bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characteristics of matched brain metastasis (BM) versus the primary breast cancer (PBC).
Authors: Duchnowska R , Jassem J , Shen C , Thorat M , Li L , Morimiya A , Zhao Q , Biernat W , Mandat T , Staszkiewicz R , Och W , Szostak W , Gugala K , Trojanowski T , Czartoryska-Arlukowicz B , Szczylik C , Nakshatri H , Steeg P , Sledge G , Badve S
Published: December 2010

Abstract #2028

Background: Brain is increasingly a site of relapse in breast cancer patients, however the molecular patho-physiology of this process is not well understood. Using a novel assay allowing high-throughput analysis of gene expression from formalin-fixed paraffin-embedded (FFPET) tumor samples, we compared molecular characteristics of BM with those of the PBC in a series of breast cancer patients who underwent excision of brain metastasis.  Methods: In each patient we performed molecular analysis of paired archived FFPET specimens from both the PBC and excised BM. Of the 37 pairs of PBC/BM samples, 24 pairs had sufficient pathology material for molecular analysis. Patient characteristics: mean age at PBC diagnosis was 46.7 years (range 27-67 years); 7 (29%) ER+, 17 (71%) ER-, 12 (50%) HER2-positive(3+). We performed cDNA-mediated annealing, selection, extension and ligation (DASL) assay (Illumina Corp) for expression of 502 known cancer genes, using 200 ng RNA. Statistical analysis for microarrays (SAM) accounting for the pairing of the primary and metastasized tumors was used to identify differentially expressed genes while controlling the false discovery rate (FDR <0.01). BeadStudio™ Absolute correlation clustering was used to cluster samples based on their expression profiles.  Results: In only 7 (29%) cases matched PBC and BM pair clustered together on unsupervised hierarchical clustering, the pairs in remaining cases clustered apart. Comparison of the two groups (PBC and BM) showed that 41 genes were up regulated in BM, including proliferation genes (CDC2, CDC25a, CCNA2 and E2F family), anti-apoptotic (BIRC5), DNA repair (RAD51, -54b, XRCC2, BRCA2, BARD1, TOP1), angiogenesis (VEGF), and development of drug resistance (ABCB1, -G2). In contrast, the 43 genes that were down regulated in BM included those associated with invasion (MMP2, -3, -14), cellular motility, and epithelial to mesenchymal transformation (CDH11). Additional analysis to validate these trends and to identify potential therapeutic targets is underway.  Conclusions: Although some BM retain remarkable similarity to the PBC, majority exhibit considerable deviation in their gene expression profile. These “adaptive” changes include greater resistance to drug therapy, increased DNA repair, a reversal back to the epithelial phenotype and decreased capacity for cell motility and invasion.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2028.
An exonic expression profile for breast cancer diagnosis of fine needle aspiration biopsies.
Authors: Delaloge S , Scott V , Michiels S , Dessen P , Suciu V , Spielmann M , Vielh P , André F
Published: December 2010

Abstract #2029

Background: Fine needle aspiration (FNA) for breast cancer diagnosis, a simple, quick and cheap procedure, is limited by a significant number of inconclusive exams. In the present study, we have analyzed whether the expression of a set of exonic probes could accurately diagnose breast cancer of FNA biopsies.  Patients and methods: cDNA from 165 breast lesions (120 cancers), obtained by FNA in 2006-2007, and for which RIN score was > 6, was hybridized on splice arrayTM in two separate series (training/validation)(data set Andre et al SABCS 2008). We focused on 703 680 evidenced probe sets. The actual diagnosis of breast cancer was established by pathological examination at surgery. Associations between exonic probes and diagnosis were assessed by correlation coefficients (using t-tests) and a nearest centroid prediction rule was determined on the training set of 94 samples (70 cancers) in order to classify lesions as malignant or benign. Using the validation set of 71 samples (50 cancers), the performance of the prediction rule was evaluated and the proportion of explained variation (PEV) estimated in a logistic regression model (including only variables significant by a likelihood ratio test).    Results: In a leave-one-out cross-validation analysis, the nearest prediction rule based on the expression of 1228 exonic probes (all probes with p<10e-9) accurately classified all samples from the training set. In the validation set, 68 out of 71 samples were accurately classified (96%) by this exonic expression profile of 1228 probes. Sensitivity and specificity were 96% (exact 95% CI: 86-100%) and 95% (76-100%). The cytological exam failed to provide a definitive diagnosis in 5 out of 71 patients. Four out of these 5 lesions were accurately classified by the nearest prediction rule. The PEV obtained by a clinical model (age and BI-RADS classification) was 75% (bootstrap standard error +/-8%) and the PEV by the exonic profile alone 81% (+/- 10%) in the validation set. When the clinical model and the exonic profile were combined, the PEV was 87% (+/-7%).  Conclusion: We have developed a profile based on the expression of 1228 exonic probes for breast cancer diagnosis using FNA samples. This exonic profile could be a complementary tool to clinical variables and to the cytologic exam to decide about surgery in post-screening programs.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2029.
A reduced qRT-PCR-based gene expression signature with prognostic value in early breast cancer.
Authors: Espinosa E , Gámez A , Sánchez I , Pinto [ , Hardisson D , Madero R , Zamora P , Redondo A , González Barón M , Fresno Vara J
Published: December 2010

