DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.

Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures.

Genomic profiling of isolated circulating tumor cells from metastatic breast cancer patients.

Molecular characterization of circulating tumor cells (CTC) from blood is technically challenging because cells are rare and difficult to isolate. We developed a novel approach to isolate CTCs from blood via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE-FACS). Isolated CTCs were subjected to genome-wide copy number analysis via array comparative genomic hybridization (aCGH).

Isolation and genomic analysis of circulating tumor cells from castration resistant metastatic prostate cancer.

Background: The number of circulating tumor cells (CTCs) in metastatic prostate cancer patients provides prognostic and predictive information. However, it is the molecular characterization of CTCs that offers insight into the biology of these tumor cells in the context of personalized treatment.

Methods: We developed a novel approach to isolate CTCs away from hematopoietic cells with high purity, enabling genomic analysis of these cells.

Two distinct routes to oral cancer differing in genome instability and risk for cervical node metastasis.

Purpose: Problems in management of oral cancers or precancers include identification of patients at risk for metastasis, tumor recurrence, and second primary tumors or risk for progression of precancers (dysplasia) to cancer. Thus, the objective of this study was to clarify the role of genomic aberrations in oral cancer progression and metastasis.

Experimental Design: The spectrum of copy number alterations in oral dysplasia and squamous cell carcinomas (SCC) was determined by array comparative genomic hybridization.

Quantum dots-based reverse phase protein microarray.

CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection.

High-efficiency microarray printer using fused-silica capillary tube printing pins.

We describe a contact printing approach for microarrays that uses fused-silica capillary tubes with tapered tips for printing pins and a pressure/vacuum system to control pin loading, printing, and cleaning. The printing process is insensitive to variable environmental factors such as humidity, and the small diameter of the pins allows routine printing from 1536 well source plates. Pin load capacity, 0.

Improving melanoma classification by integrating genetic and morphologic features.

Background: In melanoma, morphology-based classification systems have not been able to provide relevant information for selecting treatments for patients whose tumors have metastasized. The recent identification of causative genetic alterations has revealed mutations in signaling pathways that offer targets for therapy. Identifying morphologic surrogates that can identify patients whose tumors express such alterations (or functionally equivalent alterations) would be clinically useful for therapy stratification and for retrospective analysis of clinical trial data.

Comparative genomic hybridization.

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.

Distribution and significance of occult intraepidermal tumor cells surrounding primary melanoma.

Primary melanoma can recur at the excision site if not excised with a safety margin of surrounding uninvolved skin. To characterize the nature of residual melanoma in the skin surrounding primary tumors targeted by safety margins, we used array comparative genomic hybridization and fluorescent in situ hybridization to detect and spatially map aberrations in the skin adjacent to acral melanomas. Melanocytic cells with genetic amplifications in histopathologically normal skin (field cells) were detected exclusively in the epidermis in 84% of 19 cases, with a mean extension of 6.

Roadmap for new opportunities in melanoma research.

Investigators representing all major melanoma research areas present an overview of the most important challenges for the field. Four major research areas are covered plus the training of new investigators. For each area we first describe the present status, its strengths and weaknesses, and then outline specific recommendations.

Analytical description of mutational effects in competing asexual populations.

The adaptation of a population to a new environment is a result of selection operating on a suite of stochastically occurring mutations. This article presents an analytical approach to understanding the population dynamics during adaptation, specifically addressing a system in which periods of growth are separated by selection in bottlenecks. The analysis derives simple expressions for the average properties of the evolving population, including a quantitative description of progressive narrowing of the range of selection coefficients of the predominant mutant cells and of the proportion of mutant cells as a function of time.

Recurrent reciprocal genomic rearrangements of 17q12 are associated with renal disease, diabetes, and epilepsy.

Most studies of genomic disorders have focused on patients with cognitive disability and/or peripheral nervous system defects. In an effort to broaden the phenotypic spectrum of this disease model, we assessed 155 autopsy samples from fetuses with well-defined developmental pathologies in regions predisposed to recurrent rearrangement, by array-based comparative genomic hybridization. We found that 6% of fetal material showed evidence of microdeletion or microduplication, including three independent events that likely resulted from unequal crossing-over between segmental duplications.

Novel risk stratification of patients with neuroblastoma by genomic signature, which is independent of molecular signature.

Human neuroblastoma remains enigmatic because it often shows spontaneous regression and aggressive growth. The prognosis of advanced stage of sporadic neuroblastomas is still poor. Here, we investigated whether genomic and molecular signatures could categorize new therapeutic risk groups in primary neuroblastomas.

Genome position and gene amplification.

Background: Amplifications, regions of focal high-level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however, the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, we integrated a mutant form of the gene encoding dihydrofolate reductase into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells.

Detection of single clone deletions using array CGH: identification of submicroscopic deletions in the 22q11.2 deletion syndrome as a model system.

Constitutional submicroscopic DNA copy number alterations have been shown to cause numerous medical genetic syndromes, and are suspected to occur in a portion of cases for which the causal events remain undiscovered. Array comparative genomic hybridization (array CGH) allows high-throughput, high-resolution genome scanning for DNA dosage aberrations and thus offers an attractive approach for both clinical diagnosis and discovery efforts. Here we assess this capability by applying array CGH to the analysis of copy number alterations in 44 patients with a phenotype of the 22q11.

Deletion of chromosome 11q predicts response to anthracycline-based chemotherapy in early breast cancer.

Despite the recent consensus on the eligibility of adjuvant systemic therapy in patients with lymph node-negative breast cancer (NNBC) based on clinicopathologic criteria, specific biological markers are needed to predict sensitivity to the different available therapeutic options. We examined the feasibility of developing a genomic predictor of chemotherapy response and recurrence risk in 185 patients with NNBC using assembled arrays containing 2,460 bacterial artificial chromosome clones for scanning the genome for DNA copy number changes. After surgery, 90 patients received anthracycline-based chemotherapy, whereas 95 did not.

Genomic and transcriptional aberrations linked to breast cancer pathophysiologies.

This study explores the roles of genome copy number abnormalities (CNAs) in breast cancer pathophysiology by identifying associations between recurrent CNAs, gene expression, and clinical outcome in a set of aggressively treated early-stage breast tumors. It shows that the recurrent CNAs differ between tumor subtypes defined by expression pattern and that stratification of patients according to outcome can be improved by measuring both expression and copy number, especially high-level amplification. Sixty-six genes deregulated by the high-level amplifications are potential therapeutic targets.

A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes.

Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model "system" to appraise the functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented.

Regional copy number-independent deregulation of transcription in cancer.

Genetic and epigenetic alterations have been identified that lead to transcriptional deregulation in cancers. Genetic mechanisms may affect single genes or regions containing several neighboring genes, as has been shown for DNA copy number changes. It was recently reported that epigenetic suppression of gene expression can also extend to a whole region; this is known as long-range epigenetic silencing.

Chromosomal aberrations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma unspecified: A matrix-based CGH approach.

Angioimmunoblastic T-cell lymphoma (AILT) is a histopathologically well-defined entity. However, despite a number of cytogenetic studies, the genetic basis of this lymphoma entity is not clear. Moreover, there is an overlap to some cases of peripheral T-cell lymphoma unspecified (PTCL-u) in respect to morphological and genetic features.

Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas.

Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms.