bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P4-06-05: Silencing of Cyclin D1 Increases Breast Cancer Cell Migration through Increased Id1 Expression and Induction of Epithelial-Mesenchymal Transition
Authors: Tobin NP , Lundgren K , Sims A , Landberg G.
Published: April 2011

Backround: The oncogene cyclin D1 is overexpressed in many breast cancers and despite proliferation activating properties, has been linked to a less malignant phenotype. We have previously shown that down-regulation of cyclin D1 in actively cycling breast cancer cells significantly increased their migratory capacity whilst also decreasing proliferation. Gene expression analysis and animal modeling have highlighted an increase in the inhibitor of differentiation-1 (Id1) gene following cyclin D1 gene (CCND1) silencing. Notably, increased Id1 is associated not only with enhanced migratory and invasive features of breast cancer cells but also with prevention of cell differentiation and induction of an epithelial-mesenchymal-like (EMT) phenotype. Here we sought to determine if the increase in cell motility following cyclin D1 silencing was through increased Id1 expression and induction of an EMT phenotype, and confirm our findings in a large patient cohort.

Materials and Methods: Expression of cyclin D1, Id1 and SNAI2 was determined in MDA-MB-231 and ZR75 cells by qPCR and western blot, whilst cell migration was monitored by transwell assay. Gene expression of CCND1, ID1 and EMT markers was analysed from a pooled cohort of batch-mean centered publically available data sets, with data analysis performed in SPSS.

Results: Silencing of cyclin D1 increased cell migration 1.38 and 1.72 fold in MDA-MB-231 (P<0.001), and ZR75 (P<0.001) cells respectively, an effect abolished by silencing of Id1. qPCR analysis confirmed an increase in Id1 (1.73) and SNAI2 (1.50) expression following siRNA treatment of cyclin D1, an effect reversed following siRNA treatment of Id1. CCND1 gene exression was negatively, while Id1 was positively, associated with the EMT markers Vimentin (P<0.001), SNAI2 (P<0.001) and TWIST1 (P<0.001) in 1,100 breast tumors. In ER+ patients CCND1 was negatively correlated to Id1 expression (0.050) and in CCND1 low patients increasing Id1 expression was predictive of worse cumulative survival (0.027). Conclusion: Decreased CCND1 expression enhances MDA-MB-231 and ZR75 cell migration through increased Id1 expression and induction of EMT. Moreover, both genes are correlated to EMT markers in breast cancer patients and in a subset of the cohort, confer a worse survival. These results may explain the conflicting data regarding cyclin D1 expression in breast carcinomas and solidifies the rationale of the protein as a clinical target.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-05.
Abstract P4-06-07: PI3K Mutations Are More Common in Low Grade DCIS
Authors: Sakr R , Chandarlapaty S , Andrade V , Giri D , Muhsen S , Wooyul P , Morrow M , Rosen N , King T.
Published: April 2011

Background: DCIS is a non-obligate precursor to invasive breast cancer yet it remains difficult to predict which lesions will progress or over what time interval. Factors associated with a higher risk of progression include high histologic grade and HER2 amplification. HER2 signaling via the PI3K pathway is associated with accelerated invasiveness in laboratory models and mutations in the helical domain of PI3K have been associated with a worse prognosis in invasive cancer. We hypothesized that PI3K pathway mutations are associated with higher risk of progression in DCIS and therefore would be more common in high grade DCIS.

Methods: 195 cases of pure DCIS were identified from the MSKCC breast service database (1999-2003). All cases were reviewed by a single pathologist to assign histologic grade. Sections were obtained from archived formalin-fixed paraffin embedded (FFPE) blocks for manual microdissection to isolate pure DCIS lesions for DNA extraction. Multiplex array (Sequenom®) genotyping for PIK3CA was performed on prePCR amplified DNA. Comparisons were made between high and low grade DCIS using Fisher's exact test.

Results: Among 195 pure DCIS cases, 89 were classified as high grade and 106 as low grade. Sequenom® analysis was informative in all cases. PIK3CA mutations were identified in 4/89 (4%) cases of high grade DCIS and in 24/106 (23%) cases of low grade DCIS (p=0.000). All 4 mutations in high grade DCIS were located in the kinase domain, whereas in low grade DCIS, 9/24 (37%) mutations were in the kinase domain (H1047R) and 15/24 (67%) mutations were in the helical domain (E542K, E545K, N345K).

Conclusion: PI3K mutations were relatively uncommon in pure high grade DCIS as compared to low grade DCIS. PI3K mutations in low grade DCIS were observed in both the kinase and helical domain. These findings support the hypothesis that breast tumorigenesis differs by grade and PI3K mutations may be more prominent in low grade carcinogenesis. The significance of helical domain mutations in low grade lesions requires further investigation.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-07.
Abstract P4-06-09: Genetic Signature Discriminating Metastatic from Non-Metastatic Small Tumors
Authors: Ellsworth RE , Valente AL , Field LA , Kane JL , Love B , Tafra L , Shriver CD.
Published: April 2011

Background: The widespread use of mammographic screening resulted in increased diagnosis of small (<2 cm; T1) tumors. Small tumors are associated with better prognosis, including a lower likelihood of developing metastasis, than larger tumors. Although this lower propensity to metastasize suggests that less aggressive treatments may be warranted in patients with T1 tumors, a subset of patients with small tumors (10-20%) will be diagnosed with lymph node metastasis.

