bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-14-05: Final Overall Survival Analysis of the TBP Phase III Study (GBG 26/BIG 3-05): Capecitabine vs. Capecitabine + Trastuzumab in Patients with HER2-Positive Metastatic Breast Cancer Progressing during Trastuzumab Treatment
Authors: von Minckwitz G , Schwedler K , Schmidt M , Barinoff J , Mundhenke C , Cufer T , Maartense E , de Jongh F , Baumann K , Bischoff J , Harbeck N , Lück H-J , Maass N , Zielinski C , Andersson M , Stein R , Nekljudova V , Loibl S.
Published: April 2011

Background: In women with HER2-positive breast cancer progressing during trastuzumab treatment, continuation of trastuzumab plus capecitabine (XH) showed a significantly improved overall response rate and time to progression compared with capecitabine (X) alone (von Minckwitz et al, J Clin Oncol 27:1999-2006, 2009). Here we report the final analysis of overall survival (OS).

Methods: Patients (pts) with HER2-positive, locally advanced or metastatic breast cancer who progressed during treatment with trastuzumab with or without adjuvant and/or 1st-line metastatic chemotherapy were prospectively randomized to X (2500 mg/m2 on days 1-14, q3w) or XH (6 mg/kg, q3w). OS was a pre-specified secondary endpoint of the study. Results: Median follow up at June 2010 was 20.7 months. 59 of 74 and 60 of 77 pts died in the X alone and XH arm, respectively. Median OS was 20.6 and 24.9 months with X alone and XH, respectively. This difference was not statistically significant (HR=0.94 [0.65-1.35]; p=0.73). Cox proportional hazard model showed performance status, hormone receptor status and metastatic site as independent prognosticators for survival. No difference between treatment arms was observed for pts with a clinical response or clinical benefit, respectively. Pts who continued/restarted anti-HER2 treatment (trastuzumab or lapatinib) after 2nd progression (N=88) had an OS of 18.8 compared with 13.3 months for those who did not receive 3rd line treatment with anti-HER2 agents (N=52) (HR 0.63; p=0.02). Pts who continued treatment after progression as initially randomized (anti-HER2 treatment after XH (N=31) or no anti-HER2 treatment after X alone (N=53)) had an OS of 26.7 months and 20.4 months (HR 0.71; p=0.2). Conclusions: Final OS analysis of the GBG-26 study could not demonstrate a survival benefit for treatment beyond progression with trastuzumab. However in a post-hoc analysis, pts receiving anti-HER2 treatment as 3rd line therapy showed a better OS than those not receiving this targeted treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-05.
Abstract P6-14-14: Concerted Anti-Tumor/Radiosentizing Effect of Epigallocatechin Gallate, Resveratrol and Genistein by Targeting Tumor Invasion and Metastasis Molecules in Human Breast Cancer Cells
Authors: Veeraraghavan J , Kardokus K , Natarajan M , Aravindan S , Herman TS , Aravindan N.
Published: April 2011

Accomplishing the desired anti-tumor efficacy with individual dietary phytochemicals is clinically unachievable. This is due to the existence of converging and diverging pathways spanning the complex metabolic networks in cancer cells. This complexity demands the development of a multi-component approach with physiological doses of phytochemicals that could be used as “deliverable” anti-cancer agent. To address this concern, we investigated the synergistic efficacy of Genistein (GEN), Epigallocatechin gallate (EGCG) and Resveratrol (RES) in mitigating cancer progression. Human breast adenocarcinoma cells (MCF-7 and MDA-MB-231) were treated with physiological concentration of GEN, EGCG, RES, individually or all three together (GER). In parallel cultures cells were treated with GER and either exposed to radiation (2Gy;IR) or treated with β-estradiol (≥2E, positive control). The trated cells were examined after 24h. In MCF-7 cells, QPCR profiling of 93 tumor invasion and metastasis molecules revealed that EGCG, RES and GEN significantly (≥2 fold) induced 23, 36, 22 genes and completely suppressed 61, 40 and 64 genes respectively as stand-alone compounds. Conversely, GER completely knocked down the transcription of 82 genes, while another 9 genes were maintained at baseline level. More importantly, GER significantly inhibited 75 and 63 genes in ≥2E and IR exposed MCF-7 cells. Interestingly, in highly metastatic MDA-MB-231 cells, EGCG, RES, GEN significantly induced 17, 19, 2 genes and completely suppressed 41, 26 and 57 genes respectively as stand-alone compounds. Conversely, GER completely suppressed 67 genes while another 17 genes were maintained at baseline level. To that end, GER significantly inhibited 57 and 59 genes and maintained 16 genes at basal level in ≥2E and IR exposed MCF-7 cells. Taken together, these data evidently exhibit the potential synergestic effect of EGCG, GEN and RES in mitigating breast cancer progression as opposed to their stand alone effect. More importantly, these data strongly suggests that GER potentiates synergistic radiosensitization in these cells and this GER influenced radiosensitization may involve selective targeting of tumor invasion and metastatic transcription.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-14.
Abstract P6-14-17: In Vitro Evaluation of the Anti-EGFR Antibody Cetuximab and the Anti-HER2 Antibody Trastuzumab in a Panel of Human Tumor Cell Lines
Authors: Marcoe KF , Ovechkina Y , Keyser R , Bernards K , Chestnut-Speelman J , Yoshino K , O'Day C.
Published: April 2011

