bluuebunny/crossref_metadata_2025 huggingface dataset:
Truncated p95erbB2 Isoforms Are Capable of Transforming Human Mammary Epithelial Cells.
Authors: Ward T. , Iorns E. , Gallas M. , Lippman M. , Landgraf R. , Pegram M.
Published: December 2010

Background:Objective clinical response to trastuzumab monotherapy in erbB2-amplified first line metastatic breast cancer is 34% (Vogel, et al., JCO 20: 719-26, 2002). Amongst patients who respond, most develop resistance (defined by disease progression on trastuzumab). One proposed mechanism of trastuzumab resistance is proteolytic cleavage of erbB2 receptor from its full-length (p185) form into truncated, constitutively active p95. Increased expression of p95erbB2 correlates with increased nodal involvement and poor clinical outcome. Because p95erbB2 lacks the trastuzumab binding epitope, expression may designate patients who would be suitable for treatment with erbB2 kinase inhibitors.Materials and Methods:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in several cell types via retroviral vector. Additionally, an intracellular form of p95erbB2 that arises via alternative translation and an intracellular p95erbB2 construct containing two copies of a nuclear localization sequence were also expressed. Expression and proper subcellular localization of constructs were confirmed by cell fractionation, western blot analysis and confocal microscopy. Transformation of human mammary epithelial (HMEC) and NIH3T3 cells by p185erbB2 and p95erbB2 isoforms was evaluated by anchorage independent growth using a quantitative fluorescent soft agar assay, and effects on migration and invasion of these cells were investigated by wound-healing and transwell assays. Cells transfected with oncogenic Ras or empty vector were used as positive and negative controls in these experiments.Results and Discussion:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in HMEC and NIH3T3 cells, and were correctly directed to the cell membrane; nuclear targeted intracellular p95erbB2 was correctly localized to the cell nucleus. Both p185 erbB2 and membrane-bound truncated p95erbB2 were sufficient to transform HMEC cells as compared to empty vector control transfected cells [mean fluorescence intensity empty vector control 2480 ± 464 (1 standard deviation); mean fluorescence intensity p185erbB2 9208 ± 2528, p= 0.0106; mean fluorescence intensity p95erbB2 6615 ± 1588, p= 0.0124)] as was the positive control oncogenic Ras (mean fluorescence intensity 4350 ± 433, p=0.0069). Interestingly, nuclear-targeted p95erbB2 was also sufficient to transform HMEC cells (mean fluorescence intensity 6492 ± 818, p=0.0018). These data support the hypothesis that truncated p95erbB2 species may be major pathogenic drivers in erbB2-amplified cancers. P95erbB2 therefore represents an attractive target for diagnosis and treatment of erbB2+ breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3136.
Characterisation of 17q12-21 – Identification of Novel Therapeutic Targets.
Authors: Glynn R. , Miller N. , Alvarez Iglesias E. , Doolin O. , Newell J. , Kerin M.
Published: December 2010

Introduction: Over-expression of the HER2/neu oncogene is associated with poorer clinical outcome in breast cancer. Notwithstanding the success achieved with Trastuzumab in treating HER2/neu positive patients, numerous questions remain. In particular, there are concerns regarding accurate identification of HER2/neu positivity and the correct management of these patients in terms of the dosage, timing and optimal combination of Trastzumab with chemotherapeutic regimens. One approach to answering these questions has been to focus on the area of chromosome 17 in which HER2/neu is located in order to identify other genes in the vicinity. As potential therapeutic targets, we examined the amplification and expression patterns of co-localised cancer-associated genes at the HER2/neu amplicon on chromosome 17.Aims: The primary objective of this study was to examine amplification and expression patterns of co-localised genes at 17q12-21. In doing so, we aimed to establish their relationship with HER2/neu positivity, and then to quantify their relative importance in developing a gene panel predictive of HER2/neu status.Methods: Breast tissue from a cohort of patients with HER2/neu positive (n=48) and negative (n=48) tumours was retrieved at time of surgery. Protein and gene expression levels were determined using immunohistochemistry and RQ-PCR, respectively. Results were analysed to detect patterns of amplification among the genes, namely HER2/neu, STARD3, GRB7, TOP2A, TUBG1, RPL19, LASP1, RARA, and ER Alpha, and to identify associations between expression levels and clinical data. A binary logistic approach and classification tree analysis was employed to determine whether the expression levels of our target genes were useful predictors of HER2/neu status.Results: Strong correlation was noted between expression levels of HER2/neu and multiple cancer-associated genes at 17q12-21, including the novel genes LASP1 (p=0.000) and RPL19 (p=0.000). A significant relationship was also noted between TOP2A and the previously unexplored TUBG1 (p=0.000). Analysis of gene expression patterns according to clinicopathological variables revealed significant associations across intrinsic breast cancer subtypes.Conclusion: This study reaffirms the correlation between HER2/neu and its co-localised genes at 17q12-21, including novel candidate genes LASP1 and RPL19. We have also identified a novel relationship between TOP2A and TUBG1. Furthermore, our work represents a tentative step in the development of more accurate means of characterising HER2/neu status. As such, this work has again indicated the central role which this amplicon may play in selection of patients for treatment, not alone for Herceptin and anthracyclines, but also for taxane-based therapies, and as such, has revealed novel pathways for us to follow as we move towards our goal of individually tailored therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3137.
Distinctive Expression of Cancer/Testis-X Antigens in a Subset of ER Negative Breast Carcinomas.
Authors: Grigoriadis A. , Caballe O. , Hoek K. , da Silva L. , Chen Y. , Shin S. , Jungbluth A. , Miller L. , Cebon J. , Old L. , Lakhani S. , Simpson A. , Neville M.
Published: December 2010

