bluuebunny/crossref_metadata_2025 huggingface dataset:
Practical use of circulating tumor cells for the management of subsets of metastatic breast cancer patients in a community oncology practice.
Authors: Dropkin EK , Graham ML
Published: December 2010

Abstract #5024

Background : An elevated Circulating Tumor Cell Count (CTC) >5 in 7.5 ml of blood predicts worse outcome in metastatic breast cancer (MBC) patients (pt). While the FDA has approved use of this test, its systematic use in practices has been uncommon. We have measured CTC's in all MBC pts in our community oncology practice receiving hormonal therapy or chemoRx +/- biologic therapies to determine if a strategy to treat all pts to a sustained CTC count of < 5 could decrease the death rate, and if recognized molecular subsets of breast cancer [ER+, HER2 (H2)-; Triple Negative; H2+] have different patterns of CTC behavior. Methods: Initially serial CTC levels were measured by the CellSearch technology (Veridex, LLC; Warren NJ) using two commerical laboratories in all pts whether beginning a new therapy with initial recurrence or progression, or if already underway with treatment. Pts had CTC's measured from Nov 20, 2006 thr. June 10, 2008, an 18 mo. period Over time, the majority of pts have begun new treatments with progression, or with new diagnoses. If pts with elevated CTC's improved to <5 for a sustained 3-4 month period of time, we sought less morbid hormonal therapies if ER+, or lower toxicity ChemoRx +/- biologic therapy if ER- or hormone refractory. Results: 376 tests for CTC's were carried out in 72 pts for an average of 5.2 tests per pt (range 1-19). The distribution of the 72 pts according to the three molecular subtypes, the frequency of those measuring >5, >10 or >100 CTC's at any time, and the associated death rates for each group, are shown in Table 1.    High numbers of CTC's were seen in each of the three subsets of MBC, and each subtype also had pts with no elevated levels. 2/3 of deaths occurred in pts with 100 or more CTC's at some point in their treatment course. Of pts with these high levels, more than 50% eventually died, while only 3% died over 18 months if serial measurements showed no levels above 5 CTC's. Deaths from MBC in the practice have been reduced from 20 in 2005 and 15 in 2006 to 9 in the 18 mo. during which the CTC's have been uniformly applied in 2007 and 2008. Conclusion: Very high levels of CTC's > 100 constitute an oncologic emergency. The overall death rate in a community oncology practice may be reduced by uniform application of CTC technology.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5024.
Isolation of circulating tumour cells in patients with metastatic breast cancer by filter technology.
Authors: Elst H , Van Der Auwera I , Vermeulen P , Prové A , Van Dam P , Huget P , Dirix L
Published: December 2010

Abstract #5026

Introduction. Metastasis is the leading cause of cancer-related death. For several decades, numerous attempts have been made to develop reliable assays enabling the detection and enumeration of circulating tumor cells (CTC) in the peripheral blood. The LeukoLOCK™ Total RNA Isolation System (Ambion®) combines leukocyte depletion filter technology intended to isolate leukocytes from whole blood, with RNALater® as a means of stabilizing the RNA in the cells captured on the filter. We hypothesized that this innovative system would also allow for the isolation of carcinoma cells by filtration, because of their larger size in comparison to peripheral blood leukocytes. The RNA extracted from the filter device could thus be used for further quantification of CTCs by qRT-PCR.  Materials and methods. The CellSearch™ Circulating Tumor Cell Test (Veridex®) was used to enrich and enumerate CTCs in all of the blood samples. The CellSearch System™utilizes iron nano-particles (ferrofluid) conjugated to EpCAM to capture CTC. Immuno-fluorescent staining for cytokeratins (CK) 8,18,19 and the nucleus (DAPI) is used to positively identify intact CTC. CTC are defined as nuclear cells, expressing cytokeratins and lacking CD45 expression. Blood samples of 5 patients with known elevated CTC were screened again after filtration with the LeukoLOCK™filter device to ensure that CTC did not slip through the filter (Table 1). The cell count obtained with the Veridex system was compared in these 5 samples, with the quantification CK19 and mammaglobin (MAM) transcripts obtained from the cells retained by the filter. In a further study on 79 patients with metastatic breast cancer, CTCs were enumerated using LeukoLOCK™Total RNA Isolation System (Ambion®) as a means of RNA isolation and compared with the Veridex system. qRT-PCR was performed to detect the presence of CTC transcripts for CK19 in the total RNA obtained from the cells captured on the filter in all of the 79 cases.  Results. In only one sample (PAT113) could one CTC be detected in the filtered blood. The values obtained for RGE for both CK19 and MAM are in agreement with that observation.    A significant correlation was found between CTC levels and qRT-PCR normalized relative gene expression (nRGE) values for CK19 originating from the total cell population captured by the LeukoLOCK™filter device (n=79, r=0.446, p<0.001).  Conclusion. The LeukoLOCK™ system does not only enrich for leukocytes but can be considered as a reliable system for the enrichment and stabilization of CTC in patients with MBC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5026.
Cyclin overexpression inhibits Smad 3 tumor suppression in breast cancer cells.
Authors: Zelivianski S , Cooley A , Jeruss JS
Published: December 2010

