bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 476: Loss of chd5-mediated tumor suppression accelerates MYCN-driven neuroblastoma tumorigenesis in zebrafish
Authors: Zimmerman Mark W. , He Shuning , Shin Jimann , Zhu Shizhen , Mansour Marc , Joung Keith , Quan Jinhua , Yusufzai Timur , Look A. Thomas
Published: August 2015

Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system (PSNS) that originates from cells of the primitive neural crest. For the 40% of patients with high-risk disease, current therapies are often ineffective and long-term survival remains obstinately low. A major risk factor in neuroblastoma is hemizygous loss of the 1p36 chromosomal region, which has long been suspected to harbor one or more powerful tumor suppressor genes. Our studies indicate that loss of Chromodomain helicase DNA-binding protein 5 (chd5), which is in this deleted region, cooperates with MYCN overexpression to accelerate in vivo neuroblastoma tumorigenesis. Zebrafish chd5-null alleles were created utilizing both zinc-finger nuclease and TALEN technology. The resulting chd5 mutant fish exhibit abnormal development of the PSNS in the form of expansion of the superior cervical ganglia and enlargement of the interrenal gland (adrenal medulla). In order to examine the effect of chd5 haploinsuficiency on in vivo neuroblastoma tumorigenesis, chd5 mutant fish were crossed with the dbh:MYCN transgenic model resulting in neuroblastoma tumors. Consistent with a tumor suppressor function, chd5 haploinsufficient fish exhibit an accelerated neuroblastoma phenotype with tumors present beginning as early as 6 weeks compared to 15 weeks observed in wildtype fish. The chd5 protein can serve as one of two enzymatic components of the nucleosome remodeling and histone deacetylase (NuRD) complex, which is a repressor of gene expression and is reported to have diverse roles in regulating chromatin organization, developmental signaling and gene stability. Future studies will examine the mechanism and function of chd5 so that the pathways mediating tumor suppression can be elucidated and that essential proteins in these pathways can be targeted in ways that exploit the synthetic lethal relationships that are established.

Citation Format: Mark W. Zimmerman, Shuning He, Jimann Shin, Shizhen Zhu, Marc Mansour, Keith Joung, Jinhua Quan, Timur Yusufzai, A. Thomas Look. Loss of chd5-mediated tumor suppression accelerates MYCN-driven neuroblastoma tumorigenesis in zebrafish. [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 476. doi:10.1158/1538-7445.AM2015-476
Abstract 4765: A drill-down approach to identifying an ER negative-specific gene signature
Authors: Worsham Maria J. , Chitale Dhananjay , Chen Kang Mei , Datta Indrani , Stephen Josena K. , Divine George
Published: August 2015

There has been relatively little advancement in changing the management of women with ER negative (ER-) breast cancer (BC). Our objective is to examine the contribution of aberrantly methylated genes to the pathogenesis of ER- BC, determine their utility in refining classification of ER- subtypes [basal-like (BL) and Her2-enriched], in differentiating ER- BC in African American (AA) and Caucasian American (CA) women, and as new prognostic and therapeutic targets for better management of ER- BC.

The methylation status of 40 ER- and 40 ER positive (ER+) breast cancer samples was assessed using the Illumina 450K. A 3-tiered approach was used to call out genes most differentially methylated between ER- and ER+. The top 70 genes were further downsized using the TaqMan low-density array expression assays, Ingenuity Pathway Analysis (IPA) to assess their biologic significance, followed by targeted sequencing (Illumina Miseq). Further assessment of the genes in the 450K TCGA data sets (UCSC Brower 2015) was used for final downsizing.

Of the 70 highly ranked differentially methylated genes, 56 were hypermethylated and 14 hypomethylated, with positive correlation with gene expression for the majority of genes. Expression verification and significant rankings in canonical pathways and bionetworks resulted in further downsizing to 41 genes upon confirmation by targeted sequencing. Further filtering of the 41 genes with 450K TCGA data sets provided additional refinement to a 10 gene candidate panel currently being vetted in a retrospective cohort of over 1000 ER negative BC (with 40% AA).

Our iterative approach to identify an ER negative-specific gene signature has the potential to offer predictive and prognostic markers of improved classification for ER- BC in AA and CA women. Confirmation and validation of these methylation profiles in independent cohorts of BL BC defined by cDNA microarrays should further stratify poor prognosis breast cancers subgroups and serve as targets for epigenetic-modifying agents individualized to AA and CA women with ER- BC. Human therapeutic intervention trials to reverse deleterious epigenetic changes have shown promise in halting tumor growth by reactivation of the tumor suppressor genes or by reversing DNA hypomethylation.

