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Background: More than three-quarters of primary breast cancers are positive for estrogen receptor alpha (ER; encoded by the gene ESR1 ), the most important factor for directing anti-estrogenic endocrine therapy (ET). Recently, mutations in ESR1 were identified as acquired mechanisms of resistance to ET, found in 12% to 55% of metastatic breast cancers treated previously with ET., Methods: We analyzed 3217 population-based invasive primary (nonmetastatic) breast cancers (within the SCAN-B study, ClinicalTrials.gov NCT02306096), sampled from initial diagnosis prior to any treatment, for the presence of ESR1 mutations using RNA sequencing. Mutations were verified by droplet digital polymerase chain reaction on tumor and normal DNA. Patient outcomes were analyzed using Kaplan-Meier estimation and a series of 2-factor Cox regression multivariable analyses., Results: We identified ESR1 resistance mutations in 30 tumors (0.9%), of which 29 were ER positive (1.1%). In ET-treated disease, presence of ESR1 mutation was associated with poor relapse-free survival and overall survival (2-sided log-rank test P < .001 and P = .008, respectively), with hazard ratios of 3.00 (95% confidence interval = 1.56 to 5.88) and 2.51 (95% confidence interval = 1.24 to 5.07), respectively, which remained statistically significant when adjusted for other prognostic factors., Conclusions: These population-based results indicate that ESR1 mutations at diagnosis of primary breast cancer occur in about 1% of women and identify for the first time in the adjuvant setting that such preexisting mutations are associated to eventual resistance to standard hormone therapy. If replicated, tumor ESR1 screening should be considered in ER-positive primary breast cancer, and for patients with mutated disease, ER degraders such as fulvestrant or other therapeutic options may be considered as more appropriate., (© The Author(s) 2021. Published by Oxford University Press.)

Purpose: Mammographic density and tumor appearance are breast cancer prognostic factors. Conceivably, mammographic features are macroscopic reflections of tumor´s molecular composition, but to an unknown extent. Our aim was to study associations of mammographic features with molecular tumor profiles., Methods: Invasive breast cancers (2007-2016) in Malmö Diet and Cancer Study (MDCS) for which there were tumor RNA-sequencing analyses within Sweden Cancerome Analysis Network - Breast (SCAN-B) (n=102) or All Breast Cancer in Malmö (ABIM) (n=50) were identified. Density (fatty vs. dense), tumor appearance (mass vs. spiculation), and intrinsic subtypes were registered. Differences in gene/metagene expression and Microenvironment Cell Population Counter were analyzed with R. Overall survival was used as endpoint., Results: No gene expression differences between density groups was observed. In one cohort (but not the other), Luminal A tumors associated with fatty breasts. For spiculation vs. mass, (p<0.01, t-test) 86 genes were differentially expressed; only one gene was differentially expressed comparing density. Gene set enrichment analysis showed genes highly expressed in spiculated tumors were enriched for extracellular matrix-associated genes whereas genes highly expressed with masses were associated with proliferation. A spiculation metagene, based on differentially expressed genes, showed association with estrogen receptor positivity, lower grade, and improved survival, but it was not an independent prognostic factor., Conclusion: There are clear differences in molecular composition between breast tumors with a spiculated appearance vs. a mass as the dominant tumor appearance. However, there are no apparent molecular differences related to the density of the breast in which the tumor has arisen., (Copyright © 2021. Published by Elsevier Ltd.)

