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Up to 15% of women with breast cancer have locally advanced disease at diagnosis. The poor response of these patients to local therapy alone and the frequent development of disseminated disease suggest that early intensive systemic therapy may benefit these women. Twenty-four patients with non-metastatic, locally advanced, primarily inflammatory, inoperable breast cancer were treated with a 16-week dose-intense chemotherapy regimen as induction therapy. Treatment consisted of 8 repetitive 2-week cycles consisting of 100 mg/m2 cyclophosphamide orally D1-7, 40 mg/m2 doxorubicin intravenously (IV) D1, 1 mg vincristine IV D1, 100 mg/m2 methotrexate IV D1, 10 mg/m2 leucovorin every 6 hours for six oral doses D2-3, and 600 mg/m2 5-FU IV over 2 hours D2. A continuous infusion of 300 mg/m2 5-FU per day was given IV D8-9 of each 2-week cycle. After induction all patients had at least a partial clinical response and were operable; 9/24 (37%) achieved a clinical complete response. All patients underwent at least a simple mastectomy. Pathologic examination revealed no evidence of gross macroscopic tumor in 11/24 patients (46%) and no evidence of microscopic disease in 4/24 patients (17%). Seven of 24 patients (29%) had no microscopic disease in the breast. At a median follow-up of 45 months, there have been 10 relapses in the 24 patients treated with this induction regimen. The actuarial relapse-free survival at 5 years is 58%. Actuarial overall survival at 5 years is 75%. We conclude that this regimen is safe and well-tolerated and that the results of this therapy are sufficiently promising to warrant further study of this regimen in patients with locally advanced breast cancer.

The prohibitin gene, mapped to chromosome 17q21 is a candidate tumor suppressor gene. Because several somatic mutations of prohibitin were detected in breast tumors, we sought to identify potential carriers of prohibitin gene mutations in selected patients with early onset breast cancer. Seventy-six patients who developed breast cancer at or before the age of 40 were examined for mutations of prohibitin within the most conserved regions of the gene. Additionally, all exons of the prohibitin gene were examined in a subgroup of patients with a history of first-degree relatives with primary breast cancer. While several polymorphisms were noted, no inherited mutations of prohibitin were identified. This study suggests that prohibitin is not an early onset breast cancer susceptibility gene and additional studies will be necessary to understand its role in sporadic breast cancer progression.

In 1989, Hrushesky et al. (1) reported preliminary results of a retrospective study which identified the timing of breast cancer surgery in relation to the menstrual cycle as an independent predictor of disease-free survival for 41 premenopausal women. At least a dozen reports have subsequently appeared, some supporting and others refuting the claim. The debate has been played out both in the medical literature and in the lay press, resulting in much confusion for patients and physicians alike. This review will examine the biological plausibility of the hypothesis that the menstrual cycle can influence surgical outcome, the results of clinical studies done to date, and the potential methodological pitfalls inherent in performing these retrospective reviews.

PURPOSE We investigated if graft-versus-host disease (GVHD), which is associated with an antitumor effect, could be induced in women with advanced breast cancer by treatment with cyclosporine (CSA) following reinfusion of purged autologous marrow after treatment with high-dose chemotherapy and defined the toxicities of this approach. PATIENTS AND METHODS Fifty-one women with advanced breast cancer responding to therapy were treated with escalating doses of CSA (1.0, 2.5, or 3.75 mg/kg/d) for 28 days following high-dose chemotherapy and autologous bone marrow transplantation and monitored for induction of GVHD and toxicity of therapy. RESULTS GVHD was induced in a dose-dependent fashion in 14%, 68%, and 92% of patients at each dose level, respectively, a median of 15 days following autologous marrow reinfusion. GVHD was clinically mild and limited to skin. Toxicity was acceptable, with two deaths within 50 days of marrow reinfusion. Statistically significant increases in maximum creatinine and bilirubin levels were seen at all dose levels when compared with similarly treated historic controls who did not receive CSA. Time to last platelet transfusion was significantly delayed in patients treated at the highest dose. CONCLUSION GVHD can be safely induced by treatment with CSA in women with advanced breast cancer who are receiving high-dose alkylating agents and autologous bone marrow transplantation. The toxicity of this approach is acceptable. Evidence of antitumor efficacy awaits further investigation.

Many potential strategies exist for chemical protection against carcinogenesis. Multiple stages of carcinogenesis, including initiation, promotion, and progression, can serve as targets for different types of interventions [see reviews by Wattenberg (32); De Flora and Ramel (10)]. However, in the majority of experimental systems, protection has been achieved by administering the chemoprotective agent prior to and/or concurrent with the exposure to carcinogen. Given this temporal relationship between administration of anticarcinogen and carcinogen, it seems likely that these agents act principally by affecting the metabolism and disposition of carcinogens, thereby altering events critical to the initiation of carcinogenesis. Using this experimental approach, it has been possible to document protection against a diverse array of chemical carcinogens acting at many different target organ sites. Important classes of chemoprotectors that modulate the metabolic processing of carcinogens include phenolic antioxidants, indoles, organic isothiocyanates, coumarins, flavones, allyl sulfides, dithiocarbamates, and dithiolethiones.

The goal of this study is the discovery of biomarkers reflecting the pharmacodynamic action of sulforaphane (SFN), initially using normal human mammary epithelial MCF10A cells and then in tissue obtained from healthy patients undergoing reduction mammoplasty. The isothiocyanate SFN is formed by the hydrolysis of glucoraphanin, a water soluble glucosinolate found in cruciferous vegetables with especially high levels measured in 3 day old broccoli sprouts. Chemoprevention by SFN is achieved in part through the upregulation of cytoprotective enzymes via the Keap1/Nrf2 pathway. Preliminary dose response and time course studies in MCF10A cells established that SFN upregulated cytoprotective enzymes as indicated by increased levels of NQO1 transcripts, protein and activity. NQO1 induction was dependent on the Keap1/Nrf2 pathway since NQO1 transcripts, protein and activity were enhanced by KEAP1 siRNA knockdown in MCF10As. For the biomarker discovery phase SFN treated and KEAP1 knockdown MCF10A cells were analyzed by microarray to determine transcriptomic changes in response to pharmacological and genetic stimulation of the Keap1/Nrf2 pathway. The two experimental approaches were analyzed independently and then compared. The predominant class of upregulated genes common to both SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidant genes including NQO1, heme oxygenase (decycling) 1, sequestosome 1, thioredoxin reductase 1 and peroxiredoxin 1. The most highly upregulated genes in this class were the aldo-keto reductase family members including AKR1B1, AKR1B10, AKR1C1 and AKR1C3. Other classes of genes with overlapping regulation were glutathione metabolism genes such as GCLC and GCLM, molecular transport, cell growth and cell death regulation genes. Some members of the xenobiotic metabolism, molecular transport, cell growth and cell death regulation classes of genes were also regulated exclusively by SFN. Additionally histone cluster and chromatin remodeling genes were downregulated by SFN. Potential biomarker genes from the microarray study include NQO1, GCLC, SQSTM1 and AKR1C1. An accompanying proteomic study for biomarker discovery using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC) is underway. The transcriptomic and proteomic changes in response to SFN treatment and KEAP1 knockdown will be compared to determine if the changes seen on the transcriptomic level are translated to the proteomic level. Candidate biomarkers identified from microarray and SILAC studies reflecting the pharmacodynamic action of SFN in human cells will be analyzed by Western blot, qRT-PCR and enzyme assays in tissue from reduction mammoplasty patients. These women have been randomized in a clinical trial to receive either a broccoli sprout preparation containing 100 micromole SFN or a placebo beverage daily for 10 days prior to surgery. Supported by DOD grant BC073262 and NIH grants P50 CA088843 and U54 RR020839. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A47.