Methods: Frozen breast specimens were collected from women with T1 tumors and either negative (n=29) or positive (n=15) lymph node status. RNA was isolated from pure tumor cell populations after laser microdissection. Gene expression data was generated using HG U133A 2.0 arrays (Affymetrix). Differential expression was determined using Mann-Whitney testing using a P-value < 0.001 to define significance. Results for ESR1 were validated by immunohistochemistry.

Results: Tumor characteristics did not differ significantly between groups in terms of age at diagnosis, grade, HER2 or PR status; however, tumors from patients with positive lymph nodes (47%) were significantly (P<0.05) more frequently ER negative compared to node negative (14%) patients. Gene expression analysis revealed 17 genes that were differentially expressed between node negative and node positive tumors: 6 with higher expression in node positive, including AURKA, and 11 with higher expression in node negative patients, including ESR1 and EPHX2. Of note, ESR1 was expressed at >4X higher levels in tumors without metastasis, in agreement with IHC findings.

Conclusions: Small metastatic tumors differ in gene expression from those without metastasis. EPHX2 has been implicated as a metastasis suppressor while AURKA has been implicated as a metastasis promoter. These results suggest that small tumors have different propensities to metastasize and the genetic signature may serve as a new molecular tool to discriminate metastatic and non-metastatic small tumors, allowing appropriate treatment and risk assessment to be performed.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-09.
Abstract P4-06-10: Altered Activity of RhoA and RhoC GTPases Phosphorylate C-Jun N-Terminal Kinase and Induce Cellular Dormancy in Breast Cancer Cells
Authors: Chatterjee M , van Golen KL
Published: April 2011

Tumor dormancy has been a very real problem in breast cancer survivors where recurrences are common and usually fatal. Molecular mechanisms involved in this process are often poorly understood and are a “hot-spot” in breast cancer research today.

Ongoing efforts in our laboratory has shown that Rho GTPases, specifically RhoA and RhoC, play a significant role in dormancy of breast cancer. We have currently established two in vitro models of breast tumor dormancy — one involving growth of the breast cancer cell line MCF7 on fibronectin and subsequent treatment with basic fibroblast growth factor and the other involving treatment of MCF7 cells with the farnesyl transferase inhibitor FTI-L-744,832. In both these models, we find high levels of active RhoC and almost undetectable levels of active RhoA GTPases in quiescent breast tumor cells. Investigating further downstream we found c-jun N-terminal kinase (JNK) is phosphorylated in these dormant cells but not p38mitogen activated protein kinase(MAPK) or extracellular signalregulated kinase(ERK1/2). On inhibiting JNK with a pharmacological inhibitor specific for JNK, SP600125, we found dormancy could be interrupted and cell proliferation was induced. JNK has been implicated in inducing autophagy in cells by activating autophagy gene Atg5 and Beclin1 (BCN1) [Byun et al., Carcinogenesis, 2009]. Since autophagy can be a mode of survival for the dormant tumor cell [Matthew et al., Cancer Cell, 2006] we explored the possibility of autophagy being induced in these dormant tumor cells via JNK by employing the autophagy marker LC3-II. We found upregulation of the autophagy marker, thus revealing autophagy as a strategy for survival for these tumor cells in unfavorable tumor microenvironment. We are currently investigating the molecular mechanisms underlying the induction of dormancy in breast tumor cells by Rho GTPases through JNK and the dormant tumor survival strategy through autophagy.

We believe that targeting these implicated molecules like RhoA and RhoC GTPases, JNK or some of the autophagy genes might yield therapeutic benefits for breast cancer patients in remission and wipe out fears of deadly metastatic recurrences thus prolonging survival.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-10.
Abstract P4-06-15: BP1 Upregulates Twist and Induces EMT in Breast Cancer Cells
Authors: Hwang B , Rheey J , Fu SW , Berg PE.
Published: April 2011

Background: We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with estrogen receptor negativity and tumors of African American women, both associated with aggressive tumors. BP1 is also expressed in metastatic tumors, shown by immunostaining of 46 samples of inflammatory breast cancer; all cases were BP1 positive. Nine cases had metastasized, and all nine metastatic lymph nodes were BP1 positive. BP1 positive cells were observed in lymph channels and blood vessels. Here, we demonstrated BP1 induces the epithelial to mesenchymal transition (EMT), resulting in increased migratory ability. EMT, a process by which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells become more invasive, migratory and can lead to metastasis. Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT, is activated by BP1.

Materials and Methods: RNA levels of markers of EMT were verified by real-time PCR and their protein levels by Western blotting and, in some cases, by confocal microscopy. Chromatin immunoprecipitation (ChIP) was performed to verify the BP1 binding site on the Twist promoter. A scratch test was used to measure migratory ability. BP1 knockdown by siRNA transfection was also performed. Recombinant BP1 (rpBP1) was produced in E. coli.

Results: Increased expression levels of Twist were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. We demonstrated that BP1 can upregulate Twist expression in two different breast cancer cell lines, MCF-7 and Hs578T cells, by binding to the Twist promoter. BP1 upregulates mesenchymal marker expression and down-regulates epithelial marker expression, consistent with EMT. BP1 also promotes breast cancer cell migratory ability, shown by the scratch test. In addition, cells grown in medium supplemented with rpBP1 showed increased Twist expression and migration. Experiments evaluating the effects of siBP1 on EMT are underway.

