bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of the Mutant K303R Estrogen Receptor α at Serine 305 Impacts Aromatase Inhibitor Sensitivity.
Authors: Barone I. , Beyer A. , Covington K. , Tsimelzon A. , Ando' S. , Fuqua S.
Published: December 2010

Aromatase inhibitors (AIs) are challenging tamoxifen as the treatment of choice for both early and advanced breast cancer in postmenopausal women with estrogen receptor (ER) α-positive diseases. However, resistance frequently occurs. Previously, we identified a lysine to arginine transition at residue 303 (K303R) in ERα in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to the AI Anastrozole (Ana) when transfected in MCF-7 breast cancer cells. To identify genes whose expression was associated with the development of aromatase inhibitor resistance (AIR), we performed microarray analysis, and found a marked increase in the gene expression of different IGF family members in the mutant cells. Immunoblot analysis also revealed elevated constitutive phosphorylation of the IGF-1R/IRS-1/Akt pathway in K303R-expressing cells. Treatment with IGF-1R and Akt inhibitors drastically inhibited proliferation of the K303R-mutant expressing cells, while wild-type ERα-expressing clones showed little reduction of growth by these inhibitors, confirming the increased IGF signaling activation in the mutant cells.Post-translational modifications of ERα, such as phosphorylation, tightly regulate its function, and the K303R mutation resides at a major post-translational modification site, serine (S) residue 305. We found that the mutant was more efficiently phosphorylated than the WT receptor by Akt in vitro, and that the mutant cells exhibited enhanced S305 phosphorylation in vivo. Mutation of S305 to alanine (A) to destroy this phosphorylation site prevented in vitro and in vivo Akt-mediated phosphorylation.The ERα S305 residue is an important site that modifies response to tamoxifen; thus, we questioned whether the S305 site could also influence AI response. To address the role of S305 phosphorylation on AIR, we generated pools of stable transfectants expressing exogenous WT, K303R, or K303R/S305A mutant receptors, and then evaluated for the effects of Ana in soft agar assays. Expression of the K303R/S305A mutant resulted in a significant reduction in the basal non-stimulated growth and sensitivity to Ana compared to K303R ERα-expressing pools, thus mimicking the same response profile of wild-type ERα clones. A selective blocking peptide (S305 peptide, residues 298-308) able to antagonize this phosphorylation reversed AIR. Blockade of S305 phosphorylation also resulted in a specific inhibition of IGF-1R/IRS-1/Akt activation, but only in the mutant cells.Our data suggest that the K303/S305 residues of the ERα mutation may be a novel determinant of aromatase inhibitor response in breast cancer, and blockade of S305 ERα phosphorylation represents a new therapeutic strategy to overcome resistance to hormonal therapies in tumors with the ERα mutation.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5053.
Combining HSP90 Inhibition with Standard Therapies for HER2 Positive Breast Cancer.
Authors: Qadir Z. , Qadir Z. , Crown J. , Crown J. , Jensen M. , Clynes M. , Slamon D. , O'Donovan N.
Published: December 2010

Background: HSP90 is required for the stability and activity of HER2 and downstream proteins, such as Akt, which play a key role in cell survival. We aimed to assess the anti-tumor effects of the HSP90 inhibitor NVP-AUY922 in HER-2 positive breast cancer cell lines.Methods: HER2 positive breast cancer cell lines with varied sensitivity to trastuzumab (Sensitive: BT474, SKBR3, MDA-MB-361; acquired resistance: BT474Res, SKBR3Res; innate resistance: HCC1419, HCC1954, MDA-MB-453) were treated with the HSP90 inhibitor NVP-AUY922 (Novartis) and trastuzumab. IC50s were determined using the acid phosphatase assay. HER2, Akt and HSP90 levels were determined by immunoblotting after treatment with NVP-AUY922. Combinations of NVP-AUY922 with trastuzumab were tested in BT474, BT474Res, SKBR3, SKBR3Res, HCC1954, and MDA-MB-361 cells. Combinations with docetaxel, cisplatin and 5'-deoxy-5-fluorouridine (5-DFUR) were tested in BT474, SKBR3, HCC1954, MDA-MB-453 and MDA-MB-361 cells.Results: All of the HER2 positive cells were sensitive to NVP-AUY922, with IC50s ranging from 5.5 to 16.4 nM and NVP-AUY922 treatment reduced HER2 and Akt levels in a dose dependent manner. Combined treatment with NVP-AUY922 (10 nM) and trastuzumab (10 nM) showed significantly greater inhibition of growth than either trastuzumab or NVP-AUY922 alone in BT474 and BT474Res cell lines (p<0.005). In SKBR3, SKBR3Res, HCC1954, MDA-MB-453 and MDA-MB-361 cells, dual treatment with NVP-AUY922 and trastuzumab did not significantly increase response compared to NVP-AUY922 alone (Table 1). Combinations of docetaxel, cisplatin or 5-DFUR with NVP-AUY922 were antagonistic in all cell lines tested (CI values >1).Conclusions: This study demonstrates that NVP-AUY922 has anti-tumour activity in HER2 positive breast cancers regardless of sensitivity to trastuzumab. The antagonistic interactions observed for combinations of NVP-AUY922 with chemotherapy do not favour clinical evaluation of concurrent administration of NVP-AUY922 with chemotherapy. However, alternative scheduling or combinations with other targeted therapies warrants further investigation.Table 1: Percentage growth in response to NVP-AUY922 and trastuzumab. BT474BT474ResSKBR3SKBR3ResHCC1954MDA-MB-361Trastuzumab53.3 +/- 4.990.4 +/- 7.669.5 +/- 16.179.5 +/- 8.2102.9 +/- 15.558.4 +/- 3.3AUY92255.0 +/- 1.192.6 +/- 11.933.3 +/- 9.719.1 +/- 6.152.2 +/- 13.88.2 +/- 6.2Tras+AUY92222.0 +/- 2.3*59.7 +/- 17.5*30.6 +/- 7.518.3 +/- 4.347.2 +/- 3.42.5 +/- 2.2 

