Correction to: A systematic comparison of copy number alterations in four types of female cancer.

After publication of the original article [1] the authors found that the article contained an incorrect version of Fig. 4. This does not affect the results and conclusions of the article.

Whole genome resequencing reveals diagnostic markers for investigating global migration and hybridization between minke whale species.

BACKGROUND: In the marine environment, where there are few absolute physical barriers, contemporary contact between previously isolated species can occur across great distances, and in some cases, may be inter-oceanic. An example of this can be seen in the minke whale species complex. Antarctic minke whales are genetically and morphologically distinct from the common minke found in the north Atlantic and Pacific oceans, and the two species are estimated to have been isolated from each other for 5 million years or more. Recent atypical migrations from the southern to the northern hemisphere have been documented and fertile hybrids and back-crossed individuals between both species have also been identified. However, it is not known whether this represents a contemporary event, potentially driven by ecosystem changes in the Antarctic, or a sporadic occurrence happening over an evolutionary time-scale. We successfully used whole genome resequencing to identify a panel of diagnostic SNPs which now enable us address this evolutionary question. RESULTS: A large number of SNPs displaying fixed or nearly fixed allele frequency differences among the minke whale species were identified from the sequence data. Five panels of putatively diagnostic markers were established on a genotyping platform for validation of allele frequencies; two panels (26 and 24 SNPs) separating the two species of minke whale, and three panels (22, 23, and 24 SNPs) differentiating the three subspecies of common minke whale. The panels were validated against a set of reference samples, demonstrating the ability to accurately identify back-crossed whales up to three generations. CONCLUSIONS: This work has resulted in the development of a panel of novel diagnostic genetic markers to address inter-oceanic and global contact among the genetically isolated minke whale species and sub-species. These markers, including a globally relevant genetic reference data set for this species complex, are now openly available for researchers interested in identifying other potential whale hybrids in the world's oceans. The approach used here, combining whole genome resequencing and high-throughput genotyping, represents a universal approach to develop similar tools for other species and population complexes.

A systematic comparison of copy number alterations in four types of female cancer.

BACKGROUND: Detection and localization of genomic alterations and breakpoints are crucial in cancer research. The purpose of this study was to investigate, in a methodological and biological perspective, different female, hormone-dependent cancers to identify common and diverse DNA aberrations, genes, and pathways. METHODS: In this work, we analyzed tissue samples from patients with breast (n = 112), ovarian (n = 74), endometrial (n = 84), or cervical (n = 76) cancer. To identify genomic aberrations, the Circular Binary Segmentation (CBS) and Piecewise Constant Fitting (PCF) algorithms were used and segmentation thresholds optimized. The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was applied to the segmented data to identify significantly altered regions and the associated genes were analyzed by Ingenuity Pathway Analysis (IPA) to detect over-represented pathways and functions within the identified gene sets. RESULTS AND DISCUSSION: Analyses of high-resolution copy number alterations in four different female cancer types are presented. For appropriately adjusted segmentation parameters the two segmentation algorithms CBS and PCF performed similarly. We identified one region at 8q24.3 with focal aberrations that was altered at significant frequency across all four cancer types. Considering both, broad regions and focal peaks, three additional regions with gains at significant frequency were revealed at 1p21.1, 8p22, and 13q21.33, respectively. Several of these events involve known cancer-related genes, like PPP2R2A, PSCA, PTP4A3, and PTK2. In the female reproductive system (ovarian, endometrial, and cervix [OEC]), we discovered three common events: copy number gains at 5p15.33 and 15q11.2, further a copy number loss at 8p21.2. Interestingly, as many as 75% of the aberrations (75% amplifications and 86% deletions) identified by GISTIC were specific for just one cancer type and represented distinct molecular pathways. CONCLUSIONS: Our results disclose that some prominent copy number changes are shared in the four examined female, hormone-dependent cancer whereas others are definitive to specific cancer types.

Gene expression analysis supports tumor threshold over 2.0 cm for T-category breast cancer.

Tumor size, as indicated by the T-category, is known as a strong prognostic indicator for breast cancer. It is common practice to distinguish the T1 and T2 groups at a tumor size of 2.0 cm. We investigated the 2.0-cm rule from a new point of view. Here, we try to find the optimal threshold based on the differences between the gene expression profiles of the T1 and T2 groups (as defined by the threshold). We developed a numerical algorithm to measure the overall differential gene expression between patients with smaller tumors and those with larger tumors among multiple expression datasets from different studies. We confirmed the performance of the proposed algorithm by a simulation study and then applied it to three different studies conducted at two Norwegian hospitals. We found that the maximum difference in gene expression is obtained at a threshold of 2.2-2.4 cm, and we confirmed that the optimum threshold was over 2.0 cm, as indicated by a validation study using five publicly available expression datasets. Furthermore, we observed a significant differentiation between the two threshold groups in terms of time to local recurrence for the Norwegian datasets. In addition, we performed an associated network and canonical pathway analyses for the genes differentially expressed between tumors below and above the given thresholds, 2.0 and 2.4 cm, using the Norwegian datasets. The associated network function illustrated a cellular assembly of the genes for the 2.0-cm threshold: an energy production for the 2.4-cm threshold and an enrichment in lipid metabolism based on the genes in the intersection for the 2.0- and 2.4-cm thresholds.

Canine Mammary Tumours Are Affected by Frequent Copy Number Aberrations, including Amplification of MYC and Loss of PTEN.

BACKGROUND: Copy number aberrations frequently occur during the development of many cancers. Such events affect dosage of involved genes and may cause further genomic instability and progression of cancer. In this survey, canine SNP microarrays were used to study 117 canine mammary tumours from 69 dogs. RESULTS: We found a high occurrence of copy number aberrations in canine mammary tumours, losses being more frequent than gains. Increased frequency of aberrations and loss of heterozygosity were positively correlated with increased malignancy in terms of histopathological diagnosis. One of the most highly recurrently amplified regions harbored the MYC gene. PTEN was located to a frequently lost region and also homozygously deleted in five tumours. Thus, deregulation of these genes due to copy number aberrations appears to be an important event in canine mammary tumour development. Other potential contributors to canine mammary tumour pathogenesis are COL9A3, INPP5A, CYP2E1 and RB1. The present study also shows that a more detailed analysis of chromosomal aberrations associated with histopathological parameters may aid in identifying specific genes associated with canine mammary tumour progression. CONCLUSIONS: The high frequency of copy number aberrations is a prominent feature of canine mammary tumours as seen in other canine and human cancers. Our findings share several features with corresponding studies in human breast tumours and strengthen the dog as a suitable model organism for this disease.

Investigating population genetic structure in a highly mobile marine organism: the minke whale Balaenoptera acutorostrata acutorostrata in the North East Atlantic.

Inferring the number of genetically distinct populations and their levels of connectivity is of key importance for the sustainable management and conservation of wildlife. This represents an extra challenge in the marine environment where there are few physical barriers to gene-flow, and populations may overlap in time and space. Several studies have investigated the population genetic structure within the North Atlantic minke whale with contrasting results. In order to address this issue, we analyzed ten microsatellite loci and 331 bp of the mitochondrial D-loop on 2990 whales sampled in the North East Atlantic in the period 2004 and 2007-2011. The primary findings were: (1) No spatial or temporal genetic differentiations were observed for either class of genetic marker. (2) mtDNA identified three distinct mitochondrial lineages without any underlying geographical pattern. (3) Nuclear markers showed evidence of a single panmictic population in the NE Atlantic according STRUCTURE's highest average likelihood found at K = 1. (4) When K = 2 was accepted, based on the Evanno's test, whales were divided into two more or less equally sized groups that showed significant genetic differentiation between them but without any sign of underlying geographic pattern. However, mtDNA for these individuals did not corroborate the differentiation. (5) In order to further evaluate the potential for cryptic structuring, a set of 100 in silico generated panmictic populations was examined using the same procedures as above showing genetic differentiation between two artificially divided groups, similar to the aforementioned observations. This demonstrates that clustering methods may spuriously reveal cryptic genetic structure. Based upon these data, we find no evidence to support the existence of spatial or cryptic population genetic structure of minke whales within the NE Atlantic. However, in order to conclusively evaluate population structure within this highly mobile species, more markers will be required.

Integrated analysis of high-resolution DNA methylation profiles, gene expression, germline genotypes and clinical end points in breast cancer patients.

Breast cancer is a heterogeneous disease for which alterations in DNA methylation patterns have been shown to be of biological and clinical importance. Here we report on the integrated analysis of molecular alterations including the methylation status of 27 gene promoters analyzed by highly quantitative pyrosequencing, and the association to gene expression, germline genotype and clinical parameters including survival. Breast cancer specific deregulation of DNA methylation (both hyper- and hypomethylation) was found in twenty genes including ACVR1, OGG1, IL8 and TFF1. The methylation level in the promoter regions was significantly negatively correlated to gene expression for twelve genes (such as MST1R, ST6GAL1 and TFF1) indicating that a gain of aberrant methylation (hypermethylation) inhibits gene expression. Multiple associations between molecular and clinical parameters were identified, and multivariate statistical analysis demonstrated that methylation was more strongly associated to clinical parameters than gene expression for the investigated genes. The methylation level of BCAP31 and OGG1 showed significant association to survival, and these associations were validated in a larger patient cohort (The Cancer Genome Atlas). Our study provides evidence for the promise of DNA methylation alterations for clinical applications.

