bluuebunny/crossref_metadata_2025 huggingface dataset:
Prone accelerated partial breast irradiation using cone-beam CT (CBCT) guidance.
Authors: Lymberis SC , Becker SJ , Gidea-Addeo D , Jozsef G , DeWyngaert KJ , Formenti SC
Published: December 2010

Abstract #5136

Introduction: Prone external beam partial breast irradiation when used for a carefully selected subset of patients has been shown to have excellent long term control and cosmesis (Formenti et al. IJROBP 60(2): 493-504 2004). We used Cone-beam CT (CBCT) to visualize the tumor bed target prior to each daily fraction of partial breast irradiation. The purpose of this study is to measure the residual error in soft tissue positioning after alignment based on skin marks and MV portal imaging for patients treated on a prospective prone APBI study (3000cGy in 5 fractions over 5 days).  Methods: Twenty-five post-menopausal women with pT1 breast cancer have consented on our NYU partial breast protocol using CBCT. Twenty-three patients have been simulated and treated in the prone position on a specially designed mattress. After initial skin mark setup and alignment, MV portal imaging with shift using the tangent beams eye view was used to optimize setup for the first 6 patients (Group I). For all daily fractions, CBCT was performed with the Varian On-Board Imager kV imaging system with a 35cm field of view and a 2.5mm slice thickness. CBCT was compared with the planning CT to shift the patient and evaluate the residual error in setup. The residual error from CBCT setup after optimal portal imaging was recorded for each patient. For the subsequent 17 patients (Group II), portal imaging was performed without shift to calculate the residual error from CBCT setup representing skin mark setup.  Results: At the time of this report, 23 patients have been treated with all 5 fractions. The values for the residual error detected after cone-beam CT for Group I (after skin mark set up and portal imaging) and for Group II (after skin mark setup alone) are compared and shown in Table 1.  Conclusions: In this preliminary study with a small number of patients, the residual error was minimal, around 2 mm. Therefore, for patients immobilized on our customized mattress, a reproducible and accurate prone APBI setup can be achieved with use of CBCT. However, given the very small residual error detected clinicians should be weary of indiscriminant use of CBCT for PBI. Concerns over contralateral breast dose and carcinogenic risk should limit the routine use of CBCT imaging for breast radiotherapy unless larger studies demonstrate improved accuracy of treatment delivery with CBCT.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5136.
Ovarian ablation by radiotherapy; is it still an option for the ovarian function suppression in endocrine responsive premenopausal breast cancer patients?.
Authors: Bese NS , Iribas A , Oksuz DC , Atkovar G , Ober A
Published: December 2010

Abstract #5137

Background; Although surgical or medical ovarian ablation is likely to be the treatment of choice at the current time, radiation ablation (RA) can be still a resonable alternative. Data regarding the use of pelvic radiotherapy (RT) for ovarian ablation is very limited and in 10thSt Gallen consensus meeting, ovarian radiation was overwhelmingly rejected. In this retrospective study we evaluated the efficacy of RT for ovarian function suppression in 118 premenopausal breast cancer patients.  Materials and Methods; 153 breast cancer patients had RA between the years 1979 and 2002 at the single center where this study was performed. 19 patients who received chemotherapy within 12 months of RA, and 16 patients who were lost to follow-up within 6 months following RA, were not included into the analyses. All patients were premenopausal and the median age at the initiation of RA was 39 years (range, 21- 52 years). 96 of 118 patients (82%) underwent RA for metastatic disease, 17 patients (14%) received RA as the systemic treatment after locoregional recurrences and 5 patients (4%) had RA as an adjuvant treatment. 97 patients (82%) received only RA as their systemic treatment, 20 patients (17%) had RA with tamoxifen and one patient (1%) had RA with aromatase inhibitors. RT was given with either Co-60 or 15 MV photons of the linear accelerator. A modified pelvic volume was irradiated with opposing anterior and posterior fields, and the dose was prescribed to midplane. The median total dose used for RA was 15Gy in 4 fractions (range 5Gy single fraction-36Gy in 18 fractions).  Results; As the efficacy of RA, data regarding the menstrual status were collected. Amenorrhea was noted in 113 of 118 patients (96%) in 6 months following RA. 88 patients (75%) experienced amenorrhea in 2 months after pelvic RT. 5 patients (4%) who had still normal menstrual functioning after 6 months of RA had estrodiol and follicle stimulating hormone measurements. As they were found to have premenopausal levels, 3 of them received surgical and 2 of them received medical ablation. No acute Grade 3 or 4 (according to the Radiation Therapy Oncology Group radiation morbidity scoring criteria) toxicities were noted. With a median follow-up of 24,5 months (range; 6-167 months), no late severe complications that could be attributable to RT were reported. The total RT dose (more than 15Gy) were found to be significant on the rate of ovarian ablation with Mann Whithney U-test (p=0.02).  Conclusion: Ovarian ablation was achieved with a rate of 96% without any major acute and late toxicity using pelvic RT. It should be considered as an option for endocrine responsive premenopausal breast cancer patients and can be easily delivered when postoperative or palliative irradiation is given.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5137.
Six month follow up on a series of patients treated with an electronic brachytherapy technique for intracavitary accelerated partial breast irradiation.
Authors: Mehta V , Griem KL , Haile KL , Stevens R , Syzek L , Elliott K
Published: December 2010

