bluuebunny/crossref_metadata_2025 huggingface dataset:
P3-05-10: Standardized Quantitative Methods for Investigating the Intratumor Heterogeneity of HER2 in FFPE Breast Cancer Specimens Utilizing the Vectra System, inForm and AQUA Technology.
Authors: Hoyt CC , Gustavson MD , Davis WL , Lane KA , Scott CG , Graves, Jr LL
Published: June 2012

Background: While intratumoral heterogeneity of HER2 gene expression is well documented, the HER2 intratumor protein expression heterogeneity and its clinical relevance is largely unknown but generally believed to be worth of investigation to ascertain its impact on HER2 targeted treatment outcome. Methods for investigating intratumor protein expression heterogeneity must preserve tissue architecture yet provide quantitative protein measurement. Immunohistochemistry (IHC) generally lacks standardization and quantitative capabilities and furthermore, current scoring methods do not account for, or report, heterogeneity. Robust and automated methods for characterizing and quantfication of heterogeneity are needed.

Material and Methods: Whole tissue samples stained for HER2 expression by fluorescent quantitative IHC methods were analyzed by digital imaging microscopy (Vectra system with inForm software, Caliper, Hopkinton MA) providing automated pattern-recognition-based selection of area of interest and field of view sampling strategies. Quantitative image analysis (AQUA technology, HistoRx, Branford, CT) for quantitative measurement of HER2 protein expression as well as objective, quantitative determination of heterogeneity were performed. Requirements for percent tumor sampling at high-power to capture heterogeneity were assessed, to support optimization of slide scanning.

Results: Heatmap representation of HER2 expression levels, and a statistical heterogeneity score developed based on the Simpson's biodiversity index (Simpson 1949) demonstrate visually as well as quantitatively HER2 heterogeneity in breast cancer tissues. Heterogeneity as a function of % area needed for accurate determination of whole section score along with confidence intervals as a function of percentage tumor analyzed inform appropriate sampling strategies. Conclusion: This study demonstrates the suitability of standardized quantitative methods for measuring heterogeneity and the feasibility of its use in investigations exploring the clinical relevance of HER2 intratumor heterogeneity with respect to both measurement strategies as well as treatment outcome. Through characterization of heterogeneous HER2 expression in tumors and optimized measurement strategies, it should be possible to design optimized treatment strategy aimed at treating the whole tumor as a function of objective quantitative assessment of HER2 expression.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-10.
P3-09-03: Long-Chain Polyunsaturated Fatty Acid Intake and Its Relationship to Long-Chain Polyunsaturated Fatty Acids in Serum, Red Blood Cells and Breast Tissue.
Authors: Harvey KE , Li S , Carlson SE , Sullivan DK , Klemp JR , Kimler BF , Fabian CJ
Published: June 2012

Background

Long chain omega-3 (n-3) poly unsaturated fatty acids (LCPUFA) have anti-inflammatory effects and are able to counteract the effects of the pro-inflammatory omega-6 (n-6) fatty acids such as arachidonic acid (AA) by substituting for the n-6 fatty acids in triglycerides (TG) and phospholipids (PL). Several pre-clinical, observational, and case control studies suggest that intake or tissue content of n-3 LCPUFA such as eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), relative to intake or tissue content of long chain n-6 fatty acids such as AA may be associated with reduced risk of breast cancer. The goal of this study was to determine the relationships between dietary intake of fatty acids, tissue levels of fatty acids, and breast tissue biomarkers for risk of breast cancer.

Methods: Women (n=74) were recruited from a clinic in which women at increased risk for breast cancer had breast tissue acquired by random periareolar fine needle aspiration (RPFNA). Breast epithelial cells were assessed for cytomorphology and proliferation (Ki-67 immunochemistry). Fatty acid dietary intake was assessed with the National Cancer Institute Diet History Questionnaire. Plasma, erythrocyte, and breast specimens were processed for membrane PL and TG and analyzed for individual fatty acids by gas liquid chromatography.

Results: Total intake of n-3 PUFA was 1.1 ± 0.5 g/d, and the ratio of EPA+DHA:AA was 0.1:1.0 (n=66). Dietary n-3 LCPUFA correlated with n-3 LCPUFA in both plasma and erthyrocyte PL (n=62). Breast epithelial cell number, Masood cytomorphology score, and percent Ki-67 positive cells were higher in RPFNA specimens which exhibited cytologic atypia compared to those which did not (n=74; p<0.001, Mann-Whitney Test). Subjects with atypia consumed less dietary n-3 PUFA (n=66, p=0.020), had lower plasma and erythrocyte PL and plasma TG EPA, DHA, total n-3, and EPA+DHA:AA (n=70; p<0.05). In breast tissue TG, the ratio of n-3:n-6 was also lower in subjects with atypia (n=40; p=0.025).

