All Publications

  • Prediction of survival in diffuse large B-cell lymphoma based on the expression of 2 genes reflecting tumor and microenvironment BLOOD Alizadeh, A. A., Gentles, A. J., Alencar, A. J., Liu, C. L., Kohrt, H. E., Houot, R., Goldstein, M. J., Zhao, S., Natkunam, Y., Advani, R. H., Gascoyne, R. D., Briones, J., Tibshirani, R. J., Myklebust, J. H., Plevritis, S. K., Lossos, I. S., Levy, R. 2011; 118 (5): 1350-1358


    Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.

    View details for DOI 10.1182/blood-2011-03-345272

    View details for Web of Science ID 000293510000028

    View details for PubMedID 21670469

  • Lysine methylation of the NF-kappa B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappa B signaling NATURE IMMUNOLOGY Levy, D., Kuo, A. J., Chang, Y., Schaefer, U., Kitson, C., Cheung, P., Espejo, A., Zee, B. M., Liu, C. L., Tangsombatvisit, S., Tennen, R. I., Kuo, A. Y., Tanjing, S., Cheung, R., Chua, K. F., Utz, P. J., Shi, X., Prinjha, R. K., Lee, K., Garcia, B. A., Bedford, M. T., Tarakhovsky, A., Cheng, X., Gozani, O. 2011; 12 (1): 29-U47


    Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-?B subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-?B-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-? (PKC-?) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.

    View details for DOI 10.1038/ni.1968

    View details for Web of Science ID 000285465100010

    View details for PubMedID 21131967

  • IFN Regulatory Factor 5 Is Required for Disease Development in the Fc gamma RIIB(-/-)Yaa and Fc gamma RIIB-/- Mouse Models of Systemic Lupus Erythematosus JOURNAL OF IMMUNOLOGY Richez, C., Yasuda, K., Bonegio, R. G., Watkins, A. A., Aprahamian, T., Busto, P., Richards, R. J., Liu, C. L., Cheung, R., Utz, P. J., Marshak-Rothstein, A., Rifkin, I. R. 2010; 184 (2): 796-806


    Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcgammaRIIB(-/-)Yaa and FcgammaRIIB(-/-) lupus models. In contrast to IRF5-sufficient FcgammaRIIB(-/-)Yaa mice, IRF5-deficient FcgammaRIIB(-/-)Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-alpha, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcgammaRIIB(-/-)Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcgammaRIIB(-/-)Yaa mice, type I IFN receptor subunit 1-deficient FcgammaRIIB(-/-)Yaa mice maintained a substantial level of residual disease. Furthermore, in FcgammaRIIB(-/-) mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcgammaRIIB(-/-)Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

    View details for DOI 10.4049/jimmunol.0901748

    View details for Web of Science ID 000273447100030

    View details for PubMedID 20007534

  • Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks PLOS ONE Bua, D. J., Kuo, A. J., Cheung, P., Liu, C. L., Migliori, V., Espejo, A., Casadio, F., Bassi, C., Amati, B., Bedford, M. T., Guccione, E., Gozani, O. 2009; 4 (8)


    Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

    View details for DOI 10.1371/journal.pone.0006789

    View details for Web of Science ID 000269335000031

    View details for PubMedID 19956676

  • Cell Cycle- and Chaperone-Mediated Regulation of H3K56ac Incorporation in Yeast PLOS GENETICS Kaplan, T., Liu, C. L., Erkmann, J. A., Holik, J., Grunstein, M., Kaufman, P. D., Friedman, N., Rando, O. J. 2008; 4 (11)


    Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

    View details for DOI 10.1371/journal.pgen.1000270

    View details for Web of Science ID 000261481000025

    View details for PubMedID 19023413

  • The Stanford Tissue Microarray Database NUCLEIC ACIDS RESEARCH Marinelli, R. J., Montgomery, K., Liu, C. L., Shah, N. H., Prapong, W., Nitzberg, M., Zachariah, Z. K., Sherlock, G. J., Natkunam, Y., West, R. B., van de Rijn, M., Brown, P. O., Ball, C. A. 2008; 36: D871-D877


    The Stanford Tissue Microarray Database (TMAD; is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license.

