bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4753: An effective detection method for the EGFR single mutation T790M using BNA-NC clamping real-time PCR
Authors: Kim Sung-Kun , Liu Xiaoyun , Castro Aaron , Loredo Leticia , Castro Miguel
Published: August 2015

Mutations on epidermal growth factor receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on tyrosine kinase domain of EGFR signifies the response to the popular cancer drug gefitinib, which leads to the development of resistance to gefitinib. Detecting the mutation thus guide effective therapeutical options for patients who are in need of cancer drug treatments. We sought to develop a rapid, reliable detection method for the T790M mutation using bridged nucleic acids (BNA), which has been known to enhance the hybridization affinity of oligonucleotides that contain BNA bases. Oligonucleotides containing BNA bases designed to block PCR reaction against wild-type genes, called BNA-clamp, were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNA-clamping allows us to view the different levels of PCR amplifications in the degree of mixture of wild-type and mutant genes. In an effort to explore this possibility, 13mer long clamps were prepared with various numbers of BNA bases. The clamps containing 9 BNA bases appear to be most effective in blocking the PCR reactions at an optimized concentration to distinguish the mutant from the wild-type genes. In addition to PCR results, the difference in the Tm values for the 7 BNA bases in the 13mer was the largest among differently designed clamps, which is consistent with the results obtained by real-time PCR. We also examined the degree of sensitivity using the clamp containing 9 BNA bases, revealing that the clamp has the ability to determine the level of mutation as low as 1% mixture of the mutant and wild-type gene. The effectiveness of blocking the PCR reaction with only 13mers containing BNA bases allowed us to detect a single mutation, and thus, this BNA-clamping real time PCR technology may offer a promising, new avenue to detect clinically important mutations in the future.

Citation Format: Sung-Kun Kim, Xiaoyun Liu, Aaron Castro, Leticia Loredo, Miguel Castro. An effective detection method for the EGFR single mutation T790M using BNA-NC clamping real-time 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 4753. doi:10.1158/1538-7445.AM2015-4753
Abstract 4755: Maximize genomic analysis through dual recovery of DNA and RNA from the same clinical sample
Authors: Cheng Angie , Hernandez Natalie , Li Mu , San Jose Hinahon Charmaine , Magdaleno Susan
Published: August 2015

DNA and RNA provide valuable information in order to detect differences such as chromosomal mutations and imbalances in gene expression. Common sample types for these analyses include surgical and needle biopsies, which are very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can raise issues of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in a different profile so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving small samples and data consistency eliminating any sample to sample variation. Here we describe the efficient isolation of RNA and DNA from the same starting sample without splitting using the RecoverAll™ Multi-Sample RNA/DNA workflow, followed by versatile and informative downstream analysis. This workflow was applied to FFPE tissue samples, fine needle aspirates, degraded samples as well as fresh cultured cells from a number of different cancer types. As an example, we have shown that DNA and RNA can be isolated from the same non-small cell lung adenocarcinoma FFPE tumor sample and with the recovered analytes, both mutation analysis and fusion transcript detection can be performed by either qPCR or next-generation sequencing (NGS).

Citation Format: Angie Cheng, Natalie Hernandez, Mu Li, Charmaine San Jose Hinahon, Susan Magdaleno. Maximize genomic analysis through dual recovery of DNA and RNA from the same clinical sample. [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 4755. doi:10.1158/1538-7445.AM2015-4755
Abstract 4758: Genomic profiling of prostate cancer in Korean population
Authors: Park Ha Young , Kim Jong-il , Park Weon Seo , Lee Kang Hyun
Published: August 2015

Prostate cancer is one of the most common cancers for male population in the world. The search for accurate biomarkers in prostate cancer is critical for evolution of accurate management of prostate cancer. Previous studies have identified that several genomic alterations in prostate cancer including TMPRSS2-ERG fusion, PTEN deletion and SPOP single nucleotide variants (SNVs), the majority of which are from western population. To characterize the somatic mutations of prostate cancer in Korean, we sequenced the whole exome of 49 prostate cancer patient tissues and matched normal tissues. A total of 738 somatic non-silent variants affecting 692 distinct genes were identified. In addition to already known genes (SPOP, FOXA1 and PTEN), we also found previously uncharacterized genes such as GRIN2A, LRP1, LRP1B and KRT1. Of the 40 mutated genes across multiple cases, missense SNVs of SPOP (5/49 cases, 10.2%) were the most frequent, followed by missense and splice-site SNVs of GRIN2A (4/49 cases, 8.2%). Mutations in low density lipoprotein-related protein encoded by LRP1 and LRP1B affected considerable portion of all cases (7/49, 14.3%), describe the probable relation between lipid metabolism and prostate cancer. As a results, we suggested the distinct patterns of genetic variations involved in prostate cancer of Korean population.

