bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4200: Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells.
Authors: Zhu Wuling , Fan Binglin , zhang Hongge , Ji Xiaohui , Yang Wancai , Yang Wancai
Published: August 2014

The microRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate gene expression by degrading mRNA and by inhibiting translation of protein. Numerous studies have demonstrated the critical roles of miRNA in physiological and pathological procedures, including cell proliferation, differentiation and apoptosis, and in carcinogenesis. Recent studies have particularly reported that miR-100 is aberrantly expressed in multiple cancers, including colorectal, lung, prostate, ovarian, thyroid, pancreatic, liver, breast, cervical and nasopharyngeal cancers. It has also been reported that reduction of miRNA-100 is associated with poor prognosis. But the effects of miRNA-100 on human hepatocellular carcinoma cells and its potential mechanisms are largely unknown. In the present study, we transfected miRNA-100 analogue into human hepatocellular carcinoma cell line HepG2 cell, employed quantitative RT-PCR, immunoblotting, immunofluorescence, flow cytometry and other approaches, and found that the expression of miRNA-100 was markedly lower but polo-like kinase 1 (Plk1) was higher in the HCC cell line and fetal liver, compared to those in the normal hepatocytes and adult liver, respectively. Moreover, increasing miRNA-100 expression in HepG2 cells inhibited cell proliferation, caused cell cycle arrest and promoted apoptosis. Mechanistic and correlation studies revealed that the anti-cancer effects of miRNA-100 were associated with downregulation of Plk1 expression at mRNA and protein levels. Factually, bioinformatic analysis has predicted that Plk1 could be a target of miRNA-100. In conclusion, our results have shown that miRNA-100 has a tumor suppressor function in human hepatocellular carcinoma cells, which may be associated with downregulation of Plk1, and implicate the potential application of miR-100 in connection with Plk1 for targeted gene therapy of hepatocellular carcinoma.

Citation Format: Wuling Zhu, Binglin Fan, Hongge zhang, Xiaohui Ji, Wancai Yang, Wancai Yang. Effects of microRNA-100 on cell proliferation and apoptosis is associated with dysregulation of Plk1 in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4200. doi:10.1158/1538-7445.AM2013-4200
Abstract 4205: Simultaneous high resolution fluorescence imaging of cellular DNA and RNA enabled by complex oligonucleotide libraries in various sample types.
Authors: Ach Robert A. , Tsang Peter , Scheffer-Wong Alicia , Bruhn Laurakay , Powell Weston , LaSalle Janine , Yamada Alice
Published: August 2014

Fluorescence in situ Hybridization (FISH) is a powerful technique for determining the structure, organization, and localization of specific nucleic acid sequences within individual cells. However, 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 leveraged our ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length that can be used 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. Using oligonucleotide library-derived FISH probes on DNA, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we readily visualized 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 cells, 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, we have been able to simultaneously detect DNA and RNA from the same locus in the same FISH assay. We have successfully used this technique in several types of fixed tissue culture cells as well as in formalin-fixed paraffin-embedded tissue sections. Our oligo FISH methods are readily compatible with the co-detection of cellular proteins by immunocytochemistry. The ability to generate high performance FISH probes using chemically synthesized oligo libraries that can work flexibly with co-detection of other molecules yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function.

Citation Format: Robert A. Ach, Peter Tsang, Alicia Scheffer-Wong, Laurakay Bruhn, Weston Powell, Janine LaSalle, Alice Yamada. Simultaneous high resolution fluorescence imaging of cellular DNA and RNA enabled by complex oligonucleotide libraries in various sample types. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4205. doi:10.1158/1538-7445.AM2013-4205
Abstract 4209: Genotypic agreement between histological specimen and preoperative cytology in small lung adenocarcinoma.
Authors: Hirai Noriko , Takaaki Sasaki , Minami Yoshinori , Kitada Masahiro , Miyokawa Naoyuki , Ohsaki Yoshinobu
Published: August 2014

Background. Histological subtype and genotype is critical for making treatment decisions and improving patient outcomes in non-small cell lung cancer (NSCLC). Approximately 70% of lung cancers are diagnosed in advanced stages where small biopsies and cytological specimens are the only source of material for both diagnosis and genotyping. Patients with specific gene alterations in epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) likely receive more benefit from EGFR TKI or ALK TKI therapy, respectively. Although, genotyping agreement from cytological and histological samples is critical, more investigational studies need to be explored to assess the concordance of genotype. Purpose. We performed a comparing investigation on concordance rate for pathological diagnosis and EGFR gene mutation status between surgical specimen and preoperative cytological specimen in small lung cancer. Additionally we reviewed immunohistochemical (IHC) staining with two different ALK specific antibodies. Materials and Methods. 55 patients who had surgically resected small (≦3cm) lung adenocarcinoma with known preoperative bronchial brushing cytology were examined retrospectively. IHC staining was conducted by mutation-specific antibodies using E746-A750 deletion and L858R for EGFR or 5A4 and D5F3 for ALK. DNA analysis was performed by PNA-RNA PCR clamp assay. Results. 1) Diagnostic concordance rate between cytological and histological specimen was 90.9%. Disagreement cases were included one case of squamous cell carcinoma, two cases of small cell carcinoma, one case of melanoma and one case of mucoepidermoid carcinoma by cytology. 2) In IHC positive for mutation-specific EGFR antibodies, the sensitivity was 83% in E746-A750 and 100% in L858R, respectively. In EGFR IHC positive cases, well to moderate differentiation were dominant. 3) In ALK IHC, one case was observed stronger cytoplasmic staining in D5F3 than 5A4. Furthermore, the cribriform pattern was observed in H&E staining. Distinctive cytological feature was not observed. Conclusion. Our results showed that high genotype concordance rates of histology and cytology agreement in small lung adenocarcinoma.