Abstract #5033

Introduction: The transcription factor Smad 3, a member of the TGFβ signaling cascade, contributes to G1 cell cycle arrest in breast cancer cell lines. Overexpression of the cell cycle regulatory protein cyclin E, and its low molecular weight (LMW) counterparts, has been associated with poor prognosis in breast cancer. Cyclin E has been shown to mediate phosphorylation within or near the linker region of Smad 3 by cyclin dependent kinase 2 (CDK 2), and to inhibit Smad 3 activity in normal mouse fibroblasts and epithelial cells. We hypothesize that overexpression of cyclin E may exert tumorigenic effects in breast cancer cell lines through the functional inhibition of Smad 3 mediated by CDK 2 phosphorylation.  Methods: Endogenous levels of Smad 3 and phosphorylated Smad 3 in parental, vector only control, full length (FL), and two LMW forms of cyclin E overexpressing MCF-7 breast cancer cell lines were determined by western blotting. To elucidate the impact of cyclin E overexpression on Smad 3 function, constructs containing an empty vector, WT Smad 3, Smad 3 with a CDK phosphorylation site mutation within the linker region (Thr178), and outside the linker region (Thr8) were co-transfected with a Smad 3 reporter into the panel of MCF-7 cells. CDK2 or scrambled control siRNA was also transfected into the study panel of cells. Smad 3 function was then evaluated by luciferase reporter assays.  Results: MCF-7 cells overexpressing FL or LMW cyclin E had a higher level of P- SMAD 3 as compared to the parental and vector only control cells. Total SMAD 3 levels were similar in all cells examined. Transfection of WT Smad 3 elicited an increase in reporter activity in parental and vector control cells, while both FL and LMW forms of cyclin E overexpressing cells were resistant to WT Smad 3 reporter induction. In parental and vector control MCF-7 cells, expression of the Smad 3 Thr178 mutation resulted in a three-fold induction in Smad 3 reporter activity as compared to the WT Smad 3 response. In FL and LMW cyclin E overexpressing cells, induction by the Thr178 mutation was more pronounced, as seen by a 5-fold and 8-fold induction of reporter activity in FL and LMW cyclin E cells, respectively. Transient transfection of the Thr8 mutation failed to evoke a reporter response in any of the panel of MCF-7 cells. MCF-7, FL, and LMW cyclin E cells demonstrated the greatest fold induction of Smad 3 reporter activity when transfected with CDK 2 siRNA.  Conclusions: Overexpression of cyclin E may circumvent the growth inhibitory effects of Smad 3 signal transduction through the modulation of Smad 3 function. Specifically, this work indicates that cyclin E mediates Smad 3 inhibition in a CDK 2 dependent fashion in breast cancer cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5033.
Induction of cell cycle arrest and apoptosis in breast cancer cells by troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand.
Authors: Youn H , Lee B , Jung S , Park M , Yoon J , Lee J , Choi M
Published: December 2010

Abstract #5034

Background: Peroxisome proliferator-activated receptor-gamma(PPAR-γ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. And, it is reported that PPAR-γ ligands could serve as negative regulators of breast cancer development and progression, but their mechanism is still unknown. Here we wished to determine whether the PPAR-γ ligand induces cell cycle arrest and apoptosis of MDA-MB-231(ERα-negative) and MCF-7(ERα-positive) breast cancer cell.  Methods: The effect of PPAR-γ ligands on the cell viability of breast cancer cells was determined using mitochondrial tetrazolium(MTT) assay. The cell cycle distribution and apoptosis induction were evaluated by using the flow cytometry. The expression of apoptosis-related proteins were measured with Western blot analysis. Statistical analysis was performed using Student's t test, and p<0.05 was considered significant.  Results: The treatment of MDA-MB-231 cell with PPAR-γ ligand, troglitazone was shown to induce cell cycle G1 arrest and induction of apoptosis. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in phosphorylated retinoblastoma(pRb), cyclin D1, D2, D3, cyclin dependent kinase(Cdk) 2, 4, and 6 expression as well as a significant increase in Cdk inhibitor, p21 and p27. Troglitazone showed antiproliferative effect on MCF-7 cell with tamoxifen, respectively and synergically. Troglitazone and tamoxifen could induce G1 arrest and apoptosis of MCF-7 cell, through upregulation of Bax and downregulation of Bcl-2 and cyclin D1.  Conclusion: PPAR-γ ligand, troglitazone induces cell cycle arrest and apoptosis of MDA-MB-231 cell and increases the sensitivity of anti-hormonal therapy in MCF-7 cell. These results suggest that troglitazone has anticancer effect on both ERα-negative and positive breast cancer cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5034.
Prevention of primary and contralateral breast cancer by risk reducing salpingo-oophorectomy in high risk women with a BRCA1/2 mutation or familial predisposition.
Authors: Heemskerk-Gerritsen BA , Hooning MJ , Collee M , Tilanus-Linthorst MM , Bartels CC , van den Ouweland A , Ansink A , Seynaeve C
Published: December 2010