Note: This abstract was not presented at the meeting.

Citation Format: Ha Young Park, Jong-il Kim, Weon Seo Park, Kang Hyun Lee. Genomic profiling of prostate cancer in Korean population. [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 4758. doi:10.1158/1538-7445.AM2015-4758
Abstract 4760: Stromal contribution to the colorectal cancer transcriptome
Authors: Isella Claudio , Terrasi Andrea , Bellomo Sara E. , Petti Consalvo , Muratore Andrea , Mellano Alfredo , De Ridder Mark , Cassoni Paola , Storme Guy , Bertotti Andrea , Medico Enzo
Published: August 2015

Recent studies have identified a stem/serrated/mesenchymal (SSM) molecular subtype of colorectal cancer (CRC) that is associated with poor prognosis. We noted that genes upregulated in this subtype are also prominently expressed by stromal cells. This led us to hypothesize that the SSM transcripts could derive from the tumor microenvironment, rather than being intrinsic to cancer cells. To test this hypothesis, we analyzed microarray and RNAseq expression data from patient-derived xenografts (PDXs) of CRC, where human cancer cells are supported by murine stroma. Species-specific expression analysis revealed that mRNA levels of SSM genes are mostly due to stromal expression. Considering only genes exclusively expressed by stromal cells in PDXs, We then built three expression signatures specifically reporting the abundance of cancer-associated fibroblasts (CAFs), leucocytes or endothelial cells. In human CRC samples, all stromal signatures were strongly associated with the SSM subtype. A high CAF signature was associated with poor prognosis in untreated CRC patients, while in rectal cancer high stromal signatures jointly predicted radioresistance. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stroma.

Citation Format: Claudio Isella, Andrea Terrasi, Sara E. Bellomo, Consalvo Petti, Andrea Muratore, Alfredo Mellano, Mark De Ridder, Paola Cassoni, Guy Storme, Andrea Bertotti, Enzo Medico. Stromal contribution to the colorectal cancer transcriptome. [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 4760. doi:10.1158/1538-7445.AM2015-4760
Abstract 4770: Levels of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in hepatocellular carcinoma prognosis
Authors: Delgado-Cruzata Lissette , Wu Hui Chen , Thomas Tiffany , Siegel Abby B. , Shen Jing , Zhang Yu-Jing , Goyal Abhishek , Hsu Christine C. , Remotti Helen , Santella Regina M.
Published: August 2015

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world. The US incidence of HCC has tripled between 1975 and 2005 and has been accompanied by improved long term survival rates, but as a result of HCC ‘s poor prognosis, 1-year survival remains lower than fifty percent. Therefore, a better understanding of the molecular mechanisms underlying the disease process is currently needed. Epigenetic alterations have been linked to HCC and global genomic hypomethylation is a well known hallmark of the disease. Less is understood about the role hydroxymethylation plays in HCC carcinogenesis. Here, we conducted a study to determine 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels by UPLC/MS/MS in HCC tumor (T) and non-tumor (NT) adjacent tissue DNAs of sixty six individuals treated at Columbia Presbyterian Medical Center in New York. We analyzed the association between demographic and clinicopathological characteristics and 5mC and 5hmC levels, as well as performed a Cox-survival analysis to determine which factors better predicted survival. Mean levels of 5mC (5mCT = 3.15±0.49% vs.5mCNT = 3.80±0.12% (p<0.0001) and 5hmC (5hmCT = 0.11±0.04% vs. 5hmCNT = 0.23±0.07%, p<0.0001) in tumor tissues were statistically significantly lower than in non-tumor tissues. Older age was associated with lower 5mC content (5mC<60y = 3.32±0.43% vs. 5mC≥60y = 3.00±0.50%, p = 0.001), while tumor DNA of transplant (LT) patients had higher 5hmC levels compared to surgical resection (LR) patients (5hmCLR = 0.10±0.04% vs. 5hmCLT = 30.13±0.04%, p = 0.0053). However, survival was predicted by whether transplant or resection were performed (Hazard ratio LT vs. LR, 3.54 (95% confidence interval:1.34-9.29), p = 0.01). Our study had a limited sample size, but suggests that future studies of 5hmC might be informative concerning HCC prognosis.