Abstract #5149

Purpose: Phase IV prospective post market study to evaluate the safety and device performance of electronic brachytherapy treatment using the Xoft Axxent System as adjuvant therapy for early stage breast cancer.  Methods and Materials: 44 patients were enrolled between April 2007 and March 2008, and 13 patients have completed 6 months of follow up to date. Patients were eligible if they were older than 50 years, had completely resected DCIS (<2.0cm) or invasive ductal carcinoma (<2.0cm), and microscopic surgical margins of at least 1mm. The balloon applicator was placed in a closed cavity and a balloon surface to skin distance of at least 7mm was required for treatment. The radiation treatment (RT) consisted of delivering 3.4 Gy/fraction twice a day for 10 total fractions. The primary endpoint was the successful delivery of the prescribed RT. Secondary endpoint was adverse device related events.  Results: Thirteen of the forty-four patients that were enrolled and treated on this study have been followed a minimum of 6 months. The three month follow-up included cosmesis evaluation using the Harvard Cosmesis Scale, and adverse events. The three month cosmesis evaluated by physicians' showed 100% of the patients rated with excellent to good cosmesis. Related adverse events reported through three months included a total of fifteen CTC v3 Grade One AEs. Events included erythema, itching, induration, skin firmness, rash/ desquamation, fatigue. Of these events 90% were resolved. The six month cosmesis physician evaluations showed 82% of the patients had excellent to good cosmesis. Twelve of the 13 patients rated the appearance of their breast, in addition to the physician assessment, and 12 out of 12 rated their appearance good to excellent, one patient did not select any rating. Adverse events reported at six months were a total of three new CTC v3 Grade One. These AEs included occurrences of induration, skin firmness, and hyperpigmentation. A patient satisfaction survey at 6 months follow-up was completed by the patients. Certain survey questions required an answer using a scale of 0-6 for the ratings, where six was the best score. The patients' mean overall rating of satisfaction with the study participation was 5.55 out of 6 (min 3, max 6), satisfied with the radiation treatment was rated 5.54 out of 6 (min 3, max 6). All 13 patients, 100%, stated they would recommend this treatment to a friend.  Conclusion: This report provides an early look at the experience of using electronic brachytherapy as a means for intracavitary partial breast radiotherapy. The first cohort has reached a minimum of 6 months of follow up and the results appear promising. Additional patient follow up will be presented at the meeting.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5149.
Objective cosmetic analyses after breast surgery with the breast analysing tool (BAT) correlates with subjective and objective scores: a new tool for prospective trials.
Authors: Krois W , Riedl O , Dubsky P , Jakesz R , Gnant M , Wild T , Fitzal F
Published: December 2010

Abstract #5150

Objective: Objective cosmetic analyses are important to reproducibly evaluate cosmetic outcome after oncoplastic surgery, breast conserving surgery or new radiotherapy methods such as partial breast irradiation. We developed a new objective scoring software, the breast analysing tool (BAT), to measure cosmesis using a single frontal picture. Method: Images from 129 breast cancer patients after breast conserving therapy and radiotherapy were analysed with the software. Distances from the breast border to the nipple, from the Jugulum to the nipple and the breast circumference of one breast was subtracted from the other. The difference in pixels transferred to a breast symmetry index (BSI) by using a mathematic formula. This results in a BSI ranging between 0 (excellent cosmesis) and 11 (bad cosmesis). The results were correlated with the subjective Harris scale ranging from 1-4 (excellent, good, fair and poor cosmesis) and another objective tool, the breast cancer conserving treatment cosmetic result (bcct.core) software. Subjective analyses were done by students (n=4), surgeons (n=10) and experts in breast surgery (n=2) as well as by international experts using a Delphi procedure (n=13). Results: All subjective scores significantly (p<0.05) correlated with the BAT score with a Pearson correlation coefficient of 0.719 (students), 0.697 (surgeons), 0.760 (experts local) and 0.60 (experts international). There was also a significant correlation between the bcct.core and the BAT software with a correlation coefficient of 0.581.    Conclusion: The BAT software is ready to be used for prospective and retrospective trials in local breast cancer therapy.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5150.
Ipsilateral breast tumor recurrence rates in breast cancer patients treated with preoperative chemotherapy, breast conserving surgery and intraoperative radiotherapy with electrons.
Authors: Reitsamer R , Sedlmayer F , Menzel C , Glueck S , Kopp M , Peintinger F
Published: December 2010