Abstract #2030

Background: Gene profiling may improve prognostic accuracy in patients with early breast cancer, but this technology is not widely available. We used commercial assays for qRT-PCR to measure the expression of 83 genes previously found to determine breast cancer prognosis. The objective of our study was to develop a simple molecular signature predicting relapse in early breast cancer with positive hormonal receptors.  Methods: 153 patients (age 29-82 years-old) with early breast cancer and a minimum follow-up of 5 years were included. All tumours were positive for hormonal receptors and 38% had positive lymph nodes; 64% of patients received adjuvant chemotherapy (with anthracycline if N+ or high-risk N0). RNA was isolated from tissue slices of formalin-fixed paraffin-embedded samples using the MasterPure RNA Purification Kit (Epicentre Biotech). qRT-PCR amplifications were performed with TaqMan Gene Expression Assays products in an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). The reactions were carried out using the TaqMan Low Density Arrays (Applied Biosystems). A supervised analysis was done to identify a prognostic expression signature using BRB Array Tools software. Survival curves were derived from Kaplan-Meier estimates and compared by log-rank test. The association of gene expression and clinical variables with distant metastasis-free survival (DMFS) was analyzed by Cox regression models. SAS 9.1 software package was used for statistical analyses.  Results: After a median follow-up of 92 months, 28% of patients relapsed. We defined an 8-gene prognostic signature. The distant metastasis-free survival at 5 years was calculated for this profile. DMFS was 97% -good risk- versus 60% -poor risk, HR 20.3 (95% CI 6.17-67.2), p<0.001. This gene expression profile was highly informative in identifying patients with distant metastasis, even when corrected for traditional prognostic factors in Cox multivariate analysis (HR 20, 95% CI 5.9-67.4). Performance was similar to that of three validated gene profiles. The validity of this signature was verified in an independent cohort obtained at the GEO database.  Conclusions: This study identifies a new molecular signature with prognostic utility that outperforms traditional histopathologic prognostic factors. Our 8-gene qRT-PCR assay is suitable for analyzing routine formalin-fixed paraffin-embedded samples in the clinical setting.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2030.
Predicting response to RAD001 treatment in post-menopausal women with ER-positive breast cancer using the breast cancer DSA™ research tool (Almac).
Authors: Sabine VS , McCulla A , Wilson CL , Sims AH , McDyer F , McCluskey C , Halfpenny I , Oliver GR , Macaskill EJ , Renshaw L , Thomas JS , Dixon JM , Bartlett JM
Published: December 2010

Abstract #2032

Background: RAD001 (everolimus) is a rapamycin derivative that inhibits mTOR and its downstream substrates and has shown promising results in Phase I trials. The aim of this study was to assess the feasibility of predicting patient response to RAD001 treatment from pre-treatment biopsies.  Materials & Methods: 32 post-menopausal, ER-positive early breast cancer patients received 5mg RAD001 pre-operative treatment daily for 14 days prior to primary surgery. 25 patients completed the regimen, 6 patients withdrew and 1 did not start treatment. Biopsies were taken at diagnosis and at surgery from the RAD001 treated patients. Pre-treatment samples were flash frozen prior to RNA isolation. Patient response status was classified based on the % of Ki-67 positive cells determined by immunohistochemistry in the post-treatment biopsy (Macaskill et al., 2006: Breast Cancer Research & Treatment: 100: S1: S286). RNA from the pre-treatment group was analysed using the Almac Diagnostics Breast Cancer DSATM Research Tool, which represents ∼60,000 normal breast and breast cancer transcripts and Illumina Human Ref.8 chip v2 containing ∼24,500 transcripts representing well-categorised and most commonly known genes, irrespective of disease or tissue. For each array platform, modulated genes were identified using statistically robust gene selection methods. In addition, the sequence content of the two platforms was compared by probe-level analysis using BLAST.  Results & Discussion: Preliminary assessment of the expression differences between the non-responder and responder groups using the DSA™ Research Tool demonstrated 169 up-regulated and 93 down-regulated genes. This list is comprised of genes involved in chemokine activity, cell adhesion and transport. Cross-platform content comparison demonstrated that the Illumina bead arrays represented 41% of transcripts on the DSA™ Research Tool whilst 82% of the Illumina beadarray transcripts were represented on the DSA™ Research Tool. Genes of interest identified will be validated by quantitative real time PCR and the key pathways reported.  These datasets have enabled us to begin the identification of tumours most suited to RAD001 treatment and may be used as a guide for the generation of a gene expression classifier for predicting patient response to RAD001.  Acknowledgements: This work was supported by a grant from Breast Cancer Research Treatment, UK.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2032.
Prediction of chemotherapy response in breast cancer by using a four-gene panel including osteopontin, activating leukocyte cell adhesion molecule (ALCAM), HER2 and estrogen receptor (ER).
Authors: Ihnen M , Müller V , Wirtz RM , Milde-Langosch K , Witzel I , Lisboa BW , Jänicke F
Published: December 2010

