bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2-13-02: Phase II Study of Glucosamine with Chondroitin on Joint Symptoms Induced by Aromatase Inhibitors in Breast Cancer Patients
Authors: Greenlee H , Crew KD , Shao T , Kranwinkel G , Brafman L , Kalinsky K , Maurer M , Tsai WY , Hershman DL.
Published: April 2011

Background: Aromatase inhibitors (AIs) are commonly used as adjuvant endocrine therapy in postmenopausal women with hormone receptor-positive breast cancer (BC). However, many patients treated with AIs experience joint symptoms which may lead to non-adherence to treatment. We examined whether glucosamine plus chondroitin improves AI-induced arthralgias in women with early stage BC.

Methods: This is a single-arm Phase II study evaluating the efficacy of glucosamine and chondroitin in postmenopausal BC patients with moderate to severe AI-induced arthralgias. Patients took glucosamine sulfate (1500mg/d) + chondroitin sulfate (1200mg/d) for 24 weeks, and were assessed every 6 weeks. The primary endpoint was change in Outcome Measure in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) criteria at 24 weeks. Secondary endpoints include changes in Brief Pain/Stiffness Inventory-Short Form (BPI-SF), Western Ontario and McMaster Universities Osteoarthritis (WOMAC), Modified Score for the Assessment and Quantification of Chronic Rheumatoid Affections of the Hands (M-SACRAH), quality of life, grip strength and estradiol level. A Simon two-stage design was used. If 7/22 responses were observed, the trial would continue. Results: Of 36/46 patients enrolled to date, 21 were evaluable at 24 weeks. Median age: 61 (46-80); White/Hispanic/Black/Asian: 11/5/4/1. At 24 weeks, 12/21 patients (57%) had improved pain/stiffness or function in their knees or hips, and 14/21 patients (67%) had improvement in their hands, and 80% had improvement in hands and/or knees. There were also decreases in BPI pain/stiffness severity and functional interference (P<0.05). Reassuringly, there was no increase in serum estradiol levels. Conclusion: Glucosamine and chondroitin is a well-tolerated and potentially effective treatment for AI-induced arthralgia. Evaluation of these agents in a placebo controlled phase III study is warranted.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-13-02.
Abstract P2-14-04: Co-Administration of GnRH-Agonists with Adjuvant Chemotherapy in Young Breast Cancer Patients: The Effect on Ovarian Function and on Clinical Outcome
Authors: Goldberg H , Stern M , Risel S , Kuten A , Evron E , Peretz T , Katz D.
Published: April 2011

Background: Loss of fertility is a major concern for young breast cancer (BC) patients who require adjuvant chemotherapy. The risk of ovarian failure depends on patient's age and chemotherapy regimen. Use of GnRH agonists (GnRHa) has been proposed as a mean for ovarian function preservation but has been investigated mainly in small single arm studies. Objectives: To evaluate ovarian function preservation and disease free survival (DFS) in young breast cancer patients following GnRHa and adjuvant chemotherapy co-administration. Methods: In this multicenter retrospective study, we reviewed medical records of 560 breast cancer patients diagnosed before the age of 40, from 1990 through 2006. All patients were treated in one of 5 Israeli cancer centers with standard adjuvant or neoadjuvant chemotherapy regimens with curative intent. GnRHa were prescribed at the discretion of the treating physician. Ovarian function preservation was determined by resumption of menses, premenopausal hormonal profile or pregnancy during follow up. Multivariate analysis was applied in order to determine whether coadministration of GnRHa with chemotherapy affects DFS. Results: Data was available for 71 patients who received GnRHa during chemotherapy (group A), 60 of those were evaluable for fertility and 68 were evaluated for DFS. The group not receiving GnRHa included 460 patients (group B). Of those 207 were evaluable for fertility and 370 for DFS. Data concerning patients’ characteristics and outcome in each group are presented in the table 1. In a Cox regression model including age, stage, ER/PR status and GnRHa administration, the latter variable predicted a better DFS rate (RR 0.31; 95% CI 0.16-0.70; p=0.003), excluding the subgroup with ER/PR positive tumors (p=0.128). This probably reflects selection bias of prognostic factors not included in the analysis and not a real anti neoplastic hormonal effect. Conclusions: Patients who received GnRHa were younger and had less advanced disease. Fertility was preserved independent of GnRHa administration and regardless of cumulative doses of cyclophosphamide or taxans. This observation suggests that GnRHa are unnecessary for fertility preservation in patients younger than 40yrs. No detrimental effect of GnRHa during chemotherapy was observed on disease outcome.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-14-04.
Abstract P2-16-03: Sorafenib in Combination with Paclitaxel as a First-Line Therapy in Patients with Locally Recurrent or Metastatic Breast Cancer: Overall Survival Results from a Double-Blind, Randomized, Placebo-Controlled, Phase 2b Trial
Authors: Bondarde S , Kaklamani V , Prasad Sahoo T , Lokanatha D , Raina V , Jain M , Schwartzberg L , Gradishar WJ.
Published: April 2011

Background: Sorafenib (SOR) is an oral multikinase inhibitor that targets tumor growth and angiogenesis. We previously reported progression-free survival (PFS) data from the primary analysis of NU07B1 (SABCS 2009 abstr 44), a phase 2b trial assessing the combination of SOR + paclitaxel (PAC) in patients with advanced breast cancer (BC). Here we report overall survival (OS) data, a secondary endpoint.