Support: Komen Foundation: KG110218

Citation Format: Maria J. Worsham, Dhananjay Chitale, Kang Mei Chen, Indrani Datta, Josena K. Stephen, George Divine. A drill-down approach to identifying an ER negative-specific gene signature. [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 4765. doi:10.1158/1538-7445.AM2015-4765
Abstract 4771: Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast cancer
Authors: Kong Hyun Kyung , Park Sae Jeong , Kim Ye Sol , Park Jong Hoon
Published: August 2015

LY6K is associated with development of the immune response and carcinogenesis. Elevated LY6K by AP-1 activation induces invasion and metastasis by activating the ERK signaling pathway. However, the exact epigenetic mechanism of LY6K gene expression has not been clarified. The CpG island and 5′CGI shore in the region around the transcription start site of the LY6K were predicted using the UCSC genome browser and Methprimer software to elucidate the epigenetic mechanism of LY6K gene regulation and expression. We performed the methylation-specific PCR, bisulfite pyro-sequencing, and bisulfite sequencing to investigate the DNA methylation status of CpG sites from −400 to +500 on the LY6K CpG island and 5′CGI shore. ChIP and ChIP-MSP analysis were performed to confirm the histone modification and DNA methylation status. To validate the inverse correlation between LY6K 5′CGI shore methylation and LY6K expression in vivo, we performed bisulfite pyro-sequencing and tissue microarray using breast carcinoma samples. LY6K expression was inversely correlated with methylation status of CGI and 5′CGI shore in the LY6K of human breast cancer. Moreover, DNA methylation status of LY6K was correlated with histone modification. Treatment of 5-Aza-dC with low LY6K expression caused elevated LY6K expression leading to wound healing. An understanding of epigenetic changes in LY6K may contribute to diagnosing carcinogenic risk and to predict the outcome of patients with breast cancer.

Citation Format: Hyun Kyung Kong, Sae Jeong Park, Ye Sol Kim, Jong Hoon Park. Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast 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 4771. doi:10.1158/1538-7445.AM2015-4771
Abstract 4785: Deficiency of cystathionine beta synthase gene does not affect tumor formation and survival of Eμ-myc mice
Authors: Gupta Sapna , Kruger Warren D.
Published: August 2015

Aberrant DNA methylation is a hallmark of many types of cancers. S-adenosylhomocysteine (AdoHcy) is a strong inhibitor of DNA methytransferases. Deficiency of cystathionine beta synthase gene (Cbs) causes elevation of AdoHcy in variety of tissues. Here we have generated an Eμ-myc mouse model which is either wild type, heterozygous or homozygous for a deletion allele of Cbs (Eμ myc+ Cbs+/+; Eμ myc+ Cbs+/−; Eμ myc+ Cbs−/−). Homozygous Cbs deficient mice in both Eμ myc+ and Eμ myc− background were born at Mendelian frequency indicating that there is no synthetic lethality between Cbs and Eμ myc. In aging, we observed no significant difference in survival of Eμ myc+ Cbs+/+, Eμ myc+ Cbs+/− and Eμ myc+ Cbs−/− mice (Median survival of 150, 149 and 117 days respectively). However, preliminary data shows that tumor tissue from Eμ myc+ Cbs−/− animals has greatly elevated levels of AdoHcy compared to other genotypes. Our studies suggest that elevated levels of AdoHcy has no effect on tumor formation in Eμ myc mice.

Citation Format: Sapna Gupta, Warren D. Kruger. Deficiency of cystathionine beta synthase gene does not affect tumor formation and survival of Eμ-myc mice. [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 4785. doi:10.1158/1538-7445.AM2015-4785
Abstract 4822: Lack of expression of FOXO3 correlates with poor prognosis on uterine sarcomas
Authors: Almeida Thais G. , Garcia Natalia , Cunha Isabela W. , Baiocchi Glauco , Soares Fernando A. , Maciel Gustavo A. , Baracat Edmund C. , Carvalho Katia C.
Published: August 2015

Background: Uterine sarcomas are rare mesodermic tumors that represent approximately 3% of all uterine cancers. They display histological diversity and aggressive behavior, with early dissemination and high mortality when compared with epithelial tumors. Due to their diversity and rarity, there is no consensus related to risk factors for poor prognostic and appropriated treatment. The knowledge of the gene expression regulation can contribute to a better understanding of these neoplasms. Recently it was demonstrated an essential role for the forkhead transcription factor FOXO3 in the regulation of diverse cellular functions and tumors development. Our aim was to study the expression profile of the transcription factor FOXO3 in several histological types of uterine sarcoma. Method: we evaluated 100 samples of uterine sarcomas (including leiomyosarcomas, carcinosarcomas, adenosarcomas and endometrial stromal sarcomas), leiomyomas and myometrium obtained from patients during the period of 2000 to 2012. FOXO3 protein expression was assessed by immunohistochemistry. mRNA expression was evaluated by quantitative Real Time PCR. The results were submitted to statistical analyses together with patient's data. Results: We observed that carcinosarcomas (CSS) and adenosarcomas (ADS) showed the highest FOXO3 protein signal, considering exclusively the mesenchymal component. Concerning to muscular tumors (leiomyoma and leiomyosarcoma), we found an increasing enhancement of this protein expression from the myometrium to myoma and leiomiosarcoma, both in protein and transcriptional levels. Statistical analysis showed that tumors with weak or lack of FOXO3 protein present poor prognosis, regardless of their histological type. Conclusion: FOXO3 protein expression seems to be a relevant role in uterine sarcomas, however further analyses are needed.

