DNA copy number alterations and expression of relevant genes in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is defined by a lack of expression of estrogen, progesterone, and HER2 receptors, and genetically most of them fall into the basal subgroup of breast cancer. The important issue of TNBC is poorer clinical outcome and absence of effective targeted therapy. In this study, we sought to identify DNA copy number alterations and expression of relevant genes characteristic of TNBC to discover potential therapeutic targets. Frozen tissues from 114 breast cancers were analyzed using high-resolution array comparative genomic hybridization. The classification into subtype was determined by estrogen and progesterone receptor expression, and by the presence or absence of gain on the ERBB2 containing clone. The ACE algorithm was used for calling gain and loss of clones. Twenty-eight cases (25%) were classified as TNBC. Recurrent gains (> or =25%) unique to TNBC were 9p24-p21, 10p15-p13, 12p13, 13q31-q34, 18q12, 18q21-q23, and 21q22. Two published gene expression array data sets comparing basal subtype versus other subtype breast cancers were used for searching candidate genes. Of the genes upregulated in the basal subtype, 45 of 686 genes in one data set and 59 of 1,428 in the second data set were found to be located in the gained regions. Of these candidate genes, gain of NFIB (9p24.1) was specific for TNBC in a validation set by real-time PCR. In conclusion, we have identified recurrently gained regions characteristic of TNBC, and found that NFIB copy number and expression is increased in TNBC across the data sets. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

BRAF and c-kit gene copy number in mutation-positive malignant melanoma

Activating mutations in BRAF or c-kit have been reported in malignant melanoma. Because the activating mutations are dominant, it has been assumed that they are heterozygous in the affected tumors. To test this, we have carefully examined the DNA sequencing electropherograms on 43 BRAF mutation-positive and 3 c-kit mutation-positive malignant melanomas to determine the ratio of the normal to mutant allele. Of the 43 BRAF mutation-positive tumors, we classified 26 as presumptive heterozygous. Eight cases were indeterminate. Surprisingly, 9 cases appeared to contain an excess of the mutant allele. BRAF fluorescence in situ hybridization on these 9 cases suggested the increased amount of the mutant BRAF allele was due to amplification (2 cases) or chromosome 7 polysomy (7 cases). We have previously described the presence of the c-kit-activating mutation, L576P, in 2 of 100 malignant melanomas. In this report, we have evaluated an additional 53 cases and found 1 additional case that contained the L576P mutation. Evaluation of the DNA sequencing electropherograms from all 3 cases of L576P mutation-positive melanoma suggests a selective loss of the normal allele. Fluorescence in situ hybridization for c-kit on these 3 L576P mutation-positive tumors indicated that one showed slight amplification of the c-kit gene and the other 2 were present in a nonamplified diploid state. These results have important implications concerning the mechanism of oncogenesis in melanoma as well as in the response of the tumor to anticancer drugs targeting BRAF or c-kit.     

Combined genome-wide allelotyping and copy number analysis identify frequent genetic losses without copy number reduction in medulloblastoma

Detailed analysis of mechanisms of genetic loss for specific tumor suppressor genes (TSGs; e.g., RB1, APC and NF1) indicates that TSG inactivation can occur by allelic loss of heterozygosity (LOH), without any alteration in DNA copy number. However, the role and prevalence of such events in the pathogenesis of specific malignancies remains to be established on a genome-wide basis. We undertook a detailed molecular assessment of chromosomal defects in a panel of nine cell lines derived from primary medulloblastomas, the most common malignant brain tumors of childhood, by parallel genome-wide assessment of LOH (allelotyping) and copy number aberrations (comparative genomic hybridization and fluorescence in situ hybridization). The majority of genetic losses observed were detected by both copy number and LOH methods, indicating they arise through the physical deletion of chromosomal material. However, a significant proportion of losses (17/42, 40%) represented regions of allelic LOH without any associated copy number reduction; these events involved both whole chromosomes (10/17) and sub-chromosomal regions (7/17). Using this approach, we identified medulloblastoma-characteristic alterations, e.g., isochromosome for 17q, MYC amplification and losses on chromosomes 10, 11, and 16, alongside novel regions of genetic loss (e.g., 10q21.1-26.3, 11q24.1-qter). This detailed genetic characterization of the majority of medulloblastoma cell lines provides important precedent for the widespread involvement of copy number-neutral genetic losses in medulloblastoma and demonstrates that combined assessment of copy number aberrations and LOH will be necessary to accurately determine the contribution of chromosomal defects to tumor development.     

Detection of DNA copy number alterations in cancer by array comparative genomic hybridization

Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.     

Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma‐derived cell lines

A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS. If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation‐associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n = 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRAS^wt/wt, respectively) by array‐based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n = 17, 13, 13 for BRAF*, NRAS*, and BRAF/NRAS^wt/wt, respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1‐36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2‐24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS^wt/wt tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations. © 2009 Wiley‐Liss, Inc.     

Fluorescent in situ hybridization study of c-myc oncogene copy number in prostate cancer

We previously conducted a study of 88 cases of prostate cancer in an attempt to identify potential prognostic biomarkers that can distinguish aggressive cases that must be treated immediately. Prostate cancer is a serious disease affecting men worldwide and compromises the quality of life of its patients. Biomarkers studied included chromosome 7 trisomy, chromosome 8 trisomy, and HER-2/neu oncogene amplification. These biomarkers were initially studied because trisomy 8 and oncogene amplification of the HER-2/neu gene have been reported in many other cancers, including those studied in this laboratory. In view of the fact that HER-2/neu amplification was not found to play a prominent role in the group of prostate cancer specimens that we studied, an exploration of other biomarkers was felt to be warranted. Thus, we began a pilot study of c-myc oncogene copy number in prostate cancer using the same protocol for fluorescent in situ hybridization and a direct-labeled SpectrumOrange LSI c-myc probe (Vysis, Inc., Downers Grove, IL) on formalin-fixed, paraffin-embedded tissue. From a total of 36 cases of prostate cancers successfully analyzed, we found 11 (31%) tumors exhibiting 3 or more positive signals for c-myc in 15% or more of the cells. Of these, only 7 tumors (19% of the total cases studied) had >/=3 signals in 20% or more of the cells. No case had >/=3 signals in 25% or more of the cells. Compared to other molecular probes tested, the c-myc signals were more faint and the quality of the preparation was less optimal than other tumor specimens that we previously studied. Based on the information available thus far, we conclude that an increased copy number in c-myc oncogene copy number was not a prominent finding in our cohort of prostate cancer patients.     

Recurrent copy number alterations in low‐grade and anaplastic pleomorphic xanthoastrocytoma with and without BRAF V600E mutation

Pleomorphic xanthoastrocytoma (PXA) is a rare localized glioma characterized by frequent BRAF V600E mutation and CDKN2A/B deletion. We explored the association of copy‐number variants (CNVs) with BRAF mutations, tumor grade, and patient survival in a cohort of 41 PXA patients using OncoScan chromosomal microarray. Primary resection specimens were available in 38 cases, including 24 PXA and 14 anaplastic PXA (A‐PXA), 23 BRAF V600E mutant tumors (61%). CNVs were identified in all cases and most frequently involved chromosome 9 with homozygous CDKN2A/B deletion (n = 33, 87%), a higher proportion than previously detected by comparative genomic hybridization (50%–60%) (37). CDKN2A/B deletion was present in similar proportion of PXA (83%), A‐PXA (93%), BRAF V600E (87%), and wild‐type (87%) tumors. Whole chromosome gains/losses were frequent, including gains +7 (n = 15), +2 (n = 11), +5 (n = 10), +21 (n = 10), +20 (n = 9), +12 (n = 8), +15 (n = 8), and losses −22 (n = 11), −14 (n = 7), −13 (n = 5). Losses and copy‐neutral loss of heterozygosity were significantly more common in A‐PXA, involving chromosomes 22 (P = 0.009) and 14 (P = 0.03). Amplification of 8p and 12q was identified in a single tumor. Histologic grade was a robust predictor of overall survival (P = 0.003), while other copy‐number changes, including CDKN2A/B deletion, did not show significant association with survival. Distinct histologic patterns of anaplasia included increased mitotic activity in an otherwise classic PXA or associated with small cell, fibrillary, or epithelioid morphology, with loss of SMARCB1 expression in one case. In 10 cases, matched specimens were compared, including A‐PXA with areas of distinct low‐ and high‐grade morphology (n = 2), matched primary/tumor recurrence (n = 7), or both (n = 1). Copy‐number changes on recurrence/anaplastic transformation were complex and highly variable, from nearly identical profiles to numerous copy‐number changes. Overall, we confirm CDKN2A/B deletion as key a feature of PXA not associated with tumor grade or BRAF mutation, but central to the underlying genetics of PXA.     

Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration‐resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole‐exome sequencing on five CRPC/normal paired formalin‐fixed paraffin‐embedded (FFPE) samples, using the SOLiD4 next‐generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock‐down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock‐down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole‐exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next‐generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Identification of copy number variants in horses

Copy number variants (CNVs) represent a substantial source of genetic variation in mammals. However, the occurrence of CNVs in horses and their subsequent impact on phenotypic variation is unknown. We performed a study to identify CNVs in 16 horses representing 15 distinct breeds (Equus caballus) and an individual gray donkey (Equus asinus) using a whole-exome tiling array and the array comparative genomic hybridization methodology. We identified 2368 CNVs ranging in size from 197 bp to 3.5 Mb. Merging identical CNVs from each animal yielded 775 CNV regions (CNVRs), involving 1707 protein- and RNA-coding genes. The number of CNVs per animal ranged from 55 to 347, with median and mean sizes of CNVs of 5.3 kb and 99.4 kb, respectively. Approximately 6% of the genes investigated were affected by a CNV. Biological process enrichment analysis indicated CNVs primarily affected genes involved in sensory perception, signal transduction, and metabolism. CNVs also were identified in genes regulating blood group antigens, coat color, fecundity, lactation, keratin formation, neuronal homeostasis, and height in other species. Collectively, these data are the first report of copy number variation in horses and suggest that CNVs are common in the horse genome and may modulate biological processes underlying different traits observed among horses and horse breeds.     

DNA copy number alterations and expression of relevant genes in mouse thymic lymphomas induced by gamma-irradiation and N-methyl-N-nitrosourea

The genetic mechanism for the development and progression of a lymphoma is unclear. This study investigated the alterations in the DNA copy number and the expression profiles of the genes located in the altered regions in mouse thymic lymphomas that were induced by two mutagens, gamma-irradiation and N-methyl-N-nitrosourea (MNU). Microarray-based comparative genomic hybridization was used to precisely delineate the boundaries of the altered region. The copy number gains of chromosomes 4 and 5 were observed only in the radiation-induced lymphomas, and gains of chromosomes 10 and 14 were observed only in the MNU-induced lymphomas. Regional copy number losses in chromosomes 11, 16, and 19 appeared frequently in the radiation-induced lymphomas. The cancer-related genes Pten, Ikaros/Znfn1a1, Ercc4, and Top3b were located in the minimal deletion regions. In particular, the expression levels of the Pten, Top3b, and Ikaros genes were downregulated in both lymphoma groups, but the expression level of Ercc4 was downregulated only in the MNU group. This study also examined the expression levels of Sparc, Cxcl1, and Myc (synonym: c-Myc), which are located in the copy number gained chromosomes. Sparc was upregulated specifically in the radiation group, and Cxcl1 in the MNU group. c-Myc was upregulated in both groups. There was limited correlation between the DNA copy number profiles and the expression of the cancer-related genes in mouse lymphomagenesis. The chromosome aberrations and novel expression profiles of the cancer-related genes within the altered regions may provide important clues to the genetic mechanism for the development of lymphoma.     

Dietary fat-dependent transcriptional architecture and copy number alterations associated with modifiers of mammary cancer metastasis

Breast cancer is a complex disease resulting from a combination of genetic and environmental factors. Among environmental factors, body composition and intake of specific dietary components like total fat are associated with increased incidence of breast cancer and metastasis. We previously showed that mice fed a high-fat diet have shorter mammary cancer latency, increased tumor growth and more pulmonary metastases than mice fed a standard diet. Subsequent genetic analysis identified several modifiers of metastatic mammary cancer along with widespread interactions between cancer modifiers and dietary fat. To elucidate diet-dependent genetic modifiers of mammary cancer and metastasis risk, global gene expression profiles and copy number alterations from mammary cancers were measured and expression quantitative trait loci (eQTL) identified. Functional candidate genes that colocalized with previously detected metastasis modifiers were identified. Additional analyses, such as eQTL by dietary fat interaction analysis, causality and database evaluations, helped to further refine the candidate loci to produce an enriched list of genes potentially involved in the pathogenesis of metastatic mammary cancer.     

High-resolution copy number arrays in cancer and the problem of normal genome copy number variation

High-resolution techniques for analysis of genome copy number (CN) enable the analysis of complex cancer somatic genetics. However, the analysis of these data is difficult, and failure to consider a number of issues in depth may result in false leads or unnecessary rejection of true positives. First, segmental duplications may falsely generate CN breakpoints in aneuploid samples. Second, even when tumor data were each normalized to matching lymphocyte DNA, we still observed copy number polymorphisms masquerading as somatic alterations due to allelic imbalance. We investigated a number of different solutions and determined that evaluating matching normal DNA, or at least using locally derived normal baseline data, were preferable to relying on current online databases because of poor cross-platform compatibility and the likelihood of excluding genuine small somatic alterations.