Background: Cancer/testis-X antigens are a multigene family that are predominantly expressed in human germ line cells, with little or no expression in somatic adult tissues, but become aberrantly activated in various malignancies. Some such CT-X antigens represent ideal targets for cancer immunotherapy and have already been used in clinical testing. In contrast to melanomas, bladder, lung, ovarian and hepatocellular carcinomas which show higher levels of CT-X antigen expression, the reports in breast cancers have been inconclusive to date and a comprehensive gene expression and clinicopathological analysis has yet to be performed.Material and Methods: Using sequencing data as well as nine publicly available gene expression data sets, we analyzed the expression of Cancer/testis-X antigens in more than 1900 primary breast cancers. Complementary analysis was performed on three tissue microarrays comprising a total of 201 primary breast carcinomas and 53 brain metastases. Clinical information on the ER-, PR-, HER2, Ki67, p53, EGFR and basal markers was available for statistical analysis.Results: A significantly higher expression of Cancer/testis-X antigens was found in ER negative breast carcinomas over different data sets with a concordant gene expression pattern of several Cancer/testis-X antigens. Members of the MAGEA family and NY-ESO-1/CTAG1B were consistently the most prevalent. Immunohistochemical analyses confirmed a significant correlation of MAGEA family and NY-ESO-1/CTAG1B with ER negative (pValue < 0.0001), PR negative (pValue < 0.01) and Ki67 staining (pValue < 0.0001). Many of these tumors were also positive for basal markers.Discussion: Previous studies of Cancer/testis-X antigens in breast have focused on ER positive cancers, in smaller subsets and provided inconclusive results. Using comprehensive gene expression data sets and tissue microarrays, we have demonstrated a significant association of MAGEA family and NY-ESO-1/CTAG1B with ER/PR negative breast cancer. Since these cancers represent a subgroup for which less therapeutic modalities are available, we propose the use of MAGEA and NY-ESO-1/CTAG1B cancer vaccines in the adjuvant setting as an approach to restricting tumor growth and metastases. Clinical trails using MAGEA and NY-ESO-1/CTAG1B are warranted.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3139.
Role of miR-143 Regulating DNA Methyltransferases 3A in Breast Cancer.
Authors: Ng E. , Kwong A. , Tsang W. , Leung C. , Wong C. , Kwok T. , Ma E.
Published: December 2010

Background and Aims: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes including some involved in cancer development. In particular, decreased expression of miR-143 has been reported in various human cancers including colorectal cancer and B-cell lymphomas. The aim of this study was to elucidate the role of miR-143 dysregulation in breast cancer.Methods: Expression levels of human mature microRNAs (miRNAs) were compared with paired breast carcinomas and adjacent normal tissues by TaqMan real-time PCR based expression arrays. Decreased expression of miR-143 was further confirmed in breast cancer cell lines and paired breast tumors and normal adjacent tissues by qRT-PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was performed in human breast cancer cell lines to confirm target association.Results: Down-regulation of miR-143 was verified in both human breast cancer cell lines and 80% (12/15) of breast tumors (P < 0.001). DNA methyltranferase 3A (DNMT3A), one of a key enzyme involved in DNA methylation, was defined as a potential target of miR-143 by in-silico analysis. Overexpression of miR-143 in breast cancer cell lines down-regulated expression of DNMT3A, decreased tumor cell growth by MTT assay and soft agar colony formation assay. DNMT3A was demonstrated to be a direct target of miR-143 by luciferase reporter assay. Inverse correlation between DNMT3A protein and miR-143 was found in tumor and normal breast tissues.Conclusions: In this study, we show for the first time in breast cancer that miR-143 specifically targeted DNMT3A and the expression of miR-143 was inversely correlated with DNMT3A expression. Our findings demonstrated that down-regulation of miR-143 and up-regulation of DNMT3A are significant changes in breast tumors. These findings indicate a tumor suppressive role of miR-143 in epigenetic aberration of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3148.
Induction of PTEN-p53 Crosstalk in Mammary Epithelial Cells: A Novel Mechanism of Breast Cancer Prevention by the Dietary Factor Genistein.
Authors: Rahal O. , Rahal O. , Simmen R. , Simmen R. , Simmen R.
Published: December 2010

