bluuebunny/crossref_metadata_2025 huggingface dataset:
P2-01-18: Cancer Stem-Like Cells in Breast Cancer Progression.
Authors: Valejo FAM , Andrade JM , Sicchieri RD , Antonio HMR , Marana HRC , Mouro LR , Oliveira TMG , Benevides L , Silva JS , Muglia VF , Tiezzi DG
Published: June 2012

Background: increasing evidences support the presence of cancer stem-like cells (CSLCs) within breast carcinomas. A subset of cells enriched for CSLCs within the tumor can be identified by flow cytometry. It has been supposed that CSLC component is responsible for tumor resistance and metastasis. However, the analysis of such population has never been evaluated during breast cancer progression. Patients and Methods: We prospectively included 98 women with histologically proven breast carcinomas to assess the presence of CSLCs within the primary tumor. According to clinical stage (CS), five patients were CS 0 (palpable DCIS), 16 CS I, 20 CS II and negative lymph nodes (IIA-), 20 CS II and positive lymph nodes (IIA+), 6 IIIa, 17 IIIb, 1IIIc and 2 CS IV. Additionally, ten lymph node metastases from IIA+ patients were analyzed. Invasive ductal carcinoma (IDC) was observed in 79 patients. We excluded from the analysis patients with non ductal histology (n= 11). The percentage of CD44+/CD24- (n= 79), ALDH1+ (ALDEFLUOR®) (n= 29), ABCG2+ (n= 44) and CXCR4+ (n= 46) cells within ESA+ cells was determined by flow cytometry in fresh sampled tumors. The mammosphere assay was studied in 14 samples. The relationship between cytometry analyses and clinical and pathological features was analyzed.

Results: The median prevalence of CD44+/CD24- cells within ESA+ population was 1.63% in stage 0, 2.39% in stage I, 0.42% in stage IIA-, 8.15% in stage IIA+, 0.52% in stage III (p=0.005). The CD44+/CD24- cell population was analyzed in 10 lymph node metastasis and, the median prevalence of this population within ESA+ cells was 4.7%. There was no variation of median prevalence of ALDH1+, CXCR4+, ABCG2+ and the median number of spheres among clinical stages (p=0.1, p=0.8, p=0.8, p=0.8, respectively). We didn't observe any association among the expression of, ABCG2+, CXCR4+ with histological grade and the expression of ER and PgR. The median percentage of CD44+/CD24- cells was higher (2.39% vs 0.41%) in ER positive tumors (p=0.003). The median percentage of ESA+/ CXCR4+ cells was higher (42,5% vs 9,7%) in HER2− tumors. There was no correlation between the studied markers and the patients’ age and clinical tumor diameter. The results are summarized in Table 1.

Results of association between expression cells surface markers and ALDH1 activity (Aldefluor®) with clinical and pathological features in invasive ductal carcinomas.

Discussion: We found a significant variation in CSLC population during breast cancer progression. Our data show a gradual increase in CD44+/CD24- cells within ESA+ population from stage 0 to stage IIA+ and a significant decrease in stage III tumors. This observation supports the hypothesis that a population of CSLCs could be associated with breast cancer progression.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-18.
P2-01-22: Characterization of Four ER-Negative Breast Primary Tumor Dissociated Cultures as a Model for the In Vitro and In Vivo Study of Tumorigenesis, Metastasis and Angiogenesis.
Authors: Drews-Elger K , Brinkman JA , Dean SJ , El-Ashry D
Published: June 2012

