Options available--from start to finish--for obtaining data from DNA microarrays II.

Microarray technology has undergone a rapid evolution. With widespread interest in large-scale genomic research, an abundance of equipment and reagents have now become available and affordable to a large cross section of the scientific community. As protocols become more refined, careful investigators are able to obtain good quality microarray data quickly. In most recent times, however, perhaps one of the biggest obstacles researchers face is not the manufacture and use of microarrays at the bench, but storage and analysis of the array data. This review discusses the most recent equipment, reagents and protocols available to the researcher, as well as describing data analysis and storage options available from the evolving field of microarray informatics.

Cancer-associated neochromosomes: a novel mechanism of oncogenesis.

Malignant tumours are often characterised by significant rearrangement of the genome. This may be visible in the form of a deranged karyotype with both loss and gain of DNA sequences extending from chromosomal regions to whole chromosomes. In several tumour types, however, gross genomic derangements are minimal, and tumour cells contain one or more additional (supernumerary) chromosomes that may be unrecognisable in terms of a single origin. In this review we term such chromosomes cancer-associated neochromosomes (CaNCs). In the absence of other identified genomic abnormalities, and because the CaNC is a common feature of the cancer type, it is hypothesised that the genetic alterations required for cell transformation are contained within its structure. In this review, we discuss the potential impact of modern genomic technologies on our understanding of the nature and causes of CaNC formation, which is central to several cancer types, exemplified here by well-differentiated liposarcoma.

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

Gene expression analysis in absence epilepsy using a monozygotic twin design.

PURPOSE: To identify genes involved in idiopathic absence epilepsies by analyzing gene expression using a monozygotic (MZ) twin design. METHODS: Genome-wide gene expression in lymphoblastoid cell lines (LCLs) was determined using microarrays derived from five discordant and four concordant MZ twin pairs with idiopathic absence epilepsies and five unaffected MZ twin pairs. Gene expression was analyzed using three strategies: discordant MZ twins were compared as matched pairs, MZ twins concordant for epilepsy were compared to control MZ twins, and a singleton design of affected versus unaffected MZ twin individuals was used irrespective of twin pairing. An overlapping gene list was generated from these analyses. Dysregulation of genes recognized from the microarray experiment was validated using quantitative real time PCR (qRT-PCR) in the twin sample and in an independent sample of 18 sporadic absence cases and 24 healthy controls. RESULTS: Sixty-five probe sets were identified from the three combined microarray analysis strategies. Sixteen genes were chosen for validation and nine of these genes confirmed by qRT-PCR in the twin sample. Differential expression for EGR1 (an immediate early gene) and RCN2 (coding for the calcium-binding protein Reticulocalbin 2) were reconfirmed by qRT-PCR in the independent sample. DISCUSSION: Using a unique sample of discordant MZ twins, our study identified genes with altered expression, which suggests novel mechanisms in idiopathic absence epilepsy. Dysregulation of EGR1 and RCN2 is implicated in idiopathic absence epilepsy.

Cytokeratin KRT8/18 expression differentiates distinct subtypes of grade 3 invasive ductal carcinoma of the breast.

Invasive ductal carcinomas of the breast (IDC) are routinely assessed on hematoxylin and eosin stained paraffin sections, with limited use of immunohistochemistry (IHC). Most IDC are regarded as a single diagnostic entity, IDC of no special type (IDC-NST), which is subdivided further only by grading. However, recent research suggests that there is high clinical relevance in differentiating IDC subtypes. Here, we ascertain whether tumor histology alone can predict basal or luminal cell phenotype in high-grade IDC-NST, and whether IHC and molecular characteristics are associated with the observed morphologies. A total of 29 grade 3 IDC-NST samples were studied, 10 tumors from a selected pilot cohort A and 19 tumors from an unselected validation cohort B. Along with histopathological assessment, the expression of ESR1, PGR, ERBB2 (HER-2), the basal/myoepithelial marker TP73L (p63), cytokeratins 5/6 (KRT5/6) and 14 (KRT14), and the luminal-specific cytokeratins 8/18 (KRT 8/18) and 19 (KRT19) was assessed by IHC. Hierarchical cluster analysis of clinicopathological variables and, separately, microarray expression profiles showed that the phenotypically distinctive basaloid and luminal tumors of cohort A fell into two main groups, defined by heterogeneous or uniformly positive expression of KRT8/18. The 38 genes differentially expressed between these two classes included ERBB2, KRT8, and six other genes previously associated with ERBB2-positive or luminal phenotypes. Tumor histology was not predictive for validation cohort B, but quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed two molecularly defined clusters that again aligned with the KRT8/18 staining phenotypes. Metaphase comparative genomic hybridization revealed 10q, 16q, and 20q copy-number imbalances that associated recurrently with KRT8/18 staining patterns.

The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice.

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.

Generation and analysis of Siah2 mutant mice.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

A molecular diagnostic test for distinguishing lung adenocarcinoma from malignant mesothelioma using cells collected from pleural effusions.

