Lori A. Field

Identification of differentially expressed genes in breast tumors from African American compared with Caucasian women.

Breast tumors from African American women have less favorable pathological characteristics and higher mortality rates than those of Caucasian women. Although socioeconomic status may influence prognosis, biological factors are also likely to contribute to tumor behavior.

Laser Microdissection for Gene Expression Profiling

Microarray-based gene expression profiling is revolutionizing biomedical research by allowing expression profiles of thousands of genes to be interrogated in a single experiment. In cancer research, the use of laser microdissection (LM) to isolate RNA from tissues provides the ability to accurately identify molecular profiles from different cell types that comprise the tumor and its surrounding microenvironment. Because RNA is an unstable molecule, the quality of RNA extracted from tissues can be affected by sample preparation and processing. Thus, special protocols have been developed to isolate research-quality RNA after LM. This chapter provides detailed descriptions of protocols used to generate micro-array data from high-quality frozen breast tissue specimens, as well as challenges associated with formalin-fixed paraffin-embedded specimens.

Abstract B49: Molecular characterization of breast tumor-associated adipose

Background: Long thought to function only as an inert energy storage depot, the role of adipose tissue in breast tumorigenesis has been largely ignored. In light of increasing rates of obesity and use of breast conserving therapy and autologous fat grafting, improved understanding of the role of adipose in tumor etiology is crucial. Methods: Adipose adjacent to and distant from invasive breast tumors (n=20), or adjacent to non-malignant diagnoses (n=20) was laser microdissected from post-menopausal women. Gene expression data were generated using microarrays and data analyzed to identify significant patterns of differential expression between adipose groups, at the individual gene and molecular pathway level. Results: Pathway analysis revealed significant differences in immune response between non-malignant, distant and tumor adjacent adipose, with the highest response in tumor-adjacent and lowest in non-malignant adipose. Adipose from invasive breasts exhibits increased expression in anti-inflammatory genes, such as MARCO and VSIG4, while genes differentially expressed between tumor-adjacent and distant adipose such as SPP1, RRM2 and MMP9, are associated with increased cellular proliferation, invasion, and angiogenesis. Conclusions: Gene expression levels differ in breast adipose, depending on presence of or proximity to tumor cells. Heightened immunotolerance in adipose from invasive breasts provides a microenvironment favorable to tumorigenesis. In addition, tumor-adjacent adipose demonstrates expression of genes associated with tumor growth and progression. Together, these data suggest that adipose is not an inert component of the breast microenvironment but plays an active role in tumorigenesis. Citation Format: William C. Yarina, Lori A. Field, Brenda Deyarmin, Ryan van Laar, Jeffrey A. Hooke, Craig D. Shriver, Rachel E. Ellsworth. Molecular characterization of breast tumor-associated adipose. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B49. doi:10.1158/1538-7445.CHTME14-B49

Abstract A105: Identification of gene expression profiles associated with different types of breast adipose and their relationship to tumorigenesis

Background: Research over the past decade has shown the importance of the stroma in tumorigenesis, however, having long been thought to function only as an inert energy storage depot, the role of adipose tissue in tumor etiology has been largely ignored. Improved understanding of the role of adipose in tumor development and progression is crucial given the increasing rates of obesity and the use of autologous fat transfer in breast reconstruction. Methods: Adipose, adjacent to and distant from invasive breast tumors, was laser microdissected from 20 post-menopausal women, and from 20 post-menopausal women with non-malignant breast disease. Gene expression data were generated using U133 2.0 microarrays. After quality control and visualization steps, the data were analyzed to identify significant patterns of differential expression between adipose classes, at the individual gene and molecular pathway level. A subset of genes were further analyzed by qRT-PCR in out-of-sample adipose specimens. Results: Pathway analysis revealed significant differences in immune response between non-malignant, distant and tumor adjacent adipose. 141 genes were differentially expressed (FDR 2-fold difference) between tumor-adjacent and non-malignant breasts including FCGR2A, FOLR2, LGMN and NLRP3. These four genes were also differentially expressed (FDR 2-fold difference) between distant and non-malignant adipose. Within invasive breasts, no genes were differentially expressed using FDR 3-fold higher levels (P Conclusions: Gene expression levels differ in breast adipose, depending on presence of or proximity to tumor cells. Tumor-adjacent and distant adipose from invasive breasts both exhibit increased expression in genes involved in the M2 anti-inflammatory response, suggesting that the microenvironment in an invasive breast has a decreased immune response compared to the non-malignant microenvironment. Genes expressed at higher levels in tumor-adjacent compared to distant adipose are associated with increased cellular proliferation, invasion, migration, angiogenesis and metastasis, suggesting that tumor-adjacent adipose promotes the growth and progression of the tumor. Together, these data suggest that adipose is not an inert component of the breast microenvironment but plays an active role in tumorigenesis. Citation Format: Lori Field, Brenda Deyarmin, Ryan van Laar, Craig Shriver, Rachel Ellsworth. Identification of gene expression profiles associated with different types of breast adipose and their relationship to tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A105.