Abstract #2033

Background: Several prognostic factors such as TNM-stage, hormone receptor and HER2 status refer to therapeutical decisions towards adjuvant chemotherapy. Up to now it is still difficult to predict the response to chemotherapy for a single patient, which is a challenging problem in order to spare patients from cost-intensive ineffective treatment and adverse side effects. Therefore there is urgent need for additional predictive markers. Our previous findings implicated that tumor tissue expression of Activated Leucocyte Cell Adhesion Molecule (ALCAM/CD166) is associated with chemotherapy response. Osteopontin (OPN) has been described as prognostic marker in breast cancer and recently it was shown that high OPN levels in vitro mediate chemoresistance of breast cancer cells by inhibition of apoptosis. If these findings result in chemoresistance in vivo has not been investigated so far. Material and Methods: Primary breast cancer tissues from 100 patients treated with Taxane-free adjuvant standard chemotherapy regimen were collected at surgery prior to any additional therapy. The median follow-up time was 81 months. We analyzed ALCAM and OPN mRNA expression in the biological context of additional and predefined breast cancer markers, like estrogen receptor (ER), Her-2/neu (HER2), members of the urokinase plasminogen activator (uPA) system, vascular endothelial growth factor (VEGF) using oligonucleotide microarrays (Affymetrix HG-U133A). Hierarchical cluster analysis was performed to develop a gene algorithm predicting outcome after standard chemotherapy. Results: In contrast to ALCAM overexpression, which was associated with better outcome upon chemotherapy, OPN overexpression was associated with a significantly higher recurrence rate (p=0,027), but not with shorter overall survival. In addition, there were significant positive correlations of OPN with uPA, PAI-1, uPAR and VEGF-A mRNA expression. By cluster analysis based on the four markers ER, HER2, ALCAM and OPN, we identified a group of high-risk patients, which was characterized by low ER and HER2 expression, high OPN and low ALCAM levels, and with significantly shorter recurrence-free and overall survival (p<0,001). Discussion: In our cohort of patients the combination of ALCAM, OPN, ER and HER2 expression levels turned out as valuable predictive marker for response to adjuvant Taxane-free chemotherapy. The ratio of high OPN and low ALCAM levels was particularly associated with chemoresistance and a poor prognosis in tumors having low ER and HER2 expression levels. We hypothesize that this simple four-gene panel could help to reassign an optimized therapy to the single patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2033.
The predictive probability of four different breast cancer nomograms for non-sentinel axillary lymph node metastasis in positive sentinel lymph node biopsy.
Authors: Gur SA , Unal B , Johnson R , Ahrendt G , Bonaventura M , Soran A
Published: December 2010

Abstract #204

Introduction: Although completion axillary lymph node dissection (CALND) is the standard for evaluating axillary status after identification of a positive sentinel lymph node (SLN) in breast cancer (BC), almost 50% of SLN positive patients will have negative non-SLN and undergo a non-therapeutic axillary dissection. Accurate estimates of the likelihood of additional disease in the axilla can assist decision-making about further treatment. In order to predict non- sentinel lymph node metastases (NSLNM) in patients with a positive SLN biopsy, four different nomograms have been created. The Memorial Sloan Kettering Cancer Center (MSKCC) nomogram was created in 2003 and validated at sources outside that institution. The other nomograms were developed by the Tenon Hospital, the Cambridge University and the Stanford University group recently, but have not been validated yet. The aim of this study was to evaluate the accuracy of 4 different nomograms in our SLN positive BC patients. Methods: We identified 319 patients who had a positive SLN biopsy and CALND at Magee-Womens Hospital of the University of Pittsburgh Medical Center over an 8 year period. The BC nomograms developed by the MSKCC, the Tenon Hospital, the Cambridge University and the Stanford University were used to calculate the probability of NSLNM. Area Under (AUC) Receiver Operating Characteristics Curve (ROC) was calculated for each nomogram a value more than 0.70 was accepted as providing considerable discrimination. Results: One hundred-seven of 319 patients (33.5%) had positive axillary NSLNM. The mean predicted probability of positive NSLNM was 28.1 % (3-93), 67.1% (4.8-100) and, 39.1% (3.6-97.5) for the MSKCC, the Cambridge, and the Stanford nomograms, respectively. The mean point for the Tenon model was 4.45 (0-7). The AUC values were 0.70, 0.69, 0.69 and 0.64 for the MSKCC, the Tenon, the Cambridge and, the Stanford models, respectively. Conclusion: Breast nomograms can be used in the current practice to provide an accurate prediction for identifying low or high risk for NSLNM in BC. In our study, we found the MSKCC nomogram is more predictive than the other nomograms but the Cambridge model and the Tenon model reached borderline values for the good prediction. A model can provide more accurate results with its original group, but may be insufficient on the different groups. Nomograms developed at other institutions should be used with caution when counseling patients regarding the risk of additional nodal disease.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 204.
MicroRNA profiling of triple negative breast cancer: predicting outcome and targets.
Authors: Paranjape TS , Nallur SV , Keanie K , Martel M , Haffty BG , Tuck DP , Slack F , Weidhaas JB
Published: December 2010