Citation Format: Lissette Delgado-Cruzata, Hui Chen Wu, Tiffany Thomas, Abby B. Siegel, Jing Shen, Yu-Jing Zhang, Abhishek Goyal, Christine C. Hsu, Helen Remotti, Regina M. Santella. Levels of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in hepatocellular carcinoma prognosis. [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 4770. doi:10.1158/1538-7445.AM2015-4770
Abstract 4772: Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in nasopharyngeal carcinoma
Authors: PENG CHEN-CHING
Published: August 2015

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which helps explain such observation. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Our results reveal a novel mechanism wherein HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 may use as biomarkers for monitoring NPC treatment.

Citation Format: CHEN-CHING PENG. Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in nasopharyngeal carcinoma. [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 4772. doi:10.1158/1538-7445.AM2015-4772
Abstract 4797: Mutation signature and intratumor heterogeneity of esophageal squamous cell carcinoma in a Chinese cohort
Authors: Song Qingxuan , Liu Mengfei , Bai Jian , Abliz Amir , Yuan Wenqing , Liu Zhen , Liu Jingjing , Zeng Changhong , Cai Hong , Ke Yang , Li Jun
Published: August 2015

Esophageal squamous cell carcinoma (ESCC) is one of the most common and most aggressive cancers. The epidemiological features of ESCC are extremely complex, with remarkable geographic differentiation among world's populations. While rural Anyang in the Henan Province of China is a well-known high-incidence area, the causal factors in this population remain elusive. We performed exome sequencing of 81 tumor-normal pairs, identified TP53, PIK3CA and NOTCH1 as significantly mutated genes, and observed highly recurrent aberrations in several other genes previously reported for ESCC (ZNF750, MLL2, FAT1, FAT2, and FAT3). Our catalog of ∼7,000 single-nucleotide mutations revealed two main signatures: C>T transitions due to spontaneous deamination of 5-methyl-cytosine, and C>T and C>G mutations at TpCpN attributed to the APOBEC family of cytidine deaminases. Per-sample loadings of these two signatures are uncorrelated with the patient's smoking and drinking status. Since APOBEC activities are associated with exogenous viruses, the prominence of this signature suggests a role of HPV in ESCC etiology, consistent with our previous studies that detected HPV DNA in tumor samples and anti-HPV-E7 antibody in patient's blood. To characterize intratumoral heterogeneity we applied our newly developed method, CHAT, to summarize the clonal frequencies of copy number alternations and single nucleotide mutations in each tumor. Many tumors show a multi-modal distribution of the clonal frequencies, suggesting extensive within-tumor diversity. To better understand the patterns of growth, migration and metastatic potential among different cells within a tumor we performing exome sequencing to compare multiple samples in 10 ESCC patients. For each, we analyzed 4-6 sectors of the tumor, 2-4 samples of adjacent normal tissue, and 1-2 nearby lymph nodes. The spatial heterogeneity of molecular lesions within each tumor is expected to uncover major genes and pathways affected in each patient, as well as the temporal progression of tumorigenic events that may have driven the initiation and outgrowth of ESCC. By integrating the mutation signatures, introtumoral clonal heterogeneity, and clinical outcomes, we aim to gain a better understanding of the molecular bases and evolutionary path of this lethal disease.

Citation Format: Qingxuan Song, Mengfei Liu, Jian Bai, Amir Abliz, Wenqing Yuan, Zhen Liu, Jingjing Liu, Changhong Zeng, Hong Cai, Yang Ke, Jun Li. Mutation signature and intratumor heterogeneity of esophageal squamous cell carcinoma in a Chinese cohort. [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 4797. doi:10.1158/1538-7445.AM2015-4797
Abstract 48: Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells
Authors: Ohtsuka Tomoaki , Sakaguchi Masakiyo , Takata Katsuyoshi , Hashida Shinsuke , Watanabe Mototsugu , Suzawa Ken , Maki Yuho , Yamamoto Hiromasa , Soh Junichi , Asano Hiroaki , Tsukuda Kazunori , Miyoshi Shinichiro , Toyooka Shinichi
Published: August 2015

Human epidermal growth factor receptor 2 (HER2) is a member of epidermal growth factor receptor (EGFR) family. Previous studies have revealed that many kinds of malignant tumors have genetic mutations or amplification of HER2, indicating that HER2 alterations are oncogenic. Many kinds of HER2 targeted therapies are effective to HER2 positive tumors, but those treated tumors often get resistance to drugs. Thus, to elucidate HER2 related pathway in cancer biology is important to develop new therapeutic strategy for cancers.

