bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4810: Utility of promoter hypermethylation for differentiating malignant and benign effusions in liquid-based cytology specimens
Authors: Kim Ga-Eon , Lee Ji-Shin , Kim Jin-Sook , Bae Woo-Kyun , Lee Ji-Hee , Hwang Jun-Eul , Lee Jae-Hyuk
Published: June 2012

Background : The cytologic differentiation between benign and malignant effusions can be difficult. Because promoter hypermethylation of tumor suppressor genes is a frequent epigenetic event in many human cancers, it could serve as a marker for the diagnosis of cancer. The aim of this study was to investigate the feasibility of promoter hypermethylation as a diagnostic tool for the differentiation between malignant and benign effusions in liquid-based cytology samples. Methods : A multiplex, nested, methylation-specific polymerase chain reaction analysis was used to examine promoter methylation of 4 genes (RAR-β, APC, Twist and HIN-1) in malignant (n=85) and benign (n=31) liquid-based cytology samples. Results : The frequencies of hypermethylation of RAR-β, APC, Twist and HIN-1 were significantly higher in malignant effusions than in benign effusions (p<0.001 for each). In receiver-operating characteristic analysis, the area under the curve (AUC) for APC was the highest. The AUC for the best two-gene combination (APC/HIN-1) was not statistically different from the best individual tumor suppressor gene (APC). Conclusions : This study suggests that promoter methylation analysis on residual liquid-based effusion samples may be a feasible approach to detect malignant effusions, and that APC is the best marker for differentiating malignant and benign effusions.

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 4810. doi:10.1158/1538-7445.AM2011-4810
Abstract 4813: Aberrant promoter methylation of TMS1 gene in patients with colorectal carcinoma in Saudi Arabia
Authors: Uddin Shahab , Siraj Abdul K. , Al-Rasheed Maha , Husain Azhar , Bavi Prashant , Al-Sanea Nasser , Abduljabbar Alaa , Homoud Samar Al , Ashari Luai , Dayel Fouad Al , Al-Kuraya Khawla S.
Published: June 2012

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration. It serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Several genes have been shown to be epigenetically inactivated in a wide range of tumors. TMS1 is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to be hyper-methylated in various cancers. Therefore, we studied the methylation status of TMS1 using a panel of colorectal cell lines and 15 CRC cases. Methylation status of the promoter region of TMS1 gene was determined using methylation specific PCR in colorectal cancer cell lines. Seven out of 13 colorectal cell lines showed to be methylated for TMS1 gene (54%). Initial analyses with 15 clinical colorectal cases demonstrated 53% methylation. Real time PCR and western bolt analysis was employed to assess the expression levels. 5-AZA-2′-deoxycytidine was used to demethylate colorectal cancer cell lines. Four out of 7 methylated colorectal cell lines were completely methylated and 3 out of 7 showed partial methylation for TMS1 gene resulting in loss of TMS1 expression. Treating methylated colorectal cell lines with the methyltransferase inhibitor, 5-aza-2’ deoxycytidine alone resulted in partial demethylation and re-expression of TMS1 gene, suggesting the role of methylation in silencing the gene. TMS1 gene has shown tumor suppressive effects after demethylation in thyroid cancer cell. Additionally the oncogenic role of TMS1 has been demonstrated in different cancers. We will further evaluate the role of TMS1 gene after demethylation for apoptosis. After demethylation of TMS1 gene with 5-aza-2’ deoxycytidine we will treat our colorectal cancer cell lines with increasing doses of TRAIL and will evaluate its response by Annexin/PI dual staining and western blot analysis.

Our results demonstrated that TMSI gene is methylated in colorectal cancer cells and repression of methylation by 5-Aza-2′-deoxycytidine restored the expression of TMS1 gene. In conclusion, methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival in colorectal and can be utilized for targeted therapy.

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 4813. doi:10.1158/1538-7445.AM2011-4813
Abstract 4826: Identification of low prevalence somatic mutations in heterogeneous tumor samples
Authors: Harismendy Olivier , Kotsopoulos Steve , Bao Lei , Amorese Doug , Stapleton Mark , Olson Jeff , Nakano Masakazu , Cairn Brian , Pond Stephanie , Messer Karen , Chen Pengchin , Sementchenko Victor , Schwab Richard , Chee Mark , Link Darren , Frazer Kelly A.
Published: June 2012