Conclusions: BP1 stimulates Twist expression in MCF-7 and HS578T breast cancer cells, resulting in a more mesenchymal cell phenotype. We therefore hypothesize that BP1 induces EMT by upregulating Twist expression, and may result in metastasis. If BP1 is involved in EMT, it may be a good target for therapy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-15.
Abstract P4-06-16: TWEAK/Fn14 Signaling Axis as a Driver of Breast Cancer Cell Invasion and Metastasis
Authors: Stone AE , Willis AL , Niska JR , Black MA , Ramos P , Mancini CM , Cunliffe HE.
Published: April 2011

The molecular events associated with the multi-step process of cell invasion and metastasis are poorly understood. Tumor necrosis factor receptor family member fibroblast growth factor-inducible 14 (Fn14) and its ligand TWEAK (tumor necrosis factor-like weak inducer of apoptosis) have recently emerged as a pro-invasive signaling mechanism and indicator of poor prognosis in breast cancer. Our laboratory has recently reported enhanced Fn14 expression in a significant proportion of breast tumor samples, but not in normal breast epithelium. Analysis of publicly available breast cancer data indicates that high Fn14 expression positively correlates with HER2/neu positivity, estrogen receptor negativity, high tumor grade, positive lymph nodes, and metastasis. Modulation of Fn14 expression in breast cancer cell lines either by exogenous overexpression or siRNA-mediated blockade is sufficient to regulate breast cancer cell invasive behavior in vitro. However, the mechanisms leading to Fn14 overexpression, and how activation of the TWEAK/Fn14 signaling axis contributes to breast tumor malignant progression remain unclear. We hypothesized that genomic alterations and inappropriate autocrine or paracrine growth factor signaling may play a role in aberrant Fn14 regulation. Using array genomic hybridization and fluorescence in situ hybridization, we found increased Fn14 gene copy number relative to centromere 16 in two out of four Fn14-expressing, invasive breast cancer cell lines and in 28% of primary breast tumors. We also investigated the role of exogenous growth factors known to play important roles in the breast tumor microenvironment in stimulating Fn14 expression in vitro. TWEAK, EGF, IGF1, HRG and HGF/SF were sufficient to stimulate Fn14 expression de novo in a variety of biologic contexts. Many of these growth factors converge on the MAPK signaling axis. We have confirmed a functional role for the MAPK pathway in facilitating Fn14-mediated invasion. Consistent with this observation, Fn14 expression in primary breast tumors is high in the context of K-RAS mutation. We are currently evaluating the presence of important key regulatory regions within the Fn14 promoter that may be contributing Fn14 over expression in breast cancer cells. To define mechanisms by which Fn14 contributes to a malignant cell phenotype we have identified an Fn14-specific invasion signature using gene expression microarray profiling. Analysis of this data points to key malignant mechanisms likely to be impacted by Fn14 activation within different biologic contexts that warrants further investigation.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-16.
Abstract P4-06-18: Asporin Is Associated with Invasive Growth and Bone Metastasis of Breast Cancer
Authors: Bouchal J , Simkova D , Kharaishvili G.
Published: April 2011

Background: In an earlier publication we identified asporin as a novel cancer related protein (BMC Cancer 2007, 7:55), also reported among bone metastasis specific genes (Cancer Letters 2009, 276:212-220). Asporin is a member of the class I small leucine-rich repeat proteoglycan family and besides affinity to collagen, it binds calcium and initiates collagen mineralization. Asporin is abundantly expressed in tissues with high remodeling rate (e.g. peridontal ligament) whereas extremely high turnover of matrix proteins is also associated with invasive cancer. Material and methods: Formalin-fixed paraffin-embedded tissues [invasive ductal (101) and lobular (39) carcinomas classified according to hormone receptors and Her-2; 5 bone metastases from autopsies] were immunohistochemically stained with in-house polyclonal peptide antibody against asporin. The antibody was validated on uterus tissue as a positive control together with a commercial antibody (Abcam). Data were evaluated by non-parametric Mann-Whitney U test and Spearman correlation coefficient. Expression of asporin in both tissues and cell lines was also queried in databases Gene Expression Omnibus, ArrayExpress and Oncomine. Expression of asporin was further tested by RT-PCR in selected breast cancer cell lines. Migration and invasion of MDA-MB-231 cells were tested using real-time cell analysis Xcelligence system (Roche). Transwells were coated by solidifying collagen for 6 hours (with or without recombinant asporin). Starved cells were seeded on the collagen in transwells while FBS was used as a chemoattractant in bottom wells. Results: Levels of asporin were significantly higher in invasive lobularcarcinomas than ductal ones (P<0.001). Asporin was also highly expressed in all bone metastases. Asporin expression positively correlated with tumor grade within the triple negative subgroup (Rs 0.4, P<0.05) but not in luminal and Her-2 subtypes. Expression of asporin in invasive breast carcinomas was also confirmed in other public datasets (e.g. Karnoub breast and Hess breast in Oncomine database). Surprisingly, we found no breast cancer cell line overexpressing asporin, which underlines the importance of the tumor microenvironment in vivo. MDA-MB-231 cells migrated faster through collagen I with recombinant asporin.

