bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of gene expression in stromal fibroblasts of breast carcinomas and normal breast.
Authors: Bauer M , Su G , He R , Rehrauer WM , Kendziorski CM , Casper TC , Jonat W , Friedl A
Published: December 2010

Abstract #105

Background: The cancer microenvironment plays a critical role in tumor development and progression. Cancer associated fibroblasts (CAF) constitute a significant component of the tumor stroma and participate in reciprocal communication with the tumor cells. Information on differential gene expression specifically in stromal fibroblasts is sparse and data describing the variability of gene expression in CAF and normal fibroblasts (NF) is currently lacking. The purpose of this study was to identify genes differentially expressed in CAF and matched NF and to analyze the heterogeneity of gene expression profiles in the two cell types.  Materials and methods: Fibroblast cell cultures were established from 6 patients with primary invasive breast cancer. Gene expression profiles were generated using oligonucleotide microarrays (Affymetrix HG-U133 Plus 2.0). Differentially expressed genes were ranked using Empirical Bayes modeling. A cut-off value of 0.005 was chosen for the posterior probability of equivalent expression. Lists of overexpressed genes were generated after eliminating genes with less than two-fold overexpression.  Results: 17 genes were overexpressed in CAF compared to NF with known functions in paracrine and intracellular signaling, transcription regulation and extracellular matrix production. Using the same posterior probability cut-off, we identified 7 genes which were expressed at least two-fold higher in NF than in CAF. These genes have purported roles in steroid hormone metabolism, transcription, migration and cell signaling. Using semiquantitative RT-PCR and immunohistochemistry, we confirmed the over- and underexpression of a subset of 10 differentially expressed genes. The heterogeneity of gene expression in CAF vs. NF was compared with F-tests to determine variances. The estimated probability of NF gene expression variance being higher than CAF gene expression variance was 0.547 with a 95% confidence interval of 0.543 to 0.551 (p<0.0001), indicating that gene expression is more variable in NF than in CAF. By ranking the q-values of individual genes we identified 3 known genes, which show a significant difference in variance between CAF and NF (p<0.05).  Conclusion: Altered gene expression in fibroblasts likely contributes to tumor growth and progression by enhancing ECM production, promoting stromal-epithelial paracrine signaling and altering steroid hormone metabolism. The inter-individual heterogeneity of gene expression in NF may indicate that the mammary stroma varies between individuals, supporting the hypothesis that the ability of the stroma to act as a barrier to cancer development and tumor progression may also be variable. Conversely, the heterogeneous gene expression in NF may be a reflection of a relative synchronization and uniformity of gene expression in CAF.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 105.
Stromal-epithelial cell cross-talk in the breast tumour microenvironment elicits changes in tumour epithelial cell gene expression.
Authors: Potter SM , Dwyer RM , Kerin MJ
Published: December 2010

Abstract #1053

Introduction Tumour growth & progression in breast cancer is a product of autocrine & paracrine actions of factors secreted by malignant epithelial & stromal populations in the tumour microenvironment. Stromal cells, recognised as prominent modifiers of cancer progression, are thought to play a critical role in controlling neoplastic epithelial cell function. Although chemokines, which are chemotactic cytokines, have been postulated as mediators of stromal-epithelial cell signalling, the precise nature of such intricate interactions remains incompletely understood. The aim of this study was to investigate stromal cell-mediated modification of breast cancer epithelial cells.  Methods Two breast cancer cell lines with different hormone receptor profiles (ER+ MDA-MB-231 & ER- T47D) were cultured individually & on confluent monolayers of stromal cells isolated from primary breast tumours. Co-culture supernatants were harvested for chemokine quantitation by ELISA. Following direct co-culture breast cancer epithelial cells were retrieved from mixed populations with EpCAM specific magnetic beads & RNA extracted. Changes in gene expression in epithelial cells resulting from interaction with stromal cells were identified by RQ-PCR using primers for the chemokines Monocyte Chemotactic Protein-1 (MCP-1/CCL2), & Stromal Cell Derived Factor-1α (SDF-1α/CXCL12), as well as their respective receptors, CCR2 & CXCR4. Changes in expression of genes involved in invasion (MMP11 & HER-2) and proliferation (Ki67) were also analysed.  Results Expression of CCR2 & CXCR4 was confirmed in epithelial cell lines. Co-cultures of epithelial & stromal cells secreted significantly higher levels of MCP-1 than the cells cultured alone, while the reverse was true of SDF-1α secretion (Table 1). RQ-PCR revealed changes in gene expression of epithelial cells retrieved following co-culture with tumour stromal cells. The highly invasive MDA-MB-231 cells showed upregulation of both CCL2 & CXCL12 genes (2-3 fold increase) following interaction with tumour stromal cells, while the non invasive T47D cells showed downregulation of these genes. Expression of genes specifically associated with invasion, Her2 & MMP11, were elevated (1-2 fold increase) following exposure to tumour stromal cells, with little or no change observed in the proliferative marker Ki67.    Conclusion Tumour stromal cells induce altered secretion & expression of tumourigenic chemokines & invasion-associated genes in breast cancer epithelial cells, mediated through direct cell contact. These results further define the tumour promoting role of stromal cells & highlight these interactions as potential therapeutic targets.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1053.
The impact of adipose stromal cells on migration and invasion of breast cancer cells.
Authors: Walter M , Liang S , Ghosh S , Hornsby P , Li R
Published: December 2010