Citation Format: Noriko Hirai, Sasaki Takaaki, Yoshinori Minami, Masahiro Kitada, Naoyuki Miyokawa, Yoshinobu Ohsaki. Genotypic agreement between histological specimen and preoperative cytology in small lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2013-4209
Abstract 4212: Deep sequencing analysis of the ALK kinase domain identifies resistant mutations in relapse patients following crizotinib treatment.
Authors: Huang Stephen
Published: August 2014

ALK resistance mutations conferring resistance to Xalkori vary widely within the tyrosine kinase domain. So far, nine different acquired resistant mutations have been identified in the clinic, including 1151Tins, L1152R, C1156Y, F1174C, L1196M, G1202R, D1203N, S1206Y, and, G1296A. We developed a deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain in tumor biopsies. By bar-coding, we were able to sequence multiple samples in a single run. We have also developed an algorithm to determine assay noisy level and call mutations automatically. The assay sensitivity to call close to 1% mutated allele demonstrated by a titration experiment with a cell line harboring a know mutation. Genomic DNA analysis from relapsed patients revealed several known mutations. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.

Citation Format: Stephen Huang. Deep sequencing analysis of the ALK kinase domain identifies resistant mutations in relapse patients following crizotinib treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4212. doi:10.1158/1538-7445.AM2013-4212
Abstract 4217: A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC).
Authors: Schlag Peter , Hoeppner Corey , Bristol Anona , Kaur Namneet , Quashnick Stephen , Ravirala Ramani , Kramer Joyce , Negash Minet , Brophy Victoria , Lee Sung , Soviero Stephen
Published: August 2014

Somatic mutations in the PIK3CA gene encoding the p110α catalytic subunit of the PI3K protein have been linked to human cancer and have been found to occur at a high frequency in BC (25-40%). Therapeutics that target key nodes in the PI3K signaling pathway are currently being developed and stratifying patients based on their PIK3CA mutation status is becoming more important. We describe the analytical performance of the cobas® PIK3CA Mutation Test (For Research Use Only), a multiplex allele-specific PCR assay designed to detect 17 mutations in exons 1, 4, 7, 9 and 20 in the PIK3CA gene in 3 PCR wells. The test detects ∼94% of known PIK3CA mutations found in BC.

The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5 μm FFPET section with automated test results generated within 8 hours. The analytical sensitivity of the test, defined as ≥95% mutation detection rate, was found to be ∼5% mutant allele in BC FFPET tumor sections representing 4 prevalent PIK3CA mutations (H1047R, E545K, E542K, and Q546K). Additionally, the test was able to detect all 17 mutations at ≤ 5% mutant allele in plasmid/gDNA blends. The test was also compared to 2X bi-directional Sanger sequencing using 110 BC FFPET specimens. The PCR test detected mutations in 50 specimens that were Sanger (+) (100% positive agreement). Moreover, the test detected mutations in 4 Sanger (-) specimens and detected a second mutation in 1 Sanger (+) specimen that was not detected by Sanger. All 5 of the PCR (+)/Sanger (-) were confirmed mutant by 454 sequencing.

Sanger Sequencing Mutation Detected Mutation Not Detected Total cobas® PIK3CA Mutation Test Mutation Detected 50* 4 54 Mutation Not Detected 0 56 56 Total 50 60 110 *One specimen had an additional mutation.

When a panel of BC FFPET specimens was repeatedly tested over multiple days by 2 operators using 2 instruments and 2 reagent lots, a correct call was obtained in 99.5% (191/192) of tests. The results demonstrate that the cobas® PIK3CA Mutation Test is a fast, accurate and reproducible assay that is more sensitive than Sanger sequencing for the detection of PIK3CA mutations in BC tumors.