Abstract #504

Introduction: Risk reducing salpingo-oophorectomy (RRSO) has become an important preventive option against ovarian/fallopian tube cancer, while it is also risk reducing regarding breast cancer (BC) in women with a BRCA1 or BRCA2 mutation. Recently a difference in efficacy of RRSO concerning the occurrence of BC has been suggested for BRCA1 and BRCA2 mutation carriers, while no data are available on the protective effect of RRSO in women from a hereditary breast/ovarian cancer (HBOC) family without a proven BRCA mutation. Further, it is not known whether the BC risk reduction after RRSO equally applies to primary (PBC) and contralateral BC (CBC).  Methods: The occurrence of PBC and CBC was prospectively studied in 639 women ≥ 30 years (330 BRCA1 mutation carriers, 117 BRCA2 mutation carriers, and 192 HBOC women) who were enrolled in a breast/ovarian cancer surveillance program at the Rotterdam Family Cancer Clinic. Women with a history of BC without recurrent disease were eligible if BC treatment did not involve adjuvant systemic therapy. Demographics and follow-up (FU) information up to December 31, 2007, were extracted from the medical records. Ninety-eight BRCA1 mutation carriers, 33 BRCA2 mutation carriers and 15 HBOC women eventually underwent RRSO. Results were analyzed separately for women being at risk for PBC (n=489) and for CBC (n=164). The effect of RRSO on the incidence of BC was analyzed using a Cox proportional hazards model in which RRSO was modeled as a time-dependent covariate.  Results: During a mean FU of 5.5 years, RRSO was associated with a reduction in BC risk of 48% (HR=0.52; 95% CI, 0.26-1.02; p=0.058) versus 81% (HR=0.19; 95% CI, 0.02-1.95; p=0.161) in BRCA1 and BRCA2 mutation carriers, respectively. In BRCA1 mutation carriers, PBC was reduced by 74% (HR=0.26; 95% CI, 0.08-0.89; p=0.032), and CBC by 29% (HR=0.71; 95% CI, 0.23-2.16; p=0.544). In BRCA2 mutation carriers no cases of PBC occurred after RRSO (HR=0,00; 95% CI, not estimable), while 1 CBC was diagnosed (HR=0.25; 95% CI, 0.01-5.62; p=0.379). In HBOC women no BC cases were observed after RRSO.  Conclusions: RRSO is associated with a BC risk reduction in both BRCA1 and BRCA2 mutation carriers. The risk reductive effect is more pronounced regarding PBC as compared to CBC in BRCA1 mutation carriers. Although a similar trend is suggested for BRCA2 mutation carriers, larger numbers and longer FU are needed to draw firm conclusions.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 504.
Immune cells from pleural fluids of patients with inflammatory breast cancer are immunocompetent.
Authors: Cohen EN , Jimenez C , Gao H , Lee B , Li C , Krishnamurthy S , Woodward WA , Andreopoulou E , Bethancourt DL , Hortobagyi GN , Cristofanilli M , Reuben JM
Published: December 2010

Abstract #5044

Background: Malignant pleural effusion is a debilitating complication of metastatic breast cancer (MBC), including inflammatory breast cancer (IBC). The cellular components in pleural effusion are important indicators of pathologic process. Although pleural fluids from patients with IBC contain tumor cells and immune cells, little is known about the phenotype and function of the immune cell infiltrate. The objective of the study was to determine the immunophenotype and function of the cellular immune cells in pleural effusion of patients with pleural metastases.  Methods and Patients: Pleural effusions from 9 women with MBC, 6 with IBC, median age 56 years (range 44-75 yrs), were submitted for pathological review. Mononuclear cells (MNC) were reacted with a cocktail of monoclonal antibodies to detect surface antigens by 8-parameter flow cytometry. In addition, MNC were activated through the T-cell receptor (TCR) with immobilized anti-CD3 and soluble anti-CD28 to assess intracytoplasmic syntheses of pro-inflammatory [interleukin (IL)-2, tumor necrosis factor (TNF)-a and interferon-gamma (IFN-γ)] and anti-inflammatory (IL-10) cytokines by TCR-activated CD4+ and CD8+ T cells. Data are presented as the mean (±SEM) of TCR-activated CD4+ and CD8+ T-cells that synthesized inflammatory and anti-inflammatory cytokines.  Results: Upon pathological review, the mean (±SEM) number of tumor cells, MNC, and neutrophils were 20.2% ± 7.4% (range, 2%-72%), 62.4% ± 7.7% (range, 25%-96%), and 17.3% ± 5.9% (range, 0%-50.0%), respectively. The MNC consisted of 83.1% ± 8.2% CD3+ pan-T, 58.2% ± 7.0% CD4+ T, 24.2% ± 5.2% CD8+ T, 2.8% ± 0.9% CD19+ B, 4.0% ± 0.7% natural killer (NK), 3.4% ± 1.8% NKT, and 0.9% ± 0.2% dendritic cells, respectively. The CD4/CD8 T-cell ratio was 3.62 ± 0.78. The percentages of CD4+ and CD8+ T cells that synthesized inflammatory and anti-inflammatory cytokines following activation through the TCR-activated are listed in Table 1.    Conclusion: There were no statistically significant differences between pleural fluids of IBC and non-IBC subjects with respect to tumor cell involvement, MNC phenotype, or T-cell function. Moreover, the function of pleural fluid T cells of MBC patients were similar to that of normal peripheral blood T cells (historical data, not shown). These data suggest that T-cells in the pleural fluids of patients with MBC are immune competent and can be activated through the TCR to synthesize pro- and anti-inflammatory cytokines.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5044.
A new paradigm for African American breast cancer involving stem cell differentiation.
Authors: Latimer JJ , Lalanne NA , Myers NT , Courtney AB , Mock L , Grant SG
Published: December 2010

