bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 4898: Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads
Authors: Tseng Elizabeth , Clark Tyson , Ashby Meredith H. , Shenykman Gloria 
Published: August 2015

Abstract
While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. This inability to resolve full-length isoforms hinders researchers’ ability to identify key isoform changes and fusion transcripts associated with human diseases. With the PacBio® long-read technology, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso-Seq™ protocol produces reads that originate from independent observations of single molecules, and no assembly is needed if a read spans the entire length of the transcript.
In this study, we sequenced the human MCF7 cell-line using the Clontech SMARTer® cDNA preparation kit. Using the PacBio Iso-Seq bioinformatics software, we obtained 55,770 unique, full-length, high-quality transcript sequences that were subsequently mapped back to the human genome with ≥ 99% accuracy. The full-length transcripts ranged from 300 bp to 9.5 kb with a mean length of 2.1 kb. Out of 16,868 expressed loci, 149 had more than 20 isoforms, with a total average of 3 isoforms per loci. Additionally, we identified more than 100 candidate fusion transcripts, many of which were confirmed through previously published experimental validations or computation predictions. For the BCAS3-BRCA4 fusion, we detected 3 different fusion transcripts, two of which were entirely novel.
To validate the full-length isoforms, we compared the predicted peptides from the MCF-7 data against a previously published mass spectrometry dataset from the same cell line. 41,699 peptides mapped to 23,729 PacBio-predicted open reading frames (i.e. protein sequences), confirming the translation of a majority of sequenced transcripts. Notably, 251 peptides were novel and not annotated in the UniProt protein database. These peptides confirm abundant cases of single amino acid polymorphisms, cassette exon splicing, and potential alternative protein coding frames encoded in the MCF-7 proteome.
To conclude, the Iso-Seq protocol provides a new opportunity for researchers to study the complex splicing events that occur in cancer cells.
Citation Format: Elizabeth Tseng, Tyson Clark, Meredith H. Ashby, Gloria Shenykman. Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2015-4898
Abstract 4905: Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing
Authors: Nakagaki Takafumi , Sasaki Yasushi , Kobashi Kenta , Takeda Kousuke , Tamura Miyuki , Ohashi Tomoko , Ogi Kazuhiro , Idogawa Masashi , Hiratsuka Hiroyoshi , Tokino Takashi 
Published: August 2015

Abstract
Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. We performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM). We first validated a multigene sequencing screen interrogating 2855 mutational hotspots in 50 cancer-related genes using the Ion Torrent AmpliSeq cancer panel v2. Ten nanogram of DNA was used as template to amplify 207 regions in oral squamous cell carcinoma (OSCC). Approximately 1000 average coverage was obtained with more than 90% of target bases having at least 100 sequence reads. We detected mutations of the TP53 gene in 96% (22 of 23) of OSCC cell lines. Of the 62 OSCC specimens, 42 presented at least one somatic mutation among the 50 investigated genes, and 21 of these showed multiple gene somatic mutations. The most frequent mutations were detected on TP53 (26 of 62; 41.9%). CDKN2A mutations were identified in 9 cases; STK11 mutations were observed in 8 cases; and PIK3CA were mutated 5 cases. In addition, we sequenced 409 cancer-related genes in matched tumor and normal DNA from OSCC patients using the Ion Ampliseq Comprehensive Cancer Panel. This panel targets all exons of 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated (covered regions: 95.4% of total), and 15,992 amplicons amplify more than 1.2 megabases of target sequence. We piloted the use of this platform to identify somatic mutations in 37 OSCC specimens including formalin-fixed paraffin-embedded (FFPE) samples. We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data. This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings.
Citation Format: Takafumi Nakagaki, Yasushi Sasaki, Kenta Kobashi, Kousuke Takeda, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Masashi Idogawa, Hiroyoshi Hiratsuka, Takashi Tokino. Comprehensive genomic analyses of oral squamous cell carcinoma tissues by semiconductor-based next-generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4905. doi:10.1158/1538-7445.AM2015-4905
Abstract 4913: Highly complex oligonucleotide libraries for use in high-resolution RNA and RNA/DNA fluorescence in situ hybridization in various sample types
Authors: Ach Robert A. , Ghosh Mistuni , Tsang Peter , Scheffer-Wong Alicia , Bruhn Laurakay , Yamada Alice 
Published: August 2015