Conclusions: Overall, women in this high risk cohort consumed very low amounts of n-3 LCPUFAs. Dietary intake of n-3 LCPUFA was related to levels of n-3 LCPUFA in erythrocyte and plasma PL. Given the association of low levels of n-3 fatty acids with cytologic atypia (a known risk factor for breast cancer development), an intervention to increase n-3 fatty acids and n-3:n-6 ratios has merit and clinical trials in high risk women have been initiated.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-03.
P3-10-02: Gene Expression Changes in Methylnitrosourea (MNU)-Induced ER+ Mammary Cancers Following Short-Term Treatment of Rats with the Aromatase Inhibitor Vorozole.
Authors: Lubet RA , Grubbs CJ , Bode A , You M , Lu Y
Published: June 2012

Aromatase inhibitors have proven to be highly effective in both therapy and prevention of ER+ breast cancer. Vorozole (R83842), a high affinity competitive inhibitor of aromatase (similar to letrozole and anastrozole) showed strong activity in early clinical trials against ER+ breast cancer. Furthermore, vorozole was highly effective in the prevention and therapy of ER+ cancers in the MNU-mammary cancer model (Lubet, et al., Carcinogenesis 19, 1345, 1998). In the present study, rats bearing mammary cancers induced by MNU were exposed to vorozole (1.25 mg/kg BW/day) for 5 days. Global gene expression analysis showed that 162 genes were down-regulated and 180 genes up-regulated in cancers treated with vorozole (p < 0.05 and fold change > 1.5). The genes modulated by vorozole were compared with two additional sets of data. First, thirty-two genes and a number of pathways exhibited significantly concordant changes with aromatase inhibitors both in the animal model and in at least three of four published human data sets. In particular, differentially expressed genes enriched in the cell cycle pathway that were related to chromosome condensation in prometaphase [including Aurora-A, Aurora-B, Bub1B, non-SMC condensin I complex, subunit H (BRRN1), Condensin, CAP-G, CAP-G/G2, CAP-H/H2, CAP-D2/D3, CAP-E, TOP2, Cyclin A, Cyclin B, CDK1, Histone H1 and inner centromere protein (INCENP)] were downregulated after treatment with the aromatase inhibitor. These results appear to be in agreement with the strong anti-proliferative effects of aromatase inhibitors in both animal and clinical studies. A second comparison was with an in vitro study in which estrogen was removed from MCF-7 cells in culture. Decreases in genes related to the E2F1 transcription factor were observed. In our study, 13 modulated genes exhibited E2F-1 binding sites in their promoter regions, and 7 genes contained both ER binding and E2F binding sites. We were able to confirm modulation of the cell cycle related and E2F-related genes in a large independent set of human samples treated with anastrozole. The results on RNA changes for Bub 1B, Cyclin A and CDK-1 were verified by employing IHC analysis. In summary, gene changes observed in the rat closely paralleled gene changes associated with aromatase treatment and estrogen withdrawal in humans.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-02.
P3-11-02: Changing Paradigms for Breast Cancer Chemoprevention?
Authors: Chlebowski RT
Published: June 2012

The SERMs tamoxifen and raloxifene are approved for breast cancer risk reduction but their acceptance in clinical practice has been poor largely related to rare but potentially life threatening side effects which requires identification of populations at higher breast cancer risk and careful risk benefit discussion (Visvanathan 2009). Recent findings from randomized placebo-controlled clinical trials suggest potential alternative approaches. In the MAP.3 placebo-controlled trial including 4,650 postmenopausal women, exemestane decreased breast cancer incidence by 65% (P=0.002) (Goss 2011) with no significant difference between groups for fractures, CVD events, other cancers or treatment-related deaths. While the median Gail 5-yr risk score of participants was 2.3, eligibility factors included age alone (≥ 60 years) regardless of calculated breast cancer risk and accounted for 49% of entries. With extended follow-up from the Women's Health Initiative (WHI) randomized, placebo-controlled trial evaluating estrogen alone in 10,739 postmenopausal women with prior hysterectomy, a statistically significant decrease in breast cancer incidence of 23% was seen with estrogen use (P=0.02) (LaCroix 2011). Younger postmenopausal women (age 50–59 years at entry) randomized to estrogen alone had lower risk of myocardial infarction (HR 0.54, 95% CI 0.34−0.86) and death (HR 0.73, 95% CI 0.53−1.00). These findings contrast to those in the WHI trial evaluating combined estrogen plus progestin in 16,608 postmenopausal women with an intact uterus where breast cancers were increased 25% (P=0.004) as was breast cancer mortality (increased by 96%, P=0.049) (Chlebowski 2010). The apparently paradoxical findings that in postmenopausal women estrogen addition, as conjugated equine estrogen, lowers breast cancer incidence and estrogen reduction, by aromatase inhibitor use, also lowers breast cancer incidence are consistent with preclinical studies indicating condition dependent change in estrogen influence on mammary cancers (Jordan 2011). Major eligibility for the WHI trials included anticipated 3-yr survival regardless of breast cancer or CVD risk. The findings from the MAP.3 and WHI trials highlight the complex relationships between estrogen and breast cancer and suggest potential strategies for chronic disease risk reduction in select populations of postmenopausal women which may not require formal breast cancer risk assessment.

Visvanathan K, Chlebowski RT, et al. J Clin Oncol 2009;27:3235–3258.

Goss PE, Ingle JN, Ales-Martinez J, Cheung A, Chlebowski RT, et al. The NCIC CTG MAP.3 Trial. N Engl J Med 10.1056/NEJMos 1103507.

Chlebowski RT, Anderson GL, et al. JAMA 2010;304(15):1684–1692.

LaCroix AZ, Chlebowski, RT, et al. JAMA 2011;305(13):1305–14. Jordan VC, Ford LG. Cancer Prev Res. 4:633–7 (2011).

