Bio

Academic Appointments


Administrative Appointments


  • Founding Director, Stanford Tissue Bank (2001 - 2010)

Honors & Awards


  • Clinical Scientist Award in Translational Research, Burroughs Wellcome Fund (2008)
  • Elected member, American Society for Clinical Investigation (2006)
  • Amgen Outstanding Investigator Award, American Society of Investigative Pathology (2006)

Professional Education


  • M.D., University of California, San Francisco (1995)
  • Ph.D., University of California, San Francisco, Biochemistry (1993)
  • A.B., Harvard College, Biological Anthropology (1986)

Research & Scholarship

Current Research and Scholarly Interests


Our laboratory uses genomic approaches to investigate the pathogenesis and biology of human cancer, and to identify strategies for improved cancer diagnosis, prognostication and patient management. Technologies include DNA microarrays, deep sequencing, and RNAi screens, and additional insight is provided by the biocomputational integration of different data types.

Active areas of investigation include: (1) Characterizing molecular subtypes of prostate cancer to identify biomarkers for risk-assessment; (2) Identifying recurrent DNA copy number alterations and rearrangements in common epithelial tumor types (e.g. breast, prostate, lung, colon, and pancreatic cancers) to discover new cancer genes; (3) Understanding the role of altered chromatin remodeling and "lineage-dependent" oncogenes in tumorigenesis; and (4) Investigating mechanisms underlying genomic instability and the shaping of cancer genomes.

Clinical Trials


  • Microarray Analysis of Gene Expression in Prostate Tissues Not Recruiting

    The purpose of this study is to investigate gene expression profiles and biologic features of prostate tissue and how they relate to prostate cancer development and growth.

    Stanford is currently not accepting patients for this trial. For more information, please contact Cancer Clinical Trials Office, 650-498-7061.

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  • Evaluation of Pathwork Tissue of Origin (TOO) Test for Human Malignancies Not Recruiting

    The pathworks tissue of origin test is a microarray-based test with the goal of identifying the tissue of origin in patients with metastatic tumors of unknown primary site.

    Stanford is currently not accepting patients for this trial. For more information, please contact James Zehnder, (650) 723 - 9232.

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  • Biopsy of Human Tumors for Cancer Stem Cell Characterization: a Feasibility Study Not Recruiting

    To see if a limited sampling of tumor tissue from human subjects is a feasible way to gather adequate tissue for cancer stem cell quantification.

    Stanford is currently not accepting patients for this trial. For more information, please contact Ruth Lira, 650-723-1367.

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Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • EGFRvIII gene rearrangement is an early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene Del Vecchio, C. A., Giacomini, C. P., Vogel, H., Jensen, K. C., Florio, T., Merlo, A., Pollack, J. R., Wong, A. J. 2013; 32 (21): 2670-2681

    Abstract

    Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in glioblastoma multiforme (GBM). The most common variant is EGFR variant III (EGFRvIII). Research suggests that EGFRvIII could be a marker for a cancer stem cell or tumor-initiating population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM, EGFRvIII expression is focal and sporadic. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression, suggesting that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII. EGFRvIII expression was heterogeneous, with both positive and negative populations maintaining the genetic alterations, akin to primary tumors. Furthermore, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. Only cells that had recently lost EGFRvIII expression could re-express EGFRvIII, providing an important buffer for maintaining EGFRvIII-positive cell numbers. Epigenetic mechanisms had a role in maintaining heterogeneous EGFRvIII expression. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFR amplification and rearrangement are early events in tumorigenesis and EGFRvIII follows a model of hierarchical expression. Furthermore, EGFRvIII expression is restricted by epigenetic mechanisms, suggesting that drugs that modulate the epigenome might be used successfully in glioblastoma tumors.

    View details for DOI 10.1038/onc.2012.280

    View details for PubMedID 22797070

  • Breakpoint analysis of transcriptional and genomic profiles uncovers novel gene fusions spanning multiple human cancer types. PLoS genetics Giacomini, C. P., Sun, S., Varma, S., Shain, A. H., Giacomini, M. M., Balagtas, J., Sweeney, R. T., Lai, E., Del Vecchio, C. A., Forster, A. D., Clarke, N., Montgomery, K. D., Zhu, S., Wong, A. J., van de Rijn, M., West, R. B., Pollack, J. R. 2013; 9 (4)

    Abstract

    Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a "breakpoint analysis" pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.

    View details for DOI 10.1371/journal.pgen.1003464

    View details for PubMedID 23637631

  • The Spectrum of SWI/SNF Mutations, Ubiquitous in Human Cancers PLOS ONE Shain, A. H., Pollack, J. R. 2013; 8 (1)

    Abstract

    SWI/SNF is a multi-subunit chromatin remodeling complex that uses the energy of ATP hydrolysis to reposition nucleosomes, thereby modulating gene expression. Accumulating evidence suggests that SWI/SNF functions as a tumor suppressor in some cancers. However, the spectrum of SWI/SNF mutations across human cancers has not been systematically investigated. Here, we mined whole-exome sequencing data from 24 published studies representing 669 cases from 18 neoplastic diagnoses. SWI/SNF mutations were widespread across diverse human cancers, with an excess of deleterious mutations, and an overall frequency approaching TP53 mutation. Mutations occurred most commonly in the SMARCA4 enzymatic subunit, and in subunits thought to confer functional specificity (ARID1A, ARID1B, PBRM1, and ARID2). SWI/SNF mutations were not mutually-exclusive of other mutated cancer genes, including TP53 and EZH2 (both previously linked to SWI/SNF). Our findings implicate SWI/SNF as an important but under-recognized tumor suppressor in diverse human cancers, and provide a key resource to guide future investigations.

    View details for DOI 10.1371/journal.pone.0055119

    View details for Web of Science ID 000314021500156

    View details for PubMedID 23355908

  • CDX2 is an amplified lineage-survival oncogene in colorectal cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salari, K., Spulak, M. E., Cuff, J., Forster, A. D., Giacomini, C. P., Huang, S., Ko, M. E., Lin, A. Y., van de Rijn, M., Pollack, J. R. 2012; 109 (46): E3196-E3205

    Abstract

    The mutational activation of oncogenes drives cancer development and progression. Classic oncogenes, such as MYC and RAS, are active across many different cancer types. In contrast, "lineage-survival" oncogenes represent a distinct and emerging class typically comprising transcriptional regulators of a specific cell lineage that, when deregulated, support the proliferation and survival of cancers derived from that lineage. Here, in a large collection of colorectal cancer cell lines and tumors, we identify recurrent amplification of chromosome 13, an alteration highly restricted to colorectal-derived cancers. A minimal region of amplification on 13q12.2 pinpoints caudal type homeobox transcription factor 2 (CDX2), a regulator of normal intestinal lineage development and differentiation, as a target of the amplification. In contrast to its described role as a colorectal tumor suppressor, CDX2 when amplified is required for the proliferation and survival of colorectal cancer cells. Further, transcriptional profiling, binding-site analysis, and functional studies link CDX2 to Wnt/?-catenin signaling, itself a key oncogenic pathway in colorectal cancer. These data characterize CDX2 as a lineage-survival oncogene deregulated in colorectal cancer. Our findings challenge a prevailing view that CDX2 is a tumor suppressor in colorectal cancer and uncover an additional piece in the multistep model of colorectal tumorigenesis.

    View details for DOI 10.1073/pnas.1206004109

    View details for Web of Science ID 000311576300010

    View details for PubMedID 23112155

  • Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness ONCOGENE Huang, S., Gulzar, Z. G., Salari, K., Lapointe, J., Brooks, J. D., Pollack, J. R. 2012; 31 (37): 4164-4170

    Abstract

    Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

    View details for DOI 10.1038/onc.2011.590

    View details for Web of Science ID 000308688900008

    View details for PubMedID 22179824

  • Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shain, A. H., Giacomini, C. P., Matsukuma, K., Karikari, C. A., Bashyam, M. D., Hidalgo, M., Maitra, A., Pollack, J. R. 2012; 109 (5): E252-E259

    Abstract

    Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational "hills" in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational "mountain." Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

    View details for DOI 10.1073/pnas.1114817109

    View details for Web of Science ID 000299731400010

    View details for PubMedID 22233809

  • Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers GENOME BIOLOGY Brunner, A. L., Beck, A. H., Edris, B., Sweeney, R. T., Zhu, S. X., Li, R., Montgomery, K., Varma, S., Gilks, T., Guo, X., Foley, J. W., Witten, D. M., Giacomini, C. P., Flynn, R. A., Pollack, J. R., Tibshirani, R., Chang, H. Y., van de Rijn, M., West, R. B. 2012; 13 (8)
  • A fused lasso latent feature model for analyzing multi-sample aCGH data BIOSTATISTICS Nowak, G., Hastie, T., Pollack, J. R., Tibshirani, R. 2011; 12 (4): 776-791

    Abstract

    Array-based comparative genomic hybridization (aCGH) enables the measurement of DNA copy number across thousands of locations in a genome. The main goals of analyzing aCGH data are to identify the regions of copy number variation (CNV) and to quantify the amount of CNV. Although there are many methods for analyzing single-sample aCGH data, the analysis of multi-sample aCGH data is a relatively new area of research. Further, many of the current approaches for analyzing multi-sample aCGH data do not appropriately utilize the additional information present in the multiple samples. We propose a procedure called the Fused Lasso Latent Feature Model (FLLat) that provides a statistical framework for modeling multi-sample aCGH data and identifying regions of CNV. The procedure involves modeling each sample of aCGH data as a weighted sum of a fixed number of features. Regions of CNV are then identified through an application of the fused lasso penalty to each feature. Some simulation analyses show that FLLat outperforms single-sample methods when the simulated samples share common information. We also propose a method for estimating the false discovery rate. An analysis of an aCGH data set obtained from human breast tumors, focusing on chromosomes 8 and 17, shows that FLLat and Significance Testing of Aberrant Copy number (an alternative, existing approach) identify similar regions of CNV that are consistent with previous findings. However, through the estimated features and their corresponding weights, FLLat is further able to discern specific relationships between the samples, for example, identifying 3 distinct groups of samples based on their patterns of CNV for chromosome 17.

    View details for DOI 10.1093/biostatistics/kxr012

    View details for Web of Science ID 000294806800014

    View details for PubMedID 21642389

  • SMURF1 Amplification Promotes Invasiveness in Pancreatic Cancer PLOS ONE Kwei, K. A., Shain, A. H., Bair, R., Montgomery, K., Karikari, C. A., van de Rijn, M., Hidalgo, M., Maitra, A., Bashyam, M. D., Pollack, J. R. 2011; 6 (8)

    Abstract

    Pancreatic cancer is a deadly disease, and new therapeutic targets are urgently needed. We previously identified DNA amplification at 7q21-q22 in pancreatic cancer cell lines. Now, by high-resolution genomic profiling of human pancreatic cancer cell lines and human tumors (engrafted in immunodeficient mice to enrich the cancer epithelial fraction), we define a 325 Kb minimal amplicon spanning SMURF1, an E3 ubiquitin ligase and known negative regulator of transforming growth factor ? (TGF?) growth inhibitory signaling. SMURF1 amplification was confirmed in primary human pancreatic cancers by fluorescence in situ hybridization (FISH), where 4 of 95 cases (4.2%) exhibited amplification. By RNA interference (RNAi), knockdown of SMURF1 in a human pancreatic cancer line with focal amplification (AsPC-1) did not alter cell growth, but led to reduced cell invasion and anchorage-independent growth. Interestingly, this effect was not mediated through altered TGF? signaling, assayed by transcriptional reporter. Finally, overexpression of SMURF1 (but not a catalytic mutant) led to loss of contact inhibition in NIH-3T3 mouse embryo fibroblast cells. Together, these findings identify SMURF1 as an amplified oncogene driving multiple tumorigenic phenotypes in pancreatic cancer, and provide a new druggable target for molecularly directed therapy.

    View details for DOI 10.1371/journal.pone.0023924

    View details for Web of Science ID 000294251800040

    View details for PubMedID 21887346

  • A Tri-Marker Proliferation Index Predicts Biochemical Recurrence after Surgery for Prostate Cancer PLOS ONE Malhotra, S., Lapointe, J., Salari, K., Higgins, J. P., Ferrari, M., Montgomery, K., van de Rijn, M., Brooks, J. D., Pollack, J. R. 2011; 6 (5)

    Abstract

    Prostate cancer exhibits tremendous variability in clinical behavior, ranging from indolent to lethal disease. Better prognostic markers are needed to stratify patients for appropriately aggressive therapy. By expression profiling, we can identify a proliferation signature variably expressed in prostate cancers. Here, we asked whether one or more tissue biomarkers might capture that information, and provide prognostic utility. We assayed three proliferation signature genes: MKI67 (Ki-67; also a classic proliferation biomarker), TOP2A (DNA topoisomerase II, alpha), and E2F1 (E2F transcription factor 1). Immunohistochemical staining was evaluable on 139 radical prostatectomy cases (in tissue microarray format), with a median clinical follow-up of eight years. Each of the three proliferation markers was by itself prognostic. Notably, combining the three markers together as a "proliferation index" (0 or 1, vs. 2 or 3 positive markers) provided superior prognostic performance (hazard ratio = 2.6 (95% CI: 1.4-4.9); P = 0.001). In a multivariate analysis that included preoperative serum prostate specific antigen (PSA) levels, Gleason grade and pathologic tumor stage, the composite proliferation index remained a significant predictor (P = 0.005). Analysis of receiver-operating characteristic (ROC) curves confirmed the improved prognostication afforded by incorporating the proliferation index (compared to the clinicopathologic data alone). Our findings highlight the potential value of a multi-gene signature-based diagnostic, and define a tri-marker proliferation index with possible utility for improved prognostication and treatment stratification in prostate cancer.

