RNA Isolation & DASL AssayRNA was isolated from FFPE tissues, either three 5 µm sections or one core ~ 5 mm in length. Tissues were deparaffinized, extracted and purified using the commercially available RNA High Pure Kit (Roche, Mannheim, Germany) modified as previously described (Abramovitz & Kodani et al., 2008). RNA concentration and Å260/Å280 ratio were determined using the NanoDrop spectrophotometer (NanoDrop, Wilmington, DE). Quality was assessed using commercially available TaqMan RPL13a assays from 20 ng of RNA on a HT7900 real-time PCR instrument (Applied Biosystems, Foster City, CA) with a quality threshold of less than 29.5 CT required for inclusion. Samples with sufficient quantity (>0.4 μg) and quality (Ct < 29.5) were subjected to the DASL assay, which is essentially a multiplexed quantitative RT-PCR (real-time polymerase chain reaction) and hybridized to sentrix array matrices – an 8x12 plate microarray – according to the manufacturer’s protocol (Illumina, San Diego, CA). When ample RNA was available RNA replicates were run to test for reproducibility of mRNA expression from FFPE tissues.Data Quality & Normalization DASL mRNA signal intensities were interpreted in BeadStudio and quantile-normalized signal intensities were exported for meta-analysis. Samples with a signal-to-background ratio less than 3 were determined unusable and excluded from further analyses. After data QC and normalization a discrepancy in average signal intensity was apparent between the 4 sentrix array matrices utilized for this experiment. To compensate for this plate-to-plate variation sample signals were linearly scaled according to the average plate signal intensity so that the average signal across all 4 plates is the same. After normalization replicate correlations averaged r2 = 0.97.

Molecular differences in triple negative breast cancer between race/ethnicitiesMark M Bouzyk1, Benjamin G Barwick1, Mark Abramovitz2, Maja Kodani1, Gabriela Oprea1,3, Weining Tang1, Carlos S Moreno1, and Brian Leyland-Jones1,2

1Winship Cancer Institute, Emory University, Atlanta, GA, United States 2VM Institute of Research, Montreal, Canada 3Grady Memorial Hospital, Atlanta, GA, United States

Background: A disparity in prognosis of triple negative (TN) breast cancer (BC) has been observed between African American (AA) and Caucasian (CAU) race/ethnicities afflicted with this aggressive and invasive BC subtype. Etiological understanding of these differences involves accounting for several factors associated with phenotype and genotype. Here, we address the former using the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) assay to quantify mRNA expression of 512 breast cancer related genes in a cohort of 24 CAU and 56 AA TN BC tissues sourced from formalin-fixed, paraffin-embedded (FFPE) blocks.

Material and Methods: The DASL assay was used to measure mRNA expression levels from FFPE sourced tissues in both cohorts of self-identified patients. CAU BC patients were obtained from St. Mary’s Hospital, Montreal, Quebec and AA BC patients were obtained from Grady Hospital, Atlanta, Georgia. RNA extraction used the RNA High Pure Kit (Roche) and was taken from archival FFPE tissues either 5µm tissue sections or cores. Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR) less than 1% and a two fold-change criteria to determine differential regulation.

Results: In all, 33 genes were found differentially expressed between AA and CAU TN BC tumor samples, 32 of which were upregulated in the AA cohort, only 1 of which was upregulated in the CAU group. The upregulated gene in CAU TN BC was TFF1. Upregulated genes in the AA cohort (order of statistical significance according to SAM software) were KIF20A, EP300, AURKB, FGF4, C14orf155, USP22, EPOR, ZNF668, SCNN1G, MAPT, FLNB, EP400, LTA, ACOT11, RBP3, CSF3, E2F2, TGFB1, CCNE1, L1CAM, NDP, VWF, RHOB, FEN1, BIN1, KRT17, CDC42EP4, SERPINF1, CHI3L2, NES, BCL2, and RERG.

