Tumors Of Breast Cancer Patients Research Articles

Abstract IA27: Immunomodulation of breast cancer

Abstract Breast cancer is immunogenic and there is significant evidence that as breast cancer develops, tumors elicit an adaptive immune response. T-cells infiltrate most breast cancers and in several studies, the presence of elevated intratumoral CD8+ T-cells or elevated CD8+ T-cells in conjunction with a limited T-regulatory cell infiltration is associated with a favorable prognosis. Moreover, patients with breast cancer have been shown to develop immunity against specific proteins expressed by their tumors. Recent studies demonstrate that the development of humoral immunity directed against tumor associated antigens can be detected prior to a breast cancer diagnosis indicating the ability of the immune system to act as a sensor for exposure to aberrantly expressed proteins. Cellular immunity, directed against breast cancer antigens and elicited by exposure to malignancy, is present even while the disease progresses. The endogenous breast cancer specific cellular immune response is often of a Type II phenotype eliciting primarily antibody immunity rather than cytotoxic T-cells. Furthermore, type II cytokines secreted by T-cells actively dampen the development of a CD8 T-cell response. Type I cytokines, such as interferon gamma or TNF-alpha, are needed to activate antigen presenting cells allowing the presentation of tumor specific antigens via cross-priming, the primary mechanism by which cancer is recognized by T-cells. Immune cells which secrete Type I cytokines are rarely detected or found only in limited numbers in the tumors of most breast cancer patients. Several therapeutic modalities are under development and designed to increase or stimulate Type I T-cells capable of homing to breast tumors. Active immunization, targeting specific breast cancer antigens, has been shown to directly modulate the breast cancer microenvironment by enhancing cross-priming resulting in the development of epitope spreading. The identification of developing immunity against multiple tumor antigens during the course of treatment has been associated with a survival benefit after vaccination targeting HER-2/neu (HER2). High levels of vaccine induced Type I HER2 specific T-cells have been shown to be inversely associated with serum TGF-beta levels in patients with advanced stage breast cancer. Significantly increasing the number of Type I T-cells targeting breast cancer, via strategies such as adoptive T-cell therapy, has been associated with a measurable clinical response in advanced stage breast cancer. Ex vivo expansion of HER2 specific Th1 cells, after vaccine priming, and infusion of those cells into patients resulted in a 40% clinical response rate in patients with Stage IV treatment-refractory breast cancer. After infusion of Type I HER2 positive T-cells, responding patients developed multiple clonal CD4+ and CD8+ T-cell populations in their peripheral blood, which may be an indication of epitope spreading. The level of HER2 specific T-cells achieved in vivo and the development of a diversity of clonal T-cells was significantly associated with clinical response. Although breast cancer specific Type I T-cells may not be found in the malignancy at the time of diagnosis, such cells can be induced. The ability to circumvent the immune suppressive microenvironment opens the door for combination immune based therapies utilizing checkpoint blocking antibodies or specific chemotherapies shown to modulate immune suppression to further enhance the number and function of tumor specific Type I T-cells. Citation Format: Mary L. Disis. Immunomodulation of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA27.

Prognostic value of insulin-like growth factor 1 and insulin-like growth factor binding protein 3 blood levels in breast cancer

High circulating insulin-like growth factor 1 (IGF-1) levels are firmly established as a risk factor for developing breast cancer, especially estrogen positive tumors. The effect of circulating IGF-1 on prognosis once a tumor is established is unknown. The authors explored the effect of IGF-1 blood levels and of it's main binding protein, IGFBP-3, on overall survival and occurrence of second primary breast tumors in breast cancer patients, as well as reproductive and lifestyle factors that could modify this risk. Patients were accrued from six hospitals in the Netherlands between 1998 and 2003. Total IGF-1 and IGFBP-3 were measured in 582 plasma samples.No significant association between IGF-1 and IGFBP-3 plasma levels and overall survival was found. However, in a multivariate Cox regression model including standard prognostic variables high IGF-1 levels were related to worse overall survival in patients receiving endocrine therapy (HR = 1.37, 95% CI: 1.11, 1.69, P 0.004). These data at least indicate that higher IGF-1 levels, and as a consequence most likely IGF-1-induced signaling, are related to a less favorable overall survival in breast cancer patients treated with endocrine therapy. Interventions aimed at reducing circulating levels of IGF-1 in hormone receptor positive breast cancer may improve survival.

