bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-11-13: Phase I Study of Combined S-1 Plus Weekly Paclitaxel Therapy in Patients with Metastatic Breast Cancer
Authors: Watanabe J , Takahashi K , Tanaka K , Tadokoro Y , Ogiya A , Uematsu T , Kimura M.
Published: April 2011

Introduction: S-1, a novel anticancer compound, contains tegafur, a prodrug of fluorouracil (FU), 5-chloro-2.4-dihydroxypyridine, which prevents FU degradation, and potassium oxonate, which prevents drug-induced diarrhea, has a broad spectrum of activity against gastric, colorectal, head & neck, and breast cancers. While combined S-1 with cisplatin or paclitaxel (PAC) therapy is feasible and highly active against gastrointestinal cancer, the feasibility in breast cancer patients has not yet been clarified. We conducted a phase I study of combined S-1 plus weekly PAC therapy (S-1/PAC) to determine the maximum tolerated dose (MTD), recommended dose (RD) and dose-limiting toxicities (DLT) in patients (pts) with HER2-negative, taxane-naïve metastatic breast cancer (MBC).

Methods: S-1 was administered orally from day 1 through day 14 and PAC was administered intravenously on days 1, 8 and 15 of each 28-day cycle. Since S-1 is prescribed at the dose of 80mg/m2 in monotherapy, the primary dose levels (level 1) were set at 65 and 70mg/m2, respectively. The final dose (level 3) was set at 80mg/m2 for each drugs. DLT was defined over 2 cycles.

Results: Ten MBC pts were enrolled in the study. No DLTs were documented. Although grade 1 or 2 adverse events were seen in all pts, no cases of febrile neutropenia or Gr 4 neutropenia were observed. Of the 8 evaluable pts, 3 pts achieved partial response and 4 pts had stable disease.

Conclusion: Based on the results of this study, RD was determined to be 80mg/m2 for either drugs. We conclude that S-1/PAC was well-tolerated and highly active against HER2-negative MBC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-11-13.
Abstract P6-12-05: Ways To Understand Mechanism of Action for Targeted Drugs? Gene Expression Analyses from Paired Biopsies in Patients with Advanced Breast Cancer in a Substudy from the Randomized Study Docetaxel +/− Sunitinib A6181064
Authors: Lindstrom LS , Giorgetti C , Smeds J , Borg A-L , Liljegren A-L , Skoog L , Kern KA , Bergh J.
Published: April 2011

Background: The randomized SUN 1064 study, docetaxel +/− sunitinib included 593 patients with advanced breast cancer. The response rate was statistically significantly (p-value=0.001) higher in the combined arm, but no difference in progression free survival or overall survival was found, median 24.8months in combined arm and median 25.5 months in docetaxel arm (HR 1.21: 95% confidence interval 0.91-1.60).

This substudy aimed at unraveling gene expression patterns together with differentially expressed genes, contrasting gene expression before and after docetaxel +/− sunitinib treatment. The study also aimed at testing if sunitinib altered the gene expression of previously proposed sunitinib receptor tyrosine kinase targets. Material and methods:

In a substudy at Karolinska we aimed to have cytological biopsies taken; before start of therapy and repeated in both arms after 14 (+/−1 day) days of therapy, randomly consisting of either docetaxel at 100 mg/m2 q 3 w, or docetaxel 75 mg/m2 q 3w + sunitinib 37.5 mg orally day 2-15. Eighteen of 21 patients accepted participation. From 14 (7 per arm) individuals we had sufficient material to make paired comparisons. We used the HG-U133plus2 Affymetrix chip containing 54 675 probe sets in the 14 paired pre-treatment and post-treatment patient samples.

Results:

Unsupervised hierarchical clustering revealed differential gene expression patterns for the combined arm (Figure A) compared with docetaxel alone (Figure B).

The sunitinib proposed target receptor tyrosine kinases, showed no clustering patterns associated with the combined sunitinib and docetaxel treatment. The top 100 differentially expressed genes did not include the proposed sunitinib target genes. However interestingly, genes involved in the VEGF signaling pathway and apoptosis were significantly more frequently upregulated and those involved in the cell cycle were more frequently down-regulated in patients after treatment of sunitinib and docetaxel in combination, in contrast to the pre-therapy situation.

Conclusions:

Unsupervised hierarchical clustering revealed differential gene expression patterns corresponding to before and after combined sunitinib and docetaxel treatment, clearly clustering patients into the two groups. Interestingly, genes involved in the VEGF signaling pathway, apoptosis and cell cycle were modulated significantly more frequently by combined therapy treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-12-05.
Abstract P6-12-06: Oral Vinorelbine and Capecitabine Plus Bevacizumab in Recurrent Inflammatory Breast Cancer: Gene Profiling and Response to Treatment
Authors: Fumagalli L , Curigliano G , Locatelli M , Bagnardi V , Bertolini F , Mancuso P , Giudici S , Goldhirsch A.
Published: April 2011

Background: Therapies that target vasculolymphatic processes - angiogenesis, lymphangiogenesis, and vasculogenesis — have shown potential in the treatment of inflammatory breast cancer.

Methods: We randomly assigned 46 patients to receive oral vinorelbine 55 mg per square meter of body surface on days 1 and 3 and capecitabine 2000 mg per square meter on days 1 to 14 every 3 weeks either concurrent or sequential to bevacizumab 15 mg per kilogram of body weight on days 1 and 21 (BEVIX). The primary end point was progression-free survival;

the response rate and overall survival were a secondary end point. For correlative studies we collected baseline tissue and blood samples of all patients. Gene expression profiling was performed using Affymetrix HG-U133 Plus 2.0 arrays. Data were analyzed using Partek Genomic Suite 6.4 and GeneSpring 7.0. Tissue from normal breast was used as control.

