bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2151: Molecular screening of CRC from India suggests the existence of non-canonical pathway(s) of tumorigenesis
Authors: Ratheesh R , Viswakalyan K , Chaudhary A K. , Swarnalata G , Srinivasulu M , Lavanya M , Anjayneyulu V , Patnaik S , Vamsy M , Murthy S , Ravikanth K , Sastry R A. , Bashyam M D.
Published: February 2011

A major risk factor for Colorectal Cancer (CRC) is advanced age, with a majority of cases reported in the West being above 60 years of age. However, a significantly high proportion of CRC patients from India belong to a younger age group (less than 50 years) and a majority of them have no family history of cancer. A deregulated Wnt signaling pathway is the hallmark of CRC in the West and has been indicted in 70-80% of older patients. Another significant genetic aberration is Microsatellite Instability (MSI), which may contribute to 15-20% of the cases. In addition, the simultaneous occurrence of Wnt activation and p53 inactivation has been substantiated in a significantly high proportion (about 70%) of CRC cases. Sporadic CRC occurring in the young however has not been subjected to detailed analysis so far. In order to identify important deregulated pathways that drive tumor progression in young patients we have initiated a multipronged comparative study on CRC occurring in the young and older patients from India. Our results have revealed several unique features among Indian CRC patients. Firstly, Wnt activation and p53 inactivation appeared to co-exist in a significantly less proportion of samples (34%), indicating existence of alternate pathway(s) of tumor initiation. Secondly, a significantly low proportion (35%) of young patients appeared to harbor a deregulated Wnt signaling pathway, when compared to older patients (64%). Thirdly, we did not find a significant difference in other variables between the two patient age groups including gender, grade, MSI status, etc. Using array-based Comparative Genomic Hybridization (aCGH) and genome-wide transcript profiling, we identified several copy number alterations (CNA) that may harbor genes important for tumor progression in young patients. A novel amplification located at 17q11.2-21.1 was identified which included genes involved in the MAP kinase pathway. The present study is expected to yield invaluable insights into the molecular basis for sporadic CRC in the young.

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 2151.
Abstract 2154: Molecular allelo-karyotyping of pulmonary adenocarcinoma using high resolution single nucleotide polymorphism genomic arrays
Authors: Ninomiya Hironori , Kato Motohiro , Takeuchi Kengo , Kanda Hiroaki , Okumura Sakae , Ogawa Seishi , Ishikawa Yuichi
Published: February 2011

Background Lung cancer is the leading cause of cancer death in Japan. Adenocarcinoma is the most common form of lung cancer with poor-prognosis due to late-stage detection and resistance to conventional treatment. The complexity of the genetic alterations confer pulmonary adenocarcinomas with marked patterns of gene expression. It is now critical to unravel the mechanisms which underlie a subset of tumor and they should have a significant impact in selecting patients for targeted therapy. Novel SNP-chip high resolution molecular allelo-karyotyping analysis allows us to examine global DNA copy number changes and allelic loss to evaluate allele-specific gene dosage in cancer cells.

Methods A series of consecutive 111 lung adenocarcinomas, including 10 cases harboring EML4-ALK fusion, were analyzed using Affymetrix GeneChip and our robust algorithm, CNAG/AsCNAR, to perform sensitive analysis of copy number alteration and allelic composition. This robust algorithm also enabled sensitive detection of loss of heterozygosity. Clinicopathological profiles including age, gender, smoking status, WHO histology subtypes, pathological stage and clinical outcome were also examined. Genomic profiles in the selected amplified and deleted regions were obtained.

Results Chromosomal arm gains of 1q, 3q, 5p, 6p, 7p, 8q, 16p and 17q were detected, while frequent loss was found in 1p, 3p, 6q, 8p, 9p, 10q and 17p. Gene alteration profiles of known oncogenes and tumor suppressor genes were obtained with this procedure. Highly amplified regions included TERT (61.3%), MYC (45.9%), EGFR (44.1%), TNFSF6B (44.1%). Homozygous deletion was noted only in 9p23.3 (5.4%, CDKN2A, CDKN2B). Cases with EML4-ALK fusion were found to be less amplified in top 15 selected regions. Lymphatic invasion positive cases revealed distinct genomic profiles compared to negative ones.

Conclusion Global analysis of gene expression using high resolution SNP genomic arrays can provide a broader view of genomic alterations. Genomic amplification and loss profiles differ according to clinicopathological aspects and genomic background.

