bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2116: High-sensitivity K-RAS mutation detection using a plant endonuclease-based genotyping method
Authors: Legendre Ben , Shi Yanggu , Wu Grant , Gerard Gary F. , Eastlake Phil , Martin Gerald , Hild David , Kaldjian Eric
Published: February 2011

Newly developed therapeutic agents targeting the epidermal growth factor receptor (EGFR) have proven to be effective against colorectal cancer. However, in certain colorectal cancers these drugs are ineffective. Around 40% of colorectal tumors carry K-RAS gene mutations associated with poor response to EGFR antagonists. K-RAS mutation status can therefore be used to determine whether or not a tumor will respond to anti-EGFR therapy.

SURVEYOR Nuclease is a mismatch-specific plant DNA endonuclease that can scan for known and unknown mutations and polymorphisms in heteroduplex DNA by cleaving DNA with high specificity at sites of base-substitution mismatch and other alterations. This DNA endonuclease cuts both strands of a DNA heteroduplex on the 3’-side of the mismatch site, yielding fragments of smaller size that can be detected by size discrimination technologies. Small insertions/deletions as well as all base-substitution mismatches are recognized; the efficiency of cleavage varies with the sequence of the mismatch. SURVEYOR Scan K-RAS is a research-use-only kit for detecting all sequence variations in exons 2 and 3 of the K-RAS gene.

Studies using plasmid controls have demonstrated detection of all known activating exon 2 mutations in codons 12 and 13, as well as the most common mutation in exon 3, Q61H. Limit of detection experiments have demonstrated that SURVEYOR Scan is highly sensitive, capable of detecting a mixture of 1% mutant in a background of 99% non-mutant DNA. The resulting digestion patterns for codons 12, 13 and 61 are highly specific and recognizable by comparison with controls. In addition, since heteroduplex cleavage is not position specific, mutations outside of these three codons also are detected. SURVEYOR Scan has been used successfully with HPLC (WAVE® HS System) and with automated micro-fluidic capillary electrophoresis (Caliper Lab Chip GX) suitable for high-throughput analyses. Use of SURVEYOR Scan K-RAS can decrease sequencing burden and aid aggressive sequence calling where automated sequencing software fails to definitively resolve the presence of a mutation. This method has also been successfully applied to activating mutations in the BRAF and PIK3CA genes as well. The extension of SURVEYOR Scan to mutation analysis in tumor suppressor genes, such as p53, in which inactivating mutations may be present in multiple exons in numerous locations, is anticipated to be of significant utility, since mutation-specific methods are not practical and sequencing requirements are onerous.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2116.
Abstract 2117: Detection of rare mutations in patient samples by comparative allele-specific TaqMan® assay-based PCR (CastPCR)
Authors: Tan Ruoying , Brzoska Pius , Chen Caifu
Published: February 2011

Circulating tumor cells and circulating tumor DNA in the blood of cancer patient may carry cancer-associated mutations. Detection of these mutations directly from the blood of cancer patient is the key to early cancer diagnosis. Here we report a new comparative allele-specific TaqMan® assay-based PCR (CastPCR) method for rare mutation detection. CastPCR combines allele-specific TaqMan® assay with allele-specific MGB-modified blockers to suppress amplification of the wild type allele. We have demonstrated that CastPCR has TaqMan® assay-like sensitivity, linearity and dynamic range; can detect single mutant molecule in the presence of 1 million wild-type molecules; and can detect rare cancer-associated mutations in cancer patient samples. CastPCR will have broad applications in cancer research and in cancer diagnosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2117.
Abstract 2125: Somatic hypermutations in hairy cell leukemia suggest a canonical process, except in patients with the poor-prognosis VH4-34 variant
Authors: Arons Eugeny , Suntun Tara , Kreitman Robert J.
Published: February 2011