Abstract #1056

Epidemiological studies have identified obesity as a significant risk factor for developing breast cancer among postmenopausal women. Adipose tissue is an important extragonadal source of estrogen. Although circulatory estrogen levels in postmenopausal women decline, tissue-specific estrogen levels remain virtually unchanged and are even found elevated in breast tissue from obese patients and patients with breast cancer. Therefore, excessive estrogen production due to overall adiposity is considered to be a critical contributing factor to obesity-associated breast cancer risk. However, it is not clear whether adipose tissue per se can also affect breast cancer development in a hormone-independent manner. In the current study, we used adipocyte stromal cells (ASCs) isolated from lipoaspirates from cancer-free patients and studied their possible influence on tumor cell behaviors. Using the Boyden-chamber assay, we found that ASCs had a stimulatory effect on the migration of both ER-positive (MCF-7) and ER-negative breast cancer cells (MDA-MB-231), suggesting that ASCs can influence the behaviors of breast cancer cells in an estrogen-independent manner. A similar effect of ASCs on MDA-MB-231 cells was also observed in a wound-healing assay. Furthermore, in a xenograft experiment using co-transplanted MDA-MB-231 and ASCs, we observed a significant role of ASCs in promoting the invasiveness of MDA-MB-231 cells. These data indicate that, in addition to their established role in supplying local estrogen, ASCs may facilitate tumor invasion into surrounding tissue and subsequently metastasis. The possible molecular basis for this effect of ASCs on tumor cell migration and invasion will be discussed.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1056.
Fingerprinting genomic heterogeneity in primary breast carcinomas and among sentinel lymph node metastases: implications for clinical management of breast cancer patients.
Authors: Ellsworth DL , Ellsworth RE , Patney HL , Oviedo A , George A , Croft DT , Love B , Jordan RM , Deyarmin B , Becker TE , Hooke JA , Shriver CD
Published: December 2010

Abstract #1059

Background: Sentinel lymph node (SLN) status is a key prognostic factor for breast cancer patients. The SLN hypothesis postulates that cancer cells initially spread from the primary tumor to the first-draining (sentinel) node(s) before reaching higher echelon nodes. Although SLN biopsy is clinically useful for staging patients, little is known about the genomic heritage of cells that define the genetic makeup and clinical behavior of nodal metastases. We used allelic imbalance (AI) coupled with high-density SNP genotyping to examine the extent of genetic heterogeneity in primary tumors and relationships among metastases in sentinel and non-sentinel axillary lymph nodes in 30 patients with node positive breast cancer.  Material and Methods: Pathologically positive nodes were identified by H&E/IHC, while sentinel nodes were localized by standard scintigraphic techniques. For each patient, nodal metastases and 6-15 areas from multiple paraffin blocks of the primary carcinoma were isolated by microdissection. AI was assessed by microsatellite typing at 26 chromosomal regions; genome-wide copy number variation was examined using Affymetrix GeneChip Human Mapping 500K arrays and the Genotyping Consol. The genomic heritage of sentinel and non-sentinel nodal metastases in relation to multiple areas of the primary tumor was assessed by hierarchical clustering and phylogenetic analyses.  Results: Extensive genomic heterogeneity was observed in primary tumors and among nodal metastases (0-44% in primaries, 0-36% in metastases). Overall levels of variation were significantly higher (P<0.01) in primary tumors (17%) than metastases (10%), but did not differ between sentinel (10.2%) and non-sentinel (10.0%) metastases. Within patients, many (but not all) metastases appeared to be descended from different areas of the primary tumor, but patterns of descent were complex. As a group, sentinel node metastases were not genomically distinct from other axillary node metastases, and there was no evidence that SLN metastases are seeded by cells that colonize the axilla early in tumorigenesis.  Discussion: Although metastases develop in a sequential manner that maintains the known capability of SLN biopsy to accurately stage the axilla, metastases appear to develop from genomically-diverse progenitor cells descended from different areas of the primary tumor. Genomic diversity in primary tumors and metastases may be an important factor in prognosis and treatment resistance in breast cancer patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1059.
DNA repair protein biomarkers in triple negative breast cancer.
Authors: Alexander BM , Sprott K , Wang X , D'Andrea AD , Schnitt SJ , Collins LC , Weaver DT , Garber JE
Published: December 2010