The epidermal growth factor receptors, EGFR (erbB-1) and HER2 (erbB-2), and their down stream signaling events promote tumor growth and survival in a variety of epithelial tumors. Current treatment strategies used to target these receptors include mAbs directed against their extracellular domains and small molecule inhibitors of their tyrosine kinase activities. The efficacy of these types of therapeutics is increasingly being found to be contingent on the genotype of the targeted cancer. In vitro evaluation of mAbs is reliant on selection of tumor cell lines driven by the specific antigen proteins of interest and their associated pathways. We selected a panel of eight human tumor cell lines, six breast cancer (BT474, SK-BR-3, MDA-MB-453, MDA-MB-231, MDA-MB-468 and MCF-7), one colon (SW48) and one skin (A431) with varying levels of HER2, EGFR and EGFR/HER2 expression to evaluate the mAbs cetuximab and trastuzumab for antiproliferative effects, ability to induce apoptosis and the ability to block cell cycle. These findings were compared to results obtained after treatment with lapatinib, erlotinib, Ly294002 and Tamoxifen. The BT474 and SK-BR-3 breast cancer cell lines with HER2 amplified expression demonstrated cytostatic antiproliferative and G1/S cell cycle block sensitivity to the anti-HER2 antibody trastuzumab. The more prominent inhibitory effects observed in the BT474 cell line were further confirmed by measurable induced decreased levels of phospho-Akt and increased levels of the CDK2 inhibitor p27Kip1 following treatment with trastuzumab, lapatinib and Ly294002. The SW48 colon and A431 skin cancer cell lines with EGFR mutations and EGFR over expression, respectively, demonstrated cytostatic antiproliferative sensitivity to the anti-EGFR antibody cetuximab. This in vitro cell-based multiplexed approach for assessment of oncology therapeutics based on genetically directed cell line selection, demarcation of cytostatic and cytotoxic cell proliferation inhibition and assessment of receptor down stream signaling biomarkers offers greater predictive efficacious value and can be used to identify optimal candidates for combination therapies.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-17.
Abstract P6-15-03: Phase Ib Trial of the Gamma Secretase Inhibitor (GSI), MK-0752 Followed by Docetaxel in Locally Advanced or Metastatic Breast Cancer
Authors: Schott AF , Chang JC , Krop IE , Griffith KA , Layman RM , Hayes DF , Wicha MS.
Published: April 2011

Background: The cancer stem cell hypothesis asserts that there is a small population of cells within a tumor that has the ability to self renew and differentiate, and that these cells drive tumor growth and metastasis but are resistant to conventional cytotoxic chemotherapy. Pathways involved in stem cell growth and differentiation are viable targets for new anticancer therapies. One such pathway, Notch, is inhibited by GSIs which prevent translocation of Notch intracellular domain to the nucleus. Inhibition of GS concurrent with chemotherapy might improve disease control by targeting both stem cells and differentiated cells within the tumor. This Phase Ib clinical trial was designed to determine the MTD of the GSI, MK-0752, in combination with docetaxel, and to evaluate an effect on stem cell markers in serial tumor biopsies.

Methods: Eligible subjects had metastatic breast cancer or locally advanced breast cancer that did not respond to anthracycline therapy. Patients with disease that progressed on a taxane, or who had received a taxane within 6 months were excluded. MK-0752 was administered orally on days 1-3 of each 21-day cycle of therapy, in escalating doses. Dose levels (mg/day) 1=300; 2=450; 3=600; and 4=800. Docetaxel 80 mg/m2 IV was administered day 8, with pegfilgrastim day 9 each cycle. Treatment was continued until disease progression, unacceptable toxicity, or symptomatic deterioration. The trial was monitored using the Time to Event Continual Reassessment Method, targeting a 20% toxicity rate. Tumor biopsies were performed at baseline, after 1 cycle, and at treatment completion in a subset of patients.

Results: 30 patients were enrolled between Mar 2008 and Jan 2010. Dose limiting toxicities of the combination included diarrhea, hand-foot syndrome, and LFT elevation. 20/30 patients experienced Grade 1 or 2

fatigue. The final estimates and confidence intervals for the probability of dose limiting toxicity at each dose level are summarized in the table:

Probability = probability of dose-limiting toxicity 20 enrolled patients had measurable disease by RECIST criteria. Of these, 9 had PR, 8 SD, and 3 PD, for an estimated RR of 45% to the combination. 2 patients have been maintained on therapy in excess of 22 cycles. Conclusions: Dose level 3 was identified for further study in a Phase II randomized trial. Efficacy of docetaxel was not inhibited by MK-0752, as a 45% RR in patients with measurable disease was observed. There is intriguing long term disease stabilization in 2 patients. Evidence of an effect of the combination on the stem cell population was apparent on serial biopsies as presented at SABCS Dec 2009 (Abstract # 48); additional biopsy data will be presented.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-03.
Abstract P6-15-04: Targeting the PI3K Pathway Enhances the Effect of PARP-1 Inhibition in BRCA1 Deficient Breast Cancer Cells
Authors: Kimbung S , Aaltonen K , Gruvberger-Saal S , Berglund P , Hedenfalk I.
Published: April 2011

Background: Available targeted therapies have improved the prognosis for many women diagnosed with breast cancer. However, more than 15% of cases do not benefit from these targeted agents. Amongst these are tumours with Breast cancer 1 (BRCA1) dysfunction, triple-negative and basal-like breast cancers, which remain a treatment challenge and are associated with a poor outcome. New approaches to specifically target these groups of breast tumors are needed. The DNA damage repair defects associated with lack of BRCA1 confers sensitivity to poly-(ADP-ribose) polymerase 1 (PARP-1) inhibitors. Furthermore, most BRCA1 mutated tumours do not express the phosphatase and tensin homolog (PTEN) implying activation of the phosphatidylinositol-3 kinase (PI3K) pathway which promotes cell survival and resistance to apoptosis. The aim of this study was to investigate if co-targeting the PI3K pathway can improve the response of BRCA1 mutated cells to PARP-1 inhibition.