Abstract #5156

Introduction: Ipsilateral breast tumor recurrence (IBTR) rates are reported to be increased in patients treated with breast conserving surgery after preoperative chemotherapy. Aim of this study was to evaluate the IBTR rate in patients who were treated with preoperative chemotherapy followed by breast conserving surgery and intraoperative radiotherapy with electrons (IOERT).  Patients and Methods: 84 patients with clinical stage II or III breast cancer were included in the study. Patients received 3 to 6 cycles of anthracyclin/taxane containing preoperative chemotherapy. All patients had breast conserving surgery with sentinel node biopsy and axillary lymph node dissection and received intraoperative radiotherapy with 9 Gy to the 90% reference isodose as an anticipated boost radiation. Whole breast radiotherapy (WBRT) was performed after surgery for all patients.  Results: Pathologic complete response was achieved in 18/84 (21%) patients. Mean resection volume was 250 ml. After a median follow-up of 30.5 months (range 6 – 77 months) no IBTR could be observed. One patient developed a locoregional recurrence in the axilla and nine patients developed distant disease, five of those died of disease. Three patients developed contralateral breast cancer. 71 patients are alive without evidence of disease.  Conclusion: Breast conserving surgery with IOERT in boost modality after preoperative chemotherapy is a reliable tool for the prevention of IBTR. The period between IOERT and WBRT is short as no adjuvant chemotherapy is necessary. Cosmetic outcome is excellent.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5156.
Pharmacogenetic (CYP2D6) and gene expression profiles (HOXB13/IL17BR and molecular grade index) for prediction of adjuvant endocrine therapy benefit in the ABCSG 8 trial.
Authors: Goetz M , Ames M , Gnant M , Filpits M , Jakesz R , Greil R , Marth C , Samonigg H , Suman V , Safgren S , Kuffel M , Weinshilboum R , Erlander M , Ma X , Ingle J
Published: December 2010

Abstract #57

Background: ABCSG 8 is the only prospective trial examining the value of switching patients (pts) to an aromatase inhibitor after tamoxifen (Tam) in which randomization occurred prior to therapy. This trial demonstrated that 2 years (yrs) of Tam followed by 3 yrs of anastrozole (TAM to A) was superior to 5 yrs of Tam (Jakesz SABC 2005). Tam is metabolically activated by CYP2D6 to endoxifen and pts with impaired CYP2D6 metabolism have a higher risk of recurrence (Goetz JCO 2005). The HOXB13/IL17BR (H/I) index (Ma Cancer Cell 2004) is associated with Tam resistance while a 5-gene molecular grade index (MGI) distinguishes outcome in grade II tumors. H/I and MGI are complementary prognostic factors that define low (low MGI), intermediate (low H/I, high MGI) and high risk groups (high H/I and MGI) (Ma Clin Cancer Res 2008). We sought to validate these biomarkers in ABCSG Trial 8, and hypothesized that they may predict the benefit of sequencing TAM to A versus Tam alone.  Methods: From 1996-2004, ABCSG 8 randomized 3,901 pts with ER positive breast cancer. The primary endpoint was event-free survival. Assuming 40% of pts had reduced CYP2D6 metabolism, we designed a nested case control study in which 110 Tam treated cases matched to 2 controls would yield 80% power to detect a relative risk of 2.3 for pts with reduced CYP2D6 metabolism. Additionally, we sought to perform an exploratory analysis of the benefit of switching from Tam to A in pts with impaired vs non-impaired CYP2D6 metabolism. DNA and RNA derived from paraffin embedded sections were used to genotype for CYP2D6 and to obtain H/I and MGI RT-PCR profiles.  Findings: Thus far, CYP2D6 genotyping is complete in 345 pts corresponding to 53 cases/106 controls (Tam) and 62 cases/124 controls (Tam to A). DNA and RNA yields were excellent with a CYP2D6 genotype call rate of >95%. CYP2D6 allele frequencies were as expected: *3 (.01), *4, (.23), *6 (.01), *10 (.02), *17 (.00), and *41 (.10). 44% of pts had reduced CYP2D6 metabolism (at least one CYP2D6 null allele or intermediate metabolizer) and 8% were CYP2D6 poor metabolizers. For H/I and MGI, 340 pts were profiled with 64%, 20% and 16% in the low, intermediate, and high risk groups, respectively. Tissue sectioning/genotyping for additional cases/controls is ongoing.  Discussion: This is the first retrospective analysis of a large Tam versus Tam to A trial to evaluate the association between 1) CYP2D6, HOXB13/IL17BR, and MGI with TAM treatment outcome and 2) the predictive benefit of these biomarkers in pts randomized to switch from Tam to AI versus Tam alone. Final results will be presented at the meeting.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 57.
Molecular targeting of HER2 for diagnosis and therapy of breast cancer.
Authors: Capala J , Kramer-Marek G , Lee S , Hassan M , Kiesewetter DO , Puri A , Chernomordik V , Gandjbakhche A , Griffiths G , Blumenthal R
Published: December 2010

