bluuebunny/crossref_metadata_2025 huggingface dataset:
The Frequency and Determinants of Metabolic Syndrome in Patients with Operable Stage I-III Breast Cancer.
Authors: Aksoy S. , Dizdar [. , Dede D. , Arslan C. , Dogan E. , Tapan [. , Özkan [. , Hesenov S. , Cinel M. , Yaman S. , Kizilarslan M. , Özisik Y. , Altundag K.
Published: December 2010

Background: Metabolic syndrome is a cluster of multiple risk factors including obesity, insulin resistance, hypertriglyceridemia, low HDL (high density lipoprotein), and hypertension. Metabolic syndrome is associated with an increased risk of breast cancer. We evaluated the frequency and determinants of metabolic syndrome in patients with operable stage I-III breast cancer.Methods: In this cross-sectional study we analyzed the frequency and determinants of metabolic syndrome in patients with stage I-III breast cancer. Metabolic syndrome was defined according to NCEP (National Cholesterol Education Program) definitions. Data on metabolic syndrome criteria were collected along with demographic data, tumor size, grade, lymph node, estrogen, progesterone, HER2 status, grade and history of chemotherapy/endocrine therapy.Results: A total of 331 patients were analyzed. Median age was 50.0 years (range 20-84 years). Eighty-five patients were using tamoxifen, 98 patients were using aromatase inhibitors and the remaining 148 patients were not receiving hormonal therapy (newly diagnosed and/or triple-negative patients). According to the NCEP criteria 48.0% of the patients had metabolic syndrome. More patients receiving aromatase inhibitors had metabolic syndrome than those receiving tamoxifen (60.2% vs. 37.6%, p = 0.008). The frequency of MS among those not taking any hormonal therapy was 45.9%. Additionally, post-menopausal patients had higher rate of MS compared to pre-menopausal ones (53.4% vs. 25.0% p=<0.001). Patients with HER2 positive tumors and patients who had a history of oral contraceptive use had lower rates of metabolic syndrome than those with HER2 negative tumors and no oral contraceptive use, respectively (40,0% vs. 53,7% for HER2, p=0.028 and 32,6% vs. 50,3% for oral contraceptive use, p=0.034).There was no significant correlation between the presence of metabolic syndrome and estrogen/progesterone receptor status, tumor size, nodal status, stage, grade, adjuvant chemotherapy, hormone replacement therapy, smoking history.Conclusions: Nearly half of the patients with stage I-III breast cancer had metabolic syndrome. Adjuvant aromatase inhibitor therapy was found to be associated with higher rate of metabolic syndrome whereas history of oral contraceptive use and HER2 positivity were associated with lower rate. Whether the aromatase inhibitors contribute the development of metabolic syndrome or not needs to be elucidated by future prospective studies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5121.
Estrogen Deprivation Results in Altered DNA Methylation Profile in Breast Cancer Cells – Role in Endocrine Resistance?
Authors: Pathiraja T. , Xi Y. , Lee A. , Santen R. , Gannon F. , Kaipparettu B. , Chang J. , Li W. , Oesterreich S.
Published: December 2010

Postmenopausal breast cancer patients benefit from aromatase inhibitors (AIs) that reduce the levels of estrogens which are critical for tumor growth. To date, epigenetic contributions to hormonal therapy resistance have not been well characterized, with only a limited understanding of antiestrogen mediated epigenetic modification of chromatin and altered gene expression. Furthermore, no genome-wide studies have been undertaken to identify altered DNA methylation patterns due to estrogen deprivation, which mimic AI therapy. We hypothesized that estrogen deprivation results in altered methylation, and therefore altered expression of critical target genes in breast cancer cells, which ultimately helps the cell to survive in the absence of estrogen and thus would contribute to acquire AI resistance in breast cancer patients.We used previously established MCF-7 cell clones, termed C4-12 and LTED (Long Term Estrogen Deprivation) that were isolated after being cultured in estrogen-free media for 9 months and 18-24 months, respectively. A genome-wide methylation screen was done using Methyl CpG binding Domain (MBD) pull down assay followed by hybridization into Affymetrix Human Promoter 1.0R Array. Altered DNA methylations were validated by bisulfite genomic sequencing assays and gene expression of some selected genes were studied by qRT PCR.From the array, we found that long term estrogen deprivation results in widespread genomic hyper- and hypomethylation events. 267 and 301 genes were hypermethylated in C4-12 and LTED respectively. In both resistant cell lines, the number of hypermethyled genes was more prominent than hypomethylated genes since only 82 and 97 genes were hypomethylated in C4-12 and LTED respectively. While hypomethylation of genes generally correlated with increased gene expression, hypermethylation did not in many cases. A gene which was heavily methylated in both cell line system, possibly reflecting a more general and important pathway in estrogen-deprivation, was HOXC10. Transient and stable silencing of HOXC10 in MCF-7 cells resulted in increased cell proliferation in estrogen deprived medium, suggesting a potential role of HOXC10 in developing resistance to endocrine therapy.This is the first genome-wide approach to identify gene-specific epigenetic alterations, and the functional consequences of these changes in developing resistance to estrogen deprivation therapy. This approach allows the development of epigenetic signatures of therapeutic resistance to be used concurrently with genomic signatures to better understand the molecular mechanism of endocrine resistance. This study might provide the basis for future clinical trials such as combination therapy of DNA demethylating agents and aromatase inhibitors.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5127.
Circulating Epithelial Tumor Cell (CETC) Monitoring during SERM and Aromatase Inhibitor Treatment for Early Detection of Patients Resistant to Tamoxifen.
Authors: Pachmann K. , Camara O. , Rabenstein C. , Winzer H. , Runnebaum I.
Published: December 2010