Gene expression profile analysis of t1 and t2 breast cancer reveals different activation pathways.

Breast cancers today are of predominantly T1 (0.1 ≥ 2.0 cm) or T2 (>2 ≤ 5 cm) categories due to early diagnosis. Molecular profiling using microarrays has led to the notion of breast cancer as a heterogeneous disease both clinically and molecularly. Given the prognostic power and clinical use of tumor size, the purpose of this study was to search for molecular signatures characterizing clinical T1 and T2. In total 46 samples were included in the discovery dataset. After adjusting for hormone receptor status, lymph node status, grade, and tumor subclass 441 genes were differently expressed between T1 and T2 tumors. Focal adhesion and extracellular matrix receptor interaction were upregulated in the smaller tumors while p38MAPK signaling and immune-related pathways were more dominant in the larger tumors. The T-size signature was then tested on a validation set of 947 breast tumor samples. Using the T-size expression signatures instead of tumor size leads to a significant difference in risk for distant metastases (P < 0.001). If further confirmed, this molecular signature can be used to select patients with tumor category T1 who may need more aggressive treatment and patients with tumor category T2 who may have less benefit from it.

Allele-specific disparity in breast cancer.

BACKGROUND: In a cancer cell the number of copies of a locus may vary due to amplification and deletion and these variations are denoted as copy number alterations (CNAs). We focus on the disparity of CNAs in tumour samples, which were compared to those in blood in order to identify the directional loss of heterozygosity. METHODS: We propose a numerical algorithm and apply it to data from the Illumina 109K-SNP array on 112 samples from breast cancer patients. B-allele frequency (BAF) and log R ratio (LRR) of Illumina were used to estimate Euclidian distances. For each locus, we compared genotypes in blood and tumour for subset of samples being heterozygous in blood. We identified loci showing preferential disparity from heterozygous toward either the A/B-allele homozygous (allelic disparity). The chi-squared and Cochran-Armitage trend tests were used to examine whether there is an association between high levels of disparity in single nucleotide polymorphisms (SNPs) and molecular, clinical and tumour-related parameters. To identify pathways and network functions over-represented within the resulting gene sets, we used Ingenuity Pathway Analysis (IPA). RESULTS: To identify loci with a high level of disparity, we selected SNPs 1) with a substantial degree of disparity and 2) with substantial frequency (at least 50% of the samples heterozygous for the respective locus). We report the overall difference in disparity in high-grade tumours compared to low-grade tumours (p-value < 0.001) and significant associations between disparity in multiple single loci and clinical parameters. The most significantly associated network functions within the genes represented in the loci of disparity were identified, including lipid metabolism, small-molecule biochemistry, and nervous system development and function. No evidence for over-representation of directional disparity in a list of stem cell genes was obtained, however genes appeared to be more often altered by deletion than by amplification. CONCLUSIONS: Our data suggest that directional loss and amplification exist in breast cancer. These are highly associated with grade, which may indicate that they are enforced with increasing number of cell divisions. Whether there is selective pressure for some loci to be preferentially amplified or deleted remains to be confirmed.

Linear and non-linear dependencies between copy number aberrations and mRNA expression reveal distinct molecular pathways in breast cancer.