3097 Background: Epigenetic modifications contribute to breast cancer initiation and progression and may be reversible. In preclinical breast cancer models, the histone deacetylase vorinostat induc...

Gene expression can be modulated through histone methylation, which is tightly control by the balance of histone methytransferase and demethylase. Methylation (mono-, di- and tri-methylation) of lysine 4 of histone 3 is associated with active gene transcription. Lysine-specific demethylase 1 (KDM1/LSD1) can enzymatically demethylate mono- and di-methyl H3K4 that leads to suppression of gene transcription. Our previous studies demonstrated that a novel class of polyamine analogues can effectively inhibit KDM1/LSD1 and induce re-expression of candidate epigenetically silenced genes. The purpose of this study was to examine the molecular effects of polyamine analogues, 2d and PG11144, on global gene expression profile by inhibition of KDM/LSD1 in estrogen receptor-negative MDA-MB-231 breast cancer cells. The possible mechanisms of polyamine analogues mediating modulation of gene expression were also investigated. Treatment with polyamine analogous, 2d or PG11144, significantly increases global histone 3 dimethylation at lysine 4 (H3K4me2), as well as altering gene expression. There are 274 and 625 genes changed at least 2-fold after treatment with 2d and PG11144 respectively. The genes’ expression levels of twelve 2d up-regulated and fourteen PG11144 up-regulated genes were confirmed by real-time RT-PCR. Chromatin immunoprecipitation analysis demonstrated that up-regulated gene expression by polyamine analogous is associated with up-regulated active histone marks (H3K4me, H3K4me2 and ActH3K9) and down-regulated repressive histone marks (H3K9me2 and H3K27me3) in the promoter regions of the relevant target genes. Supported by NIH CA 88843, CA51085, Breast Cancer Research Foundation, and Komen for the Cure KG080923. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4877.

The goal of this study is the discovery of biomarkers reflecting the pharmacodynamic action of sulforaphane (SFN), initially in the normal human mammary epithelial MCF10A cell line and then in tissue obtained from healthy patients undergoing reduction mammoplasty surgery. SFN is formed by the hydrolysis of glucoraphanin, a water soluble glucosinolate found in cruciferous vegetables with especially high levels measured in 3 day old broccoli sprouts. Chemoprevention by SFN is achieved in part through the upregulation of cytoprotective enzymes via the Keap1/Nrf2 pathway. Preliminary dose response and time course studies in MCF10A cells established that SFN upregulated cytoprotective genes as indicated by increased levels of NQO1 transcripts, protein and activity. NQO1 induction is dependent on the Keap1/Nrf2 pathway since NQO1 transcripts, protein and activity were enhanced by KEAP1 siRNA knockdown in MCF10As. For the biomarker discovery phase isobaric tag for relative and absolute quantitation (iTRAQ) was used to quantify global protein changes using mass spectrometry. Two independent 8-plex iTRAQ experiments determined the pharmacological actions of SFN on protein expression, compared to a vehicle control, and the genetic regulation of protein expression following knockdown of KEAP1 compared to a non targeting control siRNA. In both experiments protein expression was analyzed at 12, 24, 48, and 72 hour time points after SFN treatment or KEAP1 knockdown. In addition to confirming NQO1 to be a good candidate biomarker, the iTRAQ analysis identified other upregulated candidates to be aldo-keto reductase family 1 member C1 (AKR1C1), DJ-1 protein, and thioredoxin. NQO1 and AKR1C1 transcripts were also elevated in the MCF10A cells, as assessed by microarray assay. Candidate biomarkers identified from microarray and iTRAQ studies reflecting the pharmacodynamic action of SFN in human cells will be analyzed by Western blot, qRT-PCR and enzyme assays in tissue from reduction mammoplasty patients. These women have been randomized in a clinical trial to receive either a broccoli sprout preparation containing 100 micomole SFN or a placebo beverage daily for 10 days prior to surgery. Supported by P50 CA088843, U54 RR020839 and BC073262. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5700.

The lysine deacetylase (KDAC) inhibitor, vorinostat or suberoylanilide hydroxamic acid (SAHA), is currently being used for treatment of cutaneous T cell lymphoma, however, its anti-breast cancer effects are under evaluation. SAHA's pluripotent effect on histone and non-histone lysine acetylation across cancer types raises the theoretical concern that it could both re-express epigenetically silenced tumor suppressors and activate oncogenic pathways as well. In order to investigate the potential role of SAHA in tumor suppressor, cell death and signaling pathways, we used quantitative mass spectrometric analysis of MCF10A normal mammary epithelial cells and MDA-MB-231 breast cancer cells treated with SAHA for 12 and 24 hours. We observed downregulation of EGFR, Ephrin type A receptors and cell cycle proteins including CDC2 and CDC42 protein kinase, which is consistent with anti-tumor activity. We found that SAHA treatment also promoted activation of proteins including Ephrin type B receptors, STAT3 and p120 Catenin. Such molecular effects must be characterized in greater detail in order to determine the potential efficacy of this drug in breast cancer. Our findings show that SAHA-mediated inhibition of KDACs results in a wide range of downstream consequences in mammary epithelial and breast cancer cells. Candidate molecules identified in this study can be used as biomarkers to assay the breast cancer response to SAHA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3648.

Of the 180,000 women who will be found to have breast cancer in 1991, over 90% will present with disease clinically limited to the breast and axillary lymph nodes. Unfortunately nearly one half of these women will later develop and ultimately die of metastatic breast cancer. It is now accepted that breast cancer is a systemic disease at diagnosis in many patients and therefore not curable by local treatment alone. Thus there has been considerable interest in developing effective forms of systemic adjuvant therapy with the goal of eradicating micrometastases and thereby improving relapse-free and overall survival. This chapter will review the results of major clinical trials using adjuvant chemotherapeutic and chemohormonal approaches. It will focus specifically on published randomized trials of adjuvant chemotherapy in women with axillary lymph-node-positive breast cancer. Studies of chemotherapy in node-negative breast cancer are reviewed in . The results of individual trials that evaluate perioperative chemotherapy, optimal duration of therapy, single agent vs. combination chemotherapy, utility of doxorubicin-containing regimens, combined chemohormonal therapy, and chemoimmunotherapy will be discussed. Relevant results from the Early Breast Cancer Trialists’ Overview analysis will also be highlighted, as will certain critical concepts, such as dose intensity. Finally, the recommendations of the 1985 and 1990 National Institutes of Health Consensus Conferences on Adjuvant Therapy for Breast Cancer will be reviewed, and our current recommendations regarding adjuvant chemotherapy for women with axillary lymph-node-positive breast cancer will be presented.