Methods: NU07B1 is a multinational (India, United States, Brazil) phase 2b study in HER2-negative patients with locally advanced or metastatic BC. Eligibility criteria allowed prior endocrine or radiation therapy and adjuvant use of taxanes and other chemotherapy. Patients with prior chemotherapy for advanced BC were not eligible. Patients were randomized (1:1) to receive PAC (90 mg/m2 IV weekly, 3 wk on/1 wk off) with placebo (PL) or SOR (400 mg po, twice daily). The primary endpoint was PFS. The sample size was estimated at 220 patients based on patient accrual projections and estimated rate of PFS events. OS analysis was planned after 120 events.

Results: A total of 237 patients were enrolled from June 2007 to January 2009. Study arms were balanced for age (mean, 51.9 y), ECOG performance 0 (45%) and 1 (53%), stage IV disease (85%), visceral metastatic disease (75%), and previous non-metastatic chemo (58%). In the primary analysis, PFS trended in favor of SOR+PAC vs PL+PAC with median PFS of 6.9 vs 5.6 mo (HR 0.79, 95% CI 0.56-1.11; 1-sided log rank P=0.09); Grade 3/4 toxicities included hand-foot skin reaction (30% vs 3%), neutropenia (13% vs 7%), and anemia (11% vs 6%); treatment-related deaths occurred in 2 patients (malaria and liver dysfunction), both in the SOR+PAC arm. Overall survival data are expected by 4th quarter of 2010 and will be presented, as well as updated safety data. Conclusions: Further study with SOR+PAC appears warranted but may require adjustments to the dosing algorithm and patient selection criteria to provide a more defi-nitive assessment of benefit-to-risk profile. OS data will help to determine whether this combination should be pursued further.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-16-03.
Abstract P2-16-09: Wound Healing and Catheter Thrombosis after Implantable Venous Access Device Placement in 266 Metastatic Breast Cancer Patients Treated with Bevacizumab
Authors: Kriegel I , Fourchotte V , Cottu PH , Fromantin I , Salmon RJ , Esteve M.
Published: April 2011

OBJECTIVES: To determine in a population of metastatic breast cancer the incidence of wound dehiscence after placement of an implantable venous access device (VAD) in patients treated with bevacizumab, observe the optimum interval between placement and initiation of treatment and study the risk of catheter thrombosis.

PATIENTS AND METHODS: Between 1/1/2007 and 31/12/2009, this study enrolled all VADs placed by 14 anesthetists: 273 VADs in patients treated by bevacizumab for metastatic breast cancer and 4196 VADs in patients not treated by bevacizumab. The medical charts of the 266 metastatic breast cancer patients treated with bevacizumab between 1/1/2007 and 31/12/2009 were reviewed up until 1/3/2010. A VAD was placed in all patients (goal standard in our institution). 7 patients in whom the VAD was inserted in another institution were excluded from this study. The VAD was removed and replaced in 14 patients with continuation of bevacizumab after replacement of the VAD

RESULTS: 1 patient was lost to follow-up. Thirteen cases of wound dehiscence occurred in 12 patients requiring removal of the VAD (5,1 %). All cases of dehiscence occurred when bevacizumab was administered during the first 7 days after VAD placement. Bevacizumab therapy was initiated less than 7 days after VAD placement in 150 cases, requiring removal of the device in 12 patients (8.6%). The risk of dehiscence was the same from 0 to 7 days. In parallel, the VAD wound dehiscence rate in patients not receiving bevacizumab was 8/4,197 (0.19%) (Fisher's test significant, P<0.001). No risk factors were identified: anesthetists, learning curves, irradiated patients. In particular, 70 ports were placed by juniors with 6 dehiscences, 80 ports by seniors with 7 dehiscences. VAD thrombosis occurred in 4 patients (1,5 %) 3, 5, 7 and 22 months after VAD placement. In all these cases, VAD placement was at the time of initiation of bevacizumab therapy. In parallel, VAD thrombosis occurred in 51/4,197 (1.2%) patients not receiving bevacizumab (Fisher's test not significant; p=0.43).

The other indications for VAD removal were end of treatment: 3 (0,91 %), mechanical problem other than thrombosis: 6 (1,8 %), local infection without wound dehiscence: 1 (0,31%). Bevacizumab was permanently discontinued in 5 patients related to wound dehiscence and in 1 patient due to extensive skin necrosis. Bevacizumab was continued in the other 7 patients.