Abstract #1064

Introduction: Triple negative breast tumors form a distinct, aggressive, subgroup of breast cancers that exhibit a high degree of genomic instability and have many phenotypic similarities to BRCA1 deficient tumors. These observations suggest that aberrant DNA repair may be involved in triple negative tumor carcinogenesis. We profiled tumor DNA repair capacity using immunohistochemistry in order to develop prognostic biomarkers and suggest targets for novel therapies.  Methods: We identified 143 previously treated women with triple negative breast cancers and used their archived, formalin-fixed, paraffin-embedded primary excision biopsies to create a tissue microarray (TMA). The TMA was stained using antibodies against proteins in various DNA repair pathways including XPF, FANCD2, PAR, MLH1, and MK2. Stained tissue was evaluated using machine-based image analysis and scoring that represented both the intensity and quantity of positive tumor nuclei. Biomarker scores and clinical data were assessed for correlations with outcome. Patients were randomized into training and test cohorts for the development of a multiple marker model. A set of critical threshold marker values were determined that maximally separated the training samples into low and high risk recurrence groups. Training set thresholds and marker combinations were applied towards the test set. Kaplan-Meier and Cox proportional hazards were used to test time to recurrence.  Results: Clinical data for 115 patients with primary treatment data was available with a median follow up of 58 months. There were 37 recurrences: 18 were distant first, 12 were local first and 7 were simultaneous. Low XPF (p=0.002), pMK2 (p=0.02), MLH (p<0.001) and FANCD2 (p=0.05) were associated with shorter time to recurrence. In the training cohort, the high-risk group defined by a four marker model had a relative risk of recurrence of 3.0 (p<0.00001) and shorter median time to recurrence than the low risk group (13.1 months versus not reached). This was superior to single markers and to other markers such as P53 (p = 0.02) or Ki67 (p = 0.07). In the test set, the model produced similar results with a relative risk of 2.1 (p=0.029) for the high-risk group and shorter median time to recurrence (14.1 months versus not reached).  Conclusions: Triple negative breast cancers show variable expression of proteins involved in DNA repair. Levels of four DNA repair proteins correlated significantly with recurrence free survival, and were used to develop a DNA repair profile model in a training set which was prognostic in a test set. DNA repair biomarker panels may be useful as prognostic or predictive indicators as well as suggest possible targets for novel therapies such as PARP inhibition. Further study of the model in another validation set with other clinical variables is warranted.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1064.
Local versus central HER2 immunohistochemistry correlates with kinetic RT-PCR but only central immunohistochemistry and RT-PCR predict pathological complete response: results from the neoadjuvant multicenter GeparTrio trial.
Authors: Loibl S , Müller B , Roller M , Kronenwett R , Darb-Esfahani S , Komor M , von Toerne C , Wirtz R , von Minckwitz G , Denkert C
Published: December 2010

Abstract #1070

Background: There are several methods for determination of HER2 overexpression. Immunohistochemistry (IHC) has been set up as the gold standard in routine diagnostics to identify patients eligible for anti-HER2 therapy. Nonetheless, there is still controversy about the predictive value of HER2 overexpression and pathological response to neoadjuvant anthracycline/taxane-based chemotherapy.  Methods: In this study, we investigated the HER2 expression levels in formalin-fixed, paraffin-embedded pre-treatment punch biopsies by kinetic one-step RT-PCR (kRT-PCR); local IHC performed at the pathological institutes of the participating centers; and centrally performed IHC in 150 patients from the neoadjuvant GeparTrio study (NCT00544765), where patients were treated with docetaxel, doxorubicin and cyclophosphamide (TAC) but received no trastuzumab therapy regardless of the individual HER2 status. HER2 was considered positive for IHC if expression was 3+ and for mRNA expression if normalized level was >/= 18.0 arbitrary units (as defined by bimodal expression for this cohort). We evaluated the associations by Spearman's rho correlation between the different methods as well as the best method that might predict a pathological complete response to this neoadjuvant chemotherapy.  Results: A randomly selected subset out of 150 of 2,090 patients was included in this analysis. This is a representative collective of TAC-treated patients from the GeparTrio trial with a pCR rate (ypT0; ypTis/ypN0) of 23%. The different methods revealed a HER2 positivity of 31% for the local analyses, 16% for the central HER2 testing and 20% positivity for kRT-PCR analyses for this study collective, compared to 28% for the entire trial population. Local determination of HER2 showed a correlation with central laboratory (RS=.418, P<.001, N=106) and kRT-PCR results (RS=.428, P<.001, N=120), and centrally assessed HER2 expression correlated with the results of kRT-PCR (RS=.923, P<.001, N=115). Binary logistic regression with the locally determined HER2 expression revealed no significant results in prediction of a pathological complete response (P=.802, N=121), whereas the results from central IHC (P=.038, OR 2.84, N=119) as well as the results from kRT-PCR (P=.047, OR 2.47, N=134) were significant.  Conclusions: The value of HER2 expression and response to neoadjuvant chemotherapy is still controversial. Our results on this subset of patients show that kRT-PCR of HER2 mRNA expression and centrally performed IHC but not local HER2 assessment predicts pCR for neoadjuvant TAC chemotherapy. Theses results need further confirmation on a larger group of patients.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1070.
Risk assessment of relapse in Swiss participants of BIG 1-98 with grade 2 early breast cancer by gene expression profiling using RNA from formalin-fixed, paraffin embedded (FFPE) tissue.
Authors: Aebi S , Antonov J , Popovici V , Delorenzi M , Giobbie-Hurder A , Viale G , Thürlimann B , Altermatt H , Jaggi R
Published: December 2010

