Breast Caner Research Articles

Abstract 354: The replication stress response defect is associated with tumor-initiating cell formation

Abstract Breast cancer is the most prevalent cancer in women and early diagnosis of breast cancer greatly improves successful treatment and survival rate. Therefore, it is essential to expand current understanding in breast tumorigenesis in order to find specific treatment to target breast cancer in its precancerous or earliest stage. To date, two important observations have been made in terms of precancerous and early breast caner development. First, replication stress response (RSR), also referred to as intra-S checkpoint, serves as a barrier to prevent the occurrence of precancerous lesions and tumorigenesis. When normal cells encounter DNA damages resulting from oncogenic stress, these damages will trigger RSR and provoke cell cycle arrest for repairing damaged DNA or force damaged cells to undergo senescence or apoptosis. However, when RSR is defective, the cells with DNA damage will be able to break the barrier and transform to cancer cells. Second, tumor-initiating cells, which possess stem cell-like properties, are also required for tumorigenesis. Tumor-initiating cells are on the top of the tumor development hierarchy. Therefore, they form in the early stage and then differentiate themselves into nontumorgenic cancer cell to give the heterogeneity of tumor. Even though both processes strongly regulate tumorigenesis, it remains unclear whether defective RSR is associated with tumor-initiating cell formation. To gain insight into breast tumorigenesis from this aspect, RSR-intact cells were given oncogenic stress by exogenous expression of cyclin E, a major oncogene in breast cancer. Once the cells encounter oncogenic-stress-induced DNA damage, several key RSR genes, such as ataxia telangiectasia and Rad3 related (ATR), are then suppressed in order to allow damaged cells to escape from senescence. By measuring the expression changes of transcriptional landscape in RSR-deficient model system, the molecular profile of RSR defective cells are more similar to breast mammary stem cell and breast tumor-initiating cells. Furthermore, the RSR defective cells increase ALDH1, a marker of normal and malignant human mammary stem cell, and increase the ability of mammosphere formation, an indicator of self-renewal property of mammary epithelial stem cells.Importantly, we have also identified EGFR/MEK1 signaling as a key pathway that renders RSR defective cells the ability to escape from senescence or evade apoptosis and acquire tumor-initiating properties. Our findings not only provide insight into the RSR defective function network and the association between RSR defect and tumor-initiating cell formation in early breast cancer development, but also identify the key pathway for breast cancer target therapy and prevention. Citation Format: Curtis Chun-Jen Lin, Hui Dai, Shiaw-Yih Lin. The replication stress response defect is associated with tumor-initiating cell formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 354. doi:10.1158/1538-7445.AM2014-354

Abstract P5-14-03: The impact of adjuvant radiotherapy on survival in primary breast caner: What is the role of guideline adherence in radiotherapy – A retrospective multi-center cohort study of 8935

