bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk Factor Combinations for Estimating the Probability of Breast Cancer Related Lymphedema.
Authors: Dirican A. , Wu W. , Johnson R. , Wilson J. , Soran A.
Published: December 2010

Background: LE is a common complication from BC surgery, leading to a decreased quality of life. In our previous study; both the risk and severity of LE were associated with 1) infection of the arm and breast, 2) body mass index (BMI), and 3) the level of hand use (p<0.05 for all associations). The aim of this study was to estimate the probability of LE following BC surgery by using previously published incidence rates and the risk factors of BMI, infection, and level of hand use.Method: A retrospective review was undertaken of the 2983 women treated surgically for breast cancer at MWH of the UPMC from 1990-2000. Fifty-two women were identified for whom the diagnosis of LE was established and data existed on the study variables. The design was a n:m matched case control study; data were analyzed on 52 patients with LE and 126 available controls matched on age, radiation therapy, and type of operation. In conjunction with conditional logistic regression and published estimates of LE incidence rates, Bayes' Theorem was used to estimate the probability of developing LE. This probability was estimated for 8 combinations of BMI, infection, and level of hand use.Results: LE probabilities of 8 combinations for 6 different incidence rates of LE are shown in Table 1. For example, assuming a 16% incidence rate of LE a BMI<25, no infection, and a low level of hand use; the estimated probability of LE was 6.8%. At the other extreme, assuming a LE incidence rate of 46.3%, a BMI ≥ 25, infection, and a high level of hand use led to an estimated LE probability of 93.7%. As shown in Table 1, other assumed incidence rates and risk factor combinations led to intermediate LE probabilities.Table 1 - Estimated LE probabilities for risk factor combinations according to LE incidences.  AssumedLEIncidenceRate Risk Factor Combinations16%17%19.5%20.7%25.5%46.3%BMI<25, No infection, Low level of hand use0.070.070.080.090.110.24BMI≥25, Infection, Medium/high level of hand use0.760.770.800.880.850.94 Conclusion: This study demonstrates that in both high and low risk situations for the development of LE that control of predisposing factors can have a marked effect on the probability of LE formation. Low risk patients with poor control of predisposing factors have a higher probability of developing LE than high risk patients in whom known predisposing factors are avoided.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5115.
Gene Expression Profiles of Endocrine Resistant Breast Tumours.
Authors: Larionov A. , Faratian D. , Caldwell H. , Sims A. , Fawkes A. , Murphy L. , Renshaw L. , Dixon J. , Dixon J.
Published: December 2010

AimTo identify molecular sub-classes of endocrine-resistant breast tumours.MethodsWhole genome expression profiles of 55 endocrine-resistant tumours were obtained using Illumina HT-12 BeadChip arrays. Endocrine resistance was defined as either (i) tumour increased in size during hormonal treatment or (ii) appearance of local recurrence or (iii) contra-lateral breast lesion (metastases or new cancers) during hormonal treatment. Most of the treated patients were post-menopausal. Hormonal treatment included either aromatase inhibitors (letrozole, anastrazole, arimidex) or tamoxifen.ResultsUnsupervised hierarchical clustering using 500 most variable genes split tumours into three major clusters of approximately equal size. The genes discriminating between these clusters included oestrogen receptor- and proliferation- associated set of genes (ESR1, TFF1, TFF3, FOXA1, AGR2, AGR3, XBP1, GATA3, PRAD6B, MYB, CCND1), and stroma-related genes (DCN, COL1A1, COL1A2, COL3A1, COL5A2). Initial analysis of the studied tumours was unable to identify genes associated with clinical features. Sub-division of the dataset may be required to identify these genes more clearly. Genes that tended to separate between tumours resistant to AI and Tamoxifen included MYB, GPR137B, RNF19A, FRMD6, ZNF217 and RPS12. More detailed analysis of the data is underway.DiscussionUnderstanding and overcoming of endocrine resistance will improve treatment results for the majority of breast cancer patients. This abstract reports a new gene expression dataset to compare molecular features of biopsies collected from tumours growing while on endocrine treatment. The initial analysis demonstrates diversity of the resistance mechanisms, enhancing development of individual approaches to endocrine-resistant tumours.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5132.
Redox Status Contributes to Tamoxifen-Resistant Growth of Breast Cancer.
Authors: Penney R. , Slingerland J. , Roy D.
Published: December 2010

The emergence of tamoxifen resistance is a major problem in the treatment of breast cancer. Tamoxifen-resistant growth coincides with a high intracellular oxidant state, and tamoxifen is known to increase intracellular oxidants in breast cancer cells. We postulate that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of chronic antiestrogen treatment therapy. In this study, we present data that show that modifying the redox state of thioredoxin (Trx) changes the sensitivity of cells to tamoxifen, and that these changes occur through Trx-Jab1-p27 interactions. The use of ebselen on tamoxifen-resistant LCC2 breast cancer cells increased thioredoxin expression and decreased DNA synthesis in tamoxifen-treated cells. In thioredoxin reductase 2 (TrxR2)-overexpressed cells, tamoxifen inhibited in vitro tumor formation as measured by growth of colonies, and also inhibited DNA synthesis. Probing Trx in a Jab1 immunoprecipitation showed that Jab1 interacts with Trx, and that overexpression of TrxR2 results in higher Jab1-Trx interaction. Similarly, using a p27 immunoprecipitation, we found that Jab1 interacts with p27, and p27 interacts with Trx. When we knock down Jab1, we show a decrease in both colony growth and DNA synthesis in tamoxifen-treated cells. These data support our hypothesis that altering the redox status of breast cancer contributes to the restoration of tamoxifen sensitivity, and that this restoration occurs through a Trx/Jab1/p27 mechanism. The data indicate that overexpressing TrxR2 and reducing oxidized Trx increases Jab1/Trx binding, restores the inhibitory effects of p27, and restores tamoxifen sensitivity to previously resistant breast cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5134.
Relationship between Quantitative HER2 Protein Expression and Clinical Outcomes in ER-Positive and ER-Negative Sub-Groups of Patients with Trastuzumab-Treated Metastatic Breast Cancer.
Authors: Bates M. , Sperinde J. , Leitzel K. , Ali S. , Ali S. , Fuchs E. , Koestler W. , Singer C. , Huang W. , Paquet A. , Weidler J. , Lipton A.
Published: December 2010