CONCLUSION: The risk of VAD thrombosis does not require any particular primary prevention. These data suggest the need to observe an interval of at least 7 days between VAD placement and initiation of bevacizumab therapy. Poor healing is a major complication of VAD, interfering with continuation of chemotherapy and the patient's subsequent management, resulting in a real risk of loss of chance.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-16-09.
Abstract P2-16-11: Metronomic Chemotherapy with Cyclophosphamide (CY) and Doxorubicin (DOX) Inhibits Tumor Growth and Metastases Development of Mouse Mammary Adenocarcinoma M-406
Authors: Mainetti LE , Fernandez Zenobi MV , Rozados VR , Scharovsky OG.
Published: April 2011

Metronomic chemotherapy refers to the chronic, equally spaced, deliver of low doses of different chemotherapeutic drugs, without extended interruptions. We have already demonstrated that the administration, as an intervention strategy, of metronomic CY plus Celecoxib in M-406 and M-234p mammary adenocarcinomas-bearing mice, inhibited tumor growth and metastases development, increasing the survival rate. Our present aim was to study the effect of metronomic chemotherapy with CY + DOX in the M-406 tumor-model. CBi inbred mice were s.c. and i.v. challenged with M-406 cells. On day 9 (s.c.) and day 3 (i.v.), mice were distributed in four experimental groups (n=5-6/group) that received: I) Control: 0.2ml of saline i.p. 3 times/week; II) CY (20mg/kg of body weight, daily) in the drinking water; III) Dox, i.p., 0,5mg/kg, of body weight, 3 times/week; IV) Treatment II + III. Tumors were measured and body weight was determined twice a week. Blood samples were taken at the beginning and at the end of the experiments, to determine total leukocytes count. In the M-406 s.c. model, by day 19, a significant inhibition of tumor growth was evident in group II and IV with respect to control mice (P<0.01). Moreover, on day 41 the tumor volume of animals in group IV was significantly lower than that in group II. Also in group IV, 1 mouse over 6 showed a complete remission. The survival of mice in group IV was significantly higher than in the three other groups. In fact, on day 54, when all the animals belonging to groups I, II and III had already died, 5/6 mice in group IV were alive. In the M-406 i.v. model, animals were sacrificed on day 24 when the first mouse showed metastatic illness signs. The number of lung metastasis was significantly lower in group IV when compared to the control group (P<0.05). The animals in groups II and III (with single-drug treatments) showed intervening values. The total metastatic volume (Σeach metastasis volume) in groups II, III and IV was lower than that of group I (P<0.01), the group IV showing the lowest one. In both experiments (s.c. and i.v.) the treatment was devoid of toxicity, as evaluated by body weight evolution and, importantly, by total leukocytes count. These results clearly show that metronomic chemotherapy with a combination of cyclophosphamide and doxorubicin as an intervention strategy is able to inhibit significantly tumor growth and metastasis development in the M-406 mammary adenocarcinoma tumor-model, while being devoid of toxicity. To our knowledge, this is the first time that a metronomic schedule combining cyclophosphamide and doxorubicin is employed for the treatment of a mammary adenocarcinoma. The characteristics and the effect of the therapeutic schedule herein utilized suggest its implementation at the clinical setting in the near future.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-16-11.
Abstract P2-21-01: The Potential Utility of Curcumin and/or Herceptin in the Treatment of HER-2 Overexpressed Breast Cancer — An In Vitro and In Vivo Comparison Study
Authors: Lai H-W , Kuo S-J , Chen S-T , Chen D-R.
Published: April 2011

Despite the success of herceptin, a significant proportion of HER-2 positive breast cancer patients responded poorly to the treatment. Curcumin (diferuloylmethane), derived from Curcuma longa L., can inhibit the formation of tumors, induce apoptosis, and act on a variety of signal transduction pathways. We have hypothesized that the property of curcumin to downregulate EGFR, HER-2 oncoprotein, and signal transduction pathway of PI3K/Akt, and MAPK may be used in the treatment of HER-2 over expressed breast cancer. In this preliminary study, we showed curcumin decrease the viability of various breast cancer cell lines (MCF-7, MDAMB-231, MCF-10A, BT-474 and SKBr-3(herceptin resistant, hr)). The combination of curcumin did not interfere with the action of herceptin. The herceptin sensitive BT-474 and herceptin resistant SKBr-3(hr) were all sensitive to curcumin treatment. In western blot analysis, the phosphorylation of HER-2, Akt, MAPK were reduced in BT-474 cells when treated with herceptin. In SKBr-3(hr) cells, the addition of herceptin did not downregulate the phosphorylation of HER-2, Akt, and MAPK. When treated with curcumin, the phosphorylation of HER-2, Akt, and MAPK of SKBr-3(hr) were decreased combined with the downregulation of HER-2 oncoprotein in a dose and time dependent manner.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-21-01.
Abstract P3-01-01: Cyfra 21-1 Is Correlated with Circulating Tumor Cells Detection and Patient Outcome in Metastatic Breast Cancer: Results of a Substudy of the Prospective IC 2004-06 Trial
Authors: Bidard F-C , Hajaje D , Bachelot T , Brain E , Campone M , Delaloge S , Mathiot C.
Published: April 2011

Background: The Cyfra 21-1 epitope is a polypeptide originating from cytokeratin-19 fragments possibly released by cell death and was an interesting serum marker in small and/or retrospective studies in breast cancer. On the back of circulating tumor cell (CTC) detection, the prospective IC 2006-04 study planned serum markers (CEA, Ca15.3, Cyfra 21-1) measurements.

Methods: Metastatic breast cancer patients were included in a multicentric non-interventional study before the start of the 1st line chemotherapy. Blood biomarkers, including CTC detection (CellSearch®, ≥5CTC/7.5ml), were analyzed at 4 different times: before the start of the chemotherapy, before cycle 2 (ie mostly at day 21), at first tumor evaluation (ie before cycle 3/4) and at tumor progression.