Background: MA17 showed that adjuvant letrozole after 5 years of tamoxifen markedly reduced the risk of recurrence in women with ER+ early stage breast cancer and improved overall survival in women presenting with node +ve disease. Most trials of early adjuvant aromatase inhibitor therapy required women to be postmenopausal at diagnosis to be eligible. We report here on a subset of women in the MA17 trial who were premenopausal at initial diagnosis and in whom subsequent menopause, prior to randomization, may have influenced their outcome on extended adjuvant letrozole.Methods: Women randomized to MA17 were divided into 2 groups: 1) pre-menopausal (< 50 years of age but underwent bilateral oophorectomy at time when tamoxifen treatment started or < 50 years of age at the start of tamoxifen treatment but became amenorrheic during adjuvant chemotherapy or tamoxifen treatment) and 2) post-menopausal. Disease-free survival (DFS) from time of randomization for women in these two groups were summarized by Kaplan-Meier curves and compared based on log-rank test and Cox model.Results: 889 women were identified as premenopausal and 4,277 as postmenopausal. Treatment, age, race, duration of tamoxifen, prior adjuvant chemotherapy, and mastectomy were independently associated with menopausal status on multivariate analysis. The interaction between treatment and menopausal status was statistically significant for DFS (p=0.02), indicating that women diagnosed with pre-menopausal breast cancer had significantly greater benefit (Hazard ratio=0.25, 95% CI: 0.12-0.51) with letrozole treatment in terms of DFS than those with post-menopausal status (hazard ratio=0.69 with 95% CI from 0.52 to 0.91). In comparison with women treated with placebo, pre-menopausal women on letrozole had a significantly higher incidence of arthralgia (24 vs. 16%; p=0.004) but a lower incidence of vaginal bleeding (10 vs. 16%; p=0.01). Compared with placebo post-menopausal women receiving letrozole had a higher incidence of hot flashes/flashes (55 vs. 50%; p=0.001), arthralgia (25 vs. 21%; p=0.002), myalgia (15 vs. 12%; p=0.007) and alopecia (5 vs. 3%; p=0.003).Conclusions: Letrozole after tamoxifen was more effective in improving DFS in women who were premenopausal at primary diagnosis than those who were postmenopausal. Letrozole was well tolerated in premenopausal women. These data indicate that women who are premenopausal at the time of diagnosis but become postmenopausal anytime before, or during, adjuvant tamoxifen should be considered for extended adjuvant therapy with letrozole. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 13.

Background: Black race has been associated with worse outcome in operable breast cancer, which has been attributed to a higher incidence of “triple negative” (TN) disease, disparities in care, and comorbidities. We evaluated the effect of black race on outcomes by hormone receptor (HR) and HER2 expression in patients treated with standard adjuvant therapy in trial E1199 (N Eng J Med 2008; 358: 1663)Methods: This study included 4950 eligible women with axillary lymph node positive or high-risk node-negative breast cancer, all of whom received doxorubicin and taxane-containing chemotherapy, plus standard endocrine therapy if HR-positive (but not trastuzumab if HER2-positive). Endocrine therapy included tamoxifen alone (38%) or followed by an AI (57%), or an AI alone (5%). The effect of black race was evaluated using Cox's proportional hazards model method (1) as a single variable, (2) in a model that included race, phenotype (TN vs. HER2-pos vs. HR-pos, HER2-neg/unknown [HR+]) and their interaction, and (3) in a multivariate model considering age, tumor size, number of positive lymph nodes, body mass index (BMI), and treatment arm. The stepwise method was used for model selection. The endpoints evaluated included disease free survival (DFS) and overall survival (OS). Results are expressed as hazard ratios (HR), with a HR > 1 indicating a worse outcome for black race. All p-values are two sided.Results: Of 4950 eligible patients, 416 (8.4%) were black. Black race was associated with significantly higher rates of TN disease (34% vs. 19%; p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 37.

Background: In 2007, the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) recommended new guidelines to define HER2 positivity by immunohistochemistry (3+ IHC: uniform intense membrane staining of >30% of invasive tumor cells) or fluorescent in situ hybridization (FISH+: HER2/CEP17 ratio >2.2). The original criteria used for enrollment in the pivotal N9831 trial conducted from 2000-2005 were IHC>10% (3+) or FISH ≥2.0.Purpose: To investigate the impact of the ASCO/CAP guidelines on patient (pt) eligibility and disease free survival (DFS) compared to the originally used FDA-approved definitions.Patients/Methods: This analysis included 2268 patients from the HER2+ N9831 adjuvant trastuzumab phase III trial. IHC was centrally performed at the Mayo Clinic using the DAKO HercepTest™ and was re-analyzed to determine the percent of tumor cells with 0, 1+, 2+, and 3+ staining intensities. DFS was compared between pts randomized to standard chemotherapy +/- concurrent trastuzumab (Arms A and C of the trial) within IHC/FISH subgroups using Cox proportional hazards regression stratified by hormone receptor and nodal status.Results: Based on 2268 pts, 83 pts (3.7%) were 3+ by FDA-approved guidelines but not 3+ by the ASCO/CAP guidelines. Among these pts, 77% (64/83) were FISH amplified centrally by ASCO/CAP guidelines. Only 1.5% (34/2268) of pts eligible under FDA-approved definitions from N9831 in this sample set would not meet the ASCO/CAP guidelines. In 1441 patients randomized to standard chemotherapy +/- concurrent trastuzumab, the exploratory DFS hazard ratio (HR) associated with trastuzumab in IHC/FISH subgroups were as follows: ASCO/CAP HER2+ HR=0.59 (95% CI: 0.47-0.75; p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 701.

Background: N9831 is the only randomized phase III trial comparing safety and efficacy of the addition of trastuzumab (H) to doxorubicin and cyclophosphamide then paclitaxel (Arm A: AC→T) either following (Arm B: AC→T→H) or starting concurrently with paclitaxel (Arm C: AC→T+H→H) for women with resected Stage I-III invasive HER2+ breast cancer. The 3 yr cumulative incidence of NYHA class III or IV congestive heart failure or sudden cardiac death was previously reported: 3.3% in Arm C, 2.8% in Arm B (Perez EA, et al. JCO 2008). The comparison of AC→T to AC→T+H→H was reported in a joint analysis of N9831 and NSABP B-31 in 2005 and updated in 2007, demonstrating a 52% reduction in risk of a disease event (Romond E et al., NEJM 2005; Perez EA, et al. ASCO 2007).Materials and Methods: Primary endpoint is disease-free survival (DFS). At the second planned interim analysis of Arm A vs. Arm B, the O’Brien-Fleming boundary (OFB) was crossed. NCCTG Independent Data Safety Monitoring Committee approved the release of these data as well as the data pertaining to Arm B vs. Arm C due to slow pace of events [expected 647 events in 4 yr follow-up period (f/u) vs. actual 334 events in 4.5 yr f/u]. Shortly thereafter, there were sufficient events to perform the first planned interim analysis of B vs. C. We present the results of each of these pairwise comparisons taking into account the potential for crossover to Arm C after the release of the joint analysis findings in 2005.Results: From 5/2000 to 4/2005, 2448 eligible women were enrolled for the Arm A (n=1087) vs. Arm B (n=1097) comparison. Median f/u is 5.5 yrs. with 386 events. The addition of trastuzumab sequentially to AC→T significantly improved DFS, univariately [HR(Arm B/Arm A)=0.70, 95% CI: 57-86%, logrank p=0.0005] and after adjusting for age, tumor size, number of positive nodes, and ER [PPH: HRadj=0.67 (95% CI: 0.55-0.82)]. 5 yr DFS was increased from 72% with AC→T to 80% with AC→T →H.From 5/2000 to 4/2005, 1903 eligible women were enrolled for the Arm B (n=954) vs. Arm C (n=949) comparison. Median f/u is 5.3 yrs. with 312 events. The log-rank p-value testing whether DFS differs with respect to starting time of trastuzumab was 0.019. [Not crossing pre-specified OFB for statistical significance]. After adjusting for tumor size, number of positive nodes, and ER, HRadj(Arm C/Arm B)=0.75 (95% CI: 0.60-0.94)]. 5 yr DFS was increased from 80% with AC→T →H to 84% for AC→T+H →H.Conclusions: DFS is significantly improved with the addition of 52 weeks of H (sequentially or concurrently) to AC→ T. There is a statistically significant 33% reduction in the risk of an event with the sequential addition of H following AC→T. There is a strong trend for a 25% reduction in the risk of an event with starting H concurrently with T relative to sequentially after T. Therefore, based on a positive risk/benefit ratio, we recommend that trastuzumab be incorporated in a concurrent fashion with T chemotherapy.Acknowledgements: NIH CA25224, Breast Cancer Research Foundation, Genentech. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 80. NOTE: This abstract was accepted for presentation at the Symposium after the Abstract Book went to press.