Abstract #2040

Background: Breast tumors have been categorized on their molecular basis into ER- and/or PR positive, HER2 gene amplified and triple-negative tumors. Among these, triple-negative tumors represent the most aggressive subclass of breast tumors that are most prevalent in African American women. Currently, no effective targeted therapies or potential biomarkers have been identified to predict outcome within this group.  MicroRNAs(miRNAs) are global regulatory RNAs misregulated in almost every cancer and may function as tumor suppressor genes or proto-oncogenes. Specific miRNAs are aberrantly expressed in breast tumor versus normal tissue. Although miRNA expression profiles for the ER positive and HER2 positive subtype have been reported, the triple negative subtype remains to be studied.  Hypothesis: We hypothesize that identifying the miRNA signature of the triple negative tumors as a subclass as well as differences in this signature between triple negative patients with known outcome will lead to the identification of miRNAs predictive of outcome. For these studies we will use a large, well-annotated database of triple negative breast cancer patients with long-term follow-up. We will then test the identified miRNAs in a well-established cell-culture system to determine which miRNAs can act as potential therapeutic agents in triple negative breast tumors.  Study Design: We have performed miRNA microarray analysis to identify miRNA expression patterns in over 40 triple negative breast cancers using both fresh frozen tumors and formalin-fixed paraffin embedded (FFPE) samples. We have used the Ambion miRNA Taqman Low Density Array for analysis of these samples. The potential of specific miRNAs to predict outcome is currently being determined using statistical analysis.  Results: Our initial studies indicated that the triple negative subgroup has a significantly different miRNA signature than the HER2 positive subtype. We also additionally found unique miRNA expression patterns that may define triple negative cancers as a class. The triple negative tumors are known to have low levels of p53 and our microarray data showed that miR-34a which has been shown to be co-regulated by p53 and has similar expression levels as p53 in tissues, was significantly low in the triple negative compared to HER 2 positive breast cancer. We are currently evaluating the impact of altering mir-34 expression in these cell lines in vitro.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2040.
Targeting novel microtentacles to reduce the adhesion of circulating tumor cells to blood vessel walls.
Authors: Whipple-Bettes RA , Balzer EM , Yoon JR , Cho EH , Matrone MA , Martin SS
Published: December 2010

Abstract #2044

Detached human breast tumor cell lines produce novel protrusions of the plasma membrane that promote reattachment of tumor cells to each other and extracellular matrix. We have termed these protrusions “microtentacles” due to clear mechanistic distinctions from either actin-based filopodia/invadopodia or tubulin-based cilia.    Compelling in vivo studies show that a mechanism consistent with microtentacles is responsible for the attachment of circulating tumor cells to blood vessel walls. Human breast tumor cell lines that display a disorganized actin cortex and hallmarks of epithelial-to-mesenchymal transition produce higher frequencies and greater extension of microtentacles. Conversely, some current and proposed tumor drugs that target the cytoskeleton actually increase microtentacles. While many cancer therapies are directed against the process of cell division, it remains important to consider the effects of these drugs on the invasive characteristics of tumor cells. Our current research has determined that microtentacles are composed of coordinated filaments of vimentin and detyrosinated tubulin. Previous studies indicate that elevated levels of vimentin and detyrosinated tubulin in tumors predict poor patient outcome, although the specific mechanisms remain unclear. Time-lapse video microscopy also indicates that the rapid extension and dynamic motion of microtentacles relies on kinesin activation. Again, our results suggest that the kinesin responsible is a conventional kinesin, rather than the mitotic kinesins commonly targeted by cancer therapies. We propose that the independent roles of vimentin, detyrosinated tubulin and kinesin in tumor metastasis could result from their interdependent contributions to microtentacle formation. Since large epithelial tumor cells are often fragmented in narrow capillaries, we are targeting microtentacles to promote the fragmentation of circulating tumor cells in the capillaries of the lung. We are currently using time-lapse confocal microscopy of tumor cells circulating in intact blood vessels and whole-animal optical imaging to define therapies that reduce microtentacle-dependent adhesion of circulating breast tumor cells during metastasis.    This image shows circulating tumor cells (red) adhering to the walls of an intact blood vessel (green).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2044.
A transcriptional regulatory mechanism in invasive breast cancer; role of AIB-1 and PEA-3.
Authors: Al-Hilli Z , Redmond AM , Hill AD , Young LS
Published: December 2010

Abstract #2056

The invasive potential of cancer cells is mediated in part through the action of matrix metalloproteinases (MMP), which degrade the extra-cellular membrane and promote metastasis. The transcription regulatory region of MMP genes contains binding sites for Ets transcription factors. While the underlying mechanism of MMP regulation is unclear, the Ets transcription factor PEA3 has been shown to be involved. We have previously reported that expression of PEA-3 is associated with the co-activator AIB-1, HER2 positivity, metastasis, and reduced disease free survival. Furthermore, an AIB-1 knockout mouse exhibited PEA-3 dependant MMP production, resulting in reduced metastatic potential. In this study we combined translational and molecular approaches to study the role of PEA-3 and AIB-1 in the transcriptional regulation of MMPs in breast cancer.  The expression of PEA-3, AIB-1, and MMP-9 was assessed in a tissue microarray of breast cancer patients (n=560). Strong associations were observed between both PEA-3, AIB-1, and their target gene MMP-9 (p= 0.007, p= 0.0016 respectively). In HER2 positive patients PEA-3, but not AIB-1 or MMP-9, was associated with reduced disease free survival (p<0.0044, p= 0.4491, and p= 0.0666 respectively). A stable breast cancer cell line over-expressing PEA-3 was created. Upregulation of the growth factor pathway in this cell line resulted in the increased expression of MMP-9. Furthermore, silencing AIB-1 resulted in reduced MMP-9 production in breast cancer cells in vitro.  Ets transcription factors are important mediators of breast tumour invasion. This data supports a role for the transcription factor PEA-3 and its co-activator AIB-1 in the production of MMPs in breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2056.
EGF-dependent enhancement of invasive ability in squamous cell carcinoma of the breast.
Authors: Kimura F , Iwaya K , Kawaguchi T , Yamada K , Ogata A , Kaise H , Komatsu S , Nakamura Y , Ueda N , Mukai K , Kohno N
Published: December 2010