Effector memory CD8 T cells and central memory CD4 T cells dominate a proliferation deficient T cell population in the primary tumor of breast cancer patients (P2207)

Abstract Much of our knowledge on breast cancer derives from research on animal models of the disease. Studies on human breast cancer patient samples offers potential data that is more readily translatable to clinical therapeutics. We found a significant reduction in the proliferative capability of ex vivo T cells from primary tumor tissue compared to those from paired sentinel lymph nodes (SLNs). Central memory T cells have previously been shown to have a higher proliferative capacity than effector memory T cells. We found higher proportions of effector memory cells among tumor infiltrating CD8 T cells as compared to SLN CD8 T cells. Conversely, the CD4 compartment in both tumors and SLNs was dominated by central memory CD4 T cells. We therefore considered that the diminished T cell proliferative capacity may also be explained by a CD4 compartment comprised of anergic or exhausted cells. Further examination of the tumor and SLN CD4 T cells showed only a small population producing IFNγ or IL-17 in response to TCR stimulation, which may support this possibility. We hypothesize that these observations are symptoms of an inefficient T cell response to tumor cells in breast cancer patients. Diminished T cell proliferative capacity in the tumor may be explained by the predominant effector memory status of TIL CD8s and reinforced by the presence of anergic CD4 T cells. Future characterization of these T cells will be useful to design treatments that enhance their anti-tumor ability.

HER-2/neu Status: A Neglected Marker of Prognostication and Management of Breast Cancer Patients in India

Categorizing breast tumors based on the ER, PR and HER/Neu 2 receptor status is necessary in order to predict outcome and assist in management of breast cancer. Herfe we assessed this question in South Indian patients. A total of 619 formalin fixed paraffin embedded breast tumor tissues were collected from pathology archives after receipt of ethical clearance. With the help of primary and secondary conjugated antibodies, expression status of ER, PR and HER2/neu was determined. All the experimental data were assessed for correlations with histopathological features of tumors and clinical presentation of the subjects. In the present study, the ages ranged from 20-87 years with a mean of 50.0±12.q years, and majority of the tumors (84%) were of infiltrating duct cell carcinoma type. Assessment of ER, PR and Her-2/neu expression showed that 46% were triple negative. Interestingly, an inverse relation between ER, PR and HER-2/neu was apparent in 41.2% (p<0.0001) of the tumors, of which 24.5% (p<0.0001) were ER and PR co-negative but HER-2 positive. ER and PR positive tumors are less common (i.e<30%) compared to HER-2/neu positive tumors (i.e>50%) in Indian breast cancer patients, underlining the need for effective diagnostic screening and specific therapeutic managements in order to improve the survival rate of patients in low resource countries such as India.

Abstract 5341: Clinical relevance of metastasis-related miRNAs in early breast cancer.

Abstract Introduction It was recently demonstrated that miRNAs control breast cancer metastasis by influencing multiple metastatic steps. In this study we firstly evaluated the clinical relevance of specific miRNAs -recently shown to correlate with metastasis in breast cancer by exploring their expression profile in FFPEs samples of early breast cancer patients in respect to the clinical outcome of the patients. Finally, we investigated the correlations between the expression of these selected miRNAs and other parameters such as ER and PR expression. Materials and Methods One hundred and twelve (112) FFPE blocks were obtained from breast tumors of early breast cancer patients (follow-up period of 11 years) and 13 mammoplasty specimens from healthy tissues which were used as controls. The median overall survival (OS) and disease free interval (DFI) were 84 and 68 months respectively. We have chosen to quantify the expression of six metastasis related miRNAs, namely: miR-10b, miR-2,1 miR-205, miR-210, miR-335 and let-7a by RT-qPCR in the LightCycler (Roche). HER2, ER and PR expression of the primary tumors was detected by immunohistochemistry with the monoclonal antibody CB11 and with monoclonal antibodies to ER and PR, respectively, using the OPTIMAX automated system. For all miRNAs relative expression was determined using the ΔCq approach and expression values were normalized to miR-191 which has been shown to be a suitable reference miRNA. Results Kaplan-Meier survival curves demonstrated that patients with high miR-21 expression (n=56) had a significant shorter DFI than those with low miR-21 expression (P=0.043). Moreover, patients with low expression of miR-205 (n=57) had both shorter DFI and OS time than those with high expression levels (P=0.040 and P=0.047 respectively). However, the expression levels of the other four miRNAs were not related with the DFI and OS. We also found that in the ER-negative subgroup (n=46), disease relapse was more common in patients where miR-10b was over-expressed (P=0.019), while in the PR-negative subgroup (n=47), disease relapse was more common in patients with miR-21 overexpression (P&amp;lt;0.001) or miR-10b overexpression (P =0.016). Finally, in the group of PR-negative patients we noticed that patients who overexpressed miR-21 had shorter OS (P=0.021). Conclusions Quantification of metastasis related miRNAs expression in primary tumors can give important prognostic information in patients with early breast cancer. Citation Format: Athina N. Markou, George M. Yousef, Yu Liang, E Stathopoulos, Vassilios Georgoulias, Evi S. Lianidou. Clinical relevance of metastasis-related miRNAs in early breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2013-5341

Abstract 536: Insulin-like growth factor II (IGF-II) is a potential target for the treatment of triple negative breast cancers (TNBC).

