Tumors In African American Women Research Articles

Abstract 3682: Quadruple negative breast tumors in African American women express factors associated with worse prognosis compared to triple negative tumors

Abstract Introduction: Quadruple negative breast cancer (QNBC) is defined by the absence of androgen (AR), estrogen (ER), progesterone (PR) receptors and human epidermal growth factor (HER2) expression. This subset of triple negative breast cancer (TNBC) has been described as unique to African American (AA) women and represents about 67-90% of all TNBC. Yet, there are limited clinical and therapeutic studies focused on this new subset of breast cancer (BCa) that is highly aggressive and portends a worse survival compared to AR positive Bca subtypes. The objectives of our study were to 1) determine the molecular and clinical characteristics of QNBC and compare them with other BCa subtypes and 2) determine the association of QNBC with clinicopathological factors and prognostic markers. Materials and Methods: Tissue arrays were constructed from FFPE tumor blocks of invasive ductal breast carcinomas in 202 AAs, diagnosed at Howard University from 2000 to 2010. Two separate tissue cores of IDC represented each surgical case in the TMA and was linked to an Institutional Review Board-approved database of demographic and clinical data. Using a microtome, 5-µm sections were cut from the TMA blocks and mounted onto Superfrost Plus microscope slides. Sections were stained with a mouse monoclonal antibody against AR (Clone AR 441, DAKO). Immunohistochemistry for AR was considered positive if ≥10% of tumor cells showed nuclear staining. Bivariate analysis was performed via χ2 analysis and survival data was calculated via Kaplan-Meier curves (SPSS v28.0.0). Results: The most prevalent breast cancer subtype was luminal A (ER+ or PR+, HER2−; 43.5%), followed by TNBC (33.7%). Of the 67 TNBCs on the array, 63 had AR data; 33 (52%) of the TNBCs were AR negative. Greater than 70% of all tumors were stage I and II; and Grade 3 comprised 67.4% of the tumors. QNBC tumors were more likely to be higher grade (p<0.001) and larger in size (p=0.05) compared to other subtypes. There was also a trend for increased metastases in QNBCs (Luminal and HER2 tumors=11.2%; TNBC=13.3%; AR negative= 18.8%; and QNBC=20.8% (p=0.15)). Compared to TNBCs, QNBCs were larger (p=0.048), more likely to express markers associated invasion (Fascin, p=0.004) and reduced survival (Vimentin, p=0.014). There was a trend for increased expression of markers associated with metastases: Mammaglobin (expressed in 30.8% of TNBCs compared to 52.1% of QNBCs; p=0.17) and CD105 (expressed in 6.7% of TNBCs compared to 27.1% of QNBCs; p=0.097). There was no association between QNBC and stage, recurrence-free survival, or overall survival. Conclusion: In our study of AAs, a statistically significant association was noted between QNBC tumors and poorer prognosis. The high prevalence of AR negativity of TNBCs in AAs could explain observed worse outcomes and supports the existence of a unique subtype of Bca known as QNBC, worthy of scientific attention. Citation Format: Oluwadamilola Oladeru, Yasmine Kanaan, Tammy Naab, Luisel J. Ricks-Santi. Quadruple negative breast tumors in African American women express factors associated with worse prognosis compared to triple negative tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3682.

Abstract P1-02-12: Genomic testing on breast cancer core biopsy material is feasible and provides useful treatment information

Abstract Background: Genomic profiling assays for invasive breast cancer provide useful predictive and prognostic information and are performed most commonly on surgical resection specimens. Obtaining the molecular profile at the time of initial core needle biopsy is ideal because it could provide information that could significantly alter preoperative decision-making and avoid unnecessary treatments. In January 2020, the breast center began a 3 month pilot program that would reflexively send core biopsy material on all newly diagnosed patients for genomic testing using the MammaPrint (MPT) and BluePrint (BPT) assays regardless of receptor status. The goals were to determine feasibility and examine the impact to patient care. Methods: Breast core biopsy tissue was triaged by an attending pathologist as soon as slides were available. If invasive carcinoma with at least 3 mm in linear extent of tumor with 20% cellularity was identified, materials were immediately sent to Agendia for MPT/BPT testing even before ER/PR/HER2 testing was available to the pathologist. Clinicopathologic information, turn-around time (TAT) and adequacy data were tracked. The impact was discussed regularly at breast tumor board to determine if the results led to altered decision-making that could reduce unnecessary interventions and time to initial treatment. Results: 445 core biopsy specimens were sent for genomic testing. Of those 442, (97%) yielded genomic results with an average TAT from biopsy to genomic result of 11 calendar days. MPT identified 233 (53%) cases that were low risk and 209 (47%) cases that were high risk. BPT showed that 60 (14%) cases were Basal, 18 (4%) cases were HER2, and 364 (82%) cases were Luminal. Further analysis of the Luminal subgroup demonstrated that 154 (42%) were Luminal A low risk, 78 (21%) were Luminal A Ultralow risk and 132 (36%) were Luminal B High Risk. Analysis by race demonstrated a significantly higher percentage of high risk tumors in African-American women including a higher percentage of basal cancers (26%) as compared to Caucasian women (10%). Of note, 51 patients in the pilot study had additional OncotypeDX (ODX) testing on subsequent surgical resection. There was fair correlation between the assays with the majority of the low risk MPT having low risk ODX scores (<=25) and the high risk MPT having high risk ODX scores (>25), though MPT identified more patients as being high risk. The COVID-19 pandemic altered plans to assess time to treat and treatment interventions as initially intended. However, the knowledge of genomic result enhanced the ability to triage patients, allowing those patients with low risk tumors to begin endocrine therapy and delay surgery. Use of preoperative hormone therapy was considered more often in place of neoadjuvant chemotherapy in patients with low genomic risk ER+/HER2- patients. Because of the continued feedback indicating positive impact, the pilot ultimately was extended to 11 months to allow time for formal implementation. Conclusions: Genomic testing using the MPT/BPT assay on core biopsy samples with at least 3 mm yields results 97% of the time with an average TAT of 11 days from biopsy date to result. The genomic information at the time of initial diagnosis impacted patient care most notably in the ER+ setting. The results led to the immediate implementation of direct reflex testing of all newly diagnosed ER+ and HER2- or IHC 2+ cancers by pathology after the initial pilot phase. Citation Format: Hannah Gilmore, Philip Bomeisl, Alberto Montero. Genomic testing on breast cancer core biopsy material is feasible and provides useful treatment information [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-12.