Abstract #1073

BIG 1-98 is a randomized controlled clinical trial for postmenopausal patients with early, estrogen and/or progesterone receptor (ER/PgR) positive breast cancer. It compares adjuvant therapy with 5 years of tamoxifen (T) or letrozole (L), two years of T followed by 3 years of L (T→L) or vice versa (L→T). L improved disease free survival (DFS) over T by 18% (hazard ratio 0.82, JCO 2007;25:486ff). Switzerland contributed 598/8010 patients. The patient and tumor characteristics of Swiss patients was similar to the entire BIG 1-98 population. The purpose of this study was to test a prognostic score in RNA derived from FFPE tissues.  Scores representing ER and progesterone receptor (PgR) function, proliferation (PRO14, AURKA/STK15, BIRC5/SURV, CCNB1, CCNB2, CCNE2, CDC2, ENPF, KIF20A, MKI67, MYBL2, ORC6L, PRC1, SPAG5, TOP2A), and HER-2 were built from multiple genes for each score. Genes were selected according to an analysis of publicly available cDNA array databases. In a prior set of 82 pairs of breast cancer samples the Pearson correlation coefficients of the scores between fresh frozen and FFPE tissues were at least 0.94. Cut-offs for ER and HER-2 scores were defined as the antimodes of fitted bimodal Gaussian distributions; the ER and HER-2 scores closely agreed with immunohistochemical (IHC) data (kappa(ER)=0.868, kappa(HER-2)=0.836).  FFPE tissues were available from 437 Swiss BIG 1-98 patients. RNA of sufficient quantity and quality was extracted from 388 blocks (89%). Forty-seven genes were quantified by QRTPCR on TaqMan Low Density Arrays (TLDAs) as reported previously (Proc AACR 2008, Abstract 974).  The PRO14 score was positively correlated with the histological grade. PRO14 scores greater than the median and PGR scores lower than the median were associated with shorter DFS. In patients with grade 2 tumors, PRO14 scores were significant predictors of DFS in univariate Cox regression analyses. Multivariate analyses will be presented. Our findings are in agreement with an analysis of fresh frozen tissues of Belgian participants in BIG 1-98 (European Breast Cancer Conference 2008, Abstract 481).We show that RNA can be extracted from FFPE in sufficient quantity and quality to be used in QRTPCR using TLDAs. This opens the possibility of using FFPE tumor banks from controlled clinical trials for the investigation of gene expression patterns.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1073.
Quantitative HER2 homodimer levels correlate with time to first recurrence in HER2-positive breast cancer patients who did not receive trastuzumab in the adjuvant setting.
Authors: Bates M , Koestler W , Fuchs E , Sperinde J , Leitzel K , Ali S , Weidler J , Wu Y , Chappey C , Huang W , Lipton A
Published: December 2010

Abstract #1074

BACKGROUND: HER2-positive breast cancer patients are currently treated with trastuzumab in the adjuvant setting, but prior to 2005 these patients were not routinely offered trastuzumab, creating an opportunity to examine the relationship between HER2 expression and disease progression. The VeraTag technology is a proximity-based assay system that permits quantitative measurements of total HER2 protein expression (H2T) as well as HER2 homodimers (H2D) in formalin-fixed paraffin-embedded (FFPE) tissues. We measured H2T and H2D in a cohort of patients who were HER2-positive but did not receive trastuzumab in the adjuvant setting, and correlated those measurements with time to first recurrence of disease.  METHODS: Patients were selected for study because they were treated with a trastuzumab-containing regimen for HER2+ metastatic breast cancer. HER2-positivity was defined as IHC (Herceptest) 3+ or 2+, FISH+ (Vysis). 96 HER2+ patients who had been treated (but not with trastuzumab) in the adjuvant setting were identified. The VeraTag assay was used to quantitate H2T and H2D. Kaplan-Meier and Cox proportional hazards analyses were employed.  RESULTS: The distribution of H2T ranged over approximately 135-fold, and H2D varied over approximately 100-fold. In univariate Cox proportional hazards regression analysis, H2T trended toward significance (HR=1.44, p= 0.088) while both H2D (HR=1.39, p=0.022) and the ratio H2D/H2T (HR=2.01, p=0.03) were significantly correlated with time to first recurrence. Kaplan-Meier analysis of the H2T distribution divided into tertiles showed no significant difference in time to first recurrence among the tertiles (HR=0.7, p=0.16 for low vs. high comparison) and no trend was observed (Log Rank test for trend p=0.2). However, the same analyses performed using H2D (HR=0.59, p=0.03) or H2D/H2T (HR=0.56, p=0.01) demonstrated significantly longer time to first recurrence for the lowest tertile compared with the highest tertile respectively. A trend was observed in both cases (H2D: Log Rank p=0.048; H2D/H2T: Log Rank p=0.026)  CONCLUSION: In a population of HER2+ patients who did not receive trastuzumab in the adjuvant setting, and who subsequently developed metastatic disease, higher levels of HER2 homodimers and the ratio of homodimers to HER2 total expression correlated with time to first recurrence while total HER2 expression levels did not. These data suggest that measures of the activated forms of HER2 (dimers) may be better predictors of disease progression than simple quantitation of HER2 alone.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1074.
Invasive breast carcinoma of 1 cm or smaller: risk of nodal involvement and prognosis.
Authors: Ferro A , Caldara A , Triolo R , Frisinghelli M , Micciolo R , Barbareschi M , Leonardi E , Pellegrini M , Bernardi D , Cazzolli D , Berlarnda G , Mussari S , Galligioni E
Published: December 2010

Abstract #1075

Background: Screening mammography has increased the frequency of diagnosis of small (≤1 cm) invasive breast cancer (BC), for which the prognosis after locoregional therapy remains controversial.  Patients and methods: We have performed a retrospective analysis of 882 patients with ≤1 cm BC and positive or negative-nodes, treated in our Institution from 1990 to 2005. Risks of axillary nodes involvement (ANI), relapse-free (RFS) and overall survival (OS) were analyzed according to: age, tumor size, histologic type and grading, ER and PgR status and MIB-1, HER-2 and p53 immuno histochemical expression.  Results: Median age at diagnosis was 57 years (range: 20 - 86 years). Tumor size was: 71 Tmic (a single focus of invasive carcinoma ≤ 2 mm or up to 3 foci ≤1 mm in greatest dimensions, according to Silver and Tavassoli), 225 T1a (>2 -5 mm) and 586 T1b (>5–10 mm). ANI was found in 122 cases (13,8%): only 1 inT1mic (1,4%), 21 in T1a (9,3%) and 100 in T1b (17%). Tumor size, young age, poor differentiation and ductal histology were significantly associated with ANI (p=0.01). Local relapses were: 3 in Tmic pts(2 DCIS and 1 invasive), 13 in T1a, 22 in T1b. Distant metastases were: 1 in Tmic pts, 11 in T1a, 17 in T1b. At a median follow up of 92 months, RFS was 86,4% (Tmic: 86,8%, T1a: 86,2%; T1b: 86,2%). BC-specific mortality was 2% for T1mic, 3,3% for T1a, 3,8% for T1b accounting for an OS of 96,6%. Nodal involvement was the more relevant prognostic factor for survival, followed by young age, poor differentiation, high MIB-1 and p53.  Conclusions: In pts with 1 cm or smaller Invasive Breast Carcinoma, the risk of ANI is low, but not negligible, particularly for T1b tumors. Furthermore, the prognosis of these pts is influenced by high histological grade and young age.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1075.
Identification of three-genomic regions prognostic signature in small node-negative breast carcinomas.
Authors: Gravier E , Pierron G , Vincent-Salomon A , Savignoni A , Gruel N , Raynal V , Pierga J , Fourquet A , Reyal F , Roman-Roman S , Sastre-Garau X , de Rycke Y , Asselain B , Delattre O
Published: December 2010