Results: 191 of the 267 patients included had Cyfra 21-1 assessed at inclusion. CTC level was ≥5CTC/7.5ml in 44% of the cases. Cyfra 21-1>ULNV (in 65% of pts) was not correlated with tumor ER/PR/HER2 status but strongly correlated with PS (p=8e-04), number of metastatic sites (p=0.0001), Ca15.3 (p=0.009), CEA (p=0.005), LDH (p=7e-08) and CTC detection (p=2e-06).

CTC (≥5CTC/7.5ml) and Cyfra21-1 (>ULNV) correlation (N patients)

The effectiveness of curcumin in the treatment of HER-2 overexpressed breast cancer was further confirmed through in vivo BT-474 xenograft model. The comparable ability of curcumin to inhibit cell proliferation, mobility, and angiogenesis as herceptin, combined with the potential ability to overcome the herceptin resistance, curcumin may be a novel agent in the treatment of HER-2 overexressed breast cancer.

Logistic regression showed that LDH (p=0.0005) and CTC (p=0.0004) were the two independent predictors of Cyfra 21-1 elevation. Baseline Cyfra 21-1 >ULNV was associated with early tumor progression (RECIST, p=0.03). In multivariate analysis including standard biomarkers (without CTC), baseline Cyfra 21-1 was independently associated with PFS (p=0.008, RR), together with PS (P<0.001), triple-negativity (P<0.001) and CEA (p=0.02). Multivariate analysis with CTC, subsequent Cyfra 21-1 analyses (cycles 2, 3-4 and progression) and correlation with tumor response and PFS will be will be shown at the meeting. Conclusion: Cyfra 21-1 is a commonly elevated serum marker in metastatic breast cancer and has an independent prognostic value. Although highly correlated, Cyfra 21-1 appears to be more frequently positive in metastatic breast cancer patients than CTC count. Multivariate analyses comparing CTC and Cyfra 21-1 at different time points will be shown at the meeting.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-01-01.
Abstract P3-01-02: Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast Cancer Patients
Authors: Ng EKO , Leung C , Shin VY , Chan A , Wong CLPL , Ma ESK , Jin HC , Chu KM , Kwong A.
Published: April 2011

Background: Previously, we have examined the methylation status of SLC19A3 (solute carrier family 19, member 3) promoter and found that SLC19A3 was epigenetically down-regulated in gastric cancer. Here, we aim to develop a new biomarker for cancer diagnosis using methylated SLC19A3 DNA in plasma.

Methods: SLC19A3 gene expression was examined by RT-qPCR. Methylation status of SLC19A3 promoter was evaluated by methylation-specific qPCR. A robust and simple methylation-sensitive restriction enzyme digestion and real-time quantitative PCR assay was developed to quantify SLC19A3 DNA methylation in plasma. Results: Expression of SLC19A3 was significantly down-regulated in 80% (12/15) of breast tumors (P < 0.005). Breast tumors had significant increase in methylation percentage when compared to adjacent non-tumor tissues (P < 0.05). A total of 155 independent plasma samples from participants including 60 breast cancer, 45 gastric cancer patients and 60 healthy subjects were analyzed. Plasma methylated SLC19A3 DNA yielded a ROC curve area of 77%, sensitivity of 82% and specificity of 60% in discriminating breast cancer from control subjects. This marker yielded a ROC curve area of 87%, sensitivity of 90.0% and specificity of 62% in discriminating gastric cancer from control subjects. Elevated level in plasma has been detected not only in advanced stages but also early stages of tumors. Intriguingly, of all DCIS cases from breast cancer patients this plasma marker generated a ROC value of 92%, sensitivity of 100% and specificity of 78% in discriminating DCIS cases from controls. Conclusions: These results suggested that aberrant SLC19A3 promoter hypermethylation in plasma may be a novel biomarker for early breast cancer diagnosis.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-01-02.
Abstract P3-02-05: Evaluation of Gene Transcripts in Primary Tumors at Time of Diagnosis and Circulating Tumor Cells (CTCs) at Time of Metastatic Disease
Authors: Sieuwerts AM , Mostert B , Bolt-de Vries J , Kraan J , Dirix LY , van Dam PA , van Galen A , van der Spoel P , Ramírez-Moreno R , Yu JX , Wang Y , Gratama JW , Sleijfer S , Foekens JA , Martens JWM.
Published: April 2011

Introduction: The enumeration of CTCs has already shown to bear clinical relevance as a prognostic and predictive factor in metastatic breast cancer. In addition to enumeration, isolation of CTCs enables their molecular characterization and thus holds great promise to establish association of their genetic profile with patient outcome and to identify potential drugable targets. In this study we established epithelial-specific mRNA and microRNA profiles in CTCs of patients with metastatic breast cancer, compared these profiles to the profiles measured in corresponding primary tumors, and determined their association with clinical parameters.