Molecular characterization of luminal breast tumors in African American women.

550 Background: Racial disparities in breast cancer (BC) mortality are attributed to later stage diagnoses and a higher incidence of triple-negative BC among African American (AA) women. In previous work, we showed that AA women with ER+ BC are more likely to develop biologically aggressive disease and are more likely to die from early stage, ER+ BC than non-Hispanic White women (Hoskins et al, JAMA Oncol, 2021). The underlying molecular drivers of this disparity are unknown. Here we report the molecular characterization of a series of luminal BC from AA women. Methods: Consecutive breast tumor specimens received in the Pathology Department underwent next generation sequencing (NGS). Unstained FFPE tissue sections were macro-dissected to isolate tumor cells, and nucleic acids were extracted using commercially available kits. DNA and RNA sequencing libraries were prepared with the Oncomine Comprehensive Assay v3 (OCAv3) (Thermo Fisher), which includes 161 driver genes and detects SNVs, CNVs, INDELs and gene fusions. Sequencing was performed on the Ion S5XL sequencer. Sequencing reads were mapped to the UCSC human genome build GRCh37/hg19 using Torrent Suite™ software (version 5.10; Thermo Fisher). Data analysis and variant calling was performed using the Ion Reporter analysis tool. Results: We identified 60 somatic driver gene alterations in luminal tumors from 35 AA patients (primary tumors, n = 26; metastatic tumors, n = 9). Recurrently altered genes identified in > 5% of tumors are listed in the Table. The most frequently altered gene was PIK3CA (42% of tumors). ESR1 gene fusions were seen in 25% of tumors. Interestingly, an equal frequency of ESR1 fusions were detected in primary (27%) and metastatic (22%) tumors, in contrast to activating mutations which are found in recurrent tumors following treatment with aromatase inhibitors. ARID1A alterations were identified in 17% of primary tumors. ARID1A encodes a subunit of the SWI/SNF chromatin remodeling complex. Alterations in ARID1A confer endocrine resistance, and are enriched in recurrent tumors in the literature. We also found a high number of CNVs in members of the FGF gene family (36% of tumors), which are also associated with resistance to endocrine therapy. An in silico analysis comparing our findings with publicly available datasets will be presented. Conclusions: This study of somatic driver gene alterations in a consecutive series of luminal breast tumors from AA patients found a higher than expected frequency of alterations in genes associated with endocrine resistance in untreated primary tumors, suggesting a partial explanation for racial disparities in survival.[Table: see text]

Outcome of African-American compared to White-American patients with early-stage breast cancer, stratified by phenotype.

Breast cancer mortality rates are 39% higher in the African-American (AA) women compared to White-American (WA) women despite the advances in overall breast cancer screening and treatments. Several studies have undertaken to identify the factors leading to this disparity in United States with possible effects of lower socioeconomic status and underlying aggressive biology. A retrospective analysis was done using a prospectively maintained database of a metropolitan health system. Patients were selected based on diagnosis of early-stage breast cancer between 10/1998 and 02/2017, and included women over age of 18 with clinically node-negative disease. Patients were then stratified by phenotype confirmed by pathology and patient-identified race. A total of 2,298 women were identified in the cohort with 39% AA and 61% WA women. The overall mean age at the time of diagnosis for AA women was slightly younger at 60years compared to 62years for WA women (p=0.003). Follow-up time was longer for the WA women at 95months vs. 86months in AA women. The overall 5-year survival was analyzed for the entire cohort, with the lowest survival occurring in patients with triple-negative breast cancer (TNBC). Phenotype distribution revealed a higher incidence of TNBC in AA women compared to WA women (AA 16% vs. WA 10%; p<0.0001). AA women also had higher incidence of HER2 positive cancers (AA 16.8% vs. WA 15.3%; p<0.0001). WA women had a significantly higher distribution of Non-TNBC/HER2-negative phenotype (AA 55% vs. WA 65%; p<0.0001). Furthermore, a subgroup analysis was done for a sentinel lymph node (SLN) negative cohort that showed higher rates of grade 3 tumors in AA (AA 35% vs. WA 23%; p<0.0001); and higher rates of grade 1 and grade 2 tumors in WA (30% vs. 21% and 44% vs. 40%). Despite higher grade tumors in AA women, five-year overall survival outcomes in SLN-negative cohort did not differ between AA and WA women when stratifying based on tumor subtype. Breast cancer survival disparities in AA and WA women with SLN-negative breast cancer are diminished when evaluated at early-stage cancers defined by SLN-negative tumors. Our evaluation suggests that when diagnosed early, phenotype does not contribute to racial survival outcomes. The lower survival rate in AA women with breast cancer may be attributed to later stage biology between the two races, or underlying socioeconomic disparities.