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-02.
P3-14-08: A Phase II Study of Gemcitabine and Carboplatin (GC) Plus Iniparib (BSI-201) as Neoadjuvant Therapy for Triple-Negative and BRCA1/2 Mutation-Associated Breast Cancer.
Authors: Telli ML , Kurian AW , Jensen KC , Vinayak S , Flaherty P , Lipson JA , Wapnir I , Daniel B , Carlson RW , Mills MA , Chang P-J , Schackmann E , Rocha C , Sherman B , Blackwood-Chirchir A , Bradley C , Ford JM
Published: June 2012

Background

The combination of gemcitabine, platinum and inhibition of DNA repair results in synergistic chemosensitivity in triple-negative and BRCA1-deficient breast cancer cell lines. This study was designed to assess efficacy, safety and predictors of response to iniparib, a small molecule whose mechanism of action is under investigation, in combination with GC in early-stage triple-negative and BRCA1/2 mutation-associated BC.

Methods: This single-arm, phase II study (NCT00813956) enrolled patients with clinical stage I-IIIA (T ≥1cm by MRI) estrogen receptor-negative (≤ 5%), progesterone receptor-negative (≤ 5%), and HER2−negative or BRCA1/2 mutation-associated BC. Neoadjuvant gemcitabine (1000 mg/m2; IV; D1, 8), carboplatin (AUC 2; IV; D1, 8), and iniparib (5.6 mg/kg; IV; D1, 4, 8, 11) were given every 21 days for 4 cycles, until the protocol was amended to increase the treatment duration to 6 cycles with enrollment of 36 patients. The protocol was further amended to increase the patient number with expansion to additional centers. Prior to expansion, reporting of interim safety and efficacy results was specified. The primary endpoint is pathologic complete response (pCR), defined as the absence of invasive carcinoma in the breast and axilla. Pathologic response was assessed using the residual cancer burden (RCB) index. Secondary endpoints included safety, radiographic response by MRI, breast conservation eligibility and correlation of baseline gene expression with response.

Results: The pre-expansion cohort consisted of 13 patients assigned to 4 cycles of treatment and 36 patients assigned to 6 cycles of treatment. Among these 49 patients (50 tumors), median tumor size is 35 mm, and median age is 48 years. The overall pCR rate is 34% (95% CI = 22–48); the pCR rate at 6 cycles is 41% (95% CI = 26–57). RCB 0/I was observed in 5/13 pts (38%; 95% CI = 18–64) treated with 4 cycles; 21/37 (57%; 95% CI = 41–71) treated with 6 cycles; and 9/9 pts (100%) with known BRCA1/2 mutations. Two pts experienced disease progression, two locoregional relapse, and three distant relapse. Among 45 tumors with Affymetrix-based RNA microarray gene expression data, 39/45 (87%) are classified as basal-like, 3/45 (7%) luminal B, and 3/45 (7%) normal breast-like. Most common G3/4 adverse events include neutropenia (31%), anemia (10%), and elevated ALT/AST (10%). Alopecia and chemotherapy-induced amenorrhea were uncommon. Analyses of secondary endpoints are ongoing and will be presented.

Conclusions: Preoperative GC plus iniparib is active in the treatment of early-stage triple-negative and BRCA1/2 mutation-associated breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-14-08.
P3-16-10: The Efficacy of Zoledronic Acid in Breast Cancer Adjuvant Therapy: A Meta-Analysis of Randomized Controlled Trials.
Authors: Lu J , Yan T , Yin W , Zhou Q , Zhou L , Jiang Y , Du Y , Shao Z
Published: June 2012

Background: The effect of zoledronic acid in breast cancer adjuvant therapy concerning improvement of patient survival has yet to be confirmed. We performed a meta-analysis of published and unpublished randomized controlled trials with the aim of accurate evaluation between clinical outcome and the association of the addition of zoledronic acid to adjuvant therapy.

Methods: We searched Pubmed (from 1966 to present) and online abstracts from the proceeding Annual Meetings of the American Society of Clinical Oncology (ASCO) (years 1992–2010) and online abstracts from San Antonio Breast Cancer Symposium (years 2004–2010). A total of five eligible studies including 3676 subjects and 3678 controls met our search criteria and were evaluated. Random and fixed-effects meta-analytical models were used where indicated, and between-study heterogeneity was assessed. The primary study endpoints were the DFS. Secondary endpoints were OS, distant or loco-regional recurrence free survival and bone metastasis free survival.

Results: Compared with the control arm, adjuvant breast cancer treatment with zoledronic acid did not significantly improve overall survival (OS), disease free survival (DFS), bone metastasis free survival, distant and locoregional recurrence free survival. However, in the postmenopausal subgroup, the addition of zoledronic acid to standard therapy could significantly improve DFS (RR=0.763, 95%CI 0.658−0.884, p<0.001) and reduce the risk of distant (RR=0.744, 95% CI 0.611−0.906, p=0.003) and locoregional recurrence (RR=0.508, 95% CI 0.340−0.760, p=0.001).

Discussion: Adjuvant zoledronic acid may potentially improve the prognosis of postmenopausal patients. Additional studies are needed to evaluate the value of adjuvant treatment of zoledronic acid in premenopausal couterparts, differing disease stages, and various pathological types of breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-10.
P3-16-13: Cardiac Safety of Non-Pegylated Liposomal Doxorubicin and Docetaxel as 1st Line Treatment in Metastatic HER2 Negative Breast Cancer (Myotax Study).
Authors: van Warmerdam L , Schiphorst P , Nieboer P , Dercksen M , de Jongh F , Schmidt R
Published: June 2012

Background: Doxorubicin (DOX) is an effective, but cardiotoxic agent in metastatic breast cancer (BC). Incidence of heart failure (HF) is 2–4% and increases considerably with cumulative doses over 450–550 mg/m2. In Myocet® DOX is encapsulated in liposomes. It is delivered predominantly to areas with increased capillary permeability such as tumors and reduces cardiac exposure. We conducted an open non-comparative study to assess cardiac safety of Myocet® combined with docetaxel.

