bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3063: Development of a sensitive COLD-PCR method for the detection of EGFR mutations in DNA
Authors: Richardson Katherine Richardson A. , Candau Reyes , Legendre Benjamin , Wu Grant , Shandilya Harini , Ekenkamp Karissa , Eastlake Philip , Gerard Gary 
Published: June 2012

Abstract
COLD-PCR is a method to exponentially amplify mutations in DNA while linearly amplifying wild type sequences. The method can be adapted to multiple mutation detection platforms including Sanger Sequencing and is amenable to development as a diagnostic kit.
EGFR EXONS 20 & 21: Conditions for non-biased, selective amplification of DNA with mutations in the tyrosine kinase activation regions of the EGFR gene (exon 19 deletions and the T790M and L858R point mutations) have been developed. Ice COLD-PCR methodology was used for both exon 19 deletions and the exon 21 L858R mutation. Fast COLD-PCR methodology was used for the T790M mutation in exon 20. Using control plasmids containing the T790M and the L858R mutations, COLD-PCR amplification with SURVEYOR® Nuclease/WAVE® HS confirmation, demonstrated a limit of detection (LOD) of 0.05% mutant DNA in wild type DNA. COLD-PCR amplification followed by confirmation of these mutations using Sanger sequencing had a limit of detection of 0.2%. Only 5-10 copies of amplifiable DNA containing the mutation are needed for amplification using COLD-PCR followed by downstream mutation determination. Similar results were obtained using DNA isolated from the cell lines, H1650 and A549, and serum spiked with DNA containing a T790M mutation.
EGFR EXON 19: For detection of deletions in EGFR exon 19, another series of plasmids containing 3, 6, 9, 15, and 21 base-pair deletions were constructed. The two 15 base-pair deletions were those which result in the most commonly observed mutation in non-small cell lung cancer, p.E746_A750del15 (c.2235-2249del15 or c.2236-2259del15). For these exon 19 deletions, the SURVEYOR Nuclease/WAVE HS was able to detect the presence of a mutation at 0.05% and Sanger sequencing was able to confirm these deletions at 0.2%.
CONCLUSION: These data demonstrate that COLD-PCR enriches low number and low abundance EGFR mutations including point mutations and deletions, and suggests applicability to serum/plasma. COLD-PCR coupled with mutation detection methods such as Sanger sequencing is a new model for EGFR mutation testing with potential applicability for detection of mutations in other genes.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2011-3063
Abstract 3067: Improved detection of BRAF mutation in clinical samples using PNA-mediated real-time PCR clamping techniques
Authors: Oh Miae , Cho Minhye , Choi Jae-Jin , Park Heekyung 
Published: June 2012

Abstract
The BRAF mutation is a somatic mutation with the highest pathogenic cancer-associated prevalence and a biomarker used for detecting cancer and monitoring prognosis of cancer patients. Usually, mutation in the Exon15 codons V600E mutation (1799 T&gt;A) represent more than 90% of BRAF mutations.
However, techniques using PCR and direct sequencing have limitations and are unable to detect low-level mutations in cancers to give false negative diagnoses.
We have developed a highly sensitive and simple method for detecting BRAF mutations using PNA-mediated real-time PCR clamping, PNAClampTM.
PNAClampTM method is a simple, reliable and sensitive method with a detection limit of approximately 0.1% mutant alleles. The turnaround time for this method was only 2 hours. The method was utilized to detect BRAF mutations in fine needle aspiration biopsy (FNAB) specimens from papillary thyroid cancer (PTC) patients. V600E mutation was detected in 62% (52/84) of the FNAB specimens in the PTC samples, 10% higher rate compare with PCR and direct sequencing methods. In this report, we will discuss the results of clinical studies in detail including the comparison data. In summary, PNAClampTM method can serve a rapid, reliable, and economical alternative for BRAF mutation detection in clinical settings.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2011-3067
Abstract 3069: One-step sensitive PNA mediated real-time PCR clamping assay to detect PIK3CA mutations
Authors: Kim Hyunsun , Choi Jae-Jin , Park Heekyung 
Published: June 2012

