bluuebunny/crossref_metadata_2025 huggingface dataset:
Survival of patients with HER2+ metastatic breast cancer and use of trastuzumab following progression: analysis of RegistHER.
Authors: Rugo H , Kaufman P , Tan-Chiu E , Ulcickas Yood M , Paik S , Yardley D , Brufsky A , Mayer M , Birkner M , Wang L , Brammer M , Tripathy D
Published: December 2010

Abstract #3142

Background: Most HER2+ metastatic breast cancer (MBC) patients who initially respond to trastuzumab (T)-based therapies will experience disease progression (PD). Standard practice is to discontinue the cytotoxic agent on which disease progressed, however there is evidence that tumors may remain sensitive to HER2 targeting and that there may be a potentiating effect of T on chemotherapy. We examined the treatment history of a large population of HER2+ MBC patients to assess outcomes associated with continuation of T beyond PD.  Methods: This analysis was carried out on patients enrolled in registHER, a prospective observational study of 1023 patients with newly diagnosed (within 6 months) HER2+ MBC. Median f/u from MBC diagnosis is 25 months at the time of data cut-off (12/31/07). For those treated with T prior to first PD, we compared demographics and baseline tumor characteristics for those patients treated with or without T following first PD. Treatment with T was defined as a minimum of 21 days of therapy prior to PD. In addition, we evaluated time to second PD, and overall survival post PD (both endpoints calculated from initial PD). Overall survival (OS) was also calculated from the date of treatment initiation to death for the entire treated cohort.  Results: Of 1023 evaluable patients, 873 (85%) were treated with any T-based first-line therapy. 622 T-treated patients progressed and 500 were subsequently treated with T following first PD. Patients who received T post first PD tended to be younger (<50 years of age, 45.2% vs 29.5%) and have hormone receptor positive disease (51.4% vs 42.0%) compared with patients who did not receive T post first PD. Among all treated patients the median overall survival was 35.6 mo (25th-75th-ile:18.6-63.1 mo). Median survival post PD in the 500 patients treated with trastuzumab following first PD was 21.2 mo (25th-75th-ile:11.8-35.8mo).  Conclusions: OS in patients on the prospective observational study registHER is longer than the OS of 25.1 months noted in the pivotal trial of first-line HER2+ MBC patients treated with T plus chemotherapy. We will present data on the association between various pre- and post-treatment factors, including the use of T beyond PD, and PFS and OS. This will include a multivariate analysis that specifically examines the association between T use in later lines and survival.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3142.
A novel HER2 inhibitor TAK-285 overcomes trastuzumab resistance of HER2-over-expressing breast cancer.
Authors: Hayashi A , Tamura T , Yusa T , Takagi S , Ohta Y
Published: December 2010

Abstract #3143

Background: The anti-HER2 antibody trastuzumab has been proven highly effective in the treatment of HER2-expressing breast cancers clinically. However, the continuous administration often leads to drug resistance, the mechanism of which is not well understood. Here we present a new mechanism for trastuzumab-resistance. We also show TAK-285, a novel small molecule HER2/EGFR kinase inhibitor under clinical evaluation, overcomes the resistance to trastuzumab.  Methods: A trastuzumab-resistant sub-clone (BT-474TR) of the HER2-positive human breast cancer line, BT-474, was established in house by serial passage in the presence of 5 μg/mL trastuzumab for over 5 months. Gene expression profiles of the clones were assessed using Affymetrix HG-U133 plus 2.0 human gene arrays and confirmed by real-time PCR. Target genes were depleted by siRNA-mediated knockdown and confirmed by real time-PCR and immunoblot. Effects on cell growth were assessed by the CellTiter-Glo luminescent cell viability assay (Promega).  Results: BT-474TR cells exhibited modified expression of several genes including cofilin, compared to the parental BT-474 cells. Although immunoblotting using anti-cofilin antibody revealed no clear change in protein level, BT-474TR displayed significant increase in phosphorylation of cofilin. Gene-specific knockdown of cofilin-1 led to re-sensitization of BT-474TR to trastuzumab, though the cofilin-2 knockdown did not. Likewise, knockdown of either LIMK1 or PAK1, known to regulate cofilin-phosphorylation, resulted in similar re-sensitization of BT-474TR to trastuzumab. BT-474TR also exhibited increase in phosphorylation of both LIMK1 and PAK1. TAK-285 showed almost equivalent inhibitory activity against both BT-474 and BT-474TR proliferation in cell culture and in xenograft study, while trastuzumab did not affect the proliferation of BT-474TR in either condition  Conclusions: These data suggest that activation of cofilin signaling pathway is a probable mechanism of trastuzumab resistance. PAK1/LIMK1/cofilin-1 inhibitors might therefore be useful in preventing or reversing trastuzumab resistance. The phosphorylation status of PAK1/LIMK1/cofilin might also serve as a biomarker of Trastuzumab-sensitivity. Moreover a novel HER2 inhibitor, TAK-285, might show benefit in the treatment of trastuzumab-resistant HER2-expressing breast cancers.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3143.
Incidence of hormone-receptor negative and basal type breast tumors among HLA-A2+ patients and possible implications for cancer vaccine trials: U.S. Military Cancer Institute clinical trials group study I-01, I-02, I-03, and I-04.
Authors: Holmes JP , Benavides LC , Gates JD , Mittendorf EA , Ponniah S , Peoples GE
Published: December 2010