Materials and methods: Females with locally advanced or metastatic HER2 negative BC. 6 cycles of Myocet® 60 mg/m2 and docetaxel 75 mg/m2 q3w as 1st line therapy. Left ventricular ejection fraction (LVEF) and disease status were assessed after cycle 2,4 and 6. Primary endpoint: signs and symptoms of HF (NYHA III-IV) or LVEF <50% and decrease ≥5% (with symptoms) or ≥10% (without symptoms). Results: 68 patients (pats) were included. Mean (sd;range) age 56,3 y (10,2;32-79), mean disease duration 5,5 y (5,1 y;1 mo-19 y), 31 pats had anthracyclines (AN) in the past. 49 pats completed all 6 cycles. Mean LVEF (%) over time are presented in table 1.

4 pats (3 AN pretreated) met the LVEF criteria for cardiotoxicity (no signs/symptoms of HF) after the 6th cycle. In 3 pats (all AN pretreated) a significant drop in LVEF was given as (one of the) reason(s) for premature discontinuation, but LVEF criteria for cardiotoxicity were not met. 73 grade ≥3 toxicities occurred in 32 pats; most frequent: neutropenia (±fever) 19 pats. Premature discontinuations (no. of pats): progressive disease 9, cardiotoxicity 3, other toxicity 4, death 2, other reason 1. In 10 pats the dose of Myocet was reduced. The best response was scored for 50 pats with at least 1 measurable lesion: CR 4, PR 28, SD 14, PD 4.

Mean LVEF in the entire group decreased during the study from 63 to 59 (9,2) %. The decrease in the AN pretreated group was 7% and in the not pretreated pats 3%. 4 pats met the LVEF criteria for cardiotoxicity and 3 others were withdrawn for cardiotoxicity without meeting the criteria. 6 of these pats were AN pretreated. None of them had signs/symptoms of HF. PR or better was observed in 64% of pats.

Conclusion: Liposomal DOX might provide more cardiac safety compared to conventional AN, but pats pretreated with conventional AN seem to be more prone to LVEF decrease. No signs or symptoms of HF have been observed. The combination of Myocet® and docetaxel is efficacious and sufficiently well tolerated and is currently investigated in combination with trastuzumab in HER2 positive pats.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-13.
P3-16-18: Phase 2, Open-Label Study of EZN-2208 (PEG-SN38) in Patients with Previously Treated Metastatic Breast Cancer.
Authors: O'Shaughnessy JA , Osborne CRC , Steinberg MA , Holmes FA , Kim HS , Kocs DM , Richards PD , Vukelja SJ , Berkowitz N , Buchbinder A
Published: June 2012

Background

EZN-2208 is a water-soluble PEGylated conjugate of SN38 that results in parenteral delivery, increased solubility, higher exposure, and longer apparent half-life of SN38, as well as more profound deoxyribonucleic acid (DNA) damage and inhibition of angiogenesis. EZN-2208 results in prolonged exposure of tumors to SN38 via preferential accumulation of EZN-2208 in the tumor and prolonged release of SN38 in the blood.

Methods: This trial evaluated EZN-2208 delivered as a 1-h IV infusion weekly for 3 wks in 4-wk cycles. The primary objective was to determine the overall response rate (RR) in female patients with metastatic breast cancer (MBC) who had received prior adjuvant or metastatic therapy with either 1) anthracycline and taxane (AT) or 2) anthracycline, taxane, and capecitabine (Xeloda®) (ATX). Secondary objectives included evaluation of RR based on tumor receptor status, duration of response, progression-free survival (PFS), overall survival (OS), and safety and toxicity. Response was evaluated using RECIST (v1.1).

Results: 148 patients received EZN-2208 in the AT (n=65; median age = 56 y [31-84 y]) or ATX (n=83; median age = 55 y [36-83 y]) cohorts. All 65 patients in the AT cohort had received 0–2 lines of prior cytotoxic therapy for MBC; for the ATX cohort, 31 patients (37%) had received 0–2 prior lines of cytotoxic therapy for MBC, 50 patients (60%) had received 3–4 prior lines, and 2 patients (2%) had received 5 prior lines. Preliminary results follow; final data will be presented at the meeting. Median (range) cycles of EZN-2208 was 2.3 (0.3-14) for AT and 2 (0.3-15) for ATX. Best overall response is shown in the table. RR (PR+uPR) was 22% for AT and 10% for ATX.

Median (95% CI) time to progression was 3.8 mo (3.6−7.4) for AT and 3.3 mo (1.8−3.7) for ATX. Median (95% CI) duration of response was 4.0 mo (3.7−5.6) for AT and 5.2 mo (1.9-..) for ATX. 6-mo PFS (95% CI) was 34% (19%-50%) for AT and 19% (9%-29%) for ATX. Median PFS (95% CI) was 3.8 mo (2.7−5.6) for AT and 2.9 mo (1.83.7) for ATX. Median OS (95% CI) was 9.1 mo (6.1−12.7) for AT and 7.9 mo (6.4−12.9) for ATX. Grade 3 or 4 drug-related adverse events (>10% of patients in either arm) included neutropenia (43%, 33%), diarrhea (11%, 8%), and leukopenia (11%, 6%).

