Basal-like Breast cancer DNA copy number losses identify genes involved in genomic instability, response to therapy, and patient survival.

Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher's exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11-35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype.

Integrated molecular profiles of invasive breast tumors and ductal carcinoma in situ (DCIS) reveal differential vascular and interleukin signaling.

We use an integrated approach to understand breast cancer heterogeneity by modeling mRNA, copy number alterations, microRNAs, and methylation in a pathway context utilizing the pathway recognition algorithm using data integration on genomic models (PARADIGM). We demonstrate that combining mRNA expression and DNA copy number classified the patients in groups that provide the best predictive value with respect to prognosis and identified key molecular and stromal signatures. A chronic inflammatory signature, which promotes the development and/or progression of various epithelial tumors, is uniformly present in all breast cancers. We further demonstrate that within the adaptive immune lineage, the strongest predictor of good outcome is the acquisition of a gene signature that favors a high T-helper 1 (Th1)/cytotoxic T-lymphocyte response at the expense of Th2-driven humoral immunity. Patients who have breast cancer with a basal HER2-negative molecular profile (PDGM2) are characterized by high expression of protumorigenic Th2/humoral-related genes (24-38%) and a low Th1/Th2 ratio. The luminal molecular subtypes are again differentiated by low or high FOXM1 and ERBB4 signaling. We show that the interleukin signaling profiles observed in invasive cancers are absent or weakly expressed in healthy tissue but already prominent in ductal carcinoma in situ, together with ECM and cell-cell adhesion regulating pathways. The most prominent difference between low and high mammographic density in healthy breast tissue by PARADIGM was that of STAT4 signaling. In conclusion, by means of a pathway-based modeling methodology (PARADIGM) integrating different layers of molecular data from whole-tumor samples, we demonstrate that we can stratify immune signatures that predict patient survival.

Analyzing cancer samples with SNP arrays.

Single nucleotide polymorphism (SNP) arrays are powerful tools to delineate genomic aberrations in cancer genomes. However, the analysis of these SNP array data of cancer samples is complicated by three phenomena: (a) aneuploidy: due to massive aberrations, the total DNA content of a cancer cell can differ significantly from its normal two copies; (b) nonaberrant cell admixture: samples from solid tumors do not exclusively contain aberrant tumor cells, but always contain some portion of nonaberrant cells; (c) intratumor heterogeneity: different cells in the tumor sample may have different aberrations. We describe here how these phenomena impact the SNP array profile, and how these can be accounted for in the analysis. In an extended practical example, we apply our recently developed and further improved ASCAT (allele-specific copy number analysis of tumors) suite of tools to analyze SNP array data using data from a series of breast carcinomas as an example. We first describe the structure of the data, how it can be plotted and interpreted, and how it can be segmented. The core ASCAT algorithm next determines the fraction of nonaberrant cells and the tumor ploidy (the average number of DNA copies), and calculates an ASCAT profile. We describe how these ASCAT profiles visualize both copy number aberrations as well as copy-number-neutral events. Finally, we touch upon regions showing intratumor heterogeneity, and how they can be detected in ASCAT profiles. All source code and data described here can be found at our ASCAT Web site ( http://www.ifi.uio.no/forskning/grupper/bioinf/Projects/ASCAT/).

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.

Allele-specific copy number analysis of tumors.

We present an allele-specific copy number analysis of the in vivo breast cancer genome. We describe a unique bioinformatics approach, ASCAT (allele-specific copy number analysis of tumors), to accurately dissect the allele-specific copy number of solid tumors, simultaneously estimating and adjusting for both tumor ploidy and nonaberrant cell admixture. This allows calculation of "ASCAT profiles" (genome-wide allele-specific copy-number profiles) from which gains, losses, copy number-neutral events, and loss of heterozygosity (LOH) can accurately be determined. In an early-stage breast carcinoma series, we observe aneuploidy (>2.7n) in 45% of the cases and an average nonaberrant cell admixture of 49%. By aggregation of ASCAT profiles across our series, we obtain genomic frequency distributions of gains and losses, as well as genome-wide views of LOH and copy number-neutral events in breast cancer. In addition, the ASCAT profiles reveal differences in aberrant tumor cell fraction, ploidy, gains, losses, LOH, and copy number-neutral events between the five previously identified molecular breast cancer subtypes. Basal-like breast carcinomas have a significantly higher frequency of LOH compared with other subtypes, and their ASCAT profiles show large-scale loss of genomic material during tumor development, followed by a whole-genome duplication, resulting in near-triploid genomes. Finally, from the ASCAT profiles, we construct a genome-wide map of allelic skewness in breast cancer, indicating loci where one allele is preferentially lost, whereas the other allele is preferentially gained. We hypothesize that these alternative alleles have a different influence on breast carcinoma development.