    View details for DOI 10.1093/nar/gkm861

    View details for Web of Science ID 000252545400154

    View details for PubMedID 17989087

  • TMA-combiner, a simple software tool to permit analysis of replicate cores on tissue microarrays MODERN PATHOLOGY Liu, C. L., Montgomery, K. D., Natkunam, Y., West, R. B., Nielsen, T. O., Cheang, M. C., Turbin, D. A., Marinelli, R. J., van de Rijn, M., Higgins, J. P. 2005; 18 (12): 1641-1648


    We have previously published a suite of software tools that facilitates the reformulation of tissue microarray (TMA) data so that it may be analyzed using techniques originally devised for analysis of cDNA microarray data. However, current microarray data often feature multiple scores for a given tissue sample and antibody combination. Furthermore, an efficient and systematic method for combining scores that takes into account the differing staining properties of tissue epitopes has not been described. We thus present the TMA-Combiner, a new Microsoft Excel-based macro that permits analysis of data for which tissues may have two or more scores per antibody, and permits combination of data from multiple different tissue microarrays. It accomplishes this by rendering one score per tissue per antibody from two or more scores, using one of multiple user-selectable combination rules developed to account for the differing staining properties of tissue epitopes. This greatly facilitates analysis of tissue microarrays, particularly for users with large repositories of data, and may facilitate discovery of biological trends and help refine diagnostic accuracy of tissue markers in clinical samples.

    View details for DOI 10.1038/modpathol.3800491

    View details for Web of Science ID 000233372100016

    View details for PubMedID 16258508

  • Histone variant H2A.Z marks the 5 ' ends of both active and inactive genes in euchromatin CELL Raisner, R. M., Hartley, P. D., Meneghini, M. D., Bao, M. Z., Liu, C. L., Schreiber, S. L., Rando, O. J., Madhani, H. D. 2005; 123 (2): 233-248


    In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

    View details for Web of Science ID 000232794100011

    View details for PubMedID 16239142

  • Single-nucleosome mapping of histone modifications in S-cerevisiae PLOS BIOLOGY Liu, C. L., Kaplan, T., Kim, M., Buratowski, S., Schreiber, S. L., Friedman, N., Rando, O. J. 2005; 3 (10): 1753-1769


    Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes) from those at another location (e.g., over the 3' ends of coding regions). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role.

    View details for DOI 10.1371/journal.pbio.0030328

    View details for Web of Science ID 000232404600010

    View details for PubMedID 16122352

  • Apo D in soft tissue tumors - A novel marker for dermatofibrosarcoma protuberans AMERICAN JOURNAL OF SURGICAL PATHOLOGY West, R. B., Harvell, J., Linn, S. C., Lui, C. L., Prapong, W., Hernandez-Boussard, T., Montgomery, K., Nielsen, T. O., Rubin, B. P., Patel, R., Goldblum, J. R., Brown, P. O., van de Rijn, M. 2004; 28 (8): 1063-1069


    Using gene microarray expression profiling, we previously found that apolipoprotein D (Apo D) was highly expressed in dermatofibrosarcoma protuberans (DFSP). In this study, we confirm that Apo D is highly and relatively specifically expressed in DFSP using immunohistochemistry. A tissue microarray containing 421 soft tissue tumors was constructed and stained with antibodies against Apo D and CD34. Cytoplasmic immunostaining for Apo D was found in 9 of 10 typical DFSPs. In addition, 3 of 3 Bednar tumors and 2 of 3 giant cell fibroblastomas stained in conventional sections. In contrast, Apo D was immunoreactive in only a very small subset of a diverse collection of other soft tissue tumors, including Malignant Fibrous Histiocytoma (MFH), glomus tumor, neurofibroma, and malignant peripheral nerve sheath tumors. Immunostains for Apo D were negative in conventional sections of 16 fibrous histiocytomas, and an additional 12 variants of fibrous histiocytoma. Digital images of all immunohistochemical and hematoxylin and eosin tissue microarray stains are available at the accompanying website ( We conclude that Apo D is strongly expressed in DFSPs and neural lesions and may be useful in differentiating DFSP from fibrous histiocytoma.