    View details for DOI 10.1371/journal.pone.0020293

    View details for Web of Science ID 000291005200049

    View details for PubMedID 21629784

  • Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation CANCER RESEARCH Cai, D., Shames, D. S., Raso, M. G., Xie, Y., Kim, Y. H., Pollack, J. R., Girard, L., Sullivan, J. P., Gao, B., Peyton, M., Nanjundan, M., Byers, L., Heymach, J., Mills, G., Gazdar, A. F., Wistuba, I., Kodadek, T., Minna, J. D. 2010; 70 (16): 6477-6485

    Abstract

    Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non-small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA-mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer.

    View details for DOI 10.1158/0008-5472.CAN-10-0005

    View details for Web of Science ID 000280887000010

    View details for PubMedID 20663904

  • Development of an Orthotopic Model of Invasive Pancreatic Cancer in an Immunocompetent Murine Host CLINICAL CANCER RESEARCH Tseng, W. W., Winer, D., Kenkel, J. A., Choi, O., Shain, A. H., Pollack, J. R., French, R., Lowy, A. M., Engleman, E. G. 2010; 16 (14): 3684-3695

    Abstract

    The most common preclinical models of pancreatic adenocarcinoma utilize human cells or tissues that are xenografted into immunodeficient hosts. Several immunocompetent, genetically engineered mouse models of pancreatic cancer exist; however, tumor latency and disease progression in these models are highly variable. We sought to develop an immunocompetent, orthotopic mouse model of pancreatic cancer with rapid and predictable growth kinetics.Cell lines with epithelial morphology were derived from liver metastases obtained from Kras(G12D/+);LSL-Trp53(R172H/+);Pdx-1-Cre mice. Tumor cells were implanted in the pancreas of immunocompetent, histocompatible B6/129 mice, and the mice were monitored for disease progression. Relevant tissues were harvested for histologic, genomic, and immunophenotypic analysis.All mice developed pancreatic tumors by two weeks. Invasive disease and liver metastases were noted by six to eight weeks. Histologic examination of tumors showed cytokeratin-19-positive adenocarcinoma with regions of desmoplasia. Genomic analysis revealed broad chromosomal changes along with focal gains and losses. Pancreatic tumors were infiltrated with dendritic cells, myeloid-derived suppressor cells, macrophages, and T lymphocytes. Survival was decreased in RAG(-/-) mice, which are deficient in T cells, suggesting that an adaptive immune response alters the course of disease in wild-type mice.We have developed a rapid, predictable orthotopic model of pancreatic adenocarcinoma in immunocompetent mice that mimics human pancreatic cancer with regard to genetic mutations, histologic appearance, and pattern of disease progression. This model highlights both the complexity and relevance of the immune response to invasive pancreatic cancer and may be useful for the preclinical evaluation of new therapeutic agents.

    View details for DOI 10.1158/1078-0432.CCR-09-2384

    View details for Web of Science ID 000279903100017

    View details for PubMedID 20534740

  • Genomic instability in breast cancer: Pathogenesis and clinical implications MOLECULAR ONCOLOGY Kwei, K. A., Kung, Y., Salari, K., Holcomb, I. N., Pollack, J. R. 2010; 4 (3): 255-266

    Abstract

    Breast cancer is a heterogeneous disease, appreciable by molecular markers, gene-expression profiles, and most recently, patterns of genomic alteration. In particular, genomic profiling has revealed three distinct patterns of DNA copy-number alteration: a "simple" type with few gains or losses of whole chromosome arms, an "amplifier" type with focal high-level DNA amplifications, and a "complex" type marked by numerous low-amplitude changes and copy-number transitions. The three patterns are associated with distinct gene-expression subtypes, and preferentially target different loci in the genome (implicating distinct cancer genes). Moreover, the different patterns of alteration imply distinct underlying mechanisms of genomic instability. The amplifier pattern may arise from transient telomere dysfunction, although new data suggest ongoing "amplifier" instability. The complex pattern shows similarity to breast cancers with germline BRCA1 mutation, which also exhibit "basal-like" expression profiles and complex-pattern genomes, implicating a possible defect in BRCA1-associated repair of DNA double-strand breaks. As such, targeting presumptive DNA repair defects represents a promising area of clinical investigation. Future studies should clarify the pathogenesis of breast cancers with amplifier and complex-pattern genomes, and will likely identify new therapeutic opportunities.

    View details for DOI 10.1016/j.molonc.2010.04.001

    View details for Web of Science ID 000280046100007

    View details for PubMedID 20434415

  • LYN Is a Mediator of Epithelial-Mesenchymal Transition and a Target of Dasatinib in Breast Cancer CANCER RESEARCH Choi, Y., Bocanegra, M., Kwon, M. J., Shin, Y. K., Nam, S. J., Yang, J., Kao, J., Godwin, A. K., Pollack, J. R. 2010; 70 (6): 2296-2306

    Abstract

    Epithelial-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is considered a key process driving tumor cell invasiveness and metastasis. Using breast cancer cell lines as a model system, we sought to discover gene expression signatures of EMT with clinical and mechanistic relevance. A supervised comparison of epithelial and mesenchymal breast cancer lines defined a 200-gene EMT signature that was prognostic across multiple breast cancer cohorts. The immunostaining of LYN, a top-ranked EMT signature gene and Src-family tyrosine kinase, was associated with significantly shorter overall survival (P = 0.02) and correlated with the basal-like ("triple-negative") phenotype. In mesenchymal breast cancer lines, RNAi-mediated knockdown of LYN inhibited cell migration and invasion, but not proliferation. Dasatinib, a dual-specificity tyrosine kinase inhibitor, also blocked invasion (but not proliferation) at nanomolar concentrations that inhibit LYN kinase activity, suggesting that LYN is a likely target and that invasion is a relevant end point for dasatinib therapy. Our findings define a prognostically relevant EMT signature in breast cancer and identify LYN as a mediator of invasion and a possible new therapeutic target (and theranostic marker for dasatinib response), with particular relevance to clinically aggressive basal-like breast cancer.

    View details for DOI 10.1158/0008-5472.CAN-09-3141

    View details for Web of Science ID 000278485900017

    View details for PubMedID 20215510

  • Genomic and functional analysis identifies CRKL as an oncogene amplified in lung cancer ONCOGENE Kim, Y. H., Kwei, K. A., Girard, L., Salari, K., Kao, J., Pacyna-Gengelbach, M., Wang, P., Hernandez-Boussard, T., Gazdar, A. F., Petersen, I., Minna, J. D., Pollack, J. R. 2010; 29 (10): 1421-1430

    Abstract

    DNA amplifications, leading to the overexpression of oncogenes, are a cardinal feature of lung cancer and directly contribute to its pathogenesis. To uncover such novel alterations, we performed an array-based comparative genomic hybridization survey of 128 non-small-cell lung cancer cell lines and tumors. Prominent among our findings, we identified recurrent high-level amplification at cytoband 22q11.21 in 3% of lung cancer specimens, with another 11% of specimens exhibiting low-level gain spanning that locus. The 22q11.21 amplicon core contained eight named genes, only four of which were overexpressed (by transcript profiling) when amplified. Among these, CRKL encodes an adapter protein functioning in signal transduction, best known as a substrate of the BCR-ABL kinase in chronic myelogenous leukemia. RNA-interference-mediated knockdown of CRKL in lung cancer cell lines with (but not without) amplification led to significantly decreased cell proliferation, cell-cycle progression, cell survival, and cell motility and invasion. In addition, overexpression of CRKL in immortalized human bronchial epithelial cells led to enhanced growth factor-independent cell growth. Our findings indicate that amplification and resultant overexpression of CRKL contribute to diverse oncogenic phenotypes in lung cancer, with implications for targeted therapy, and highlight a role of adapter proteins as primary genetic drivers of tumorigenesis.

    View details for DOI 10.1038/onc.2009.437

    View details for Web of Science ID 000275392400002

    View details for PubMedID 19966867

  • KIT mutations confer a distinct gene expression signature in core binding factor leukaemia BRITISH JOURNAL OF HAEMATOLOGY Lueck, S. C., Russ, A. C., Du, J., Gaidzik, V., Schlenk, R. F., Pollack, J. R., Doehner, K., Doehner, H., Bullinger, L. 2010; 148 (6): 925-937

    Abstract

    Core binding factor (CBF) leukaemias, characterized by either inv(16)(p13.1q22) or t(8;21)(q22;q22), constitute acute myeloid leukaemia (AML) subgroups with favourable prognosis. However, 40-50% of patients relapse, emphasizing the need for risk-adapted treatment approaches. In this regard, studying secondary genetic aberrations, such as mutations of the KIT gene, is of great interest, particularly as they can be targeted by receptor tyrosine kinase inhibitors (TKI). However, so far little is known about the biology underlying KIT-mutated CBF leukaemias. We analysed gene expression profiles of 83 CBF AML cases with known KIT mutation status in order to gain novel insights in KIT-mutated CBF pathogenesis. KIT-mutated cases were characterized by deregulation of genes belonging to the NFkB signalling complex suggesting impaired control of apoptosis. Notably, a subgroup of KIT wildtype cases was also characterized by the KIT mutation signature due to yet unknown aberrations. Our data suggest that this CBF leukaemia subgroup might profit from TKI therapy, however, the relevance of the KIT mutation-associated signature remains to be validated prior to clinical implementation. Nevertheless, the existence of such a signature supports the notion of relevant biological differences in CBF leukaemia and might serve as diagnostic tool in the future.

    View details for DOI 10.1111/j.1365-2141.2009.08035.x

    View details for Web of Science ID 000275094900012

    View details for PubMedID 20064158

  • Focal amplification and oncogene dependency of GAB2 in breast cancer ONCOGENE Bocanegra, M., Bergamaschi, A., Kim, Y. H., Miller, M. A., Rajput, A. B., Kao, J., Langerod, A., Han, W., Noh, D., Jeffrey, S. S., Huntsman, D. G., Borresen-Dale, A., Pollack, J. R. 2010; 29 (5): 774-779

    Abstract

    DNA amplifications in breast cancer are frequent on chromosome 11q, in which multiple driver oncogenes likely reside in addition to cyclin D1 (CCND1). One such candidate, the scaffolding adapter protein, GRB2-associated binding protein 2 (GAB2), functions in ErbB signaling and was recently shown to enhance mammary epithelial cell proliferation, and metastasis of ERBB2 (HER2/neu)-driven murine breast cancer. However, the amplification status and function of GAB2 in the context of amplification remain undefined. In this study, by genomic profiling of 172 breast tumors, and fluorescence in situ hybridization validation in an independent set of 210 scorable cases, we observed focal amplification spanning GAB2 (11q14.1) independent of CCND1 (11q13.2) amplification, consistent with a driver role. Further, small interfering RNA (siRNA)-mediated knockdown of GAB2 in breast cancer lines (SUM52, SUM44PE and MDA468) with GAB2 amplification revealed a dependency on GAB2 for cell proliferation, cell-cycle progression, survival and invasion, likely mediated through altered phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling. GAB2 knockdown also reduced proliferation and survival in a cell line (BT474) with ERBB2 amplification, consistent with the possibility that GAB2 can function downstream of ERBB2. Our studies implicate focal amplification of GAB2 in breast carcinogenesis, and underscore an oncogenic role of scaffolding adapter proteins, and a potential new point of therapeutic intervention.

    View details for DOI 10.1038/onc.2009.364

    View details for Web of Science ID 000274223700013

    View details for PubMedID 19881546

  • DR-Integrator: a new analytic tool for integrating DNA copy number and gene expression data BIOINFORMATICS Salari, K., Tibshirani, R., Pollack, J. R. 2010; 26 (3): 414-416

    Abstract

    DNA copy number alterations (CNA) frequently underlie gene expression changes by increasing or decreasing gene dosage. However, only a subset of genes with altered dosage exhibit concordant changes in gene expression. This subset is likely to be enriched for oncogenes and tumor suppressor genes, and can be identified by integrating these two layers of genome-scale data. We introduce DNA/RNA-Integrator (DR-Integrator), a statistical software tool to perform integrative analyses on paired DNA copy number and gene expression data. DR-Integrator identifies genes with significant correlations between DNA copy number and gene expression, and implements a supervised analysis that captures genes with significant alterations in both DNA copy number and gene expression between two sample classes.DR-Integrator is freely available for non-commercial use from the Pollack Lab at http://pollacklab.stanford.edu/ and can be downloaded as a plug-in application to Microsoft Excel and as a package for the R statistical computing environment. The R package is available under the name 'DRI' at http://cran.r-project.org/. An example analysis using DR-Integrator is included as supplemental material.Supplementary data are available at Bioinformatics online.