Abstract In the United States, African American (AA) women have a lower incidence of breast cancer compared to Caucasian (CA) women, but higher overall mortality. Our lab have demonstrated that Breast Cancer from AA patients express significantly higher levels of IGF-II and show a higher activation of the IGF signaling system in African American (AA) when compared to Caucasian (CA) women. We showed that IGF-II promotes breast cancer progression and metastasis by inhibiting tumor cell death and promoting resistance to chemotherapy, thus contributing to increased breast cancer (BC) mortality. This study was designed to determine how IGF-II contributes to the aggressiveness of TNBC, the most deadly of breast tumors, which is highly prevalent among AA. To analyze IGF-II in a TNBC cell model, we chose cell lines established from tumors of AA and CA breast cancer patients. Expression of IGF-II and IGF-II regulated proteins mRNA expression was probed using the CFX96 real time PCR system. Similarly, IGF-II and related proteins expression was analyzed by western blots and chemiluminescence. We also characterized the epigenetic activation of IGF-II in these cell lines since variation in the expression of IGF-II alleles regulates a signature of proteins associated with tumor aggressiveness. Our exciting results demonstrated that higher IGF-II levels in TNBC cells from AA was associated with distinct epigenetic changes that included different promoter activation and higher levels of IGF-II translation which are inheritable traits. In contrast, TNBC from CA had higher IGF-II levels due to different molecular IGF-II regulation. Thus, IGF-II is an important biomarker and can be used as therapeutic target for treatment of TNBC. Citation Format: Vinodh Kumar Radhakrishnan, Qianwei Tan, Marino A. De León, Daisy D. De León. Insulin-like growth factor II (IGF-II) is a potential target for the treatment of triple negative breast cancers (TNBC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 536. doi:10.1158/1538-7445.AM2013-536

Abstract 504: ERα36 as a potential target in breast cancer that promotes an aggressive tumor phenotype in vitro and predicts outcome in vivo.

Abstract Steroid hormones exhibit dynamic effects on cancer progression. 17β-estradiol (E2), in particular, enhances tumor growth and development through a non-classical estrogen receptor (ER) found in the plasma membrane of triple negative breast cancer (TNBC) cells. Our lab has previously found that E2 rapidly increases protein kinase C (PKC) activity, which is associated with increased tumorigenicity, and promotes cancer cell survival and metastatic potential by acting through the ERα variant, ERα36. The aims of this study were to elucidate the mechanism by which ERα36 promotes cancer cell survivability and to determine if ERα36 levels in human cancer tissue can predict clinical outcome. Using specific inhibitors against signaling molecules we hypothesized to as part of the proposed anti-apoptotic pathway, we determined the effect of pathway inhibition on the protective effect of E2 against taxol-induced apoptosis in HCC38 TNBC cells. In order to evaluate these effects, we used MTT to measure cell viability, TUNEL to measure cell death, and caspase-3 activity as a marker of apoptosis. Our results show that the anti-apoptotic effect of E2 against taxol-induced apoptosis is mediated through ERα36 at the cell membrane, which activates a signaling cascade including phosphatidylcholine specific phospholipase D, lysophosphatidic acid, and phosphoinositide-3 kinase. In order to determine if ERα36 is a potential target for either diagnosis or treatment in breast cancer, tissue microarrays containing breast cancer patient tumor tissue were assayed for presence of ERα36 by immunohistochemistry (IHC) and blindly scored by three independent observers to quantify the number of positive cells and intensity. We also performed IHC for VEGF, which is associated with aggressive tumor formation. We performed categorical analysis using either chi-square test or fisher's exact test to obtain p-values for correlations of several variables with either ERα36 or VEGF, as well as correlation of ERα36 to VEGF. Our analysis included TNM classification, stage, age, receptor status(ER, PR, HER2), p53 status, and VEGF. Our analyses indicate ERα36 intensity was significantly correlated to age (p=.0450 for patients &amp;lt;50 years vs. patients &amp;gt;50) and VEGF (p=.0226) in our breast cancer cohort. Interestingly, receptor status was not correlated to ERα36, suggesting that ERα36 may be a possible target regardless of clinical ER status and classification of the cancer. The results of this study indicate that ERα36 mediates the anti-apoptotic effect of E2 in breast cancer and that it is also correlated to variables that may be associated with adverse outcome, such as tumor angiogenesis. This study therefore suggests that ERα36 may serve as a potential biomarker for further classification of breast cancer patients, as well as a potential target for therapeutic intervention in the development of adjuvant therapies. Citation Format: Reyhaan A. Chaudhri, Agreen Hadadi, Zvi Schwartz, Ruth O'Regan, Barbara D. Boyan. ERα36 as a potential target in breast cancer that promotes an aggressive tumor phenotype in vitro and predicts outcome in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 504. doi:10.1158/1538-7445.AM2013-504