Results: From July 2007 through January 2010, a total of 46 patients were enrolled. Concurrent oral vinorelbine and capecitabine plus bevacizumab significantly increased the objective response rate as compared to the sequential treatment (11.1% vs. 46.4%, p=0.025). No difference in progression-free survival was observed between the two arms (median time to progression 4.3 vs. 4.7 months, p=0.69; Hazard Ratio: 0.86, 95% CI: 0.41-1.80). The median overall survival was 9.1 months in concurrent arm and 15.8 months in sequential arm, (p=0.43). Global gene expression was analyzed in tumor samples from 18 pts bearing a triple negative phenotype. This cohort displayed a specific gene expression profile, including high expression levels of several cytokeratins, Aurora kinase A, Cyclin E and others, similar to the basal-like subtype. This profile did not include genes previously reported as differentially expressed in inflammatory breast cancers. We identified a set of 75 genes whose expression correlates with response to bevacizumab base therapy.

Conclusions: BEVIX is an active treatment for patients with recurrent inflammatory breast cancer. These tumors showed a gene expression pattern similar to that of basal-like type. We identified a set of 75 genes whose expression levels predict the response to BEVIX. The validity of this expression signature is currently being assessed. Supported by Roche and Associazione Italiana per la Ricerca sul Cancro (AIRC) grants. Data partially submitted to 2010 ASCO Meeting

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-12-06.
Abstract P6-12-07: Phase II Trial of Vinorelbine and Sorafenib in Metastatic Breast Cancer (MBC)
Authors: Luu T , Frankel P , Chung C , Mortimer J , Hurria A , Somlo G.
Published: April 2011

BACKGROUND

Vinorelbine, a semisynthetic vinca alkaloid, at 20-25mg/m2 has a response rate of 15-30% for second line therapy of MBC. Sorafenib is an oral multi-kinase inhibitor against the tyrosine kinases for vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), Flt-3 and c-Kit receptors. In preclinical studies, sorafenib demonstrated anti-tumor activity by inducing complete tumor stasis in a variety of tumor types through the Raf/MEK/ERK pathway. In the NCI H460 cell line for NSCLC tumor xenograft, synergy between vinorelbine and sorafenib were observed. We established the phase II recommended dose in MBC for the combination of vinorelbine (d1,18,15 on a 28 day cycle) with daily sorafenib at vinorelbine 20mg/m2 and sorafenib 200mg oral twice daily (reported at ASCO 2010). Phase II trial will complete in 6/2010. METHODS

Eligibility include: MBC with adequate end-organ function and performance status 0-2 who have up to two lines of prior chemotherapy regimens. Patients were treated at the recommended phase II dose as above. Tumor response was evaluated every two cycles by RECIST criteria. N=39 (target accrual 41 by 6/10) patients enrolled at the above recommended dose from 6/09 to 6/10. Treatment was continued until disease progression or unacceptable toxicity. RESULTS

Sites of metastatic disease included liver, lung, bone, bone marrow and skin. A total of 22/39 patients were enrolled prior to 2/17/2010 and are therefore evaluable for 4-month progression free survival (PFS) evaluation at this time. Median age of these patients was 56 yrs (range 39-72). ECOG performance status is 0 in 8 and 1 in 14 participating patients. Prior number of chemo regimens for MBC was 0 (n=4), 1 (n=10), and 2 (n=8). There was 1 partial response (PR) with 2 prior, and 8 stable disease (SD) with PFS for more than 4 months with 6 with 1 prior and 2 with 2 prior regimens for MBC. A total clinical benefit rate (PR+ SD) of 9/22 (41 %). SD patients included 1 who progressed at 11 months (1 prior), and 3 still on therapy.

The patient with SD for 11 months previously progressed in 2 months on abraxane. Toxicities resulted in treatment discontinuation in only 2 patients. Toxicities include: Grade 3 hand-foot reaction (n=3), Gr 4 ANC (n=3), Gr 3 fatigue (n==3), Gr 3 Diarrhea (n=2), Gr 3 hypertension (n=2), Gr 3 infection (n=3), 2 skin), Gr 3 elevated lipase (n= 1), Gr 3 hypophosphatemia (n=2), Gr 3 hyponatremia (n=2), Gr 3 Somnolence (n=1), Gr 4 PLT (n=1), Gr 3 back pain (n=1), and Gr 3 cough (n=1). CONCLUSIONS

The addition of sorafenib to vinorelbine to date showed a promising 41% clinical benefit rate in this heavily pretreated MBC population with a tolerable toxicity profile. Follow-up is continuing, we will report on the entire cohort of 41 patients at SABCS.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-12-07.
Abstract P6-12-09: A Phase I-II Trial of Dasatinib (D) in Combination with Weekly (w) Paclitaxel (P) for Patients (Pts) with Metastatic Breast Carcinoma (MBC)
Authors: Morris PG , Abbruzzi A , D'Andrea G , Gilewski T , Lake D , Bromberg J , Dang C , Dickler M , Modi S , Seidman AD , Sklarin N , Chang J , Patil S , Norton L , Hudis CA , Fornier MN.
Published: April 2011

Background: Inhibition of SRC is a novel approach for MBC. D is an inhibitor of multiple tyrosine kinases, including the SRC family. Pre-clinical data show D inhibits multiple breast cancer cell lines, including those of “basal-like” subtype. In preclinical models D + P had superior antitumor activity to either agent alone. We designed this phase I-II study to translate this observation.