    View details for Web of Science ID 000222891400012

    View details for PubMedID 15252314

  • The use of chromatin immunoprecipitation assays in genome-wide analyses of histone modifications CHROMATIN AND CHROMATIN REMODELING ENZYMES, PT B Bernstein, B. E., Humphrey, E. L., Liu, C. L., Schreiber, S. L. 2004; 376: 349-360

    View details for Web of Science ID 000189439600023

    View details for PubMedID 14975317

  • Global nucleosome occupancy in yeast GENOME BIOLOGY Bernstein, B. E., Liu, C. L., Humphrey, E. L., Perlstein, E. O., Schreiber, S. L. 2004; 5 (9)


    Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays.Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the RPS11B promoter, by removing the Rap1 consensus sites.The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues.

    View details for Web of Science ID 000223656100007

    View details for PubMedID 15345046

  • Development and validation of a T7 based linear amplification for genomic DNA BMC GENOMICS Liu, C. L., Schreiber, S. L., Bernstein, B. E. 2003; 4


    Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail.We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach.We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.

    View details for Web of Science ID 000183542600001

    View details for PubMedID 12740028

  • Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Arava, Y., Wang, Y. L., Storey, J. D., Liu, C. L., Brown, P. O., Herschlag, D. 2003; 100 (7): 3889-3894


    We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.

    View details for DOI 10.1073/pnas.0635171100

    View details for Web of Science ID 000182058400066

    View details for PubMedID 12660367

  • Immunoprofile of cervical and endometrial adenocarcinomas using a tissue microarray VIRCHOWS ARCHIV Alkushi, A., Irving, J., Hsu, F., Dupuis, B., Liu, C. L., Rijn, M., Gilks, C. B. 2003; 442 (3): 271-277


    Adenocarcinomas of the uterine cervix show a wide range of morphological features, and can be confused with endometrial adenocarcinoma in biopsy or curetting specimens. The objective of this study was to use tissue microarray technology to evaluate the immunoprofile of a large set of uterine adenocarcinomas with an extended panel of antibodies, comparing the profile of primary cervical and endometrial adenocarcinomas. A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 141 hysterectomy specimens. Duplicate 0.6-mm cores were obtained from 57 cervical adenocarcinomas (16 in situ and 41 invasive) and 84 endometrial adenocarcinomas. Tissue array sections were immunostained with 21 commercially available antibodies [B72.3, CD 99, carcinoembryonic antigen (CEA), c-kit, pancytokeratin, CK 5/6, CK 7, CK8/18, CK19, CK 20, CK 22, EMA, estrogen receptor (ER), KP-1, melan-A, p53, PLAP, S-100, synaptophysin, TTF-1, and vimentin] utilizing the avidin-biotin (ABC) technique. Hierarchical clustering analysis of the tumors was done based on the immunostaining results. Only ER ( P<0.001), CEA ( P=0.04), vimentin ( P<0.001), and CK 8/18 ( P=0.002) showed a significantly different frequency of positivity in endometrial relative to cervical adenocarcinomas. ER, vimentin, and CK 8/18 were more likely to be expressed in endometrial adenocarcinomas, while cervical adenocarcinomas more frequently expressed CEA. We were able to identify immunoprofiles that were highly specific for endocervical adenocarcinoma (ER(-), vimentin(-), CK 8/18(-), CEA(+)) or endometrial adenocarcinoma (ER(+), vimentin(+), CK 8/18(+), CEA(-)), but most tumors showed an intermediate, non-specific immunophenotype. Hierarchical clustering analysis was useful in the interpretation of these intermediate immunophenotypes. Papillary serous adenocarcinoma of the endometrium was less likely to express vimentin ( P=0.002) than endometrioid carcinoma of the endometrium.