Citation Format: Thais G. Almeida, Natalia Garcia, Isabela W. Cunha, Glauco Baiocchi, Fernando A. Soares, Gustavo A. Maciel, Edmund C. Baracat, Katia C. Carvalho. Lack of expression of FOXO3 correlates with poor prognosis on uterine sarcomas. [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 4822. doi:10.1158/1538-7445.AM2015-4822
Abstract 4823: Serum level of urokinase plasminogen activator receptor is suggested as diagnostic and prognostic biomarker in prostate cancer
Authors: Wach Sven , Al-Janabi Omar , Weigelt Katrin , Fischer Kersten , Greither Thomas , Marcou Marios , Theil Gerit , Nolte Elke , Holzhausen Hans-Juergen , Stoehr Robert , Huppert Verena , Hartmann Arndt , Fornara Paolo , Wullich Bernd , Taubert Helge W.
Published: August 2015

Background: This study aimed to assess the applicability of the soluble urokinase plasminogen activator receptor (suPAR) protein as a diagnostic and/or prognostic biomarker for prostate cancer (PCa) patients.

Material and Methods: SuPAR levels were evaluated by ELISA in serum samples from 146 PCa patients, 35 benign prostate hyperplasia (BPH) patients and 18 healthy controls.

Results: Antigen levels of suPAR differed between healthy controls and PCa or BPH patients (P<0.001). Additionally, suPAR levels differed between the Gleason sum groups GS = 7 vs. GS>7, with higher levels in the latter group (P = 0.011). For further statistical analysis, patients and proband cohorts were separated according to their serum expression of suPAR in a group with low/intermediate level (≤66% percentile) and a group with high levels (>66% percentile). A significant difference in the disease-specific survival was detected between the low/intermediate and the high suPAR level groups (191.4 vs. 144.5 months; P = 0.039; log rank test). The high level group showed a 3.91-fold increased risk of tumor-related death compared to the low/intermediate suPAR level group but this was not significant (P = 0.055; univariate Cox's regression hazard analysis). Therefore, future studies with larger cohorts of PCa patients as well as prospective surveys are necessary.

Conclusion: The differences between the PCa/BPH/healthy control cohorts for suPAR in conjunction with the association of high suPAR antigen levels in the serum with a poor prognosis suggest a diagnostic and prognostic impact of this biomarker for PCa patients.

Citation Format: Sven Wach, Omar Al-Janabi, Katrin Weigelt, Kersten Fischer, Thomas Greither, Marios Marcou, Gerit Theil, Elke Nolte, Hans-Juergen Holzhausen, Robert Stoehr, Verena Huppert, Arndt Hartmann, Paolo Fornara, Bernd Wullich, Helge W. Taubert. Serum level of urokinase plasminogen activator receptor is suggested as diagnostic and prognostic biomarker in prostate 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 4823. doi:10.1158/1538-7445.AM2015-4823
Abstract 4824: Pancreatic adenocarcinoma: Comparative analysis of cancer stem cell markers between neoadjuvant and treatment naïve specimens
Authors: Horsley Whitney , Stanley Melissa , O'Neil Bert , Shahda Safi , Sandusky George
Published: August 2015

To better understand the effect of neoadjuvant therapy on the presence of stem cell within pancreatic adenocarcinoma, CD24 and ALDH1 expression levels were compared between surgical specimens that received neoadjuvant therapy and those that did not. ALDH1+ and CD24+ cells have been implicated as markers for cancer stem cells in various cancers. For this study, paraffin-embedded tissues slides were obtain from Indiana University Simon Cancer Center Tissue Procurement and Indiana University Health Pathology Laboratory under approved IRB protocols. The tissues were then stained with immunohistochemical stains for CD24 and ALDH1, scanned using Aperio Imaging System and analyzed using the positive-pixel algorithm in ImageScope giving a percentage of tumor tissue expressing each marker. The results showed that the neoadjuvant tissues (avg. 42.1%) expressed a higher level of ALDH1 (p<0.001) than the treatment naïve tissue (avg. 24.9%); and that the treatment naïve tissues (avg. 30.1%) expressed a higher level of CD24 (p = 0.01) than the neoadjuvant tissue (avg. 17.9%). This indicates that neoadjuvant therapy may be effectively eliminating non-stem cells but not as effective at targeting stem cells expressing ALDH1, though somewhat effective at targeting CD24+ cells.