Consumption of soy foods either at an early age or for lifetime has been associated with reduced risk for developing breast cancer in humans and in animal models. However, this association continues to be controversial and the precise mechanisms for protection remain elusive. Among the soy products, the isoflavone genistein (GEN) has been widely suggested to confer mammary tumor protection. Previously we demonstrated the increased expression of tumor suppressors PTEN and p53 in mammary epithelial cells (MECs) isolated from young adult female rats fed dietary soy protein isolate (SPI) or casein (CAS) supplemented with GEN, when compared to MECs from rats fed the control (CAS) diet. Since NMU-administered rats fed SPI had reduced tumor incidence and increased tumor latency than those fed CAS, PTEN and p53 likely mediate the observed tumor resistance with SPI in vivo. We hypothesized that GEN induction of PTEN and p53 in MECs results in the formation of a PTEN/p53 functional complex to negatively regulate breast cancer development. Here, we used the human non-tumorigenic, ER-negative mammary epithelial cell line, MCF-10A, as an in vitro system to mechanistically dissect ER-independent actions of GEN involving PTEN and p53. GEN (40 nM, 2μM) augmented PTEN and p53 expression in treated relative to control cells. GEN also induced nuclear co-localization and physical association of PTEN and p53. To test a functional consequence of GEN-induced PTEN/p53 cross-talk on mammary epithelial phenotype, we analyzed GEN effects on cell cycle progression and acini formation in 3D cultures. Our results showed attenuated cell proliferation and lower cyclin D1 and pleiotrophin transcript levels in GEN-treated cells, which were abrogated by small interfering RNA to PTEN, indicating PTEN-dependence. Using FACS analysis, we showed that GEN induced cell cycle arrest at G0-G1 phase. Treatment with GEN promoted early acini formation of MECs grown in Matrigel, which temporally coincided with PTEN-dependent suppression of p21 and p27 transcript levels. Further analyses of GEN effects on MECs demonstrated induction by GEN of PTEN promoter-luc reporter activity as measured by dual-luciferase assay. Interestingly, treatment with siRNA to either PTEN or p53 reduced basal and GEN-induced PTEN promoter activity. Given that p53 binds to the PTEN promoter, our results suggest a feed-forward cycle in which dietary factor (GEN) induction of nuclear PTEN leads to PTEN promotion of its own signaling. By maintaining a stable pool of nuclear p53 to boost its transcription, PTEN ensures its continuous expression in MECs to favor cell differentiation. These data elucidate a novel mechanism by which dietary factors with PTEN-inducing activity may attenuate breast cancer risk and development. Funding by USDA-CRIS 6251-5100002-06S and the Department of Defense Breast Cancer Program (0810548).

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3149.
Downregulation of the Novel Candidate Tumor Suppressor Gene NCOR2/SMRT in Human Breast Cancer Progression.
Authors: Geradts J. , Saxena R. , Groth J. , Desouki M. , Burchette J. , Liao S.
Published: December 2010

Background: We recently described a number of DNA copy number changes that differentiated low grade ductal carcinoma in situ (DCIS) lesions with and without associated invasive carcinoma. One of the invasion-associated candidate genes was Nuclear Corepressor 2/Silencing Mediator of Retinoid and Thyroid Receptors (NCOR2/SMRT). This gene is a component of a repressing nuclear receptor complex and a mediator of ER activity. It facilitates recovery from DNA double strand breaks. Previous studies showed that downregulation of the gene was associated with transformation of non-Hodgkin lymphomas.Materials and Methods: Genomic DNA was extracted from microdissected paraffin sections of formalin fixed low grade DCIS lesions with an invasive component (n=42), as well as morphologically similar DCIS lesions without associated invasion within at least 5 years (n=38). Comparative PCR was performed with GAPDH as the reference gene. In addition, we assayed tissue microarrays (TMAs) by immunohistochemistry using two anti-Smrt antibodies. Three TMAs included 162 interpretable cases of invasive breast carcinoma from Roswell Park Cancer Institute (RPCI). One additional TMA included 73 interpretable breast cancers from a previously described cohort from Vienna, Austria. Nuclear staining in neoplastic cells was scored as positive, reduced (significantly weaker compared to admixed benign cells), and negative. Ncor2/Smrt expression was correlated with various patho-biologic variables.Results: Allelic loss of NCOR2/SMRT was observed in 79% (33/42) of DCIS lesions with associated invasion, but in only 26% (10/38) of pure DCIS cases (p<0.01). In the RPCI breast cancer cohort, nuclear Ncor2/Smrt protein was absent or downregulated in 73% (119/162) of tumors. In the Vienna cohort, there was no or markedly reduced nuclear staining in 48% (35/73) of cases. In both cohorts, loss of nuclear reactivity was more common in ER-negative breast cancers. In the Vienna series, Ncor2/Smrt loss was more frequent in PR-negative tumors as well. In the RPCI cohort, Ncor2/Smrt downregulation was more common in ductal (81%, 109/135) than in lobular (37%, 10/27) carcinomas. Neither series showed a significant association between Ncor2/Smrt expression and tumor grade or HER2/neu status.Discussion: Allelic loss of NCOR2/SMRT was significantly more common in DCIS lesions associated with stromal invasion. In invasive tumors, the protein downregulation rate was between 48% and 73%. The gene was more commonly downregulated in ER-negative carcinomas, consistent with its role as a modulator of ER function. Our data provide new evidence that NCOR2/SMRT may play an important part in early breast cancer progression.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3150.
Constitutive Activation of Smoothened (SMO) Enhances MMTV-c-ErbB2-Induced, but Not MMTV-Wnt1-Induced, Mammary Tumorigenesis.
Authors: Landua J. , Moraes R. , Li Y. , Lewis M.
Published: December 2010

