bluuebunny/crossref_metadata_2025 huggingface dataset:
Does the Level of Axillary Node Clearance Influence Adjuvant Treatment in Patients with Breast Cancer?
Authors: Cannings E. , Nicol A. , Hansell D.
Published: December 2010

Background: The SIGN (Scottish Intercollegiate Guidelines Network) guidelines on axillary surgery do not advise as to whether level 1/2 or level 3 clearances should be performed when axillary clearance is required. Although there is a theoretical risk of increased morbidity, in experienced hands a level 3 clearance reduces the chance of residual tumour and allows for full staging.Materials and Methods: 124 consecutive axillary clearances (level 1, 2 and 3) were performed by a single operator. Level 1 and 2 nodes were removed as a single specimen, with level 3 nodes removed separately. Patient case notes, operation notes and pathology reports were used in the collection of data and all data were recorded in an Excel databaseResults: Of the 124 axillary clearances performed, 64 (52%) had level 1/2 lymph nodes positive for carcinoma. Of these 64 patients, 14 (11%) also had positive level 3 nodes. One patient (1%) had negative level 1/2 nodes but positive level 3 nodes. The Nottingham Prognostic Index (NPI) value increased in two patients when the level 3 nodes were included (6.6 to 7.6 and 4.3 to 5.3).Discussion: While it is generally accepted that axillary radiotherapy deals with residual level 3 nodal involvement, basic surgical oncologic principles would suggest that removal of as much tumour as possible should be performed. The patient with positive level 3 nodes but negative level 1/2 nodes would have been under-treated, with viable tumour remaining. Furthermore, the under-estimate of the NPI when level 3 nodes are not included could lead to sub-optimal adjuvant therapy being offered. The performance of a level 3 clearance can preclude the need for axillary radiotherapy, thereby reducing morbidity.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3114.
Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blockers May Reduce Breast Cancer Recurrence.
Authors: Chae Y. , Valsecchi M. , Lucca A. , Kim J. , Desai A.
Published: December 2010

There have been epidemiologic and biochemical findings alluding to the role of angiotensin converting enzyme inhibitors(ACEIs) and angiotensin receptor blockers(ARBs) in reducing the risk of developing cancer. To date, there exists no study that reported any relationship between their use and the risk of cancer recurrence. Thus, we investigated the associations between the use of ACEI/ARB and the risk of tumor recurrence among breast cancer patients without any evidence of disease.We reviewed the medical records of female patients diagnosed with stage II/III breast cancer in Albert Einstein Medical Center between 1999 and 2005 and later reached no evident disease (NED) stage after curative therapy. Follow up period was from 1999 to 2008 with mean and maximum being 4.4 years and 9.8 years, respectively. ACEI/ARB user was defined as patients taking the medication in NED stage for at least 6 months. Multivariate analyses were performed using logistic regression and Cox proportional hazard model.Of 707 cancer patients and of 335 cancer patients with hypertension, 164, representing 23.3% of total and 49.0% of those with hypertension were prescribed ACEI/ARB. ACEI/ARB use was associated with older age (P<0.001), postmenopausal status (P<0.001), and diabetes (P<0.01). Fourteen percent (23/164) of ACEI/ARB users developed cancer recurrence, while 23.3% (125/541) of nonusers developed a recurrence (odds ratio [OR], 0.54; 95% confidence interval [CI], 0.33-0.97; P=0.01). Kaplan-Meier survival analysis revealed that ACEI/ARB use was related to disease-free survival benefit (log-rank test P<0.01). Five year disease-free survival for ACEI/ARB users was 0.85 compared with 0.76 for nonusers (P<0.01). Adjusting for statistically significant variables above, ACEI/ARB use was associated with reduced risk of developing recurrence not only in all breast cancer patients but also when they were compared to a subset of the nonusers who had hypertension (AHR, 0.41; 0.23-0.80; P<0.01). While ACEI/ARB use was not associated with reduced mortality in all breast cancer patients (AHR, 1.01; 95% CI: 0.61-1.65; P=0.36; log-rank test P=0.52), it was when compared to the subset of patients with hypertension (AHR, 0.43; 95% CI, 0.25-0.68; P<0.01; log-rank test P<0.01).The use of ACEI/ARB is related to reduced risk of developing recurrence in patients with breast cancer. This result provides clinical evidence, in accordance with existing biochemical and epidemiologic data, that antagonizing angiotensin system is inversely associated with tumor recurrence. However, prospective randomized trials are needed to validate this finding.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3121.
An Inhibitor of ADAMs (a Distintegrin and Metalloproteinase) Overcomes HER3-Mediated Resistance to Trastuzumab and Lapatinib.
Authors: Scherle P. , Marando C. , Covington M. , Yao W. , Newton R. , Friedman S.
Published: December 2010