Citation Format: Peter Schlag, Corey Hoeppner, Anona Bristol, Namneet Kaur, Stephen Quashnick, Ramani Ravirala, Joyce Kramer, Minet Negash, Victoria Brophy, Sung Lee, Stephen Soviero. A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2013-4217
Abstract 4220: Establishment of a rapid and automated detection system for BRAF mutations in malignant melanoma.
Authors: Ide Masaru , Koba Shinichi , Nagano Yumi , Aragane-Sueoka Naoko , Sato Akemi , Inoue Takuya , Kobayashi Naomi , Misago Noriyuki , Narisawa Yutaka , Kimura Shinya , Sueoka Eisaburo
Published: August 2014

BRAF gene mutations have been observed in 30-70% of malignant melanoma patients. Recent development of therapeutic intervention using BRAF inhibitors requires an accurate and rapid detection system for BRAF mutations. In addition, there are only a few reports that investigated the clinical characteristics of the melanoma associated with BRAF mutations in Japanese patients on a large scale evaluation. We recently established quenching probe system (QP) for detection of an activating BRAF mutation, V600E and evaluated 113 melanoma samples diagnosed in Saga University Hospital from 1982 to 2011. The QP system includes fully automated genotyping, based on analysis of the probe DNA melting curve, which binds the target mutated site using a fluorescent guanine quenched probe. BRAF mutations were detected in 54 of 115 (47%) including 51 of V600E and 3 of V600K in Japanese melanoma cases. Among clinical subtypes of melanoma, nodular melanoma showed high frequency (12 of 15; 80%) of mutation followed by superficial spreading melanoma (13 of 26; 50%). The QP system is a simple and sensitive method to determine BRAF V600E mutation, and will be useful tool for patient-oriented therapy with BRAF inhibitors.

Citation Format: Masaru Ide, Shinichi Koba, Yumi Nagano, Naoko Aragane-Sueoka, Akemi Sato, Takuya Inoue, Naomi Kobayashi, Noriyuki Misago, Yutaka Narisawa, Shinya Kimura, Eisaburo Sueoka. Establishment of a rapid and automated detection system for BRAF mutations in malignant melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4220. doi:10.1158/1538-7445.AM2013-4220
Abstract 4225: PNA mediated real-time PCR clamping assay to detect IDH1 mutations .
Authors: Yoon Jihye , Choi jae jin , Choi Woohyung , Park Heekyung
Published: August 2014

The hotspot mutations in codon 132 of isocitrate dehydrogenase 1 (IDH1) is a frequent DNA alteration in gliomas and astrocytomas. IDH mutations are prognostic and diagnostic for tumors with mutation, associated with a proneural subclass, and have longer survival compared with those without the mutation.

We have developed a highly sensitive and simple method to detect hotspot mutations in codon 132 of IDH1 using PNA-mediated real-time PCR clamping, PNAClampTM.

PNAClampTM is based on unique properties of PNA probes that PNA oligomers are not recognized by DNA that can serve as sequence-selective clamps during PCR amplification. This method can also detect 5 mutations through only one tube reactions simultaneously, which requires only 10 ng of the target DNA each.

Data analysis was much faster and easier to perform than analysis of the sequencing data, and results could be obtained in 3hr from DNA extraction to analysis.

Furthermore, we could readily identify a mixture of 1% mutant allele vs. 99% wild type allele with our PNAClamp assay, in comparison to direct sequencing assay which have detection limit of approximately 20% mutant allele.

We performed the assay on 191 paraffin-embedded glioma specimens in Korea for diagnosing IDH mutation.

The results showed that the PNAClampTM IDH1 detected higher number of mutation (27.2%, 52/191) than of direct sequencing (20.9%, 40/191) in 191 giloma samples in Korea. The result was not in agreement with the sequencing in12 (6.3%) samples. This result was likely due to the high sensitivity of PNAClampTM.

PNAClampTM is a fast, sensitive and easy to perform method which can reliably detect common mutations of IDH genes in gliomas or other tumors.

Citation Format: Jihye Yoon, jae jin Choi, Woohyung Choi, Heekyung Park. PNA mediated real-time PCR clamping assay to detect IDH1 mutations . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4225. doi:10.1158/1538-7445.AM2013-4225
Abstract 4229: Study on the germline mutation and protein expression of hMLH1 and hMSH2 gene in Chinese HNPCC probands.
Authors: Sheng Xia
Published: August 2014