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 2154.
Abstract 2158: Loss of heterozygosity at chromosome 3p in ductal and lobular breast carcinomas
Authors: de Oliveira Marcia Maria Costa , Cavalli Luciane R. , Urban Cícero Andrade , de Lima Rubens Silveira , Ribeiro Enilze Maria de Souza Fonseca , Cavalli Iglenir João
Published: February 2011

The two main histological types of infiltrating breast cancer, lobular (ILC) and the more common ductal (IDC) carcinoma are morphologically and clinically distinct revealing different patterns of chromosomal aberrations and gene expression. Loss of heterozygosity (LOH) at chromosome 3p is frequently observed in breast tumors and a whole genome study utilizing a SNP array assay found a significantly higher frequency of LOH on 3p in ductal than lobular tumors. In order to investigate the molecular alterations at 3p associated with these breast cancer subtypes we analyzed LOH in 118 IDC and 18 ILC samples using seven microsatellite markers. Genomic DNA was obtained from frozen tumor tissue and peripheral blood was used as a normal reference. All samples were genotyped on a MegaBace® system and analyzed using Fragment Profiler® Version 2.1 software. Significance was determined using Chi-square test (X2). A significant higher frequency of LOH was observed in IDC (62.7%) when compared with ILC (38.9%) (X2=4.60 P<0.05). When the markers were analyzed separately, LOH at three of them, D3S1307 in 3p26.3, D3S1286 in 3p24.3 and D3S1300 (intragenic FHIT) in 3p14.2, were significantly more frequent in ductal than lobular tumors (X2= 4.66 P<0.05; X2= 3.99 P<0.05 and X2= 5.43 P<0.025, respectively). D3S1307 marker showed the highest frequency of LOH in IDC (47.8%) and associations between loss of this marker and loss of estrogen and progesterone receptors were found in these samples (X2= 4.61 P<0.05 and X2= 4.49 P<0.05, respectively) suggesting that there are genes on this region involved in the control of differentiation of ductal tumors. For lobular tumors, the highest loss (53.3%) was seen for D3S1274 on 3p12.3 (intragenic DUTT1). In addition to confirm previous studies these findings suggest that specific losses on 3p (3p26.3, 3p24.3 and 3p14.2) are more common in IDC when compared with ILC breast tumors.

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 2158.
Abstract 216: Role of Akt isoforms in chemoresistance of human endometrial carcinoma cells
Authors: Girouard Julie , Asselin Eric
Published: February 2011

AKT (also known as PKB) is a crucial survival kinase that is normally activated by growth factors but is constitutively activated at a high frequency in tumors through oncogenic events affecting its upstream regulation. Since studies have demonstrated that the three Akt isoforms exhibit distinct physiological functions, the aim of this study was to determine the isotype-specific functions for Akt in chemoresistance of endometrial carcinoma cells. In order to determine the role of Akt isoforms, we have stably produced transfectants in which the expression of Akt is downregulated by specific shRNAs for each isoforms using the KLE endometrial carcinoma cell line. This endometrial cell line is known to constitutively express the three Akt isoforms and to highly resist to chemotherapeutic treatment. In this study we have demonstrated that stably downregulation of Akt2 by RNA interference severely sensitized KLE cells to cisplatin and induced apoptosis. Moreover, cisplatin treatment of these cells induced the activation of pro-apoptotic factors such as caspase −3 and −9 but also inhibited the expression of anti-apoptotic factors such as XIAP and bcl-2. Alternatively, we used constitutively active Akt expression vectors for each isoform to produce stable transfectants using the chemosensitive Hec 1A endometrial cancer cell lines in which no Akt activity is detected. Constitutive Akt2 expression leads to increase cisplatin resistance in this stable transfectant. Our findings highlight the contribution of Akt2 isoform in the molecular mechanisms that govern chemoresistance of endometrial carcinoma. Furthermore, Akt isoform-specific transfectants will provide a strong model to determine the involvement of each Akt isoforms in chemoresistance of endometrial carcinoma 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 216.
Abstract 2161: Network-based inference of ALK oncogenic signaling in T-cell lymphoproliferative disorders
Authors: Piva Roberto , Pellegrino Elisa , Agnelli Luca , Cantarella Daniela , Ferreri Cristina , Ferracin Manuela , Cuccuru Giuditta , Voena Claudia , Provero Paolo , Negrini Massimo , Cunto Ferdinando Di , Neri Antonino , Medico Enzo , Inghirami Giorgio
Published: February 2011

Anaplastic large cell lymphomas (ALCL) are a subset of T-cell Non-Hodgkin Lymphomas (T-NHL) in which the intracytoplasmic domain of the anaplastic lymphoma kinase (ALK) gene is frequently fused to nucleophosmin (NPM1). It has been previously shown that the transcription factor STAT3 is a major substrate of ALK enzymatic activity and it is strictly required for the growth and survival of ALK-transformed cells.