Background: Inhibitors of HER-2/neu and EGF receptors such as trastuzumab, lapatinib, and erlotinib have demonstrated clinical efficacy but not all HER-2/neu or EGFR positive tumors respond and many that initially respond develop resistance. Ligand mediated HER-3 signaling results in tumor growth and survival and is a proposed mechanism of resistance to trastuzumab and lapatinib. Proteolytic cleavage of both ErbB ligands and receptors has been shown to be a critical event that results in ErbB pathway activation. Cleavage is necessary for the generation of soluble, functionally active forms of ErbB ligands and in the case of HER-2/neu, cleavage results in a shed extracellular domain (ECD) and a membrane bound fragment (p95) that contains a kinase domain with significant constitutive activity. In addition, it has been shown that the preferential association between HER3 and p95 can further activate the pathway. Both ErbB ligand and HER-2/neu cleavage are mediated by the ADAM family of proteases. Further, we have previously shown that the ADAM protease inhibitor, INCB7839, provides synergistic inhibition of HER2+ breast cancer cell line growth when combined with either trastuzumab or lapatinib. Materials and Methods: The HER-2 overexpressing BT-474 human breast cancer cell line was treated with either lapatinib or trastuzumab in the presence or absence of the HER-3 ligand, heregulin, and the effects on cell growth measured. The effects of the ADAM protease inhibitor INCB7839 in this system were also examined. Results: The addition of heregulin overcame the anti-proliferative effects of both lapatinib and trastuzumab on the growth of the BT-474 cell line in vitro. Addition of INCB7839 synergized with lapatinib or trastuzumab and importantly, restored the anti-proliferative effects of these agents in the presence of heregulin. Further, pretreatment of BT-474 cells with INCB7839 for 6 days, which we have previously shown completely eliminates the presence of the p95 form of HER2, amplified these effects, presumably by eliminating p95 before stimulating the cells with heregulin. Discussion: Together, these results confirm that heregulin can overcome the anti-proliferative effects of both trastuzumab and lapatinib as previously reported. Prevention of p95 formation by ADAM protease inhibitors appears to restore the anti-proliferative effects of both trastuzumab and lapatinib when heregulin/HER3 signaling occurs, negating a proposed mechanism of resistance to these agents in the clinic. These results suggest that combining an ADAM inhibitor with targeted inhibitors of the ErbB family can overcome HER-3-mediated resistance and enhance the clinical efficacy of approved HER2-targeted agents in the clinical setting. INCB7839 is currently under clinical investigation in combination with trastuzumab for women with metastatic breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3138.
Novel Cytotoxic Proteins of Bacillus thuringiensis Isolated from Peninsular India.
Authors: Devassy B. , Chubicka T. , Devaki G. , Nair H. , Padikala J.
Published: December 2010

Bacillus thuringiensis (Bt) is a versatile soil born bacterium with potential insecticidal activity over a wide range of species of agriculture pests. These unusual properties are due to the crystal (Cry) inclusion proteins largely produced during the sporulation. Recent findings indicate that some non-insecticidal crystal inclusions are selectively cytotoxic to cancer cells. The toxicity is due to the induction of membrane permeability and apoptosis in susceptible cells. Recently, we have reported the cytotoxic effect of proteins extracted from three native strains of Bacillus thuringiensis from the Western Ghats of Kerala (India). Studies have shown that these potent toxins exhibit selective cytotoxicity against some human breast cancer cells including MCF-7, MDA-MB-231, ZR-75 and SKBR3. Presently, we report the cytotoxic properties of more number of Bacillus thuringiensis isolates from Peninsular India, which are already assessed for their diversity. The cytotoxic properties of isolates were primarily analysed by the amplification of DNA using specific primers designed for cytotoxic parasporal genes. The strains showing positive result were subjected to in vitro cytotoxic analysis using cancer cell lines. The crystal proteins were harvested from 72h old cultures in sporulating medium, after confirming the lysis of the cells and release of the protein. The cytotoxic proteins were isolated by aqueous biphasic (sodium dextran sulfate/polyethylene glycol) separation method and purified by various chromatographic (sephadex/nickel coated columns) and electrophoretic procedures. The molecular weight of proteins isolated from different isolates was varying and ranged from 15 to 80 kDa. The pro-protein isolated was activated and subjected in vitro cytotoxicity assay on various normal as well as breast cancer cell lines. The protein was found to have strong cytocidal activities against tumour cells and the treated cells showed remarkable morphological alterations and apoptotic cell death. Thus, a new 14 isolates were found to be cytotoxic by the study. The study revealed that the diversity of cytotoxic Cry genes in Peninsular India is perplexingly high and specific to certain cancers. Considering these results, we suggest that the receptor-mediated action of crystal proteins against tumour cells may find a new place in the therapeutic as well as diagnostic field of cancer therapy and the bacteria and its genetic potential may be utilized as versatile tools against cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3143.
The Expression of Phosphorylated mTOR in Breast Cancer from BRCA1-Mutation Carriers.
Authors: Korski K. , Malicka A. , Wysocki P.
Published: December 2010