Citation Format: Whitney Horsley, Melissa Stanley, Bert O'Neil, Safi Shahda, George Sandusky. Pancreatic adenocarcinoma: Comparative analysis of cancer stem cell markers between neoadjuvant and treatment naïve specimens. [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 4824. doi:10.1158/1538-7445.AM2015-4824
Abstract 4825: Association between genetic profiles and histological classifications of 18 Japanese pseudomyxoma peritonei
Authors: Noguchi Rei , Yano Hideaki , Gohda Yoshimasa , Suda Ryuichiro , Igari Toru , Ohta Yasunori , Yamashita Naohide , Yamaguchi Kiyoshi , Terakado Yumi , Ikenoue Tsuneo , Furukawa Yoichi
Published: August 2015

Pseudomyxoma peritonei (PMP) is a rare disease occurring in 1-2 / 1000,000 and shows a distinct clinical feature caused by cancerous cells that produce mucin or gelatinous fluid in the abdominal cavity. Tumor cells are the most frequently originated from appendiceal mucinous cancer, and less frequently from mucinous tumors of the ovary. PMP is histologically classified into two types; disseminated peritoneal adenomucinosis (DPAM) showing milder phenotype, and peritoneal mucinous adenocarcinomas (PMCA) exhibiting more aggressive phenotype. Several molecular studies have shown frequent mutations in the KRAS and GNAS genes. However molecular profiles associated with different histological and/or clinical characteristics remain largely unknown. To elucidate molecular mechanisms underlying PMP, we analyzed 18 Japanese PMP tumors comprising of 10 DPAMs and 8 PMCAs. DNA was extracted from tumor tissues and their matched normal samples, and was subjected to amplicon sequencing using Ion AmpliSeq Cancer Panel v2 (Life Technologies) containing 49 cancer-related genes. In the 18 tumors, we identified a total of 42 somatic mutations (34 missense, 1 deletion, 1 insertion, 2 intron, 1 splice site mutations, and 3 synonymous mutations) in 13 genes. Of the 42 mutations, 35 were judged as pathogenic, and the remaining seven as non-pathogenic by the search of public databases and reports, or by in silico analysis. KRAS and GNAS mutations were found in 14 (77.8%) and 8 (44.4%) tumors, respectively. Interestingly, no mutations were detected in APC or CTNNB1. Notably, TP53 mutation was found in 3 of the 8 PMCAs, but not in the 10 DPAMs. Two of the 8 PMCAs carried mutation in either PIK3CA or AKT1. These data suggest that KRAS and/or GNAS mutations play a vital role in the development of both types of PMP, and that inactivation of Wnt signaling pathway may not be involved in the tumorigenesis. Furthermore, mutations in TP53 and/or genes related to PIK3CA-AKT pathway may be associated with malignant properties of PMCA. Further studies will facilitate the elucidation of molecular mechanism underlying PMP, and contribute to the development of effective therapeutic and preventive strategies to this disease.

Citation Format: Rei Noguchi, Hideaki Yano, Yoshimasa Gohda, Ryuichiro Suda, Toru Igari, Yasunori Ohta, Naohide Yamashita, Kiyoshi Yamaguchi, Yumi Terakado, Tsuneo Ikenoue, Yoichi Furukawa. Association between genetic profiles and histological classifications of 18 Japanese pseudomyxoma peritonei. [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 4825. doi:10.1158/1538-7445.AM2015-4825
Abstract 4832: Independent prognostic role of chromosome 12p deletion in prostate cancer
Authors: Minner Sarah Jane Pauline , Ahray Ramin , Kluth Martina , Schlomm Thorsten , Simon Ronald , Sauter Guido , Tsourlakis Maria Christina
Published: August 2015

Deletion of chromosome 12p is a recurrent alteration in prostate cancer, but the prevalence and clinical significance of this alteration is unclear. More than 7,000 prostate cancers with clinical follow-up data in a tissue microarray (TMA) format were analyzed by dual labeling fluorescence in situ hybridization (FISH) using probes for 12p13 (CDKN1B; p27) and for centromere 12 as a reference. CDKN1B was selected as a probe because this gene is located in the center of the deletion, which spans >10 Mb and includes >50 genes in 80% of cancers with 12p deletion. In 13.7% of prostate cancers a deletion of 12p was found (13.5% hemizygeous; 0.2% homozygeous deletions). 12p deletions were linked to advanced tumor stage (p<0.0001), high Gleason grade (p<0.0001), lymph node metastasis (p = 0.0004), rapid tumor cell proliferation (p<0.0001), and early biochemical recurrence (BCR) (p = 0.0027). Multivariate analysis including pT stage (p<0.0001), Gleason grade (p<0.0001), pN status (p = 0.0001), preoperative PSA levels (p = 0.0001), and resection margin status (p = 0.0001) revealed an independent prognostic value of 12p deletion (p = 0.0014). There was no association with ERG fusion status. Deletion of 12p deletion was only minimally linked to p27 expression status, which by itself was unrelated to clinical outcome. This argues against p27 as the key target gene of 12p deletions. Since none of the involved 12p genes is recurrently mutated in next generation sequencing studies, simultaneous dosage reduction of multiple genes may exert a relevant biological effect in 12p deleted cancers. In summary, the results of our study demonstrate that 12p deletion is frequent and provides independent prognostic information in prostate cancer.