    View details for DOI 10.1093/bioinformatics/btp702

    View details for Web of Science ID 000274342800021

    View details for PubMedID 20031972

  • Molecular Profiling of Breast Cancer Cell Lines Defines Relevant Tumor Models and Provides a Resource for Cancer Gene Discovery PLOS ONE Kao, J., Salari, K., Bocanegra, M., Choi, Y., Girard, L., Gandhi, J., Kwei, K. A., Hernandez-Boussard, T., Wang, P., Gazdar, A. F., Minna, J. D., Pollack, J. R. 2009; 4 (7)

    Abstract

    Breast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes-luminal A, luminal B, ERBB2-associated, basal-like and normal-like-with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes.Whole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression.Transcriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem/progenitor-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 multi-copy deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes.Overall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, cancer stem cell biology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes.

    View details for DOI 10.1371/journal.pone.0006146

    View details for Web of Science ID 000267806300015

    View details for PubMedID 19582160

  • Integration of diverse microarray data types. Methods in molecular biology (Clifton, N.J.) Salari, K., Pollack, J. R. 2009; 556: 205-216

    Abstract

    Over the past decade, DNA microarrays have proven to be a powerful tool in biological research for the molecular surveillance of cells and tissues. The expansive utility of DNA microarrays owes its nascence to the development of a multitude of microarray platforms that enable the systematic and comprehensive exploration of diverse genomic properties and processes. Concomitant with the explosive generation of microarray data over the last several years has been an increasing interest in the integration of such diverse data types, thus spurring the development of novel statistical techniques and integrative bioinformatics tools. This chapter will outline general approaches to microarray data integration and provide an introduction to DR-Integrator, a broadly useful analysis tool for the integration of DNA copy number and gene-expression microarray data.

    View details for DOI 10.1007/978-1-60327-192-9_15

    View details for PubMedID 19488881

  • DNA microarray technology. Introduction. Methods in molecular biology (Clifton, N.J.) Pollack, J. R. 2009; 556: 1-6

    Abstract

    DNA microarray technology has revolutionized biological research by enabling genome-scale explorations. This chapter provides an overview of DNA microarray technology and its application to characterizing the physical genome, with a focus on cancer genomes. Specific areas discussed include investigations of DNA copy number alteration (and loss of heterozygosity), DNA methylation, DNA-protein (i.e., chromatin and transcription factor) interactions, DNA replication, and the integration of diverse genome-scale data types. Also provided is a perspective on recent advances and future directions in characterizing the physical genome.

    View details for DOI 10.1007/978-1-60327-192-9_1

    View details for PubMedID 19488867

  • Comparative genomic hybridization on spotted oligonucleotide microarrays. Methods in molecular biology (Clifton, N.J.) Kim, Y. H., Pollack, J. R. 2009; 556: 21-32

    Abstract

    Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used for detecting DNA copy number alterations on a genomic scale, where the mapping resolution is limited only by the number of probes on the DNA microarray. In this chapter, we present a validated protocol utilizing print-tip spotted HEEBO (Human Exonic Evidence Based Oligonucleotide) microarrays for conducting array-CGH using as little as 25 ng of genomic DNA from a wide variety of sources, including cultured cell lines and clinical specimens, with high spatial resolution and array-to-array reproducibility.

    View details for DOI 10.1007/978-1-60327-192-9_3

    View details for PubMedID 19488869

  • CAMK1D amplification implicated in epithelial-mesenchymal transition in basal-like breast cancer MOLECULAR ONCOLOGY Bergamaschi, A., Kim, Y. H., Kwei, K. A., Choi, Y. L., Bocanegra, M., Langerod, A., Han, W., Noh, D., Huntsman, D. G., Jeffrey, S. S., Borresen-Dale, A., Pollack, J. R. 2008; 2 (4): 327-339

    Abstract

    Breast cancer exhibits clinical and molecular heterogeneity, where expression profiling studies have identified five major molecular subtypes. The basal-like subtype, expressing basal epithelial markers and negative for estrogen receptor (ER), progesterone receptor (PR) and HER2, is associated with higher overall levels of DNA copy number alteration (CNA), specific CNAs (like gain on chromosome 10p), and poor prognosis. Discovering the molecular genetic basis of tumor subtypes may provide new opportunities for therapy. To identify the driver oncogene on 10p associated with basal-like tumors, we analyzed genomic profiles of 172 breast carcinomas. The smallest shared region of gain spanned just seven genes at 10p13, including calcium/calmodulin-dependent protein kinase ID (CAMK1D), functioning in intracellular signaling but not previously linked to cancer. By microarray, CAMK1D was overexpressed when amplified, and by immunohistochemistry exhibited elevated expression in invasive carcinomas compared to carcinoma in situ. Engineered overexpression of CAMK1D in non-tumorigenic breast epithelial cells led to increased cell proliferation, and molecular and phenotypic alterations indicative of epithelial-mesenchymal transition (EMT), including loss of cell-cell adhesions and increased cell migration and invasion. Our findings identify CAMK1D as a novel amplified oncogene linked to EMT in breast cancer, and as a potential therapeutic target with particular relevance to clinically unfavorable basal-like tumors.

    View details for DOI 10.1016/j.molonc.2008.09.004

    View details for Web of Science ID 000264062400005

    View details for PubMedID 19383354

  • MYC stimulates EZH2 expression by repression of its negative regulator miR-26a BLOOD Sander, S., Bullinger, L., Klapproth, K., Fiedler, K., Kestler, H. A., Barth, T. F., Moeller, P., Stilgenbauer, S., Pollack, J. R., Wirth, T. 2008; 112 (10): 4202-4212

    Abstract

    The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.

    View details for DOI 10.1182/blood-2008-03-147645

    View details for Web of Science ID 000260691300041

    View details for PubMedID 18713946

  • Identification of candidate prostate cancer genes through comparative expression-profiling of seminal vesicle PROSTATE Thompson, M., Lapointe, J., Choi, Y., Ong, D. E., Higgins, J. P., Brooks, J. D., Pollack, J. R. 2008; 68 (11): 1248-1256

    Abstract

    Prostate cancer is the most frequently diagnosed cancer among men in the United States. In contrast, cancer of the seminal vesicle is exceedingly rare, despite that the prostate and seminal vesicle share similar histology, secretory function, androgen dependency, blood supply, and (in part) embryonic origin. We hypothesized that gene-expression differences between prostate and seminal vesicle might inform mechanisms underlying the higher incidence of prostate cancer.Whole-genome DNA microarrays were used to profile gene expression of 11 normal prostate and 7 seminal vesicle specimens (including six matched pairs) obtained from radical prostatectomy. Supervised analysis was used to identify genes differentially expressed between normal prostate and seminal vesicle, and this list was then cross-referenced to genes differentially expressed between normal and cancerous prostate. Expression patterns of selected genes were confirmed by immunohistochemistry using a tissue microarray.We identified 32 genes that displayed a highly statistically significant expression pattern with highest levels in seminal vesicle, lower levels in normal prostate, and lowest levels in prostate cancer. Among these genes was the known candidate prostate tumor suppressor GSTP1 (involved in xenobiotic detoxification). The expression pattern of GSTP1 and four other genes, ABCG2 (xenobiotic transport), CRABP2 (retinoic acid signaling), GATA3 (lineage-specific transcription), and SLPI (immune response), was confirmed by immunohistochemistry.Our findings identify candidate prostate cancer genes whose reduced expression in prostate (compared to seminal vesicle) may be permissive to prostate cancer initiation. Such genes and their pathways may inform mechanisms of prostate carcinogenesis, and suggest new opportunities for prostate cancer prevention.

    View details for DOI 10.1002/pros.20792

    View details for Web of Science ID 000258021300012

    View details for PubMedID 18500686

  • ESR1 gene amplification in breast cancer: a common phenomenon? NATURE GENETICS Horlings, H. M., Bergamaschi, A., Nordgard, S. H., Kim, Y. H., Han, W., Noh, D., Salari, K., Joosse, S. A., Reyal, F., Lingjaerde, O. C., Kristensen, V. N., Borresen-Dale, A., Pollack, J., van de Vijver, M. J. 2008; 40 (7): 807-808

    View details for DOI 10.1038/ng0708-807

    View details for Web of Science ID 000257166500003

    View details for PubMedID 18583965

  • Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer ONCOGENE Kwei, K. A., Kim, Y. H., Girard, L., Kao, J., Pacyna-Gengelbach, M., Salari, K., Lee, J., Choi, Y., Sato, M., Wang, P., Hernandez-Boussard, T., Gazdar, A. F., Petersen, I., Minna, J. D., Pollack, J. R. 2008; 27 (25): 3635-3640

    Abstract

    Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.

    View details for DOI 10.1038/sj.onc.1211012

    View details for Web of Science ID 000256468500015

    View details for PubMedID 18212743

  • An FLT3 gene-expression signature predicts clinical outcome in normal karyotype AML BLOOD Bullinger, L., Doehner, K., Kranz, R., Stirner, C., Froeling, S., Scholl, C., Kim, Y. H., Schlenk, R. F., Tibshirani, R., Doehner, H., Pollack, J. R. 2008; 111 (9): 4490-4495

    Abstract

    Acute myeloid leukemia with normal karyotype (NK-AML) represents a cytogenetic grouping with intermediate prognosis but substantial molecular and clinical heterogeneity. Within this subgroup, presence of FLT3 (FMS-like tyrosine kinase 3) internal tandem duplication (ITD) mutation predicts less favorable outcome. The goal of our study was to discover gene-expression patterns correlated with FLT3-ITD mutation and to evaluate the utility of a FLT3 signature for prognostication. DNA microarrays were used to profile gene expression in a training set of 65 NK-AML cases, and supervised analysis, using the Prediction Analysis of Microarrays method, was applied to build a gene expression-based predictor of FLT3-ITD mutation status. The optimal predictor, composed of 20 genes, was then evaluated by classifying expression profiles from an independent test set of 72 NK-AML cases. The predictor exhibited modest performance (73% sensitivity; 85% specificity) in classifying FLT3-ITD status. Remarkably, however, the signature outperformed FLT3-ITD mutation status in predicting clinical outcome. The signature may better define clinically relevant FLT3 signaling and/or alternative changes that phenocopy FLT3-ITD, whereas the signature genes provide a starting point to dissect these pathways. Our findings support the potential clinical utility of a gene expression-based measure of FLT3 pathway activation in AML.

    View details for DOI 10.1182/blood-2007-09-115055

    View details for Web of Science ID 000255387400016

    View details for PubMedID 18309032

  • Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer PLOS GENETICS Kwei, K. A., Bashyam, M. D., Kao, J., Ratheesh, R., Reddy, E. C., Kim, Y. H., Montgomery, K., Giacomini, C. P., Choi, Y., Chatterjee, S., Karikari, C. A., Salari, K., Wang, P., Hernandez-Boussard, T., Swarnalata, G., van de Rijn, M., Maitra, A., Pollack, J. R. 2008; 4 (5)

    Abstract

    Pancreatobiliary cancers have among the highest mortality rates of any cancer type. Discovering the full spectrum of molecular genetic alterations may suggest new avenues for therapy. To catalogue genomic alterations, we carried out array-based genomic profiling of 31 exocrine pancreatic cancers and 6 distal bile duct cancers, expanded as xenografts to enrich the tumor cell fraction. We identified numerous focal DNA amplifications and deletions, including in 19% of pancreatobiliary cases gain at cytoband 18q11.2, a locus uncommonly amplified in other tumor types. The smallest shared amplification at 18q11.2 included GATA6, a transcriptional regulator previously linked to normal pancreas development. When amplified, GATA6 was overexpressed at both the mRNA and protein levels, and strong immunostaining was observed in 25 of 54 (46%) primary pancreatic cancers compared to 0 of 33 normal pancreas specimens surveyed. GATA6 expression in xenografts was associated with specific microarray gene-expression patterns, enriched for GATA binding sites and mitochondrial oxidative phosphorylation activity. siRNA mediated knockdown of GATA6 in pancreatic cancer cell lines with amplification led to reduced cell proliferation, cell cycle progression, and colony formation. Our findings indicate that GATA6 amplification and overexpression contribute to the oncogenic phenotypes of pancreatic cancer cells, and identify GATA6 as a candidate lineage-specific oncogene in pancreatobiliary cancer, with implications for novel treatment strategies.