Background: The mammalian target of rapamycin (mTOR) is a serine-threonine kinase coupling growth stimuli and metabolic signals to cell cycle progression and protein synthesis. mTOR represents also a novel therapeutic target in various types of cancer. The role of mTOR in development and progression of breast cancer is not fully understood. There are studies suggesting that mTOR is responsible for the resistance of breast cancer to tamoxifen, trastuzumab and various chemotherapeutic agents. Since there are no studies evaluating the activation (phosphorylation) of mTOR in breast cancer cells carrying a germinal mutation of BRCA1, we have decided to assess activation of this kinase in tumor specimens in BRCA1-mutation carriers.Materials and methods: Formalin fixed paraffin-embedded tumor samples were obtained from 19 patients carrying BRCA1 germinal mutation and 24 patients with intact BRCA1 gene. All patients have been treated in the Great Poland Cancer Center between 2000-2008. The blocks were retrieved from the archives of the Department of Pathology and immunostained for phospho mTOR (Phospho-mTOR (Ser 2448), rabbit antibody, Cell Signaling).Results: Phosphorylation of mTOR was not detected in any of 19 patients carrying the mutated BRCA1 gene. Most of the BRCA1-mutated cancers were ER and HER2 negative (18 (95%) and 16 (94%), respectively). In the tumors with a wildtype BRCA1 gene, mTOR activation was detected in 12 samples (50%). Lack of mTOR phosphorylation was again observed mostly in ER and HER2 negative tissue samples (10 (83%) and 11 (92%), respectively).Discussion: Our findings have demonstrated differential activation of mTOR between tumors carrying a wild-type and mutated BRCA1 gene. We have also observed a negative correlation between ER and HER2 status and phosphorylation of mTOR. Although our study included a relatively low number of cases, the results indicate that activation of mTOR and probably also of its upstream targets do not play a crucial role in biology of tumors with a BRCA1 mutation or with the triple-negative phenotype.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3144.
Integrative Molecular Profiling of Triple Negative Breast Cancers Identifies Potential Therapeutic Targets Including Amplifications of FGFR2.
Authors: Turner N. , Turner N. , Lambros M. , Horlings H. , Horlings H. , Pearson A. , Sharpe R. , Mackay A. , Natrajan R. , Geyer F. , van Kouwenhove M. , Kreike B. , Ashworth A. , van de Vijver M. , van de Vijver M. , Reis-Filho J.
Published: December 2010

Triple negative breast cancers (TNBCs) have a relatively poor prognosis emphasising the need to identify new subtype specific target therapies. Based on the concept of oncogene addiction, we searched for potential therapeutic targets by identifying genes consistently over-expressed when amplified in TNBC. Fifty six TNBCs were subjected to high resolution tiling path microarray-based comparative genomic hybridisation (aCGH); out of these cases, 24 were also subjected to genome-wide microarray-based mRNA expression analysis. TNBCs showed a high level of genetic instability, with recurrent regions of amplification (>4 copies) included multiple regions on 1q and 8q, 3q25, 10p14, 10q26, 13q34, 15q26 and 19q12-19q13. Integration of aCGH and expression data revealed 38 genes that were significantly overexpressed when amplified. This list includes known oncogenes and potential therapeutic targets, such as MCL1 (1q21.2), FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34), PKN1 (19p13.12), and NOTCH3 (19p13.12). To validate FGFR2 as a therapeutic target, we screened a panel of cell lines, by western blotting and aCGH, and identified two TNBC cell lines with FGFR2 amplification. In these cell lines FGFR2 was constitutively active in a ligand independent manner, and RNA interference-mediated silencing of FGFR2 selectively decreased survival of cell lines harbouring FGFR2 amplification. Likewise FGFR2 amplified cell lines were highly sensitive to FGFR tyrosine kinase inhibitor PD173074 (IC50 <20nM). Treatment with PD173074 induced apoptosis in amplified cell lines, as did treatment with PI3 kinase inhibitors LY294002 and BEZ-235 suggesting that apoptosis resulted from inhibition of AKT signalling. Examination of publically available CGH data sets confirmed FGFR2 amplification in 4% (5/124 95%CI 1.3-9.2%) of TNBC, with no cases of FGFR2 amplification in other subtypes (0/150, p=0.02). Our results suggest that FGFR2 amplification is a therapeutic target in a small subset of TNBCs.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3147.
Induction of Aggressive Breast Cancer in p53 Wild Type Cells by BRCA1-IRIS Overexpression.
Authors: ElShamy W.
Published: December 2010

Background: Agents that introduce genotoxic stresses in mammalian cells, such as paclitaxel (Taxol) or ionizing radiation activate a complex array of cellular responses, including p53 activation. As a component of the response to acute stress, p53 has a well-established role in protecting against cancer development. It is not surprising that half of all human tumors have mutated p53. We asked how then tumors are initiated in the cells that carry wild-type p53 allele?Material and Methods: Here we report the identification of a potential novel mechanism that induces tumor formation in p53 wild-type cells. Overexpression of the proto-oncogene, BRCA1-IRIS in human mammary epithelial (HME) cells not only upregulates the expression of the serine/threonine phosphatase; Wild-type p53-Induced Phosphatase (WIP1) through induction of p53 expression but also increases the WIP1 gene copy number in these cells. WIP1 is a negative regulator of p53 as well as p38MAPK. Both proteins are involved in arresting and/or killing cells with extensive genotoxic stresses. Failure to arrest or die in response to massive genotoxic stress leads to the propagation of damaged cells and the development of cancer. BRCA1-IRIS overexpressing cells failed to arrest or die when exposed to UV radiation or Taxol. In fact, treatment of normal HME cells with UV or Taxol upregulated the expression of BRCA1-IRIS and thus WIP-1, prevented p53 and p38MAPK activation and their death.Results: This data suggest that BRCA1-IRIS is perhaps involved in the acquisition of cellular resistance to these agents upon prolonged exposure. On the other hand, depletion of BRCA1-IRIS from tumor cell lines induced apoptosis even in cells with functional p53 and p38MAPK.Discussion: We propose that in p53 wild type cells, whether endogenous or UV- or chemotherapy-induced BRCA1-IRIS overexpression leads to the survival and proliferation of what most likely are severely damaged cells, leading to the transformation of mammary cells in vitro and the induction of breast tumors in vivo. We also propose that BRCA1-IRIS and WIP1 overexpression abrogates the homeostatic balance maintained by p38-p53-WIP1 pathway. Together with the ability of BRCA1-IRIS to upregulate Cyclin D1 expression increases the likelihood that BRCA1-IRIS is an oncogene that induces breast cancer progression when overexpressed, hence making it an attractive target for novel breast cancer therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3154.
GCIP Is a Tumor Suppressor in Breast Cancer.
Authors: Chang T. , Chang M.
Published: December 2010