Study design:

For this study we included 50 breast cancer patients, of which 32 presented themselves with over 5 CTCs at the time of metastatic disease. From 14 of these patients with more than 5 CTCs at the time of metastatic disease also the primary tumor at time of breast cancer diagnosis was evaluated. Total RNA was extracted 1) from blood of the 50 patients with metastatic disease after EpCAM-based enrichment of 7.5 mL whole blood with the CellSearch™ Profile Kit [Veridex LCC], 2) from 14 unprocessed whole blood preparations from healthy blood donors, and 3) from 14 primary tumors. Gene transcript levels of CTC-specific and potentially clinically relevant mRNAs and microRNAs were compared in CTCs isolated at time of metastatic disease and the corresponding primary tumors. In addition, the association of these transcript levels with clinical data was assessed.

Results:

We identified 24 mRNA and 14 microRNAs more abundantly expressed in CellSearch-enriched fractions from patients with at least 5 CTCs compared with those without CTCs and/or compared with unprocessed whole blood prior to CellSearch enrichment (Mann-Whitney U-test P<0.05). In addition, when comparing transcript levels present in CTCs during metastatic disease and those measured in the corresponding primary tumor, potentially clinically relevant discrepancies were observed. Findings of interest included changes in transcript levels of genes such as ESR1, ERBB2, TOP2A and MGB1, and in genes associated with proliferation and EMT. Finally, associations were observed between transcript levels measured in CTC preparations and clinical data like nodal status and size of the primary tumor.

Conclusion:

Our results show that molecular characterization of CTCs is feasible and has potential for a more tailored clinical approach above CTC enumeration in the treatment of metastatic breast cancer patients.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-05.
Abstract P3-03-01: Minimal Residual Disease Monitoring in Breast Cancer Patients
Authors: Mikulova V , Janatkova I , Cabinakova M , Tesarova P , Pavlista D , Kolostova K , Pinterova D , Romzova M , Rusnakova V , Kubista M , Zima T.
Published: April 2011

BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed.

METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332,

MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level.

This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.
Abstract P3-04-02: Promoter CpG Island Hypermethylation along Multistep Carcinogenesis of Breast Cancer
Authors: Park SY , Kwon HJ , Lee HE , Kim S-W , Kim JH , Kim IA , Cho N-Y , Kang GH.
Published: April 2011

Background: In breast cancer, promoter CpG island hypermethylation has been described for various genes in all aspects of cellular function. However, most studies focused on the methylation status in tumor-related genes in invasive breast cancer, compared to normal breast tissue, and methylation studies of potential precursor lesions of breast cancer were sparse. We investigated the changes in promoter CpG islands hypermethylation during breast cancer progression from intraductal proliferative lesions [usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC) of breast, and tried to find the specific changes in each step of tumorigenesis. Materials and Methods: We performed MethyLight analyses for methylation status of 57 CpG island loci in 20 IDCs and its paired normal breast tissues. After selecting 15 CpG island loci showing cancer-specific DNA methylation, another sets of normal breast tissue from non-cancer patients (n=10), UDH (n=10), ADH (n=20), DCIS (n=30) and IDC (n=30) were analyzed for these loci.

Results: By this approach, we found six new methylation markers of breast cancer including DLEC1, GRIN2B, HOXA1, MT1G, SFRP4 and TMEFF2, as well as APC, GSTP1, HOXAIO, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1) and SFRP1. The number of methylated genes showed stepwise increase from normal breast to ducal hyperplasia (DH) and

DCIS, while IDC showed no difference with DCIS (median, 1 for normal breast, 2 for DH, 5 for DCIS, 6 for IDC; P<0.001). Of the 15 CpG island loci tested, nine loci already showed methylation in normal breast tissue. Methylation of RASSF1A, DLEC1 and SCGB3A1 (HIN-1) promoter CpG islands was significantly higher in DH than in normal breast tissues. Eight CpG island loci (APC, GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1 and TMEFF2) showed higher methylation frequency and levels in DCIS compared to DH. DICS and IDC showed no difference in the methylation levels or frequency in the 14 CpG islands except HOXA10. Discussion: Our results indicated that CpG island methylation of tumor-related genes is an early event and accumulate with breast cancer progression. CpG island methylation was significantly changed during the progression of DH to DCIS, and DCIS showed similar CpG island methylation with IDC, suggesting that the transition of DH to DCIS is a critical step for breast cancer tumorigenesis associated with promoter CpG islands hypermethylation.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-02.
Abstract P3-04-05: Hypermethylation and Downregulation of Glutathione Peroxidase-3 in Inflammatory Breast Carcinogenesis
Authors: Sabet SF , Mostafa MM , El-Shinawi M , Peng D , Nouh MA , El-Rifai W.
Published: April 2011

Background: The breast tumor microenvironment is characterized by the release of endogenous reactive oxygen species (ROS), and has been suggested as being associated with disease aggressiveness. Normal cells have an antioxidant system that controls the balance between production and removal of oxygen radicals, thereby protecting against oxidative damage, such as the Glutathione peroxidases (GPXs) enzymes. Epigenetics and DNA methylation play important roles in several inflammatory disorders. We therefore, analyzed the promoter of GPX3 and found a dense CpG island closest to the transcription start-site. The aim of the present study is to 1) investigate GPX3 expression in breast carcinoma and normal breast tissues, 2) identify whether GPX3 is epigenetically regulated in breast carcinoma tissues versus normal tissue, and 3) compare the levels of expression of GPX3 in Inflammatory Breast Cancer (IBC) versus non-IBC tissues.