The observation, that part of breast cancer patients benefit from ovarectomy led to the realization that in these patients the estrogen receptor crucially contributes to the growth potential of malignant tumors of the female reproductive system. This has led to the development of compounds specifically binding to the estrogen receptor but without activating activity. Although this has led to an impressive improvement in relapse free survival a considerable part of patients some of which being carriers of the CYP2D6 polymorphism will not respond to this targeted treatment. Therefore, in addition to defining the subpopulation with a high probability of responding to the targeted therapy only an immediate control of the response to treatment will ultimately improve outcome.Epithelial tumor cells circulating in peripheral blood are easily accessible and have been shown to reflect the response of the tumor to therapy. They can, therefore, be used to monitor the effect of therapy also in the adjuvant situation when the tumor has been removed by surgery.Here we report on the application of therapy monitoring using CETCs during adjuvant hormone blocking therapy. ER positive breast cancer were prospectively analysed for the number of CETC before therapy, before each new cycle of chemotherapy and during maintenance therapy with tamoxifen of aromatase inhibitor or during switch from one principle to the other. 1ml of blood was drawn into EDTA vials, red blood cells lysed and the white blood cell pellet stained with FITC-labelled anti-Epcam. Green fluorescent cells were detected by image analysis and dead cells excluded due to red PI fluorescence.178 patients were followed initially at quarterly intervals and later at annual intervals for their numbers of CETCs. 131 patients remained disease free during the observation interval of up to 7 years and 47 (26%) suffered distant relapse. Of the patients in complete continuous remission, 58% showed decreasing or stable CETCs whereas in the patients with relapses 73% showed increasing CETCs.45 patients were subsequently switched to aromatase inhibitor treatment, 17 relapsed patients and 28 patients without evidence of disease. Of the relapsed patients 35% have remained progress free all with a re-decrease in CETCs during AI treatment.Thus, gauging of CETCs can help to monitor hormone treatment and in the future contribute to early switch patients to AI if an increase in CETCs indicates resistance to tamoxifen.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5129.
CD44-Activated HER-2 Signalling in Tamoxifen Resistant Breast Cancer Cells Promotes a Migratory Phenotype.
Authors: Baruah B. , Barrett-Lee P. , Smith C. , Nicholson R. , Hiscox S.
Published: December 2010

Background: Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling. In tamoxifen-resistant ('TamR') MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein known to interact with, and modulate the function of, a number of growth factor receptors including members of the erbB family and plays a major role in tumor metastasis. Here, we have explored the role of CD44 as a modulator of erbB activity in TamR cells.Methods: Western blotting and immunocytochemistry were used to investigate expression of CD44 together with the activity of erbB signalling pathways following stimulation of CD44 by its ligand, hyaluronan (HA), or stimulation with erbB ligands (heregulin β1) after knockdown of CD44 expression using siRNA. Immunofluorescence microscopy was used to determine association between CD44 and erbB family members. Cell migration was assessed by seeding cells onto fibronectin-coated microporous inserts for 24 hours and also by performing scratch wound assays. Cell proliferation was determined by WST assay in response to trastuzumab (0-100nM) ± HAResults: CD44 was found to be over-expressed in TamR cells where it co-localized with HER-2. Whilst stimulation of HER-2 with heregulin beta-1 promoted an increase in cell migration, heregulin action was significantly attenuated in the absence of CD44 using siRNA. Moreover, treatment of TamR cells with the natural CD44 ligand, HA, promoted HER-2 signalling through MAPK and an increase in cell migration. Furthermore, whilst TamR cells were sensitive to the growth-inhibitory effects of trastuzumab, these effects were diminished in the presence of HA.Conclusions: Overexpression of CD44 sensitizes tamoxifen-resistant cells to heregulin and HA, factors commonly found within the stromal environment, with a resultant increase in migratory behaviour. Interestingly, activation of CD44 by HA appears to suppress the effects of trastuzumab on cell proliferation. Thus, CD44 may prove a valuable therapeutic target in breast cancer that expresses HER-2 and CD44.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5137.
Different Treatment Benefit for Luminal A and Luminal B Advanced Breast Cancinomas Receiving Aromatase Inhibitors.
Authors: Ruiz-Borrego M. , Moreno A. , Salvador J. , Carabantes F. , Torregrosa M. , Olmos T. , Martinez J. , de la Haba J. , Valero M. , Martinez E. , Santaballa A. , Hernández R. , Murias A. , Tortorella A. , Guzmán C. , Palacios J.
Published: December 2010