With the aim to unravel the regulatory network underlying NPM-ALK-mediated transformation and tumor maintenance, we timely dissected the mRNA and miRNA expression signature of ALK-positive ALCL cell lines following ALK or STAT3 silencing. We observed that the majority of ALK-regulated genes were concordantly dependent on STAT3, including known and novel STAT3 target genes. Additional transcription factors (C/EBP beta, LEF1, and others) emerged as STAT3-dependent initiators and master regulators of ALK oncogenic properties in ALCL. However, STAT3 signaling was inconsistent and completely dispensable in non small cell cancer cell lines expressing EML4-ALK fusion proteins, suggesting a T-cell specific STAT3 regulatory network.

Finally, we analyzed the gene expression profiling of 90 systemic peripheral T cell lymphomas, including a set of ALK positive and negative ALCL (n=36). We observed that ALK positive ALCL were significantly clustered in a separate subgroup, characterized by a relevant STAT3 signature. Using prediction analyses, we identified a set of genomic classifiers able to a clearly separate ALK positive and ALK negative ALCL from other T-NHL.

In conclusion we proved that inference of ALK oncogenic transcriptional networks is a powerful tool to identify meaningful signaling molecules that regulate the transition into a pathologic cellular state. Our molecular classification suggests that ALCL may represent a unique entity discernible from other T-NHL, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, to address the therapy of patients.

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 2161.
Abstract 2163: Global gene expression profiles of the Egyptian hepatocellular carcinoma associated with HCV-Genotype-4
Authors: Zekri Abdel-Rahman N. , Bahnassy Abeer , Hafez Mohamed , Hassan Zeinab
Published: February 2011

Background: Hepatocellular carcinoma (HCC) is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory.

Methods: Forty cases of HCV genotype 4-associated HCC patients from Egypt were studied for gene expression profile by 41 K c-DNA microarray.

Result: Out of the 41,000 genes, 958 were differentially expressed; 503 were up regulated (58 genes are involved in 65 different pathways) and 455 were down regulated (49 genes are involved in 59 different pathways). Only 19 pathways were specifically up regulated by 27 genes (ABCB10, ABCG2, RXRA, PRKAB2, IKBKG, ELOVL2, RAF1, ATP6V1A, FLJ21865, GUSB, IL7, EPO, CD1B, FGFR1, PDPK1, KPS6KB1, NOTCH4, CDK7, MNAT1, PRKACB, RXRA, PRKACB, ACCN1, RPS6KB1, BMP8A, SMAD6, CYCLIN E2) and 13 pathways are specifically down regulated by 19 genes (ARHGEF2,WASL, BACE1, A2M, PPP3CA, SLC1A, ITGAV, NRXN2, NCAM1, PER1, PIGX, ST3GAL2, ST3GAL4, DGKQ, ATG5, ENPEP, POLR2B, LTA). Sixty seven genes showed significant difference in their expression between HCC cases with and without cirrhosis. Confirmation of array data by RT-PCR was performed on a subset of genes (PPP3CA [75%], ATG-5 [85%], BACE [62%] which showed down regulation and ABCG2 [90%], RXRA [80%], ELOVL2 [65%], CXR3 [80%]) which showed up regulation.

Conclusion: This is the first study on the global gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a panel of new prognostic and diagnostic markers for HCV- associated HCC. Also, our data identified some genes which are related to hepatic encephalopathy like ACCN1, BACE1, apoB, GABA-A AND ATG5 which could play an important role in the cross talk between liver and brain. They could also be used to identify candidate genes for molecular target therapy.

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 2163.
Abstract 2167: Unbiased genomic approaches identify 2 major subclasses of Gastric Cancer with prognostic and predictive value superior to Lauren's and also reveals subtype-specific treatment opportunities
Authors: Tan Iain B. , Lee Julian , Ooi ChiaHuey , Lee MingHui , Boussioutas Alex , Tallez Manuel S. , Ngeow Joanne , Lim KiatHon , So Jimmy , Choo SuPin , Wong WaiKeong , Tan Patrick
Published: February 2011

Introduction: The Lauren's classification of gastric cancer (GC) is widely used with 2 subtypes, intestinal and diffuses having distinct histo-pathological and epidemiological characteristics. Beyond diagnosis, Lauren's classes have limited prognostic and no predictive value.

Methods: We performed gene expression profiling on 37 GC cell lines (GCCL) and 2 independent cohorts of 200 (SG) and 68 (AU) primary GC tumors. We used integrative molecular profiling to discover and characterise molecular subtypes of GC.