High throughput sequencing enables the digital measurement of each allele in a DNA sample. This provides an ideal method to interrogate mutations present in heterogeneous samples such as solid tumors in which clonal selection or contamination with stroma can hinder the identification of important somatic mutations. We developed an ultra-deep targeted sequencing (UDT-Seq) assay to screen 46 cancer genes via microdroplet PCR (RainDance Technologies) and direct sequencing of the amplicons on the Illumina GA. This UDT-Seq library interrogates ∼86 kb of DNA located in cancer mutational hotspots (87% of all COSMIC mutations) and ∼29 kb located in exons sequenced in HapMap samples for the assay calibration and performance evaluation. We devised a statistical filtering of the mutations by using both experimental estimation and statistical modeling of the sequencing error rate. We measured the performance of our assay by processing 4 blends of 4 HapMap samples, interrogating 160 SNPs with known prevalence in each blend. The sensitivity and specificity of our method is >90% and >99% respectively for variants present at 5% prevalence or greater. We next interrogated 4 cancer samples (xenografts, 2 of which with matching primary samples) from 4 different tissues. We were able to detect low-prevalence somatic mutations in all samples of which some are well-known driver mutations. Interestingly, the mutational profile from primary to xenograft is conserved for one sample but different for another. Finally, we analyzed the robustness of the detection and prevalence measurement after performing whole-genome amplification on DNA extracted from fresh frozen and FFPE tissue using Single Primer Isothermal Amplification technology (SPIA® -NuGEN Technologies). Featuring a streamlined sample preparation to interrogate a large number of bases, this assay is well suited for clinical applications to study clonal selection in cancer progression or treatment with sub-optimal heterogeneous cancer samples.

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 4826. doi:10.1158/1538-7445.AM2011-4826
Abstract 4827: Loss of 11p in primary breast cancer and brain metastases
Authors: Wikman Harriet Anna-Liisa , Lamszus Katrin , Sielaff-Frimpong Bettina , Wiztel Isabell , Westphal Manfred , Pantel Klaus
Published: June 2012

Metastatic breast cancer remains essentially incurable, with mortality being especially high in patients who develop brain metastases. Approximately 15% of all epithelial tumors metastasize to the brain, with incidence rates highly dependent on the primary tumor type. Apparently, the brain microenvironment is specifically permissive for the out-growth of tumor cells from some carcinomas but not others.

In a previous study we screened by array CGH primary breast tumors and brain metastases. A 10MBp loss at 11pter-p15.4 was found in 70 % of the brain metastases whereas only 15% of the early stage primary tumors showed loss of 11p. In this study the aberration pattern of 11p was verified by microsatellite analysis in primary breast tumors and brain metastases. Six markers with an average spacing of 2 MBp were analyzed at 11p15.5-p15.4. Allelic imbalance (AI) at 11p was found in 26% (11/42) of the primary tumors whereas 71.4% (15/21) of brain metastases showed AI (p=0.001). In primary tumors the occurrence of AI was associated with high grade (p=0.03) and positive lymph node status (p=0.02). Interestingly, primary tumors with a brain relapse showed an equally high AI rate (75%) as the brain metastases (71%) in contrast to primary tumors without a relapse (26%). Furthermore, two matched pairs of primary tumors and brain metastases showed equal aberrations patterns and the third matched pair showed an enlarged AI of 11p, indicating that loss of 11p is defining a specific subgroup of breast cancer patients with a high risk of brain relapse.

In summary, these results indicate an accumulation of chromosomal imbalances during the carcinoma-metastasis sequence and the possible involvement of loss of 11p in brain metastasis formation.

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 4827. doi:10.1158/1538-7445.AM2011-4827
Abstract 4845: Tumor cell line profiling of eighteen cancer therapeutics to evaluate relationships between tumor genotypes and cancer cell sensitivities
Authors: Ovechkina Yulia Y. , O'Day Christine , Marcoe Karen , Keyser Robert , Bernards Karen , Chesnut-Speelman Jessica , Nguyen Phuong , Mulligan Janine , Lin Teddy , Shively Rodney , Hnilo Jim , Nelson Brian
Published: June 2012