Abstract #3153

Background: Breast cancer prognosis is significantly impacted by expression levels of steroid hormone receptors for estrogen (ER) and progesterone (PR), as well as HER2/neu, a member of the epidermal growth factor receptor family of tyrosine kinases. Recent advances in molecular typing of breast tumors have defined a poor risk subtype of tumor called basal cell. These basal cell breast tumors are “triple negative,” meaning ER and PR negative, and none to low HER2/neu expression (immunohistochemistry [IHC] 0, 1+, or 2+ tumors). Basal cell tumors have a poorer prognosis and do not respond to conventional hormonal or targeted therapy aimed at HER2/neu overexpressing tumors (IHC 3+). Our Cancer Vaccine Development Program (CVDP) has performed several phase I and II clinical trials using immunogenic peptides from the HER2 protein to include E75, GP2, and AE37. Here we present data analysis of ER, PR, and basal cell tumors in HLA-A2+ and A2- patients. Methods: Vaccine trials utilizing E75, GP2, and AE37 were performed separately with similar designs targeting patients with all levels of HER2 expression (IHC 1-3+). Upon completion of standard breast cancer therapy and prior to HLA-typing, disease-free patients were enrolled in the various vaccine trials. HLA-A2 status was determined via flow cytometry of peripheral blood mononuclear cells as E75 and GP2 are HLA-A2+ restricted whereas AE37 is a promiscuous HLA Class II binder. Standard demographic data (including ER, PR and HER2/neu status) were collected, and patients vaccinated according to their respective trial protocol. Data from all trials were pooled for this analysis. Results: To date, of 254 patients enrolled in the trials 54.3% (n=138) were HLA-A2+. Age, tumor size and nodal status were similar between the A2+ and A2- patients. Significantly more A2+ patients had hormone receptor negative (ER-PR-) tumors (n=49) compared to A2- patients (n=27) (35.5% vs. 23.3%; p= 0.03). There was a trend towards increased basal cell tumors among A2+ patients compared to A2- (15.2% vs. 9.5%; p=0.17). Conclusions: Across these clinical trials conducted by the CVDP, HLA-A2+ patients have an increased incidence of ER-PR- tumors and trend toward increased basal cell tumors. ER-PR- and basal cell tumors have a worse prognosis and fewer treatment options than ER+PR+ tumors. As previously reorted, peptide vaccines targeting the HER2/neu protein are safe and effective in eliciting an immune response in patients with tumors expressing various levels of HER2/neu,to include the low levels associated with basal cell tumors. Therefore, peptide cancer vaccines may provide an excellent adjunctive to current treatment for basal cell tumors.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3153.
A phase II study of trastuzumab-DM1, a first-in-class HER2 antibody-drug conjugate, in patients with HER2+ metastatic breast cancer.
Authors: Vukelja S , Rugo H , Vogel C , Borson R , Tan-Chiu E , Birkner M , Holden SN , Klencke B , O'Shaughnessy J , Burris HA
Published: December 2010

Abstract #33

Background  Trastuzumab-DM1 (T-DM1), an antibody drug conjugate (ADC) designed to combine the biological activity of trastuzumab with the targeted delivery of a highly potent anti-microtubule agent (DM1) to HER2-expressing cells, has demonstrated activity in a Phase I study. The maximally tolerated dose (MTD) for T-DM1 given every 3 wks was 3.6 mg/kg.  Methods  This is a multi-institutional, open-label, single-arm, Phase II study of T-DM1, 3.6 mg/kg administered by IV infusion every 3 wks, to pts with HER2+ MBC; 100 efficacy-evaluable pts who have progressed on prior T and chemotherapy given in the metastatic setting will be enrolled. The primary objective is to assess objective response rate (ORR) and safety and tolerability in this patient population. Key secondary objectives include measurement of duration of objective response and progression-free survival; to characterize the pharmacokinetics of this T-DM1 regimen; and to assess the formation of antibodies to T-DM1. A preplanned, protocol specified interim analysis of efficacy data was performed after the first 30 efficacy evaluable pts had completed 4 cycles (12 wks) of treatment. A final analysis will be conducted 26 wks after the last patient has been enrolled.  Results  As of June 6, 2008, 92/100 pts have been enrolled. Demographic data from the first 31 enrolled pts include: median age 57 (range 38-79); PS 0-1, 29; PS 2, 2; median number prior metastatic chemo agents 2 (range 1-9); median duration of prior T is 76.1 wks (range 12-379); 13 pts (42%) received prior lapatinib. Of the 31 pts, 30 were evaluable for efficacy per protocol. Based on investigator assessments, 12/30 (40%) pts have had a partial (11) or complete (1) response reported. The IRF has reported a partial response in 9/30 (30%) pts to date. To date, these 31 pts have received a median of 4 T-DM1cycles (range 1-11). Gr2 adverse events (AEs) include thrombocytopenia (10%), fatigue (13%), nausea/vomiting (10%), infusion reaction/fever/chills (10%). Gr3 thrombocytopenia occurred in 10% of pts and Gr4 AEs occurred in 2 (6%) pts (thrombocytopenia and transaminase elevation). Sixteen (52%) pts have discontinued therapy, 11 for progressive disease.  Conclusions  T-DM1 has shown single-agent clinical activity in pts who have progressed on prior HER2-directed therapy. The safety profile of T-DM1 appears tolerable at the recommended dose. To date, preliminary data show a 40% investigator-determined response rate and a 30% IRF-reported response rate. Updated results for the entire study population will be presented.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 33.
Nanoporous silicon delivery vectors for breast cancer imaging.
Authors: Serda RE , Godin B , Bankson J , Decuzzi P , Ferrari M
Published: December 2010