The predictive and prognostic significance of pre- and post-treatment topoisomerase IIα in anthracycline-based neoadjuvant chemotherapy for local advanced breast cancer

To investigate the predictive and prognostic value of topoisomerase IIα (Topo IIα, Topo II) expression in the primary tumors and residual tumors of local advanced breast cancer (LABC) patients being treated with anthracycline-based neoadjuvant chemotherapy (NCT). The data from 283 LABC patients who had been treated with anthracycline-based neoadjuvant chemotherapy were collected. The expression of Topo IIα, HER-2 and other biomarkers was determined via immunohistochemical analysis in pre- and post-chemotherapy specimens. The status of pre-treatment biomarkers was correlated with the clinical response determined by the RECIST 1.1 criteria, whereas the post-treatment biomarkers were studied for prognostic value using the Cox model. By analyzing the complete data from 99 patients, the co-expression of HER-2/Topo IIα was found to be significantly correlated with the clinical response to chemotherapy (Logistic regression P=0.042). Notably, a 20% alteration in the Topo IIα status during neoadjuvant chemotherapy was found, which could also influence the sensitivity to treatment. With a survival analysis performed in 245 patients with residual tumors after NCT, node metastasis, HER-2 and Ki-67 were independent predictors of patient outcome. However, post-treatment Topo IIα expression demonstrated significant prognostic value in HER-2+ patients (P=0.002). A relatively lower disease-free survival and overall survival was observed in HER-2+/Topo- patients (log rank P=0.010 for DFS and P<0.001 for OS). Topo IIα, together with HER-2, might help to select for patients who could benefit from anthracycline-based neoadjuvant chemotherapy and identify non-complete responders at a higher risk of disease recurrence or death.

Abstract PR2: Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells

Abstract Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer cells and patient tumor specimens (LBC “instigators”) establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors (“responders”). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells (BMCs) enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGFR2+ tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically. This abstract is also presented as Poster B6. Citation Format: Timothy Marsh, Hanna Kuznetsov, Beth Markens, Zafira Castano, April Greene-Colozzi, Samantha Hay, Victoria Brown, Andrea Richardson, Sabina Signoretti, Elisabeth Battinelli, Sandra McAllister. Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR2.

Abstract P2-10-26: Association between circulating tumor cells and molecular breast cancer subtypes

Abstract Background and Objective: Circulating tumor cells (CTCs) provide crucial information for the management of breast cancer patients. CTCs analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. Several contradictory results presented about the breast cancer subtypes of CTCs recently. The aim of this study was therefore to analysis the association between the CTCs and molecular breast cancer subtypes. Methods: Blood samples were obtained from 143 breast cancer patients admitted to Breast Surgery Department, Affiliated Hospital of Medical College, Qingdao University. CTCs were detected by using immunomagnetic enrichment and a commercial kit, based on RT-PCR, was used to characterize CTCs. The expression of ER, PR, HER2 was detected by immunohistochemistry. The molecular subtypes were defined as: luminal A (ER+ and/or PR+, HER2−), luminal B (ER+ and/or PR+, HER2+), TNBC (ER−, PR−, HER2−), and HER2+, (ER−, PR−, and HER2+). The relations between the positive rate of CTCs and clinicopathological characteristics of primary tumor and molecular subtypes of breast cancer patients were analyzed. Results: Overall positive rate of CTCs was 39.8% (57/143). The positive rate of CTCs in patients with Luminal A, Luminal B, HER2+ and TNBC subtype was 33.8% (26/77), 47.1% (8/17),50.0% (6/12) and 45.9%(17/37). There was significant difference in positive rate of CTCs by different molecular subtypes (P = 0.0415), especially at different AJCC stages (P = 0.004). The positive rate of CTCs in patients with Luminal A subtype was significantly lower than patients with the others breast cancer subtypes (p = 0.0387). Conclusion: Our study suggests that presence of CTCs was more frequently found in patients with HER2+ and Luminal B subtypes. The presence of CTCs in patients with TNBC subtype was not as high as we wished. Theoretically, the positive rate of CTCs in TNBC subtype should be higher than that of others subtypes for being more aggressive histologically features and having increased potential to be invasive. One possible explanation for this might because of lacking epithelial markers expression and epithelial-mesenchyme transition (EMT) characteristics, CTCs of TNBC subtype are missed when using the standard EpCAM-based method. Shortage of patient's number was another possible reason. Further research on the Association between CTCs and molecular breast cancer subtypes will contribute to a better understanding of the biology of metastatic development in cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-26.