Citation Format: Sarah Jane Pauline Minner, Ramin Ahray, Martina Kluth, Thorsten Schlomm, Ronald Simon, Guido Sauter, Maria Christina Tsourlakis. Independent prognostic role of chromosome 12p deletion in prostate 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 4832. doi:10.1158/1538-7445.AM2015-4832
Abstract 4846: Automated RNA and DNA purification from FFPE samples
Authors: Mandrekar Michelle , Horejsh Douglas , Lewis Samantha , Moreland Chris , Urh Marjeta
Published: August 2015

Formalin-fixed, paraffin-embedded (FFPE) samples are commonly used for archiving pathology samples for use by many researchers including clinical research labs. These samples provide a valuable tool for retrospective studies of diseases such as cancer. Traditional methods for the purification of DNA or RNA from FFPE tissue samples are often labor intensive, include the use of hazardous organic reagents, and involve difficult pre-processing steps. Here, we describe development of automated methods for the purification of RNA or DNA from FFPE tissue sections using the Maxwell® RSC Instrument, which can process between 1 and 16 samples. DNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human colon. RNA was purified from FFPE mouse brain, liver, lung, skin and spleen as well as FFPE human breast tissue. Eluates were tested for inhibition in qPCR or RT-qPCR. A comparison with another automated method was performed with FFPE mouse spleen and skin and a human tissue. The Maxwell RSC methods produced high quality amplifiable DNA or RNA from a variety of sample types with little or no inhibition. In addition, these methods simplified pre-processing, minimized hands-on time, and did not use hazardous organics.

Citation Format: Michelle Mandrekar, Douglas Horejsh, Samantha Lewis, Chris Moreland, Marjeta Urh. Automated RNA and DNA purification from FFPE samples. [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 4846. doi:10.1158/1538-7445.AM2015-4846
Abstract 4855: DBGC: database of gastric cancer
Authors: Yu Yingyan
Published: August 2015

DBGC is a comprehensive database, integrating various gastric cancer related data resources. In this database, gastric cancer related transcriptomics projects, proteomics projects, mutations, biomarkers and drug sensitive genes from different sources were collected and unified. Besides, follow-up information and clinicopathological information of gastric cancer patients in Shanghai, China, were also integrated through ID-mapping. We believe that this database will greatly facilitate the research work of gastric cancer researchers.

Database URL: http://bminfor.tongji.edu.cn/dbgc/index.do

Note: This abstract was not presented at the meeting.

Citation Format: Yingyan Yu. DBGC: database of gastric 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 4855. doi:10.1158/1538-7445.AM2015-4855
Abstract 4856: ITD Assembler: An algorithm for internal tandem duplication discovery from short-read sequencing data
Authors: Hampton Oliver A. , Rustagi Navin , Li Jie , Xi Liu , Gibbs Richard A. , Plon Sharon E. , Kimmel Marek , Wheeler David A.
Published: August 2015

Tandem duplication of coding sequence is an important mechanism of somatic mutation capable of activating oncogenes such as fms-related tyrosine kinase 3 (FLT3) as well as the potential for underlying constitutional or germline disease. Detection of internal tandem duplication (ITD) in short-read sequencing data remains challenging due to deficiencies in short-read alignment where reads carrying signatures of ITD are often unmapped. Assembly based approaches collapse repetitive sequences, resulting in reduced or lost genomic complexity. The majority of structural variation detection tools are limited to finding insertions that are contained entirely within individual read alignments, and are unable to detect read-spanning duplications. Tools designed to detect large structural variation from paired-end reads that map discordantly based on expected fragment size and orientation do not provide the precision needed to detect ITDs. Only tools that consider the characteristic alignment features that signal the presence of ITDs will be able to detect them; these alignment features include: an excess of reads with unaligned, soft-clipped bases, read pairs where one end is unmapped located near the site of insertion, and read pairs where both ends are unmapped.

We introduce ITD Assembler, a novel approach that searches all unmapped reads from exome capture or whole genome sequencing data to identify duplications. From the entire set of unmapped reads plus subset of mapped reads harboring soft-clipped bases, de Bruijn graph assembly is applied to select reads that form cycles, indicative of duplicated sequence structures. Reads from de Bruijn graph cycles are then assembled using an Overlap Layout Consensus (OLC) methodology thereby alleviating the collapse of repeat sequences affecting de Bruijn graph assembly approaches. Resulting OLC assembled contigs are locally aligned to the reference sequence to annotate the position of ITDs.

ITD Assembler was run on The Cancer Genome Atlas (TCGA) acute myeloid leukemia (AML) dataset, and FLT3-ITD detection rates were compared against orthogonal algorithms. ITD Assembler identified the highest percentage of TCGA FLT3-ITDs, reported significantly higher allele fractions, and discovered additional ITDs in the KIT, CEBPA and WT1 cancer genes.