Background. Smoothened (Smo) is a key effector protein in the hedgehog signaling pathway, a developmental pathway involved in the regulation of pattern formation, proliferation, cell fate, and stem/progenitor cell function in the mammary gland. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development with SMO ectopically expressed in 70% of DCIS and 30% of IBC. Coupled with our genetic studies in mice, these clinical observations suggest a causal or contributory role in breast cancer.Materials and Methods. To determine whether activation of SMO is oncogenic in the mammary gland, and to study possible interactions between hedgehog signaling and other oncogenic signaling pathways, we crossed transgenic mice expressing a constitutively active form of Smo (SmoM2) under the Mouse Mammary Tumor Virus promoter (MMTV-SmoM2) with mice expressing either MMTV-Wnt1 or MMTV-c-ErbB2. Time to tumor onset, tumor growth, and tumor multiplicity were measured, and resulting tumors evaluated histologically.Results. Expression of MMTV-SmoM2 was not oncogenic as a single genetic alteration. In genetic crosses, simultaneous expression of MMTV-Wnt1 and MMTV-SmoM2 within mammary epithelium did not affect tumor formation. However, simultaneous expression of MMTV-c-ErbB2 and MMTV-SmoM2 resulted in significantly accelerated median time to tumor formation, with an overall tumor formation rate of 100% compared to MMTV-c-ErbB2 alone (74%).Discussion. Activated SMO is not oncogenic under the conditions tested. However, MMTV-SmoM2 interacts genetically with MMTV-c-ErbB2, but not MMTV-Wnt1 to promote mammary tumorigenesis. Together, our data indicate a contributory role for Smo in breast cancer progression and suggest that hedgehog signaling inhibitors may be useful for treatment of a subset of human breast cancers.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3152.
Cellular Location and Expression Pattern of NHERF1 in Human Breast Cancer and the Impact of NHERF1 on the Growth of Breast Cancer Cells.
Authors: Cheng S. , Cheng S. , He J. , Martin T. , Ye L. , Mansel R. , Jiang W.
Published: December 2010

BACKGROUND: The Na+/H+ exchange regulatory factor 1 (NHERF1) is an adapter protein that, when present in the cytoplasmic region, regulates trafficking and signaling of several G protein-coupled receptors (GPCRs). However, it has been recently suggested that, when located in the nucleus of cells, NHERF1 may have an oncogenic role in cancer. Recent work has shown that NHERF1 may be involved in the progression of certain solid tumours including breast cancer. The aim of the current study were: first to evaluate the spatial and cellular location of the NHEREF1 protein in mammary tissues, mammary epithelial and cancer cells, second to determine the expression pattern of the NHERF1 transcript in human breast cancer and deduce a possible association with clinical and pathological factors and, finally, to investigate the cellular impact of NHERF1 expression on breast cancer cells.MATERIALS AND METHODS: Expression of NHERF1 was examined in a cohort of breast tissue samples. The protein levels and distributions were assessed using immunohistochemical staining (IHC) and imaging analysis tools. The distribution of the NHERF1 protein in nucleus and cytoplasm was calculated using the protein staining intensity ratio between the two compartments. The transcript level was determined using quantitative real time-PCR. Constructed ribozyme transgenes were used to knock-down NHERF1 in MCF-7 cells, and the effect this had on in vitro cell growth was examined using in vitro methods.RESULTS: NHERF1 protein staining was seen in both normal epithelial cells and cancer cells in tissues. However, the staining pattern in cancer cells and normal epithelial cells was different. The protein was seen at a higher level in the nucleaus of cancer cells, as shown by a higher nuclear/cytoplasmic ratio of NHERF1 staining in breast cancer cells when compared with that in normal mammary epithelial cells (p=0.038). NHERF1 expression was increased in high grade tumours compared with low grade tumours (p=0.0016, grade-3 vs grade-1). Quantitative analysis of the NHERF1 transcript revealed a higher level expression in samples from patients with poor prognosis and that this was linked to the long term survival: mean survival for patients with high NHERF1 was 102 (55-148.8, 95%CI) months compared with 136 (126.6-145.9) months for patients with low NHERF1 transcripts. Using human breast cancer cell line, MCF7, we created NHERF1 knockdown subline. Loss of NHERF1 in the MCF-7 subline resulted in an increase in the growth rate, in vitro.CONCLUSION: This study shows that NHERF1 influences the growth of breast cancer cells. However, the effect of NHERF1 is dependent upon the levels of expression of NHERF1 in breast cancer tissues and most importantly determined by the cellular location of this protein in cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3155.
Suppressors of Cytokine Signaling and Their Role in Human Breast Cancer.
Authors: Sasi W. , Jiang W. , Sharma A. , Mokbel K.
Published: December 2010

BACKGROUND:Suppressors of cytokine signaling (SOCS) are important negative feedback regulators of the JAK/STAT signaling pathway, and have been recently investigated for their role in the development of different cancers. In this study, we examined the expression of SOCS 1-7 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors.PATIENTS AND METHODS:SOCS 1-7 mRNA extraction and reverse transcription were performed on breast cancer tissue samples (n= 127) and normal background breast tissue (n= 31). Transcript levels of expression were determined using real-time PCR and analyzed against TNM stage, tumour grade and clinical outcome over a 10 year follow-up period.RESULTS:SOCS 1,4,5,6 and 7 expression decreased with increased TNM stage (TNM1 vs. TNM3 p= 0.039, TNM1 vs. TNM4 p= 0.016, TNM1 vs. TNM3 p= 0.012 and TNM1 vs. TNM3 p= 0.044 respectively). SOCS 2 and 3 expression decreased with increased Nottingham Prognostic Index (NPI) (NPI1 vs. NPI3 p=0.033 and NPI2 vs. NPI3 p=0.041 respectively). SOCS-7 expression decreased with higher tumour grade (Grade 3 vs. Grade 2 p=0.037). After a median follow up period of 10 years, we found higher levels of SOCS 1, 2 and 7 expression among those patients who remained disease-free compared to those who developed local recurrence (p= 0.0073, p= 0.021 and p= 0.039 respectively). Similarly, we found higher levels of SOCS 2, 4 and 7 expression in those who remained disease-free compared to those who developed distant recurrence (p= 0.022, p=0.024 and p= 0.033 respectively). Patients who remained disease-free had higher levels of SOCS 1 and 2 expression compared to those who died from breast cancer (p= 0.02 and p=0.033 respectively). The disease free survival (DFS) and overall survival (OS) curves showed that higher levels of SOCS 1, 3 and 7 were significant predictors of higher DFS (p=0.015, p=0.024 and p=0.03 respectively) and OS (p=0.005, p=0.013 and p= 0.035 respectively). Higher levels of SOCS 4 were significant in predicting better OS (p=0.007) but not DFS.CONCLUSIONHigher mRNA expression levels of SOCS 1, 3, 4 and 7 are significantly associated with earlier tumour stage and better clinical outcomes in human breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3157.
Transforming Acidic Coiled-Coil-Containing Protein 2 (TACC2) in Human Breast Cancer, Expression Pattern and Clinical/Prognostic Relevance.
Authors: Cheng S. , Cheng S. , Douglas-Jones A. , Mansel R. , Jiang W.
Published: December 2010