Breast cancer is a disease of genomic alterations, of which the panorama of somatic mutations and how these relate to subtypes and therapy response is incompletely understood. Within SCAN-B (ClinicalTrials.gov: NCT02306096), a prospective study elucidating the transcriptomic profiles for thousands of breast cancers, we developed a RNA-seq pipeline for detection of SNVs/indels and profiled a real-world cohort of 3,217 breast tumors. We describe the mutational landscape of primary breast cancer viewed through the transcriptome of a large population-based cohort and relate it to patient survival. We demonstrate that RNA-seq can be used to call mutations in genes such as PIK3CA, TP53, and ERBB2, as well as the status of molecular pathways and mutational burden, and identify potentially druggable mutations in 86.8% of tumors. To make this rich dataset available for the research community, we developed an open source web application, the SCAN-B MutationExplorer (http://oncogenomics.bmc.lu.se/MutationExplorer). These results add another dimension to the use of RNA-seq as a clinical tool, where both gene expression- and mutation-based biomarkers can be interrogated in real-time within 1 week of tumor sampling., (©2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: More than 70% of patients with breast cancer present with node-negative disease, yet all undergo surgical axillary staging. We aimed to define predictors of nodal metastasis using clinicopathological characteristics (CLINICAL), gene expression data (GEX), and mixed features (MIXED) and to identify patients at low risk of metastasis who might be spared sentinel lymph node biopsy (SLNB). Experimental Design: Breast tumors ( n = 3,023) from the population-based Sweden Cancerome Analysis Network-Breast initiative were profiled by RNA sequencing and linked to clinicopathologic characteristics. Seven machine-learning models present the discriminative ability of N0/N+ in development ( n = 2,278) and independent validation cohorts ( n = 745) stratified as ER + HER2 - , HER2 + , and TNBC. Possible SLNB reduction rates are proposed by applying CLINICAL and MIXED predictors., Results: In the validation cohort, the MIXED predictor showed the highest area under ROC curves to assess nodal metastasis; AUC = 0.72. For the subgroups, the AUCs for MIXED, CLINICAL, and GEX predictors ranged from 0.66 to 0.72, 0.65 to 0.73, and 0.58 to 0.67, respectively. Enriched proliferation metagene and luminal B features were noticed in node-positive ER + HER2 - and HER2 + tumors, while upregulated basal-like features were observed in node-negative TNBC tumors. The SLNB reduction rates in patients with ER + HER2 - tumors were 6% to 7% higher for the MIXED predictor compared with the CLINICAL predictor accepting false negative rates of 5% to 10%., Conclusions: Although CLINICAL and MIXED predictors of nodal metastasis had comparable accuracy, the MIXED predictor identified more node-negative patients. This translational approach holds promise for development of classifiers to reduce the rates of SLNB for patients at low risk of nodal involvement., (©2019 American Association for Cancer Research.)

Purpose: Oestrogen receptor-positive (ER+) and human epidermal receptor 2-negative (HER2-) breast cancers are classified as Luminal A or B based on gene expression, but immunohistochemical markers are used for surrogate subtyping. The aims of this study were to examine the agreement between molecular subtyping (MS) and surrogate subtyping and to identify subgroups consisting mainly of Luminal A or B tumours., Methods: The cohort consisted of 2063 patients diagnosed between 2013-2017, with primary ER+/HER2- breast cancer, analysed by RNA sequencing. Surrogate subtyping was performed according to three algorithms (St. Gallen 2013, Maisonneuve and our proposed Grade-based classification). Agreement (%) and kappa statistics (κ) were used as concordance measures and ROC analysis for luminal distinction. Ki67, progesterone receptor (PR) and histological grade (HG) were further investigated as surrogate markers., Results: The agreement rates between the MS and St. Gallen 2013, Maisonneuve and Grade-based classifications were 62% (κ = 0.30), 66% (κ = 0.35) and 70% (κ = 0.41), respectively. PR did not contribute to distinguishing Luminal A from B tumours (auROC = 0.56). By classifying HG1-2 tumours as Luminal A-like and HG3 as Luminal B-like, agreement with MS was 80% (κ = 0.46). Moreover, by combining HG and Ki67 status, a large subgroup of patients (51% of the cohort) having > 90% Luminal A tumours could be identified., Conclusions: Agreement between MS and surrogate classifications was generally poor. However, a post hoc analysis showed that a combination of HG and Ki67 could identify patients very likely to have Luminal A tumours according to MS.

