bluuebunny/crossref_metadata_2025 huggingface dataset:
3-Tesla MRI for the evaluation of spreading area of ductal carcinoma in situ: a comparative study with 1.5-Tesla MRI.
Authors: Kubo M , Shirahane K , Tanaka M
Published: December 2010

Abstract #4028

Background: High quality of space and temporal resolution is demanded from the diagnosis for intraductal spreading area of breast cancer. In our hospital, from April 2007, 3-Tesra (T) MRI was transduced and high resolution dynamic studies for bilateral mammary have been performed. However, superiority of 3-T MRI to 1.5-T MRI remains almost unproven in the evaluation of intraductal spreading area of breast cancer.  Methods: We assessed 46 patients with primary ductal carcinoma in situ who underwent the 3-T or 1.5-T breast MRI as pre-operative examination between July, 2007 and March, 2008. Concerning the diagnosis of intraductal spreading area of breast cancer, we weighed 3T MRI against conventional 1.5T MRI. Using 1.5-T or 3-T MRI (Achieva, Philips), we injected contrast medium (Magnepist) with 0.2ml/kg body weight in 1ml/sec and photographed five aspects before injection and after injection for every 90 seconds. We obtained fat-sat T1-weighted magnetic resonance image (T1WI), multi-planar reconstruction (MPR) image and standard maximum intensity projection (MIP) image based on T1WI by coronal section, and furthermore slab MIP image with Aquerius net (Terarecon), and we evaluated the enhanced area.  Results: 32 cases were examed with 1.5-T MRI, and 14 cases with 3-T MRI. In 1.5-T group, 10 cases (31%) were underestimated, additional resection were required intraoperativelly with cancer-cells positive surgical margin in 7 cases, and surgical margin was positive with carcinoma cells by final pathological findings in 4 cases. In one patient we were forced to changes from breast-conserving surgery to mastectomy, and two cases underwent reoperation (mastectomy) with the diagnosis of widely positive surgical margin.In comparison with 1.5 T MRI group, three (21%) were underestimated in 3-T MRI group, intraoperative addition resection in two cases, and surgical margin positive by the final pathological findings in two cases, respectively.  Conclusions: The concern to enhance an artifact is known because 3-T MRI has high resolution, but it may be more useful in the evaluation of intraductal spreading area of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4028.
Clinical significance of bone marrow micrometastasis detected by nested RT-PCR for cytokeratin-19 in breast cancer patients.
Authors: Hur L , Jung Y , Kim K
Published: December 2010

Abstract #4032

Purpose: Breast cancers frequently undergo distant metastasis during the early phase, on which the survival of patients is greatly dependent. It has been suggested that the occurrence of micrometastasis relates with other prognostic features of breast cancer, such as lymph node metastasis and the presence of vascular invasion. The aim of this study was to examine the presence of cytokeratin-19 mRNA in bone marrow aspirates obtained from breast cancer patients, and their possible correlation with distant disease free survival.  Method: Between 1999 and 2003, bone marrow samples were obtained from 254 breast cancer patients during primary surgery. We separated the mononuclear fraction from the samples and carried out nested reverse transcriptase polymerase reaction (RT-PCR) for the detection of cytokeratin-19 mRNA using two different pairs of primers. The clinical and pathological data and the results of the bone marrow cytokeratin-19 nested RT- PCR were reviewed and analyzed together with clinical results on distant recurrence and survival. We also studied the possible correlations between CK-19 and the tumor size, nodal involvement, stage, and distant metastasis. The median follow up time was 57 months. The median age of patients was 46 years old.  Results: 66(26%) of the 254 samples were cytokeratin-19 positive. And four stage VI patients and two patients lost during follow up period were excluded. 16 cases (24%) of in the cytokeratin-19 positive group showed a recurrent disease in distant organs. Whereas, 23(12.5%) out of 182 cytokeratin-19 negative patients had distant recurrences. Therefore CK-19 positive group have more distant recurrence than CK-19 negative group (p=0.024). And we found that CK-19 positive group have lower disease free survival rate than CK-19 negative group (p=0.017). However, there was no statistically significant difference in overall survival (p=0.083). The stage, nodal status, and estrogen receptor were independent of bone marrow micrometastasis.  Conclusion: Bone marrow micrometastasis, detected by nested RT-PCR for cytokeratin-19, could be a useful predictive marker for the distant metastasis of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4032.
Validation of breast cancer biomarkers between a field immunohistochemistry laboratory in Nigeria and its US-based counterpart.
Authors: Malaka DO , Khramtsov A , Oluwasola OA , Odetunde A , Falusi AG , Grushko T , Huo D , Olopade OI
Published: December 2010