Methods: For phase I: pts with MBC, ECOG PS 0-1, normal hepatic, renal, marrow function were eligible. Pts with pleural/pericardial effusions were excluded. For phase II: pts had measurable, HER2-negative MBC, ≥2 prior rx for MBC. Prior taxanes, stable brain metastases and baseline neuropathy grade ≥1 were allowed. Cycle (C) consisted of wP 80 mg/m2 IV 3/4 weeks + D 70mg orally daily; escalating to 100 mg, 120 mg and 150 mg in cohorts of 3pts. Toxicity was assessed by CTCAE v3.0, response by RECIST.

Results: 17 pts enrolled (15 phase I; 2 phase II); median age 54 (range 35-74), median PS=1 (range 0-1). 12 (71%) pts rcvd prior adjuvant chemoRx. Pts rcvd a median of 3 prior rx for MBC (range 0-12). Pts rcvd median of 2 C of D + P (range 1-14). One DLT occurred at 150mg (G3 fatigue); this cohort was expanded with no further DLTs. However 3 pts on this dose level withdrew;1 pt delayed hypersensitivity rash (grade 1), 1 pt febrile neutropenia (grade 3), 1 pt paclitaxel pneumonitis (grade 3). Therefore the phase II dose is D 120mg. Overall the most common toxicities have been hematologic and low G (table). 13 pts are assessable for response; 4 patients (31%) had a PR, including 3 patients previously treated with taxanes. 5 pts (29%) had stable disease.

Toxicities > Grade 1

Conclusion: Treatment with wP and D is feasible in pts with MBC. In the phase I study, 1 DLT occurred at D 150mg but due to cumulative toxicities the recommended dose for the ongoing phase II study is 120mg. Preliminary evidence of activity has been seen in taxane-pretreated pts at the phase II dose. Identification of biomarkers to select appropriate pts for this therapeutic approach is the subject of ongoing correlative studies.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-12-09.
Abstract P6-14-02: A Phase 3 Randomized, Controlled Trial of Myocet, Trastuzumab and Paclitaxel vs. Trastuzumab and Paclitaxel for 1st Line Therapy of HER2+ Metastatic Breast Cancer
Authors: Baselga J , Manikhas A , Roman L , Semiglazov V , Goldfarb RH , Forenza S , Matera J , Azarnia N , Hudis C , Rozencweig M
Published: April 2011

Background: Myocet™ (M) is a non-pegylated liposomal doxorubicin which exhibits reduced cardiotoxicity as compared to conventional doxorubicin. A phase 1/2 non-randomized multi-site trial combining M, trastuzumab (H) and paclitaxel (T) has resulted in high response rate, prolonged time to progression and improved overall survival, with no clinical cardiac toxicity in patients (pts) with first line metastatic breast cancer (MBC) over-expressing human epidermal growth factor-2neu (HER2+) (Cortes et al Clin Cancer res 2009:15:307).

Methods: This multicenter study was designed to demonstrate the efficacy and cardiac safety of M+ H + T vs. the standard of care, H + T, in pts with HER2+ MBC with no prior chemotherapy for MBC. After stratification by hormone receptor status, prior or no prior anthracycline (A) therapy, age ≥50 or >50 y and geography, patients were randomly allocated to receive either M (50 mg/m2 q 3 wks for 6 cycles), H (4 mg/kg loading dose followed by 2 mg/kg q wk) and T (80 mg/m2 q wk) or H + T until progression or toxicity. The primary efficacy outcome is progression-free survival (PFS), as determined by a blinded independent review committee (IRC). The primary safety outcome is the incidence of NYHA class 3 or 4 heart failure or cardiac death, independently adjudicated. Secondary endpoints are overall survival and tumor response, also determined by the IRC according to RECIST. 332 pts were needed to detect a 30% difference in PFS with 80% power.

Results: Between 2006 and 2009, 363 HER2+ women from 12 countries were enrolled. Median age is 53 yr (range 22-79); 80% are Caucasian; with ECOG performance status of 0 (62%) and 1 (38%); 42% are ER and/or PR positive; 40% received adjuvant chemotherapy, 24% hormone therapy, and 37% radiotherapy; 33% received prior A, median lifetime cumulativedoxorubicin and epirubicin dose (range) were 240 (45-360) mg/m2 and 360 (69-600) mg/m2, respectively. The final analysis of PFS is scheduled for 3Q10.

Conclusions: The safety and efficacy data will be presented at the meeting. Clinical Trials.Gov Identifier: NCT00294996

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-02.
Abstract P6-14-09: Acute-Phase Reactions Following Treatment with Zoledronic Acid or Denosumab: Results from a Randomized, Controlled Phase 3 Study in Patients with Breast Cancer and Bone Metastases
Authors: Stopeck AT , Lipton AA , Campbell-Baird C , von Moos R , Fan M , Haddock B , Braun A.
Published: April 2011

Background: Intravenous (IV) bisphosphonates (BP) are currently used to treat bone metastases and prevent skeletal-related events (SRE) in patients with advanced breast cancer. In a phase 3 study, denosumab, a fully human monoclonal antibody against RANKL, was shown to be superior to zoledronic acid (ZA) in delaying or preventing SREs in patients with breast cancer and bone metastases. This prespecified analysis compares ZA and denosumab for the incidence of acute-phase reactions (flu-like syndrome including pyrexia, chills, flushing, bone pain, arthralgias, and myalgias) during the first 3 days after initial treatment in that study.