PURPOSE: Patients with malignant mesothelioma or adenocarcinoma of the lung often present with respiratory complications associated with a malignant pleural effusion. Distinguishing between these malignancies is frequently problematic, as many of the clinical, cytologic, and histologic features of the diseases overlap. Following cytologic analysis of pleural effusions, subsequent confirmatory tissue biopsies involve increased patient morbidity and expense. We have therefore designed a gene expression-based test to classify the primary tumor causing a malignant pleural effusion, using cells collected from the effusion itself. EXPERIMENTAL DESIGN: We have used microarray data for 190 lung adenocarcinomas and 33 malignant mesotheliomas to identify genes differentially expressed between the two diseases. Genes expressed in normal mesothelial cells were removed, allowing the development of a PCR-based test to measure the expression of genes that discriminate between mesothelioma and lung adenocarcinoma from cytology specimens. RESULTS: Applying an real-time PCR-based assay involving 17 genes to 13 independent samples from biopsy-proven malignant mesothelioma and lung adenocarcinomas resulted in the correct identification of all samples. CONCLUSIONS: We have developed a test that is able to distinguish between lung adenocarcinoma and mesothelioma in cells collected from pleural effusions.

An expression-based site of origin diagnostic method designed for clinical application to cancer of unknown origin.

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.

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis.

BACKGROUND: Concerns are often raised about the accuracy of microarray technologies and the degree of cross-platform agreement, but there are yet no methods which can unambiguously evaluate precision and sensitivity for these technologies on a whole-array basis. RESULTS: A methodology is described for evaluating the precision and sensitivity of whole-genome gene expression technologies such as microarrays. The method consists of an easy-to-construct titration series of RNA samples and an associated statistical analysis using non-linear regression. The method evaluates the precision and responsiveness of each microarray platform on a whole-array basis, i.e., using all the probes, without the need to match probes across platforms. An experiment is conducted to assess and compare four widely used microarray platforms. All four platforms are shown to have satisfactory precision but the commercial platforms are superior for resolving differential expression for genes at lower expression levels. The effective precision of the two-color platforms is improved by allowing for probe-specific dye-effects in the statistical model. The methodology is used to compare three data extraction algorithms for the Affymetrix platforms, demonstrating poor performance for the commonly used proprietary algorithm relative to the other algorithms. For probes which can be matched across platforms, the cross-platform variability is decomposed into within-platform and between-platform components, showing that platform disagreement is almost entirely systematic rather than due to measurement variability. CONCLUSION: The results demonstrate good precision and sensitivity for all the platforms, but highlight the need for improved probe annotation. They quantify the extent to which cross-platform measures can be expected to be less accurate than within-platform comparisons for predicting disease progression or outcome.

Distinctive patterns of gene expression in premalignant gastric mucosa and gastric cancer.

Current epidemiological evidence supports a pathogenetic model of gastric cancer involving intermediate stages that include chronic gastritis and intestinal metaplasia. This study explores the molecular features of gastric cancer and premalignant stages using DNA microarray-based gene expression profiling and relates these findings to clinical, pathological, and ethnic parameters. A total of 124 tumor and adjacent mucosa samples were analyzed using spotted cDNA microarrays containing 9381 nonredundant gene elements. Tumor specimens were diffuse, intestinal, or mixed gastric cancer and adjacent mucosa, which generally displayed signs of chronic gastritis or intestinal metaplasia. Expression patterns could be discerned that readily defined premalignant and tumor subtypes. Chronic gastritis exhibits a pronounced mitochondrial gene expression signature, which may be linked to Helicobacter pylori pathogenesis. Intestinal metaplasia was associated with increased expression of many intestinal differentiation genes, many of which were not overexpressed in tumors. Samples were obtained from 91 Australian and 33 Chinese patients to explore potential variation in gene expression between these populations. Despite differences in the incidence, and potentially the etiology, of gastric cancer between these ethnic groups, we found the tumors to be molecularly similar. The identification of molecular signatures that are characteristic of subtypes of gastric cancer and associated premalignant changes should enable further analysis of the steps involved in the initiation and progression of this disease.

The architecture and evolution of cancer neochromosomes.

We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.

Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACis) inhibit tumor cell growth and survival, possibly through their ability to regulate the expression of specific proliferative and/or apoptotic genes. However, the HDACi-regulated genes necessary and/or sufficient for their biological effects remain undefined. We demonstrate that the HDACis suberoylanilide hydroxamic acid (SAHA) and depsipeptide regulate a highly overlapping gene set with at least 22% of genes showing altered expression over a 16-h culture period. SAHA and depsipeptide coordinately regulated the expression of several genes within distinct apoptosis and cell cycle pathways. Multiple genes within the Myc, type beta TGF, cyclin/cyclin-dependent kinase, TNF, Bcl-2, and caspase pathways were regulated in a manner that favored induction of apoptosis and decreased cellular proliferation. APAF-1, a gene central to the intrinsic apoptotic pathway, was induced by SAHA and depsipeptide and shown to be important, but not essential, for HDACi-induced cell death. Overexpression of p16(INK4A) and arrest of cells in G(1) can suppress HDACi-mediated apoptosis. Although p16(INK4A) did not affect the genome-wide transcription changes mediated by SAHA, a small number of apoptotic genes, including BCLXL and B-MYB, were differentially regulated in a manner consistent with attenuated HDACi-mediated apoptosis in arrested cells. We demonstrate that different HDACi alter transcription of a large and common set of genes that control diverse molecular pathways important for cell survival and proliferation. The ability of HDACi to target multiple apoptotic and cell proliferation pathways may provide a competitive advantage over other chemotherapeutic agents because suppression/loss of a single pathway may not confer resistance to these agents.

Molecular profiling of giant cell tumor of bone and the osteoclastic localization of ligand for receptor activator of nuclear factor kappaB.

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.