Background: GCIP protein is a HLH-leucine zipper (HLH-LZ) proteins without basic DNA-binding region, which was identified to be a cyclin D1-binding protein and also was recognized as a human homologue of MAID protein (HHM). Recently, GCIP was found to function as a tumor suppressor in liver and colon tumorigenesis. In this study, we examined in vitro the functional role of GCIP in various breast cancer cell lines.Material and Methods: GCIP-directed siRNA construct was prepared and stable downregulation of GCIP in epithelial-like and nonaggressive/differentiated breast cancer cell line, MCF-7, were selected. In addition, two aggressive/ invasive breast cancer cells (MDA-MB231 and MDA-MB435s) that exhibit low levels of endogenous GCIP expression were transfected with p3x-GCIP-flag, respectively and stable GCIP-re-expressing transfectants were selected. The phalloidin staining cell morphology and functional assays with these stable GCIP downregulating or GCIP re-expressing were performed to characterize the biological effects of GCIP.Results: Stable MCF-7 cells with down-regulated GCIP expression acquired a mesenchymal-like shape and exhibited increased colony formation, migration and invasion capabilities. Furthermore, this was found to be associated with the loss of membrane E-cadherin and increased expression of the mescenchymal marker vimentin. In addition, both the stable MDA-MB231 and MDA-MB435s with GCIP-re-expression acquired an epithelial-like shape and exhibited decreased colony formation, migration and invasion capabilities.Conclusion: our previous study found that decreased expression of GCIP in breast carcinomas compared with normal ductal epithelium (P < 0.001). In this study, we present evidence that GCIP inactivation contributes to the enhanced migratory and invasive capacity of MCF-7 and that GCIP re-expression inhibits a wild range of cellular functions related to the malignant properties of MDA-MB231 and MDA-MB435s, confirming that GCIP is a tumor suppressor in breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3158.
Immunohistochemically Defined T1N0 Breast Cancer Subgroups Have Different Clinical Outcomes. A Retrospective Turkish Oncology Group Study.
Authors: Basaran G. , Okkan S. , Caglar H. , Benekli M. , Gumus M. , Haydaroglu A. , Eralp Y. , Abali H. , Yildiz F. , Bese N. , Gursel B. , Guney N. , Derin D.
Published: December 2010

Introduction: T1N0 breast cancer (BC) has a very favorable outcome therefore the need of adjuvant systemic therapy is controversial. Molecular profiling studies identified BC subtypes with distinct clinical outcomes. We planned to determine whether immunohistochemically (IHC) defined subgroups of T1N0 BC patients have different clinical outcomes. Methods: Medical records of pts with T1N0 BC and known ER, PR and Her-2 status who have been treated between 1998-2008 were reviewed. Patients with bilateral disease, who had adjuvant trastuzumab therapy were excluded. Clinical/pathological, treatment characteristics were recorded and patients were subdivided into four BC subtypes: triple negative (ER, PR, HER2 negative), HER2 (ER and PR-negative, HER2-positive), luminal B (ER-positive and/or PR-positive, HER2-positive), and luminal A (ER and/or PR-positive and HER2-negative). Her-2 positivity was defined as IHC 3+ or FISH positive IHC2 2+. Survival outcome of the IHC defined subgroups were analyzed the Kaplan-Meier method and compared with the log-rank test. Results: A total of 1035 pts have been identified from 17 Medical/Radiation Oncology centers in Turkey. Median age was 51 and median follow up was 39 months. Forty-four % of patients were premenopausal, 55% had left breast involved, 73% had breast conserving surgery, 81% had invasive ductal carcinoma. T1a,b,c tumors were 7%, 30%, 63% ; grade 1,2,3 tumors were 20%, 53%, 27% respectively. HER2 was 2 + in 59 (6%) and 3 + in 111 (11%) tumors. Twelve % tumors were triple negative. Thirty-seven % patients had adjuvant chemotherapy; 80 % had adjuvant endocrine therapy 73% had adjuvant radiotherapy. Five-yr DFS and OS of the whole group were 92% and 99% respectively. So far, 19 pts had local and 24 pts had distant metastases, 4 pts had secondary cancers, 6 patients contralateral BC and 11 patients died (7/11 pts died from breast cancer). Classification based on ER, PR and Her-2 status had significant impact on 5-yr DFS (p:0.00) and OS (p:0.00). The 5-yr DFS and OS were 84%;74%;%;94% and 96%;83%;100%;99% respectively for triple negative, HER2, luminal B, and luminal A subgroups respectively. Hormone receptor and triple negative status were the two other parameters affecting survival outcome significantly (p:0.00, p: 0.007) in the univariate analyses. Conclusion: Most patients with T1N0 disease have generally a favorable clinical outcome, however even within this very early stage of BC there are subgroups with diverse clinical outcomes. Despite our short follow up with low number of events, we found that Her-2 positive and triple negative T1N0 BC have poor clinical outcomes compared to luminal T1N0 subgroups and therefore they might need special concern with respect to adjuvant systemic therapy.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3167.
Breast Cancer Subtypes in Northern Chinese Women.
Authors: Gao J. , Zhang X. , Zhang B. , Wang J. , Liang X.
Published: December 2010