    View details for DOI 10.1371/journal.pgen.1000081

    View details for Web of Science ID 000256869100012

    View details for PubMedID 18535672

  • hCAP-D3 expression marks a prostate cancer subtype with favorable clinical behavior and androgen signaling signature AMERICAN JOURNAL OF SURGICAL PATHOLOGY Lapointe, J., Malhotra, S., Higgins, J. P., Bair, E., Thompson, M., Salari, K., Giacomini, C. P., Ferrari, M., Montgomery, K., Tibshirani, R., van de Rijn, M., Brooks, J. D., Pollack, J. R. 2008; 32 (2): 205-209

    Abstract

    Growing evidence suggests that only a fraction of prostate cancers detected clinically are potentially lethal. An important clinical issue is identifying men with indolent cancer who might be spared aggressive therapies with associated morbidities. Previously, using microarray analysis we defined 3 molecular subtypes of prostate cancer with different gene-expression patterns. One, subtype-1, displayed features consistent with more indolent behavior, where an immunohistochemical marker (AZGP1) for subtype-1 predicted favorable outcome after radical prostatectomy. Here we characterize a second candidate tissue biomarker, hCAP-D3, expressed in subtype-1 prostate tumors. hCAP-D3 expression, assayed by RNA in situ hybridization on a tissue microarray comprising 225 cases, was associated with decreased tumor recurrence after radical prostatectomy (P=0.004), independent of pathologic tumor stage, Gleason grade, and preoperative prostate-specific antigen levels. Simultaneous assessment of hCAP-D3 and AZGP1 expression in this tumor set improved outcome prediction. We have previously demonstrated that hCAP-D3 is induced by androgen in prostate cells. Extending this finding, Gene Set Enrichment Analysis revealed enrichment of androgen-responsive genes in subtype-1 tumors (P=0.019). Our findings identify hCAP-D3 as a new biomarker for subtype-1 tumors that improves prognostication, and reveal androgen signaling as an important biologic feature of this potentially clinically favorable molecular subtype.

    View details for Web of Science ID 000252759900004

    View details for PubMedID 18223322

  • Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers JOURNAL OF MOLECULAR DIAGNOSTICS Dumur, C. I., Lyons-Weiler, M., Sciulli, C., Garrett, C. T., Schrijver, I., Holley, T. K., Rodriguez-Paris, J., Pollack, J. R., Zehnder, J. L., Price, M., Hagenkord, J. M., Rigl, C. T., Buturovic, L. J., Anderson, G. G., Monzon, F. A. 2008; 10 (1): 67-77

    Abstract

    Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa >0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.

    View details for DOI 10.2353/jmoldx.2008.070099

    View details for Web of Science ID 000252521200009

    View details for PubMedID 18083688

  • Genomic profiling reveals alternative genetic pathways of prostate tumorigenesis CANCER RESEARCH Lapointe, J., Li, C., Giacomini, C. P., Salari, K., Huang, S., Wang, P., Ferrari, M., Hernandez-Boussard, T., Brooks, J. D., Pollack, J. R. 2007; 67 (18): 8504-8510

    Abstract

    Prostate cancer is clinically heterogeneous, ranging from indolent to lethal disease. Expression profiling previously defined three subtypes of prostate cancer, one (subtype-1) linked to clinically favorable behavior, and the others (subtypes-2 and -3) linked with a more aggressive form of the disease. To explore disease heterogeneity at the genomic level, we carried out array-based comparative genomic hybridization (array CGH) on 64 prostate tumor specimens, including 55 primary tumors and 9 pelvic lymph node metastases. Unsupervised cluster analysis of DNA copy number alterations (CNA) identified recurrent aberrations, including a 6q15-deletion group associated with subtype-1 gene expression patterns and decreased tumor recurrence. Supervised analysis further disclosed distinct patterns of CNA among gene-expression subtypes, where subtype-1 tumors exhibited characteristic deletions at 5q21 and 6q15, and subtype-2 cases harbored deletions at 8p21 (NKX3-1) and 21q22 (resulting in TMPRSS2-ERG fusion). Lymph node metastases, predominantly subtype-3, displayed overall higher frequencies of CNA, and in particular gains at 8q24 (MYC) and 16p13, and loss at 10q23 (PTEN) and 16q23. Our findings reveal that prostate cancers develop via a limited number of alternative preferred genetic pathways. The resultant molecular genetic subtypes provide a new framework for investigating prostate cancer biology and explain in part the clinical heterogeneity of the disease.

    View details for DOI 10.1158/0008-5472.CAN-07-0673

    View details for Web of Science ID 000249679500013

    View details for PubMedID 17875689

  • Gene copy number variation spanning 60 million years of human and primate evolution GENOME RESEARCH Dumas, L., Kim, Y. H., Karimpour-Fard, A., Cox, M., Hopkins, J., Pollack, J. R., Sikela, J. M. 2007; 17 (9): 1266-1277

    Abstract

    Given the evolutionary importance of gene duplication to the emergence of species-specific traits, we have extended the application of cDNA array-based comparative genomic hybridization (aCGH) to survey gene duplications and losses genome-wide across 10 primate species, including human. Using human cDNA arrays that contained 41,126 cDNAs, corresponding to 24,473 unique human genes, we identified 4159 genes that likely represent most of the major lineage-specific gene copy number gains and losses that have occurred in these species over the past 60 million years. We analyzed 1,233,780 gene-to-gene data points and found that gene gains typically outnumbered losses (ratio of gains/losses = 2.34) and these frequently cluster in complex and dynamic genomic regions that are likely to serve as gene nurseries. Almost one-third of all human genes (6696) exhibit an aCGH- predicted change in copy number in one or more of these species, and within-species gene amplification is also evident. Many of the genes identified here are likely to be important to lineage-specific traits including, for example, human-specific duplications of the AQP7 gene, which represent intriguing candidates to underlie the key physiological adaptations in thermoregulation and energy utilization that permitted human endurance running.

    View details for DOI 10.1101/gr.6557307

    View details for Web of Science ID 000249236900003

    View details for PubMedID 17666543

  • Gene-expression profiling identifies distinct subclasses of core binding factor acute myeloid leukemia BLOOD Bullinger, L., Ruecker, F. G., Kurz, S., Du, J., Scholl, C., Sander, S., Corbacioglu, A., Lottaz, C., Froehling, J., Ganser, A., Schlenk, R. F., Doehner, K., Pollack, J. R., Doehner, H. 2007; 110 (4): 1291-1300

    Abstract

    Core binding factor (CBF) leukemias, characterized by either inv(16)/t(16;16) or t(8;21), constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, there exists substantial biologic and clinical heterogeneity within these cytogenetic groups that is not fully reflected by the current classification system. To improve the molecular characterization we profiled gene expression in a large series (n = 93) of AML patients with CBF leukemia [(inv (16), n = 55; t(8;21), n = 38)]. By unsupervised hierarchical clustering we were able to define a subgroup of CBF cases (n = 35) characterized by shorter overall survival times (P = .03). While there was no obvious correlation with fusion gene transcript levels, FLT3 tyrosine kinase domain, KIT, and NRAS mutations, the newly defined inv(16)/t(8;21) subgroup was associated with elevated white blood cell counts and FLT3 internal tandem duplications (P = .011 and P = .026, respectively). Supervised analyses of gene expression suggested alternative cooperating pathways leading to transformation. In the "favorable" CBF leukemias, antiapoptotic mechanisms and deregulated mTOR signaling and, in the newly defined "unfavorable" subgroup, aberrant MAPK signaling and chemotherapy-resistance mechanisms might play a role. While the leukemogenic relevance of these signatures remains to be validated, their existence nevertheless supports a prognostically relevant biologic basis for the heterogeneity observed in CBF leukemia.

    View details for DOI 10.1182/blood-2006-10-049783

    View details for Web of Science ID 000248655300035

    View details for PubMedID 17485551

  • A perspective on DNA microarrays in pathology research and practice AMERICAN JOURNAL OF PATHOLOGY Pollack, J. R. 2007; 171 (2): 375-385

    Abstract

    DNA microarray technology matured in the mid-1990s, and the past decade has witnessed a tremendous growth in its application. DNA microarrays have provided powerful tools for pathology researchers seeking to describe, classify, and understand human disease. There has also been great expectation that the technology would advance the practice of pathology. This review highlights some of the key contributions of DNA microarrays to experimental pathology, focusing in the area of cancer research. Also discussed are some of the current challenges in translating utility to clinical practice.

    View details for DOI 10.2353/ajpath.2007.070342

    View details for Web of Science ID 000248496300001

    View details for PubMedID 17600117

  • c-Jun N-terminal kinase is activated in non-small-cell lung cancer and promotes neoplastic transformation in human bronchial epithelial cells ONCOGENE Khatlani, T. S., Wislez, M., Sun, M., SRINIVAS, H., Iwanaga, K., Ma, L., Hanna, A. E., Liu, D., Girard, L., Kim, Y. H., Pollack, J. R., Minna, J. D., WISTUBA, I. I., Kurie, J. M. 2007; 26 (18): 2658-2666

    Abstract

    c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.

    View details for DOI 10.1038/sj.onc.1210050

    View details for Web of Science ID 000245831200013

    View details for PubMedID 17057737

  • Evolutionary and biomedical insights from the rhesus macaque genome SCIENCE Gibbs, R. A., Rogers, J., Katze, M. G., Bumgarner, R., Weinstock, G. M., Mardis, E. R., Remington, K. A., Strausberg, R. L., Venter, J. C., Wilson, R. K., Batzer, M. A., Bustamante, C. D., Eichler, E. E., Hahn, M. W., Hardison, R. C., Makova, K. D., Miller, W., Milosavljevic, A., Palermo, R. E., Siepel, A., Sikela, J. M., Attaway, T., Bell, S., Bernard, K. E., Buhay, C. J., Chandrabose, M. N., Dao, M., Davis, C., Delehaunty, K. D., Ding, Y., Dinh, H. H., Dugan-Rocha, S., Fulton, L. A., Gabisi, R. A., Garner, T. T., Godfrey, J., Hawes, A. C., Hernandez, J., Hines, S., Holder, M., Hume, J., Jhangiani, S. N., Joshi, V., Khan, Z. M., Kirkness, E. F., Cree, A., Fowler, R. G., Lee, S., Lewis, L. R., Li, Z., Liu, Y., Moore, S. M., Muzny, D., Nazareth, L. V., Ngo, D. N., Okwuonu, G. O., Pai, G., Parker, D., Paul, H. A., Pfannkoch, C., Pohl, C. S., Rogers, Y., Ruiz, S. J., Sabo, A., Santibanez, J., Schneider, B. W., Smith, S. M., Sodergren, E., Svatek, A. F., Utterback, T. R., Vattathil, S., Warren, W., White, C. S., Chinwalla, A. T., Feng, Y., Halpern, A. L., Hillier, L. W., Huang, X., Minx, P., Nelson, J. O., Pepin, K. H., Qin, X., Sutton, G. G., Venter, E., Walenz, B. P., Wallis, J. W., Worley, K. C., Yang, S., Jones, S. M., Marra, M. A., Rocchi, M., Schein, J. E., Baertsch, R., Clarke, L., Csuros, M., Glasscock, J., Harris, R. A., Haviak, P., Jackson, A. R., Jiang, H., Liu, Y., Messina, D. N., Shen, Y., Song, H. X., Wylie, T., Zhang, L., Birney, E., Han, K., Konkel, M. K., Lee, J., Smit, A. F., Ullmer, B., Wang, H., Xing, J., Burhans, R., Cheng, Z., Karro, J. E., Ma, J., Raney, B., She, X., Cox, M. J., Demuth, J. P., Dumas, L. J., Han, S., Hopkins, J., Karimpour-Fard, A., Kim, Y. H., Pollack, J. R., Vinar, T., Addo-Quaye, C., Degenhardt, J., Denby, A., Hubisz, M. J., Indap, A., Kosiol, C., Lahn, B. T., Lawson, H. A., Marklein, A., Nielsen, R., Vallender, E. J., Clark, A. G., Ferguson, B., Hernandez, R. D., Hirani, K., Kehrer-Sawatzki, H., Kolb, J., Patil, S., Pu, L., Ren, Y., Smith, D. G., Wheeler, D. A., Schenck, I., Ball, E. V., Chen, R., Cooper, D. N., Giardine, B., Hsu, F., Kent, W. J., Lesk, A., Nelson, D. L., O'Brien, W. E., Prufer, K., Stenson, P. D., Wallace, J. C., Ke, H., Liu, X., Wang, P., Xiang, A. P., Yang, F., Barber, G. P., Haussler, D., Karolchik, D., Kern, A. D., Kuhn, R. M., Smith, K. E., Zwieg, A. S. 2007; 316 (5822): 222-234

    Abstract

    The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.