Abstract #1081

Background: The purpose of this study was to identify a genomic signature of early metastatic recurrence, in order to predict accurately breast carcinomas clinical outcome and to select patients with node negative and small tumor size (<3cm) who would benefit from adjuvant chemotherapy.  Patients and methods: Using genome-wide BAC-PAC Genomic Comparative Hybridization (CGH) array (1kb), we analyzed a training set of 78 patients. All patients had invasive ductal carcinomas and were initially treated by surgery and radiotherapy, without chemotherapy. The validation was performed on an independent test set of 90 patients. The training and tests sets were composed of respectively 53 and 58 patients disease-free survivors at 60 months (good prognosis group), and by 25 and 32 patients with distant metastatic recurrence before 48 months (poor prognosis group). In the training set, a signature was established as a logistic multivariate model of regions containing contiguous BAC clones with statistically different ratios and median frequencies of gains and losses between the poor and the good prognosis groups. This signature was then validated using the independent test set to evaluate its accuracy to classify T0T1T2N0 patients according to their outcome.  Results: The training test identified a prognostic signature defined by 3 genomic regions, located on the 2p (38.3 to 40.9Mb), 3p (32 to 80.3Mb), and 8q (78.8 to 128.9Mb) chromosomes. In the test set, 90% of patients of favourable outcome were ER +ve and 88% were PR +ve, compared to 62% and 55% in the poor outcome group, respectively. In the test set, our signature was highly informative to identify patients that developed distant metastases before 48 months: the rate of patients well classified was 0.74, CI (95%): [0.64; 0.83], with a specificity of 95%, CI (95%): [86%; 99%]. On Kaplan-Meier analysis, the poor-prognosis genomic signature group of patients had a RR of 3.5 of metastatic relapse (log rank test p<0.001).  Conclusions: Our signature, validated on an independent series of small T0T1T2N0 and on a majority of ER/PR positive tumors, may provide a robust and accurate tool to identify, in addition to classical parameters, patients who would benefit from adjuvant medical treatments. The comparison of this genomic signature with RNA based signatures and clinico-pathological parameters, is currently being investigated.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1081.
Triple negative phenotype: not affecting survival in young patients with operable breast cancer.
Authors: Kim E , Han W , Park I , Lee J , Ko E , Yu J , Moon H , Noh D
Published: December 2010

Abstract #1083

Purpose: To investigate the clinicopathological characteristics and outcomes of triple negative breast cancer in young patients.  Experimental Design: We analyzed 1,498 patients with primary invasive breast cancer who underwent surgery between January 2000 and December 2003. Patients were divided into those < 40 and ≥ 40 years old and into triple negative and non-triple negative groups.  Results: A total of 326 (21.8%) were aged < 40; relative to patients ≥ 40 years old, the younger group showed larger tumor size, higher lymph node positivity, and elevated Ki-67 index. Recurrence-free survival (RFS; P = 0.0002) and overall survival (OS; P = 0.0483) were significantly lower in the younger group. Three hundred seventy four patients (25.0%) had the triple negative phenotype, and of which 93 (24.9%) were < 40 years old. Univariate survival analysis showed that the triple negative phenotype was not a prognostic factor for either RFS or OS in the younger group. In the older group, however, the triple negative phenotype was a significant prognostic factor for both RFS (P = 0.0103) and OS (P = 0.0081) by univariate analysis, and it remained a significant prognostic factor for OS by multivariate analysis (hazard ratio, 1.9; 95% confidence interval, 1.09-3.44; P = 0.025). When the non-triple negative group was subdivided into luminal and HER2 subtypes, the HER2 subtype showed the worst OS in both age groups (P < 0.001), followed by the triple negative group. However, the difference between the HER2 subtype and the triple negative group was significant only in the younger group (P = 0.0064), but not in the older group (P = 0.1446).  Conclusions: Triple negative phenotype did not affect survival in patients < 40 years old, because of the markedly poorer survival of non-triple negative tumors of the HER2 subtype in this age group.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1083.
Telephone disclosure of BRCA1/2 test results: a survey of genetic counselors.
Authors: Bradbury AR , Patrick-Miller L , Cummings SA , Fetzer D , Daly M
Published: December 2010