Abstract #6004

Background: Expression of HER2 receptors in breast cancer is correlated with poor prognosis and may be different in distant metastases as compared to the primary tumor. We are developing methods to assess global expression of HER2 in vivo and to deliver therapeutic agents specifically to HER2-positve cells.  Materials and Methods: As the targeting agent we use an Affibody molecule (http://www.affibody.com). These very stable and highly soluble proteins are relatively small (8.3 kDa) and bind to HER2 receptors with high affinity (22 pM). For imaging with PET, SPECT, or optical methods, an appropriate group containing the imaging beacon can be attached by a selective chemical reaction to a unique C-terminal cysteine residue of Affibody. For therapeutic purposes, they can be conjugated to multifunctional nanoparticles containing both imaging and therapeutic agents. We have conjugated affibody molecules with thermo-sensitive liposomes or gold nanoparticles (subsequently activated with neutrons) and characterized their biodistribution using optical imaging and SPECT, respectively. We used PET imaging with 18F-ZHER2-Affibody to monitor the down-regulation of HER2 following four doses (50 mg/kg) of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin, 17-DMAG, an inhibitor of Hsp90 known to decrease HER2 expression. Animals were scanned before and after treatment. Immediately after the last scan, the mice were euthanized and tumors were frozen for ex-vivo analysis of receptor expression. For optical imaging, we used AlexaFluor dyes conjugated with affibody molecules containing an albumin binding domain that extended their circulation time. We have attached nanoparticles (liposomes and gold) to Affibody molecules using the same type of maleimide chemistry.  Results: Our results showed that Affibody molecules do not affect the targeted cells and that their binding does not interfere with either the binding or the effectiveness of trastuzumab. 18F-ZHER2-Affibody was eliminated quickly from blood and normal tissues, providing high tumor/blood and tumor/muscle ratios by 1h post injection. The signal obtained from PET and optical imaging correlated well with the number of receptors expressed in the studied tumors as assessed by western blot, ELISA, and IHC. Following 17-DMAG treatment, the level of HER2 expression, estimated by PET imaging, in BT474 and Mcf-7/clone18 tumors decreased 70% and 30%. This change was confirmed by the biodistribution studies, ELISA and western blot.  Discussion: This strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6004.
Multi-modality imaging of N-methyl-N-nitrosourea-induced mammary tumors by MRI and small animal PET.
Authors: Shoghi KI , He J , Su Y , Yan Y , Rowland D , Garbow J , Mach RH , Lubet RA , You M
Published: December 2010

Abstract #6008

Background: We evaluated the use of high resolution Magnetic Resonance Imaging (MRI) and small animal Positron Emission Tomography (PET) imaging in assessing tumor proliferation and response to therapy in N-methyl-N-nitrosourea (MNU)-induced mammary tumors.  Materials and Methods: The study comprised of 30 untreated MNU rats, 6 Targretin-treated MNU rats, and 6 Vorozole-treated MNU rats. Rats received a baseline imaging session when they developed their first palpable mammary tumor and were subsequently randomized into a control group or a treatment group with either Targretin (220mg/kg in the diet) or Vorozole (1.25mg/kg body weight by gavage). Treatment lasted for 8 weeks following the baseline imaging session. Each rat was imaged for 10-weeks at 2-week intervals with a) FDG to assess the metabolic state of tumors, b) MRI to monitor tumor volume, and c) [18F]ISO-1, a Sigma-2 radiolabeled ligand, to assess the proliferative status of MNU-induced tumors. Static images (10-minute) were obtained 60-minutes post-injection of FDG and [18F]ISO-1. In untreated tumors, a minimum of 40 PET outcome measures were assessed for their ability to predict changes in MRI-derived volume measurements between consecutive imaging sessions. In addition, regression analysis was performed to assess the correlation (R) between the measures. In treated tumors, the time-course of changes in PET outcome measures relative to baseline was evaluated to assess the efficacy of Targretin and Vorozole, in particular in characterizing short-term response (initial 2-weeks) and response to treatment withdrawal (weeks 8-10).  Results: An index characterizing a MRI-normalized [18F]ISO-1 uptake significantly correlated (R=0.7, P<0.0003) with changes in tumor volume between consecutive imaging sessions in untreated tumors. In contrast, there was no correlation between FDG outcome measures and changes in tumor volume. Targretin had the strongest short-term efficacy with a reduction in tumor load, on average, by as much as 60% compared with Vorozole's 20% reduction in tumor load. We observed higher rate of tumor resurgence in rats treated with Vorozole compared with Targretin, suggesting a residual effect of Targretin. In general, PET outcome measures of both FDG and [18F]ISO-1 delineated short-term response to treatment and non-responding tumors. [18F]ISO-1 was more sensitive than FDG in capturing tumor growth following treatment withdrawal at week 8.  Conclusion: MRI and small animal PET provide a platform to assess MNU-induced tumor proliferation and the efficacy of therapeutic interventions non-invasively, as demonstrated in this work.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6008.
Prognostic and predictive value of enzymes of the sphingolipid metabolism in breast cancer.
Authors: Ruckhäberle E , Rody A , Gaetje R , Engels K , Holtrich U , Karn T , Kaufmann M
Published: December 2010

