bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Nucleotide Polymorphisms of CYP19A1, rs10459592 and rs4775936 Predict Clinical Benefit of Letrozole in Patients with Metastatic Breast Cancer.
Authors: Park I. , Ro J. , Lee K. , Kim S. , Lee Y.
Published: December 2010

BackgroundThe CYP19A1 gene encodes the enzyme aromatase, which is involved in the production of estrogen in various tissues. We evaluated the efficacy of an aromatase inhibitor, letrozole in metastatic breast cancer patients according to DNA polymorphisms of the aromatase gene CYP19A1.Patients and methodsPatients (n=113) with hormone receptor positive metastatic breast cancer were treated with letrozole. Monthly GnRH agonist was added in premenopausal patients. DNA was isolated from peripheral blood samples and was genotyped for 50 polymorphisms of CYP19A1 by iPLEX Gold assay based on matrix-associated laser desorptional ionization, time-of-flight mass spectrometry.ResultsAmong 50 examined single nucleotide polymorphisms (SNP), 10 SNPs were revealed as monomorphic for Korean population. After trimming of the data using HWE test, rs10459592 (T/G) and rs4775936 (C/T), which were located in exon I.6 of CYP19A, were significantly associated in an over-dominant model with higher disease control rate (CR+PR+SD≥ 6months) (HR=2.82 [95% CI 1.21-6.59], p=0.0149; HR=2.62 [95% CI 1.12-6.10], p=0.0238, respectively). In linkage disequilibrium analysis, these were strongly linked (D'=1.0, r2=0.936), and subsequent haplotype analysis showed a statistical significance with regard to higher disease control rate (HR=2.61 [95% CI 1.12-6.1], p=0.0238). Median time to progression (TTP) with a median follow-up of 85.7 months (range 9.7-107.4 months) was improved in patients having over-dominant form of rs10459592 without statistical significance (median 7.74 months [95% CI 6.48-9.00] vs. median 12.07 months [95% CI 8.87-15.26], p=0.147). In multivariate analysis, rs10459592 (HR=2.81 [95% CI 1.12-7.06], p=0.027) was an independent factor affecting disease control rate besides age (HR=1.06 [95% CI 1.01-1.12], p=0.019) and HER2 positivity (HR=0.25 [95% CI 1.12-7.06], p=0.027).ConclusionIn patients with hormone responsive metastatic breast cancer treated with aromatase inhibitor, letrozole, the presence of SNPs in the exon I.6 of CYP19A1, rs10459592 and rs4775936 were significantly associated with improved treatment efficacy. Although further study is needed, our data present possible candidate markers for the letrozole treatment in metastatic breast cancer. (NCC grant number 0910320-1)

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2117.
Microtubule Associated Protein-Tau (MAPT) Is Influenced by ER: ICI182,780, a Selective ER Inhibitor, Down-Regulates MAPT Expression and Reverses Resistance to Taxanes in MAPT- and ER-Positive Breast Cancer Cells.
Authors: Ikeda H. , Taira N. , Hara F. , Nogami T. , Shien T. , Doihara H.
Published: December 2010

Background: Taxanes are important drugs in treatment of breast cancer. These drugs bind to tubulin and suppress spindle microtubule dynamics, which leads to cell cycle arrest in the G2/M phase followed by apoptosis. However, resistance to taxane therapy prevents some patients from benefiting from these drugs. Several mechanisms of taxane resistance have been described, including the involvement of microtubule-associated protein-tau (MAPT). MAPT binds to the same pocket as taxanes in microtubules and obstructs the function of the drug. Estrogen receptors (ER) are transcriptional factors that play an important role in the development and progression of breast cancer. However, the relationship between ER and MAPT in breast cancer is not entirely clear. In this study we examined the correlation between MAPT expression and the sensitivity of human breast cancer cells to taxanes, and the relationship between ER and MAPT at the protein level in these cells. We also examined combination therapy with hormone drugs and taxanes.Methods: The correlation between MAPT expression and sensitivity to taxanes was examined in 12 human breast cancer cell lines using real time PCR, western blotting analysis and an MTS assay. Following small interfering RNA (siRNA) knockdown of MAPT expression, the alteration of cellular sensitivity to taxanes was examined by MTS assay, flow cytometry and immunofluorescence. To examine the relationship between ER and MAPT, ER expression was knocked down with siRNA or stimulated with 17-β-estradiol in MAPT- and ER-positive cell lines (MCF-7 and ZR75.1, respectively). The cells were also treated with hormone drugs (tamoxifen and ICI182,780) and changes in MAPT protein expression were examined.Results: Six cell lines showed high MAPT mRNA expression and four showed high MAPT protein expression; that is, expression at the mRNA level did not always correlate with that at the protein level. MAPT mRNA expression did not correlate with taxane resistance, but expression of MAPT protein isoforms under 70 kDa correlated with taxane resistance. Downregulation of MAPT increased sensitivity to taxanes. MAPT protein expression was decreased by ER knockdown and increased by 17-β-estradiol stimulation. The MAPT protein level was also increased by tamoxifen, but decreased by ICI182,780. Combination treatment of taxanes with ICI182,780 showed a strong synergistic effect, but similar treatment with tamoxifen had an antagonistic effect in both cell lines.Conclusions: Expression of MAPT protein isoforms under 70 kDa correlates with taxane resistance in breast cancer cells. MAPT expression is influenced by ER in breast cancer and ICI182,780, a selective ER inhibitor, can reverse the resistance to taxanes in both MAPT- and ER-positive breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2118.
Aberrations of HER2 and TOP2A Genes in Breast Cancer.
Authors: Nielsen K. , Müller S. , Møller S. , Schønau A. , Balslev E. , Knoop A. , Ejlertsen B.
Published: December 2010