Abstract #1098

Disclosure of BRCA1/2 test results has historically been conducted in person by a certified genetic counselor (GC) and/or other health care professional. Due to increasing demand for and access to BRCA1/2 testing, there has been interest in providing genetic counseling services, including disclosure of test results, by telephone and internet. The practice of telephone disclosure among certified genetic counselors has not been described. We conducted semi-structured interviews with GCs to determine current prevalence of, and future interest in, telephone disclosure (TD) of BRCA1/2 test results. Surveys were self-administered and completed via a secure web site. Participants were recruited through the NSGC Cancer Special Interest Group. 195 GCs completed the survey (25% response rate). 23% of respondents have provided pre-test counseling by telephone. 98% reported having provided genetic test results by telephone, although many (48%) conduct TD rarely (<25% of the time). 33% reported TD experiences that had made them question TD as a practice. Comfort with TD varied by test result (37% comfortable with TD for positive test results, 77% for true negative results, 49% for indeterminate results and 33% for VUS results). The majority of GCs do not include a physician (85%) in the TD. Many report encouraging in-person follow-up with a GC (44%) and/or physician (30%). Overall, 42% reported being very interested in TD and 73% felt TD could be an acceptable standard practice. These results suggest many genetic counselors have provided genetic test results by phone and are interested in including telephone disclosure of BRCA1/2 test results in their practice. Given provider interest and the expansion of testing for BRCA1/2 and other hereditary cancer syndromes, further research evaluating the cognitive, affective and behavioral responses to telephone disclosure is warranted. Understanding the impact of telephone disclosure on factors such as, comprehension, risk perception, communication and performance of risk reducing behaviors and their mediators will be critical for the development of telephone disclosure policy and procedures that will optimize adaptive responses to receiving genetic test results via telephone or internet.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1098.
Effects of 5 years of treatment with lasofoxifene on incidence of breast cancer in older women.
Authors: LaCroix AZ , Cummings SR , Delmas P , Eastell R , Ensrud K , Reid DM , Vukicevic S , Armstrong RA , Thompson J , Thompson D , Wolter K , Allred C , Goss P , Osborne KC , Powles TJ
Published: December 2010

Abstract #11

The Postmenopausal Evaluation and Risk-reduction with Lasofoxifene (PEARL) Trial evaluated the effects of 5 years of a new potent SERM on the incidence of estrogen receptor positive (ER+) breast cancer (BC) (co-primary endpoints, invasive alone and invasive plus ductal carcinoma in situ [DCIS] together). 8556 women ages 5980 with femoral neck or spine T-score ≤ –2.5 and > –4.5 were enrolled in 32 countries. Women were randomly assigned to receive 0.25 mg/d or 0.5 mg/d of lasofoxifene (LASO), or placebo (PBO). BC events were ascertained through annual clinical breast exam and mammographic screening and adjudicated by physician oncologists and a BC pathologist blinded to treatment assignment. 77% of women completed 5 years of follow-up and 62–64% remained on study medication until planned trial closure. ITT analyses were conducted using Cox Proportional Hazards models. The protocol-defined primary analysis for all ER+ BCs compared each of the LASO groups to PBO. In the PBO group, 21 women developed invasive or non-invasive ER+ BC (17.3/10,000 person-years (P-Y)) compared to 10 in the LASO 0.25 mg/d group (9.0/10,000 P-Y; HR=0.52, 95%CI 0.25–1.08) and 5 in the 0.5 mg/d LASO group (3.3/10,000 P-Y; HR=0.19, 95% CI 0.07–0.56). For all invasive BCs (ER+ve and ve), 20 women in the PBO group were affected (16.4/10,000) compared to 16 in the LASO 0.25 mg/d group (13.2/10,000 P-Y; HR=0.79, 95%CI 0.41–1.52) and 3 in the 0.5 mg/d LASO group (2.5/10,000 P-Y; HR=0.15, 95% CI 0.04–0.50). For total BC, 24 women in the PBO group were affected (19.8/10,000 P-Y) compared to 20 in the LASO 0.25 mg/d group (16.4/10,000 P-Y; HR=0.82, 95%CI 0.45–1.49) and 5 in the 0.5 mg/d LASO group (4.1/10,000 P-Y; HR=0.21, 95% CI 0.08–0.55). Too few cases of DCIS occurred to determine effects of LASO on noninvasive breast tumors. No significant differences among treatment groups were observed for breast density. LASO also reduced the incidence of vertebral (primary at 3 years) and nonvertebral clinical fractures (co-primary at 5-years). LASO increased risk of venous thromboembolic events, but not stroke, endometrial cancer or endometrial hyperplasia and decreased risk of major coronary events at 0.5 mg/d. Endometrial hypertrophy, uterine polyps, and fibroids were more common with LASO while hypertension and hyperlipidemia was less frequent compared to PBO. We conclude that LASO reduces the incidence of ER+ BC and appears to have a favorable benefit-risk profile for prevention of clinical fractures in postmenopausal women with osteoporosis.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 11.
Sharing BRCA results: are there disparities?.
Authors: Beattie MS , Han PZ , Yu T , Chan S , Wilcox C , Crawford B
Published: December 2010

