bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting Survival Time in Women Starting First Line Chemotherapy for Advanced Breast Cancer.
Authors: Lee C. , Stockler M. , Malcolm H. , Coates A. , Val G. , Sally L. , Richard S.
Published: December 2010

Purpose: Women starting first line chemotherapy for advanced breast cancer have widely differing baseline characteristics and ultimate survival times. We sought to develop and validate a pragmatic prognostic index for clinical practice and research based on simple clinical and laboratory data.Methods: Prognostic models were developed in 378 women treated with CMF in the control groups of 3 randomized trials, and validated in 564 women treated with AC or mitoxantrone or capecitabine in the experimental groups of these trials. Multivariable censored linear regression of log-transformed survival times was used to select prognostic variables, which were then dichotomised to define negative prognostic features (NPFs). The effects of NPFs on survival time are expressed with hazard ratios (HR) and p-values from multivariable Cox models.Results: There were 7 independently significant NPFs (see table). In the development dataset, an elevated neutrophil count or history of brain metastasis approximately halved survival time, while the other NPFs reduced survival time by about one third. Women with 0 NPFs (15%) had a median survival of 18 months, about 1.4 times as long as those with 1 or 2 NPFs (63%, 13 months), and about 2.6 times as long as those with 3 or more NPFs (22%, 7 months). In the validation dataset, the effects of brain metastasis and elevated neutrophils were more modest, and most NPFs reduced survival by about one third. The median survivals of women with 0 NPFs (17%), 1 or 2 NPFs (60%) and 3 or more NPFs (23%) were 21 months, 13 months and 8 months respectively.Conclusions: A prognostic index using widely available baseline characteristics could improve prognostication in clinical practice. Stratification of patients using the prognostic index could improve the efficiency of randomised trials. Development (n=378)  Validation (n=564)  Negative prognostic feature% of womenHRp-value% of womenHRp-valueBrain metastasis32.410.00641.730.002Neutrophil count >ULN121.910.001151.350.03ALP >2 x ULN221.660.001201.380.008Age >60451.490.001451.210.05Hb <120g/L351.480.03291.230.05ER negative401.360.02281.52<0.001ECOG 2 or 3271.320.03251.500.001ULN = upper limit of normal Development (n=378)  Validation (n=564)  Number of NPFs% of womenHRp-value% of womenHRp-value 150.850.30170.670.0021 or 2631.00-601.00-3 or more222.11<0.001231.97<0.001NPF = negative prognostic feature

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4066.
Immunoglobulin Kappa C Has Independent Prognostic Significance in Node-Negative Breast Cancer.
Authors: Schmidt M. , Boehm D. , Lebrecht A. , Gehrman M. , Koelbl H. , Hengstler J.
Published: December 2010

Background: Utilizing microarray based gene-expression analysis of fresh-frozen tissue we could recently demonstrate the prognostic impact of a B cell metagene in node-negative breast cancer (Cancer Res 68: 5405-5413, 2008). In the present study we investigated the prognostic significance of immunoglobulin kappa c (IGKC) in formalin-fixed paraffin embedded (FFPE) breast cancer specimens of 363 node-negative breast cancer patients which were not treated in the adjuvant setting.Methods: RT-PCR was used to analyze mRNA expression of IGKC in FFPE tissue of 363 patients with node-negative breast cancer. Additionally to IGKC we examined the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER) and progesterone receptor (PR). Metastasis-free survival (MFS) was analyzed with uni- and multivariate Cox regression.Results: Median follow-up was 10 years. 69 patients (19%) developed distant metastasis. Univariate analysis showed significant results for histological grade (HR 2.2, 95% CI 1.54-3.19, p<0.0001), tumor size (HR 1.48, 95% CI 1.22-1.77, p=0.0002) and IGKC. Patients with a higher expression of IGKC showed better MFS (HR 0.84, 95% CI 0.76-0.92, p= 0.0004). Age, ER and PR failed to show an association with MFS in univariate Cox regression. Using multivariate Cox regression, IGKC retained its independent prognostic significance (HR 0.85, 95% CI 0.77-0.93, p=0.0005). Besides IGKC histological grade (HR 2.5, 95% CI 1.62-3.77, p<0.0001) and ER (HR 1.08, 95% CI 1.01-1.16, p=0.031) showed an independent association with MFS.Conclusion: Utilizing FFPE we could prove the independent prognostic significance of IGKC in 363 node-negative breast cancer patients. This should aid in incorporating the prognostic impact of the immune system into prognostic algorithms based on FFPE tissue.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4067.
A Quantitative Immunofluorescence Assay (AQUA) Suggests Significant Misclassification (15%) of Estrogen Receptor Status in Breast Cancer.
Authors: Welsh A. , Moeder C. , Alarid E. , Rimm D.
Published: December 2010

