Citations

Abstract Bisphosphonates reduce the incidence of skeletal-related events (fractures, pain, hypercalcemia) in patients (pts) with bone metastases from breast cancer. By inhibiting osteoclast function and subsequent bone turnover they may inhibit the growth of bone (and other) metastases. Their role in preventing or delaying the development of bone (or other) metastases in pts with early breast cancer is uncertain. Three previous trials of oral clodronate have given mixed results. The largest trial (placebo-controlled) and a smaller open-label trial suggested that oral clodronate benefitted pts with improved bone metastases-free survival (BMFS) and overall survival (OS), but a third open-label study showed no benefit with an apparent detrimental effect on survival. Studies of IV zoledronate in open-label trials in early breast cancer have also shown mixed results: the AZURE trial in pts with node positive breast cancer showed no benefit in disease-free survival (DFS) or OS with a possible effect in pts aged over 60. The ABCSG-12 trial in GnRH (plus tamoxifen or anastrozole) treated women with early breast cancer showed a small DFS benefit for those receiving IV zoledronate but no BMFS or OS benefit. Methods: B-34 is a prospective, randomized, double-blind, phase III clinical trial in pts with stage 1, 2 or 3 breast cancer assessing oral clodronate 1600mg daily for 3 years compared to placebo given alone or in addition to adjuvant chemo- or hormone therapy. Stratification is by age (<50, +50), number of positive nodes (0, 1–3, 4+) and ER/PR status. The primary end-point is DFS. Secondary endpoints are the incidence of skeletal metastases, OS, relapse-free survival, incidence of non-skeletal metastases, and incidence of skeletal morbid events. Results: 3323 patients were accrued — 1662 in Group A and 1661 in Group B. As of March 31, 2011, 54 pts were declared ineligible: 23 in Group A and 31 in Group B. The average time on study is 101.3 months. Patient characteristics were evenly distributed throughout both groups. Approximately 75% of pts in each group had negative axillary lymph nodes and some 78% in each group were ER and/or PR positive. About 64% of pts were age 50 or over. Oral clodronate was generally tolerable, toxicities observed being mainly due to the concomitant systemic chemotherapy. One pt in Group A had a 1 mm area of osteonecrosis on the palatal taurus. Compliance, as expected with trials of oral medications, has been a problem. 1910 pts completed 3 years of therapy. Event rates were slower than anticipated. This final analysis assesses a total of 598 events in the two arms. Conclusions: This trial is the largest placebo-controlled study of an oral bisphosphonate in patients with early breast cancer and will provide further information on the role of bisphosphonates in breast cancer management. Supported for NSABP by NCI U10CA12027, −37377, 69651, 69974; and Bayer Schering Pharma Oy; for ECOG by U10CA021115; for NCCTG by U10CA25224; for SWOG by U10CA38926. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-3.

Abstract Background: Unstained recuts from formalin-fixed paraffin-embedded tissues are commonly collected for cooperative group studies. There is concern among pathologists that improper storage conditions can lead to antigen degradation. In an effort to quantify this effect, we compared the expression of HER1 and HER2 on two sets of identical cohort tissue microarrays (TMAs) from the N9831 HER2+ adjuvant phase III trial (NCT00005970; www.clinicaltrials.gov); one freshly cut set (cut April 18, 2011) and a second set stored at 4 degrees for over two years (cut between Nov, 2007 and Jan, 2008). Methods: The two sets of TMA slides containing 1580 tumor samples from the N9831 cohort were treated identically using the AQUA method of quantitative immunofluorescence. HER1 was tested with D38B1 (rabbit monoclonal, Cell Signaling Technology, Inc.) and HER2 with CB11 (mouse monoclonal, Biocare, Inc.) on tumors from 695 patients (712 specimens) in the fresh TMAs and 779 patients (800 specimens) in the old TMAs in up to three-fold redundancy per specimen. Results: Frequency distributions of the expression of HER2 revealed bimodality in the fresh TMAs compared to an attenuated distribution of the old cases. The average score of the entire cohort was significantly lower in old TMAs compared to fresh cuts (paired t-test, p<0.0001). Linear regression of the average HER2 scores from new TMAs versus the average scores from old TMAs showed a slope term of 0.52, which is statistically significantly different from the hypothetical value of 1 (p<0.0001). Regressions between any two fresh slides showed slopes close to 1.0. Similar results were seen for HER1, but fewer positive cases made the changes less dramatic. Conclusions: The storage condition of tissue slides is a critical pre-analytical variable that can dramatically lower the score of HER1 and HER2, artificially. Thus, studies done on inadequately stored slides, either whole sections or TMAs, must be interpreted with caution. Tissue collection and analysis of biomarkers for cooperative group studies should not include unstained recuts, but rather, entire blocks or large cores from tissue blocks. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-04.