Background: HER-2/neu over-expression is associated with poor prognosis and with improved outcomes on trastuzumab in patients with metastatic breast cancer (MBC). Estrogen receptor (ER)-mediated signaling in breast cancer cells has been linked to HER2 gene expression, and some ER+/HER2- patients fail tamoxifen with HER2+ disease. These data suggest an interaction between the ER and HER2 signaling mechanisms that may complicate trastuzumab responsiveness in MBC. We have previously reported that quantitative measurements of HER2 protein expression (H2T) using the HERmark assay identifies sub-populations of previously characterized “HER2-positive” patients (by IHC and/or FISH) that have different clinical outcomes on trastuzumab (Leitzel, ASCO 2008; Lipton, SABCS 2008). Here we investigate whether the relationship between quantitative HER2 protein expression and clinical outcomes on trastuzumab are the same in ER+ and ER- sub-populations.Methods: 100 patients who had received trastuzumab in the metastatic setting and for whom ER status was known were tested by the HERmark assay and H2T levels were determined. Only 6 of 36 ER+ patients that had received hormonal therapy in the adjuvant setting subsequently received hormonal therapy (exemestane) concomitantly with trastuzumab in the metastatic setting. The HERmark measurements were correlated with TTP and OS using Kaplan-Meier and Cox proportional hazards regression analyses stratified by ER status.Results: 64 patients in the cohort were ER-negative and 36 were ER-positive. Considering all patients and using an optimized cutoff defined by positional scanning to discriminate high from low H2T, patients with high H2T levels experienced longer TTP than those with lower H2T levels (median TTP 11.3 vs. 4.5 mos., HR=0.5, p=0.002). Those patients that were ER-negative showed similar results (median TTP = 11.3 vs. 4.5 mos, HR=0.42, p=0.002) whereas there was no significant relationship between H2T and TTP in ER+ tumors (median TTP 9.6 vs. 6.7 mos, HR=0.58, p=0.15). For OS, there was no statistically significant relationship with H2T in the whole population (median OS 35.5 vs. 28.7 mos, HR=0.74, p=0.26) or the ER+ sub-group (HR=0.9, p=0.84), but there was in the ER-negative sub-group favoring high H2T (median OS 31.7 vs. 26.6 mos, HR=0.52, p=0.047).Conclusions: These data suggest that HER2 levels in ER+ breast tumors may change in response to hormonal treatment and such changes may alter the predictive value of HER2 assessments made prior to treatment. Studies comparing quantitative HER2 levels in ER+ tumors before and after exposure to hormonal therapy are needed. These data are not conclusive given the small number of ER+ tumors studied, and require confirmation in larger datasets.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5136.
2-(4-Hydroxyphenyl)-Benzo[b]thiophen-6-ol, an Estrogen-Like Compound, Induces Apoptosis in T47D/PKCα Breast Cancer Cells.
Authors: Molloy M. , Thatcher G. , Bolton J. , Tonetti D.
Published: December 2010

Background: Tamoxifen (TAM) treatment failure is a major obstacle encountered in the clinical setting. Our lab has previously shown that constitutive overexpression of PKCα imparts a TAM resistant/ hormone independent phenotype in the T47D:A18 breast cancer cell line. Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, 2009). Before TAM treatment was introduced, breast cancer patients received high-dose E2 and diesthystilbesterol (DES) treatment. DES treated patients may have a survival advantage over TAM treated patients in the long term (Peethambaram,1999). Others have shown that E2 can have inhibitory effects on MCF-7 cells both in vitro and in vivo (Lewis-Wambi, 2009). Taken together, this suggests an estrogenic compound may be efficacious for TAM-resistant breast cancer. The present study was designed to evaluate the effect of an estrogenic compound, 2-(4-hydroxyphenyl)-benzo[b]thiophen-6-ol (BTC), related to the SERMs raloxifene and arzoxifene, on the T47D:A18/PKCα breast cancer cell line.Methods: To determine if BTC can activate estrogen regulated genes we preformed an ERE-luciferase reporter assay 24 hours after BTC treatment. 3D Matrigel colony formation assay was performed to determine if BTC could inhibit colony formation. Colonies were stained and counted on day 10. To determine if apoptosis was occurring, cells were grown in Matrigel and the TUNEL Assay was performed on Day 6.Results: T47D:A18/PKCα cells showed a 31-fold induction in ERE-luciferase reporter activity when treated with BTC (10-7M) compared to 7-fold induction when treated with E2. In the T47D:A18/Neo parental cell line, BTC induced a 40-fold induction whereas estradiol induced a 96-fold induction. In the Matrigel colony formation assay T47D:A18/PKCα cells formed significantly fewer colonies when treated with BTC. Conversely, in the T47D:A18/Neo parental cell line BTC enhanced colony formation similar to E2 treatment. Our results indicate that BTC was able to induce significant apoptosis on day 6 (P= 0.038) as determined by the TUNEL Assay. Therefore BTC acts similarly to estradiol by inhibiting T47D:A18/PKCα colony formation and inducing apoptosis when cells are grown in Matrigel.Conclusion: These findings suggest that BTC is a potential lead compound in the treatment of PKCα overexpressing breast cancer. Furthermore, BTC was shown to be a potent inducer of the cytoprotective enzyme NQO1 in the Hepa 1c1c7 cell system as well as a strong activator of antioxidant responsive elements (Yu, 2007) indicating it may have the added benefit of chemoprotective effects which are absent in estradiol treatment. Further understanding of the mechanism in which BTC induces apoptosis in the T47D:A18/PKCα cell line may result in a clinical advantage to estradiol treatment including fewer side effects.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5140.
CYR61, Estrogen Receptor and FOXO3a: The Interplay in Aggressive Breast Carcinomas.
Authors: Espinoza I. , Liu H. , Busby R. , Lupu R.
Published: December 2010

Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. More than 60% of human breast cancer tumors express ER and many of those are Estrogen (E2) dependent. ER positive tumors are considered to have a good prognosis; however some tumors escape to E2 requirements. Thus, the identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The pro-angiogenic factor CYR61 induces breast cancer cells to bypass the normal estrogen requirements for growth and acquire an estrogen-independent and antiestrogen resistant phenotype. However, the mechanism by which CYR61 regulates the hormonal response in breast cancer is not well understood. We over-expressed CYR61 in the ER positive cells MCF-7 (MCF-7/CYR61). Our results demonstrated that CYR61 induces constitutive transcriptional activity of the ER, which is independent of E2. Moreover, we demonstrated that CYR61 co-localizes with the ER in the nucleus and casually CYR61 transcriptional activity is regulated, at least in part by FOXO3a binding site. In search of the mechanism by which CYR61 that modulates hormonal response we discovered that CYR61 and FOXO3a interact in the presence of E2. This functional interplay among CYR61/ER/FOXO3a most likely plays a critical role in blocking hormone-response of breast cancer growth in vitro and in vivo. Furthermore, these studies demonstrate that CYR61 and FOXO3a are novel therapeutic targets to halt hormone-independent breast carcinomas.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5145.
Intermittent Hypoxia: Potential Factor of Resistance to Endocrine Therapy.
Authors: Yin W. , Liu G. , Di G. , Shen Z. , Shao Z.
Published: December 2010

Background: It has been reported that intermittent hypoxia, which is likely more prevalent than acute hypoxia in breast cancers, can cause persistent depression of estrogen receptor alpha (ER-α) expression in breast cell lines. However, it remains suspension whether intermittent hypoxia may be associated with resistance to endocrine therapy.Material and methods: The role of intermittent hypoxia in resistance to endocrine therapy was investigated in ER-α positive breast cell lines and animal models.Results: We observed that intermittent hypoxia promoted cancer cell proliferation in vitro and in vivo compared with normoxia. At both mRNA and protein levels, intermittent hypoxia induced down-regulation of ER-α and factor inhibiting HIF-1 (FIH) as well as up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) and carbonic anhydrase IX (CA-IX). ER-α positive breast cancer cells under normoxic condition were capable of an intact response to ICI 182,780 (Tocris), whereas the inhibitory effect of ICI 182,780 (Tocris) was significantly reduced in those after intermittent hypoxia. Such effect was further validated with fulvestrant (Astrazeneca) in the animal models. To clarify whether intermittent hypoxia was associated with resistance to endocrine therapy, the expression of HIF-1α, a critical regulator of hypoxia-related pathway, was blocked by a reformed type of small interfering RNA (siRNA), stealth RNAi (Invitrogen), which can reduce the cytotoxic interferon response unlike conventional siRNA. Interestingly, knock-down of HIF-1α did result in the restoration of not only the ER-α expression but also the response to endocrine therapy, which was reproducible with administration of Bortezomib, a proteasome inhibitor of HIF-1α.Discussion: These data provide functional evidence that intermittent hypoxia may confer resistance to endocrine therapy in breast cancers through the crosstalk between HIF-1α and ER-α signaling, which holds a promise to overcome endocrine resistance.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5148.
Metabolic Syndrome in Breast Cancer: Paracrine Mediators in Mammary Adipose Tissue Mediated by Metabolic Dysfunction Influence Tumour Cell Proliferation.
Authors: Carroll P. , Carroll P. , Healy L. , Lysaght J. , Ennis D. , Dunne B. , Griffin M. , Boyle T. , Kennedy J. , Pidgeon G. , Pidgeon G. , Connolly E. , Connolly E.
Published: December 2010