BACKGROUND&AIMS: Compare with sporadic colorectal cancer (SCRC), Hereditary nonpolyposis colorectal cancer (HNPCC) shows its own characteristcs associated with the molecular mechanism,clinical features,the methods for treatment and management of HNPCC kindreds. The molecular genetics basis of HNPCC was the mutation of mismatch repair (MMR) gene. Some HNPCC families which can found the mutation of MMR gene didn't fulfil any clinical criteria while more than fifty percent suspected HNPCC patients can't found mutation of MMR gene. Therefore, the tumorigenesis of HNPCC must be associated with other disease genes which have not been credited. The expression of hMLH1 and hMSH2 protein in 50 HNPCC probands fulfilling defferent clinical criteria and the germline mutation of hMLH1 and hMSH2 gene in 26 probands were analysed. Methods: The peripheral blood was collected from the probands of 26 HNPCC families fulfilling different clinical criterial, of which 7 families fulfilled AC, 19 kindreds fit BG. Genomic DNA was extracted following the manafacturer's protocol and used as the template to amplify 35 exons of the 2 genes by PCR. PCR products were purified and used as the template for direct DNA sequencing. The results of sequencing were analysed by different bioanalysis software. To further investigate the pathological effects of detected missense mutations, we analyse the same exon of hPMS2 gene using PCR-based sequencing in 130 healthy persons without family history. IHC envision two step method was performed.Results: 8 germline mutation of hMLH1 and hMSH2 was found in 26 HNPCC probands families. 4 fulfilled AC and 4 fulfilled BG..17 probands was found to be hMLH1 and hMSH2 protein deficient. Conclusion: The rate of germline mutation and protein expression was 30.8% and 34%. AC are the most sensitve clinical creteria to predict mutations. Immunohistochemical staining are reliable screening method with high predictive value for the detection of mutation. Some suspected HNPCC patients can't found mutation of MMR gene.

Citation Format: Xia Sheng. Study on the germline mutation and protein expression of hMLH1 and hMSH2 gene in Chinese HNPCC probands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2013-4229
Abstract 423: Metabolomic analysis of urine from patients undergoing total body irradiation.
Authors: Laiakis Evagelia C. , Mak Tytus D. , Anizan Sebastien , Amundson Sally A. , Barker Christopher A. , Wolden Suzanne L. , Brenner David J. , Fornace Albert J.
Published: August 2014

Development of biomarkers for the responses to radiation has relevance to assessment of patient course and outcome after radiation therapy, as well as to other radiologic events including accidental and terrorist. The purpose of this study was to extend the biomarker discovery to humans, as previous studies have focused on mice. Urine was collected from patients undergoing total body irradiation (TBI) at Memorial Sloan Kettering prior to bone marrow transplantation at 4-6h post irradiation (a single dose of 125cGy) and 24h (three fractions of 125cGy each). Patient diagnoses include leukemias (ALL, AML, and CML), non-hodgkins lymphoma, myelodysplastic syndrome, aplastic anemia, and essential thrombocytopenia. Global metabolomic profiling was obtained through analysis with Ultra Performance Liquid Chromatography (UPLC) coupled to time-of-flight mass spectrometry (TOFMS) (Waters). Peak alignment and deconvolution of the chromatographic data were conducted with MarkerLynx software (Waters, MA). Prior to further analysis, each sample was normalized to its respective creatinine. Ions with non-zero abundance values in at least 75% of samples in both groups were analyzed for statistical significance via the nonparametric Kolmogorov-Smirnov test for equality of probability distributions on the log-transformed dataset. Statistical significance for ions with non-zero abundance values in at least 75% of samples in only one group were analyzed categorically for presence (i.e. non-zero abundance) via the Fisher's exact test. Biomarker candidates were verified by tandem mass spectrometry (MS/MS) by comparison to pure chemicals and quantification of the markers was conducted with the triple quadrupole mass spectrometer XEVO TQ MS (Waters). Validated and quantified biomarkers include L-Octanoylcarnitine, Hypoxanthine, Trimethyl-L-Lysine, Acetyl-carnitine, Decanoylcarnitine, Pantothenic acid, Hippuric acid, Uric acid, and Xanthine, and thus contribute to the development of a human metabolomic signature following exposure to radiation, and can now be integrated with features including underlying disease status, gender, age, and outcome.

Citation Format: Evagelia C. Laiakis, Tytus D. Mak, Sebastien Anizan, Sally A. Amundson, Christopher A. Barker, Suzanne L. Wolden, David J. Brenner, Albert J. Fornace. Metabolomic analysis of urine from patients undergoing total body irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2013-423
Abstract 4230: A novel method for sequence- and strand-specific, genome-wide 5-hmC profiling.
Authors: Sun XueGuang , Petterson Adam , Chung Hunter , Jia Xi Yu , Van Eden Marc E.
Published: August 2014