Conclusion: Asporin is expressed by both invasive breast carcinomas and bone metastases. Mechanistically, asporin may affect extracellular matrix structure and promote invasive growth of breast cancer cells. Further investigation of the role of asporin in metastasizing to bone is additionally justified given its affinity for calcium. Acknowledgements

This work was supported by grants NS 9956-4 from the Czech Ministry of Health and MSM 6198959216 from the Czech Ministry of Education.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-18.
Abstract P4-06-22: Persistent Triple-Negative Phenotype Is Associated with Poorest Outcome among Patients with Metastatic Breast Cancer (BC) - Results of the Retrospective Multicenter PriMet Study Comparing Molecular BC Phenotypes in Primary Tumors and Corresponding Recurrences
Authors: Liedtke C , Gluz O , Heitz F , Freudenberger M , Würstlein R , Hungermann D , Ortmann M , Kates RE , Nitz U , du Bois A , Fehm TN , Harbeck N.
Published: April 2011

Background: Patients with triple negative (TN) breast cancer (BC), defined by lack of both ER/PR expression and HER2 overexpression, have an unfavorable prognosis. Although phenotype changes between primary tumor (PT) and disease recurrence (DR) have been described, their clinical significance is still unclear.

Methods: We conducted PriMet, a retrospective multicenter (n=11) study to compare BC phenotype in PT and corresponding DR. Inclusion criteria comprised (1) unilateral BC with subsequent/synchronous local/regional/distant DR and (2) immunohistochemical confirmation of DR. Our aim was to (a) evaluate discordance rates between PT/DR, (b) find predictors for discordance, and (c) analyze the impact of discordance on patient outcome.

Results: 436 patients were entered into PriMet; 414 had no evidence of primary metastatic disease (M0). Median follow-up in patients alive at time of analysis was 73. 1 (4.4-293.6) months. Triple receptor status for PT and DR was available in 377 patients; 68 patients (18.0 %) showed TNBC in PT, 40 patients (10.6 %) had TNBC in both PT and DR (i.e. TNBC persistence); 28 patients (7.4 %) changed from TNBC to non-TNBC (15 became HER2 positive (4.0 %)). Status changes for ER and PR were significantly positively associated (P<0.001). Patients with either persistent TNBC or non-TNBC had a median age at diagnosis of 52.0 compared to 55.5 yrs for patients with discordant TN status (p=0.04). Compared to differing constellations, persistent TNBC was associated with higher tumor grade (p=0.018) as well as with both decreased disease-free survival (DFS) (see figure) and post-recurrence survival (PRS) in M0 patients. In multivariate analysis containing pT stage, nodal stage, tumor grade in PT, TNBC in PT and TNBC persistence, TNBC persistence remained significantly associated with decreased DFS and PRS.

Conclusion: PriMet provides substantial evidence regarding potential phenotype changes in PT vs. DR and underlines the importance of immunohistochemical DR verification even in initially TN disease. TNBC persistence was significantly associated with adverse patient outcome.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-22.
Abstract P4-07-02: Expression of Cell Adhesion Molecules Predicts Prognosis in Early Breast Cancer Patients
Authors: de Kruijf EM , Saadatman S , Sajet A , Boer W , van Velzen N , Putter H , Smit VTHBM , Liefers GJ , van de Velde CJH , Kuppen PJK.
Published: April 2011

Purpose: New prognostic and predictive factors are sought for improvement of tailored treatment in early breast cancer. We examined the clinical impact of cell adhesion molecules (CAM): E-cadherin, N-cadherin, Ep-CAM and CEA.

Patients and Methods: Our study population (n=574) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. A tissue micro array (TMA) of formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained for expression of mentioned CAM. The percentage of membranous stained cells was microscopically analyzed. Based on the median score, all CAM were classified in two groups: low expression versus high expression. For CEA, high expression was further subdivided based on the intensity of staining: high expression and highest expression. Results: High expression was seen for E-cadherin, N-cadherin and Ep-CAM in 49%, 46%, 27% of patients respectively. Low expression, high expression and highest expression were found in respectively 48%, 45% and 8% of cases for CEA. Low expression of E-cadherin (p=0.015) and higher expression levels of N-cadherin, Ep-CAM, CEA (p=0.004; 0.046; 0.001 respectively) all resulted in a worse relapse free period (RFP) of patients. Multivariate analysis revealed only E-cadherin and CEA to be independent prognostic variables. A combination variable was created with expression of both markers: (1) E-cadherin high expression, (2) E-cadherin low or CEA low or high expression (3) CEA highest expression. This variable revealed to be an independent prognostic parameter with high discriminative power for RFP (P<0.001, E-cadherin low or CEA low or high expression versus E-cadherin high expression: Hazard Ratio (HR)= 1.9; CEA highest expression versus E-cadherin high expression: HR= 3.6). A statistically significant interaction was found between expression of both CAM (P<0.001), suggesting a biological connection in their functioning.

Conclusion: We have demonstrated that E-cadherin, N-cadherin, Ep-CAM and CEA are of prognostic influence on outcome concerning RFP in breast cancer patients. A combined variable of E-cadherin and CEA expression revealed to have prognostic influence on RFP with high discriminative power and therefore is a candidate parameter for future outcome prediction of patients.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-02.
Abstract P4-07-06: Yes-Associated Protein, a Proposed Tumour Suppressor, Is Inversely Correlated to Cyclin D1 in Breast Tumours and Associated with a Worse Prognosis
Authors: Lehn S , Tobin NP , Sims AH , Jirström K , Landberg G.
Published: April 2011

Background: In breast cancer, cyclin D1 over-expression and CCND1 (11q13) gene amplification have been linked to a worse prognosis. Conversely, lower levels of cyclin D1 have also been correlated to an unfavourable outcome. In a microarray analysis of cyclin D1 silenced MDA-MB-231 cells we identified Yes-associated protein (YAP), located at 11q22, as one of the most down-regulated genes. As such, we aimed to determine the importance of YAP in relation to cyclin D1 in breast cancer biology.