Introduction:Obesity and it's associated metabolic syndrome are known epidemiological risk factors for breast cancer.¹ Adipose tissue is a potent endocrine organ secreting many factors that can potentially regulate tumour growth. The breast constitutes an excellent model to determine the paracrine mediators of adiposity on breast cancer. The aim of this preliminary study was thus to describe the adipocytokine milieu in the breast and determine what influence adiposity and metabolic dysfunction played in tumour progression.Methods:Adipose conditioned media (ACM) was produced by harvesting adipose tissue from sites adjacent to (peritumoural PT) and distal (D) to tumour in 20 fresh mastectomy specimens and cultured in serum free media for 72hrs. MDA-MB 231 and MCF-7 cell lines were then cultured with this ACM for 24-48hrs. Cell survival/proliferation was determined by BrDU assay. An in depth characterisation of adipocytokine production from each adipose depot was performed using multiplex bead arrays.Results:ACM from peritumoural/distal adipose tissue promoted cell proliferation in both cell lines. The greatest proliferative effect was observed in ER+ve MCF-7 cell line relative to controls (151.9±7.3% and 150.8±9.8% in peritumoural and distal ACM respectively), compared to ER-ve MDA-MB-231 cells (111.4±5.0, 112.0±2.5%).PT ACM from metabolically unhealthy/obese patients significantly increased proliferation in MCF-7 cells, compared to normal weight cancer patients (171±12.6% vs 135.3±9.3%, p=0.034). Similarly, adipocytokine expression reflected these changes with expression differences in IL-6 (6.72±0.09, 6.31±0.16 ng/ml p=0.028), HGF (7.5±1.3, 3.98±0.43 ng/ml p=0.028).Conclusion:Peritumoural adipose tissue from metabolically unhealthy and obese patients increased cell proliferation compared to normal weight patients. Metabolic dysfunction influenced expression of several adipocytokines in ACM. A thorough analysis of this milieu should lead to a greater understanding of how obesity/metabolic dysfunction regulate tumourigenesis in breast cancer.References:1: Lahmann PH et al: Body size and breast cancer risk: findings from the European Prospective Investigation into cancer and Nutrition (EPIC) International J Cancer 111: 762-771

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5150.
Stage-Specific Expressions of GLUT-1, GLUT-3 and GLUT-12 in Estrogen-Induced Breast Cancer.
Authors: Kocdor M. , Kocdor H. , Pereira J. , Vanegas J. , Russo I. , Russo J.
Published: December 2010

Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. The glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12 have been shown in breast cancer cells in which estrogen play a critical role on its regulation and also associated with poor prognosis. However. the stage-specific GLUT regulation during the process of mammary carcinogenesis is unclear. In the present study we are aiming to study the GLUT expression pattern in an in vitro-in vivo model of estrogen induced carcinogenesis in which the human breast epithelial cell line MCF-10F is considered the normal counterpart E2 the transformed, C5 the invasive and T4 the tumorigenic stage. The gene expression of GLUTs was correlated with the ductulogenic pattern in a collagen matrix and the invasive properties using the Boyden chamber The RT-PCR data showed that the GLUT1 expression was downregulated in MCF10F after the treatment with 17β-estradiol (E2) and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasion capacity. Whereas, GLUT-12 is downregulated in invasive and tumorigenic cells. We conclude that the estrogen-induced malignant transformation is associated with remarkably GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation for transformed MCF-10F cells. (This work was supported by grant R21 ES015894from the NIEHS)

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5152.
Mir-128 Regulates the Self-Renewal of Breast Tumor-Initiating Cells through HTERT.
Authors: Yu F. , Zhu Y. , Gong C. , Jiao Y. , Song E.
Published: December 2010

Background: hTERT (human telomerase reverse transcriptase), which maintains telomere ends by addittion of the telomere repeat sequence TTAGGG, is increased in cancer cells and embryonic stem cells. However, whether hTERT has a role in regulating breast tumor-initiating cells remains unknown. Therefore, the regulation of hTERT in breast tumor initiating cells (BT-IC) would potentially facilitate our understanding of the etiology of breast cancer.Material and Methods: Enrichment for BT-ICs was performed by mammospheres from suspension cultures. The capacity of self-renewal and differentiation of BT-ICs were determined by mammosphere formation, immunohistochemical analysis of epithelial markers and FACS, respectively. TRAP assay was used to detect telomerase activity. The expression levels of hTERT in BT-ICs, with or without lentivirus-mediated mir-128 transduction, were determined by qRT-PCR and western blot. hTERT siRNA or lentivirus-shTERT were used to silence the expression of hTERT in BT-ICs. Differentially expressed mir-128 in BT-ICs was quantified by qRT-PCR and Northern Blot. The expression of BMI1 was determined by Western Blot.Results: hTERT, as well as telomerase activity, had a higher expression in BT-ICs, compared to the differentiated breast cancer cells. BMI1, the target of mir-128, is increased in BT-ICs. Conversely, the expression level of mir-128 is lower in BT-ICs, compared to the differentiated cells. Mir-128 over-expressing reduced the level of BMI1,as well as hTERT, in BT-ICs. Silencing BMI1 by lentivirus-shRNA transfection in BT-ICs leads to the decrease of hTERT protein level. Furthermore, BT-ICs, with hTERT knockdown, lost its ability to form mammospheres and proliferated poorly under differentiating conditions.Conclutions: mir-128 miRNA is reduced in mammospheric BT-ICs, resulting in up-regulation of BMI1, which further brought on the up-regulation of hTERT. And hTERT promotes self-renewal and blocks epithelial differentiation of BT-ICs. Therefore, mir-128 regulates BT-IC stem-cell like properties by BMI1 and hTERT.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5157.
Quantitative Copy Number Analysis of Onco- and Tumor Suppressor Genes in Invasive Breast Cancer by Dedicated Multiplex Ligation-Dependent Probe Amplification.
Authors: van Diest P. , Moelans C. , Monsuur H. , de Weger R.
Published: December 2010