    View details for DOI 10.1126/science.1139247

    View details for Web of Science ID 000245654500037

    View details for PubMedID 17431167

  • A variant TMPRSS2 isoform and ERG fusion product in prostate cancer with implications for molecular diagnosis MODERN PATHOLOGY Lapointe, J., Kim, Y. H., Miller, M. A., Li, C., Kaygusuz, G., van de Rijn, M., Huntsman, D. G., Brooks, J. D., Pollack, J. R. 2007; 20 (4): 467-473

    Abstract

    Prostate cancer is the most commonly diagnosed cancer among men in the United States. Recently, fusion of TMPRSS2 with ETS family oncogenic transcription factors has been identified as a common molecular alteration in prostate cancer, where most often the rearrangement places ERG under the androgen-regulated transcriptional control of TMPRSS2. Here, we carried out rapid amplification of cDNA ends (RACE) on a prostate cancer specimen carrying an atypical aberration discovered by array-based comparative genomic hybridization (array CGH), suggesting an alternative fusion partner of ERG. We identified novel transcribed sequences fused to ERG, mapping 4 kb upstream of the TMPRSS2 start site. The sequences derive from an apparent second TMPRSS2 isoform, which we found also expressed in some prostate tumors, suggesting similar androgen-regulated control. In a reverse transcription-polymerase chain reaction (RT-PCR)-based survey of 63 prostate tumor specimens (54 primary and nine lymph node metastases), 44 (70%) cases expressed either the known or novel variant TMPRSS2-ERG fusion, 28 (44%) expressed both, 10 (16%) expressed only the known, and notably six (10%) expressed only the variant isoform fusion. In this specimen set, the presence of a TMPRSS2-ERG fusion showed no statistical association with tumor stage, Gleason grade or recurrence-free survival. Nonetheless, the discovery of a novel variant TMPRSS2 isoform-ERG fusion adds to the characterization of ETS-family rearrangements in prostate cancer, and has important implications for the accurate molecular diagnosis of TMPRSS2-ETS fusions.

    View details for DOI 10.1038/modpathol.3800759

    View details for Web of Science ID 000245271000007

    View details for PubMedID 17334351

  • Target discovery and validation in pancreatic cancer. Methods in molecular biology (Clifton, N.J.) Beaty, R. M., Gronborg, M., Pollack, J. R., Maitra, A. 2007; 360: 57-89

    Abstract

    Pancreatic cancer is a lethal disease and rational strategies for early detection and targeted therapies are urgently required to alleviate the dismal prognosis of this neoplasm. The use of global RNA and protein expression-profiling technologies, such as DNA microarrays, serial analysis of gene expression, and mass spectrometric analysis of proteins, have led to identification of cellular targets with considerable potential for clinical application and patient care. These studies underscore the importance of pursuing large-scale profiling of human cancers not only for furthering our understanding of the pathogenesis of these malignancies but also for developing strategies to improve patient outcomes.

    View details for PubMedID 17172725

  • Expression of tumor-associated antigens in acute myeloid leukemia: implications for specific immunotherapeutic approaches BLOOD Greiner, J., Schmitt, M., Li, L., Giannopoulos, K., Bosch, K., Schmitt, A., Dohner, K., Schlenk, R. F., Pollack, J. R., Dohner, H., Bullinger, L. 2006; 108 (13): 4109-4117

    Abstract

    The expression of tumor-associated antigens (TAAs) might play a critical role in the control of minimal residual disease (MRD) in acute myeloid leukemia (AML), and therefore might be associated with clinical outcome in AML. In a DNA microarray analysis of 116 AML samples, we found a significant correlation between high mRNA levels of G250/CA9 and longer overall survival (P = .022), a similar trend with high mRNA levels of PRAME (P = .103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1, we found no correlation with clinical outcome. High expression of at least 1 of the 3 TAAs, RHAMM/HMMR, PRAME, or G250/CA9, provided the strongest favorable prognostic effect (P = .005). Specific T-cell responses were detected in 8 (47%) of 17 patients with AML in complete remission for RHAMM/HMMR-R3 peptide, in 7 (70%) of 10 for PRAME-P3 peptide, and in 6 (60%) of 10 for newly characterized G250/CA9-G2 peptide, a significant increased immune response compared with patients with AML patients who had refractory disease (P < .001). Furthermore, we could demonstrate specific lysis of T2 cells presenting these epitope peptides. In conclusion, expression of the TAAs RHAMM/HMMR, PRAME, and G250/CA9 can induce strong antileukemic immune responses, possibly enabling MRD control. Thus, these TAAs represent interesting targets for polyvalent immunotherapeutic approaches in AML.

    View details for DOI 10.1182/blood-2006-01-023127

    View details for Web of Science ID 000242675300030

    View details for PubMedID 16931630

  • A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies PLOS MEDICINE Shames, D. S., Girard, L., Gao, B., Sato, M., Lewis, C. M., Shivapurkar, N., Jiang, A., Perou, C. M., Kim, Y. H., Pollack, J. R., Fong, K. M., Lam, C., Wong, M., Shyr, Y., Nanda, R., Olopade, O. I., Gerald, W., Euhus, D. M., Shay, J. W., Gazdar, A. F., Minna, J. D. 2006; 3 (12): 2244-2263

    Abstract

    Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

    View details for DOI 10.1371/journal.pmed.0030486

    View details for Web of Science ID 000243482500018

    View details for PubMedID 17194187

  • Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer GENES CHROMOSOMES & CANCER Bergamaschi, A., Kim, Y. H., Wang, P., Sorlie, T., Hernandez-Boussard, T., Lonning, P. E., Tibshirani, R., Borresen-Dale, A., Pollack, J. R. 2006; 45 (11): 1033-1040

    Abstract

    Breast cancer is a leading cause of cancer-death among women, where the clinicopathological features of tumors are used to prognosticate and guide therapy. DNA copy number alterations (CNAs), which occur frequently in breast cancer and define key pathogenetic events, are also potentially useful prognostic or predictive factors. Here, we report a genome-wide array-based comparative genomic hybridization (array CGH) survey of CNAs in 89 breast tumors from a patient cohort with locally advanced disease. Statistical analysis links distinct cytoband loci harboring CNAs to specific clinicopathological parameters, including tumor grade, estrogen receptor status, presence of TP53 mutation, and overall survival. Notably, distinct spectra of CNAs also underlie the different subtypes of breast cancer recently defined by expression-profiling, implying these subtypes develop along distinct genetic pathways. In addition, higher numbers of gains/losses are associated with the "basal-like" tumor subtype, while high-level DNA amplification is more frequent in "luminal-B" subtype tumors, suggesting also that distinct mechanisms of genomic instability might underlie their pathogenesis. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat

    View details for DOI 10.1002/gcc.20366

    View details for Web of Science ID 000240601400005

    View details for PubMedID 16897746

  • Application of genomic technologies to human prostate cancer OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY Li, S., Bhamre, S., Lapointe, J., Pollack, J. R., Brooks, J. D. 2006; 10 (3): 261-275

    Abstract

    Prostate cancer is the most commonly diagnosed non-cutaneous malignancy in U.S. males and has a broad spectrum of clinical behavior ranging from indolent to lethal. Microarray technology has provided unprecedented opportunity to explore the genetic processes underlying prostate cancer by providing a comprehensive survey of a cell's transcriptional landscape. Prostate cancer, however, has posed significant challenges that have contributed to inconsistent results between studies and difficulty replicating findings. Despite these challenges, several important insights have been gained along with new clinical biomarkers of diagnosis and prognosis. Continued improvements in methods of tissue preparation, microarray technology and data analysis will overcome existing challenges and fuel future discoveries.

    View details for Web of Science ID 000241666200002

    View details for PubMedID 17069507

  • Chromosome instability leaves its mark NATURE GENETICS Pollack, J. R. 2006; 38 (9): 973-974

    View details for Web of Science ID 000240112100008

    View details for PubMedID 16941002

  • Disclosure of candidate genes in acute myeloid leukemia with complex karyotypes using microarray-based molecular characterization JOURNAL OF CLINICAL ONCOLOGY Ruecker, F. G., Bullinger, L., Schwaenen, C., Lipka, D. B., Wessendorf, S., Froehling, S., Bentz, M., Miller, S., Scholl, C., Schlenk, R. F., Radlwimmer, B., Kestler, H. A., Pollack, J. R., Lichter, P., Doehner, K., Doehner, H. 2006; 24 (24): 3887-3894

    Abstract

    To identify novel genomic regions of interest in acute myeloid leukemia (AML) with complex karyotypes, we applied comparative genomic hybridization to microarrays (array-CGH), allowing high-resolution genome-wide screening of genomic imbalances.Sixty AML cases with complex karyotypes were analyzed using array-CGH; parallel analysis of gene expression was performed in a subset of cases.Genomic losses were found more frequently than gains. The most frequent losses affected 5q (77%), 17p (55%), and 7q (45%), and the most frequent genomic gains 11q (40%) and 8q (38%). Critical segments could be delineated to genomic fragments of only 0.8 to a few megabase-pairs of DNA. In lost/gained regions, gene expression profiling detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, high-level DNA amplifications were identified in several regions: 11q23.3-q24.1 (n = 7), 21q22 (n = 6), 11q23.3 (n = 5), 13q12 (n = 3), 8q24 (n = 3), 9p24 (n = 2), 12p13 (n = 2), and 20q11 (n = 2). Parallel analysis of gene expression in critical amplicons displayed overexpressed candidate genes (eg, C8FW and MYC in 8q24).In conclusion, a large spectrum of genomic imbalances, including novel recurring changes in AML with complex karyotypes, was identified using array-CGH. In addition, the combined analysis of array-CGH data with gene expression profiles allowed the detection of candidate genes involved in the pathogenesis of AML.

    View details for DOI 10.1200/JCO.2005.04.5450

    View details for Web of Science ID 000240052300014

    View details for PubMedID 16864856

  • RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes GENES CHROMOSOMES & CANCER Kao, J., Pollack, J. R. 2006; 45 (8): 761-769

    Abstract

    DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes "driving" amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20-30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon "core") at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes.

    View details for DOI 10.1002/gcc.20339

    View details for Web of Science ID 000238774400005

    View details for PubMedID 16708353

  • Molecular profiling reveals myeloid leukemia cell lines to be faithful model systems characterized by distinct genomic aberrations LEUKEMIA Ruecker, F. G., Sander, S., Doehner, K., Doehner, H., Pollack, J. R., Bullinger, L. 2006; 20 (6): 994-1001

    Abstract

    To model and investigate different facets of leukemia pathogenesis, a widely accepted approach is to use immortalized leukemia cell lines. Although these provide powerful tools to our knowledge, few studies have addressed the question whether hematopoietic cell lines represent accurate and reliable model systems. To improve the molecular characterization of these model systems, we analyzed 17 myeloid leukemia cell lines using DNA microarray technology. By array-based comparative genomic hybridization, we identified recurrent genomic DNA gains and losses, as well as high-level amplifications. Parallel analysis of gene expression helped delineate potential candidate genes, and unsupervised analysis of gene expression data revealed cell lines to cluster in part based on underlying cytogenetic abnormalities. Comparison with clinical leukemia specimens showed that key signatures were retained, as myeloid cell lines with characteristic cytogenetic aberrations co-clustered with leukemia samples carrying the respective abnormality. Signatures were also quite robust, as expression data from cell lines correlated highly with published data. Thus, our analyses demonstrate myeloid cell lines to exhibit conserved and stable signatures reflecting the underlying primary cytogenetic aberrations. Our refined molecular characterization of myeloid cell lines supports the utility of cell lines as faithful and powerful model systems and provides additional insights into the molecular mechanisms of leukemogenesis.

    View details for DOI 10.1038/sj.leu.2404235

    View details for Web of Science ID 000237806500012

    View details for PubMedID 16721385

  • DNA microarray and proteomic strategies for understanding alcohol action ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH Sikela, J. M., MacLaren, E. J., Kim, Y., Karimpour-Fard, A., Cai, W. W., Pollack, J., Hitzemann, R., Belknap, J., McWeeney, S., Kerns, R. T., Downing, C., Johnson, T. E., Grant, K. J., Tabakoff, B., Hoffman, P., Wu, C. C., Miles, M. F. 2006; 30 (4): 700-708

    Abstract

    This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.

    View details for DOI 10.1111/j.1530-0277.2006.00081.x

    View details for Web of Science ID 000236756300015

    View details for PubMedID 16573589

  • Amplification of EMSY, a novel oncogene on 11q13, in high grade ovarian surface epithelial carcinomas GYNECOLOGIC ONCOLOGY Brown, L. A., Irving, J., Parker, R., Kim, H., Press, J. Z., Longacre, T. A., Chia, S., Magliocco, A., Makretsov, N., Gilks, B., Pollack, J., Huntsman, D. 2006; 100 (2): 264-270

    Abstract

    Amplification of the 11q13 locus is commonly observed in a number of human cancers including both breast and ovarian cancer. Cyclin D1 and EMS1 have been implicated as candidate oncogenes involved in the emergence of amplification at this locus. Detailed analysis of the 11q13 amplicon in breast cancer led to the discovery of four regions of amplification suggesting the involvement of other genes. Here, we investigate the role of EMSY, a recently described BRCA2 interacting protein, as a key element of the 11q13 amplicon in ovarian cancer. EMSY maps to 11q13.5 and is amplified in 13% of breast and 17% of ovarian carcinomas.EMSY amplification was assessed by fluorescent in-situ hybridization (FISH) in 674 ovarian cancers in a tissue microarray and correlated with histopathological subtype and tumor grade. A detailed map of the 11q13 amplicon in 51 cases of ovarian cancer was obtained using cDNA-array-based comparative genomic hybridization (aCGH). To further characterize the role of EMSY within this amplicon, we evaluated both the amplification profiles and RNA expression levels of EMSY and two other genes from the 11q13 amplicon in an additional series of 22 ovarian carcinomas.EMSY amplification was seen in 52/285 (18%) high grade papillary serous carcinomas, 4/27 (15%) high grade endometrioid carcinomas, 3/38 (8%) clear cell carcinomas, and 3/10 (30%) undifferentiated carcinomas. aCGH mapping of 11q13 in ovarian cancer showed that EMSY localized to the region with the highest frequency of copy number gain. Cyclin D1 and EMS1 showed a lower frequency of copy number gain. A highly significant correlation between EMSY gene amplification and RNA expression was also observed (P = 0.0001). This was a stronger correlation than for other genes at 11q13 including Cyclin D1 and PAK1.These findings support the role of EMSY as a key oncogene within the 11q13 amplicon in ovarian cancer.