Hairy cell leukemia (HCL) cells express an immunoglobulin (Ig) heavy chain variable (VH) gene with different degrees of somatic mutation. We recently reported that of 82 patients with HCL, 14 expressed VH4-34 and that these patients had a poorer outcome compared to the other 68 patients. Moreover, poor prognosis in the VH4-34 patients was independent of the known poor-prognosis variant of HCL, called HCLv. To confirm these findings, and to more extensively characterize the somatic mutations occurring in the non-VH4-34 patients, we studied mutations from these and an additional 32 patients. A total of 118 rearranged VH sequences from 114 HCL patients were analyzed. Four patients had 2 rearrangements each, none of them VH4-34. Seventeen (15%) of the 114 patients had VH4-34 rearrangements, comprising 10 (38%) of 26 HCLv and 7 (8%) of 88 classic HCL patients (p=0.0005). Sixteen (94%) of 17 VH4-34 rearrangements were unmutated (>98% homologous to germline), compared to 15 (15%) of the other 101 (p=2 × 10-10). Median homology to germline was 99.6% in VH4-34 vs 95.4% in the other rearrangements (p<0.0001). Previous clinical findings that VH4-34+ HCL is associated with high white blood cell count at diagnosis, inferior response and progression-free survival after initial cladribine therapy, and poor overall survival from diagnosis independent of HCLv status, were confirmed. Characterization of the locations of somatic VH mutations in lymphocytic leukemias is rare. One published study reported that in 172 chronic lymphocytic leukemia (CLL) rearrangements, mutations were concentrated in the complementarity determining (CDR) regions, with higher replacement/silent (R/S) mutation ratios for CDRs (4.60) vs framework (FR) regions (1.72) (Blood, 103:3490-3495, 2004). We found that in our 118 HCL rearrangements containing 1385 mutations, R/S was 2.40 overall, 4.29 for CDRs, and 1.83 for FRs, nearly identical to the reported CLL data. Other features reported in CLL, including mutations preferentially in RGYW hot spots and consisting predominantly of transitions rather than transversions, were also observed in HCL. These data suggest that like CLL, VH mutations in HCL result from canonical somatic hypermutation and select for stable antigen receptors, similar to the mutation process taking place in the normal germinal center. However, the origin of VH4-34+ HCL appears different and requires further study.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2125.
Abstract 2127: Screening of the PALB2 gene in 57 Chilean patients with hereditary breast cancer
Authors: Alvarez Carolina , Gajardo Patricia , Sánchez Alejandro , Tapia Teresa , Herrera Cristobal , Benavides Felipe , Camus Mauricio , Alvarez Manuel , Carvallo Pilar
Published: February 2011

PALB2 is essential for the nuclear localization and function of BRCA2. Together, these proteins participate in DNA damage repair by Homologous Recombination (HR). It has been described, that women carrying mutations in PALB2 gene have greater susceptibility to develop breast cancer than non-carriers. In order to determine the presence of PALB2 mutations among Chilean breast cancer patients, we analyzed through Single Strand Conformomer Polymorphisms analysis (SSCP) and Direct Sequencing, 57 Chilean families with breast cancer, that are not carriers of BRCA1 or BRCA2 mutations. We found 11 sequence alterations: 8 exonic, 2 intronic, and 1 in 5′UTR region. Among these, 2 are novel and found for the first time in our study: an intronic deletion c.3351-66delT and an exonic silent variant p.G752G. With the purpose of assessing the effect of each aminoacidic variant on PALB2, we use the software Polyphen, showing that the missense variant p.G998E may affect the function and/or structure of PALB2 protein. We also evaluated a possible effect of some silent variants on splicing using the software ESEfinder, finding that the 5′UTR nucleotide substitution c.1-47G>A, and the silent variant p.T1100T affect the binding of SR proteins to Exonic Splicing Enhancer sequences. The 5′UTR variant and two aminoacidic substitutions, p.G998E and p.Q559R, were screened in 200 healthy controls, finding no association to hereditary breast cancer. Our work is the first mutation screening study for PALB2 gene among families with breast cancer in a Latin American population.

Financed by FONDECYT 1080595

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2127.
Abstract 2128: Whole transcriptome sequencing in normal and cancer prostate tissue
Authors: Tang Wei , Prokunina-Olsson Ludmila
Published: February 2011

Prostate cancer (PrCa) is the second most common type of cancer in males. Established risk factors for PrCa are age, ethnicity and family history. PrCa shows consistent evidence of familial aggregation and heritable factors have been estimated to explain 42% (29-50%) of disease risk in individuals of European ancestry. More than 20 regions harboring genetic risk variants associated with increased risk of PrCa have been identified through Genome-wide association studies (GWAS) to date (MSMB, HNF1B, SLC22A3, LMTK2, KLK2-KLK3, NUDT11, THADA, ITGA6, PDLIM5, TET2, NKX3.1, TH, TTLL1, EHBP1, CTBP2, JAZF1 and 3p12.1, 7p15.3, 8q24, 11q13.3 and 12q13.13 genomic regions). We began to investigate and characterize the entire transcriptome of normal and cancer prostate tissue through a massively parallel sequencing technology (Illumina /Solexa Genome Analyze II). We generated transcriptome profiles based on 36 or 72 bp paired-end reads for cell lines established from cancer and matching normal tissue from two patients. To analyze the data, we used BWA, Bowtie, Tophat packages and Partek Genomics Suite software.