Several genes have been shown to be implicated in the development, progression and response to therapy of invasive breast cancer. The phenotypic uniqueness (and thus the heterogeneity of clinical behavior) among patients' tumors may be traceable to the underlying variation in DNA copy number. In the present work, we sought to obtain a more complete view of copy numbers of breast cancer related genes by multiplex ligation-dependent probe amplification (MLPA) in 104 invasive breast cancers, and to assess phenotypic correlations.There was a significant correlation between the number of amplifications on the one hand and histological grade and mitotic index on the other. MLPA revealed the following frequent amplifications: MYC in 48%, PRDM14 in 34%, TOP2A in 32%, ADAM9 in 32%, HER2 in 28%, CCND1 in 26%, EMSY in 25%, IKBKB in 21%, AURKA in 17%, FGFR1 in 17%, ESR1 in 16%, CCNE1 in 12% and EGFR in 9%. Amplifications of EGFR, CCNE1 and HER2 were negatively associated with ER while FGFR1, ADAM9, IKBKB and TOP2A were positively associated with ER. Amplifications of ESR1, PRDM14, MYC and HER2 were associated with a higher mitotic index and PRDM14 and HER2 amplifications with higher grade. MYC amplification was more frequent in ductal tumors and high level MYC amplifications were significantly associated with larger tumor size. HER2-MYC, HER2-CCNE1 and EGFR-MYC co-amplified tumors were significantly larger than tumors with either of both amplifications. CDH1 (20%) and FGFR1 (10%) were the most frequently lost genes.In conclusion, gene copy number analysis with this “breast cancer MLPA kit” allowed to simultaneously assess copy numbers of 20 important breast cancer genes, providing an overview of the most frequent (co)amplifications as well as interesting phenotypic correlations, and thereby data on the potential importance of these genes in breast carcinogenesis and progression.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5168.
Multiplexed Tumor Marker Profiling at Single-Cell Resolution: Quantifying Breast Cancer Molecular Signatures.
Authors: Liu H. , Huebschman M. , Garner H. , Lane N. , Cheng L. , Rana D. , Lin A. , Hughes G. , Uhr J.
Published: December 2010

Excluding skin cancers, about 1.5 million new cases of cancer and about 450,000 cancer related deaths occur each year in the United States. The validation and molecular profiling of tumor markers are contributing to new paradigms in cancer research. In this presentation, we will discuss the information that can be obtained by using an Intelligent Single-Cell Optical Profiling Engine (I-SCOPE), consisting of hyperspectral imaging, multiplexed biomarker kits, and bioinformatics tools, to markedly expand the molecular profiling of tumor markers on individual carcinoma cells.Using biomarker antibodies conjugated to particular fluorochromes, we developed a multiplex of 10 different fluorochrome conjugates, one for each different tumor marker plus a nuclear dye. Staining and scanning using the I-SCOPE system showed that it can presently distinguish 10 different fluorochromes in a single pass, resulting in the identification of spatial location and relative intensity of each biomarker being interrogated. By using this system, we analyzed thousands of tumor cells from touch preparations of tumor tissue, circulating tumor cells (CTCs) in pleural fluid and blood, and disseminated tumor cells (DTCs) from bone marrow with 2 multiplex kits for a total of 17 different tumor markers. We have utilized clinically-accepted markers such as estrogen receptor (ER), progesterone receptor (PR), and HER2 in addition to biomarkers undergoing investigation such as B-cell lymphoma 2 (Bcl-2) and urokinase-type plasminogen receptor (uPAR). Furthermore, with the ability to simultaneously image 10 markers, each panel can also contain desired positive and negative controls such as CD45 (positive for white blood cells, negative for CTCs) and cytokeratin (positive for epithelial cells).The results of immunophenotyping of individual tumor cells with 17 different tumor markers indicated that the average expression of each tumor marker in 25 tumor cells was a reproducible representation of the tumor. However, comparison of individual CTCs' expression levels revealed remarkable heterogeneity in primary breast cancer cases.We conclude that hyperspectral microscope imaging together with individual tumor cell analysis represents a major step forward in developing a far more detailed and precise molecular signature of a patient's cancer. Further studies might demonstrate how it can facilitate treatment tailored to the individual patient's tumor, an important objective as new oncology drugs with specific molecular targets become more prevalent.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6002.
Grade 3 Status Dominates Prognosis in Breast Cancer.
Authors: Oliveros S. , Unger K. , Morishita M. , Davies C. , Gudi M. , Thomas G. , Leonard R.
Published: December 2010

