[Show abstract][Hide abstract] ABSTRACT: Periostin (POSTN), a secreted homodimeric protein that binds integrins αvβ3, αvβ5 and α6β4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop 6 mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvβ3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status. This article is protected by copyright. All rights reserved.
Full-text · Article · Dec 2015 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon, which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology - the tumour cells display an invasive growth pattern, with detached, single cells or single-files of cells, and a canonical feature is the loss of E-cadherin expression. These characteristics are indicative of an EMT, or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (Fibronectin, Vimentin, N-cadherin, Smooth Muscle Actin, Osteonectin, Snail, Twist) in 148 ILC and performed a meta-analysis of publically available molecular data from 155 ILC. Three out of 148 (2%) ILC demonstrated an early and coordinated alteration of multiple EMT markers (downregulation of E-cadherin, nuclear TWIST and upregulation of vimentin, osteonectin and smooth muscle actin). However the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILC.
No preview · Article · Oct 2015 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Treatment options for patients with brain metastases (BM) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilised, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BM (from breast, lung, melanoma and oesophageal cancers) using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were: (1) identification of novel candidates with possible roles in brain metastasis development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5; and the DNA repair, ERBB/HER signalling, axon guidance and protein kinase-A signalling pathways. (2) Mutational signature analysis was applied to successfully identify the primary cancer type for two BM with unknown origins. (3) Actionable genomic alterations were identified in 31/36 BM (86%). In one case we retrospectively identified ERBB2-amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (4) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p<0.0001), and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq (with NRG1 (8p12) genomic loss in 63.6% breast cancer-BM), suggesting a microenvironmental source of ligand. In summary, this is the first study to characterise the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BM, and highlight the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BM independent of primary site and systemic therapy.
This article is protected by copyright. All rights reserved.
Full-text · Article · Jul 2015 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Calcium pumps and channels modulate cell proliferation and apoptosis by regulating intracellular calcium (Ca2+). The plasma membrane Ca2+ ATPase isoform, PMCA2, is a calcium efflux mechanism that extrudes Ca2+ from the cytosol into the extracellular space. PMCA2 has a restricted expression, including expression in cochlear hair cells and cerebellar Purkinje cells. PMCA2 expression is increased in mouse mammary glands during lactation where it plays a major role in the excretion of Ca2+ into milk; however, PMCA2 expression has not been assessed in human breast tissue exhibiting lactational changes. Our previous studies have shown that PMCA2 mRNA levels are elevated in some breast cancer cell lines and that pan-PMCA antisense attenuates the proliferation of MCF-7 breast cancer cells. However, the consequences of silencing PMCA2 in breast cancer cells are still not well understood. Our study assessed PMCA2 expression in breast tissue exhibiting lactational change and in human malignant breast tissue samples. The role of PMCA2 in the proliferation of breast cancer cells was also evaluated. Immunohistochemistry using a rabbit anti-PMCA2 antibody showed membranous PMCA2 expression in the luminal epithelium of breast tissue exhibiting lactational change. PMCA2 expression was assessed in human breast tumor samples assembled into tissue microarrays. Nine of 96 breast tumours (9.4%) showed membranous PMCA2 staining. PMCA2 expression did not significantly correlate with the breast cancer pathological markers, estrogen, progesterone or HER2 receptor status. High-content imaging demonstrated that PMCA2 silencing in MDA-MB-231 breast cancer cells is associated with a reduction in cell number and an inhibition of the percentage of S-phase positive cells. The effect of PMCA2 silencing combined with various cytotoxics (cisplatin, doxorubicin or mitomycin C) on cell proliferation was assessed in MDA-MB-231 cells using a kinetic imaging system (IncuCyte). The results showed that PMCA2 silencing promotes the effects of some cytotoxics. These findings indicate that PMCA2 protein expression is elevated during human lactation and in some breast cancers. Inhibitors of PMCA2 may represent a novel therapeutic strategy for some breast cancers.