Copy number changes in TOP2A are frequently observed in HER2 amplified breast cancers, and amplification of a whole amplicon containing both genes has been suggested as the underling mechanism. Here, we describe copy number changes of HER2 and TOP2A in two cell lines; one characterized by having amplification of both genes and the other by having amplification of HER2 and deletion of TOP2A. The characteristics are compared to findings in patients with invasive breast cancer.Material and methods: Fluorescence in situ hybridization (FISH) with HER2, TOP2A and centromere 17 (CEN-17) probes was performed on metaphases, interphases and cut sections from breast cancer cell lines. Paired HER2/CEN-17 and TOP2A/CEN-17 data was available from 649 patients from a previously published biomarker study (Knoop et al. J Clin Oncol 2005;23:7483-90).Results: The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. HER2 and TOP2A aberrations seem to be due to different mechanisms of amplifications. Most gene signals are translocated to abnormal marker chromosomes. HER2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher HER2 ratio. This observation was confirmed by patient FISH data: 373 tumors (57%) had normal status for both genes. Among the 276 abnormal tumors, 67% had different HER2 and TOP2A status. Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while deletion of both genes was observed in 5% of the cases (2% of all patients). The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for HER2 in the amplified tumors (P<0.001; χ2-test). In the HER2 assay 398 (61%) of the 649 tumors had more than 4 gene signals per nucleus while only 96 (15%) of the tumors had more than 4 TOP2A signals per nucleus.Conclusion: In the majority of breast cancer patients, simultaneous aberration of HER2 and TOP2A is not explained by simple co-amplification. Amplification of the two genes may be caused by different mechanisms, leading to higher level of amplification for HER2 compared to TOP2A.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2119.
Crosstalk between Estrogen Receptor alpha 36, a Dominant Extra-Nuclear ER, and HER2 Promotes Tumor Cell Survival in Breast Cancer.
Authors: Jiang L. , Huang J.
Published: December 2010

The ER-α36 protein is a 36kDa novel splice variant of the full length 66kDa ER-α. ER-α36 lacks both transcriptional activation domains of ER-α66 but retains the DNA-binding domain and partial dimerization and ligand-binding domains. It has been shown that ER-α36 could trigger membrane-initiated signaling in ER-α36 over-expressing 293 cells, suggesting that ER-α36 might have functions linked with activation of intracellular signaling.To further explore the mechanism(s) of ER-α36 activation of intracellular signaling, in particular the possible relationship between ER-α36 and HER2 in breast cancer, we performed a pilot study. First, we examined the correlation between ER-α36 and HER2 by immunohistochemistry (IHC) using a specific anti-ER-α36 antibody designed against a unique epitope located in the C-tail of ER-a36 which is not present in ER-α66. The results showed that the expression of ER-α36 was highly associated with HER2 in a cohort of 249 breast tumors (P<0.05). Then, we examined whether ER-α36 physically interacts with HER2 in breast cancer cell lines using co-immunoprecipitation (Co-IP) and Western blotting (WB) experiments. We found that ER-α36 interacts with HER2 in both MCF7/HER2 and BT474 cell lines. Second, we explored whether ER-α36 influences the activation of HER2 and its downstream signaling by knocking down ER-α36 expression in both MCF7/HER2 and BT474 cell lines. We found that knockdown of ER-α36 by siRNA abrogated the activation of P-HER2, as well as Akt and MAPK, in both MCF7/HER2 and BT474 cells. Finally, we asked the key question whether ER-α36 was associated with cell proliferation/survival. Strikingly, we found that when both MCF7/HER2 and BT474 cells were deprived of ER-α36 by siRNA, they died quickly, whereas control cells grew normally.In conclusion, our preliminary data suggests that there might be a crosstalk between ER-α36 and HER2 signaling, and that this crosstalk might promote tumor cell survival through HER2/Akt and/or HER2/MAPK signaling pathways in breast cancer. Thus, ER-α36 may be a valuable biomarker and a novel therapeutic target in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2121.
Development of a Novel GREB1 Monoclonal Antibody and Applications in Breast Cancer Research.
Authors: Hnatyszyn H. , Liu M. , Hilger A. , Thomas D. , Rae J. , Lippman M.
Published: December 2010