Abstract Introduction: Breast cancer remains the most common malignancy in women in industrialized countries. Over the last decades improved adjuvant therapy strategies were the key for a favorable prognosis in patients. Among these, radiotherapy is one of the important backbones in adjuvant therapy strategies. This study tries to answer the following questions: 1. Does guideline adherent radiotherapy improve survival in primary breast cancer patients? 2. Is breast conserving surgery followed by RT equal to mastectomy in outcome parameters? 3. Is there a difference in survival between patients receiving BCT followed by RT with non guideline conform incomplete tumor resection (R1)? Does adjuvant RT compensate incomplete resection of the tumor? Material and Methods: In this German retrospective multi-center cohort study called BRENDA (breast cancer care under evidence based guidelines) we investigate data of 8.935 primary breast cancer patients recruited from 17 participating breast cancer centers in Germany (all certified breast cancer centers by the German Cancer Society). Guideline adherence is established in all adjuvant treatment modalities based on internationally validated guidelines. Results: Patients who received guideline adherent RT in primary breast cancer were associated with significantly improved survival parameters [RFS: p < 0.001; HR = 0.28 (95% CI: (0.24 – 0.33)] [OAS: p < 0.001; HR = 0.26 (95% CI: 0.19 – 0.36)] compared to patients who did not receive guideline adherent adjuvant RT. Compared to other adjuvant treatment modalities (surgery/chemotherapy/endocrine therapy) guideline violations concerning radiotherapy and chemotherapy have the most important impact on survival parameters. Furthermore, the results of Fisher et al. and Veronesi et al. were confirmed by demonstrating that BCT followed by RT is equal to mastectomy [RFS: p = 0.293; HR = 1.20 (95% CI:0.85-1.70)] [OAS: p = 0.104; HR = 1.31 (95% CI: 0.95-1.81)]. Adjuvant RT could not improve the outcome in patients with non-guideline conform incomplete tumor resection via BCT (R1) showing a significantly impaired RFS [p<0.001; HR = 2.87 (95% CI: 2.00-4.12)] and OAS [p<0.001; HR = 2.06 (95% CI: 1.43-2.98)] compared to guideline conform complete tumor resection via BCT (R0). Additionally, non guideline adherent RT after mastectomy (only T1/T2) (overtherapy) did not influence survival significantly [RFS: p = 0.838; HR = 1.07 (95% CI: 0.57-1.98)] [OAS: p = 0.613; HR = 1.18 (95% CI: 0.63-2.20)]. Discussion: Guideline adherent adjuvant RT is associated with an improvement of survival parameters in primary breast cancer. Patients undergoing guideline conform BCT (R0) followed by RT present an equal outcome as patients undergoing mastectomy. However adjuvant RT can not compensate an inadequate tumor resection via BCT and RT overtherapy after mastectomy does not improve survival parameters. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-03.

Abstract P6-05-12: Comprehensive molecular analysis of estrogen receptor positive breast cancer to determine clinically actionable alterations

Abstract Introduction. Understanding the genetic landscape of estrogen receptor positive breast caner is critical for defining clinically actionable alleles that may be targeted using next generation biologics. PIK3CA mutations have previously been reported to be the most common mutation within estrogen receptor positive breast cancer, however the overlap and mutual exclusivity with other key driving alleles is poorly understood, especially how these biomarkers change following treatment failure. We custom designed a mutation, copy number variation and RNA expression panels to profile biomarkers from low quality formalin fixed paraffin embedded extracted material. Data relating to key pathways, such as PI3K and immune modulatory pathways, and overlap with other biomarkers will be presented. Methods. Formalin fixed paraffin embedded material was available from 195 primary cases and 95 paired metastatic estrogen receptor positive breast cancer patients. Samples were assayed for the expression of PTEN by IHC, hotspot mutations in 11 oncogenic driving genes using Q-PCR based technology, copy number alterations in 42 genes using Q-PCR based technology and RNA expression using a custom designed 400 breast cancer specific NanoString gene panel. Results. PTEN loss, as defined by H-score of 0, was found in 4.5% of primary samples and in 4.3% of metastatic samples. PIK3CA mutations were found in 43% of primary samples and in 38% of metastatic samples and were largely found to be mutually exclusive with PTEN loss. AKT1 mutations were found in 5.4% of primary samples and in 4.1% of metastatic samples. Less frequent mutations in KRAS (7.0%) and BRAF (1.7%) were found in the primary sample, and some were co-existing with PIK3CA mutations. Copy number gains were found in CCND1 (23.5%), ZNF703 (19.5%), FGFR1 (16.8%) and PAK1 (11.4%) and were largely concordant with the paired metastatic sample. Analysis with clinical outcome is ongoing. Conclusions. PIK3CA was the most frequently altered gene detected and was mutually exclusive with other key driving mutations within the PI3K pathway (AKT1 and PTEN). A strong concordance was observed between genetic alterations found in the primary sample and the paired metastatic sample. Mutations and expression gains within clinically actionable targets were found less frequently, but may provide alternative treatment strategies for these patients following failure of endocrine therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-12.