Introduction: Estrogen Receptor (ER) is arguably the most powerful predictive marker in breast cancer. However, a recent incident in Canada revealed a strikingly high false-negative rate and raised awareness of the current limitations in our measurement of ER. The current clinical standard is both subjective and qualitative. Even though guidelines are about to be issued to standardize this assay, there is very little data on the misclassification rate in current practice in the US.Hypothesis: Our hypothesis is that the use of a quantitative assay for ER on a series of retrospective collections may reveal both the level and significance of the misclassification rate.Method: Cell lines with a range of ER expression levels were analyzed by quantitative western blotting in parallel with IF/AQUA analysis (r2 = 0.865), in order to create standard curves for assessment of absolute ER protein concentration in tissue. The optimized assay was then used to quantify ER protein expression in a large cohort of archival breast cancer samples from Yale (1962-1982, n =617).Results: Using a set of standard curves with recombinant ER and cell line controls, we developed a standardized method for quantifying ER as an absolute concentration (pg ER per μg total protein) in formalin-fixed tissue on TMAs. This ER AQUA assay has a range in sensitivity from 50 pg/μg to 1500pg/μg total protein. Quantification of ER protein expression on the Yale archival cohort revealed a unimodal distribution with 49.8% of cases above the 50pg/ug threshold and thus defined as positive. When compared to pathologist performed conventional ER classification, we found a false negative rate of 6.65% and a false positive rate of 10.2%, for a total misclassification rate of 16.6%. Although no response data is available on that cohort, data is under analysis on 4 other cohorts with endocrine therapy treatment information, including an independent Yale cohort, SWOG 9313, NSABP B14, and the TEAM trial.Conclusion: We have developed a quantitative method to measure absolute levels of ER in breast tissue. Use of this assay on a series of cohorts suggests a misclassification rate in the 15% range. The significance of this level of misclassification with respect to response to endocrine therapy is currently under study.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4068.
Aromatase Inhibitors (AIs) Versus Not (Placebo/Observation) as Late Extended Adjuvant Therapy for Postmenopausal Women with Early Stage Breast Cancer (BC): Overviews of Randomized Trials of AIs after ∼ 5 Years of Tamoxifen.
Authors: Goss P. , Mamounas E. , Jakesz R. , Markopoulos C. , Dowsett M. , Peto R. , Godwin J. , Davies C.
Published: December 2010

Backround: 5 yrs of adjuvant tamoxifen (Tam) substantially reduces recurrence and mortality in ER+ breast cancer. Two to five years of late extended AI (anastrozole, exemestane or letrozole) adjuvant therapy after ∼ 5 years of Tam has been studied in prospective trials in postmenopausal patients (pts) in comparison with placebo or observation.Methods: A meta-analysis of outcomes data submitted to EBCTCG was conducted for four clinical trial data sets: NCIC CTG MA17; NSABP B33; ABCSG VIA; ATENA. The primary analysis involved patients with tumors reported to be ER positive with endpoints of first BC recurrence (all, local only, contralateral BC only, distant), death with and without recurrence, any death. All p-values were 2-sided. Follow-up for MA.17 was censored at the date of un-blinding; this was not possible with B-33, which consequently includes some events following subsequent treatment crossovers.Results: Preliminary results are available; updated and sub-group analyses including progesterone receptor, age, grade and nodal status will be presented. Highlights include: at a median follow-up of 2.5 years, AI therapy was associated with an absolute 2.9% [standard error (SE) 0.5] decrease in BC recurrence [relative decrease, 43% (SE 8), p<0.00001] and an absolute 0.5% (SE 0.3) decrease in BC mortality [relative decrease 27% (SE 17), 2p=0.11].Conclusions: A meta-analysis of 4 clinical trials, two of which were stopped early showed that ∼2-5 years late extended adjuvant therapy beyond 5 years from diagnosis produced a highly significant reduction in BC recurrence compared with no therapy. A trend toward improved survival is also seen. The magnitude of the effect seen on disease free and overall survival in these analyses is likely under estimated because of some cross-over after un-blinding. Longer than five years of adjuvant endocrine therapy is superior to just 5 years in ER+ breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4081.
The Effect of Breast Cancer Diagnose on Cognitive Function.
Authors: Hedayati E. , Schedin A. , Albertsson M. , Nyman H.
Published: December 2010

Background: Women with breast cancer treatment complain about cognitive impairment. Previous studies indicate separately that receiving a breast cancer diagnosis has negative psychological impact and adjuvant treatment impairs cognitive functions. To investigate the impact on cognitive function is of great importance, since breast cancer is the most common cancer among women at working age in Sweden. We have started a longitudinal study to investigate if the effects of receiving a breast cancer diagnosis may affect cognitive functions. Materials and methods: The study is dimensioned for 148 subjects. The subjects were recruited consecutively and prospectively from the mammography-screening program at South Hospital, Stockholm. Those women who were recalled for further triple diagnosis assessment were invited to participate in our study. According to the results of the diagnostic process the patients were grouped in to: healthy women (n=69) and women with breast cancer (n=79). All participants were examined by a web-based battery of neuropsychological tests at inclusion (T1) and after diagnosis and subsequent surgery (T2). We measured four cognitive domains (Response speed, Processing speed, Memory and Attention). Results: Our results show that neither the healthy women nor patients with a recent breast cancer diagnosis with its subsequent surgery demonstrate any tendency toward a cognitive decline in any measured domains. The result did not differ significantly with regard to age, educational level or other demographic variables. Conclusions: The results may be interpreted to suggest that receiving a breast cancer diagnosis and/or surgery may not be associated to a substantial cognitive decline. Cognitive functions are not related with secondary depression and anxiety due to cancer diagnosis.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4091.
Validating CYP2D6 Genotype-Guided Tamoxifen Therapy for a Multiracial U.S. Population.
Authors: Irvin W. , Carey L. , Dees E. , Lange L. , Chiu W. , Evans J. , Anderson S. , Freidman K. , Weck K. , Desta Z. , Walko C. , Olajide O. , Raab R. , Corso S. , Peppercorn J. , Flockhart D. , McLeod H.
Published: December 2010