Material and methods:, Two BRCA1 and PTEN deficient breast cancer cell lines; MDA-MB-436 and SUM149, were treated with the PARP-1 inhibitor AG14361 (1-(4-dimethylaminomethyl-phenyl)-8-9-dihydro-7H-2,7,9a-benzo[cd]azulen-6-one) and the PI3K inhibitor LY294002 (2-(4-morpholinyl)-8-phenyl-chromone), as single agents and in combination. Cytotoxicity was estimated using the sulpho-rhodamine B assay while cell cycle phase distribution and DNA integrity were analyzed by flow cytometry.

Results: AG14361 induced DNA damage leading to G2/M arrest and decreased viability. LY294002, on the other hand, affected the cell cycle by arresting cells in G1. Importantly, combining AG14361 with LY294002 further decreased survival (P<0.001) with a concomitant decrease in G2/M (P<0.05).

Discussion: This observation may indicate a combination specific up-regulation of apoptosis and suggests that LY294002 potentiates the effects of AG14361. Our results may represent an improved selective targeted treatment strategy for BRCA1 deficient breast cancers and other cancers with DNA damage repair defects and activated PI3K signalling.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-04.
Abstract P6-15-05: A Cyclin-Dependent Kinase Inhibitor P276 Inhibits Growth of Breast Cancer Cell Lines Including Basal-Type/Triple-Negative Alone and in Combination with Chemotherapeutic Drugs
Authors: Sharma S , Rathos M , Manohar S , Joshi K , Badhwar A , Kharas F , Sonawane V , Chaudhuri U , Joshi K.
Published: April 2011

Background: Development of novel agents and drug combinations are urgently needed for the treatment of breast cancer especially triple negative breast cancer (TNBC). P276 is a potent cyclin-dependent kinase inhibitor with excellent antiproliferative activity against various human cancer cell lines. Here, we report the activity of P276, as a single agent and in combination with paclitaxel or gemcitabine/carboplatin in TNBC cell lines.

Material and Methods: In vitro effect of P276 on breast cancer cell lines was determined by cytotoxicity assay, flow cytometry, western blotting and immunofluorescence studies. The antiangiogenic potential of P276 was evaluated using HIF-1 a and VEGF inhibition assay, wound healing and tube formation. In vivo efficacy was studied using human xenograft models in SCID and nude mice.

Results: Human breast cancer cell lines including TNBC, treated with P276 were found to be highly susceptible with IC50 ranging from 0.3 to 1.0 mM. In MCF-7 (Her2-, BRCA+/−) and MDA-MB-231 (ER-, PR-, Her2-). P276 significantly down regulated cell cycle proteins pRbser780, cyclin D1, Cdk4 and antiapoptotic protein Bcl-2 followed by 80-85% apoptosis. P276 treatment also lead to the inhibition of PARP enzyme activity or PARP cleavage depending on the cell type. Interestingly, P276 inhibited the key angiogenic mediators HIF-1 a and VEGF with IC50 of 0.095mM and 0.31mM respectively in reporter gene based assays. These antiangiogenic effects were confirmed by immunofluorescence and migration studies in TNBC cell lines and endothelial tube formation. In breast cancer patient derived tumor cell line MAXF 401 (basal like) xenografts in nude mice, cyclical dosing of P276 showed growth inhibition of 71%. Moreover, combination studies of P276 with paclitaxel or gemcitabine plus carboplatin showed synergistic effect in TNBC cell lines. Discussion: Our studies provide compelling evidence for the clinical development of P276 for the treatment of triple negative breast cancer either as monotherapy or in combination with paclitaxel or gemcitabine/carboplatin.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-05.
Abstract P6-15-10: Targeting Delta-Like 4 Ligand (DLL4)/Notch Signaling by a Novel Anti-DLL4 Antibody Inhibits Tumor Growth through Altering Cancer Stem Cell and Epithelial-to-Mesenchymal Transition-Associated Genes in Triple Negative Breast Cancer
Authors: Yen W-C , Fischer M , Beviglia L , Yeung P , Kapoun A , Wang M , Lewicki J , Gurney A , Hoey T.
Published: April 2011