Background: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction., Results: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures., Conclusions: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

Background: Cancer stem cells (CSCs) have been invoked in resistance, recurrence and metastasis of cancer. Consequently, curative cancer treatments may be contingent on CSC selective approaches. Of particular interest in this respect is the ionophore salinomycin, a natural product shown to be 100-fold more active against CSCs than clinically used paclitaxel. We have previously reported that synthetic salinomycin derivatives display increased activity against breast cancer cell lines. Herein we specifically investigate the CSC selectivity of the most active member in each class of C20-O-acylated analogs as well as a C1-methyl ester analog incapable of charge-neutral metal ion transport., Methods: JIMT-1 breast cancer cells were treated with three C20-O-acylated analogs, the C1-methyl ester of salinomycin, and salinomycin. The effects of treatment on the CSC-related CD44(+)/CD24(-) and the aldehyde dehydrogenase positive (ALDH(+)) populations were determined using flow cytometry. The survival ability of CSCs after treatment was investigated with a colony formation assay under serum free conditions. The effect of the compounds on cell migration was evaluated using wound-healing and Boyden chamber assays. The expression of vimentin, related to mesenchymal traits and expression of E-cadherin and β-catenin, related to the epithelial traits, were investigated using immunofluorescence microscopy., Results: Treatment with each of the three C20-acylated analogs efficiently decreased the putative CSC population as reflected by reduction of the CD44(+)/CD24(-) and ALDH(+) populations already at a 50 nM concentration. In addition, colony forming efficiency and cell migration were reduced, and the expression of the epithelial markers E-cadherin and β-catenin at the cell surface were increased. In contrast, salinomycin used at the same concentration did not significantly influence the CSC population and the C1-methyl ester was inactive even at a 20 μM concentration., Conclusions: Synthetic structural analogs of salinomycin, previously shown to exhibit increased activity against cancer cells, also exhibited improved activity against CSCs across several assays even at nanomolar concentrations where salinomycin was found inactive. The methyl ester analog of salinomycin, incapable of charge-neutral metal ion transport, did not show activity in CSC assays, lending experimental support to ionophoric stress as the molecular initiating event for the CSC effects of salinomycin and related structures.

Aims: Aldehyde dehydrogenase family 1 member A1 (ALDH1A1) is a putative marker of breast cancer stem cells (CSCs) with prognostic implications when expressed in cancer or stroma. However, previous results are contradictory and we therefore aimed to further evaluate the impact of ALDH1A1 on distant disease-free survival (DDFS) and correlation with clinicopathological variables in breast cancer, specifically by evaluating different cut-offs., Methods: Two breast cancer cohorts (N=216 and N=210) were evaluated with immunohistochemistry for ALDH1A1 on tissue microarrays with three different cut-offs in cancer cells and in stromal cells. The association of ALDH1A1 with DDFS and other clinicopathological variables was assessed. As further validation, gene expression levels of ALDH1A1 and association with survival were analysed in one of the cohorts and a separate cohort., Results: ALDH1A1 expression in cancer cells was associated with either a better or a worse prognosis, depending on cut-off. Considering weakly stained cancer cells as positive, ALDH1A1+ was associated with a better prognosis in both cohorts. Considering only strongly stained cells as positive, ALDH1A1+ was associated with oestrogen receptor and progesterone receptor negativity in both cohorts and worse prognosis in one of the cohorts. Stromal ALDH1A1 staining was associated with improved DDFS in one cohort. Gene expression analysis showed that a high ALDH1A1 expression was associated with a better prognosis., Conclusions: ALDH1A1 is associated with DDFS and clinicopathological variables, both in cancer cells and stroma, but is highly cut-off dependent. Only the strongly ALDH1A1-stained cells show a more aggressive phenotype typical for CSCs., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Salinomycin, a naturally occurring polyether ionophore was recently found to selectively reduce the proportion of CD44(+)/CD24(-) cells, a phenotype associated with breast cancer stem cells. Subsequent studies from our group showed that chemical modification of the allylic C20 hydroxyl of salinomycin, located at the C-ring, can enhance the activity of derivatives against breast cancer cells over 5-fold compared to the native structure. Access to C-ring-modified salinomycin analogues is thus of interest from both a mechanistic and a synthetic perspective. Here, we report efficient strategies for gram scale synthesis of the natural product SY-1 (20-deoxy salinomycin), and a saturated analogue, 18,19-dihydro SY-1, for a comparative in vitro investigation of the biological profiles of these compounds with that of salinomycin. Across several assays, the deoxygenated structures required higher concentrations to elicit similar cellular responses to that of salinomycin. Similarly to salinomycin, SY-1 or 18,19-dihydro SY-1 treatment was found to reduce the proportion of CD44(+)/CD24(-) cells with essentially complete selectivity up to ∼IC25. Importantly, the proportion of CD44(+)/CD24(-) cells showed a pronounced U-shaped dose response curve for salinomycin and its derivatives, but not for paclitaxel. The concentration for maximum response in this assay followed differences in IC50 for salinomycin and its analogues, which emphasizes the importance of taking concentration dependence into account when comparing effects on the CD44(+)/CD24(-) phenotype. Small differences in the global conformation within the triad of compounds investigated together with differences in activity across assays emphasize the importance of substitution at C20 for the activity of salinomycin and its derivatives.