Conclusions: EZN-2208 is active in patients with previously treated MBC. The activity is similar regardless of ER status and is promising in the TNBC population. The safety profile of EZN-2208 is acceptable with good tolerability in most patients. Further evaluation of EZN-2208 in this population is warranted.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-18.
P3-17-06: Final Results of a Controlled, Randomized 3-Arm Phase II Trial of EndoTAG”-1, a Cationic Liposomal Formulation of Paclitaxel Targeting Tumor Endothelial Cells, in Advanced Triple-Negative Breast Cancer (TNBC).
Authors: Awada A , Bondarenko IN , Tarasova O , Bonneterre J , Nowara E , Ferrero JM , Bakshi AV , Weidenthaler H , Wilke C , Piccart MJ
Published: June 2012

Background

EndoTAG™-1 (ET) is an innovative therapy of paclitaxel (P) embedded in cationic liposomes displaying antitumor activity by targeting negatively charged activated endothelial cells of tumor vessels.

Methods: 140 patients (pts) with locally relapsed (r) or metastatic (m) centrally verified TNBC were randomized to i.v. weekly ET (22mg/m2 ) plus P (70mg/m2) (ET+P), biweekly ET alone (44mg/m2) or weekly P (90mg/m2) in a 2:2:1 ratio. Pts had ≤ 1 prior chemotherapy (CT) for r or m disease and ≥ 6 months (mo) after taxane. A cycle (cy) comprised 3 weeks(w) of therapy and 1 w rest. Pts were treated for a minimum of 4 cy or until disease progression/unacceptable toxicity.

Primary endpoint was progression-free survival (PFS) rate at week 16 based on blinded central imaging and local assessment. Secondary endpoints included median overall survival (mOS), tumor response (RECIST), Quality of Life (QoL) and safety. The study was not powered for intergroup comparisons.

Results: Pts baseline characteristics were overall well distributed. Appr. 80% of all pts were treated 1st line for r or m TNBC. Results of PFS rates at week 16 are shown in table. Based on central review, median PFS at week 16 was 4.2 mo for ET+P [95% CI: 3.5−9.1], 3.4 on ET [2.0−3.8] and 3.7 on P [1.9−6.7]. Antitumor activity was highest for ET+P with a clinical benefit rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥6 mo at data cut off date of week 41) of 53% (26/49 evaluable pts), 31% (15/49) on ET and 36% (9/25) on P. The best overall response (CR, PR, SD at any time) on ET+P was 80% (39/49), on ET 65% (32/49) and 68% (17/25) on P.

Analysis of median OS is shown in table. Analysis of the not predefined subgroup (centrally TNBC + ECOG 0/1 + first line population) showed an impressive median OS of 17.8 mo for ET+P. No differences were reported for QoL. No additional toxicities to those known for ET and P were observed except uncomplicated grade 3/4 neutropenia in the ET + P arm.

Conclusions: At cut-off date week 41, the final analysis demonstrated ET+P as a promising combination compared to single agent arms. ET+P was well tolerated. These data will be the basis for a confirmatory phase III study.

Acknowlegments: B. Glasschroeder, U. Elsasser

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-06.
P4-01-11: Autocrine Human Growth Hormone (hGH) Reduces the Sensitivity of Breast Cancer Cells to Treatment with Tamoxifen and Fulvestrant.
Authors: Perry JK , Chen H , Bougen NM , Liu D-X , Lobie PE
Published: June 2012

Despite recent advances in breast cancer treatment regimes, intrinsic or development of resistance to conventional breast cancer therapies is still a significant clinical challenge. Expression of growth factors by tumour cells has been proposed as one mechanism for developing resistance to radiotherapy and chemotherapeutic agents.

The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development in human breast cancer. Recent studies have demonstrated that humans with growth hormone receptor deficiency are protected from developing cancer. Studies investigating human growth hormone (hGH) expression in human breast cancer have demonstrated that hGH expression is associated with specific histopathological features and survival outcomes for patients. Increased hGH expression is significantly associated with lymph node metastasis, tumour stage, proliferative index, worse relapse free survival and reduced overall survival in mammary carcinoma. In recent studies we have demonstrated that autocrine hGH promotes cell proliferation, cell survival and oncogenicity of human mammary carcinoma cells, enhancing anchorage independent cell growth, and supporting tumour formation in immunodeficient mice. Autocrine hGH also promotes mammary carcinoma cell migration/invasion and epithelial to mesenchymal transition and tumour vascularisation in xenograft studies.

In this study we investigated whether autocrine hGH promotes resistance to anti-estrogen therapeutic drugs, sensitivity to tamoxifen and fulvestrent using the human breast cancer cell lines MCF-7, T47D and BT474. Stable transfection with an expression vector containing the hGH gene enhanced MCF-7, T47D and BT474 cell viability, total cell number and anchorage independent growth following treatment with tamoxifen or fulvestrant when compared to control transfected cells. Conversely, treatment of BT474 cells with an hGH receptor antagonist (B2036), increased BT474 cell sensitivity to treatment with tamoxifen or fulvestrant. Using an estrogen response element (ERE) luciferase assay, we observed that autocrine hGH enhanced basal estrogen receptor (ER)-mediated transcriptional activity.