Abstract P1-03-11: Harnessing the distinctive properties of tumor-adjacent tissues to develop ethnicity-dependent biomarkers of breast cancer

Abstract Recent data demonstrating a correlation between lymph node positivity at the time of detection, and the probability of disease recurrence even decades post detection only solidifies the principle that the detection of breast cancer prior to lymph node metastasis can appreciably better clinical outcomes. Although radiologic methods have greatly improved early detection and remain the mainstay for detection, molecular assays to complement existing strategies will reduce number of false positives as well as enhance detection in cases that preclude conclusive diagnosis with radiologic techniques. Normal breast biology is routinely studied using tissues from reduction mammoplasty or normal tissues adjacent to tumor (NATs). However, studies have shown histologic abnormalities in reduction mammoplasty samples and DNA methylation and gene expression abnormalities in NATs due to “field” effects of the tumor. To interrogate the differences between normal breast and NATs as potential early detection markers, we created a tissue microarray (TMA) comprising breast tissues of 100 age-matched healthy women from the Komen Tissue Bank (KTB) and tumor-NAT pairs from 100 women (a total of 300 samples). Approximately 50% of women in each set were of African American (AA) ancestry and the remaining was of European decent. The TMAs was curated as such, because of our recent findings on ethnicity-dependent differences in breast stem-luminal progenitor-mature cell hierarchy. TMA was analyzed for ZEB1, an oncogenic transcription factor that is central to cell fate and stemness, and estrogen receptor alpha (ERα) and FOXA1, which are expressed predominantly in hormone-responsive mature luminal cells. ZEB1 expressing cells were localized to surrounding ductal structures of the normal breast, whereas ERα+ and FOXA1+ cells were located within the ductal compartment. KTB-normal of AA women contained significantly higher levels of ZEB1+ cells compared to KTB-normal of Caucasian women (CA). We observed only marginal increases in ZEB1+ cells in NATs or tumors of AA women. By contrast, in CA women, both NATs and tumors compared to KTB-normal contained higher levels of ZEB1+ cells. The unique localization pattern external to the ductal structures, as well as intrinsically higher expression in AA women suggest that ZEB1+ cells serve not only as stem cells from which cancers may originate but could also contribute to the microenvironment conducive for ductal tumor progression leading to aggressive and early onset of breast cancer as observed in AA women. Conversely, KTB-normal of AA showed modestly higher FOXA1 expression compared to CA women, and further, FOXA1 levels were declined in NATs of AA but not CA women. ERα levels did not change in any of our analyses, pointing to the specificity of ethnicity-dependent changes in this TMA. We also noted ethnicity-dependent variations in the levels of CD8+ T cells, PD-1+ immune cells and PD-L1+ cells but not CD68+ macrophages in NATs, suggesting distinctive immune environment in NATs. This comprehensive approach will not only serve as a platform to develop tumor-adjacent “normal” tissues as molecular markers for early detection but also provides a molecular basis for aggressive breast tumor in AA women. Citation Format: Nakshatri H, Kumar B, Burney H, Cox ML, Jacobson M, Sandusky G, D'Souza-Schorey C, Storniolo AM. Harnessing the distinctive properties of tumor-adjacent tissues to develop ethnicity-dependent biomarkers of breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-11.

Abstract B41: BP1 is an important biomarker in breast cancer

Abstract Beta protein 1 (BP1), a transcription factor (TF) we identified and cloned, is encoded by a homeobox gene called DLX4. BP1 is overexpressed in breast cancer, prostate cancer, ovarian cancer, acute myeloid leukemia, non-small cell lung cancer, and possibly other malignancies as well. Important characteristics of BP1 in breast cancer, a focus in our laboratory, include findings that: (1) BP1 is expressed in 80% of invasive ductal breast tumors, including 89% of the tumors of African American women. These data are based on both RNA and protein data. (2) BP1 expression correlates with the progression of breast tumors, from 0% in normal breast tissue to 21% in hyperplasia and 46% in ductal carcinoma in situ. (3) BP1, which maps to 17q21, can be activated by DNA amplification. (4) BP1 appears to be associated with metastasis. Forty-six cases of inflammatory breast cancer were examined; all were positive for BP1 expression. Nine cases had metastasized; lymph nodes from all nine were also BP1 positive. Moreover, BP1 activates a known trigger of metastasis, the Twist gene. (5) BP1 overexpression induces oncogene expression. BP1 activates the BCL-2 gene; high BCL-2 protein levels are associated with resistance to drug and radiation therapy. BP1 also activates VEGF and c-MYC, as well as other genes important in angiogenesis, invasion, and metastasis. (6) Cells overexpressing BP1, when injected into the fat pads of nude mice, were associated with larger and more frequent tumors than found in control mice receiving cells with low BP1. In summary, BP1 appears to confer properties on breast cancer cells that lead to a more invasive and aggressive phenotype. Since the functions of homeotic transcription factors are highly conserved, it is possible that BP1 regulates many of the same processes and genes in other malignancies in which it is active. Citation Format: Patricia E. Berg, Yan-gao Man, Samuels Simmens, Sidney W. Fu, Lucianne Cavalli, Farhad Vesuna, Saurabh Kirolikar, Arnold Schwartz. BP1 is an important biomarker in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B41.