Background. Aromatase inhibitors (AIs) are indicated for postmenopausal women with hormone receptor (HR) positive metastatic breast cancer. However, at present the only predictive markers associated to AI response is estrogen receptor (ER) expression. Gene expression profiling of breast cancer has identified two biologically ER-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. In this study, we analyzed a clinically practical immunohistochemistry assay based on ER, progesterone receptor (PR), Ki67 and HER2 expression to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to AI response.Methods and Results. 41 women with HR positive metastatic breast cancer receiving AIs for their advanced disease have been enrolled in this study. Median age was 67 years (range, 39-84). 71% (29 of 41) of patients have received prior adjuvant therapy for their primary breast cancer. 41% of patients developed visceral disease and 58% of patients non visceral disease. 66% (27 of 41) of patients did not receive other prior treatment to the AI for their metastatic disease. 38 patients have received Exemestane and 3 patients Letrozol. Median duration of treatment was.7.9 months (range 1.2-31). Median follow up of patients was more than one year (14 months (range 1.2- 39.5).Best response to the treatment in 37 evaluated patients was: 11% PRs (CI95%: 0.3-21.3), 2.7% CR (CI95%: 0.0-8.2) and 76% of SD (CI95%: 61.2-90.2); 70% of SD ³ 6 months. Clinical benefit (PR+CR+SD) was 89.2% (CI95%: 78.7-99.7).Treatment benefit depending on the molecular subtype has been evaluated at present in 26 patients. 17 cases were classified as luminal B defined as ER and/or PR low expression levels (Allred score for ER or PR equal or less than 4) and/or Ki67 equal or higher than 15%, and/or HER2-positive. 9 cases were classified as luminal A (Allred score for ER and PR equal or higer.than 5, KI67 lower than 15% and HER2-negative). Although this number of cases is still small, median time to progression seems to be lower in those patients belonging to the luminal B subgroup (303 vs. 496 days) (p=0.1785; RR=2.396)Conclusion: Although analysis of more cases is still awaited, preliminary data from this small exploratory study suggest that the benefit with AIs is lower in those patients whose tumors belong to the luminal B molecular subtype.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5141.
Bortezomib Enhances the Efficacy of Fulvestrant by Promoting the Aggregation of the Estrogen Receptor in the Cytoplasm.
Authors: Germain D. , Bahadur U. , Halpern M. , Adelson K. , Raptis G. , Waxman S. , Ishii Y.
Published: December 2010

Background: Aromatase inhibitors (AIs) are standard treatment for Estrogen Receptor (ER)+ breast cancers in post-menopausal women where the main source of estradiol comes from adipose tissue when the aromatase enzyme converts androgens to estrogens. However, in premenopausal women, functioning ovaries flood the body with estrogens, and aromatase inhibitors used alone offer no therapeutic benefit. In addition to tamoxifen and aromatase inhibitors, estrogen receptor down-regulators, are a third type of anti-estrogen. The first of this class to be FDA approved is Fulvestrant, which acts by promoting the proteosomal degradation of the ER. Like tamoxifen, fulvestrant binds directly to the ER but while tamoxifen has both antagonist and agonist effects on the ER, fulvestrant is a pure antagonist. Other important advantages of fulvestrant over tamoxifen are that 1) fulvestrant prevents the ER from binding DNA, 2) fulvestrant is not linked to increased risk of endometrial cancer, 3) fulvestrant promotes permanent degradation of the ER.The molecular machinery leading to the degradation of the ER in the nucleus following fulvestrant treatment is well described and correlates with its ubiquitination in the nucleus. A less well-recognized mechanism, is fulvestrant’s ability to promote the aggregation of the newly synthesized ER in the cytoplasm. Understanding that protein aggregates are toxic when not eliminated by the proteasome, we took advantage of this effect of fulvestrant to ask whether combining fulvestrant with the proteasome inhibitor Bortezomib could enhance the efficacy of Fulvestrant.Results: We found that bortezomib enhances the aggregation of the ER in the cytoplasm following treatment with fulvestrant. Further, these aggregates were found to be insoluble and to activate the unfolded protein response (UPR), a stress response that leads to cell death. Further, bortezomib is able to prevent the activation of cytoprotective responses linked to the acquisition of fulvestrant resistance. Furthermore, in a breast cancer mouse model of tamoxifen resistance, the combination induced tumor regression.Conclusion: We conclude that adding bortezomib to fulvestrant enhances its efficacy by taking advantage of a previously poorly recognized mechanism–fulvestrant’s induction of ER aggregation in the cytoplasm. Further, our data suggest that this strategy will block the ability of cells to acquired resistance to fulvestrant. Our group has developed a clinical trial that will test this combination.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5142.
Genes Regulated by miRNA in 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

RationaleChange in genes expression is one of the major molecular drivers of tumour growth. Mechanisms controlling genes expression include transcriptional factors, mutations, aberrations in gene copy number, epigenetic mechanisms and micro-RNA (miRNA) regulation. The last mechanism has been discovered recently and there is a need to investigate its involvement in genes regulation in endocrine resistant breast tumours.Methods and ResultsTo identify genes regulated by miRNA in endocrine resistant breast tumours we collected frozen biopsies from a series of 51 tumours growing on endocrine treatment (aromatase inhibitors or tamoxifen, post-menopausal patients). Gene expression profiles were obtained using Illumina HT-12 micro-arrays; miRNA expression profiles were obtained using Illumina DASL® Sentrix Array Matrix.Unsupervised hierarchical clustering using 50 most variable miRNAs split tumours into two major clusters (25 and 26 tumours each). We suggest that genes differentially expressed between these two clusters can be potentially regulated by miRNA. The top differentially expressed genes included SAA1, VTCN1, SLC40A1, FBLN1, COL17A1, HDC and CD59.An alternative bioinformatics approach was based on correlations between mRNA and miRNA in the studied tumours. Initially we identified 66 genes negatively correlated with at least one miRNA (r<-0.6). Then we screened the correlated pairs to identify those in which miRNA has target sequence within the corresponding gene. This approach yielded just 2 genes: RAG1 and USP19: RAG1 being potentially regulated by mir92 and USP19 regulated by miR15a.DiscussionEndocrine resistance is a major obstacle in treatment of many breast cancer patients. Molecular mechanisms of the resistance are yet unclear. The abstract presents a sub-group analysis within a larger study, aimed to investigate molecular mechanisms underlying endocrine resistance at both mRNA and miRNA levels. Most of the identified potential miRNA-regulated genes have not been previously reported in the context of breast cancer biology. On our knowledge, this study represents the first systematic attempt to identify genes regulated by miRNA in endocrine-resistant breast cancers.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5149.
The Antiproliferative Effect of Calcitriol on Breast Cancer Cell Lines Depends on Vitamin D Receptor Status and the Expression of Prostaglandin Metabolizing Enzymes.
Authors: Thill M. , Friedericke H. , Katharina K. , Dittmer C. , Dorothea F. , Klaus D. , Michael F. , Becker S.
Published: December 2010