Nuclear loss of protein arginine N-methyltransferase 2 in breast carcinoma is associated with tumor grade and overexpression of cyclin D1 protein

Human protein arginine N-methyltransferase 2 (PRMT2, HRMT1L1) is a protein that belongs to the arginine methyltransferase family, and it has diverse roles in transcriptional regulation through different mechanisms depending on its binding partners. In this study, we provide evidences for the negative effect of PRMT2 on breast cancer cell proliferation in vitro and in vivo. Morever, cyclin D1, one of the key modulators of cell cycle, was found to be downregulated by PRMT2, and PRMT2 was further shown to suppress the estrogen receptor α-binding affinity to the activator protein-1 (AP-1) site in cyclin D1 promoter through indirect binding with AP-1 site, resulting in the inhibition of cyclin D1 promoter activity in MCF-7 cells. Furthermore, a positive correlation between the expression of PRMT2 and cyclin D1 was confirmed in the breast cancer tissues by using tissue microarray assay. In addition, PRMT2 was found to show a high absent percentage in breast caner cell nuclei and the nuclear loss ratio of PRMT2 was demonstrated to positively correlate with cyclin D1 expression and the increasing tumor grade of invasive ductal carcinoma. Those results offer an essential insight into the effect of PRMT2 on breast carcinogenesis, and PRMT2 nuclear loss might be an important biological marker for the diagnosis of breast cancer.

Clinicopathological features and prognosis of triple-negative breast cancer

This study was conducted to analyze the clinicopathological characteristics and patient survival factors in triple-negative breast cancer (TNBC). A retrospective analysis was performed on 14 506 breast cancer patients admitted to the Departmrnt of Breast Surgery, Tianjin Medical University Cancer Hospital from January 2004 to June 2010. The correlation of pathological characteristics, recurrence time and patterns, and prognosis with TNBC was analyzed. Among the 14 506 cases, there were 1886 (13.0%) cases of triple-negative breast cancer, 7282 (50.2%) cases of luminal A breast caner, 3380 (23.3%) cases of luminal B breast caner, and 1958 (13.5%) cases of HER-2 overexpressing breast cancer. Compared with the other groups, the triple-negative breast cancer patients had significantly higher histological grade, lower percentage of invasive breast ductal carcinoma and higher pathological stage (P < 0.001) . The 5-year disease-free survival rate of the triple negative breast cancer was 79.5%, significantly lower than that of the other subtype breast cancer (P < 0.001), but the difference in 5-year overall survival rate was not significant (P = 0.113). The independent factors of DFS in TNBC including: age, tumor size, clinical stage, surgery and chemotherapy (P < 0.05). Compared with the other subtype breast cancers, the patients with triple-negative breast cancer have higher histological grade, lower percentage of invasive breast ductal carcinoma and higher pathological stage, and they also have a poor prognosis.

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

Abstract P5-10-10: The miR-34a is down-regulated in breast cancer and breast stem cells and a potential to eradicating breast cancer via a systemic delivery of a VISA –miR-34a nanoparticle system

Abstract Breast cancer is the most common disease in women around the world and the current treatment strategies are not potent enough for the patients, especially those who are the triple-negative type of molecular classifications. Therefore, novel and more effective treatments are pressingly needed. Of the current methods, target therapy, which not only retains cancer-specific expression but also limits toxicity, is a new strategy for treatment of cancers. In this study, it was to investigate miR-34a expression in breast caner specimen and its relationship with patient's clinical status, and develop targeted miR-34a delivery system as a potential method for breast cancer therapy. miR-34a expression was investigated by qRT-PCR and related to clinicopathologic significance and found to be down-regulated in breast cancer as compared with normal adjacent tissues and involved in breast cancer stem cell through down-regulation of CD44, ZEB1, and Bmi1. We designed targeted miR-34a expression using T-VISA system (hTERT promoter driven VP16-Gal4-WPRE Integrated Systemic Amplifier) liposomed-based nanoparticles and evaluated the antitumor effect in breast cancer cells in vitro and in orthotopic animal model as well as its systemic toxicity. The T-VISA-miR-34a system robust targeted to breast cancer cells and breast cancer stem cells and prolonged duration expression of miR-34a. Furthermore, a systemic delivery of a VISA –miR-34a nanoparticle system targeted efficiently expression of miR-34a to tumors and could significantly inhibit tumor growth and prolong mouse survival in multiple living imaging xenograft and syngeneic models of orthotopic breast cancer and without toxicity in intact mice. Our study demonstrated that miR-34a expression was down-regulated in breast cancer and breast cancer stem cells through down-regulation of CD44, ZEB1, and Bmi1. The T-VISA-miR-34a nanoparticle system showed robust antitumor effects in breast cancer and could be a potential therapeutic approach to eradicating breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-10.