More than 80% of the sequence reads were mapped to the human reference sequence (hg19). The aligned sequences were examined for presence of alternative splicing forms, novel exons, non-coding transcripts and chimeric events compared with a reference transcript annotation from RefSeq Genes of UCSC genome browser database. Based on RPKM algorithm, we also evaluated the expression level in the cancer and normal tissues. We detected over 70,000 transcripts and of ∼ 5, 000 of them were novel splicing isoforms not present in the UCSC database. We examined transcriptome landscape in GWAS-identified loci associated with PrCa. Within the 8q24 region we observed differential expression or evidence of alternative transcripts for SRRM1L, POU5F1B and LOC72767. Within 11q13.3 we observed a novel transcript found only in normal prostate tissue. Many of the risk candidate genes such as ITGA6, PDLIM5, THADA, TET2, SLC22A3, CTBP2 and EHBP1 had multiple splicing isoforms and some of these isoforms were differentially expressed between normal and tumor samples. The expression of alternative transcripts will be confirmed by qPCR in independent samples and correlated with cancer -associated genetic variants identified by GWAS. In conclusion, we generated a resource for studies of PrCa. This reference set will be useful for functional exploration of genetic association signals detected through GWAS.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2128.
Abstract 2129: Pediatric diffuse intrinsic pontine gliomas are genetically distinct from high and low grade astrocytomas
Authors: Pawel Buczkowicz , Bartels Ute , Morrison Andrew , Zarghooni Maryam , Bouffet Eric , Hawkins Cynthia
Published: February 2011

Tumors of the central nervous system constitute the most common type of solid pediatric tumors and account for the majority of mortality and morbidity in pediatric oncology. Brainstem glioma (BSG) is a type of brain tumor that arises in the medulla, pons or midbrain and accounts for approximately 10-20% of pediatric brain tumors. Diffuse gliomas of the pontine region, diffuse intrinsic pontine glioma (DIPG) are the most common type of brainstem tumor, accounting for about 58-78% of BSG and the number one cause of brain tumor related death in children with a 1-2 year survival. Despite this very little is understood about the biology of these tumors. To try to address this lack of knowledge we have undertaken genomic analysis of a series of DIPGs and compared their copy number changes to those seen in pediatric supratentorial high grade astrocytomas as well as low grade astrocytomas. Post mortem tumor and matched normal brain samples (n= 9) and surgical samples (n= 4) were collected for a series of DIPG patients. We performed high-resolution genetic analysis using whole-genome single-nucleotide polymorphism (SNP) arrays (Affymetrix 500K and 6.0). Data analysis was conducted using Partek Genome Suite and Copy Number Analysis Tool (Affymetrix). Analysis of copy number alterations returned hits in several cancer related pathways including MAPK, Wnt, prostate cancer and gliomagenesis. DIPG copy number was also compared to that of pediatric high grade (n= 20) and low grade (n= 30) astrocytomas. Unsupervised Pearson's Dissimilarity clustering resulted in 3 distinct groups. One contained 6 DIPGs; the second contained mostly LGA with a few HGA, while the third group contained mostly HGA with the remaining DIPGs clustering with this group. Although there were some similarities, DIPG are genetically distinct from supratentorial high grade and low grade astrocytomas. A better understanding of DIPG biology is needed in order to develop agents targeted more specifically towards this disease particularly given the dismal prognosis and unresponsive nature of these tumors to conventional treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2129.
Abstract 2130: Genomic profiling identifies CDX2 as a lineage-dependent oncogene amplified in colorectal cancer
Authors: Salari Keyan , Cuff Justin , Giacomini Craig P. , Lin Albert , van de Rijn Matt , Pollack Jonathan R.
Published: February 2011

Somatic DNA alteration underlies tumor development and progression, and gives rise to tumors with diverse genetic contexts. Characterizing these contexts helps identify driving genetic lesions and potential therapeutic vulnerabilities. Here, we surveyed a collection of 29 colorectal cancer cell lines for DNA copy number alterations and identified in a subset of samples gain/amplification on chromosome 13, which was well represented among the genomic profiles of 344 primary colorectal tumors examined, but not in other tumor types. A minimal region of high-level amplification on 13q12.2, containing the caudal type homeobox transcription factor CDX2, was identified in one cell line and 7 of 123 (6%) primary colorectal tumors. A tissue microarray consisting of an independent set of 83 colorectal tumors confirmed the presence of a subset of tumors harboring copy number gain/amplification of 13q with increased CDX2 protein expression. In the context of genomic amplification, RNA interference experiments showed CDX2 is required for proliferation of colorectal cancer cells and promotes cell cycle progression without an effect on apoptosis. CDX2 is a known transcriptional regulator of normal gut development and intestinal epithelial cell differentiation and maintenance. Here we have shown that for a subset of lineage-derived colorectal tumors, cell proliferation is dependent on the abnormal amplification and overexpression of CDX2. Thus, we nominate CDX2 as a novel lineage-dependent oncogene deregulated in colorectal cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2130.
Abstract 2134: Copy number variation in low- and high-grade breast tumors
Authors: Ellsworth Rachel E. , Patney Heather L. , Kane Jennifer L. , Hooke Jeffrey A. , Shriver Craig D.
Published: February 2011