Abstract
Fluorescence in situ Hybridization (FISH) is a powerful technique for determining the localization specific nucleic acid sequences within individual cells. Previously, the use of FISH has often been dependent upon access to cloned template DNA for the generation of probes, which can be difficult if clones for specific regions are unavailable, or if the genomic region of interest contains repetitive and/or other problematic sequences. We have developed the ability to chemically synthesize DNA in massively parallel reactions, which we have used to produce libraries of oligonucleotides up to 200 bases in length that can be utilized for the generation of FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci. We have found that these oligonucleotide library-derived FISH probes can detect human genomic regions as small as 1.8 kb and as large as whole chromosomes in both metaphase and interphase cells, using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we can readily visualize regions rich in repeats and/or GC content. We have also used these oligonucleotide library-derived FISH probes to detect the localization of a variety of both coding and non-coding RNAs in fixed tissue culture cells and formalin-fixed paraffin-embedded tissue sections, using both conventional fluorescence and structured illumination microscopy. Simultaneous hybridization of FISH probes labeled with different fluorophores enables visualization of multiple sequences at once. Using probes designed specifically to transcribed vs. non-transcribed regions has enabled the simultaneously detect DNA and RNA from the same locus, or from two different loci, in the same FISH assay. The ability to generate high performance FISH probes using chemically synthesized oligo libraries that can simultaneously detect DNA and RNA yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function.
Citation Format: Robert A. Ach, Mistuni Ghosh, Peter Tsang, Alicia Scheffer-Wong, Laurakay Bruhn, Alice Yamada. Highly complex oligonucleotide libraries for use in high-resolution RNA and RNA/DNA fluorescence in situ hybridization in various sample types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2015-4913
Abstract 4920: The detection of circulating tumor DNA using Quantstudio 3D digital PCR
Authors: Greer Adam H. , Mills Glenn , Yin Hong 
Published: August 2015

Abstract
Purpose: The purpose of this study is to employ the Quantstudio 3D (Q3D) system to detect circulating tumor DNA in cancer patients’ plasma.
Background: Circulating tumor DNA (ctDNA) as a non-invasive biomarker plays roles in monitoring solid tumor progression, metastasis, and therapeutic effects. The identification of ctDNA depends on mutant determination. The digital PCR technology is a powerful tool to identify ctDNA. The Q3D platform is a relatively new digital PCR systemwhcih has not been extensively tested in the detection of ctDNA. In this study, we tested the capacity of Q3D in detecting ctDNA.
Methods: Genomic DNA was extracted from tumor tissue of patients. Variants were identified in tumor tissue DNA by Illumina's TruSeq Amplicon Cancer Panel. To detect the circulating tumor DNA, DNA was extracted from the plasma of the patient with known variants identified by targeted-sequencing. The circulating DNA was isolated from 400μl of plasma from 12 patients using QIAamp DNA Blood kit. The DNA concentration was measured using the Qubit 2.0. The quality and size of the circulating DNA was determined using the Agilent 2100. The circulating DNA size was approximately 170-180 nucleotides in length, as reported in literatures. The variant determination by Q3D was tested with custom TagMan SNP Genotyping Assays. The absolute copy number of variant was determined with QuantStudio® 3D AnalysisSuite™ Software.
Results: We first tested several assays with known-variant tumor tissue DNA samples in Q3D. The mutants identified by assays are BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA E542K. The results showed that data points of mutant call, wild type call were well separated from non-signal data points. We examined the sensitivity of these assays using serial dilution of tumor DNA sample in non-tumor human tissue DNA. 0.1% to 1% of mutants were detectable depending specific assays. We collected circulating DNA from XX Patients’ plasma. The DNA concentrations ranged from 5 ng/ml to 30 ng/ml of plasma. KRAS G12D and G12V was detected with circulating DNA from patients carrying KRAS mutant. The mutant frequency of KRAS G12D and G12V determined by Q3D is 3.2% and 3.7%, respectively. The absolute mutant copy number for KRAS G12D is 9 and for KRAS G12V is 8.
Conclusions: Q3D with validated custom TagMan SNP Genotyping Assays is applicable in detecting ctDNA. We will expect to utilize the technique to evaluate ctDNA.
Citation Format: Adam H. Greer, Glenn Mills, Hong Yin. The detection of circulating tumor DNA using Quantstudio 3D digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4920. doi:10.1158/1538-7445.AM2015-4920
Abstract 4931: A circulating cell free DNA workflow for targeted sequencing - from isolation through validation
Authors: Dhingra Dalia , Fang Xingwang , Carter Matt , Brinza Dumitru , Scafe Charles , Davies Christopher , Hyland Fiona 
Published: August 2015