Abstract #4039

Introduction: The importance of hormone receptor status in assigning treatment and the potential use of HER2 targeted therapy have made it imperative for laboratories to improve detection techniques. As inter-laboratory variability in immunohistochemical (IHC) tests may also affect epidemiologic studies of breast cancer subtypes in different countries, we conducted a validation study of breast cancer biomarkers between a well-established laboratory in the US and a field laboratory at the Institute for Medical Research and Training at the University College Hospital in Ibadan Nigeria.  Method: 232 breast tumor blocks were evaluated for ER, PR, and HER2 at both laboratories using tissue micro arrays (TMA) technique. Web-based conferences were held periodically to discuss IHC staining protocols, standardize scoring systems and to resolve discrepant cases. Pathologists used whole slide imaging for joint review and kappa statistic (κ) was used to indicate concordance between the two laboratories. Fluorescence in situ hybridization was carried out to confirm HER2 status in all cases.  Results: Initially, concordance analysis revealed an agreement of 91% (κ=0.52) for ER, 85% (κ=0.49) for PR, and 80% (κ=0.39) for HER2 between the two labs. Antigen retrieval techniques and scoring methods were identified as important reasons for discrepancy. After quality assurance and training, the agreement improved to 92% (κ=0.53) for ER, 88% (κ=0.64) for PR, and 94% (κ=0.75) for HER2. To date, florescence in situ hybridization (FISH) has been completed for 67 cases to confirm HER2 status, out of which 16 (24%) were shown to amplify the HER2 gene, 6 out of the 12 discordant HER2 results were resolved by FISH.  Conclusion: We found web-based conference with TMA and digital microscopy a useful and cost-effective tool for quality assurance of IHC, consultation and collaboration between distant laboratories. Quality improvement exercises in testing of tumor biomarkers will reduce misclassification in epidemiologic study of breast cancer subtypes and provide much needed capacity building in resource poor field sites.  Acknowledgement  This study was carried out with the support of Breast SPORE NCI P50 CA125183, Breast Cancer Research Foundation and the Lee Jeans Entertainment Industry Fund.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4039.
Phase I-II study of the histone deacetylase inhibitor (HDACi) vorinostat plus paclitaxel and bevacizumab in metastatic breast cancer (MBC): New York Cancer Consortium trial P7703.
Authors: Ramaswamy B , Bhalla K , Cohen B , Pellegrino C , Hershman D , Chuang E , Somlo G , Goetz M , Swaby R , Hopkins U , Christos P , Espinoza-Delgado I , Sparano JA
Published: December 2010

Abstract #404

Background: The oral HDACi vorinostat (V) sensitizes breast cancer cells to tubulin polymerizing agents and anti-VEGF directed therapies in vitro. We sought to determine the safety and efficacy of V plus paclitaxel (P) and bevacizumab (B) as first-line therapy for MBC.  Methods: Eligible patients (pts) with measurable disease, ECOG PS 0 or 1, and no prior chemotherapy for MBC, received P (90mg/m2) on days 1, 8, 15 and B (10mg/kg) on days 1,15 every 28 days, plus V 200 mg (N=3) or 300 mg (N=28) BID orally for 3 days given the day before, day of, and day after each P dose. Response was assessed after every 3 cycles (RECIST). The trial was designed to distinguish between a response rate of < 40% vs. > 60% (alpha=0.10, beta=0.10) using Simon's optimal 2-stage design. At least 12 responses were required in the first 28 evaluable pts in the 1st stage and 21 of 41 in the 2nd stage.  Results: There were no dose-limiting toxicities among the first 6 pts treated at the 1st (200 mg BID; N=3) and 2nd (300 mg BID; N=3) dose levels. Among the 26 evaluable pts treated at the recommended phase II dose of 300mg bid in the 1st stage, 14 patients had a partial response (PR-54%; 90% confidence interval [C.I.] 36%-71%), 6 had stable disease (SD) at 3 months and remain on treatment, 2 had SD as the best response with progressive disease (PD) at 11.8 and 15 months, and 4 had PD as their best response. Additionally, 2 of 3 pts treated at the first V dose level had a PR, indicating PR in 16 of 29 overall (55%, 90% C.I 38%-71%). Only 11 of 29 evaluable pts (38%) have progressed after a median followup of 10.5 months (R-2.3-14.9). Two pts had tumor biopsies and peripheral blood mononuclear cells (PBMC) collected before and 4 hours after the 3rd V dose (300 mg BID) which showed increased acetylation of K69 lysine residue of the chaperone protein Hsp 90, upregulation of Hsp70, and downregulation of AKT by Western Blot, providing evidence of Hsp90 inhibition in tumor and PBMC.    Gr 3-4 toxicities included neutropenia (26%), neuropathy (23%), fatigue (16%), thrombosis (10%), vomiting (6%), hypertension (3%), and diarrhea (3%).  Conclusions: Our preliminary findings indicate that the HDACi V inhibits Hsp90 and downregulates AKT in breast cancer in vivo, and enhances the effectiveness of P plus B in MBC. Accrual is nearly complete and the final efficacy analysis will be presented.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 404.
Role of extracellular signal-regulated kinase 5 (ERK5) in human breast cancer.
Authors: Watson CM , Kipgen D , Orange C , Tovey S , Cooke TG , Edwards J , Leung HY
Published: December 2010