Objective Histological grade classifies breast carcinomas into low-, intermediate- and high-grade. Developmental pathways for high-grade disease remain controversial; clinical data have been used to describe both linear models, in which high-grade tumors evolve from low-grade precursors, as well as models that depict low- and high-grade carcinomas as distinct molecular diseases. Genome-wide copy number alterations were examined in low- and high-grade breast tumors to identify genetic changes associated with the etiology of low- and high-grade breast carcinomas.

Methods Tumors were diagnosed and characterized by a single, dedicated breast pathologist. DNA was isolated after laser microdissection from low- (n= 53) and high-grade (n=68) tumors. Genotype data were generated using Human Mapping 250K Sty arrays (Affymetrix). Copy number alterations and LOH were detected using GenomeConsole 3.0.2 (Affymetrix).

Results The most common alterations in low-grade carcinomas were gain of chromosomes 1q (75%) and 16p (58%) and loss of chromosome 16q (86%). These alterations were frequently simple and included the entire chromosomal arm. In contrast, patterns of alterations were complex in high-grade tumors, frequently with alternating gains and losses on a single chromosomal arm. In high-grade tumors, the most frequent alterations included gain of chromosome 1q (85%), 8q (84%), and loss of chromosomes 8p (54%), 14q (57%) and 17p (69%). Loss of 16q (P<0.0000005) and gain of 16p (P<0.005) occurred significantly more frequently in low- compared to high-grade tumors, while gain of 8q24 (P<0.000005) and 10p15-p14 (P<0.00005) and loss of chromosome 17p13 (P<0.00005) occurred significantly more frequently in high- compared to low-grade tumors. Only two samples, one low- and one high-grade, had a “flat” profile with no detectable chromosomal alterations.

Conclusion Low- and high-grade breast tumors are genetically different. Gain of chromosome 1q is common in both low- and high-grade tumors, and may be an early event in tumorigenesis. Given the significantly different patterns of alterations, especially significantly higher loss of chromosome 16q in high- compared to low-grade tumors, high-grade disease may not arise from dedifferentiation of low-grade but rather represent a separate disease defined by unique genetic alterations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2134.
Abstract 2149: Analysis of the cancer-specific correlation between genomic copy number aberration and gene expression in colorectal cancer
Authors: Iwatsuki Masaaki , Mimori Koshi , Baba Hideo , Mori Masaki
Published: February 2011

Background: Recently, the technical innovation in molecular biological fields such as laser micro-dissection (LMD), cDNA microarray and array-based comparative genomic hybridization (array-CGH) have made it possible to analysis both genomic alteration and aberrant gene expression simultaneously at the level of cancer cell obtained from human cancer tissues. The aim of current study is to identify the novel genes to predict disease recurrence or prognosis with the concordant relationship between genomic alterations and aberrant gene expression during the progression of colorectal cancer (CRC).

Materials and Methods: Candidate gene was found using cDNA microarray and array-CGH after LMD in 133 Japanese CRC. We picked up the several genes among the overexpressing genes with genomic amplification on the responsible loci in CRC. The expression of representative gene and its clinicopathlogical significance, including prognosis were evaluated another subset of 98 CRC cases.

Results: We demonstrated that a correlation between chromosomal aberrations and gene expression in CRC was found in 8q, 13q and 20q. We identified 185 genes overexpressig in specific CRC cells among the genes on these chromosomes. We focused on SUGT1 (13q), which is associated with the assembling of kinetochore proteins at the metaphase of the cell cycle. SUGT1 mRNA expression was evaluated in 98 CRC cases to determine the clinicopathological significance of SUGT1 expression. The mean level of SUGT1 mRNA expression in tumor tissue specimens was significantly higher than in non-tumor tissue. The high SUGT1 expression group was characterized by a significantly elevated frequency of recurrence and a significantly poorer prognosis than the low expression group. Furthermore, SUGT1 expression was found to be a significant factor affecting five year overall survival following surgery in multivariate analysis (p = 0.04). Conclusion: We performed cDNA microarray and array-CGH analysis after LMD in a large scale study of Japanese CRC patients to identify new genomically amplified genes and determine the impact of their presence. Our results indicate that identified genes are not influenced by epigenetic alteration or microRNA. Furthermore, from a technical point of view, it is easier to handle genomic DNA than mRNA, as the latter is fragile, and unstable. Therefore, our findings suggest that this method of analysis might uncover additional genes indicative of patient prognosis and sensitivity to anticancer drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2149.
Abstract 2156: Integration of whole genome exon array and array CGH analysis in gastric and colorectal cancer cell lines
Authors: Furuta Kazuyuki , Arao Tokuzo , Sakai Kazuko , Nagai Tomoyuki , Tamura Daisuke , Aomatsu Keiichi , Kudo Kanae , Kaneda Hiroyasu , Fujita Yoshihiko , Matsumoto Kazuko , Yamada Yasuhide , Yanagihara Kazuyoshi , Sekijima Masaru , Nishio Kazuto
Published: February 2011