Abstract #1101

Background: BRCA is a unique breast cancer biomarker; it defines risk not only for an individual, but also for a family. Little is known about sharing BRCA results with relatives. This study aims to determine which relatives are told of BRCA results and which relatives pursue BRCA testing. This study also explores whether patient and relative characteristics can account for differences in sharing BRCA results and BRCA testing of relatives.  Methods: As part of the UCSF Cancer Risk Program Cohort, all women who receive genetic counseling and BRCA testing at the UCSF/Mt. Zion Comprehensive Cancer Center (CCC) and at San Francisco General Hospital (SFGH) are sent a survey regarding their health habits and their genetic testing experience. Women surveyed are asked: whether they have shared BRCA results; if so, with whom; and whether other family members have pursued BRCA testing. Patient-level variables examined include: cancer status, hospital site, and BRCA results. Relative-level variables examined include: gender and degree of relationship (first- or second-degree).  Results: Over 80% of women surveyed have responded to date (n = 151). Overall, 95% report sharing BRCA results with at least one relative, and 41% indicate that other relatives have received BRCA testing. Tables 1 and 2 show rates of telling relatives and of BRCA testing in relatives based on patient and relative characteristics. Although the proportion of BRCA carriers is similar at both sites (22% at the CCC and 20% at SFGH), relatives of SFGH patients are significantly less likely to pursue BRCA testing (p=0.001).        Conclusions: After BRCA testing, women are less likely to share results with male and second-degree relatives. The proportion of BRCA carriers is similar at SFGH and at the CCC. Rates of telling relatives of BRCA results are also similar at both sites. A large and significant disparity exists, however, in BRCA testing of relatives of women from SFGH (8% from SFGH vs. 47% from the CCC, p = 0.001). Future research will examine the reasons for low uptake of BRCA testing in this population, barriers to BRCA testing, and potential interventions.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1101.
Enhanced mismatch mutation analysis: a new high throughput screening method for simultaneous detection of point mutation and gross gene rearrangement designed for BRCA1 and BRCA2 genes.
Authors: Gastaldello B , Champ J , Lafon D , Fraud A , Longy M , Viovy J , Weber J , Bonnet F , Sevenet N
Published: December 2010

Abstract #1102

Background : Recent studies showed that large genetic rearrangements (duplication or deletion of one or several exons) are of great importance in genetic diseases. Only few methods such as QMPSF and MLPA fit a routine use for these large deletions and duplications. They have to be performed additionally to a point mutation detection method (usually sequencing or dHPLC), which is labor intensive, time consuming and expensive. We present here a method called Enhanced Mismatch Mutation Analysis (EMMA) based on heteroduplex analysis that offers great sensitivity for point mutation detection and simultaneously detection of gross gene rearrangement by capillary electrophoresis.  Material and Methods : EMMA is able to detect point mutations in PCR fragment ranging from 200 bp to 550 bp. It is thus possible to perform size multiplexing and analyze up to 6 fragments in the same run. PCR multiplex fragments of different sizes are prepared by semi-quantitative PCR. Separations are performed in multi-capillary electrophoresis. Poly(acrylamide-g-poly(dimethylacrylamide)) is used as a self coating sieving matrix; this separation matrix contains also additives like free nucleotides for a better sensitivity. One of the fragment is chosen as a reference for peak area normalization.  Results : A series of 30 patients has been screened for BRCA1 and BRCA2 genes simultaneously by EMMA and the classical package dHPLC-QMPSF. All mutations and polymorphism detected by dHPLC have been detected by EMMA and new polymorphisms have been characterized for those genes. Furthermore, we will present the whole results of the positive mutation controls of BRCA1 and BRCA2 of the Institut Bergonie molecular genetic lab database screened by EMMA.  Discussion : Using a unique condition of capillary electrophoresis separation for a lower number of PCR reaction for each gene (9 for BRCA1 and 15 for BRCA2), EMMA demonstrates a 5 times saving of lab time for technician, and displays a large easiness to use it in a routine way for molecular diagnosis. Finally this technology will benefit of a grant of French Ministry of Industry to be spread in molecular lab of Comprehensive Cancer Centers.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1102.
Characterization of estrogen receptor-positive breast cancers in BRCA1 mutation carriers.
Authors: Wang Y , Collins L , Schnitt S , Garber J , Tung N
Published: December 2010

Abstract #1105

Background: Invasive breast cancers (IBC) in BRCA1 mutation carriers are usually estrogen receptor (ER) negative (-) and more than 80% have a basal-like molecular phenotype. These tumors are typically poorly differentiated invasive ductal carcinomas with a high mitotic rate and frequently show a prominent lymphoid infiltrate, pushing or circumscribed margins, and geographic necrosis or a central fibrotic focus. However, some women with BRCA1 germline mutations develop ER positive (+) cancers; little is known about the characteristics of the ER+ tumors in this group.  Design: We identified 41 ER+ IBC that developed in women with BRCA1 germline mutations with available pathologic material for review. The histologic features were analyzed in detail and compared with those of 45 ER- IBC that developed among BRCA1 mutation carriers.  Results: Mean patient age was 46y for ER+ and 45y for ER- cases. Ninety percent of the ER+ cases and all the ER- cases were invasive ductal carcinomas or invasive carcinomas with ductal and lobular features. There were 2 mucinous and 2 tubular carcinomas in the ER+ group. The ER+ cancers exhibited a range of histologic grades: 12 (29.3%) were grade I, 10 (24.4%) grade II, and 18 (43.9%) grade III (1 case of microinvasive carcinoma could not be graded). In contrast, 43 of the 45 ER- cancers were grade III (95.6%) and 1 (2.2%) grade II (1 case of microinvasive carcinoma could not be graded). Histologic features commonly seen in association with ER- BRCA1 mutation-associated IBC were compared between the two groups and the results are summarized in the table.    Of note, a brisk mitotic rate, pushing margin, and the presence of geographic necrosis/central fibrosis were all significantly more common in ER- than in ER+ tumors.  Conclusions: To our knowledge, this study is the first to document in detail the histologic features of the uncommon ER+ IBC occurring in BRCA1 mutation carriers. Our observations suggest that ER+ IBC in BRCA1 mutation carriers represent a morphologically diverse group. This raises the possibility that at least some ER+ IBC that develop in women with germline BRCA1 mutations may be sporadic rather than BRCA1-associated. We are currently analyzing these lesions with a panel of biomarkers and assays for loss of heterozygosity at the BRCA1 mutation sites to further address this important issue.  This work was supported by a grant from the Breast Cancer Research Foundation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1105.
Effects of methyl derivatives of the rexinoid UAB30 on methylnitrosourea (MNU) induced mammary cancers and on various indicators of toxicity.
Authors: Muccio DD , Lubet RA , Atigadda VR , Brouillette WJ , Grubbs CJ
Published: December 2010