Abstract #6014

Backround:  Sphingolipids do not only have structural roles for the cell membrane, but also act as effector molecules and second messengers in signal transduction with links to cancer initiation, progression and treatment response. Knowledge of sphingolipid's impact on breast cancer and its prognosis is limited to in vitro data. Clinical data is rare but needed.  Material and Methods: We analyzed gene expression of 43 proteins from the sphingolipid pathways in different subtypes of breast cancer using microarray data of 1269 tumor samples (test set n=171; validation sets n=1098). Kaplan Meier analysis of disease free survival was performed to examine the prognostic value of different markers. In addition immunohistochemistry was done to determine whether the genes of the sphingolipid rheostat are expressed in cancer or stroma cells of the tumor.  Results: Significant relationships of several genes of the sphingolipid metabolism with clinical parameters were obtained. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidesynthase (GCS), dihydroceramidesynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a significant worse outcome of patients with high SPHK1 expression. LASS 6 and GCS gene expression had no prognostic impact when the estrogen receptor status was taken into account in multivariate analyses. Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor.  Conclusions: These studies of a large cohort of clinical breast cancer samples reveal that sphingolipids do play a role in breast cancer. Enzymes of the sphingolipid metabolism have impact on the prognosis of breast cancer patients and targeting the sphingolipid rheostat can open new treatment options.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6014.
Notch-1 activates ErbB-2 through a PEA3-dependent mechansim.
Authors: Clementz AG , Osipo C
Published: December 2010

Abstract #6015

Over-expression or gene amplification of ErbB-2 occurs in 25% of breast cancer which is associated with malignancy, poor overall survival, and resistance to some anti-hormonal therapies. Although trastuzumab has had a tremondous impact on survival, resistance results within the first year of treatment in 15% of cases and remains a major problem in the metastatic setting. We have recently published (Osipo et al., 2008. Oncogene) that the expression and activity of Notch-1, a potent breast oncogene and cell fate determinant is increased in ErbB-2 positive breast cancer cells in response to trastuzumab. Furthermore, we have shown that Notch-1 might contribute to trastuzumab resistance. In the current study, we asked whether Notch-1 could activate ErbB-2. Results demonstrated that a Notch inhibitor, γ-secretase inhibitor (GSI) or a Notch-1 siRNA decreased tyrosine phosphorylated and total ErbB-2 expression in SKBR3, MCF-7, and MDA-MB-231 cells. Furthermore, overexpression of constitutively active Notch-1 increased tyrosine phosphorylated ErbB-2 and reversed the inhibition by a GSI. ErbB-2 promoter activity using a luciferase reporter construct was decreased by Notch-1 siRNA only when the PEA3 Ets binding site was mutated. These results suggest that Notch-1 is an activator of ErbB-2 in breast cancer cells that express low or inactive PEA3.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6015.
Involvement of notch and RUNX pathways in human breast cancer.
Authors: Yong T , Meyers S , Davis N , Sun A
Published: December 2010

Abstract #6021

Background: Recent studies have demonstrated that the Notch signaling and the RUNX-mediated transcription pathways converge in mammalian hematopoiesis where they regulate proliferation and development. Evidence from in vitro experiments, mouse models and human tumors demonstrate that Notch signaling plays an oncogenic role in human breast cancer. Studies of RUNX factors involvement in human breast cancer suggest that the Core Binding Factor [CBF, a heterodimeric transcription factor composed of a RUNX DNA binding protein (RUNX1, RUNX2, or RUNX3) and a single non-DNA binding component termed CBFβ] participates in regulating the malignant phenotype of the tumor. Thus, the RUNX factors are primarily important to Notch signaling, and it seems likely that Notch:RUNX cross-talk will be important to cellular function and disease in a variety of tissue types. Here, we present our findings on cross-talk between Notch and RUNX pathways, which will lead to a better understanding of Notch and RUNX regulated transcription in breast carcinogenesis.  Material and Methods: The expression and localization of the Notch1 NICD and RUNX2 in normal breast tissue and breast tumor were determined by immunohistochemistry on human breast tissue microarray obtained from Feist-Weiller Cancer Center. The expression profiles of Notch and RUNX were evaluated by real-time RT-PCR, Western Immunoblot, and electrophoretic mobility shift assay (EMSA).  Results and Discussion: We found activated Notch1 and RUNX2 predominantly expressed in the cytoplasm and nucleus of cells in human normal breast tissues, respectively. However, in human breast cancer tissue samples, activated Notch1 was found in the nucleus in a high proportion of the cancer cells, while RUNX2 presence in the nucleus was much reduced and this could be due to mislocalization, over-expression or delayed degradation of the proteins. The major transcript in the breast cancer cell lines is RUNX1, but by EMSA, RUNX2 factor is the predominant biologically active and functional form of RUNX factors associated with breast cancer cell line-derived CBF. Transcript degradation and protein stability may play significant role in the regulation of RUNX factors in breast carcinogenesis. Down-regulation of CBFβ was found to affect Notch signaling in breast cancer cells and this may lead to the identification of new therapeutic targets for the successful treatment of breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6021.
Cytotoxic doses of curcuminoids provoke a down-regulation of phospholipid biosynthesis – a focused metabolomic profiling.
Authors: Bayet-Robert M , Morvan D , Chollet P , Barthomeuf C
Published: December 2010