Citation Format: Oliver A. Hampton, Navin Rustagi, Jie Li, Liu Xi, Richard A. Gibbs, Sharon E. Plon, Marek Kimmel, David A. Wheeler. ITD Assembler: An algorithm for internal tandem duplication discovery from short-read sequencing data. [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 4856. doi:10.1158/1538-7445.AM2015-4856
Abstract 4875: HIVE Proteomics: Integrated, cloud-based RNA-Seq and proteomics analysis of prostate adenocarcinoma samples
Authors: Dingerdissen Hayley , Mazumder Raja
Published: August 2015

Automated bottom-up proteomics workflows implemented with modern mass-spectrometry instrumentation can readily generate millions of peptide fragmentation spectra from cell-lines and clinically derived samples. The tandem mass-spectra promise to reveal wild-type and somatic mutations, insertions, and deletions and alternatively spliced isoforms translated to functional protein isoforms in tumor samples. However, the available reference proteomes represent a poor analysis substrate for the observation of protein evidence of genomic and transcript variation. The advent of cheap and fast RNA sequencing (RNA-Seq) provides an elegant solution to the lack of sample-specific reference proteomes. We anticipate that the falling cost of RNA-Seq will prompt an increasing number of proteomics labs to use contract next-gen sequencing (NGS) services to obtain RNA-Seq data to derive sample-specific reference proteomes. In contrast to public repositories of genomic variation, sample-specific RNA-Seq data captures transcribed rare, individual, cell-type, and sample-specific genomic variation. RNA-Seq-based transcripts also provide sample-specific information on observable proteins. Furthermore, paired RNA-Seq and proteomics data links gene expression and protein abundance, enabling the study of gene regulation linked to protein abundance dynamics. However, the analysis of multi-gigabyte paired mass-spectra and RNA-Seq datasets pose significant scientific and logistical challenges. Few proteomics labs have the personnel, archival data storage, computational resources, or informatics pipelines needed. The cloud-based genomics analysis platform High-performance Integrated Virtual Environment (HIVE) will provide turn-key integrated proteomics and RNA-Seq analyses to the wider proteomics community in a secure, trackable, sharable, and scalable computing platform. We will then use this integrated proteomics_RNA-Seq analysis pipeline to identify high-value mutations in prostate adenocarcinoma samples, thereby demonstrating the utility of the platform while also generating key data for future investigations.

Citation Format: Hayley Dingerdissen, Raja Mazumder. HIVE Proteomics: Integrated, cloud-based RNA-Seq and proteomics analysis of prostate adenocarcinoma samples. [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 4875. doi:10.1158/1538-7445.AM2015-4875
Abstract 4882: Megabase-scale phased haplotypes of genetic aberrations from whole cancer genome sequencing of primary colorectal tumors
Authors: Lau Billy , Bell John M. , Schnall-Levin Michael , Jarosz Mirna , Hopmans Erik , Wood Christina M. , Zheng Grace X. , Giorda Kristina , Ji Hanlee P.
Published: August 2015

Cancer genomes contain multiple types of genetic aberrations that include mutations, deletions, copy number variants and chromosomal rearrangements. Despite advances in next generation sequencing, it remains a major challenge to delineate many of these somatic genomic alterations because of intrinsic complexity of cancer genomes. Haplotyping involves the assignment of genetic variants such as mutations and structural variants to specific segment of homologous chromosomes. Experimentally determined phasing of cancer genomes offers an opportunity to resolve complex genomic structures such as somatic rearrangements, aneuploidy composition and ongoing evolutionary changes. However, contiguous phasing of cancer genomes on a megabase (Mb) scale remains difficult to achieve with sequencing-based approaches.

In this proof-of-concept study, we experimentally determined Mb-scale haplotypes of primary tumor samples via whole genome sequencing. To generate haplotypes, we employed an automated instrument that partitions long DNA fragments into hundreds of thousands of reactions, each of which incorporates a unique, nonrandom barcode into indexed sequencing libraries. Given need to amplify from sparse numbers of molecules and the high efficiency of the automated sequencing library construction process, the DNA requirements for each sample are less than 5 ng.

We sequenced the genomes of primary colorectal cancer samples and their matched normal diploid DNA with an Illumina sequencer. We used the single nucleotide variants to generate Mb-scale haplotype blocks (N50 of 1.2 Mb) with phased haplotype block size of up to 11.3 Mb. We were able to delineate cancer genome haplotypes that cover allelic imbalances, copy number variations such as deletions and other genomic instability events. Structural variants were identified in the context of their position in specific chromosome homologues. Thus, we improved the characterization of somatic genetic aberrations using contiguity mapping and cancer genome haplotypes in the context of whole cancer genome sequencing. Overall, we demonstrated the feasibility and potential utility of conducting contiguous phased haplotypes in whole cancer genome sequencing from primary tumor samples.