5-Hydroxymethylcytosine (5-hmC) is a new epigenetic hallmark rapidly getting much interest as the subject of mapping and sequencing work. While the exact function of 5-hmC is not fully understood, it is likely to regulate gene expression via active DNA de-methylation. Previous studies have shown it may play an important role in cell differentiation and carcinogenesis. Cells that are more stem- and progenitor-like have greatly reduced levels of 5-hmC compared with more differentiated cells. Similarly, tumor cells display less 5-hmC than their normal counterparts. This was not associated with either grade or stage, suggesting that global loss of 5-hmC may be an early event in carcinogenesis. To date, several methods have been developed to profile 5-hmC at the genomic level: most are enrichment-based and utilize antibodies to 5-hmC or its modified forms or make use of bioorthogonal labeling and pull-down of glycosylated 5-hmC. The caveat is that these approaches are low resolution and that they require large amounts of input genomic DNA. Initial efforts for detection of 5-hmC at single-base resolution require several micrograms of DNA, require parallel or subtractive sequencing, and employ successive chemical treatments that can degrade DNA and hinder sequencing. Here we report on a new method that by combining modification-sensitive restriction enzymes with massively parallel (“next-generation”) sequencing approaches, genome-wide 5-hmC can be mapped at single site resolution from low (100 ng) DNA inputs. Importantly, this method can be used for strand-specific localization of 5-hmC as well as direct identification of single nucleotide polymorphisms (SNPs) within the sequencing reads. Data can also be compared directly with single-base resolution DNA methylation data from reduced-representation bisulfite sequencing (RRBS) for simultaneous 5-mC and 5-hmC profiling from the same sample. 5-mC and 5-hmC of human brain DNA using this combined method indicates unique distributions of the 5-hmC modification. This new method should provide a unique tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in carcinogenesis.

Citation Format: XueGuang Sun, Adam Petterson, Hunter Chung, Xi Yu Jia, Marc E. Van Eden. A novel method for sequence- and strand-specific, genome-wide 5-hmC profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4230. doi:10.1158/1538-7445.AM2013-4230
Abstract 4232: Increased sensitivity in whole-genome bisulfite sequencing (WGBS): A novel “post-bisulfite conversion” library construction method from sub-nanogram DNA inputs.
Authors: Sooknanan Roy , Hitchen John , Caruccio Nick
Published: August 2014

Genome-wide analysis of 5-methylcytosines is possible with whole-genome bisulfite sequencing (WGBS), where unmethylated cytosine residues are converted to uracil. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under conditions required for complete conversion. Typically, ∼90% of input DNA is degraded and thus, is especially problematic with limited starting amounts of DNA. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite “post-library construction” can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment resulting in incomplete coverage and bias when performing whole genome bisulfite sequencing.

Here, we describe a novel “post-bisulfite conversion” library construction method for preparing NGS libraries from genomic DNA prior to the addition of one or both adapters. This “post-bisulfite conversion” library construction method uses the resulting untagged or mono-tagged single-stranded DNA as template for the subsequent addition of adapter sequences required for NGS. Thus, single-stranded DNA fragments independent of size and position of strand breaks remain as viable templates for library construction, eliminating the loss of fragments and the selection bias associated with a “post-library construction” bisulfite conversion strategy. This novel “post-bisulfite conversion” library construction method exhibits high diversity, increased efficiency and sensitivity (500 picograms human genomic DNA input), and improved coverage required for WGBS.

Citation Format: Roy Sooknanan, John Hitchen, Nick Caruccio. Increased sensitivity in whole-genome bisulfite sequencing (WGBS): A novel “post-bisulfite conversion” library construction method from sub-nanogram DNA inputs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2013-4232
Abstract 4234: The use of a synthetic peptide-DNA complex as an internal reference for chromatin immunoprecipitation.
Authors: Li Zirong , Thyagarajan Bhaskar , Rosenfeld John , Solache Alejandra , Sturges Michael
Published: August 2014

Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate interaction of proteins with specific genomic DNA regions in vivo to provide a better understanding of mechanisms of gene regulation, DNA replication and DNA repair. However, variations in the efficiency of immunoprecipitation, background signals from carryover in immunoprecipitation, and loss of material during immunoprecipitation and purification of the ChIP DNA are sources of variability that restrict the use of ChIP as a quantitative tool. To address these limitations, we have developed a simple spike-in control that enables labs to generate results that are more consistent and more quantitative. The approach we have developed uses a synthetic peptide-DNA reagent that is spiked into chromatin immunoprecipitation reactions. Because this reagent mimics the behavior of cross-linked chromatin and can be quantitatively recovered by immunoprecipitation, it can be readily incorporated in to virtually any ChIP protocol. To utilize this internal control, a fixed amount of the synthetic peptide-DNA complex is spiked into the chromatin and co-ChIPed simultaneously with the antibody of interest. The recovered synthetic DNA is then used to normalize the results obtained with the antibody of interest. This spike-in control can be designed using a peptide specific to the ChIP antibody of interest and recovered using a single antibody. Alternatively, a universal spike-in control can be designed using a peptide that is recognized by an epitope tag antibody. This universal spike-in control and epitope tagged antibody are used along with the antibody of interest in a standard ChIP reaction. Our data show that the inclusion of our internal control significantly reduces variability between ChIP assays, thus increasing the accuracy of the ChIP data. Because of the universal feature, this method can also be used to compare ChIP data between different antibodies or to normalize data variations that often result between users and multiple experiments.