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5055.
Hyperthermia Sensitizes Breast Cancer Stem Cells to Radiation Therapy.
Authors: Atkinson R. , Zhang M. , Diagaradjane P. , Krishnan S. , Rosen J. , Rosen J. , Chang J. , Chang J.
Published: December 2010

Background In cancer, metastatic disease and recurrence are hypothesized to result, at least in part, from the residual cancer stem cells (CSCs), also referred to as tumor-initiating cells(TICs), which evade initial treatment. In several genetically engineered mouse models that resemble human breast cancer, the tumor subpopulation CD29+/CD24+/lin- has been shown to be more tumorigenic as compared to other subpopulations by in vitro and in vivo assays. We and others have demonstrated that the tumorigenic CD29+/CD24+/lin- subpopulation repairs radiation-induced DNA strand breaks more efficiently than the other subpopulations. Therefore, we hypothesized that hyperthermia, a documented means of inhibiting DNA repair, may radiosensitize CSCs to radiation therapy(RT) by delaying and inhibiting the repair of DNA strand breaks. Methods To test this hypothesis, we assayed for tumorigenic CD29+/CD24+/lin- cells in p53null mouse mammary tumor model with and without radiation, and in combination with hyperthermia (42°C) administered as optically-activated gold nanoparticles. We then determined if there was a decrease in functional CSCs by FACS analysis and limiting dilution transplantation assays. Results These studies confirmed that CD29+/CD24+/lin- cells were more radiation resistant and that their relative proportion was increased after RT, as compared to the other cell populations. However, the addition of hyperthermia increased the sensitivity of the CD29+/CD24+/lin- subpopulation to radiation. Radiation treatment alone in vivo resulted in a decrease in tumor size, but the percentage of tumorigenic cells increased as compared to untreated controls. In contrast, following post-treatment with 20 minutes of local hyperthermia, using intravenously administered optically activated gold nanoparticles, a larger regression in tumor size was observed, without a concomitant increase in the percentage of CD29+/CD24+/lin- tumorigenic cells. Using the functional limiting dilution transplantation assay to assay for CSCs, we showed that 48 hours after treatment cells derived from tumors treated with RT exhibited an increased frequency of TICs as compared to untreated controls, and tumors treated with radiation and hyperthermia. In addition, clonogenic survival assays of this tumorigenic subpopulation had consistent results with the in vivo data. Conclusion These results suggest that localized hyperthermia serves as a simple strategy to selectively sensitize the radioresistant subpopulation of CSCs to enhance their response to radiation therapy. The mechanisms responsible for these effects of hyperthermia are currently under investigation. Supported by grant NCI R01 CA112305 and CA16303.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 506.
Marked Synergism In Vitro with the Histone Deacetylase Inhibitor LBH-589 and Classical Agents: Potential New Leads for Combination Therapy.
Authors: Budman D. , Calabro A. , Tai J.
Published: December 2010

Background: Clinical studies of new agents with targeted effects have been tempered by the poor single agent activity in solid tumors of most of these agents with the exception of trastuzumab and lapatinib. In addition, off-target effects may also play a role in their anti-cancer activities so their putative mechanism of action may not be applicable. As a consequence, a combinatorial approach of combining agents has been employed in the clinic with little regard of whether or not the combination may display synergy or antagonism. As over 400 drugs are in development, the clinical trials mechanism can only evaluate a paucity of agents. Methods: We have employed median effect analysis of drugs applied to human cancer cells in short term log phase culture, combined with curve shift analysis, to identify potential interesting combinations(such as tyrosine kinase dual Her2/EGFR inhibitors with capecitabine)and to discard antagonistic combinations (AntiCancer Drugs. 17: 921-928, 2006). This technique allows schedule to be examined and up to three agents combined simultaneously. The following human breast cancer cell lines were used: MCF7/wt (ER+), MCF7/adr (ER-, Her2+ multiply resistant), BT474 (ER+, Her2+), SK-BR3 (ER-, P53 mutant, Her2+), HCC-1395 (triple negative), and HTB-25 (triple negative). LBH-589 (gift of Novartis USA)is a histone deacetylase inhibitor in clinical trials with broad biological effects including perturbation of survivin, pro-apoptotic effects, induction of differentiation, and non cycle dependent effects (Int J Ca 121: 1138, 2007). Results: This agent was combined with more classical agents. Synergism (CI at Fa50 < 1.0) was noted for doublets combined with either carboplatin (6/6 cell lines) docetaxel(5/6 cell lines), 5'DFUR (6/6 cell lines), doxorubicin (5/6 cell lines), and the triplets examined. Her2 antagonists such as trastuzumab or GW282974X (lapatinib analogue) were not enhanced by co-treatments with LBH-589. CI values are shown for the triplets. Only SK-BR3 demonstrated resistance with one combination.Conclusions: This data suggests several innovative combinations which might have greater efficacy than traditional therapy. A doublet of LBH589 with gemcitabine or a triplet are most attractive. Supported in part by the Don Monti Foundation, the Donna Dennis Foundation, and NCI CA 35279.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5061.
BIBW 2992, a Novel Irreversible EGFR/HER1 and HER2 Tyrosine Kinase Inhibitor for the Treatment of Patients with HER2-Negative Metastatic Breast Cancer after Failure of No More Than Two Prior Chemotherapies.
Authors: Harbeck N. , Schmidt M. , Harter P. , Possinger K. , Jonat W. , Lück H. , Beckmann M. , Fasching P. , Schütte J. , Solca F. , Uttenreuther-Fischer M. , Taton M. , Lahogue A. , Awada A. , De Grève J. , Canon J. , Dirix L. , Fleischer F. , Neven P. , Gottschalk N. , Piccart M. , Schuler M.
Published: December 2010