Results: Unsupervised hierarchal clustering in GCCLs identified 2 distinct major molecular subtypes termed “G1”and “G2”. Linear models for microarray data (LIMMA) identified 171 distinguishing genes. These were mapped into 2 independent datasets of primary tumors where G1 and G2 subclasses were again identified. G1/G2 subtypes, discovered by unbiased techniques, were found to be related to intestinal and diffuse histology respectively (p < 0.001, chi square test) with 69% concordance. We found G1/G2 but not Lauren's classes to be prognostic (G1 vs G2: median survival: 41 vs 25 mths, HR 1.5, 95% CI: 1.1-2.0 p=0.02; Laurens: p=0.6). Among discordant cases, survival approximated G1/G2 classes rather than Laurens. Survival was superior in “G1 but diffuse” compared to “G2 but intestinal” (median 36 vs 17 months, p=0.08). We next examined the interaction of the subclasses with 5-fluorouracil (5-FU), the only chemotherapy approved for adjuvant treatment of GC. 5 -FU sensitivity as measured by GI-50 in a previous publication was greater in G1 lines (p=0.04). In primary tumours, G1 patients had improved progression free survival with 5FU/RT of borderline significance (test for interaction, p=0.09). We next applied genomic pathway analysis using an approach we previously published (Ooi et al. Plos Genetics 2009). Her2/Ras/ER/PR/BRCA pathways and VEGF/p53/EGFR pathways were deregulated in G1 and G2 subtype respectively suggesting possible subtype specific treatment opportunities.

Conclusion: We describe an unbiased genomic approach that improves upon Lauren's to identify the two major subclasses of gastric cancer. G1/G2 classes have superior prognostic and predictive value and subclass specific therapeutic opportunities.

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 2167.
Abstract 2168: Molecular pathway analysis of esophageal squamous cell differentiation versus carcinoma formation
Authors: Yan Wusheng , Zhu Liang , Shih Joanna , Rodriguez-Canales Jaime , Hipp Jason , Player Audrey , Hu Nan , Goldstein Alisa M. , Taylor Philip R. , Emmert-Buck Michael R. , Erickson Heidi S.
Published: February 2011

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer and is characterized by high mortality. Our previous work analyzing candidate tumor associated genes in ESCC highlighted the important differences in mRNA expression between the normal esophageal and tumor epithelial compartments. However, to date there are limited studies examining expression changes that occur during esophageal squamous cell differentiation versus those associated with ESCC tumorigenesis.

To address this issue, we microdissected the basal cell layer and differentiated cell layer from normal epithelium and compared these expression profiles with matched tumor samples in twelve patients using gene expression microarrays (Hu133A 2.0, Affymetrix, Inc.) and qRT-PCR for selected miRNAs. Genes and pathways of interest were determined using class comparison and pathway analyses.

Pathway expression of BRCA1 in the DNA damage response pathway presented the most significant difference (P <0.00001) in normal differentiated cells compared to tumor differentiated cells, but no difference was present between normal basal cells and tumor peripheral cells. PDGF signaling was significant only in the normal basal vs. tumor comparison. Taken together, BRCA1 and PDGF expression suggest oncogenetic process differences in esophageal epithelial compartments. Basal cells presented with CCND1 and CDKN2A high expression and p53 low expression, implying that basal cells gene expression is closer to a pattern of tumorigenesis than that of differentiated cells. A typical “stem cell” pattern of CD44high/CD24low expression in normal basal cells vs. normal differentiated cells and tumor vs. normal comparisons indicated that the basal cell compartment may house progenitor or stem-like cells and tumor cells expression is indicative of this stem-like origin. Converse to our expectations, tumor peripheral vs. tumor center comparison revealed that the ESCC tumor clusters were essentially homogenous on the transcriptomic level.

Overall, the preliminary results indicate that tumor profiles were closer to basal cells than differentiated cells, consistent with their rapid growth rate and less well differentiated morphology. Investigation of gene expression patterns in normal cell types and ESCC provides novel insight into the transcript profiles associated with normal development versus carcinogenesis.

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 2168.
Abstract 2169: Integrated proteomic and genomic profiling data from a tongue squamous cell carcinoma cell line separates normal and tumor tongue tissue
Authors: Ceder Rebecca , Staab Claudia A. , Roberg Karin , Jörnvall Hans , Höög Jan-Olov , Grafström Roland C.
Published: February 2011

In depth characterization of individual human tumor cell lines might serve to explore the concept of future personalized cancer management. To address this question, standardized serum-free cultures of normal oral keratinocytes (NOK) were compared relative to the tongue squamous cell carcinoma line LK0412 via multiple bioinformatics-based assessments of transcript and protein profiles. Two-dimensional polyacrylamide electrophoresis identified 27 differentially expressed abundant proteins in LK0412. Gene Ontology enrichment analysis indicated alterations of the regulation of cell death, protein metabolism, energy pathways and various binding activities. Based on the protein-enriched GO-categories, the web-based program AffyAnnotator conducted the selection of 119 differently expressed transcripts from the microarray data in LK0412 vs. NOK. Ingenuity Pathway Analysis of these pertubated transcripts then revealed associations to cancer, cellular assembly and organization as well as cellular movement, and moreover, identified an 11-gene profile composed of central regulators within the networks. Notably, the differently expressed proteins and transcripts, as well as the 11-gene profile, variably classified two independent public microarray data sets of totally 38 normal and 57 tumor tongue samples, including a power of 95% accuracy from the 11-gene profile. Additional evaluation of the derived signatures by so called “signature evaluation tool” (implementing the Golub's weighted voting algorithm), identified reduced gene number signatures that similarly discriminated the normal from the tongue tissues in both data sets up to an accuracy rate of 95%. The overall results suggest that combined bioinformatics processing of transcript and protein data might serve as a constructive strategy for identification of potential candidate biomarkers in individual tumor samples, that may then serve to aid in cancer management. Additionally, gene signature profiles, generated as described, may serve to improve current efforts to accurately classify normal and tumor tongue tissues.

