Breast Cancer Tumor Tissues Research Articles

P4-19-04: Evaluation of Histopathological Parameters in Male Breast Cancer Reveals Differences Compared with Female Breast Cancer.

Abstract Purpose: To characterize male breast cancer (MBC) by evaluating established histopathological parameters and their prognostic value. Methods: 197 male patients with invasive breast cancer and available paraffin-embedded tumor tissue were retrospectively assessed. Patient files were reviewed for clinicopathological data. Tumors were re-evaluated for histologic grading on conventional sections. Immunohistochemical (IHC) stainings for estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), silver-enhanced in situ hybridization (SISH), Ki-67, CK5/6 and epidermal growth factor receptor (EGFR) were performed on tissue micro arrays. Data on vital status and cause of death were retrieved from the Cause of Death Registry. Cox proportional regression models were used for uni- and multivariate analyses. Results: ER and PgR positivity was demonstrated in 93 and 78 % of patients, respectively. Nottingham histologic grade III was seen in 41% and HER2 positivity in 11% of all patients. Defining molecular phenotypes using IHC markers revealed luminal (ER+ and/or PgR+, and HER2−) in 83%, luminal/HER2+ (ER+ and/or PgR+, and HER2+) in 11%, basal-like (ER-,PgR-,HER2−,EGFR+ and/or CK5/6 +) in 1%, but no cases of HER2-like or unclassified. Node-positivity (HR 4.6; 95% CI 1.9−11.4), tumor size > 20 mm (HR 2.3; 95% CI 1.1−5.0) and ER-negativity (HR 5.2; 95% CI 1.9−14.2) were significant risk factors for breast cancer death. Grade, HER2 status, Ki-67, or age did not demonstrate independent prognostic information. No difference in breast cancer deaths was demonstrated between luminal and luminal/HER2. Conclusion: Male breast cancer tumors seem to be of grade III more often than female breast cancer, whereas HER2 expression appears equally common. In our study, the most important predictors for breast cancer death in male breast cancer were lymph nodes, tumor size and ER status. The most common molecular phenotype was luminal. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-19-04.

P5-13-20: TOP2A Amplification Have Associated with Response to Anthracycline-Based Preoperative Chemotherapy in Primary Breast Cancer.

Abstract Background: Recent studies have suggested that HER2 gene amplification or overexpression have association with sensitivity of anthracycline-based chemotherapy. Preclinical studies found that TOP2A amplification or deletion only could be detected in HER2 amplification or over-expressive cases. And TOP2A protein is the target of anthracycline. In fact, the value of HER2 for predicting response to anthracycline-based chemotherapy in breast cancer may be more likely related to the concomitant amplification of the TOP2A gene. In this study we studied the association between TOP2A gene amplification and response to anthracycline-based preoperative chemotherapy. Methods: 309 early and local advanced breast cancer cases were enrolled in our study. HER2 proteins were qualitatively analysed by IHC, and TOP2A gene alterations were quantified by real-time polymerase chain reaction (RT-PCR) in primary tumor core biopsies from all HER2 over expressive cases. The enrolled patients received an intense dose dense (IDD) (CE, Cyclophosphamide + Epirubicin) or conventionally (TE, Paclitaxel+Epirubicin) scheduled anthracycline- based preoperative chemotherapy. The tumor was evaluated every two cycles. Median time on study for 309 patients with follow-up was 38 months. Results: The pCR was 14.3%. HER2 overexpression was found in 80/309 (25.9%) breast cancer cases, of which 61/80 cases have been tested for TOP2A status. 19/80 cases have not been tested for TOP2A status because of lacking core biopsies tumor tissue after pathological diagnosis. HER2 overexpression was associated with a significantly higher pCR rate compared to HER2 lower expression (27.5% vs. 9.6%, P<0.001). Further analysis was carried on and found the significantly higher pCR rate in TOP2A co-amplified cases compared to TOP2A deleted or normal cases (56.3% vs. 13.8%, P=0.001). HER2 overexpression was associated with a significantly higher pathologic complete response (pCR) rate only when TOP2A was co-amplified (56.3% vs. 9.6%, P<0.001), but not when deleted or normal (13.8% vs. 9.6%, P=0.183), compared to HER2 lower expression tumors. The interaction between HER2 or TOP2A and anthracycline-based regiment was observed not only in IDD but conventionally scheduled preoperative chemotherapy. Discussion: Previous studies demonstrate that TOP2A gene amplification may define a subtype of HER2−positive breast cancer, This subtype of breast cancer may be highly sensitive to anthracycline-based chemotherapy, which may improve progression in HER2 and TOP2A co-amplification cases. Howerver, this viewpoint needs suppose from preoperative chemotherapy. Using RT-PCR to test TOP2A statue from 61 primary breast cancer tumor tissue, we demonstrate that TOP2A amplification breast cancer have highly sensitive to anthracycline-based preoperative chemotherapy. This finding suggests that TOP2A as a predictive marker in breast cancer should be included in future studies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-20.