ER negative breast cancers comprise a subtype of tumors for which there are few therapies with successful response. Currently, the use of established cell lines has allowed detailed molecular and cellular studies on cancer mechanisms ranging form stem cells to proliferation, from tumorigenesis to invasion and metastases, and from tumor suppressors to oncogenes. But while cell lines provide a source of homogeneous, propagatable material, a drawback of this model is that most breast cancer cell lines are derived from metastatic lesions that may harbor additional mutations and have little resemblance with the primary tumor of origin. Additionally, the continuous passage of these cells over the last 20-plus years has given rise to well-established differences in growth rate, hormone receptor status, karyotype and clonogenicity. Our purpose here was to characterize four primary dissociated tumor (DT) cell cultures from ER- breast tumors, the establishment of which we had previously described, to determine if they could provide more accurate models for both in vitro and in vivo studies. These four DT cultures (DT16, DT22, DT25 and DT28) display a triple negative phenotype and were characterized by their genomic profile by the PAM50 predictor analysis as belonging to the basal (DT22, DT25 and DT28) and luminal B (DT16) subtypes. Of those cultures for which primary tumor sample was available, microarray gene expression profiling and cell line clustering analysis show the primary tumor and dissociated culture cluster together and to similar breast tumors. Interestingly, of the three basal DT cultures, DT22 clusters with Claudin-low tumors and cell lines. We have established that each of these 4 DTs are tumorigenic in the NOD/SCID mouse model, and are now assessing their metastatic potential. DT16 generated metastases in 25% of the mice (6 out of 24; two in liver, four in lymph node). DT22 generated highly vascularized tumors, which may provide a relevant model for study of the in vivo vascular recruitment process. In addition, we have analyzed and characterized the role of pivotal signaling pathways often deregulated in breast cancer such as EGFR, P-MAPK, PI3k/mTOR, p53, TGFb, Hedgehog, Notch, and Wnt in both the 2D culture as well as in sections of xenograft samples. Given that the scope of primary tumor heterogeneity and consequently the early stages of breast tumorigenesis are under-represented by current in vitro models in breast cancer research, the use of these DT cell cultures could more accurately recapitulate the disease in both the in vitro and in vivo settings.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-22.
P2-01-25: Truncated p110 ERBB2 (CTF611) Increases Migration and Invasion of Breast Epithelial Cells by Inhibiting STAT5b Activation.
Authors: Ward TM , Iorns E , Hoe N , Kim P , Singh S , Ernani V , Liu X , Jegg A-M , Gallas M , Lippman ME , Pegram MD
Published: June 2012

Background: Truncated ERBB2 receptors are present in a subset of human ERBB2+ amplified/overexpressing breast tumors, and are associated with trastuzumab resistance, metastasis, and poor clinical prognosis. However, whether truncated ERBB2 receptors are drivers of metastasis has not been well defined. In this study, we examined effects of full-length (p185) and truncated (p110) ERBB2 on the migration and invasion of human mammary epithelial cells, including HMLE and MCF10A cells.

Material and Methods: Recombinant p185 and p110 ERBB2 were stably expressed in human mammary epithelial cells (HMLE) and MCF10A cells via retroviral vector. Expression of comparable levels of p185 and p110 in cells was confirmed by western blot. The phosphorylation states of downstream signaling proteins including STAT5 were assayed via phosphoproteomics and Collaborative Enzyme Enhanced Reactive (CEER™) immunoassay. The effects of the p110 constructs on cell migration and invasion were investigated by transwell assays. shRNA-encoding lentivirus was used for specific silencing of STAT5b in HMLE cells, and STAT5b silencing was confirmed at the protein level using western blot.

Results and Discussion: Expression of p110 ERBB2 increased cell migration (HMLE, p = 0.04; MCF10A, p< 0.01) and invasion (HMLE, p= 0.03) when compared to expression of p185. Furthermore, expression of p110 in HMLE cells was associated with reduced phosphorylation of STAT5b. shRNA mediated silencing of STAT5b was sufficient to increase the migration (p < 0.01) and invasion of HMLE cells, phenocopying the p110 driven effects on HMLE cells. In clinical studies, loss of activated STAT5 protein correlates with breast cancer progression and is a negative predictor of survival. By analyzing publicly available gene expression datasets, we found that STAT5b mRNA expression is also significantly decreased in breast cancer compared to normal breast tissues in several studies, as well as in ERBB2 amplified vs. nonamplified samples. To our knowledge, this is the first reported perturbation of STAT signaling by truncated ERBB2 receptor, and suggests a mechanism by which truncated p110 ERBB2 (CTF611) increases migration and invasion of breast epithelial cells. This study extends the available data regarding STAT5 loss in breast cancer progression.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-25.
P2-01-26: TIMPv: a novel downstream transcriptional target gene that underpins CD146-suppressed breast tumor invasion
Authors: Ouhtit A , Abdelmageed Z , Fernando A , Gaur RL , Raj MHG
Published: June 2012

Background: CD146, a marker of endothelial cells, promotes tumor progression of many cancers including melanoma and prostate. Strikingly, several lines of evidence suggest that it is a suppressor of breast cancer (BC) progression. Not only the ligand(s) has not been identified, but CD146-downstream signaling mechanisms remain unknown.