    View details for DOI 10.1016/j.ygyno.2005.08.026

    View details for Web of Science ID 000235022800009

    View details for PubMedID 16236351

  • Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification ONCOGENE Kim, Y. H., Girard, L., Giacomini, C. P., Wang, P., Hernandez-Boussard, T., Tibshirani, R., Minna, J. D., Pollack, J. R. 2006; 25 (1): 130-138

    Abstract

    DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22,000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.

    View details for DOI 10.1038/sj.onc.1208997

    View details for Web of Science ID 000234406400014

    View details for PubMedID 16116477

  • A gene expression signature of genetic instability in colon cancer CANCER RESEARCH Giacomini, C. P., Leung, S. Y., Chen, X., Yuen, S. T., Kim, Y. H., Bair, E., Pollack, J. R. 2005; 65 (20): 9200-9205

    Abstract

    Genetic instability plays a central role in the development and progression of human cancer. Two major classes of genetic instability, microsatellite instability (MSI) and chromosome instability (microsatellite stable; MSS), are best understood in the context of colon cancer, where MSI tumors represent approximately 15% of cases, and compared with MSS tumors, more often arise in the proximal colon and display favorable clinical outcome. To further explore molecular differences, we profiled gene expression in a set of 18 colon cancer cell lines using cDNA microarrays representing approximately 21,000 different genes. Supervised analysis identified a robust expression signature distinguishing MSI and MSS samples. As few as eight genes predicted with high accuracy the underlying genetic instability in the original and in three independent sample sets, comprising 13 colon cancer cell lines, 61 colorectal tumors, and 87 gastric tumors. Notably, the MSI signature was retained despite genetically correcting the underlying instability, suggesting the signature reflects a legacy of the tumor having arisen from MSI, rather than sensing the ongoing state of MSI. Our findings support a model in which MSI and MSS preferentially target different genes and pathways in cancer. Further, among the MSI signature genes, our findings implicate a role of elevated metallothionein expression in the clinical behavior of MSI cancers.

    View details for DOI 10.1158/0008-5472.CAN-04-4163

    View details for Web of Science ID 000232566800019

    View details for PubMedID 16230380

  • The retinoic acid synthesis gene ALDH1a2 is a candidate tumor suppressor in prostate cancer CANCER RESEARCH Kim, H., Lapointe, J., Kaygusuz, G., Ong, D. E., Li, C. D., van de Rijn, M., Brooks, J. D., Pollack, J. R. 2005; 65 (18): 8118-8124

    Abstract

    Prostate cancer is the most common cancer among men in the United States, and aberrant DNA methylation is known to be an early molecular event in its development. Here, we have used expression profiling to identify novel hypermethylated genes whose expression is induced by treatment of prostate cancer cell lines with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-dC). Of the 271 genes that were induced by 5-aza-dC treatment, 25 also displayed reduced expression in primary prostate tumors compared with normal prostate tissue, and the decreased expression of only one gene, aldehyde dehydrogenase 1 family, member A2 (ALDH1a2), was also associated with shorter recurrence-free survival. ALDH1a2 encodes an enzyme responsible for synthesis of retinoic acid (RA), a compound with prodifferentiation properties. By immunohistochemistry, we observed that ALDH1a2 was expressed in epithelia from normal prostate but not prostate cancer. Using bisulfite sequencing, we determined that the ALDH1a2 promoter region was significantly hypermethylated in primary prostate tumors compared with normal prostate specimens (P = 0.01). Finally, transfection-mediated reexpression of wild-type ALDH1a2 (but not a presumptive catalytically dead mutant) in the prostate cancer cell line DU145 resulted in decreased colony growth (P < 0.0001), comparable with treatment with either 5-aza-dC or RA. Taken together, our findings implicate ALDH1a2 as a candidate tumor suppressor gene in prostate cancer and further support a role of retinoids in the prevention or treatment of prostate cancer.

    View details for DOI 10.1158/0008-5472.CAN-04-4562

    View details for Web of Science ID 000231848800010

    View details for PubMedID 16166285

  • Comparative genomic hybridization on mouse cDNA microarrays and its application to a murine lymphoma model ONCOGENE Sander, S., Bullinger, L., Karlsson, A., Giuriato, S., Hernandez-Boussard, T., Felsher, D. W., Pollack, J. R. 2005; 24 (40): 6101-6107

    Abstract

    Microarray-based formats offer a high-resolution alternative to conventional, chromosome-based comparative genomic hybridization (CGH) methods for assessing DNA copy number alteration (CNA) genome-wide in human cancer. For murine tumors, array CGH should provide even greater advantage, since murine chromosomes are more difficult to individually discern. We report here the adaptation and evaluation of a cDNA microarray-based CGH method for the routine characterization of CNAs in murine tumors, using mouse cDNA microarrays representing approximately 14,000 different genes, thereby providing an average mapping resolution of 109 kb. As a first application, we have characterized CNAs in a set of 10 primary and recurrent lymphomas derived from a Myc-induced murine lymphoma model. In primary lymphomas and more commonly in Myc-independent relapses, we identified a recurrent genomic DNA loss at chromosome 3G3-3H4, and recurrent amplifications at chromosome 3F2.1-3G3 and chromosome 15E1/E2-15F3, the boundaries of which we defined with high resolution. Further, by profiling gene expression using the same microarray platform, we identified within CNAs the relevant subset of candidate cancer genes displaying comparably altered expression, including Mcl1 (myeloid cell leukemia sequence 1), a highly expressed antiapoptotic gene residing within the chr 3 amplicon peak. CGH on mouse cDNA microarrays therefore represents a reliable method for the high-resolution characterization of CNAs in murine tumors, and a powerful approach for elucidating the molecular events in tumor development and progression in murine models.

    View details for DOI 10.1038/sj.onc.1208751

    View details for Web of Science ID 000231718100004

    View details for PubMedID 16007205

  • Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer NEOPLASIA Bashyam, M. D., Bair, R., Kim, Y. H., Wang, P., Hernandez-Boussard, T., Karikari, C. A., Tibshirani, R., Maitra, A., Pollack, J. R. 2005; 7 (6): 556-562

    Abstract

    Pancreatic cancer, the fourth leading cause of cancer death in the United States, is frequently associated with the amplification and deletion of specific oncogenes and tumor-suppressor genes (TSGs), respectively. To identify such novel alterations and to discover the underlying genes, we performed comparative genomic hybridization on a set of 22 human pancreatic cancer cell lines, using cDNA microarrays measuring approximately 26,000 human genes (thereby providing an average mapping resolution of <60 kb). To define the subset of amplified and deleted genes with correspondingly altered expression, we also profiled mRNA levels in parallel using the same cDNA microarray platform. In total, we identified 14 high-level amplifications (38-4934 kb in size) and 15 homozygous deletions (46-725 kb). We discovered novel localized amplicons, suggesting previously unrecognized candidate oncogenes at 6p21, 7q21 (SMURF1, TRRAP), 11q22 (BIRC2, BIRC3), 12p12, 14q24 (TGFB3), 17q12, and 19q13. Likewise, we identified novel polymerase chain reaction-validated homozygous deletions indicating new candidate TSGs at 6q25, 8p23, 8p22 (TUSC3), 9q33 (TNC, TNFSF15), 10q22, 10q24 (CHUK), 11p15 (DKK3), 16q23, 18q23, 21q22 (PRDM15, ANKRD3), and Xp11. Our findings suggest candidate genes and pathways, which may contribute to the development or progression of pancreatic cancer.

    View details for DOI 10.1593/neo.04586

    View details for Web of Science ID 000230209600002

    View details for PubMedID 16036106

  • Genomics in myeloid leukemias: an array of possibilities. Reviews in clinical and experimental hematology Bullinger, L., Döner, H., Pollack, J. R. 2005; 9 (1): E2-?

    Abstract

    Myeloid leukemias are clonal hematopoietic stem cell disorders characterized either by proliferation of one or more of the myeloid lineages (chronic myelogenous leukemia) or by clonal expansion of myeloid blasts (acute myeloid leukemia). Over the past several years our knowledge of these hematologic malignancies has increased tremendously. The result is a classification that incorporates morphologic, immunophenotypic, genetic and clinical features in an attempt to define biologically and clinically relevant entities. Nevertheless, in many tumor subtypes the pathogenic event is still unknown. Furthermore, well-defined leukemia subgroups exhibit considerable heterogeneity, arousing the suspicion that several molecularly distinct subtypes might exist within the same cytogenetic category. Therefore, an ideal classification system would ultimately be based on the underlying molecular pathogenesis, but such knowledge is not yet available. However, by surveying the expression levels of thousands of genes in parallel, DNA microarrays have recently contributed to an increasingly refined molecular taxonomy of myeloid disorders. This powerful technology is becoming well established and has been used to diagnosis cancer and predict clinical outcome, to discover novel tumor subclasses, to gain insights into pathogenesis, and to identify new therapeutic targets. While many challenges remain ahead, genomic technologies have already demonstrated tremendous potential. We expect whole genome approaches will significantly contribute to a better understanding of the pathogenesis and result in a refined molecular classification of myeloid leukemias.

    View details for PubMedID 16027104

  • An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin CANCER RESEARCH Tothill, R. W., Kowalczyk, A., Rischin, D., Bousioutas, A., Haviv, I., Van Laar, R. K., Waring, P. M., Zalcberg, J., Ward, R., Biankin, A. V., Sutherland, R. L., Henshall, S. M., Fong, K., Pollack, J. R., Bowtell, D. D., Holloway, A. J. 2005; 65 (10): 4031-4040

    Abstract

    Gene expression profiling offers a promising new technique for the diagnosis and prognosis of cancer. We have applied this technology to build a clinically robust site of origin classifier with the ultimate aim of applying it to determine the origin of cancer of unknown primary (CUP). A single cDNA microarray platform was used to profile 229 primary and metastatic tumors representing 14 tumor types and multiple histologic subtypes. This data set was subsequently used for training and validation of a support vector machine (SVM) classifier, demonstrating 89% accuracy using a 13-class model. Further, we show the translation of a five-class classifier to a quantitative PCR-based platform. Selecting 79 optimal gene markers, we generated a quantitative-PCR low-density array, allowing the assay of both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue. Data generated using both quantitative PCR and microarray were subsequently used to train and validate a cross-platform SVM model with high prediction accuracy. Finally, we applied our SVM classifiers to 13 cases of CUP. We show that the microarray SVM classifier was capable of making high confidence predictions in 11 of 13 cases. These predictions were supported by comprehensive review of the patients' clinical histories.

    View details for Web of Science ID 000229062000009

    View details for PubMedID 15899792

  • Amplification of whole tumor genomes and geneby-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA JOURNAL OF MOLECULAR DIAGNOSTICS Bredel, M., Bredel, C., Juric, D., Kim, Y., Vogel, H., Harsh, G. R., Recht, L. D., Pollack, J. R., Sikic, B. I. 2005; 7 (2): 171-182

    Abstract

    Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens.

    View details for Web of Science ID 000228736900004

    View details for PubMedID 15858140

  • Genome-wide characterization of gene expression variations and DNA copy number changes in prostate cancer cell lines PROSTATE Zhao, H. J., Kim, Y., Wang, P., Lapointe, J., Tibshirani, R., Pollack, J. R., Brooks, J. D. 2005; 63 (2): 187-197

    Abstract

    The aim of this study was to characterize gene expression and DNA copy number profiles in androgen sensitive (AS) and androgen insensitive (AI) prostate cancer cell lines on a genome-wide scale.Gene expression profiles and DNA copy number changes were examined using DNA microarrays in eight commonly used prostate cancer cell lines. Chromosomal regions with DNA copy number changes were identified using cluster along chromosome (CLAC).There were discrete differences in gene expression patterns between AS and AI cells that were not limited to androgen-responsive genes. AI cells displayed more DNA copy number changes, especially amplifications, than AS cells. The gene expression profiles of cell lines showed limited similarities to prostate tumors harvested at surgery.AS and AI cell lines are different in their transcriptional programs and degree of DNA copy number alterations. This dataset provides a context for the use of prostate cancer cell lines as models for clinical cancers.