Citation Format: Billy Lau, John M. Bell, Michael Schnall-Levin, Mirna Jarosz, Erik Hopmans, Christina M. Wood, Grace X. Zheng, Kristina Giorda, Hanlee P. Ji. Megabase-scale phased haplotypes of genetic aberrations from whole cancer genome sequencing of primary colorectal tumors. [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 4882. doi:10.1158/1538-7445.AM2015-4882
Abstract 4888: Clinical sequencing in the National Institute of Health's Clinical Sequencing Exploratory Research (CSER) Program
Authors: Crawford Catherine , Caga-Anan Charlisse , Kaufman Dave , Lee Alexander , Mcewen Jean , Schully Sheri , Hutter Carolyn , Hindorff Lucia
Published: August 2015

To evaluate best uses of and support the integration of genomic sequencing into the clinical workflow, the National Human Genome Research Institute and National Cancer Institute co-fund the Clinical Sequencing Exploratory Research (CSER) project. This initiative aims to 1) leverage NHGRI's experience in genomic sequencing and analysis to facilitate adoption of these methods into patient care, 2) develop and disseminate best practices for the integration of genomic sequencing into clinical care, and 3) research the ethical, legal, and psychosocial (ELSI) implications of bringing broad genomic data into the clinic. The CSER Consortium includes nine multi-disciplinary projects, nine ELSI-specific projects, a Coordinating Center and NHGRI's Intramural ClinSeq program. Four of the multi-disciplinary projects and one ELSI project have a specific focus on cancer settings and outcomes: CanSeq (Dana-Farber Cancer Inst.), BASIC3 (Baylor College of Med.), ONCOSEQ (Univ. of Michigan), and NEXT Medicine (Univ. of Washington) are developing robust frameworks for the integration of whole exome sequencing (WXS) in tumor and germline samples. By measuring the effectiveness of novel platforms for returning results, analyzing the costs and benefits of using sequencing technology to identify rare variants, and evaluating patient experiences, these projects will help define best practices for incorporating WXS into cancer management. As of August 2014, these projects had collected WXS data from 415 patients (269 adults and 146 children). The CSER consortium also has nine working groups that strive to advance best practices and communicate lessons learned to the community regarding the use of genomic sequence data in the clinical workflow. The Tumor Working Group explores the unique technical, interpretive, and ethical challenges involved in clinical sequencing of both somatic and germline cancer genomes. This presentation will highlight progress to date, anticipated products and results, and the relationship between these cancer sequencing projects and broader CSER consortium efforts.

Citation Format: Catherine Crawford, Charlisse Caga-Anan, Dave Kaufman, Alexander Lee, Jean Mcewen, Sheri Schully, Carolyn Hutter, Lucia Hindorff. Clinical sequencing in the National Institute of Health's Clinical Sequencing Exploratory Research (CSER) Program. [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 4888. doi:10.1158/1538-7445.AM2015-4888
Abstract 4893: High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries
Authors: Wang Jun , Li Guoli , Chen Ming , Heng Jianfu , Guo Xinwu , Peng Limin , Zhang Fan , Li Zibo , Wang Shouman , Xiao Zhi , Dai Lizhong , Yi Wenjun , Tang Lili
Published: August 2015

Breast cancer is one of the most common cancers and also the principal cause of cancer-related death among women in the world. BRCA1 and BRCA2 genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Mutations of BRCA1/2 genes greatly increase lifetime risk to develop breast cancer and these mutations are frequently observed in hereditary breast cancer. In addition, recent studies suggested that breast cancer patients who carry a BRCA1 or BRCA2 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, it would be of great interest to develop a high-throughput approach for mutation screening of the full exon regions in BRCA1 and BRCA2 gene.

We have developed a targeted gene exon-sequencing approach using a nanofluidic PCR platform, the Access Array™ system, which enables simultaneous amplification of 48 genetic regions (amplicons) from 48 samples in parallel. We have applied this system to generate the libraries of BRCA1 and BRCA2 coding regions. Total 120 target-specific primer pairs were designed covering all 22 exons of BRCA1 and 26 exons of BRCA2. A primer design strategy was applied to incorporate sample-specific barcodes and Illumina sequencing adaptors into each amplicon, allowing direct sequencing without further library preparation. The exon regions of these 2 genes have been amplified on Access Arrays using 48 multiplex mixes of the 120 primer sets in paired tumor/normal tissues from 144 breast cancer patients. We will present the next-generation sequencing data of BRCA1 and BRCA2 mutation screening collected from amplicon libraries generated using the Access Array system. Our results indicate the excellent utilization of this approach in the mutation screening of BRCA1 and BRCA2 in clinical samples.

Note: This abstract was not presented at the meeting.

Citation Format: Jun Wang, Guoli Li, Ming Chen, Jianfu Heng, Xinwu Guo, Limin Peng, Fan Zhang, Zibo Li, Shouman Wang, Zhi Xiao, Lizhong Dai, Wenjun Yi, Lili Tang. High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries. [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 4893. doi:10.1158/1538-7445.AM2015-4893
Abstract 4921: Next-generation sequencing as a potential tool in the diagnostics of APC mosaicism in FAP patient
Authors: Yamaguchi Kiyoshi , Komura Mitsuhiro , Yamaguchi Rui , Imoto Seiya , Shimizu Eigo , Kasuya Shinichi , Shibuya Tetsuo , Hatakeyama Seira , Takahashi Norihiko , Ikenoue Tsuneo , Hata Keisuke , Tsurita Giichiro , Shinozaki Masaru , Suzuki Yutaka , Sugano Sumio , Miyano Satoru , Furukawa Yoichi
Published: August 2015