Methods: Eligible patients were randomized in a double-blind, double-dummy fashion to receive IV ZA 4 mg (adjusted for creatinine clearance as specified by the Zometa label) or subcutaneous denosumab 120 mg every 4 weeks. Most patients (99%) were women; mean (SD) age was 57 (12) years, and baseline characteristics were balanced between groups. Safety analyses were conducted in patients who received ≥1 dose of denosumab (N=1020) or ZA (N=1013). Patient records were searched for adverse events (AEs) and serious AEs that occurred during the first 3 days after the first administration of study drug, using 37 prespecified MedDRA 12.0 preferred terms potentially indicating acute-phase reactions. Per study protocol, AEs were considered serious if they were fatal, life-threatening, required or prolonged in-patient hospitalization, resulted in a persistent or significant disability, or were considered to present a significant medical hazard.

Results: AEs associated with acute-phase reactions in the first 3 days after treatment occurred in fewer patients in the denosumab group (10.4%) than in the ZA group (27.3%; P<0.0001), and no events were attributed to denosumab. The most common acute-phase reaction AEs included pyrexia (0.9% denosumab, 11.5% ZA), fatigue (2.4% denosumab, 4.0% ZA), bone pain (1.3% denosumab, 3.6% ZA), chills (0.3% denosumab, 3.6% ZA), and arthralgia (1.5% denosumab, 3.2% ZA). No patients (0%) in the denosumab group and 10 patients (1%) in the ZA group reported serious AEs associated with acute-phase reactions during the first 3 days. These events included pyrexia (n=7); bone pain (n=2); and asthenia, back pain, chest pain, chills, headache, and malaise (n=1 each). For 6 of the 10 patients, events of pyrexia, chest pain, chills, and bone pain were resolved within 4 days. Three patients with serious acute-phase reaction AEs discontinued ZA treatment after the first dose. Conclusion: Patients treated with denosumab experienced no serious AEs of acute-phase reaction and significantly fewer overall AEs of acute-phase reaction than patients receiving ZA.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-09.
Abstract P6-14-13: A Flavone from Traditional East Asian Natural Extract Inhibits STAT3 and Tumor Growth in a Syngeneic Mouse Breast Cancer Model
Authors: Senthil M , Akmal YM , Xing Q , Yan J , Yim JH.
Published: April 2011

Background: Recent studies have shown that increased dietary consumption of flavones and isoflavones is associated with reduced risk of developing cancer. Natural plants/herbs used in Traditional East Asian

Medicine (TEAM) have been recognized as potentially efficacious natural treatments against cancer and are enriched with isoflavones and flavones, as well as other interesting compounds. We used a novel high throughput screening method to identify natural extracts that may have a potential anti-cancer effect and identified the TEAM natural product Scutellaria baicalensis and a known active component, the flavone, baicalein. The objective of our current study is to investigate the anti-cancer effects of baicalein in vitro and in vivo.

Methods: In vitro testing was performed in mouse (C3L5) and human (SKBR3) breast cancer cell lines. Cell viability was assessed by MTT assay. Apoptosis was assessed using FITC-Annexin V and PI staining with bivariate reading by flow cytometry at 48 h. A syngeneic mouse breast cancer model (C3H/HeJ — C3L5) was used for in vivo testing. Mice bearing subcutaneous tumors were either treated with daily intraperitoneal injection of vehicle or baicalein (20 mg/kg) five days a week for three weeks (n=6/group). Tumor size and weights were measured.

Results: Baicalein had a marked cytotoxic effect in C3L5 cells with an IC50 of less than 10mM (data not shown). FITC-Annexin V and PI analysis with flow cytometry for C3L5 cells treated at 10μM for 48h showed a marked increase in the Annexin V positive fraction indicative of apoptosis.

In vivo studies demonstrated a significantly decreased tumor growth in mice treated with intraperitoneal baicalein when compared to the control group (P<0.05).

Baicalein treated cells demonstrated STAT3 inhibition on western blot analysis by decreased presence of phospho-STAT3 (data not shown).

Conclusions: Baicalein, a flavone from TEAM demonstrates significant anti-cancer effects due to apoptosis in breast cancer. STAT3, a well-known anti-apoptotic signaling intermediate, is inhibited in activation as reflected by decreased phosphoSTAT3 which may account for these effects.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-13.
Abstract P6-15-01: A Phase 1b Study To Assess the Safety and Tolerability of the PARP Inhibitor Iniparib (BSI-201) in Combination with Irinotecan for the Treatment of Patients with Metastatic Breast Cancer (MBC)
Authors: Moulder S , Mita M , Bradley C , Rocha C , Harris L.
Published: April 2011

Background: Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a key regulator of DNA repair and cell proliferation, potentiates the anti-tumor activity of DNA-damaging chemotherapy agents, including irinotecan, a topoisomerase inhibitor (Ossovskaya et al. AACR 2009). Combination of gemcitabine and carboplatin with the PARP inhibitor iniparib has demonstrated promising efficacy and safety in patients with triple-negative MBC (O'Shaughnessy et al. SABCS 2009). Single agent irinotecan is active in heavily pre-treated metastatic breast cancer (MBC), with response rates (RR) up to 23% (Perez et al. JCO 2004;22:2849). A Phase 1 dose-escalation study of iniparib in advanced solid tumors showed no dose-limiting toxicities (DLTs) at iniparib doses of 0.5 to 8.0 mg/kg (Kopetz et al. ASCO 2008). That study was expanded here to determine the maximum-tolerated dose (MTD) of irinotecan that can be used in combination with fixed dose iniparib in patients with locally advanced or MBC.