Abstract #4003

Nano- and micro-sized delivery systems are emerging as powerful tools for the systemic delivery of therapeutic and imaging agents in different biomedical applications, from cancer to cardiovascular diseases. These delivery systems can be loaded with a multitude of drug molecules and contrast agents to simultaneously provide therapeutic and imaging capabilities. Following intravenous injection, these particles are transported by the blood stream into different vascular districts. We have recently introduced a multi-stage delivery system designed to overcome sequential biological barriers. The first stage, comprised of porous silicon microparticles, targets tumor-associated endothelium. Once arrested at the tumor site, second stage nanoparticles, carrying therapeutic and imaging agents are released into the tumor microenvironment by time-dependent degradation of the silicon vehicle. Free nanoparticles cross the endothelial barrier by the inherent leaky nature of the tumor vessel, a phenomenon known as enhanced permeation and retention. In this study, iron oxide nanoparticles serve as contrast agents for visualizing distribution in murine mammary tumors using magnetic resonance imaging.  The silicon microparticles are hemispherical in shape, a design that facilitates margination and interaction of particles with the endothelium. Loading of iron oxide nanoparticles into the silicon microparticles was accomplished by covalent attachment. The loaded particles were characterized by Prussian Blue quantitation of iron, as well as SEM and TEM analysis. Human breast cancer xenografts were established in nude mice by injection of 4T1 mouse mammary carcinoma cells into fatpads of Crl Foxnu nude mice. At day 15, mice were intraveneously administered either free iron oxide nanoparticles, or 1 x 108 silicon microparticles, non-loaded or loaded with iron oxide nanoparticles. Magnetic resonance imaging was carried out using a 7 Telsa magnetic resonance imager. Serial sagittal T2-weighted images revealed tumors with hypointense contrast for animals injected with free and multi-stage encapsulated iron oxide nanoparticles at both 3 and 5 hr. A separate quantitative study compared iron and silicon content in lungs, liver, spleen, kidney, and tumor by inductive coupled plasma atomic emission spectroscopy analysis of organs from 25 mice 5 hrs following tail vein injections of the following systems: 1) saline control, 2) silicon microparticles (Si), 3) iron oxide nanoparticles, and 4) iron oxide loaded silicon particles.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4003.
Factors influencing false negative diagnosis of 3D-MRI in pre-operative breast cancer patients.
Authors: Hisamatsu K , Takiyama W , Hirabayashi N , Kaneko M , Ono T , Sakabe R , Stou Y , Saeki S , Mukaida H , Yamashita Y
Published: December 2010

Abstract #4005

Introduction: We presented 3D-MRI will be the useful breast imaging tool in diagnosing intraductal spread and tumor size in breast cancer patients (ASCO 2008). But, false negative cases were very important in breast conserving surgery and careful management was needed in 3D-MRI guided surgery. This study was to review imaging diagnosis using 3D-MRI and to evaluate factors influencing false negative diagnosis of 3D-MRI, retrospectively. Material and methods: From July 2000, pre-operative 3D-MRI was underwent 433 cases of breast cancer patients. Images were obtained 426 cases (98%). Tumor size between 3D-MRI and resected specimens were evaluated retrospectively. Cases with tumor size difference between 3D-MRI and resected specimens less than 1cm were defined related cases and cases with tumor size in 3D-MRI was 1cm more smaller than resected specimens were defined false negative cases. Investigated factors influencing false negative diagnosis of 3D-MRI were age, patterns of 3D-MRI images, presence of pre-operative chemotherapy, histological type, histological grade, microscopical invasive tumor size (mm) and presence of ductal spreading. From April 2005, the entire specimen was serially step cut and evaluated. Results: Tumor diameter from nipple side to lateral margin were measured by MRI and specimens, tumor size was correlated between MRI and specimens, significantly (n=118, r=07186, P < 0.001). On Multivariate analysis revealed that factors influencing 3D-MRI diagnosis were presence of ductal spreading (p=0.0002), patterns of 3D-MRI images (p=0.06) and presence of pre-operative chemotherapy (P=0.04). Factors influencing false negative diagnosis of 3D-MRI were presence of pre-operative chemotherapy (P<0.05), presence of ductal spreading (P<0.01) and infiltration of lymphocyte in the edge of ductal spreading (P<0.05). Selection for breast conserving surgery and margin width were based by 3D-MRI images. The proportion of breast-conserved surgery were obtained 89% (380/426 cases) and local failure seen only 6 cases (local failure rate 1.4%) in 3D-MRI guided surgery. (6-94 months, median follow up 51 months).  Conclusion: These results suggest that MRI will be the useful breast imaging tool in diagnosing intraductal spread and tumor size in breast cancer patients. But, caution will be needed in cases with pre-operative systemic therapy and cases expected ductal spreading by other imaging tools.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4005.
Inflammatory breast cancer: defining breast magnetic resonance imaging features.
Authors: Le-Petross HT , Cristofanilli M , Qing Y , Jackson EF , Gong Y , Reed B , Hortobagyi GN , Yang WT
Published: December 2010