Alternative splicing is a major source of protein diversity and cancer is driven by mutations in genes that control the proliferation of cells. The specific exon abnormality may contribute to the etiology of cancer and may provide selective drug targets. However, most studies of alternative splicing in human disease have used a targeted approach and focused on individual genes among whole genome.

In this study, we evaluated an integrated approach of whole genome scale exon array and SNP array to detect cancer-associated alternative splicing, especially focusing on the protein kinase family. We investigated exon-level expression profiling against 22 gastric and colorectal cancer cell lines using Affymetrix Human Exon 1.0 ST Array. Array-based comparative genomic hybridization (CGH) analysis using high-density SNP Array (Affymetrix 250K Nsp /SNP6.0 Array) was also performed to explore genomic amplification/deletion. Several candidate alternative splicing or abnormalities of specific exon expression were detected in cancer-associated genes which detected genomic amplification/deletion frequently; exampling FGFR2, CDKN2A, and CDH13. We found the gene amplification of FGFR2 with C-terminus truncation. We also detected the existence of short CDH13 transcript resulted from small genomic deletion including consecutive 3 exons. In addition, exon-level expression pattern was compared between gastric and colorectal cancer cell lines. In conclusion, integration of whole genome exon array analysis and array CGH analysis may be a promising approach to explore the alternative exon abnormalities.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2156.
Abstract 217: Vitamin E forms induce apoptosis, necrosis, and autophagy and caused accumulation of sphingolipid intermediates in human colon cancer cells
Authors: Rao Xiayu , Jiang Qing
Published: February 2011

Emerging evidence by us and others suggests that certain forms of vitamin E may be promising chemoprevention agents. Here we systemically investigated the effect and mechanism of different forms of vitamin E on the growth and death of human colon cancer HT29 and HCT116 cells. γ-tocopherol (γT), δ-tocopherol (δT), and γ-tocotrienol (γTE) inhibited the proliferation of HT29 and HCT116 cells in a time- and dose-dependent manner, with the potency of γTE > δT > γT. HCT116 cells appeared to be more sensitive to the treatment than HT29 cells. Studies using annexin V and propidium iodide staining revealed that γT (50 µM) and γTE (20 µM) induced externalization of phosphatidylserine and enhanced permeability of the plasma membrane, indicating apoptosis and necrosis. On the other hand, none of the treatments activate caspase-3 and PARP except in HCT116 by γTE at 20 µM. Interestingly, γT, δT and γTE enhanced expression of microtubule-associated protein light chain-3 II (LC3 II) in both HT29 and HCT116 cells, suggesting that these vitamin E forms may induce autophagy. Consistently, electromicroscopic results showed that after 14-h treatment, γTE (20 µM) induced extensive formation of autophagic vacuoles with intact cytoplasmic membrane and little induction of chromatin condensation in HCT116. Myriocin, a specific inhibitor of serine palmitoyltransferase, a key enzyme in de novo synthesis of sphingolipids, partially protected HT29 and HCT116 cells from γTE-induced cell death. Importantly, γTE caused marked accumulation of dihydrosphingosine and dihydroceramide but not ceramide or sphingosine as early as after 8-hour incubation. Because dihydrosphingosine is shown to be a potent inducer of cell death, these findings demonstrated that vitamin E may induce apoptosis, autophagy and necrosis in colon cancer cells by modulation of sphingolipid synthesis pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 217.
Abstract 2171: Viral infection as an etiological risk factor for diffuse large B cell lymphoma (DLBCL): A comparative study between Egypt and Sweden
Authors: Jacobson Therese AS , Bahnassy Abeer A. , Moshfeg Ali , Zekri Abdel Rahman N. , Porwit Anna Porwit , Lundahl Joachim , Mokhtar Nadia M. , Abdel-Hamid Nayera A. , Khaled hussein M. , Mellstedt Håkan
Published: February 2011