[Show abstract][Hide abstract] ABSTRACT: Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.
Full-text · Article · Oct 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Background:
Stratification of patients for treatment of ductal carcinoma in situ (DCIS) is suboptimal, with high systemic overtreatment rates.
A training set of 95 tumours from women with pure DCIS were immunostained for proteins involved in cell survival, hypoxia, growth factor and hormone signalling. A generalised linear regression with regularisation and variable selection was applied to a multiple covariate Cox survival analysis with recurrence-free survival 10-fold cross-validation and leave-one-out iterative approach were used to build and test the model that was validated using an independent cohort of 58 patients with pure DCIS. The clinical role of a COX-2-targeting agent was then tested in a proof-of-concept neoadjuvant randomised trial in ER-positive DCIS treated with exemestane 25 mg day−1±celecoxib 800 mg day−1.
The COX-2 expression was an independent prognostic factor for early relapse in the training (HR 37.47 (95% CI: 5.56–252.74) P=0.0001) and independent validation cohort (HR 3.9 (95% CI: 1.8–8.3) P=0.002). There was no significant interaction with other clinicopathological variables. A statistically significant reduction of Ki-67 expression after treatment with exemestane±celecoxib was observed (P<0.02) with greater reduction in the combination arm (P<0.004). Concomitant reduction in COX-2 expression was statistically significant in the exemestane and celecoxib arm (P<0.03) only.
In patients with DCIS, COX-2 may predict recurrence, aiding clinical decision making. A combination of an aromatase inhibitor and celecoxib has significant biological effect and may be integrated into treatment of COX2-positive DCIS at high risk of recurrence.
Full-text · Article · May 2014 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This 'aggressiveness gene list' was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an 'aggressiveness score' as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.
[Show abstract][Hide abstract] ABSTRACT: There remain no clear guidelines for the optimal management of patients with metastatic breast cancer. To better understand its natural history, we undertook a detailed examination of 197 autopsies performed on women who died of breast cancer. We reviewed clinical, treatment, and pathology aspects of all cases and additionally, pathology features and biomarker expression (ER, PgR, HER2, EGFR, p53, Ki67, c-Kit, CK AE1/AE3) were assessed in detail for the primary tumour and matched metastases for 55 of the cases. Genomes of the primary tumour and multiple metastases were analyzed by array-based comparative genomic hybridization for six cases. 945 metastatic deposits were identified with a median of four per patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%), and non-axillary lymph nodes (55%). Major findings included: i) patients with CNS metastases were more likely to have bone metastases (p<0·013); ii) younger age was associated with metastasis to the liver (≤49 years; p<0·001) and to gynaecological organs (≤49 years; p=0·001); iii) surgical excision of the primary tumour was associated with metastasis to the liver (p=0·002) and iv) ER and PgR showed down-regulation during progression in a non-random manner, particularly in lung/pleura (ER: p<0·001), liver, and bone metastases. Genomic analysis revealed DNA copy number variation between the primary tumour and metastases (e.g. amplification of 2q11.2-q12.1 and 10q22.2-q22.3) but little variation between metastases from the same patient. In summary, the association of CNS and bone metastases, liver and gynaecological metastases in young women and the risk of liver metastases following surgery have important implications for management of patients with breast cancer. Clonal heterogeneity of the primary tumour is important in developing metastatic propensity and the change in tumour phenotype during progression/colonization highlights the importance of sampling metastatic disease for optimal treatment strategies.