Abstract #4008

PURPOSE:   Inflammatory breast carcinoma (IBC) is an aggressive cancer with radiological and clinical features of a heterogeneous disease. The clinical presentation of IBC is characterized by the sudden onset of breast erythema and edema, often without an associated palpable breast mass. Mammography is associated with variable, not descriptive findings and it is often not diagnostic. We planned to address the role of Magnetic Resonance Imaging (MRI) of the breast in women with newly diagnosed IBC.  MATERIALS AND METHODS:  We performed a retrospective analysis of newly diagnosed IBC cases evaluated at the University of Texas. M D Anderson Cancer Center between December 2003 and February 2008. Baseline breast MRI exams were performed on 1.5-Tesla (T) or 3.0-T GE scanners and included dynamic three-dimensional T1-weighted fast spoiled gradient echo sequences with parallel imaging after a bolus injection of gadolinium based IV contrast. Exams were reviewed in batches and the findings rated in accordance with the ACR BI-RADS MRI Lexicon. The following parameters were evaluated; presence and characteristics of breast masses and skin description. All patients had concomitant mammograms and ultrasound exams.  RESULTS:   Eighty women with a clinical diagnosis of IBC were included in the study with a median age of 52 years, (range, 25 to 78). MRI detected a primary breast lesion in 80 of 82 symptomatic breasts (98%), compared to only 53 of 78 (68%) with mammogram (p < 0.0001). The most common MRI morphologic characteristic of the lesions was irregular margins (72%) with heterogeneous internal enhancement (82%). Qualitative evaluation of the enhancement pattern demonstrated wash-out in 85% of the cases.  MRI detected skin thickening in 90% of the patients (average skin thickness of 8 mm [range, 4-17 mm]), compared to 69% with mammogram. Heterogeneous skin enhancement (82%) and nodular or irregular skin lesions (43%) were identified only with MRI. These skin lesions demonstrated a progressive enhancing pattern, in contrast to the rapid wash-out pattern expected for malignant breast lesion.  CONCLUSIONS:   This study establishes breast MRI as the most sensitive and accurate imaging modality in assessing the breast of IBC patients. The most common radiological features are represented by the presence of a primary breast lesion and global skin thickening with heterogeneous skin enhancement. The combination of morphology and enhancing pattern may provide information to understand the biology of this disease and accurately monitor residual disease during neoadjuvant therapies.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4008.
A novel combination therapy with histone deacetylase inhibitor and Aurora kinase inhibitor for “triple negative” breast cancers.
Authors: Samuel T , Fiskus W , Buser C , Bhalla K
Published: December 2010

Abstract #401

Background: Aurora A and B are essential for normal mitosis. Aurora A (AA) is amplified and/or co-overexpressed with Aurora B (AB) in over 50% of breast cancers, including the basaloid variety, “Triple Negative” breast cancers. MK-0457 (M) is a pan-Aurora kinase inhibitor that inhibits in vitro and in vivo tumor growth, as well as induces apoptosis of cancer cells. Aurora kinases are chaperoned by hsp90, and their down regulation is known to sensitize transformed cells to paclitaxel. Vorinostat (V) is a pan-histone deacetylase inhibitor (HA-HDI) that induces in vitro growth arrest, differentiation and apoptosis of human breast cancer cells. By inhibiting HDAC6 and inducing hyper-acetylation and inhibition of hsp90, V depletes the levels of hsp90 chaperoned proteins, including AA and AB kinase. Methods: AA amplification in MB-231 (“Triple Negative”) and BT-474 (HER-2 amplified) cells were determined by FISH and array-CGH. The cells were treated with M (50 to 250 nM) and/or V (0.5 to 2.0 μM) for 24 to 48 hours. Cell cycle status, DNA endoreduplication (PI staining) and apoptosis (Annexin V/PI staining) were determined by flow cytometry. Intracellular protein levels of p-AA or p-AB, AA and AB, p-survivin, survivin, were determined by immunoblot analyses. Results: Treatment with M or V induces cell cycle G2/M phase accumulation and apoptosis of the breast cancer cells. Dose-dependently, treatment with M depleted phosphorylated (p)-AA, -survivin, and -Ser10 H3 levels. V induced hsp90 acetylation and depleted the levels of AA, AB and survivin. Both M and V induced formation of multi-polar aberrant mitotic spindles. Co-treatment with M and V mediated more depletion of p-survivin, AA and AB levels, as well as synergistically induced apoptosis of MB-231 and BT-474 cells. In the orthotopic MB-231 xenograft breast cancer model, co-treatment with M and V caused significantly more tumor growth delay, lung metastases and survival than treatment with either agent alone. Treatment with V induced in vivo hsp90 hyper-acetylation in the mouse xenografts, as well as in the accessible tumors from patients with breast cancer. This was determined by immunoblot analysis of post-treatment versus pre-treatment tumors with the anti-acetylated-K69-hsp90 antibody, which was developed for these studies. Hsp90 hyper-acetylation was associated with in vivo depletion of AA and AB levels in the mouse xenografts. Co-treatment with M and V, versus treatment with either M or V, also led to greater in vivo attenuation of AA, AB and p-survivin. Conclusion: These studies support the rationale to evaluate the safety and efficacy of the combination of M and V, without or with paclitaxel, against breast cancers with AA amplification and/or overexpression.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 401.
MRI findings in women with triple negative breast cancer.
Authors: Hines SL , McLaughlin SA , Vallow LA
Published: December 2010