Abstract Background: Prediction of benefit from trastuzumab in patients (pts) with HER2+ breast cancer remains an important goal. We sought to investigate the predictive value of quantitative measurement of HER2, HER3, HER4, EGFR, ER and PTEN protein expression on the benefit of trastuzumab in the phase III HER2+ adjuvant N9831 study for pts randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C). Methods: For each marker, we evaluated quantitative expression, relationship with demographic data, and association with disease-free survival (DFS) of pts. Freshly cut tissue microarray slides with up to three-fold redundancy per specimen from the N9831 cohort were treated identically using the AQUA (Camp, et al; Nat Med 2002, JCO 2008) method of quantitative immunofluorescence for each marker. HER2 was tested with CB11 (mouse monoclonal, Biocare, Inc.) and preliminary results were available for 698 of nearly 1400 pt specimens to be tested. The minimum value per pt was used in statistical analysis. Specimens were classified with high versus low expression based on a median value cutpoint for each marker. Median follow-up was 7.0 yrs. Results: Quantitative HER2 was compared with centrally performed HER2 testing by IHC and FISH. Median quantitative HER2 via AQUA was 10,017 units for the HER2 IHC 3+ group (n=607) versus 1058, 831, and 970 for the HER2 IHC 2+ (n=68), 1+ (n=11), and 0 (n=11) groups, respectively. The Spearman correlation between quantitative HER2 and FISH HER2/CEP17 ratio was 0.32 (p<0.001). High quantitative HER2 was associated with lower percentage of hormone receptor positivity (48% vs 59%, chi-sq p=0.003) but not associated with age, race, nodal positivity, tumor histology, grade, or size. High HER2 did not impact DFS in any arm of the study (See Table). Data for additional HER2 testing, HER3, HER4, EGFR, ER and PTEN are in process and will be ready by September, 2011. Conclusions: Similar to results based on standard HER2 testing by IHC and FISH in N9831, quantitative HER2 did not impact benefit from adjuvant trastuzumab. Results for additional markers will be presented. Our complete quantitative results for a second epitope on HER2, HER3, HER4, ER and EGFR will be the first report of these markers in a large patient cohort in the adjuvant setting. Disease Free Survival Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-03.

Abstract Background: The role of fusion genes and associated fusion transcripts has long been recognized in hematopoietic malignancies. Until quite recently it has been difficult to detect such events on a genomic scale in solid tumors. Consequently, little is known about the potential role of fusion genes, transcripts, and proteins as driver mutations, biomarkers, or therapeutic targets in breast cancer. Methods: We have developed a novel analytical pipeline, Snowshoes-FTD, for detection of fusion transcripts in breast cancer cell lines and tumor samples (Asmann, et al. NAR 2011; May 27 ePub ahead of print). Preliminary analyses have been carried out with a panel of 8 each ER+, HER2+, and triple negative (TN) primary breast tumors, 8 primary human mammary epithelial cell (HMEC) lines from biopsy samples, plus 16 normal tissues from the Illumina Body Map dataset. Results: We have identified 120 redundant, tumor-specific fusion transcripts, expressed in two or more tumors and in no non-transformed samples. Sixteen of these represent intrachromosomal fusions and 104 arise from fusion of transcripts that map to two different chromosomes. Every breast tumor expressed one or more fusion transcripts. Twenty-nine fusion transcripts appeared to be tumor subtype specific. Among these, we have identified 2 HER2+, 10 ER+, and 17 triple negative specific redundant transcripts. In general, HER2+ tumors expressed fewer fusion transcripts (range 4 to 28/tumor) compared to TN (range11 to 44/tumor). Chromosomal distribution patterns were also markedly different among the tumor subtypes. For example, ER+ tumors expressed a preponderance of redundant fusion transcripts that involve chr1 and 2, whereas TN tumors had no fusion transcripts that map to either chromosome. Conversely, the predominant locus for TN fusion transcripts was chr19, which contains only one HER2+ fusion and no ER+ fusion transcripts. Conclusions: Primary breast tumors express many chimaeric transcripts, which we presume to arise primarily from genomic rearrangements. The majority of these transcripts are redundant, and a subset are tumor subtype specific. These transcripts may mark regions of chromosomal instability. HER2+ tumors, in general, appear to evidence less chromosomal instability, as inferred from fusion transcripts; although some HER2+ tumors appear to be quite unstable. TN tumors contain many more redundant fusion transcripts, implying increased genomic instability, particularly in chr19. We conclude that these fusion transcripts represent a class of heretofore unrecognized biomarkers that may be used for sub-classification of breast tumors. Some of these transcripts appear to encode proteins that may function as tumor-subtype-specific driver mutations and may have potential as therapeutic targets in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-02.