It has been well established that GREB1 mRNA expression is induced by estrogen in breast cancers and that GREB1 is critically involved in the estrogen-induced growth of breast cancer. To date, all studies of GREB1 have focused on mRNA levels using PCR and array technologies with very little known about GREB1 protein expression in normal and breast cancer cells. The lack of a specific antibody to GREB1 has inhibited protein-based investigations in target cells. Our collaborative research group has generated a novel monoclonal GREB1 antibody (GREB1ab) for research use in Western blotting as well as immunohistochemical (IHC) applications.Our team created a hybridoma expressing a murine monoclonal antibody against a 119 amino acid peptide specific to human GREB1. By Western Blot Analysis, GREB1ab detects a 216 kD protein corresponding to GREB1a in ER+ breast cancer cells expressing GREB1 as well as cells transfected with a GREB1a expression plasmid. GREB1ab specificity was verified using ICI 182,780, an estrogen receptor antagonist to prevent GREB1 induction, as well as silencing siRNA targeting GREB1 mRNA expression. GREB1 protein expression was reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared with that of estrogen treatment. There was no detectable GREB1 protein expression when GREB1 mRNA is silenced by siRNA at 48 hours. In a time course study, inhibition of GREB1 protein occurred as early as 24 hours and lasted up to 72 hours. Thus, by Western Blot Analysis, the monoclonal GREB1ab is specific for GREB1 and can be employed to detect changes in GREB1 expression in breast cancer cell lines under various experimental conditions.GREB1ab was validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). IHC staining with GREB1ab revealed GREB1 was strongly expressed in the ERα-positive breast cancer cell line, MCF-7, with weak GREB1 expression in ERα-negative MDA-231 cells. A panel of breast cancer cell lines was screened for endogenous expression of GREB1 protein in a TMA format using IHC. As expected, ER-positive cell lines (n=5) were observed to express GREB1 while ER-negative cell lines (n=11) did not express detectable levels. Using breast cancer tissue whole sections, IHC with the GREB1ab indicated protein expression in ERα positive breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα negative breast cancer tissue.The monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution and function of GREB1 in normal breast and breast cancer tissues.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2126.
Discordant HER2 Total and HER2 Homodimer Levels by HERmark Analysis in Matched Primary and Metastatic Breast Cancer FFPE Specimens.
Authors: Leitzel K. , Conte P. , Guarneri V. , Barbieri E. , Huang W. , Ali S. , Ali S. , Haddad M. , Sperinde J. , Lie Y. , Weidler J. , Bates M. , Lipton A.
Published: December 2010

Background: HER-2/neu is over-expressed in approximately 25% of primary invasive breast cancers and selection of patients for consideration of trastuzumab is a critical step in defining the treatment plan. We have previously reported that quantitative measurements of HER2 protein expression (H2T) and HER2 homodimers (H2D) using the HERmark assay identifies sub-populations of “HER2-positive” patients (by IHC and/or FISH) that have different clinical outcomes on trastuzumab (Leitzel, ASCO 2008; Lipton, SABCS 2008). Previous studies report up to a 20 % discordance in HER2 status using conventional IHC or FISH analysis between the primary and metastatic breast tumors. Here we correlate HER2 total and homodimer levels in matched primary and metastatic tissue from the same patient.Methods: 27 patients had matched primary and metastatic FFPE (formalin-fixed, paraffin-embedded) specimens tested in the HERmark assay to quantitate and compare their H2T and H2D expression levels.Results: FFPE tissue was available from 27 primary breast cancers and metachronous metastatic sites. Metastatic lesions included 7 skin, 5 lymph node, 3 bone, 3 pleura, 2 brain, 2 chest wall, and 5 other soft tissue lesions. The median elapsed time between matched primary and metastatic sites was 71 mo. (range 9-137 mo). During the time period between the primary specimen harvest and the metastatic biopsy, 6 patients were treated with chemotherapy alone, 10 received hormonal therapy without trastuzumab, 3 patients received trastuzumab, and 3 received no treatment. Treatment was not known for 5 patients. For the whole population, there was a weak to moderate positive correlation between primary and metastatic cancers with H2T (r2=0.36, p<0.001) and for H2D (r2=0.27, p<0.006). Using the optimized time to progression (TTP) positional scanning cutpoints for H2T and H2D defined in our previous reports, 4/20 patients (20%) converted from low to high, and 1/7 (14%) converted from high to low H2T. Using the H2D cutpoint, 7/15 patients (47%) converted from low to high, and 3/12 (25%) converted from high to low H2D. Overall discordance between primary and metastatic sites was 19% for H2T, and 37% for H2D.Conclusions: HERmark analysis of matching primary and metastatic breast cancers revealed 19% discordance for H2T, and 37% for H2D. The most frequent conversion was from low HER2 in the primary tissue to high HER2 in the metastatic site.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2131.
Analysis of Topoisomerase IIa and HER2 Status in 126 Patients from the US Oncology 9735 Trial of Adjuvant Chemotherapy with Docetaxel/Cyclophosphamide (TC) vs Doxorubicin/Cyclophosphamide (AC) in Early Breast Cancer.
Authors: Aitelli C. , Aitelli C. , Saad R. , Mackey D. , Asmar L. , Jones S. , Jones S. , Jones S. , Pippen J. , Pippen J. , Pippen J.
Published: December 2010

Background: HER2/neu (HER2) positive breast cancers are associated with worse survival, resistance to cyclophosphamide/methotrexate/fluorouracil, and hypothesized to be sensitive to anthracyclines. It has been postulated that only a subset of HER2 positive breast cancers are sensitive to anthracyclines. Investigators have been working on how to identify this subset of patients, thus sparing patients from anthracyclines that have potential cardiac and bone marrow toxicities. Overexpression of the topoisomerase IIa gene (TOP2A) is the putative biomarker for sensitivity to anthracyclines. Testing tumors for overexpression of this gene may help identify patients who will not benefit from anthracylines. TOP2A is located in the same amplicon as the HER2 gene and can be assessed for overexpression by a FISH assay.Materials and Methods: Paraffin tissue blocks were obtained in 126 patients entered on US Oncology trial 9735 which recruited patients between 1997 and 2000. Data on HER2 status and outcome was previously reported (JCO 27:1177-1183, 2009). TOP2A status was then assessed by fluorescent in situ hybridization (FISH) using the LSI TOP2A Spectrum Orange Probe (Vysis). A total of 20 cells were counted and a gene ratio of greater than 2.0 was considered positive. Clinical data and outcome were available for statistical analysis.Results: We found that none of the 97 HER2 negative cases demonstrated overexpression of TOP2A by FISH analysis. TOP2A was overexpressed in 43% of the 29 HER2 positive cases. An analysis of outcome will be presented at the meeting, although the number of cases limits this observation.Discussion: Our study supports the observation that TOP2A is not overexpressed in HER2 negative disease and is only observed in a subset of cancers overexpressing HER2. Clinical correlation of outcome will be necessary to confirm whether or not TOP2A is a reliable biomarker of sensitivity to anthracyclines. The ongoing US Oncology trial of TC vs. TAC in HER2 negative breast cancer (USOR Trial 06-090) should provide that evidence.Supported in part by a grant from sanofi-aventis.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2134.
Biomarkers of Oxidative Stress in Breast Cancer.
Authors: Jassal R. , Talwar D. , Mcmillan D. , Tovey S.
Published: December 2010