Triple negative breast cancer (TNBC, ER-, PR-Her2-) accounts for 10% of all types of breast cancer and is known to be particularly aggressive and refractory to current therapies. Chemotherapeutic agents, such as taxanes, are currently the only treatment option for this type of breast cancer. However, these treatments often result in local and systemic relapse. While several promising targeted agents have improved the relapse rate in the combination with chemotherapy in patients with pathologic complete response, those who do not achieve pathologic complete response still have a poor prognosis. We previously demonstrated that interfering with Deltalike 4 ligand (DLL4)/Notch signaling by an anti-DLL4 antibody decreased tumor growth and delayed tumor recurrence following paclitaxel treatment through reducing breast cancer stem cell fraction and tumorigenicity (Proc Am Asso Cancer Res. 51:4, 2010). CD44+/CD24low cells have been demonstrated to be highly tumorigenic in breast tumors and are associated with treatment resistance (PNAS U S A, 106: 13820-13825, 2009). To gain insights into the mechanism of action of anti-DLL4-mediated TNBC growth inhibition, we evaluated the effect of anti-DLL4 on breast cancer CD44+/CD24low the expression patterns of a list of genes involved in sternness, differentiation, and key stem cell signaling pathways in TNBC xenograft tumors following paclitaxel with and without antibody treatment in vivo. Our data showed that paclitaxel treatment resulted in significant changes of gene expression patterns in the CD44+/CD24low cells in the residual tumors. We further analyzed these genes and identified several genes related to Notch signaling, stem cell maintenance and epithelial-to-mesenchymal transition that were modulated differentially by treatment with paclitaxel and/or anti-DLL4. In a metastatic TNBC orthotopic tumor model, the anti-DLL4 treatment resulted in tumor growth inhibition at the primary site and distant organs. Taken together, our findings suggest that targeting DLL4 may improve the efficacy of current treatments, delay tumor recurrence, and reduce metastatic growth in TNBC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-10.
Abstract P6-15-14: Inhibit SIAH E3 Ligase, a Downstream Signaling Module Required for the HER2/EGFR/RAS Signal Transduction, To Block Tumorigenesis and Metastasis of Highly Invasive Human Breast Cancers
Authors: Tang AH , Park CH , Liao Y , Ahmed AU , Reed NR , Hesse SE , Lingle WL , Jimenez RE , Tang AH.
Published: April 2011

The central importance of the HER2/Neu/EGFR/RAS signaling pathway has been well established in the growth, invasion and metastasis of human breast cancer, even though oncogenic RAS mutations are rare in breast cancer, activation of the tumor-promoting ERBB/RAS/MEK/MAPK pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated ERBB/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the RAS pathway — the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We then asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These findings suggest that SIAH2 may be an attractive new therapeutic target for novel breast cancer therapy and developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. More effective breast cancer treatments may be obtained by multi-pronged synergistic inhibitions at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel. Thus, by attacking the growth-promoting ERBB/RAS pathway at multiple signaling modules simultaneously, we may be in a great position to halt the genesis, progression and metastasis of human breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-14.
Abstract P6-15-16: LIV-1 Antibody-Drug Conjugate: A Novel Therapeutic Agent for Breast Cancer
Authors: Smith LM , Sussman D , Nesterova A , Westendorf L , Duniho S , Anderson M , Mbow B , Gregoire A , Hunter J , Whiting N , Law CL , Benjamin DR.
Published: April 2011

LIV-1, also known as SLC39A6 or ZIP6, is a member of the zinc transporter family and was first identified as an estrogen-inducible gene in breast cancer. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. Consistent with its role in cancer, we determined by immunohistochemical (IHC) analysis that LIV-1 is expressed in 86% (n=22) of estrogen receptor-positive (ER+), hormone-treated tumors (both primary and metastatic sites), and in 65% (n=20) of ER-/PR-/Her2- (triple-negative) breast cancers. In healthy human tissues, LIV-1 expression is limited to hormonally-regulated organs (prostate, uterus, and breast). We generated an antibody-drug conjugate (ADC) consisting of a humanized anti-LIV-1 mAb conjugated to the antitubulin agent monomethyl auristatin E (MMAE), via a plasma stable, enzyme-cleavable linker (vc). The humanized LIV-1 mAb bound with high affinity to both human and cynomolgous LIV-1 (2-4 nM). In vitro, anti-LIV-1-vcMMAE showed specific cytotoxic activity against a MCF-7 cell line with an IC50= 78 ng/ml. In vivo studies also demonstrated antitumor activity of anti-LIV-1-vcMMAE in preclinical xenograft models, including MCF-7 breast cancer cell line, with significant delay of tumor growth compared to control groups. The broad expression of LIV-1 in both hormonally-treated and triple-negative tumors and the limited expression in vital organs make LIV-1 an excellent target for an ADC. These findings support the further development of anti-LIV-1-vcMMAE as a promising therapeutic agent for the treatment of ER+ and triple-negative breast cancers.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-16.
Abstract P6-15-20: Development of PEA-15-Based Therapy in Breast Cancer in a Preclinical Study
Authors: Xie X , Bartholomeusz C , Maxwell DS , Peng Z , Kazansky A , Bornmann WG , Ueno NT.
Published: April 2011

Background: PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) has tumor suppressor properties in both breast and ovarian cancer cells. Overexpression of PEA-15 in MDA-MB-468 triple-negative breast cancer cells using adenoviral vector (Ad.PEA-15) inhibited cell growth by inducing apoptosis. Treatment with Ad.PEA-15 led to significant regression of tumors in mice, indicating that PEA-15 could have therapeutic potential in breast cancer. PEA-15 exerts its antitumor activity by binding to ERK in the cytoplasm and preventing its translocation into the nucleus, thereby inhibiting ERK-dependent transcription and cell proliferation. Because ERK inhibitors have been shown to be toxic, in this study, we developed PEA-15 mimetic peptides as a therapeutic agent in breast cancer. These peptides, which are expected to be less toxic because of their ability to mimic PEA-15, are proposed to inhibit cell proliferation by sequestering ERK in the cytoplasm.