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 2169.
Abstract 2173: Cytokeratin-19, a predictive marker and an early determinant of progenitor-derived hepatocellular carcinoma
Authors: Andersen Jesper B. , Loi Roberto , Perra Andrea , Factor Valentina M. , Ledda-Columbano Giovanna M. , Columbano Amedeo , Thorgeirsson Snorri S.
Published: February 2011

Background: Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin which in liver includes hepatocyte and/or adult stem/progenitor cells. Objective: To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Methods: Sixty samples classified as focal lesions, adenoma, early and advanced HCCs were micro-dissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. Results: The analysis of progression of the persistent GSTP+ focal lesions to fully developed HCC showed that about 50% of persistent nodules and all HCCs expressed CK19 whereas 14% of remodeling nodules were CK19+. Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Further, supervised analysis using the differentially expressed genes in each cluster combined with the gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19+ lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes vs. normal liver) consistent with remodeling towards differentiated phenotype. Finally, comparative functional genomics revealed a stringent clustering of CK19+ early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19 associated gene expression signature accurately predicted the patient survival (P<0.009) and tumor recurrence (P<0.006). Conclusion: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of the CK19 associated gene signature to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.

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 2173.
Abstract 2177: Long-term storage in TRIzol has little effect on microarray-based gene expression profiling in primary myeloma or acute myeloid leukemia cells
Authors: Wang Michael , Wang Zhi-Qiang , Sun Luhong , Ma Wencai , Graber David , Matthews Jairo , Champlin Richard , Yi Qing , Orlowski Robert Z. , Kwak Larry , Weber Donna M. , Thomas Sheeba K. , Shah Jatin , Kornblau Steven , Davis Richard E.
Published: February 2011

We used paired samples to address whether long-term storage of cells in TRIzol has a significant effect on microarray-based gene expression profiling (GEP). The TRIzol reagent is widely used for the preservation and isolation of RNA, but there is suspicion that prolonged storage of tissues prior to RNA isolation, even at −80 C., can cause chemical modification (depurination) of RNA. This could result in early termination during reverse transcription (RT) of mRNA molecules, potentially affecting GEP more strongly for transcripts with probes located farther from the 3’ end. The Myeloma and Leukemia Tissue Banks at M. D. Anderson Cancer Center routinely collect and store primary tumor samples for research under informed consent. From these banks we selected primary tumor samples of multiple myeloma (MM; CD138+) and acute myeloid leukemia (AML; Ficoll-purified, CD3- and CD19-depleted), for which paired aliquots had been stored frozen in TRIzol (“Tri”) or cryopreservation medium (RPMI + 20% FCS + 10% DMSO, “Cryo”) since the time of initial isolation (range, 2-9 years). Cryo aliquots were quickly thawed, washed, and placed in TRIzol, then total RNA was isolated from all aliquots as per the TRIzol manufacturer's instructions. RNA quality was assessed with a Bioanalyzer 2100 (Agilent); the RNA integrity number (RIN) was > 9 for 12/12 AML Tri, 6/12 AML Cryo, 6/6 MM Tri, and 3/6 MM Cryo samples. RNA from 12 sample pairs (6 AML, 6 MM, all with RIN > 7) was further purified with Qiagen RNAeasy columns, and the Illumina® TotalPrep RNA Amplification Kit (Ambion) was used to generate amplified, biotinylated cRNA after RT by the Eberwine procedure. Bioanalyzer-generated histograms of cRNA were similar for all sample aliquots, suggesting no early termination of RT due to TRIzol. GEP was performed with cRNA on Illumina HT-12 BeadArrays. Analysis of “probe-level” data with GenomeStudio software (Illumina), after median normalization, showed excellent concordance between sample Tri/Cryo pairs for all but probes with low intensities (<50). For the 48795 probes on the arrays, no more than 320 gave DiffScores < −13 or > 13 for any sample pair, and most pairs had <50 probes that differed. After exclusion of low-intensity values, hierarchical clustering associated all Tri aliquots with their corresponding Cryo aliquot. We conclude that long-term TRIzol storage has little effect on microarray-based GEP, and preserves RNA quality better than does cryopreservation.