Abstract Background Obesity, as defined by body mass index (BMI), has been associated with increased recurrence rate, shorter DFS and increased death rates due to breast cancer (BC). Most of the studies to date have examined the relationship of BMI and DFS in patients with hormone receptor positive disease. To our knowledge, BMI and its relationship with outcome in early stage HER2 positive breast cancer has not previously been examined. The N9831 is a large phase III trial testing the role of trastuzumab in the adjuvant setting of high risk patients with early stage HER2+ BC. We hypothesized that the occurrence of overweight and obesity may correlate with outcome. Methods: This analysis presents BMI and its relation to tumor characteristics and DFS in patients (pts) enrolled in the N9831 clinical trial. Pts were categorized as normal weight, overweight or obese using the WHO BMI classification parameters of < 25%, 25–29% and ≥ 30% respectively. For patient characteristics, patients were grouped into non-obese (BMI< 30) and obese (≥ 30) cohorts. DFS was estimated by the Kaplan-Meier method. Comparisons between arms A (chemotherapy alone), B (chemotherapy plus sequential trastuzumab) and C (chemotherapy plus concurrent trastuzumab) were performed using the Cox proportional hazards model, stratified by BMI. Results: Analysis was completed on 3,017 eligible pts. Obese pts were more likely to be older and postmenopausal (p<0.0001 for both). There was no significant association between BMI and ER/PR status (p=0.07) or histologic tumor grade (p=0.33). Obese pts were found to have significantly larger tumors ≥ 2 cm (p=0.002) and more positive lymph nodes (p=0.02). There was no significant difference in DFS within each intrinsic arm (A, B and C) between the obese and non-obese pts at 3, 5 or 7 yrs of follow up. However, pts in the non-obese group had significantly improved DFS in arm B and C compared to arm A (p=0.001 and p<0.0001 respectively). Also obese pts in arm C had significantly improved DFS compared to obese pts in arm A (p=0.008). There was a trend of improved DFS in the obese group in arm B compared to arm A, but this was not statistically significant (p=0.09). Pts in the normal weight and overweight groups did significantly better in arm B (p=0.02 for both) and arm C (p=0.01 and p=0.002 respectively) compared to arm A. Conclusions: This analysis of data from the N9831 study confirms that obese pts with early stage HER2+ tumors have worse clinical outcome than pts with BMI < 30%. Adjuvant trastuzumab improved clinical outcome regardless of BMI. This study supports weight loss intervention for obese women with early stage HER2+ BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-02.