Background: BIBW 2992 (Tovok™*) is a novel, oral, irreversible inhibitor of the epidermal growth factor receptor (EGFR)/ human epidermal growth factor receptor (HER)1 and HER2 inhibitor. It has promising preclinical activity in HER2-negative MDA-MB-453 and triple-negative SUM 149-PT cell lines, and showed clinical Phase I activity. First results of a Phase II study of BIBW 2992 in patients with HER2-negative, hormone receptor (HR)-positive or triple-negative breast cancer who have progressed following no more than two prior lines of chemotherapy are presented. The trial was conducted as a multi-institutional, open-label study in Germany and Belgium.Methods: Eligible patients had stage IIIB or IV HER2-negative metastatic breast cancer (MBC), with progression following no more than two prior lines of chemotherapy. No prior EGFR-targeted therapy was allowed and measurable disease, Eastern Cooperative Oncology Group performance status of 0–2 and an adequate organ function were required. Patients received 50 mg BIBW 2992 once-daily until disease progression. Tumor assessment was performed every other treatment course (one course is 28 days). The primary endpoint was objective response rate by Response Evaluation Criteria In Solid Tumors criteria in patients with HR-positive and triple-negative MBC. The latter was changed to clinical benefit, i.e. objective response (OR) or stable disease (SD) on treatment for at least four cycles for patients with triple-negative disease. Safety data were collected and analyses are still ongoing.Results: A total of 56 patients were enrolled with 50 actually starting treatment on the trial, including 29 patients with triple-negative and 21 patients with HER2-negative and HR-positive MBC. No ORs were observed in either cohort. Three patients in the triple-negative cohort had SD for at least 4 treatment cycles, one of them for 12 cycles. The most frequently observed side-effects at snapshot date (on 49 patients) are diarrhea (Common Terminology Criteria for Adverse Events Grade 3 in 18 patients [36.7%]) and Grade 3 skin rash in two patients [4.1%]). These were managed by symptomatic treatments and dose reductions.Conclusion: Long-term oral administration of BIBW 2992 was safe in HER2-negative MBC patients. Side effects mainly affected the skin and gastrointestinal tract, and were manageable. Clinical benefit was achieved in a fraction of triple-negative MBC patients.*Trade name not FDA approved.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5062.
Vimentin: A Novel Chemopreventive Target for Breast Cancer Metastasis.
Authors: Thaiparambil J. , Bender L. , Kline E. , Ganesh T. , Snyder J. , Liotta D. , Marcus A.
Published: December 2010

Background: Cancer metastasis is the major cause of death in nearly all tumor types; however, most treatments target the primary tumor and not the highly invasive metastatic cells. We propose to prevent cancer metastasis by precisely targeting the cancer invasion machinery. To do this we disrupt a novel target-the vimentin cytoskeleton. Vimentin is overexpressed in nearly all invasive solid tumors and is critical for cell migration. Here we show that Withania sominifera root extracts (WRE) and one of its primary constituent, Withaferin A (WFA), targets vimentin to inhibit cancer cell motility and migration while having negligible effects on viability. WFA is a steroidal natural product with 4-rings (A-D) connected to a lactone ring. We test the hypothesis that WFA is an anti-invasive compound that disrupts vimentin function with limited toxicity.Material and Methods: We employ cutting-edge live cell confocal imaging studies in combination with traditional molecular biology techniques to dissect the precise mechanism of how WRE or WFA inhibits cancer cell migration and invasion. We have employed a wounding model and Matrigel invasion assay to determine the anti-migratory and anti-invasive properties of WRE and WFA using MDAMB-231 cells. We have used GFP-vimentin to visualize the changes in vimentin dynamics. Effect of WRE and WFA on cell cycle was analyzed by Flow cytometry.Results: Our results show WFA has weak anti-proliferative activity at low concentrations but potently inhibits breast cancer migration and invasion in a dose-dependent manner. High resolution confocal images reveal that in WFA-treated cells, vimentin fails to invade the lamellipodia resulting in decreased cell migration. This is supported by live cell confocal imaging showing that at high doses WFA treatment depolymerizes GFP: vimentin. Furthermore, treatment with WFA results in aberrant vimentin phosphorylation, likely resulting in a net depolymerized phenotype. In order to determine if the vimentin-binding A ring of WFA is critical for its anti-invasive efficacy, we synthesized and tested A-ring modified analogs of WFA. All three analogs showed severely reduced potency in inhibiting cancer cell migration and invasion, suggesting that the A ring is critical for its anti-invasive activity.Discussion: The present study shows that WRE or WFA inhibits breast cancer cell migration at nanomolar concentrations by disrupting vimentin at concentrations that have minimal effect on proliferation. We believe its strong anti-migratory activity make this an appropriate compound as an anti-metastatic chemopreventative. Ultimately, we envision WFA can be used as a vimentin-targeting chemopreventative in high-risk metastatic patients and has the potential to be used with traditional cytotoxics.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5063.
Constitutive Activation of Src Kinase as a Mechanism of Acquired Resistance to Dasatinib in Triple Negative Breast Cancer.
Authors: Corkery B. , Corkery B. , Crown J. , Crown J. , Roche S. , O'Connor R. , Tryfanopoulos D. , Tryfanopoulos D. , Clynes M. , O'Donovan N.
Published: December 2010