Salinomycin has found renewed interest as an agent for prevention of cancer recurrence through selectively targeting cancer stem cells. Strategies for generation of improved salinomycin analogs by individual modification of its hydroxyl groups are presented. An evaluation of the dose-response effects of the resulting library on breast cancer cell lines shows that acylation of the C20 hydroxyl can be used to improve IC50 values down to one fifth that of salinomycin.

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Breast tumors from BRCA1 germ line mutation carriers typically exhibit features of the basal-like molecular subtype. However, the specific genes recurrently mutated as a consequence of BRCA1 dysfunction have not been fully elucidated. In this study, we used gene expression profiling to molecularly subtype 577 breast tumors, including 73 breast tumors from BRCA1/2 mutation carriers. Focusing on the RB1 locus, we analyzed 33 BRCA1-mutated, 36 BRCA2-mutated, and 48 non-BRCA1/2-mutated breast tumors using a custom-designed high-density oligomicroarray covering the RB1 gene. We found a strong association between the basal-like subtype and BRCA1-mutated breast tumors and the luminal B subtype and BRCA2-mutated breast tumors. RB1 was identified as a major target for genomic disruption in tumors arising in BRCA1 mutation carriers and in sporadic tumors with BRCA1 promoter methylation but rarely in other breast cancers. Homozygous deletions, intragenic breaks, or microdeletions were found in 33% of BRCA1-mutant tumors, 36% of BRCA1 promoter-methylated basal-like tumors, 13% of non-BRCA1-deficient basal-like tumors, and 3% of BRCA2-mutated tumors. In conclusion, RB1 was frequently inactivated by gross gene disruption in BRCA1 hereditary breast cancer and BRCA1-methylated sporadic basal-like breast cancer but rarely in BRCA2 hereditary breast cancer and non-BRCA1-deficient sporadic breast cancers. Together, our findings show the existence of genetic heterogeneity within the basal-like breast cancer subtype that is based upon BRCA1 status., (©2012 AACR.)

Amplification of chromosomal region 11q13, containing the cell cycle regulatory gene CCND1, is frequently found in breast cancer and other malignancies. It is associated with the favourable oestrogen receptor (ER)-positive breast tumour phenotype, but also with poor prognosis and treatment failure. 11q13 spans almost 14 Mb and contains more than 200 genes and is affected by various patterns of copy number gains, suggesting complex mechanisms and selective pressure during tumour progression. In this study, we used 32 k tiling BAC array CGH to analyse 94 CCND1-amplified breast tumours from sporadic, hereditary, and familial breast cancers to fine map chromosome 11q13. A set containing 281 CCND1-non-amplified breast tumours was used for comparisons. We used gene expression data to further validate the functional effect of gene amplification. We identified six core regions covering 11q13.1-q14.1 that were amplified in different combinations. The major core contained CCND1, whereas two cores were found proximal of CCND1 and three distal. The majority of the CCND1-amplified tumours were ER-positive and classified as luminal B. Furthermore, we found that CCND1 amplification is associated with a more aggressive phenotype within histological grade 2 tumours and luminal A subtype tumours. Amplification was equally prevalent in familial and sporadic tumours, but strikingly rare in BRCA1- and BRCA2-mutated tumours. We conclude that 11q13 includes many potential target genes in addition to CCND1.

Background: The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes., Methods: We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA., Results: Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival., Conclusions: We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to CSCs and tumor progression should consider the expression of various CD44 isoforms.