Identification of Luminal Breast Cancers That Establish a Tumor-Supportive Macroenvironment Defined by Proangiogenic Platelets and Bone Marrow–Derived Cells

Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer (LBC) cells and patient tumor specimens (LBC "instigators") establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors ("responders"). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGF receptor 2(+) tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically. Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy.

Lactate dehydrogenase, Catalase, and Superoxide dismutase in Tumor Tissue of Breast Cancer Patients in Respect to Mammographic Findings

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.

The prognostic and predictive value of mRNA expression of vascular endothelial growth factor family members in breast cancer: a study in primary tumors of high-risk early breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial

IntroductionThe main prognostic variables in early breast cancer are tumor size, histological grade, estrogen receptor/progesterone receptor (ER/PgR) status, number of positive nodes and human epidermal growth factor receptor 2 (HER2) status. The present study evaluated the prognostic and/or predictive value of vascular endothelial growth factor (VEGF) family members in high-risk early breast cancer patients treated with adjuvant chemo-hormonotherapy.MethodsRNA was isolated from 308 formalin-fixed paraffin-embedded primary tumor samples from breast cancer patients enrolled in the HE10/97 trial, evaluating adjuvant dose-dense sequential chemotherapy with epirubicin followed by cyclophosphamide, methotrexate, fluorouracil (CMF) with or without paclitaxel (E-T-CMF versus E-CMF). A fully automated method based on magnetic beads was applied for RNA extraction, followed by one-step quantitative RT-PCR for mRNA analysis of VEGF-A, -B, -C and vascular endothelial growth factor receptor (VEGFR) 1, 2, 3.ResultsWith a median follow-up of 8 years, 109 patients (35%) developed a relapse and 80 patients (26%) died. In high VEGF-C and VEGFR1 mRNA expressing tumors, ER/PgR-negative tumors (Fisher's exact test, P = 0.001 and P = 0.021, respectively) and HER2-positive tumors (P <0.001 and P = 0.028, respectively) were more frequent than in low VEGF-C and VEGFR1 expressing tumors, respectively. From the VEGF family members evaluated, high VEGFR1 mRNA expression (above the 75th percentile) emerged as a significant negative prognostic factor for overall survival (OS; hazard ratio (HR) = 1.60, 95% confidence interval (CI): 1.01 to 2.55, Wald's P = 0.047) and disease-free survival (DFS; HR = 1.67, 95% CI: 1.13 to 2.48, P = 0.010), when adjusting for treatment group. High VEGF-C mRNA expression was predictive for benefit from adjuvant treatment with paclitaxel (E-T-CMF arm) for OS (test for interaction, Wald's P = 0.038), while in multivariate analysis the interaction of VEGF-C with taxane treatment was significant for both OS (Wald's P = 0.019) and DFS (P = 0.041) and continuous VEGF-B mRNA expression values for OS (P = 0.019).ConclusionsThe present study reports, for the first time, that VEGF-C mRNA overexpression, as assessed by qRT-PCR, has a strong predictive value in high-risk early breast cancer patients undergoing adjuvant paclitaxel-containing treatment. Further studies are warranted to validate the prognostic and/or predictive value of VEGF-B, VEGF-C and VEGFR1 in patients treated with adjuvant therapies and to reveal which members of the VEGF family could possibly be useful markers in identifying patients who will benefit most from anti-VEGF strategies.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998

Prognostic significance of RACGAP1 mRNA expression in high-risk early breast cancer: a study in primary tumors of breast cancer patients participating in a randomized Hellenic Cooperative Oncology Group trial

RACGAP1 is a Rac GTPase-activating protein involved in cell growth regulation, cell transformation and metastasis. The aim of the present study was to explore the prognostic and/or predictive significance of RACGAP1 mRNA expression on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients and compare it to that of Ki67 protein expression and to the Nottingham prognostic index (NPI). A total of 595 high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative dose-dense sequential chemotherapy with epirubicin followed by CMF with or without paclitaxel. RNA was extracted from 314 formalin-fixed paraffin-embedded primary tumor tissue samples followed by one-step quantitative RT-PCR for assessing RACGAP1 mRNA expression. High RACGAP1 mRNA expression (above the median) was associated with poor DFS (log-rank, p = 0.002) and OS (p < 0.001). High histological grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of RACGAP1. Results of the Cox multivariate regression analysis revealed that high RACGAP1 mRNA expression independently predicted poor overall survival (Wald's p = 0.008). High Ki67 protein expression was also an adverse prognostic factor for death (p = 0.016), while high NPI score values were not. High RACGAP1 mRNA expression, as assessed by qRT-PCR, was found to be of adverse prognostic significance in high-risk early breast cancer patients treated with dose-dense sequential chemotherapy. The utility of RACGAP1 mRNA expression in patient selection for treatment with aggressive chemotherapy regimens should be further explored and validated in larger cohorts.