Abstract #6029

Background: Curcuminoids of which curcumin have proved efficacy in breast cancer prevention [Aggarwal et al, Anticancer Res (2003);23(1A):363]. In addition, there is growing evidence that these compounds may treat cancer [Karunagaran et al, Curr Cancer Drug Targets (2005);5(2):117]. However, the mechanisms of cytotoxicity of curcuminoids remain partially unknown. Functional genomics include several tools increasingly applied in pharmacology, like DNA and RNA microarrays, and proteomics. It was recently shown that a novel post-genomics technology, NMR spectroscopy-based metabolomics, was able to unravel metabolic pathways of the response to anticancer drugs [Morvan et al, Cancer Res. (2007);67(5):2150 ; Bayet-Robert et al, AACR Proceedings (2008);abstract 3938]. We applied this technique to investigate the metabolic features of the response of MCF7 human breast carcinoma cells to curcuminoids. We here report the analysis of the phospholipid metabolism subset of our data.  Material and Methods: MCF7 cells were exposed to 10 µg/mL curcuminoids and followed at 12, 36 and 72 h intervals of time. Cytotoxicity assay was performed using Hoescht 33342. A novel high resolution magic angle spinning 1H-NMR spectroscopy-based metabolomics technique was used to obtain a quantitative metabolite profiling of about thirty metabolites, among which 8 phospholipid metabolism derivatives, in intact treated and untreated MCF7 cells.  Results: A 72 h-exposure to 10 µg/mL curcuminoids decreased MCF7 cell survival to 4% (p=0.0039). Polyunsaturated fatty acids increased by +120% at 36 h and +62% at 72 h (both p=NS, Mann-Whitney test), phosphoethanolamine by +24% at 36 h (p=NS) and +120% at 72 h (p=0.0062), cytidinediphosphocholine (CDP-Cho) by +22% at 12 h, +132% at 36 h (both p=NS), and +547% at 72 h (p=0.0062). There was a decrease of phosphatidylcholine (PtdCho) by -30% at 36 h (p=NS), and -42% at 72 h (p=NS). Glycerophosphoethanolamine decreased by -28% at 12 h (p=0.0446) and glycerophosphocholine by -53% (p=0.0106). The PtdCho/CDP-Cho ratio decreased from +93% to +8% between 12 and 72 h, which indicates a blockade of PtdCho biosynthesis.  Discussion and Conclusion: Our results are consistent with a rapidly progressive down-regulation of cholinephosphotransferase activity in response to curcuminoids, a biochemical mechanism closely linked to apoptosis [Miquel et al, J Biol Chem. (1998);273(40):26179 ; Cui et al, Biochim Biophys Acta. (2002);1585(2-3):87].

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6029.
Protein expression comparison and physiological profiling of matched pairs DCIS/normal and invasive/normal primary epithelial cells.
Authors: Minkeu A , Quong JN , Rosenberg A , Brill KL , Quong AA
Published: December 2010

Abstract #6031

Background: Approximately 50% of patients who are diagnosed with Ductal Carcinoma In-Situ (DCIS) develop invasive breast cancer. The progression from DCIS to invasive cancer is not well understood. To better understand the range of biological processes involved in this progression, we have successfully isolated primary epithelial cell cultures from patient breast tissue and are using mass spectrometry-based methods to identify differentially expressed proteins in these different cell types. Bioinformatics analysis was used to identify differences in the underlying pathways.  Methods: Primary Human Mammary epithelial cells from DCIS and Stage 3 Invasive tumors and normal tissue from the same patient were isolated immediately after surgery. Cells were maintained in defined CNT27 media for up to 2 passages and were characterized for epithelial and breast cancer markers by immunohistochemistry and western blot. Tamoxifen response was measured by cell proliferation assay. Protein lysates were labeled with the ITRAQ isobaric tags. Two-dimensional high-pressure liquid chromatography and mass spectrometry were used to identify differentially expressed proteins.  Results: The proliferation rate of cells from DCIS tissue was 33% higher than the matched control cells and 50% higher from invasive tissue. Furthermore, Tamoxifen was more effective at inhibiting DCIS cell proliferation than the invasive cells in a dose- and time-dependent manner. We identified 50-100 proteins differentially expressed between the different tissue types. Validated tumor markers such as members of protein S100 family were significantly different between the tumor and normal samples. Thrombospondin, a known angiogenesis inhibitor, had similar concentrations in normal and DCIS tissues but was down-regulated (ratio=0.4) in the invasive tumor samples.  Discussion: Using the analogous methods from gene expression studies, we found that pathways associated with processes such as differentiation, angiogenesis, and invasion are enriched in the proteome of the invasive tumors. Further comparisons of additional samples is continuing with the goals of better understanding the underlying biology that drives DCIS to invasive breast cancer and ultimately defining markers that could be used in better prognosis for DCIS patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6031.
Uniformly positive (>80%) HER2 expression maximizes sensitivity and specificity for prediction of response to trastuzumab in CALGB 9840.
Authors: Rimm DL , Broadwater G , Friedman P , Berry D , Seidman A , Hudis C , Winer E , Harris L , Thor A
Published: December 2010