SGK3 is associated with estrogen receptor expression in breast cancer

While breast cancer mortality rate has seen a steady decline in the last few decades, advances in better treatment and diagnostic tools remain important as we come into the age of personalized therapy. In this report, we describe our studies of SGK3's role in breast cancer. SGK3 (also known as CISK) is a member of the AGC family of kinases. Our previous work indicates that SGK3 functions downstream of the PI 3-kinase cascade and shares molecular and biochemical similarities with Akt. Here, we show that SGK3 expression is linked to estrogen receptor (ER) both in breast caner cell lines and in primary tumor samples. Our analysis also indicated a positive correlation between SGK3 expression and tumor prognosis. Importantly, our immunochemistry analysis of human tumor samples established a clinical link between SGK3 expression and ER+ tumors. These findings implicate SGK3 as an additional component to a complex and heterogeneous disease, and point to the potential benefits of incorporating SGK3 into the process of breast cancer diagnosis and treatment.

The in vivo Therapeutic Effect of Free Wanderer Powder (逍 遙 散 xiāo yáo sǎn, Xiaoyaosan) on Mice with 4T1 Cell Induced Breast Cancer Model

In the present study, we investigated the therapeutic effect of a classical TCM formula, Free Wanderer Powder ( xiāo yáo sǎn), in a breast cancer mouse model induced with estrogen-insensitive breast cancer 4T1 cells. Ovariectomized Balb/c mice (6-8 weeks) or sham mice were injected into the fourth mammary fat pad with 4T1 cells in which tumors were palpable 7 days after injection. On the eighth day, the mice were divided into 4 groups and tubefed daily with vehicle, Free Wanderer Powder ( xiāo yáo sǎn) formula or tamoxifen for 28 days. Tumor growth inhibition and the decrease of the average tumor mass were most evident in mice treated with Free Wanderer Powder ( xiāo yáo sǎn). Free Wanderer Powder ( xiāo yáo sǎn) treatment significantly reduced Bcl-2 and elevated Bax and p53 protein expressions in breast cancer tumor. These results were further confirmed by immunohistochemisty. Tamoxifen could decrease spleen mass and Bcl-2 protein expression, increase the Bax protein expression as well as exert uterotrophic effects by increasing uterus index and inducing the gene expressions in the uterus. Taken together, these results show that Free Wanderer Powder ( xiāo yáo sǎn) treatment induced apoptosis at protein level and inhibited the tumor growth in 4T1-induced ovariectomized Balb/c female mice, indicating the possibility of its future use for treatment of estrogen-insensitive breast caner.

D-Pinitol a low-molecular cyclitol prevents 7,12-Dimethylbenz [a] anthracene induced experimental breast cancer through regulating anti-apoptotic protein Bcl-2, mitochondrial and carbohydrate key metabolizing enzymes

Compounds of dietary origin have always been considered as a paramount entity due to their popular belief of the absence of adverse side effects and also for their extraordinary efficacy against many diseases among which cancer is most important. The present investigation was aimed to evaluate the modulatory effect of d-Pinitol a compound from dietary origin, on the key mitochondrial tricarboxylic acid cycle, carbohydrate metabolizing enzymes and anti-apoptotic protein Bcl-2 against 7, 12-Dimethylbenz [a] anthracene (DMBA) induced mammary carcinoma in female Sprague Dawley rats. Breast caner was induced by intragastric administration of single dose of 20mg/kg body weight of DMBA. The results revealed that the levels of TCA cycle enzymes and carbohydrate metabolizing enzymes were drastically altered in both breast and liver tissues of cancer-bearing animals when compared to controls. Further, the anti-apoptotic protein Bcl-2 was found to be expressed during breast cancer. Interestingly, the oral administration of d-Pinitol at a dosage of 200mg/kg body weight for 45days to breast cancer-bearing rats were found to be significantly brought back the altered enzymes status to near normal range. This therapeutic activity may be due to the antioxidant property of d-Pinitol, which ultimately results in the mitochondrial compartment. Thus, our results clearly confirmed that d-Pinitol has some key modulatory property in mitochondrial and carbohydrate metabolizing enzymes through its antioxidant nature and also regulatory role in the proteins of anti-apoptotic cascade against the free radicals involved oxidative stress-mediated diseases, and which results in the consideration of the compound as safe and more effective in the treatment of mammary carcinoma in the near future.