Is the prevalence of ER-negative breast cancer in the US higher among Africa-born than US-born black women?

Previous studies have reported that the prevalence of ER-negative tumors in breast cancer patients is much higher in black women than in white women in the US. Herein, we examine whether the proportion (prevalence) in Africa-born black breast cancer patients residing in the US is similar to those in US-born black patients. We obtained information on invasive female breast cancers diagnosed during 1996-2008 in 17 Surveillance Epidemiology and End Results cancer registries according to select place of birth: Western-Africa-born, Eastern-Africa-born, Jamaica-born, and US-born blacks and US-born whites. The majority of Western-Africa-born and Eastern-Africa-born blacks were from Nigeria (64 %) and Ethiopia (74 %), respectively. We examined group variations in ER status using Chi-squared tests and the prevalence of ER-negative tumors in Africa-born blacks compared to US-born blacks, expressed as prevalence ratio (PRR), using multivariable regression models. The prevalence of ER-negative tumors significantly varied from 22.0 % (n = 41/186) in Eastern-Africa-born to 32.9 % (n = 47/143) in Western-Africa-born blacks. After adjustment for differences in age at diagnosis and other covariates, compared to US-born blacks, the prevalence was similar in Western-Africa-born (PRR = 0.87; 95 % CI 0.70-1.08) and Jamaica-born blacks (PRR = 0.88; 95 % CI 0.74-1.03), but significantly lower in Eastern-Africa-born blacks (PRR = 0.58; 95 % CI 0.44-0.75). Notably, the ER-negative prevalence in Eastern-Africa-born black was comparable to the US-born whites with breast cancer. Our findings highlight the heterogeneity of breast cancer among black women in the US, which should be considered in future studies of hormone receptor status in these women.

Breast cancer stem cells: A novel therapy for breast cancer.

e13030 Background: Simple BRCA screening is insufficient for ‘event-free survival’ as breast cancer is clinically and pathologically an extremely heterogeneous disease. Targeting Breast Cancer Stem Cells (BCSCs) present in bone marrow and breast tissues is a lucrative alternative. Identification of BCSCs is salient aspect of our research. Invasive and mesenchymal property of BCSCs with CD44+/CD24low/ALDH1+ phenotype has made them a promising target for eliminating metastatic capacity of primary tumors. We hypothesize that ability to therapeutically attack stem cell hinges upon identifying unique targets like cell surface markers and this will decide development of specific target therapies. Methods: A total of 10 early chemo-naive patients with biopsy proven triple-negative metastatic breast cancer in the age group of 18-36 yr.s (mean age 28 yr.s) were selected randomly and tested for CD44/CD24 cell surface markers following immunosorting using magnetic cell sorter and immunophenotyping by flowcytometric analysis. Isolated BCSCs were cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel. Correlation was drawn between cell differentiation, % of stem cells and drug response. Accordingly chemotherapy was designed for a particular patient. % of BCSCs in pre- and post-chemotherapeutic condition was further compared. Results: We have detected BCSCs in 90% of cases. Among positive samples, 89% patients showed platinum sensitivity and rest were found to be anthracycline sensitive. No sensitivity to docetaxel was observed. In lieu of this, cisplatin was applied in vivo and % of BCSCs came down to 6.58% from initial 11.16% (for a representative case). Conclusions: Thus primary aim to target BCSCs at the onset of tumors in breast cancer patients to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival. Finally, if no BCSCs prevail after chemotherapy, then patients would be kept under observation and if traces are found, we would proceed to targeted therapy trial like PARP inhibitor or autologous stem cell replacement.