INTRODUCTIONDespite significant improvements in cancer diagnosis and treatment, approximately one-quarter of breast cancer patients will die of their disease. Oxidative stress has been implicated as a contributory factor in the development of breast cancer. Reactive oxygen species can initiate lipid peroxidation, resulting in generation of reactive secondary products which can induce oxidative DNA damage and mutations in the p53 tumour suppressor gene, which is one of the most frequent mutations associated with breast cancer development. Oxidative stress has also been associated with activation of transcription factors and tamoxifen resistance. The aim of the present study was to identify the levels of lipid peroxdation products; malondialdehyde and 8-iso-Prostaglandin F2α (8-iso-PGF2α), in patients with primary operable breast cancer, and to correlate these levels with pathological factors of disease including tumour grade, ER and HER2 status and also Nottingham prognostic index (NPI) status.METHODS:89 patients with primary operable breast cancer and 10 healthy subjects were studied. Plasma levels of MDA (total and free) and 8-iso-PGF2α were measured.RESULTS:The majority of patients were over 50 years (87%), had grade II/III tumours (68%), were axillary lymph node positive (41%) and had ER-positive tumours (82%). Compared to the control, patients had higher levels of total MDA (p<0.001), free MDA (p<0.001), and 8-iso-PGF2α (p<0.001). Both free (p<0.05) and total MDA (p=0.001) were significantly correlated with increasing tumour grade. Furthermore, total MDA levels were also associated with increased tumour size (p<0.05) and ER-negative tumours (p<0.05). Taken together, both total (p<0.001) and free (p <0.05) MDA were significantly associated with higher NPI categories. 8-iso-PGF2α showed only a correlation with increasing age (p<0.05)CONCLUSION:The present study shows that patients with breast cancer have evidence of increased levels of oxidative stress. Furthermore, MDA concentration was significantly associated with conventional markers of poor prognosis. This data suggests oxidative stress may be involved in tumour progression to a more aggressive phenotype. Thus MDA maybe a useful marker of tumour associated oxidative damage and may provide a promising marker for risk prediction and targets for preventative measures in breast cancer patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2141.
Decreased Phosphorylation of RSK May Predict Response for Neoadjuvant Chemotherapy.
Authors: Yi J. , Yu J. , Yu J. , Ko E. , Ko E. , Han W. , Han W. , Noh D. , Noh D.
Published: December 2010

Neo-adjuvant chemotherapy is an essential part of the management of locally advanced breast cancer. The resistance in breast cancer towards chemotherapeutic agents makes chemotherapy an incommodious problem. Current clinical factors cannot predict therapy response sufficiently in the treated patients. Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. The resistance mechanisms responsible for doxorubicin-resistance of breast cancer have been the subject of numerous investigations. Identification of proteins that are associated with doxorubicin resistance is a first step toward better response prediction and tailored treatment of patients.In this study, we compared the expression of core proteins and their phosphorylated forms of several canonical pathways (MAPK signaling, Apoptosis, PI3K/Akt Signaling, Cell Cycle/Checkpoint and TGF-β/Smad signaling) in breast cancer tissues which were divided into two classes based on the type of response to doxorubicin therapy by immunoblotting. As a result, we found that the phosphorylated form of ribosomal protein S6 kinase (RSK) was low in doxorubicin-resistant breast cancer tissues compared with doxorubicin-sensitive breast cancer tissues.We also investigated RSK's phosphorylation rate and doxorubicin sensitivity of various breast cancer cell lines including MCF-7, MCF-7s (sphere form), MDA-MB-231, MDA-MB-231s, MDA-MB-436, MDA-MB-453, MDA-MB-468, ZR75-1, BT474, Hs578T, T47D and MCF-10A to determine the phosphorylation rate of RSK in relation to the drug sensitivity of the breast cancer cell lines. Expectedly, the phosphorylation of RSK was low in doxorubicin resistant breast cancer cell line such as MDA-MB-453, ZR75-1 and Hs578T. Additionally, MCF-7s and MDA-MB-231s showed decreased phosphorylation rates compared with MCF-7 and MDA-MB-231.In conclusion, decreased phosphorylation of RSK may predict response and further clinical studies should evaluate for molecular function of phosphorylated RSK during doxorubicin therapy. Furthermore, immunohistochemistry for the detection of RSK's phosphorylation rate in breast cancer tissues is being developed for the analysis of increased and independent group of patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2146.
Mouse Mammary Tumor Expressing MMTV-SmoM2 as a Tool To Study Lung Metastasis.
Authors: Moraes R. , Landua J. , Lewis M.
Published: December 2010