Material and Methods: PEA-15 mimetic peptides were designed and synthesized based on structural analysis of linear segments from the C-terminal end of PEA-15, which is one of the two regions that are presumably critical for ERK binding, using nuclear magnetic resonance spectroscopy. These synthetic peptides were labeled with FAM to enable determination of their subcellular localization. The PULSin reagent was used as a delivery reagent for peptide penetration into cancer cells. The penetration ability of these peptides into HeLa and MDA-MB-468 cells was analyzed by fluorescence microscope and fluorescence-activated cell sorting. Their effect on ERK subcellular localization was examined by cell-fractionation analysis. Their impact on cell survival was determined by trypan blue viability assay and propidium iodide staining. Results: Ten peptides were designed and synthesized based on the C-terminal segments of PEA-15. Three of these FAM-labeled synthetic peptides delivered with the PULSin reagent were able to penetrate HeLa and MDA-MB-468 cells with 80-90% and 30-40% efficiency, respectively. Further, these peptides redirected ERK into the cytoplasm from the nucleus in MDA-MB-468 cells and caused 31% reduction in viability of HeLa cells as compared to the untreated control. Ongoing studies are evaluating PEA-15 peptides for their ability to interact with ERK using ELISA. In the future, we will also test their therapeutic efficacy and safety using a xenograft breast cancer model.

Discussion: Previous studies revealed that PEA-15 has antitumor activity against breast and ovarian cancer cells by sequestering ERK in the cytoplasm. Due to the toxicity of ERK enzymatic inhibitors, we developed PEA-15 mimetic peptides and demonstrated their therapeutic potential for breast cancer, which may provide opportunities for breast cancer patients who currently have limited treatment options.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-20.
Abstract PD01-05: A Combination of HDAC Inhibitor Entinostat (MS-275), All Trans Retinoic Acid (ATRA) and Chemotherapy Drug(s) Causes Regression of Established Xenografts of Triple Negative Breast Cancer
Authors: Nguyen NK , Korangath P , Sabnis G , Brodie A , Ordentlich P , Stearns V , Sukumar S.
Published: April 2011

Triple negative breast cancer (TNBC) is a subgroup of breast cancer that rapidly develops resistance to chemotherapy. Currently, TNBC patients are treated with anthracycline and taxane-based regimens. These treatments result in some improvements in the clinical outcomes; however, they are associated with poor prognosis. Also, endocrine therapies are ineffective since the tumors do not express the estrogen receptor. Recent studies have shown that histone deacetylase (HDAC) inhibitors could reverse the epigenetic profile of some genes, including ER, EGFR, and RAR≥2. We have recently shown that treatments that combine the HDAC inhibitor, Entinostat (MS-275), with the aromatase inhibitor, Letrozole, result in regression of xenografts of MDA-MB-231 breast cancer cells. Since RAR≥2 is re-expressed as well, we reasoned that combining epigenetic therapy using MS-275, with differentiation therapy using a RAR≥2 agonist (ATRA) will provide an effective combination of drugs against TNBC. We also examined possible potentiation of anticancer activity of these agents by low nontoxic doses of chemotherapy (doxorubicin and paclitaxel). In vitro, human TNBC cell lines, MDA-MB-231, HCC 1143, SUM159, SUM149 and BT20 responded significantly better to the triple combination compared to the drugs used singly or in combination with MS-275. Upon treatment, re-expression of the silenced RAR≥2 and downstream effectors was observed in the cell lines in vitro and in tumor xenografts of MDA-MB-231 cells. Subcutaneous tumor growth in immunodeficient mice was strongly inhibited (10/14 significant regression) using a combination of MS-275, ATRA and low dose Doxorubicin, compared to the drugs administered singly or in combination with MS-275. No significant weight loss was observed in any of the groups. This combination was then used to treat tumor xenografts of SUM159, another TNBC cell line, and gave a promising result. Furthermore, combination of MS-275, ATRA, and a clinical dose of Paclitaxel can effectively inhibit growth of MDA-MB-231 and SUM159 cells. These results suggest that combination of HDAC inhibitor and RAR≥2 agonist with low dose chemotherapy has the potential to be an effective treatment against TNBC. This study provides encouraging new information for a nontoxic, and novel combination of drugs which could be effective against TNBC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD01-05.
Abstract PD02-01: Human Breast Cancer Lymphovascular Tumoral Emboli and Micrometastasis Recapitulate an In Vitro Mammosphere Stem Cell Phenotype
Authors: Barsky SH , Graham CE , Llewellyn AL , Ye Y , Yearsley K.
Published: April 2011

Background: The existence of stem cells in human cancers has been inferred by clonality experiments and marker studies in vitro and clinical observations in vivo concerning tumor recurrences and emerging drug resistance. We have studied the existence of stem cells in a human model of inflammatory breast cancer, termed MARY-X, a model which forms spheroids which are similar to normal tissue stem cell-derived mammospheres. Comparing MARY-X with common non-IBC breast carcinoma and normal cell lines, we found specific embryonal stem cell markers within the MARY-X spheroids. RT-PCR analyses of MARY-X also revealed the expression of transcriptional determinants essential for the pluripotency and self-renewal of human embryonal stem cells. Since MARY-X spheroids, when injected into mice, form tumors with florid lymphovascular emboli that give rise to micrometastases in numerous organs, we wondered whether tumor emboli and micrometastases from actual human cancers also recapitulated a mammosphere stem cell phenotype.