Abstract C63: Elucidating the role of AIB1 in triple-negative breast cancer cell lines

Abstract Triple negative breast cancer (TNBC) patients do not benefit from directed therapy because these tumors do not express targetable molecules; such as, hormone or growth factor receptors (ER, PR and HER2). This subtype represents 15-20% of all breast cancer cases diagnosed in women in the United States. Higher incidence of TNBC affects, particularly, young African American women compared with other women. Clinically, TNBC patients often present an advanced stage disease. These tumors exhibit an increased inflammatory signal and an aggressive phenotype that leads to an increased frequency of metastasis. These patients have a poor survival outcome. Coincidentally, expression of the amplified in breast cancer 1 (AIB1) oncogene has also been reported in higher grade, inflammatory and invasive breast cancer. AIB1 is a nuclear coactivator that regulates transcription of several genes through hormone and/or growth factor signaling. Here we demonstrate that cell lines derived from tumors in African American women representing different TNBC subtypes have varying degree of sensitive to AIB1 shRNA by lentiviral infection. AIB1 knockdown has a considerable effect on viability of all the TNBC subtypes; however, we observed the presence of a small fraction of cells that proliferate independent of AIB1. Interestingly, these cells that retain the AIB1 knockdown following sub-culture for 3-4 passages are comparable to the initial TNBC cells as regards to cell cycle and apoptosis. Intriguingly, these TNBC cells that proliferate independent of AIB1 show increase in the percentage of cells expressing CD44+/CD24- cell surface markers indicating a selection of tumor initiating cells. Concomitantly, there is a decrease in the percentage of CD44-/CD24- cells suggesting that AIB1 may regulate tumor initiating cell differentiation upon external stresses such as those caused by chemotherapeutic agents during treatment. We will further study the chemo sensitivity of TNBC cells resistant to AIB1 loss and assess whether these cells represent a model of cancer cell dormancy associated to recurrence in TNBC patients. Citation Format: Francisco R. Saenz, Anna T. Riegel. Elucidating the role of AIB1 in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C63.

Abstract B25: Triple-negative breast tumors exhibit distinctive patterns of expression and subcellular localization of Insulin-like Growth Factor Binding Protein 6

Abstract African-American women of all ages have higher breast cancer mortality rates than Caucasian women, despite lower breast cancer incidence among African-American women over 40. While differences in socioeconomic status and access to healthcare may play a role in this disparity, the disproportionately high occurrence of aggressive, triple-negative breast tumors in African-American women suggests a biological link between ancestry and breast cancer prognosis. Interestingly, both African ancestry and breast tumor aggressiveness are associated with distinct transcript and protein expression levels of Insulin-like growth factor-II (IGF-II) and its binding partner, Insulin-like growth factor binding protein 6 (IGFBP6), and we have previously observed higher levels of IGFBP6 transcript and protein expression in African-American and Yoruba (African) HapMap cell lines when compared to Amish cell lines. To gain a better understanding of potential mechanisms underlying these associations, we have investigated the expression and subcellular localization of these proteins and their transcripts using qPCR and immunofluorescence (IF). Our qPCR data indicate lower IGFBP6 transcript expression in breast cancer cells relative to normal cells, with lower transcript levels in the ER positive breast cancer cells than in triple negative cells. Conversely, our IF data revealed higher levels of IGFBP6 protein in ER positive breast cancer cells than in triple negative breast cancer cells, suggesting that breast tumor subtypes may be associated with unique patterns of transcript and protein regulation in these genes. We also observed a distinctive pattern of IGFBP6 subcellular localization in the triple-negative cells in which aggregates of this protein are localized at the edge of the nucleus, and co-localized with IGF-II. We hypothesize that IGF-II will be more dispersed throughout the nucleus following si-RNA knockdown of IGFBP6. Taken together, these data suggest the possibility of a subtype-specific IGF-II/IGFBP6 complex in triple negative breast cancer. Further studies will assess how this complex may promote tumor aggression in triple-negative breast tumors by inhibiting IGFBP6's IGF-independent, pro-apoptotic qualities within the nucleus and/or prolonging the half-life of IGF-II, thus promoting cancer cell growth. Citation Format: DeJuana Ford, Rupali Hire, Elizabeth Howerth, Briana Bennett, Krissean Lea, Jillian Hood, Melissa Davis. Triple-negative breast tumors exhibit distinctive patterns of expression and subcellular localization of Insulin-like Growth Factor Binding Protein 6. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B25.

Phyllodes tumors in African American women

BackgroundPhyllodes is a rare tumor accounting for less than 1% of all breast neoplasms. Studies defining clinical predictors of malignant phyllodes (MP) are rare and inconsistent. Furthermore, MP occurrence in African American (AA) women has never been analyzed. This study will delineate clinical and pathologic features in AA patients that may reasonably predict the probability of malignancy. MethodsA retrospective study of clinical records was carried out for 50 AA patients diagnosed with phyllodes tumors (PT) and treated between 1982 and 2012. Patients' charts were analyzed regarding demographics, pathology findings, and treatment. ResultsThe diagnosis of benign disease was made in 40 (78%), borderline in 3 (6%), and malignancy in 7 (14%) patients; however, 1 patient (2%) had mixed phyllodes with ductal carcinoma in situ. The mean age was significantly different for patients with benign disease (33 years) compared with those with malignancy (54 years; P < .001). The average tumor size was twice as large (11.8 vs 4.1 cm; P = .029) and mitoses were higher with 50% of MPs having greater than 5 per 10 high power fields. Although rare, nodal metastasis, ulceration, and multicentric disease occurred only in MP. ConclusionsAmong AA patients with phyllodes tumors, those with malignant tumors were older and had larger tumors and higher mitotic indices than those with benign disease. AA patients also displayed some of the more rare features of advanced disease and presented with malignancy near the highest reported frequency.