Decreased expression of Numb, resulting in activation of the proto-oncogene Notch1 and reduction in the tumor suppressor p53, has been demonstrated in mammary carcinomas. The aim of this study was to investigate the relationship between Numb protein expression and clinicopathological characteristics, tumor biological subtypes and putative cancer stem cell markers in a well-characterized cohort of primary human breast cancers. Immunohistochemistry was performed on tissue microarrays of primary invasive breast tumors using a polyclonal anti-Numb primary antibody. Of the 241 tumors evaluated, 50 (21%) displayed deficient or reduced Numb immunoreactivity. Retained Numb expression was significantly correlated to estrogen (ER) and progesterone receptor (PR) positivity (P < 0.001 and P = 0.004, respectively). Interestingly, we found that a higher percentage of the tumors with deficient or reduced Numb expression belonged to the triple-negative (ER-/PR-/HER2-) subgroup compared to tumors with retained Numb expression (P = 0.004). Transcriptional profiling of a subset of these tumors linked NOTCH1 and BIRC5, both downstream targets of Numb, to the triple-negative subgroup in an inverse manner. Typically, subgroups characterized by the low expression of Numb expressed higher levels of NOTCH1 and BIRC5 (encoding survivin). We also found deficient expression of Numb in a significantly higher proportion of BRCA1 dependent tumors, which are usually triple-negative, compared to sporadic tumors. The expression of Numb in 14 breast cancer cell lines correlated similarly to their respective molecular subtypes. We further established an inverse correlation between the Numb expression levels and the CD44+/CD24- cancer stem cell phenotype (P = 0.05) in primary tumors. Finally, decreased Numb expression was associated with poorer distant disease-free survival (P = 0.01). Taken together, our results indicate that loss of Numb expression is a marker of tumor aggressiveness, potentially linked to BRCA1 status and a cancer stem cell phenotype in primary breast cancer.

PURPOSE Human epidermal growth factor receptor 2 (HER2) gene amplification or protein overexpression (HER2 positivity) defines a clinically challenging subgroup of patients with breast cancer (BC) with variable prognosis and response to therapy. We aimed to investigate the heterogeneous biologic appearance and clinical behavior of HER2-positive tumors using molecular profiling. PATIENTS AND METHODS Hierarchical clustering of gene expression data from 58 HER2-amplified tumors of various stage, histologic grade, and estrogen receptor (ER) status was used to construct a HER2-derived prognostic predictor that was further evaluated in several large independent BC data sets. RESULTS Unsupervised analysis identified three subtypes of HER2-positive tumors with mixed stage, histologic grade, and ER status. One subtype had a significantly worse clinical outcome. A prognostic predictor was created based on differentially expressed genes between the subtype with worse outcome and the other subtypes. The predictor was able to define patient groups with better and worse outcome in HER2-positive BC across multiple independent BC data sets and identify a sizable HER2-positive group with long disease-free survival and low mortality. Significant correlation to prognosis was also observed in basal-like, ER-negative, lymph node-positive, and high-grade tumors, irrespective of HER2 status. The predictor included genes associated with immune response, tumor invasion, and metastasis. CONCLUSION The HER2-derived prognostic predictor provides further insight into the heterogeneous biology of HER2-positive tumors and may become useful for improved selection of patients who need additional treatment with new drugs targeting the HER2 pathway.