Abstract #5051

Background: African American (AA) women have a higher incidence of breast cancer (BC) than white women before age 40, and are more likely to die from BC at every age. These differences are often attributed to socio-economic factors, however, there may be intrinsic biological differences in the breast tissue of AA and white women. For example, triple negative breast tumors are more prevalent in AA women than in non-Hispanic white women, although neither population is monolithic in its breast cancer phenotypes. We hypothesis that intrinsic biological differences between women of AA and European-white ancestry might be revealed by differences in growth and differentiation in vitro, reflecting underlying differences in the proportions and/or potency of breast epithelial stem cells.  Methods: Our laboratory has developed a novel tissue engineering system for Human Mammary Epithelial Cells (HMEC), both normal and malignant. This system allows for long-term establishment of non-diseased primary cultures that begin as 3-dimensional attached “epispheres”, that are structures made up of 40-100 epithelial cells. These non-diseased epispheres subsequently differentiate into functional, organotypic branching ducts and lobules that demonstrate ESA and CK -18,-19 staining, lumen, polarized nuclei, desmosomes, microvilli on apical surfaces and casein secretion.  Results: We have established primary HMEC cultures from 36/36 breast reduction mammoplasty tissues, including 9 AA donors matched in socioeconomic status. We have found: 1) The more children a woman had, the less likely her breast tissue was to form ductal structures in vitro. This finding is consistent with the idea that lactational differentiation decreases the number of pluripotent stem cells in the breast. 2) Pre-menopausal breast tissue was more likely to form ductal structures than post-menopausal tissue. 3) Race was a modifying factor in the ability to form ductal architecture in culture, significantly affecting the rate of differentiation. Putative mammary stem cells were analyzed by flow cytometry (CD44+, CD24-, MUC1-, α6 integrin+) and cloning. Stem cell proportions varied widely (range 0.5-15%), with no obvious association with race.  Discussion: One interpretation of the hormonal risk factors for BC involves the susceptibility of undifferentiated breast tissue to transformation. In other systems, especially the gonads, interference with terminal differentiation is also associated with susceptibility to transformation. If AA breast tissue has more robust differentiation potential than that of white women, it may also contain stem cells more likely to transform into an aggressive tumor type.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5051.
Detection of tumor stem cells among circulating epithelial tumor cells (CETC) and relationship to therapy response.
Authors: Hekimian K , Stein EL , Pachmann U , Pachmann K
Published: December 2010

Abstract #5053

Background: It has been shown that the metastatic process is highly inefficient. Thus in the experimental setting, using breast cancer cell lines, only 0,1 to 0.01% of the injected cells are capable of forming metastases. With respect to cells released form primary tumors it is neither known, how many cells survive in the circulation nor what proportion is capable of forming metastases. Therefore we have attempted to determine the fraction of stem cells among the CETC and to correlate their frequency to relapse.  Material and Methods: Tumor cells were quantified with an automated microscope (Laser Scanning Cytometer®, Icis or ScanR) from anticoagulated blood drawn before each new therapy cycle from patients treated with systemic chemotherapy after surgery and subsequent maintenance therapy. After red blood cell lysis leucocytes were stained with PE-anti-CD45 and tumor cells with FITC-anti-EpCAM. Tumor stem cells were tried to define in 2 ways: 1. Using PE-conjugated anti-CD24 Epithelial antigen positive cells were counterstained and the CD24 negative cells determined. 2. Using the Aldefluor method epithelial cells staining intracellularly positive were determined and changes registered as % increase or decrease. The response to therapy was correlated to outcome.  Results: Both methods were well applicable. With the anti-CD24 counter stain, most CETC stained positively. A minor fraction of less than 1% of CETC was lacking CD24 staining and was regarded as the CD24 negative stem cell population. With regard to the Aldefluor method, both green fluorescing FITC anti-CD326 and red fluorescing Dye-anti-CD326 were used for screening of CETC and subsequently green intracellular fluorescence was determined inside the CETC. Aldefluor positive CETC also were in the range of 1%. Patients with CETC increasing during systemic chemotherapy which are defined as poor prognosis patients are now investigated for their proportion of stem cells during CETC increase.  Discussion: These analyses will help to clarify whether an increase in CETC during systemic chemotherapy is due to tumor stem cells, surviving conventional chemotherapeutic agents and whether they might be susceptible to new agents.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5053.
Low proteasome activity as a means to track and target breast cancer stem cells in-vivo.
Authors: Lagadec CH , Vlashi E , Dolla Donna L , McBride W , Pajonk F
Published: December 2010

Abstract #5055

Based on clinical observations, the existence of cancer stem cells (CSCs)/cancer initiating cells (CICs) in solid cancers has been postulated by radiation biologists and oncologists for decades. According to the stem cell hypothesis, only a small number of CSCs/CISs within a tumor have the ability to repopulate an entire tumor while their progeny does not. CSCs/CISs in solid tumors can now be identified prospectively in gliomas, head & neck cancers, pancreatic cancers, colon cancers, prostate cancers, melanomas, and in breast cancers. CSCs/CISs are thought to be mostly quiescent and relatively resistant to conventional anti-cancer therapy.  Breast CSCs/CICs are enriched in cell populations with high CD44 and low or absent CD24 expression. Unfortunately, detection of breast CSCs/CISs, using CD44 and CD24 expression analysis, requires dissociation of the tumors and antibody staining for surface markers. We recently discovered that cancer stem cells, and specially breast CSCs/CICs, have low proteasome activity. In order to utilize this novel marker for the identification of breast CSCs/CICs, we developed retroviral vectors coding for fusion proteins between thymidine kinase (TK), the fluorescent protein ZsGreen and the c-terminal degron of murine ornithine decarboxylase (cODC). We stably transfected human breast cancer cells lines (MCF-7, MDA-MB-231, T47-D) with this reporter gene construct for 26S proteasome activity.  Our reporter construct allowed us to track CSCs/CICs responses to radiation therapy in vivo via fluorescent imaging, but also to target them selectively using suicide gene (TK) therapy. Thus, the use of thymidine kinase in our fusion constructs allows for specific elimination of this cell population by ganciclovir preventing self-renewal in-vitro and causing T47-D and MDA-MB-231 tumor xenograft regression in vivo.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5055.
ALDH1 expression in human breast carcinoma.
Authors: Bhargava R , Beriwal S , McManus K , Dabbs DJ
Published: December 2010