Introduction: Breast cancer is currently regarded as a heterogeneous disease. Gene expression analysis has identified several subtypes that differ in prognosis and therapy response. Little is known about the distribution of these subtypes among native Chinese women.Patientsand Method: From December 2006 to November 2008, 1,133 women were diagnosed with invasive breast cancer in Shanxi province cancer hospital and 818 had assessable information for ER, PR, and Her-2/neu status. Based on immunohistochemical surrogates for ER, PR, and Her-2/neu status obtained from the department of pathology, the molecular subtypes were defined as: luminal A (ER+ and/or PR+, HER2−), luminal B (ER+ and/or PR+, HER2+), basal-like (ER−, PR−, HER2−), and Her-2/neu (ER−, PR−, and HER2+).Results: Luminal A was the most prevalent breast cancer subtype (70.6%), compared with the luminal B breast cancer subtype (15%), basal-like breast cancer subtype (9.6%), and Her-2/neu breast cancer subtype (4.8%). The frequency of Her2 positive breast cancer was 19.88% in the cohort studied. The luminal B breast cancer subtype was largely presented in patients aged less than 50 years old (58.8%) compared with the luminal A subtype (48.5%), basal-like subtype (52.6%) and Her-2/neu breast cancer subtypes (47.4%). The largest tumor size was basal-like breast cancer (28.1mm), followed by luminal B (25.8mm), Her-2/neu (25.7mm) and luminal A (23mm) (P = 0.003). The lymph node status did not vary among the four groups, however, the mean number of lymph nodes metastasis was significantly different with 3.2, 2.9, 5.7 and 4.9 for luminal A, luminal B, Her-2/neu and basal-like subtypes respectively (P = 0.005).Conclusions: The distribution of breast cancer subtypes in Northern Chinese women was similar to other ethnicities. The basal-like and Her-2/neu breast cancer subtypes were associated with unfavorable clinical features. Further studies will identify the difference in treatment response and prognosis among the four subtypes in this cohort.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3168.
Biomarker Discovery and Evaluation of Response to Anti-Cancer Therapeutics in Breast Cancer Using a Novel Nanofluidic Immunoassay Platform.
Authors: Iacovides D. , Johnson A. , Boge A. , Nguyen U. , Shah S. , Gentalen E. , Gray J.
Published: December 2010

Our research efforts focus on the identification and detection of fundamental molecular differences between normal and tumor cells in breast, as well as differences among distinct breast cancer subtypes, especially in terms of signal transduction pathways that control cell cycle, apoptosis and cell growth. Cancer subtype specific molecular variations dramatically affect patient responses to already existing treatments. For example, the phosphorylation status of many proteins that are involved in signal transduction pathways perturbed in cancer cells is extremely important in determining whether these cells are susceptible to killing by available cancer therapeutics. Therefore, differentially phosphorylated protein isoforms can be a particularly useful prognostic biomarker of drug response in the clinic. However, accurate detection and quantitative analysis of cancer-related phosphoproteins in tumors is limited by current technologies.Using a novel, fully automated nanocapillary electrophoresis technology (FireFlyTM) designed to separate protein molecules based on their isoelectric point (pI), we are currently developing highly sensitive assays for reliable assessment of the phosphorylation status of cancer-related phosphoproteins in tumors, before and during drug treatment. We have already developed and optimized assays measuring AKT1, AKT2, AKT3, ERK1 and ERK2, and their respective phosphoisoforms. Using these assays, we were able to measure levels of activated ERK1/2 and AKT1/2/3 in a breast cancer cell line panel developed in our lab, using protein extracted from as few as 125 cells. Based on RNA expression data, cell lines in this panel have previously been categorized in two distinct subtypes (Basal and Luminal) and their molecular phenotypes closely resemble the respective profiles of tumors obtained from breast cancer patients (CITATION). This cell line panel is extensively used to measure cellular responses to breast cancer therapeutics, including drugs that target MEK, ERK, PI3K and AKT. Using FireFly assays, we are currently measuring changes in the phosphorylation states of these targets during drug treatment, in order to completely characterize pharmacodynamic changes in these cells during treatment, and develop molecular profiles that predict response in breast cancer tumors. Since this technology enables accurate detection and quantification of protein isoforms and post-translational modifications from only very small amounts of tumor samples or serum, it promises to propel cancer biomarker discovery and enable the development of clinically useful prognostic and diagnostic assays that predict responses to drugs targeting cancer-specific molecular networks.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3172.
Breast Cancer Risk Estimation Using the OncoVue® Model Compared to Combined GWAS Single Nucleotide Polymorphisms.
Authors: Jupe E. , Pugh T. , Knowlton N. , Defreese D.
Published: December 2010