Introduction: Breast cancer is a profoundly heterogeneous disease with respect to biologic and clinical behavior. Gene-expression profiling has been used to dissect this complexity and to stratify tumors into intrinsic gene-expression subtypes, associated with distinct biology, patient outcome, and genomic alterations. Additionally, breast tumors occurring in individuals with germline BRCA1 or BRCA2 mutations typically fall into distinct subtypes., Methods: We applied global DNA copy number and gene-expression profiling in 359 breast tumors. All tumors were classified according to intrinsic gene-expression subtypes and included cases from genetically predisposed women. The Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm was used to identify significant DNA copy-number aberrations and genomic subgroups of breast cancer., Results: We identified 31 genomic regions that were highly amplified in > 1% of the 359 breast tumors. Several amplicons were found to co-occur, the 8p12 and 11q13.3 regions being the most frequent combination besides amplicons on the same chromosomal arm. Unsupervised hierarchical clustering with 133 significant GISTIC regions revealed six genomic subtypes, termed 17q12, basal-complex, luminal-simple, luminal-complex, amplifier, and mixed subtypes. Four of them had striking similarity to intrinsic gene-expression subtypes and showed associations to conventional tumor biomarkers and clinical outcome. However, luminal A-classified tumors were distributed in two main genomic subtypes, luminal-simple and luminal-complex, the former group having a better prognosis, whereas the latter group included also luminal B and the majority of BRCA2-mutated tumors. The basal-complex subtype displayed extensive genomic homogeneity and harbored the majority of BRCA1-mutated tumors. The 17q12 subtype comprised mostly HER2-amplified and HER2-enriched subtype tumors and had the worst prognosis. The amplifier and mixed subtypes contained tumors from all gene-expression subtypes, the former being enriched for 8p12-amplified cases, whereas the mixed subtype included many tumors with predominantly DNA copy-number losses and poor prognosis., Conclusions: Global DNA copy-number analysis integrated with gene-expression data can be used to dissect the complexity of breast cancer. This revealed six genomic subtypes with different clinical behavior and a striking concordance to the intrinsic subtypes. These genomic subtypes may prove useful for understanding the mechanisms of tumor development and for prognostic and treatment prediction purposes.

Introduction: Five different molecular subtypes of breast cancer have been identified through gene expression profiling. Each subtype has a characteristic expression pattern suggested to partly depend on cellular origin. We aimed to investigate whether the molecular subtypes also display distinct methylation profiles., Methods: We analysed methylation status of 807 cancer-related genes in 189 fresh frozen primary breast tumours and four normal breast tissue samples using an array-based methylation assay., Results: Unsupervised analysis revealed three groups of breast cancer with characteristic methylation patterns. The three groups were associated with the luminal A, luminal B and basal-like molecular subtypes of breast cancer, respectively, whereas cancers of the HER2-enriched and normal-like subtypes were distributed among the three groups. The methylation frequencies were significantly different between subtypes, with luminal B and basal-like tumours being most and least frequently methylated, respectively. Moreover, targets of the polycomb repressor complex in breast cancer and embryonic stem cells were more methylated in luminal B tumours than in other tumours. BRCA2-mutated tumours had a particularly high degree of methylation. Finally, by utilizing gene expression data, we observed that a large fraction of genes reported as having subtype-specific expression patterns might be regulated through methylation., Conclusions: We have found that breast cancers of the basal-like, luminal A and luminal B molecular subtypes harbour specific methylation profiles. Our results suggest that methylation may play an important role in the development of breast cancers.

Polyamine analogue treatment results in growth inhibition and sometimes in cell death. Therefore, polyamine analogues are considered in the treatment of cancer; however, the cellular properties that govern sensitivity are not known. The objective of this study was to elucidate molecular mechanisms behind apoptosis induced by the polyamine analogue N, N-diethylnorspermine (DENSPM). Four different breast cancer cell lines were treated with DENSPM. Cell death was evaluated with flow cytometry and a caspase 3 assay. The levels of a number of proapoptotic and antiapoptotic proteins in subcellular compartments were evaluated with western blot. In the most sensitive cell line, DENSPM treatment induced the release of cytochrome c from mitochondria, resulting in activation of caspase 3 but without decreasing the mitochondrial transmembrane potential. However, in the three other cell lines DENSPM treatment did not induce extensive cell death. This is partly explained by the high levels of antiapoptotic proteins Bcl-2 and Bad and low levels of proapoptotic proteins Bax and procaspase 3 in these three cell lines. The results are also partly explained by the degree of activation of the catabolic enzyme spermidine/spermine-N-acetyltransferase and polyamine pool reduction achieved by DENSPM treatment. Our results show that the protein profile of proapoptotic and antiapoptotic proteins may contribute to the outcome to treatment with the polyamine analogue DENSPM. The results also indicate that it should be possible to find molecular markers for sensitivity to DENSPM that could be used in the clinic to predict sensitivity to a polyamine analogue.