Abstract #2061

The factors promoting the clinical aggressiveness of squamous cell carcinoma of the breast is still not well understood. In order to examine whether the mechanism of cell motility is involved in the aggressive behavior of squamous cell carcinoma, we established a squamous cell carcinoma cell line from breast primary (HBC 9) and assayed its invasiveness. The number of HBC9 cells migrating through matrigel was significantly increased by stimulation with epidermal growth factor (EGF). This enhancement of cell motility was distinctly detected in comparison with four ductal carcinoma cell lines (MCF7, BT474, HBC5, and MDA-MB-231). EGF stimulation also induced surface protrusions of HBC9 cells. To examine the interaction between Arp2/3 complex and N-WASP which is the final intracellular signal of invadopodia formation that is downstream from EGF stimulation, we observed surface protrusion of HBC 9 by laser microscopy. Co-localization of Arp2 and N-WASP was detected with actin polymerization visualized by phalloidin. Furthermore, the localization of cortactin was found to agree with that of N-WASP and Arp2/3 complex in the invadopodia. The cortactin gene copy number was evaluated using real time PCR by comparison between 12 DNA samples extracted from primary mammary squamous cell cancer tissue and those from corresponding normal breast tissue. A higher level of cortactin was detected in cancer tissue than in normal tissue in 10 cases (83%). At a protein level, intense immunostaining of cortactin was detected in all 12 cases, among which expression was confirmed in 2 cases by Western blot analysis. These results suggest that one of the causes of clinical aggressiveness of squamous cell carcinoma of the breast is the enhancement of cell motility by invadopodial formation. Overexpression of cortactin may result in the formation of invadopodium.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2061.
Quantitative gene expression analysis using Oncotype DX in ductal carcinoma in situ that is adjacent to invasive ductal carcinoma.
Authors: Baehner FL , Sangli C , Millward C , Cherbavaz D , Goddard A , Shak S
Published: December 2010

Abstract #2066

Background: It is common for ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) to be observed adjacent to each other in the same tissue section. To explore the quantitative biology of DCIS, we measured ER, PR, HER2, proliferation gene expression and Recurrence Score (RS) using Oncotype DX in DCIS adjacent to IDC.  Material and Methods: Thirty cases of ER positive tumors were identified with adjacent DCIS and IDC of similar nuclear grade. Manual microdissection was performed to separate DCIS and IDC tissues from each case. Using the Oncotype DX assay, standardized quantitative expression of 16 individual cancer related genes including ER, PR and HER2 and the proliferation index (the average of the 5 proliferations genes) was measured on a scale from 0 to 15 (relative to reference genes), where a one unit increment is associated with a 2-fold change in expression. The RS was also calculated. Descriptive statistics and Pearson's R correlation coefficients were determined to compare DCIS to IDC.  Results: 27 of the 30 samples had sufficient RNA for analyses of the DCIS component (8 with low grade, 10 with intermediate grade, and 9 with high grade DCIS). The correlation coefficients for the comparison of DCIS and adjacent IDC for quantitative ER, PR, HER2, and the proliferation index was 0.78, 0.73, 0.76, and 0.83, respectively. For ER, IDC and DCIS measures were within 2 units for all 27 cases and within 1 unit for 22/27 (81%). For PR, IDC and DCIS measures were within 2 units for 25 of 27 cases and within 1 unit for 19/27 (70%). For HER2, IDC and DCIS measures were within 2 units for 26 of 27 cases and within 1 unit for 24/27 (89%). For proliferation index, IDC and DCIS measures were within 2 units for all 27 cases and within 1 unit for 26/27 (96%). There was a wide range of RS in DCIS (mean 13.9; range 0 to 56). Although for RS there was a significant correlation between DCIS and IDC (R=0.84), on average the RS was 4 units lower for DCIS.  Discussion: Quantitative RT-PCR analysis of DCIS is technically feasible and sufficient RNA for Oncotype DX analysis is obtained in 90% of cases. As has been described previously for invasive breast cancer, there is considerable expression heterogeneity in DCIS between patients. DCIS adjacent to invasive breast cancer demonstrates very similar, but not identical, quantitative biology for the genes in the assay. Further DCIS studies to evaluate Oncotype DX and recurrence rates would be of great interest.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2066.
Bone-specific alkaline phosphatase (BSAP) and serum N-telopeptide (sNTX) as predictors of bone loss in postmenopausal women with early breast cancer receiving letrozole as adjuvant therapy: a 5-year study (Z-FAST).
Authors: Brufsky A , Harker G , Beck T , Carroll R , Warsi G , Jin L , Argonza-Aviles E , Ericson S
Published: December 2010