    View details for DOI 10.1007/s00428-002-0752-4

    View details for Web of Science ID 000182395600010

    View details for PubMedID 12647218

  • Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry MODERN PATHOLOGY Hsu, F. D., Nielsen, T. O., Alkushi, A., Dupuis, B., Huntsman, D., Liu, C. L., van de Rijn, M., Gilks, C. B. 2002; 15 (12): 1374-1380


    There has been considerable variability in the reported results of immunohistochemical staining for some diagnostically relevant antigens. Our objectives in this study were to (1) use a multitumor tissue microarray with tissue from 351 cases received in our department, representing 16 normal tissues and 47 different tumor types, to compare immunohistochemical staining results in our laboratory with published data, using a panel of 22 antibodies; (2) assess interlaboratory variability of immunohistochemical staining for S-100 using this microarray; and (3) test the ability of hierarchical clustering analysis to group tumors by primary site, based on their immunostaining profile. Tissue microarrays consisting of duplicate 0.6-mm cores from blocks identified in the hospital archives were constructed and stained according to our usual protocols. Antibodies directed against the following antigens were used: B72.3, bcl-2, carcinoembryonic antigen, c-kit, pankeratin, CD 68, CD 99, CK 5/6, CK 7, CK 8/18, CK19, CK 20, CK 22, epithelial membrane antigen, estrogen receptor, melan-A, p53, placental alkaline phosphatase, S-100, synaptophysin, thyroid transcription factor-1, and vimentin. Staining results on the array cases were compared with published results, and hierarchical clustering analysis was performed based on the immunohistochemical staining results. Unstained slides of the multitumor tissue microarray were sent to five other diagnostic immunohistochemistry laboratories and stained for S-100 protein. The staining results from the different laboratories were compared. Staining results using our current methods and samples from our laboratory were compatible with those described in the literature for most antigens. Placental alkaline phosphatase staining was not specific with our protocol, showing staining of a broad spectrum of different tumors; this finding initiated a review of our recent requests for placental alkaline phosphatase immunostaining and revealed two instances in which placental alkaline phosphatase positivity was incorrectly interpreted as evidence of a germ cell tumor. S-100 staining was less sensitive but more specific for the diagnosis of melanoma or neural tumor in our laboratory, compared to some published reports. Assessment of interlaboratory variability of S-100 immunostaining showed that there was more frequent staining of carcinomas in some laboratories, resulting in decreased specificity of S-100 staining in distinguishing melanoma from carcinoma. Hierarchical clustering analysis showed a strong trend for tumors to cluster by tissue of origin, but there were significant exceptions. We conclude that multiple-tumor microarrays are an efficient method for assessing the sensitivity and specificity of staining with any antibody used diagnostically. As a tool for quality assurance, they offer the advantage of taking into account local differences in tissue fixation, processing, and staining. They also allow cost-effective assessment of interlaboratory variability in immunohistochemical staining. Results of hierarchical clustering analysis show the potential for panels of immunohistochemical stains to identify the primary site of metastatic carcinomas but also confirm the limitations of currently available antibodies in giving unequivocal tissue-specific staining patterns.

    View details for DOI 10.1097/01.MP.0000039571.02827.CE

    View details for Web of Science ID 000180034300018

    View details for PubMedID 12481020

  • Software tools for high-throughput analysis and archiving of immunohistochemistry staining data obtained with tissue microarrays AMERICAN JOURNAL OF PATHOLOGY Liu, C. L., Prapong, W., Natkunam, Y., Alizadeh, A., Montgomery, K., Gilks, C. B., van de Rijn, M. 2002; 161 (5): 1557-1565