Abstract 4531 Introduction According to the Surveillance, Epidemiology, and End Results (SEER) database 12,810 patients will be diagnosed with acute myeloid leukemia (AML) in 2009 with an incidence rate of 3.5 per 100,000 persons in the United States. The SEER data estimates that 55% of those diagnosed are 65 years or older. Standard first line therapy for the treatment of AML consists of at least one round of chemotherapy commonly referred to as induction. A significant portion of those patients who receive standard induction chemotherapy will have leukemia that fails to show a complete response as defined by less than 5% leukemia blasts in the bone marrow with normal tri-lineage hematopoiesis and peripheral blood count recovery (Mueller S, et al. BMC Cancer 2006, 6:143). Age, patient performance status, the presence of secondary AML, cytogenetics, and molecular markers are prognostic for particular cohorts of patients, however, there is currently no means to predict whether an individual's leukemia will or will not undergo a complete response (CR) after the administration of standard AML induction chemotherapy. Methods A budget impact model was designed to assess the incremental societal cost and incremental cost to a typical national health plan of treating patients who will fail induction therapy. The analysis only evaluated elderly patients over the age of sixty five. Data were gathered from the peer reviewed literature. All costs have been updated to 2009 dollars using the medical care component of the consumer price index. The patient population was determined to be 6,981 patients in 2009 with 30% receiving induction therapy (Menzin J, et al. Arch Intern Med. 2002; 162:1597-1603) and only 38% demonstrating a CR (Appelbaum FR, et al. Blood 2006 107:3481-5) resulting in 1,303 patients failing during induction therapy. The costs were taken from Menzin et al., which used Medicare claims data to determine the associated costs of AML patients during the first two years post diagnosis. In the model Medicare payments are used as a proxy for direct costs to society and a national health plan. These reimbursements are able to capture the costs of the initial hospital visit, lab/diagnostic/radiology, supportive care, drugs, and adverse events through the observation of payments across all appropriate settings of care (inpatient hospitalization, skilled nursing facility, outpatient hospital/clinical, physician's office, home health, and hospice). A patient undergoing chemotherapy incurred costs of $120,468 over two years, while a patient avoiding chemotherapy only incurred costs of $40,720. Results The incremental cost of a patient receiving induction therapy was calculated to be $79,748. For a national health care plan with an assumed average of one million members results in a cost of $283,856 to treat patients whose leukemia would not respond to induction chemotherapy. The overall societal impact of treating this patient population with ineffective therapy is $104 million. Conclusions Patients who are subjected to ineffective chemotherapy face the cytotoxic effects of the treatment, none of the benefits of treatment response, and impose a significant cost burden to themselves and the healthcare payment system. Diagnostics currently in development that can identify patients at the time of diagnosis with disease unresponsive to therapy may have the ability to alleviate unnecessary costs, while steering patients to better tailored and more effective therapies. Disclosures: Cleary Cohen: Nodality, Inc: Employment. Middlebrook:Nodality Inc: Employment.

520 Background: Targeted therapy using trastuzumab (an anti-HER-2 receptor monoclonal antibody) has significantly improved survival in breast carcinoma patients (pts), but determining which pts will respond to this therapy remains a challenge. We previously reported (Miller DV, ASCO 2004 abstract #568) a subset of pts with breast cancers demonstrating focal HER-2 amplified clones (FHAC) amidst otherwise nonamplified tumor cells by fluorescence in-situ hybridization. These accounted for 21% of the HER-2 amplified but immunohistochemistry (IHC) negative cases and 30% of the HER-2 amplified but IHC equivocal cases. The clinical significance of this phenomenon was unclear at that time. We now report the disease-free survival (DFS) data on 91 FHAC pts with a comparison to the diffusely amplified (DA) cases in this trial group. Methods: Breast tumors were evaluated for HER-2 gene amplification using PathVysion™. FHAC cases demonstrated 2–40% of cells with >10 HER-2 signals and HER-2:CEP17 ratio >5.0, regardless of the overall HER-2:CEP17 ratio. Patient and disease characteristics were compared using chi-square tests. Cox regression models compared DFS between pts randomized to arms A (standard chemotherapy) and C (standard chemotherapy with concurrent trastuzumab) within 91 FHAC and 1571 DA cases. Median follow up was 4.0 years. Results: Age, race, menopausal status, surgical procedure, nodal status, histologic type and grade, and tumor size, were not significantly different between pateints with FHAC and DA. Pateints with FHAC had more frequent hormone receptor positivity compared to DA cases (66% vs 50%; p = 0.004). Hazard ratios between pts with FHAC and DA showed that both groups of pts had similar DFS (A: HR = 0.86, p = 0.65; C: HR = 0.72, p = 0.57). Hazard ratios between arms within FHAC and DA groups demonstrated similar benefit from trastuzumab in each group (FHAC: HR = 0.50, p = 0.30; DA: HR = 0.59, p < 0.0001). Results remained consistent when including hormone receptor status in the model. Conclusions: Based on a small number (n = 91) of pts with FHAC, benefit from trastuzumab appears to be similar whether the population of HER-2 amplified cells with breast carcinomas is focal or diffuse. [Table: see text]

500 Background: Triple negative breast cancer (TNBC) is associated with a higher risk of recurrence and earlier recurrences than other breast cancer phenotypes. We evaluated the genotypic features of TNBC compared with hormone receptor (HR)-positive disease, and also evaluated genotypic features associated with recurrence. Methods: RNA extracted from tumor samples obtained from 764 patients with stage I-III breast cancer was analyzed by RT-PCR for 371 genes. All patients received adjuvant chemotherapy (plus hormonal therapy in HR-positive disease) in trial E2197; HR and HER2 expression were evaluated by immunohistochemistry (IHC) in a central lab (J Clin Oncol 26:2473–2481). An unsupervised clustering analysis was performed in all samples (N=764). Cox proportional hazard models were used to identify differences in gene expression in TNBC versus HR-positive disease, and with recurrence in phenotypically defined (by IHC) TNBC (N=246) and HR-positive (N=465) disease. Results: Unsupervised analysis revealed two major clusters that differed with regard to HR expression by IHC. Supervised analysis comparing the TNBC vs. HR-positive phenotypes revealed 269 genes (73%) with significantly different expression (p [Table: see text]

Abstract #56 Background: Recent findings from NSABP B-31 suggested that breast cancer (BC) patients (pts) with c-MYC (MYC)/HER2 co-amplification in their primary tumors may benefit more in terms of disease-free survival (DFS) from adjuvant trastuzumab (n=471; p0.05); however, MYC amplified pts were significantly younger than MYC non-amplified pts (mean 46.4 vs. 51.0, t-test p=0.02) and MYC/HER2 co-amplified pts were significantly younger than MYC/HER2 non-co-amplified pts (mean 45.9 vs. 51.0, t-test p=0.01). Conclusions: In our initial analyses of 239 pts, we observed a lower frequency of MYC/HER2 co-amplification than the previously reported 30% for NSABP B-31. Complete FISH analyses of 868 pts in N9831 and association with DFS will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 56.