Abstract
Circulating cell free DNA has been shown to have the potential as a non-invasive substrate for the detection of cancer and its progression as well as the determination of therapeutic resistance. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing is an optimal tool for mutation detection. Here, we demonstrate a complete workflow from isolation through validation of circulating tumor DNA.
We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation.
We then completed molecular characterization of the isolated circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples complete with TaqMan® validation.
Our results validate the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing and validation, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.
Citation Format: Dalia Dhingra, Xingwang Fang, Matt Carter, Dumitru Brinza, Charles Scafe, Christopher Davies, Fiona Hyland. A circulating cell free DNA workflow for targeted sequencing - from isolation through validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4931. doi:10.1158/1538-7445.AM2015-4931
Abstract 4938: APC in breast cancer: the ABCs of gene expression
Authors: Fernandez Soto Katia , VanKlompenberg Monica K. , Cole Jennifer , Prosperi Jenifer R. 
Published: August 2015

Abstract
Breast cancer is the most common malignancy and cancer-related cause of death in non-smoking women in the United States. Due to the heterogeneity of this disease, the oncogenic and signaling pathways contributing to these tumors are distinct. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70% of sporadic breast cancers; however, the mechanism by which APC mutation impacts tumorigenesis remains unexplored. Up-regulation of the zinc finger transcription factor EGR-1 has been associated with cell invasion and enhanced drug resistance, which is an important property of cancer stem cells (CSCs). ALDH1 and STAT3 have been useful in the identification of CSCs, which are associated with poor clinical outcomes including increased tumor survival and invasion. We utilized two human breast cancer cell lines, MDA-MB-157 and DU4475, and cells isolated from the Mouse Mammary Tumor Virus-Polyoma middle T (MMTV-PyMT);ApcMin/+ mouse model. APC was knocked down in the MDA-MB-157 cells through shRNA lentiviral transduction, or transfected into DU4475 cells. First we looked at the expression of the transcription factor EGR-1 through Real-Time PCR in the MDA-MB-157 and DU4475 cell lines. Next, ALDH1 and STAT3 were measured in the MMTV-PyMT;ApcMin/+ mouse model and in MDA-MB-157 cells through western blots. In addition, an ALDEFLUOR assay was used to measure ALDH1 activity in the mouse model. To translate our findings to an in vivo model, we injected MMTV-PyMT;ApcMin/+ cells into the mammary fat pads of FVB-N syngeneic mice. EGR-1 expression was increased in both MDA-MB-157 APC-knockdown and DU4475 APC-mutant cells compared to cells expressing APC. In addition, STAT3 expression was found to be increased in MDA-MB-157 APC-knockdown and MMTV-PyMT;ApcMin/+ cells compared to MDA-MB-157 APC-wildtype and MMTV-PyMT;Apc+/+ cells. ALDH1 activity and tumor development were enhanced in ApcMin/+ cells compared to control cells. Combined, these data suggest that APC knockdown increases tumorigenic properties of cancer cells through EGR-1 overexpression and modulation of known cancer stem cell markers, ALDH1 and STAT3. Future studies will include measuring the expression of EGR-1 in the MMTV-PyMT;ApcMin/+ cells as well as ALDH1 in the human cell lines. Furthermore, because of the overexpression of these genes and markers, strategies designed to target positive populations might lead to more effective therapies of APC-mutant breast cancers.
Citation Format: Katia Fernandez Soto, Monica K. VanKlompenberg, Jennifer Cole, Jenifer R. Prosperi. APC in breast cancer: the ABCs of gene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4938. doi:10.1158/1538-7445.AM2015-4938
Abstract 4940: Role of Smad3 linker phosphorylation in breast cancer progression
Authors: Liu Fang , Xie Wen , Liu Zhengxue , Roy Tanima , Bae Eunjin , Wakefield Lalage M. , Kim Seong-Jin , Ooshima Akira , Reiss Michael , Matsuura Isao 
Published: August 2015