Background: Dasatinib, a multi-target inhibitor of tyrosine kinases including Src, Bcr-Abl, PDGFR, and ephrin A receptor kinases, preferentially inhibits growth in triple negative breast cancer (TNBC) cell lines compared to other breast cancer subtypes. Previous studies have shown that dasatinib is a substrate for the drug efflux pumps, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We sought to develop a model of acquired resistance to dasatinib to investigate potential mechanisms of resistance.Materials and Methods: Resistance to dasatinib in MDA-MB-231 was achieved by continuous exposure to incrementally increasing concentrations of dasatinib (200 - 500 nM). IC50 values and combination assays were measured using the acid phosphatase assay. Parental and resistant cells were treated with dasatinib (1 µM) for 2 hours and accumulation of dasatinib in cells was quantified by liquid chromatography–mass spectrometry (LC-MS). The effect of the P-gp/BCRP inhibitor, elacridar, on dasatinib sensitivity was also assessed. Src and phospho-Src (P-Src) levels were assessed by western blot.Results: The MDA-MB-231-Das cell line displayed significant resistance to dasatinib (IC50 > 5 µM) compared to the parental MDA-MB-231 cells (IC50 39.3 ± 14.2 nM). There was no significant difference in the doubling time of the parental and MDA-MB-231-Das cells (Table 1). In addition, the growth rate of the resistant cells did not significantly decrease in response to dasatinib treatment, in contrast to the parental cell line. No difference in accumulation of dasatinib was observed in the parental and resistant cells. Furthermore, inhibition of P-gp and BCRP with elacridar did not enhance response to dasatinib in either cell line. P-Src levels were not altered in the resistant cell line. However, P-Src levels did not decrease in response to dasatinib treatment in the MDA-MB-231-Das cells, whereas a dose dependent decrease in P-Src levels was observed for the parental cells.Discussion: Src kinase has been implicated in aberrant signalling through multiple receptors involved in tumorigenesis, and is a putative target for response/resistance to dasatinib. We developed a novel triple negative breast cancer model of acquired resistance to dasatinib, and examination of Src phosphorylation suggests that constitutive activation of Src is a potential mechanism of resistance.Table 1 Doubling time (± standard deviation (hrs)) and growth rate of MDA-MB-231 and MDA-MB-231-Das cells with and without dasatinib (D) treatment. ControlD 50 nMD 100 nMMDA-MB-231 (parental)17.6 ± 1.232.2 ± 3.3*46.8 ± 5.1*Growth rate (%)100.054.737.6MDA-MB-231-Das (Dasatinib-resistant)19.1 ± 2.421.0 ± 0.221.2 ± 3.1Growth rate (%)100.091.090.1 

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5066.
ABT-869 in Combination with Paclitaxel (P) as First-Line Treatment in Patients (Pts) with Advanced Breast Cancer.
Authors: Rugo H. , Lopez-Hernandez J. , Gomez-Villanueva A. , Gupta N. , Qian J. , Qin Q. , Ricker J. , Carlson D. , Kozloff M.
Published: December 2010

Background: ABT-869 is a novel orally active, potent and selective inhibitor of VEGF and PDGF receptor tyrosine kinases. ABT-869 potentiates the action of P in a number of preclinical tumor models including breast carcinoma (BC). This study was conducted to assess pharmacokinetic (PK) interaction and safety of ABT-869 combined with weekly P, and to guide ABT-869 dosing in the randomized portion of the trial.Methods: Pts with measurable chemotherapy naïve metastatic or unresectable locally advanced BC were eligible for this trial. Treatment consisted of weekly P (90 mg/m2) on Day (D) 1, 8 and 15 of every 28-day cycle (C) and ABT-869 0.20 orally once daily (QD) starting on D3 of C1. Treatment continued until progressive disease (PD) or unacceptable toxicity was observed. CT scans were conducted at baseline (within 21 days prior to C1 D1) and at the end of every 3 cycles. Adverse events (AE) were graded by NCI CTCAE V3.0; efficacy was evaluated by RECIST.Results: As of May 2009, 8 pts have enrolled in this study: 5 at 0.20 mg/kg and, based on the frequent need for dose reductions in this and other ongoing trials, a second cohort has enrolled 3 out of 6 planned pts at 0.15 mg/kg. Preliminary safety and efficacy results are shown (table). Of the 5 pts dosed at 0.20 mg/kg, 2 pts had confirmed partial responses (PR) and continued on drug at reduced dose for 12 and 10 cycles, and 3 discontinued (DC); 1 due to PD, 1 withdrew consent, and 1 for AE (pulmonary embolism, cycle 2). Three pts have been dosed at 0.15 mg/kg; one is early and two have PR. The most common AEs were neutropenia (n=3), stomatitis, vomiting, ALT increased, hyperglycemia, hypokalemia and hypertension (n=2 each). These events were generally grade 1 or 2 except for neutropenia.Conclusion: The combination of ABT-869 0.20 mg/kg QD with P weekly resulted in antitumor activity (2 PR) but required frequent dose reductions due to AEs. A reduced dose of 0.15 mg/kg with weekly P is being evaluated (2 PR). Updated results of this ongoing study will be presented.Pt #Initial ABT-869 Dose (mg)Current ABT-869 Dose (mg)Cycle of Dose ReductionReason for Dose ReductionEnd Cycle 3 AssessmentsEnd Cycle 6 AssessmentsCurrent status   0.20 mg/kg    1001107.54G3 hand-footPRPRCycle 121041105.03,5G3 fatiguePRPRCycle 1010022020----DC in Cycle 2 (PD)102112.512.5--SD-DC in Cycle 4 (withdrew consent due to headaches)10221515----DC in Cycle 2 (Pulmonary embolism)   0.15 mg/kg    10231052,4G2 hand painPR-Cycle 510247.57.5--PR-Cycle 410251010----Cycle 1G= grade

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5076.
Effects of Rapamycin on Breast Cancer Cell Migration through the Cross-Talk of MAPK Pathway.
Authors: Zhao H. , Cui K. , Nie F. , Jin G. , Li F. , Wu L. , Wang L. , Brandl M. , Yilidirim N. , Zhang S. , Sun A. , Wong S.
Published: December 2010