Background: Tamoxifen is a selective estrogen receptor modulator that is the most commonly used and cost effective agent for hormone sensitive breast cancer with documented efficacy in prevention, treatment of preneoplasia, and treatment in both the adjuvant and metastatic settings. However, tamoxifen is a prodrug, and up to half of those taking it may not receive the full benefit because of genetic differences in CYP2D6 that affect metabolism to its active form, endoxifen. Tamoxifen is FDA approved for use at both 20mg/day and 40 mg/day, however by convention is dosed at 20mg. We aimed to determine if endoxifen levels can be manipulated by genotype-guided dosing of tamoxifen.Methods: 118 patients on tamoxifen ≥ 4 months and not on potent CYP2D6 inhibiting medications enrolled in Lineberger Comprehensive Cancer Center (LCCC) trial 0801. Genotyping was performed using the CYP450 Amplichip® (Roche Diagnostics) for 2D6 alleles: *1-11, *15, *17, *19, *20, *29, *35, *36, *40, *41, *1XN, *2XN, *4XN, *10XN, *17XN, *35XN and *41 XN. Tamoxifen dose was increased from 20mg to 40mg in patients with any intermediate or poor metabolizing (IM or PM) alleles [but not in patients homozygous for extensive metabolizing (EM) alleles]. Endoxifen levels were drawn at baseline and 4 months later. Assuming that endoxifen levels in IM pts are 40% lower than EM at baseline (Jin et al., 2005) and with a one-sided significance level of 0.025 and a sample size of 40 patients with intermediate metabolizing CYP2D6 genotypes, this study would have 84% power to detect a 40% increase in endoxifen.Results: Of the 118 patients, 25 withdrew or were removed from study, leaving 93 who have completed this study and who are evaluable for the primary analysis. Genotyping results were presented at 2009 ASCO meeting (Irvin et al.). For this analysis, 19 (20%) are African-American, 69 (74%) are non-Hispanic white, 2 are Hispanic, and 3 are Asian. For the 93 evauable patients, genotyping revealed 31 (33%) EM/EM, 1 EM/UM (ultra-rapid), 20 (22%) EM/IM, 19 (20%) EM/PM, 4 (4%) IM/IM, 9 (10%) IM/PM, 9 (10%) PM/PM and 1 unknown. The primary outcome, the tamoxifen metabolite levels, including endoxifen, will be available in July.(Supported by NC University Cancer Research Fund, NCI SPORE, Laboratory Corporation of America, Roche Diagnostics)

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 410.
Evaluation of Fiducial Marker Migration and Respiratory-Induced Motion for Image Guided Radiotherapy in Whole Breast Irradiation.
Authors: Latifi K. , Forster K. , Harris E.
Published: December 2010

Purpose: Image guided radiation therapy (IGRT) may be beneficial to improve set up accuracy and reduce treatment margins, therefore improving efficacy while decreasing long term toxicity of whole breast irradiation (WBI). In order to assess if intraparenchymal fiducials can facilitate the use of IGRT, this study quantifies fiducial motion over the normal course of whole breast irradiation.Materials and Methods: On a prospective IRB-approved protocol, fiducials were placed in fourteen patients who were then treated with 3D conformal WBI. Three or four intraparenchymal gold fiducial markers were placed in each patient at the periphery of the surgical bed intraoperatively. Free breathing 4D CT image sets were obtained at pre-treatment simulation and then every two weeks over a six week course of treatment. Each fiducial marker was contoured at end-inspiration and end-expiration on all 4D CT image sets. The effects of respiration induced motion were assessed by comparing the positions of the fiducials between inspiration and expiration. Overall fiducial migration was determined by comparing the relative positions of each marker to one another from the pre-treatment and post-treatment 4D CT image sets.Results: The 4D CT image sets were acquired over a period of 55±4 days with a range of 49 to 60 days. The respiration induced fiducial motion, as measured by the average change in the fiducials' center of mass, was 0.8±0.6 mm with a range of 0 to 2.2 mm. The average fiducial displacement over the course of treatment as determined from relative seed position from the image sets was 1.5±1.2 mm with a range of 0.1 to 3.3 mm for all individual fiducials with 71% of the fiducials moving closer to each other and 29% away from each other.Conclusions: The preliminary results of using gold fiducials for breast IGRT are promising. Fiducial position was very stable during treatment, and there was very little respiration induced motion. Using IGRT, there is the potential to improve the accuracy of daily set-up and to reduce margins in WBI. Better tumor bed localization and reduced margin size will decrease the volume of normal tissue treated, which may translate into improved local control by ensuring accurate coverage of the target volumes throughout the treatment course and improvement in cosmesis and other long term toxicities.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4113.
The Effect of Radiotherapy on Local Regional Recurrence among Patients with Pathologic Complete Response to Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer.
Authors: Fasola C. , Godette K. , McDonald M. , O'Regan R. , Zelnak A. , Landry J. , Torres M.
Published: December 2010