Material and Methods: Here, we report the functional importance of CD146 and that of a novel transcriptional target of CD146-signaling, variant of tissue inhibitor of metalloproteinases (TIMPv) identified by microarray analysis, in underpinning the suppression of BC invasion, using novel validated Enhanced Green Fluorescent Protein (EGFP)-inducible systems of CD146 expression both in vitro and in vivo. Results: In functional experiments, induction of CD146 inhibited BC cell migration and invasion. TIMPv was identified by expression profiling as a novel transcriptional target of CD146-signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast and melanoma cancer cells. However, siRNA inhibition of CD146 in the SKMel-28 melanoma cell line increased TIMPv expression, suggesting that while TIMPv is a positive transcriptional target of CD146 in BC cells, it is negatively regulated in melanoma cells. Furthermore, the functional relevance of TIMPv to CD146-suppressed metastasis was demonstrated by selective suppression of TIMPv in CD146-expressing BC inducible cells using RNAi. More interestingly, induction of CD146 expression in vivo using mouse EGFP-inducible system of CD146 expression resulted in suppression of breast tumor growth.

Discussion: Our study is the first report to provide a functional molecular link of TIMPv to cancer via unique axis that underpin CD146-suppressed BC progression, thus identifying TIMPv as a potential target for guiding the development of novel therapeutic strategies for BC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-26.
P2-05-01: Acquired Tamoxifen Resistance Promotes Angiogenic Responses in ER+ Breast Cancer Cells.
Authors: Hayes E , Smith C , Nicholson RI , Gee J , Hiscox S
Published: June 2012

Background: In the treatment of pre-menopausal women with oestrogen positive (ER+) breast cancer, tamoxifen represents a first line of adjuvant treatment with demonstrable benefits. Despite this, resistance is frequently acquired to tamoxifen with an associated poor prognosis. Breast cancer cell models have revealed the importance of growth factor signalling networks in sustaining growth of endocrine-resistant cancers and, more recently, their ability to promote a highly migratory and invasive phenotype, together with the expression of genes with pro-angiogenic ontology. The potential of endocrine resistant cells to elicit angiogenic responses, however, remains unknown.

Materials and Methods: Real-time PCR was used to validate results from preliminary Affymetrix-based gene profiling of pro-angiogenic gene expression in endocrine-sensitive MCF7 cells and their tamoxifen-resistant (TamR) counterparts. The expression of pro-angiogenic factors in conditioned media (CM) from these cells was assessed by ELISA. The proliferative and migratory effects of MCF7 and TamR CM on vascular endothelial cells (HUVEC and HECV cells), was determined by MTS cell proliferation assay, wound closure assays and Matrigel tubule formation assays. Changes in endothelial cell migration following co-culture with endocrine-resistant cells were examined using Boyden-chamber chemotaxis assays. Growth factor signalling and migration pathway activation in endothelial cells in response to CM was determined by Western blotting. Results: TamR cells were found to express high levels of HIF-1α, IL-8 and VEGF-A at an mRNA level compared with expression in MCF7 cells. High levels of VEGF-A protein were also confirmed in the conditioned media from TamR cells versus their endocrine-sensitive counterparts. TamR conditioned media promoted endothelial cell proliferation, migration and the formation of tubules to a greater extent than that seen in MCF7 CM treated cells. TamR conditioned media was found to stimulate VEGFR2 phosphorylation and downstream activation of MAPK in endothelial cells compared to MCF7 CM. Pharmacological inhibition of VEGFR2 activity in endothelial cells suppressed TamR-induced endothelial cell proliferation and VEGFR phosphorylation. Further pharmacological manipulation of erbB receptors and intracellular kinases in TamR cells revealed an ERGF/Her2-Src kinase dependent mechanism of VEGF-A production in these cells.