Background: We have developed a new breast cancer risk assessment model (OncoVue) that integrates information from 22 single nucleotide polymorphisms (SNPs) and 5 personal risk factors. It has been shown to effectively stratify risk in three independent validation populations. Genome wide association studies (GWAS) have identified seven candidate SNPs independently associated with breast cancer risk. Using estimates of relative risks and allele frequencies derived from these studies, a theoretical examination of these SNPs has concluded that they have the potential to modestly improve clinical risk estimation. We have examined both the OncoVue model and these GWAS SNPs in the same case-control population to determine their utility in risk estimation.Materials and Methods: A randomly selected subset of participants (376 cases and 982 controls) that had enrolled in a larger case-control study conducted in six distinct geographic regions of the United States were examined. DNAs were genotyped for the 22 SNP variants in OncoVue and combined with personal factors to calculate the risk scores for the individual participants. DNAs were also genotyped for the following GWAS SNPs: rs2981582 (FGFR2), rs3817198 (LSP1), rs889312 (MAP3K), rs4415084 (MRPS30), rs13281615 (POU5F1P1), rs13387042 (TNP1), and rs3803662 (TOX3, formerly TNRCR9). Association of individual GWAS SNP genotypes with breast cancer risk was evaluated by calculating odds ratios (ORs). Assuming independent contribution of each SNP to risk, a combined GWAS risk score was calculated using a multiplicative model. Positive likelihood ratios (PLRs) were calculated using a risk threshold of 1.5-fold the control population mean risk to evaluate the proportion of individuals placed at elevated risk by OncoVue compared to the combined GWAS risk scores.Results: OncoVue exhibited significant ability to stratify risk at the 1.5-fold mean with a PLR of 2.2 and at a risk level of 2.5-fold mean the PLR increased to 5.0. In analyses of individual GWAS SNPs, statistically significant associations with breast cancer were identified for homozygous carriers of the rare allele for the FGFR2, MAP3K and TOX3 genes. However, the PLR for the combined GWAS risk scores at 1.5-fold mean was 1.0 indicating no ability to discriminate risk in this study population.Conclusions: In this case-control population OncoVue effectively stratified risk by accurately assigning elevated risk to breast cancer cases. In the same population, a combined risk score produced from seven GWAS SNPs did not effectively stratify risk. These results indicate that the OncoVue model has clinical utility for identifying elevated risk women who might benefit from additional screening and prevention.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3177.
Selective Tri-Nucleotide Spacers Modulate the Regulatory Potential of Estrogen Response Elements.
Authors: Kallen C. , Yang D. , Sidell N.
Published: December 2010

Estrogens bound to Estrogen Receptors (ERs) play critical roles in breast cancers through their binding to certain DNA motifs (i.e. estrogen response elements or EREs) in the promoters of target genes to regulate their transcriptional activity. The consensus ERE has the 15 base sequence AGGTCAnnnTGACCT where nnn are known as the tri-nucleotide spacer. The base specificity or sequence of this spacer is thought to be unimportant in terms of its effect on ERα-ERE binding affinity or transactivation efficiency. In the present experiments, whole genome sequence analysis of ERα-DNA binding sites was performed in MCF-7 breast cancer cells using CHIP-on-chip assays to determine frequency variations in EREs that bind ERα. Here, we present evidence that ERα binds to widely variant ERE sequences, both in terms of half-site specificity and tri-nucleotide spacer identity. Overall, our analysis indicated that whole genome ERα-ERE binding highly favored specific C(A/T)G enriched tri-nucleotide spacers, suggesting some evolutionary advantage to this three base configuration. To address this question in MCF-7 cells, we designed consensus EREs containing different nnn spacers (CTG, GCC, TTT) to test as binding probes in Electrophoretic Mobility Shift Assays and as enhancer elements in luciferase reporter vectors. Our results indicated differences in the ERα-ERE binding affinity depending on the nnn spacer sequence with the order of affinity being CTG > GCC > TTT. To determine the potential functional significance of the tri-nucleotide spacers, luciferase reporter assays in MCF-7 cells were performed. Results showed the same dependency on the sequence of the nnn spacer to induce luciferase activity in response to estrogen treatment (CTG>GCC>TTT). These experiments indicate that the identity of the three base spacer elements in the ERE are important for influencing ERα-ERE interactions and functional activity. These data support the results of the whole genome CHIP-on-chip analysis and suggest that increased frequency of ERα binding to ERE motifs with C(A/T)G spacers is due to preferential binding characteristics resulting in efficient transactivation of estrogen target genes.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3179.
Systemic MiRNA Levels as Novel Breast Cancer Biomarkers.
Authors: Heneghan H. , Miller N. , Newell J. , Kerin M.
Published: December 2010