Methods: Patients were treated with a 3+3 study design to determine safety of iniparib (8 mg/kg IV twice-weekly on Days 1, 4, 8, and 11 every 21 days) in combination with escalating doses of irinotecan (80 — 125 mg/m2 IV on Days 1 and 8 every 21 days). RRs were measured per modified RECIST criteria. Serial pharmacokinetic (PK) assessments were performed during Cycle 1, and archived paraffin-embedded tumor samples were collected to correlate markers of hypoxia (CAIX) with PARP1 expression (as measured by immunohistochemistry [IHC]) and response. Results: The median age of 34 patients who received therapy was 50 years (range, 32-84), and the median number of prior therapies was 2 (range, 0-6). Preliminary analysis show that tumors from 22 (65%) patients were ER-/PR-/HER2- (triple-negative), 10 (29%) were ER+/HER2-, and 1 (3%) was HER2+. One DLT of Grade 3 diarrhea with lower GI bleed was observed, thus establishing the optimal regimen from this study at 8 mg/kg iniparib + 125 mg/m2 irinotecan. Other toxicities included neutropenia, anemia and diarrhea. Of 26 patients evaluable for response, 5 (19%) had partial response (PR), 10 (38%) had stable disease (SD) for >4 cycles of therapy, and 9 (35%) developed progressive disease (PD) as best response. Of 10 patients who had reached 6 cycles at time of analysis, 5 (50%) had SD. Median follow-up time was 11 weeks. PK and IHC evaluations are ongoing and will be presented, along with mature response data. Conclusions: An MTD of 8 mg/kg iniparib and 125 mg/m2 irinotecan was identified in patients with MBC. Iniparib in combination with irinotecan was well tolerated and showed promising efficacy, with evidence of clinical benefit (RR + SD ≥6 cycles) in 50% in patients with treatment-refractory MBC. (ClinicalTrials.gov number, NCT00298675)

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-01.
Abstract P6-15-08: KGFR Tyrosine Kinase Inhibitors for the Treatment of Breast Cancer
Authors: Pento T , Meghna MM , Kesinger JW , Brueggemeier RW , Hackett J , Xiao Z , Lerner MR , Brackett DJ , Awasthi V , Li P-K.
Published: April 2011

Background: Keratinocyte growth factor (KGF) produces a rapid increase in the proliferation and motility of estrogen receptor (ER)-positive breast cancer cells (Zang, XP and Pento, JT, Clin. & Exptl. Metastasis 18: 573-80, 2001). It has been demonstrated that the KGF receptor (KGFR) is up-regulated in breast cancer; thus, KGF/KGFR signaling appears to be an early event in breast cancer progression (Zang, XP, Lerner, ML, Bahr, SJ, Brackett, DJ and Pento, JT, Cancer Genomics & Proteomics, 3: 369-72, 2006). Molecular modeling was used to create group of KGFR selective tyrosine kinase inhibitors (TKI) (Pento, JT and Li, PK, Drugs of the Future, 32: 965-72, 2007). The present study examined the effect of a group of these compounds on KGF-mediated proliferation and Erk signaling in MCF-7 breast cancer cells. In addition, the oncolytic potential of compound L-27, a potent KGFR TKI, was examined in an orthotopic xenograft model of human breast cancer.

Methods: A group of high affinity KGFR TKI were tested for their ability to inhibit KGF-mediated proliferation and Erk phosphorylation in MCF-7 cells using MTS and ELISA assays. In addition, immuno-cytochemistry was used to examine the influence of the 5 most potent inhibitor compounds on the density of cell membrane KGFR. The effects of a KGFR TKI on breast cancer growth and progression in vivo was examined using an orthotopic xenograft model, with clones of MCF-7 cells which were transfected with a KGF-producing plasmid to enhance cell growth and metastasis and a GFP reporter to quantify tumor growth and metastasis. PET imaging was also used to identify metastatic development.

Results: From the initial group of 53 compounds, 5 compounds were observed to produce greater than 50% inhibition of KGF-mediated cancer cell proliferation, reduce Erk phosphorylation and the density of the KGFR on the membrane surface of the cancer cells. Compound L-27, one of the most potent KGFR inhibitors, reduced the growth of MCF-7 tumors and metastatic development in the xenograft model in a dose-related fashion at doses of 4, 10 and 25 mg/kg. Animal weights in the treatment groups remained the same as the control group; indicating a lack of toxicity at the doses employed.

Discussion: The novel compounds tested in this project reduce or abolish KGF-mediated proliferation, Erk signaling and KGFR membrane density in vitro. In addition, compound L-27, a potent KGFR inhibitor in vitro, was found to reduce the progression of MCF-7 tumor xenografts in vivo. In conclusion, the KGFR TKI represent a novel class of anticancer agents for the prevention of metastatic progression and may provide a new therapeutic approach for the treatment of breast and other cancers. Acknowledgement: This work was supported by the grants from DoD (DAMD17-01-1-0591) and the NCI (CA-89740) and (CA-125493).

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-08.
Abstract P6-15-11: Role of Proteasome Activators in the Inhibition Breast Tumors and Suppression of Metastasis
Authors: Asea A , Kaur P , Neumann W , Asea E , Nagaraja G.
Published: April 2011

Background: Breast cancer is the second leading cause of cancer-related death of women in the U.S. A number of genes involved in the process of growth, proliferation, apoptosis and DNA repair have aberrant expression in tumor cells. Metastasis is major cause of the decline in the quality of life breast cancer patients. Heat shock protein, Hsp25 is known to participate in tumorigenesis, and is highly expressed in breast tumors biopsies. However, the exact role of Hsp25 is incompletely understood. Materials and Methods: We have silenced the expression of Hsp25 using lentiviral constructs of short interfering RNA in highly metastatic mouse 4T1 cell line. Mouse tumor formation and metastasis assay was done by subcutaneous injection of 4T1-controlshRNA and 4T1-Hsp25shRNA cells into the mammary pad of female BALB/c mice. Tumor progression/regression was monitored by using an in vivo animal imaging system (MaesteroTM In-vivo imaging system, CRI, Woburn, MA, USA). Microarray analysis was done by using protocols of Applied microarray, Inc, USA. In vivo antitumor assay and adoptive transfer of CD8+ T cells was done by using standard protocol.