Background: Histological grading (HG) is probably the most important clinico-pathological parameter that will provide information about tumour intrinsic characteristics and aggressiveness. HG is included in prognostic guidelines for treatment decision making such as St Gallen, Nottingham Prognostic Index and Adjuvant!Online. Grade 3 (G3) Breast cancer (BC) is associated with poor prognosis in terms of survival and recurrence. G3 BC reflects independent biological entities rather than being the end of the spectrum of cancer progression. Our aim is to establish the clinical relevance of HG as prognostic factor in a specific cohort of BC patients and define its relation with survival and recurrence endpoints.Method: This is a retrospective cohort analysis from 1489 operable Breast Cancer patients attending the Oncology Service at Singleton Hospital, Wales, UK between 1996 and 2002.HG was recorded for 1339 cases and outcome data were available on 1213 cases. Correlation of clinical variables with Overall Survival (OS), Distant Disease Free Survival (DDFS) and Disease Free Survival (DFS) was performed using Cox regression model.Results:The median follow-up for the cohort was 5.43 years (range 0.09 to 10.14 years) Patients were characterised according to age, HG, tumour size (TNM-T), nodal status, ER status and systemic treatment received (polychemotherapy/tamoxifen). Independent Cox regression models for each endpoint were developed based on univariate analyses. Multivariate analysis results are presented in Figure 1.Subgroup analysis of G3 cases showed a younger population: median age G3 59.4y vs G1/G2 62.9 y p:< 0.001); 47.5% of G3 cases were ER positive against 94.5% of Grade 1/2 (p < 0.001). Patients with G3 tumours died more rapidly from all causes (median time to death 2.22 vs 3.42 y, p <0.001) and from BC (median time to death from BC 2.22 y vs 3.82 y in G1/G2, p <0.001) than other HG. Similarly, G3 also relapsed quicker both distantly, (median time 1.64 vs 2.54 y G1/G2 (p<0.04) and locally, median time to relapse 1.6 vs 2.35 y for G1/G2 (p<0.036). There was also a significant association between grade and tumour size (median G1/G2 1.7cm vs G3 2.2cm(p< 0.001); and nodal involvement and tumour size, median 1.5cm N-ve, 2.2 cm N+ve (p< 0.001).Conclusion: Patients with G3 tumours tended to be younger, had a more advanced TNM staging at diagnosis and relapsed and died more rapidly than other histological grades. In our cohort, G3 tumours were adversely associated with OS, DDFS and DFS with a stronger association with these endpoints than node positivity. It is perhaps time to reconsider the HG as a prognostic factor as important as nodal status for the selection of adjuvant chemotherapy regimens.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6005.
Prelminary Results of a NEQAS Ring Study Evaluating the Ventana INFORM™ HER2 Dual Colour Single Slide ISH Assay.
Authors: Bartlett J. , Campbell F. , Ibrahim M. , Kay E. , Jasani B. , Morgan J. , O'Grady A. , Di Palma S. , Starczynski J. , Miller K.
Published: December 2010

Background: Recent innovations in HER2 testing include the development of novel chromogenic in situ hybridisation tests. Most countries use FISH or ISH based testing for HER2 gene amplification in equivocal (IHC 2+) cases. Data on the reproducibility and portability of novel and even established HER2 ISH methods is sparse. We report initial results from the multicentre evaluation of intra and inter-observer/site variation in scoring HER2 results from 30 breast cancers measured in 5 laboratories using the Ventana Dual colour ISH assay.Methods: A commercially available tissue micro-array (TMA; Stretton Scientific UK) containing 2 replicate cores from 30 breast cancers was circulated to 5 UK NEQAS reference laboratories (numbered 1-5). All laboratories performed chromogenic ISH using a 60 core TMA and reported results for all assessable cores. For all cases 20 cells were scored and a further 20 cells scored on a separate occasion to check intra-observer variation. Results were collated centrally and compared with FISH peformed in a single laboratory. Concordance between FISH and CISH is reported on a core by core and case by case analysis. Intra and inter-observer variation between laboratories is also assessed. Finally regression analysis comparing results was performed. Results from a single laboratory are presented here and full results will be available for the meeting.Results: 93% of cores were successfully analysed by the Ventana dual colour ISH assay. 87% of cores were successfully analysed by PathyVision® FISH CISH was concordant with FISH in 96.4% of cores evaluated. In one sample FISH results were 2.05 & 1.93 vs CISH of 2.03 and 2.14. In a second sample FISH results were 1.60 & 1.53 versus CISH results of 2.00 & 1.65. When mean results were calculated (2 cores per case) concordance between FISH and CISH results for this laboratories was 96.7% (1 discordant case – mean FISH – 1.99, mean CISH 2.09). 48 cores were scored twice (20 cells x 2) by a single observer. Mean intra-observer variation (for HER2 ratio) was 1.72% (Range 0.0-7.42%). No difference in scores was observed for the first vs second 20 cells. Scoring 40 cells produced no advantage. For results from a single observer for 2 cores (intra-site variation) from 42 core pairs (each core counted twice) variation was 7.5%.Conclusions: Preliminary evidence from the current study suggest that the Ventana INFORM™ HER2 dual colour single slide ISH assay can be robustly evaluated using conventional scoring approaches (20 cells per case). Concordance with FISH was excellent in the single laboratory reporting results to date. Further evidence will be reported from the other 4 participating laboratories.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6011.
Molecular Characterization of Breast Cancer Core Biopsy Specimens by Gene Expression Analysis Using Standardized Quantitative RT-PCR.
Authors: Anderson J. , Shak S. , Millward C. , Labusch M. , Chen I. , Watson D. , Baehner F.
Published: December 2010