Material and Methods: We enrolled 40 breast cancer patients with tumor mass range from 1.8-9cm (with median size 4.77 ± 3.9). Patients were sub-grouped as IBC (n = 20) or non-IBC (n = 20), healthy breast tissues from the same patients were used as control. Using real-time PCR and immunohistochemistry, we assessed the level of expression of GPX3 at mRNA and protein levels in breast cancer tissues versus control. To identify whether the CpG island of the GPX3 gene was epigenetically regulated, we analyzed the methylation profile of GPX3 in breast carcinoma versus normal tissues using DNA bisulfate treatment and methylation-specific PCR (MSP).

Results: GPX3-mRNA expression was down regulated in breast cancer samples compared to control tissues. There was a significant decrease (P = 0.01) in the mRNA expression level of IBC compared to non-IBC carcinoma tissues. MSP showed that the GPX3 gene was hypermethylated in breast carcinoma tissues compared to control. Moreover, confirmatory semiquantitative immunohistochemical scoring revealed weak or negative immunostain of carcinoma tissues compared to healthy breast tissues. Conclusion: These preliminary data suggest that epigenetic inactivation of GPX3 is a frequent finding in inflammatory breast cancer. Silencing of GPX3 in IBC versus non-IBC carcinoma cells suggested that GPX3 may be critical in the development and progression of IBC. To our knowledge, this is first study to test the role of GPX3 in breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-05.
Abstract P3-04-06: A Functional Investigation of MAL: A Frequently Hypermethylated Gene Marker in Breast Cancer
Authors: Hillocks YMN , Lo P-K , Teo WW , Fackler MJ , Sukumar S.
Published: April 2011

Hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a common event in cancer, and is an important mechanism in inactivating tumor suppressor gene function in breast cancer. Computational searches of existing large databases for genes underexpressed in multiple cancers, combined with CpG island analysis of those genes, identified a novel potential methylation marker gene in breast cancer called MAL (Maturation-Associated protein for T-Lymphocyte). MAL encodes a 17kD protein that is found in mature T-cells and participates in T-cell differentiation and T-cell receptor signaling. Recent studies have reported gene promoter hypermethylation of MAL in breast tumors and breast cancer cell lines. Furthermore, MAL inactivation has been shown to be predictive of benefit from adjuvant chemotherapy. We proposed that alteration of MAL expression has functional implications in breast cancer. In addition, we proposed that MAL has the potential to serve as a methylation marker for early detection of breast cancer. MAL gene methylation as assessed in breast cancer cell lines and primary tumors by quantitative multiplex methylation specific PCR (QM-MSP) showed that it is frequently methylated in cell lines (40/48; 83%), in primary tumors (11/22; 50%) and in distant metastasis (16/32; 50%), but not in normal breast tissues or human mammary epithelial cells (HMECs). The high frequency of MAL methylation was common to all of the subtypes of breast cancer cells tested (Basal A, Basal B, and Luminal). Q-RT-PCR analysis of MAL expression and QM-MSP analysis of MAL methylation showed that 71% (20/28) of breast cancer cell lines show an inverse correlation between methylation and mRNA expression. This suggests that epigenetic alteration is the major mechanism of silencing since pharmacologic intervention with demethylating agents and histone deacetylase inhibitors resulted in re-expression of MAL in the breast cancer cell lines tested. MAL methylation is being assessed in a panel of 150 primary tumor samples with corresponding clinical data such as histological subtype, tumor grade and tumor specific survival. This will allow for correlations to be made between MAL methylation status and survival data etc. to see if MAL can serve as a predictive marker for breast cancer. This analysis will be useful in further validating MAL as a DNA methylation marker for breast cancer detection in tissue samples. The function of MAL is being deciphered at both the biological and biochemical level by performing overexpression and knockdown studies in breast cells using already constructed expression vectors. The characterization and validation of MAL, a lipid raft protein, as a potential DNA methylation marker in breast cancer could provide a valuable early detection tool for breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-06.
Abstract P3-05-01: “VISA” Nanopaticles to Breast Cancer In Vitro and in Living Imaging Animal Models
Authors: Xie X , Wei W , Guo J , Xiao X , Xie X , Kong Y , Tang J , Liu P , Wang X , Lv N.
Published: April 2011