Abstract #6048 Introduction. Basal-like breast cancer (BBC) has low expression of ER, PR, and HER2, and is often called triple negative (TN) BrCa. TBCRC 001 is a multicenter randomized phase II study of cetuximab (C) alone or combined with carboplatin (P) in TN BrCa. Pts randomized to Arm 1 received C alone (400 mg/m2, then 250 mg/m2 weekly) with P (AUC 2, 3 of 4 weeks) added upon progression (PD). Pts on Arm 2 received C+P throughout. The primary endpoint was objective response. To explore markers of activity and response, we measured circulating tumor cells (CTCs) in pts on study and directly compared two methods of CTC enumeration; CellSearch (Veridex) and immunomagnetic enrichment followed by flow cytometry (IE/FC). A subset of samples were isolated for molecular profiling. Methods. Blood was obtained from consenting pts at baseline, 7 to 14 days after the first infusion, then monthly until the end of the study. CellSearch assay was performed as previously described using 7.5 cc blood in a CellSave tube and the CellSpotter analyzer, and the percent of CTCs staining for EGFR was also measured. For IE/FC, 20 ml of blood was subjected to IE using anti-EpCAM MAb-conjugated iron particles, followed by multiparameter FC for EpCAM, CD45, and nucleic acid content. CTC results were correlated by method, and to time on study treatment. In this preliminary analysis, the endpoint is time on study treatment (to progression/toxicity, TST). Results: Safety and efficacy data from arms 1 and 2 have been presented (Carey et al; SABCS 2007, ASCO 2008). Of 102 TN pts enrolled in this study, 65 had CTC measurements by both methods on at least one time point. CTC levels by the two assays were highly correlated at all time points. At baseline, Spearman's rank correlation coefficient was 0.67 (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6048.

Abstract #25 Background: Breast cancer lacking expression of the estrogen and progesterone receptor and overexpression of HER2/neu (ie, "triple-negative” disease) accounts for about 10-15% of all breast cancer and is characterized by a higher risk of recurrence, early recurrence, resistance to cytotoxic therapy, and lack of any specific targeted therapy. Methods: We extracted RNA from primary tumor samples of 246 patients with stage I-III triple-negative breast cancer (confirmed in a central lab) treated with 4 cycles of adjuvant doxorubicin (60 mg/m2) plus cyclophosphamide (600 mg/m2) or docetaxel (60 mg/m2) who were enrolled on trial E2197, and correlated RNA expression (by quantitative RT-PCR using a panel of 371 rationally selected genes) with recurrence. There was no difference in recurrence between the two treatment arms in the entire study population, nor in the 246 patients in this analysis (of whom 59 recurred) after a median followup of 76 months. Results: Higher expression of GRB7 was the only gene significantly associated with an increased risk of recurrence (nominal p value 0.0000853, Korn's adjusted p value controlling false discovery at 10% (KP10) p=0.0359), but did not correlate with any clinicopathologic features except age (low expression associated with age > 65 years, p=0.03). In a Cox proportional hazards model adjusted for age, nodal status, tumor size, and grade, higher GRB7 expression was associated with an increased risk of recurrence when evaluated as a continuous variable (hazard ratio 3.41; p = 0.001) or as a dichotomous variable (hazard ratio 2.24 above vs. below median; p=0.006). The 5-year recurrence rates were 10.5% (95% C.I.7.8%, 14.1%) in the low and 20.4% (95% C.I. 16.5%, 25.0%) in the high GRB7 groups. There were only six genes whose expression correlated with GRB7 (r> 0.4), including ERBB2 (r=0.70), DDR1 (discoidin domain receptor tyrosine kinase 1; r=0.53), KRT19 (keratin 19; r=0.49), ERBB3 (r=0.48), GPR56 (G protein-coupled receptor 56; r=0.48) and PHB (prohibitin; r=0.42). Conclusions: GRB7 is a calmodulin-binding protein which has an SH2 (Src homology 2) domain that binds to phosphorylated tyrosine residues and other specific protein targets, and which plays a critical role in signaling (EGFR, HER2), motility (eprhins), migration (focal adhesion kinase), and cell-matrix/cell-cell interactions (integrins). Higher GRB7 RNA expression is associated with a significantly higher risk of recurrence in triple-negative breast cancer, indicating that GRB7 or GRB7-dependent pathways are potential therapeutic targets in triple-negative disease. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 25.

There are several established prognostic factors for women who have early breast cancer. These include extent of axillary lymph node involvement and tumor size, histology, and steroid receptor studies. Although clinically quite useful, these factors unfortunately are not perfect predictors of individual outcome. This review summarizes recent advances in the field of breast cancer biology that may ultimately enhance our ability to predict more accurately the prognosis for established breast cancer, select therapy more appropriately, and perhaps even identify prospectively women who are at high risk for breast cancer development.

Since the discovery of the aflatoxins in 1960 there has been increasing concern about the possible health hazards engendered by naturally-occurring toxic, and possibly carcinogenic, compounds in foods by molds. This concern arises from the extreme potency of aflatoxins, particularly aflatoxin B1 [AFB1], as toxins and hepatocarcinogens in animals coupled with their wide distribution in the human food supply (1). Furthermore, primary hepatocellular carcinoma is one of the most lethal and common cancers in Africa and Asia and is associated with dietary exposure to AFB1. The extent of aflatoxin contamination in foods and feed products is a function of the ecology of molds and is not completely preventable. Thus, chemoprotective interventions may offer a useful approach for reducing the incidence of human liver cancer in high-risk areas.

B104 Cancer chemoprevention may be achieved through induction of phase II detoxifying enzymes, a process mediated by the antioxidant response element (ARE) in the promoter region of these genes. A key transcription factor involved in induction of phase II enzymes is nuclear factor erythoid-2 related factor (Nrf2). Although cellular function of Nrf2-Keap1 pathway in human carcinogenesis has been extensively investigated, majority of these studies focus on the negative regulator Keap1, which is responsible for cytoplasmic retention of the Keap1-Nrf2 complex, thereby preventing Nrf2-mediated gene transcription. Given the critical role of lysine acetylation in regulating diverse cellular pathways, we use clinically relevant histone deacetylase (HDAC) inhibitors to explore novel mechanisms that govern Nrf2/ARE pathway. By a global proteomic survey, we have learned that the HDAC inhibitor, vorinostat, induces lysine acetylation of 73 proteins in MDA-MB-231 human breast cancer cells. These proteins include chromatin-associated proteins, transcriptional factors, transcriptional regulators, chaperone proteins, DNA repair enzymes, structural proteins and kinase mediators. Among these proteins, SQSTM1 is of particular interest as a recent study has confirmed its critical function in activation of the Nrf2/ARE pathway. Our work shows that vorinostat down-regulates Keap1 protein and elevates protein expression of Nrf2 and NQO1. Induction of Nrf2 by vorinostat is mediated through SQSTM1 as depletion of SQSTM1 by siRNA diminishes the effect of vorinostat on Nrf2 and NQO1. These findings suggest that therapeutic HDAC inhibitors may be found to be useful as chemopreventive agents for breast cancer and support work to elucidate the roles of lysine acetylation in the Nrf2/ARE dependent gene transcription as an underlying mechanism. Supported by FAMRI, Department of Defense Era of Hope, and NIH CA88843. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B104.