Abstract #6046

Purpose: The recent ACO/CAP guidelines revised the cut-point for HER2 expression by immunohistochemistry (IHC) from 10% up to 30%. Recently we showed that heterogeneity of expression is a function of the assessed biomarker and, for HER2, >80% area of expression was most prognostic and predictive. Here we test this hypothesis in CALGB 9840, a prospective cooperative group clinical trial of Paclitaxel (P) and Trastuzumab (T).  Experimental Design: Tissue from 274 cases selected from cases with available slides from the TH arm were first scored by pathologists as 0-3+, but then again scored to assess percentage of tissue staining with prospectively chosen cut points of >10%, >30% or >80%. Response was assessed by modified RECIST criteria, including complete and partial response. Stable disease and progressive disease were considered non-response.  Results: Amongst the 176 cases treated with P+T there were 90 (52%) responses (PR+CR), 3 cases were unevaluable for response. In the subset of cases treated with T, there was inconsistent differences in the >10% vs the >30% categories but the >80% category was uniformly statistically significantly predictive of response. Receiver Operator Characteristic (ROC) curves constructed to assess prediction of response showed areas under the curves (AUC) of 0.59 for IHC and 0.60 for FISH suggesting both are failed tests for defining an optimal cut-point. However, selecting only the cases scored as 3+ shows an AUC of 0.72 and the optimal cut-point is for the test is at 95% positive 3+ HER2 staining. This modification of the cut-point would have denied T to 6 of the 49 patients who responded to therapy in the 3+ group. However, there were also 41 (of 86) patients in the 0,1+ and 2+ groups that responded to therapy, raising the possibility that the response in those 6 patients may have been unrelated to T therapy.  Conclusions: When using clinical outcome to assess the value of the current tests for prediction of response to T, ROC curves suggest that both the old and new HER2 tests fail at defining an optimal cut-point. However, using only strongly positive cases (3+), an acceptable test can be achieved which is maximized at 95% area of expression. This cut-point, while statistically rigorous, is limited by the fact that the patients were treated with both P and T. This result suggests a similar analysis is warranted on a larger cohort of T treated patients, perhaps in the adjuvant or neoadjuvant setting. The availability of other options for HER2 amplified cases (ie lapatinib) and the increased use of T in the adjuvant setting warrant optimization of tests for the best possible patient selection.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6046.
p53 mutational status, but not immunohistochemical staining (IHC), is associated with a clinical response of the primary tumor in women receiving neoadjuvant docetaxel-capecitabine chemotherapy for locally advanced breast cancer.
Authors: Ross JS , Perou CM , Aki N , Patten N , Wu L , McKenna EF , Lawrence H , Royce M , Avisar E , Gluck S
Published: December 2010

Abstract #6047

Background: The XeNa trial enrolled 157 patients in a multicenter Phase II study of neoadjuvant capecitabine and docetaxel in women with operable early stage breast cancer (T2-T3, N0-N1). HER2-positive patients also received trastuzumab. The primary endpoint was the rate of pathological complete response (pCR; residual cancer burden [RCB] = 0) and near-complete response (npCR; RCB = 1). A secondary endpoint of the study was to evaluate the association between p53 alterations and response to chemotherapy.  Methods: p53 status was assessed in by mutational analysis on all cases using a microarray-based resequencing assay (AmpliChip p53 – Roche Molecular Systems, Pleasanton, CA); and also by IHC (using the Bp-53-11 antibody (CONFIRM™, Ventana, Tucson, AZ) in 120/157 (76%) of the cases. Gene expression profiling for breast cancer subtypes was also performed using a new 50-gene centroid-based method.  Results: p53 mutations were detected in 78/157 (50%) of cases with 70% missense, 17% nonsense, 11% frame shift and 1% splice site. Silent mutations were rare (1%). Concordance of AmpliChip mutation status with IHC + staining was 65% (78/120 samples). Of the 16/60 (27%) cases which were IHC - and AmpliChip p53 mutation +, 10 (63%) had non-missense mutations which are typically IHC -. In the 60 mutation negative cases with IHC data, there were 26/60 (46%) IHC + cases. p53 mutation + status by AmpliChip was highest in Basal-like (75%) and HER2-enriched (57%) subtypes and lower in Luminal A (29%), Luminal B (12%) and Normal-like (39%). p53 IHC + staining varied little among the subtypes (35 to 50%) and did not correlate with p53 mutational status. The frequency of pCR and npCR was significantly higher in cases with p53 sequence mutations (20/58 - 35%) than in patients without mutations (7/52 - 13%; p = 0.014; 2-tailed Fisher's Exact Test), and this trend was apparent in both HER2+ and HER2- cases. IHC status did not correlate with treatment response (18% pCR and npCR in IHC- cases vs. 27% in IHC + cases; p = 0.35). Notably 3 cases achieving pCR had non-missense p53 mutations and were IHC -.  Conclusion: P53 mutations were found in 50% of locally advanced breast cancers and were more frequent in Basal-like and HER2-enriched subtypes and less common in other subtypes. IHC staining lacks sensitivity and specificity for the presence of p53 mutations. In contrast to studies of anthracycline-based therapy, tumors harboring p53 mutations, especially missense mutations, respond better to this taxane-based regimen than wild-type tumors. P53 mutational analysis is a more accurate predictor of treatment response than IHC.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6047.
Comparison of MRI tumor volumetric and diameter measurements for predicting recurrence free survival in breast cancer patients undergoing preoperative chemotherapy.
Authors: Newitt DC , Gibbs J , Partridge SC , Li KL , Lobo EC , Esserman L , Hylton NM
Published: December 2010