Abstract #4042

Background  ERK5 is a novel member of the mitogen-activated protein kinase (MAPK) family. MAPKs regulate multiple cellular functions, including proliferation, differentiation and survival. Recent studies have implicated the ERK5 pathway in human breast cancer. As breast cancer is now the most common type of cancer in the UK, research into this potential biomarker is essential.  Method  An optimised immunohistochemistry protocol (McCracken S. Oncogene 2008) was applied to examine expression of ERK5 in a cohort of 10 normal breast samples and 402 estrogen receptor (ER) positive breast cancer specimens from patients treated with Tamoxifen. ERK immunoreactivity was quantified using a weighted histoscore method (Kirkegaard T. Histopathology 2006), and the data was corroborated to clinico-pathologic parameters including the HER family (Tovey S. Clin Can Res. 2005).  Results  Of 402 tumours on the TMA, 346 cases were informative. Follow-up was available for a minimum of 10 years: 239 with stable disease, 94 cases recurred (13 with bilateral recurrence) and 84 received adjuvant chemotherapy.  ERK5 expression in the tumour cohort was observed in the cytoplasm with 78% of samples having weak staining with a histoscore of <100, 20% with moderate staining with a score between 100-200 and 2% with strong staining, histoscore>200. ERK5 expression in the nucleus demonstrated 85% of cores with a histoscore <100 (weak) and 15% between 100-200 (moderate). Almost all ERK5 membrane staining was weak (99% had a histoscore of <100). ERK5 cytoplasmic and nuclear expression were directly associated (c.c.=0.682, p<0.001) and ERK5 nuclear expression showed a trend for association with tumour size (p=0.069). In addition, ERK5 cytoplasmic expression was weakly associated with HER1-3 positivity (p=0.022) and nodal status (p=0.039). Interestingly, both cytoplasmic and nuclear ERK5 expression correlated with cytoplasmic and nuclear ERK1/2 expression (p=0.001 and p=0.003, respectively).  All of the benign breast cases expressed cytoplasmic ERK5 at a relatively weak level (100% with a histoscore<100). Again cytoplasmic and nuclear expression in the benign cohort correlated (c.c. =0.879 and p=0.001). Benign and tumour breast tissue expressed significantly different levels of nuclear ERK5 (p=0.002).  Discussion   This study demonstrates that ERK5 is commonly expressed in breast cancer specimens. ERK5 nuclear expression was shown to be related to tumour size, suggesting a potential role in proliferation. In support of this observation ERK5 expression also strongly correlated with expression of ERK1/2 in this cohort. ERK5 was also shown to be associated with nodal status and HER1-3 expression. Therefore our data supports the need for further investigation into the role of ERK5 in breast cancer, including ER negative patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4042.
Combination of carboplatin and the mTor inhibitor RAD001 is a new therapeutic approach for p53-mutated breast cancer.
Authors: Liu H , Possinger K , Eucker J
Published: December 2010

Abstract #405

Platinum agents are very effective and commonly used chemotherapeutics for the treatment of many solid tumors. However, acquired and intrinsic resistance remains a major obstacle for platinum-based therapy. Recent evidence indicates that mTOR inhibition is a promising strategy to sensitize some p53-wild type solid tumors to conventional chemotherapies. Since p53-mutation is a common phenomenon in breast cancer, we investigated in this study the possibilities of the combined application of carboplatin and the mTOR inhibitor RAD001 in these human breast cancer cells in vitro.  Combination of carboplatin and RAD001 resulted in synergistic inhibitions of cell proliferation both in p53-wild type (MCF-7, ZR-75), and in p53-mutated (SKBR-3, BT-474, BT-20), as well as drug resistant (tamoxifen-resistant MCF-7, adriamycin-resistant MCF-7) breast cancer cell lines as analyzed with Calcusyn software. Higher doses of the two drugs resulted in synergistically apoptosis after 24- or 48-hours treatment independent of p53-status of breast cancer cells. Investigation of several cell survival signaling pathways revealed an additively decreased phosphorylation of Erk, STAT3 and Akt after co-treatment suggesting that interference of cell survival signaling may be one of the mechanisms responsible for growth inhibition mediated by this combination.  With low doses of the two drugs, we observed impaired cell cycle progression, additively reduced cyclins and CDKs, and enhanced p27 level by flow cytometry analysis and western blot. No rapid apoptosis and caspases-involved cell death were observed. Immunohistological analysis after prolonged treatment with low doses of the two drugs demonstrated that carboplatin alone resulted in the formation of endopolyploid cells in p53-mutated breast cancer cells. RAD001 alone had not such an effect, but strengthened this effect of carboplatin. In addition, decreased level of cyclin B1, the main driver of mitosis, was detected by the co-treatment of the two drugs. These data collectively suggest that the combination use of low doses of carboplatin and RAD001 may present a strategy to overcome the resistance of breast cancer to chemotherapy. It is a promising combination that warrants clinical evaluation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 405.
BRCA1 methylation contributes to the triple-negative breast cancer phenotype.
Authors: Nwachukwu C , Grushko TA , Xu J , Khramtsov A , Olopade OI
Published: December 2010

Abstract #4050

Triple-negative breast cancers are tumors characterized by their lack of hormone receptors (ER and PR) and HER2. They are the most aggressive form and account for 10–17% of all breast carcinomas. A subgroup of triple negative tumors with a basal-like phenotype share morphological features and similar gene expression profiles with tumors from BRCA1 mutation carriers. It has recently been shown that inactivation of BRCA1 in breast epithelial stem cells restricts subsequent progenitor cells to a basal cell phenotype and promotes expansion of ER negative cells (Liu et al 2008, PNAS). While mechanisms of BRCA1 inactivation in sporadic tumors are not completely elucidated, methylation of the BRCA1 promoter is one important mechanism that contributes to loss of BRCA1 expression in sporadic breast cancer. We hypothesize that inactivation of BRCA1 by epigenetic mechanisms such as promoter methylation contributes to the triple negative phenotype.  Materials and Methods: Using a combination of methylation specific PCR and Immunohistochemistry, 120 primary breast cancers were analyzed for methylation of the BRCA1 promoter and protein expression of ER, PR and HER2. All tumors with negative staining for ER, PR and HER2 were classified as triple negative and all others were classified as non triple-negative.  Results: We were able to classify 111 of the 120 (%) tumors by IHC. We found that 30 out of 111 (27%) tumors were ER, PR, and HER2 negative (triple negative). BRCA1 methylation was detected in 14 out of 30 (47%) triple negative tumors, compared with 10 out of 81 (12%) other tumor phenotypes (p<0.001).  Conclusion: These data suggest that BRCA1 inactivation by mutation or methylation may in part drive breast tumor progression toward the triple negative phenotype. Ongoing work will evaluate epigenetic mechanisms regulating gene expression in triple negative breast cancers  This abstract is supported by Breast Cancer Research Foundation and the Lee Jeans Entertainment Industry Fund.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4050.
MicroRNAs and epigenetics in human breast cancer.
Authors: Miyamoto K , Ushijima T
Published: December 2010