Full-text · Article · Jan 2014 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Basal-like and triple-negative breast cancers usually display a high level of genomic instability and often carry TP53 mutations. Mutations in EGFR have been reported in about 10 % triple-negative tumours from Chinese women, and there is some evidence that triple-negative and basal-like tumours might carry additional mutations against which targeted therapies are available. We, therefore, sought to determine the frequency of 238 targetable mutations in 19 oncogenes (including EGFR) in a panel of basal-like and triple-negative breast cancers from Caucasian women. We used the OncoCarta panel to screen for 238 mutations across 19 common oncogenes in 107 basal-like and triple-negative breast cancers from Caucasian women. Mutations were then verified using Sanger sequencing or primer extension by iPLEX. We identified and validated 10 mutations across five genes. Most of the mutations were observed in the PIK3CA gene (18/107, 16.8 %), while mutations in KRAS, NRAS, MET and AKT1 were present in only one tumour each (1/107, 0.9 %). Among the missense substitutions in PIK3CA the point mutation resulting in the amino acid change H1047R was the most frequent (8/18, 44 %). All mutations were mutually exclusive, apart from one basal-like breast tumour which harboured mutations in both MET (p.T992I) and PIK3CA (p.H1047R). We did not identify any mutations in the EGFR gene. In conclusion, we found that with the exception of mutations in PIK3CA, these actionable oncogenic mutations on the Oncocarta panel are rare in basal-like and triple-negative breast cancers from Caucasian women. Custom panels, designed to detect mutations identified by exome sequencing of basal-like and triple-negative breast cancers, are, therefore, needed to identify women who might be eligible for targeted treatment.
No preview · Article · Dec 2013 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: The thromobospondins are a family of extracellular glycoproteins that are activated during tissue remodeling processes such as embryogenesis, wound healing and cancer. Thrombospondin-4 (THBS4) is known to have roles in cellular migration, adhesion and attachment, as well as proliferation in different contexts. Data to support a role in cancer biology is increasing, including for gastrointestinal and prostate tumours. Here, using a combination of immunohistochemistry, immunofluorescence and analysis of publicly available genomic and expression data, we present the first study describing the pattern of expression of THBS4 in normal breast and breast cancer. THBS4 was located to the basement membrane of large ducts and vessels in normal breast tissue, but was absent from epithelium and extracellular matrix. There was a significant induction in expression in cancer-associated stroma relative to normal stroma (P = 0.0033), neoplastic epithelium (P < 0.0001) and normal epithelium (P < 0.0001). There was no difference in stromal expression of THBS4 between invasive ductal carcinomas (IDC) and invasive lobular carcinomas (ILC). The THBS4 mRNA levels were variable yet were generally highest in tumours typically rich in stromal content (ILC, ER positive low grade IDC; luminal A and normal-like subtypes). Genomic alterations of the THBS4 gene (somatic mutations and gene copy number) are rare suggesting this dramatic activation in expression is most likely dynamically regulated through the interaction between invading tumour cells and stromal fibroblasts in the local microenvironment. In summary, THBS4 expression in breast cancer-associated extracellular matrix contributes to the activated stromal response exhibited during tumour progression and this may facilitate invasion of tumour cells.
Full-text · Article · Aug 2013 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.
[Show abstract][Hide abstract] ABSTRACT: Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high-level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.Laboratory Investigation advance online publication, 8 April 2013; doi:10.1038/labinvest.2013.54.
No preview · Article · Apr 2013 · Laboratory Investigation
[Show abstract][Hide abstract] ABSTRACT: The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S (<3 mm from IDC), DCIS-S (<3 mm from DCIS) and breast cancer associated-normal stroma (BC-NS; >10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development.
Preview · Article · Jun 2012 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular
subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast
tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism–comparative genomic hybridization
(SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could
not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent
gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous
loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours
(5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical
consequences for treatment and prognosis.
BRCA1 and BRCA2
-Molecular subtypes-Familial breast cancer-Gene expression-Copy number aberrations
No preview · Article · Oct 2010 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin-fixed, paraffin-embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumour banks. Once samples are fixed in formalin, the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study, we have evaluated the ability of the whole genome DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay from Illumina to perform transcriptomic analysis of archived breast tumour tissue in formalin-fixed, paraffin-embedded (FFPE) blocks. We profiled 76 familial breast tumours from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r(2) = 0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumour molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterization of many diseases.