Integrated study of copy number states and genotype calls using high-density SNP arrays.

We propose a statistical framework, named genoCN, to simultaneously dissect copy number states and genotypes using high-density SNP (single nucleotide polymorphism) arrays. There are at least two types of genomic DNA copy number differences: copy number variations (CNVs) and copy number aberrations (CNAs). While CNVs are naturally occurring and inheritable, CNAs are acquired somatic alterations most often observed in tumor tissues only. CNVs tend to be short and more sparsely located in the genome compared with CNAs. GenoCN consists of two components, genoCNV and genoCNA, designed for CNV and CNA studies, respectively. In contrast to most existing methods, genoCN is more flexible in that the model parameters are estimated from the data instead of being decided a priori. GenoCNA also incorporates two important strategies for CNA studies. First, the effects of tissue contamination are explicitly modeled. Second, if SNP arrays are performed for both tumor and normal tissues of one individual, the genotype calls from normal tissue are used to study CNAs in tumor tissue. We evaluated genoCN by applications to 162 HapMap individuals and a brain tumor (glioblastoma) dataset and showed that our method can successfully identify both types of copy number differences and produce high-quality genotype calls.

Pathway based analysis of SNPs with relevance to 5-FU therapy: relation to intratumoral mRNA expression and survival.

Genetic factors are thought to play a role in resistance towards chemotherapeutic agents such as 5-fluorouracil (5-FU). Approximately 30 genes are directly or indirectly involved in 5-FU metabolism, and genetic variation in any of these may contribute to anti-tumor response. Polymorphisms in these genes were analyzed in relation to tumoral mRNA levels of 5-FU metabolizing genes, response to chemotherapy and survival. A total of 21 genetic variants were studied in 35 breast cancer patients treated with 5-FluoroUracil, mitomycin (FUMI) and in a similar cohort of 90 doxorubicin treated breast cancer patients. Genotype distributions were compared using 109 healthy controls. No significant association was found between any polymorphisms and response to chemotherapy as measured by shrinkage of tumor. However, carriers of 3 copies of the TYMS 5'UTR repeat had shorter survival than noncarriers (p = .048) in the FUMI treatment group, but not in the doxorubicin treated group. Carriers of 3 copies of the repeat were also more frequently observed in both patients groups than in healthy controls (p = .034). Several highly significant associations were observed between genotypes and expression levels of 5-FU metabolizing genes. A SNP in codon 72 of TP53 was revealed to be a key regulator of 5-FU metabolizing genes such as DHFR and MTHFR, constituting 50% of all significant associations observed after FUMI therapy. These data suggest that 3 copies of the TYMS 5'UTR repeat may give a treatment specific reduced survival in breast cancer patients, and that TP53 may have a direct, allele specific, role in 5-FU mediated response.

Genome-wide analysis identifies 16q deletion associated with survival, molecular subtypes, mRNA expression, and germline haplotypes in breast cancer patients.