Background: Core biopsies (CBx) are increasingly the initial diagnostic procedure of choice for breast masses. Clinical studies of the 21 gene assay, which was optimized for small amounts of fixed paraffin-embedded tumor tissue, have been performed on core biopsies in the neoadjuvant setting (Gianni et al, JCO 2005 and Chang et al, Br Cancer Res Treat 2008). We report here the results of pathology review and quantitative RT-PCR analysis of a large number of CBx in clinical practice.Methods: Cases included all Oncotype DX tests reported by the Genomic Health laboratory from July 15, 2005 through May 31, 2009. Pathology review of H and E stained sections, manual microdissection, RNA quantification, and quantitative RT-PCR analysis for each gene were performed according to the pre-specified eligibility criteria and methods used in the NSABP and Kaiser Permanente clinical validation studies. Descriptive analysis was performed on de-identified data with IRB approval.Results: There were 103,863 submissions to the clinical laboratory, of which 11,757 (11.3%) were identified on pathology review as CBx. The initial submission success rate was 94.3% on all cases, and was slightly lower in the CBx (91.6%) than in surgical resections (SRx) (95.7%). The most common reason for initial failure was insufficient tumor or no tumor found in 5.6% of CBx and 3.0% of SRx. Insufficient RNA (<275 ng) was found in 2.6% of CBx and 0.3% of SRx. Other causes of failure, such as RT-PCR process failures, were uncommon. In 80% of cases of initial failure another specimen was submitted by the site; the success rate on the resubmissions was 80% for both initial CBx and initial SRx. Thus, the overall success rate was >97%. As expected, the rate of manual microdissection to remove non-tumor elements was higher in SRx than CBx (33.0 vs 3.9% of cases). In addition, the total yield of RNA, on average, was greater in SRx than CBx (4.2 µg vs 2.5 µg). The average Recurrence Score (RS) in CBx and SRx was similar - RS mean (±SD) = 18.9 (±12.4) in CBx and 19.7 (±11.3) in SRx. A wide range of RS was observed in CBx. The proportion of tumors with RS <18, 18 - 30, and ≥ 31 was 58.8%, 28.6%, and 12.6% in CBx and 52.3%, 35.6%, and 12.1% in SRx. The distributions of quantitative ER, PR, and HER2 in CBx were also similar to SRx.Conclusions: This large study indicates that quantitative RT-PCR analysis can be successfully performed on breast cancer core biopsies. Rigorous pathology review prior to gene expression analysis to identify cases with insufficient or no tumor is required to ensure the quality of gene expression analysis.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6021.
Factors, Predicting Failure of Adjuvant Hormonotherapy of Breast Carcinoma. A Study in Tamoxifen Treated Patients.
Authors: Petrakova K. , Nenutil R. , Grell P. , Palacova M. , Vyzula R.
Published: December 2010

Background: The latest clinical trials indicate better performance of aromatase inhibitors, compared to tamoxifen, in adjuvant hormonotherapy of breast carcinoma. The identification of molecular markers, predicting resistance to tamoxifen, could help to identify patients, which are most likely to benefit from aromatase inhibitors in up-front adjuvant hormonotherapy.Material and methods: Tissue microarrays were constructed from archival paraffin blocks of primary tumors of 179 patients with estrogen receptor positive operable breast carcinoma in stage I-III, subsequently treated with tamoxifen for five years or until relapse, with at least 7 years follow up available. The amplifications of Her-2 and cyclin D1 genes were evaluated by fluorescence in-situ hybridization. The level of progesterone receptor (PR) and Ki67 were estimated by immunohistochemistry.Results: 54 of above patients recurred during follow up. In univariate analysis of disease free survival, the presence of more than three nodal metastases (RR=4,5 p<0,001), grade 3 (RR=2,3 p=0,035), cyclin D1 (RR=3,06 p<0,001) and Her-2 (RR=3,06 p<0,001) amplifications were identified as significant risk factors, together with the negativity of PR (RR=2,1 p=0,013). In multivariate analysis, only clinical stage III (RR=2,6 p=0,003), cyclin D1 (RR=2,7 p=0,001) and Her-2 (RR=2,1 p=0,014) amplifications proved significant. In 77 patients who received adjuvant chemotherapy no statistically significant risk factor was identified. In multivariate analysis of 102 patients without adjuvant chemotherapy only stage III (RR=6,9 p=0,001) and Her-2 amplification (RR=4,5 p=0,001) were confirmed.Discussion: The advanced clinical stage, cyclin D1 and Her-2 gene amplifications represent factors, predicting the failure of adjuvant tamoxifen treatment, but their predictive value is much lower in patients receiving adjuvant chemotherapy. This fact indicates, they can reflect the common biological aggressiveness of tumor and need not to be tamoxifen specific.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6031.
The Relationship between Pre-Operative Biomarkers of Inflammation, Clinicopathological Characteristics and Cancer Specific Survival in Early Breast Cancer.
Authors: Obondo C. , Mansell J. , Afra A. , Doughty J. , Lannigan A. , McMillan D.
Published: December 2010

Breast cancer is the second most common malignancy in women. Established prognostic factors include tumour size, grade, nodal involvement, and hormonal receptor status. Recently, the systemic inflammatory response (as evidenced by elevated C-reactive protein and low serum albumin) has been established as an independent predictor of survival in patients with metastatic breast cancer (1). However, the relationship between these systemic inflammatory markers, clinicopathological characteristics and cancer specific survival has not been established in early breast cancer.During the period June 2001 to May 2008, patients with early breast cancer presenting to two hospitals in the West of Scotland were prospectively included into this study (n=959). Preoperative C-reactive protein, albumin and clinico-pathological data were recorded for each patient. The thresholds for normal C-reactive protein and albumin were taken as <6 mg/l and >43g/l respectively.The median follow-up of the survivors was 4.1yrs. During this period, 93 patients died of their cancer. On multivariate analysis, tumour size (HR 2.03; 95%CI 1.41-2.91, P<0.001), lymph node status (HR 2.23; 95%CI 1.45-3.41, P<0.001), hormone receptor status (HR 1.58; 95%CI 1.24-2.00, P<0.001) and albumin <43g/l (HR 1.97; 95%CI 1.28-3.01, P=0.002) were significant independent predictors of cancer-specific survival. Lower serum albumin concentrations (<43g/l) were associated with deprivation (P=0.019) and significantly poorer 5-year cancer-specific survival (85 vs 92% P=0.005).The results of the present study show that lower preoperative albumin concentrations, but not elevated C-reactive protein concentrations, predict cancer-specific survival, independent of clinico-pathologic status in early breast cancer. Further, evaluation of the pre-operative systemic inflammatory response and outcome in patient subsets is warranted.1. Al Murri AM, Wilson C, Lannigan A, Doughty JC, Angerson WJ, McArdle CS, McMillan DC. Evaluation of the relationship between the systemic inflammatory response and cancer-specific survival in patients with primary operable breast cancer.Br J Cancer. 2007;96:891-5