Abstract #4051

MicroRNAs (miRNAs), which regulate gene expression at the post-transcriptional level, are suggested to play important roles in carcinogenesis. To clarify the miRNAs dysregulation and the relation to epigenetic mechanisms in human breast cancer, we analyzed a breast cancer cell line, MDA-MB-468 that has previously been used for the genome-wide epigenetic analysis: methylation-sensitive-representational difference analysis (MS-RDA). Normal human mammary epithelial cells (HMEC) were compared with MDA-MB-468. The global expression profile (471 miRNAs) of these samples was examined using 3D-Gene Human miRNA oligo chip (Toray, Tokyo, Japan). We identified 4 miRNAs (0.8%), hsa-miR-200a, hsa-miR-429, hsa-miR-652 and hsa-miR-200b, which were expressed at ten-times higher levels in cancer cells and, conversely, 12 miRNAs (2.5%), hsa-miR-137, hsa-miR-138, hsa-miR-34b, hsa-miR-34c, hsa-miR-374, hsa-miR-376a, hsa-miR-422b, hsa-miR-452, hsa-miR-520e, hsa-miR-455, hsa-miR-554 and hsa-miR-574 that were silenced in the cancer cells. Expression analyses using other breast cancer cells (MCF-7, YMB-1-E, T-47D, MDA-MB-231) revealed a significant overexpression of 4 miRNAs, hsa-miR-200a, hsa-miR-429, hsa-miR-652 and hsa-miR-200b and frequent silencing of 5 miRNAs, hsa-miR-138, hsa-miR-34c, hsa-miR-376a, hsa-miR-452, and hsa-miR-455 in breast cancer cells. As for epigenetic mechanisms, overexpressed miRNAs were found to target silenced genes in the MDA-MB-468 cell line, and treatment of MDA-MB-468 with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 5 miRNAs, hsa-miR-137, hsa-miR-374, hsa-miR-520e, hsa-miR-554 and hsa-miR-574. Our results show the existence of a novel miRNA signature in breast cancer, suggesting that epigenetic mechanisms may be involved in the regulation of miRNA expression.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4051.
Role of the insulin receptor in IGF-system signaling and biology.
Authors: Oberg DH , Zhang H , Yee D
Published: December 2010

Abstract #4055

The role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in breast cancer has been extensively studied, and has led to the development of several IGF1R inhibitors currently in clinical trials. The closely related insulin receptor (IR) has been shown to contribute to IGF signaling through the formation of IGF1R/IR-hybrid receptors and through a homodimer of an alternatively spliced form of the IR (IR-A). Understanding the role of the IR in cancer biology will help determine which receptor(s) in the IGF system should be targeted to efficiently inhibit IGF signaling. Our previous work has shown that selective downregulation of IGF1R by siRNA resulted in enhanced sensitivity to insulin through increasing the numbers of insulin receptor homodimers. To determine how downregulation of IR affected IGF1R signaling, we used T47D cells, a breast cancer cell line expressing both the IR and IGF1R, as our model system. We transiently downregulated IR levels using siRNA and evaluated signaling. 5 nM of insulin activated downstream signaling molecules, such as insulin receptor substrate 1(IRS-1) and Akt in control cells, but this response was diminished in the IR-downregulated cells. In contrast, 5 nM of IGF-I did not result in differences in signaling between IR-downregulated cells and control cells. Using IR shRNA, we created stable clones with IR levels reduced by more than 72%. IR shRNA clones demonstrated decreased insulin binding, as measured by 125I-insulin binding assay. Additionally, clones showed decreased response to insulin signaling. 5 nM of insulin activated IRS-1 and Akt in parental T47D cells, but this response was attenuated in the IR-downregulated clones. Proliferation was also decreased in response to 5 nM insulin in the clones compared to parental cells. In contrast, IGF-I mediated signaling was not altered in clones when compared to vector control cells. Further, no change in proliferation was observed in response to IGF-I between wild-type and IR shRNA clones. We also generated stable IR shRNA clones using LCC6 cells. Insulin signaling was reduced in LCC6 clones when compared to wild-type LCC6 cells. However, in contrast to the T47D clones, IGF-I signaling was increased in LCC6 IR shRNA clones. Thus, the role of the insulin receptor in cancer biology may be dependent upon the ratio of IR, IGF1R, and the hybrid receptor. If cells contain mostly homodimers of IGF1R and IR, then downregulation of IR may affect only insulin response. However, if there are high levels of hybrid receptor, then downregulation of IR could affect, and even augment, IGF1R signaling. Our data suggest that the ratio and confirmation states of both IR and IGF1R should be understood to predict the effect of receptor downregulation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4055.
Recombinant human G-CSF increases invasiveness of breast cancer cells.
Authors: Webster DJ , Wilde ZJ , Orzol AR , Davis JL , Troxell ML , Bae D
Published: December 2010