Background: The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, ductal dysplasias, and increase the proportion of mammosphere-forming cells, with reduction of stem cell pool, in virgin mice.Material and Methods: We isolated and established a tumor line from this mouse line (named TU505). To investigate the role of hedgehog signaling in mammary tumorigenesis and metastasis we transplanted TU505 fragments (1-2mm3) to a cleared fat pad of 3 week-old mice. After 10 days we removed the primary tumors and let the mice recover for another 3 weeks, which at that time the animals were sacrificed and the lungs removed under stereomicroscope. The lung metastases visible at naked eyes were snap frozen for RNA isolation, and the remaining lungs fixed in 4%PFA.Results: The TU505 is a tumor expressing MMTV-SmoM2, triple-negative (ER-, PR-, and HEB2-), hormone independent, highly metastatic to the lung, and with a robust expression of Ptch1, Ptch2, Smo, Gli2, and Gli3. The gene expression of primary tumor and metastasis are compared.Discussion: The hedgehog signaling networks has been associated with metastasis. The TU505 is a strong tool to study mammary gland tumorigenesis and metastasis.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2165.
Use of Bisphosphonates and Risk of Postmenopausal Breast Cancer.
Authors: Rennert G. , Pinchev M. , Rennert H.
Published: December 2010

Background Bisphosphonates are commonly used for the treatment of osteoporosis and for prevention and treatment of skeletal lesions due to malignancy. However the association between the use of bisphosphonates and the risk of developing breast cancer has not been reported.Methods The Breast Cancer in Northern Israel Study (BCINIS) is a population-based case-control study in northern Israel of breast cancer cases and age/clinic/ethnic-group matched controls. Use of bisphosphonates for at least 5 years was assessed in 4,575 postmenopausal cases and controls using a structured interview. It was further validated by data from prescription records among participants for whom they were available.Results The self-reported long-term use of bisphosphonates prior to diagnosis was associated with a significantly reduced relative risk of breast cancer (Odds Ratio=0.66, 95% CI: 0.47-0.93). This association remained significant in a pharmacy records based analysis after adjustment for age, fruit and vegetable consumption, sports activity, family history of breast cancer, ethnic group, BMI, use of calcium supplements, HRT use, number of pregnancies, months of breast feeding and age at first pregnancy (OR=0.71, 0.57-0.90). A significant dose response association between length of use of bisphosphonates and breast cancer risk was found. Breast tumors identified in bisphosphonates users were more often ER positive and less often poorly differentiated.Conclusions The use of bisphosphonates for more than 1 year was associated with a 29% relative reduction in the risk of postmenopausal breast cancer. Tumors developing under bisphosphonates treatment tended to have a favorable prognostic factors profile.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 27.
Transcriptomic Profiles and Circulating Tumor Cell Detection in Primary Breast Cancer.
Authors: Bidard F. , Reyal F. , Valet F. , Mathiot C. , Brain E. , André F. , Espié M. , Salmon R. , De Cremoux P. , Spyratos F. , Marty M. , Pierga J.
Published: December 2010

Background: We have previously demonstrated that circulating tumor cells (CTC) detection by the CellSearch system is strongly associated with metastatic outcome in non-metastatic breast cancer (BC) treated by neoadjuvant chemotherapy (NACT) [Pierga, Clin Cancer Res 2008]. Transcriptomic analysis of primary tumors may uncover molecular phenotypes associated with CTC detection.Methods: Both CTC detection in blood at diagnosis and transcriptomic analysis of the primary tumor have been performed prospectively in 58 non-metastatic BC patients (pts) treated by NACT in a phase II trial (REMAGUS02). We searched for an association between CTC detection and (i) intrinsic molecular subtypes, (ii) stemness signature, (iii) other published signatures, and (iv) expression of molecular markers involved in CTC detection. CTC-associated genes were also studied (v).Results: (i) CTC detection was not statistically associated with an intrinsic molecular subtype: 29% in basal-like (n=5/17pts), 33% in HER2+ (3/9pts), 13% in luminal A (2/13pts), 20% in luminal B (2/10pts) and 29% in normal-like (2/7pts) BC. (ii) No association was found between CTC detection and the stemness signature. (iv) CTC detection was also not related to the “stemness profile”; it was independent of EpCAM, CK8 and CK18 transcriptomic expression. Other results (iii, v) will be disclosed at the meeting.Conclusion: Our study is the first to compare tumor gene expression profiles and blood dissemination of cancer cells in early BC pts. In these pts, CTC detection by the CellSearch system does not depend on intrinsic molecular subtypes, contrary to a published report based on in vitro-grown cells lines [Sieuwerts, J Natl Cancer Inst 2009]. Being independent from molecular prognostic factors, CTC detection is likely to play a critical role in early BC management.Supported by PHRC AOM/2OO2/02117, Pfizer inc., Roche, sanofi-aventis.ISRCTN10059974

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3005.
Characterization of Metastatic Breast Cancer Patients with Non-Detectable Circulating Tumor Cells.
Authors: Mego M. , Mego M. , Mego M. , Dawood S. , De Giorgi U. , De Giorgi U. , Valero V. , Andreoupolou E. , Handy B. , Ueno N. , Reuben J. , Cristofanilli M.
Published: December 2010