Reciprocal expression of survivin and SMAC/DIABLO in primary breast cancer

There is an intimate relation between disturbance in survivin and SMAC/DIABLO expressions and cancer formation in many tissues; however, this was not confirmed in the primary breast cancer. The aim of this study was to evaluate the relationship between survivin and SMAC/DIABLO mRNA expressions in primary breast cancer using RT-PCR and their relationship to some of the risk factors (age, family history, breast-feeding, use of contraceptive pills and hormonal receptors). Breast cancer tissues were studied for the detection of the mRNA expression of survivin and SMAC/DIABLO. This was done by qualitative reverse transcription-polymerase chain reaction (RT-PCR). SMAC/DIABLO mRNA was detected by RT-PCR only in 1 sample in breast cancer tissues (6.25%). However, it was expressed in 16 out of 25 (64%) of the benign tumor tissues (P=0.00). In contrast, survivin mRNA was highly expressed in breast cancer tissues, 36 out of 48 (75%) (P=0.00), while its expression was very low in benign tumor tissues, only 1 out of 37 (2.7%) (P=0.00). Expressions of SMAC/DIABLO and survivin were significantly reciprocal in breast cancer and benign tumor tissues. SMAC/DIABLO inhibits apoptosis of breast cancer cells by suppression of survivin. These two genes probably form an important link in the signaling pathway of formation of breast cancer cells, which may be chosen as therapeutic targets in the primary breast cancer.

Nuclear Targeting Dynamics of Gold Nanoclusters for Enhanced Therapy of HER2+ Breast Cancer

Recent advances in fluorescent metal nanoclusters have spurred tremendous interest in nanomedicine due to the ease of fabrication, excellent biocompatibility, and, more importantly, excellent wavelength-dependent tunability. Herein, we report our findings on fluorescent BSA-protected gold nanoclusters (AuNCs), ∼2 nm in size conjugated with Herceptin (AuNCs-Her), for specific targeting and nuclear localization in ErbB2 over-expressing breast cancer cells and tumor tissue as a novel fluorescent agent for simultaneous imaging and cancer therapy. More interestingly, we found that AuNCs-Her could escape the endolysosomal pathway and enter the nucleus of cancer cells to enhance the therapeutic efficacy of Herceptin. We elucidate the diffusion characteristics (diffusion time and number of diffusers) and concentration of the fluorescing clusters in the nucleus of live cells. Our findings also suggest that the nuclear localization effect of AuNCs-Her enhances the anticancer therapeutic efficacy of Herceptin as evidenced by the induction of DNA damage. This study not only discusses a new nanomaterial platform for nuclear delivery of drugs but also provides important insights on nuclear targeting for enhanced therapy.

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most “dietary sensitive” genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B2 and B6) were more “epigenetically active” as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention.

LSINCT5 is over expressed in breast and ovarian cancer and affects cellular proliferation

More than 98% of the human genome is comprised of non-protein coding sequences. Interestingly, a considerable fraction of these sequences is transcribed into non-protein coding RNA transcripts. These transcripts range in size from very small RNAs such as the miRNAs (20–25 base pairs) to transcripts that can range up to 100 kb or more. Some longer non-coding RNAs (lncRNAs) have been found to play important regulatory roles within cells. In this report, we demonstrate that LSINCT5 is a 2.6 Kb polyadenylated, long stress-induced non-coding transcript that is on the negative strand, localized in the nucleus and potentially transcribed by RNA polymerase III. LSINCT5 is overexpressed in breast and ovarian cancer cell lines and tumor tissues, relative to their normal counterpart. In addition, knocking down the expression of LSINCT5 in cancer-derived cell lines causes a decrease in cellular proliferation. Finally, we identified 95 genes with more than 2-fold changes when knocking down LSINCT5 expression by using the Affymetrix U133 Plus 2 array. We chose a subset of these genes to validate using qPCR and found that ten of these genes were indeed significantly affected by the LSINCT5 knockdown. Interestingly, two genes that were significantly downregulated were the lncRNA NEAT-1 and a protein coding gene PSPC1. We have therefore characterized a novel lncRNA that is overexpressed in breast and ovarian cancers, enhances cellular proliferation and may play a significant role in multiple processes.