Abstract #5057

Aldehyde dehydrogenase 1 (ALDH1) is an enzyme responsible for oxidation of intracellular aldehydes. It has recently been shown to be a marker of "stem cells". It is hypothesized that breast cancer response to chemotherapy and breast cancer specific survival (BCSS) may be related to the presence and/or amount of self-renewing "stem cells".  Since breast cancer molecular classes have distinct prognosis and show variability in response to chemotherapy, ALDH1 immunohistochemical (IHC) expression was analyzed in 189 invasive breast carcinomas (IBCs) to study its correlation to breast cancer molecular classes. The IBCs were classified using immunohistochemical (IHC) surrogate (to molecular classes) markers-estrogen receptors (ER), progesterone receptors (PR) and HER2. ER and PR were scored using a semi-quantitative scoring method modified from previously described “H-score” method with a dynamic range of 0-300. HER2 was considered positive only if 3+ by IHC or amplified (HER2 to chromosome 17 ratio of >2.2) by FISH. The tumors were divided in 6 groups as follows: luminal A (LUMA; ER score 200 or higher, HER2 negative); ERBB2 (ER and PR score 10 or less, HER2+); triple negative (TN; ER and PR score 10 or less, HER2 negative); luminal A-HER2 hybrid (LAHH; ER score 200 or higher, HER2+); luminal unclassified HER2 negative (LUHN; ER or PR score 11-199, HER2 negative); luminal unclassified HER2 positive (LUHP; ER or PR score 11-199, HER2+). THE IBCs were represented with 3-fold redundancy on tissue microarrays (TMAs). The TMAs were stained with ALDH1 antibody (clone 44, dilution 1:150; BD Biosciences). Any staining for ALDH1 was considered as positive and results were analyzed based on %age of positive cells and semiquantitative IHC score with a dynamic range from 0-300 (as described above).  ALDH1 was differentially expressed in various molecular classes (p=0.0007).    Our results suggest that strong receptor positive tumors (LUMA) have the most committed or differentiated cells, while receptor negative or low receptor positive tumors (TN, LUHN) have higher number of progenitor or "stem cells". These differences likely account for tumor chemotherapeutic response and BCSS between ER+ and ER negative tumors. HER2+ tumors have intermediate range of "stem cells" and likely have mechanisms other than presence of "stem cells" that explains aggressive tumor growth and response to chemotherapy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5057.
Reduced self-renewal ability and drug resistance by inhibition of notch-4 and ABCG2 in anoikis-resistant MDA-MB-231 breast cancer cells.
Authors: Ko E , Han W , Noh D
Published: December 2010

Abstract #5059

Background In this study, we aimed to investigate the effect of anoikis resistance on drug responsiveness and tumor initiating ability in MDA-MB-231 breast cancer cell line, and to determine whether ABCG2 inhibitor and notch-4 inhibitor will modulate the drug resistance and self renewal ability of anoikis-resistant cells. Methods Anoikis-resistant MDA-MB-231 cells isolated from the MDA-MB-231 cell line using sequential passages through the floating culture system and tested the tumor initiating ability (mammosphere-forming efficacy in vitro and limiting dilution transplantation in vivo), tumor growth rate, and drug responsiveness. Results Anoikis resistant cells were shown to have a greater mammosphere forming efficacy than parent cells (19% versus 2%, P<.001). Similarly, both tumor formation rate and growth rate of anoikis-resistant cells were greater than parent cells when injected into NOD/SCID mice. Anoikis resistant cells were also more resistant to doxorubicin and docetaxel than parent cells. Immunoblots shown that expression of ABCG2 and notch-4 increased in anoikis-resistant cells as compared to parent cells. ABCG2 inhibitior (fumitremorgin C) and notch-4 inhibitor (gamma secretase) were found to preferentially reduce drug resistance and mammosphere-forming efficacy of anoikis resistant cells, respectively. Conclusion Acquisition of anoikis resistance in human breast cancer cells results in increased tumor initiating ability, tumor growth rate and resistance to chemotherapeutic agents. The increased expression of notch-4 and ABCG2 may provide an explanation for the increased tumor initiating ability and drug resistance in anoikis resistant cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5059.
Exploratory study of clonality determined by expression of tumor markers in intrinsic subtypes of breast cancers.
Authors: Kashiwaba M , Inaba T , Takeda Y , Takiyama I , Komatsu H , Uesugi N , Wakabayashi G
Published: December 2010