Abstract 4266: Identification of gene expression profiles associated with different types of breast adipose and their relationship to tumorigenesis

Background: Research over the past decade has shown the importance of the stroma in tumorigenesis, however, having long been thought to function only as an inert energy storage depot, the role of adipose tissue in tumorigenesis has been largely ignored. Improved understanding of the role of adipose in tumorigenesis is crucial given the increasing rates of obesity and the use of autologous fat transfer in breast reconstruction. Methods: Adipose, adjacent to and distant from invasive breast tumors, was laser microdissected from 20 post-menopausal women, and from 20 post-menopausal women with non-malignant breast disease. Gene expression data were generated using U133 2.0 microarrays. After quality control and visualization steps, the data were analyzed to identify significant patterns of differential expression between adipose classes, at the individual gene and molecular pathway level. Results: Pathway analysis revealed that immune response differs between non-malignant, distant and tumor adjacent adipose; this response is seen as a gradient with the largest response closest to the tumor. Gene expression differed significantly in adipose from invasive compared to non-malignant breasts with FCGR2A, FOLR2, LGMN, MARCO and NLRP3 expressed at significantly higher levels and HLA-DQB1 and HLA-DQA1 at significantly lower levels in adipose from invasive breasts. Within the invasive breasts, MMP9, PLA2G7, RRM2 and SPP1 were expressed at >3-fold higher levels in adjacent compared to distant adipose. Conclusions: Gene expression levels differ in breast adipose, depending on presence of or proximity to tumor cells. Adipose adjacent to the tumor demonstrated the largest immune response; this response may reflect a reaction to surgical insult from the original biopsy; however, response to surgical injury has been associated with increased ability to metastasize. In addition, within breasts with invasive breast cancer, genes involved in cellular proliferation, degradation of the extracellular matrix and angiogenesis were expressed at higher levels in adjacent compared to distant adipose. Together, these data suggest that adipose is not an inert component of the breast microenvironment but plays an active role in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4266. doi:1538-7445.AM2012-4266

Abstract P3-04-07: Comparison of breast tumors with HER2 amplification and polysomy 17

Background: Approximately 20% of invasive breast cancers overexpress the human epidermal growth factor receptor, Her2, and are associated with poor prognosis including decreased relapse-free and overall survival. Overexpression of the Her2 protein is primarily due to amplification of the HER2 oncogene on chromosome 17, and these patients are typically treated with targeted therapies such as trastuzumab or lapatinib. However, increased HER2 copies may also result from chromosome 17 polysomy in which the entire chromosome has been duplicated one or more times. The clinical benefit of treating patients with polysomy 17 in the absence of HER2 amplification with targeted therapy remains unclear. Herein, we compared the clinical characteristics as well as the gene expression profiles of breast tumors with increased HER2 copy numbers due to HER2 amplification or chromosome 17 polysomy. Methods: Patients for this study were enrolled in the Clinical Breast Care Project. HER2 and CEP17 copy numbers were determined using the PathVysion HER-2 DNA Probe Kit (Abbott Laboratories). Only those patients with at least 4 HER2 copies per cell were included in the analysis; those with a HER2/CEP17 ratio 2.2 (N = 44) were considered amplified. Microarray data were generated on HG U133A 2.0 arrays (Affymetrix) for those samples with tissue available (14 polysomy and 18 amplified cases) and analyzed using Partek Genomics Suite. Results: Tumor grade and size did not differ between polysomic and amplified samples. Although amplified cases tended to be younger at diagnosis and less likely to be diagnosed with Stage I tumors, this difference did not reach statistical significance. HER2 amplified tumors were more likely to be ER negative ( P = 0.027) and have lymph node metastases ( P = 0.028) than polysomic tumors. Comparison of polysomic and amplified cases by expression microarray identified 67 genes that were differentially expressed (P 2) between the two groups. Thirty-nine genes were more highly expressed and 28 genes more lowly expressed in tumors with HER2 gene amplification when compared to tumors from patients with chromosome 17 polysomy. Discussion: ER and lymph node status of breast tumors from women with increased HER2 copy number differed depending on whether the increased copy number was due to HER2 gene amplification or conversely, polysomy of chromosome 17. Likewise, gene expression profiles also differed depending on whether increased HER2 was due to gene amplification or chromosomal gain. Those genes with significant levels of differential expression between these two groups are involved in cell cycle control, cell adhesion and migration, cell differentiation, cytoskeleton organization, signal transduction, protein synthesis and degradation, and ion transport. Interestingly, the top 4 most significant genes with higher expression in the polysomic samples, including TUBG1, CPD, BLMH, and JUP, are all located on 17q. Currently, it is not clear whether treatment with targeted Her2 therapy is beneficial in patients with chromosome 17 polysomy. However, the genes identified here may represent novel targets for developing new treatments that are effective in these women and require further study. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-07.