Our results demonstrate that forced expression of hGH in mammary carcinoma cells enhances ER-mediated signal tranduction and promotes resistance to tamoxifen and fulvestrant, while functional antagnonism of hGH increased cell sensitivity. Consequently, antagonism of the hGH receptor may help maintain sensitivity to anti-estrogen therapy and improve the prognosis of patients with hormone sensitive breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-11.
P4-01-13: Biology of Aggressiveness and Tamoxifen Resistance in Hormone Receptor-Positive Very Young Age Breast Cancer.
Authors: Han W , Ro J , Jung S
Published: June 2012

It has been known that young breast cancer patients have worse outcomes compared with older premenopausal or postmenopausal patients. Survival difference between young and older breast cancer patients is evident only in hormone receptor positive breast cancer regardless of tamoxifen treatment according to our previous report. However, the biology of aggressiveness and endocrine resistance of breast cancer in very young women is largely unknown. The purpose of this study is to find molecular characteristics of hormone receptor positive breast cancers of very young age women (<35) compared with those of older premenopausal women.

We extracted mRNA from fresh frozen hormone receptor positive primary breast cancer tissues of 24 young age breast cancer patients (<35 years) and 31 older premenopausal women (40 to 49 years) by standard method. All tumor specimens analyzed contained more than 50% tumor cells. Hormone receptor status was determined by immunohistochemistry (IHC). Gene expression microarray experiment was done in the 55 samples using Illumina HumanRef-8 v3 Expression BeadChip (Illumina, Inc., San Diego, CA). Functional and pathway analysis of differentially expressed genes were done using DAVID (http://david.abcc.ncifcrf.gov/home.jsp) and Ingenuity pathway analysis (IPA, http://www.ingenuity.com). Ki-67 assay was done using IHC in 4,957 ER+ breast cancer patients of Seoul National University cohort and 1,863 ER+ patients from National Cancer Center, Korea.

355 genes were upregulated (>1.5 fold) and 209 genes were down-regulated in breast cancer tissues of young age patients (<35) compared with those of older patients. In pathway analysis of the highly expressed genes in young patients, cell cycle function and pathway was significantly activated. The genes of central role in this pathway were MYC and CCND1. In IHC assay for 4,957 ER+ Seoul National University dataset, the proportion of high Ki-67 expression (>=10%) was positively correlated with decreasing age: 26.4%, 24.0%, 21.3%, 14.8%, 12.0% of women aged <30, 30–34, 35–39, 40–49, 50–59, respectively (p<0.001). The same correlation pattern between younger age and high Ki-67 expression was also seen in another 1,863 patient cohort.

In conclusion, we showed that genes involved in cell cycle pathway were upregulated in very young age (<35) ER+ breast cancer using microarray study. It was validated in large data set using Ki-67 IHC assay. High proliferation and fast cell cycle could be a mechanism of worse outcome and tamoxifen resistance of ER+ very young age breast cancer patients.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-13.
P4-01-21: An Estrogen-Inducible Transcription Factor FOXP1 Promotes Estrogen-Dependent Cell Proliferation of Breast Cancer Cells and Is Associated with 5-Year Disease-Free Survival in Patients with Tamoxifen-Treated Breast Cancer.
Authors: Shigekawa T , Ijichi N , Ikeda K , Horie-Inoue K , Shimizu C , Saji S , Aogi K , Tsuda H , Osaki A , Saeki T , Inoue S
Published: June 2012

Estrogen signaling pathways are involved in the growth and development of breast tumors through the activation of estrogen receptor a (ERα). ERα is expressed in most breast cancers and involves in estrogen-dependent proliferation by acting as transcription factors activating the expression of target genes. Various coregulators and transcription factors are associated with ERα-mediated transcriptional control of target genes. Therefore, a comprehensive understanding of estrogen signaling pathways in breast cancer is required for both treatment and diagnosis of the disease. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. Here, we investigated the expression pattern of FOXP1 by immunohistochemistry in a series of 133 invasive breast cancers and compared it with clinicopathological factors. The expression of FOXP1 was detected in nuclei in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including ERα and progesterone receptor (PgR), and negatively with pathological tumor size (pT). And in ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and that ERα recruitment to ER binding sites within the FOXP1 gene region identified by ChIP-chip analysis was increased. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Moreover, in MCF-7 cells, FOXP1 enhanced estrogen response element (ERE)-driven transcription. Finally, FOXP1 immunoreactivity was significantly more elevated in relapse-free breast cancer patients treated with tamoxifen than in relapse patients treated with it. Taken together, these results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity might be associated with the estrogen dependency of breast cancer clinically, which may predict favorable prognosis in the patients treated with tamoxifen.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-21.
P4-02-08: Obesity-Induced Aromatase Expression in the Breast Microenvironment Promotes Estrogen Receptor Activity Independent of Circulating Estradiol Levels.
Authors: Bowers LW , Brenner AJ , Li R , Tekmal RR , deGraffenried LA
Published: June 2012

Epidemiological studies indicate that obesity increases the risk of postmenopausal breast cancer by approximately 50%. In the past, researchers have hypothesized that elevated estrogen synthesis by the peripheral adipose tissue may be the principal mediator of breast tumorigenesis in this population, which primarily develops estrogen receptor alpha (ERα) positive breast cancer. However, obesity is also accompanied by an elevation in growth factor and cytokine signaling, and these pathways have been linked to tumorigenesis. In addition, certain growth factor and cytokine family members can promote aromatase expression in both the epithelial and stromal tumor compartments. Consequently, we hypothesized that obesity increases the risk of postmenopausal breast cancer via elevated aromatase expression and/or activity in the local mammary tissue.