No preview · Article · Aug 2010 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: Breast cancer currently accounts for more than one-quarter of all female cancers and, despite the great progress in treatment observed in the past few years, the need for identification of new gene targets that can be used for diagnosis, prognosis and therapy is evident. A previous study identified the transcription factor NR4A1 as a gene upregulated in primary breast cancer compared with normal tissue by microarray analysis and sequencing technologies. The purpose of the study was to identify the role of NR4A1 in normal mammary epithelial and breast cancer cell biology.
NR4A1 expression in breast tumours was assessed by semiquantitative and real-time PCR using RNA from normal and tumour samples or breast cancer cell lines. Immunohistochemistry on tissue microarrays was performed to check NR4A1 protein expression in breast tumours. MCF-10A and 226L normal mammary epithelial cells as well as the tumour lines PMC42, ZR-75-1 and MDA-MB-231 were transduced with full-length NR4A1, and the ability of NR4A1-overexpressing cells to migrate was tested using scratch wound or transwell migration assays. Proliferation was measured using the MTT and BrdU assays, while apoptosis was determined by the Annexin V assay. The ability of the cells to adhere to extracellular matrix was tested by adhesion assays and integrin cell surface expression was measured by flow cytometry. Activation of the FAK as well as ERK1/2 and PI3K pathways was checked by western blotting.
Breast tissue microarray analysis showed NR4A1 expression in primary tumours, which was reduced in higher grade and metastatic tumours. Ectopic expression of NR4A1 in MCF-10A, 226L, PMC42 and ZR-75-1 cells led to reduced ability of the cells to migrate, while no differences were observed in their proliferation and apoptotic index. NR4A1 expression altered the ability of the MCF-10A cells to adhere to the extracellular matrix and affected cell surface expression of integrins.
NR4A1 acts as an antimigratory factor in two normal mammary epithelial and two breast cancer cell lines tested. It is therefore possible that NR4A1 acts as an antimigratory factor in breast tumours, and further studies should be conducted to understand the mechanisms involved.
Full-text · Article · Jul 2010 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.
We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.
Most brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.
Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.
Full-text · Article · Jul 2010 · Breast cancer research: BCR
[Show abstract][Hide abstract] ABSTRACT: Microglandular adenosis (MGA) is an uncommon, benign breast lesion that is characterized by a proliferation of small uniform. round glands lined by a single layer of epithelial cells around open lumina with haphazard infiltrative growth in fibrous and fatty breast tissue. Although MGA usually has an indolent course, there is morphologic evidence that MGA can be,I precursor for the development of intraductal and invasive ductal carcinoma. To investigate the possibility of such a transition, we studied 17 cases of MGA or atypical MGA some of which had given rise to carcinoma in situ (CIS) and/or invasive ductal carcinoma using the reticulin stain, immunohistochemistry (S-100, p63, Ki-67, and p53), and a molecular approach involving microdissection and high-resolution comparative genomic hybridization and MYC chromogenic in situ hybridization. MGA and carcinomas arising from MGA were typically negative for p63 and positive for S-100 and Ki-67 and occasionally positive for p53. High-resolution comparative genomic hybridization identified recurrent gains and losses in MGA (2q +, 5q - 8q +, and 14q -) and atypical MGA (1q +, 5q - 8q +, 14q - and 15q -). Some examples of MGA and carcinomas arising from MGA harbored few, gross chromosomal abnormalities whereas others had considerable genetic instability with widespread aberrations affecting numerous chromosomal arms. Such widespread genetic changes, together with recurrent loss of 5q and gain of 8q were reminiscent of those reported specifically for basal-like, estrogen receptor-negative, and BRCA1-associated breast tumors. Concordant genetic alterations were identified between MGA, atypical MGA, and higher risk lesions (CIS and invasive ductal carcinoma) and in some cases there was an accumulation of
No preview · Article · Dec 2008 · The American journal of surgical pathology