Daniel P. Gaile

Loss of Breast Cancer Metastasis Suppressor 1 Protein Expression Predicts Reduced Disease-Free Survival in Subsets of Breast Cancer Patients

Purpose: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. Experimental Design: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. Results: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P = 0.006) or HER2 positive (P = 0.039) and <50 years old at diagnosis (P = 0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P = 0.008) or PR (P = 0.029) or HER2 overexpression (P = 0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. Conclusions: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.

Genomic profiling of myeloid sarcoma by array comparative genomic hybridization.

Myeloid sarcoma (MS) is a tumor mass of myeloblasts or immature myeloid cells occurring in an extramedullary site. In this study, seven cases of MS [stomach (1), testis (1), skin (2), and lymph node (3)] and 3 synchronous and 1 follow‐up bone marrow (BM) samples were studied for genomic abnormalities using array comparative genomic hybridization (array‐CGH). Array‐CGH construction used ∼5,400 bacterial artificial chromosome clones from the RPCI‐11 library, spanning the human genome. Data were analyzed using the DNAcopy software and custom heuristics. All MS cases had genomic abnormalities detected by array‐CGH. Unbalanced genomic abnormalities in five MS cases were confirmed by conventional cytogenetics (CC) and/or fluorescence in situ hybridization (FISH); these abnormalities included loss of 4q32.1‐q35.2, 6q16.1‐q21, and 12p12.2‐p13.2 and gain of 8q21.2‐q24.3, 8, 11q21‐q25, 13q21.32‐q34, 19, and 21. Array‐CGH was also invaluable in identifying possible deletions, partner translocations, and breakpoints that were questionable by CC. The remaining two MS cases had genomic aberrations detected by array‐CGH, but were not studied further by CC/FISH. Chromosome 8 was most commonly abnormal (3/7 cases). Identical genomic abnormalities were demonstrated in MS and in synchronous BM in two cases. These results demonstrate that array‐CGH is a powerful tool to screen MS tissue for unbalanced genomic abnormalities, allowing identification of chromosome abnormalities when concurrent BM is nonanalyzable or nonleukemic.

Differential Copy Number Aberrations in Novel Candidate Genes Associated with Progression from In Situ to Invasive Ductal Carcinoma of the Breast

Only a minority of intraductal carcinomas of the breast give rise to stromally invasive disease. We microdissected 206 paraffin blocks representing 116 different cases of low‐grade ductal carcinoma in situ (DCIS). Fifty‐five were pure DCIS (PD) cases without progression to invasive carcinoma. Sixty‐one cases had a small invasive component. DNA was extracted from microdissected sections and hybridized to high‐density bacterial artificial chromosome arrays. Array comparative genomic hybridization analysis of 118 hybridized DNA samples yielded data on 69 samples that were suitable for further statistical analysis. This cohort included 20 pure DCIS cases, 25 mixed DCIS (MD), and 24 mixed invasive carcinoma samples. PD cases had a higher frequency of DNA copy number changes than MD cases, and the latter had similar DNA profiles compared to paired invasive carcinomas. Copy number changes on 13 chromosomal arms occurred at different rates in PD versus MD lesions. Eight of 19 candidate genes residing at those loci were confirmed to have differential copy number changes by quantitative PCR. NCOR2/SMRT and NR4A1 (both on 12q), DYNLRB2 (16q), CELSR1, UPK3A, and ST13 (all on 22q) were more frequently amplified in PD. Moreover, NCOR2, NR4A1, and DYNLRB2 showed more frequent copy number losses in MD. GRAP2 (22q) was more often amplified in MD, whereas TAF1C (16q) was more commonly deleted in PD. A multigene model comprising these candidate genes discriminated between PD and MD lesions with high accuracy. These findings suggest that the propensity to invade the stroma may be encoded in the genome of intraductal carcinomas.

Long tandem repeats as a form of genomic copy number variation: structure and length polymorphism of a chromosome 5p repeat in control and schizophrenia populations

Objectives Genomic copy number variations (CNVs) are a major form of variation in the human genome and play an etiologic role in several neuropsychiatric diseases. Tandem repeats, particularly with long (>50 bp) repeat units, are a relatively common yet underexplored type of CNV that may significantly contribute to human genomic variation and disease risk. We therefore carried out a pilot experiment to explore the potential role of long tandem repeats as risk factors in psychiatric disorders. Methods A bacterial artificial chromosome-based array comparative genomic hybridization (aCGH) platform was used to examine CNVs in genomic DNA from 34 probands with schizophrenia or schizoaffective disorder. Results The aCGH screen detected an apparent deletion on 5p15.1 in two probands, caused by the presence in each proband of two low copy number (short) alleles of a tandem repeat that ranges in length from fewer than 10 to greater than 50 3.4 kb units in the population examined. Short alleles partially segregate with schizophrenia in a small number of families, though linkage was not significant. An association study showed no significant difference in repeat length between 406 schizophrenia cases and 392 controls. Conclusion Although we did not demonstrate a relationship between the 5p15.1 repeat and schizophrenia, our results illustrate that long tandem repeats represent an intriguing type of genetic variation that have not been studied in earlier connection with psychiatric illness. aCGH can detect a small subset of these repeats, but systematic investigation will require the development of specific arrays and improved analytical methods.

Arsenic responsive microRNAs in vivo and their potential involvement in arsenic-induced oxidative stress.

