Successive extreme climatic events lead to immediate, large-scale, and diverse responses from fish in the Arctic.

The warming trend of the Arctic is punctuated by several record-breaking warm years with very low sea ice concentrations. The nature and reversibility of marine ecosystem responses to these multiple extreme climatic events (ECEs) are poorly understood. Here, we investigate the ecological signatures of three successive bottom temperature maxima concomitant with surface ECEs between 2004 and 2017 in the Barents Sea across spatial and organizational scales. We observed community-level redistributions of fish concurrent with ECEs at the scale of the whole Barents Sea. Three groups, characterized by different sets of traits describing their capacity to cope with short-term perturbations, reacted with different timing and intensity to each ECE. Arctic species co-occurred more frequently with large predators and incoming boreal taxa during ECEs, potentially affecting food web structures and functional diversity, accelerating the impacts of long-term climate change. On the species level, responses were highly diversified, with different ECEs impacting different species, and species responses (expansion, geographical shift) varying from one ECE to another, despite the environmental perturbations being similar. Past ECEs impacts, with potential legacy effects, lagged responses, thresholds, and interactions with the underlying warming pressure, could constantly set up new initial conditions that drive the unique ecological signature of each ECE. These results highlight the complexity of ecological reactions to multiple ECEs and give prominence to several sources of process uncertainty in the predictions of climate change impact and risk for ecosystem management. Long-term monitoring and studies to characterize the vertical extent of each ECE are necessary to statistically link demersal species and environmental spatial-temporal patterns. In the future, regular monitoring will be crucial to detect early signals of change and understand the determinism of ECEs, but we need to adapt our models and management to better integrate risk and stochasticity from the complex impacts of global change.

Correction: An improved methodology for quantifying causality in complex ecological systems.

[This corrects the article DOI: 10.1371/journal.pone.0208078.].

An improved methodology for quantifying causality in complex ecological systems.

This paper provides a statistical methodology for quantifying causality in complex dynamical systems, based on analysis of multidimensional time series data of the state variables. The methodology integrates Granger's causality analysis based on the log-likelihood function expansion (Partial pair-wise causality), and Akaike's power contribution approach over the whole frequency domain (Total causality). The proposed methodology addresses a major drawback of existing methodologies namely, their inability to use time series observation of state variables to quantify causality in complex systems. We first perform a simulation study to verify the efficacy of the methodology using data generated by several multivariate autoregressive processes, and its sensitivity to data sample size. We demonstrate application of the methodology to real data by deriving inter-species relationships that define key food web drivers of the Barents Sea ecosystem. Our results show that the proposed methodology is a useful tool in early stage causality analysis of complex feedback systems.

Evolution of root apical meristem structures in vascular plants: plasmodesmatal networks.

PREMISE OF THE STUDY: The apical meristem generates indeterminate apical growth of the stem and root of vascular plants. Our previous examination showed that shoot apical meristems (SAMs) can be classified into two types based on plasmodesmatal networks (PNs), which are important elements in symplasmic signaling pathways within the apical meristem. Here, we examined the PNs of root apical meristems (RAMs) in comparison with those of SAMs. METHODS: Root apical meristems of 18 families and 22 species of lycophytes and euphyllophytes were analyzed. Plasmodesmata (PD) in cell walls in median longitudinal sections of RAMs were enumerated using transmission electron micrographs, and the PD density per 1 µm2 of each cell wall was calculated. KEY RESULTS: Root apical meristems with prominent apical cells of monilophytes (euphyllophytes) and Selaginellaceae (lycophytes) had high PD densities, while RAMs with plural initial cells of gymnosperms and angiosperms (euphyllophytes), and of Lycopodiaceae and Isoetaceae (lycophytes) had low PD densities. This correlation between structures of apical meristems and PD densities is identical to that in SAMs already described. CONCLUSIONS: Irrespective of their diversified structures, the RAMs of vascular plants can be classified into two types with respect to PNs: the fern (monilophyte) type, which has a lineage-specific PN with only primary PD, and the seed-plant type, which has an interspecific PN with secondary PD in addition to primary PD. PNs may have played a key role in the evolution of apical meristems in vascular plants.

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.

Treating lateral epicondylitis with corticosteroid injections or non-electrotherapeutical physiotherapy: a systematic review.

OBJECTIVES: To evaluate the current evidence for the efficacy of corticosteroid injection and non-electrotherapeutic physiotherapy compared with control for treating lateral epicondylitis. DESIGN: Systematic review. PARTICIPANTS: We searched five databases in September 2012 for randomised controlled studies with a minimum quality rating. Of the 640 studies retrieved, 11 were included, representing 1161 patients of both sexes and all ages. INTERVENTIONS: Corticosteroid injection and non-electrotherapeutic physiotherapy. OUTCOME MEASURES: Relative risk (RR) or standardised mean difference (SMD) for overall improvement, pain and grip strength at 4-12, 26 and 52 weeks of follow-up. RESULTS: Corticosteroid injection gave a short-term reduction in pain versus no intervention or non-steroidal anti-inflammatory drugs (SMD -1.43, 95% CI -1.64 to -1.23). At intermediate follow-up, we found an increase in pain (SMD 0.32, 95% CI 0.13 to 0.51), reduction in grip strength (SMD -0.48, 95% CI -0.73 to -0.24) and negative effect on the overall improvement effect (RR 0.66 (0.53 to 0.81)). For corticosteroid injection versus lidocaine injection, the evidence was conflicting. At long-term follow-up, there was no difference on overall improvement and grip strength, with conflicting evidence for pain. Manipulation and exercise versus no intervention showed beneficial effect at short-term follow-up (overall improvement RR 2.75, 95% CI 1.30 to 5.82), but no significant difference at intermediate or long-term follow-up. We found moderate evidence for short-term and long-term effects of eccentric exercise and stretching versus no intervention. For exercise versus no intervention and eccentric or concentric exercise and stretching versus stretching alone, we found moderate evidence of no short-term effect. CONCLUSIONS: Corticosteroid injections have a short-term beneficial effect on lateral epicondylitis, but a negative effect in the intermediate term. Evidence on the long-term effect is conflicting. Manipulation and exercise and exercise and stretching have a short-term effect, with the latter also having a long-term effect.

Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors.

BACKGROUND: Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. METHODS: Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. RESULTS: Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. CONCLUSIONS: In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

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.

Let-7 miRNA-binding site polymorphism in the KRAS 3'UTR; colorectal cancer screening population prevalence and influence on clinical outcome in patients with metastatic colorectal cancer treated with 5-fluorouracil and oxaliplatin +/- cetuximab.

BACKGROUND: Recent studies have reported associations between a variant allele in a let-7 microRNA complementary site (LCS6) within the 3'untranslated region (3'UTR) of KRAS (rs61764370) and clinical outcome in metastatic colorectal cancer (mCRC) patients receiving cetuximab. The variant allele has also been associated with increased cancer risk. We aimed to reveal the incidence of the variant allele in a colorectal cancer screening population and to investigate the clinical relevance of the variant allele in mCRC patients treated with 1st line Nordic FLOX (bolus 5-fluorouracil/folinic acid and oxaliplatin) +/- cetuximab. METHODS: The feasibility of the variant allele as a risk factor for CRC was investigated by comparing the LCS6 gene frequencies in 197 CRC patients, 1060 individuals with colorectal polyps, and 358 healthy controls. The relationship between clinical outcome and LCS6 genotype was analyzed in 180 mCRC patients receiving Nordic FLOX and 355 patients receiving Nordic FLOX + cetuximab in the NORDIC-VII trial (NCT00145314). RESULTS: LCS6 frequencies did not vary between CRC patients (23%), individuals with polyps (20%), and healthy controls (20%) (P = 0.50). No statistically significant differences were demonstrated in the NORDIC-VII cohort even if numerically increased progression-free survival (PFS) and overall survival (OS) were found in patients with the LCS6 variant allele (8.5 (95% CI: 7.3-9.7 months) versus 7.8 months (95% CI: 7.4-8.3 months), P = 0.16 and 23.5 (95% CI: 21.6-25.4 months) versus 19.5 months (95% CI: 17.8-21.2 months), P = 0.31, respectively). Addition of cetuximab seemed to improve response rate more in variant carriers than in wild-type carriers (from 35% to 57% versus 44% to 47%), however the difference was not statistically significant (interaction P = 0.16). CONCLUSIONS: The LCS6 variant allele does not seem to be a risk factor for development of colorectal polyps or CRC. No statistically significant effect of the LCS6 variant allele on response rate, PFS or OS was found in mCRC patients treated with 1st line Nordic FLOX +/- cetuximab.

Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity.

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminal-like cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple "stem-like" cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.

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.

Methylation profiling with a panel of cancer related genes: association with estrogen receptor, TP53 mutation status and expression subtypes in sporadic breast cancer.

Breast cancer is a heterogeneous disease that can be divided in subtypes based on histology, gene expression profiles as well as differences in genomic aberrations. Distinct global DNA methylation profiles have been reported in normal breast epithelial cells as well as in breast tumors. However, the influence of the tumor methylome on the previously described subgroups of breast cancer is not fully understood. Here we report the DNA methylation profiles of 80 breast tumors using a panel of 807 cancer related genes interrogating 1505 CpG sites. We identified three major clusters based on the methylation profiles; one consisting of mainly tumors of myoepithelial origin and two other clusters with tumors of predominantly luminal epithelial origin. The clusters were different with respect to estrogen receptor status, TP53 status, ErbB2 status and grade. The most significantly differentially methylated genes including HDAC1, TFF1, OGG1, BMP3, FZD9 and HOXA11 were confirmed by pyrosequencing. Gene Ontology analysis revealed enrichment for genes involved in developmental processes including homeobox domain genes (HOXA9, HOXA11, PAX6, MYBL2, ISL1 and IPF1) and (ETS1, HDAC1, CREBBP, GAS7, SPI1 and TBX1). Extensive correlation to mRNA expression was observed. Pathway analyses identified a significant association with canonical (curated) pathways such as hepatic fibrosis including genes like EGF, NGFR and TNF, dendritic cell maturation and the NF-κB signaling pathway. Our results show that breast tumor expression subtypes harbor major epigenetic differences and tumors with similar gene expression profiles might belong to epigenetically different subtypes. Some of the transcription factors identified, with key roles in differentiation and development might play a role in inducing and maintaining the different phenotypes.

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.