Abstract #2067

Background: Changes in biochemical markers of bone turnover are expected to be predictors of long-term changes in bone mineral density (BMD). We studied the potential of bone-specific alkaline phosphatase (BSAP) and serum N-telopeptide (sNTX) at 6 and 12 months (mo) in predicting BMD of the lumber spine (LS) and total hip (TH) at 48 mo in Z-FAST study.  Methods: Postmenopausal women with ER+ and/or PR+ early stage breast cancer were randomized 1:1 into Up-front (U) or Delayed (D) arms (N=301 in each). Patients (pts) in U received zoledronic acid (ZOL) 4 mg IV q6 mo starting at baseline; pts in D started ZOL q6 mo when their LS or TH T-score fell below -2. All pts started Letrozole 2.5 mg/day for 5 years. Levels of BSAP and sNTX were assessed at screening and every 6 mo on a subset of pts. LS and TH BMD were assessed by dual-energy x-ray absorptiometry (DEXA) at screening, 6 mo and every 12 mo thereafter for all available pts. This is an exploratory analysis to investigate the correlation between short-term % changes from baseline in BSAP and sNTX and long-term % changes from baseline in BMD. Pearson correlations and Receiver Operating Characteristics (ROC) analysis were performed. We report the results based on 48 mo data.  Results: The Pearson correlations between % change in BSAP at 6 mo and LS BMD at 48 mo were -0.13 [(N=38, not significant (NS)] and -0.17 (N=39, NS) in arms U and D respectively. The correlations between % change in BSAP at 12 mo and LS BMD at 48 mo were -0.15 (N=39, NS) in U and -0.43 (N=38, p=0.0077) in D. Very similar correlations were obtained for % change in sNTX at 6, 12 mo and LS BMD at 48 mo in D, but in U the correlations were smaller and positive. The correlations between % change of both markers at 6, 12 mo and TH BMD at 48 mo were all NS at 0.05. ROC analysis showed that at best performance (maximum of sensitivity plus specificity), for any increase in sNTX at 12 mo, the sensitivity (SE), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) to predict LS BMD loss at 48 mo was 74%, 77%, 85% and 63% in D. For BSAP change of 3%, the four ROC parameters were 71%, 71%, 81% and 59% respectively. For prediction of TH BMD loss, the SE, SP and NPV were lower for both markers with higher PPV. In U, the SE, SP and PPV were all much lower with very high NPV for both markers and BMD loss.  Conclusion: Interim results of Z-FAST data show that short-term changes in BSAP and sNTX have the potential for predicting long-term bone loss. The weaker correlations in U also suggest that zoledronic acid may have a direct preventive effect on bone loss independent of changes in the markers.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2067.
Fluorescence in situ hybridization (FISH) technique by reference centers for HER2 status determination in metastatic breast cancer: quality assurance results of FISH 2002 study.
Authors: Penault-Llorca F , Lannes B , Vincent-Salomon A , MacGrogan G , Vilain M , Fermeaux V , Treilleux I , Blanc-Fournier C , Leroux A , Antoine M , Ettore F , Charpin C , Jacquemier J , Escourrou G , Lacroix-Triki M , Gosset P , Sagan C , Arnould L , Couturier J
Published: December 2010

Abstract #2069

Background: Concordance between IHC and FISH in local and regional centers was evaluated in the FISH 2002 Study. Primary metastatic breast cancer (MBC) tumor samples were tested by IHC for HER2 status according to in-house techniques at 18 regional (n=871) and 81 peripheral (n=670) centers in France between 2002 and 2006. All samples were tested by FISH at regional centers [Penault-Llorca et al. SABCS 2006]. This second part of the study comprises quality assurance evaluation of FISH techniques.  Methods: 464 cases were selected according to the protocol and were either re-assessed using archived pictures or retested by 3 reference centers (Clermont-Ferrand, Strasbourg, Paris-Curie). 246 samples were re-assessed and 166 were retested.  Results:Global discordance combining re-assessment and retesting results was 10.7% in the first half of the study and 5.1% in the second part (p=0.041).    Discussion: Concordance was excellent for negative (100%) and IHC 2+ cases (97%). Among low amplification cases (≥6 and <8 copies), 70% were concordant: discordant cases were mainly interpretation pitfalls of polysomic cases, emphasizing the importance of double-stained FISH analyses. These quality assurance results demonstrate that overall concordance and quality of HER2 status determined by FISH has improved significantly over time. This improvement is likely to be related to introduction of rigorous quality control procedures, training, and education, all of which should be continued.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2069.
Phosphorylated c-Src predicts clinical outcome in triple negative breast cancers.
Authors: Elsberger B , Tovey SM , Tan BA , Brown SB , Brunton VG , Mallon EA , Cooke TG , Edwards J
Published: December 2010