Abstract #4057

Background: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) is commonly given to breast cancer patients to prevent neutropenia during chemotherapy. It binds to the G-CSF receptor (G-CSFR) on neutrophil precursors, promoting their proliferation within the bone marrow. We observed breast cancer growth in patients with advanced breast cancer during rhG-CSF administration. The G-CSFR is present on some solid tumors, but has not been examined in breast cancer. We asked whether breast cancer cells express the G-CSFR, and whether rhG-CSF has any direct effects on breast cancer proliferation or invasion in vitro.  Material and Methods: MDA MB231 is a highly aggressive ER–PR–Her2/neu– breast cancer cell line derived from a human pleural effusion. We analyzed this cell line for the presence of the G-CSFR by immunohistochemistry, reverse-transcriptase PCR (RT-PCR), Western Blot analysis and flow cytometry. We sequenced DNA products of RT-PCR to verify results. We examined the effects of physiologic doses of rhG-CSF on breast cancer cell proliferation and invasion using MTT proliferation and Matrigel transwell assays, respectively.  Results: MDA MB231 breast cancer cells express G-CSFR protein and mRNA. Of seven known G-CSFR isoforms, we found evidence of isoforms 1 and 4. Treating MDA MB231 breast cancer cells with rhG-CSF at 50ng/ml did not increase cell proliferation, but did increase invasion across a Matrigel-coated transwell membrane. Cells treated with rhG-CSF appeared more stellate than untreated cells. Studies are ongoing to evaluate which signal transduction pathways and genes are involved in this increased invasion response. The effects of rhG-CSF on breast cancer growth in vivo are also being examined in murine models.  Discussion: rhG-CSF is given routinely as part of many adjuvant chemotherapy regimens for breast cancer. Although dose-dense regimens using rhG-CSF have shown equivalent or improved survival when compared to standard dosing regimens, it is possible that rhG-CSF may increase the growth or invasive potential of some breast cancers. It is also possible that rhG-CSF could mobilize breast cancer cells from the bone marrow, making these cells more susceptible to chemotherapy. These possibilities should promote thoughful use of rhG-CSF in the clinical setting, and should factor into the design and implementation of clinical trials using rhG-CSF.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4057.
Multicenter phase I clinical trial of daily and weekly RAD001 (everolimus) in combination with vinorelbine and trastuzumab in patients with HER-2-overexpressing metastatic breast cancer with prior resistance to trastuzumab.
Authors: Fasolo A , Gianni L , Rorive A , Bergh J , Dieras V , Cardoso F , Vittori L , Pylvaenaeinen I , Sahmoud T , Jerusalem G
Published: December 2010

Abstract #406

Background: Resistance to trastuzumab (H) is associated with loss of PTEN and AKT/mTOR activation. Preclinically, RAD001, an oral inhibitor of mTOR, enhances the efficacy of H suggesting that RAD001 could reverse resistance to H.  Methods: Vinorelbine (V) was administered at a dose of 25 mg/m2, IV over 10-15 min on days 1 and 8 q3w. H 4 mg/kg loading dose administered IV over 90 min on Day 1 (if pt was not already receiving H), followed by weekly H 2 mg/kg IV over 30 min. In the daily RAD001 arm, 3 cohorts were planned (2.5, 5 and 10mg) and in the weekly RAD001 arm, 4 cohorts were planned (20, 30, 50 and 70mg). Treatment continued until progression or unacceptable toxicity.  Results: As of June 10 2008, 28 heavily pretreated pts were enrolled: 15 to 5mg/d, 6 to 20mg/w and 7 to 30mg/w cohorts. All pts received prior taxanes. Median number of prior CT regimens was 3 (range: 1-5). The main safety events were grade 2 and 3 stomatitis which led to dose reduction of RAD001 in 3 pts and dose interruption in 3. Grade 4 and 3 Neutropenia were seen in 24 pts (leading to dose reduction of V in 11 pts). Twenty two pts have been evaluated for efficacy (table 1). 1 pt had a CR (45+ weeks), 2 pts had PR (35+, 38 weeks), 15 pts SD (Median 26+, range 8+ to 58 weeks) and 4 pts PD. Clinical benefit rate (CR+PR+SD > 24 wks) is 55%+.  Conclusions: Initial findings show that RAD001 is generally well tolerated in combination with V and H and shows promising anticancer activity and clinical benefit in heavily pretreated patients with HER2+ advanced breast cancer. Updated results will be presented.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 406.
Novel egg white based 3D cell culture system for breast cancer research.
Authors: Kaipparettu BA , Kuiatse I , Tak-Yee Chan B , Kaipparettu MB , Lee AV , Oesterreich S
Published: December 2010