Abstract #4014

Background  Preoperative magnetic resonance imaging (MRI) of the breasts in women with breast cancer (BC) has improved the detection of occult ipsilateral (I/L) and contralateral (C/L) malignancies. Breast MRI findings in patients with estrogen receptor (ER), progesterone receptor (PR), and HER2 negative (triple negative (TN)) disease have not been previously reported. Women with TN BC are at increased risk of distant metastases and a reduced survival vs. BC patients as a whole. One potential explanation may be the presence of occult disease. The role of bilateral breast MRI in these patients remains to be determined.  Hypothesis  Patients with TN BC are more likely to have occult malignancy identified by preoperative MRI vs. those with non-TN BC.  Methods  All newly diagnosed BC patients from 2003-2007 were reviewed. Information on age, race, histology, ER, PR, HER2, staging, treatment, MRI findings, subsequent investigations, and additional cancer diagnoses was recorded. The incidence of additional I/L or C/L malignany was calculated. The percentage of total patients initially felt to be candidates for partial breast irradiation who required a change in their cancer management was calculated.  Results  55 (11%) were diagnosed with TN BC, of whom 52 women completed preoperative MRI. 456 (89%) had non-TN BC; 421 completed preoperative MRI. The median age among TN and non-TN patients was 62 years (Table 1). A majority of women in both groups were Caucasian. African-American women were more likely to be diagnosed with TN vs. non-TN disease (11% vs. 6%).    I/L BC was documented in 13/52 (25%) of TN patients vs. 25% of non-TN patients. No C/L cancers were detected in the TN group (6% in the non-TN group). Of the TN group, 15 patients required a change in their treatment (29%) based on findings from MRI vs. 32% of non-TN patients.  Conclusions  Patients with a new diagnosis of TN BC are not at increased risk of occult malignancy than those with non-TN BC. 25% of patients with TN BC had additional I/L disease not previously detected on clinical examination, mammogram, or ultrasound. Of those with non-TN BC, 25% were diagnosed with an additional I/L and 6% with C/L malignancy. The benefit of MRI was limited by false positives, with a number of patients in both groups undergoing a benign biopsy.  These results suggest that patients with TN BC are not more likely than those with non-TN disease to have additional occult I/L or C/L cancer. The incidence of additional disease remains high and preoperative MRI may be beneficial.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4014.
Proximity-based assays for the detection of activated HER3, HER2/HER3 heterodimers and HER3/PI3K complexes in formalin-fixed, paraffin-embedded cell line controls and tumors.
Authors: Mukherjee A , Badal Y , Nguyen X , Miller J , Pidaparthi S , Chen L , Newton A , Cheung J , Parry G , Williams S
Published: December 2010

Abstract #4040

Although kinase-defective, the role of the HER3 receptor in driving tumor growth through dimerization with other HER family members is well recognized. There is increasing interest in developing therapeutic strategies to interfere with signaling through this receptor, either directly or indirectly. At the same time, there is recognition that the activation of HER3 could be an important biomarker of drug response and resistance. In particular the HER2/HER3 heterodimer has been extensively studied and, among all the possible homo and heterodimers formed by HER receptors, is considered the most mitogenic because of the ability of HER3 to effectively couple to the pro-survival PI3K/Akt pathway.  Using the VeraTag™ technology, we have developed a suite of assays designed to detect quantitatively the activation status of the HER3 receptor by measuring HER3-specific phosphorylation and formation of HER3 signaling complexes in formalin-fixed, paraffin-embedded (FFPE) cell line controls and tissue samples.These assays are dual antibody-based and measure complexes of proteins in close association since signal is generated only when the antibodies are bound to target-specific epitope sites proximal to one another. A capillary electrophoresis instrument equipped with laser-induced fluorescence provides a highly sensitive and quantitative read-out of the assay. We report data on assays which detect a ligand-induced, 2-5-fold change in HER2/HER3 heterodimer formation, a 10-20 fold change in phosphorylated HER3 signal and as high as a 10 fold change in HER3/PI3K complex formation in a series of cell line controls, spanning a wide range of HER2 and HER3 receptor levels. FFPE data were verified with results from co-immunoprecipitation using antibodies identical to those used in the VeraTag assay. The influence of the HER2 and HER3 levels on the formation of HER2/HER3 heterodimers and correlation with HER3 phosphorylation and formation of the PI3K adaptor complex will be discussed.  In addition, we have used these assays to landscape HER3 activation in a number of tissues, including breast and ovarian tumors, focusing particularly on HER2 positive breast cancer, where the HER2/HER3 heterodimer is thought to play a particularly significant role.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4040.
The relationship between tumour NF-kB expression, hormone status and survival in primary invasive breast cancer.
Authors: Tannahill CT , Obondo C , Tovey S , Orange C , Doughty JD , Wilson CR , Cooke TG , McMillan DC , Edwards J
Published: December 2010

Abstract #4041

Breast cancer is one of the most common and lethal malignancies in the UK. Conventionally, prognosis is based on hormone status, tumours size, grade and lymph node involvement. We have recently observed that NF-kB expression and activation was associated with progesterone receptor (PR) expression irrespective of oestrogen receptor (ER) status in primary invasive breast cancer (Tannahill et al., unpublished observations). The aim of the present study was to examine the relationship between NF-kB, PR and survival in a selected cohort of ER positive tamoxifen treated primary invasive breast cancer.  Immunohistochemistry was performed on 372 breast tumours, with full clinicopathological characteristics. Antibodies were directed total NF-kB and NF-kB phosphorylated at serine 536 (phospho NF-kB). Two observers independently scored expression using the weighted histoscore technique, and grouped expression as tertiles. There was a minimum follow-up period of 4.9 years and a median follow-up of 6.8 years. During follow-up 104 patients died, 65 of which died of their disease, 96 patients recurred, 70 of which was while on tamoxifen.  Nuclear NF-kB, either total or phosphorylated, expression was not associated with recurrence or survival. However, when the cohort was subdivided into PR positive (n=220) and PR negative tumours (n=140), increased nuclear phospho NF-kB expression was associated with increased recurrence (p<0.05) and increased recurrence on tamoxifen therapy (p<0.05) in PR positive tumours. In contrast, increased nuclear phospho NF-kB expression was associated with decreased recurrence (p<0.05) and decreased recurrence on tamoxifen therapy (p<0.05) in PR negative tumours.  The results of the present study suggest that the effect of nuclear expression of phospho NF-kb on outcome in ER positive primary invasive breast cancer is dependent on PR status. The study highlights the importance of patient selection for the evaluation of the use of NF-kb inhibitors in patients with breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4041.
CAPC expression correlates with breast tumor progression and invasion.
Authors: Man Y , Zhang Z , Wang C , Gao L , Zhang X
Published: December 2010