Abstract
Phosphatidylinositol 3-kinases (PI3K) are important regulators of cellular growth, transformation, adhesion, apoptosis, survival and motility. Mutations in the PIK3CA oncogene encoding PI3K have been frequently found in various human cancers. The presence of PIK3CA mutations in a tumor has been suggested as a prognostic factor and may predict responses to select treatments. Therefore, detection of PIK3CA mutations may help predict drug responses and prognosis of cancer patients.
We have developed a highly sensitive and simple method to detect 11 mutations in exons 9 and 20 of PIK3CA using one-step PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on unique properties of PNA probes that PNA oligomers are not recognized by DNA serving as sequence-selective clamps during PCR amplification. This method can also detect 11 mutations through only three tube reactions each with 10 ng of the target DNA. PNAClampTM is simple and reliable with a detection limit of approximately 0.1% mutant alleles using 10 to 50 ng of normal DNA as a template. The turnaround time for performing this assay was only 2 hours.
In clinical studies with breast cancer patients in Korea, PIK3CA mutations were detected utilizing PNAClampTM in 12 (35.3%) of clinical samples, and the results were compared with direct sequencing method. Among 34 patients, 1 (2.94%) had mutations in codon 542, 6 (17.65%) had mutations in codon 545 and 5 (14.71%) had mutations in codon 1047. The results will be discussed in detail in this report including the method development and optimization.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2011-3069
Abstract 3084: The role of RNA-binding protein HuR in lung metastasis of triple-negative breast cancer
Authors: Techasintana Patsharaporn , Gubin Matthew Michael , Magee Joseph David , Dahm Garrett MacKenzie , Atasoy Ulus 
Published: June 2012

Abstract
Breast cancer is the second most common cancer among women worldwide. It is estimated that approximately one million women are diagnosed with breast cancer annually. Furthermore, more than 410,000 women will die each year from the disease primarily due to tumor metastasis. Currently, even the most effective treatments often result in recurrence and metastasis, in part due to genetic factors. Therefore, understanding the mechanism of breast cancer related gene regulation is crucial for the development of novel treatments and preventive strategies to those who are predisposed to developing breast cancer. Previously, we have shown that the RNA-binding protein HuR posttranscriptionally regulates various proto-oncogenes by stabilizing their mRNAs and facilitating their translation into proteins. Additionally, HuR upregulation and cytoplasmic localization has been associated with invasive cancer progression and poor prognosis. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. HuR regulates genes involved in angiogenesis, cell growth and cell cycle regulation including VEGFα, TSP1, HIF1α, CDKN1A(p21) and β-catenin. In this study, we investigated the role of HuR in an aggressive triple-negative (estrogen receptor ER-, human epidermal growth factor receptor 2 (HER2)- and progesterone receptor (PR)-) breast cancer cell subline LM-2. The LM-2 cells were derived from triple-negative breast cancer cells MDA-MB-231 which were isolated from two rounds of in vivo selection of the cancer cells which metastasized to the lungs. The LM-2 cells were retrovirally transduced with triple-fusion protein reporter construct encoding thymidine kinase1, green fluorescent protein (GFP) and firefly luciferase in order to obtain nuclear imaging, fluorescent and bioluminescent properties for in vivo and in vitro tracking. LM2 cells were further transfected with a plasmid containing HA-HuR or empty vector control to investigate the function of HuR in LM2 cells. Two clones of HuR overexpressing LM2 cells were shown to grow faster in vitro compared to the empty vector control. Also, HuR overexpression significantly facilitates tumor invasion in vitro by matrigel invasion assay. Further analysis of HuR overexpressing LM2 cells using in vivo imaging system (IVIS) will reveal the role of HuR in breast cancer growth and metastasis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2011-3084
Abstract 31: Isoform level gene expression signature is a better discriminator of cancer from normal cells than gene level expression signature
Authors: Zhang ZhongFa , Davuluri Ramana V. 
Published: June 2012