Discussion: TFF1 upregulated in the CAU population, has been indicated as biomarker of favorable prognosis in endocrine therapy in clinical studies which is consistent with race/ethnicity disparities. The remaining genes upregulated in the AA cohort include transcription factors E2F2 and RBP3/E2F1 both with cyclin binding domains which may interact with CCNE1, extracellular and adhesion related genes KRT17, L1CAM and FGF4, genes associated with cell cycle AURKB, EP400, and EP300 (activator of HIF-1A). Several RAS related genes were also found differentially expressed in the AA cohort including RHOB, RERG, BIN1, and EPOR. Moreover, it is worth mentioning that BCL2 which is expressed in the aggressive mammary cancer cell line MCF-7 was also found upregulated in the AA cohort. These initial findings suggest that several differentially regulated genes between AA and CAU race/ethnicities may account for the disparity in outcomes resultant in these populations. These initial data warrant further investigation which is currently ongoing.

Abstract

Introduction

1. Lund MJ, Trivers KF, Porter PL, Coates RJ, Leyland-Jones B, Brawley OW, Flagg EW, O'Regan RM, Gabram SG, Eley JW. Race and triple negative threats to breast cancer survival: a population-based study in Atlanta, GA. Breast Cancer Res Treat 2008 Mar 7

2. Bibikova M, Yeakley JM, Wang-Rodriguez J, and Fan JB. Quantitative Expression Profiling of RNA from Formalin-Fixed, Paraffin-Embedded Tissues Using Randomly Assembled Bead Arrays. Methods Mol Biol. 2008;439:159-77.

3. Abramovitz, M., M. Ordanic-Kodani, Y. Wang, Z. Li, C. Catzavelos, M. Bouzyk, G. W. Sledge Jr., C. S. Moreno, and B. Leyland-Jones. Biotechniques:44:417-423, 2008.

4. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5116-21.

5. Ukomadu C, Dutta A. p21-dependent inhibition of colon cancer cell growth by mevastatin is independent of inhibition of G1 cyclin-dependent kinases. J Biol Chem. 2003 Oct 31;278(44):43586-94.

6. Trock BJ. Breast cancer in African American women: epidemiology and tumor biology. Breast Cancer Res Treat. 1996;40(1):11-24.

Background182,460 women are estimated to be diagnosed with breast cancer (BC) in 2008, which will result in over 40,000 mortalities from disease, and account for 26% of new cancer cases among women in the United States. While BC incidence is higher in Caucasian (CAU) women, the mortality rate is substantially higher in African America (AA) women (33.5 vs. 24.4 per 100,000). A major component of this health disparity is triple negative (TN) disease, a particularly aggressive and invasive BC with little to no targeted therapies composing 15-25% of breast cancers and defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This disease differentially affects AA women for which a disparity in outcomes has been shown to exist (Lund et al). TN breast cancer patients experience the worst prognoses as compared to the HER2 and hormone (ER or PR) positive breast cancer subtypes. Compounding the tragedy of this disease is the predisposition for it to afflict younger and pre-menopausal women. Study Aim & ExpansionTo investigate potential molecular and etiological differences between ethnicities in TN breast cancers we have compared mRNA expression levels of 87 African Americans and 26 Caucasians expanded from the original cohort of 56 AAs and 24 CAUs with TN breast cancers sourced from formalin-fixed, paraffin-embedded (FFPE) tissues. This expanded cohort yielded expanded yet highly overlapping results to those identified in the original abstract. More robust normalization yielded a higher replicate correlation (r2 = 0.97) and more balanced results in terms of up and down regulated genes. Criteria for inclusion was relaxed to a differential fold change of 1.5 but still imposed a false discovery rate (FDR) of less than 1%. This yielded an expanded set of 113 genes differentially regulated between AA and CAU cohorts.

Methods Results

ReferencesFigure 1. A, Average raw signal intensity and standard error. B, Signal distribution by plate. C, Quantile normalised and plate scaled signal intensity by plate. D, Normalised signal distributions have slow less plate-to-plate variance.