In vitro cellular response profiling of tumor human cell lines has become a widely used approach for the targeted cancer therapeutics development. Correlation of the drug sensitivity and resistance cellular response with genomic data offers a robust and sensitive system for predicting clinical efficacy and identifying more efficacious patient populations. We have developed a high throughput cellular approach to evaluate the relationship between tumor genotypes and drug sensitivity over 240 human tumor cell lines. A panel of the cancer therapeutic agents was tested for proliferative, apoptotic and cell cycle arrest responses using multiplexed high content screening with automated fluorescence microscopy and image analysis based technology. Growth index was measured using nuclear dye. Activated caspase 3 antibodies were used for the apoptosis induction detection. Phospho-histone H3 antibodies were used to measure the cell cycle block. The eighteen cancer therapeutics were used in this study: BMS-387032 (SNS-032), PD0325901, PD173074, API-2 (Triciribine), Lapatinib (Tykerb), BMS-536924, Geldanamycin, Sorafenib, Dasatinib, Erlotinib, VX-680, Sunitinib, Everolimus, Tandutinib, Cl 1040, Doxorubicin, Paclitaxel and Staurosporine. We generated cell line profiles to reveal drug sensitivity and resistance patterns and identified markers associated with a specific response. It was found that Raf/Ras mutations confer sensitivity to MEK inhibitors, PD0325901 and Cl 1040, while PIK3CA and RB mutations were associated with resistance. Resistance to IGF-1R, FGFR, and EGFR inhibitors correlated with PI3K/PTEN/Akt or Raf/Ras pathway activation. EGFR mRNA overexpression was associated with resistance to FGFR and IGF-1R inhibitors. Resistance to AKT inhibitor, API-2 (Triciribine) was associated with PTEN mutations and amplification of AKT. APC mutations were associated with resistance to Aurora kinase inhibitor, VX-680, while mutations in the beta-catenin gene were linked with a sensitive phenotype. This approach could provide insight into the mechanisms of enhanced susceptibility or resistance which in turn could be used for the optimization of the targeted cancer therapeutics.

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 4845. doi:10.1158/1538-7445.AM2011-4845
Abstract 4850: Paired-end RNA-sequencing based identification of 24 novel fusion genes in breast cancer
Authors: Edgren Henrik , Murumägi Astrid , Kangaspeska Sara , Nicorici Daniel , Hongisto Vesa , Kleivi Kristine , Rye Inga Hansine , Wolf Maija , Børresen-Dale Anne-Lise , Kallioniemi Olli
Published: June 2012

Fusion genes have recently been described in common solid tumors, revealing an important class of mutations that contribute to cancer development and progression. Next-generation sequencing technologies allow systematic characterization of cancer cell transcriptomes, including the fusion genes resulting from underlying chromosomal translocations. Using paired-end RNA-sequencing in conjunction with high-resolution chromosomal copy number analyses we characterized 24 novel and 3 previously known fusion genes in breast cancer cell lines. An improved bioinformatic approach allowed fusion gene validation with a 95% success rate by RT-PCR, Sanger sequencing and FISH. Among the identified fusions, partner genes were found to contribute both promoters (5′UTR; e.g. TATDN1-GSDMB), coding sequences (e.g. ACACA-STAC2) as well as 3′UTRs (e.g. CSE1L-ENSG00000236127). Most fusion genes (82%) were associated with copy number transitions, such as high level amplifications at e.g. 8q21, 17q12, 17q23 and 20q13, implying that fusion gene formation may contribute to the selective advantage provided by copy number amplifications. Although no prevalent single fusion gene was detected, the functional importance of the multiple fusion genes is supported by several lines of evidence. First, some of the fusion partner genes were expressed exclusively as fusion transcripts, e.g. TATDN1-GSDMB, indicating that the fusion event lead to the activation of a latent gene. Second, several fusion gene partners, including ACACA, NOTCH1 and RARA, have been previously found to be involved in oncogenic fusions in leukemias and lymphomas. Third, functional analysis using RNAi-mediated knockdown suggested a potential role for two fusion genes in controlling cancer cell proliferation. In conclusion, using RNA-sequencing together with improved bioinformatics and genomic copy number profiling, we have discovered multiple novel fusion genes in breast cancer, which may provide new insights on breast cancer, and provide therapeutic and diagnostic clues.

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 4850. doi:10.1158/1538-7445.AM2011-4850
Abstract 4854: Mutations activating proliferation pathways in ovarian cancer
Authors: Uhrhammer Nancy A. , Bidet Yannick , Chene Gautier , Viala Sandrine , Communal Yves , Raoelfils Inès , Penault-Llorca Frédérique , Bignon Yves-Jean
Published: June 2012

Ovarian malignancies may arise through different mechanisms of oncogenesis, which influence their treatment and are important for prognosis. Essential to carcinogenesis is the activation of proliferation pathways such as those directed by PI3 kinase or KRAS. New sequencing technologies may improve the detection of somatic mutations in tumor tissue, which is complicated by the low representation of a mutation within a tissue sample inevitably containing normal cells and subpopulations of malignant cells.

We analyzed selected exons in PIK3CA, PIK3R, KRAS, BRAF, EGFR and ERBB2 in 34 primary malignant ovarian tumors, including 10 serous, 4 borderline, 11 endometrioid, and 9 mixed or rare subtypes. DNA was extracted from flash-frozen tumor tissues, amplified, and sequenced on a 454-GS-Flx Genome Sequencer. Expression of PTEN, p53, Bcl2, ALDH and Ki67 were assessed by immunohistochemistry on formalin-fixed tissues.