Development of cancer gene therapy has been hampered by the fact that there are no effective cancer-specific expression vectors available, which is critical for improving therapeutic efficacy and reducing toxicity in clinics. To overcome this problem, we recently developed a safe and effective strategy fro targeting a potent pro-apoptotic gene (BikDD) to the pancreatic tumors (Xie et al. Cancer Cell, 2007), lung cancer (Sher et al. Oncogene, 2009) and ovarian cancer (Xie et al. Molecular Cancer Therapeutics, 2009). A VISA system (VP16-Gal4-WPRE integrated systemic amplifier) was engineered by using the two-step transcriptional amplification (TSTA) system and the posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE). The VISA system can boost the activity of cancer-specific promoters by an average of 600-800-folds compared to their basal levels. C-VISA (CCKAR-VISA) nanoparticles transcriptionally targets transgene expression effectively to pancreatic tumors in vivo. C-VISA-BikDD nanoparticles was shown to be highly effective in reducing tumor burden and increasing animal survival rate in orthotopic pancreatic cancer models which is moving into clinical trials at University of Texas M.D. Anderson Cancer Center, USA. In the current study, we identified hTERT(human telomerase reverse transcriptase), survivin, β-cateinin, claudin-4, and FASN (fatty acid synthase) promoters as breast cancer-selective promoters, and amplified their activity to hundreds of folds greater by the “VISA” system(called x-VISA) without loss of their specificity. Very importantly, the x-VISA promoters were also robust in the primary cultured breast cancer cells, with 42.5 %-65% of the CMV promoter, while retaining stringent pancreatic cancer specificity. x-VISA was also shown to target expression of firefly luciferase to MDA-MB-468 xenografts in an orthotopic mouse model, confirmed by noninvasive imaging with an Xenogen IVIS™ imaging system and measuring with a luminometer. The cancer-specific index was 35-60 for x-VISA-Luc and 0.01 for CMV-Luc in vivo. We further demonstrated significant antitumor activity of targeted BikDD expression driven by nanoparticles of the hTERT-VISA-BikDD vectors in Her-2-negative and-positive and triple-negative breast cancer cell lines in vitro, and in multiple breast cancer models of living imaging by the Xenogen IVIS imaging system with limited toxicity. In addition, treatment with hTERT-VISA-BikDD nanoparticles plus lapatinib or produces evident combinational therapeutic efficacy, which is likely due to the ability of lapatinib to promote apoptosis of Her-2-overexpressing breast cancer cells. Thus, our newly developed hTERT-VISA-BikDD nanoparticles are an innovative strategy for targeted antitumor effects of Her-2-negative, Her-2-positive and triple-negative breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-05-01.
Abstract P3-07-01: Clonal Relatedness between Lobular Carcinoma In Situ and Synchronous Malignant Lesions: Analysis of Fresh Frozen Tissue Using SNP Array
Authors: Andrade VP , Morrogh M , Giri D , Ostrovnaya I , Seshan V , Olvera N , Begg C , Morrow M , King TA.
Published: April 2011

INTRODUCTION: Lobular carcinoma in situ (LCIS) confers one of the greatest risks for subsequent development of invasive breast carcinoma yet historical data suggests that LCIS is not a precursor to invasive disease. Emerging evidence of shared molecular alterations between LCIS and co-existing invasive lesions in a small number of archival samples have reopened the debate regarding the true significance of LCIS and its malignant potential.

METHODS: We investigated clonality between LCIS and adjacent malignant lesions in 26 patients using fresh frozen samples harvested from mastectomy specimens. Laser Capture Microdissection was used to isolate pure cell populations and DNA was subject to Affymetrix SNP 6.0 array. Data were converted for copy number analysis and the closeness of identified paired copy number changes was measured to determine the probability of clonality.

RESULTS: After excluding cases with low DNA yield/quality and noise signals, 14 LCIS with seven patient matched invasive lobular carcinomas (ILC), 6 matched ductal carcinoma in situ (DCIS) and 3 matched invasive ductal carcinomas with lobular features (IDCLF) were considered adequate for statistical comparison. The immunohistochemical profile of invasive cancer showed: 10 cases ER+/PR+/Her-2(-), 2 cases ER-/PR-/Her-2(+), 1 ER-/PR-/Her2(-) and 1 case non-available for staining (microinvasive carcinoma). The results suggest that the tumor pairs are more likely to be of clonal than independent origin between LCIS and DCIS in 4/6 samples (66.6%), between LCIS and low-grade IDCLF in 3/3 (100%) samples and between LCIS and classic ILC in 5/7 (71.4%) samples. Independent LCIS/DCIS samples were seen in association with Her2+ cancers. In LCIS/DCIS samples favoring a clonal origin the DCIS were low-grade cribriform type associated with ER+ cancers.

CONCLUSION: Clonal relatedness was detected in the majority of LCIS and adjacent malignancies, including cases of low-grade DCIS and IDCLF. These results support a true precursor role for LCIS and provide additional evidence to the concept of a low-grade malignancy pathway as opposed to the classic lobular versus ductal dichotomy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-07-01.
Abstract P3-08-01: The Influence of the BAD Apoptosis Pathway on Breast Cancer Progression and Relapse-Free Survival after Adjuvant Treatment
Authors: Ismail-Khan RR , Marichion DC , Xiong Y , Fulp WJ , Kamath SG , Eschrich SA , Chen D-T , Lancaster JM.
Published: April 2011

Objective. The phosphorylation status of the BCL2 Antagonist of Cell Death (BAD) protein influences cell survival and apoptosis. BAD phosphorylation is determined by the relative activity of a series of kinases and phosphatases within the BAD pathway. We therefore sought to evaluate the influence that expression of the BAD apoptosis pathway has on breast cancer progression and on clinical outcome in a series of breast cancer patients.

Methods. We used Principal components analysis to derive a BAD-pathway gene expression signature with a corresponding “pathway score” representing overall gene expression levels for BAD pathway genes. The BAD-pathway signature score was evaluated in clinical-genomic breast cancer datasets obtained from a total of 502 patients, including intra-ductal hyperplasia (n=8), ductal carcinoma in-situ (n=30), and three separate data sets of invasive ductal carcinoma patient samples (n=23, n=286, n=155). The influence of the BAD-pathway signature score on breast cancer progression and relapse-free survival was evaluated by student's t-test and Kaplan Meier/log-rank test, respectively.