    View details for DOI 10.1002/pros.20158

    View details for Web of Science ID 000228400100007

    View details for PubMedID 15486987

  • Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer CANCER RESEARCH Amann, J., Kalyankrishna, S., Massion, P. P., Ohm, J. E., Girard, L., Shigematsu, H., Peyton, N., Juroske, D., Huang, Y., Salmon, J. S., Kim, Y. H., Pollack, J. R., Yanagisawa, K., Gazdar, A., Minna, J. D., Kurie, J. M., Carbone, D. P. 2005; 65 (1): 226-235

    Abstract

    Epidermal growth factor receptor (EGFR) is occasionally amplified and/or mutated in non-small cell lung cancer (NSCLC) and can be coexpressed with other members of the HER receptor family to form functional heterodimers. We therefore investigated lung cancer cell lines for alterations in EGFR gene copy number, enhanced expression of EGFR and other HER family members, and EGFR coding sequence mutations and correlated these findings with response to treatment with the EGFR inhibitors and the kinetics of ligand-induced signaling. We show here that somatic deletions in the tyrosine kinase domain of EGFR were associated with increased EGFR gene copy number in NSCLC. Treatment with the specific EGFR tyrosine kinase inhibitors (TKI) gefitinib or erlotinib or the EGFR inhibitory antibody cetuximab induced apoptosis of HCC827, a NSCLC cell line with EGFR gene amplification and an exon 19 deletion. H1819, a NSCLC cell line that expresses high levels of EGFR, ErbB2, and ErbB3 but has wild-type EGFR, showed intermediate sensitivity to TKIs. In both cell lines, ligand-induced receptor tyrosine phosphorylation was delayed and prolonged and AKT was constitutively phosphorylated (but remained inhibitable by EGFR TKI). Thus, in addition to EGFR mutations, other factors in NSCLC cells, such as high expression of ErbB family members, may constitutively activate AKT and sensitize cells to EGFR inhibitors.

    View details for Web of Science ID 000226080200029

    View details for PubMedID 15665299

  • A DNA microarray survey of gene expression in normal human tissues GENOME BIOLOGY Shyamsundar, R., Kim, Y. H., Higgins, J. P., Montgomery, K., Jorden, M., Sethuraman, A., van de Rijn, M., Botstein, D., Brown, P. O., Pollack, J. R. 2005; 6 (3)

    Abstract

    Numerous studies have used DNA microarrays to survey gene expression in cancer and other disease states. Comparatively little is known about the genes expressed across the gamut of normal human tissues. Systematic studies of global gene-expression patterns, by linking variation in the expression of specific genes to phenotypic variation in the cells or tissues in which they are expressed, provide clues to the molecular organization of diverse cells and to the potential roles of the genes.Here we describe a systematic survey of gene expression in 115 human tissue samples representing 35 different tissue types, using cDNA microarrays representing approximately 26,000 different human genes. Unsupervised hierarchical cluster analysis of the gene-expression patterns in these tissues identified clusters of genes with related biological functions and grouped the tissue specimens in a pattern that reflected their anatomic locations, cellular compositions or physiologic functions. In unsupervised and supervised analyses, tissue-specific patterns of gene expression were readily discernable. By comparative hybridization to normal genomic DNA, we were also able to estimate transcript abundances for expressed genes.Our dataset provides a baseline for comparison to diseased tissues, and will aid in the identification of tissue-specific functions. In addition, our analysis identifies potential molecular markers for detection of injury to specific organs and tissues, and provides a foundation for selection of potential targets for selective anticancer therapy.

    View details for Web of Science ID 000228246400007

    View details for PubMedID 15774023

  • A method for calling gains and losses in array CGH data BIOSTATISTICS Wang, P., Kim, Y., Pollack, J., Narasimhan, B., Tibshirani, R. 2005; 6 (1): 45-58

    Abstract

    Array CGH is a powerful technique for genomic studies of cancer. It enables one to carry out genome-wide screening for regions of genetic alterations, such as chromosome gains and losses, or localized amplifications and deletions. In this paper, we propose a new algorithm 'Cluster along chromosomes' (CLAC) for the analysis of array CGH data. CLAC builds hierarchical clustering-style trees along each chromosome arm (or chromosome), and then selects the 'interesting' clusters by controlling the False Discovery Rate (FDR) at a certain level. In addition, it provides a consensus summary across a set of arrays, as well as an estimate of the corresponding FDR. We illustrate the method using an application of CLAC on a lung cancer microarray CGH data set as well as a BAC array CGH data set of aneuploid cell strains.

    View details for DOI 10.1093/biostatistics/kxh017

    View details for Web of Science ID 000226346300005

    View details for PubMedID 15618527

  • Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins CANCER RESEARCH Ramirez, R. D., Sheridan, S., Girard, L., Sato, M., Kim, Y., Pollack, J., Peyton, M., Zou, Y., Kurie, J. M., DiMaio, J. M., Milchgrub, S., Smith, A. L., Souza, R. F., Gilbey, L., Zhang, X., Gandia, K., Vaughan, M. B., Wright, W. E., Gazdar, A. F., Shay, J. W., Minna, J. D. 2004; 64 (24): 9027-9034

    Abstract

    By expressing two genes (hTERT and Cdk4), we have developed a method to reproducibly generate continuously replicating human bronchial epithelial cell (HBEC) lines that provide a novel resource to study the molecular pathogenesis of lung cancer and the differentiation of bronchial epithelial cells. Twelve human bronchial epithelial biopsy specimens obtained from persons with and without lung cancer were placed into short-term culture and serially transfected with retroviral constructs containing cyclin-dependent kinase (Cdk) 4 and human telomerase reverse transcriptase (hTERT), resulting in continuously growing cultures. The order of introduction of Cdk4 and hTERT did not appear to be important; however, transfection of either gene alone did not result in immortalization. Although they could be cloned, the immortalized bronchial cells did not form colonies in soft agar or tumors in nude mice. The immortalized HBECs have epithelial morphology; express epithelial markers cytokeratins 7, 14, 17, and 19, the stem cell marker p63, and high levels of p16(INK4a); and have an intact p53 checkpoint pathway. Cytogenetic analysis and array comparative genomic hybridization profiling show immortalized HBECs to have duplication of parts of chromosomes 5 and 20. Microarray gene expression profiling demonstrates that the Cdk4/hTERT-immortalized bronchial cell lines clustered together and with nonimmortalized bronchial cells, distinct from lung cancer cell lines. We also immortalized several parental cultures with viral oncoproteins human papilloma virus type 16 E6/E7 with and without hTERT, and these cells exhibited loss of the p53 checkpoint and significantly different gene expression profiles compared with Cdk4/hTERT-immortalized HBECs. These HBEC lines are a valuable new tool for studying of the pathogenesis of lung cancer.

    View details for Web of Science ID 000225809200036

    View details for PubMedID 15604268

  • Lineage-specific gene duplication and loss in human and great ape evolution PLOS BIOLOGY Fortna, A., Kim, Y., MacLaren, E., Marshall, K., Hahn, G., Meltesen, L., Brenton, M., Hink, R., Burgers, S., Hernandez-Boussard, T., Karimpour-Fard, A., Glueck, D., McGavran, L., Berry, R., Pollack, J., Sikela, J. M. 2004; 2 (7): 937-954
  • Lineage-specific gene duplication and loss in human and great ape evolution. PLoS biology Fortna, A., Kim, Y., MacLaren, E., Marshall, K., Hahn, G., Meltesen, L., Brenton, M., Hink, R., Burgers, S., Hernandez-Boussard, T., Karimpour-Fard, A., Glueck, D., McGavran, L., Berry, R., Pollack, J., Sikela, J. M. 2004; 2 (7): E207-?

    Abstract

    Given that gene duplication is a major driving force of evolutionary change and the key mechanism underlying the emergence of new genes and biological processes, this study sought to use a novel genome-wide approach to identify genes that have undergone lineage-specific duplications or contractions among several hominoid lineages. Interspecies cDNA array-based comparative genomic hybridization was used to individually compare copy number variation for 39,711 cDNAs, representing 29,619 human genes, across five hominoid species, including human. We identified 1,005 genes, either as isolated genes or in clusters positionally biased toward rearrangement-prone genomic regions, that produced relative hybridization signals unique to one or more of the hominoid lineages. Measured as a function of the evolutionary age of each lineage, genes showing copy number expansions were most pronounced in human (134) and include a number of genes thought to be involved in the structure and function of the brain. This work represents, to our knowledge, the first genome-wide gene-based survey of gene duplication across hominoid species. The genes identified here likely represent a significant majority of the major gene copy number changes that have occurred over the past 15 million years of human and great ape evolution and are likely to underlie some of the key phenotypic characteristics that distinguish these species.

    View details for PubMedID 15252450

  • Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia NEW ENGLAND JOURNAL OF MEDICINE Bullinger, L., Dohner, K., Bair, E., Frohling, S., Schlenk, R. F., Tibshirani, R., Dohner, H., Pollack, J. R. 2004; 350 (16): 1605-1616

    Abstract

    In patients with acute myeloid leukemia (AML), the presence or absence of recurrent cytogenetic aberrations is used to identify the appropriate therapy. However, the current classification system does not fully reflect the molecular heterogeneity of the disease, and treatment stratification is difficult, especially for patients with intermediate-risk AML with a normal karyotype.We used complementary-DNA microarrays to determine the levels of gene expression in peripheral-blood samples or bone marrow samples from 116 adults with AML (including 45 with a normal karyotype). We used unsupervised hierarchical clustering analysis to identify molecular subgroups with distinct gene-expression signatures. Using a training set of samples from 59 patients, we applied a novel supervised learning algorithm to devise a gene-expression-based clinical-outcome predictor, which we then tested using an independent validation group comprising the 57 remaining patients.Unsupervised analysis identified new molecular subtypes of AML, including two prognostically relevant subgroups in AML with a normal karyotype. Using the supervised learning algorithm, we constructed an optimal 133-gene clinical-outcome predictor, which accurately predicted overall survival among patients in the independent validation group (P=0.006), including the subgroup of patients with AML with a normal karyotype (P=0.046). In multivariate analysis, the gene-expression predictor was a strong independent prognostic factor (odds ratio, 8.8; 95 percent confidence interval, 2.6 to 29.3; P<0.001).The use of gene-expression profiling improves the molecular classification of adult AML.

    View details for Web of Science ID 000220819800005

    View details for PubMedID 15084693

  • High-resolution array-based comparative genomic hybridization for distinguishing paraffin-embedded Spitz nevi and melanomas DIAGNOSTIC MOLECULAR PATHOLOGY Harvell, J. D., Kohler, S., Zhu, S., Hernandez-Boussard, T., Pollack, J. R., van de Rijn, M. 2004; 13 (1): 22-25

    Abstract

    Distinguishing between Spitz nevus and melanoma presents a challenging task for clinicians and pathologists. Most of these lesions are submitted entirely in formalin for histologic analysis by conventional hematoxylin and eosin-stained sections, and fresh-frozen material for ancillary studies is rarely collected. Molecular techniques, such as comparative genomic hybridization (CGH), can detect chromosomal alterations in tumor DNA that differ between these 2 lesions. This study investigated the ability of high-resolution array-based CGH to serve as a diagnostic test in distinguishing Spitz nevus and melanoma using DNA isolated from formalin-fixed and paraffin-embedded samples. Two of 3 Spitz nevi exhibited no significant chromosomal alterations, while the third showed gain of the short arm of chromosome 11p. The latter finding has previously been described as characteristic of a subset of Spitz nevi. The 2 melanomas showed multiple copy number alterations characteristic of melanoma such as 1q amplification and chromosome 9 deletion. This study has shown the utility of array-based CGH as a potential molecular test in distinguishing Spitz nevus from melanoma. The assay is capable of using archival paraffin-embedded, formalin-fixed material; is technically easier to perform as compared with conventional CGH; is more sensitive than conventional CGH in being able to detect focal alterations; and can detect copy number alterations even with relatively small amounts of lesional tissue as is typical of many skin tumors.

    View details for Web of Science ID 000189276600004

    View details for PubMedID 15163005

  • Gene expression profiling identifies clinically relevant subtypes of prostate cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lapointe, J., Li, C., Higgins, J. P., van de Rijn, M., Bair, E., Montgomery, K., Ferrari, M., Egevad, L., Rayford, W., Bergerheim, U., Ekman, P., DeMarzo, A. M., Tibshirani, R., Botstein, D., Brown, P. O., Brooks, J. D., Pollack, J. R. 2004; 101 (3): 811-816

    Abstract

    Prostate cancer, a leading cause of cancer death, displays a broad range of clinical behavior from relatively indolent to aggressive metastatic disease. To explore potential molecular variation underlying this clinical heterogeneity, we profiled gene expression in 62 primary prostate tumors, as well as 41 normal prostate specimens and nine lymph node metastases, using cDNA microarrays containing approximately 26,000 genes. Unsupervised hierarchical clustering readily distinguished tumors from normal samples, and further identified three subclasses of prostate tumors based on distinct patterns of gene expression. High-grade and advanced stage tumors, as well as tumors associated with recurrence, were disproportionately represented among two of the three subtypes, one of which also included most lymph node metastases. To further characterize the clinical relevance of tumor subtypes, we evaluated as surrogate markers two genes differentially expressed among tumor subgroups by using immunohistochemistry on tissue microarrays representing an independent set of 225 prostate tumors. Positive staining for MUC1, a gene highly expressed in the subgroups with "aggressive" clinicopathological features, was associated with an elevated risk of recurrence (P = 0.003), whereas strong staining for AZGP1, a gene highly expressed in the other subgroup, was associated with a decreased risk of recurrence (P = 0.0008). In multivariate analysis, MUC1 and AZGP1 staining were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative prostate-specific antigen levels. Our results suggest that prostate tumors can be usefully classified according to their gene expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification.