Background: Circulating tumor cells (CTC) are independent predictor of progression free and overall survival in metastatic breast cancer patients, with superior prognosis for patients with CTC <5 per 7.5mL of peripheral blood. However, 30-50% of patients have non-detectable CTC. The aim of this study was to assess the prognostic factors in MBC patients with non-detectable CTC.Methods: This retrospective study included 271 MBC patients evaluated between January 2004 and December 2007. Median age of patients was 56 years (range: 23-82 years). CTCs were enumerated before patients started a new line of treatment using the CellSearchTM. Overall survival (OS) was calculated from the date of CTC measurement, estimated by the Kaplan-Meier product limit method, and compared between groups with the log-rank test.Results: Median CTC count was 2 (range: 0-1780) per 7.5 mL. CTCs were not detected in 99 (36.5%) patients, while 112 (41.3%) patients had CTC ≥ 5. Median OS for patients with 0 CTC; 1-4 CTC and ≥ 5 CTC was 29.3; 26.4; and 19.4 months (p = 0.04), respectively. Patients with brain metastasis have the highest probability of non-detectable CTC (71.4% vs. 33.6%; p = 0.001), while patients with bone metastasis are more likely to have CTC ≥ 1 (48.2% vs. 31.8%; p = 0.01). There was no association between other tumor characteristics (ER, PR, HER2, number and localization of tumor metastasis) and non-detectable CTC status. Patients with CTC = 0 have non-significantly better OS compared to patients with CTC ≥ 1 (29.3 vs. 23.3 months; p = 0.09) and have superior survival in all but one analyzed subgroups; only inflammatory breast cancer (IBC) patients with CTC = 0 have inferior OS compared with patients with CTC ≥ 1 (26 vs. 37 months; p = 0.67). In a subgroup of patients with non-detectable CTC, triple negative MBC has the poorest survival (median OS = 18.8 months), while hormone receptor positive MBC, without visceral metastases and non-inflammatory breast cancer has the best survival (median OS = 36.9 months). In multivariate analysis, hormone receptor status and line of therapy were only independent prognostic factors for OS in patients with non-detectable CTC (Table 1).Conclusion: Patients with non-detectable CTC before start of new line of therapy comprise a heterogeneous group of patients with substantially different prognosis. Triple negative and IBC patients represent poor prognosis subgroups. These data suggest heterogeneity of microscopic disease in advanced cancer and indicate the need to investigate combining additional detection technologies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3006.
Circulating Tumor Cells (CTCs) and Epithelial Mesenchymal Transition (EMT) in Breast Cancer: Describing the Heterogeneity of Microscopic Disease.
Authors: Mego M. , Mego M. , Mego M. , Mani S. , Li C. , Andreoupolou E. , Tin S. , Jackson S. , Cohen E. , Gao H. , Cristofanilli M. , Reuben J.
Published: December 2010

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer (BC) patients. CTC are readily detected by CellSearch System based on their expression of EpCAM. Epithelial-mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased chemotherapy resistance. Human mammary epithelial cells (HMEC) transformed by the EMT transcription factor TWIST1 and spiked into normal peripheral blood (PB) are not detected by EpCAM enrichment based conventional detection methods compared to non-transformed HMECs. We hypothesize that CTCs undergoing EMT and resultant loss of epithelial markers may escape detection by conventional detection methods. The aim of this study was to detect CTCs based on expression of EMT genes in breast cancer patient's peripheral blood.Methods: This prospective ongoing study of breast cancer patients consisted of 16 (57.1%) patients with metastatic disease, 19 (67.9%) patients with inflammatory breast cancer (IBC) and 12 (42.9%) patients with primary, non-IBC breast cancer, respectively. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using anti-CD45 coated magnetic beads. RNA extracted from CD45-depleted (CD45-) PBMC were interrogated for expression of TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 and EpCAM gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of healthy donors (HD). Expression of one or more gene transcripts was considered a positive result. Concurrently, a 7.5 mL PB sample was collected for determination of CTC by CellSearch.Results: Median age was 54 year (range: 34-72 years). Overall, the median CTC count by CellSearch was 2 (range; 0-750) per 7.5 mL of PB. TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 were overexpressed in CD45- PBMC in 7%, 4 %, 4%, 0% and 14 % of patients, respectively. At least one of the EMT genes was overexpressed in 6 (21%) of patients. TWIST1 and SLUG were overexpressed only in IBC patients (10.5% and 5.3% of patients, respectively). Patients with triple negative breast cancer more commonly overexpressed EMT genes compared to non-triple negative patients (30.8% vs. 13.3%). There was no correlation between expression of EMT genes, EpCAM expression or CTC count measured by CellSearch, respectively.Conclusions: These data suggest that EMT genes may be involved in the dissemination of CTCs. Loss of epithelial antigen on CTC due to EMT, triggered by high expression of these genes, may be responsible for their undetection by conventional methods in a fraction of patients with early or advanced breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3011.
Effect of Trastuzumab and Chemotherapy on Circulating Tumor Cells in Patients with Poor Prognosis Metastatic Breast Cancer.
Authors: De Giorgi U. , De Giorgi U. , Mego M. , Ueno N. , Handy B. , Jackson S. , Reuben J. , Valero V. , Cristofanilli M.
Published: December 2010