Phase I/II clinical studies with rapamycin analogs in breast and other cancers have demonstrated favorable responses. However, little is known on the effects of the mTOR inhibitor on breast cancer cell metastasis, which is a major cause of morbidity and death. We developed a highly sensitive 3-dimensional (3D) proliferation/invasion assay using quantitative bioluminescence (BL) imaging and applied this assay to evaluate the effects of rapamycin on the triple negative breast cancer cell line MDA-MB231. Without cytotoxicity of rapamycin on this cell line, rapamycin at 10nM inhibited the cell migration/invasion, but not at 1nM and 100nM, which was confirmed by the time-lapse single cell tracking analysis. The quantification of cytoskeleton changes showed most potent effects of 10nM rapamycin on the MDA-MB231 cells, with the formation and rearrangement of specialized cell membrane structures and actin fiber implicated in cell motility. Then, the Panorama Cell Signaling Antibody Microarray, enabling the global comparative analysis of cell signal proteins simultaneously, was exploited to analyze the effects of rapamycin on the cellular signaling network of the MDA-MB231 breast cancer cell line. 100nM rapamycin activated the MAPK pathway obviously, through the attenuated negative feedback of activated S6K1 to PI3K-Raf, which increased the expressions of activated Jun N-terminus kinase (JNK), Erk1/2, MEK-1, Raf-pS621, and MAPK-activated protein kinase 2 (MAPKAPK2) in the cells exposed to 100nM rapamycin. MEK inhibitor U0126 or PD98059 could restore the anti-migration effects of 100nM rapamycin on the MDA-MB231 cells. Furthermore, the combination of MEK inhibitors and rapamycin performed synergism on inhibiting the cell proliferation and migration/invasion. Accordingly, rapamycin at a certain dose suppresses MDA-MB231 cell migration/invasion, and the co-targeting of mTOR and MAPK pathways enhances the inhibition on cell proliferation and migration/invasion, underscoring the potential therapeutic utility of rapamycin, and rapamycin combining with MAPK inhibitors in triple negative breast cancer progression, and the results highlight the cross-talk homeostasis of mTOR and MAPK pathways in cancer treatment.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5080.
Pooled Analysis of Cardiac Safety in Patients Treated with Pertuzumab.
Authors: Suter T. , Brammer M. , Ross G. , Lenihan D.
Published: December 2010

Background: Pertuzumab, a monoclonal antibody, is a human epidermal growth factor receptor 2 (HER2)-dimerisation inhibitor directed to the dimerisation epitope of HER2, with promising activity when given with trastuzumab to patients with HER2-positive metastatic breast cancer following progression on trastuzumab.1 As anti-HER2 treatment has been associated with cardiac dysfunction, we investigated the incidence of reduced left ventricular ejection fraction (LVEF) and congestive heart failure (CHF) in patients treated with pertuzumab as single agent or in combination with cytotoxic or anti-HER therapy.Methods: We analysed patients in completed Phase II studies with pertuzumab. Cardiac dysfunction was defined as: 1) reduced LVEF (baseline normal: LVEF reduced ≥10% absolute and to below 50%); 2) symptomatic CHF: any symptoms suggestive of CHF counted (severity then classified according to New York Heart Association).Results: In 554 patients analysed, the overall incidence of reduced LVEF was 5.8% and of CHF 0.7%. Individual data for patients in different treatment protocols are shown in Table 1. PatientsReduced LVEFCHF (n)(n/%)(n/%)Pertuzumab as single agent (24% with prior anthracyclines)30221/7.01/0.3Pertuzumab with cytotoxic therapy1756/3.42/1.1Pertuzumab with trastuzumab (71% with prior anthracyclines)775/6.51/1.3 The majority of patients with reduced LVEF or symptomatic CHF had significant improvement or return to baseline function on follow-up or with standard cardiac medication.Conclusions: Overall, the incidence of cardiac dysfunction in Phase II studies of patients treated with pertuzumab was low. When combined with trastuzumab in selected patients, there is no apparent indication that pertuzumab exacerbates the known risk of reduced LVEF and CHF with trastuzumab.2 These data suggest that the combination of pertuzumab with trastuzumab or chemotherapy for HER2-positive breast cancer appears safe from a cardiac perspective with appropriate monitoring in ongoing studies.References1. Gelmon K et al. Poster 1026 presented at the 44th ASCO Annual Meeting, Chicago, Illinois, USA, 30 May-3 June, 20082. Muehlbauer S et al. Abstract presented at SABCS 2008; abs 6136

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5088.
Population Pharmacokinetics of Trastuzumab-DM1, a First-in-Class HER2 Antibody-Drug Conjugate Given Every 3 Weeks (q3w) and Weekly (qw) to Patients with HER2-Positive Metastatic Breast Cancer (MBC).
Authors: LoRusso P. , Girish S. , Burris H. , Beeram M. , Vukelja S. , Modi S. , Yi J. , Wang B. , Saad O. , Gupta M.
Published: December 2010