Abstract 788: Uptake and cross-presentation of the leukemia associated antigens neutrophil elastase (NE) and proteinase-3 (P3) increases susceptibility of solid tumors to PR1-targeted immunotherapy

Abstract We have shown that the HLA-A2-restricted nonapeptide PR1 (VLQELNVTV) is a leukemia-associated peptide derived from P3 and NE. Furthermore, immunologic and clinical responses to PR1 peptide vaccination occur in patients with acute (AML), chronic (CML) myeloid leukemia and myelodysplastic syndrome. We have also shown that PR1 expression results from NE and P3 cross-presentation by B-cells and dendritic cells. Because NE and P3 are present in lung and breast cancer tumor tissue, we investigated whether uptake of NE and P3 might result in PR1 expression on solid tumors, which could induce susceptibility of non-hematopoietic tumors to PR1-targeted immunotherapy. We studied melanoma, breast, ovarian and lung cancer cell lines to determine whether NE and P3 are cross-presented. Using flow cytometry, we show that numerous malignant cell lines take up 5 to 10 °g/ml of soluble NE and P3 and was dose- and time-dependent, consistent with a receptor-mediated mechanism. In addition to soluble NE and P3, uptake of granulocyte-associated NE and P3 was also demonstrated; however, uptake of soluble NE and P3 were 3- and 2-fold higher, when compared to granulocyte-associated proteins. Within 4 hours of uptake, confocal microscopy showed that soluble P3 localized to endosomes and lysosomes, suggesting the possibility of lysosomal degradation of P3, a common pathway for MHC-I processing of cross-presented proteins. With the use of 8F4, a monoclonal antibody that is specific for a PR1/HLA-A2 conformational epitope, we show that PR1/HLA-A2 is expressed by 12-hours on P3- and NE-pulsed HLA-A2+ melanoma and breast cancer cell lines. Because 8F4 mediates complement-dependent cytotoxicity (CDC) of AML, we studied whether PR1 expression induced susceptibility of P3- and NE-pulsed tumor cells to killing by 8F4 and PR1-CTL. Twelve hours after pulsing the HLA-A2+ breast cancer cell lines MDA-MB-453 and MDA-MB-231 with P3 or NE, up to 40% and 60% of pulsed breast cancer cells were lysed by PR1-CTL and 8F4, respectively. We conclude that PR1 is cross-presented by solid tumors following receptor-mediated uptake of soluble NE and P3 at concentrations observed in tumors and inflammatory sites. Importantly, this cross-presentation increased susceptibility of embryological distinct tumors to PR1-specific immunotherapy. Together, these data suggest that cross-presentation of extracellular inflammatory proteins that are normally not expressed in the tumor may be a mechanism shared by many tumors, which could be exploited by immunotherapy strategies targeting these proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2011-788

Abstract 5610: Air pollution exposure and promoter methylation of E-cadherin, p16, and RAR-β in breast cancer tumors from cases in the WEB study

Abstract Purpose: Aberrant methylation has been shown to be associated with breast cancer tumor tissue samples and ambient air pollution exposure has been shown to be associated with aberrant methylation of gene promoter regions; we investigated potential associations between air pollution exposure in early life and promoter methylation of genes among breast cancer tumors. Methods: Paraffin embedded, formalin fixed tumor tissue blocks were obtained for 803 breast cancer cases participating in the WEB study and assayed for DNA methylation status of three promoters regions of E-cadherin, p16 and RAR-β. Methylation specific PCR was used to determine methylation status. Historical annual ambient total suspended particulate matter (TSP) concentration (μg/m3) was collected from 87 air monitoring sites in Erie and Niagara Counties dating as far back as the late 1950's. Residential addresses were obtained for the time of birth, time of menarche, time of first birth if applicable, and 10 years prior to diagnosis with breast cancer. Using Arc View software, the addresses were geocoded and overlaid with the air pollution information to estimate exposure to TSP at each time point. TSP exposures varied greatly over time, therefore they were categorized into high and low based on the mean exposure among the non-breast cancer controls in the WEB study. This analysis consists of case-case comparisons, comparing TSP exposure to DNA methylation status among breast cancer patients. Odds ratios and 95% confidence limits were calculated with unconditional logistic regression, adjusting for potential confounders. We stratified all analyses by menopausal status. Results: Among pre- but not postmenopausal women, there was an increased likelihood of a case having methylation of the p16 promoter region for those exposed to >109.03 μg/m3 of TSP at first birth compared to those exposed to ≤109.03 μg/m3 (n=123; OR=6.5, 95% CI: 1.2-34. 1). E-cadherin promoter region methylation was inversely associated with high TSP at birth (> 137.88 μg/m3) among the pre-menopausal women (n=138; OR=0.2, 95% CI: 0.1-0.8) but not for post-menopausal women (n=289; OR=0.8, 95% CI: 0.4-1.6) nor for the other genes. There was no evidence of association between TSP exposures and RAR-β promoter region methylation for any of the time periods. Conclusion: Air pollution exposure at birth was much higher than exposure at later times due to increasing regulation. Exposure to higher levels of TSP at birth was associated with decreased likelihood of promoter region methylation for E-cadherin, particularly for premenopausal cases. TSP exposure at the time of a first birth was associated with increased likelihood of p16 promoter methylation in pre- but not postmenopausal breast tumors. While our results are necessarily preliminary, they suggest that exposure to air pollutants in early life may affect promoter methylation of E-cadherin and p16 genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2011-5610