Abstract Background: T-DM1, an antibody-drug conjugate composed of trastuzumab, a stable linker, and the cytotoxic agent DM1, is in development for the treatment of HER2−positive cancers. Single-agent T-DM1 3.6 mg/kg every 3 weeks (q3w) has demonstrated clinical activity in 2 phase II studies (TDM4258g and TDM4374g) in patients with pretreated HER2−positive MBC. The efficacy and safety of T-DM1 vs trastuzumab + docetaxel was investigated in patients with no prior MBC treatment in the randomized phase II study TDM4450g/BO21976. Here we report the PK of T-DM1 from that study and compare these data with those from studies that enrolled pretreated patients. Methods: In all 3 studies, PK parameters, including maximum concentration (Cmax), area under the concentration-time curve (AUC), terminal half-life (t½), steady-state volume of distribution (Vss), and clearance (CL) were estimated by noncompartmental analysis (NCA) for serum T-DM1, serum total trastuzumab (conjugated and unconjugated), and plasma DM1. The effects of baseline trastuzumab and HER2 extracellular domain (ECD) concentration on T-DM1 exposure were explored and the relationship between T-DM1 exposure and clinical response (objective response rate [ORR] and progression-free survival [PFS]) was modeled. Results: T-DM1 PK from evaluable patients enrolled in 3 studies are shown in Table 1. No significant correlations were observed between efficacy (as measured by ORR) and T-DM1 exposure (AUC, Cmax) after administration of T-DM1 to pretreated patients; efficacy-exposure analyses (ORR and PFS) for previously untreated patients will be presented. Patients with measurable concentrations of trastuzumab at baseline had a greater AUC during cycle 1; however, this did not impact ORR. Baseline circulating HER2 ECD concentrations also had no effect on ORR for pretreated patients. The impact of baseline trastuzumab and HER2 ECD concentrations on ORR and PFS in previously untreated patients will be presented. Conclusions: Single-agent T-DM1 has similar PK in patients who have received prior therapy for MBC and in those who have not. The PK of T-DM1 was not affected by prior trastuzumab treatment or by circulating HER2 ECD, and no significant correlations were observed between efficacy (ORR) and T-DM1 exposure (AUC, Cmax) or HER2 ECD for pretreated patients. The relationships between efficacy and T-DM1 exposure and HER2 ECD concentrations will be presented for patients without prior MBC treatment. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-13.

Abstract Background Tumors that are negative for estrogen and progesterone receptors and do not over express HER2 are referred as “triple negative” breast cancer (TNBC). These tumors are characterized by unique molecular profiles on gene expression arrays, aggressive behavior with a high recurrence rate, an increased risk of visceral metastases, poor prognosis and lack of targeted therapies. Brostallicin is a novel synthetic compound from the class of DNA minor groove binding (MGB) anti-cancer agents. It retains activity in cancer cells resistant to alkylating agents, topoisomerase I inhibitors and is fully active against DNA-mismatch repair deficient tumor cells. Cells expressing relatively high glutathione/glutathione S-transferase (GSH/GST) levels are more susceptible to brostallicin antitumor efficacy. Cisplatin administration increases expression of GST in tumor cells leading to an increased anti-tumor efficacy of brostallicin. Trial design: Single-stage phase II study — based on the effects of cisplatin on GSH/GST levels in preclinical models, the most reasonable sequence to explore was cisplatin on Day 1 followed by brostallicin on Day 2 repeated every 21 days. Eligibility criteria: Women or men ≥18 years of age with confirmed adenocarcinoma of the breast with clinical evidence of measurable metastatic disease and triple negative subtype according to current ASCO CAP guidelines [ER/PR ≤1%; HER2 negative), who received 0–4 prior chemotherapy regimens in the metastatic setting; with adequate hematologic, renal and hepatic functions; and no active CNS metastases. Aims: To study the efficacy of the novel drug, brostallicin, as well as to serve as proof of concept of its mechanism of action in TNBC. The primary endpoint is to evaluate clinical efficacy of the combination of brostallicin and cisplatin in the treatment of patients with metastatic TNBC, as measured by progression-free survival (PFS) at 3 months with 89% power (0.10 significance level) to detect an absolute difference of 20%. Secondary endpoints include ORR by RECIST, duration of response, 6-month PFS, overall survival (OS) and adverse event profile. Tertiary endpoints include assessment of 1) GSH levels prior to the administration of cisplatin and of brostallicin; and 2) the prevalence of BCRA-1 mutation by IHC in the primary or metastatic tumor. Statistical methods: The largest 3-month PFS proportion where the proposed treatment regimen would be considered ineffective in this population was estimated at 35% based on the median PFS of 60 days in patients with metastatic TNBC enrolled in the N0234 trial (erlotinib and gemcitabine as 1st/2nd line), and the smallest 3-month PFS success proportion that may warrant subsequent studies with the proposed regimen in this patient population was estimated at 55%. The interim analysis will be reported when the 20th eligible patient has been followed for 3 months. Present accrual and target accrual: 21 patients have been accrued at the time of abstract submission (June 2011). Target accrual is 42 evaluable patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-14.