Nine mutations were observed in PIK3CA, 5 in BRAF, 4 in KRAS, and 2 in EGFR. SNPs present in the amplicons analyzed allowed allelic imbalance to be estimated, showing possible amplification of EGFR in 9 of 20 informative samples, and of PIK3CA in 6 of 19 informative samples, none of which contained a point mutation.

Endometrioid tumors frequently showed anomalies in PIK3CA, Bcl2 expression, and not p53 overexpression. P53 overexpression was typical of serous tumors, and was associated with the absence of mutations in any of the genes sequenced. Borderline and low grade tumors were characterized by BRAF mutation, no imbalance at EGFR, no anomalies at PIK3CA, and less frequent p53 overexpression. The absence of BRAF mutations in high grade tumors suggested that they do not evolve from borderline tumors. PIK3CA mutations were not mutually exclusive with KRAS mutations, confirming the non-redundancy of these intertwined signaling pathways. PTEN extinction was not significantly negatively associated with either point mutations or allelic imbalance at PIK3CA, consistent with different roles for PTEN loss and PI3-kinase activation at different stages of tumorogenesis, in spite of the close association of PTEN with the PI3-kinase pathway. No significant associations were observed between ALDH and Ki67 and any parameters measured.

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 4854. doi:10.1158/1538-7445.AM2011-4854
Abstract 4862: Whole genome amplification allows for testing of valuable specimens by array comparative genomic hybridization
Authors: Edelman Daniel C. , Stevenson Holly , Suuriniemi Miiia , Singh Parvati , Rodriguez-Canales Jamie , Hanson Jeffery C. , Walker Robert , Emmert-Buck Michael R. , Meltzer Paul
Published: June 2012

Many cancer clinical specimens are obtained in minute quantities and in varying states of degradation; for example, when they are formalin or ethanol fixed. In addition, many investigators seek tests on multiple genomic analytes and frequently insufficient DNA remains for all inquiries after the initial priority testing. To accommodate all clinical scientific inquiries, the Sigma GenomePlex whole genome amplification (WGA) protocol has been evaluated as a means to produce sufficient genomic material for array comparative genomic hybridization (aCGH) downstream testing. However previous studies, including our own, have shown that testing degraded samples with WGA coupled to oligo CGH arrays resulted in decreased data quality. We contend this could be due to unequal genomic representation in the WGA product. Our objective was to assess the importance and efficacy of using correctly matched reference DNA and to compensate for the WGA amplification bias, potentially improving aCGH results.

We obtained DNA from ethanol-fixed prostate tissues in which both tumor and normal tissue compartments were separately isolated by laser capture microdissection or by directed needle-core sampling from formalin-fixed paraffin embedded blocks. We sought to optimize the Sigma WGA protocol by testing the following parameters: source of reference DNA (commercial versus matched normal), WGA cycle number, and the use of size-exclusion columns post-WGA. Quality control measures for the purified WGA product included electrophoresis (gel and Agilent Bioanalyzer), and a multiplex PCR to determine the extent of DNA genomic representation across the WGA product. ChromaSpin columns were used to reduce the number of smaller WGA amplicons prior to aCGH. WGA products were assayed with Agilent 8×60K CGH microarrays and chromosomal aberrations and data quality were assessed using BioDiscovery Nexus software.

Data will be presented comparing the effectiveness of using sheared reference DNA as opposed to matched normal DNA, the use of QC methods prior to hybridization, any benefit in removing small WGA DNA products, and varying the WGA cycle number. We continue to evaluate how these modifications can bring improvement to WGA when coupled to aCGH, because there is promise that this method could expand the genomic testing opportunities for valuable and rare clinical specimens.

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 4862. doi:10.1158/1538-7445.AM2011-4862
Abstract 4864: Antibody detection of translocations (ADOT) in Ewing's sarcoma
Authors: Luo Wen , Milash Brett , Dalley Brian , Smith Richard , Dutrow Natalie , Cairns Brad , Lessnick Stephen
Published: June 2012

Objective: The detection and identification of chromosomal translocations has important implications in the diagnosis, prognosis, and treatment of patients with cancer. Current approaches to translocation detection often have significant shortcomings, including limited sensitivity and/or specificity, and can be difficult to apply to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach, antibody detection of translocations (ADOT), that avoids many of the shortcomings of current techniques.

Methods: ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. Microarrays were designed that probe for every possible fusion transcript in Ewing's sarcoma, both previously identified and theoretical. These include EWS/FLI, EWS/ERG, EWS/ETV1, EWS/ETV4, EWS/FEV and others. Total RNA, purified from Ewing's sarcoma cells or tumors (that express different types of translocations), is hybridized to the custom microarrays. A high-affinity antibody (S9.6) that binds to RNA-DNA duplexes formed during hybridization is used as a detection reagent.