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 2177.
Abstract 2181: Rapid screening of molecular targets using RT-PCR for solid tumor profiling
Authors: Pouliot Kimberly L. , Cadet Rudolph , Watkins Brynmor , Sonis Stephen T. , Fey Edward
Published: February 2011

Purpose: The identification of molecular targets will be an essential step in developing mechanistically-based approaches to cancer treatment, as well as for determining tumor response and/or resistance to therapy. Not only will identification of these targets in solid tumors, but in the subsequent metastasis as well. The purpose of this study was to evaluate the utility of a large-scale tumor bank, using both xenograft and orthotopic models, as a source of material for quantification of gene expression as a tumor profiling and screening tool.

Experimental Design: Solid tumors representing nine different indications were grown as a xenograft in nude mice. The panel of cell lines included: epidermoid carcinoma (A-431), lung carcinoma (A549, NCI-H460, small cell lung carcinoma: NCI-H82, NCI-H69), pancreatic carcinoma (AsPC-1, BxPC-3 and PANC-1), prostate carcinoma (Du145), breast carcinoma (MDA-MB-231, MDA-MB-435, MCF-7), colon carcincoma (HCT-15, HCT-116 and HT-29), ovarian carcinoma (OVCAR-3 and SK-OV-3) and glioma (U87-MG and U118-MG). In addition, five cell lines representing three indications were grown orthotopically (A549, NCI-H460, MDA-MB-231, MCF-7 and HT-29) in the lung, breast or colon, respectively. When subcutaneously implanted tumors reached a volume of 1500-2000mm3 samples were harvested by aseptic technique. Orthotopically implanted tumors were excised after 25 to 45 days post-implantation and tissues harvested. Animals with mammary fat pad tumors, also had lymph nodes and lungs removed. RNA was purified under sterile conditions. Expression of Akt, phosphoinositide-3 kinase (PI3K), epidermal growth factor receptor (EGFR), p53 and BCL-2 was determined using RT-PCR (Applied BioSystems TaqMan System).

Results: Differential expression of target genes within solid tumors was determined using RT-PCR. An emphasis on gene expression in orthotopic and metastatic models will be discussed.

Conclusion: Unlike semi-quantitiative methods such as immunohistochemistry (IHC) or Western Blotting, RT-PCR is ideal as screening tool as it is rapid, robust, quantitative and only requires a small amount of starting material. We describe a rapid screening method of gene expression in both xenograft and orthotopic models.

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 2181.
Abstract 2187: Optimization free oncogene mutation screening work flow
Authors: Wang Jonathan T. , Lee Michael K. , Janaway Gordon A. , Koch Nathalie , Geuns Elke , Stevens Junko F.
Published: February 2011

BRCA1 and BRCA2 are the two primary breast cancer susceptibility genes in which identifying mutations is important to access cancer risk and decision for treatment. Prescreening of exons by High Resolution Melt (HRM) prior to sequencing can reduce effort in mutation screening in large transcripts such as the BRCA genes. Published methods for BRCA1 and 2 HRM screening have relied on complicated touchdown cycling protocols and custom reagent mixes that can lead to variable result. We have validated a streamlined workflow for adaptability in a clinical diagnostic environment. First, genomic DNA template was extracted on an automated sample processor using magnetic bead technology to purify genomic DNA directly from blood sample. Then, the PCR was performed using a PCR master mix optimized for HRM coupled with universal cycling conditions. We have successfully amplified all 120 amplicons covering exons of both BRCA genes using universal PCR condition. Potential variations identified by HRM screening were confirmed by direct sequencing of HRM PCR amplicons with M13 universal tags. By using unified protocol for PCR and universal sequencing for all amplicons the risk of incorrect procedure is greatly reduced. This simplified workflow for oncogene mutation screening using automated sample extraction and universal protocol for PCR and sequencing provides significant advantages for application in diagnostic settings.

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 2187.
Abstract 22: Cyclin E amplification, a novel mechanism of resistance to trastuzumab in HER2 amplified breast cancer
Authors: Scaltriti Maurizio , Eichhorn Pieter , Gili Magui , Guzman Marta , Aura Claudia , Jimenez José , Prudkin Ludmila , Green Simon R. , Cortés Javier , Chandarlapaty Sarat , Mai Sabine , Hudis Clifford A. , Rosen Neal , Baselga José
Published: February 2011

HER2 amplification/overexpression occurs in 20% of breast cancers and is associated with poor prognosis. Therapeutic agents against HER2, including monoclonal antibodies and tyrosine kinase inhibitors, have shown to improve the survival of these patients. However, primary and acquired resistance to these agents is a major barrier to the effective treatment of this disease.