52P Breast Cancer Stem Cells: A Novel Therapy for Breast Cancer

ABSTRACT Simple BRCA screening is insufficient for ‘event-free survival’ as breast cancer is clinically and pathologically an extremely heterogeneous disease. Targeting breast cancer stem cells (BCSCs) present in bone marrow and breast tissues is an attractive alternative. Identification of BCSCs is the salient aspect of our research. The invasive and mesenchymal properties of BCSCs with CD44+/CD24low/ALDH1+ phenotype has made them a promising target for eliminating the metastatic capacity of primary tumors. We hypothesize that the ability to therapeutically attack stem cells hinges upon identifying unique targets such as cell surface markers which will be decisive for the development of specific target therapies. A total of 10 early chemo-naive patients with biopsy-proven triple-negative metastatic breast cancer in age group 18-36 years (mean age 28 years) were selected randomly and tested for CD44/CD24 cell surface markers following immunosorting using a magnetic cell sorter and immunophenotyping by flow cytometric analysis. Isolated BCSCs were cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel and correlation was drawn between cell differentiation, percentage of stem cells and drug response. Accordingly chemotherapy was designed for a particular patient. The percentage of BCSCs in pre- and post-chemotherapeutic conditions were further compared. We have detected BCSCs in 90% of the cases. Among positive samples, 89% of patients showed platinum sensitivity and the rest were found to be anthracycline sensitive. No sensitivity to docetaxel was observed. In lieu of this, cisplatin was applied in vivo and the percentage of BCSCs came down to 6.58% from an initial 11.16% (for a representative case). Thus the primary aim to target BCSCs at onset of tumors in breast cancer patients to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate the ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival. Finally, if no BCSCs were to prevail after chemotherapy, then patients would be kept under observation and if traces were found, we would proceed to stem cell replacement. Disclosure All authors have declared no conflicts of interest.

Abstract 701: TUMOR lymphocyte infiltration, expression of CD4, CD8, FOXP3, CD34 molecules and their relationship with tumor evolution after primary treatment in breast cancer patients

Abstract Considering that, approximately, only half of breast cancer patients respond to present therapies, a better knowledge of breast cancer biology, one of the most common neoplasms in women, will enable to design better therapeutic approaches. It has been proposed that different tumor characteristics like inflammatory infiltrate intensity, number of T regulatory (Tregs) cells and density of tumor vasculature could be useful to anticipate the response to therapy. Our aim was: a) to analyze the lymphocytic infiltrate and the expression of CD4, CD8, CD34 (endothelial marker) and Foxp3 (Treg marker) in primary tumors of breast cancer patients, and b) to relate them with clinical evolution (disease free: DF or relapsed: R) after 5 years from the primary treatment (surgery). There were analyzed archive samples of ductal mammary tumors (n=27), mostly in stages I and II. The expression of CD4 and CD8 in intra- and peri-tumoral lymphocytes was determined by immunohistochemistry (IHC) and confirmed by confocal microcopy; also the expression of Foxp3 in lymphocytes and tumor cells and CD34 in endothelial cells was evaluated by IHC. The lymphocytic infiltrate was assessed in histological sections stained with hematoxylin-eosin. The quantification was done in 20 high magnification fields and a score ranging from 0 (null) to 6 (high) was utilized. In DF patients (n=22) the intratumoral lymphocytic infiltrate (median [range]: 3 [0-6]) was more intense than that of R patients (n=5), (2 [0-2]), (P=0.05). For CD4, CD8, CD34 and Foxp3 molecules no differences between groups of patients were observed, being their expression highly variable: the score for CD34 varied from 1 to 6 and for the other three molecules from 0 to 6, in either group. In conclusion, the presence of an intense intratumoral lymphocytic infiltrate could be a prognostic marker of good response to treatment, at least in the first 5 years after surgery. These results warrant the study of higher number of tumor samples, since its confirmation could give support for its use in the clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 701. doi:1538-7445.AM2012-701

Abstract 4158: MEKK1 regulates DMP1 transcriptional activity via phosphorylation and predicts breast cancer patient outcome