Breast carcinomas are characterized by DNA copy number alterations (CNAs) with biological and clinical significance. This explorative study integrated CNA, expression, and germline genotype data of 112 early-stage breast cancer patients. Recurrent CNAs differed substantially between tumor subtypes classified according to expression pattern. Deletion of 16q was overrepresented in Luminal A, and a predictor of good prognosis, both overall and for the nonluminal A subgroups. The deleted region most significantly associated with survival mapped to 16q22.2, harboring the genes TXNL4B and DXH38, whose expression was strongly correlated with the deletion. The area most frequently deleted resided on 16q23.1, 3.5 MB downstream of the area most significantly associated with survival, and included the tumor suppressor gene ADAMTS18 and the cell recognition gene CNTNAP4. Whole-genome association analysis identified germline single nucleotide polymorphisms (SNPs) and their corresponding haplotypes, residing on several different chromosomes, to be associated with deletion of 16q. The genes where these SNPs reside encode proteins involved in the extracellular matrix (CHST3 and SPOCK2), in regulation of the cell cycle (JMY, PTPRN2, and Cwf19L2) and chromosome stability (KPNB1).

Single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1): effects on its expression and clinicopathological characteristics in breast cancer patients.

OBJECTIVES: Resistance of tumor cells to multiple cytostatic agents is one of the major impediments of successful cancer chemotherapy. A large part of resistance of tumors to chemotherapy is caused by the ABC transporter P-glycoprotein encoded by the ABCB1 gene. The main aim of this study was to assess the prognostic value of ABCB1 genotype and phenotype in breast cancer. METHODS: Six ABCB1 single nucleotide polymorphisms (SNPs) were determined in 90 Czech breast cancer patients by a novel method that allows simultaneous assessment of multiple polymorphisms on a single electronic microarray. Expression levels of ABCB1 were quantified in tumor and nontumor samples of breast cancer patients by real-time PCR. T-test, analysis of variance and Fisher's exact test were used to analyze the effect of ABCB1 polymorphisms on ABCB1 expression levels and for the analysis of associations between ABCB1 expression, genotype and clinical and pathological characteristics. RESULTS: ABCB1 was expressed in 98.9% of the tumor and in 97.5% of the nontumor samples. ABCB1 was downregulated in 79.5% of tumors compared with the nontumor samples. No significant correlation was observed between ABCB1 mRNA expression levels and clinical and pathological characteristics. High frequencies of the variant alleles in ABCB1 exon 12 (1236C>T, 38.3%) and exon 26 (3435C>T, 54.0%) were observed. Individuals with variant alleles in exons 12 and 26 had significantly lower ABCB1 expression levels in their tumors. SNPs in exons 12 and 26 also correlated with estrogen receptor status of patients. CONCLUSION: ABCB1 SNPs may affect function of P-glycoprotein by influencing the expression level and modify breast cancer prognosis.

Transcriptional networks inferred from molecular signatures of breast cancer.

Global genomic approaches in cancer research have provided new and innovative strategies for the identification of signatures that differentiate various types of human cancers. Computational analysis of the promoter composition of the genes within these signatures may provide a powerful method for deducing the regulatory transcriptional networks that mediate their collective function. In this study we have systematically analyzed the promoter composition of gene classes derived from previously established genetic signatures that recently have been shown to reliably and reproducibly distinguish five molecular subtypes of breast cancer associated with distinct clinical outcomes. Inferences made from the trends of transcription factor binding site enrichment in the promoters of these gene groups led to the identification of regulatory pathways that implicate discrete transcriptional networks associated with specific molecular subtypes of breast cancer. One of these inferred pathways predicted a role for nuclear factor-kappaB in a novel feed-forward, self-amplifying, autoregulatory module regulated by the ERBB family of growth factor receptors. The existence of this pathway was verified in vivo by chromatin immunoprecipitation and shown to be deregulated in breast cancer cells overexpressing ERBB2. This analysis indicates that approaches of this type can provide unique insights into the differential regulatory molecular programs associated with breast cancer and will aid in identifying specific transcriptional networks and pathways as potential targets for tumor subtype-specific therapeutic intervention.

Genes harbouring susceptibility SNPs are differentially expressed in the breast cancer subtypes.