Background: Both first studies in prostate cancer and our results in breast cancer suggest a link between vitamin D and prostaglandin metabolism. There is a growing body of evidence that COX-2 expression is a fundamental step in breast cancer pathogenesis acting through prostaglandin-dependent and independent mechanisms. The antiproliferative effects of calcitriol (1,25(OH)2D3) mediated via the vitamin D receptor (VDR), render the biologically active form of vitamin D a promising target in breast cancer therapy.Materials and methods: The expression of VDR, COX-2 and 15 PGDH was determined by Western blot in MCF-10F cells, a human benign epithelial cell line, in MCF-7 cells, a moderate invasive and in MDA-MB-231 cells, a highly invasive breast cancer cell line. Measurement of basal PGE2 secretion was performed with ELISA. The influence of calcitriol on cell proliferation was determined by 5-bromo-2-deoxyuridine (BrDU) incorporation (BrDU ELISA). In addition we examined the effect of calcitriol on COX-2, 15 PGDH and VDR protein levels compared to untreated cells.Results: After 72 hours the proliferation of MCF-10F cells was dose-dependent inhibited to 0.83± 0.1 by 1 nM calcitriol. Concurrently, we observed a reduction of COX-2 expression to 0.63± 0.05 as compared to the untreated control group, but we also found a reduction of 15 PGDH to 0.73± 0.16. These effects were associated with a higher expression of VDR to 1.64± 0.16. Interestingly, MCF-7 cell growth was reduced to 0.52± 0.04 by 10 nM calcitriol and to 0.7± 0.06 by 1 nM. These effects do not seem to be associated with COX-2 and 15 PGDH and are independently of VDR. VDR was detected neither in stimulated nor in unstimulated cells. A PGE2 secretion could not be detected by ELISA. In contrast, we found no effect on cell growth, COX-2 and 15-PGDH expression of the MDA-MB-231 cells when treated by calcitriol (10 nM and 1 nM). Although PGE2 was synthesized 2.3-fold more by MDA-MB-231 cells than MCF-10F cells; the COX-2 expression was consistent with the MCF-10F cells, but 15-PDGH was expressed about 50 % less. As we have shown in MCF-7 cells, an expression of the VDR could not be detected.Conclusions: The growth inhibiting effect of calcitriol in MCF10F cells is associated with the increasing expression of VDR. and a remaining question is whether the loss of VDR goes along with the invasiveness of the breast cancer cell lines. Additionally, we suggest that the invasiveness of the breast cells is abetted by the increased COX-2 expression (MCF-7) or by the decreased expression of 15 PGDH (MDA-MB-231). These results suggest even more a link between vitamin D and prostaglandin metabolism and therefore a possible synergism between COX-2 inhibition and calcitriol in breast cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5155.
Genetic Characterization of Columnar Cellular Lesions and Atypical Ductal Hyperplasia of the Breast.
Authors: Ellsworth R. , Weyandt J. , Fantacone-Campbell J. , Deyarmin B. , Ellsworth D. , Hooke J. , Shriver C.
Published: December 2010

Aims: Columnar cell lesions (CCL) and atypical ductal hyperplasia (ADH) frequently coexist and share molecular changes with in situ and invasive components, suggesting that CCL and ADH may be precursors to breast cancer. These conclusions are, however, largely based on studies examining CCL and/or ADH from patients diagnosed with more advanced disease. Thus, we assessed allelic imbalance (AI) in pure CCL or ADH specimens to characterize molecular changes in early breast lesions.Methods and results: DNA samples were obtained from laser microdissected lesions from CCL with (n=42) or with ADH (n=31) without concurrent in situ or invasive disease. AI was assessed at 26 chromosomal regions commonly altered in breast cancer. The average AI frequency was 6.2% (range 0-20%) in CCL and 6.1% (range 0-25%) in ADH with no significant difference in levels of AI between CCL and ADH. The highest levels of AI were on chromosomes 8q24 and 17q21 in ADH (23%) and CCL (15%), respectively.Conclusions: In conjunction with low overall levels of AI, chromosomal alterations that characterize low- and high-grade in situ and invasive disease were not frequent in pure CCL and ADH. Thus, pure lesions are not genetically advanced and are molecularly distinct from synchronous lesions.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5156.
“VISA” Nanoparticles to Breast Cancer.
Authors: Xie X. , Deng W. , Yang M. , Tang J. , Wang X. , Wei W. , Xie Z. , Xiao X. , Liu P. , Lang J. , Hung M.
Published: December 2010