Objective: Previous studies have reported an association between increased risk of diffuse large B cell lymphoma (DLBCL) and hepatitis B, hepatitis C, Epstein Barr viruses infection in some countries including Egypt. We therefore assume that the progression of DLBCL might be enhanced by chronic viral infection and that specific pathways can be targeted for the prevention and therapy of DLBCL. Our aim is to assess the possible role of viral infections in the development and progression of DLBCL by determining the genetic profile of DLBCL cases from areas with different prevalence of viral infection. Methods: Gene expression profile of well-characterized cases of DLBCL (30 from Egypt and 16 from Sweden) together with their normal matched controls was studied using the Affymetrix Human GeneShip U133 plus 2.0. Data sets were assigned to two groups (Swedish and Egyptian) and the expression profiles were compared to identify genes that were differentially expressed between the groups. Lists were compiled of all genes for which expression was differentially either induced or suppressed >2-fold between groups. The gene lists were categorized according to their biological functions as described in GeneSpring and Ingenuity. Validation studies were done using RT-PCR, in situ hybridization and immunohistovhemistry. Results: After statistical analysis and filtration by fold change more than 2 times, 2790 genes entities were differentially expressed in the two groups. 1) STAT 2 was highly expressed in Swedish patients and the difference between the two patient groups is clearly indicated in the IFN signaling pathway, 2) higher expression of antigen presentation gene in Egyptian patients compared to Swedish patients (HLA-DQA1, HLA-B, MHCIIα, MHCIα), and 3) higher expression genes in wnt signaling pathway in Egyptian patients compared to Swedish patients (wnt7A, β catenine, CK1). Conclusions: Chronic viral infection could be a risk factor for DLBCL in Egypt (which represents an area with high prevalence of viral infection) but not in Sweden.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2171.
Abstract 2182: Differential transfection on a cell chip for high throughput analysis of paracrine gene effects in angiogenesis and tumor invasion
Authors: Kuhn Elisabeth , Naschberger Elisabeth , Konrad Andreas , Croner Roland S. , Münstedt Helmut , Stürzl Michael
Published: February 2011

Introduction: Tumor invasion and angiogenesis are key processes in tumor progression. Previously, transcriptome analyses identified numerous genes which are associated with tumor progression. The role of these genes in tumor invasion and angiogenesis is mostly unknown. Here we describe for the first time a cell chip-based high throughput analysis of paracrine gene functions in tumor invasion and angiogenesis.

Method: Expression plasmids of tumor progression-associated genes, gelatin and transfection reagent were spotted onto slides using a microarray robot. Subsequently mixtures of tumor cells and target cells (e.g. endothelial cells for angiogenesis, fibroblasts for tumor invasion) were seeded onto the slides. The conditions were adjusted that exclusively the tumor cells but not the target cells got transfected. Restriction of paracrine activities on the different transfection spots was achieved by an overlay with appropriate polymer matrices to decrease spot to spot diffusion. Immunocytochemical detection of the proliferation marker Ki67 and matrix metalloproteinases in the target cells indicated paracrine activities of transfected genes on angiogenesis (increased endothelial cell proliferation) and invasion (increased expression of matrix metalloproteinases in fibroblasts).

Results: Using epithelial tumor cells and fibroblasts the differential transfection on the cell chip could be successfully established. Model experiments with ectopic expression of IFN-gamma in the tumor cells and the detection of IFN-gamma-induced genes in fibroblasts showed that paracrine gene effects could be sensitively detected. An overlay with polymeric matrices restricted locally the diffusion of IFN-gamma on the respective transfection spots. With this method 150 transfections could be carried out on a single chip in order to investigate paracrine gene functions.

Conclusion: Differential transfection on a cell chip provides a novel and efficient high throughput technology to determine paracrine gene functions in angiogenesis and tumor invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2182.
Abstract 2183: High-throughput screening, identification and characterization of novel kinases involved in chemosensitization of breast cancer cells
Authors: Hsieh Charline , Lin Asha , Hsiao Michael
Published: February 2011