To test our hypothesis, we investigated how ERa activity in mammary epithelial cells was influenced by adipose stromal cells (ASC) cultured under obesity-associated conditions, including high cell density and exposure to elevated levels of circulating growth factors and cytokines. For the latter condition, sera was obtained from postmenopausal women, pooled by BMI category (lean: 18.5−24.9; obese: ≥30), and applied to the ASC, which were originally derived from women undergoing reduction mammoplasty. High ASC density was achieved via the protocol previously published by Dr. Li. Preliminary data indicated that both elevated cell density and sera from obese postmenopausal women induces greater aromatase expression in ASC, indicating that multiple factors may be contributing to the increased local aromatase expression seen with obesity. We are currently exploring the signaling pathways responsible for obesity's upregulation of ASC aromatase expression and will present these results at the meeting. Intriguingly, exposure to conditioned media from both the high density and obese sera-exposed ASC enhanced ERa activity in MCF-7 mammary epithelial cells, independent from exogenous estradiol but dependent on the presence of androgens, suggesting an important role for the aromatase enzyme in this observation. To expand on this finding, we plan to investigate the effect of ASC conditioned media on different markers of cancer aggression, including proliferation and survival, and assess the degree to which these effects depend on estradiol. Through further examination of obesity's impact on signaling pathways in both the epithelial and stromal tumor compartments, we ultimately hope to identify more effective chemopreventive and therapeutic regimens for the high-risk obese postmenopausal population.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-08.
P4-05-01: Rab25 Alters Cellular Energetics and Growth Signaling during Breast Oncogenesis.
Authors: Mitra S , Cheng KW , Dennison JB , Mills GB
Published: June 2012

Background: Conservation and consumption of cellular energy is centrally regulated by growth and metabolism of the whole organism. The cancer cell gains autonomy from this regulation to become an organism with indeterminate growth potential. Plasma membrane receptors including growth factors and nutrient receptors are sensors for microenvironmental cues that dictate the intracellular energetic pathways to recalibrate ATP production based on available resources. So altercations in both growth and metabolic pathways are common features of cancers. A unifying component between receptor mediated growth signaling and metabolic pathways within a cell could be the vesicular trafficking system that imports, transports and exports a wide array of cargo from both these pathways.

In fact, derailed endocytosis is a hallmark of cancer. The endocytic pathway, as demonstrated by our laboratory, is a frequent target of genomic aberrations in cancer, and plays a critical role in maintenance of cellular polarity, stem cell fnction, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. The Rab GTPases along with their effectors are critical regulators of this endocytic machinary and can have a huge impact on the cellular iterinary of growth and metabolism.

Methods and results: A broad spectrum analysis of distribution of all the Rab family members across 52 breast cancer cell lines reflected a dichotomous distribution of Rab25, with higher mRNA and protein expression in ER+ cell lines. Rab 25 is of immense interest to us because of its unique substitution of leucine for glutamine at position 71which typically decreases the intrinsic GTPase activity, resulting in a dominant, constitutively active protein, akin to a transforming RAS mutation.

Using high throughput mRNA data analysis, we report that Rab25 and its effector Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER +ve breast cancers. Specifically in the luminal B subtypes, they are significantly associated with a markedly worsened outcome. In vitro, in MCF7 cell line, we observe an increased EGFR stability and MAPK signaling in Rab25 overexpressing stable clones which could be reversed with shRNA knockdown of Rab25. Loss of either Rab25 or RCP inhibited survival and growth of MCF7 lines under serum free conditions suggesting that Rab25 can potentially facilitate growth factor independence in cancer cells. Most importantly, we report that Rab25 enhances mitochondrial ATP production in breast and ***ovarian cancer cells and significantly reduces acidification of the ECM. Our ongoing experiments are directed towards understanding the mechanistic link between Rab25 mediated effects on EGFR/pERK signaling and its effects on cellular metabolism.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-01.
P4-05-03: Mechanism Underlying Metabolic Heterogeneity in Breast Cancer Subtypes.
Authors: Molina JR , Dennison JB , Zhang F , Mills GB
Published: June 2012

Background: Several distinct subtypes of breast cancer (Luminal, Basal-like and Her-2+) have been identified by gene expression profiling of breast cancers and cell lines. Although much is known about the regulation of cell signaling in each breast cancer subtype, little is known about subtype-specific energy metabolism and its regulation. The majority of cancers, including breast cancer, acquire an accelerated metabolic index as part of the transformation process. One of the most studied metabolic changes in cancer, referred to as the Warburg effect, is the increased uptake of glucose and its conversion to lactate; which is released from the cell, thus creating an astringent tumor microenvironment with high lactate and low pH. Accumulation of lactate in the tumor microenvironment presents cancer cells with a potential rich carbon source that could be exploited when the preferred nutrient sources, glucose and glutamine, are not abundant or available. Thus uptake and conversion of lactate to pyruvate and then entry into the TCA cycle and oxidative phosphorylation could generate energy that could potentially allow cancer cells to survive until other nutrients become available or until the cancer cells can invade and migrate toward nutrient rich environments, in other words, until they spread locally and metastasize.