Development of cancer gene therapy has been hampered by the fact that there are no effective cancer-specific expression vectors available, which is critical for improving therapeutic efficacy and reducing toxicity in clinics. To overcome this problem, we recently developed a safe and effective strategy fro targeting a potent pro-apoptotic gene (BikDD) to the pancreatic tumors (Xie et al. Cancer Cell, 2007). A VISA system (VP16-Gal4-WPRE integrated systemic amplifier) was engineered by using the two-step transcriptional amplification (TSTA) system and the posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE). The VISA system can boost the activity of cancer-specific promoters by an average of 600 folds compared to their basal levels. C-VISA (CCKAR-VISA) nanoparticles transcriptionally targets transgene expression effectively to pancreatic tumors in vivo. C-VISA-BikDD nanoparticles was shown to be highly effective in reducing tumor burden and increasing animal survival rate in orthotopic pancreatic cancer models which is moving into clinical trials at University of Texas M.D. Anderson Cancer Center, USA. In the current study, we identified hTERT(human telomerase reverse transcriptase), ß-cateinin, claudin-4, and FASN (fatty acid synthase) promoters as breast cancer-selective promoters, and amplified their activity to hundreds of folds greater by the “VISA” system without loss of their specificity. We further demonstrated significant antitumor activity of targeted BikDD expression driven by nanoparticles of the hTERT-VISA-BikDD vectors in Her-2-negative and -positive breast cancer cell lines and in multiple breast cancer models of living imaging by the Xenogen IVIS imaging system with limited toxicity. In addition, treatment with hTERT-VISA-BikDD nanoparticles plus lapatinib or produces evident combinational therapeutic efficacy, which is likely due to the ability of lapatinib to promote apoptosis of Her-2-overexpressing breast cancer cells. Thus, our newly developed hTERT-VISA-BikDD nanoparticles are an innovative strategy for targeted antitumor effects of both Her-2-negative and Her-2-positive breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5159.
Genetic Contribution of GADD45A to Susceptibility to Sporadic and Non-BRCA1/2 Familial Breast Cancers: A Systemic Evaluation in Chinese Populations.
Authors: Yu K. , Di G. , Shao Z.
Published: December 2010

GADD45A is a candidate breast cancer susceptibility gene because its product participates in DNA repair and it is a downstream gene of p53 and BRCA1, both of which are breast cancer susceptibility genes. The genetic contribution of GADD45A to sporadic and familial breast cancer is still ambiguous. We screened germline mutation of GADD45A in 185 non-BRCA1/2 familial breast cancer patients, but no deleterious mutation was found. Seven single-nucleotide-polymorphisms were identified in a sub-sample. Five common variants (minor allele frequency (MAF)>10%) were genotyped for association analyses to scrutinize the relationship between breast cancer and polymorphisms in GADD45A in two independent population sets. In the first case-control study, a comparison of genotype frequencies between sporadic breast cancer patients and controls indicated the CT/TT-genotypes of +1506C>T and CG/CC-genotypes of +3204G>C were associated with decreased breast cancer risk (adjusted odds ratio (OR), 0.77; 95% confidence interval (CI), 0.62-0.96; and adjusted OR, 0.71; 95%CI, 0.57-0.88, respectively) compared with their wild-type homozygotes. A common hyplotype CGTCC was also associated with reduced risk (P=1.0×10-4). In a second familial breast cancer patient-based case-control study, although +1506C>T and +3204G>C failed to be validated, the haplotype CGTCC showed a borderline significance. Notably, the combined P-values were robust for +3204G>C (P=3.1×10-4) and CGTCC (P=1.6×10-5). Moreover, CGTCC was correlated with a higher GADD45A expression in normal breast tissues. In conclusion, although germline mutation of GADD45A is not common in familial breast cancer patients, polymorphisms/haplotypes in GADD45A contribute to breast cancer risk, at least to sporadic breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5163.
Gene Expression Profiling of Phenotypically-Defined Hormone-Receptor Positive Breast Cancer: Evidence for Increased Transcriptional Activity of the Insulin Growth Factor Receptor Pathway and Other Pathways.
Authors: Sparano J. , Gray R. , Goldstein L. , Childs B. , Brassard D. , Bugarini R. , Rowley S. , Baker J. , Shak S. , Badve S. , Baehner F. , Kenny P. , Perez E. , Shulman L. , Martino S. , Sledge G. , Davidson N.
Published: December 2010