Abstract #2076

Background: Recent in vitro evidence suggests that c-Src inhibitors may be effective in inhibiting growth of basal-type/"triple-negative" cell lines. However there is little evidence to support this in clinical specimens. Development of treatment for women with these aggressive ER, PR and HER2 negative cancers is particularly important with the lack of current effective therapies. Activation of Src family members are associated with phosphorylation at two sites Y419: the classical activation site and Y215, known to induce a 50-fold increase in activation.  The aim of the current study is to assess if expression levels and/or activation of Src kinase is associated with clinical outcome measures in ER and HER2 negative human breast cancers.  Methods: Tissue microarrays consisting of 172 ER and HER2 negative breast cancer tumors were constructed. Median clinical follow up was 5.9 years with 64 deaths attributed to breast cancer. Immunohistochemistry was performed using antibodies to c-Src, pSrc419 and pSrc215 (antibodies to Y419 and Y215 will detect phosphorylation of c-Src and other family members). Expression was assessed by two independent scorers. All statistical calculations were performed using SPSS 15, including Kaplan-Meier life table analysis with log rank testing of differences in breast cancer related survival.  Results: Membrane expression of c-Src, pSrc419 and pSrc215 was rarely observed and was not associated with outcome. However cytoplasmic expression was frequently observed. There was a trend towards reduced survival seen with c-Src and pSrc419 overexpression. In direct contrast pSrc215 was significantly associated with increased disease specific survival and was independent on multivariate analysis.  We then hypothesised that activation of c-Src via Y419 but not Y215 was driving the observed association with outcome. Indeed c-Src positive, pSrc419 positive and pSrc215 negative patients have a 25% 5yr survival rate, in comparison all other patients in the cohort had a 72% 5yr survival rate.    Discussion: These results support the in vitro studies suggesting Src family members are associated with progression of triple negative breast cancers. If confirmed in a larger cohort, these results suggest that Src family members may offer a therapeutic target for triple negative cancers. However care must be taken to appropriately select patients with phosphorylation only at Y419 and not Y215. The reason why Y215 phosphorylation is associated with improved outcome requires further investigation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2076.
Time distributions and prognosis factors of breast cancer recurrence in different sites after operation.
Authors: Zhou L , Yin W , Lu J , Di G , Wu J , Shen K , Shen Z , Shao Z
Published: December 2010

Abstract #2080

Objective Our study aims to analyze the breast caner patients with different sites of recurrence, and to have knowledge of the regular patterns and influence factors of metastastic breast cancer.  Methods We performed a retrospective study of 210 female breast cancer patients who were continuously followed up in our hospital from January 2005 to May 2007 and had complete data of metastasis. The clinical and biological characteristics were compared between the patients with organ metastases (lung, liver, brain) and non-viscera metastases (lymph node, bone).  Results Cox regression analysis-hazard function showed that patients with elder age (RR=0.927 95%CI 0.877∼0.981, P=0.008) and HER-2 negative(RR=0.253 95%CI 0.076∼0.836, P=0.024)tended to develop non-viscera metastases, while patients with larger tumors (RR=3.832 95%CI 1.073∼13.687, P=0.039) tended to develop organ metastasesp.    When stratified by the prognostic factors, the annual risk hazard curve of the patients with high risk of recurrence had double peaks at the second year and the ninth years after mastectomy respectively, and it was significantly higher than those of the patients with middle and low risk.[figure1]Annual risk hazard curves of both local relapse and metastasis also showed a double-peaked pattern. The curves for lymph node, bone, lung and liver metastasis showed a similar pattern, but all the peaks of curve emerged later than that of the local relapse.    Conclusion Patients with different clinical and biological characteristics may develop recurrence in different sites. There are certain regular patterns of time distribution for different metastasis sites after mastectomy. The local recurrence may be the foreboding for the metastasis and we should pay attention to the local relapse signs especially in the patients with high risk.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2080.
Noggin, impact on in vitro cell growth, expression pattern and the prognostic value in breast cancer.
Authors: Ye L , Mansel RE , Jiang WG
Published: December 2010