Abstract #4060

Introduction: Over the last few years, much attention has been paid to the development and utilization of three-dimensional (3D) cell culture to replace classical two-dimensional (2D) monolayer cell culture systems grown as a flat layer on plastic. Not completely surprisingly, there are tremendous differences between cells in 2D and 3D culture. However, currently available 3D cell culture media are expensive for regular use by the majority of research laboratories and thus large scale use of 3D cell culture system is presently cost prohibited. In our search for a reliable and economically viable replacement for the expensive commercial 3D media, we hypothesized that avian egg white could be a potential alternative.  Methods: Egg white-based chamber slide: Separated egg white from the chicken eggs and coated each wells of an eight well culture chamber with 80μl of egg white by careful heating at 600C on a heating block. After 30-60 minutes, the egg white becomes a semi-solid that adheres to the bottom of the well. The unstuck egg white is washed out by adding 500µl of growth medium and removing it slowly using a Pasteur pipette.  Cell culture and analysis: To each well 2x103 immortalized human breast epithelial cells (MCF10A), were added in 0.5ml of appropriate culture medium. We have analyzed the growth curve of the acini, lumen formation, apoptosis and cell proliferation at different days post-culture. Apico/basal polarization of the acini was analyzed using appropriate antibodies. We have also cultured IGFR1-transformed MCF10A cells, different established human cell lines (MCF7, HEK293, HeLa, LNCaP, and Saos-2), and MMTV-PyMT transformed mouse mammary epithelial cells in their appropriate growth media. For comparative studies, we used a well-established reconstituted basement membrane matrix preparation (BM).  Results: Our analysis shows that this simple avian egg white based system supports growth of cells in 3D, with significantly decreased cost. Specifically, the growth of MCF10A in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apico/basal polarization comparable to what has been described using established 3D culture media. There was no significant difference in MCF10A proliferation and acini size between egg white and BM. We have also observed similar morphology for different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells between egg white and BM.  Conclusion: Our data convincingly argue that egg white can be used as a suitable alternative model for 3D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3D cell culture on a regular basis, and thus result in a dramatic increase of utilization of the 3D cell culture in research.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4060.
Biological significance of allele specific loss of the p53 gene in breast carcinomas.
Authors: Wegman P , Paulmalakkaran B , Wingren S
Published: December 2010

Abstract #4064

Background: The p53 tumour suppressor gene has a central role in the defence against cancer, including breast cancer, and contains a polymorphic variant (Arg/Pro) at codon 72 that has been shown to have different biological properties regarding apoptosis and cell cycle arrest. Earlier studies have shown allele specific loss of heterozygosity (LOH) at this particular site and we aimed to investigate its biological relevance in codon 72 heterozygous breast cancer patients (i.e. survival and age of disease onset).  Method: 199 postmenopausal cases were analyzed for LOH using MegaBACE1000 and statistics was performed using Statistical Package for Social Sciences. As reference for loss/retention of heterozygosity at the Arg or Pro allele DNA from 30 heterozygous blood donors included in a population based DNA bank was used.  Results: LOH was found in totally 124 (62.3%) patients and the Pro allele (n = 103) was significantly more often deleted compared to the Arg allele (n = 21) (p = 0.001). Patients with LOH of the Arg allele were diagnosed at an earlier age (mean age 62.5 years) than those with loss of the Pro allele (mean age 69.2 years) (p = 0.011). LOH of the Arg allele was also associated with worse survival (p = 0.05). Tumour size or number of positive lymph nodes was not associated with LOH.  Conclusion: We found a non-random loss of the codon 72 Pro allele, implying the existence of a selective pressure during carcinogenesis that confers a growth advantage to cells lacking the Pro allele. We also found that earlier age of onset was associated with loss of the Arg allele. These results were achieved from primary tumors without any influence of adjuvant treatment, possibly showing a picture of the natural history of carcinogenesis. The worsen prognosis associated with loss of the Arg allele is somewhat more complex to interpret since recurrence free survival could be influenced by genotype, mutational status and adjuvant treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4064.
p53 transcriptional activity is selectively suppressed by estrogen in SNP309 mdm2 overexpressing breast cancer cells.
Authors: Brekman A , Talbott K , Bargonetti J
Published: December 2010

Abstract #4065

p53 is a tumor suppressor frequently inactivated in cancers. Mdm2 targets p53 for proteasomal degradation via E3 ubiquitin ligase activity and represses p53 transcriptional activity by co-localization with p53 on the chromatin. Mdm2 overexpression is associated with estrogen signaling in breast cancer. To investigate estrogen-associated tumorigenesis and p53 pathway suppression, we examined estrogen receptor positive and p53 wild-type breast cancer cell lines, MCF-7 and ZR-75-1. MCF-7 cells are heterozygous for the mdm2 gene single nucleotide polymorphism from T to G at position 309 (SNP309 T/G) and ZR-75-1 are SNP309 homozygous T/T. Cells that carry the G-allele overexpress Mdm2. We found that in the presence of estrogen, the transcriptional activity of p53 in MCF-7, but not in ZR-75-1 cells, was selectively compromised. When MCF-7 cells were treated with the DNA-damaging drug etoposide or the Mdm2 antagonist Nutlin-3, transcription of p53 target genes p21, mdm2, puma and gadd45 was upregulated. However in the presence of estrogen, transcription of puma and gadd45, and not of mdm2 or p21, was downregulated. In ZR-75-1 cells, we did not observe selective inhibition of p53 transcriptional activity in the presence of estrogen. Importantly, we could potentiate p53 transcriptional activity by adding mdm2 siRNA in the presence of estrogen. The coordination between suppression of p53 transcriptional activity and mdm2 SNP309 genotype suggests that estrogen downregulates the p53 response in breast cancer cells by selecting for cells that overexpress Mdm2. This suggests that downregulating Mdm2 in ER+ breast cancer cells could be a beneficial therapeutic approach.  This work was supported by The Breast Cancer Research Foundation and was facilitated by grant number RR-03037 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4065.
Estrogen regulation of p53 isoforms in the human breast cancer cell line MCF7.
Authors: Vendrell JA , Bourdon J , Cohen PA , Thompson AM
Published: December 2010