Abstract
Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis in multiple tissues, including the breast tissue. At late stages, Smad3 promotes breast cancer progression and metastasis. We previously showed that the proline-rich linker region of Smad3 is phosphorylated by several kinases, such as by CDKs and MAP kinases. We have mapped these phosphorylation sites. We show that mutation of the phosphorylation sites in the Smad3 linker region increases the ability of Smad3 to inhibit cell proliferation and promote apoptosis. Interestingly, mutation of the Smad3 linker phosphorylation sites also increases the ability of Smad3 to activate genes that promote metastasis. Accordingly, mutation of the Smad3 linker phosphorylation sites markedly inhibits tumorigenicity but promotes metastasis of breast cancer cell lines. Using human breast cancer tissue microarrays, we further show that Smad3 linker phosphorylation is progressively reduced along breast cancer progression. Taken together, our findings strongly suggest that Smad3 linker phosphorylation promotes tumorigenesis but inhibits metastasis. Our findings have important implications for cancer therapy.
Citation Format: Fang Liu, Wen Xie, Zhengxue Liu, Tanima Roy, Eunjin Bae, Lalage M. Wakefield, Seong-Jin Kim, Akira Ooshima, Michael Reiss, Isao Matsuura. Role of Smad3 linker phosphorylation in breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4940. doi:10.1158/1538-7445.AM2015-4940
Abstract 4951: Expression analysis of the LOT1 gene in head and neck cancer
Authors: EKIZOGLU ERATAK SEDA , Karaman Emin , BUYRU NUR A. 
Published: August 2015

Abstract
Head and neck cancer (HNC) which is the sixth most common type of cancer worlwide, comprise a diverse group of tumors including neoplasias of the paranasal sinuses, the oral cavity, the trachea, the pharynx and the larynx. The most well known environmental risk factors for developing HNC are tobacca, alcohol and betel nut use and sexually transmitted viral pathogens such as human papilloma viruses (HPV). In addition to environmental factors, genetic factors may play a key role in the development of HNC. Therofore, genetic abnormalities have been studied extensively.
The LOT1 (Lost-on-transformation 1) gene is localized on human chromosome 6q24-25, a chromosomal region that is frequently deleted in many types of human cancer including squamous cell carcinomas of the head and neck. The product of the LOT1 gene is a 463 amino acid protein which contains 7 C2H2-type zinc-finger motifs at the amino terminal, as well as proline and glutamine rich areas at the carboxyl terminal. Functional analysis of LOT1 demonstrated that it may play a significant role as a transcription factor modulating growth suppression through mitogenic signaling pathways.
To elucidate the role of the LOT1 gene in tumorigenesis, many studies analyzing alterations of the gene expression were performed. As a result of these studies, the absence or reduced expression of LOT1 gene was reported in many types of tumors including breast, ovarian, pituitary and lung tumors.
In this study, we analyzed the role of the LOT1 gene in head and neck carcinogenesis. For this purpose, we investigated the expression level of LOT1 gene in tumor tissue and adjacent non-cancerous tissue samples from 81 patients with HNC by quantitative RT-PCR.
We found that LOT1 gene expression was decreased in 64 of 81 tumor tissues (79%) when compared to normal tissue. Statistically, downregulation of the LOT1 gene mRNA expression was significant (p&lt;0.001). No significant correlation was found between LOT1 gene expression and clinicopathological parameters including age, sex, stage, histological grade and tumor location (p&gt;0.05).
Our data indicate that loss of the LOT1 gene function may play a role in the etiology of head and neck carcinogenesis.
Citation Format: SEDA EKIZOGLU ERATAK, Emin Karaman, NUR A. BUYRU. Expression analysis of the LOT1 gene in head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4951. doi:10.1158/1538-7445.AM2015-4951