Abstract Breast cancer remains a significant public health issue in the industrialized countries. Physicians depend on surgery, cytotoxic adjuvant chemotherapy, or radiation for treatment of breast cancer patients. Few novel therapies are approved for clinical use. Although existing therapies are effective in controling the disease in many patients, the cytotoxic side-effects make them limited. This is especially the case in the patients with early stage breast cancer whose prognosis with surgery alone is good and avoiding adjuvant chemotherapy would spare them toxic side effects. On the other hand, significant fraction of patients relapses and develops metastasis. Therefore, it is critical to identify markers for better breast cancer patient stratification based on their prognosis that would guide physicians in selection and aggressiveness of therapies. Recently, we linked transcription factor DMP1 (cyclin D binding protein 1, Dmtf1) as a critical tumor suppressor that blocks proliferative signals from ErbB2 and oncogenic Ras by activating p14Arf-p53 pathway. hDMP1 is hemizygously deleted in ∼50% of human breast tumors that retain wild-type ARF and p53. In this study we identified an upstream kinase, MEKK1, which cooperates with Dmp1 to activate Arf transcription. In constitutively active form (C-terminal kinase domain or CA-MEKK1), MEKK1 increased transcriptional activity of Dmp1 via direct phosphorylation, while the full length (regulatory and kinase domain) form behaved as a feedback inhibitor by increasing Dmp1 ubiquitination. Phosphorylation sites on DMP1 that increase its transcriptional activity were mapped to the C-terminal transactivation domain. Expression of CA-MEKK1 in breast cancer cell lines activated endogenous Arf-p53 pathway. hMEKK1 is located on human 5q11 chromosome, a locus frequently deleted in breast and lung cancer. Since MEKK1 appeared to be an activator of p53 tumor suppressor pathway, we wondered if MEKK1 is involved in human cancer. Using DNA from breast cancer patient tumors and matched normal tissue, we analyzed hMEKK1 gene deletion using specific loss of heterozygosity (LOH) primers. MEKK1 was found hemizygously deleted in ∼20% of patients. Immunohistochemistry confirmed reduction of MEKK1 protein intensity to LOH(+) compared to LOH (-) patients. In the independent cohort of breast cancer patients, we show that low MEKK1 mRNA expression is associated with adverse outcome. Here we show that constitutively active MEKK1 is a novel activator of Dmp1-Arf-p53 pathway via Dmp1 phosphorylation. The MEKK1 gene was frequently deleted in breast tumor tissue and its expression was correlated with disease outcome. Overall, MEKK1 is a potential prognostic/predictive indicator for breast cancer and could be used by physicians to better stratify patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4158. doi:1538-7445.AM2012-4158

Abstract LB-193: Ganglioside GD2 identifies cancer stem cells and inhibition of GD2 biosynthesis by targeting GD3 synthase exerts antitumor effects

Abstract Currently, only very few markers either as single or in combination are available to identify cancer stem cells (CSCs). In this report, we identified that the ganglioside GD2, a marker known to express on mesenchymal stromal cells (MSCs) is expressed on a small fraction (5.5% ± 3.4%) of transformed human mammary epithelial cells (HMLER) and breast cancer patient tumors. FACS sorted GD2+ cells appear spindle shaped and proliferate 5 fold slower compared to GD2- cells in-vitro. Analysis of breast cancer cell lines (n=12) indicated that GD2 expression varies and that the basal breast cancer cell lines have a higher percentage of GD2+ cells (median 9%, range 1.2-17%, n=6), as compared to their luminal counterparts (median 0.2%, range 0-3%, n=6, p&amp;lt;0.001). Functional analysis revealed that GD2+ HMLER and MDA-MB-231 cells produced 2-5 fold more mammospheres in-vitro (p&amp;lt;0.003) and tumors in-vivo than GD2- cells (p&amp;lt;0.04). Recent reports suggest that breast tumor cells with CD44highCD24low phenotype exhibit CSCs characteristics. Interestingly, the majority (94% ± 3.5%) of GD2+ cells were characterized by the CD44highCD24low phenotype in HMLER cells. Analysis of primary breast cancer samples (n=10) revealed that GD2 is variably expressed in these samples (median 4.35%, range 0.5%-35.8%) and 95.5% ± 2.7% of GD2+ cells co-segregated with CD44highCD24lowCD45- phenotype. In contrast, only 2.4%± 0.4% of GD2- cells displayed this phenotype. Gene-chip and quantitative RT-PCR analysis revealed that GD3 synthase (GD3S), the enzyme responsible for synthesis of GD3, the precursor of GD2, was expressed 10-fold higher in GD2+ cells compared to GD2- HMLER and MDA-MB-231 cells. Analysis of GD2 expression in HMLER cells induced to undergo EMT revealed that the percentage of GD2+ cells increased 6-7 fold and the expression of GD3S &amp;gt; 10 fold. Moreover, we observed spontaneous generation of GD2+ from GD2- cells and vice versa in both in-vitro and in-vivo, suggesting a role of EMT in this process. Stable knock-down of GD3S in MDA-MB-231 cells using shRNA impaired in-vitro matrigel invasion by more than 10-fold and completely abolished tumor growth in-vivo. Importantly, Triptolide, an anti-inflammatory and anti-cancer drug, which was recently shown to inhibit GD3S expression in melanoma cells, also inhibited GD3S expression in MDA-MB-231 and SUM159 cells by &amp;gt;95% in a dose dependent manner and thereby inhibited growth of GD2+ cells in a time dependent manner. Intra-peritoneal administration of Triptolide (0.15mg/Kg/day) in NOD/SCID mice bearing MDA-MB-231 breast tumors completely eliminated tumors in 50% and reduced the tumor volume 7- to 8-fold in 25% of the mice. In conclusion, we identified GD2 as a new CSC specific cell surface marker and GD3 synthase as a potential therapeutic target for CSCs, with the potential of improving survival and cure rates of patients with breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-193. doi:1538-7445.AM2012-LB-193