Background: Approximately 70% of all breast cancers are hormone receptor (HR)-positive tumors that are sensitive to endocrine therapy, but some patients have recurrence despite adjuvant endocrine therapy. We performed an exploratory analysis of gene expression in HR-pos operable breast cancer in order to identify potential novel therapeutic targets and biomarkers associated with recurrence. Methods: RNA was extracted from primary tumor samples obtained from 776 patients with stage I-III breast cancer treated with adjuvant chemohormonal therapy in trial E2197 (JCO 2008; 26: 4092-4099), of whom 458 had HR-pos disease (defined in a central lab; JCO 2008; 26: 2473). We evaluated RNA expression patterns (by quantitative RT-PCR using a panel of 371 rationally selected genes) in HR-pos cases compared with the HR-neg cases using weighted T statistics, and determined which genes in the HR-pos, HER2-neg group were associated with recurrence (using Cox proportional hazards model score test, Korn's adjusted P value <5% with false discovery rate < 10%).Results: The top 10 genes exhibiting significantly higher expression in the HR-pos group (p≤ 6.17e-160) included ESR1 plus 5 estrogen regulated genes, confirming our approach of evaluating gene expression in phenotypically-defined subsets. Other pathways that exhibited higher expression in the HR-pos group (among the 40 top genes with higher expression, p<8.66e-53) included the insulin growth factor (IGF) (IRS1, IGFR1, IGFB2), Ras (RhoB, RhoC, RAB27B, GGPS1), and HER pathways (ERBB2, ERBB3, ERBB4), and other genes involved in apoptosis (BCL2, BCL2L1, BAG1, NME6, BBC3), signaling (MAPK3, SEMA3F, RXRA), mismatch repair (MSH3), cell cycle regulation (CCND1), stress response (HSPB1), and tumor suppressor genes (TP53BP1, APC). These patterns were similar in HER2-pos cases. Pathway analysis (Ingenuity) revealed substantial interconnectivity among these genes, especially between IGFR1, ERB2/3/4, MAPK3, BCL2, and CCND1, but not RhoB/RhoC. Genes for which increased expression was associated with increased recurrence included those associated with proliferation (TOP2A, AURKB, PLK1) and apoptosis (BIRC5 - survivin).Conclusions: This exploratory analysis reveals several pathways that exhibit higher transcriptional expression in HR-pos disease, some of which are also associated with a higher risk of recurrence, suggesting that they may be potential therapeutic targets. This provides rationale for testing agents currently available in the clinic that inhibit the IGF and other pathways.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5165.
Should Liver Metastases of Breast Cancer Be Biopsied To Improve Treatment Choice?
Authors: Fumagalli L. , Curigliano G. , Bagnardi V. , Locatelli M. , Aurilio G. , Della Vigna P. , Monfardini L. , Giudici S. , Viale G. , Toppo L. , Goldhirsch A.
Published: December 2010

Background: Decision making on systemic treatment of women with metastatic breast cancer is based on features like estrogen receptor (ER), progesterone receptor (PgR), and HER2 status assessed on the primary tumor. We evaluated the concordance of receptor status between primary tumor and liver metastases and estimated the potential change of these pathological features and its impact on treatment choice.Materials and methods: We retrospectively analyzed a database including ultrasound guided liver biopsies performed from 1995 to 2008. All tissue samples, both from primary tumor and liver metastases, were analyzed for ER, PgR and HER2 status. Clinical and biological data were obtained from medical charts (including treatments before and after liver biopsy). Differences between proportions were evaluated using the Pearson chi-square test.Results: We identified 215 consecutive patients with matched primary and liver tissue samples. Median time from primary diagnosis to liver biopsy was 4.1 years (range 0-18.3 years). Changes in ER status were observed in 38/215 patients (17.7%). 15/47 patients (31.9%) changed from ER negative to positive and 23/168 patients (13.7%) changed from ER positive to negative [p= 0.0038]. Changes in PgR status were observed in 111/215 patients (51.6%). 16/74 patients (21.6%) changed from PgR negative to positive and 95/141 patients (67.4%) from PgR positive to negative [p <0.0001]. 11/41 patients (26.8%) changed from ER and PgR negative to ER or PgR positive [Group A] and 25/174 patients (14.4%) changed from ER or PgR positive to ER and PgR negative [Group B] [p=0.054]. In the Group A the treatment of 4/11 patients (36.4%) was changed after biopsy: 2/4 started endocrine treatment (HT) and 2/4 stopped it. In Group B the treatment of 16/25 patients (64%) was changed after biopsy: 15/16 stopped HT. Changes in HER2 status were observed in 19/131 patients (14.5%): 5/93 patients (5.4%) changed from HER2 negative to positive and 14/38 patients (36.8%) changed from HER2 positive to negative [p= <0.0001]. In this group 5/5 patients started a trastuzumab containing treatment after biopsy.Conclusions: There was a substantial discordance in receptor status between primary tumor and liver metastases, which led to change in therapy for 21.8 % of patients. Biopsy of metastases for reassessment of biological features should be considered in all patients when safe and easy to perform, since it might impact treatment choice for many.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6003.
Pregnancy Related Breast Carcinoma: Comparison of Receptor Status with Age-Matched Controls.
Authors: Bhargava R. , Sukumvanich P. , Beriwal S.
Published: December 2010