Abstract P2-05-22: Preferential Activation of BP1 and c-Myc in Breast Cancer of African American Women Compared with Caucasian American Women

Abstract Introduction: BP1 is a member of the homeobox gene family of transcription factors. BP1 is activated in 80% of breast tumors, including 89% of the tumors of African American women (AAW) compared with 57% of the tumors of Caucasian American women (CAW). AAW with breast cancer have larger tumors and an almost 50% higher mortality rate than CAW. BP1 expression is also associated with larger and more aggressive tumors, suggesting BP1 may contribute to the aggressiveness of tumors of AAW. Our goal is to identify molecular pathways underlying the discrepancy of breast cancer aggressiveness in AAW and CAW with particular attention to BP1 regulated pathways. Materials and Methods: Gene expression analysis using RNA from cell lines derived from tumors of AAW or CAW was performed using Illumina microarrays. Changes in c-Myc mRNA were measured by real-time PCR and levels of c-Myc protein by immunoblotting. Knock down of c-Myc and BP1 expression was performed using their respective siRNA. Chromatin immunoprecipation (ChIP) analysis was used to determine potential BP1 and c-Myc binding sites in vivo. Immunohistochemistry was performed using a commercial rabbit polyclonal antibody to c-Myc and a rabbit polyclonal antibody to BP1 developed by the laboratory of one of the authors (PB). Results: To study differential gene expression related to racial disparities and BP1 expression, we used breast cancer cell lines derived from tumors of AAW and cell lines derived from tumors of CAW. Microarray analysis was performed on three cell lines from AAW and two cell lines from CAW. The oncogene c-Myc was overly represented in the cell lines derived from AAW compared to their CAW derived counterparts; these cell lines also overexpress BP1. The data was verified by real-time PCR and Western blot analysis. Knock down of BP1 and c-Myc in separate experiments using siRNA showed a significant decrease of BP1 protein and c-Myc protein, respectively. ChIP analysis revealed binding of BP1 protein to DNA upstream of the c-Myc gene and of BP1 DNA by c-Myc protein, suggesting reciprocal activation. Clinical studies. Age and stage matched cases of ductal breast cancers from AAW (n = 15) and CAW (n = 15) were immunostained and evaluated for nuclear c-Myc and nuclear BP1. In CAW, there was a linear correlation between BP1 and c-Myc staining. However, in AAW there was not a linear correlation due to extensive variability in individual results. Conclusions: c-Myc is a potent oncogene known to increase transformation and proliferation. We show here that c-Myc is upregulated by BP1, a gene which is preferentially activated in breast tumors of AAW. Furthermore, we have identified an interesting mechanism by which BP1 and c-Myc may co-activate transcription involving a positive feedback loop, an attractive therapeutic target. The proposed mechanism could partially explain the aggressiveness of tumors of AAW. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-22.

Abstract A58: Role of insulin-like growth factor binding protein acid labile complex in ER-PR- Her2+ breast cancer in African American women.

Abstract The emergence of the obesity epidemic worldwide has been associated with increases in breast cancer and type 2 diabetes. Symptoms associated with obesity collectively known as metabolic syndrome have been associated with triple negative breast tumors that are common among premenopausal women, especially those of African American (AA) descent. The insulin-leptin adiponectin axis has been implicated in these breast tumors that are insensitive to estrogen. It is suggested that insulin, insulin like growth factors and epidermal growth factors mediate the interactions between these hormones. Previous studies have shown that insulin like growth factor 2 (IGF-2) synergistically cross-talks with estrogen receptor (ER) alpha and ER-beta to promote estrogen independent breast cancer progression. This suggests that IGF-2 can activate insulin like growth factor I (IGF-1) receptor and insulin receptor to activate both ER alpha and ER beta in breast cancer cells leading to proliferation and tumorigenesis. The purpose of this study was to find the the role of insulin-like growth factor binding protein acid labile complex (IGFBP-ALS) identified in our previous serum proteomic pilot study that included six AA breast cancer women and six healthy AA controls. Methods: The serum samples were processed on IgY12 (Beckman) antibody column to remove the top 12 proteins. The flow through fraction was digested using trypsin for LC/MS analysis. The protein digest serum samples were analyzed in triplicate on a high performance LTQFT (Thermo Electron) mass spectrometer coupled to an online Surveyor LC system equipped with an autosampler. Samples were loaded onto a trap column using a sample pump. An MS pump was used for eluting the peptides onto an analytical column for further separation and online nano-ESI LC/MS/MS analysis. The raw MS data files were imported into DeCyderMS (GE Healthcare) module and data were processed for peptide/protein expression analysis. Results: DeCyderMS analysis revealed significant differences in the expression of 215 peptides between the two groups (t-test: &amp;lt; 0.05; ave fold ratio: &amp;lt;0.5 or &amp;gt;2). Most of these peptides were mapped to 28 proteins in the Swiss-Prot database. DecyderMS analyses on all the replicates from each sample are underway to identify the peptide pattern differences between the healthy and cancer samples. Among other differentially expressed proteins IGFBP-ALS was significantly low in sera from African American women with breast cancer with tumors possessing the ER-PR-Her2+receptor status. In a previous study, 60% of the women from the same cohort experienced metabolic syndrome. Conclusions: It is suggested that IGFBP-ALS is necessary for the binding of IGF-1 or IGF-2 to the plasma IGF protein BP-53. Low levels of IGFBP-ALS subunit in the sera of ER-PR-HER2+ AA cancer patients could serve as biomarker for early-stage, breast cancer with aggressive features. Additionally this protein could reveal an association between obesity and breast cancer. Efforts are currently underway to validate this biomarker to gain insight into the etiology of aggressive breast tumors in AA women. Funded by The Susan G. Komen For the Cure Breast Cancer Foundation. Citation Format: Padma Uppala. Role of insulin-like growth factor binding protein acid labile complex in ER-PR- Her2+ breast cancer in African American women. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A58.