BACKGROUND: TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Recent work has shown that members of this family, including TACC2, may be involved in the progression of certain solid tumours. The aim of the current study was to identify the role of TACC2 in breast cancer and to establish if a prognostic relevance exists.MATERIALS AND METHODS: Fresh frozen primary human breast cancer tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were used. The distribution and location of TACC2 was assessed using immunohistochemical staining (IHC) and imaging analysis tools. The transcript levels of TACC2 were determined using quantitative real time-PCR. The results were analyzed against the clinical, pathological and follow-up (10 years) data. Statistical analysis was by Mann-Whitney U test and Kaplan-Meier method. Shown are median transcript level.RESULTS: TACC2 protein staining was seen in both normal epithelial cells and cancer cells in mammary tissues. Increased staining of TACC2 was seen in most of the breast tumours examined when compared with normal breast tissues. Quantitative analysis of the TACC2 transcript also revealed a higher level expression in tumours compared with normal tissues (p=0.027, tumours vs normal). TACC2 expression was significantly increased in higher grade tumours compared to those in lower grade tumours (grade-3 vs grade-1, p=0.046). Using the Nottingham Prognostic Index (NPI), TACC2 transcript was significantly higher in tumours from patients with a moderate prognosis than from those with a good prognosis (p = 0.019). The expression in samples from patients with poor clinical outcome (with metastasis, recurrence and breast cancer related death) was significantly higher than that from patients who remained disease free (median (IQR) 33.8 (3.0-110.2) copies vs 6 (1.6-28.9) copies, p=0.038). This was reflected by the shorter disease-free survival for patients with high TACC2 (107 (91-122.8, 95% CI) months compared with 137 (125-150.6) months for patients with low TACC2 transcripts (p=0.019).CONCLUSION: This study shows that increased expression of TACC2 correlates with poor prognosis in patients with breast cancer. This suggests that TACC2 mediates an oncogenic effect on breast cancer cells. The findings also suggest that TACC2 may be a potential therapeutic target.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3159.
Consistent Principal Components from Triple Negative Breast Cancer Expression Profiles Yields Agent Specific Predictors of Chemotherapy Response.
Authors: Li Q. , Eklund A. , Juul N. , Desmedt C. , Haibe-Kain B. , Haibe-Kain B. , Sotiriou C. , Symmans W. , Pusztai L. , Richardson A. , Szallasi Z. , Szallasi Z.
Published: December 2010

Genome scale expression profiling is likely to yield improved methods for optimizing therapy for individual breast cancer patients. Microarray based predictors of response to chemotherapy have been described for ER-positive tumors, but supervised methods haven't yielded highly accurate predictors of response in triple-negative breast cancer (TNBC). The problem of deriving predictive or prognostic classifiers is not trivial, and care must be taken to avoid pitfalls such as data set-specific noise and overfitting.To overcome these problems, we developed a two-stage unsupervised method to derive consistent metagenes from multiple data sets. In the first stage, we used principal component analysis to identify aspects of gene expression profiles that are consistently observed in multiple starting data sets. We then identified a subset of genes that best captured the information derived from these components. In the second stage, we factorized this subset of genes and derived metagenes applicable to any external data set. With this method, overfitting is eliminated by virtue of the unsupervised approach, and data set-specific noise is reduced by selecting only components presented in multiple data sets.We first applied our method to four TNBC data sets not obtained in a neoadjuvant setting and derived four metagenes. We then applied these metagenes to another four independent data sets annotated with response to various neoadjuvant therapies in order to assess their predictive performances. Two of the metagenes were associated with response: one predicted pathological complete response (pCR) in the two TFAC cohorts but not in the other two cohorts (Fig. a); the other was associated with pCR to TFAC, residual disease (RD) to epirubicin single agent, but not with response to FEC (Fig. b). Notably, these metagenes derived in an unsupervised manner were more strongly predictive than were classifiers derived using conventional supervised methods.As conclusion, we report and validate an unsupervised strategy as a robust and powerful alternative to supervised methods for deriving predictors for response to chemotherapy from microarray data. If confirmed in additional independent cohorts, these metagenes may lead to clinical tests to optimize therapy for individual TNBC patients.Figure 1. ROC curves indicate the association between two metagenes and agent-specific pathological complete response to neoadjuvant therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3163.
The Luminal B Marker NHERF1 Predicts Endocrine Resistance.
Authors: Rody A. , Karn T. , Solbach C. , Ruckhaeberle E. , Hanker L. , Mueller V. , Schmidt M. , Gaetje R. , Holtrich U. , Kaufmann M.
Published: December 2010