Abstract #4067

Background: p53, a transcription factor activated in response to different cellular stresses, is involved in the majority of cancers. Recently, we have shown that the p53 gene, due to alternative splicing, encodes for 9 different p53 proteins (isoforms) expressed in a tissue-dependent manner. Moreover, p53 isoforms bind preferentially some p53 target gene promoters, increasing the complexity of the p53 pathway. Preliminary results indicate that p53 isoforms are differentially expressed in breast tumours and are associated with survival.  Methods and Results: As some p53 isoform expression is also associated with ERa status in breast cancer, we aimed to investigate the effect of 17b-estradiol (E2) on the expression of p53 isoforms in MCF7 cells. We observed that E2 regulated the expression of the p53b isoforms at the protein level but not at the mRNA level. To investigate whether E2 confers resistance to genotoxic stress, we treated cells with doxorubicin, a commonly used chemotherapeutic agent. E2 sensitises MCF7 cells to doxorubicin treatment after a long exposure (3 days). However, we observed that E2 protects MCF7 cells from cell death following short exposure (1 hour) to doxorubicin in a p53-dependent manner. p53 isoforms are differentially expressed after 1h or 3 days treatment with doxorubicin, suggesting their involvement in the response to doxorubicin. These data are consistent with our findings in primary breast cancer, linking p53 isoforms to prognosis.  Conclusion: Our data demonstrated that E2 may influence p53 isoforms and response to doxorubicin in breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4067.
Tumor suppressor PTEN signaling is up-regulated in mammary epithelial cells by soy isoflavone genistein: implications for breast cancer protection.
Authors: Rahal OM , Simmen RC
Published: December 2010

Abstract #4069

Breast cancer is the second leading cause of death in the Western hemisphere affecting one of eight women in their lifetime. In addition to chromosomal and genetic alterations, nutrition is considered a risk determinant for breast cancer. Prevailing evidence suggests a negative correlation between intake of soy-rich foods and breast cancer incidence. Epidemiological and case control studies have shown a two-to eight fold lower occurrence of the disease in Asian women whose early intake of soy products is 20-25 times higher than their American counterparts. Previous work from our group has shown diet-induced protection against DMBA- and NMU-induced mammary tumors in vivo, with decreased tumor incidence and increased tumor latency in rats exposed to dietary soy protein isolate (SPI) or casein (CAS) supplemented with the major soy isoflavone genistein (GEN) when compared to those fed the control casein (CAS) diet. We also have shown that GEN enhanced apoptosis in mammary epithelial cells in vivo and in vitro (MCF-7 cells), attendant with increased expression of the tumor suppressor PTEN and several pro-apoptotic genes Bax, Bok, and p21. Here, we test the hypothesis that GEN induction of PTEN expression and nuclear localization in mammary epithelial cells result in decreased cellular proliferation and enhanced cellular differentiation, to confer protection from mammary tumorigenesis. Using the non-tumor human mammary epithelial cell line MCF-10A, we show that GEN at physiologically relevant concentrations (40 nM, 2 µM) increased PTEN mRNA and protein levels and enhanced nuclear accumulation of active (non-phosphorylated) PTEN. GEN induction of PTEN transcript levels was lost with the inclusion of the transcription inhibitor actinomycin D, suggesting PTEN is a direct GEN target. Further, cells treated with GEN for 6 days had lower cell viability when compared to control cells, concomitant with decreased expression of cyclin D1 and of the PTEN regulated gene pleiotrophin. Interestingly, GEN up-regulation of PTEN expression was accompanied by similar effects on another tumor suppressor p53, which co-localized with PTEN in the nuclei of GEN-treated cells. PTEN siRNA targeting, which decreased basal PTEN expression by 60%, significantly increased cyclin D1 but not p53 expression. Our findings provide support to the hypothesis that the protective effects of GEN in the mammary epithelium may be mediated by PTEN to enhance differentiation and inhibit cellular proliferation, and suggest the participation of another key tumor suppressor p53. Funding by USDA-CRIS 6251-5100002-06S.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4069.
Krupple-like factor 5 promotes breast cancer proliferation through activating fibroblast growth factor-binding protein transcription.
Authors: Zheng H , Zhou Z , Chen C
Published: December 2010

Abstract #4071

Breast cancer is the second leading cause of cancer death in U.S. women. The estrogen receptor negative basal breast cancer is usually invasive and the prognosis is poor. Kruppel-like Factor 5 (KLF5) is a zinc-finger transcription factor promoting cell proliferation, survival, cell cycle, and angiogenesis. High expression level of KLF5 has been shown to be associated with shorter breast cancer patient survival time. In our previous microarray studies, fibroblast growth factor binding protein (FGF-BP) is suggested to be one of the KLF5's downstream target genes in the TSU-Pr1 bladder cancer cell line. FGF-BP has been appreciated to be overexpressed in breast tumors and promotes tumorigenesis. To test whether KLF5 promotes cell proliferation through inducing FGF-BP gene transcription in breast cells, we first examined the KLF5 and FGF-BP protein levels in a panel of breast cell lines and found that KLF5 and FGF-BP are co-expressed in ER-negative basal breast cell lines. Over-expression of KLF5 by adenovirus dramatically increased the FGF-BP mRNA and protein expression in MCF7 and BT474. In contrast, KLF5 knockdown decreased the FGF-BP mRNA and protein expression in HCC1937, BT20, and SW527. Importantly, we found that FGF-BP, like KLF5, promotes cell proliferation in MCF10A, SW527 and TSU-Pr1 cells by H3-thymidine incorporation assay. Furthermore, we demonstrated that KLF5 activates the FGF-BP promoter through a putative KLF5 binding site by luciferase reporter assays. We further showed that KLF5 binds to the FGF-BP promoter in vitro by oligo pull down assays and in vivo by chromatin immunoprecipetation (CHIP) assays. These findings suggest that KLF5 may promote breast cancer cell proliferation through directly activating the FGF-BP mRNA transcription. This study will yield valuable insights into the mechanism of KLF5 induced cancer pathogenesis, and will result in useful diagnostic and therapeutic targets.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4071.
Characterization of PTEN allelic status in a series of sporadic breast carcinomas demonstrating loss of 10q23.3 region by array-CGH.
Authors: Bonnet F , Lafitte M , MacGrogan G , Longy M , Sevenet N
Published: December 2010