Recently, we newly identified a protein X (a temporary name) as a novel binding protein to HER2 with immunoprecipitation and following LC-Ms/Ms analysis. The protein generally expressed in lung and breast cancers at remarkable level.

We constructed plasmid vectors carrying wild type HER2 and gene X. These vectors were simultaneously introduced to HEK293T cells to examine the binding ability of protein X and HER2 as well as the effect of gene X on HER2-mediated signal-transduction pathway. The approach clearly showed that the expression of gene X, resulted in phosphorylation of HER2 and subsequent activation of oncogenic effector molecules.

We next constructed several kinds of gene X-truncated variants and subjected to the binding assay to look for the binding domain of gene X to HER2. The analysis showed that N-terminal head domain of gene X was essential for the HER2 binding. This domain has an ability to induce HER2 phosphorylation and subsequent activation of the effector kinase, ERK.

In conclusion, we found that gene X is a novel binding protein to HER2 and has a role in HER2 activation.

Citation Format: Tomoaki Ohtsuka, Masakiyo Sakaguchi, Katsuyoshi Takata, Shinsuke Hashida, Mototsugu Watanabe, Ken Suzawa, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Shinichi Toyooka. Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells. [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 48. doi:10.1158/1538-7445.AM2015-48
Abstract 4803: BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma
Authors: Harismendy Olivier , Alakus Hakan , Yost Shawn , Woo Brian , French Randall , Lin Grace Y. , Jepsen Kristen , Frazer Kelly A. , Lowy Andrew M.
Published: August 2015

Malignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ∼10% cases. With only ∼300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown. We analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12). Peritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 non-silent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median 1.3 10-6). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a non-functional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level. Our findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention.

Citation Format: Olivier Harismendy, Hakan Alakus, Shawn Yost, Brian Woo, Randall French, Grace Y. Lin, Kristen Jepsen, Kelly A. Frazer, Andrew M. Lowy. BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma. [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 4803. doi:10.1158/1538-7445.AM2015-4803
Abstract 4860: Detection of genetically distinct subclones in cancer: a novel NGS algorithm with high spatial and genetic resolution
Authors: Karagiannis Konstantinos , Chumakov Konstantin , Simonyan Vahan , Mazumder Raja
Published: August 2015

Despite the large number of genome data banks, reference sequences are not always the best match for high-throughput analysis. This is especially true in studies of intratumor heterogeneity where increased mutation rates are observed; the use of a reference genome can complicate the interpretation of the results. Current techniques attempt to circumvent this obstacle by applying de novo assembly algorithms, but the process is slow and often without viable results. Here, we introduce a new technique to decrease the dependency of these pipelines on reference sequences. Our clone discovery algorithm takes advantage of existing alignment data and correlates distant mutations, reported by an alignment algorithm. The algorithm makes no statistical assumption; it extracts information from the overlapping alignments in a step-wise fashion, using reference coordinates of aligned reads. Due to the randomness of noise, the algorithm can be very sensitive and identify clones of low frequency.

Citation Format: Konstantinos Karagiannis, Konstantin Chumakov, Vahan Simonyan, Raja Mazumder. Detection of genetically distinct subclones in cancer: a novel NGS algorithm with high spatial and genetic resolution. [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 4860. doi:10.1158/1538-7445.AM2015-4860
Abstract 487: Whole genome screen to identify genes targeting MYCN-driven embryonal tumors
Authors: Thiele Carol J. , Liu Zhihui , Veschi Veronica , Buehler Eugene , Martin Scott
Published: August 2015