Abstract #5060

[Background] However expression profile-based intrinsic subtypes revealed heterogeneity of breast cancer, several biological issues are not clarified yet, especially in tumor development. [Objectives] In order to clarify the relationship between intrinsic subtypes and positivity of tumor markers (TMs), CEA, CA15-3, BCA225 and NCC-ST439 were measured from sera taken from patients at the relapse retrospectively. In addition to the analysis of positivity of TMs, migration of TMs were analyzed in each intrinsic subtype, especially focused on changes of CA15-3 (equivalent to soluble MUC1) to identify the heterogeneity and development to other subtypes in the meaning of TMs expression. [Methods] Sera were collected from seventy five patients of newly diagnosed metastatic breast cancer. Intrinsic subtypes were simply determined by immunohistochemical staining as Luminal A (LA) ,B (LB), HER2 and TN. The positivity of TM was defined if it elevated 20% higher than normal upper limit. [Results] The positive rate of any TMs were higher in order of LA, LB, HER2 and TN. There was significant difference between Luminal (LA+LB) and TN in positive rate of TMs(p<0.05). If focused on mammary epithelium development marker MUC1 (CA15-3 as a soluble form), it was significantly elevated in Luminal (LA+LB) than HER2 and TN(p<0.05). Interestingly, 18 of 23 (72.3%) TN tumors showed all TMs negative at the relapse, although 4 of 18 (22.2%) TN tumors developed to TMs positive after palliative chemotherapies. [Conclusion] Luminal type showed higher expression of TMs at the relapse. It might be explained by the fact that common TMs were selected as responsive serum proteins to the tumor volume of dominant population of breast cancer by chance. But if we integrate the idea that CA15-3 as a development marker, luminal cancer was developed from ER+ highly developed progenitor and HER2 and TN might be developed from ER- progenitor lineage. The migration of TMs of TN after palliative chemotherapies might be illustrated with clonal selection by chemosensitivity. These phenomenon will be understood by further biological experiments and prospective clinical trials to improve the outcome of metastatic breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5060.
Differential transcriptional response to cisplatinum in BRCA1-defective versus BRCA1-reconstituted breast cancer cells by microarrays.
Authors: Di Martino MT , Ventura M , Guzzi PH , Pietragalla A , Neri P , Bulotta A , Calimeri T , Barbieri V , Caraglia M , Veltri P , Cannataro M , Tassone P , Tagliaferri P
Published: December 2010

Abstract #5062

BRCA1-related breast cancer represents a distinct clinical entity characterized by basal-like molecular phenotype. HCC1937 is a BRCA1-defective breast cancer cell line which discloses higher sensitivity to cisplatinum (CDDP) as compared to the BRCA1 full-length cDNA transfected clone HCC1937/wtBRCA1. To study the molecular bases of BRCA1-related differential sensitivity to the drug, we have analyzed the whole gene expression profile of HCC1937 and HCC1937/wtBRCA1 cells following in vivo and in vitro exposure of tumour cells to CDDP.  Differential in vivo sensitivity was evaluated in xenografted tumors in SCID mice after 4 weeks of treatment with CDDP (5 mg/kg) or vehicle. Tumors were retrieved from animals 4 hours after the last dose of drug or vehicle. In vitro data were obtained exposing HCC1937 and HCC1937/wtBRCA1 cell lines to CDDP at IC50 doses of 30 and 70µM, or at 60 or 140µM respectively, for 3, 12 and 24 hours. Gene expression profiling was performed by Affymetrix® technology using GeneArray 1.0ST. Array data were analyzed using Gene Expression Console, GeneSpring software and Ingenuity Pathway Analysis (IPA)®.  We found up-regulation of G2/M DNA damage checkpoint pathway, in a dose- and time-dependent manner, as well as down-regulation of Notch signaling pathway in HCC1937 cells, after treatment with CDDP in vitro. Both pathways were not affected in BRCA1-reconstituted cells. Notch signaling pathway plays a key role in normal development of several tissues and its aberrant activation is linked with breast cancer tumourigenesis. Our findings suggest that sensitivity of the BRCA1-defective cells to CDDP exposure is related to the DNA-damage repair machinery due to BRCA1-deficiency but also to the modulation of survival pathways triggered by BRCA1. In vivo data analysis of HCC1937/wtBRCA1 tumours exposed to CDDP identified modulation of pathways involving AKT3 and PRKAR2B, also known as RII-BETA, a cAMP-dependent protein kinase, whereas no Notch signaling pathway modulation was detected.  In conclusion, reconstituted expression of BRCA1 induces resistance to CDDP in vitro and in vivo but produces a different transcriptional response to drug exposure. This effect could be related to the different growth and environmental milieu as well as to the different exposure conditions. Signaling pathways involving Notch, AKT and RII-BETA are involved in these events. Our data reinforce the concept that BRCA1 offers a molecular marker for individualized therapeutical options in breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5062.
Cell cycle phase progression analysis identifies unique replication phenotypes of major prognostic and predictive significance in cancer.
Authors: Loddo M , Kingsbury SR , Rashid M , Proctor I , Holt C , Young J , El Sheikh S , Falzon M , Eward KL , Prevost T , Sainsbury R , Stoeber K , Williams GH
Published: December 2010