Material and Methods: We carried out laser capture microdissection of tumor emboli and lymph node micrometastases in 200 cases of human breast cancer including cases of both infiltrating ductal as well as lobular cancer. These cases included all of the common molecular classes of breast cancer including luminal A, luminal B, luminal C, Her-2/neu positive and triple negative including the basal subtype. The common denominator which was studied in all of these cases was the lymphovascular embolus and lymph node micrometastasis.

Results: By both RT-PCR and IHC, lymphovascular emboli and micrometastases exhibited five-ten fold higher stem cell markers including Stellar, H19, Rex-1, Nestin, CD133 and Aldehyde Dehydrogenase 1 (ALDH1) as well as stem cell transcriptional determinants including OCT4, SOX2, and Nanog than the non-embolic tumoral areas. In addition, stem cell signaling pathways specifically involved in self-renewal and pluripotency including Bmi-1, Hedgehog and Notch 3 were activated selectively within the lymphovascular tumor emboli and micrometastases.

These observations held true irrespective of the molecular class of breast cancer from which the embolus was derived and irrespective of the adhesion status (presence or absence of E-cadherin) of the embolus or micrometastasis.

Discussion: Our findings indicate that the lymphovascular embolus and micrometastasis is not simply a cellular fragment that detaches from the main tumor but rather represents a selection for a stem cell phenotype. This finding may explain the increased resistance of lymphovascular tumor emboli and micrometastases to chemotherapy and the decreased disease-free survival and poorer prognosis exhibited by patients with significant lymphovascular emboli and micrometastases.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-01.
Abstract PD02-05: Low Immunogenicity of Breast Cancer Stem Cells Leads to Selective Survival When Challenged with Trastuzumab and Natural Killer Cells
Authors: Honig A , Reim F , Becker K , Engel JB , Junker M , Dietl J , Wischhusen J.
Published: April 2011

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Considering that clinical efficacy of trastuzumab correlates with the infiltration of natural killer (NK) cells into the tumor site, we established a cell-culture-system to select for ovarian cancer and mammary carcinoma cells that survive a challenge by trastuzumab and NK cells. Under these conditions, NK cells can eliminate tumor cells either via antibody-dependent-cell-mediatedcytotoxicity (ADCC) or by direct recognition of tumor-associated ligands for NKG2D or DNAM-1. The most striking phenotypic alteration observed in immunoselected ovarian cancer cells was a down-regulation of HER2 expression, leading to an ADCC-resistant phenotype. All breast cancer cells tested (MCF7, SK-BR-3, MDA-MB231, BT474), however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK-cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44highCD24low ‘'cancer-stem-celllike” phenotype (CSC) and expressed significantly less HER2 compared with non-stem cells. Moreover, the molecular determinants for the direct recognition of transformed cells, most notably the NKG2D ligands MICA, MICB, ULBP1-4 and the DNAM-1 ligands CD112 and CD155, were virtually absent from CSC.

When immunoseleced breast cancer cells were then re-expanded, they mostly lost the observed phenotype and regenerated a tumor cell culture that displayed initial HER2 surface expression and ADCC susceptibility, but was enriched in CD44highCD24low CSC. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. We provide evidence that recruition of NK-cells and induction of ADCC by trastuzumab may spare actual tumor-initiating-cells, which could explain clinical relapse. Moreover, our observation that “relapsed” in vitro cultures show identical HER2 surface expression and susceptibility toward ADCC suggests that administration of trastuzumab beyond relapse might be considered, especially when combined with proteasome inhibition which increase the expression of NKG2D ligands.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-05.
Abstract PD03-01: Significant Reduction of Triple-Negative Breast Cancers in Diabetic Women on Metformin
Authors: Meiers PJ , Sharon E , DongYu W , Pooni A , McCready DR , Leong WL.
Published: April 2011

Background: Diabetes is an increasingly prevalent chronic disease. Many of these patients may develop breast cancer (BC). A meta-analysis by Larsson (2007)demonstrated that diabetic women have an increased risk (RR: 1.2) of BC particularly for estrogen receptor positive (ER+) subtypes (RR: 1.22). However, a recent study by Bodmer (2010) showed that women on long-term metformin have a reduced incidence of BC (OR 0.44, 95% CI 0.24-0.82). Metformin has antiproliferative effects on BC based on studies using proliferative marker Ki67. BC patients on metformin have better cancer-specific survival based on Landman (2010). We hypothesize that when compared to other diabetic medications; including exogenous insulin and other oral hypoglycemics; metformin specifically reduces the incidence of triple negative BCs (TNBC or ER-/PR-/Her2-). Methods: We conducted a retrospective chart review of an unselected cohort of patients who underwent surgical interventions for their primary BC to correlate the history of pre-existing diabetes and metformin use to the BC subtypes based on the immunohistochemistry of the surgical specimens. P-values were calculated using Chi-square and Fisher exact tests.