Recently, genome-wide association studies of breast cancer revealed single nucleotide polymorphisms (SNPs) in five genes with novel association to susceptibility. While there is little doubt that the novel susceptibility markers produced from such highly powered studies are true, the mechanisms by which they cause the susceptibility remain undetermined. We have looked at the expression levels of the identified genes in tumours and found that they are highly significantly differentially expressed between the five established breast cancer subtypes. Also, a significant association between SNPs in these genes and their expression in tumours was seen as well as a significantly different frequency of the SNPs between the subtypes. This suggests that the observed genes are associated with different breast cancer subtypes, and may exert their effect through their expression in the tumours. Thus, future studies stratifying patients by their molecular subtypes may give much more power to classic case control studies, and genes of no or borderline significance may appear to be high-penetrant for certain subtypes and, therefore, be identifiable.

ABCB1 and GST polymorphisms associated with TP53 status in breast cancer.

BACKGROUND AND OBJECTIVE: Many environmental and genetic factors influence the development of chemoresistance. The goal of this study was to characterize the genetic variation in the ABCB1, GSTM1, GSTT1 and GSTP1 genes, as well as the haplotype structure in the ABCB1 gene. METHODS: Variants in these genes were studied in 109 healthy controls and 93 breast cancer cases, both of Caucasian origin. The cases were analyzed in relation to TP53 mutation status and response to doxorubicin. Both single and multiple single nucleotide polymorphism analyses were performed. RESULTS: Chi-square analyses revealed a significant association between TP53 mutation status and both the GA genotype of ABCB1 exon 11 (Ser400Asn) and the GG genotype of GSTP1 (Ile105Val; P<0.01 and P<0.05, respectively). Multifactor dimensionality reduction showed that carriers of the combined GG genotype for GSTP1 and the GG for ABCB1 exon 11 had the highest chance of acquiring a mutation in the TP53 gene (P<0.02). Haplotype analysis of ABCB1 revealed a significantly different distribution of haplotypes between the breast cancer cases and the controls (P<0.01). A specific haplotype association to TP53 mutation (P<0.01) distant metastases (P<0.05) and estrogen receptor status (P<0.05) was also observed in the case group. CONCLUSION: An association between polymorphisms in GSTP1 and ABCB1 and risk of acquiring intratumoral TP53 mutations suggests the existence of putative predisposing genotype backgrounds. The degree of linkage disequilibrium in the ABCB1 gene was higher in healthy individuals, whereas haplotypes in the cases seemed degenerated by a number of low frequency variants. This observation may either point to the existence of a protective haplotype in the controls or may underline the importance of the accumulation of low frequency variants as susceptibility factors.

The novel p21 polymorphism p21G251A is associated with locally advanced breast cancer.

PURPOSE: p21 is a main effector of growth arrest induced by p53. In addition, a second transcript from the same gene (p21B) has been linked to apoptosis. We previously analyzed p21 status in breast cancer and reported two novel polymorphisms of the p21 gene. In the present study, we present a larger study designed to explore a possible association between these novel polymorphisms and breast cancer. EXPERIMENTAL DESIGN: The p21/p21B polymorphisms were analyzed in 507 breast cancer patients and 1,017 healthy individuals using cDNA or genomic DNA from tumor and/or blood samples. RESULTS: We detected five polymorphisms of the p21 gene. Three of these polymorphisms are earlier reported by others, whereas two were reported for the first time in a recent study by us. The presence of the A allele of the p21G251A polymorphism was observed more frequently among patients with primary stage III breast cancer (4.5%) compared with stage I and II tumors (1.5%) and healthy female controls (1.4%; P = 0.007, comparing the three groups; P = 0.0049 and P = 0.0057, comparing locally advanced to stage I/II and healthy controls, or to healthy controls alone, respectively). The allele frequencies of the remaining four polymorphisms were evenly distributed among patients and healthy individuals. DISCUSSION: The finding of an association between locally advanced breast cancer and one particular polymorphism of the p21 gene suggests this polymorphism to be related to tumor behavior, including enhanced growth rate. If confirmed in other studies, this may add significant information to our understanding of the biology as well as of the clinical behaviour of locally advanced breast cancers.

Genetic variation in putative regulatory loci controlling gene expression in breast cancer.