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Data AnalysisDifferential mRNA expression was determined using significance analysis of microarrays (SAM) (Tusher et al., 2001) for probe and gene-level data, for which we imposed a false discovery rate (FDR) of less than 1%. Heatmaps were generated using Gene Cluster 3.0, which clustered patients and genes using a complete linkage method (Eisen et al., 1998), heatmaps were generated with Java Treeview (Saldanha, 2004). Pathway analysis used Ingenuity Pathway Analysis (IPA), Gene Set Enrichment Analysis (GSEA), and KEGG.

breast cancer patients (89 TN). Discordant records were precluded from further analysis. The final analysis included 113 TN patients composed of 87 African Americans and 26 Caucasians.

Figure 3. Contrasting race/ethnicities yielded 138 differentially regulated probes across 113 genes with a fold change > 1.5 and a FDR < 1%. Patients are clustered across the top with blue indicating African American (AA) and purple representing Caucasian (CAU).

Figure 4. Fold change of mRNA transcripts superimposed over canonical cell cycle signaling

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Figure 2. TN population cohort size

Immunohistochemical (IHC) staining for ER, PR, and HER2 was obtained from the respective coordinating pathology office as was race which was self-identified. Surveillance, epidemiology and end results (SEER) data was also obtained for samples acquired from Grady Memorial Hospital. Race recorded in the SEER registry was compared with race identified from the pathology records and was concordant in 132 of the 134

Results Discussion

Cell Cycle SignalingAA v. CAU

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Expression & Pathway AnalysisPathway analysis tools GSEA and IPA yielded a similar theme in results, specifically indicating at least three interrelated molecular mechanisms: 1) DNA damage and repair, 2) BRCA1 signaling in DNA damage response, and 3) the subsequent impact on cell cycle regulation. The major components of cell cycle signaling showed a divergence in regulation between race/ethnicities. We observed significant upregulation of BRCA1 signaling in the AA cohort. This signaling was apparent in down stream targets E2F transcription factors needed to initiate the G1/S phase transition, SWI/SNF which is involved in chromatin remodeling and polo-like kinase PLK1 a mitotic kinase highly expressed in G2/M cell cycle transition. Retinoblastoma 1 (RB1) a negative regulator of cell cycle progression was downregulated in AA patients. In the opposing p53 signaling arm of cell cycle regulation we observed subtle downregulation in the AA cohort. This included attenuation of the CDK inhibitor p21/CDKN1A, the growth arrest and DNA damage-inducible gene GADD45A, proliferating cell nuclear antigen PCNA, and increased expression of BCL-XL, a proliferation gene negatively associated with p53 signaling. Counterintuitively we observed abrogated expression of cyclin D1 as well as the cyclin dependent kinase CDK4. However, cyclin D2 was upregulated (1.6 fold change) in AA cohort and acts as a surrogate in the absence of cyclin D1. Cyclin E1 was also upregulated (1.5 fold change) which activates CDK2 to drive G1/S transition in a p21/CDKN1A independent manner (Ukomadu et al). Finally, we also observed upregulation of CDC25B a phosphatase which facilitates cell cycle progression by activating cyclin dependent kinases, including CDK1 allowing cyclin B1 – upregulated in the AA cohort – to bind and activate components needed for G2/M transition including the mitotic kinase PLK1. Taken together, these data suggest upregulation of cell cycle components through BRCA1 and cyclin signaling in the AA cohort.

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Major health inequities exist between AA and CAU race/ethnicities emphasized by an unadjusted mortality rate almost 40% higher in African Americans. A major component of this disparity is TN disease for which a lack of therapeutic targets is driving urgent need for increased pathological understanding and subsequent clinical translation of treatment modalities. Admittedly, there are confounding factors in this study, specifically that the majority of TN CAU were sourced from one hospital while the majority of AA from another, introducing influences such as diet and standard of healthcare. However, it is encouraging that dysregulation was indicated in BRCA1 / p53 biology as previous studies have found p53 mutational spectra to differ among race (Trock). Increased expression down the BRCA1 signaling cascade contrasted with slight attenuation of p53 arm –canonically activated by BRCA – suggests a dysfunctional BRCA / p53 interaction. Thus, we have highlighted a well studied pathway, that shows differential expression between CAU and AA populations and we further hypothesize that ablation of cyclin and E2F transcription factors will be an effective modality for treating TN disease in African Americans.