Abstract #1108

UAB30 is a RXR selective retinoid that has shown excellent activity in preventing chemically-induced mammary cancers with minimal toxicity (specifically not increasing serum triglycerides), and is currently undergoing a Phase I clinical trial. Several methyl analogs were synthesized in an attempt to obtain a more active preventive agent and to better understand the mechanisms of activity/toxicity of this class of agents. The retinoids included 4-methyl-UAB30, 5-methyl-UAB30, 6-methyl-UAB30, 7-methyl-UAB30, and 8-methyl-UAB30. The compounds were evaluated in the MNU-induced mammary cancer model using female Sprague-Dawley rats. MNU (75 mg/kg BW) was administered at 50 days of age and the animals observed for 120 days afterward for the development of ER+ mammary cancers. The retinoids were given at a dose of 200 mg/kg diet, except 7-methyl-UAB30 (given at 100 mg/kg diet). 4-Methyl-UAB30 and 7-methyl-UAB30 were highly active; reducing mammary cancer multiplicity by 74 and 61%, respectively. The retinoids 5-methyl-UAB30, 6-methyl-UAB30, and 8-methyl-UAB30 did not have chemopreventive activity in this model. The 8-methyl-UAB30 derivative actually caused a 108% increase in growth of the mammary cancers. As we have previously shown (Carcinogenesis 27, 1232-1239, 2006), serum triglycerides correlated with cancer preventive activity; i.e., high levels were observed with active retinoids. 4-methyl-UAB30 caused an initial large increase in body weight gain of the rats. Of interest, serum levels of 6-methyl-UAB30 and 7-methyl-UAB30 were high, while the other agents were low or could not be detected. All methyl derivatives caused varying decreases in liver retinyl palmitate levels. Structure-activity relationships are also being evaluated using crystallography of RXR/UAB-rexinoid complexes as a guide. Structure-based and dynamic-based approaches are used to facilitate the design of new rexinoids that fit into the LBD of RXRs. For example, 4-methyl-UAB30 had a 5-fold greater binding affinity to hRXR alpha LBD than 9-cis-retinoic-acid. These studies emphasize that minor modifications of a retinoid molecule can drastically change its absorption, metabolism, toxicity, binding affinities to receptors, and activity in preventing mammary cancers. (Supported by NCI Breast SPORE CA089019 and contract HHSN261200433001C).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1108.
A potential role of EGFR-inhibitors for the prevention of BRCA1-related breast cancer.
Authors: Burga LN , Tung N , Troyan SL , Lee BT , Houlihan M , Pories SE , Staradub V , Tobias A , Come SE , Coffey K , Wulf GM
Published: December 2010

Abstract #1110

Background: The majority of women who develop a BRCA1-related breast cancer develop an ER-/PR-/HER2- breast cancer. Endocrine prophylaxis does not prevent these ER- breast cancers. Therefore, there is a need to develop novel chemoprevention agents in this population. 67% of BRCA1-associated ER- breast cancers also overexpress epidermal growth factor receptor (EGFR). We therefore examined EGFR as a potential target for chemoprevention  Methods: We isolated primary mammary epithelial cells (HMECs) from women with a germline BRCA1 mutation who underwent prophylactic mastectomies and from age-matched controls without a mutation who underwent reduction mammoplasties. We used a three-dimensional matrigel-based colony formation assay to assess clonality and proliferative capacity of these cells as well as their response to EGFR-inhibition. Flow cytometry was used to determine the number of EGF binding sites per cell. As a corresponding mouse model we used conditional MMTV-Cre BRCA1-/-p53+/- mice. Results: HMECs from BRCA1 mutation carriers and from controls express EGFR to a similar extent as HCC1937 BRCA1-associated triple-negative breast cancer cells (5x103 binding sites/cell). In ex vivo 3D-cultures we observed that HMECs derived from BRCA1 mutation carriers showed greater clonal and proliferative capacity when compared to normal controls. However while the HMECs derived from BRCA1 mutation carriers and normal controls were equally sensitive to the growth-inhibitory effect of Erlotinib at concentrations as low as 0.2 μM, the ID50 for HCC1937 breast cancer cells was > 10 μM. Similar findings were observed in murine MECs derived from normal control mice as well as BRCA1-/-p53+/- mice which develop breast cancer at the age of 7 to 8 months. Both groups of murine MECs were equally sensitive to Erlotinib growth inhibition. Conclusion: MECs derived from breast tissue of women and mice with a germline BRCA1 mutation express EGFR and are highly sensitive to growth inhibition with the EGFR inhibitor Erlotinib. In contrast, BRCA1-associated HCC 1937 breast cancer cells are more resistant to Erlotinib inhibition despite EGFR expression. We are now studying whether Erlotinib given via oral gavage daily has the potential to delay or prevent breast cancer in this mouse model of BRCA1-related breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1110.