Abstract #4044

Background: Recent studies identified a uncharacterized gene that encodes a molecule named as cytokeratin-associated protein in cancer (CAPC). RT-PCR analysis detected significant elevation of CAPC mRNA in 26 of 33 human breast cancers. Our current study attempted to elucidate CAPC protein expression profile in human breast tissues, to assess: [1] if CAPC expression is associated with tumor progression and invasion, and [2] if CAPC is co-expressed with BP1 and c-erbB2, two known invasiveness and aggressiveness-related proteins.  Materials and Methods: Consecutive sections were prepared from paraffin-embedded breast tissues from 100 female patients with breast tumors that harbored co-existing normal, hyperplastic, in situ, and invasive components. Sets of four adjacent sections from each case were subjected to immunohistochemistry for CAPC, BP1, c-erbB2, and Ki-67. The expression status of these molecules in different tissue components were statistically compared.  Results: In normal breasts and hyperplastic or in situ breast lesions, CAPC positive cells were generally distributed as duct or acinar clusters with a distinct boundary with their CAPC negative counterparts. Under low magnification of H&E stained sections, these CAPC positive duct or acinar clusters were morphologically indistinguishable from their CAPC negative counterparts. Under high magnification, however, the myoepithelial cell layers of these CAPC positive clusters were focally disrupted, and the associated epithelial cells often showed malignancy-associated nuclear alterations. Both the number of CAPC positive cells and the intensity of CAPC immunostaining linearly increased with tumor progression and invasion. In invasive lesions, a vast majority of cancer cells in over 80% of the cases expressed high levels of CAPC. A majority of the normal, benign, and malignant tumors with high levels of CAPC showed substantially higher expression of BP1 and c-erbB2, along with a higher proliferation index, compared to their morphologically similar CAPC negative counterparts . In non-mitotic cells, CAPC was localized mainly in the cytoplasm. In malignant mitotic cells, high levels of CAPC expression were localized close to the cell membrane, forming a ring-like structure surrounding condensed chromosomes. In contrast, no distinct CAPC was detected in normal or hyperplastic mitotic cells.  Discussion: These findings suggest CAPC might be a tumor specific marker that could be used for the development of diagnostic and/or therapeutic agents.  Acknowledgment: Supported in part by grant BCTR0706983 from The Susan G. Komen Breast Cancer Foundation and grant 2006CB910505 from the Ministry of Chinese Science and Technology Department.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4044.
Tumor necrosis factor transactivates ErbB2 in breast cancer cells.
Authors: Rivas MA , Tkach M , Proietti CJ , Rosemblit C , Beguelin W , Sundblad V , Díaz Flaqué MC , Charreau EH , Elizalde PV , Schillaci R
Published: December 2010

Abstract #4056

We have previously shown that TNFα induces proliferation of the murine mammary adenocarcinoma C4HD through activation of the PI-3K/Akt signaling pathway that converges on the transcriptional activation of NF-κB. Since C4HD tumor overexpresses ErbB2 and given that this tyrosine kinase plays a critical role in C4HD cell proliferation, we wondered whether interactions between TNFα and ErbB2 could be taking place. Our findings revealed that treatment of C4HD cells with the ErbB2 inhibitor AG825 blocked TNFα-induced proliferation. Similar results were obtained using the human ErbB2-overexpressing cell line SK-BR-3. Then, we studied the effect of TNFα on ErbB2 phosphorylation in C4HD and SK-BR-3 cells. We found that TNFα increased total ErbB2 tyrosine phosphorylation in C4HD and SK-BR-3 cells, as well as on the specific residues tyrosines 927 and 1172 in murine cells and on its human homologues 877 and 1222. These effects where not caused by the release of ErbBs ligands from the cell membrane by TNFα, since treatment with the metalloprotease inhibitor GM6001 did not affect TNFα-induced ErbB2 phosphorylation. We then studied if c-Src was involved in the above effect given that it is known to directly phosphorylate ErbB2 in the Tyr 877 residue. We found that addition of PP2, a Src family inhibitor, completely inhibited phosphorylation of Tyr 927/877 ErbB2, and that to a lesser degree it inhibited Tyr 1172/1222 ErbB2 in both cell types. We also performed an in vitro cold phosphorylation assay where we observed that c-Src immunoprecipitated from C4HD cells treated with TNFα was able to phosphorylate ErbB2 on Tyr 927 residue. Taken together, these results indicate that TNFα induces phosphorylation of ErbB2 at Tyr877/927 residue and that c-Src is the tyrosine kinase involved in this effect. In addition, we observed that upon TNFα stimulation, ErbB2 associated with ErbB3 leading to PI-3K/Akt pathway activation. Treatment of cells with AG825 inhibited Akt and NF-κB activation by TNFα, as evidenced by western blots of phospho proteins and reporter gene studies, respectively. The above data for the first time identify TNFα as a cytokine able to transactivate ErbB2, disclosing c-Src involvement in such effect. Also we demonstrated that TNFα ability to activate Akt and NF-κB transcriptional activation is dependent on ErbB2 phosphorylation in breast cancer cells that overexpress ErbB2. Interestingly, TNFα appears as a new player in ErbB2-overexpressing breast tumors, and its eventual worth as a prognostic factor in anti ErbB2 therapy is yet to be determined.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4056.
Bone morphogenetic protein 10 (BMP-10) inhibits growth of breast cancer cells and correlates with poor prognosis.
Authors: Ye L , Kynaston HG , Mansel RE , Jiang WG
Published: December 2010