Background:Tumors of the luminal B subtype of ER postive breast cancer are characterized by high proliferation as compared to the luminal A subtype. The luminal B have a worse prognosis. We aimed to identify genes specifically expressed in the luminal B subtype of breast cancers and analyze the prognostic impact of these genes and their relationship to endocrine therapy.Methods:121 genes overexpressed in LumB tumors were identified in a test set of 171 Breast cancer samples and reproducibly obtained in four independent validation datasets. The scaffold protein NHERF1 was analyzed in a large scale meta-analysis of microarray datasets encompassing n=3030 breast cancer samples.Results:NHERF1 is an ER regulated gene located on chromosome 17 coding for a scaffold protein involved in growth factor signal transduction. NHERF1 expression among ER positive tumors is associated with larger tumor size, higher histolocigal grading, and HER2 expression. A prognostic value of NHERF1 was observed among ER positive tumors (univariate HR 1.49, 95% CI 1.23-1.80, P<0.001) but not among ER negative samples. NHERF1 remained significant in multivariate analysis (HR 1.37, 95% CI 1.05-1.79, P=0.020) and is not a surrogate marker for high proliferation. A benefit of endocrine treatment seems to be restricted to NHERF1 negative tumors.Conclusions:Markers like NHERF1 specific for the luminal B subtype of breast cancer correlate with poor prognosis and seem to be predictive for endocrine treatment response.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3164.
High Resolution Comparative Genomic Hybridization (CGH) Indicates That Genomic Profiles Are Very Heterogeneous for HER2 and TOP2A in FISH-Amplified Human Breast Cancer Specimens.
Authors: McArthur H. , Brogi E. , Patil S. , Wigler M. , Norton L. , Hicks J. , Hudis C.
Published: December 2010

Background: Clinical studies relating TOP2A and HER2 status by FISH and response to specific drug therapies, including anthracyclines, have yielded inconsistent results. The discordance across studies appears to reflect, in part, the well-documented limitations of FISH testing, including the use of large FISH probes to evaluate the relatively small genes of interest. However, it is also likely that the dichotomization of gene status into “amplified” or “non-amplified” categories inadequately describes complex changes across the gene areas of interest. Identification and delineation of specific amplification-deletion patterns in this region may be of significant clinical interest and could provide deeper insight into mechanisms of drug resistance. Consequently, we selected a subset of HER2 and TOP2A amplified specimens (by FISH) from an ongoing study of CGH, and examined individual amplification-deletion patterns by CGH.Methods: 471 consecutive, archived, formalin-fixed paraffin-embedded, >1cm, HER2 2+ and 3+ by immunohistochemistry primary breast cancer specimens diagnosed between January 2000 and 2006 were selected. HER2 status was evaluated by FISH at MSKCC and CGH at Cold Spring Harbor Laboratory (CSHL) for all 471 specimens in a double-blinded experiment and clinical correlates evaluated. TOP2A status was also evaluated by FISH at MSKCC and CGH at CSHL in the subset of 64 HER2 2+ by IHC, HER2 FISH-positive specimens. Individual genome profiles by CGH were examined for 20 HER2 and TOP2A FISH co-amplified specimens.Results: 45 specimens had results available for analysis, 20 of which were amplified for both HER2 and TOP2A by FISH. By CGH: 6/20 (30%) were not amplified for HER2 or TOP2A; 4/20 (20%) had homogeneous amplification of HER2 and TOP2A; 8/20 (40%) had HER2 > TOP2A amplification; and 2/20 (10%) had TOP2A > HER2 amplification. Individual genome profiles around HER and TOP2A were examined by CGH. Marked heterogeneity was observed across specimens.Conclusions: CGH at the genome level suggests that current dichotomization strategies for classifying HER2 and TOP2A status appear to incompletely describe and represent the heterogeneity of genomic lability in these regions. The dichotomization of gene amplification results by FISH may contribute to the discordance demonstrated in reported some clinical studies. Studies exploring clinical correlates are underway.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3165.
Evolution of Genomic Diversity in the Breast Cancer Cell Line MCF-7.
Authors: den Hollander P. , Osadchey I. , Hampton O. , Hampton O. , Coarfa C. , Milosavljevic A. , Milosavljevic A. , Lee A.
Published: December 2010