Abstract 2703: Leukocyte complexity in human breast cancer and response to chemotherapy

Abstract Despite the use of immune based biomarkers that predict outcomes for women with breast cancer (BC), leukocyte complexity in BCs remains poorly described, with scant information available regarding effects of cytotoxic therapy on leukocytic infiltrates. To evaluate leukocyte complexity and nature of the immune microenvironment in BCs, and to determine if chemotherapy (CTX) alters leukocyte density or phenotype, we evaluated BC tumor tissue, as compared to “normal” adjacent breast tissue, from women who had either received neoadjuvant CTX or had not received CTX prior to surgery. Tissues were evaluated immediately following surgical resection by polychromatic flow cytometry, as well as by immunohistochemical evaluation of tissue sections, or quantitative real-time PCR for mRNA expression. Independent of neoadjuvant CTX, myeloid-lineage leukocytes represented the dominant immune population in adjacent normal tissue. Tumor tissue from treated- and untreated patients contained significantly higher infiltration of activated CD4+ and CD8+ T cells, as compared to adjacent normal tissue. CD8+ T cells in treated tumors comprised a larger percent of the T cell infiltrate, corresponding to increased expression of CD3∈, CD8α, and cytotoxic molecules (granzyme B, perforin, granulysin) indicating higher infiltration by functional cytotoxic T cells. Importantly, adjacent normal and tumor tissue from women that had received neoadjuvant CTX contained significantly higher levels of infiltrating myeloid cells (macrophages, dendritic cells, neutrophils and mast cells), as compared to untreated women. Furthermore, mRNA expression analysis revealed corresponding increases in expression of myeloid-associated genes CD68, CD86, and HLA-DR, and evidence of induction of an immunosuppressive microenvironment, e.g., increased expression of IL-10 and -12β, and reduced expression of IL-12α. To determine if these changes were a direct response to CTX, or instead a response to CTX-induced cell death, we evaluated multiple human BC cell lines in culture following exposure to either Paclitaxel or Cisplatin and found CTX induced mRNA expression of cytokines and chemokines involved in myeloid recruitment. Using a murine model of mammary carcinogenesis, we revealed that, as predicted from these clinical data, CTX induced myeloid recruitment into mammary tumors, and that these tumor-promoting leukocytes in part regulated the response to cytotoxic therapy. Together, these data indicate that neoadjuvant chemotherapy leads to simultaneous induction of both a T cell-mediated anti-tumor immune response and recruitment of immunosuppressive and protumor myeloid cells that blunt response to cytotoxic therapy. Therapeutic strategies targeting these immunosuppressive and tumor-promoting immune cells may improve treatment efficacy and extend survival for women with BC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2703. doi:10.1158/1538-7445.AM2011-2703