Results: Microarray investigation on Hsp25 silenced cells demonstrated an upregulation of tumor suppressor gene (N-myc downstream regulated gene 2) and down regulation of metastasis gene (urokinase-type plasminogen activator) signatures. Subcutaneous injection of Hsp25 silenced 4T1 tumor (4T1-Hsp25shRNA) cells into immunocompetent female BALB/c mice results in complete tumor regression, absence of pulmonary metastasis and enhanced CD8+ CTL-dependent tumor killing. In vivo depletion of CD8+ T cells allowed the 4T1-Hsp25shRNA tumors to grow; however, there was no observable metastasis to the lungs. These results were confirmed by in vitro cytotoxicity assays in which CD8+ T cells recovered from mice injected with 4T1 -Hsp25shRNA cells, but not 4T1-controlshRNA cells effectively lysed 4T1 targets. Furthermore, the adoptive transfer of 4T1-Hsp25shRNA reactive CD8+ T cells into mice injected with 4T1-controlshRNA cells enabled them to effectively reject 4T1 tumors.

Discussion: Our study strongly indicate Hsp25 as a therapeutic target for breast cancers.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-11.
Abstract P6-15-12: Preclinical Evaluation of a STAT3 Inhibitor in Breast Cancer
Authors: Walker SR , Nelson EA , Chaudhury M , Madoux F , Subramaniam P , Chase P , Koenig M , Roush WR , Hodder P , Frank DA.
Published: April 2011

Breast cancer often arises from inappropriate activation of transcription factors involved in normal mammary development, such as the signal transducers and activators of transcription (STAT) family of transcription factors. STAT3, which plays an important role in mammary remodeling, is activated (as assessed by tyrosine phosphorylation) in approximately 70% of primary breast cancers, with many of these tumors being highgrade. Furthermore, inhibition of STAT3 leads to a loss of tumorigenicity of breast cancer cells. Therefore, targeting STAT3 in breast cancer would be a potentially beneficial form of therapy.

To identify small molecule STAT3 inhibitors, we developed a high throughput cell-based assay to identify compounds that inhibit STAT3- dependent transcriptional activity. We screened approximately 200,000 compounds, and specificity for STAT3 was ensured by eliminating molecules that inhibited either STAT1- or NF-kappaB-dependent transcriptional activity. From this screen, we identified 4-[4-(phenylmethyl) piperidin-1-yl] thieno[2,3-d]pyrimidine (PMPTP) (Probe ID ML116) as a potent inhibitor of STAT3 transcriptional activity, which has no effect on STAT1 or NF-kappaB-dependent transcriptional activity. Treatment with PMPTP of MDA-MB-468 breast cancer cells, which contain constitutively activated STAT3, leads to inhibition of expression of STAT3 target genes including bcl-x and mcl1. Furthermore, PMPTP is a potent inhibitor of viability of MDA-MB-468 breast cancer cells. By contrast, SK-BR-3 cells, which do not contain constitutively active STAT3, were only minimally affected. This suggests that PMPTP reduces the viability of cells that depend on constitutively active STAT3. PMPTP has no effect on STAT3 tyrosine or serine phosphorylation or STAT3 nuclear translocation, suggesting that PMPTP may be inhibiting STAT3 at the level of DNA binding or co-factor recruitment.

Importantly, PMPTP lowers the apoptotic potential of breast cancer cells thereby making them more susceptible to other forms of therapy. Building on this observation, we identified a number of beneficial combinations of PMPTP with cytotoxic drugs currently used in breast cancer therapy as well as novel anti-cancer agents. Additionally, we analyzed a number of structural analogues of PMPTP, and we identified two compounds that had approximately 4-fold increased potency in inhibiting the viability of breast cancer cells containing STAT3 activation.

Taken together, these findings identify PMPTP as a potential new form of therapy for breast cancers containing activated STAT3, which can be enhanced using conventional and non-conventional chemotherapy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-12.
Abstract P6-15-15: MM-111 - A Novel Bispecific Antibody Targeting HER-2/HER-3 Z Heterodimer: Safety and Tolerability in a First-In Human Phase I/II Study in Patients with Refractory HER2-Positive (HER-2+) Cancers
Authors: Beeram M , Denlinger CS , Tolcher AW , Goldstein LJ , Patnaik A , Papadopoulos K , Murray J , McDonagh C , Andreas K , Niyikiza C , Moyo VM.
Published: April 2011

Background: The ErbB2/ErbB3 oncogenic heterodimer is the most potent ErbB receptor pairing with respect to strength of interaction, receptor tyrosine phosphorylation, and downstream signaling through mitogen activated protein kinase and phosphoinositide-3 kinase pathways. ErbB3 signaling has emerged as an important mechanism of resistance to ErbB2 (HER-2) targeted agents in clinical use (Sergina et al Nature 2007, Garrett et al SABCS 2009 abstract 63). MM-111 is a novel bispecific antibody fusion protein that specifically targets the ErbB2/ErbB3 heterodimer and abrogates ligand binding. In preclinical models of HER-2+ gastric, breast, ovarian and lung cancers, MM-111 inhibits ligand-induced ErbB3 phosphorylation, cell cycle progression, and tumor growth. This first-inhuman phase 1-2 study evaluates the safety and tolerability of MM-111 and preliminarily explores efficacy in HER-2+ advanced breast cancer (ABC). The safety data obtained during the Phase 1 dose escalation portion of this study provide the basis of this report.