BackgroundTrastuzumab-DM1 (T-DM1) is a first-in-class HER2 antibody–drug conjugate (ADC) in development for the treatment of HER2-postive MBC. T-DM1 is composed of trastuzumab (Herceptin®), DM1, an inhibitor of tubulin polymerization derived from maytansine, and the stable MCC linker that conjugates DM1 and trastuzumab. T-DM1 has been tested at multiple dose levels in a Phase I trial: q3w (0.3–4.8 mg/kg) and weekly (1.2–2.9 mg/kg), and in two subsequent Phase II trials with T-DM1 administered as a single agent at 3.6 mg/kg q3w. A population pharmacokinetic (PK) model for T-DM1 conjugate was developed using pooled PK data from these trials to estimate typical PK parameter values and inter-patient variability in patients with HER2-positive MBC. The model was further developed to explore and quantify the effect of body size and pathophysiologic covariates on the pharmacokinetics of T-DM1 to better understand the clinical factors that might affect safety for individual patients.MethodsFor the current analysis, 2935 T-DM1 serum concentration–time datapoints from 167 patients were simultaneously fitted using NONMEM® software. T-DM1 concentration-time data to date are best described using a two-compartment linear model. All relevant and plausible covariates likely to have an effect on T-DM1 systemic exposure, e.g., body size covariates (body weight [BW], body surface area [BSA], and body mass index [BMI]), albumin, liver enzymes, creatinine clearance, tumor burden, baseline trastuzumab levels, HER2ECD, concomitant medications, etc., will be explored for possible correlation with the parameters in the model. In addition, the effect of selected covariates on the pharmacokinetics of DM1 is also explored.ResultsEstimated PK parameters for T-DM1 from the base population PK model with data available to date are: clearance (CL) 0.75 L/day, central distribution volume (V1) 3.37 L, peripheral volume (V2) 1.04 L, and inter-compartmental clearance (Q) 0.92 L/day. Inter-individual variability on CL, V1, and V2 was 26%, 17%, and 66%, respectively. Preliminary univariate covariate analysis suggests that body size metric (BSA, WT, BMI) has an influence on exposure of T-DM1. Albumin and tumor burden also appear to influence the clearance of T-DM1. Additional covariate analysis is ongoing. Results of the updated PK model and covariate analysis based on ongoing Phase II trials will be presented at the meeting.ConclusionsModerate inter-individual variability for the estimated PK parameters was observed. The model incorporating all available PK data from Phase I and II trials will help identify clinical factors, from the covariates studied, that affect the pharmacokinetics of T-DM1 and thus potentially, the safety of T-DM1 in individual patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5099.
Phase II Study of a HER-2/Neu (HER2) Intracellular Domain (ICD) Vaccine Given Concurrently with Trastuzumab in Patients with Newly Diagnosed Advanced Stage Breast Cancer.
Authors: Disis M. , Dang Y. , Bates N. , Higgins D. , Childs J. , Slota M. , Coveler A. , Jackson E. , Waisman J. , Salazar L.
Published: December 2010

HER2 is a tumor antigen in breast cancer and several trials have demonstrated that breast cancer patients can be immunized against this protein. We have developed HER2 peptide based vaccines that are aimed at eliciting CD4+ Th1 tumor antigen specific T cell responses. Th1 effectors provide immunologic memory, enhance cross priming which will allow the elaboration of tumor specific CD8+ T cells, and stimulate epitope spreading which we have shown to be a potential biomarker of clinical response. 52 patients will be enrolled with the primary objective to determine relapse free survival after active immunization. Eligible patients are newly diagnosed with Stage III (B or C) or Stage IV breast cancer and begin vaccination within 6 months of starting maintenance trastuzumab. This interim report will present data on the first 25 patients enrolled; 21 stage IV and 4 locally advanced patients. The vaccine is well tolerated with all adverse events (AE) being Grade I or 2. The most common AE is injection site reaction. Moreover, the combination of HER2 vaccination with trastuzumab did not result in additive cardiac toxicity in these patients. Immune responses were evaluated by IFN-gamma ELISPOT. To date, 88% of patients immunized developed significant immunity to the components of the ICD vaccine. The majority, 75%, developed robust immunity to the HER2 protein. Our group has recently demonstrated that a broadening of immunity throughout the HER2 protein, to components of the protein that weren't in the vaccine, i.e. epitope spreading, may be associated with improved survival in vaccinated patients. 63% of immunized patients demonstrated evidence of intramolecular epitope spreading. We questioned whether such high frequencies of homing Type 1 T cells might modulate the immunosuppressive tumor microenvironment, so we evaluated whether circulating serum immunosuppressive cytokines were impacted by immunization. TGF-beta is an immunosuppressive cytokine secreted by tumor stroma and regulatory T cells. We found that the levels of serum TGF-beta decreased significantly in the majority of patients after vaccination. We further analyzed the correlation between the change of serum levels of TGF-beta post vaccination and HER2 ICD vaccine-induced T cell responses. We found that the greater the magnitude of the HER2 specific T cell response, as demonstrated by IFN-gamma secretion, the greater the decrease in serum TGF-beta (p=0.0045, r=0.742). The correlation between the increased epitope spreading T cell response and decreased levels of TGF-beta was even more significant (p=0.0003). The median overall survival has not been reached with 100% of patients alive at this time. Relapse free survival data will be presented.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5102.
Response Rates in Non-Anthracycline Versus Anthracycline Exposed Patients with Metastatic Breast Cancer Treated with Lapatinib.
Authors: Rugo H. , Johnston S. , Preston A. , Kemner A. , Stein S. , Gomez H.
Published: December 2010