Results: We demonstrate the feasibility of ADOT by examples in which both known, and unknown, translocation fusion transcripts are identified from cell lines, tumor xenografts, and primary tumor samples. Translocation fusion points identified by ADOT were confirmed using traditional RT-PCR and sequencing. Furthermore, ADOT is able to detect translocations in FFPE tumor samples in which case RT-PCR fails. The sensitivity of ADOT to detection of translocations is also determined.

Conclusion: ADOT allows for the accurate and sensitive identification of translocations and also provides exon-level information about the specific fusion transcript produced. Furthermore, the technique is readily generalizable to any potential fusion transcript, and can also detect previously undescribed fusions. This suggests that ADOT may be used as a diagnostic tool for pediatric sarcoma or other translocation-based cancers.

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 4864. doi:10.1158/1538-7445.AM2011-4864
Abstract 4867: Whole transcript expression microarray profiling of normal colon and adenocarcinoma total RNA
Authors: Mullinax Becky , Bergstrom-Lucas Anne , Venneri Jennifer , Kulkarni Vinayak , Janis Michael , Ghosh Jayati , Gao Jing , Fulmer-Smentek Stephanie
Published: June 2012

Whole transcript expression profiling by microarray analysis is an important tool for understanding biological mechanisms and development, classifying tissue and tumor types, and identifying indicators for diagnosis and prognosis. This analysis measures the alternative splicing of exons from a given RNA transcript leading to the translation of a variety of proteins from a single RNA transcript. To address the need for whole transcriptome expression profiling from low quantities of total RNA, we have developed an exon expression workflow which includes custom and catalog human, mouse, and rat exon microarrays, a whole transcript labeling method, and analysis software. This fast and simple microarray-based method employs a modified linear amplification procedure to generate Cy-labeled cRNA representing the whole transcript. The labeling method, available in the Low Input Quick Amp Labeling WT Kit (LIQA WT), employs a mixture of oligo dT- and random nucleotide-based T7 promoter primers (WT Primer Mix) resulting in high cRNA yields and specific activities from low RNA inputs. The probes on the human, mouse, and rat SurePrint G3 exon microarrays were designed from the high quality content of the public databases, primarily RefSeq and Ensembl. Exon array results are analyzed for gene and exon-level expression using GeneSpring GX 11.5 software. The exon gene expression workflow allowed whole transcript gene expression profile comparisons to be made in fewer than two days. Comparisons of probe signals from technical replicate samples demonstrated high reproducibility with a wide dynamic range and comparable signals across a broad range of input amounts. High correlations were demonstrated in platform comparisons to both qRT-PCR and RNA-Seq. This new workflow was used to detect alternative splicing of exons between cancerous and normal cells resulting in gene and exon expression profiles consistent with the current literature. These results demonstrate the value of the new exon expression workflow in identifying known exons, alternative start and stop sites, mutually exclusive exons, and cassette exons in cancer research.

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 4867. doi:10.1158/1538-7445.AM2011-4867
Abstract 4873: Protein complexes in cancer: A novel strategy to identify protein complexes through reduction of false discovery rate
Authors: Das Sudipto , Xiao Zhen , Bosley Allen D. , Chan King C. , Issaq Haleem J. , Veenstra Timothy D. , Andresson Thorkell , Green Jeffery E.
Published: June 2012

Most cellular processes are controlled by protein-protein interactions, and aberrant protein-protein interactions can lead to human diseases such as cancer. Hence a comprehensive understanding of a protein interaction network and its dynamics response to different physiological conditions is essential for understanding a protein's function and may reveal novel therapeutic opportunities for fighting cancer.

Affinity purification coupled with mass spectrometry has become the method of choice to study in vivo protein interactions and formation of protein complexes. However, the methodology has some limitations that reduce it application such as, under sampling of low abundant proteins and high background leading to an elevated false discovery rate (FDR).

Herein we describe a strategy utilizing differential expression of the target protein followed by analysis using quantitative proteomics. By performing affinity purification on cells expressing increasing amounts of the polycomb complex protein Bmi-1, we show that known interacting proteins could be distinguished very efficiently from the background proteins based on their abundance profile. Applying this method to the data set significantly reduced the FDR and allowed us to identify potentially new interacting proteins for Bmi-1 that otherwise would have been considered non-specifically bound proteins.