Material and methods: Validation of cyclin D1, YAP antibodies and protein levels were performed in MDA-MB-231, MDA-MB-468 and MCF-7 by western blot. YAP protein expression was examined by screening 144 breast tumours and gene expression assessed by meta-analysis of six previously published breast cancer datasets totalling 1107 tumours with clinical follow-up. Subsequent data analysis was performed in SPSS. Results: YAP protein levels were down-regulated in cyclin D1 silenced MDA-MB-231, MDA-MB-468 and MCF-7 cell lines as predicted by microarray analysis. Surprisingly, YAP and cyclin D1 were inversely correlated at both protein and gene level in primary tumours (p=0.004 and P<0.001). Amplification of the CCND1 gene was correlated to low YAP protein expression (P<0.001) in the screening tumour material. Low YAP mRNA was an independent prognostic factor of recurrence (HR= 1.30, 95% CI 1.009-1.678, p=0.043) in the gene expression dataset. This effect was particularly pronounced in the luminal A subtype of breast tumours. Discussion: The independent prognostic value of YAP strengthens the notion of this protein as a tumour suppressor in breast cancer. The regulation of YAP by cyclin D1 appears to occur on both the protein level as seen in cell lines, and on the chromosomal level in terms of 11q aberrations. The inverse correlation observed in the tumour materials between cyclin D1 and YAP may be partly explained by chromosomal rearrangements; the amplification of the CCND1 gene (11q13) is often accompanied by loss of the 11q22 region of the chromosome, harbouring the YAP locus. Further studies are ongoing to clarify the link between YAP and cyclin D1 in breast cancer biology.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-06.
Abstract P4-07-08: High Level of Nuclear S100P Is Associated with Poor Survival in Early Breast Cancer Patients
Authors: Maciejczyk A , Lacko A , Ekiert M , Halon A , Matkowski R , Surowiak P.
Published: April 2011

Background: S100P — low molecule acidic protein, which belong to the EF-hand superfamily of calcium binding proteins has been shown to be involved in processes of proliferation, survival, angiognesis and metastasis in various human malignancies. In breast cancer S100P expression is associated with immortalization of neoplastic cells, progression and aggressive tumor behaviour indicating that this protein may have prognostic value. Several studies also revealed relationship between S100P level and resistance to cytotoxic drugs, however these observations are conflicting. Materials and methods: We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer (BC) patients with 17 years follow up. Immunohistochemical (IHC) reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. Semi-quantitative scale of ImmunoReactive Score (IRS) was applied (score 1-12) to evaluate the S100P expression level. Results were correlated with clinical and pathologic data. The Kaplan-Meier method was used to estimate the survival probabilities.

Results: IHC staining for S100P was predominantly nuclear (S100Pn). Of the 85 specimens evaluated, 23 (27,0%) were not stained, 35 (41,2%) exhibit borderline or weak expression (IRS: 1-4) and 27 (31,8%) strong expression (IRS: 6-12). Patients with strong S100Pn expression had significantly lower overall survival (OS) (P<0,00001) and disease free survival (DFS) (p = 0,00002). Cytoplasmic S100P (S100Pc) expression did not appear to impact prognosis. When stratifying for lymph node status, S100Pn was a significant prognostic factor only in node-positive (N/+/) group and had borderline significance in N/-/(p =0,05777 and p=0,04105, for DFS and OS respectively). In contrast to other studies we did not find statistically significant correlation between S100P expression and tumor variables such as: lymph node involvement, estrogen receptor (ER), progesterone receptor (PR) and HER-2 status. Discussion (albo Conclusion): We demonstrated that high S100Pn expression level is associated with poor survival in early stage BC patients. Since preliminary data indicate that expression of S100P is up-regulated by activation of glucocorticoid receptor and other agents have potential to activate or inhibit S100P promoter activity, this protein may become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value. Key words: breast cancer, prognostic biomarker, S100P

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-08.
Abstract P4-08-02: A Comparison between AQUA Quantitative Fluorescent Immunohistochemistry and Conventional Immunohistochemistry for Hormone Receptors
Authors: Bartlett JMS , Gustavson M , Stocken D , Rimm D , Christiansen J , van de Velde CJH , Hasenburg A , Kieback D , Putter H , Brookes C , Markopoulos C , Dirix L , Robson T , Seynaeve C , Dolled-Filhart M , Jones C , Graves L , McGuire J , Rea D.
Published: April 2011

Background: We have previous data showing that quantitation of hormone receptors can be highly informative in determining risk of early relapse in ER positive early breast cancer treated with tamoxifen or exemestane. Both quantitative immunohistochemistry (QIHC) and flouresecent immunohistochemistry (F-IHC as measured by AQUA technology) are highly prognostic over a wide expression range. We have explored the results of both assays to determine if current assays provide maximum information using current approaches.

Patients &

Methods: Pathology blocks from 4598 TEAM patients were collected and tissue microarrays constructed. Quantitative AQUA and IHC analysis (using image quantitation) of ER and PgR was performed centrally (Edinburgh & HistoRx). Results from both assays were compared and their prognostic impact on DFS at 2.75 years examined.

Results: Both AQUA and QIHC demonstrated linear relationships between intensity of staining for either ER or PgR and DFS at 2.75 years. For both PgR and ER AQUA provided significantly greater prognostic information that QIHC. However AQUA staining explained only 29% and 68% of the variability in ER and PgR QIHC results by logistic regression. Using both AQUA and QIHC data in a forward stepwise selection survival model demonstrated that AQUA and QIHC provided similar prognostic information over 70% and 50% of the range for ER and PgR respectively. High ER QIHC and low ER AQUA scores, and low PgR IHC and high PgR AQUA scores provided prognostic information unique to either platform.