Abstract #4068

Background: Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and are known to be involved in the development of bone metastasis from solid tumours. Recent studies have shown that BMP-10 plays some critical roles during the fetal or postnatal development, particularly during the morphogenesis of heart. However, its role in cancer is largely unknown. The present study investigated the role of BMP-10 in breast cancer cells and pattern of expression in clinical breast cancers.  Methods: The expression of BMP-10 was examined in a cohort of human breast cancer specimen (normal, n=23; cancer, n=97), using both quantitative real time PCR and immunohistochemical staining. Levels of BMP-10 transcripts were correlated with clinical and pathological, together with clinical outcome information of the patients. A human BMP-10 expression construct was used to transfect breast cancer cells (MDA-MB-231, negative in BMP-10 expression). The expression of BMP-10 in the transfected cells was verified using RT-PCR and western blot. The effect of BMP-10 expression on growth of MDA-MB-231 cells was investigated using an in vitro growth assay.  Results: Immunohistochemical staining revealed a decreased protein expression of BMP-10 in breast cancer cells, compared with normal mammary epithelial cells. The quantitative real time PCR further demonstrated that low levels of the BMP-10 transcripts correlated to poor prognosis. Tumours form patients with good prognostic index (NPI<3.4) had significantly higher levels of BMP-10 transcripts, compared with those from poor prognostic index (NPI>5.4) (p=0.045). In addition, patients who died of breast cancer showed a significantly lower levels of the transcript compared with those who remained disease free (p=0.027). Mean disease free survival of the patients with higher level of BMP-10 was 132.8 (122.0-143.5, 95% CI) months, which was significantly longer compared to 93.7 (60.3-127.2) months of patients with lower level of BMP-10 expression, p=0.043 using Kaplan-Meirer survival analysis. An inhibitory effect on cell growth by over-expression of BMP-10 was seen in the breast cancer cells. The absorbance of MDA-MB-231 BMP-10 over-expressing (MDA-MB-231BMP-10exp) cells of Day 5 is 1.40±0.19, p<0.01 vs both MDA-MB-231 wild type (MDA-MB-231WT, 2.17±0.11) and empty vector control (MDA-MB-231pEF/His, 1.83±0.17).  Conclusions: Taken together, BMP-10 can inhibit the cell growth of breast cancer cells. A decrease in BMP-10 expression correlates with poor prognosis of patients with breast cancer. BMP-10 may function as tumor suppressor in breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4068.
Consistent TP53 mutations in BRCA1 and sporadic basal-like breast tumors, while infrequent in luminal BRCA1 tumors.
Authors: Manié E , Vincent-Salomon A , Lehmann-Che J , Pierron G , Turpin E , Warcoin M , Gruel N , Lebigot I , Sastre-Garau X , Delattre O , de Thè H , Stoppa-Lyonnet D , Stern M
Published: December 2010

Abstract #4070

Breast tumors with a germline mutation of BRCA1 (BRCA1 tumors) and Basal-Like Carcinoma (BLC) have been shown to be associated with a high rate of TP53 mutations. Because it has also been shown that BRCA1 tumors frequently display a basal-like phenotype, we sought to determine whether TP53 mutations were correlated to the hereditary BRCA1 mutated status or to the particular histological type of these tumors. We first explored the status of the TP53 gene in a series of 28 BRCA1 BLCs using immunohistochemistry, direct sequencing of the coding sequence, and functional assay in yeast (FASAY), and compared it to the status of TP53 in 27 series of sporadic (non hereditary) BLCs. With this sensitive approach, TP53 was found consistently mutated in BRCA1 (28/28) and sporadic (26/27) BLCs. However, mutation spectrum was different, in particular with a much higher rate of insertion/deletion in BRCA1 BLCs (12/27) than in the sporadic group (2/25). Secondly, we analyzed the incidence of TP53 mutations in 10 BRCA1 luminal tumors, using the same strategy. Surprisingly, only 3 of these 10 tumors were mutated, a frequency similar to that found in matched sporadic luminal tumors. In conclusion, TP53 mutation is a mandatory event in BRCA1 or sporadic BLCs, but not a common feature of BRCA1 luminal tumors.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4070.
Breast cancer patients unknowingly dosing themselves with estrogen by using topical moisturizers.
Authors: Olson AC , Link JS , Kupiec TC
Published: December 2010