Familial adenomatous polyposis (FAP) is a hereditary disease characterized by the development of multiple adenomatous polyps ranging from hundreds to thousands in the large intestine, and extra-colonic tumors. Patients are predisposed to colorectal cancer that accounts for ∼1% of newly diagnosed colorectal cancer cases. Most of FAP cases result from germline mutations in the tumor suppressor gene APC (adenomatous polyposis coli), but some cases are caused by germline mutations in MUTYH, POLD1, or POLE. The rate of mutation detection depends on the methods used for genetic testing and the genes analyzed in the patients. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing (NGS) has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic adenomatous polyposis patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. As a result, whole-genome sequencing and subsequent amplicon-based targeted sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in approximately 12% of his peripheral leucocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data suggest that genetic testing by NGS facilitates the identification of genetic mosaicism responsible for hereditary diseases. NGS will improve genetic diagnosis of hereditary diseases whose mutations have been overlooked by conventional direct sequencing.

Citation Format: Kiyoshi Yamaguchi, Mitsuhiro Komura, Rui Yamaguchi, Seiya Imoto, Eigo Shimizu, Shinichi Kasuya, Tetsuo Shibuya, Seira Hatakeyama, Norihiko Takahashi, Tsuneo Ikenoue, Keisuke Hata, Giichiro Tsurita, Masaru Shinozaki, Yutaka Suzuki, Sumio Sugano, Satoru Miyano, Yoichi Furukawa. Next-generation sequencing as a potential tool in the diagnostics of APC mosaicism in FAP patient. [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 4921. doi:10.1158/1538-7445.AM2015-4921
Abstract 4932: Development of a highly sensitive library prep method for identifying pathogenic variants from FFPE tumor samples
Authors: Young Amanda G. , Lin Charles
Published: August 2015

The application of genomics in cancer has led to an improved understanding of the disease. To date, 125 known driver genes have been discovered*. These genes and their pathways have become the focus of small-molecule drug development. While the number of available targeted therapies is limited, an estimated 800 oncology drugs are in development*, many of which target specific mutations. Single-analyte companion diagnostics exist for the approved therapies, and more might be developed as new therapeutics become available, but the need to assay each variant individually can be ineffective.. An additional, well-known challenge is the degradation and damage caused to DNA in formalin-fixed, paraffin-embedded (FFPE) samples. For these reasons, Illumina is developing a novel method for simultaneous detection of multiple somatic variants from FFPE specimens. This method leverages a multiplexed PCR approach with amplicons designed to a 15-gene panel that can detect somatic variants in 14 genes, and copy number changes in 3 genes. In our experiments, this multiplexed PCR assay has been demonstrated to be highly sensitive, yielding results from as little as 20 ng of DNA extracted from FFPE tissue, even if the DNA is highly damaged and degraded. Somatic variants can be detected down to 5% allele fraction, and gene amplifications can be detected for ≥ 2.2-fold change in copy number. This method, incorporating newly designed software, will be deployed as part of the Illumina Universal Oncology Test System on the MiSeqDx™ (under development), analyzing the sequencing results, and generating reports of mutations and copy number changes in cancer samples.

Citation Format: Amanda G. Young, Charles Lin. Development of a highly sensitive library prep method for identifying pathogenic variants from FFPE tumor samples. [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 4932. doi:10.1158/1538-7445.AM2015-4932
Abstract 4936: The interaction and effects of hypoxia, surrounding fibroblasts and p16 expression on breast cancer cell migration and invasion
Authors: Lu Yi , Zhang Jun
Published: August 2015

Cell migration and invasion play essential roles in the metastatic cascade of tumor cells. Accumulated evidences suggest that intratumoral hypoxia, a characteristic of fast-growing solid tumors, promotes cancer cell motile and invasive abilities. In this study, we investigated the effects and interaction of hypoxia, surrounding fibroblasts, and p16 expression on the migration and invasion of breast cancer (BCa) cells MDA-MB-231, JygMC(A) and LM2. We found that hypoxia promoted BCa cell migration and invasion, and cocultured MEF fibroblasts stimulated invasiveness of BCa cells. Moreover, by using a Tet-on inducible system, we found that p16 is capable of inhibiting hypoxia-induced BCa cell migration and invasion (e.g., 36.0% and 85.8% inhibition on migration and invasion, respectively, in MDA-MB-231 cells), and suppressing cocultured fibroblast-stimulated invasiveness of BCa cells. These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia-mediated cancer cell migration and invasion. This study may provide important validation for p16-mediated cancer therapy either by gene therapy or pharmacological activation of internal p16 gene that is usually inactive due to hypermethylation in the tumor cells.

Note: This abstract was not presented at the meeting.

Citation Format: Yi Lu, Jun Zhang. The interaction and effects of hypoxia, surrounding fibroblasts and p16 expression on breast cancer cell migration and invasion. [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 4936. doi:10.1158/1538-7445.AM2015-4936