The method was also applied to characterize the interactome for Snail I, II and III a family of Zn-finger proteins thought to be involved in transcriptional repression. These proteins have been implicated in diverse biological function such as regulation of epithelial to mesenchymal transition, formation and maintenance of embryonic mesoderm, generation and migration of neural crest cells 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 4873. doi:10.1158/1538-7445.AM2011-4873
Abstract 4877: High throughput proteomic applications using protein A/G magnetic beads
Authors: Doyle Joanna , Ray Matthew , Ouyang Anlong , Benton Betsy , Bell Peter A.
Published: June 2012

Speed and sample purity are key issues for proteomics researchers. Sample and data-processing throughputs are current bottlenecks in proteomic analysis. Magnetic separations can provide greater consistency and automation of proteomic sample preparation. We report here the development of a novel 1µm protein A/G magnetic bead that enables fast and convenient isolation of proteins via immunoenrichment. Thermo Scientific Pierce Protein A/G Magnetic Beads use a recombinant protein A/G that combines the IgG binding domains of both protein A and protein G. Protein A/G contains four Fc-binding domains from protein A and two from protein G, making it a convenient tool for capture of immunoglobulins from a variety of species and isotypes. Recombinant protein A/G was covalently coupled to 1 μm, blocked double-shelled magnetic particles with a polymeric core. The beads had a binding capacity of >60 μg rabbit IgG per mg bead along with low non-specific binding. Using a Thermo Scientific KingFisher Flex Magnetic Particle Processor, antibodies were purified from serum or ascites with high yield using the Pierce Protein A/G Magnetic Beads. The beads were also used to immunoprecipitate and co-immunoprecipitate multiple proteins significant to different phases of the cell cycle, including cyclin B, Cdk1, cyclin E, and cyclin D. U2OS and HeLa cell lines were first synchronized in serum starved media. The media was replaced with enriched media and cells were harvested at 4 hours (beginning of G1 phase), 6 hours (end of G1 phase), and 18 hours (end of G2 phase). Proteins were effectively isolated from the cell lysates using Pierce Protein A/G Magnetic Beads and identified by Western blot. In addition, these beads were successfully used in a chromatin-immunoprecipitation (ChIP) assay to measure binding of RNA Polymerase II to the proximal myc promoter in A431 lung carcinoma cells treated with and without EGF. In Conclusion, the results indicate that Pierce Protein A/G Magnetic Beads can effectively isolate protein targets in a variety of proteomic applications.

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 4877. doi:10.1158/1538-7445.AM2011-4877
Abstract 4880: Monitoring tumor response to chemotherapy by in vivo real-time imaging of phosphatidylserine targeting antibodies
Authors: Gong Jian , Him Linda , Hughes Christopher , Freimark Bruce
Published: June 2012

Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane that becomes exposed on tumor vascular endothelial cells and tumor cells in response to chemotherapy, irradiation and oxidative stresses in the tumor microenvironment. Binding of antibodies targeting PS on the tumor endothelial cells recruit immune cells and engage the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric anti-PS antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in randomized Phase IIb clinical trials. In the present study, we demonstrate in vivo tumor imaging of PS expression using real-time, near infrared fluorescence imaging of antibodies that specifically target PS. Human prostate PC-3 tumors expressing luciferase were orthotopically transplanted in SCID mice and tumor growth was monitored by bioluminescence. Targeting antibodies used in this study bind PS through the interaction of beta-2-glycoprotein 1 (β2GP1) in the same manner as bavituximab binding to β2GP1 in humans. Specific localization of fluorescence-labeled PS targeting antibodies after a single dose was observed in tumors compared to an isotype control antibody. Persistence of antibody localization was prolonged using intact IgG compared to F(ab’)2 fragment. Chemotherapy was shown to enhance the binding of PS targeting antibodies to tumors. These data provide a rationale to image PS expression to localize tumors or metastases and to monitor chemotherapy-induced PS expression during the course of bavituximab therapy.

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 4880. doi:10.1158/1538-7445.AM2011-4880
Abstract 4883: Peptide-based PET imaging agents using novel bifunctional chelates and 64Cu
Authors: Gill Rajanvir K. , Wong May , Sutherland Brent , Ng Sylvia , Bensimon Corrine , Jurek Paul , Kiefer Garry , Yapp Donald T.T. , Ferreira Cara
Published: June 2012

Introduction: Developing peptide-based PET imaging agents would be highly advantageous due to their high affinity and specificity. Two novel bifunctional chelates (BFC), PCTA and Oxo-DO3A, have previously been used to label antibodies with 64Cu. Complexation of 64Cu with PCTA and Oxo-DO3A was more efficient than with DOTA; the PCTA and OXO-DO3A complexes were also more stable and had more favourable pharmacokinetics[1]. Similarly, PCTA was also found to complex 68Ga more efficiently than DOTA[2]. The utility of PCTA and Oxo-DO3A for radiolabeling peptides specific for tumour angiogenesis processes were further investigated.