Background. Trastuzumab has significant activity in HER2 neu amplified metastatic breast cancer (MBC). We hypothesized that it may selectively act against circulating tumor cells (CTC) in HER2-positiveMBC. We assessed the effect of trastuzumab-based regimens on CTC in HER2-positive MBC with poor prognosis (≥ 5 CTC).Patients and Methods. We retrospectively evaluated patients with poor prognosis MBC (baseline ≥ 5 CTC) treated with a first-line regimen consisting of trastuzumab+antimitotic agents in 11 HER2-positive patients not previously pretreated with trastuzumab, chemotherapy with antimitotic agents in 24 HER2-normal patients, and other chemotherapeutic drugs (mainly capecitabine) in other 16 HER2-normal patients. CTC were detected and enumerated using the CellSearch system (Veridex LLC, Warren NJ, USA). We evaluated the effect on CTC counts and on progression-free survival (PFS) and overall survival (OS).Results. At a median follow-up of 16 months (range, 4 to 48), 24 patients (47%) died. All 11 HER2-positive patients treated with trastuzumab+antimitotic agents had <5 CTC during the treatment. Only 16 (67%) with HER2-normal MBC had <5 CTC with antimitotic agents (p = 0.037), and 28 (70%) with other chemotherapeutic regimens (p = 0.048). No statistically significant difference was observed between patients treated with antimitotic agents and those treated with other chemotherapeutic agents (p = 0.73). There was no difference between patients receiving polychemotherapy and monochemotherapy (p = 0.73). The median PFS was 12 months in HER2-positive patients treated with trastuzumab+antimitotic agents compared with 7 months for those with HER2-normal (p = 0.09). The median OS was not reached (>20 months) and 19 months (p = 0.034), respectively. The median PFS was 8 months in HER2-normal patients with ≥5 CTC and 4 months in those with <5 CTC (p = 0.01). The median OS was not reached (>17 months) and 9 months (p = 0.01), respectively.Conclusions. Trastuzumab is highly effective in patients with HER2-positive MBC with poor prognosis (≥ 5 CTC). Antimitotic agents and other chemotherapy agents did not show a similar effect in HER2-normal MBC. CTC might be useful in the monitoring of poor prognosis MBC patients undergoing therapy with trastuzumab.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3014.
Clinical Relevance of Early Disseminated Breast Cancer Cells Depends on Their Cytokeratin Expression Pattern.
Authors: Effenberger K. , Borgen E. , Bartkowiak K. , zu Eulenburg C. , Brors B. , Kaaresen R. , Nesland J. , Pantel K. , Naume B.
Published: December 2010

Background: The factors determining the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients are largely unknown. Here we compared the specificity and clinical performance of two anti-cytokeratin(CK) antibodies frequently used for DTC detection, namely A45-B/B3 (A45) and AE1/AE3 (AE). Additionally we explored the CK gene expression patterns in primary breast tumors.Methods: Specificities of antibodies A45 and AE for selected CKs were assessed by 2-DE western blot analysis. Using these antibodies bone marrow aspirates from 391 breast cancer patients (M0, pT1-3, pN0-3) were screened for the presence of DTC. To obtain prognostic information, patients were followed up over a median of 83 months for time to relapse and 99 months for time to death. Two breast cancer patient datasets (Oslo (n=123), and van de Vijver et al. (NEJM 2002; n=295)) were analysed for CK primary tumor gene expression patterns and grouped by hormone receptor (HR) status of the primary tumors. T-tests and hierarchial clustering analyses were applied.Results: AE detected CK5, CK7, CK8, and CK19, whereas A45 recognized CK7 and CK18. In total, 24 of 391 patients (6.1%) were DTC-positive for A45, and 41 (10.5%) for AE. Although concordance between the two antibodies was 84.4%, overlap among positive cases was only 3.2%. DTC-positivity with AE and A45 was more frequent in patients of higher nodal status (p=0.019 and p=0.036, respectively). Nearly all patients with A45-positive DTC had hormone receptor-positive tumors (23/24), while detection of AE-positive DTC was more frequent among hormone receptor negative patients (p=0.006). Clear differences in primary tumor CK expression patterns between HR+ and HR- tumors were detected in both datasets. Compared to HR- tumors, CKs up-regulated in HR+ tumors were CK8 and 18, in addition CK19 in two datasets, whereas e.g. CK5, CK6, CK7, CK14, CK15, CK16, and CK17 were significantly down-regulated.Survival analyses of all patients revealed shorter distant disease-free survival (p=0.039) for patients with A45-positive DTC, whereas the prognostic relevance of AE-positive DTC was restricted to node positive patients.Conclusion: The clinical utility of immunocytochemical DTC detection depends on the anti-CK antibody used, which may reflect the complex CK composition of DTC. Tumor CK expression patterns and their correlation to antibody-sprecific DTC detection will further be explored by A45- and AE- staining of corresponding patient samples.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3021.
Clinical and Prognostic Features of Infiltrating Lobular Carcinoma of the Breast in a Korean Series.
Authors: Lee J. , Park B. , Park S.
Published: December 2010