557 Background: Although E2197 showed no difference in recurrence or overall survival, an exploratory analysis suggested differential benefit for AC in PR-pos disease and AT in PR-neg disease (Proc...

11521 Background: The addition of trastuzumab to adjuvant chemotherapy has markedly improved survival outcomes of women with HER2+ breast cancer. We hypothesized that the addition of trastuzumab to the taxane portion of preoperative AC-T chemotherapy will be associated with a significantly improved pCR, a surrogate marker for long-term survival. Methods: We conducted a retrospective review of treatment outcomes of women with stage II or III HER2+ breast cancer treated with preoperative sequential AC-T or AC-TH. We compared patient characteristics and pCR of the treatment groups using Fisher’s exact tests. Results: We identified 37 women who received preoperative therapy for HER2+ breast cancer from May 2002 to January 2007. Of those, 14 women received AC-T and 17 received AC-TH (Table). Six women who did not receive sequential anthracycline and taxane based therapy were not included in the analysis. pCR was observed in 3 (21%) women in the AC-T group and in 11 (65%) AC-TH treated women (P=0.029). Trastuzu...

3009 Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines have shown promise, but their activity is limited by immune tolerance. We tested the hypothesis that integrating vaccination with cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine activity. Methods: We conducted a 3-by-3 factorial dose-ranging study of CY and DOX with an allogeneic, HER-2+ GM-CSF-secreting breast tumor vaccine in patients with stable metastatic breast cancer. CY was given at doses of 200, 250, and 350 mg/m2, and DOX at 15, 25, and 35 mg/m2. Subjects received three monthly immunizations, and a boost 6 to 8 months later. CY was given Day -1, vaccination Day 0, and DOX Day +7. Vaccine-induced HER-2-specific immunity was measured by delayed type hypersensitivity (DTH) to the HER-2-derived MHC Class II peptide epitopes p369 and p776. HER-2-specific antibody was measured by ELISA. Results: Twenty-seven individuals received at least 1 immunization with vaccine alone (6 subjects), or ...

529 Background: sHER2 is a circulating oncoprotein cleaved from full-length, membrane-bound HER2. Previous data support that increased sHER concentration is an indicator of poor prognosis and predictor of poor response to chemotherapeutic and hormone treatment in metastatic breast cancer. The role of sHER2 level on the outcome of patients in the adjuvant setting has not been defined, which we evaluated in the context of N9831. Methods: The objectives were to analyze the association between baseline sHER2 and disease-free survival (DFS) in patients (pts) randomized to Arms A (standard chemotherapy) and C (standard chemotherapy with concurrent trastuzumab) of N9831. Baseline serum samples of 1,423 pts from both arms were analyzed for sHER2 using the ADVIA Centaur HER2 assay (Siemens Diagnostics; Tarrytown, NY). Cut off value of 15 ng/mL was used as recommended by the manufacturer/FDA. Patients who cancelled prior to receiving therapy, ineligible pts, and pts whose HER2 status was not corroborated by central...

1036 Background: E2100 was an open-label, multi-center, randomized, Phase III trial comparing P to P+B as initial chemotherapy for MBC. The original analysis based on investigator-assessed progress...

22009 Background: Laboratory assessment of HER2 is of marked clinical importance. Accurate quantification remains problematic within and between laboratories. Here we report central laboratory HER2 results comparing IHC and quantitative RT-PCR using Oncotype DX in patients enrolled in a large adjuvant breast cancer trial. Methods: 755 patients with 0 to 3 positive lymph nodes from Intergroup study E2197 were studied. Central IHC for HER2 was performed using duplicate 1.0 mm microarrays (HercepTest; Dako). Percent positive cells and staining intensity (0–3+) was assessed, where IHC positive cases exhibited 3+ staining in >30% cells, IHC equivocal cases exhibited 3+ staining in 10.7

512 Background: The joint efficacy analysis of NCCTG N9831 and NSABP B-31 demonstrated improved outcomes with the addition of trastuzumab (H) to doxorubicin and cyclophosphamide (AC) followed by paclitaxel (T) in women with surgically removed HER2- positive breast cancer (NEJM 2005). Following this report, patients randomized to AC→T and less than 6 months from completion of chemotherapy were eligible to receive H. We have updated the initial combined results of these two trials. Methods: Primary and secondary efficacy endpoint were disease free survival (DFS) and overall survival (OS). Cox modeling was performed. Hazard ratios are adjusted for nodal status, tumor size, T schedule, hormone receptor status, and trial (also age for OS). Results: Among the 3,969 women enrolled (ages: 22 to 80yrs), there have been 619 events (H group: 222, non-H: 397). First events were: recurrence (511), contralateral breast disease (18), other second primary cancers (48), and death without recurrence or second primary cancers (42). The median follow-up among the 3,711 women still alive is 2.9 years (range up to 6,4 years). The 4 yr DFS rate and 4 yr OS rate respectively were: 85.9% (95%CI: 84.0–87.8%) and 92.6% (95%CI: 91.2–94.2%) in H group and 73.1% (95%CI: 70.6–75.8%) and 89.4% (95%CI: 87.6–91.2%) in non- H group. Hazard ratio for adjuvant (H/non-H) was 0.49 (P [Table: see text]

516 Background: Evidence suggests that docetaxel is more effective than paclitaxel, and paclitaxel is more effective when given weekly than every 3 weeks in metastatic breast cancer (BC). Methods: Eligibility included axillary lymph node positive or high-risk (tumor at least 2 cm) node-negative BC. All patients received 4 cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) every 3 weeks, followed by either: (1) paclitaxel 175 mg/m2 every 3 weeks × 4 (P3), (2) paclitaxel 80 mg/m2 weekly × 12 (P1), (3) docetaxel 100 mg/m2 every 3 weeks × 4 (D3), or (4) docetaxel 35 mg/m2 weekly × 12 (D1). The primary comparisons included taxane (P vs. D) and schedule (every 3 weeks vs. weekly), and secondary comparisons included P3 vs. other arms. The trial had 86% power to detect a 17.5% decrease in disease-free survival (DFS) for either primary comparison, and 80% power to detect a 22% decrease for the secondary comparisons (2-sided nomimal 5% level tests corrected for multiple comparisons). Results: A total of 4,950 eligible patients were accrued. There was no difference in the primary comparisons afer 856 DFS events and 483 deaths after a median follow-up of 46.5 months at the 4th interim analysis ( www.sabcs.org , abstract 48). This is the final pre-specified analysis for the primary comparisons after 1,042 DFS events and 650 deaths (with 1,020 DFS events at this time, to be updated at the meeting). After a median followup of 60.2 months, there remains no significant difference in the hazard ratio (HR) for the taxane (1.02; p=0.73) or schedule (1.07; p=0.30) (as in the first analysis). In secondary comparisons of the standard arm (P3) with the other arms (HR > 1 favoring the experimental arms), the HRs were 1.30 (p = 0.003) for arm P1, 1.24 (p=0.02) for arm D3, and 1.09 (p=0.33) for arm D1. Analysis of interaction by hormone-receptor status will be presented. The incidence of worst grade toxicity (grade 3/4) was 24%/6% for arm P3, 24%/3% for arm P1, 21%/50% for arm D3, and 38%/6% for arm D1. Conclusions: There were no differences in DFS when comparing taxane or schedule overall. DFS was significantly improved in the weekly paclitaxel and every 3-week docetaxel arms compared with the every 3-week paclitaxel arm. [Table: see text]