Abstract B32: The importance of BP1 and EMT in breast cancer of African American women

Abstract Introduction: Our laboratory has discovered a protein, BP1, which is a transcription factor of the homeobox gene family activated in 89% of the tumors of African American women (AAW) compared with 57% of the tumors of Caucasian American women (CW). High level BP1 expression has been shown to be associated with aggressive and larger tumors. Tumors of AAW are generally more aggressive – higher grade, larger, more frequently estrogen receptor (ER) negative, and more proliferative – than those of CW, resulting in an almost 50% higher mortality rate. Preliminary data suggest that BP1 is involved in the epithelial to mesenchymal transition (EMT) which is associated with the transdifferentiation of malignant epithelial cells to mesenchymal cells; BP1 up-regulates the Twist gene, an inducer of EMT. We hypothesize that EMT may be more frequent in tumors of AAW than CW, and that an important mechanism of EMT is via activation of BP1. Materials and Methods: Cell lines engineered to overexpress BP1 were compared with controls with respect to EMT characteristics using Western blot analysis, confocal microscopy, and migration assays. Breast cancer cell lines derived from the tumors of AAW and CW were characterized with respect to BP1 and EMT markers using Western blots. Selected AA or CW cell lines are being analyzed using microarrays to attempt to identify pathways that may differ between them. Several cell lines will be treated with siBP1 to reduce BP1 or used to overexpress BP1, followed by microarray analysis to identify EMT-related and other pathways dysregulated by BP1 in AAW or CW. Tumor tissues from six AAW and from six CW, matched for age and stage of breast cancer, were immunostained to determine differential expression of BP1 and/or EMT markers, including E-cadherin (ECA), vimentin (VIM), and smooth muscle actin (SMA). Results: (a) EMT. MCF7 cells overexpressing BP1 exhibit increased Twist and decreased E-cadherin (an epithelial marker), while Hs578T cells overexpressing BP1 show increased Twist, as well as vimentin and fibronectin (mesenchymal markers). Overexpression of BP1 also leads to increased migration of Hs578T cells. (b) Cell line analysis. We have analyzed five AAW cell lines and five CW cell lines; interestingly, in both types of cells, two were epithelial, one was mesenchymal, and two were not classifiable. Microarray analysis is underway. MDA-MB 157 and HCC70 cells, derived from tumors of AAW, and Hs578T, MCF7, and MDA-MB-231cells, derived from tumors of CW, were identified as candidates to perform siBP1 knock-down and microarray analysis. Upon siBP1 treatment of MDA-MB-231cells, vimentin expression decreased, further support for regulation of EMT by BP1. (c) Clinical Studies. The nuclear membrane area was stained most intensely for BP1, nucleus &amp;gt; cellular membrane &amp;gt; cytoplasm. Nuclear and cytoplasmic staining for BP1 tended to be less for CW than AAW and membrane ECD was less in CW than AAW. VIM and SMA were not informative. Additional cases are being studied. Conclusions: Our data support the hypothesis that BP1 overexpression upregulates EMT in breast cancer cells by directly stimulating Twist expression. If EMT is more prevalent in tumors of AAW than CW, genes expressed during the transition would make good targets for therapy, with the possibility of reducing or preventing metastases. If BP1 is a regulator of EMT in patients, there would be a strong rationale for targeting BP1 using small molecule inhibitors, siBP1 or other approaches. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B32.

Abstract P3-13-04: The Role of PSPHL in Breast Cancer in African American Women

Abstract Background: Younger age at diagnosis and poorer prognosis in African American women (AAW) with breast cancer have led to a number of studies evaluating gene expression differences in tumors from AAW compared to those from Caucasian women (CW). Studies have found that PSPHL expression is significantly higher not only in breast but also in prostate and endometrial tumors from African Americans compared to Caucasians. SNP rs6700 in the 3’ UTR of PSPHL has been found to correlate with expression. Here, we investigated how rs6700 affects PSPHL expression levels in AAW and CW with and without breast cancer. Methods: Genomic DNA was isolated from 74 women with (35 AAW and 39 CW) and 37 women without breast cancer (23 AAW and 14 CW). rs6700 was genotyped using TaqMan SNP Genotyping assay C__314922_10. PSPHL expression levels in breast tissue were measured in these same women using TaqMan Gene Expression assay Hs00863464_m1 using RNA isolated from laser microdissected breast tumor cells or normal breast tissue from women with or without breast cancer, respectively. Results: PSPHL expression in breast tumors from AAW was significantly higher than in CW (P = 6 x 10-6). The median fold change in expression in AAW was 42.2; while only 2 (6%) AAW had no detectable expression of PSPHL, 21/39 (54%) CW did not express PSPHL. The minor allele frequency (T) in AAW with breast cancer was 0.41 compared to 0.06 in CW with breast cancer. In both AAW and CW, median levels of PSPHL expression were lowest (15.45 in AAW, 0 in CW) in women with the C/Cgenotype. In addition, all patients whose tumors had no detectable PSPHL expression had the C/C genotype. In women without breast cancer, the C/C genotype was also correlated with lower PSPHL expression and was more frequent among CW compared to AAW (79% and 39%, respectively). Conclusions: PSPHL is more highly expressed in both diseased and normal breast tissue from AAW compared to CW and expression of PSPHL correlated with rs6700 genotypes. Although the function of PSPHL is unknown, it may function in cellular proliferation. Higher expression in AAW may, therefore, promote the development of tumors in AAW. It must be noted, however, that the frequency of the C allele is significantly lower in the African American population as a whole (53% vs. 92%); therefore, differences described here may reflect population differences rather than any specific contribution to breast cancer etiology. However, due to the higher expression of PSPHL in African Americans with other hormone-dependent cancers, its role in tumorigenesis requires further study. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-13-04.