Conclusion: Both QIHC and AQUA analysis of HR expression provides significant and highly important information on DFS risk in early breast cancer. It appears that these two platforms provide overlapping prognostic information and that the range of ER and PgR expression which impacts patient outcome is wider than measured by either system alone. Further investigation of the clinical significance of this broader range of hormone receptor expression in treatment decisions is warranted.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-02.
Abstract P4-08-06: Companion Diagnostic Test for Expression of β-III tubulin (ß3T) in Breast Cancer (BC) Using an Immunohistochemical (IHC) Assay in Studies of Ixabepilone (Ixa)
Authors: Roach C , Tanna V , Welcher R , Dumontet C , Sève P , Trifan O.
Published: April 2011

Background: Preclinical and retrospective clinical data suggest that ≥3T is a biomarker for poor prognosis and resistance to paclitaxel in breast cancer (BC). Preclinical studies have demonstrated that Ixa has activity in ≥3T over-expressing xenograft models resistant to taxanes and Ixa has shown clinical activity in BC patients with high ≥3T mRNA levels [Horak et al., J Clin Oncol 27:15s, 2009 (suppl; abstr 3587]. Dako and BMS have developed an IHC assay for detection of ≥3T to explore the potential of ≥3T as a biomarker to select BC patients that may benefit from Ixa. A neoadjuvant BC study comparing ixa to weekly paclitaxel employs this assay to assess ≥3T expression levels in patients’ tumor samples. Here we describe the application and initial validation testing of the assay in BC tumor samples.

Methods: An IHC assay employing a murine monoclonal anti-≥3T antibody (TUJ1) was developed to assess ≥3T expression in formalin-fixed paraffin-embedded tissue specimens. Banked specimens for initial testing included 233 cases of invasive BC.

Results: Patterns of ≥3T staining were typically homogeneous cytoplasmic staining with normal tissue elements such as endothelial cells and nerve cells serving as internal positive controls. Staining intensity was scored as 0, 1+, 2+ or 3+ with 0 being no visible staining and 2+ being equal to that of the internal positive control. Tumors with ≥50% cells staining ≥2+ were defined as positive for ≥3T based on previous published studies. Of the 233 breast tumors, 81 (35%) were positive and 152 (65%) were negative for ≥3T expression. HER2 status was defined for 128 of these specimens, with 37 of them (29%) found to be HER2 positive. Comparison of the samples with defined HER2 status demonstrated similar patterns of ≥3T expression, with 15 (40.5%) of the HER2+ samples being positive for ≥3T and 31 (34.1%) of HER2- specimens expressing elevated levels of ≥3T. In pathologist training exercises the inter-reader and intra-reader agreement of ≥3T scoring exceeded 85%. Antigen stability has been confirmed and other assay validation efforts are ongoing.

Conclusions: These findings are consistent with previously reported data on ≥3T expression in breast cancer, and support the use of this ≥3T IHC assay in studies with ixabepilone.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-06.
Abstract P4-08-11: Surface Plasmon Resonance (SPR) Analysis of COX-2 Expression for Early Detection and Progression of Breast Cancer and Evaluation of a Novel Class of Peptides as Its Inhibitor
Authors: Singh AK , Parshad R , Das SN , Dalal K , Dey S
Published: April 2011

Background: Cyclooxygenase-2 (COX-2) over expression has been observed in different cancers. Till date, COX-2 expression in breast cancer has been studied using RT-PCR and immunohistochemistry. The aim of this study was to use surface plasmon resonance (SPR) technology for determining the expression levels of COX-2 in breast cancer and design peptide inhibitors to evaluate their enzyme inhibition in vitro. Patients and methods: This case-controlled study was performed on 84 biopsy proven breast cancer patients having invasive ductal carcinoma and 40 age, sex and ethnicity matched healthy subjects. Blood and tissue samples were collected and serum and tissue lysate was prepared. Serum and tissue levels of COX-2 were evaluated using SPR where samples were allowed to flow over anti-COX-2 antibody immobilized sensor chip to obtain the respective RUs. Similarly, standard curve of COX-2 was prepared from cloned, expressed and purified protein to deduce the concentrations in different samples. To evaluate enzyme inhibition, fifteen peptides were synthesized using the solid and solution-phase peptide synthesis based on the active site structure of COX-2 and enzyme activity was evaluated spectrophotometrically.

Results: A significant increase in COX-2 levels in breast cancer patients 11.23-30.77μg/ml (Mean±SD=14.55±5.74) as compared to normal controls 2.13-5.16µg/ml (Mean±SD=3.71±0.86) (P>0.0001) was observed. Serum COX-2 levels were significantly higher (P<0.0061) in patients with lymph node involvement. More than 65% patients showed significantly (P<0.0025) reduced COX-2 levels after chemotherapy. This was confirmed by western blot to check tissue level expression and spectrophotometrically to determine the activity of COX-2 at different stages. In addition, the peptides showed significant in vitro enzyme inhibition, and had a KD ranging from 1 X 10-8 to 1X10-10.