Abstract #2084

Background: Noggin, a BMP (bone morphogenetic proteins) antagonist, has been demonstrated to play critical roles in regulation of biological functions of BMPs, members of the TGF-β superfamily and known regulators of bone metastasis in solid tumours including breast cancer. We have recently reported that the presence of endogenous noggin in cancer cells is a pivotal factor that governs the cells response to stimulation by bone morphogenetic protein, for example BMP-7. However, the role of noggin in breast cancer remains unclear. We presently investigated the role of Noggin in breast cancer cells and the pattern of expression in clinical breast tumours.  Methods: The expression of Noggin mRNA and presence of Noggin protein was examined in a cohort of human breast cancer specimens (normal, n=33; cancer, n=112), using both quantitative real time PCR and immunohitochemical staining, respectively. The full-length human Noggin was cloned into a mammalian expression plasmid vector (pEF/His TOPO TA plasmid vector). The constructed Noggin expressing constructs were then transfected into breast cancer cells (MDA-MB-231). The expression of Noggin in the transfected cells was determined using RT-PCR and western blot. The effect on growth of MDA-MB-231 cells by Noggin was investigated using an in vitro growth assay.  Results: Immunohistochemical staining revealed that Noggin protein was abundant in the cytoplasmic region of normal epithelial cells. Reduced staining of Noggin in breast cancer cells was seen when compared to normal breast tissue. Quantitative real time PCR further demonstrated that the decreasing Noggin expression correlated to poor prognosis, in that patients who remained disease-free showed a higher levels of the transcript (11113±3612) compared with those who had a poor clinical outcome (with metastasis and mortality, 6420±2241). Kaplan-Meier survival analysis showed that patients with high Noggin level had a longer survival (134.4 (121.3-147.5 - 95%CI) months) that those with low levels (102.8 (80.8-124.8) months). The absorbance of MDA-MB-231 Noggin over-expressing (MDA-MB-231Nogginexp) cells of Day 5 is 1.07±0.13, p<0.01 vs both MDA-MB-231 wild type (MDA-MB-231WT, 1.54±0.08) and empty vector control (MDA-MB-231pEF/His, 1.72±0.17).  Conclusions: Noggin inhibits the growth of breast cancer cells and has a pattern of expression in that decreased Noggin expression correlates to poor prognosis of breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2084.
Good news for a change: receipt of adjuvant chemotherapy in diverse populations.
Authors: Griggs JJ , Abrahamse PH , Hawley ST , Hamilton AS , Graff JJ , Katz SJ
Published: December 2010

Abstract #2092

Background: Racial, ethnic, and social disparities in the use of chemotherapy have been demonstrated in women with breast cancer in numerous clinical settings. The purpose of this study was to investigate patterns and correlates of chemotherapy use in a recently-treated population-based sample of black, Latina, and non-Hispanic white women with invasive breast cancer. Methods: A sample of women diagnosed with breast cancer between August 2005 and March 2007 and reported to the Los Angeles County and Detroit Metropolitan Surveillance Epidemiology and End Results (SEER) Registries was identified and invited to complete a survey about their breast cancer treatment. Patient responses were linked to SEER data. Black women and Latinas were oversampled. Bivariate and multivariate analyses examined the use of adjuvant chemotherapy according to clinical, disease, and sociodemographic characteristics, including self-reported height and weight, comorbidity, educational attainment, income, insurance, marital status, and employment factors. Results: Of the 1655 respondents, 1041 (60.8%) received chemotherapy. Black women (65.4%) and Latinas (69.5%) were more likely than non-Hispanic white women (57.1%) to receive adjuvant chemotherapy (p = 0.0001). In multivariate models, only younger age (p < 0.0001), increasing tumor size (p < 0.0001), increasing number of involved lymph nodes (p < 0.0001), and negative hormone receptor status (p < 0.0001) were significantly associated with receipt of chemotherapy. In addition, severely obese women were significantly more likely to receive adjuvant chemotherapy than healthy weight women (odds ratio 1.63, 95% confidence interval 1.04, 2.57). Comorbidity, education, income, insurance, marital status, and employment status were not associated with receipt of chemotherapy. Conclusion: Use of adjuvant chemotherapy in this population-based sample was higher among patients who were more likely to benefit from chemotherapy. In contrast to practice-based studies of chemotherapy use in cohorts treated in older time periods, this study using patient report of chemotherapy and of sociodemographic information demonstrates no evidence of racial, ethnic, or social disparities in chemotherapy use in this diverse population-based sample of recently-treated women.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2092.
Communication of treatment goals in breast cancer: a comparison of perceptions of physicians, nurses and patients.
Authors: West HF , Cohn WF , Guterbock TM , Gurka MJ , Back AL , Blackhall LJ
Published: December 2010

Abstract #2099

Background: Previous evidence suggests that 20-50% of patients do not have a clear understanding of the goal of their cancer treatment, using the provider's goal as the standard for comparison. This lack of alignment between patients and providers may undermine the concept of informed decision making. We aimed to determine the current level of understanding of the goal of chemotherapy in patients with breast cancer by comparing treatment goals of the patients with those of their providers.  Patients and methods: Forty – three women undergoing chemotherapy for breast cancer and their providers (nine physicians and three nurses) were surveyed. The primary outcome was the level agreement between the patient and the physician regarding the goal of chemotherapy. Additionally, to examine the extent to which disagreement was recognized by the participants, physicians and nurses were asked to give their perception of the patient's goal, and patients and nurses were asked to give their perception of the physician's goal. Agreement was estimated using generalized estimating equations, which allowed for correlation among patients seen by the same physician.  Results: Agreement between the physicians and the patients regarding goal of chemotherapy was 38% (95% CI: 22-57%). The patient's goal and the nurse's perception of the patient's goal agreed 36% of the time (95% CI: 35-37%), while the patient's goal and the physician's perception of the patient's goal agreed 33% of the time (95% CI: 21-49%). When response categories were collapsed into groups with similar broad treatment intent (palliative versus curative/adjuvant), then the level of agreement improved dramatically (91% -100%).  Discussion: While broad agreement regarding treatment intent as curative or palliative was higher in this study than in previous research, agreement of the goal on a more detailed level was still poor across varying combinations of the triad of patient, physician and nurse. Future communication studies should focus on interventions to align patients and providers around a common understanding of the goal of chemotherapy, which will improve the quality of care through the provision of truly informed decision making.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2099.