Citation Format: Zirong Li, Bhaskar Thyagarajan, John Rosenfeld, Alejandra Solache, Michael Sturges. The use of a synthetic peptide-DNA complex as an internal reference for chromatin immunoprecipitation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4234. doi:10.1158/1538-7445.AM2013-4234
Abstract 4242: Methylation, SNP and expression analysis of the Vitamin D receptor (VDR) gene in different tumor tissues.
Authors: Poulin Matthew L. , Meyer Ann , Gonzalez Gregory , Bi Kevin , Yan Liying
Published: August 2014

The vitamin D receptor gene encodes the nuclear hormone receptor for vitamin D3 and belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. Downstream targets of this nuclear hormone receptor are principally involved in mineral metabolism though the receptor regulates a variety of other metabolic pathways, such as those involved in the immune response and cancer. In light of evidence for promoter methylation of the vitamin D receptor in the control of the expression of this gene, we have designed methylation assays that cover many regions of this gene in order to determine its methylation profile in normal individuals, ovarian tumors and ovarian cell lines. We have isolated the DNA from ten tumor tissues and their adjacent normal tissue from four different tumor types; ovarian, breast, colon and cervical. By utilizing a combination of bisulfite sequencing, SNP genotyping, and expression analysis, we show that there are distinct patterns of methylation levels between different regions of the VDR gene in each of the cancers observed. We have also analyzed a number of SNPs within these genes and show a possible correlation with the methylation and /or expression of the genes and the SNP genotypes. These data may provide information that could lead to the development of biomarkers for different cancers.

Citation Format: Matthew L. Poulin, Ann Meyer, Gregory Gonzalez, Kevin Bi, Liying Yan. Methylation, SNP and expression analysis of the Vitamin D receptor (VDR) gene in different tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4242. doi:10.1158/1538-7445.AM2013-4242
Abstract 4249: Landscape of DNA methylation status of human hepatocellular carcinoma revealed by Human Methylation BeadChip 450K.
Authors: Nishimura Takafumi , Mori Yukiko , Nishida Naoshi
Published: August 2014

 Global DNA hypomethylation and regional hypermethylation in CpG island has been recognized as common feature of cancer DNA, including that of hepatocellular carcinoma (HCC). However, precise mechanism underlying the alteration of methylation is still unclear. We conducted a genome-wide methylation analysis to elucidate distribution and characteristics of genes that subject to cancer-related DNA methylation in human HCC.  With written informed consent, 20 HCC and 3 corresponding matched non-cancerous liver tissues were obtained at the time of hepatectomy (2 tumors) or orthotopic living-donor liver transplantation (17 tumors and 3 non-tumors). After bisulfite modification, the converted DNA was subject to hybridization on Infinium Human Methylation BeadChip 450K (Illumina, Inc., San Diego, CA) that contained 485,577 CpGs of the human genome including gene promoters and repetitive DNA elements. The methylation level for each CpG was represented as a beta value according to the fluorescent intensity ratio. In order to identify alteration of methylation profile between HCC and non-tumor liver, difference of average beta values between tumor and non-tumor was calculated.  Of 485,577 CpG loci, differences of average beta values of 460,036 loci (94.7%) were within 30%. These were considered as constitutively methylated or unmethylated. Tumor-specific hypomethylation was found at 18,807 loci. Of these, 65.1% were located outside CpG islands, shore or shelf (so-called ‘open sea’). Tumor-specific demethylation rarely occurred within CpG islands (6.5%). On the other hand, tumor-specific DNA methylation was found at 6734 loci. Of these, 77.6% were located within CpG islands. Interestingly, normally methylated CpGs (average beta value < 0.20) were mainly located in CpG islands (95,943 of 138,081, 69.0%), and many of normally unmethylated or variably methylated CpGs were located in the open sea (165,209 of 347,496, 47.5%).  Tumor-specific methylation occurring at CpG islands, may simply be dependent on the distribution of normally unmethylated CpGs. This finding may help us target epigenetically silenced tumor suppressor genes.

Citation Format: Takafumi Nishimura, Yukiko Mori, Naoshi Nishida. Landscape of DNA methylation status of human hepatocellular carcinoma revealed by Human Methylation BeadChip 450K. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4249. doi:10.1158/1538-7445.AM2013-4249
Abstract 425: Fractionated radiation-induced tumor suppressor microRNAs in human prostate carcinoma cells.
Authors: Coleman C. Norman , John-Aryankalayil Molykutty , Makinde Adeola Y. , Palayoor Sanjeewani T.
Published: August 2014