Abstract #6053

Background: Assessment of tumor response to chemotherapy has traditionally relied on the bidimensional tumor measurement guidelines proposed by the World Health Organization (WHO, 1979) and more recently on the unidimensional Response Evaluation Criteria in Solid Tumors (RECIST, Therasse et al., 2000). MRI is being used increasingly to monitor breast cancer response to preoperative chemotherapy and allows both linear and volumetric assessment of tumor size. The purpose of this study was to compare pre- and post-treatment RECIST, WHO, and volumetric measures of tumor size on MRI for predicting recurrence free survival (RFS) in patients undergoing preoperative chemotherapy.  Materials & Methods: 56 patients with locally advanced breast cancer were imaged with MRI (1.5T GE scanner) before and after 4 cycles of preoperative chemotherapy. Tumor longest diameter (LD) at physical exam (ClinLD) was recorded before (N=56) and after (N=48) treatment. Fat suppressed, contrast enhanced, T1-weighted sagittal 3DFGRE images (TE/TR=8/4.2ms, flip=20°, 2mm thick, 18-20cm FOV, 256x192 matrix) were acquired for tumor size measurements.  All MRI LD measurements were made manually following RECIST and WHO guidelines. Tumor volume was measured with a semi-automated tumor segmentation algorithm based on a specific enhancement ratio calculation. Univariate Cox proportional hazards analysis was used to assess the value of clinical, pathology, RECIST, WHO, and volume measurements for predicting RFS. Variables with p<0.15 were combined in a stepwise multivariate model to determine the greatest predictive value.  Results: 23 patients have recurred since surgery (mean time 132 weeks). The mean RFS in the non-recurrent group is 330 weeks. Results for univariate and multivariate analysis are show in the Table. Final tumor volume was most predictive of recurrence free survival, and was the only variable found to be an independent significant predictor in the multivariate analysis. Age, tumor grade, and positive lymph node status were not significant predictors.  Discussion: Post-chemotherapy tumor volume calculated via a semi-automatic algorithm was found to be a significant predictor of RFS for patients undergoing preoperative chemotherapy, out-performing manual 1D RECIST, 2D WHO, and clinical measurements. The results support previous work demonstrating the value of MRI tumor volume for predicting patient outcome.   

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6053.
Tumor tissue levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) and survival following adjuvant chemotherapy in pre-menopausal lymph node-positive breast cancer patients (N=525).
Authors: Schrohl A , Look MP , Meijer-van Gelder ME , Foekens JA , Brünner N
Published: December 2010

Abstract #6054

Predictive markers are needed to guide planning of adjuvant therapy for breast cancer patients. We have recently shown that high tumor tissue levels of TIMP-1 are associated with decreased response to chemotherapy in metastatic breast cancer (Schrohl et al, Clin Cancer Res, 2006) suggesting that TIMP-1 may be a predictive marker in breast cancer patients.  Purpose: This study investigates the association of tumor tissue TIMP-1 levels with response to adjuvant chemotherapy with CMF (cyclophosphamide/methotrexate/5-fluorouracil) or an anthracycline-based regimen.  Patients and Methods: 525 pre-menopausal lymph node-positive patients were included; 324 patients received adjuvant CMF, 99 received an adjuvant anthracycline-containing regimen and 102 had no adjuvant chemotherapy. Total TIMP-1 levels were measured using ELISA in cytosolic extracts of frozen primary tumors. Using the untreated patient group as a reference group we analyzed the benefit of adjuvant CMF and anthracyclines in TIMP-1 high and low patients, respectively. The median TIMP-1 concentration was used to dichotomize patients into high and low TIMP-1 groups. End points were disease-free and overall survival (DFS, OS).  Results: The median TIMP-1 level in the total patient group was 12,54 ng/mg of total protein (range, 0 – 112,9 ng/mg). TIMP-1 levels in subgroups according to adjuvant therapy were not significantly different (P=0,20). In a multivariate model including basic clinico-pathological parameters, TIMP-1 low and high patients benefited differentially from adjuvant CMF and anthracyclines when compared to untreated patients. In particular, patients with high tumor levels of TIMP-1 had little benefit from adjuvant anthracyclines. Hazard ratios (HR) and 95% confidence intervals (CI) are given in the table for the analysis of DFS. A similar pattern was seen in the analyses of OS. In the CMF-treated group, both TIMP-1 low and high patients had significantly better survival than untreated patients (P<0,01). Among anthracycline-treated patients those with TIMP-1 low tumors appeared to benefit more from the adjuvant therapy than TIMP-1 high patients although these results did not reach significance in the present analyses.    Conclusion: This study suggests that high tumor tissue TIMP-1 levels are associated with decreased benefit from adjuvant chemotherapy. Especially in the group treated with anthracycline-based therapy there is a strong tendency for TIMP-1 high tumors to be less sensitive to the treatment. This group, however, is small and should be enlarged to confirm our results. In the group treated with CMF all patients benefit from the therapy with TIMP-1 high patients having slightly less benefit than TIMP-1 low patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6054.