Pregnancy related breast cancer (PRBC) is defined as breast cancer that is diagnosed during a pregnancy or within 1 year post-partum. These are generally considered as biologically aggressive tumors but the recent data suggests their prognosis to be similar to that of non-pregnant patients matched for age and stage. In the last few years, gene-expression profiling has been used to define breast cancer molecular classes with prognostic significance. The determination of tumor molecular class by immunohistochemistry is a reasonable alternative to molecular profiling. Therefore, we used immunohistochemical surrogate markers, estrogen receptors (ER), progesterone receptors (PR) and HER2 to determine breast cancer molecular classes in PRBC and compared them to age matched controls. ER and PR were scored using a semi-quantitative H-score like method that take into account both intensity and percentage cellular staining, and the score has a dynamic range of 0-300. An ER and PR score >10 was considered a positive result. An ER/PR score of 200 or higher in a tumor was considered as strongly positive for the hormone receptor. HER2 was considered positive only if 3+ by IHC or unequivocally amplified by FISH. The tumors were classified as luminal A (LUMA), Luminal B (LUMB), Triple Negative (TN), ERBB2, Luminal A-HER2 Hybrid (LAHH), and Luminal B-HER2 Hybrid (LBHH). The immunohistochemical criteria, prevalence of molecular subtypes in PRBC and age matched controls (age less than 40 years) is shown in the table below. Statistical analysis was performed using SPSS software version 16.0. Chi-square test was performed to compare the differences in percentages between PRBC and the non-pregnant control group. A p-value <0.05 was considered as significant.Tumor ClassCriteriaPregnancy related breast cancerAge matched controls (less than or equal to 40 years of age)LUMAStrong ER+; any PR; HER2 negative10/45 (22%)31/99 (31%)LUMBWeak to moderate ER+ or PR+; HER2 negative9/45 (20%)11/99 (11%)TNNegative for ER, PR and HER220/45 (25%)34/99 (34%)ERBB2Negative for ER and PR; HER2+4/45 (9%)3/99 (3%)LAHHStrong ER+; any PR; HER2+1/45 (2%)7/99 (8%)LBHHWeak to moderate ER+ or PR+; HER2 positive1/45 (2%)13/99 (13%) Comparison of tumor molecular classes using semi-quantitative ER, PR and HER2 results between PRBC and age matched controls shows no statistical difference between the 2 groups (p value of 0.06).The results suggest that PRBCs are not unique and rather represents aggressive biologic tumor types (i.e. triple negative tumors) that are characteristic for young age of the patients.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6012.
Histological Characterization of 47 Breast Cancers with Neoplastic Meningitis Evolution.
Authors: Le Rhun E. , Devos P. , Baranzelli M. , Faivre-Pierret M. , Bonneterre J. , Bonneterre J.
Published: December 2010

IntroductionAround 5% of patients with breast cancer develop neoplastic meningitis (NM). The prognosis of NM in this population is supposed to be better with a median survival time of 7 months if treated with intrathecal therapy, systemic treatment and adapted supportive care (versus 4-6 weeks if untreated). This evolution needs to be detected as early as possible and it is necessary to identify tumors with such a risk. We report histological characteristics in 47 breast cancers with leptomeningeal treated in our institution for the last 2 years.Aim of the studyTo identify histopathological characteristics of breast cancer predictive of leptomeningeal evolution.Patients and methods47 consecutive female breast cancer patients were diagnosed with neoplastic meningitis on the basis of cerebrospinal fluid (CSF) cytology and/or cerebrospinal MRI plus clinical symptoms. These patients were matched with 47 breast cancer's patients without leptomeningeal evolution, according to age at breast cancer diagnosis (+/- 2 years), year of breast cancer diagnosis (+/- 2 years) and use of systemic chemotherapy as initial treatment of breast cancer. Differences between the 2 groups of patients were analyzed using usual statistical methods (t-test, Chi square test).ResultsAt the time of NM diagnosis, the median (min-max) age of patients with NM was 51 (26-76) years. Median delay after breast cancer diagnosis was 33 months (0-193). Brain, hepatic, bone, lung metastases were present in respectively 47%, 43%, 47% and 19%. CSF cytology was positive in 72%, median proteinorachia was 0,75 g/l (0,17–16,12); cerebrospinal MRI was also positive in 87%. At the time of breast cancer, median age was 49 (25-70) years. Initial breast samples were tumor biopsy in 43%, partial mastectomy in 31% and total mastectomy in 27%. Breast tumors were invasive ductal carcinoma in 84% with histoprognostic grade II/III in 49/42%, MSBR grade I/II in 52/47%. Grade 3 of differentiation was observed in 68%, grade 3 of anisonucleosis in 59% and grade 1 of mitosis in 48%. In the tumors, estrogen/progesterone receptors (ER/PR) were detected in 67/48% and HER2 overexpression was observed in 24%. 19% of the tumors were triple negative. Vascular emboli were detected in 58% of the cases. Tumors were T2 in 43%, N+ in 66% and M+ in 14%. Significant differences between initial breast cancers with or without NM evolution concerned histological type (p = 0,043), ER (p = 0,004) and PR (p = 0,023), triple negative status (p = 0,009) and initial metastatic status (p < 0,007).ConclusionsCarcinomatous meningitis preferentially occurs in invasive lobular tumors without hormonal receptors, particularly in triple negative tumors and in tumors metastatic upfront. HER2 overexpression is not a risk factor of NM, as is observed with brain metastases.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6013.
Automated Intra-Operative Analysis of Breast Cancer.
Authors: Martin D. , Ruidiaz M. , Ta C. , Sandoval S. , Cortes-Mateos M. , Messmer D. , Wang-Rodriguez J. , Kummel A. , Blair S.
Published: December 2010