Background: Radiotherapy has been shown to reduce local recurrence rates of breast cancer after treatment with surgery and adjuvant chemotherapy. Indications for postmastectomy radiation have historically been based on pathology, but indications for radiation in the setting of pathologic complete response (pCR) to neoadjuvant chemotherapy are not well defined. The aim of this study was to evaluate the rates of local regional recurrence (LRR) among patients with pCR to neoadjuvant chemotherapy followed by surgery with or without radiation (XRT).Patients and Methods: The case histories of 337 patients with locally advanced breast cancer treated with neoadjuvant chemotherapy, surgery with or without radiation from October 1997 to December 2006 were analyzed. Median age at diagnosis was 50.6 years (range: 25-84). The clinical stage at diagnosis was I in 3 (1%), IIA in 105 (31%), IIB in 94 (28%), and III in 135 (40%) patients. All patients received preoperative systemic therapy with a doxorubicin-based regimen (92%) or a single-agent taxane regimen (7%) or CMF (1%). A total of 272 patients received radiation following surgery and 65 did not receive any radiation. Median follow-up time was 40 months among all patients. The Kaplan-Meier method and log rank test were used to evaluate LRR rates and overall survival among both groups.Results: Patients treated with neoadjuvant chemotherapy followed by surgery and XRT had a lower incidence of LRR at 3 years with 24 cases of local recurrence among 272 patients who received XRT compared with 12 cases of local recurrence among the 65 patients who did not receive XRT (8.9% v 18.5%, P=0.02). Radiation also reduced local regional recurrence at 3 years among a subset of patients with clinical stage T2N1 disease (4.9% v 28.6%, P=0.03). There was no difference in overall survival or disease free survival among patients who received XRT compared to patients who did not receive XRT. There were 62 (18%) patients who achieved a pCR to neoadjuvant chemotherapy. Among patients with a pathologic complete response, radiation appeared to reduce the risk of LRR compared to patients who did not receive XRT (7.4% v 25%, P=0.17).Conclusion: Radiotherapy significantly reduced local regional recurrence rate among patients with locally advanced breast cancer treated with neoadjuvant chemotherapy and surgery. Of particular interest, patients who achieve a pathologic complete response to neoadjuvant chemotherapy still had high LRR rates and may benefit from radiotherapy by lowering the risk of local regional recurrence. Further studies on the significance of LRR and overall survival among these patients are needed.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4114.
RFA for Small Breast Cancer — What Tumor Can Be Treated Safely?
Authors: Takahashi M. , Hosoda M. , Takahashi H. , Matsuno Y. , Todo S.
Published: December 2010

Breast conserving surgery for small breast cancer has been acceptable as one of standard treatments. However, there are some patients who do not be satisfied with the cosmesis. The aim of this study is to verify the clinical safety and usefulness of the RadioFrequency Ablation (RFA) for small breast cancer without resection. Between January 2006 and Octobar 2007, 20 breast cancer patients were performed RFA with cool-tip RF system after sentinel LN biopsy under general anesthesia. All patients has consented as a clinical research, beforehand. MR mammography was performed after 3 and 6 months of follow-up. All patients were performed postoperative histological examination by 8G-needle mammotome biopsy after 6 months MRI evaluation.Tumor viability after RFA was assessed by hematoxylin -eosin and ssDNA staining. QOL after RFA was assessed both subjectively(QOL-ACD score) and objectively(JBCS cosmesis criterion). Preoperative mammotome revealed 4 patients had DCIS and 16 patients had IDC. The patients median age was 53 years old. The mean greatest dimension of tumors was 1.0 cm (range, 0.5-1.5 cm). MRI showed the dimention of coagulation necrotic area by RFA treatment was 3.5cm (range, 2.5-5.4 cm).All patients have been performed postoperative histlogical examination by 8G mammtome biopsy 6 month after RFA treatment. Coagulation necrosis of the tumor was complete in 20of 20 patients (100%).No patient was troubled skin burn. Neither local and distant recurrence has occurred in all patients (median follow up was 30 month). To treat RFA for breast cancer more safely, we analyzed factors which influenced the size of necrosis. Patient age, body mass index (BMI), background concentration measured by MMG, Fat involvement measured by CT did not influence with the size of nectosis (P=0.21,0.17,0.64,0.40). The state of posterior echo detected by ultrasonography was associated with the size of necrosis (p=0.045). Hence, RFA for the tumor with rich fiber such as scirrhous type tend to make necrosis wider than one with poor fiber such as solid tubular type.Most of patients were satisfied with the cosmesis of the RFA treated breast. Objective QOL score (JBCS cosmesis criterion) showed splendid cosmetic outcome. RFA is one of the effective local treatment for small breast cancer (< 1.5 cm in diameter), and bring breast cancer patients cosmetic satisfaction.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4119.
Locoregional Recurrence with Breast Conservation Surgery and Radiotherapy after Neoadjuvant Chemotherapy.
Authors: Lee S. , Shin K.
Published: December 2010