21022 Background: Central and local laboratory concordance for hormone and HER2 receptor measurement is of national interest. This study compares ER/PR/HER2 by local laboratories using immunohistochemistry (IHC) and central laboratories (IHC & quantitative RT-PCR). Methods: Of 2952 patients in E2197, a case-cohort sample of 776 patients who either did (N=179) or did not recur was studied. Central IHC for ER/PR/HER2 was performed using single 0.6 mm microarrays; Allred score (AS) was used for ER/PR (AS>2 = positive). Positive HER2 was 3+ staining in >10% cells for Central IHC and 2+ or 3+ for Local IHC. RT-PCR analysis by Oncotype DX™ for ER/PR/HER2 was performed using pre-defined cutoffs of 6.5, 5.5 and 11.5 units, respectively. Hormone receptor (HR) pos was defined as ER &/or PR pos. Results: Results from Local IHC (ER/PR in 776 & HER2 in 517 pts) were compared with Central IHC (760 pts) and RT-PCR results (776 pts). The discordance between HR positivity by Local IHC and RT-PCR was very low. However, 12% of HR neg pts by Local IHC (38/321) & Central IHC (39/326) were HR pos by RT-PCR. The relationship between ER and recurrence as a function of AS was examined. Patients with AS of 3–4 were found to be closer to the AS=2 group than to the AS>4 group Patients with AS of 3–4 were found to be closer to the AS ÿ 2 group than to the AS > 4 group (Est.HR for ER 0.97 for AS 3–4 vs. 0–2 and 0.46 for AS 5–8 vs. 0–2, and for PR were 0.84 for AS 3–4 vs. 0–2 and 0.41 for AS 5–8 vs. 0–2). Conclusions: There is a high degree of overall concordance among Local IHC, Central IHC, and Central RT-PCR for ER and PR. The degree of concordance is even greater for HR compared to ER or PR alone. Although the concordance with local labs for HER2 testing was poor, the concordance between Central IHC and RT-PCR was very high. The relatively high incidence (12%) of IHC HR neg pts who are HR pos by RT- PCR is notable. [Table: see text] [Table: see text]

579 Background: N9831 and other clinical trials have demonstrated the additive survival benefit of trastuzumab (H) therapy with chemotherapy for patients with resected HER-2 positive breast cancer. Cardiac toxicity (CTox) is increased by this strategy. Prediction of or early detection of CTox is important. Methods: 95 patients provided informed consent. Brain natriuretic peptide (BNP), C-reactive protein (CRP), troponin T (TnT) and I (TnI), TNF-alpha, IL-1b, and IL-6 were to be drawn at baseline, after initiation of doxorubicin/cyclophosphamide (AC), after initiation of paclitaxel (T), and after the initiation of H (may have been given concurrently with T). 67 patients with at least a baseline sample, a post-treatment sample, and a baseline and subsequent evaluation of LVEF were studied. No patient in this group had a grade 3 decline in LVEF. Only 3 of these patients did not receive H. Values above the 99th percentile were considered abnormal for troponin. 40 ng/ml was considered elevated for BNP. Results: Values of CRP, TNF-alpha, IL- 6 and IL1b changed in very few patients and without a discernible pattern. For further evaluation of BNP, TnI, and TnT, 2 definitions of CTox were tested: 1. a >10% drop in LVEF from baseline (n=27) and 2. a drop in LVEF of 10–15% to less than the institutional lower limit of normal (LLN) OR a >15% decline in LVEF (n=14). No pre-treatment marker appeared to predict a > 10% decline in LVEF (definition 1). For patients fulfilling definition 2, pre-treatment BNP > 40 was seen in 21% of patients vs 8% of normals (nls) (p= 0.18) and an abnormal TnI (>0.04) was seen in 21% vs 6% of nls (p=0.10). Only doubling of BNP appeared promising as a serial marker of CTox using definition 2 (27% vs 7% for nls (p = 0.09). Conclusions: Baseline BNP and TnI and serial measures of BNP may be promising for further investigation for the prediction and detection of treatment-related CTox. Due to limitations of the study including the small numbers of patients analyzed, the difficulty in defining CTox, and the variation in the timing of sample procurement and assessment, these data are considered exploratory. Supported by CA-25224, CA-2115, CA 38926, CA31946. No significant financial relationships to disclose.

1033 Background: Activation of EGF receptor has been associated with resistance to trastuzumab in breast cancer cell lines. EGFR tyrosine kinase inhibitors inhibit HER2 phosphorylation and synergize with trastuzumab in HER2+ cell lines that co-express EGFR. Methods: Pts with MBC and HER2 overexpression by immunohistochemistry (3+) and/or HER2 gene-amplification by FISH, 0–2 prior chemotherapy regimens for met disease, LVEF 50%, and no prior trastuzumab were treated with trastuzumab 2 mg/kg/wk and gefitinib 250- 500 mg/day until disease progression, unacceptable toxicity or withdrawal of consent. The phase I portion of the trial used a 3+3 design to determine MTD. In the phase II portion of the trial, patients were stratified based upon prior chemotherapy exposure (Group 1= no prior exposure to chemotherapy, Group 2= prior exposure to 1–2 chemotherapy regimens). Response measured using RECIST criteria. The primary endpoint was to increase proportion progression free from 50 to 65% at 6 months in Group 1 and from 50 to 70% at 3 months in Group 2. Results: Phase I: DLT (Grade 3 diarrhea) occurred in 2/3 patients treated at the 500 mg/day dose level of gefitinib in combination with weekly trastuzumab. 0/3 patients treated at the 250 mg/day dose level experienced DLT. This was considered MTD and was the dose selected for the Phase II portion of the trial. Phase II: 36 eligible pts were enrolled. Most patients were ECOG PS of 0 and had visceral organ involvement. Of the patients enrolled in Group 1, one pt achieved a CR, one PR and 7 had SD (≥ 24 weeks). Median time to progression (TTP) was 2.9 months (95% CI, 2.5–4). In Group 2 no responses were observed with a median TTP of 2.5 months (95% CI, 1.5- 2.7). Most common severe toxicities were rash (grade 3, 14%) and diarrhea (grade 3, 30%). No grade 3 cardiac toxicity was encountered. Conclusions: Trastuzumab in combination with gefitinib at doses of 250 mg/day demonstrated an acceptable toxicity profile; however, during planned interim analysis, the TTP did not meet predetermined statistical endpoints required for study continuation. These results do not support the further use of this combination and have implications for other trials using trastuzumab and EGFR TK inhibitors simultaneously. [Table: see text]

New clinical reports demonstrate that targeting the biological pathways of hormone and peptide growth factor receptors holds promise for the prevention and treatment of breast cancer.