Discussion: These data suggest acquired tamoxifen resistance is accompanied by development of an erbB receptor/Src kinase-dependant pro-angiogenic phenotype which, if recapitulated in vivo, may promote tumour progression. Therapeutic targeting of erbB/Src axis may prove beneficial in such cases.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-01.
P2-05-08: Expression of VE-Statin/egfl7 in Breast Cancer.
Authors: Lauridant-Philippin G , Baranzelli M-C , Fournier C , Samson C , Soncin F , Bonneterre J
Published: June 2012

Patients and methods: Specimens of breast cancer (174 invasive and 37 in situ) were obtained from 205 patients (pts) treated in our institution in 2005.Their median age was 57 years (39-92). VE-statin expression was studied by immunohistochemistry (IHC) using anti-hVE-statin/egfl7 (AF3638, R&D) and by RT-qPCR on formalin-fixed paraffin sections. 174 invasive carcinomas (141 ductular and 30 lobular) and 37 DCIS were studied. Determination was done twice by two different observers. Statistics used Chi2 and Fisher's exact tests for categorical data and Kruskall-Wallis test for continuous variables.

Results: Pathological characteristics of the patients with invasive tumors were as follows: 81% ductular, 17% lobular; median tumor size 18 mm (2-100); SBR grade 1 in 27%, 2 in 58% and 3 in 15% of the tumors; estradiol receptor (ER) + in 90%, progesterone receptor (PR) + in 66%; 6,5% HER2 positive. In 105 cases there was DCIS together with invasive tumors. For invasive tumors and DCIS, progression-free survival (PFS) at 5 years were 85% and 100% and overall survival (OS) 96% and 95%, respectively. Characteristics of the DCIS were median tumor size 18 mm (2-60), ER+ in 88%, PR+ in 62%. IHC results were obtained in 159 invasive carcinoma and 34 DCIS, RT-qPCR results in 96 samples. In the group of invasive tumors, a cytoplasmic VE-statin/egfl7 signal was obtained in 80% of invasive cells, more frequently than in normal glandular cells (71% vs 41%, p=0,003) but significantly less than in peritumoral DCIS (71% vs 89%, p=0,001). The signal was higher in ductular than in lobular carcinoma (85% vs 54%, p=0,003). In RT-qPCR analyses, RNA levels were higher in SBR1 grade tumors (p=0,008), in ER+ invasive tumors (p=0,02) and in pN- tumors (p=0,003). In the group of DCIS, a cytoplasmic signal was obtained in 82% of the cells. It was more frequently observed in tumor than in normal glandular cells (82% vs 52%, p=0,02). A 3+ cytoplasmic signal was more frequent in DCIS alone than in DCIS associated with invasive carcinoma (23% vs 6%, p=0,02). No prognostic significance differences were observed on PFS or on OS.

Discussion: This work is the first expression study of VE-statin/egfl7 in breast cancer. The results were reproduced by two different observers. IHC signal was cytoplasmic and higher in DCIS than in invasive carcinomas. Furthermore the signal was even higher when there was only DCIS than when it was associated with invasive lesion. The signal was higher in ductular than lobular carcinoma, consistent with previous publications on other angiogenesis mediators. Similarly, the signal was higher in low SBR grade, as well in ER positive tumors. Phase 1 studies are ongoing in order to evaluate the effects of inhibition of VE-statin/egfl7.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-08.
P2-07-01: Copy Number Variants in Early-Onset-Breast Cancer.
Authors: Ludwig S , Ivanovich J , Graubert TA , Goodfellow PJ
Published: June 2012

Background: Copy number variants (CNVs) are known to contribute to the risk for development of immune diseases, neurological disorders and to a lesser extent cancer. Many CNVs do not directly involve gene sequences. CHL1, a cell adhesion molecule with homology to L1CAM, is down-regulated in a large proportion of breast cancer. A deletion CNV immediately 5’ of the CHL1 transcriptional start site has been described. The CHL1 CNV's role in breast cancer risk has not previously been explored.

Material and Methods: 50 research subjects recruited to the Washington University/Siteman Cancer Center Young Women's Breast Cancer Program (YWBCP) were selected for a CNV discovery study. These women were diagnosed breast cancer < age 40, tested negative for BRCA1/2 mutations and have a negative family history for breast cancer. The selection criteria enriched for de novo mutations. Comparative genomic hybridization (CGH) analyses were undertaken using the NimbleGen 2.1 million oligonucleotide array. DNA copy number was compared between proband and each of her parents to identify potential deletions or amplifications in the daughter relative to both parents (de novo changes). PCR was used to confirm a subset of the CGH-predicted differences in copy number. Chi-square test was used to determine statistical significances of CNV occurrences near the CHL1 gene between women with breast cancer and control cases.

