Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. Conclusion We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies

Proteomic Analyses Reveal High Expression of Decorin and Endoplasmin (HSP90B1) Are Associated with Breast Cancer Metastasis and Decreased Survival

BACKGROUND: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays. METHODS AND FINDINGS: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment. CONCLUSIONS: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. Conclusion We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies.</p

Close or positive resection margins are not associated with an increased risk of chest wall recurrence in women with DCIS treated by mastectomy: a population-based analysis

Abstract Mastectomy is effective treatment for ductal carcinoma in situ (DCIS) but some women will develop chest wall recurrence. Most chest wall recurrences that develop after mastectomy are invasive cancer and are associated with poorer prognosis. Past studies have been unable to identify factors predictive of chest wall recurrence. Therefore, it remains unclear if a subset exists of women with DCIS treated by mastectomy experience a high rate of recurrence in whom more aggressive treatment may be of benefit. We report outcomes of all women in Ontario (N = 1,546) diagnosed with pure DCIS from 1994 to 2003 treated with mastectomy without radiotherapy and evaluate factors associated with the development of chest wall recurrence. Treatments and outcomes were validated by chart review. Proportional differences were compared using Chi square analyses. Survival analyses were used to study the development of chest wall recurrence in relation to patient and tumor characteristics. Median follow-up was 10.1 years. Median age was 57.1 years. 36 patients (2.3%) developed chest wall recurrence. The 10-year actuarial chest wall recurrence-free survival rates and invasive chest wall recurrence-free survival rates were 97.6 and 98.6%, respectively. There was no difference in cumulative 10 year rates of chest wall recurrence by age at diagnosis (50 years = 2.1%; p = 0.19), nuclear grade (high = 3.0%, intermediate = 1.4%, low = 1.0%, unreported = 2.5%; p = 0.41), or among women with close or positive resection margins (positive = 3.0%, 2 mm or less = 1.4%, >2 mm = 1.5%, unreported = 2.8%; p = 0.51). On univariate and multivariable analysis, none of the factors were significantly associated with the development of chest wall recurrence. In this population cohort, individuals treated by mastectomy experienced low rates of chest wall recurrence. We did not identify a subset of patients with a high rate of chest wall recurrence, including those with positive margins

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development