Aiming to identify the molecular pathways responsible for resistance to anti-HER2 therapy we have established HER2 positive breast cancer cell lines refractory to the anti-proliferative effects of the therapeutic antibody trastuzumab. We have studied their acquired genetic aberrations by performing gene expression microarray and genome single nucleotide polymorphisms analyses. In trastuzumab resistant cells we identified an increase in copy number variation in locus 19q12. This chromosome region comprises seven genes, among them cyclin E1 (CCNE1). We further confirmed Cyclin E over-expression in trastuzumab resistant cells by western blot.

To test the hypothesis that increased cyclin E expression could play a role in the acquisition of trastuzumab resistance we have demonstrated that ablation of cyclin E expression by siRNA restores trastuzumab sensitivity in HER2 positive resistant cells. Furthermore, we have determined that trastuzumab resistant cells are highly sensitive to the specific CDK inhibitor seliciclib and its more potent derivative Cmpd. 5 (Cyclacel) both in vitro and in vivo. In addition, we have analyzed a cohort of HER2 positive breast cancer patients treated with trastuzumab-based therapy and identified a proportion of patients refractory to trastuzumab that harbor amplification of the cyclin E gene. Our results suggest that cyclin E amplification decreases trastuzumab sensitivity and may provide the rationale to test the efficacy of CDK2 inhibitors in breast cancer patients with cyclin E amplification that escaped trastuzumab therapy.

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 22.
Abstract 220: 14-3-3 zeta downregulation sensitizes head and neck cancer cells to chemotherapeutic agents
Authors: Ajay Matta , Desouza Leroi V. , Ralhan Ranju , Siu KW Michael
Published: February 2011

Introduction. Advancements in treatment modalities have not translated into marked improvement in the prognosis of Head and Neck squamous cell carcinoma (HNSCC) patients. Major emphasis is being laid on designing new therapeutic strategies for more effective disease management. Recently, using iTRAQ-labeling and liquid chromatography-mass spectrometry (MS), we identified a panel of biomarkers, including 14-3-3 zeta and 14-3-3 sigma in HNSCC and oral pre-malignant lesions. Retrospective study of HNSCC revealed correlation of increased expression of both these biomarkers with poor disease prognosis. Herein, we report preliminary results on determining the potential of 14-3-3 zeta as a molecular target for HNSCC therapy.

Methods. Small interfering RNA (siRNA) targeting 14-3-3 zeta was used to down-regulate its expression in head and neck cancer cell lines (SCC4 / HSC2). Transient transfection of siRNAs targeting 14-3-3 zeta in these cultured cells was done using LipofectAMINE 2000. The chemosensitivity of transfected cells was determined by measuring the cell death using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Flow cytometry-based cell cycle analysis, Annexin V dye binding assay and TUNEL assay.

Results: Knockdown of 14-3-3 zeta showed reduced cell viability and G2/M arrest in head and neck cancer cells transfected with 14-3-3 zeta siRNA (100 nM, 48 hours). Importantly, doxorubicin / cisplatin treatment of 14-3-3 zeta siRNA transfected head and neck cancer cells (SCC4 / HSC2), showed increased cell death at lower doses in comparison to cells treated with doxorubicin or cisplatin alone. Increased sub-G0 phase was observed in SCC4 cells (showing reduced 14-3-3 zeta expression) on treatment with any of these chemotherapeutic agents. Annexin V staining and TUNEL assay revealed increased apoptosis. Western blot analysis showed cleaved caspase 9 and caspase 3, confirming the induction of apoptosis.

Conclusions: Our results showed that 14-3-3 zeta downregulation sensitizes head and neck cancer cells to chemotherapeutic agents by induction of apoptosis. We propose that down-regulation of 14-3-3 zeta may serve as a novel strategy to improve the efficacy of chemotherapeutic agents in the management of head and neck 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 220.
Abstract 2201: Pathway analysis of breast cancer genome wide association study highlights three pathways and one canonical signaling cascade
Authors: Menashe Idan , Maeder Dennis , Garcia-Closas Montserrat , Figueroa Jonine D. , Bhattacharjee Samsiddhi , Rotunno Melissa , Kraft Peter , Hunter David J. , Chanock Stephen J. , Rosenberg Philip S. , Chatterjee Nilanjan
Published: February 2011