Interleukin 13 expression in the primary breast cancer tumor tissue

The aim of this study was to investigate the presence and the expression levels of the interleukin 13 (I1-13) in the primary breast cancer tumour tissue in relation to the unchanged breast tissue in the same patients and to the breast tissue in the patients with benign breast disease, and to investigate the correlation between the IL-13 expression levels and the pathohistological factors, and between IL-13 expression and estrogens and progesterone receptor status. 50 patients with invasive ductal breast cancer and 20 patients with benign breast diseases were included in this prospective case-control study. The three-step immunohistochemical staining was used for testing the levels of IL-13 expression and hormone receptor status. IL-13 was present in breast cancer tumour tissue, and in the surrounding unchanged tissue in the same patients, and in breast tissue in patients with benign breast disease. The expression of IL-13 was significantly higher in breast cancer tumour compared with surrounding tissue (P < 0.05) of the same, lymph node-positive patients. In addition, IL-13 expression was significantly higher in breast cancer tumour compared with breast tissue in patients with benign breast diseases (P < 0.01). There was significant correlation between IL-13 expression and tumour size in patients with lymph node-negative breast cancer (r = 0.405, P = 0.050). There was no significant correlation between IL-13 expression and the other pathohistological factors, and no significant correlation between IL-13 expression and the lymph node status. Obtained results suggest possible involvement of IL-13 in breast carcinogenesis.

Abstract P3-09-01: Higher Serum miR-21 Expression Correlates with Higher Levels of VEGF and bFGF in Patients with Metastatic Breast Cancer

Abstract Background MicroRNAs (miRNAs) play an important role in the regulation of tumorigenesis and metastatic process. MiR-21 is an oncomiR that is overexpressed by breast cancer tumor tissue and is associated with high tumor grade and negative hormone receptor status. MiR-21 can control angiogenesis by regulating vascular endothelial growth factor (VEGF) as well as the expression and secretion of basic fibroblast growth factor (bFGF). MiR-19a is a member of the miR-17-92 family that was shown to suppress breast cancer cell proliferation. As angiogenesis factors act as attractants of circulating tumor cells (CTCs), a strong predictor of overall survival in metastatic breast cancer (MBC), we investigated the relationship between CTC count, levels of VEGF and bFGF, and the expression of miR-21 and miR-19a in sera of MBC patients. Methods RNA was extracted from sera collected from 33 MBC patients and 10 healthy donors (HD) using Total RNA purification Kit (Norgene Biotek Corporation, ON, Canada). The expression levels of miR-21, miR-19a and miR-192 were evaluated in triplicate by qRT-PCR using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of miR-21 and miR-19a. The fold-change values were calculated using 2-DCt, where DCt= mean CTtarget-miRNA - mean CTmiR-192. CTCs were detected by CellSearch System (Veridex LLC, Warren, NJ). Serum VEGF and bFGF were determined using a multiplex bead assay (Millipore, Billerica, MA). Mann-Whitney U test was used to compare the miRNAs expression level between MBC patients and HD. Spearman's correlation was used to determine the association between the expression of miRNAs and growth factors. Results There was a significantly higher median level of miR-21 in MBC patients compared with HD (22.8 versus 7.2, P=0.003), while the expression level of miR-19a was similar for both groups. On average, the relative abundance of miR-21 in sera (ratio of MBC versus HD) is 3.1-fold overexpression. Furthermore, levels of miR-21 was positively correlated with the serum levels of VEGF (Spearman Rho= 0.364; P= 0.038) and bFGF (Spearman Rho= 0.367; P = 0.036). CTCs were detected in 23 patients, ranged from 1 to 134 per 7.5 mL of peripheral blood. Patients with no CTCs had a significantly higher median level of miR-19a than patients with no CTC (6.5 vs 2.8, P =0.047). Conclusion Our results support the notion that miR-21 overexpression may play a role in angiogenesis and metastasis by upregulating VEGF and bFGF in MBC. Undetectable CTC count may be related to overexpression of miR-19a, an epigenetic suppressor of breast tumor proliferation. Whereas overexpression of miR-19a may limit the tumor cells from entering the peripheral circulation, increasing miR-21 in the serum could accelerate the disease progression by promoting angiogenesis in MBC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-01.

Adiponectin and breast cancer

Adiponectin, an adipose tissue-derived hormone, has been studied intensively for the past decade because of its anti-inflammatory, anti-atherogenic, and anti-diabetic properties. Recent advances suggest that adiponectin also plays an important role in the development and progression of various cancers, especially obesity-related cancers. In this review, the authors focus on the potential role of adiponectin in breast cancer, an obesity- and endocrine-associated tumor. Epidemiological studies have shown that plasma adiponectin level is a risk factor for breast cancer in post-menopausal women. Adiponectin and its receptors are expressed on both breast cancer line cells and tumor tissues. Furthermore, exogenous adiponectin has exhibited therapeutic potential in animal models. Underlying mechanisms include the inhibition of cell proliferation and promotion of apoptosis, the regulation of tumorigenic-related factors, and the suppression of angiogenesis. The signaling pathways linking adiponectin with tumorigenesis might provide potential drug targets for the future. However, more convincing evidence is needed to fully elucidate the exact role of adiponectin in breast cancer, since both its beneficial effects and possible mechanisms remain controversial.