Background: Tumors arise from an accumulation of genetic changes via a multi-step process. Genetic rearrangements can potentially lead to fusions with consequent deregulation of a gene, or the generation of new genes (through the fusion of two genes) that maybe functionally important in tumor development and progression. Our knowledge of rearrangements in solid tumors is limited and it is likely that solid tumors are defined by fusion genes that are not yet discovered. We recently reported 157 genomic breakpoints that were identified in a BAC library generated from MCF-7 cells (termed MCF-7/BAC), and could be confirmed by PCR across breakpoint joins as likely somatic mutations. To gain insight into the heterogeneity of genomic breakpoints in breast cancer cell lines, we studied these 157 validated breakpoints in a two common MCF-7 sublines.Methods: DNA was isolated from MCF-7/ATCC and MCF-7/B and genomic breakpoints were amplified by PCR and visualized by gel electrophoresis. Breakpoints were called present or absent based upon amplification of a product at the predicted size. By mapping the breakpoints to specific stages in the evolution of MCF-7 we reconstructed the sequence of chromosomal rearrangement events in the cancer genomesResults: MCF-7/B harbored only 91 of the breakpoints from the original 157 identified from MCF-7/BAC, and MCF-7/ATCC only shared 40. MCF-7 had 3 breakpoints that were found in MCF-7/BAC but not in MCF-7/B. Thus, of the original 157 breakpoints, 37 (23.6%) were shared all three sublines. MCF-7/ATCC contained the least breakpoints, which might indicate that MCF-7/ATCC is ancestral to MCF-7/B and MCF-7/BAC. Consistent with this, of the 157 genomic breakpoints in MCF-7/BAC, 50.3% contained genes, while this was enriched in the MCF-7/ATCC to 72.5%. Interestingly, two breakpoints involving genes in double strand break repair are retained in all three sublines, indicating that these breakpoints might originate from an ancestral cell line, and that deficient double strand break repair might be the underlying cause of genomic instability in these cell lines.Discussion: These preliminary results imply that MCF-7/ATCC maybe ancestral to other MCF-7 sublines. We are now expanding our data set with six other MCF-7 sublines to reconstruct the phylogeny of this breast cancer cell lines. With this information we may get a better understanding of the evolution and heterogeneity of genomic instability and rearrangements in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3171.
Childbirth Following Breast Cancer: An Evaluation of the 'Healthy Mothers' Excess Mortality Risk.
Authors: Hartman M. , Hartman M. , Lim G. , Czene K. , Bhalla V. , Chow K. , Yap K. , Chia K. , Chia K. , Verkooijen H. , Verkooijen H. , Verkooijen H.
Published: December 2010

Background: The favorable outcome of mothers giving birth after breast cancer compared to those who do not, has been attributed to a healthy mother effect. Mainly former breast cancer patients with excellent health status and outlook proceed to having children after their disease. This study evaluates the excess mortality risk among these 'healthy mothers' and factors modifying this risk.Methods: By linking the Singapore Birth Registry (319'437 individuals) and Swedish Multi-Generation Cohort (11 million women) with the respective national cancer registries we identified all 509 women who gave birth >12 months after a breast cancer diagnosis. We calculated excess absolute mortality risks and Standardized Mortality Ratios (SMRs). We used Poisson regression analysis to calculate relative risks of death by country, period of follow up and interval between cancer diagnosis and subsequent birth.Results: The overall 15-year cumulative mortality was 18.2% (95% CI: 13.3 – 20.7%) which is substantially higher than that of the background population (SMR 13.3, 95%CI: 10.4-16.9). Excess mortality risks remained constant over follow up time and were comparable between Sweden and Singapore. Mortality risks decreased significantly with increasing interval between diagnosis and subsequent birth.Conclusion: Despite being 'healthy mothers', women who give birth after a diagnosis of breast cancer have a substantially higher risk of death than the background population. This information may be useful in a woman's decision whether to have a(another) child after breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3173.
Bone Health in Postmenopausal Women Treated with Adjuvant Aromatase Inhibitors for Early Breast Cancer: 12 Months Follow-Up.
Authors: Nogués X. , Servitja S. , Peña M. , Martinez M. , Nadal R. , Garrigos L. , Diez Perez A. , Albanell J. , Tusquets I.
Published: December 2010

Background:The use of aromatase inhibitors (AI) in the adjuvant setting for postmenopausal women with receptor-positive breast cancer is well established. The AI treatment has been associated with decline in bone mineral density (BMD), and increased bone turnover markers. We designed a prospective study to evaluate the effect of oral bisphosphonates (BP) on vertebral and non vertebral fractures (VF), bone turnover markers (bTM), BMD, and quality of life (QoL) in postmenopausal women receiving adjuvant AI for hormone receptor positive early breast cancer.Material and methods:Postmenopausal, receptor-positive breast cancer women treated with AI as initial therapy [Letrozol (LET)] or after 2 or 5 years of tamoxifen [switched to exemestane (EXE)] were included. Patients were stratified by lumbar spine, femoral neck and total hip BMD to receive open-label treatment with BP, alendronate or risedronate, (if osteoporosis [T-score<-2,5] or osteopenia [T-score<-2.0] and 1 major risk factor, defined as group of high risk of fracture) or calcium and vitamin D supplementation (if normal BMD or T-score>-2.0 and no major risk factor). Levels of 25-OH vitamin D, N-telopeptide (NTx), bone alkaline phosphatase (bALP) and osteocalcin (OC) were measured at baseline, 3, 12 and 24 moths, and BMD and thoracic and lumbar spine X-rays were assesed at baseline, 1 and 2 years. QoL was evaluated by ECOS-16 questionnaire and a visual analogue scale used to evaluate arthralgia. Results:from 03/2006 to 03/2009, 261 patients have been included and 117 have completed >12 month follow up. All the bTM increased after 12 months of AI treatment and a significant decrease on BMD was also observed (Table 1). This effect is reverted with oral BP treatment, maintaining the BMD level and reducing the bTM. With the AI introduction QoL of patients became worse, and the BP treatment did not improve neither arthralgia nor bone pain. Osteoporotic fractures were recorded at baseline in 12.3% of women (4.2% VF and 7.7% non VF).Conclusions:AI treatment increased bone turnover, decreased BMD and QoL. Of note, BP use decreased bone turnover, has neutral effect in BMD and does not improve QoL. This study suggests that the current recommendation of the use of BP should be revisited to consider including all postmenopausal women treated with adjuvant AI to avoid increased bone turnover and bone mass decrease.Table 1. BMD at baseline, 12 months and changes  Without BP (n=76)  With BP (n=41)  BMD value (g/cm2)AIBasal12 month% change / pBasal12 month% change / pFemoral NeckLET0.7650.751- 1.96 / 0.020.6480.657+ 1.61 / NS EXE0.7560.749- 0.6 / NS0.6260.644+ 2.63 / 0.013Lumbar spineLET0.9900.969- 2.36 / 0.00010.8150.813- 0.17 / NS EXE0.9570.940- 1.57 / 0.0430.8110.814+ 0.86 / NS 