Purpose; Breast conservation surgery (BCS) and radiotherapy(RT) after neoadjuvant chemotherapy (NEO) has concerns of high locoregional recurrence (LRR) rates. This analysis was done to know LRR rates and the variables increasing LRRs.Patients and Methods; 242 breast cancer patients treated with BCS and RT after NEO between 2001 and 2006 were included in this study. All patients were clinically node positive by positron emission tomography. Clinical stage at diagnosis (2003 AJCC) was IIA in 22.7%, IIB in 43.0%, IIIA in 33.5% and IIIB in 0.8% of patients. Of the patients, 51.4% received anthracycline-based NEO, 34.2% received a taxane, and 14.4% received anthracycline combined taxane regimen. RT was done to all patients.Results; At a median follow-up of 56 months (range, 14 - 86 months), 24 patients (9.9%) had developed LRR. Among these, 18 (7.4%) were ipsilateral breast tumor recurrences (IBTR), 2(0.8%) axilla, 3 (1.2%) supraclavicular and 5 (2.1%) internal mammary recurrences. Five-year actuarial rates of IBTR-free and LRR-free survival were 86.9% and 84.4%, respectively. Distant metastases were detected in 36 patients (14.9%). On multivariate analyses of LRR, adjuvant hormone therapy, pathological lymph node status and clinical stage were associated with higher rates of LRR. The hazard ratios for lack of hormone therapy, pathological nodal stage N2-3, and clinical stage III were 8.9 (95% CI, 3.5 to 23.1; p<0.0001), 4.3 (95% CI, 1.7 to 11.2; p=0.003) and 2.7 (95% CI, 1.1 to 6.5; p=0.02), respectively. 5-year actuarial LRR-free survival rate was 86.5% in patients with adjuvant hormone therapy while it was 76.8% in patients without adjuvant hormone therapy.Conclusion; BCS with RT after NEO results in acceptably low rates of IBTR and LRR in clinical node positive patients. The adjuvant hormone therapy, pathologic lymph node status and clinical stages predict higher rates of LRR.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4120.
miR-92 Is a Novel Regulator of ERβ1 Expression in Breast Cancer.
Authors: Al-Nakhle H. , Burns P. , Cummings M. , Hanby A. , Hughes T. , Satheesha S. , Shaaban A. , Smith L. , Speirs V.
Published: December 2010

Down-regulation of ERβ1 expression is thought to contribute to carcinogenesis in the breast and consequently is seen in many breast cancers. The molecular mechanisms responsible for the down-regulation of ERβ1 remain unclear. microRNAs are a novel family of regulators of gene expression that have been shown to act on the expression of many critical cancer genes but their relationship with ERβ1 has not so far been demonstrated. The aims of this study were to establish whether miR-92 regulates ERβ1 expression, and whether this regulation plays a role in defining ERβ1 expression levels in breast cancers.Using a bioinformatics approach we initially identified potential binding sites for miR-92 within the 3' untranslated regions of ERβ transcripts using RNAhybrid software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). Two conserved target-sites for miR-92 were identified within the ERβ1 3'-UTR sequence contained within Genebank. We confirmed the expression of these UTRs in MCF7 cells. Next, we performed 3' RACE reactions to determine the 3'-UTR sequence of ERβ1 in MCF7 cellsWe used qPCR analyses of expression in paired normal breast and breast tumour samples (n=6) to examine the relative expression of miR-92 and ERβ1. Upregulation of miR-92 expression was observed in breast tumours compared with normal breast. An inverse relationship with ERβ1 expression was observed in these samples. In a separate cohort of breast tumours (n=36), a significant negative correlation between ERβ1 mRNA and miR-92 was observed (Spearman's correlation coefficient, r = -0.5, p=0.001). Elevated ratios of ERβ1 mRNA /miR92 were also observed in ERβ1 positive compared to ERβ1 negative cells lines.Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose–dependent manner at RNA levels. Enhanced GFP reporter constructs containing miR-92 binding sites from the 3'-UTR of ERβ1 were used to determine whether miR-92 downregulates ERβ1 via the direct targeting of this 3'-UTR. Inhibition of miR-92 increased the translational efficiency (protein produced per unit of mRNA) of the GFP reporter, confirming that the miR-92 binding sites are a critical regulatory region. Finally, we showed that miR-92 expression was upregulated by 17β-estradiol and downregulated by tamoxifen in MCF7 cells (ERα+ ERβ+) but not in ERβ negative cells (BT20 and MDAMB453), suggesting ERs can mediate miR-92 regulation. Our results demonstrate that ERβ1 expression in breast cancer is regulated by miRNA-92.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4139.
Phosphorylation of Estrogen Receptor β at Serine 105 in Primary Breast Cancer.
Authors: Hamilton-Burke W. , Shaaban A. , Cummings M. , Holliday D. , Pollock S. , Horgan K. , Speirs V.
Published: December 2010

Background: Estrogen receptor (ER) α predicts response to hormonal therapy in breast carcinoma. The role of ERβ is less well understood. ER activities can be modulated by post-translational modifications including phosphorylation and phosphorylated ERα has been correlated with patient outcome. We investigated whether ERβ phosphorylated at Serine 105 (P-S105-ERβ) is expressed in breast carcinoma and assessed its potential clinical implications.Material and Methods: Tissue microarrays comprising 427 breast tumours (23% grade 1, 43% grade 2, 34% grade 3) with median follow up 118 months and 106 endocrine resistant breast tumours (15% grade 1, 41% grade 2, 44% grade 3) with median follow up 71 months were used in this study. Expression of P-S105-ERβ was studied by immunohistochemistry and analysed against clinical data. Regulation of P-S105-ERβ in vitro was assessed by immunofluorescence and Western blotting.Results: Expression of P-S105-ERβ was mainly nuclear and could be abolished by phosphatase pre-incubation, indicating specificity. In the first cohort Allred scoring ranged from 0 to 8 (median 6). Nuclear speckling was observed in 45% of cases. P-S105-ERβ correlated with ERβ1 (Allred score ≥ 3) and was associated with better overall survival (OS) and disease free survival (DFS) (p=0.028 and p=0.027, respectively). In a multivariate Cox hazard analysis, P-S105-ERβ overexpression was a significant predictor of better survival independent of tumor grade, lymph node status, size or ERα. In the endocrine resistant cohort Allred scoring ranged from 0 to 8 (median of 4) and the overexpression of P-S105-ERβ was also associated with improved OS and DFS (p=0.044 and p=0.033, respectively). Nuclear speckling was present in only 11% of cases. The difference in expression of P-S105-ERβ and association with survival outcome between the two cohorts was statistically significant. P-S105-ERβ was expressed in MCF-7 cells and in response to 17β-estradiol (E2); levels were raised within 30 min, and sustained for 24 hours. ERβ1 expression was unaffected by this treatment. Nuclear speckling was also observed and was markedly increased at 24 hours following E2 but not Tamoxifen.Conclusion: This is the first time to our knowledge that the expression of P-S105-ERβ in breast carcinoma has been investigated. Its presence in breast carcinoma and association with improved survival suggest P-S105-ERβ might be a useful additional prognostic marker in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4141.
Receptor Conversion in Breast Cancer Metastases.
Authors: Hoefnagel L. , van der Wall E. , van de Vijver M. , van Diest P.
Published: December 2010