Results: There were 99 TNBC in the 1005 patients reviewed. Of the 90 diabetic patients, none of the 44 patients took metformin had TNBC, compared to 7 of the 46 diabetic patients not taking metformin had TNBC (0% vs. 15.2% p=0.007). Discussion: Studies that examined the incidence of BC in diabetics were not able to differentiate the effects of various diabetic treatments on the subtypes of BC. The reported increase in ER+ BC in diabetics is likely multifactorial but may be attributed to the proliferative effects of hyperinsulinemia. Interesting, of all the diabetic medications, Bodmer (2010) reported only metformin reduced the incidence of BC. In this study, we reported a statistical significant reduction in the incidence of TNBC. In fact, in our study population, there was no TNBC in patients who took metformin. Hirsch (2009) reported a selective inhibition of BC stem cells with metformin. Of interest, many studies have shown that BC stem cells were ER-/PR-/Her2-. This study supports the view that the phenotype of the BCs can be influenced by drug. At this point, we cannot differentiate if metformin can “protect” patients from developing TNBC, or “convert” TNBC to other subtypes of BC, further study is underway to address this question.

Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-01.
Abstract PD03-07: Evaluation of the Anti-Diabetic Drug Metformin in the Methylnitrosourea (MNU)-Induced Model of ER+ Mammary Cancer and the MMTV-Neu/p53KO Model of ER-Mammary Cancer
Authors: Grubbs CJ , Bode AM , Steele VE , Lubet RA.
Published: April 2011

Metformin, an anti-diabetic drug, activates AMP-activated protein kinase (AMPK), and has generated a great deal of interest as a potential preventive agent for a variety of cancers, including breast. This interest is based both on epidemiologic studies showing that diabetics treated with metformin have a lower incidence of a variety of cancers, and on mechanistic considerations arguing that activation of AMPK is likely to be relevant to cancer treatment and prevention. We are examining the preventive efficacy of metformin in ER+ and ER-rodent mammary cancer models. Treatment of Sprague-Dawley female rats at 50 days of age with a single dose of MNU (75 mg/kg BW) resulted in the development of multiple ER+ mammary cancers beginning approximately 7 weeks after MNU treatment. This model has been highly responsive to a wide variety of agents that affect ER+ tumors in humans (SERMs, aromatase inhibitors, pregnancy, ovariectomy). Metformin was administered by gavage (7x/week) at either 150 or 50 mg/kg BW/day beginning 7 days after MNU dosing, and continuing for the duration of the study. Body weights were not altered by these dose levels and other signs of toxicity were not observed. Metformin failed to decrease mammary tumor incidence and, in fact, increased tumor multiplicity in this model by approximately 35%. In addition, we are evaluating the ability of metformin to inhibit mammary cancer in MMTV-Neu/p53KO mice (which spontaneously develop ER-cancers). In this study, metformin is being administered in the diet at a dose level of 1500 mg/kg diet. Initial supplementation of the diet with this agent began when the mice were 60 days of age, and will continue for 10 months. Preliminary results examined at 6 months into the study imply no efficacy of metformin in this model as well (average tumors/mouse: controls, 0.9; metformin, 1.6). Thus, these initial studies fail to observe any chemopreventive efficacy of metformin in two commonly employed models of ER+ or ER-mammary cancer in rodents. Supported by NCI contract number HHSN261200433001C.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-07.
Abstract PD04-03: Circulating Tumor Cells as Predictor of Metastatic Disease Spread in Patients with Breast Cancer
Authors: Giuliano M , Giordano A , Handy BC , Ueno NT , Alvarez RH , Hortobagyi GN , Reuben JM , Valero V , Cristofanilli M.
Published: April 2011

Background. The formation of distant metastases in each tissue is a tightly regulated biological event. The characteristic organ distribution of metastasis in the various cancers was first recognized in 1889 by Paget and gave rise to the “ seed and soil “ hypothesis. Several reports demonstrate that the presence of more than 5 circulating tumor cells (CTC) in peripheral blood is associated with short survival in patients (pts) with metastatic breast cancer (MBC). Interestingly, pts with bone metastasis had higher absolute number and percentage of ≥5 CTC (De Giorgi et al. 2010). Because of the correlation between CTC detection, metastatic site and prognosis, we hypothesized that the baseline detection of ≥5 CTC at the time of initial recurrence can predict the development of new metastatic sites, when progression of disease occurs.

Methods. We retrospectively evaluated a cohort of 516 MBC pts who had a CTC assessment before starting systemic treatment at the M.D. Anderson Cancer Center between September 2004 and November 2009. CTC were enumerated using the CellSearch™ technology. Among all pts, we selected 408 women with documented radiological progression of disease defined according to RECIST. Pts were divided in 2 categories: subjects who experienced progression of the pre-existing metastatic site(s) and those who developed new metastatic site(s). The rate of development of new metastatic sites was compared between groups according to baseline value of CTC (<5 vs of ≥5), by Fisher Exact Test.

Results. At a median follow-up of 16 months (range, 1-58), 168 (41%) pts had died. The estimated overall survival was 28.6 months for pts with <5 CTC and 19.1 months for those with ≥5 CTC (log-rank P=.001). The rates of development of new metastatic sites according to baseline value of CTC, and stratified by the original sites of disease are shown in table 1. Fifty-three (22%) pts with <5 CTC and 52 (32%) with ≥5 CTC developed new metastatic sites when progression of disease was documented (P=.028). Pts presenting with baseline CTC ≥5, and bone metastases without visceral involvement had the highest rate of development of new metastatic sites.