Background - Development of drug resistance is considered to be one of the most challenging issues in chemotherapy for cancer patients. As one of the conventional chemotherapeutic agent, paclitaxel was originally extracted from Pacific yew tree and has been applied widely in ovarian cancer, breast cancer and lung cancer. Resistance to paclitaxel has posed severe problems in cancer treatment. We are therefore interested in identifying the genes that render paclitaxel-resistant breast cancer cells to become more sensitive with the drug treatment. The mechanisms and signaling pathways of these kinases in relations to paclitaxel resistance and their molecular targets have been investigated. Methods - We have screened a shRNA library containing 592 novel kinase genes with 1306 clones for the chemosensitizer loci. DNA was extracted in a high throughput manner, and cell transfection/transduction was carried out on 384 well plates using a unique solid-phase DNA delivery method. The positive hits were selected according to the alteration on their IC50 values for the drug using cell viability assays. Quantitative PCR, Western blot, and caspase assays have been employed to elucidate the signaling pathways of these kinases in response to paclitaxel. Protein purification and interaction studies have also been carried out in attempt to elucidate the putative substrates for the kinases and their interaction partners. Results and Conclusions - We have established a high throughput screening platform for identification of the chemosensitizer loci. High transfection/transduction efficiencies can be achieved using our solid-phase gene delivery method. Several novel unknown kinase genes that render drug-resistant cancer cells to become sensitive have been selected. The positive shRNA hits were further verified by quantitative real-time PCR. Among the 20 hits that showed diminished IC50 values of paclitaxel in breast cancer cells, we have selected two candidates for further studies. Knocking down of TAOK3 or VRK3 in breast cancer cells resulted in reduction of IC50 of paclitaxel to half of the original value. Furthermore, we have investigated the signaling pathways of these 2 kinases in relation to paclitaxel resistance. The different signaling pathways elucidated have revealed the complex nature of drug resistance development in cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2183.
Abstract 2184: SUMO-binding proteins as effectors and regulators of SUMO modification
Authors: Guzzo Catherine M. , Uzoma Ijeoma , Zhu Heng , Cox Eric , Blackshaw Seth , Matunis Michael J.
Published: February 2011

SUMOs (Small Ubiquitin-related Modifiers) are reversibly conjugated to other proteins in the cell, thereby regulating a wide range of essential functions. Many of the functions regulated by SUMOylation are intimately tied to processes directly relevant to cancer, including maintenance of genome integrity and cell cycle regulation. Studies in the past several years have in fact linked SUMOylation to a variety of human cancers, including leukemia, prostrate, breast, and colon cancers. Thus, developing a fundamental understanding of the signals and factors that regulate SUMOylation, as well as how SUMOylation affects protein function, has the potential to lead to important new insights into cancer development, diagnosis, and treatment. We hypothesize that the three SUMO paralogs expressed in vertebrate cells (SUMO-1, SUMO-2 and SUMO-3), as well as their polymeric forms, represent unique signals recognized by unique proteins. Moreover, we propose that SUMOylation often promotes interactions between modified proteins and downstream factors containing SUMO-interacting motifs (SIMs). To test this hypothesis, we have initiated high throughput studies to identify novel SUMO-1, SUMO-2 and SUMO-2 polymeric chain binding proteins. Using microarray analysis, we identified ∼400 mostly nuclear proteins, many with preferential affinity for specific SUMO paralogs. Among these 400 putative SUMO-binding proteins was a family of zinc finger proteins that we have determined to possess a novel SIM. This family of proteins contains a stretch of unique tandem zinc fingers called MYM (myeloproliferative and mental retardation) domains. Although the exact functions of MYM-type zinc finger domains are not known, proteins containing these domains are linked to myeloproliferative syndromes, X-linked mental retardation, and transcriptional regulation. We have found that the MYM-type zinc finger domains are both necessary and sufficient for SUMO-binding. MYM-type zinc finger domains represent only the second known class of SUMO-binding motifs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2184.
Abstract 2189: Efficacy of combination therapies in lung cancer
Authors: Sos Martin L. , Weiss Jonathan , Peifer Martin , Thomas Roman K.
Published: February 2011

Genetically activated oncogenes induce dependency on the constitutively activated oncoprotein in cancer cells. Targeted therapeutics that shut down the oncogenic signaling, induce apoptosis and thus lead to tumor shrinkage in patients can exploit this dependency. However, efficacy of single-agent inhibition often is limited by release of negative feedback loops or incomplete suppression of its primary target at clinically relevant concentrations.

The lack of preclinical models that capture the genomic diversity of human tumors impedes the transition from preclinical drug discovery to clinical trials lies in and this diversity. We sought to overcome these limitations and therefore we established a panel of 84 non-small cell lung cancer cell lines and characterized in depth in gene copy number, gene expression and mutation space.