Background: Since breast cancer metastases are not usually biopsied due to their often inaccessible locations, choice of systemic treatment for metastatic breast cancer is usually based on tissue characteristics of the primary tumor, as determined by immunohistochemistry. However, pilot studies have shown that the immunophenotype of breast cancer metastases may be different from that of the primary tumor (“receptor conversion”). This means that many metastatic patients may not get the appropriate systemic treatment. The pilot studies published so far are however small and methodologically suboptimal as original staining data of the primary tumors were often used, and decalcified bone metastases were included that may give rise to false negative immunohistochemistry. Therefore, definite conclusions that may chance clinical practice could not be drawn. The aim of this study was therefore to study receptor conversion for ERα, PR, and HER2 in a large group of (non-bone) breast cancer metastases, restaining all primary tumors and metastases with current optimal immuno-histochemical methods.Methods: A group of 150 distant breast cancer metastases from different distant sites (79 skin, 33 liver, 22 lung, and 16 brain) was immunohistochemically stained for ERα, PR, and HER2, and receptor expression was compared to that of the primary tumor.Results: Receptor conversion for ERα, PR and HER2 occurred in 6%, 27.4% and 2.7% of the 150 patients, respectively. Conversion was mainly from positive to negative for ER and PR, and from negative to positive for HER2. Receptor conversion seemed to occur especially in liver metastases (ER 9.1%, PR 36.4%, HER2 6.1%) while brain metastases showed no conversion for ER and HER2.Conclusion: Receptor conversion in breast cancer metastases indeed occurs, is rare for ER and HER2, but is more frequent for PR, and may be most outspoken in liver metastases. This underlines the importance of having breast cancer metastases frequently biopsied when possible. Alternatively, molecular imaging strategies are warranted for sites that are difficult to biopsy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4145.
FOXP1 as a Potential ER Coregulator in Human Breast Cancer.
Authors: Shigekawa T. , Shigekawa T. , Ijichi N. , Takayama S. , Tsuda H. , Ikeda K. , Horie K. , Osaki A. , Saeki T. , Inoue S. , Inoue S.
Published: December 2010

Estrogen activates the estrogen receptor (ER) by recruiting various coregulators and cofactors, resulting in transcriptional regulation of target genes. Recently, we have shown that the FOXP1 forkhead transcription factor is a novel androgen-regulated coregulator for the androgen receptor (Biochem Biophys Res Commun, 374, 388-93, 2008). However, its role in breast cancer is still unknown.Here, we investigated the expression pattern of FOXP1 by immunohistochemistry in a series of 110 invasive breast cancers and compared it with clinicopathological factors. The expression of FOXP1 was detected in nuclei in 76 cases (69.1%) and correlated with expressions of ERα (P=0.001) and progesterone receptor (PgR) (P=0.003), while there was no association with age, lymph node status, size, or grade. Expression levels of FOXP1 mRNA are investigated by qRT-PCR using samples selectively obtained from breast cancer specimens with laser microdissection system (Zeiss Axiovert + P.A.L.M laser microdisection system). ERα, ERβ, and PgR mRNA levels were also measured.Then, we examined expression and function of FOXP1 in human breast cancer MCF-7 cells that possess functional ERα. The expression level of FOXP1 mRNA was up-regulated by estrogen treatment. In addition, estrogen-responsive element-driven luciferase reporter analysis showed that exogenously transfected FOXP1 increased estrogen-dependent transcriptional activity. We also observed that FOXP1 overexpression and knockdown could modulate the growth of MCF-7 cells.Taken together, these findings suggest that FOXP1 plays an important role as a potential ER coregulator in human breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4149.
Relationships between Progesterone Receptor Isoforms and the HER Receptors and Ligands Network in 299 Primary Breast Cancers.
Authors: Révillion F. , Lindet C. , Lhotellier V. , Hornez L. , Bonneterre J. , Bonneterre J. , Peyrat J.
Published: December 2010