BACKGROUND: Elucidating the exact relationship between gene copy number and expression would enable identification of regulatory mechanisms of abnormal gene expression and biological pathways of regulation. Most current approaches either depend on linear correlation or on nonparametric tests of association that are insensitive to the exact shape of the relationship. Based on knowledge of enzyme kinetics and gene regulation, we would expect the functional shape of the relationship to be gene dependent and to be related to the gene regulatory mechanisms involved. Here, we propose a statistical approach to investigate and distinguish between linear and nonlinear dependences between DNA copy number alteration and mRNA expression. RESULTS: We applied the proposed method to DNA copy numbers derived from Illumina 109 K SNP-CGH arrays (using the log R values) and expression data from Agilent 44 K mRNA arrays, focusing on commonly aberrated genomic loci in a collection of 102 breast tumors. Regression analysis was used to identify the type of relationship (linear or nonlinear), and subsequent pathway analysis revealed that genes displaying a linear relationship were overall associated with substantially different biological processes than genes displaying a nonlinear relationship. In the group of genes with a linear relationship, we found significant association to canonical pathways, including purine and pyrimidine metabolism (for both deletions and amplifications) as well as estrogen metabolism (linear amplification) and BRCA-related response to damage (linear deletion). In the group of genes displaying a nonlinear relationship, the top canonical pathways were specific pathways like PTEN and PI13K/AKT (nonlinear amplification) and Wnt(B) and IL-2 signalling (nonlinear deletion). Both amplifications and deletions pointed to the same affected pathways and identified cancer as the top significant disease and cell cycle, cell signaling and cellular development as significant networks. CONCLUSIONS: This paper presents a novel approach to assessing the validity of the dependence of expression data on copy number data, and this approach may help in identifying the drivers of carcinogenesis.

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density.

INTRODUCTION: Mammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. METHODS: Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. RESULTS: SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. CONCLUSIONS: Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer.

Genotypes and haplotypes in the insulin-like growth factors, their receptors and binding proteins in relation to plasma metabolic levels and mammographic density.

BACKGROUND: Increased mammographic density is one of the strongest independent risk factors for breast cancer. It is believed that one third of breast cancers are derived from breasts with more than 50% density. Mammographic density is affected by age, BMI, parity, and genetic predisposition. It is also greatly influenced by hormonal and growth factor changes in a woman's life cycle, spanning from puberty through adult to menopause. Genetic variations in genes coding for hormones and growth factors involved in development of the breast are therefore of great interest. The associations between genetic polymorphisms in genes from the IGF pathway on mammographic density and circulating levels of IGF1, its binding protein IGFBP3, and their ratio in postmenopausal women are reported here. METHODS: Samples from 964 postmenopausal Norwegian women aged 55-71 years were collected as a part of the Tromsø Mammography and Breast Cancer Study. All samples were genotyped for 25 SNPs in IGF1, IGF2, IGF1R, IGF2R, IGFALS and IGFBP3 using Taqman (ABI). The main statistical analyses were conducted with the PROC HAPLOTYPE procedure within SAS/GENETICS (SAS 9.1.3). RESULTS: The haplotype analysis revealed six haploblocks within the studied genes. Of those, four had significant associations with circulating levels of IGF1 or IGFBP3 and/or mammographic density. One haplotype variant in the IGF1 gene was found to be associated with mammographic density. Within the IGF2 gene one haplotype variant was associated with levels of both IGF1 and IGFBP3. Two haplotype variants in the IGF2R were associated with the level of IGF1. Both variants of the IGFBP3 haplotype were associated with the IGFBP3 level and indicate regulation in cis. CONCLUSION: Polymorphisms within the IGF1 gene and related genes were associated with plasma levels of IGF1, IGFBP3 and mammographic density in this study of postmenopausal women.

GSTP1 promoter haplotypes affect DNA methylation levels and promoter activity in breast carcinomas.

The CpG island spanning the transcription start of the glutathione S-transferase P1 becomes methylated in a variety of human cancers including breast cancer. To study the effect of sequence variation on hypermethylation of the GSTP1 promoter, we analyzed the genetic and epigenetic variability in 90 tumors from patients with locally advanced breast cancer. High-resolution quantitative analysis revealed large variability in the DNA methylation levels. Lack of methylation was more often observed in the basal and normal-like estrogen receptor (ER)-negative tumors, and methylated GSTP1 was associated with better overall survival (P = 0.00063). Studies of the genetic variation identified 14 different haplotypes. The distribution of methylation levels of tumors homozygous for the most frequent haplotype was significantly different from other haplotype combinations (P = 0.011), the difference being more pronounced in ER-positive (P = 0.005) and progesterone receptor-positive (P = 0.008) tumors. Regression modeling identified the ER status and haplotype as the main determinants of DNA methylation variability. We identified a putative c-Myb response element (MRE) that was present in one of two minimal promoter haplotypes. In vitro analysis showed that c-Myb binds to the MRE, but binding was weakened by the two polymorphisms. Transient cotransfections in luminal-type and basal-like breast cancer cell lines confirmed cell-specific differential binding of c-Myb to the polymorphic sites, leading to a change in the expression from the GSTP1 promoter in vivo. GSTP1 expression was moderately but significantly (P = 0.01) reduced after siRNA-mediated knockdown of c-Myb. Our results indicate that haplotype structure of a promoter is important for the extent of DNA methylation.