Abstract
The majority of mammalian genes generate multiple transcript variants and protein isoforms, which usually are differentially expressed in different cellular and disease conditions. We hypothesize that the isoform level gene expression profiles can better discriminate normal and cancer cells with different sub-types than gene-level expression profiles. We analyzed Affymetrix exon array data sets, collected from public domain (ENCODE and TCGA datasets from NCBI GEO database), comprising 160 cell lines of either normal or tumor tissues of origin. The unsupervised clustering of genome-wide gene expression estimates at isoform-level effectively grouped the normal and cancer cell lines with 97.5% homogeneity, regardless of the tissue origins of the cell lines, with only 4 exceptions out of 160 samples. However, at gene level, this homogeneity grouping rate was much lower, and was about 75% at best. The supervised method revealed genes and isoforms that are differentially expressed between pairs of tumor and normal samples, which can serve as signatures to effectively differentiate tumor and normal cell lines. Finally, the pathway analyses both at gene and isoforms-level indicate that there are subtle differences of derived pathways. The study reveals that there exists a common isoform-level signature that can be used not only for effective discrimination of oncogenic cell lines from non-oncogenic cell lines, but may also have important biological implications for a better understanding of common defining characteristics of tumors of varied types. Finally, we present the identified isoform-level gene signatures and associated pathways for melanoma, breast cancer and glioblastoma.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 31. doi:10.1158/1538-7445.AM2011-31
Abstract 3104: Stromal fibroblasts promote progression of esophageal squamous cell carcinoma cells both in vitro and in vivo
Authors: Saito Shin , Morishima Kazue , Matsubara Daisuke , Hosoya Yoshinori , Sata Naohiro , Yasuda Yoshikazu , Ishikawa Shumpei , Niki Toshiro 
Published: June 2012

Abstract
Objectives: The purpose of the study was to clarify the role of fibroblasts in the proliferation and growth pattern of esophageal squamous cell carcinoma and to experimentally study interactions between cancer cells and fibroblasts.
Methods: We used 22 esophageal squamous cell carcinoma cell lines (mainly, TE series and KYSE series obtained from the RIKEN Cell Bank and the Japanese Collection of Research Bioresources, respectively) and human esophageal fibroblasts that had been established in our laboratory from surgical specimens. Cancer cells were cultured on Matrigel in the presence or absence of fibroblasts in the Matrigels, and their growth patterns were examined. Cell count assays were performed to examine the effects of the fibroblast culture supernatant, growth factors, cytokines, and various inhibitors on cancer cell growth. We also examined the influence of co-injection of fibroblasts on tumor growth in immunodeficient mice.
Results: When plated on Matrigel, nearly all cell lines spread and grew in an invasive manner in the presence of fibroblasts, while in the absence of fibroblasts, most cell lines formed round or spheroidal colonies. Cell-count assay showed that in most cancer cells, cell proliferation was stimulated by addition of fibroblast supernatant. In particular, cell proliferation increased by more than 50% in 11 cell lines. Addition of hepatocyte growth factor (HGF) or interleukin-6 elicited a definitive proliferative response in only 2 and 3 cell lines, respectively. Both KYSE-220 and KYSE-50 proliferated in the presence of HGF, but the addition of MET inhibitor (PHA665752) suppressed the proliferation of only the former. In vivo tumor growth was stimulated by co-injection of fibroblasts in 9 of 22 cancer cell lines.
Conclusions: Fibroblast had a profound influence on cell growth pattern on Matrigel. In many esophageal cancer cell lines, cell proliferation was promoted by the presence of fibroblasts both in vivo and in vitro. The stimulatory effect of fibroblast could be accounted for by HGF and/or interleukin-6 in some, but not in all, esophageal cancer cells.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2011-3104
Abstract 3105: Extracellular matrix density and the development of breast acini and ducts in 3D cultures
Authors: Swamydas Muthulekha , Ricci Krista , Burg Karen J.L. , Dreau Didier 
Published: June 2012