Results. We developed a 48-gene BAD-pathway signature, the expression score of which decreased with progression from intra-ductal hyperplasia through ductal carcinoma in-situ and invasive ductal carcinoma (p=0.0004). Furthermore, BAD-pathway signature score was associated with relapse free survival from breast cancer in two independent clinical-genomic datasets (n=286, p=0.01; n=155, p=0.02). Conclusions.BAD pathway gene expression is associated with breast cancer progression and disease-free survival. The pathway represents an appealing human cancer prognostic biomarker and therapeutic target. Figures available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-08-01.
Abstract P3-09-03: microRNAs and Target Gene Networks in Breast Cancer Cell Lines
Authors: Santarpia L , Diao L , Baggerly K , Boulbes D , Di Leo A , Calin GA , Symmans FW , Esteva F , Hortobagyi GN , Pusztai L.
Published: April 2011

Background: MicroRNAs (miRNAs) are a class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Several miRNAs have been involved in the regulation of several cellular processes during oncogenesis.

Objective: We simultaneously analyzed 18 different breast cancer cell lines (BCC) for miRNA and mRNA expression profiles in order to identify regulatory miRNA target gene networks and novel miRNA-directed therapeutic targets.

Materials and Methods: We preprocessed the mRNA and miRNA expression data using R software (justRMA) and performed quantile normalization using background-subtracted log ratio distributions across arrays. We identified 39 hsa-miRNAs with the largest variation across samples and examined the Spearman correlations of miRNAs with the Affymetrix probesets. We calculated false discovery rates for probe sets with stringent correlation coefficients. We performed hierarchical clustering using the 39 miRNAs and correlated dendograms branches with basal/luminal classes. Potential miRNA target genes were identified. We performed pathway analyses by using all 39 miRNA target genes. Western blot analyses were performed to confirm regulation of specific proteins. Results: We demonstrated 39 deregulated miRNAs and 10612 mRNA targets genes in BCC. Twenty-three miRNAs were upregulated in basal and downregulated in the luminal groups targeting 6790 genes, and 16 miRNAs downregulated in basal and upregulated in luminal cells, targeting 3822 genes. The first direct comparison screening for the 39 miRNAs and all mRNAs identified 29 direct pairs where the correlation magnitude was above 0.9. MiR-221/222, -31, -143/145, -34a, -200 were the most and consistently deregulated miRNAs in all luminal and basal like BCC. We identified genes, such as CD59, PDGF-β and discoidin domain receptor tyrosine kinase 1-DDR1 and different cancer network pathways, such as cell cycle and cell surface markers, potentially regulated by a subset of these miRNAs. Interestingly, we found that the expression of single genes might be regulated by several miRNAs.

Conclusions: We found a specific miRNA signature and respective genome targets that well defined different clusters of BCC. Breast cancer cell lines are associated with changes in expression of multiple miRNAs that, in turn, disrupt a network of genes that either activate or inhibit important cellular functions. A better understanding of the network of genes and cellular pathways regulated by these miRNAs will enable to understand breast cancer pathogenesis and miRNA based breast cancer therapy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-03.
Abstract P3-09-06: microRNA Signature of Columnar Cell Hyperplasia from Patients with High Risk of Breast Cancer
Authors: Nilsson S , Landberg G
Published: April 2011

Background: Columnar cell hyperplasia (CCH) is considered to originate in normal terminal duct lobular units (TDLUs) and is potentially the first histologically identifiable alteration in the development of breast cancer. In order to detect early pathological changes in breast tissue, which could reveal the underlying mechanism to cancer progression, we investigated the microRNA (miRNA) expression profile in TDLUs and CCH. Material and Methods: Epithelial cells and surrounding stroma of TDLUs and CCH from formalin-fixed paraffin embedded tissue (FFPE) from patients with high risk of developing breast cancer were laser capture microdissected and RNA was extracted. MiRNA expression profiles were obtained using a qRT-PCR-based array analyzing 667 human miRNAs. Results: Increased levels of ERα were present in the epithelial cells of CCH as previously reported. Immunohistochemical staining for the stromal activation marker smooth muscle actin (SMA) was negative in fibroblasts surrounding both TDLUs and CCH, suggesting lack of major activation of the stroma. Interestingly, array analysis revealed changes in miRNA expression pattern in both epithelial and stromal compartments. Correlation analyses of the array results indicated a high reproducibility (r2:0.82-0.94) supporting stable miRNA profiles in TDLUa and CCH. MiRNA array analysis of paired samples from specimens with both TDLUs and CCH resulted in significant (P<0.05) decreased levels of 15 miRNAs in epithelial cells of CCH and downregulation of 18 miRNAs in the stroma. Examples of altered miRNAs in the epithelial cells are miR-532-3p, miR-886-3p, let-7c, miR-491-5p, miR-365, and example miRNAs in the stroma are miR-195, miR-99a, miR-224, miR-19b, miR-423-5p. Further validation

of a selection of miRNAs in a patient material using in situ hybridization is currently being performed to verify the array results. Discussion: This data will potentially link these miRNAs to the development of TDLUs towards CCH and possibly lead to the discovery of new markers for precancerous conditions and potentially new therapeutic targets.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-06.