Abstract P4-06-15: BP1 Upregulates Twist and Induces EMT in Breast Cancer Cells

Abstract Background: We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with estrogen receptor negativity and tumors of African American women, both associated with aggressive tumors. BP1 is also expressed in metastatic tumors, shown by immunostaining of 46 samples of inflammatory breast cancer; all cases were BP1 positive. Nine cases had metastasized, and all nine metastatic lymph nodes were BP1 positive. BP1 positive cells were observed in lymph channels and blood vessels. Here, we demonstrated BP1 induces the epithelial to mesenchymal transition (EMT), resulting in increased migratory ability. EMT, a process by which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells become more invasive, migratory and can lead to metastasis. Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT, is activated by BP1. Materials and Methods: RNA levels of markers of EMT were verified by real-time PCR and their protein levels by Western blotting and, in some cases, by confocal microscopy. Chromatin immunoprecipitation (ChIP) was performed to verify the BP1 binding site on the Twist promoter. A scratch test was used to measure migratory ability. BP1 knockdown by siRNA transfection was also performed. Recombinant BP1 (rpBP1) was produced in E. coli. Results: Increased expression levels of Twist were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. We demonstrated that BP1 can upregulate Twist expression in two different breast cancer cell lines, MCF-7 and Hs578T cells, by binding to the Twist promoter. BP1 upregulates mesenchymal marker expression and down-regulates epithelial marker expression, consistent with EMT. BP1 also promotes breast cancer cell migratory ability, shown by the scratch test. In addition, cells grown in medium supplemented with rpBP1 showed increased Twist expression and migration. Experiments evaluating the effects of siBP1 on EMT are underway. Conclusions: BP1 stimulates Twist expression in MCF-7 and HS578T breast cancer cells, resulting in a more mesenchymal cell phenotype. We therefore hypothesize that BP1 induces EMT by upregulating Twist expression, and may result in metastasis. If BP1 is involved in EMT, it may be a good target for therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-15.

Abstract 2298: BP1 upregulates Twist, a trigger of EMT and cancer metastasis

Abstract BP1 Upregulates Twist, a Trigger of EMT and Cancer Metastasis Bin-Jin Hwang, Jinguen Rheey, and Patricia E. Berg George Washington University, Department of Biochemistry and Molecular Biology, Washington, D.C. Background. We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Increasing evidence suggests that homeobox genes are important in malignant transformation and tumorigenesis, and our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with breast cancer aggressiveness: 100% of estrogen receptor negative tumors and 89% of the tumors of African American women are BP1 positive. Now we have evidence that BP1 may regulate the epithelial to mesenchymal transition (EMT). EMT, a process in which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells to become more invasive and migratory, and can lead to metastasis. BP1 upregulates Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT. Our objective is to determine whether BP1 plays a role in induction of EMT. Materials and Methods. The expression levels of Twist, E-cadherin and vimentin were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. RNA levels were verified by real-time PCR and protein levels by Western blotting and, in some cases, by confocal microscopy. Summary. Twist mRNA expression on microarrays is higher in MCF-7 cells overexpressing BP1 compared with cells containing an empty vector. Real-time PCR confirmed that Twist mRNA is increased five-fold in cells with high levels of BP1, and Western blots demonstrated that Twist protein is also elevated in the same cells. We have discovered a potential DNA binding site for pBP1 in the Twist promoter, suggesting that BP1 may directly regulate Twist. This will be verified by ChIP. Expression of two genes regulated by Twist was also examined: Twist activates vimentin and represses E-cadherin. Cells overexpressing pBP1 also show increased vimentin and decreased E-cadherin. The effect of increased pBP1 on cell migration is under investigation. Conclusions: BP1 stimulates Twist expression in MCF-7 breast cancer cells, resulting in increased vimentin and decreased E-cadherin levels. We therefore hypothesize that BP1 is upstream of Twist and induces EMT. If BP1 is required for EMT, it may be a good target for therapy of metastatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2298.

“your darkness also/rich and beyond fear”: Community Performance, Somatic Poetics and the Vessels of Self and Other

“your darkness also/rich and beyond fear”: Community Performance, Somatic Poetics and the Vessels of Self and Other

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

Abstract 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.&amp;#x2028; 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 Ct Method. A Wilcoxon test was used to determine significance.&amp;#x2028; 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&amp;lt;0.000001). Similarly, primary tumors from AAW also had higher levels of PSPHL expression (median = 42.22) compared to those from CW (median = 0; P&amp;lt;0.0005).&amp;#x2028; 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.

Molecular differences between the triple negative tumors of African-American women and white women.