Background: The development of clinically validated biomarkers for breast cancer has remained an insurmountable task despite other advances in the field of molecular biology. Mi(cro)RNAs have many characteristics of an ideal biomarker most notably their inherent stability, resilience, and ease of detection. Recent blood based miRNA profiling studies, reporting their presence in serum and plasma, have generated the concept that circulating miRNAs hold much potential as novel non-invasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of circulating microRNAs as novel breast cancer biomarkers.Materials and Methods: Using a real-time quantitative PCR approach, expression levels of a panel of 7 oncogenic miRNAs were quantified in tissue and blood specimens of 148 patients with breast cancer and 24 age-matched disease free control individuals. Advanced QBase Plus software and SPSS were used for biostatistical analysis of the data, and correlation with clinicopathological variables.Results: Cancer specific miRNAs were detected and significantly altered in the circulation of breast cancer patients preoperatively, most notably miR-195 (12 fold increase) and let-7a (5-fold increase), (p < 0.001 and p<0.001 respectively). Increased systemic miR-195 levels in breast cancer patients were also reflected in breast tumors. Systemic let-7a levels varied inversely with disease stage, being highest in carcinoma in-situ patients and getting progressively lower through to stage IV invasive disease. Furthermore we identified that circulating levels of miR-195 and let-7a decreased in cancer patients post-operatively following curative tumor resection (p<0.001 and p<0.001 respectively). Finally we found that specific circulating miRNAs correlated with certain clinicopathological variables, namely nodal status and estrogen receptor statusDiscussion/Conclusion: Our results demonstrates the novel finding that miRNAs are detectable in the circulation of breast cancer patients, that expression profiles differ between patients and controls, and that the circulating miRNA expression profile correlates with various biopathologic parameters of breast cancer. Also, systemic miRNA levels revert to normal levels following tumour resection. In this, lies the expectation that systemic miRNAs will be clinically useful as a novel minimally invasive tool to aid in the early diagnosis and perioperative management of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 32.
Adjuvant Tamoxifen Treatment Outcome According to Cytochrome P450 2D6 (CYP2D6) Phenotype in Early Stage Breast Cancer: Findings from the International Tamoxifen Pharmacogenomics Consortium.
Authors: Goetz M. , Berry D. , Klein T. ,   International Tamoxifen Pharmacogenomics Consortium
Published: December 2010

Background: An estimated 1.3 million women worldwide will be diagnosed this year with early stage breast cancer. The majority of women have estrogen receptor (ER) positive breast cancer and are candidates for endocrine therapy. Tamoxifen remains the most commonly employed agent worldwide and substantially reduces recurrence and mortality. Tamoxifen is a pro-drug. CYP2D6 converts tamoxifen to endoxifen, which is primarily responsible for inhibition of estrogen-stimulated breast cancer growth in vitro. There have been conflicting reports regarding the relationship between CYP2D6 genotype/phenotype and tamoxifen clinical outcome. The ITPC was established to collect the worldwide experience relating to genetic variation in CYP2D6 and the outcomes of women with ER positive breast cancer treated with adjuvant tamoxifen. Establishment of the predictive value of CYP2D6 would enable the a priori identification of patients that could be spared ineffective therapy.Methods: Individual patient data including tumor characteristics, genotype, medication data, and recurrence information were requested from all published and non-published reports of clinical outcomes related to CYP2D6 and deposited at PharmGKB. CYP2D6 genotype was available for the following null (*3, 4, 5 and 6) and IM (*10, 17 and 41) alleles. CYP2D6 inhibiting medication data was requested for weak and potent CYP2D6 inhibitors. The primary analysis was to determine the association of CYP2D6 phenotype (combined genotype and medication) with breast cancer recurrence in tamoxifen treated early stage, ER positive invasive breast cancer.Results: At this time, the ITPC has received patient data on 3432 patients from the U.S., Sweden, UK, Germany, Belgium, and Japan. Median age at diagnosis was 62 years. CYP2D6 genotype was available in 3,392 (99%). The allele frequency for CYP2D6*4, the most common allele associated with the CYP2D6 poor metabolizer phenotype, was q=0.23. A total of 1152 recurrences representing first events (either local, regional, distant, or contralateral breast cancer) were recorded. At the time of abstract submission, patient and genotype data from an additional 1300 tamoxifen treated patients were pending submission.Conclusions: The ITPC represents the largest collection of individual patient data in regard to the potential importance of CYP2D6 phenotype in adjuvant tamoxifen therapy. The findings from this consortium are expected to substantially improve the global understanding of CYP2D6 and tamoxifen clinical outcome. Final results will be presented at the meeting

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 33.
Breast MRI Screening of Women with a Prior High-Risk Lesion: Preliminary Results.
Authors: Brennan S. , Liberman L. , Dershaw D. , Morris E.
Published: December 2010

Purpose: To determine the frequency of breast cancer detection in women at increased risk due to a prior high-risk lesion.Materials and methods: An IRB approved retrospective review was performed of the records of 2184 breast MRIs starting from 1999, to identify patients who commenced screening breast MRI who had a history of a prior high risk lesion. Eighty-three women were identified who had a prior biopsy demonstrating a high-risk lesion such as atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH) or lobular carcinoma in situ (LCIS). History, imaging, and pathologic findings were reviewed.Results: Histologies of prior high-risk lesions in these 83 women were ADH in 24, LCIS in 54 and ALH in 5. The median length of MRI follow-up was four (range, 1-9) years. Biopsies were recommended based on MRI findings in 39 (47%) of 83 women, yielding cancer in seven (8%) women (four of whom also had benign biopsies), and benign findings only in 32 (39%) women. Six of the seven women who developed a breast cancer also had a family history of breast cancer. Histologic findings in these seven cancers were ductal carcinoma in situ (DCIS) in 4 (57%) and invasive cancer in three (43%). Invasive cancer histologies (n=3) were ductal in one and lobular in two; maximal histologic size was 2.0 cm. This large invasive cancer had a mammographic, sonographic, and palpable correlate, and was node-positive; the other cancers were identified by MRI only. Among these seven cancers, two (29%) were detected in screening year 1, two (29%) in screening year 2, two (29%) in screening year 3 and one in screening year 4 (14%). Three (43%) of the seven cancers were in the contralateral breast. In these 83 women, the likelihood of detecting cancer by MRI was higher in those with, rather than without, a family history of breast cancer (6/54=11% vs. 1/29=3%) and in women with prior LCIS rather than prior ADH (6/54=11% vs. 1/24=4%).Conclusion: Breast MRI detected cancer in 7 (8%) of 83 women with a prior high-risk lesion, six (86%) of whom also had a family history of breast cancer. Among these cancers, all were detected during the first four years of screening and less than half were invasive (43%). Further work with more patients is needed to determine the utility of breast MRI screening in women with specific prior high-risk lesions, with and without additional risk factors.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4002.
Randomized Double-Blind Placebo-Controlled Clinical Trial Testing Ketorolac Tromethamine for Reducing the Pain and Discomfort Experienced during Mammography.
Authors: Freitas-Junior R. , Freitas-Junior R. , Silveira-Junior L. , Tokaski W. , Ximenes C. , Rahal R. , Santos A. , Tomazini M. , Martins E. , Freitas N.
Published: December 2010