Abstract
Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here we present the effects of increasing density of stroma composition on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures in in vitro 3D cultures. Normal murine mammary gland (NMuMG) cells were cultured in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices composed of increasing concentrations (0.8 to 8 mg/ml) of collagen I and growth-factor reduced Matrigel® (1:1). The organization of the breast-like structures was assessed by immunohistochemistry. After 3-5 days in culture, the organization and the presence of acinus-like and tubule-like structures were determined. By immunohistochemical analysis, NMuMG cells co-cultured with D1 cells formed acinar and tubular structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral and separated by laminin-positive basement membrane. Collagen I /Matrigel® matrices with density of 1.6 and 2.4 mg/ml were associated with significantly higher concentrations of acinus-like structures compared to other densities (p&lt;0.05). In contrast, the high concentration (8mg/ml) matrix prevented the development of acinus-like structures (p&lt;0.05). The number of tube-like structures was drastically reduced as the density of the collagen I / Matrigel matrix increased from 0.8 to 3.2 mg/ml (p&lt;0.05). These results indicate that the density of the matrix influences the organization and the generation of acinus-like and duct-like structures in the mammary tissues. Whether the biomechanical and density properties of the breast extracellular matrix can be harnessed to build improved tissue culture systems to benefit breast cancer patients remains to be determined.
This work was supported by grants from the Department of Defense Era of Hope program (BC044778) and the National Science Foundation EFRI program (CBE0736007).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2011-3105
Abstract 3110: Microglia-stimulation of glioma cell invasion is dependent on EGFR and CSF1R signaling
Authors: Coniglio Salvatore J. , Dobrenis Kostantin , Stanley Richard , Symons Marc H. , Segall Jeffrey E. 
Published: June 2012

Abstract
Glioblastoma multiforme is a deadly cancer whose current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma makes complete removal of these tumors nearly impossible. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. The two cell populations were differentially labeled with cell tracker dyes and invasion was quantified by confocal imaging. The effect of inhibiting EGFR and CSF1R (c-fms) on microglia-stimulated glioblastoma invasion using pharmacological agents was tested. We also determined the level of EGF and CSF1 ligand expression by glioblastoma and microglial cells using ELISA, western blotting, and immunofluorescence. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately 8 fold in an in vitro invasion assay. Pharmacological inhibition of EGFR strongly inhibited microglia-stimulated invasion. Furthermore, using a pharmacological CSF-1R kinase domain inhibitor, we showed that CSF-1R signaling is also required for microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which are unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. We also demonstrated that microglia express EGF which is largely associated with the plasma membrane. These data indicate that during invasion, glioblastoma and microglia cells signal to each other using EGF and CSF-1. The results of this study indicate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3110. doi:10.1158/1538-7445.AM2011-3110
Abstract 3115: Role of nuclear coactivator AIB1 in endothelial cell function and angiogenesis
Authors: Al-Otaiby Maram A. , Zhangzhi Hu. , Riegel Anna , Wellstein Anton 
Published: June 2012

Abstract
The nuclear receptor coactivator oncogene AIB1 (Amplified In Breast Cancer 1) has a clearly defined role in steroid and growth factor signaling in epithelial cells of cancers. Recently, we have demonstrated that AIB1 is functionally required for HER2-mediated mammary tumorigenesis in MMTV/Neu mouse model for breast cancer through regulation of HER2 phosphorylation and signaling. Generation of neu/HER2 transgenic mice with either loss of one or both copies of the AIB1 gene resulted in reduced or complete abolition of mammary tumor development, respectively. One very interesting aspect of this study was that the tumors that arose in HER2 AIB1 HET (+/-) mice were phenotypically different from those in HER2 AIB1 WT (+/+) mice in that they were smaller, more necrotic and significantly less vascularized. This data indicated to us the possibility that the role of AIB1 was not confined to the mammary epithelium proliferation but also had major effects on the surrounding stromal cells. In addition, recent expression array data indicated that AIB1 is also overexpressed in human breast cancer stroma which includes the endothelium. In here, we examined the role of AIB1 in endothelial cell function. We demonstrate that reduction of AIB1 levels in endothelial cells cultured in vitro prevents their ability to form endothelial tubes, in conjuction with reduced proliferation and motility. Furthermore, in ECIS (Electric Cell-substrate Impedance Sensing) assay, HUVEC cells with low AIB1 expression loss the ability to form a monolayer on the surface of gold-film electrodes. Consistent with the reduced proliferation that we observed, cDNA array analysis that compared gene expression patterns of HUVEC cells +/- AIB1 shRNA indicates that a number of key cell cycle regulatory genes and cytokines were regulated by reduction in AIB1 levels. Overall these data describe a new role for nuclear receptor coactivator AIB1 in the endothelium.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2011-3115