We applied a chemo-genomics approach in order to determine the efficacy of single-agent and combination treatment with PI3K-, MAPK- and receptor tyrosine kinase (RTK-) inhibitors in genotypically defined subsets of tumor cells. We found RTK-driven tumors to largely depend on PI3K and RAS-/ RAF-driven tumors to be addicted to the MAPK signaling pathways, respectively. However, release of negative feedback loops led to activation of the alternate pathway. Combined inhibition of PI3K- and MAPK-pathways potently suppressed release of negative feedback loops, thereby resulting in enhanced induction of apoptosis in tumor cells and tumor shrinkage in vivo. In the subset of EGFR mutated cells harboring the gatekeeper mutation T790M we found limited efficacy of single-agent treatment with irreversible EGFR inhibitors. This limited activity could be linked to decreased binding velocity to the mutant kinase. However combined treatment of T790M-mutant tumor cells with irreversible ERBB inhibitors, and PI3K/mTOR inhibitors led to induction of apoptosis. Recently we established a systematic screening pipeline for the study of combined inhibition of key nodules of oncogenic signaling pathways and preliminary results suggest a critical role of PI3K/mTOR-signaling for efficacy of combination therapies. Overall our approach builds a robust framework for the preclinical study of combination therapies and might help to direct future drug development and patient stratification in clinical trials.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2189.
Abstract 219: Treatment with survivin siRNA augments cytotoxicity of paclitaxel in primary ovarian cancer cells
Authors: Kar Rajarshi , Palanichamy Jayanth K. , Sharma Chandresh , Chattopadhyay Parthaprasad , Jain Sunesh K. , Kumar Lalit , Singh Neeta
Published: February 2011

Introduction

Chemoresistance is a major problem encountered during the treatment of ovarian cancer. Paclitaxel one of the first line drugs is less effective in patients whose cancer cells overexpress survivin. This study investigates whether reducing expression of survivin by siRNA treatment of primary cultures of ovarian cancer cells can increase sensitivity to paclitaxel.

Methodology

Five primary cultures were established from ascites obtained from untreated ovarian cancer patients. Ascites mixed with MCDB 105 and M199 medium (ratio 1:1) was plated in 75 mm2 flasks for establishment of primary cultures. Survivin siRNA was synthesized using in-vitro transcription technique and was quantified using pico green. A dose of 15 nM of Survivin siRNA was used for further experiments. Primary cultures were treated with survivin siRNA using olgofectamine as the transfecting agent. After 24 hours Survivin expression was quantified by RT-PCR. Cells plated in 96 well plates were treated with survivin siRNA (15nM). After 24 hours, paclitaxel treatment was given at four different doses (0.1, 1, 5 and 25 µg/ml) in separate wells. Untransfected cells, cells transfected with oligofectamine alone (mock transfection) and with universal control siRNA were also treated with same doses of paclitaxel after 24 hours. Cell survival was quantified using MTT assay. All experiments were done in triplicates.

Results

On treating the primary cultures with survivin siRNA there was a 6 fold decrease in Survivin expression. Survivin siRNA alone caused 23 ±3% cell deaths in the 5 primary cultures. When these cultures were treated with paclitaxel alone (untansfected cells) at different doses (0.1, 1, 5 and 25 µg/ml) the average cell death was 21.2%, 22.4%, 26% and 37.5% respectively. There was no significant difference in cell death in mock transfected and control siRNA transfected cells when compared to untransfected cells. However when the cells were pretreated with survivin siRNA and then followed by same doses of paclitaxel after 24 hours, cell death increased to 52.8%, 55.4%, 60% and 70% respectively. All the primary cultures were resistant to paclitaxel treatment as 62.5% of the cells survived the highest dose of paclitaxel. Survivin siRNA treatment could significantly increase the sensitivity of the primary ovarian cancer cell cultures to paclitaxel.

Conclusion

Survivin appears to play a major role in the response of ovarian cancer cells to paclitaxel treatment and knocking off survivin expression may prove beneficial in the treatment of ovarian cancer patients with paclitaxel whose tumors overexpress Survivin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 219.
Abstract 2190: Yeast-based pharmacogenomics approaches to identify Cisplatin responsive genes
Authors: Ferrari Elisa , Lucca Chiara , Spitaleri Gianluca , de Braud Filippo , Foiani Marco
Published: February 2011

Platinum-based drugs are largely used in cancer treatment. Using genome-wide approaches we aimed at identifying cisplatin primary and secondary targets and at characterizing cisplatin responsive genes.

The yeast Saccharomyces cerevisiae is widely used to study a variety of eukaryotic cellular processes including those that are relevant for human health.

Yeast can be used for genetic, genomic and mechanistic studies as well as for pharmacogenomic approaches. Using yeast-based pharmacogenomic screens we identified novel genes relevant for the cisplatin response. In particular, we have identified genes involved in DNA repair pathways such as Nucleotide Excision Repair, Homologous Recombination and Post Replicative Repair, as well other genes.

We also used mechanistic approaches to further characterize those factors that have obvious human ortologues and whose functions are still elusive.

Altogether our approaches allowed us to unmask novel factors that contribute to cell viability and genome integrity in response to cisplatin treatment. Complementary studies on the human ortologues are ongoing.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2190.