    The creation of tissue microarrays (TMAs) allows for the rapid immunohistochemical analysis of thousands of tissue samples, with numerous different antibodies per sample. This technical development has created a need for tools to aid in the analysis and archival storage of the large amounts of data generated. We have developed a comprehensive system for high-throughput analysis and storage of TMA immunostaining data, using a combination of commercially available systems and novel software applications developed in our laboratory specifically for this purpose. Staining results are recorded directly into an Excel worksheet and are reformatted by a novel program (TMA-Deconvoluter) into a format suitable for hierarchical clustering analysis or other statistical analysis. Hierarchical clustering analysis is a powerful means of assessing relatedness within groups of tumors, based on their immunostaining with a panel of antibodies. Other analyses, such as generation of survival curves, construction of Cox regression models, or assessment of intra- or interobserver variation, can also be done readily on the reformatted data. Finally, the immunoprofile of a specific case can be rapidly retrieved from the archives and reviewed through the use of Stainfinder, a novel web-based program that creates a direct link between the clustered data and a digital image database. An on-line demonstration of this system is available at

    View details for Web of Science ID 000179197000006

    View details for PubMedID 12414504

  • Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Diehn, M., Alizadeh, A. A., Rando, O. J., Liu, C. L., Stankunas, K., Botstein, D., Crabtree, G. R., Brown, P. O. 2002; 99 (18): 11796-11801


    Optimal activation of T cells requires effective occupancy of both the antigen-specific T cell receptor and a second coreceptor such as CD28. We used cDNA microarrays to characterize the genomic expression program in human peripheral T cells responding to stimulation of these receptors. We found that CD28 agonists alone elicited few, but reproducible, changes in gene expression, whereas CD3 agonists elicited a multifaceted temporally choreographed gene expression program. The principal effect of simultaneous engagement of CD28 was to increase the amplitude of the CD3 transcriptional response. The induced genes whose expression was most enhanced by costimulation were significantly enriched for known targets of nuclear factor of activated T cells (NFAT) transcription factors. This enhancement was nearly abolished by blocking the nuclear translocation of NFATc by using the calcineurin inhibitor FK506. CD28 signaling promoted phosphorylation, and thus inactivation, of the NFAT nuclear export kinase glycogen synthase kinase-3 (GSK3), coincident with enhanced dephosphorylation of NFATc proteins. These results provide a detailed picture of the transcriptional program of T cell activation and suggest that enhancement of transcriptional activation by NFAT, through inhibition of its nuclear export, plays a key role in mediating the CD28 costimulatory signal.

    View details for DOI 10.1073/pnas.092284399

    View details for Web of Science ID 000177843100048

    View details for PubMedID 12195013

  • Precision and functional specificity in mRNA decay PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, Y. L., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., Brown, P. O. 2002; 99 (9): 5860-5865


    Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from approximately 3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

    View details for DOI 10.1073/pnas.092538799

    View details for Web of Science ID 000175377800023

    View details for PubMedID 11972065

  • Stereotyped and specific gene expression programs in human innate immune responses to bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boldrick, J. C., Alizadeh, A. A., Diehn, M., Dudoit, S., Liu, C. L., Belcher, C. E., Botstein, D., Staudt, L. M., Brown, P. O., Relman, D. A. 2002; 99 (2): 972-977


    The innate immune response is crucial for defense against microbial pathogens. To investigate the molecular choreography of this response, we carried out a systematic examination of the gene expression program in human peripheral blood mononuclear cells responding to bacteria and bacterial products. We found a remarkably stereotyped program of gene expression induced by bacterial lipopolysaccharide and diverse killed bacteria. An intricately choreographed expression program devoted to communication between cells was a prominent feature of the response. Other features suggested a molecular program for commitment of antigen-presenting cells to antigens captured in the context of bacterial infection. Despite the striking similarities, there were qualitative and quantitative differences in the responses to different bacteria. Modulation of this host-response program by bacterial virulence mechanisms was an important source of variation in the response to different bacteria.

    View details for Web of Science ID 000173450100078

    View details for PubMedID 11805339

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