Abstract B31: Molecular signature discriminating triple-negative breast tumors between African American and Caucasian women

Background: Triple negative (TN) tumors are characterized by aggressive behavior and, the negative hormone receptor and HER2 status precludes the use of personalized therapies such as tamoxifen and trastuzumab. African American women have higher rates of both breast cancer mortality as well as TN breast tumors. What remains unknown is how much of these disparities can be attributed to molecular differences between populations. Methods: The Clinical Breast Care Project database was queried to identify African American women with triple negative, high-grade tumors. Caucasian women with highgrade TN tumors were then matched by age at diagnosis, tumor stage, size and lymph node status. RNA was isolated after laser microdissection of the tumor and hybridized to HG U133A 2.0 microarrays. Data was analyzed using Partek Genomics Suite. Results: Principal Component Analysis accurately clustered the triple negative specimens by ethnicity. Using a false-discovery rate (FDR) p-value Discussion: Despite matching of tumors by clinicopathological characteristics, molecular profiles of triple negative breast tumors differed between AAW and CW. These differences may serve as molecular targets for the development of novel prevention strategies and therapeutics to reduce the risk of developing and improve treatment of African American women with triple negative breast cancer. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B31.

Role of phosphoserine phosphatase-like in breast disease in African American women.

Abstract #2095 Background: Although breast cancer incidence is higher among Caucasian women (CW), mortality is higher in African American women (AAW). AAW also tend to be diagnosed at a younger age and with larger and more aggressive tumors. Previously, we have found that gene expression patterns in AAW and CW differ in both breast tissues from women without breast disease as well as those from women with invasive breast cancer. Characterization of these differentially expressed genes is critical to understanding breast pathogenesis in AAW.
 Methods: Total RNA was isolated from breast tissue from 29 AAW and 26 CW without evidence of breast disease using the RNeasy Lipid Tissue Midi kit (Qiagen) and from laser microdissected primary breast tumors from 23 AAW and 24 CW using the RNAqueous Micro kit (Ambion). Based on earlier microarray results, genes were selected for validation by quantitative real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems). All TaqMan assays were performed on duplicate samples using the iCycler Real-Time Detection System (Bio-Rad). Relative expression levels were determined using the Comparative C t Method. A Wilcoxon test was used to determine significance.
 Results: Among those genes that were confirmed to be differentially expressed in breast tissues from AAW and CW was phosphoserine phosphatase-like, or PSPHL. PSPHL showed significantly higher expression in both disease-free samples as well as primary tumors from AAW when compared to the same sample type from CW. In tissues from disease-free women, the relative level of PSPHL expression was significantly higher in AAW (median = 17.15) compared to CW (median = 0; P P Discussion: The current study has confirmed that expression of PSPHL is significantly higher in both disease-free and invasive breast tissues from AAW than those from CW. Recently, PSPHL has also been shown to have higher expression in prostate tumors from African American men than those from Caucasian men. The function of PSPHL is unknown; however, its increased expression has been associated with aberrant cellular proliferation and cell cycle progression in Fanconi anemia. Higher expression of PSPHL in both normal and diseased breast tissues may, therefore, increase cellular proliferation, contributing to the initial development and/or aggressiveness of breast tumors in AAW. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2095.