OBJECTIVE: To study the possibility of reducing the pain and discomfort experienced during mammography, by means of using an analgesic as premedication. PATIENTS AND METHODS: This was a randomized double-blind placebo-controlled clinical trial testing ketorolac tromethamine for reducing the pain and discomfort experienced during mammography. There were 308 participants in the study, who all signed a free and informed consent statement. Of these, 153 patients (group 1) received 10 mg of ketorolac tromethamine orally and 155 (group 2) received placebo orally, 60 minutes before undergoing mammographic examination. After taking the medication and while waiting to undergo the procedure, the patients themselves filled out a form asking about personal data and possible factors relating to pain and discomfort while undergoing mammography. After the examination, the patients subjectively assessed the pain and discomfort experienced during the examination using a visual analog scale (VAS) graded from zero to ten. The statistical analysis consisted of characterizing the groups using absolute and relative frequencies and comparing the dependent and independent variables between the groups using the chi-squared association test, with Yates correction when necessary. RESULTS: The mean VAS pain level presented by the patients was 3.36 and the median was 2.90. It was observed that 51 (16.6%) of the patients did not present any pain, 106 (34.4%) presented mild pain, 94 (30.5%) moderate pain, 40 (13%) severe pain and 17 (5.5%) intolerable pain. With regard to discomfort, 68 (54.9%) reported tolerable discomfort, 63 (20.5%) a lot of discomfort and 8 (2.6%) intolerable discomfort. Among the factors related to pain, only the presence of previous mastalgia was significant (p < 0.05). Somnolence was the adverse event most present: 2.6 and 5.8% in the ketorolac and placebo groups, respectively. CONCLUSIONS: The use of 10 mg of ketorolac tromethamine did not have any influence regarding reduction of the pain and discomfort experienced by the patients during mammography, when compared with placebo. Among the factors studied, the only one associated with greater possibility of pain and discomfort while undergoing mammography was a history of mastalgia.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4014.
Invasive Lobular Carcinoma: Imaging Characteristics, Size at Presentation and Bilaterality.
Authors: Hu J. , Lanitis S. , Dimopoulos N. , Bright-Thomas R. , Cunningham D. , Faissola B. , Al-Mufti R. , Rice A. , Cleator S. , Hadjiminas D.
Published: December 2010

Aims: Invasive Lobular Carcinoma (ILC) is often described as elusive to standard imaging modalities and usually requiring mastectomy to obtain clear margins. Its reputation of being bilateral has been disputed by most recent studies. The aim of this study is to investigate whether the radiological diagnosis of ILC at first symptomatic presentation is more difficult than that of Invasive Ductal Carcinoma (IDC) in a modern breast clinic; also whether its size, surgical treatment at presentation and bilaterality is different from IDC and its subtypes.Materials & Methods: The radiological codes given to all new symptomatic patients with invasive breast cancer, presenting at the breast clinic of St Mary's Hospital and undergoing surgery as first treatment, (n=754), were collected prospectively between 1998 and 2005 and final mastectomy rates were also recorded. Histological reports of all synchronous and metachronous bilateral cancers presenting with at least one tumour between 1998 and 2007 were analyzed (n=65).Results: ILC was given suspicious radiological codes as often as IDC (90% vs 93%). On final histology, ILC measured on average 3.6cm compared to 2.5cm for IDC (p<0.05). There was a trend of ILC requiring mastectomy more often (51% vs 43%). 12.7% of patients with at least one ILC diagnosis have developed bilateral breast cancer compared to 4.5% of patients who have never had an ILC diagnosis (p<0.05).Results IDCILCMixedSpecialUnknownTotalNo of patients551 (73%)86 (11%)52 (7%)62 (8%)3 (0.4%)754Age (mean)5861596157.659Mastectomy191 (43%)36 (51%)23 (49%)13 (25%) 263Breast conservation257 (58%)36 (51%)24 (51%)41 (77%) 358Size cm (mean)2.53.62.52.0-2.7USS suspicious473 (93%)72 (90%)41 (90%)42 (76%)2 (100%)630MMG suspicious412 (81%)58 (73%)39 (80%)43 (80&)2 (100%)554 Conclusions: ILC is as easy to diagnose radiologically as IDC in symptomatic breast patients. However, ILC is larger at first presentation. In our database, ILC identified a subgroup of breast cancer patients who have a 3-fold higher risk of developing bilateral breast cancer of various histological types.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4015.