MYCN is a driver of neuroblastoma (NB) tumorigenesis and is over-expressed in a number of tumors of embryonal origin, including rhabdomyosarcoma, medulloblastoma and diffuse intrinsic pontine gliomas. We sought to identify regulators of MYCN transcription by performing a whole genome screen (WGS) for regulators of MYCN promoter activity using a NB cell model. A plasmid containing the MYCN promoter (1.3kb upstream of MYCN TSS) fused to luciferase and stably integrated into the genome of NGP NB cells was the readout system. NGP-MYCNpluc, was selected based on MYCN luciferase activity inhibition by ATRA and HDAC inhibitors to a similar extent as endogenous MYCN mRNA levels. After assay optimization, siRNAs targeting 11,000 genes using 3 siRNAs/gene were evaluated. The siRNA library encompassed the druggable genome and the majority of human transcription factors. Using a 384-well format, siRNAs were reverse transfected in duplicate into NGP-MYCNpluc cells, cultured for 3 days at 37oC and analyzed for luciferase activity and cell viability using a ONE-Glo and Cell Titer-Glo assays (Promega), respectively. A robust statistical measure of median absolute deviation (MAD) was used to standardize siRNA activities in the screen. This identified 36 “high-confidence” genes in which all 3 siRNAs significantly decreased NGP-MYCNpluc luciferase activity, and 49 genes which significantly decrease cell viability. Most drugs associated with the essential viability genes have shown activity in NB cells (bortizamab, gemcitabine/paclitaxel, erlotinib/gemcitabine, UCN-01 and BI 2536). A number of MYCN reporter hits also decreased a CMV luciferase promoter in HEK293 cells, suggesting these hits did not specifically regulate the MYCN promoter. However, several hits did not affect CMV reporter activity, suggesting specificity to the MYCN reporter system. Low-throughput secondary screens are being utilized for assay confirmation and mechanistic evaluation. A 23,000 unique gene set is currently under evaluation and more elegant informatics tools are being used to further illuminate actives within the data

Citation Format: Carol J. Thiele, Zhihui Liu, Veronica Veschi, Eugene Buehler, Scott Martin. Whole genome screen to identify genes targeting MYCN-driven embryonal 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 487. doi:10.1158/1538-7445.AM2015-487
Abstract 4881: Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP)
Authors: Saito Kan , Taganov Konstantin , Rosenfeld John M. , Asbrock Nick , Chu Vi
Published: August 2015

Gene regulation plays a critical role during development and progression of Cancer. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non-coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored.

Chromatin Isolation by RNA Purification (ChIRP) is one of the methods, which allows discovery of the sites of interaction of chromatin-associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe-based hybridization to target RNA molecules in chromatin. To perform ChIRP multiple biotinylated oligonucleotide probes complementary to the RNA of interest are used. To help eliminate non-specific signals two different pools of probes are used (even and odd probe sets). These probe sets are combined with chromatin and hybridized to the chromatin-associated RNA. Complexes containing biotinylated-probes bound to the chromatin-associated RNA are then isolated using streptavidin magnetic beads. DNA can then be recovered and analyzed by quantitative PCR or next generation sequencing (ChIRP-seq). Alternatively, RNA may also be isolated from an aliquot of the recovered chromatin to detect other RNA molecules that may be associated with the RNA of interest.

To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19). The peaks were called separately for odd and even reactions, and only common peaks were selected. We successfully identified several NEAT1 binding sites in the genomic DNA sequence.

In summary the methods developed allow unbiased discovery of RNA-associated genomic DNA sequences, RNA sequences, and potentially proteins.

Citation Format: Kan Saito, Konstantin Taganov, John M. Rosenfeld, Nick Asbrock, Vi Chu. Analysis of long-noncoding RNA interaction at chromatin by chromatin isolation by RNA purification (ChIRP). [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 4881. doi:10.1158/1538-7445.AM2015-4881
Abstract 4886: Repair of challenging FFPE DNA improves library success rate and sequencing quality
Authors: Liu Pingfang , Chen Lixin , Ettwiller Laurence , Sumner Christine , Stewart Fiona , Dimalanta Eileen , Davis Theodore , Evans Thomas
Published: August 2015

Formalin-fixed, paraffin-embedded (FFPE) clinical samples are an invaluable source of information about genetic alterations in human disease, especially cancer. Next generation sequencing is a powerful tool to mine that information. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. In this study, we investigated the effects of DNA repair on library preparation and sequencing from FFPE samples. We evaluated a repair enzyme mix that is designed to work on a broad range of DNA damage including modified bases, nicks, gaps, and blocked 3′ ends. Results from FFPE DNA samples of varying quality show that DNA repair generally increases library yields by 10-170%, with the largest improvement on samples of poor quality. Careful analysis of sequencing data shows that base calling qualities for all 4 bases are improved upon DNA repair. Aberrant G:C to A:T mutation were significantly reduced, consistent with cytosine deamination being induced during fixation and storage at room temperature. Interestingly, sequencing miscalls for base pair changes not typically associated with fixation were also reduced. We are currently investigating the source of these mutations. With DNA repair a noticeably positive impact on read mapping and read pairing was observed, resulting in the generation of more useable data. Pretreatment of FFPE DNA by the repair enzyme mix is easily implemented upstream of library construction, allowing fast turnaround time and easy automation. We expect that these improvements will enable the analysis of many FFPE samples that would otherwise not be accessible.

Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis, Thomas Evans. Repair of challenging FFPE DNA improves library success rate and sequencing quality. [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 4886. doi:10.1158/1538-7445.AM2015-4886
Abstract 4887: TCR profiling of T lymphocytes in ovarian tumors and malignant ascites using next-generation sequencing
Authors: Jang Miran , Yew Poh-Yin , Hasegawa Kosei , Ikeda Yuji , Fujiwara Keiichi , Fleming Gini F. , Nakamura Yusuke , Park Jae-Hyun
Published: August 2015

Tumor-infiltrating lymphocytes (TILs) are often observed in many types of solid tumors and considered to reflect the host immune system that eliminates or protects tumor cells. Many studies indicated that higher TIL infiltration is correlated with better prognosis of patients. In addition, some studies suggested that T cells in malignant ascites are associated with clinical outcome. Thus, it is important to define the immune microenvironment in malignant effusions through characterization of T cell repertoire. We here applied a high-throughput next generation sequencing method to characterize T cell receptor (TCR) sequences of a set of primary cancer, ascites, and blood from 12 ovarian cancer patients. We also analyzed the TCR cDNAs of the T cell subtype populations in malignant fluids from 3 ovarian cancer patients. We observed the clonal expansion of certain T cells in tumor and ascites, and found the enriched TCR sequences representing the distinct profiles. Moreover, we investigated TCR sequence signature of CD4+, CD8+, and CD4+CD25+ regulatory T cells in malignant effusion and identified clonally expanded T cell populations. Interestingly TCR sequences among three T cell subsets were almost mutually exclusive. Although the functional studies of clonally expanded T cell populations are definitely required, our approach offers the detailed characterization of T cell immune microenvironment for investigating a beneficial immune response in the host immune system and further contributes to the improvement of cancer immunotherapy.

Citation Format: Miran Jang, Poh-Yin Yew, Kosei Hasegawa, Yuji Ikeda, Keiichi Fujiwara, Gini F. Fleming, Yusuke Nakamura, Jae-Hyun Park. TCR profiling of T lymphocytes in ovarian tumors and malignant ascites using 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 4887. doi:10.1158/1538-7445.AM2015-4887
Abstract 4897: Accurate variant detection using molecular barcodes
Authors: Cher Charmian , Johansson Henrik , Chi Javelin , Zobeck Katie , Forsmark Linus , Isaksson Magnus , Hogrefe Holly
Published: August 2015

HaloPlex is an amplicon based method for targeted sequencing. The protocol utilizes specificity gained from restriction enzyme recognition, hybridization and DNA ligation to capture molecules originating from the target region to be sequenced. The target region is fully customizable from a single gene up to several thousand discrete regions. Amplicon based methods for multiplex target enrichment are, in general, convenient methods for capturing a wide range of target region sizes. However, in contrast to hybridization capture methods where random shearing is deployed, it is not possible for Haloplex or other amplicon based techniques to use the start point of paired end reads to identify duplicate reads. Duplicate read information can be useful for improving base calling accuracy and to monitor sampling to determine the degree of confidence to assign calls at different presumed allelic fractions. For somatic variants, which are generally present at a lower than 50% allelic fraction, it is even more advantageous to know how many molecules have been sampled when calling a particular base. To enable identification of duplicate reads from libraries prepared with Haloplex, we have added a molecular barcode to the introduced primer cassette. The molecular barcode consists of ten degenerate bases allowing for over one million unique sequences to be present for tagging of molecules. Using information derived from the molecular barcode sequences we demonstrate observation of variants down to 5% allelic fraction in multiple molecules tagged with different molecular barcodes. The new protocol has, besides the introduction of molecular barcodes, been optimized in a few additional aspects. Due to improved reagent formulations and streamlining of workflow, complete target enrichment can now be completed in less than 5 hours. Using 50 ng input we demonstrate >85% specificity and above 90% of target regions being covered at >10% of average depth.

Citation Format: Charmian Cher, Henrik Johansson, Javelin Chi, Katie Zobeck, Linus Forsmark, Magnus Isaksson, Holly Hogrefe. Accurate variant detection using molecular barcodes. [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 4897. doi:10.1158/1538-7445.AM2015-4897
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

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

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

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

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