Breast cancer incidence has risen steadily over most of the last 50 years. Multiple explanations have been invoked for this rise and include increased mammographic screening, an aging population, and changes in the prevalence of accepted risk factors associated with prolonged exposure to unopposed estrogen (e.g., delayed childbearing and earlier menarche). However, no explanation is more contentious than the putative role of weakly estrogenic environmental contaminants such as the pesticide DDT and the industrial products polychlorinated biphenyls (PCBs). Although widespread use of both types of compounds effectively ceased in the United States during the 1970s, their ability to accumulate and to persist in adipose tissues has raised fears about their contribution to breast cancer development in recent years. This has not been an easy area to study. One need only recall two conflicting reports (1,2) on the association between breast cancer risk and blood levels of organochlorines published in this Journal. Wolff et al. (1) initially reported higher levels of the DDT metabolite DDE but not of PCBs in serum specimens taken from women around the time of breast cancer diagnosis than in serum specimens taken from matched, unaffected control subjects, whereas Krieger et al. (2) were unable to document any relationship between DDE or PCB levels in serum specimens obtained some years before breast cancer diagnosis and subsequent breast cancer development. A 1994 meta-analysis of available data (3) appears to confirm the lack of an association. However, the small number of women (fewer than 750) in the aggregated studies, the uncertain value of serum organochlorine levels as a surrogate marker for tissue levels, and the unanswered question of the appropriate interval between serum or tissue sampling and breast cancer development leave this an open area of research (3). Similar controversies abound in the laboratory. A report (4) that combinations of environmental chemicals, including PCBs, can synergistically activate the human estrogen receptor in a yeast reporter system could not be confirmed (5) and was recently withdrawn (6). The relevance of some findings in rodent systems to humans was challenged when species differences were documented in the ability of certain DDT isomers and metabolites to bind competitively to the estrogen receptor (7). In this issue of the Journal, Shekhar et al. (8) enter the fray with an analysis of the ability of members of the DDT and PCB families, alone and in combination, to affect estrogen receptormediated events in preneoplastic MCF10AT and malignant MCF-7 human mammary epithelial cells. They suggest that both o,p8-DDT and p,p8-DDT can stimulate cell growth and can increase expression of a reporter construct containing an estrogen response element as well as of the endogenous estrogenresponsive pS2 gene. Shekhar et al. found that p,p8-DDT was more effective than o,p8-DDT in eliciting these responses, even though o,p8-DDT was more active in competitive binding assays for the human estrogen receptor. Most importantly, the potential for interaction between different organochlorines was suggested by their studies showing that equimolar mixtures of the two DDT isomers were more effective than either isomer used alone at concentrations some 10to 100-fold higher. Blocking studies with the antiestrogen ICI 182,780 as well as negative results using similar reporter assays in estrogen receptor-negative MCF10A cells (the latter data not shown) were said to imply that interaction between organochlorines and the estrogen receptor is a critical event (8). The strength of the analysis by Shekhar et al. (8) lies in its use of a relevant model system—both premalignant and malignant mammary cells of human origin. However, their conclusions about organochlorine regulation of estrogen receptor-mediated cellular responses must be tempered somewhat in the face of the data as presented. First, it is surprising that p,p8-DDT, the more effective DDT isomer in the functional assays, had limited ability to compete with tritiated 17b-estradiol for estrogen receptor binding. It is noteworthy that a separate study by Chen et al. (7) documented that p,p8-DDT and o,p8-DDT bound equally well to recombinant human estrogen receptor and activated to a similar extent a yeast cell human estrogen receptor-reporter system. However, in MCF-7 cells, o,p8-DDT was a more effective inducer than p,p8-DDT of another endogenous estrogenresponsive gene, the progesterone receptor gene. Taken together, these findings raise the possibility that the effects of organochlorines are mediated, at least in part, through pathways other than the estrogen receptor pathway. Second, the strategy of using the pure antiestrogen ICI 182,780 at 100-fold molar excess could be flawed; it seems unlikely that this drug at concentrations of 100 mM to 1 mM will function solely as an estrogen receptor antagonist. Rather, nonspecific and unexpected effects

512 Background: AT is one of the most active regimens in ABC (response rates=44–83%). Methods: A Phase III Intergroup trial was completed to test adjuvant AT vs. AC. Women with 1–3 N + or N - and T-size > 1cm were randomized to four cycles of AT (60 mg/m2/60 mg/ m2) or AC (60 mg/m2/600 mg/m2) q 3 wk x 4. After chemotherapy, pts with ER + and/ or PR + received tam for 5 years. Patients were stratified by nodal, hormone receptor (ER+ PR+, ER+PR-, ER-PR+, ER-PR-, ER/PR unk) and menopausal status. The primary endpoint was DFS. With 2778 patients (including 10% ineligible), this study had 83% power to detect a 25% reduction in DFS failure hazard rate using AT(assuming a 78% 5-yr DFS on the AC arm). Results: 2952 pts were randomized between 7/30/98 and 1/21/00. 2889 were eligible and analyzable. Arms were balanced for age, hormone receptor, menopause, nodes, surgery, grade and T-size: median age 51; 64% ER +; 65% LN-; grade: 10% low, 38% int., 46% high; and median T-size - 2.0 cm. Febrile neutropenia occurred i...

556 Background: NCCTG N9831 is a 3-arm randomized Phase III trial comparing safety and efficacy of AC followed by weekly paclitaxel (T) (Arm A) vs the same chemotherapy with 1 yr of trastuzumab (H) either after (Arm B) or starting with T (Arm C). Clinical and asymptomatic cardiac tolerability is the focus of this abstract. Methods: Women with normal left ventricular ejection fraction (LVEF) assessed by MUGA or echocardiogram scan were eligible. LVEF evaluation was repeated post-AC, 6, 9 and 18 months post entry. Clinical cardiac evaluation occurred as a continuum. A Cardiac Safety DMC reviewed asymptomatic and symptomatic LVEF changes monthly. Interim analyses were planned after 100, 300, and 500 evaluable pts per arm began post-AC therapy and were followed for additional 6 months. Primary endpoint was proportion of cardiovascular (CV) events in each arm (congestive heart failure (CHF) and definite or probable cardiac deaths). A difference of 4% would trigger suspension or closure of the H-containing stud...

568 Background: Accurately and reliably identifying HER2/neu status is a critical priority in the treatment decision process. Current HER2 testing strategies include immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), but discrepancies between them (3+IHC and FISH-, 0–2+ IHC and FISH+) interfere with interpretation and patient management. We postulate that some of these discrepancies may reflect tumor heterogeneity, derived from focal HER2 amplified clones (FHAC) within a tumor. Methods: As part of the ongoing N9831 “Phase III trial of doxorubicin and cyclophosphamide followed by weekly paclitaxel with or without trastuzumab treatment for women with HER2 overexpressing or amplified node positive or high–risk node negative breast cancer” adjuvant Intergroup trial, breast tumors were evaluated for HER2 status by both IHC and FISH using HercepTest™ and PathVysion™ assays. Samples with FHAC were defined as containing 2% to 40% of cells demonstrating unequivocal amplification (cells with ...

Thanks to many public health advances, in the developed countries the average woman will be postmenopausal for about one third of her life. As a consequence, she and her physician will ultimately need to make a decision about postmenopausal estrogen-replacement therapy. This treatment has been used for several decades, but there is a surprising lack of consensus about when and how to use it. The initial goal of estrogen-replacement therapy was to alleviate menopausal symptoms. More recently, the possibility has emerged that estrogen can reduce morbidity and mortality from ischemic heart disease and osteoporosis. However, women and their physicians must . . .