Introduction: Infiltrating lobular carcinoma (ILC) is the second most common histological type of invasive breast cancer (IBC) and accounts for 10% of all IBCs. ILC differs from IDC in many respects including age of patients, tumor size and increased frequency of multifocality and bilaterality. But, the outcome of ILC remains unclear. Therefore, we studied the clinical features and prognosis of patients with ILC and infiltrating ductal carcinoma (IDC). Methods: We analyzed data from 3716 breast cancer pts who were were treated at the Yonsei University Health System between 1984 and 2005. Median follow time is 74 months. The patients were categorized as having ILC or IDC. Results: 95(2.3%) tumors were classified as ILC, 3621 (89.4%) as IDC. In analysis there were no differences of age, tumor size, nodal status and stage between ILC and IDC. However, patients with ILC were significantly more likely to have estrogen positive (79.7% for ILC vs 62.6% for IDC, p=0.003) and HER-2 negative tumors (83.6% for ILC vs 70.8% for IDC, p=0.038) compared to patients with IDC. The frequency of bilateral breast cancers was higher for patients with ILC than for patients with IDC (ILC: 6.8% vs. IDC: 3.5%, p=0.013). The systemic recurrence and local recurrence rates for ILC were similar to IDC. However, the sites of systemic recurrence were different. IDC more commonly metastasized lung than IDC (36.5% for IDC vs 9.1% for ILC). ILC more commonly metastasized to bone and the ovaries than IDC (bone: 72.7% for ILC vs 36.5 % for IDC, ovary: 18.2% for ILC vs 0% for IDC). there were no statistically significant differences in DFS and OS between ILC and IDC. The 5-year DFS was 87.6% for ILC and 78.7% for IDC.(p=0.174) The 5-year OS was 93.6% for ILC and 85.5% for IDC.(p=0.104) Conclusion: In the results of this study, the incidence of ILC was quite low in Korean breast cancer patients. ILC showed different histologic features as bilateral involvement, ER positivity, HER-2 negativity and systemic recurrence sites compared to IDC. But the prognosis of ILC was similar to that of IDC.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3036.
High Nuclear Expression of CXCR4 Is Associated with Metastases and Decreased Survival in Hormone Sensitive Breast Cancer.
Authors: Cronin P. , O'Connell F. , Wang J. , Redmond H.
Published: December 2010

Introduction: CXCR4 chemokine receptor and its specific ligand SDF-1alpha are thought to play an important role in breast cancer metastases. High levels of SDF-1alpha in the common destination organs of metastasis are thought to attract CXCR4-positive tumour cells.Aim: The aim of the study was to test whether CXCR4 expression in the primary tumour can indicate breast cancer with high likelihood of metastasising.Methods: Tissue slides were prepared from the paraffin embedded primary tumours of breast cancer patients. CXCR4 expression was detected using immunohistochemistry assessed by a pathologist. Disseminated tumour cells (DTC's) were detected in the bone marrow samples of patients taken at the time of surgery using anti-HEA/FITC-conjugated antibody and flow cytometry. All other clinicopathological features of the patients were also compiled.Results: CXCR4 expression was assessed in one hundred and ten patients. They had a mean age of 57.18 years and a median follow up of 84 months (range 4 – 108). 72.6% of patients had a T2 or a T3 tumour. The majority were hormone sensitive, 80.9% and Her2/neu negative, 76.1%. Disseminated tumour cells were found in the majority of patients, 85.5%, but 57.2% had greater that 1000 DTC's/100,000. There was no correlation found between cytoplasmic staining for CXCR4 and the clinicopathological features of the tumours including DTC's. High nuclear staining was associated significantly with decreased overall and disease free survival, p<0.041 and p<0.025. Tumours with high nuclear staining were significantly more likely to be hormone sensitive, tubulo-lobular tumours [p<0.008, p<0.0349], from older, post-menopausal women [p<0.081, p<0.018]. There was no correlation between her2/neu and CXCR4 expression. Disseminated tumour cells and lymph node metastases were not associated with nuclear staining but these tumours were more likely to develop distant metastases, p<0.034.Conclusion: High nuclear expression of CXCR4 was significantly associated with hormone sensitive breast cancers and development of distant metastases. These patients had decreased overall and disease free survival. Our results show CXCR4 has an important role in breast cancer metastases and has both prognostic and therapeutic potential.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3037.
Association between the Thr431asn Polymorphism of the Rock2 Gene and Breast Cancer.
Authors: Kalender M. , Demiryürek S. , Oztuzcu S. , Kizilyer A. , Demiryürek A. , Sevinc A. , Dikilitas M. , Yildiz R. , Camci C.
Published: December 2010

Background: The objective of this study was to analyze the genotype distributions and allele frequencies for Threonine431Asparagine (Thr431Asn) and Arginine83Lysine (Arg83Lys) polymorphisms of the Rho kinase2 (ROCK2) gene among the breast cancer patients.Material and Methods: In this case-control study, 223 patients with breast cancer were recruited and divided into two groups according to metastases (n=128) and without metastases (n=95). Genomic DNA from the patients and the control cases (n=150) was analyzed by real-time PCR using a Light-Cycler.Results: Neither genotype distributions nor the allele frequencies for the Arg83Lys polymorphism showed a significant difference between the groups. Although no marked changes was observed with no metastasis group, a statistically significant association was found between the control and metastatic group for the Thr431Asn polymorphism of the ROCK2 gene. Further analysis revealed that there were marked associations of this polymorphism with bone, liver, and lung metastases. Homozygous carriers of the Thr431Thr genotype were more frequent among the metastatic patients (39.8%) than among controls (26.0%; P =0.0485). There was also an increase in Thr431 allele (60.5% in patients vs 51.7% in controls) and decrease in Asn431 allele frequencies (48.3% in control vs 39.5% in patients) in metastatic groups (P =0.0356). Progesterone and estrogen receptor negativity was more frequent among metastatic patients carrying a Thr431 allele when compared to the nonmetastatic group.Discussion: Our resulsts are the first to demonstrate that Thr431Asn polymorphism of the ROCK2 gene could be a risk factor for the metastases of the breast cancer, and may help in predicting the prognosis.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3038.