Background: Multiple operations are a major problem for breast conservation therapy. Accurate interpretation of intra-operative tumor margins can limit multiple re-excision procedures in order to obtain negative margins while maintaining a good cosmetic result from BCT. Intra-operative touch preparations have been used in the past but can be difficult to interpret without an experienced cytopathologist.Objective: To examine the reliability of fluorescently labeled intra-operative touch preps in conjunction with automated analysis in correctly identifying cancer cells compared to final pathologic diagnosis.Methods: Touch preps were propctively performed on 38 consecutive cases of patients undergoing breast surgery. The surgical cases included 15 invasive tumors, 9 ductal carcinoma in situ, 5 prophylactic mastectomies, and 9 normal tissues from breast reduction operations. Tumors were grossly located by a surgical pathologist and a cross sectional cut was made at the suspected tumor center. Touch preps with poly-l-lysine coated slides were then taken on the tissue surfaces exposed from the cross sectional cut. Additionally, the location where the touch preps were performed was isolated in a cassette and separated for later comparison. In cases of benign disease, touch preps were performed at the center of the excised tissue. The slides were fixed in 95% ethanol and stained with a Cytokeratin, an epithelial cell marker, and Hoechst, a nuclear stain. The prepared slides were then imaged with an automated fluorescent microscope, and suspected cancer cells were detected through standard image processing and statistical techniques.Results: The overall accuracy of our automated fluorescence analysis was 95% for identifying invasive or pre-invasive cancer compared final pathologic diagnosis. The overall specificity was 100% (there were no false positives). The sensitivity for ductal carcinoma in situ was 75% and invasive cancer was 90%. Overall sensitivity was 87% which is comparable to the best reported results from manual examination of touch preparations.Conclusion: Automated analysis of fluorescently stained touch prep slides is highly accurate in identifying cancer with no false positives. The immunofluorescence stains and automated microscopy may help the pathologist to quickly identify cancer cells in fresh breast tissue during BCT operations, and, thus, limit breast re-excisions in the future.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6016.
Lymphovascular Invasion in BRCA Related Breast Cancer Compared to Sporadic Controls.
Authors: van der Groep P. , Heerma van Voss M. , Bart J. , van der Wall E. , van Diest P.
Published: December 2010

Background: Germline mutations in the BRCA1 and BRCA2 genes predispose to the development of breast cancer, exhibiting a specific histological phenotype. Identification of possible hallmarks of these tumours serves several purposes. Biomarkers can contribute to the selection of high-risk patients for genetic screening, help to identify the pathogenicity of so called “unclassified variant” mutations, and insight in the carcinogenetic pathways allows to develop new targeted therapies. Since BRCA1-associated breast cancers have pushing borders that prevent them from easily reaching vessels, and are often of the medullary (like) type that has a low rate of lymphovascular invasion (LVI), we hypothesized that absence of LVI could characterize BRCA1 related breast cancer.Methods: A population of 68 BRCA1 and 10 BRCA2 related invasive breast cancers was evaluated for LVI by an experienced breast pathologist blinded to mutation status, and compared to a control group matched for age, grade and tumour type.Results: LVI was present in 25.0% of BRCA1 related cases, compared to 20.6% of controls (P=0.54, OR=1.29, CI 0.58-2.78). No significant differences were found for the BRCA2 group either (P=1).Conclusion: LVI seems to occur as much in BRCA1 germline mutation related breast cancers as in sporadic controls. Apparently, these hereditary cancers find their way to the blood and lymph vessels despite their well demarcation and often medullary differentiation, indicating that these cancers may have specific vasculoinvasive properties.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6019.
Preliminary Study of Pre-Metastatic Niche (PMN) in Nonmetastatic Nodes for the Assessment of Metastasis Risk in Breast Cancer (BC).
Authors: Adamo V. , Ieni A. , Caristi N. , Scarfì R. , Barresi V. , Giuffrè G. , Adamo B. , Colonese F. , Ferraro G. , Ricciardi G. , Tuccari G.
Published: December 2010

Bone marrow-derived hematopoietic progenitor cells (HPCs) seem to participate in early spread of cancer cells by forming a peculiar and highly organized micro-environment called PMN. Identifying such HPCs in the PMNs is emerging as a key step in assessing risk of metastasis.In this preliminary study we analysed 294 axillar nodes (271 pN0 and 23 pN1a) obtained from 30 pts undergone surgery for breast ductal invasive carcinoma in the period 1998-2000.Pts mean age was 61.2 years (range 45-79); 9 pts (30%) were premenopausal and 21 (70%) postmenopausal; 13 pts (43.3%) had ER+, 10 (33.3%) PR+; MIB1 was ≤ 25% in 22 pts (73.3%), >25% in 6 (20%), unknown in 2 pts. After a median follow-up of 81.27 mos (range 12–136), 14 pts are disease-free, 9 pts dead of metastatic disease. Involved sites were: 1 loco-regional, 7 bone, 3 lung, 4 liver, 1 brain.Samples were fixed in 10% neutral buffered formalin for 12-72 hrs and included in paraffin at 56°C; 4 mm thick sections were pre-treated in microwave owen in 10 mM citric acid, pH 6.0 and incubated overnight with the following poly/mono-clonal antisera: VEGF-R1 (Santa Cruz Biothechnology 1:400),CD 133(Abgent 1:80),CD 117 (DAKO 1:500),CD-34 (DAKO 1:50).Clusters of immunoreactive cells were evident with CD 117 and CD 34 in all nodes.A different degree of immunopositivity was found with VEGR-R1 and CD 133 antibodies for different sites of metastasis; in particular, the cases spread in lungs, liver and central nervous system were more immunoreactive, compared to those with bone localization. All nodes classified as pN1a showed no immunoreactivity.The prognostic significance of HPCs in PMN needs to be further investigated, especially in node-negative BCs. The immunomorphologic aspect of the lymph node microenvironment could have significant clinical implications, in order to optimize BC therapeutic strategies.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6024.