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6033.
Sequential Treatment with Epirubicin/Cyclophosphamide, Followed by Docetaxel Is Equieffective, but Less Toxic Than FEC120 in the Adjuvant Treatment of Breast Cancer Patients with Extensive Lymph Node Involvement: The German ADEBAR Phase III Study.
Authors: Janni W. , Harbeck N. , Sommer H. , Rack B. , Augustin D. , Simon W. , Jueckstock J. , Wischnik A. , Annecke K. , Hepp P. , Friese K. , Kiechle M.
Published: December 2010

Background: Based on meta-analytic evidence, taxane containing adjuvant chemotherapy has been established as standard treatment in node-positive breast cancer. However, in the MA-21 study, adriamycin-cyclophosphamide, followed by paclitaxel (AC-P) was significantly inferior to the gold standard of anthracycline treatment, FEC120 (Burnell, SABCS 2006). We prospectively compared a sequential epirubicin-docetaxel chemotherapy regimen to FEC120.Patients and Methods:The ADEBAR study was a multicenter phase III trial (n=1502) to evaluate whether breast cancer (BC) pts with > 3 axillary lymph node metastases benefit from a sequential anthracycline-docetaxel regimen (E90C–D: 4 cycles epirubicin [E] 90 mg/m2 plus cyclophosphamide [C] 600 mg/m2 q21 days followed by 4 cycles docetaxel [D] 100mg/m2 q21 days) compared to dose-intensive anthracycline-containing polychemotherapy (FE120C: 6 cycles E 60 mg/m² d 1+8, 5-FU 500mg/m² d 1+8 and C 75 mg/m² d 1-14, q4 weeks). The median follow-up time was 47 mts.(range 2-83 mts).Results:Treatment was stopped prematurely in 3.7% of the pts in the E90C–D arm and in 8.0% in the FE120C arm due to toxicity (p=0.0009). Antibiotic treatment was given in 10.4% (E90C–D) vs. 19.7% (FE120C), G-CSF support in 39.2% vs 61.4 % and erythropoietin stimulation in 8.7% vs. 20.0%, respectively (p<0.0001). Haematological toxicity (leucopenia, neutropenic fever, thrombocytopenia, anemia) was significantly higher in the FE120C-arm.At the time of the current analysis, 281 events of recurrence of breast cancer, were observed: 128 events in the FE120C group and 153 in the E90C–D group. The unadjusted hazard ratio (HR) was 0.88 (95 percent confidence interval, 0.694 to 1.115; p=0.2197, log-rank test). Overall survival in the two groups was not significantly different: (84 deaths with FE120C vs. 88 with E90C–D (HR 0.999, 0.738-1.352, p=0.99). Subgroup analyses, stratifying for tumor size, lymph node involvement, hormone receptor and HER2-neu status showed no significant difference between the two treatment arms.Conclusion:Different toxicity profiles given, hematological toxicity in the FE120C group was more severe than in the E90C–D.In contrast to AC-P in earlier studies, EC-Doc provides a feasible and effective alternative option to dose-intensified FEC with different saftey profile in this high risk breast cancer cohort.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 604.
Cyclin B1 Is an Independent Prognostic Proliferation Marker with a High Reproducibility in a Population-Based Lymph Node Negative Breast Cancer Cohort.
Authors: Niméus-Malmström E. , Koliadi A. , Ahlin C. , Holmqvist M. , Holmberg L. , Amini R. , Jirström K. , Wärnberg F. , Blomqvist C. , Fernö M. , Fjällskog M.
Published: December 2010

Purpose: A large proportion of women with lymph node negative breast cancer treated with chemotherapy do not benefit from such treatment. Proliferation markers can recognize patients at high risk for recurrence. However, no proliferation marker is generally accepted for clinical use. We investigated the independent prognostic importance of cyclin B1 in node negative breast cancer and included a study of the reproducibility.Study Design: In a population-based, case-control study, 190 women who died from breast cancer were defined as cases and 190 women alive at the time for the corresponding case's death as controls. Inclusion criteria were tumor size ≤ 50 mm, no lymph node metastases, and no adjuvant chemotherapy. Tumor tissue was immunostained for cyclin B1. Two investigators evaluated the staining independently by counting approximately 100, 200, 500, and 1000 cells.Results: Cyclin B1 was statistically significantly associated to breast cancer death, in both uni- and multivariate analyses (adjusted for tumor size, age, and endocrine therapy), with odds ratios 2-3 for both investigators. The reproducibility between the two investigators was good to very good, regardless of the number of counted cells (kappa values between 0.74 and 0.82).Conclusions: Cyclin B1 is a prognostic factor for breast cancer death in a population-based and node negative patient cohort and can identify high-risk node negative patients with a good to very good reproducibility.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6051.