Phase II trial of preoperative paclitaxel, gemcitabine, and trastuzumab combination therapy in HER2 positive stage II/III breast cancer: The Korean Cancer Study Group BR 07-01

An addition of trastuzumab preoperatively to chemotherapy for human epidermal growth factor receptor 2 (HER2) positive breast cancer improved relapse-free survival (RFS). This study was designed to evaluate the efficacy and safety of preoperative paclitaxel, gemcitabine, and trastuzumab (PGH) combination for HER2-positive breast caner. Pathologically, proven node positive stage II/III breast cancer patients with adequate organ function and no history of anti-cancer therapy were eligible. Patients received weekly trastuzumab with paclitaxel 80 mg/m(2) and gemcitabine 1,200 mg/m(2) on days 1 and 8, every 3 weeks for 6 cycles. Postoperatively, patients completed trastuzumab for 1 year and hormone therapy for 5 years if indicated. All patients received postoperative radiation therapy. Of 53 enrolled patients with a median tumor of 5.3 (range, 2.0 to >12) cm; 43.4%, T3/T4; 75.4%, N2/N3; and 45.3%, positive hormone receptors. The pathologic complete response (pCR) rate was 58.5% in both tumor and lymph nodes. Grade 3/4 adverse events were neutropenia (32%), febrile neutropenia (0.6%), and transient elevation of AST/ALT (1.6%) during a total of 318 cycles. All patients maintained normal cardiac function. With a median follow-up of 40 months, 3-year RFS rate was 84% with 91.7% distant metastasis-free survival rates. Remarkable pCR rate was obtained with non-anthracycline-based PGH therapy for HER2-positive stage II/III breast cancer. Adverse events were mild with few incidences of febrile neutropenia.

Epigenetic silencing of the tumor suppressor klotho in human breast cancer

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

Abnormal expression of RSK-4 and its clinical significance in breast cancer

To study the expression and clinical significance of ribosomal S6 kinase-4 (RSK-4) in breast cancer and explore the role of RSK-4 in the genesis and development of breast cancer. The expression levels of RSK-4 mRNA and protein were detected in 56 cases of breast cancer and the normal breast tissues, as well as in 20 cases of breast benign lesions, by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression rates of RSK-4 mRNA in breast cancer, the normal breast tissues and breast benign lesions were 48.2%, 76.8% and 75.0%, respectively. The expression level of RSK-4 mRNA in breast cancer was significantly lower than those in normal breast tissues and breast benign lesions tissues (P < 0.05). The expression level of RSK-4 significantly correlated with tumor size and clinical stage (P < 0.05).The expression rate of RSK-4 protein was 39.3% in breast cancer tissues, which was significantly lower than that of normal breast tissues (71.4%) and breast benign lesions (75.0%, P < 0.01). The expression level of RSK-4 protein was lower in breast cancer with large tumor, high clinical stage and lymph node metastasis. In 56 cases of breast cancer samples, the consistency rate of RSK-4 mRNA and protein was 73.2%. A significant correlation was found between RSK-4 mRNA and protein (χ² = 10.254, P < 0.05). The down-regulation of RSK-4 expression in breast caner suggests that it is a breast cancer suppressor gene, and the lack or down-regulation of RSK-4 expression is involved in the genesis and progression of breast cancer.