Introduction: Anthracycline free regimens are increasingly used as adjuvant therapy for HER2 positive breast cancer due to concerns related to cardiac toxicity. Lapatinib plus capecitabine is approved for the treatment of patients with prior exposure to anthracyclines, taxanes and trastuzumab. With this recent shift in treatment practice, an exploratory analysis was performed to evaluate whether prior anthracycline exposure had an effect on clinical benefit in patients treated with lapatinib.Methods: The activity of lapatinib was examined in 7 completed studies (2 Phase III, 5 Phase II) in which lapatinib was administered as monotherapy or in combination with paclitaxel, bevacizumab, or letrozole for the treatment of HER2 positive metastatic breast cancer. In each study, patients were grouped by prior anthracycline exposure to evaluate the clinical activity of lapatinib containing regimens, as measured by overall response rate (RR).Results: In 7 studies, 192 of 593 patients were exposed to prior anthracycline. 401 patients were not. Lapatinib was administered as monotherapy in 3 studies and in combination in 4 studies. Within in each study, RR were similar in both groups (no prior anthracycline and prior anthracycline). In the combination of lapatinib plus letrozole, overall RR was higher in patients not exposed to prior anthracyclines (36.1% versus 18%). Cardiac data to be presented. Recognizing the limitations of patient numbers and cross study comparisons, as a whole, the results of this analysis indicate that patients had similar overall response rates to lapatinib regardless of exposure to prior anthracycline.Summary of Response Rate  Response Rate by Prior Exposure to Prior AnthracyclineStudyTreatment ArmsNo Prior Anthracycline, % (n/N)Prior Anthracycline, % (n/N)Total, % (n/N)EGF30001 (HER2+)lapatinib+paclitaxel56.7 (17/30)63.6 (14/22)59.6 (31/52)EGF30008 (HER2+)lapatinib+letrozole36.1 (22/61)18.0 (9/50)27.9 (31/111)EGF20009lapatinib QD22.4 (11/49)30.0 (6/20)24.6 (17/69) lapatinib BID30.2 (13/43)34.6 (9/26)31.9 (22/69)EGF102580lapatinib42.9 (18/42)N/A42.9 (18/42)EGF103009lapatinib10.6 (15/141)N/A10.6 (15/141)EGF103890lapatinib+bevacizumab16.7 (3/18)8.8 (3/34)11.5 (6/52)EGF105764lapatinib+paclitaxel76.5 (13/17)77.5 (31/40)77.2 (44/57) Conclusions: With the increasing availability of agents to treat breast cancer, it is common that many patients do not receive prior anthracycline treatment for their disease. In this exploratory analysis, it appears the clinical activity of lapatinib is maintained in patients with no prior anthracycline exposure; and as observed in the lapatinib plus letrozole combination study, may even increase the overall response rate.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5105.
HER-2 Status May Change in Recurrent Breast Cancer.
Authors: Arnaout K. , Khalife M. , Younes M. , Hanbali A.
Published: December 2010

Introduction:The human epidermal growth factor receptor (HER-2) is a cell membrane surface-bound receptor tyrosine kinase which has been associated with increased breast cancer recurrence and a worse prognosis. Its presence also determines which patients are candidates for targeted therapy with trastuzumab. The purpose of this retrospective study was to determine the percentage of HER-2 status change in recurrent breast cancer.Methods:In this study, we retrospectively reviewed the charts of 2,652 patients diagnosed with breast cancer in our institutution between 2003 and 2009. Forty-four patients with recurrent breast cancer and available HER-2 status at the time of diagnosis and at recurrence were identified. HER-2 status was assessed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). When both methods were available, we used FISH as our reference.Results:A change in HER-2 status at recurrence was observed in six patients (13.6%). One patient (2.3%) changed from negative to positive. Five patients (11.3%) had a change from positive to negative. These five patients represented 50% of the patients with positive HER-2 at the time of initial diagnosis. HER-2 status remained unchanged in thirty-eight patients (86.4%).Conclusion:A change in HER-2 status can be noted at the time of recurrence. The exact mechanism is not yet well understood, and further studies are needed to identify it. Our data support the fact that patients with recurrent breast cancer should have their HER-2 status re-evaluated, since this affects therapeutic options.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5109.
A Phase 1 Study To Assess the Safety, Tolerability and Pharmacokinetics of ARRY-380 – An Oral Inhibitor of HER2.
Authors: Chia S. , Baetz T. , D'Aloisio S. , Fernetich G. , Freeman B. , Barrett E. , Kass C. , Kang J. , Sajan B. , Moulder S. , Garrus J.
Published: December 2010

Background: ARRY-380 is a potent, orally active small molecule that selectively inhibits HER2 signaling in vitro and in vivo and significantly inhibits tumor growth in a variety of HER2-dependent tumor xenograft models. Based on its preclinical activity, tolerability and pharmacokinetic (PK) profile, a Phase 1 study was initiated to evaluate the safety, tolerability and PK profile of ARRY-380 as a single agent in patients with solid tumors and to establish the maximum tolerated dose (MTD).Methods: Patients with HER2 positive breast cancer or other tumor types for which published evidence of HER2 expression exists were treated with ARRY-380 as a single oral dose on Cycle 1 Day 1, followed by continuous twice-daily (BID) oral dosing starting on Cycle 1 Day 3. ARRY-380 was escalated in cohorts of 3 to 4 patients, using standard dose-limiting toxicity (DLT) criteria during Cycle 1 to determine dose escalation. Serial PK assessments were made during Cycle 1 on Days 1, 2, 3 and at steady-state on Day 15.Preliminary Results: As of June 1, 2009, 15 patients have been treated in 5 dosing cohorts at doses of 25 to 300 mg BID. Patients had a median age of 61 years (range, 37-77 years) and ECOG PS of 0 to 2. Cancer types included HER2+ breast cancer (8), colorectal (6) and salivary gland (1). No DLTs have been observed and drug-related adverse events have included Grade 1 nausea, rash and fatigue and Grade 2 fatigue in 2 patients at the 200 mg BID dose level. Preliminary PK analyses indicate a trend for increasing Cmax and AUC with increasing dose, a median Tmax of 2 hours and a mean t1/2 of 4.6 hours across all cohorts. Two patients with HER2+ breast cancer have had stable disease for ≥ 4 months with no significant toxicity. One of these two patients had a notable reduction in liver metastases (28%) after 2 cycles of ARRY-380 and is currently on study.Conclusions: ARRY-380 has demonstrated an acceptable safety and PK profile and preliminary signs of clinical benefit. Dose escalation continues to determine the MTD.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5111.
Phase II Evaluation of a Novel Capecitabine Schedule in Combination with Lapatinib for Patients with HER2-Positive Metastatic Breast Cancer Refractory to Trastuzumab.
Authors: Gajria D. , Theodoulou M. , Feigin K. , Patil S. , Geneus S. , Drullinsky P. , D'Andrea G. , Lake D. , Norton L. , Hudis C. , Traina T.
Published: December 2010