Abstract #5066

BACKGROUND: The cell cycle machinery acts as an integration point for information transduced through complex and redundant upstream oncogenic signalling pathways. Multiparameter analysis of core regulatory proteins involved in G1-S and G2-M cell cycle phase transitions provides a powerful biomarker readout for assessment of the cell cycle state. We have applied this novel algorithm to breast cancer, investigating its cell cycle kinetics, and determining how this impacts on pathobiology and disease progression in-vivo.  METHODS AND FINDINGS: Protein expression profiles of key constituents of the DNA replication licensing pathway (Mcm2, geminin) and mitotic machinery (Plk1, Aurora A, Aurora substrate Histone H3S10ph), mediators of G1-S and G2-M transition respectively, were generated for a cohort of 182 patients. Arrested differentiation and development of genomic instability was associated with increased engagement of cells into the cell division cycle (p<0.0001). Three unique cell cycle phenotypes were identified; (I) well differentiated tumours composed predominantly of Mcm2 negative cells indicative of an out-of-cycle state (18% of cases), (II) high Mcm2 expressing tumours but with low geminin, Aurora A, Plk1 and H3S10ph levels (S-G2-M progression markers) indicative of a G1 delayed/arrested state (24% cases), (III) high expressing Mcm2 tumours, but also expressing high levels of the S-G2-M progression markers, indicative of accelerated cell cycle progression (58% of cases). The accelerated cell cycle phenotype had a significantly higher risk of relapse when compared with out-of-cycle and G1 delayed/arrested tumour phenotypes (HR=3.90 [1.81-8.40], p<0.001). Notably high grade tumours with the G1 delayed/arrested phenotype showed an identical low risk of relapse to well differentiated out-of-cycle tumours (HR=1.00 [0.22-4.46], p=0.99), suggesting that many patients are receiving inappropriate S-G2-M phase directed adjuvant chemotherapy.  CONCLUSIONS: This biomarker algorithm provides novel insights into the cell cycle state of dynamic tumour cell populations in-vivo, information that impacts on individualised therapeutic decisions. The cell cycle phenotype has a major influence on disease progression, identifying those patients at most risk of relapse. Importantly, it is only patients displaying an accelerated phenotype, tumours that show S-G2-M phase transit, that are likely to derive benefit from S and G2-M phase specific adjuvant chemotherapeutic agents and mechanistic drugs.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5066.
GATA3, P53, Ki67 and vascular peritumoral invasion are strongly predictive of hormonal resistance in luminal breast cancer.
Authors: Jacquemier JJ , Charafe Jauffret EE , Monville FF , Esterni BB , Extra J , Houvenaeghel GG , Xerri LL , Birnbaum DD
Published: December 2010

Abstract #5068

Background: Breast cancers are traditionally divided into hormone receptor positive and negative cases that guide patient management. The two luminal subtypes in gene expression profiling , A and B, are defined by high expression of ER, GATA3, and FOXA1. There is a little doubt that ER status is a strong predictor of response to endocrine therapy. Prognostic immunohistochemical biomarkers for ER-positive breast cancer include progesterone (PR) and androgen (AR) receptors, proteins related to proliferation or apoptotic resistance. The aim of this study was to identify the best predictors of success of hormonal therapy among immunohistochemical and classical factors.  Methods: We studied 10 markers in a consecutive series of 832 cases of breast carcinoma treated at the Paoli-Calmettes Institute from 1990 to 2002 and deposited onto tissue-micro arrays (TMA): luminal-related ER, PR, AR, FOXA1, and GATA3 factors, proliferation-related KI67 and CCND1, ERBB2, anti-apoptotic BCL2, and p53. We also measured the vascular peritumoral invasion (VPI) size, Grade and lymph nodes involvement (N). On these series 145 cases had been profiled on Affymetrix microarrays. The patients had been treated with adjuvant chemotherapy and/or hormonal therapy . The 132 events observed and taken into account were metastases (MF).  Results: Of the 67 luminal A cases of this series 63 were ER-positive, 98% were FOXA1-positive; only half of them expressed the five luminal-related markers. In 584 ER-positive patients, 200 did not receive adjuvant hormonal therapy with a highly significant difference in MF with those who received Tamoxifen (p=0.017). For the 240 cases who received hormonal therapy, the factors associated to the MF in multivariate analysis were:    A prognostic score can be established summing the impact of each four parameters with a significant Logrank test : p= 0.0003.    Discussion: A panel of three antibodies (Ki67, P53 and GATA3), associated with VPI can significantly improve the traditional prognosticators in predicting outcome for ER-positive breast cancer patients receiving hormonal therapy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5068.
Sirtuin expression is decreased in malignant breast tissue and is associated with patient outcome.
Authors: Zino S , Ashraf N , Kingsmore DB , Payne AP , George W , Shiels PG
Published: December 2010

Abstract #5073

Background: Age is associated with increased incidence of cancer; therefore exploring factors associated with biological ageing in cancer may improve our understanding of the disease and result in new prognostic markers or therapeutic targets. Sirtuins belong to a family of genes that are involved in cell processes including DNA repair, cell-cycle regulation and ageing. SIRT6 modulates telomeric chromatin, is involved in DNA repair and the suppression of genomic instability, aberrations of which are key features of cancer cells. SIRT7 is a positive regulator of RNA polymerase I (Pol I) transcription. Recently, altered sirtuins expression has been linked with lung cancer, gliomas and thyroid cancer. This study aimed to investigate whether SIRT6 and SIRT7 expression is altered in breast tumour biopsies and to determine any association between sirtuin expression and patient outcome.  Materials and Methods: The transcriptional expression of SIRT6 and SIRT7 was determined using real-time PCR in archival breast biopsies (73 non-malignant and 70 malignant). SIRT6 and SIRT7 expression relative to HPRT (house keeping gene) was then analysed with respect to histopathology, disease recurrence and survival.  Results: We observed a significant decrease in the relative transcriptional expression of SIRT6 and SIRT7 in breast cancer biopsies (median for SIRT6 = 0.799 and SIRT7= 0.482) when compared with non-malignant tissue (median for SIRT6 = 1.823 and SIRT7= 0.607) (Mann-Whitney test, p <0.001 and p= 0.043 respectively). There was a significant association between SIRT6 expression and tumour grade (Kruskal Wallis Test, p=0.002), whereas only a trend was observed for SIRT7 (Kruskal Wallis Test, p=0.067). Kaplan-Meier survival analysis showed that mean survival was significantly shorter in patients with lower SIRT6 expression when compared with those with higher levels of expression (9.32 years vs 13.06 years, p=0.01). A trend was observed between levels of SIRT7 expression and survival (p=0.083). Furthermore, multivariate Cox-regression analysis revealed that SIRT6 expression was independent of tumour size, grade, nodal status, and oestrogen receptor status as a survival predictor (p =0.036).  Discussion: These results demonstrate an association between SIRT6 expression and breast cancer pathogenesis and suggest possible SIRT7 involvement. Decreased SIRT6 expression is consistent with genomic instability associated with tumourigenesis and might indicated telomeric dysfunction. Lower levels of SIRT7 are an indication of increased biological ageing in cancerous cells. Furthermore, the association between SIRT6 and both tumour grade and survival, indicates that SIRT6 might prove to be an additional prognostic marker for breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5073.
Quantitative proteomic analysis of radioresistant breast cancer cell lines.
Authors: Smith L , Potts D , Qutob O , Watson MB , Beavis AW , Garimella V , Lind MJ , Drew PJ , Cawkwell L
Published: December 2010