Tryptophan degradation in women with breast cancer: a pilot study

BackgroundAltered tryptophan metabolism and indoleamine 2,3-dioxygenase activity are linked to cancer development and progression. In addition, these biological factors have been associated with the development and severity of neuropsychiatric syndromes, including major depressive disorder. However, this biological mechanism associated with both poor disease outcomes and adverse neuropsychiatric symptoms has received little attention in women with breast cancer. Therefore, a pilot study was undertaken to compare levels of tryptophan and other proteins involved in tryptophan degradation in women with breast cancer to women without cancer, and secondarily, to examine levels in women with breast caner over the course of chemotherapy.FindingsBlood samples were collected from women with a recent diagnosis of breast cancer (n = 33) before their first cycle of chemotherapy and after their last cycle of chemotherapy. The comparison group (n = 24) provided a blood sample prior to breast biopsy. Plasma concentrations of tryptophan, kynurenine, and tyrosine were determined. The kynurenine to tryptophan ratio (KYN/TRP) was used to estimate indoleamine 2,3-dioxygenase activity. On average, the women with breast cancer had lower levels of tryptophan, elevated levels of kynurenine and tyrosine and an increased KYN/TRP ratio compared to women without breast cancer. There was a statistically significant difference between the two groups in the KYN/TRP ratio (p = 0.036), which remained elevated in women with breast cancer throughout the treatment trajectory.ConclusionsThe findings of this pilot study suggest that increased tryptophan degradation may occur in women with early-stage breast cancer. Given the multifactorial consequences of increased tryptophan degradation in cancer outcomes and neuropsychiatric symptom manifestation, this biological mechanism deserves broader attention in women with breast cancer.

Abstract 4908: Deep sequence analysis of the relationship between gene expression, CpG island methylation, and gene copy number in breast cancer cells

Abstract Background: Estrogen receptor (ER) expression in breast caner is an important biomarker for targeted therapy and outcome prediction. Nevertheless, the molecular mechanism that controls the phenotypic difference is little known. We used deep sequence technology to profile the transcriptome, gene copy number, and CpG island methylation simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in ER+ and ER- breast cancer. Materials and methods: Total mRNA sequence (mRNA-seq), gene copy number (DNA-seq), and genomic CpG island methylation (Methyl-seq) were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome (hg18) to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands; differentially expressed genes between ER+ and ER- cell lines were selected and then correlated with methylation status of these genes’ CpG islands within 5kb of transcript start or copy number changes. The genes that appeared controlled by methylation or copy number changes in cell lines were further evaluated for their expression in the dataset of 129 primary breast tumors. Results: ER+ breast cancer cells had very different gene expression pattern from ER- cancer cells. The two cell line types formed two distinct clusters in unsupervised cluster and 1,873 genes were differentially expressed by moderated t statistics. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5kb of the 5’ end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. Eighty nine of these 149 genes were also differentially expressed in the primary tumor samples and 84 of them were consistent with cell line gene expression and methylation data. The set of 149 genes were significantly enriched in the ER+ tumors by Gene Set Enrichment Analysis. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; however, only 9 of these genes were overexpressed in ER+ tumors. Many of the methylation or CNA affected genes were functionally significant and some are known to be associated with breast cancer outcome such as GATA3 and LYN. Conclusions: A global pattern of differential CpG island methylation influences expression of a cohort of genes that contribute to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion in defining the transcriptome landscape of ER+ and ER- cells appears to more modest, although several potentially significant genes appear to be globally regulated by copy number aberrations. 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 4908. doi:10.1158/1538-7445.AM2011-4908

Abstract 2430: Aromatase expression inhibits anoikis and promotes bone metastasis of ERα positive breast cancer cells

Abstract Aromatase is the rate-limiting enzyme that catalyzes the final step in estrogen biosynthesis. In postmenopausal women the ovary ceases to make estrogen. Although it has been shown that estrogen is produced locally in breast cancer (BCa) tissue of postmenopausal women for the proliferation and survival of ERα (+) BCa cells, there is little information on the role and the regulation of aromatase in breast cancer cells for their survival when they intravasate into the circulation where estrogen level is very low. Human breast cancer CAMA-1 cell along with aromatase-transfected MCF-7 (MCF-7/Aro) and ZR-75-1 (ZR-75-1/Aro/CL10) cells were cultured in suspension to mimic circulating tumor cells. Interestingly, all the three ERα(+) BCa cell lines expressed more aromatase in suspension culture compare to the adhesion culture. Moreover both endogenous and transfected aromatase were upregulated in suspension culture. Additionally, a higher expression of ERα was also observed in suspension culture, which could increase the sensitivity of the breast caner cells to intracrine estrogen. We found that anoikis was shown to be inhibited by the supplementation of testosterone, which is converted to estradiol by aromatase. Conversely, addition of the aromatase inhibitor letrozole blocked the testosterone-induced anoikis inhibition. The increase of pro-apoptotic protein BAX and BAD were observed in suspension culture, while the treatment of estrogen inhibited the expression of BAX and BAD in both adhesion culture and suspension culture. More interestingly, mice with intracardiacally inoculated ZR-75-1/Aro cells presented distant bone metastasis in the mandible and tibiae/femora after 2-weeks of inoculation without estrogen supplement, while the control ZR-75-1 cells were incapable of forming metastasis without estrogen. Our findings provide an important foundation for future investigation on how hormone-dependent BCa cells survive in circulation during the process of metastasis. 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 2430. doi:10.1158/1538-7445.AM2011-2430