Genomic alterations of primary tumor and blood in invasive ductal carcinoma of breast

BackgroundGenomic alterations are important events in the origin and progression of various cancers, with DNA copy number changes associated with progression and treatment response in cancer. Array CGH is potentially useful in the identification of genomic alterations from primary tumor and blood in breast cancer patients. The aim of our study was to compare differences of DNA copy number changes in blood and tumor tissue in breast cancer.MethodsDNA copy number changes in blood were compared to those in tumor tissue using array-comparative genomic hybridization in samples obtained from 30 breast cancer patients. The relative degree of chromosomal changes was analyzed using log2 ratios and data was validated by real-time polymerase chain reaction.ResultsForty-six regions of gains present in more than 30% of the tissues and 70 regions of gains present in more than 30% of blood were identified. The most frequently gained region was chromosome 8q24. In total, agreement of DNA copy numbers between primary tumor and blood was minimal (Kappa = 0.138, p < 0.001).ConclusionAlthough there was only a slight agreement of DNA copy number alterations between the primary tumor and the blood samples, the blood cell copy number variation may have some clinical significance as compared to the primary tumor in IDC breast cancer patients.

Enhanced Resistance to Tamoxifen by the c-ABL Proto-oncogene in Breast Cancer

Targeting the estrogen receptor is an important strategy in breast cancer therapy. However, although inhibiting estrogen receptor function with specific estrogen receptor modulators can achieve a primary response in cancer patients, intrinsic or subsequently acquired resistance to the therapy remains a major obstacle in the clinic. Thus, it is critical to gain amore thorough understanding of howestrogen receptor functions are regulated in breast cancer.Here, we demonstrate that the non-receptor tyrosine kinase c-ABL is a functional partner of the estrogen receptor, as expression of c-ABL sustained transcriptional activity of the estrogen receptor. More importantly, inhibition of c-ABL resulted in sensitization to treatment by tamoxifen (TAM) in estrogen receptor-positive breast cancer cells, as manifested by inhibition of cell survival and suppression of anchorage-independent growth. We found that c-ABL interacts with estrogen receptor in breast cancer cells and that expression of c-ABL is a frequent event in primary breast cancer tumor tissues. In estrogen receptor-positive tumors, the expression of c-ABL significantly correlated with disease progression and metastasis. This study shows that c-ABL regulates the cellular response to TAM through functional interaction with the estrogen receptor, which suggests c-ABL as a therapeutic target and a prognostic tumor marker for breast cancer.

Undetectable and Decreased Expression of KIAA1949 (Phostensin) Encoded on Chromosome 6p21.33 in Human Breast Cancers Revealed by Transcriptome Analysis

Cytogenetic aberration and loss of heterozygosity (LOH) are documented on chromosome 6 in many cancers and the introduction of a neo-tagged chromosome 6 into breast cancer cell lines mediates suppression of tumorigenicity. In this study, we described the identification of KIAA1949 (phostensin) as a putative tumor suppressor gene. Our microarray analysis screened 25,985 cDNAs between a tumorigenic and metastatic breast cancer cell line MDA-MB-231 and the chromosome 6-mediated suppressed, non-tumorigenic and non-metastatic derivative cell line MDA/H6, resulting in the identification of 651 differentially expressed genes. Using customized microarrays containing these 651 cDNAs and 117 controls, we identified 200 frequently dysregulated genes in 10 breast cancer cell lines and 5 tumor tissues using MDA/H6 as reference. Our bioinformatics analysis revealed that chromosome 6 encodes 25 of these 200 genes, with 4 downregulation and 21 upergulation. Northern analysis validated microarray results and was used to detect the size and number of RNA species of 2 downregulated (KIAA1949, PTK7) and 2 upregulated (SFRS3, HMGN3) genes in the cell lines. Particularly, the KIAA1949 gene at 6p21.33, a band region with the frequent cytogenetic aberration and LOH encodes 4 poly(A)-RNA species (4.6-, 4-, 3- and 1.5-kb) in multiple normal and breast cancer samples. Microarray analysis revealed KIAA1949 downregulation in 86.7% (n=15) of breast cancer cell lines and tumor tissues. Northern analysis demonstrated undetectable and decreased expression of KIAA1949 in 100% (n=10) of breast cancer cell lines. Taken together, these results strongly suggest KIAA1949 is a novel candidate breast cancer suppressor gene.