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3175.
Beneficial Effect of Tamoxifen for Women with DCIS: Long-Term Results from the UK/ANZ DCIS Trial in Women with Locally Excised DCIS.
Authors: Cuzick J. , Sestak I. , Pinder S. , Ellis I. , Hackshaw A. , Bundred N. , Forbes J. , Bishop H. , Fentiman I. , George W.
Published: December 2010

Background: Initial results of the UK/ANZ DCIS trial found that adjuvant radiotherapy reduced the recurrence of ipsilateral invasive breast cancer (BC) and ductal carcinoma in situ (DCIS) by 55% and 64% respectively, but the effect of tamoxifen was smaller (Lancet 2003, 362: 95-102). Here, we report an updated analysis of this trial.Methods: 1701 women with completely locally excised DCIS were enrolled in a randomised 2x2 trial of radiotherapy, tamoxifen or both. Elective decision to withhold or provide one of the treatments was also permitted and long-term follow-up was performed by treating hospitals. Seven patients were excluded due to protocol violations leaving 1694 patients available for analysis.Results: After a median follow-up of 12.7 years, a total of 376 breast cancers were diagnosed; 162 invasive (122 ipsilateral; 39 contralateral) and 197 DCIS (173 ipsilateral; 17 contralateral). Radiotherapy continued to reduce the incidence of ipsilateral invasive disease (HR=0.32 (0.19-0.56), P<0.0001) and ipsilateral DCIS (HR=0.38 (0.22-0.63), P<0.0001), but had no significant effect on contralateral breast cancer (HR=0.84 (0.45-1.58)). Tamoxifen significantly reduced all recurrences by 29% (HR=0.71 (0.58-0.88), P=0.002) (Figure 1). This consisted of a 30% (HR=0.70 (0.51-0.86)) reduction in recurrent ipsilateral DCIS, a small 5% (HR=0.95 (0.66-1.38)) reduction in ipsilateral invasive disease, and a 56% (HR=0.44 (0.25-0.77)) reduction in contralateral tumours. Overall, women randomised to radiotherapy and not receiving tamoxifen had a 59% reduction in recurrence (HR=0.41 (0.30-0.57), P<0.0001), whereas those randomised to tamoxifen and not receiving radiotherapy had an overall reduction of 29% (HR=0.71 (0.57-0.87), P=0.001).Conclusion: This updated analysis confirms the long-term benefit of radiotherapy in women with DCIS treated by complete local excision and also established a benefit for tamoxifen. Ongoing work will examine in detail which subsets of tumours (e.g. high grade, oestrogen receptor positive) will benefit most from these treatments.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 34.
Elevation of Serum NT-Pro BNP Correlates with Asymptomatic Cardiac Dysfunction Induced by Doxorubicin.
Authors: Kittiwarawut A. , Vorasatekarnkit Y. , Tanasanwimon S. , Manusnayakorn S. , Sriuranpong V.
Published: December 2010

Background: Cardiac dysfunction is one of the major limitations of anthracycline treatment in cancer patients. Current reliable investigation to detect early cardiac damage from anthracycline is not available. There are several serum cardiac markers useful in other types of cardiomyopathy including NT-pro BNP, Troponin-T, and CK-MB. We chose to investigate potential application of these serum biomarkers in the cancer patients receiving anthracycline.Patients and methods: We prospectively collected data from a cohort of 52 female breast cancer patients who receiving doxorubicin and cyclophosphamide (AC) regimen every 3 weeks for 4 cycles for preoperative or postoperative adjuvant treatment. Cardiac function evaluations by echocardiography were done at baseline and post 4th cycle chemotherapy. Patients' blood samplings were serially measured for cardiac biomarkers at baseline, after 1st cycle, and after 4th cycle chemotherapy.Results: All patients were in good performance status with ECOG scale 0-1.The mean cumulative dose of doxorubicin in this study was 236.69 mg/m2. There was no symptomatic heart failure detected during study period. However, there were significant asymptomatic reductions of left ventricular ejection fraction (LVEF) from 70.73 ± 5.97% at baseline to 66.96 ± 5.48% (p<0.001). The severities of LVEF declination were 17.4% in grade1 and 3.8% in grade 2 by clinical toxicity criteria of NCI version 2. We found significant elevation of serum NT-proBNP (p = 0.044) following the first dose of chemotherapy when compared the measurements in patients who had reduction of LVEF to those with normal LVEF. Measurement of serum Troponin-T and CK-MB showed no significant correlation with changes in LVEF. Generalized-estimating-equations correlation analysis demonstrated that the reduction of fractional shortening (FS) was significantly associated with the elevation NT-pro BNP (r = -0.016, p = 0.014). At cutoff level 45 pg/mL, serum NT-proBNP had 100% sensitivity and 66.67% specificity for the detection of cardiotoxicity in patients under 50 year-old.Conclusion: Asymptomatic reductions in cardiac function are common in patients treated with doxorubicin. NT-pro BNP may serve as a potential convenient serum biomarker for early detection of cardiotoxicity induced by anthracycline.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4003.