Methods: Patients (pts) with histologically confirmed HER-2+ advanced solid tumors progressing or recurring on standard therapy; aged ≥18 years; ECOG PS ≥2 and adequate organ function were eligible for phase I. Pts with stable CNS lesions were also eligible. A Modified Fibonacci schema was used for dose escalation in Phase I. Primary endpoints for Phase 1 were determination of maximum tolerated dose/maximum feasible dose while secondary endpoints included determination of dose-limiting toxicity, adverse events, and pharmacokinetic (PK) and immunogenicity profiles of MM-111. MM-111 was administered intravenously weekly in 4-week cycles.

Results: 12 pts (11 ABC and 1 gastric cancer) were treated: median age 59 (range 36-82), median PS 1 (range 0-1), 11♂:1♀, median numberof prior therapies 7 (3-12). A total 19 courses (median 2; range 1-2) of therapy was administered at 4 dose-levels (3 mg/kg, 6mg/kg, 12mg/kg and 20 mg/kg respectively). Common adverse events reported included pain (n=7) fatigue (n=4), dyspepsia (n = 3) and pyrexia (n = 3). There were no treatment interruptions due to adverse events. No dose limiting toxicities were observed and there has been no evidence of cardiotoxicity or immunogenicity to date. PK data indicate dose proportionality at the dose-levels explored and supports weekly dosing.

Conclusions: MM-111 is well tolerated at the doses levels studied and the PK profile suggests that weekly dosing is feasible. Enrollment to phase II is anticipated to commence shortly.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-15.
Abstract P6-15-17: Synergy of Ixabepilone and Dasatinib in Triple Negative Breast Cancer Cell Lines
Authors: Ismail-Khan RR , Marichion DC , Cubitt CL , Lancaster JM.
Published: April 2011

Background: Treatment options are very limited for breast cancers that do not express HER2 or estrogen and progesterone receptors (Triple Negative Breast Cancer; TNBC). Dasatinib is an orally active small molecule inhibitor of the src kinase. It has been proposed that dasatinib inhibits growth of breast cancer cells by modulating epidermal growth factor receptor (EGFR) signaling. Approximately 70% of breast cancers over express EGFR family members as well as c-Src. Src is co-expressed with at least one of the EGFRs. Both src and EGFR1 expression have been associated with TNBC. Dasatinib has previously shown activity as a single agent in TNBC cell lines. However, dasatinib has limited single agent activity in a cohort of chemotherapy refractory TNBC patients according to a phase I/II study. We believe that dasatinib may sensitize breast cancer cells to anti-mitotic agents. Ixabepilone is a potent anti-mitotic agent that has shown significant response in TNBC patients, both in the neoadjuvant setting and in the metastatic setting, including those cancers that are taxane refractory. Therefore, combining these two drugs may have utility in this

setting and may lead to inhibition of tumor cell proliferation, invasion, metastasis, angiogenesis, and tumor cell survival. Objectives: To study the activity of dasatinib combined with Ixabepilone in breast cancer cell lines.

Methods: A panel of 8 breast cancer cell lines was used in these experiments. These included MCF-7(ER+), T-47D (ER +), SKBR3 (HER2+), and five triple negative cell lines: BT-549, HS578T, MDA-MB-157, MDA-MB-231, MDA-MB-436. First, these cell lines were evaluated for src expression by Western blot as well as sensitivity to dasatinib alone, ixabepilone alone and then the combination of these two drugs using a high-throughput, 384- well CellTiter-Blue cell viability assay. Percent viability was calculated from four replicate cell wells after 72-hour drug incubation and IC50s were determined. Experiments were repeated a minimum of three times. Synergistic, additive, or antagonistic effects were determined using the combination index (CI) method developed by Chou and Talalay.

Results: TNBC cell lines expressed higher levels of src protein than cell lines with positive expression of HER2, ER and/or PR by Western blot analysis. Viability assays suggested a correlation between src expression and dasatinib IC50. Combinations of dasatinib and ixabepilone were determined to have combinational indexes <1 in all cell lines tested, suggestive of synergistic activity. Conclusion: This study demonstrates that in a range of breast cell lines with varying levels of src expression, dasatinib combined with ixabepilone is a synergistic combination and may prove to be beneficial to patients with very limited treatment options.

Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-17.
Abstract PD02-02: Characterization of Mammary Stem Cells during Embryogenesis
Authors: Engle DD , Lin J , Spike B , Cheung S , Wahl G.
Published: April 2011

Mammary stem cells (MaSCs) play a key role in the development and maintenance of the breast. MaSCs self-renew and generate the different cell types of the breast such that a single MaSC can regenerate an entire functional mouse mammary gland. Similarly, tumors are heterogeneous, with some cells exhibiting potent tumor forming ability in transplantation studies. These cells exhibit the stem cell properties of self-renewal and multi-lineage differentiation in that they can serially regenerate the cellular complexity of the original tumor. These cells, referred to as cancer stem cells (CSCs), have in some cases been shown to be more resistant to cancer therapies. Therefore, if even a single CSC survives chemotherapy, it could result in cancer relapse. Consequently, an important goal is to be able to prospectively identify CSCs and elucidate critical growth control pathways to enable development of effective targeted therapies. Unfortunately, pure MaSCs or breast CSCs have not been isolated, precluding the generation of reagents to detect or eliminate them. My research takes a new direction to identify, isolate, and characterize MaSCs using properties central to mammary gland development and stem cell function. We isolated MaSCs during development and found that they are naturally enriched late in embryogenesis. We sorted the mammary cells at E18.5 using cell surface proteins we previously found to be expressed in the rudiment at E18.5 and were able to obtain MaSCs purified to a significantly higher degree than reported in the literature. These fetal MaSCs express several genes implicated in cancer, including erbB2, which is found in a subset of dangerous human cancers. In addition, like human cancers that over-express erbB2, fetal MaSC growth is inhibited by the erbB2 antagonist Lapatinib.

