis. We observed that depletion of TET2 significantly inhibited the proliferation of MLL-AF9 positive MOLM14 leukemia cells. Similarly, knockdown of TET2 markedly reduced proliferation in murine Ba/F3 cells stably expressing MLL-AF9. Conversely, overexpression of TET2 significantly increased proliferation in MLL-AF9 expressing MOLM14 and Ba/F3 cells. In addition, deletion of TET2 significantly inhibited MLL-AF9 mediated myeloid colony formation in the bone marrow (BM) cells ex vivo.
Next, we assessed the in vivo effects of TET2 deletion in MLL-AF9-mediated leukemogenesis using retroviral bone marrow transduction and transplantation assays. Whereas recipients of MLL-AF9-transduced wild type BM exhibited marked increase in white blood cells (WBC) and neutrophils in their peripheral blood and significant increase in the spleen size, recipients of MLL-AF9-transduced TET2-deficient BM showed significantly reduced WBC and neutrophil counts and reduced spleen size. Flow cytometric analysis showed significantly increased myeloid precursors (Gr-1+/Mac1+) and granulocyte macrophage progenitors (GMP) in the BM of transplanted animals receiving MLL-AF9 expressing wild type BM compared with recipients of MLL-AF9 expressing TET2-deficient BM. Furthermore, transplanted animals receiving MLL-AF9-transduced wild type BM showed marked increase in hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+), which was significantly reduced upon TET2 deletion. Deficiency of TET2 also significantly reduced MLL-AF9-mediated increase in hematopoietic progenitor colony formation in the BM. In addition, deletion of TET2 resulted in increased overall survival in mice expressing MLL-AF9. Together, these data indicate that TET2 is required for leukemic transformation mediated by MLL-AF9. Our results suggest a novel tumor promoter function for TET2 in MLL rearranged leukemia.
Citation Format: Dipmoy Nath, Golam Mohi. Requirement of TET2 in MLL-AF9 mediated leukemogenesis [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 5376. doi:10.1158/1538-7445.AM2017-5376