To understand the role of microRNAs (miRNA) in regulation of radiation-induced gene expression and to help define potential radiation-inducible targets, miRNA expression was studied in wild type p53 LNCaP and p53-mutated PC3 and DU145 cells. We have previously investigated the changes in the molecular profiles of tumor cells that were exposed to single dose (SD) versus fractionated radiation (MF) in vitro, and identified immune and stress response pathways that were induced by fractionated but not single dose radiation. Methods: Cells were exposed to 5Gy and 10Gy either as SD or MF radiation radiation. Microarray analyses were done using human Agilent miRNA Microarray Kit (V2). Data were analyzed using Gene Spring software. Validation of the miRNA expression and gene expression of miRNA targets was evaluated by real-time RT-PCR analysis. Target filter analysis of differentially expressed miRNAs (>1.5 fold change and p value<0.05) and their mRNA targets were done. Results: Microarray analyses revealed that radiation differentially expressed 84, 68 and 8 miRNAs with high confidence (>1.5fold change, p<0.05) in LNCaP, PC3 and DU145 cells, respectively. MF radiation affected more miRNAs than SD radiation in all cell lines. In LNCaP and PC3 cells, MF radiation upregulated tumor suppressor microRNAs miR-34a, miR-200, miR-135, miR-221 and let7. Baseline expression of miR-34a was markedly reduced in PC3 cells and DU145 cells compared to LNCaP cells. However, miR-34a was upregulated by fractionated irradiation in LNCaP and PC3 cells, but not in DU145 cells. RT_PCR analysis of 22 experimentally verified miR-34a targets, showed distinct expression patterns in PC3 and LNCaP cells 6 and 24 hours after radiation. An inverse correlation of NOTCH1, E2F5 and MDM4 expression was observed in PC3 cells 6 and 24 hours after MF but not in LNCaP cells. On the other hand, IFNB1 showed an inverse correlation only in LNCaP cells. Majority of the differences in the expression patterns of miR-34a targets were at 24 hours after MF. Conclusion: Differences in the miRNA expression exist between cell lines and after varying radiation regimens. Among the three prostate carcinoma cell lines, tumor suppressor miRNAs are upregulated after MF radiation in LNCaP and PC3 cells. Currently, we are in the process of modulating miRNAs and their targets to examine their effects on radio sensitivity.

This work was supported by the Intramural Research Program of the NIH, NCI, CCR.

Citation Format: C. Norman Coleman, Molykutty John-Aryankalayil, Adeola Y. Makinde, Sanjeewani T. Palayoor. Fractionated radiation-induced tumor suppressor microRNAs in human prostate carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2013-425
Abstract 4265: Derivation and characterization of endometrial cancer cells resistant to phosphatidylinositol-3-kinase (PI3K) pathway inhibitors.
Authors: Klempner Samuel J. , Myers Andrea P. , Li Jane , Mills Gordon , Cantley Lewis C.
Published: August 2014

Endometrial cancer remains the most common gynecologic malignancy in the United States with an estimated 43,000 cases annually. Limited options exist for patients with recurrent endometrial cancer after tumor progression following platinum/taxane combinations, and newer therapies are needed. Endometrial cancers have among the highest rates of oncogenic aberrations in the phosphatidylinositol-3-kinase (PI3K) pathway with frequencies approaching 75%, and clinical trials targeting components on the PI3K pathway are ongoing. Despite the clinical observation that many responses to PI3K inhibitors are short lived an understanding of the molecular mechanisms of resistance is lacking. We have derived endometrial cancer cell lines resistant to a panel of PI3K/AKT/mTOR inhibitors in clinical development in an effort to understand molecular mechanisms and signaling changes underlying resistance to PI3K-targeted therapy. All lines have previously undergone mutational analysis of the PI3K and RAS-ERK pathways. Representative compounds inhibiting each node in the canonical class I PI3K signaling cascade have been chosen. These agents have been synthesized base on the published structures and include a pan-p110 inhibitor, an allosteric AKT inhibitor, a dual p110-mTOR, and a MEK inhibitor. Using parallel high-throughput intracellular phospho-kinase and phospho-receptor tyrosine kinase (RTK) arrays for screening we have identified candidate common resistance mechanisms to inhibition of signaling nodes in the PI3K pathway. The phospho-screening approach has highlighted the interplay between PI3K/AKT/mTOR signaling and the Ras/Raf/MEK/ERK pathways in the development of resistance. We have begun to validate ERK upregulation as an activated escape pathway in patient samples from an AKT inhibitor clinical trial. Conserved resistance profiles may ultimately be used to define a subset of endometrial cancer patients unlikely to respond to PI3K/Akt/mTOR inhibitors and may inform more rational early phase combination strategies.