Arsenic exposure is postulated to modify microRNA (miRNA) expression, leading to changes of gene expression and toxicities, but studies relating the responses of miRNAs to arsenic exposure are lacking, especially with respect to in vivo studies. We utilized high-throughput sequencing technology and generated miRNA expression profiles of liver tissues from Sprague Dawley (SD) rats exposed to various concentrations of sodium arsenite (0, 0.1, 1, 10 and 100mg/L) for 60days. Unsupervised hierarchical clustering analysis of the miRNA expression profiles clustered the SD rats into different groups based on the arsenic exposure status, indicating a highly significant association between arsenic exposure and cluster membership (p-value of 0.0012). Multiple miRNA expressions were altered by arsenic in an exposure concentration-dependent manner. Among the identified arsenic-responsive miRNAs, several are predicted to target Nfe2l2-regulated antioxidant genes, including glutamate-cysteine ligase (GCL) catalytic subunit (GCLC) and modifier subunit (GCLM) which are involved in glutathione (GSH) synthesis. Exposure to low concentrations of arsenic increased mRNA expression for Gclc and Gclm, while high concentrations significantly reduced their expression, which were correlated to changes in hepatic GCL activity and GSH level. Moreover, our data suggested that other mechanisms, e.g., miRNAs, rather than Nfe2l2-signaling pathway, could be involved in the regulation of mRNA expression of Gclc and Gclm post-arsenic exposure in vivo. Together, our findings show that arsenic exposure disrupts the genome-wide expression of miRNAs in vivo, which could lead to the biological consequence, such as an altered balance of antioxidant defense and oxidative stress.

Wide-scale Alterations in Interchromosomal Organization in Breast Cancer Cells: Defining a Network of Interacting Chromosomes

The interchromosomal spatial positionings of a subset of human chromosomes was examined in the human breast cell line MCF10A (10A) and its malignant counterpart MCF10CA1a (CA1a). The nine chromosomes selected (#1, 4, 11, 12, 15, 16, 18, 21 and X) cover a wide range in size and gene density and compose ∼40% of the total human genome. Radial positioning of the chromosome territories (CT) was size dependent with certain of the CT more peripheral in CA1a. Each CT was in close proximity (interaction) with a similar number of other CT except the inactive CTXi. It had lower levels of interchromosomal partners in 10A which increased strikingly in CA1a. Major alterations from 10A to CA1a were detected in the pairwise interaction profiles which were subdivided into five types of altered interaction profiles: overall increase, overall decrease, switching from 1 to ≥2, vice versa or no change. A global data mining program termed the chromatic median calculated the most probable overall association network for the entire subset of CT. This interchromosomal network was drastically altered in CA1a with only 1 of 20 shared connections. We conclude that CT undergo multiple and preferred interactions with other CT in the cell nucleus and form preferred-albeit probabilistic-interchromosomal networks. This network of interactions is highly altered in malignant human breast cells. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program of these malignant cancer cells.

The genomic relationship between primary breast carcinomas and their nodal metastases.

We screened the whole tumor genome to identify DNA copy number gains and losses that discriminate between primary breast carcinomas (MP) and their nodal metastases (ML). Six candidate genes were confirmed by quantitative PCR to have differentially distributed copy number changes. Three of the genes (ERRγ, DDX6, and TIAM1) were more commonly amplified in nodal metastases. Principal component analysis revealed that MP-ML pairs varied markedly in their genomic divergence. The latter was larger in PR-negative tumors. Nodal metastases may form early or late in the development of breast carcinomas and PR-negative tumors may metastasize earlier or are genomically less stable.

A New Normalizing Algorithm for BAC CGH Arrays with Quality Control Metrics

The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose “SmoothArray”, a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package “aCGHplus,” this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

Myd88 is required for disease development in a primary Sjögren’s syndrome mouse model

Sjögren’s syndrome (SS) is an autoimmune disease that often results in diminished exocrine gland function. SS patients also experience systemic disease manifestations, including hypergammaglobulinemia and pulmonary and renal pathoses. MyD88 is a ubiquitously expressed adaptor molecule used by all immune cells that is required for IL‐1 receptor (IL‐1R), IL‐18R, and most TLR signaling. The precise role of MyD88 in SS has not been evaluated, although this adaptor is critical for development of lupus, a related autoimmune disease. This study tested the hypothesis that Myd88‐mediated signaling is required for local and systemic SS manifestations. To this end, we generated NOD.B10Sn‐H2b/J (NOD.B10) mice that are deficient in Myd88 (NOD.B10Myd88−/−). We found that NOD.B10 animals that lack Myd88 show reduced exocrine and extraglandular inflammation. Moreover, these animals are protected from loss of salivary flow. Splenocytes from NOD.B10Myd88−/− mice did not up‐regulate activation markers or secrete IL‐6 in response to a Myd88‐dependent agonist, although BCR signaling remained intact. Finally, IgM, IgG, and anti‐nuclear autoantibodies were reduced in NOD.B10Myd88−/− mice compared with the parental strain. These data demonstrate that Myd88 is a crucial mediator of local and systemic SS disease manifestations.

iGenomicViewer: R package for visualisation of high dimension genomic data

While the technologies for high dimensional data have been advancing, a lack of adequate visualisation tools to accommodate the results and inability to integrate multiple sources of data has emerged. The move towards multi-disciplinary work and collaborative research impresses the need for visualisation and analysis tools that are platform independent and customisable. iGenomicViewer through the use of customisable tool-tips that may include links and images, allows for a greater level of data integration for genomic data in a variety of formats. The iGenomicViewer is a freely available R software which allows users to generate interactive, platform-independent plots of genomic data.