Methods: The receptor number and affinity of RGD peptides were determined in mouse vascular endothelial cells. Mouse vascular endothelial cells were grown and varying amounts of I-125 echistatin were added to 1 million cells until receptors were saturated. A competitive binding assay, where a set amount of I-125 echistatin was added to the cells in the presence of unlabeled peptides, confirmed the specificity of the peptides for the target. In vivo studies were carried out in HT29 human colorectal tumors grown subcutaneously in RAG2M mice. 64Cu radiolabeled bioconjugates of PCTA, Oxo-DO3A and DOTA were injected with and without excess cyclo-(RGDyK) peptide. The amount of activity in the blood, tumor, liver, kidney and muscle was measured and compared to each of the BFC.

Results: The binding assay revealed the saturation of mouse vascular endothelial cells was achieved when 0.8µci of I-125 echistatin was added. The competitive binding assay indicated an IC50 at a concentration of 9pM of DOTA-RGD. In vivo biodistribution studies showed good tumor to blood ratios for all of the 64Cu radiolabeled bioconjugates and better tumor to muscle ratios for the 64Cu radiolabeled bioconjugates of PCTA and Oxo-DO3A.

Conclusion: The bifunctional chelates PCTA and Oxo-DO3A were found to be effective for radiolabeling peptides with 64Cu; the resulting complexes appear to be more stable than those of DOTA. The data show that PCTA and Oxo-DO3A radiolabel peptides more efficiently than DOTA, and that the resulting complexes are more stable. Angiogenesis in HT-29 tumours was also imaged successfully using the peptides labeled with PCTA and Oxo-DO3A.

References:

1. C.L. Ferreira, D.T.T. Yapp. E. Lamsa, M. Gleave, C. Bensimon, P. Jurek, G.E. Kiefer, Nucl. Med. Biol. 35: 875-882 (2008)

2. C.L. Ferreira, E. Lamsa, M. Woods, Y. Duan, P. Fernando, C. Bensimon, M. Kordos, K. Guenther, P. Jurek, G.E. Kiefer, Bioconj. Chem. 21: 531-536 (2010)

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 4883. doi:10.1158/1538-7445.AM2011-4883
Abstract 4899: Development of a quantitative measurement of p53 – p53 antibody interactions
Authors: Atha Donald H. , Reipa Vytas
Published: June 2012

We are developing methods to quantify antibody interactions that includes a quartz crystal microbalance (QCM) system to measure, on a molecular basis, the interaction of p53 and anti-p53 antibodies. Previously, as a model system, we developed a reference device consisting of p53 protein (human wild type), characterized by mass spectroscopy and immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with immunohistochemical (IHC) assays that incorporate commercially available anti-p53 antibodies and probes. In the current study, we compared three different monoclonal antibodies (clones: BP53.12, PAb 1801 and DO-1) for their ability to detect p53 in HT1080 cells. Both visible microscopy (diaminobenzidine probe) and flow cytometry (AlexaFluor 488 probe) showed significant increases in staining intensity compared to controls without primary antibody. However, quantification of these observations requires direct measurement of the concentration of the p53 and the concentrations and specificities of the antibodies. For this the p53 protein is covalently immobilized on glass coated quartz crystals and the resonance frequency shift is followed in-situ. The functionalized sensor is then incubated with the anti-p53 antibody, which provides a direct gravimetric measure of antibody-antigen binding. This allows the comparison of the surface immobilized protein concentrations with those previously obtained by fluorescence measurement. The p53 functionalized QCM system offers an independent measure of surface immobilized protein concentration and is essential in the development of our IHC calibration prototypes. These results also provide a benchmark for comparing systems that may be used in developing molecular diagnostic assays such as aspiration biopsy analysis and the detection of biomarker proteins in blood and urine.

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 4899. doi:10.1158/1538-7445.AM2011-4899
Abstract 4900: Development of a method for metabolomics and histology of tissue biopsies
Authors: Brown Meredith V. , Adam Klaus-Peter , Shuster Jeffrey R. , Troyer Dean A. , Lawton Kay A.
Published: June 2012