NCCN Task Force Report: Estrogen Receptor and Progesterone Receptor Testing in Breast Cancer by Immunohistochemistry

The NCCN Task Force on Estrogen Receptor and Progesterone Receptor Testing in Breast Cancer by Immunohistochemistry was convened to critically evaluate the extent to which the presence of the estrogen receptor (ER) and progesterone receptor (PgR) biomarkers in breast cancer serve as prognostic and predictive factors in the adjuvant and metastatic settings, and the ability of immunohistochemical (IHC) detection of ER and PgR to provide an accurate assessment of the expression of these biomarkers in breast cancer tumor tissue. The task force is a multidisciplinary panel of 13 experts in breast cancer who are affiliated with NCCN member institutions and represent the disciplines of pathology, medical oncology, radiation oncology, surgical oncology, and biostatistics. The main overall conclusions of the task force are ER is a strong predictor of response to endocrine therapy; ER status of all samples of invasive breast cancer or ductal carcinoma in situ (DCIS) should be evaluated by IHC; IHC measurements of PgR, although not as important clinically as ER, can provide useful information and should also be performed on all samples of invasive breast cancer or DCIS; IHC is the main testing strategy for evaluating ER and PgR in breast cancer and priority should be given to improve the quality of IHC testing methodologies; all laboratories performing IHC assays of ER and PgR should undertake formal validation studies to show both technical and clinical validation of the assay in use; and all laboratories performing IHC assays of hormone receptors in breast cancer should follow additional quality control and assurance measures as outlined in the upcoming guidelines from the American Society of Clinical Oncology and College of American Pathologists.

Detection of autoantibodies against cyclophilin A and triosephosphate isomerase in sera from breast cancer patients by proteomic analysis

Much interest is presently being shown toward identifying markers for the detection of breast cancer. To detect autoantibodies that could represent diagnostic markers for breast cancer, we comprehensively analyzed serum autoantibodies showing immunoreactivity to proteins in tumor tissues of breast cancer. Tumor tissues were obtained from 40 patients with breast cancer, along with sera from 30 other patients with breast cancer and 22 healthy donors. Proteins from tumor tissues were separated by 2-DE. After blotting onto PVDF membranes, tissue proteins were immunoblotted with sera from patients or healthy donors. By comparing each immunoblot pattern, three immunoreactive spots displayed stronger staining intensity with patient sera than with sera from healthy donors. The matched protein spots on 2-DE gels were digested and used for LC-MS/MS analysis, and identified as cyclophilin A (peptidyl-prolyl cis-trans isomerase A), triosephosphate isomerase and ubiquitin-conjugating enzyme E2N. Immunoblot analysis was then performed using commercially available purified proteins, confirming the specificity of anti-cyclophilin A and anti-triosephosphate isomerase antibodies in sera from patients.

Up-regulation of bone marrow stromal protein 2 (BST2) in breast cancer with bone metastasis

BackgroundBone metastases are frequent complications of breast cancer. Recent literature implicates multiple chemokines in the formation of bone metastases in breast cancer. However, the molecular mechanism of metastatic bone disease in breast cancer remains unknown. We have recently made the novel observation of the BST2 protein expression in human breast cancer cell lines. The purpose of our present study is to investigate the expression and the role of BST2 in bone metastatic breast cancer.MethodscDNA microarray analysis was used to compare the BST2 gene expression between a metastatic to bone human breast cancer cell line (MDA-231BO) and a primary human breast cancer cell line (MDA-231). The BST2 expression in one bone metastatic breast cancer and seven non-bone metastatic breast cancer cell lines were also determined using real-time RT-PCR and Western blot assays. We then employed tissue array to further study the BST2 expression in human breast cancer using array slides containing 20 independent breast cancer tumors that formed metastatic bone lesions, 30 non-metastasis-forming breast cancer tumors, and 8 normal breast tissues. In order to test the feasibility of utilizing BST2 as a serum marker for the presence of bone metastasis in breast cancer, we had measured the BST2 expression levels in human serums by using ELISA on 43 breast cancer patients with bone metastasis, 43 breast cancer patients without bone metastasis, and 14 normal healthy controls. The relationship between cell migration and proliferation and BST2 expression was also studied in a human breast recombinant model system using migration and FACS analysis.ResultsThe microarray demonstrated over expression of the BST2 gene in the bone metastatic breast cancer cell line (MDA-231BO) compared to the primary human breast cancer cell line (MDA-231). The expression of the BST2 gene was significantly increased in the bone metastatic breast cancer cell lines and tumor tissues compared to non-bone metastatic breast cancer cell lines and tumor tissues by real time RT-PCR, Western blot and TMA. Furthermore, serum levels of BST2 measured by ELISA were also significantly higher among patients with breast cancer metastatic to bone compared to breast cancer patients without metastatic to bone (P < .0001). Most importantly, the breast cancer cell line that transfected with BST2 demonstrated increased BST2 expressions, which was associated with increased cancer cell migration and cell proliferation.ConclusionThese results provide novel data indicating the BST2 protein expression is associated with the formation of bone metastases in human breast cancer. We believe that BST2 may be a potential biomarker in breast cancer with bone metastasis.

