Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer.

BACKGROUND: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance. MATERIALS AND METHODS: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations. RESULTS: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80). CONCLUSIONS: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.

Microarray analysis has become a widely used tool for the generation of gene expression data on a genomic scale. Although many significant results have been derived from microarray studies, one limitation has been the lack of standards for presenting and exchanging such data. Here we present a proposal, the Minimum Information About a Microarray Experiment (MIAME), that describes the minimum information required to ensure that microarray data can be easily interpreted and that results derived from its analysis can be independently verified. The ultimate goal of this work is to establish a standard for recording and reporting microarray-based gene expression data, which will in turn facilitate the establishment of databases and public repositories and enable the development of data analysis tools. With respect to MIAME, we concentrate on defining the content and structure of the necessary information rather than the technical format for capturing it.

Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays.

To identify new Drosophila genes involved in the immune response, we monitored the gene expression profile of adult flies in response to microbial infection by using high-density oligonucleotide microarrays encompassing nearly the full Drosophila genome. Of 13,197 genes tested, we have characterized 230 induced and 170 repressed by microbial infection, most of which had not previously been associated with the immune response. Many of these genes can be assigned to specific aspects of the immune response, including recognition, phagocytosis, coagulation, melanization, activation of NF-kappaB transcription factors, synthesis of antimicrobial peptides, production of reactive oxygen species, and regulation of iron metabolism. Additionally, we found a large number of genes with unknown function that may be involved in control and execution of the immune response. Determining the function of these genes represents an important challenge for improving our knowledge of innate immunity. Complete results may be found at http://www.fruitfly.org/expression/immunity/.

Ceramic capillaries for use in microarray fabrication.

We have used ceramic capillary tips generally used in the microelectronics industry for the production of DNA microarrays. The ceramic tips improve the morphology of microarray elements, allow higher element density, and increase printing tip life over the customary slotted stainless-steel pins. Ceramic tips are less expensive than steel pins and allow printing from 1536-well sample source plates. In this work, we describe experiments that establish printing performance of the ceramic tips and hybridization experiments that show that DNA hybridization is unaffected by the choice of tip material.

The Stanford Microarray Database.

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.

Genomic expression programs in the response of yeast cells to environmental changes.

We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth.

There are about 800 genes in Saccharomyces cerevisiae whose transcription is cell-cycle regulated. Some of these form clusters of co-regulated genes. The 'CLB2' cluster contains 33 genes whose transcription peaks early in mitosis, including CLB1, CLB2, SWI5, ACE2, CDC5, CDC20 and other genes important for mitosis. Here we find that the genes in this cluster lose their cell cycle regulation in a mutant that lacks two forkhead transcription factors, Fkh1 and Fkh2. Fkh2 protein is associated with the promoters of CLB2, SWI5 and other genes of the cluster. These results indicate that Fkh proteins are transcription factors for the CLB2 cluster. The fkh1 fkh2 mutant also displays aberrant regulation of the 'SIC1' cluster, whose member genes are expressed in the M-G1 interval and are involved in mitotic exit. This aberrant regulation may be due to aberrant expression of the transcription factors Swi5 and Ace2, which are members of the CLB2 cluster and controllers of the SIC1 cluster. Thus, a cascade of transcription factors operates late in the cell cycle. Finally, the fkh1 fkh2 mutant displays a constitutive pseudohyphal morphology, indicating that Fkh1 and Fkh2 may help control the switch to this mode of growth.

Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu

Cluster analysis and display of genome-wide expression patterns.

A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.

Effect of the (alpha 2-->8)-linked polysialic acid capsule on adherence of Neisseria meningitidis to human mucosal cells.

The effect of (alpha 2-->8)-linked polysialic acid on the adherence of Neisseria meningitidis to human mucosal cells was examined using a serogroup B-encapsulated strain and a capsule-defective (Cap-) mutant of this strain. The Cap- mutant contains a single truncated insert of Tn916 in a 3.8-kb HaeIII chromosomal fragment. The Tn916 insert was shown to be responsible for the phenotype by linkage studies and by demonstration that loss of the insert restored encapsulation. The Cap- mutant consistently adhered to human buccal epithelial cell in greater numbers than the encapsulated parent, but the increase in adherence was less than twofold. Adherence of the Cap- mutant during infection of human nasopharyngeal organ cultures was 1.3- to 6.5-fold greater than that of the encapsulated parent. However, specificity of adherence of meningococci for nonciliated nasopharyngeal epithelial cells and the ability to be internalized by these cells was not due to the (alpha 2-->8)-linked polysialic acid capsule.

Evidence for functionally distinct pili expressed by Neisseria meningitidis.

In order to investigate possible functional consequences of phase and antigenic variation of meningococci, the attachment of 15 strains of Neisseria meningitidis to human erythrocytes was studied by a nitrocellulose hemadsorption assay. This assay allows the study of individual meningococcal colonies with respect to erythrocyte attachment. Of the 15 strains studied, 7 demonstrated binding of human erythrocytes (HA+). Among these seven strains, the percentage of colonies that were HA+ ranged from 0.2 to 97%. Meningococcal colonies that did not produce pilin (the major structural subunit of pili) did not demonstrate erythrocyte binding (HA-). The HA+ colony phenotype was correlated with assembly of pilin into pili and expression of pili on the meningococcal surface. However, only some piliated colonies bound human erythrocytes. This could not be explained by differences between piliated HA+ and HA- colonies in the amount of pilin produced or by differences in number of pili expressed per diplococcus. Pili of five of the meningococcal strains with HA+ colonies were antigenically related to gonococcal pili (class I meningococcal pili), but HA+ colonies were also seen in two meningococcal strains expressing class II meningococcal pili. Changes from HA+ to HA- and from HA- to HA+, in the presence of continuing pilin production and pilus assembly, occurred at frequencies of up to 10(-2)/CFU per generation. Such frequencies resemble those of phase and antigenic variation described previously for Neisseria species pilin. These studies indicate that phase variation influences the ability of meningococci to attach to human cells and suggest that meningococci may express functionally different pili.

Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis.

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916-carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.