Metabolomics, the global study of small molecules in a biological sample, is performed with the goal of extracting, identifying, and quantifying metabolites. As such, the technology is ideal for identifying biomarkers for diagnostics and drug therapy. One of the limitations with the current practice for metabolite extraction is that sample destruction precludes additional uses of the sample. This is particularly troublesome for high value samples with limited availability, such as clinical tumor biopsies. Clinical biopsies are taken in order to histologically diagnose and gauge cancer aggressiveness. Thus, physically disrupting these valuable samples is not ideal, even though quantitative metabolomic data could enhance histopathologic evaluation. In order to improve the amount of information obtained from patient biopsies, we have developed a method (termed PReservation by Extraction and Fixation, PREF) by which metabolite extraction simultaneously preserves the tissue for histological analysis. Briefly, upon sample collection, the biopsy is placed directly in methanol. Methanol incubation serves to recover metabolites, precipitate proteins, and fix the tissue. Following overnight incubation, the solvent is removed, evaporated to dryness, and reconstituted in a proprietary mixture of recovery standards. The needle biopsies are placed in biopsy bags and cassettes and transferred to Molecular Fixative until processed for histology. Solvent sample aliquots are taken for analysis on GC/MS and LC/MS platforms. Raw mass spectrometry data files are automatically extracted using a proprietary informatics system that includes peak identification and compound identification software. Compared to the current state of the art method for metabolite extraction, PREF recovers equivalent numbers and types of biochemicals from the tissue specimen. Furthermore, PREF preserves the tissue biopsy sample such that cellular architecture is retained, allowing for pathological analysis by chemical staining (hematoxylin & eosin) and antibody binding (immunohistochemistry). We have developed a non-destructive method for efficient metabolite extraction from tumor biopsies combined with preservation of the tissue for histological analysis. It is our hope that this novel method will augment the current histopathologic diagnosis and classification of tumors through quantitative measures of biochemicals in the same tissue. Given that certain biochemicals have been shown to correlate with disease aggressiveness, it is likely that this method will be valuable in helping to differentiate cancer aggressiveness.

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 4900. doi:10.1158/1538-7445.AM2011-4900
Abstract 491: Defining the origins of ras/p53 mediated squamous cell carcinoma
Authors: White Andrew C. , Tran Kathy , Khuu Joan , Lowry Bill
Published: June 2012

The precise identity of cancer cells of origin and the molecular events of tumor initiation in epidermal squamous cell carcinoma (SCC) are unknown. Here we show that malignancy potential is related to the developmental capacity of the initiating cancer cell in a genetically defined, intact, and inducible in vivo model. Specifically, these data demonstrate that hair follicle stem cells (SCs) serve as cancer cells of origin when oncogenic Kras is expressed and p53 is ablated, whereas their more developmentally restricted progeny, the transit amplifying (TA) cells, are unable to generate even benign tumors in the same genetic context. Employing a temporal model of tumorigenesis in situ, we highlight the phenotypes of cancer progression from hair follicle SCs including hyperplasia, dysplasia, epithelial to mesenchymal transition, and SCC formation. Furthermore, we provide insights into the inability of hair follicle TA cells to respond to tumorigenic stimuli.

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 491. doi:10.1158/1538-7445.AM2011-491
Abstract 4910: Unveiling the role of network and systems biology in pancreatic cancer drug discovery
Authors: Azmi Asfar S. , Philip Philip A. , Choi Minsig , Shields Anthony F. , Sarkar Fazlul H. , Mohammad Ramzi M.
Published: June 2012

Over the last half decade our laboratory has extensively worked on the preclinical development of small molecule inhibitor of HDM2 (MI-219-Ascenta Therapeutics) in multiple tumor models with especial emphasis on the intractable pancreatic cancer (PC). During this discourse we have observed significant anti-tumor activity when MI-219 was given with oxaliplatin that lead to tumor free survival in 50% of the animals. However, not much is known about the exact mechanism behind this synergy. Here we applied a network-centric approach to identify the global expression changes and the networks involved in this two drug combination without losing the key molecular details. Gene analysis of wt-p53 capan-2 cells shows that MI-219 alone induces a minimal alteration (only 48 genes). Oxaliplatin (a cytotoxic drug) treatment, on the other hand showed alterations of 700 genes. However, the combination of MI-219-oxaliplatin resulted in emergence of 286 synergy unique genes. Principal component analysis revealed that each drug treatment had unique global gene signatures. Further analysis of the synergy unique genes revealed the presence of 14 statistically enriched functional pathways. Overall, the central p53 activity hub was found to be supported by 5 crucial players; CREBBP which is ubiquitously expressed in PC and is involved in the transcriptional co-activation of p53. CARF that is responsible for p53 stability along with NF-κB and EGR1 tumor suppressor modules, all positively affecting p53 re-activation that drives cells towards apoptosis. Additionally, our analysis revealed a novel role of hepatocyte nuclear factor alpha (HNF4α) in aiding this synergy. In addition to PC, the gene signatures could be verified and validated at the RNA and protein level in multiple wt-p53 solid tumor cell lines like MCF-7 for breast and HCT-116 for Colon. siRNA knockdown of key network genes abrogated apoptotic potential of MI-219-oxaliplatin in all three cell lines and confirmed their key role in the observed anticancer effects of this combination. Our findings show that Network and Systems biology can be utilized in identification of crucial driver genes and help in the design of drug combinations in a genetically pre-defined subset of patients that would positively impact treatment outcome.

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 4910. doi:10.1158/1538-7445.AM2011-4910