P34SEI-1 Inhibits Apoptosis through the Stabilization of the X-Linked Inhibitor of Apoptosis Protein: p34SEI-1 as a Novel Target for Anti–Breast Cancer Strategies

The p34(SEI-1) protein exerts oncogenic effects via regulation of the cell cycle, which occurs through a direct interaction with cyclin-dependent kinase 4. Such regulation can increase the survival of various types of tumor cells. Here, we show that the antiapoptotic function of p34(SEI-1) increases tumor cell survival by protecting the X-linked inhibitor of apoptosis protein (XIAP) from degradation. Our findings show that p34(SEI-1) inhibits apoptosis. This antiapoptotic effect was eliminated by the suppression of p34(SEI-1) expression. We also determined that direct binding of p34(SEI-1) to the BIR2 domain prevents ubiquitination of XIAP. Interestingly, p34(SEI-1) expression is absent or weak in normal tissues but is strongly expressed in tissues obtained from patients with breast cancer. Furthermore, the expression levels of p34(SEI-1) and XIAP seem to be coordinated in human breast cancer cell lines and tumor tissues. Thus, our findings reveal that p34(SEI-1) uses a novel apoptosis-inhibiting mechanism to stabilize XIAP.

Overexpression of LMW cyclin E results in faster M phase progression via Cdc25C interaction leading to chromosome missegragation in breast cancer cells.

Abstract Abstract #5032 Cyclin E is a G (1) cyclin essential for S-phase entry and has a profound role in oncogenesis. Cyclin E is overexpressed and present in low-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. We have previously shown that ectopic expression of low molecular weight of Cyclin E (LMW-E) in breast cancer cell lines induces genomic instability by promoting aneuploidy and the formation of structural chromosomal lesion. To determine the mechanism of induction of genomic instability by ectopic expression of LMW-E, we generated the MCF-7 cells inducibly expressing either the full length (EL) or low molecular weight (LMW-E) form of cyclin E. We observed 5.5 fold higher number of cells with centrosome defects in LMW-E induced versus EL induced cells. Notably, elevated levels of LMW-E led to a significant increase in fragmented nuclei and multinucleated cells. Immunofluorescence analysis also showed a 4-5 fold increase in the number of cells containing lagging chromosomes and anaphase bridges. Karyotype analysis for gross chromosomal aberrations metaphase preparation of the uninduced and EL and LMW induced cells showed a significantly higher percentage of chromosomal defects including dicentric chromosome and chromosomal breaks associated with LMW-E overexpression. We also show that while overexpression of EL leads to accumulation of cells in S and G2/M phases of the cell cycle, the overexpression of LMW of cyclin E causes a faster M phase progression. As a consequence, LMW-cyclin E overexpressing cells are resistant to nocodazole-induced M phase arrest. Using biochemical assays, we show that EL and LMW-E interact with the phosphatase Cdc25C and when either EL or LMW are in complex with CDK2 that they phosphorylate Cdc25C. Such association of LMW-E with Cdc25C results premature inactivation of cyclin B/CDK1 upon treatment of these cells with nocodazole. Additionally, continued induction of LMW-E results in premature inactivation of PLK1 in mitotic cells and deregulated localization of PLK1 such that it is no longer associated to central spindles during anaphase and telophase. Collectively, these results suggest that overexpression of LMW-E interferes with the activation of CDK1 during mitosis. The relatively low activity of CDK1 during the passage through prophase, prometaphase, and metaphase interferes with the regulation of the spindle checkpoint and facilitates the premature transition of dividing cells from metaphase to anaphase resulting in chromosomal instability and aneuploidy. This is the first observation of how mechanistically the overexpression of a proto-oncogene can lead to genomic instability in breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5032.