Conclusion: SPR is a useful proteomic technique for early detection and monitoring progession of breast cancer and peptides are a novel class of COX-2 inhibitors that can be further developed for anti-tumor therapy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-11.
Abstract P4-09-02: Prognostic and Predictive Value of Centrally Reviewed Ki67 Labeling Index in Postmenopausal Women with Endocrine-Responsive Breast Cancer: Results from Austrian Breast and Colorectal Cancer Study Group Trial 8
Authors: Bago-Horvath Z , Rudas M , Jakesz R , Dubsky P , Singer CF , Dietze O , Greil R , Jelen A , Offner F , Lang A , Gruber C , Pöstlberger S , Gnant MFX , Filipits M.
Published: April 2011

Purpose: The aim of the present study was to assess the prognostic and predictive value of Ki67 labeling index in postmenopausal hormone receptor-positive early breast cancer patients who were treated with adjuvant tamoxifen or anastrozole after tamoxifen. Patients and Methods: We determined the expression of Ki67 by immunohistochemistry on whole tissue sections of breast carcinoma patients who had been enrolled in Austrian Breast and Colorectal Cancer Study Group (ABCSG) Trial 8 and received tamoxifen for 5 years or tamoxifen for 2 years followed by anastrozole for 3 years. Ki67 labeling index was evaluated as continuous variable or dichotomized at 10%. Distant recurrence and death were analyzed using Cox models adjusted for clinical and pathological factors.

Results: High Ki67 labeling index was observed in 394 of 1587 (23%) tumors and was associated with poor outcome. Patients with high Ki67 labeling index had a significantly shorter distant recurrence-free survival (adjusted hazard ratio [HR] for distant recurrence 2.16, 95% confidence interval [CI] 1.43-3.25, P < 0.001) and overall survival (adjusted HR for death 1.77, 95% CI 1.30-2.42, P < 0.001) as compared to patients with low Ki67 labeling index. No interaction between Ki67 labeling index and treatment was observed (P = 0.84).

Conclusion: High Ki67 labeling index is an independent poor prognostic factor for distant recurrence and death in postmenopausal women with early-stage, hormone receptor-positive breast cancer but is not predictive for outcome of adjuvant treatment with either tamoxifen or tamoxifen followed by anastrozole.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-02.
Abstract P4-09-09: Serum SDF-1: Biomarker of Bone Relapse in the NCIC MA.14 Adjuvant Breast Cancer Trial
Authors: Ali SM , Aguilar-Mahecha A , Chapman J-AW , Lipton A , Leitzel K , Jahan K , Hassan S , Shepherd LE , Han L , Wilson CF , Pritchard KI , Pollak MN , Basik M.
Published: April 2011

Background: Massague et al have shown that breast cancer cell line subpopulations with elevated bone metastatic activity overexpress chemokine receptor 4 (CXCR4), interleukin 11 (IL11), osteopontin (OPN) and connective tissue growth factor (CTGF) (Cancer Cell 3:537, 2003). CXCR4 overexpression results in bone-homing and extravasation of tumor cells in bone. In MA.14, we found that serum β-CTx was associated with bone-only relapse while Basik et.al showed that higher serum stromal cell-derived factor 1 (SDF-1) (ligand for CXCR-4) levels were associated with worse overall event-free survival (EFS) (ASCO 2010). In this study, we examined concurrently the association of both β-CTx and serum SDF-1 with bone relapse.

Methods: Serum β-CTx (Serum CrossLaps, Nordic Biosciences, Copenhagen, DN) was determined in pretreatment sera from 621 of 667 NCIC CTG MA.14 patients. SDF-1 (CXCL12) (R&D Systems, Minneapolis, MN) levels were successfully determined in the 4 month post-treatment serum (SDF-1) for 508 (76%) of the patients. Trial stratification was by administration of adjuvant chemotherapy, axillary lymph node status, and ER and/or PR status. Recurrence-free survival (RFS) was defined as the time from randomization to the time of recurrence of the primary disease. Adjusted and unadjusted Cox step-wise forward multivariate analyses were used to assess the effects of β-CTx, SDF-1, trial therapy and baseline patient characteristics on non-bone, all bone and bone-only RFS; a factor was added if p<=0.05.

Results: Joint assessment of β-CTx and SDF-1 was possible for 493 (74%) of the 667 patients. Imbalances in who was, or was not, included in this subset led to the trial arm of Tamoxifen + Octreotide LAR having a significant longer unadjusted ITT non-bone RFS (p=0.03-0.06). There was shorter time to bone metastasis of any type with higher lymph node involvement (p=0.001), larger T (p=0.02), and higher log SDF-1 (p=0.03). Meanwhile, high categorical and continuous β-CTx was associated multivariately with shorter bone-only RFS (p=0.04 and 0.01, respectively); higher log SDF-1 was only associated with shorter bone-only RFS (p=0.02) when the number of strata were reduced to 2 categories per factor.

Conclusions: Higher serum SDF-1 level may be associated with bone metastasis, although there is less evidence of its relevance in bone-only relapse than there is for the biomarker β-CTx. Serum SDF-1 deserves further study as a promising predictive factor of bone relapse in breast cancer.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-09.
Abstract P4-09-11: Variables of Worse Outcome in Premenopausal ≥35 Years old Patients (pts)
Authors: Vallejos CS , Gómez HL , Cotrina JM , Marcelo MJ , Doimi FF , Neciosup SP , Suazo JF , Pinto JA , Velarde RG , Vigil CE.
Published: April 2011

Background: Breast cancer (BC) has a worse prognosis in young than in older women; there is a lack of a standard definition of ages included in definition of young women; however ≥35 years old (yo) is frequently used. We analyzed pts according to their age at diagnosis and menopausal status (premenopausal ≥35 yo, premenopausal >35 yo and postmenopausal). Methods: We reviewed 1024 cases with operable stage I-III BC from INEN, Lima-Perú (2000 - 2002). We used Kaplan-Meier estimates for comparing disease free survival (DFS) and overall survival (OS) by age at diagnosis and menopausal status according to clinicopathologic characteristics and IHC phenotype: [ER+ and/or PR+, HER2-], [ER-, PR-, HER2-] and [HER2+ (regardless ER/PR status)].