Background: The effects of progesterone are mediated by two distinct nuclear receptors, called PR-A and PR-B, which are encoded by a single gene. In the normal human breast, these two isoforms are co-expressed at similar levels of expression whereas breast cancer seems to be characterized by an unbalanced expression of PR-A and PR-B, with a predominance of either one of these two isoforms. In the present study, we analyzed the relationships between the PR isoforms and the HER receptors and ligands network in primary breast cancer.Materials and Methods: This study involves 299 breast cancer samples from patients operated in our institute for locoregional disease between May 1989 and December 1991. Since PR-A has no specific sequence to distinguish it from PR-B, the mRNA expression of total PR-(A+B) isoforms as well as those of PR-B were assessed. They were quantified by real time quantitative RT-PCR using TaqMan probes, as previously described for HER receptors (Epidermal Growth Factor Receptor EGFR, HER2, HER3, and HER4) (Pawlowski et al, Clin Cancer Res 2000;6:4217-25) and HER ligands (EGF, Transforming Growth Factor alpha (TGFa), amphiregulin (AREG), betacellulin (BTC), Heparin-Binding EGF (HB-EGF), Epiregulin (EREG), and neuregulins 1-4 (NRG1-4)) (Révillion et al, Ann Oncol 2008;19:73-80).Results: PR-(A+B) and PR-B mRNA levels strongly correlated with PR and ER protein levels determined by radioligand assay. PR-(A+B) were found to correlate positively to IGF1 receptor, and both isoforms correlated negatively to histoprognostic grading. Considering the HER receptor and ligands network, PR-(A+B) were positively correlated with HER3, HER4, EGF, AREG, while they were negatively correlated with EGFR, TGFa, HB-EGF, EREG, and NRG2. Similarly, a positive correlation was found between PR-B and HER3, HER4, EGF and AREG while PR-B correlated negatively to EREG, NRG3 and NRG4. Finally, PR-(A+B) expression (higher than the lower quartile) was significantly associated with a longer overall survival (p= 0.02, Kaplan-Meier analysis).Conclusion: Our results, demonstrating strong correlations between PR isoforms and HER receptors and ligands network, reinforce the observations (Proietti et al, Mol Cell Biol 2009;29:1249-65) demonstrating that cross-talk between PR and the HER family appears to be a hallmark of breast cancer growth.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4150.
Amplification of the Prolactin Receptor Gene in Mammary Lobular Neoplasia.
Authors: Tran-Thanh D. , Tran-Thanh D. , Arneson N. , Pintilie M. , Warren K. , Bane A. , O'Malley F. , O'Malley F. , Done S. , Done S.
Published: December 2010

Background: Lobular carcinoma in situ (LCIS) has long been considered a marker of increased risk of cancer in both breasts. However, recent studies have shown that it can behave as a non-obligate precursor lesion as well. The biology and natural history of LCIS still remains ill defined in part because it is a challenging lesion to study as it usually does not have an identifiable gross appearance and is only recognized in fixed tissue specimens.Material and Methods: Using array comparative genomic hybridization (aCGH) we have analyzed regions of amplification found in LCIS and adjacent invasive lobular carcinoma (ILC) in a series of thirteen cases of archival patient samples from our institution. Degenerate oligonucleotide primed (DOP) PCR was performed for whole genome amplification of the extracted DNA from microdissected tissue samples prior to microarray analysis. Analysis of microarray data was performed using Significance Analysis of Microarrays (SAM). Of the ten most amplified genes in LCIS (highest SAM scores), one was selected for quantitative real time PCR (Q-PCR) validation due to the limited amount of material available from these cases. Q-PCR validation was performed on samples from 8 cases of LCIS and invasive lobular carcinoma and 12 archival cases of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) for comparison.Results: Amongst the 10 genes with the highest SAM scores, the prolactin receptor gene (PRLR) was selected for further Q-PCR validation in our limited samples. Amplification of PRLR was confirmed by Q-PCR in 4/8 (50%) of cases of LCIS and 4/8 (50%) cases of ILC, compared to 0/12 (0%) cases of DCIS and 3/12 (25%) cases of IDC. When LCIS and ILC were combined into one group, there was more amplification of the prolactin receptor gene when compared to the DCIS and IDC group (p= 0.01). The level of amplification between the two groups also differed in the range of copy number values, which was lower in the ductal group (0.78-1.58, n=24) compared to the lobular group (0.92-3.68, n=16) (p<0.05).Conclusion: We have identified the prolactin receptor as a potential molecular target in lobular neoplasia using array comparative genomic hybridization. In contrast, we have shown that the prolactin receptor may not be as important for the progression of ductal lesions. These results support the view that lobular and ductal carcinoma evolve along separate pathways. Validation of the expression of PRLR in a larger number of LCIS cases is warranted.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4151.
Expression Levels of Enzymes Related to In Situ Estrogen Synthesis and Clinicopathological Parameters in Breast Cancer Patients.
Authors: Saeki T. , Suzuki M. , Nakata T. , Shiotsu Y. , Ishida H. , Akinaga S. , Aogi K. , Utsumi T. , Harada N.
Published: December 2010