Cell morphology observation of breast cancer on fine needle aspiration biopsy

Objective To summarize the cytological features of breast carcinoma by fine needle aspiration and differential diagonsis of breast hyperplasia and breast flbroadenoma. Methods Cytological features about 175 cases of breast cancer were analyzed,contrasted with 76 cases of breast hyperplasia and 93 eases of fibroadenoma. And 20 cases of low-diagnosis breast cancer were analyzed, compared with 13 cases of excessive diagnosis. Results The followings were found:disorderly arranged cells in 169 cases of breast cancer, about 96.9% in 175 cases,loose distance between the nucleus in 125 cases of all cases (about 71.4%). The percentage of small groups or scattered cells, medium size cells, round nucleus and irregular border nucleus was 34.3% (60/175) ,81.1% (142/175), 88.6% (155/175) and 28.6% (50/175) respectively. The nucleus/cytoplasm ratio was increased in 105 cases of all (about 60%). The nucleus/cytoplasm ratio was obviously reduced in 15 cases of all (about 8.6%). Big nucleolis were observed in 49 cases of all (about 28%). There was significant difference between nucleus size in 136 cases of all (about 77.7%). There was not myoepithelial cell in 168 cases of all (about 96%). The percentage of visible necrosis, scsttered round nucleus and integrity of cytoplasmic was 13.7% (24/175) ,89.7% (157/175), 66.9% (117/175) respectively. The cases of all above features were significantly more than those of breast hyperplasia and breast fibroadenoma (P <0.05). The percentage of large number of cells was 52.6% in 175 breast cancer cases,higher than 5.3% in 76 breast hyperplasia cases. There was a significant difference(P <0.05) between two groups. In 20 cases of low-diagnosis breast cancer the percentage of round nucleus with integrity of cytoplasmic, special arrangement cells was 70% (14 cases) ,35% (7 cases) ,higher than the cases in 13 excessive diagnosis cases. There was a significant difference(P < 0.05) between two groups. Conclusion The cell morphological characteristics of breast caner by fine needle aspiration are large number of cells, loose distance between the nucleus, disorderly arranged cells, small guoups or scattered cells, integrity of cytoplasmic, increased nuclear, round nuclear, irregular border nucleus,increased or decreased nucleus/cytoplasm ratio, big nucleoli, significant difference in nuclear size, no myoepithelial cell, visible necrosis. Most of breast cancer can be distinguished from breast hyperplasia and breast fibroadenoma by above characteristics. All those cytological characteristics are helpful to identify difficult case. Key words: Breast neoplasms; Fine needle aspiration biopsy; Cytopathology; Diagonsis, differential

Abstract PD02-08: Development of DNA Aptamers That Inhibit Invasion, Migration, and Growth of Breast Cancer Cells

Abstract Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer. We utilized three TN breast cancer cell lines (HCC38, HCC1806, HCC1937) as model systems to characterize mechanisms of growth, migration, and invasion. FACS analyses demonstrated that these TN cell lines showed a phenotype of CD44+CD24-/lowepithelial specific antigen (ESA)+. An inhibitory antibody against exon v10 of CD44 significantly inhibited three-dimensional (3D) growth of tumor cells. Importantly, this antibody also inhibited alpha2beta1/alpha3beta1 integrins-mediated migration and invasion into matrigel. These results suggest that CD44 harboring exon 10 facilitates progression and metastasis of TN breast cancer cells in vivo by enhancing growth, migration, and invasion. We also demonstrated that this exon interacts with a cell surface tyrosine kinase, EphA2. Thus, we hypothesize that CD44 would form a molecular complex with EphA2 through exon v10 on TN breast cancer cells that plays a key role in facilitating invasion and metastasis. In order to develop novel reagents to inhibit TN breast cancer invasion and metastasis, we have utilized SELEX (systematic evolution of ligands by exponential enrichment) to isolate DNA aptamers that specifically recognize exon v10 of CD44. DNA aptamers are functional molecules with the appropriate sequence and structure to form a complex with a target molecule. Typically, dissociation constants for these aptamer-target complexes are in the high pico-molar to low nano-molar range, which is comparable to antibodies. Given the fact of the ease of chemical synthesis and modifications of oligonucleotides, DNA aptamers that recognize cancer cells will be easier to develop and have a great impact for the next generation of strategies for cancer detection, diagnosis, and therapy. We describe the development of “function-blocking” DNA aptamers specific for exon v10 of CD44 that inhibits TN breast cancer invasion and metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-08.