The MaSC population isolated during fetal development is enriched for epithelial cells expressing both myoepithelial and luminal keratin markers as well as epithelial cells expressing neither of these markers. It is possible that either of these sub-populations contains the stem cell activity. We have focused on using high throughput single cell QPCR analysis to cluster the fetal MaSCs by their keratin marker expression pattern and identify cell surface markers that can be used to isolate viable cells for stem cell activity assays. Altogether, these experiments enabled isolation of highly enriched MaSC populations for elucidation of key growth pathways and set the stage for future in vivo analyses.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-02.
Abstract PD02-10: Radiation Induces Notch-Dependent De Novo Generation of Breast Cancer Stem Cells
Authors: Lagadec C , Della Donna L , Vlashi E , Dekmezian C , Pajonk F.
Published: April 2011

Like in normal tissues, the self-renewal of cancer stem cells (CSCs) might be also under tight control of developmental pathway like the Notch, Wnt, Sonic Hedgehog or TGFβ pathways. The Notch pathway plays an important role in normal breast development, cell fate, and normal stem cell self-renewal, and its deregulation has been shown to play a role in cancer. Aberrant Notch signaling has been implicated in the development and progression of both preinvasive ductal carcinomas in situ and invasive. Interestingly, in breast cancer, the Notch pathway plays major role for CSCs maintenance. We previously published that BCSCs (Breast Cancer Stem Cells) are more resistant to radiation. Here we demonstrate a link between BCSCs radio-resistance and the Notch pathway. We show that irradiation of MCF-7 cells increases CSC numbers and that this correlates with an induction of Notch signaling proteins expression in a dose and time specific manner. Jagged 1 was quickly (1h) increased 28-fold after 2Gy, DLL1 was increased 15-fold after 3 to 6h of 2 and 4 Gy irradiation, and Notch 2 was increased 16-fold 6h after 2, 4, 6 or 8 Gy, while DLL3 was increased 10-fold after the highest doses (6 to 12 Gy). Inhibition of Notch signaling pathway by the γ-secretase inhibitor prevents enrichment for CSCs and reduces radiation-induced overexpression of Notch proteins. More interestingly, we demonstrated that radiation-induced Notch signaling contributes to the phenotype plasticity of BCSCs and their progeny. Therefore, ionizing radiation induces de novo generation of BCSCs from non-tumorigenic cells. Moreover, we also identified, by FACS analysis, an induction of polyploid CSCs. expressed a relatively higher level of Oct4 and Sox2 than the unirradiated non-CSCs (two key transcription factors involved in stemness maintenance). In summary, we provide evidence that radiation-induced Notch signaling contributes to the plasticity of the BCSC phenotype, thereby generating BCSCs de novo from non-tumorigenic cells. Our data suggest that stochastic as well hierarchical CSC models apply to breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-10.
Abstract PD03-02: A Randomized Pre-Surgical Trial of Metformin in Breast Cancer. Preliminary Feasibility and Safety Results
Authors: Bonanni B , Cazzaniga M , Puntoni M , Pruneri G , Serrano D , Lazzeroni M , Guerrieri-Gonzaga A , Macis D , Luini A , Veronesi P , Galimberti V , DeCensi A. E.O.
Published: April 2011

Background: Metformin has been associated with decreased breast cancer (BC) incidence in diabetic patients in epidemiological studies. Moreover, this drug results in initiation of an AMPK-dependent energy stress response which can adversely affect survival of breast cancer cell lines and inhibition of PI3K/Akt/mTOR signaling leading to reduced proliferation of BC cell lines.

Methods: We conducted a randomized, pre-surgical, phase IIb, placebo-controlled, biomarker trial in women with stage I-III BC candidate to elective surgery. The primary endpoint was the change in cell proliferation in malignant, dysplastic and hyperplastic tissue as measured by Ki-67 labeling index (LI). With 150 subjects, the study was 80% powered to test for the interaction between metformin activity and ER status. Two interim analyses are being planned after 100 and 200 women enrolled. Results: As of June 18, 2010, 175 subjects have been randomized and 162 have completed treatment. Here we report data on the first 100 women enrolled. At the symposium, full data on the first 200 women enrolled will be available. As of December 31, 2009, a total of 163 women were screened, 26 were not eligible and 40 refused to participate, 6 dropped out during the study for AEs (n=2) or refusal to continue treatment (n=4), thus leaving 95 subjects assessable for the primary endpoint. The main subject and tumor characteristics blinded as to the allocated arm were mean age 52 (31-77), Pre/postmenopause, 54/41, mean BMI, 23.6 (18.0-40.2). At baseline, median Ki-67LI was 18% (range 4%-65%) at biopsy and 19% (4%-70%) after 4 weeks at surgery. All adverse events except for 1 SAE were grade 1 or 2, consisting of G2 nausea and G2 diarrhea in 4% and 6% of the cases, respectively. The prevalence of ductal intraepithelial neoplasia was 91% (median Ki-67 LI, 10%) in samples both adjacent and distant from the tumor, whereas the prevalence of ductal hyperplasia was 77% (median Ki-67LI, 2%) in samples distant from the tumor. Conclusions: Our preliminary results show the feasibility, high compliance and safety of a metformin trial in breast cancer patients. Assessment of tissue and circulating biomarkers is currently ongoing to characterize the whole spectrum of metformin activity in malignant and dysplastic tissue.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-02.