Background: Local estrogen synthesis may play an important role of progression of breast cancer with hormone receptors. Local progression of breast cancer may depend on high concentration of estrogens and estrogen receptor status. To clarify correlation between expression levels of enzymes in breast cancer and clinicopathological parameters, we analyzed expression of steroid sulfatase(STS), estrogen sulfatase(EST), 17b-hydroxyl steroid dehydrogenase type I & II(17b-HSDI & II) and aromatase. In addition, mRNA expression of estrogens receptor alpha(ERa), estrogen receptor beta(ERb), cycline D1 and erbB2 were measured. Material and Methods: The mRNA expression levels of genes in 196 breast cancer tissues removed from surgery were assayed by RT-PCR. Results: The level of STS were significantly higher in patients with clinical stage. STS expressed none in stage 0 or I, 0.011 in stage II, and 0.013 amol/1 amol b-actin in stage 3 or 4. In addition, EST expressed 58.5 amol/1 amol b-actin in stage 2 and 36.8 in stage 3 or 4, otherwise, the expression level of EST in stage 0 or 1 revealed 15.3 amol/1 amol b-actin. There were no correlation between clinical stage and aromatase expression. Expression levels of ERa and cyclineD1 significantly correlated with protein levels of ER, and there were significant correlation between erbB2 expression and prognosis of patients. Conclusion: Up-regulation of STS play an important role in tumor progression of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4155.
MMP-3 and MMP-9 Gene Transfer Decrease Growth and Angiogenesis in Breast Cancer Xenografts In Vivo.
Authors: Bendrik C. , Dabrosin C.
Published: December 2010

Matrix metalloproteinases (MMPs) are largely implicated in tumor angiogenesis and metastasis due to their extracellular matrix (ECM) degrading capacities. Although MMP activity generally is discussed in terms of facilitating tumor invasion, MMP inhibition in clinical trials has failed. Moreover, increasing amounts of data show that MMPs may inhibit tumor progression by generation of potent anti-angiogenic factors such as endostatin from the tumoral stroma. We have previously shown that intratumoral gene transfer of MMP-9 induced tumor regression and reduced angiogenesis of breast cancer in vivo. If MMP activity induces tumor progression or regression may depend on type of MMP or expression levels of MMP in the tumor tissue. In this study we treated established breast cancers in nude mice with adenovirus vectors carrying the human genes of MMP-3 or MMP-9 in a dose response manner. Microdialysis was used to sample endostatin in situ and tumor growth was monitored for 35 days. Tumors in mice treated with low-dose of either MMP-3 or MMP-9 vectors exhibited tumor stasis throughout the experiment whereas high-dose gene transfer of either MMP-3 or MMP-9 induced significant tumor regression compared to controls treated with empty adenovirus vectors. The extracellular in vivo levels of endostatin were significantly increased in tumors treated with adenovirus carrying the MMP genes compared to controls. Tumors treated with high-dose MMP vectors also exhibited decreased microvessel area. Our results propose that increased expression of MMP-3 and MMP-9 have therapeutic effects of established breast cancer in a dose dependent manner where a slight increase of MMP expression results in tumor stasis and a high expression of either MMP-3 or MMP-9 by gene transfer results in a potent tumor regression.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4164.
YB-1 Mediates a Crosstalk between ERα and TGFβ Signaling in Breast Cancer Cells.
Authors: Popp S. , Stope M. , Buck M. , Joffroy C. , Fritz P. , Knabbe C.
Published: December 2010

The multifunctional Y-box binding protein-1 (YB-1) belongs to the highly conserved family of cold shock proteins being involved in transcriptional/translational regulation, DNA repair as well as stress response. Additionally, it is highly expressed in many malignant tissues which seems to correlate with a high proliferation level, drug resistance and a poor prognosis in a variety of tumors including breast cancer.Using quantitative immunohistochemistry we now confirm the previously described negative prognostic impact of YB-1 on survival in a clinical study with tumor samples from 199 breast cancer patients (p=0.0146; median follow up 79 months).Treatment of the estrogen receptor α (ERα) positive human breast cancer cell line MCF-7 with antiestrogens (4-hydroxytamoxifen, faslodex) resulted in a time and dose dependent decrease of YB-1 expression on the RNA- as well as on the protein level. In contrast, antiestrogen treatment of the ERα negative cell line MDA-MB-231 does not have any effect indicating that the antiestrogen effect on YB-1 is mediated by ERα. We have already demonstrated that the action of all antiestrogens is at least partially mediated by transforming growth factor β (TGFβ) (e.g. Breast Cancer Res. Treat. 107: 15-24, 2008). Using TGFβ-responsive reporter gene assays we now show that overexpression of YB-1 in MCF-7 cells enhances the induction of p3TPlux activity by TGFβ suggesting a crosstalk between YB-1 and TGFβ signaling pathways. Moreover, YB-1 also increases the antiestrogen induction of the TGFβ2-mRNA level coming along with a YB-1 regulated enhancement of the antiestrogen effect on cell growth.Very recent findings of our group point towards a ligand-independent influence of ERα on TGFβ signaling (Breast Cancer Res. Treat., in press). Due to the antiestrogen regulation of YB-1 via ERα and its crosstalk with the TGFβ pathway, the potential role of YB-1 as a mediator of the interaction between ERα and TGFβ signaling was analyzed. Performing pull-down assays with recombinant GST-ERα and whole cell lysates of MCF-7 cells confirms a protein-protein interaction between YB-1 and ERα. The physiological relevance of this interaction could be demonstrated, as the TGFβ-induced p3TPlux activity is clearly modulated by both, YB-1 and ERα.In conclusion, we present evidence that YB-1 and TGFβ signaling are functionally linked in MCF-7 cells. Combined with the detection of a physical interaction between YB-1 and ERα, our findings shed new light on a potential role of YB-1 as a part of the mechanism responsible for switching TGFβ from its anti-proliferative role in early tumor stages to tumor promoting effects in late-stage disease, most likely via a YB-1 regulated shift of TGFβ-signaling to pro-invasive pathways. This would also explain why high levels of YB-1 correlate with a poor prognosis in breast cancer patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4172.