Methods and Results: Examination of 59 breast cancer cell lines shows differences in expression of numerous proteins and enzymes involved in cellular metabolism, including the lactate transporters (MCT), the enzyme lactate dehydrogenase (LDH), and glucose transporter proteins (GLUT). We found that MCT1 expression is lost in the Luminal subtype and correlates with loss of LDHB and the regulator and chaperone of MCT1, CD147 (Basigin). Conversely, MCT1 is highly expressed in Basal-like and normal cell lines, along with CD147 and LDHB. While monocarboxylate transporter proteins can transport bi-directionally, MCT1 preferentially transports lactate into the cell, while MCT4 transports lactate out of the cell. The loss of MCT1 in luminal cells suggests a distinct energy metabolism in this subtype versus basal-like cells. The basal-like subtype has been further categorized into Basal and Claudin-low subtypes. Claudin-low cells express stem-like markers such as CD44+/CD24-, EMT markers such as vimentin, and are low expressers of the Claudin proteins. We found that MCT1 and CD147 are highly expressed and differentially regulated in Claudin-low cell lines as compared to normal cells. We have evidence that high lactate (10mM) as a sole energy source delays ATP loss and apoptosis in MCT1 expressing cells, but not in luminal cell lines that lack MCT1. Thus, claudin-low tumors may benefit from local lactate production providing an unexpected energy source.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-03.
P4-07-17: Isolation of Highly Pure Circulating Tumor Cells (CTCs) from Metastatic Breast Cancer (MBC) Patients for Gene Expression Analysis.
Authors: Magbanua MJ , Hauranieh L , Pendyala P , Sosa E , Scott J , Rugo H , Park J
Published: June 2012

Background: The paucity of information regarding the molecular nature of CTCs can be largely attributed to the difficulty of isolating these rare cells. As such, efforts on RNA profiling of CTCs have been limited to studies using immuno-enriched samples containing a high background of leukocytes.

Methods: We developed a two-step process: immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) to isolate CTCs away from leukocytes and performed expression analysis on a limited panel of genes. Magnetic beads coated with EpCAM mAb were used to enrich for tumor cells. Enriched samples were then subjected to FACS using fluorescently labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. CTCs and matched leukocytes were isolated from 67 sequential patients with MBC. Total RNA from isolated 20–50 cells was reverse-transcribed into cDNA. Sixty four (64) CTC-related genes from previous published reports were chosen for expression analysis. cDNA of these genes were pre-amplified and analyzed in triplicate via Taqman®-based RT-PCR in a low density array format. Statistical analysis of gene expression data was performed using Realtime Statminer®.

Results: Unsupervised hierarchical clustering analysis showed that CTCs clustered away from the leukocytes. Differential gene expression analysis revealed the up-regulation of several genes including EPCAM, MUC1, and KRT19 in CTCs (adjusted p <0.05). In addition, CTCs showed a significant down-regulation of the leukocyte-specific marker PTPRC (CD45) as well as CD44 and VIM, markers associated with stem cellness and epithelial to mesenchymal transition, respectively.

Discussion: We demonstrate the feasibility of isolation and gene expression analysis of CTCs. Molecular profiling confirmed that isolated CTCs were highly pure with little or no evidence of leukocyte contamination. This approach can serve as a non-invasive source of tumor tissue for further molecular characterization of CTCs and for monitoring of therapeutic efficacy of targeted therapies in clinical trials. This study was supported by CALGB and BCRF.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-17.
P4-08-01: Predictive Value of HER2 Serum Levels in Patients Treated with Lapatinib or Trastuzumab – A Translational Project in the Neoadjuvant “Geparquinto” Trial.
Authors: Witzel ID , Loibl S , von Minckwitz G , Abdallah A , Kühn T , Overkamp F , Fehm T , Schrader I , Uleer C , Kohls A , Stirnberg S , zu Eulenburg C , Untch M , Müller V
Published: June 2012

Background

Neoadjuvant chemotherapy (NT) has become an important approach to assess therapeutic efficacy of new treatment strategies. We observed a predictive role of of serum HER2 (sHER2) with neoadjuvant trastuzumab treatment in the previous trial “Geparquattro”. Predictive markers for response to new HER2−targeted therapies are still lacking. Therefore, we investigated the role of sHER2 in the context of trastuzumab and lapatinib treatment.

Methods: The clinical trial Geparquinto incorporated either trastuzumab or lapatinib treatment combined with chemotherapy for HER2−positive breast cancer patients. Serum samples were taken at three different time points: before initiation of NT, after 4 cycles of chemotherapy with epirubicin and cyclophophamide and after finalization of NT with 4 cycles of docetaxel (pre-surgery). Only those patients were included in the analysis with serum available at all three time points. sHER2 levels were measured by a commercially available ELISA in 159 patients with a HER2−positive primary tumor, 77 (48%) patients were treated with trastuzumab, 82 (52%) with lapatinib. Pathological complete remission (pCR) was defined as no microscopic evidence of invasive residual tumor cells in the breast and lymph nodes.

Results: Overall pCR rate in this cohort was 28%. sHER2 levels were higher in patients with larger tumors (> 5cm, p=0.012) and positive nodal status (p=0.001). Higher pre-chemotherapy sHER2 levels were associated with higher pCR rates in the entire study cohort (OR 2.9, 95% CI 1.3−6.7, p=0.012). Additionally, in the lapatinib treated patient group, a decrease of serum levels of more than 20% after 4 cycles of chemotherapy showed a tendency to be associated with higher pCR rates (OR 7.7, 95% CI 0.8- 70, p=0.071). In contrast to the finding of the previous trial Geparquattro, this was not the case in the trastuzumab-treated patient group (p=n.s.).

Conclusion: Results of this study demonstrate pre-chemotherapy sHER2 levels to be an independent predictor of response to NT with both trastuzumab and lapatinib treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-08-01.