BackgroundCapecitabine (C) administered for 14 days followed by a 7 day rest (14—7) in combination with lapatinib (L) improved TTP in patients with HER2(+) metastatic breast cancer (MBC) that progressed following trastuzumab (T). We applied mathematical methods to C dosing which predicted 7 days of treatment (Tx) followed by 7 days of rest (7—7) to be optimal. In animal models, C(7—7) was better tolerated than C(14—7) at higher doses and led to improved survival. A phase I study in patients (pts) with MBC concluded the MTD of C(7—7) is 2,000mg BID. To optimize the combination of C+L, we are testing C(7—7) + L.MethodsEligible pts have measurable, HER2(+) MBC with progression following trastuzumab. HER2(+)=3+ or FISH-amplified. Pts have normal LVEF by MUGA, ECOG performance status (PS) ≤2 and normal organ function. Prior therapy is limited to <3 chemotherapy (CRx) regimens for MBC; prior fluoropyrimidine is not permitted. Tx = C (2,000mg orally BID, 7—7) plus L (1,250mg orally daily). Cycle length=4 weeks (wks). Pts were evaluated for toxicity every 4 wks, for response every 12 wks and for LVEF by MUGA every 12 wks. Primary endpoint: Response Rate. Secondary endpoints: toxicity by NCI CTCv3, stable disease (SD)>6 months and PFS. Using a Simon optimal 2-stage design, with alpha 10% and power 90% to discriminate between RR 10% and 25%, 21 pts will be accrued to the 1st stage. If >2 patients respond, 29 additional pts will be enrolled. If >7/40 pts respond, then C(7—7) + L will be considered worthy of further study.ResultsAs of 5/28/09, 12 pts are enrolled; 6 are evaluable for response. Median (med) age 61 years (41-71), med ECOG PS 0 (0-2), ER/PR(+) 5, HER2(+) 12. Sites of MBC: Viscera 5, Brain 2. Med LVEF 64% (51-71%). Prior Tx: Adjuvant: CRx (7), hormone Tx (3), T (4); MBC: CRx (6), hormone Tx (4), T (8). After a med of 3 cycles (1-8), 6 pts are evaluable for response: CR 0, PR 1, SD>6mo 2, SD<6 mo 3. Five pts are too early for response assessment; 1 pt discontinued Tx during 1st cycle for palliative radiation without documented progression. There were no treatment-related grade 4/5 events or unexpected toxicity. Tx-related Grade (Gr) 2 or 3 toxicity in >10% of pts includes: hand-foot syndrome (Gr 3: 8%, Gr 2: 17%) and diarrhea (Gr 2: 17%). There were no declines in LVEF.ConclusionCapecitabine (7—7) plus lapatinib appears well-tolerated and demonstrates activity in pts with HER2(+) MBC that has progressed following trastuzumab based therapy. Additional efficacy and safety data is expected prior to this meeting.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5113.
Post-Progression Survival (PPS) and Overall Survival (OS) According to Treatment Type in Contemporary Phase III Trials in Advanced Breast Cancer (ABC).
Authors: Saad E. , Katz A. , Machado K. , Buyse M.
Published: December 2010

Objective: Progression-free survival (PFS) and time to tumor progression (TTP) have been the most frequent primary endpoints in recent phase III trials in ABC. OS, the most frequent secondary endpoint (Katz ASCO 2009), is largely influenced by subsequent-line therapy, and the role of PFS/TTP as surrogates for OS is still uncertain, perhaps due to the influence of post-progression therapy. Thus, PPS is of interest as a determinant of OS, and may vary according to treatment type.Methods: Using the medical subject headings “breast neoplasms” and “drug therapy”, we searched PubMed for phase III trials on systemic antineoplastic therapies published between January 1, 1998, and December 31, 2007 in one of 11 leading medical journals (Ann Oncol, BCRT, Br J Cancer, Cancer, Clin Cancer Res, Eur J Cancer, JCO, JNCI, Lancet Oncol, Lancet and NEJM). We excluded trials with factorial design, studies on high-dose chemotherapy, papers reporting combined analyses of two or more trials, and companion studies on correlative biology or prognostic factors. PPS was calculated as median OS minus median PFS/TTP for each trial arm. The proportion of OS accounted for by PPS (PPS%OS) was calculated as 100 - [median PFS/TTP/median OS]% for each trial arm.Results: The search yielded 75 trials enrolling a total of 28,973 evaluable patients in 159 trial arms. The following table displays the results, considering trial arms with available medians for both PFS/TTP and OS. PPS%OS is larger in hormone-therapy trials than in chemotherapy trials (P<0.01). Overall, PPS has a stronger correlation with OS than do PFS/TTP (R-squares of 0.83 and 0.24, respectively; both P<0.01), at least in part because PPS is longer than PFS/TTP.Main results  Mean ofmedians(months) Trial typeN armsPFS/TTPOSPPSMedian PPS%OSOVERALL1276.920.513.665.7%1. Chemotherapy96718.311.463.7%1.1 Chemo 1st line only597.820.712.964.4%1.2 Chemo 2nd line only146.715.28.559.8%1.3 Other (mixed lines)235.014.19.164.4%2. Hormone therapy296.927.420.574.4%2.1 Hormone 1st line only139.331.121.869.0%2.2 Hormone 2nd line only135.023.218.177.9% Conclusion: The contemporary average median OS in ABC is 20.7 months for first-line chemotherapy ± targeted agents and 31.1 months for first-line hormone therapy ± chemotherapy trials. Subsequent lines of therapy play a major role in determining OS in ABC, and further studies are needed to understand the role of PFS/TTP versus PPS in determining OS, since first-line PFS/TTP have not been convincingly shown to be good surrogates for OS in the literature on ABC.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5116.