Abstract Abstract #2087 Purpose. We recently reported associations between triple negative tumors (TNTs) and several key cell-cycle proteins. TNTs have also been associated with: 1) younger age and African-American (AA) race, 2) a relative lack of therapeutic targets, and 3) higher recurrence and poorer survival relative to other tumor subtypes. Furthermore, AA women diagnosed with TNTs experience worse outcomes than white women. TNTs are a mixed group of tumors that includes both basal and non-basal types. Markers that could help differentiate tumors with the poorest outcome would be of great use in determing how to effectively treat women with TNT. This study examines whether there are racial differences in key cell-cycle and apoptotic proteins among women with TNTs and could help to identify potential molecular mechanisms underlying racial disparities in these aggressive tumors.&amp;#x2028; Methods. Invasive TNTs from a population-based cohort of 136 Atlanta women (56 AA and 79 white) aged 20–54, diagnosed between 1990 and 1992, were centrally reviewed and immuno-histochemically analyzed for ER, PR, HER2, Bcl2, Cyclins D1 and E, pRb, p16, p21, p27, p53, p130, Ki67, and apoptotic index (AI). Weighted frequency distributions, Chi-square tests, and logistic regression were used to assess racial differences. Age and stage-adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated.&amp;#x2028; Results. Among the 136 women diagnosed with TNTs, AA women were significantly more likely than white women to present at a later stage, with larger tumors, and increased grade, mitotic activity, tumor necrosis, and apoptotic index. AA women were also significantly younger, poorer, and obese. Cell cycle protein differences between AA and white women diagnosed with TNTs included higher expression of Cyclin E [OR = 4.0 (95% CI=1.9, 8.5)], pRB [OR = 2.8 (95% CI=1.6, 4.9)], p16 [OR = 2.2 (95% CI=1.3,3.8)], p21 [OR = 2.0 (95% CI=1.1, 3.5)], AI [OR = 2.1 (95% CI=1.3, 3.9)], and lower expression of p130 [OR = 0.6 (95% CI=0.3, 1.0)].&amp;#x2028; Conclusions. Exclusively among triple negative tumors, we identified racial differences in expression levels of several key cell-cycle and apoptotic related proteins. Combined with other poor prognostic factors, these differences may account for racial disparities in outcome. Future etiologic studies and clinical trials dedicated to TNTs should consider underlying molecular differences. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2087.

Abstract 555: Gene expression differences in primary breast tumors from African American and Caucasian women.

Abstract Background: Incidence of breast cancer varies across ethnic groups. Overall incidence is highest among Caucasian women (CW); however, incidence in women younger than 40 as well as mortality is highest among African American women (AAW). Although socioeconomic disparities are often blamed for these differences, AAW are more likely than CW to present with large, aggressive tumors with poor prognostic features such as estrogen receptor negativity, triple negative phenotype, and lymph node involvement, suggesting that molecular factors may also contribute to the younger age of onset and worse outcomes in AAW. In the present study, we have compared primary breast tumors from AAW and CW by gene expression profiling to identify differentially expressed genes that may explain why diagnosis at a young age, aggressive tumor biology and lower survival are more prevalent in AAW than CW with breast cancer. Materials and Methods: Breast tumor specimens were obtained from 26 pairs of age and stage-matched AAW and CW enrolled in the Clinical Breast Care Project (CBCP). Tumor cells were isolated by laser microdissection (Leica) and RNA isolated using the RNAqueous Micro kit (Ambion). RNA was amplified and labeled with biotin-11-UTP using two rounds of amplification with the MessageAmp II aRNA Amplification kit (Ambion). Fragmented aRNA was hybridized to the HG U133A 2.0 array (Affymetrix). A Mann-Whitney U test was used to identify differentially expressed genes (p ≤ 0.001). Results: Sixty-five genes were differentially expressed between AAW and CW. Twenty-eight genes were more highly expressed in AAW, including SOS1, TSPO, and PSPH, while 37genes had lower expression in AAW, including ZNF228, SCAMP4, and PSD3. Discussion: These results support a model in which molecular factors influence breast cancer onset, progression and prognosis in AAW. Genes with higher expression in tumors from AAW function in steroid, fatty acid, and serine biosynthesis, signal transduction, cell proliferation and adhesion, and cytoskeleton organization and biogenesis. Functions of genes with lower expression in AAW tumors include regulation of transcription, vesicular transport, iron metabolism, signal transduction, and extracellular matrix organization and biogenesis. These results suggest that breast tumors from AAW and CW differ at the gene expression level and that these differences may be responsible for the more aggressive clinical features of breast tumors in AAW compared to CW.

African-American/White differences in breast carcinoma: p53 alterations and other tumor characteristics.

Despite mounting evidence that breast tumors in African-American (AA) women are more aggressive compared with breast tumors in white (W) women, little is known regarding racial/ethnic differences in genetic alterations that may be of prognostic importance. In this population-based cohort of 322 AA women (45%) and W women (55%) who were diagnosed with breast carcinoma between 1987-1989, the authors evaluated available archived tumor tissue (n = 247 samples) for racial differences in selected genetic alterations and other prognostic indicators. Tumor characteristics were assessed by immunohistochemistry and/or expert review. Alterations in p53 were significantly more common in AA women compared with W women (odds ratio, 4.00; 95% confidence interval, 1.77-9.01) and remained statistically significant in models that were adjusted for disease stage at diagnosis, according to American Joint Committee on Cancer (AJCC) criteria, and for other prognostic indicators. No racial difference with regard to HER-2/neu status was observed, but alterations in c-met were more common in AA women once the model was adjusted for negative confounders (not significant). Among other tumor characteristics, significant findings included later AJCC stage and higher histologic and nuclear grade tumors in AA women. In addition, the burden of aggressive tumor characteristics was greater in AA women because they were more likely to be at high risk on multiple factors (e.g., both high histologic grade and high nuclear grade [P = 0.03] and negative status for both estrogen receptors and progesterone receptors [P = 0.01]). Data from this population-based cohort confirmed that breast tumors in AA women most likely are more aggressive compared with breast tumors in W women and offer new evidence for possible racial/ethnic differences with regard to p53 alterations.