The clinical research of local recurrence after breast-conserving therapy for breast cancer

To compare and analyze the data of breast cancer recurrence after breast-conserving therapy (BCT), and to find high risk factors that can affect local recurrence. A total of 1034 patients in the data base between January 2000 and June 2008 were analyzed retrospectively. The patients aged 23 to 94 years when diagnosed (median age, 48 years). The data was investigated to compare of two different groups in local recurrence rate and survival rate. The high risk factors of recurrence after BCT [estrogen receptor (ER)/progesterone receptor (PR), human epidermal growth factor receptor (HER-2), age, lymph node involvement, tumor diameter, neoadjuvant chemotherapy, pathological status] were studied. The patients were followed-up to June 2010, and the median period was 42 months (range, 3-126 months). During the period, 35 patients developed ipsilateral breast tumor recurrence (3.3%), 47 patients had metastasis to distant organs (4.5%). The 5-year disease-free survival was 87.7%, 5-year distant disease-free survival was 94.0%. The lymph node status, HER-2 status and age were significant risk factors for ipsilateral breast tumor recurrence on univariate analysis. One peak recurrence period was from the 2nd to 3rd year, and the other was from the 5th to 6th year after the operation. The HER-2 status was independent factors of ipsilateral breast tumor recurrence on multivariate analysis. The recurrence happens primarily in the 2nd to 3rd and the 5th to 6th year after the breast-conserving therapy. HER-2 status is an independent factor of ipsilateral breast tumor recurrence. The patients with high risk factors of recurrence should be treated more aggressively.

Association analysis between polymorphism of dopamine transporter variable number tandem repeat and breast cancer with abnormal Hilit

To explore the association between polymorphism of dopamine 1 transporter variable number tandem repeat (DAT1 VNTR) and breast cancer with abnormal Hilit in Chinese Han population from Xinjiang. The breast caner patients were divided into four body fluids according to Uighur medicine theory. And polymerase chain reaction and VNTR polymorphism technique were employed to detect genotypic and allelic frequencies of a 40 bp VNTR polymorphism situated in 3' untranslated region of DAT1 gene in 144 breast cancer patients with abnormal Hilit and 104 normal control subjects in Han population of Xinjiang province. In our sample, the repeat numbers of 40 bp were 7 and 9 - 11 (PCR product length of 360 bp and 440 bp to 520 bp) and 10-repeat allele (480 bp) detected was the dominant allele of DAT1 gene polymorphisms with an allelic frequency of 90.9%; Seven kinds of genotype were detected and genotype 10/10 was the most common genotype with a genotypic frequency of 83.1%. The frequency of 10-repeats allele and 10/10 genotype was significantly higher in breast caner patients with abnormal balgham Hilit than in controls (OR = 0.127, 95%CI 0.016 - 0.988, P = 0.026; OR = 0.134, 95%CI 0.018 - 1.016, P = 0.020) and breast caner patients with abnormal Sapra Hilit (OR = 0.132, 95%CI 0.016 - 1.075, P = 0.049; OR = 0.132, 95%CI 0.017 - 1.042, P = 0.033). The 10-repeats allele and 10/10 genotype of 40 bp VNTR polymorphism of DAT1 may increase the risk of breast cancer with Uighur medicine abnormal Hilit in Chinese Han population from Xinjiang province and it is not correlated with the susceptibility to breast cancer with Uighur medicine abnormal Sapra Hilit.