    View details for DOI 10.1073/pnas.0304146101

    View details for Web of Science ID 000188555400023

    View details for PubMedID 14711987

  • Boosted PRIM with application to searching for oncogenic pathway of lung cancer 2004 IEEE COMPUTATIONAL SYSTEMS BIOINFORMATICS CONFERENCE, PROCEEDINGS Wang, P., Kim, Y., Pollack, J., Tibshirani, R. 2004: 604-609
  • Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans AMERICAN JOURNAL OF PATHOLOGY Linn, S. C., West, R. B., Pollack, J. R., Zhu, S., Hernandez-Boussard, T., Nielsen, T. O., Rubin, B. P., Patel, R., Goldblum, J. R., Siegmund, D., Botstein, D., Brown, P. O., Gilks, C. B., van de Rijn, M. 2003; 163 (6): 2383-2395

    Abstract

    Dermatofibrosarcoma protuberans (DFSP) is an aggressive spindle cell neoplasm. It is associated with the chromosomal translocation, t(17:22), which fuses the COL1A1 and PDGFbeta genes. We determined the characteristic gene expression profile of DFSP and characterized DNA copy number changes in DFSP by array-based comparative genomic hybridization (array CGH). Fresh frozen and formalin-fixed, paraffin-embedded samples of DFSP were analyzed by array CGH (four cases) and DNA microarray analysis of global gene expression (nine cases). The nine DFSPs were readily distinguished from 27 other diverse soft tissue tumors based on their gene expression patterns. Genes characteristically expressed in the DFSPs included PDGF beta and its receptor, PDGFRB, APOD, MEOX1, PLA2R, and PRKCA. Array CGH of DNA extracted either from frozen tumor samples or from paraffin blocks yielded equivalent results. Large areas of chromosomes 17q and 22q, bounded by COL1A1 and PDGF beta, respectively, were amplified in DFSP. Expression of genes in the amplified regions was significantly elevated. Our data shows that: 1) DFSP has a distinctive gene expression profile; 2) array CGH can be applied successfully to frozen or formalin-fixed, paraffin-embedded tumor samples; 3) a characteristic amplification of sequences from chromosomes 17q and 22q, demarcated by the COL1A1 and PDGF beta genes, respectively, was associated with elevated expression of the amplified genes.

    View details for Web of Science ID 000186769800024

    View details for PubMedID 14633610

  • Gene expression profiling in prostate cancer cells with Akt activation reveals Fra-1 as an Akt-inducible gene MOLECULAR CANCER RESEARCH Tiwari, G., Sakaue, H., Pollack, J. R., Roth, R. A. 2003; 1 (6): 475-484

    Abstract

    To determine which genes may be regulated by Akt and participate in the transformation of cells, we have examined by microarray analyses genes turned on in the prostate cancer cell line, PC3, when Akt activity was induced. PC3 cells, which lack the lipid phosphatase PTEN, were treated overnight with a reversible inhibitor of the phosphatidylinositol 3-kinase, LY294002 (a treatment which was found to reversibly decrease Akt enzymatic activity). The inhibitor was then washed out and mRNA collected 2, 6, and 10 h later and compared by microarray analyses with mRNAs present immediately after removal of the inhibitor. One of the identified induced mRNAs, Fra-1, was further studied by transient transfections of a reporter construct containing its 5' regulatory region. This construct was found to be directly induced 4- to 5-fold by co-transfection with constitutively active Akt3 but not kinase dead Akt. The regulation by Akt3 was found to be due to two specific regions in the Fra-1 regulatory sequence which match Sp1 consensus sites. Finally, gel shift studies showed that the binding of Sp1 to one of these sites was dependent on the PI 3-kinase pathway. These results indicate that LY294002 treatment and washout is a useful method to study the activation of Akt in the context of a tumor cell. Moreover, the identification of Fra-1 as an Akt-regulated gene may have implications for the ability of Akt to transform cells since Fra-1 has been implicated in cell growth and the aggressiveness of tumors.

    View details for Web of Science ID 000182325600008

    View details for PubMedID 12692267

  • Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray AMERICAN JOURNAL OF PATHOLOGY Higgins, J. P., Shinghal, R., Gill, H., Reese, J. H., Terris, M., Cohen, R. J., Fero, M., Pollack, J. R., van de Rijn, M., Brooks, J. D. 2003; 162 (3): 925-932

    Abstract

    Renal cell carcinoma comprises several histological types with different clinical behavior. Accurate pathological characterization is important in the clinical management of these tumors. We describe gene expression profiles in 41 renal tumors determined by using DNA microarrays containing 22,648 unique cDNAs representing 17,083 different UniGene Clusters, including 7230 characterized human genes. Differences in the patterns of gene expression among the different tumor types were readily apparent; hierarchical cluster analysis of the tumor samples segregated histologically distinct tumor types solely based on their gene expression patterns. Conventional renal cell carcinomas with clear cells showed a highly distinctive pattern of gene expression. Papillary carcinomas formed a tightly clustered group, as did tumors arising from the distal nephron and the normal kidney samples. Surprisingly, conventional renal cell carcinomas with granular cytoplasm were heterogeneous, and did not resemble any of the conventional carcinomas with clear cytoplasm in their pattern of gene expression. Characterization of renal cell carcinomas based on gene expression patterns provides a revised classification of these tumors and has the potential to supply significant biological and clinical insights.

    View details for Web of Science ID 000181215000023

    View details for PubMedID 12598325

  • Parallel analysis of gene copy number and expression using cDNA microarrays. Methods in molecular biology (Clifton, N.J.) Pollack, J. R. 2003; 224: 89-97

    View details for PubMedID 12710668

  • The diagnosis and management of pre-invasive breast disease - Promise of new technologies in understanding pre-invasive breast lesions BREAST CANCER RESEARCH Jeffrey, S. S., Pollack, J. R. 2003; 5 (6): 320-328

    Abstract

    Array-based comparative genomic hybridization, RNA expression profiling, and proteomic analyses are new molecular technologies used to study breast cancer. Invasive breast cancers were originally evaluated because they provided ample quantities of DNA, RNA, and protein. The application of these technologies to pre-invasive breast lesions is discussed, including methods that facilitate their implementation. Data indicate that atypical ductal hyperplasia and ductal carcinoma in situ are precursor lesions molecularly similar to adjacent invasive breast cancer. It is expected that molecular technologies will identify breast tissue at risk for the development of unfavorable subtypes of invasive breast cancer and reveal strategies for targeted chemoprevention or eradication.

    View details for DOI 10.1186/bcr655

    View details for Web of Science ID 000185946100009

    View details for PubMedID 14580250

  • Characterizing the physical genome NATURE GENETICS Pollack, J. R., Iyer, V. R. 2002; 32: 515-521

    Abstract

    The genome of an organism is a dynamic physical entity, comprising genomic DNA bound to many different proteins and organized into chromosomes. A thorough characterization of the physical genome is relevant to our understanding of processes such as the regulation of gene expression, DNA replication and repair, recombination, chromosome segregation, epigenetic inheritance and genomic instability. Methods based on microarrays are beginning to provide a detailed picture of this physical genome, and they complement the genome-wide studies of mRNA expression profiling that have previously been so successful.

    View details for DOI 10.1038/ng1035

    View details for Web of Science ID 000179742900012

    View details for PubMedID 12454647

  • Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pollack, J. R., Sorlie, T., Perou, C. M., Rees, C. A., Jeffrey, S. S., Lonning, P. E., Tibshirani, R., Botstein, D., Borresen-Dale, A. L., Brown, P. O. 2002; 99 (20): 12963-12968

    Abstract

    Genomic DNA copy number alterations are key genetic events in the development and progression of human cancers. Here we report a genome-wide microarray comparative genomic hybridization (array CGH) analysis of DNA copy number variation in a series of primary human breast tumors. We have profiled DNA copy number alteration across 6,691 mapped human genes, in 44 predominantly advanced, primary breast tumors and 10 breast cancer cell lines. While the overall patterns of DNA amplification and deletion corroborate previous cytogenetic studies, the high-resolution (gene-by-gene) mapping of amplicon boundaries and the quantitative analysis of amplicon shape provide significant improvement in the localization of candidate oncogenes. Parallel microarray measurements of mRNA levels reveal the remarkable degree to which variation in gene copy number contributes to variation in gene expression in tumor cells. Specifically, we find that 62% of highly amplified genes show moderately or highly elevated expression, that DNA copy number influences gene expression across a wide range of DNA copy number alterations (deletion, low-, mid- and high-level amplification), that on average, a 2-fold change in DNA copy number is associated with a corresponding 1.5-fold change in mRNA levels, and that overall, at least 12% of all the variation in gene expression among the breast tumors is directly attributable to underlying variation in gene copy number. These findings provide evidence that widespread DNA copy number alteration can lead directly to global deregulation of gene expression, which may contribute to the development or progression of cancer.

    View details for DOI 10.1073/pnas.162471999

    View details for Web of Science ID 000178391700085

    View details for PubMedID 12297621

  • A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC developmental biology Willert, J., Epping, M., Pollack, J. R., Brown, P. O., Nusse, R. 2002; 2: 8-?

    Abstract

    Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

    View details for PubMedID 12095419

  • Challenges in developing a molecular characterization of cancer SEMINARS IN ONCOLOGY Pollack, J. R., van de Rijn, M., Botstein, D. 2002; 29 (3): 280-285

    Abstract

    DNA microarrays are widely used to measure gene expression across thousands of genes in parallel. Recently, considerable efforts have been made to utilize this technology to improve our understanding of cancer and to identify novel diagnostic markers and therapeutic targets. Here, we detail some of the challenges in developing a molecular characterization of cancer and in translating these new discoveries towards clinical utility.

    View details for DOI 10.1053/sonc.2002.32903

    View details for Web of Science ID 000176156500009

    View details for PubMedID 12063681

  • Molecular characterisation of soft tissue tumours: a gene expression study LANCET Nielsen, T. O., West, R. B., Linn, S. C., Alter, O., Knowling, M. A., O'Connell, J. X., Zhu, S., Fero, M., Sherlock, G., Pollack, J. R., Brown, P. O., Botstein, D., van de Rijn, M. 2002; 359 (9314): 1301-1307

    Abstract

    Soft-tissue tumours are derived from mesenchymal cells such as fibroblasts, muscle cells, or adipocytes, but for many such tumours the histogenesis is controversial. We aimed to start molecular characterisation of these rare neoplasms and to do a genome-wide search for new diagnostic markers.We analysed gene-expression patterns of 41 soft-tissue tumours with spotted cDNA microarrays. After removal of errors introduced by use of different microarray batches, the expression patterns of 5520 genes that were well defined were used to separate tumours into discrete groups by hierarchical clustering and singular value decomposition.Synovial sarcomas, gastrointestinal stromal tumours, neural tumours, and a subset of the leiomyosarcomas, showed strikingly distinct gene-expression patterns. Other tumour categories--malignant fibrous histiocytoma, liposarcoma, and the remaining leiomyosarcomas--shared molecular profiles that were not predicted by histological features or immunohistochemistry. Strong expression of known genes, such as KIT in gastrointestinal stromal tumours, was noted within gene sets that distinguished the different sarcomas. However, many uncharacterised genes also contributed to the distinction between tumour types.These results suggest a new method for classification of soft-tissue tumours, which could improve on the method based on histological findings. Large numbers of uncharacterised genes contributed to distinctions between the tumours, and some of these could be useful markers for diagnosis, have prognostic significance, or prove possible targets for treatment.

    View details for Web of Science ID 000174989700013

    View details for PubMedID 11965276

  • Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays GENOME BIOLOGY Lin, J. Y., Pollack, J. R., Chou, F., Rees, C. A., Christian, A. T., Bedford, J. S., Brown, P. O., Ginsberg, M. H. 2002; 3 (6)

    Abstract

    Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology.CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT.The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.

    View details for Web of Science ID 000207581100008

    View details for PubMedID 12093373

  • Molecular portraits of human breast tumours NATURE Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., Pollack, J. R., Ross, D. T., Johnsen, H., Akslen, L. A., Fluge, O., Pergamenschikov, A., Williams, C., Zhu, S. X., Lonning, P. E., Borresen-Dale, A. L., Brown, P. O., Botstein, D. 2000; 406 (6797): 747-752

    Abstract

    Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.

    View details for Web of Science ID 000088767700049

    View details for PubMedID 10963602

  • Genome-wide analysis of DNA copy-number changes using cDNA microarrays NATURE GENETICS Pollack, J. R., Perou, C. M., Alizadeh, A. A., Eisen, M. B., Pergamenschikov, A., Williams, C. F., Jeffrey, S. S., Botstein, D., Brown, P. O. 1999; 23 (1): 41-46

    Abstract

    Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.

    View details for Web of Science ID 000082337300013

    View details for PubMedID 10471496

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