Abstract #4087

Background: Topically applied estrogens are more efficiently absorbed into the body than estrogens administered orally, due to the first pass effect by the liver. Estrogen receptor positive breast cancer patients need to avoid exogenously administered estrogen to minimize chance of recurrence. This is especially significant for women on aromatase inhibitor treatment,where topical estrogen may overcome the therapeutic effect. Also, non breast cancer patients using estrogen containing topical moisturizers may be unknowingly dosing themselves daily with estrogen, often for years, increasing their chances of developing breast cancer.  Materials and Methods: Sixteen different topical moisturizers were purchased or donated from department stores or chain drugstores. None of the creams noted any estrogen content in their list of ingredients. Each of the sixteen commercial products was analyzed for estradiol, estriol and estrone utilizing High Performance Liquid Chromatography (HPLC). HPLC is commonly used in the pharmaceutical industry to provide component analysis. Detection was carried out through use of a Variable Wavelength Detector (VWD) and a Diode Array Detector.  Results: Four samples contained greater than 0.40% estriol, one contained 0.17% estriol, and one contained 0.05% estrone, despite having no listing on the ingredient list.  Discussion: Topical estrogens are considered effective in enhancing the “youthful look” of aging skin. Manufacturers are marketing incompletely labeled moisturizers containing estrogens. Consumers are unaware their moisturizer may be providing them with unwanted systemic estrogen exposure. The FDA cosmetics division is not presently testing for this potential risk for breast cancer patients. Other breast cancer advocacy groups and the FDA need to promptly repeat these procedures and also check for exotic "designer" estrogens.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4087.
Pre operative assessment enables the early detection and treatment of shoulder impairments related to breast cancer treatment.
Authors: Stout Gergich NL , Levy E , Springer B , Pfalzer L , McGarvey C , Gerber L , Soballe P
Published: December 2010

Abstract #4091

Background: Early detection and management of impairments and limb dysfunction after breast cancer treatment contribute to more successful functional outcomes. Health care providers should be encouraged to provide a pre-operative screening visit to ascertain the patient's baseline physical function measures. Patients should then be followed prospectively at regular intervals during and after treatment for early identification of impairments and to provide intervention while the functional limitations are minimal, thereby preventing long term loss of function.  Materials and Methods: A prospective, observational study (n=196) with interval follow up over one year assessed the effects of an early identification and intervention model of physical therapy care on women after breast cancer treatment. Measurement of upper extremity (UE) function included; active range of motion (ROM), strength, and limb volume as measured by the optoelectronic volumeter (Perometer®), and self-reported physical activity disability. Individuals were seen for an initial visit prior to surgical treatment and followed-up at approximately one month post-operatively and at three month intervals thereafter, up to 18 months. Patients received a general home exercise program pre-operatively and instructed to initiate at 2 weeks post op. If impairments were identified at any of the follow up visits, physical therapy intervention was prescribed.  Results: A significant decrease in active shoulder ROM was noted at one month post-op as compared to baseline measurement in 102 patients. This significance resolved at the 12+ month follow up visit. Loss of shoulder ROM correlated with other UE treatment related morbidities including; axillary web syndrome, seroma and pain.  Discussion: Incidence of upper extremity morbidity after breast cancer treatment is documented as high as 70%. This study clearly demonstrates the potential for substantial reduction in UE dysfunction related to breast cancer treatment when using an early identification and intervention model. Many morbidities such as pain, reduced range of motion, decreased strength and sub-clinical lymphedema can be detected early and managed with minimal, cost-effective intervention. Additional research is needed to assess ways of maintaining this cost effective model of care in the long-term.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4091.
Adjuvant dose-dense doxorubicin-cyclophosphamide x 4 with or without bevacizumab is safe: no evidence of short-term changes in left ventricular ejection fraction.
Authors: Morris PG , Dickler M , McArthur H , Traina T , Godfrey L , Steingart R , Rugo H , Lin N , Moy B , Come S , Sugarman S , Norton L , Hudis CA , Dang C
Published: December 2010

Abstract #4102

Background  Dose-Dense (dd) doxorubicin-cyclophosphamide (AC)-paclitaxel (P) is superior to q3w AC-P. Both are associated with a <1% incidence of congestive heart failure (CHF). The advent of targeted therapies potentially raises the risks of cardiac toxicity. We have, therefore, conducted 3 sequential safety studies using ddAC → P, one of which included bevacizumab (B) concurrently with ddAC. Here we report the cardiac safety of ddAC alone or with B.  Methods  Patients with HER2-positive breast cancer were treated on successive trials of ddAC → P and trastuzumab (T) (left ventricular ejection fraction (LVEF) > 55%) and ddAC → PT-lapatinib (LVEF > 50%). Patients with HER2-normal breast cancer were treated with B-ddAC → B-nanoparticle albumin-bound-P (LVEF > 50%). In all 3 trials LVEF was measured by MUGA scan at enrolment and at month 2 following completion of ddAC x 4. Prospective cardiac safety assessments before and after q 2 week AC x 4 were aggregated to determine the acute risks of cardiac dysfunction.  Results  From Jan '05 to May '08 236 pts were enrolled in 3 trials. Median age was 47yrs (range 27-75). Median LVEF pre-ddAC was 68% (range 52-82%). LVEF post-ddAC is available in 233 pts (99%) (1 pt withdrew consent, 1 progressive disease, 1 died from viral pneumonia). Median LVEF was unchanged at 68% (range 47-81%). No pt developed symptomatic CHF. Two pts had asymptomatic LVEF decline >15%: 1 pt (post B-ddAC) had decline from 71 to 53%, which improved to 64% in 4 wks and 1 pt (post ddAC) had decline 68% to 47%, which improved to 64% in 4 wks. No other pt had asymptomatic LVEF decline below 50%. Five of six pts with LVEF declines of 10-15% had improvement in LVEFs and continued treatment while 1 withdrew consent. Post-ddAC LVEF was similar whether or not pts received B.    Conclusions  Previously reported high rates of asymptomatic LVEF post q3w AC leading to pt dropout were not seen in these studies of ddAC or B-ddAC. Post-ddAC LVEFs were similar with or without concurrent bevacizumab. Dose-dense AC is safe and not associated with frequent acute changes in LVEF regardless of the concurrent use of B.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4102.