Abstract #4072

Background: Since its characterization, PTEN gene seems to play an increasing major role in the breast tumor development. Recently, various studies demonstrated that inactivation of PTEN is more and more detected in sporadic breast carcinomas and that the way of inactivation seems to be gross gene rearrangement rather than deleterious point mutation. Furthermore, because PTEN is an essential component of the PI3K signal transduction pathway, its complete molecular exploration should be done, since it has been demonstrated that PTEN loss-of-function is associated with herceptin resistance in HER2+-breast tumours.  Material and methods: A series of 135 sporadic invasive ductal carcinomas resected between 1989 and 1992 in Institut Bergonie, has been extensively studied for genomic profiling using array-CGH. A subgroup of 27 tumours demonstrated allelic imbalance at the locus 10q23.3, targeting the BAC containing the genomic sequence of PTEN was characterized. Genomic DNA and total RNA from those 27 tumours, and 11 other tumours demonstrating IHC PTEN intensity decreased or absent, were used for PTEN point mutation (dHPLC following by sequence of the variants) and gross gene rearrangement (QMP-SF) screening. Total RNA was used for qRT-PCR with specific assays for PTEN, and/or confirmation of the impact of point mutation at the transcript level.  Results: 6 PTEN gene point mutations for 5 tumours, 15 biallelic and 10 monoallelic gross PTEN gene rearrangements were detected. All but one of the 27 tumours demonstrating deletion of 10q23.3 by array-CGH were confirmed with other molecular techniques. Some of the tumours with IHC PTEN decreased or absent demonstrated decreased quantity of transcript regarding to a tumour control. All the results of the extensive molecular PTEN exploration will be presented.  Discussion : We clearly characterized a subgroup of sporadic breast carcinomas demonstrating biallelic inactivation of PTEN gene either by gross gene rearrangement alone or by the combination of gross gene rearrangement and point mutation. All these molecular events are deleterious leading to a loss-of-function of PTEN protein. In our study, a high percentage of sporadic breast carcinomas demonstrated PTEN inactivation (80% of the subgroup; 15% of the 135 breast carcinomas). Those results demonstrated that inactivation of PTEN is a key step in overall breast carcinogenesis and not only restricted to BRCA1-mutated tumours.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4072.
Disparity between serum and nipple aspirate fluid (NAF) estradiol concentrations during the menstrual cycle.
Authors: Chatterton RT , Heinz RE , Habe-Evans M , Khan SA
Published: December 2010

Abstract #4077

Background: NAF, elicited from healthy women, may provide information about the hormonal environment of the breast and the potential risk for breast cancer. We have studied NAF sex hormone changes across the menstrual cycle and after menopause to determine the relationship to serum concentrations.  Methods: Subjects were women attending the Lynn Sage Breast Center at Northwestern Memorial Hospital. Samples of NAF from 20 premenopausal and 23 postmenopausal women were evaluated. Premenopausal women were divided into groups 1 through 4, backdating from the day of menstruation, -28 through -19, -18 through -13, -12 through -6 and -5 through 0, respectively. NAF and venous blood were collected using standard methods. Total protein was measured by a fluorometric procedure. Unconjugated steroids, estradiol (E2), estrone, progesterone (P), and testosterone (T), were extracted into ethyl acetate-hexane (3:2) and were separated by HPLC on a C-18 column with gradient elution before assay. DHEA sulfate, estrone sulfate, and cathepsin D were measured in the aqueous fraction. All analytes were measured by immunoassays. Transformation of the data of all analytes to their natural logarithms normalized the data. Data were analyzed by ANOVA.  Results: The protein concentration in the samples was similar among all groups, mean = 128 mg/ml (P=0.904). In NAF only E2 and P differed during the menstrual cycle, P = 0.064 and 0.007, respectively, but the pattern of NAF E2 followed that of NAF P and not serum E2. The figure shows the geometric mean values at each phase of the menstrual cycle. Ovulation should occur in group 2 (-18 through -13 days from menstruation). With the exception of P and DHEA sulfate, which were lower, analytes in samples from postmenopausal women were not significantly different from those from premenopausal women.    Comment: The most significant finding is that NAF E2 does not parallel serum levels but follows closely NAF P levels. NAF E2 was actually lowest when serum E2 was highest. Changes in other analytes during the menstrual cycle were minimal. Estradiol, its direct precursors, and the E2-related protein, cathepsin D, were not decreased in NAF after menopause. (Supported by NIH R01 CA 120555-01).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4077.