Abstract #5074

Background: Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. We aimed to develop a novel in vitro model of radioresistance using a breast cancer cell line and to subsequently identify molecular biomarkers which may be associated with the radioresistant phenotype. We utilised a quantitative proteomics technique (iTRAQ) based on MALDI-TOF/TOF mass spectrometry (MS) to identify differentially expressed proteins.  Material and Methods: We established 3 novel breast cancer cell sublines which were significantly resistant to radiotherapy when compared with the parental cells. The radioresistant sublines were created by irradiating cells in fractionated doses of 2Gy up to a total dose of 40Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for each novel cell subline when compared with parental cells. One radioresistant/parental cell pair was first analysed using in-solution digestion and liquid chromatographic separation with protein identification by MALDI-TOF/TOF (LC-MALDI analysis) on an Applied Biosystems 4800 Plus instrument. Quantitative iTRAQ (Applied Biosystems) was then performed on the same instrument for all 3 radioresistant/parental cell pairs.  Results: A total of 586 and 652 proteins were identified in T47D and T47DRR cells respectively by LC-MALDI. Those proteins identified in both cell lines and any redundant entries were removed to reveal those proteins which were unique to each cell line. In total 244 unique proteins were identified in T47D cells and 311 unique proteins were identified in T47DRR cells.  Comparison of the 3 pairs of radioresistant/parental cell samples by iTRAQ revealed a number of differentially expressed proteins. Using a standard ≥2-fold change in expression, these iTRAQ analyses revealed significant changes in the expression of 51 proteins in one or more of the radio-resistant derivatives.  Further confirmation by immunoblotting is underway. Currently the decrease in expression of 26S proteasome associated subunits has been confirmed by this method.  Conclusion: LC-MALDI and iTRAQ analysis has revealed a large number of candidate proteins which may be associated with a radioresistant phenotype. These now require further confirmatory studies. These MS-based techniques offer a powerful proteomic approach to identify candidate biomarkers which may be involved in radioresistance.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5074.
Enterolactone but not genistein decreases estradiol-induced tumor growth, angiogenesis, and secreted VEGF in vivo in human breast cancer.
Authors: Saarinen NM , Abrahamsson A , Dabrosin C
Published: December 2010

Abstract #5081

Phytoestrogens have been shown to be potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. There are two major classes of phytoestrogens in the diet; lignans and isoflavones. One main phytoestrogen in Western diet is the mammalian lignan enterolactone (ENL) whereas soy is the major source of the isoflavone genistein (GEN) usually found in Eastern diets. In this paper we investigated the effects of ENL and GEN on estradiol induced breast cancer growth in nude mice. Ovariectomized mice treated with a continuous release of estradiol were used. MCF-7 tumors were established and mice were fed with basal diet (BD), or BD with added ENL, GEN, or a combination of ENL and GEN. Tumor growth was followed and microdialysis was used to sample extracellular VEGF and FGF-2 in vivo after 2-3 weeks of treatment. We show that ENL alone or in combination with GEN, counteracted estradiol-induced growth and angiogenesis of solid tumors. GEN alone did not affect tumor growth or angiogenesis compared with BD. The pro-angiogenic extracellular levels of VEGF in vivo were significantly decreased by ENL and ENL+GEN diets compared with tumors in the BD group whereas GEN diet alone did not affect the levels compared with BD. FGF-2 levels were similar in all groups. Our in vivo findings were confirmed in vitro. By adding ENL and/or GEN, estradiol-induced VEGF secretion in MCF-7 cells decreased significantly in the ENL and ENL+GEN groups whereas GEN alone had no effects compared with estradiol exposed cells. FGF-2 was unaffected by estrogen and phytoestrogen exposure compared with control cells without added hormones. Estradiol up-regulated VEGFR-2 in human umbilical vein endothelial cells and this up-regulation was inhibited by ENL suggesting a pro-angiogenic effect by estradiol by two different mechanisms, both of which were inhibited by the addition of ENL. Our results suggest that ENL has potent anti-estrogenic effects on ER+ breast cancer whereas GEN failed to inhibit estradiol-induced tumor growth and VEGF secretion. Various phytoestrogens may have very different effects on breast cancer. ENL may prove to be beneficial in breast cancer prevention strategies in the future while GEN may cause opposite effects.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5081.