Conclusions. Our analysis indicates that the presence of a high number of CTC before starting systemic treatment can predict for a higher probability to develop new metastatic sites on progression. This finding seems to be largely limited to pts with bone involvement at baseline. Our results may suggest the need to evaluate more effective bone-directed therapies for MBC pts with ≥5 CTC in order to prevent further progression of disease.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-03.
Abstract PD04-08: Circulating tumor cells (CTC) in peripheral blood of breast cancer patients two years after adjuvant chemotherapy depending on endocrine treatment — The German SUCCESS-Trial
Authors: Janni W , Zwingers T , Hepp P , Andergassen U , Schneeweiss A , Lichtenegger W , Beckmann MW , Sommer H , Pantel K , Salmen J , Jueckstock J , Rack BK.
Published: April 2011

Background: The prognostic significance of CTC in metastatic, as well as in primary breast cancer has been demonstrated (Rack et al., ASCO 2010). The optimal endocrine treatment strategy for postmenopausal patients (pts) with hormone sensitive breast cancer remains unclear.We analyzed the prevalence of CTC two years after primary diagnosis in patients with tamoxifen or anastrozole treatment.

Methods: As part of the translational research project of the German SUCCESS-trial, we analyzed 23ml of peripheral blood from 307 N+ and high risk N-postmenopausal pts with hormone sensitive breast cancer two years after adjuvant taxane based chemotherapy and with tamoxifen or anastrozole treatment. The presence of CTCs was assessed with the CellSearchSystem (Veridex, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labelled with anti-cytokeratin (8,18,19) and anti-CD45 antibodies to distinguish between epithelial cells and leukocytes. Standard within the study was early switch treatment (tamoxifen for 2 years, followed by anastrozle), while pts with contraindications against tamoxifen were allowed to receive anastrozole up-front.

Results: In 10.1% of pts (n=31) >1 CTC was detected after the completion of chemotherapy (range 2-33), while 7.8% (n=24) presented with >1 CTC (range 2-99) two years after completion of chemotherapy. The median age in the tamoxifen group was 59.9 years and 59.8 in the anastrozole group. In the tamoxifen group, 33.0% of the pts had a pT1 tumor, 5.3% G1 grading and 21.6% of the pts were node negative. In the anastrozole group, 30.0% of the pts had a pT1 tumor, 7.5% G1 grading and 22.5% of the pts were node negative, respectively. None of these differences were statistically significant. After the completion of chemotherapy, 9.7% of the pts were CTC positive in the tamoxifen group (range number of cells: 2-33) and 11.3% in the anastrozole group (range of cells: 2-24), p=0.69. Two years after primary diagnosis, 7.9% of the pts were CTC positive in the tamoxifen group (range number of cells:2-99) and 7.5% in the anastrozole group (range cells: 2-5), p=0.90. Actuarial disease free and overall survival will be presented at the meeting.

Conclusions: The prognostic relevance of CTC in peripheral blood of early breast cancer patients both before and after chemotherapy has been demonstrated. The presented data will add information on the monitoring potential of CTC during adjuvant endocrine treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-08.
Abstract PD04-09: Multiple Biomarker Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients
Authors: Bruce RH , Hsieh HB , Bennis R , Krivacic RT , Liu X , Frankel P , Lau S , Somlo G.
Published: April 2011

Background: Numeration of circulating tumor cells, CTCs, from metastatic breast cancer (MBC) patients (pts) is predictive of outcome. Biomarker characterization of CTCs may be a useful adjunctive guide for personalized targeted and systemic treatment (Rx) selection.

Method: A multimarker assay was used to simultaneously quantify expression of HER2, ER and ERCC1. A fast laser scanning instrument was used for sensitive location of CTCs on large glass substrates. CTCs are identified using automated digital microscopy by morphology, the presence of cytokeratin and a nucleus, and the absence of CD45. At the COHCC pts with newly diagnosed/progressing MBC were accrued. Blood samples (10 ml) were procured prior to initiating systemic Rx and at subsequent 3 month intervals and sent to PARC for analysis. Cell lines with expression of each marker were used for normalization of the cell intensities. Sample scores were derived from the percentage of CTCs expressing the marker and the average expression level.

Results: The multiple-marker assay was done on CTCs at repeat time points and results were compared to findings from the original primary BCs (P) and biopsied metastases (M) in 30 and 20 MBC pts respectively. While P and M tissue scores were concordant for HER2, the CTC score was discordant in 58% of the samples; HER2 expression changed during Rx in 19% of pts. While the status for ERCC1 was discordant between P and M tissue in 13% of the pts, CTCs scores were discordant with P and M tumors in 63% and 67% of the patients respectively, and CTC expression status changed during Rx in 15% and 7% of pts respectively. While the status for ER was discordant between P and M tissue in 15% of the pts, CTC scores were discordant from the P and M tumors in 42% and 71% of pts respectively, and CTC ER status changed in 7% and 17% respectively of pts during Rx.

Conclusions: Significant discordances in expression level of ER, HER2 andERCC 1 was observed between CTCs, and both primary and metastatic BC tissue. Changes in CTC expression patterns were also observed during the course of Rx for all three markers. Correlation of CTC biomarker expression patterns and changes with response to Rx therapy is ongoing to validate medical significance. Multimarker testing may ultimately lead to improvements in personalized Rx for pts with MBC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-09.