Amy E. McCart Reed

Metastatic progression of breast cancer: insights from 50 years of autopsies

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 pathological aspects of all cases and, additionally, pathological 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 analysed by array‐based comparative genomic hybridization for six cases##. 945 metastatic deposits were identified, with a median of four/patient. The most common organs involved were lung/pleura (80%), bone (74%), liver (71%) and non‐axillary lymph nodes (55%). Major findings included: (a) patients with CNS metastases were more likely to have bone metastases (p < 0.013); (b) younger age was associated with metastasis to the liver (≤ 49 years; p < 0.001) and to gynaecological organs (≤ 49 years; p = 0.001); (c) surgical excision of the primary tumour was associated with metastasis to the liver (p = 0.002); and (d) 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 the 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.

Invasive lobular carcinoma of the breast: morphology, biomarkers and ’omics

Invasive lobular carcinoma of the breast is the most common ‘special’ morphological subtype of breast cancer, comprising up to 15% of all cases. Tumours are generally of a good prognostic phenotype, being low histological grade and low mitotic index, hormone receptor positive and HER2, p53 and basal marker negative, and with a generally good response to endocrine therapy. Despite this, clinicians face countless challenges in the diagnosis and long-term management of patients, as they encounter a tumour that can be difficult to detect through screening, elicits a very invasive nature, a propensity for widespread metastatic colonisation and, consequently, in some studies a worse long-term poor outcome compared with invasive carcinoma of no special type. Here we review the morphological and molecular features that underpin the disparate biological and clinical characteristics of this fascinating tumour type.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under‐utilized, 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 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy‐number analysis and exome‐ and RNA‐sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM 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. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (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. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r2 = 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‐HER3Y1222 or phospho‐HER4Y1162 membrane‐positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer‐BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over‐expressed and activated in BMs, independent of primary site and systemic therapy. Copyright

Expression profiling of archival tumors for long-term health studies

Purpose: More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate. Experimental Design: We present the two largest whole-genome expression studies from FFPE tissues to date, comprising 1,003 colorectal cancer (CRC) and 168 breast cancer samples, combined with a meta-analysis of 14 new and published FFPE microarray datasets. We develop and validate quality control (QC) methods through technical replication, independent samples, comparison to results from fresh-frozen tissue, and recovery of expected associations between gene expression and protein abundance. Results: Archival tissues from large, multicenter studies showed a much wider range of transcriptional data quality relative to smaller or frozen tissue studies and required stringent QC for subsequent analysis. We developed novel methods for such QC of archival tissue expression profiles based on sample dynamic range and per-study median profile. This enabled validated identification of gene signatures of microsatellite instability and additional features of CRC, and improved recovery of associations between gene expression and protein abundance of MLH1, FASN, CDX2, MGMT, and SIRT1 in CRC tumors. Conclusions: These methods for large-scale QC of FFPE expression profiles enable study of the cancer transcriptome in relation to extensive clinicopathological information, tumor molecular biomarkers, and long-term lifestyle and outcome data. Clin Cancer Res; 18(22); 6136–46. ©2012 AACR.

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP-CGH analysis

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.

An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas

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 ILCs and performed a meta‐analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down‐regulation of E‐cadherin, nuclear TWIST, and up‐regulation 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 ILCs. Copyright

Omics approaches in breast cancer research and clinical practice

Our understanding of the natural history of breast cancer has evolved alongside technologies to study its genomic, transcriptomic, proteomic, and metabolomics landscapes. These technologies have helped decipher multiple molecular pathways dysregulated in breast cancer. First-generation ‘omics analyses considered each of these dimensions individually, but it is becoming increasingly clear that more holistic, integrative approaches are required to fully understand complex biological systems. The ‘omics represent an exciting era of discovery in breast cancer research, although important issues need to be addressed to realize the clinical utility of these data through precision cancer care. How can the data be applied to predict response to molecular-targeted therapies? When should treatment decisions be based on tumor genetics rather than histology? And with the sudden explosion of “big data” from large ‘omics consortia and new precision clinical trials, how do we now negotiate evidence-based pathways to clinical translation through this apparent sea of opportunity? The aim of this review is to provide a broad overview of ‘omics technologies used in breast cancer research today, the current state-of-play in terms of applying this new knowledge in the clinic, and the practical and ethical issues that will be central to the public discussion on the future of precision cancer care.

Molecular signatures in breast cancer

The use of molecular signatures to add value to standard clinical and pathological parameters has impacted clinical practice in many cancer types, but perhaps most notably in the breast cancer field. This is, in part, due to the considerable complexity of the disease at the clinical, morphological and molecular levels. The adoption of molecular profiling of DNA, RNA and protein continues to reveal important differences in the intrinsic biology between molecular subtypes and has begun to impact the way patients are managed. Several bioinformatic tools have been developed using DNA or RNA-based signatures to stratify the disease into biologically and/or clinically meaningful subgroups. Here, we review the approaches that have been used to develop gene expression signatures into currently available diagnostic assays (e.g., OncotypeDX® and Mammaprint®), plus we describe the latest work on genome sequencing, the methodologies used in the discovery process of mutational signatures, and the potential of these signatures to impact the clinic.

High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations

BackgroundThe complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior.MethodsWe have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the ‘in-cell’ analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks.ResultsThe ‘in-cell’ analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response.ConclusionsWe demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.