Genome-wide association studies (GWAS) focus on relatively few highly significant loci while less attention is given to other genotyped markers. Employing pathway analysis to existing GWAS data may shed light on relevant biological processes, and illuminate new candidate genes. We employed a pathway-based approach to the breast cancer GWAS data of the National Cancer Institute (NCI) Cancer Genetic Markers of Susceptibility (CGEMS) project that includes 1145 cases and 1142 controls. Pathways were retrieved from three databases: KEGG, BioCarta, and the NCI's Protein Interaction Database (PID). Genes were represented by their most strongly associated SNP, and an enrichment score (ES) reflecting the overrepresentation of gene-based association signals in each pathway was calculated using a weighted Kolmogorov-Smirnov procedure. Finally, hierarchical clustering was used to identify pathways with overlapping genes, and clusters with excess of association signals were determined by the adaptive rank-truncated product (ARTP) method. A total of 421 pathways containing 3962 genes were included in our study. Of these, three pathways (‘Syndecan-1-mediated signaling ‘, ‘Signaling of Hepatocyte Growth Factor Receptor’ and ‘Growth Hormone Signaling’) were highly enriched with association signals (PES < 0.001, False Discovery Rate (FDR) = 0.118). Our clustering analysis revealed that pathways containing key components of the RAS/RAF/MAPK canonical signaling cascade, were significantly more likely to have excess of association signals than expected by chance (PARTP = 0.0051, FDR = 0.07). These results suggest that genetic alterations associated with these three pathways and one canonical signaling cascade may contribute to breast cancer susceptibility.

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 2201.
Abstract 2203: Integrative analysis for efficient candidate oncogene discovery in cancer cell lines
Authors: Yee Andrew J. , Wittner Ben S. , Gentry Jeff , Mahoney Crystal , McCutcheon Kaitlin , Singh Anurag , Greninger Patricia , Ma Xiao-Jun , Erlander Mark G. , Sharma Sreenath V. , Haber Daniel A. , Shioda Toshi , Settleman Jeffrey , Ramaswamy Sridhar
Published: February 2011

A fundamental challenge in cancer genomics is to efficiently identify biologically relevant candidate oncogenes from the large amount of molecular information generated in human cancer studies from various high throughput modalities. METHODS. We developed the DELTA (Difference in Expression Lined to Amplification) measurement to systematically identify candidate oncogenes in human cancer cell lines. We applied this metric to a large, diverse collection of 365 cancer cell lines densely profiled at both the expression and genomic level using the Affymetrix U133X3P and 500K Mapping arrays. RESULTS. We first identified recurrent cancer-driving amplification events using a GISTIC-inspired approach. This algorithmic analysis revealed 170 amplicons across 18 different cancer types. The majority of these recurrent amplicons do not contain known oncogenes. We then used the DELTA metric to further prioritize candidate oncogenes within these recurrent amplicons. Systematic shRNA knock down of these candidates was then used to identify SOX2 and S100A8 as candidate oncogenes in esophageal and lung cancer cell lines, respectively. CONCLUSION. Differential gene expression analysis with the DELTA metric may be useful for the functional analysis of recurrent genomic amplicons across human 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 2203.
Abstract 2208: High-resolution analysis of insertional mutagenesis screens to study genetic interactions in heterogeneous tumors
Authors: Koudijs Marco J. , Kool Jaap , Sie Daoud , Prasetyanti Pramudita , Cuppen Edwin , Berns Anton , Hilkens John , van Lohuizen Maarten , Adams David , Jonkers Jos
Published: February 2011

Insertional mutagenesis (IM) screens in genetically engineered mouse models of cancer are a powerful tool to identify genes or regions in the genome contributing to tumorigenesis. Current methods to identify the integration sites are not quantitative and do not give information about the clonality of a given insertion in a tumor. Consequently, the results of IM screens are hard to interpret since the obtained data are incomplete and highly biased due to technical variation.

We have developed and optimized a next-generation sequencing-based method for ultra high-throughput analysis of insertional mutations in a less biased and quantitative manner. Integration sites of retroviral or transposon based insertional mutagens are amplified using Splinkerette-mediated PCR and subsequent 454 sequencing. In contrast to current protocols, based on restriction enzyme (RE) fragmentation, we randomly shear gDNA, which allows a random ligation of the Splinkerette. This enables us to generate randomly sized amplicons per integration, which reduces amplification- and sequencing bias compared to amplicons with a fixed length per integration using RE based methods. Since Splinkerette ligation can occur at every base pair, we can determine the amount of cells containing the given insertion by counting unique ligation points, each representing a unique chromosome, and thereby a cell, in the tumor. This latter feature, addressing clonality of an insertional mutagen, will allow the identification of both clonal and sub-clonal mutations in genetically heterogeneous tumors. More importantly, we are now able to study co-occurring mutations, e.g. underlying synthetic lethality, which cannot be performed reliably using the current method.

The insertional mutagenesis data obtained with the improved method will represent the biological situation of a given integration within a tumor, thereby facilitating subsequent validation experiments. Additionally, data obtained from our retroviral- and transposon-based screens in various mouse models of cancer will serve as a catalogue of cancer-relevant mutations that can be used for cross-species comparative analysis of the large numbers of mutations found in human breast cancer.

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 2208.