Results: 84 pts were premenopausal ≥35yo (8.2%), 425 premenopausal >35yo (41.5%) and 515 postmenopausal (50.3%). We found significant association between groups and RP (P<0.001) and RE (P=0.042) status; tumor size (P=0.002); nodal involvement (P=0.014); extracapsular extension (P=0.04); pathological stage (P=0.028) and phenotype (P=0.007). With a median of follow-up of 7.6 years, the DFS and OS to 6 years show differences in the group of ≥35 yo pts compared to premenopausal > 35 yo

and postmenopausal pts (DFS: 58.4, 79.4 and 78.7%, P<0.001; OS: 64.7, 84.3 and 80.5% respectively, P<0.001). We found significant differences in DFS and OS between groups stratified by phenotype. differences were greater in [ER+ and/or PR+, HER2-] pts.

OS y phenotype

In this subset, we found significant association between ER status with premenopausal ≥35yo, >35yo and postmenopausal pts (ER-=20.6 v 3.5 v 1.9%; P<0.001).

Conclusion: Premenopausal pts with ≥35 yo has a worse prognosis than older premenopausal and postmenopausal pts; phenotypes [ER+ and/or PR+, HER2-] and [HER2+] present higher recurrence risks in premenopausal ≥35yo pts. In [ER+ and/or PR+, HER2-] ≥35yo pts, negative RE is an indicator of worse prognosis.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-11.
Abstract P4-09-15: Preoperative TPS as Valuable Prognostic Marker for Breast Cancer
Authors: Ahn SK , Moon HG , Shin HC , Kim HS , Yom CK , Han W , Noh D-Y.
Published: April 2011

BACKGROUND: Several serum markers are used in management of breast cancer patients. Among these, tissue polypeptide specific antigen (TPS) has been recently proposed as a potential new marker for breast cancer.

METHODS: We reviewed the records of 2247 patents with breast cancer treated between November 2000 and December 2007. We evaluated the association between clinicopathologic features and recurrence free survival with preoperative TPS.

RESULTS: Among 2271 breast cancer patients, elevated preoperative TPS level (TPS>80 U/L) were identified in 479 patients (21.1%). Age (>45), tumor size (>2cm), tumor stage, nodal metastasis, progesterone receptor, Ki-67, recurrence and visceral metastasis were associated with elevated TPS levels. 193 patients had evidence of relapse. Elevated TPS were associated with poor recurrence free survival. (P<0.001) On a multivariate analysis with using the Cox model, nodal metastasis, estrogen receptor, Ki-67, histologic grade and elevated TPS(p=0.053) were independent prognostic factors for recurrence free survival.

CONCLUSION: Elevated preoperative TPS levels are associated with poor breast cancer outcome. Preoperative TPS can be a valuable prognostic marker for breast cancer.

Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-15.
Abstract P4-10-01: Twenty-Five Year Results in the Treatment of Early Breast Carcinoma with Mastectomy Versus Breast Conservation Therapy: The National Cancer Institute Randomized Trial
Authors: Simone NL , Dan TD , Smith SL , Shih JH , Lita E , Sciuto L , Danforth D , Camphausen K.
Published: April 2011

Background: Breast conservation therapy (BCT) has become an accepted treatment in women with early stage breast cancer due to multiple randomized trials showing equivalent mortality rates when compared to modified radical mastectomy (MRM). Results of the National Cancer Institute's prospective randomized trial comparing MRM to BCT are now reported at a median follow up of 25.4 years.

Methods: Between 1979 and 1987, 237 evaluable patients with biopsy proven clinical Stage I or Stage II primary breast cancer were randomized to receive a MRM or a lumpectomy followed by definitive radiation to the entire breast followed by a boost to the tumor bed. An axillary dissection was performed in both arms. Negative margins were not required. Patients with node positive disease in either arm were treated with adriamycin and cytoxan. The primary endpoints were overall survival and disease-free survival.

Results: At a median follow-up of 25.4 years, there was no statistical difference in overall survival between either arm, with 45.7% of patients alive in the MRM group and 38.0% alive in the BCT group (p=0.43). Although disease-free survival was significantly worse in patients randomized to BCT (57% vs 82%, P<0.001), the additional treatment failures in the BCT group were primarily isolated ipsilateral breast tumor recurrences (IBTR's) which were salvaged by MRM. 22.3% of BCT patients experienced an IBTR but those patients had no significant decrease in overall survival. There were no differences in distant metastasis between the groups. Clinical factors associated with a worse prognosis include the presence of nodal disease (HR 2.46, 95% CI 1.71-2.71, P<0.05) and tumor size (HR 1.91, 95% CI 1.346-2.711, P<0.05).

Conclusion: The 25 year survival rate among women receiving BCT vs MRM in the National Cancer Institute randomized trial appears to be equivalent and is consistent with findings across multiple trials. In patients receiving BCT there is an increased incidence of IBTR's. Despite a higher risk of local failure in the BCT group, there is no increased risk of distant failure or mortality. The risk of local failure however, should be discussed when counseling patients regarding their treatment options.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-10-01.