Abstract Background NKTR-102 is a next-generation topoisomerase I inhibitor-polymer conjugate with a markedly reduced Cmax and a continuous exposure profile compared to irinotecan. A phase 2 trial of single-agent NKTR-102 compared a dose of 145 mg/m2 every 2w or every 3w in 3rd-line MBC (Awada et al, ASCO 2011). Overall the ORR was 29% (including 3% CR) with the prior ATC subset demonstrating an ORR of 31%. Dosing every 3w was better tolerated; in this arm, median PFS equaled 5.3m and median OS equaled 13.1m. Trial Design: NKTR-102 will be compared to TPC in an open-label, randomized, parallel, two arm multicenter Phase 3 pivotal study in pts with previously treated locally recurrent or metastatic breast cancer. Key Entry Criteria: Adult females, with ECOG 0 or 1 with adequate liver, kidney and marrow function. All patients must have received prior therapy with an anthracycline (in neo/adjuvant or metastatic setting or both), a taxane (in neo/adjuvant or metastatic setting or both) and capecitabine (in neo/adjuvant or locally advanced/metastatic setting or both) unless not medically appropriate or explicitly contraindicated for the patient. All chemotherapy- and radiation-related toxicities must have resolved to ≤ Grade 1, except for stable sensory neuropathy ≤ Grade 2 and alopecia. Pts with brain metastases may be eligible, if stable for prior 4 weeks without steroids. Pts with Grade ≥ 2 pre-existing diarrhea or receiving chronic anti-diarrheal supportive care are not eligible. Statistical Methods: The primary efficacy endpoint is OS. Secondary endpoints include ORR by RECIST v1.1 and PFS. Pts will be randomized 1:1 to NKTR-102 given IV at 145 mg/m2 over 90-min every 21 days or TPC. Patients randomized to TPC will receive single agent chemotherapy, limited to choice of one of the following 5 agents: eribulin, ixabepilone, vinorelbine, gemcitabine, or a taxane. The investigator must decide which agent will be given to the patient prior to randomization. Pts will be stratified by geographical region and disease characteristics. Target Accrual: Approximately 840 patients (420 pts per treatment arm) will be randomized in order to obtain 615 deaths (alpha = 0.05; power 90% with one interim analysis scheduled when 50% of the deaths have occurred). PK sampling will be performed in a subset of pts and blood and/or tumor samples are planned for potential predictive markers of response and toxicity. Enrollment is expected to remain open until the end of 2013. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-07.

Abstract Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p<0.01). Imputation using HapMap2 data was performed within 200 kb on either side of regions containing SNPs with p<E-05. Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p<E-06 were observed in this same region, with the lowest p value for rs9773022 (p=8.1E-08). We then performed functional genomic studies, and observed that two of the SNPs with low p values (rs4736312, p=1.49E-05 and rs4736350, p=6.34E-06) created apparent Oct-1 transcription factor binding sites (TFBS). Differential nuclear protein binding of Oct-1 to probes for these SNPs was verified with EMS and supershift assays using nuclear extract from A549 and NCI-H295R cells. Treatment of these two cell lines with 10 nM androstenedione led to the disruption of Oct-1 binding with the A (wild type) allele on rs4736312. rs4736312 is located in the 3'-UTR of CYP11B1 15 bp distant from response elements for the progesterone receptor and the glucocorticoid receptor. We also demonstrated that CYP11B1 and CYP11B2 were induced by estradiol in U2OS cell lines that express ER receptor and by androstenedione in H295R cells that express the androgen receptor. Transfection of a reporter gene construct with the rs4736312 SNP sequences into COS-1 and H295R cells suppressed reporter gene activity for the variant SNP sequence as compared to wild type, an observation that may correlate with the higher plasma levels of androstenedione in women (549× 328 vs 410×208 mean×SD, M/ml) with the variant allele. Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.

Abstract —Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca 2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca 2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca 2+ homeostasis.