Citation Format: Samuel J. Klempner, Andrea P. Myers, Jane Li, Gordon Mills, Lewis C. Cantley. Derivation and characterization of endometrial cancer cells resistant to phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2013-4265
Abstract 4267: MUC1 increases epidermal growth factor receptor levels and signaling in endometrial cancer cell lines.
Authors: Engel Brian J. , Carson Daniel D.
Published: August 2014

Endometrial cancer is the most common gynecological cancer, affecting over 600,000 women in the United States. Furthermore, increased incidence of endometrial cancer is observed among obese and premenopausal women. Currently, the standard of care is hysterectomy with the addition of radiation and chemotherapy for late stage cancers. Novel therapies are required to avoid surgical complications associated with obese patients and to preserve the fertility of young patients. MUC1 is a large, heavily glycosylated transmembrane protein that functions to lubricate mucosal surfaces, protect cells from external insult and modulates the embryo implantation process. Recent evidence suggests that the C-terminus of MUC1 is involved in intracellular signaling through interaction with several cancer-associated proteins. One of these proteins is the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that influences cellular proliferation, migration and apoptosis. Elevated MUC1 levels are associated with increased levels and signaling of EGFR in breast cancer; however, this phenomenon has not been described in the context of endometrial cancer and the mechanisms involved are not well understood in any context. To this end, human endometrial cancer cell lines were subjected to siRNA knockdown of MUC1 followed by qRT-PCR, western blotting, EGFR promoter-driven luciferase assays, proliferation and migration assays. These assays demonstrate a correlation of MUC1 levels with EGFR mRNA levels, protein levels, signaling, gene expression and cellular proliferation and migration. These results demonstrate that MUC1 control of EGFR levels and signaling also occurs in endometrial cancer-derived cell lines. Future experiments will elucidate the mechanisms by which MUC1 promotes EGFR gene expression and signaling in endometrial cancer. These insights may provide novel MUC1-based targets for endometrial cancer therapies which when used in conjunction with current non-surgical treatments may provide a therapeutic strategy that avoids surgical complications and preserves fertility. (Work supported by NIH grants RO1 HD 29963 and NCI 2P5098258-06 as well as Rice University funds to DDC).

Citation Format: Brian J. Engel, Daniel D. Carson. MUC1 increases epidermal growth factor receptor levels and signaling in endometrial cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2013-4267
Abstract 4269: Human Epidermal Growth Factor (EGF) supresses Tumor Necrosis Factor-a (TNF-a)-mediated induction of Caspase-1, STAT1 and Apoptosis in human cervical carcinoma cells.
Authors: Ozes Osman N.
Published: August 2014

Background: Members of Human Epidermal Growth

Factor Receptors are over-expressed in variety of cancers. The EGFR-mediated activation of signal transduction pathways ultimately induce cellular growth, supress apoptosis and can cause tumor malignancy. On the contrary, TNF-α induces the expression of numerous genes which contribute to the inhibition of growth and the induction of apoptosis. Therefore, we tested the impact of EGF on TNF-α-induced expression of Caspase-1, activation of STAT1 and induction of apoptosis on human cervical carcinoma cells, (ME180).

Results: Treatment of ME180 cells with TNF-α (10 ng/ml) induced the expression of Caspase-1 in a time dependent manner. Also, pre-treatment of these cells with EGF (10ng/ml) completely inhibited

TNF-induced Caspase-1 induction. Since Caspase-1 induction is mediated by

STAT1, the levels of STAT1 activation by

TNF was determined. It was found that TNF induces STAT1 activation in these cells. However, pre-treatment of ME180 cells with EGF completely inhibited TNF-α-mediated activation of STAT1. Parallel to these findings, pre-treatment of ME180 cells with EGF for 1 hour completely inhibited TNF-induced apoptosis in these cells.

Conclusion: These results suggest that tumor microenvironment created by the tumor or surrounding cells are important in controlling the growth and apoptosis of cancer cells. Therefore, abundance of growth factors and pro-apoptotic cytokines may determine the fate of tumor behavior.

Citation Format: Osman N. Ozes. Human Epidermal Growth Factor (EGF) supresses Tumor Necrosis Factor-a (TNF-a)-mediated induction of Caspase-1, STAT1 and Apoptosis in human cervical carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4269. doi:10.1158/1538-7445.AM2013-4269
Abstract 4272: NanoLucTM as an improved BRET donor to monitor EGFR interactions within living cells.
Authors: Robers Matthew B. , Machleidt Thomas , Kirkland Thomas , Woodroofe Carolyn , Fan Frank , Cong Mei , Wood Keith
Published: August 2014

As a small, versatile protein fusion reporter, NanoLuc™ luciferase offers superior brightness and sensitivity compared to any luciferase described to date. The small size and enhanced brightness of NanoLuc™ makes it ideally suited as an energy donor for Bioluminescence Resonance Energy Transfer (BRET) to a variety of fluorescent acceptors within living cells. By combining genetic reporters such as NanoLuc™ luciferase and HaloTag™ fluorescent labelling technology, it is possible to monitor the interactions of cell surface receptors such as EGFR with adapter proteins in response to growth factor stimulation. In addition to demonstrating inducible protein interactions, it may also be possible to apply this foundational BRET system to the measurement of ligand or small molecule binding events within living cells.

Citation Format: Matthew B. Robers, Thomas Machleidt, Thomas Kirkland, Carolyn Woodroofe, Frank Fan, Mei Cong, Keith Wood. NanoLucTM as an improved BRET donor to monitor EGFR interactions within living cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2013-4272