Introduction: Human breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44+/CD24- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes., Methods: Double-staining immunohistochemistry was used to quantify CD44 and CD24 expression in 240 human breast tumors for which information on other tumor markers and clinical characteristics was available. Gene expression data were also accessible for a cohort of the material., Results: A considerable heterogeneity in CD44 and CD24 expression was seen both between and within tumors. A complete lack of both proteins was evident in 35% of the tumors, while 13% contained cells of more than one of the CD44+/CD24-, CD44-/CD24+ and CD44+/CD24+ phenotypes. CD44+/CD24- cells were detected in 31% of the tumors, ranging in proportion from only a few to close to 100% of tumor cells. The CD44+/CD24- phenotype was most common in the basal-like subgroup--characterized as negative for the estrogen and progesterone receptors as well as for HER2, and as positive for cytokeratin 5/14 and/or epidermal growth factor receptor, and particularly common in BRCA1 hereditary tumors, of which 94% contained CD44+/CD24- cells. The CD44+/CD24- phenotype was surprisingly scarce in HER2+ tumors, which had a predominantly CD24+ status. A CD44+/CD24- gene expression signature was generated, which included CD44 and alpha6-integrin (CD49f) among the top-ranked overexpressed genes., Conclusion: We demonstrate an association between basal-like and particularly BRCA1 hereditary breast cancer and the presence of CD44+/CD24- cells. Not all basal-like tumors and very few HER2+ tumors, however, contain CD44+/CD24- cells, emphasizing that a putative tumorigenic ability may not be confined to cells of this phenotype and that other breast cancer stem cell markers remain to be identified.

Purpose: Endocrine therapies, such as tamoxifen, are commonly given to most patients with estrogen receptor (ERalpha)-positive breast carcinoma but are not indicated for persons with ERalpha-negative cancer. The factors responsible for response to tamoxifen in 5% to 10% of patients with ERalpha-negative tumors are not clear. The aim of the present study was to elucidate the biology and prognostic role of the second ER, ERbeta, in patients treated with adjuvant tamoxifen., Experimental Design: We investigated ERbeta by immunohistochemistry in 353 stage II primary breast tumors from patients treated with 2 years adjuvant tamoxifen, and generated gene expression profiles for a representative subset of 88 tumors., Results: ERbeta was associated with increased survival (distant disease-free survival, P = 0.01; overall survival, P = 0.22), and in particular within ERalpha-negative patients (P = 0.003; P = 0.04), but not in the ERalpha-positive subgroup (P = 0.49; P = 0.88). Lack of ERbeta conferred early relapse (hazard ratio, 14; 95% confidence interval, 1.8-106; P = 0.01) within the ERalpha-negative subgroup even after adjustment for other markers. ERalpha was an independent marker only within the ERbeta-negative tumors (hazard ratio, 0.44; 95% confidence interval, 0.21-0.89; P = 0.02). An ERbeta gene expression profile was identified and was markedly different from the ERalpha signature., Conclusion: Expression of ERbeta is an independent marker for favorable prognosis after adjuvant tamoxifen treatment in ERalpha-negative breast cancer patients and involves a gene expression program distinct from ERalpha. These results may be highly clinically significant, because in the United States alone, approximately 10,000 women are diagnosed annually with ERalpha-negative/ERbeta-positive breast carcinoma and may benefit from adjuvant tamoxifen.

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the cellular pools of putrescine, spermidine and spermine by down-regulating the activity of the polyamine biosynthetic enzymes and up-regulating the activity of the catabolic enzyme spermidine/ spermine N(1)-acetyltransferase (SSAT). In the breast cancer cell line L56Br-C1, treatment with 10 microm DENSPM induced SSAT activity 60 and 240-fold at 24 and 48 h after seeding, respectively, which resulted in polyamine depletion. Cell proliferation appeared to be totally inhibited and within 48 h of treatment, there was an extensive apoptotic response. Fifty percent of the cells were found in the sub-G(1) region, as determined by flow cytometry, and the presence of apoptotic nuclei was morphologically assessed by fluorescence microscopy. Caspase-3 and caspase-9 activities were significantly elevated 24 h after seeding. At 48 h after seeding, caspase-3 and caspase-9 activities were further elevated and at this time point a significant activation of caspase-8 was also found. The DENSPM-induced cell death was dependent on the activation of the caspases as it was inhibited by the general caspase inhibitor Z-Val-Ala-Asp fluoromethyl ketone. The results are discussed in the light of the L56Br-C1 cells containing mutated BRCA1 and p53, two genes involved in DNA repair.