Candidate single-nucleotide polymorphisms (SNPs) were analyzed for associations to an unselected whole genome pool of tumor mRNA transcripts in 50 unrelated patients with breast cancer. SNPs were selected from 203 candidate genes of the reactive oxygen species pathway. We describe a general statistical framework for the simultaneous analysis of gene expression data and SNP genotype data measured for the same cohort, which revealed significant associations between subsets of SNPs and transcripts, shedding light on the underlying biology. We identified SNPs in EGF, IL1A, MAPK8, XPC, SOD2, and ALOX12 that are associated with the expression patterns of a significant number of transcripts, indicating the presence of regulatory SNPs in these genes. SNPs were found to act in trans in a total of 115 genes. SNPs in 43 of these 115 genes were found to act both in cis and in trans. Finally, subsets of SNPs that share significantly many common associations with a set of transcripts (biclusters) were identified. The subsets of transcripts that are significantly associated with the same set of SNPs or to a single SNP were shown to be functionally coherent in Gene Ontology and pathway analyses and coexpressed in other independent data sets, suggesting that many of the observed associations are within the same functional pathways. To our knowledge, this article is the first study to correlate SNP genotype data in the germ line with somatic gene expression data in breast tumors. It provides the statistical framework for further genotype expression correlation studies in cancer data sets.

Direct isolation of recombinant human antibodies against group B Neisseria meningitidis from scFv expression libraries.

The successful generation of human antibodies from large nai;ve antibody libraries requires iterative selection steps. Here, we describe a new and fast method for the isolation of high affinity antibodies directly from human single chain Fv antibody (scFv) expression libraries. Escherichia coli scFv expression libraries were made from peripheral blood lymphocytes from four individuals vaccinated with group B Neisseria meningitidis outer membrane vesicle (OMV) vaccine. Forty thousand clones were directly screened for antibodies binding N. meningitidis strain 44/76 (B:15:P1.7,16). Of 430 specific clones detected, 225 candidates were isolated and re-screened against the N. meningitidis strains NZ-98/254 (B:4:P1.7b,4) giving 4% cross-reactive clones. Antibodies were further characterized by DNA sequencing, ELISA and surface plasmon resonance (SPR) analysis, showing broad V-gene diversity and nanomolar scFv affinities. Antibodies derived by this method may assist in the discovery and development of new vaccine antigens as well as therapeutic antibody agents for the treatment of meningococcal diseases.

Seed 1-cysteine peroxiredoxin antioxidants are not involved in dormancy, but contribute to inhibition of germination during stress.

Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.

ABI3 mediates expression of the peroxiredoxin antioxidant AtPER1 gene and induction by oxidative stress.

The peroxiredoxin antioxidant gene AtPER1 has been considered to be specifically expressed in the embryo and aleurone layer during maturation and desiccation stages of development, and in the mature seed, typically for late embryogenesis-abundant (lea) transcripts. In the abscisic acid-insensitive abi3-1 mutant, the AtPER1 transcript level is strongly reduced, suggesting ABI3 to be a prime regulator of AtPER1. We have studied the expression pattern and regulation of AtPER1 with a series of nine promoter::GUS constructs with deletions and/or mutations in putative regulatory elements. Arabidopsis lines harbouring these constructs revealed AtPER1 promoter activity in the endosperm, especially the chalazal cyst, already when the embryo is in the late globular stage, in the embryo from the late torpedo stage, and in distinct cells of unfertilized and fertilized ovules. Early expression seems to be dependent on a putative antioxidant-responsive promoter element (ARE), while from the bent cotyledon stage endosperm and embryo expression is dependent on an ABA-responsive element (ABRE) likely to bind ABI5. The shortest promoter fragment (113 bp), devoid of ARE, ABRE and without an intact RY/Sph element thought to bind ABI3 did not drive GUS expression. The AtPER1::GUS construct also revealed expression in cotyledons, meristems and stem branching points. In general, seed and vegetative expression coincided with the expression pattern of ABI3. In plants ectopically expressing ABI3, AtPER1::GUS expression was found in true leaves, and AtPER1 could be induced by exogenous ABA and oxidative stress (H2O2 and hydroquinone). ABI3-mediated oxidative stress induction was dependent on the presence of an intact ARE element.