Results: aCGH data analysis has been completed for 25 YWBC trios (patients and parents). 247 differences in copy number in the breast cancer cases relative to their parents were identified (1-18 putative de novo CNVs per case). 91% of the observed changes were deletions and 9% were amplifications. One proband showed an 11,560 bp deletion 5’ of the CHL1 gene that was not seen in her parents. Conventional PCR confirmed a homozygous deletion of a ∼3 kb region in the patient, in the region of a known CNV with a reported frequency of ∼3%. Testing an additional 368 early-onset breast cancer cases for the CHL1 deletion revealed 25 additional homozygous deletions (6.8%). The deletion was present in 9 of 197 controls (4.5%). qPCR studies revealed the parents of the patient with the CHL1 deletion each carried a single copy of the CNV, making de novo copy number change in the proband very unlikely. Deletion specific assays for the CHL1 CNV are being used to test CNV genotypes in an extended case-control study.

Discussion: CHL1 expression has previously been shown to be down-regulated in primary breast tumors. Our preliminary analysis of YWBC cases and controls indicates the CHL1 regulatory region CNV is unlikely to be associated with risk for early-onset breast cancer. Additional candidate de novo copy number changes seen in the genomes of patients with early-onset breast cancer are being evaluated to determine their roles as genetic risk factors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-07-01.
P2-08-08: Benefit of Preoperative Breast MRI in Patients with Newly Diagnosed Breast Cancer.
Authors: Miesbauer M , Sheu H , Schoissengeier A , Kirchweger B , Waldenberger P
Published: June 2012

Objective: The aim of our study was to evaluate the effect of routinely performed preoperative Breast MRI (pMRI) on the planned therapeutical management of all patients with newly diagnosed Breast cancer.

Subjects and Methods: 180 patients with newly diagnosed Breast Cancer between 9/2009 - 12/2010 were enrolled in this retrospective study performed in a certified European Breast Cancer Unit; mean age 60,6 years; 1,5 T MRI ( TIM-Symphony, Siemens Germany).

Results: In 53/180 (29,4%) cases the preoperative MRI changed the planned therapeutical management concerning surgical and chemotherapeutical options. In 42/180(23,3%) patients additional lesions were identified only by pMRI, 33/42(78,6%) have been malignant and 8/42(19%) were benign. The real extent of the tumor was detected in 43/53 (81%) cases — group A; a bilateral carcinoma was detected in 4/53 (7,5%) cases — group B; in 6/53 (11,5%) cases the malignant lesion could only be detected with preoperative MRI — group C; for group A the pMRI led to wider excisions with 15 segmental -or quadrant resections and 28 mastectomies; for group B the pMRI resulted in 4 additional lumpectomies; for group C the pMRI caused 4 lumpectomies, 1 segemental resection and 2 mastecomies .5 of 6 patients got neoadjuvant chemotherapy only due to results of pMRI; in 3 of these 5 patiens breast conserving surgery was possible. Histology: DCIS 23/53 (43,4%), invasive cancer 23/53 (43,4%), multicentric/multifocal malignant lesions 7/53 (13,2%). The sensitivity of the pMRI was not influenced by the histology of the lesion, the age of the patient or menopausal status. The maximum periode between initial presentation of the patient and breast surgery was 21 days.

Conclusion: In 29,4% of our patients the pMRI resulted in a significant change of the planned therapeutical regime. Histology of the lesion, patients age or menopausal status did not influence the sensitivity of pMRI. There was no relevant delay in the initiation of treatment. Therefore we recommend performing pMRI in breast cancer staging routinely.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-08-08.
P2-09-14: Evaluation of Angiogenesis Using Synchrotron Radiation in Xenograft Mouse Models of Breast Cancer.
Authors: Gu S-M , Zhang X , Li R-M , Jin W , Shen Z-Z , Shao Z-M , Xu X-M , Wu J
Published: June 2012

Visualization of microvasculatures is an important step in understanding the mechanism of early vessel disorders in breast cancer and developing effective therapeutic strategies. However, quantitative evaluation of breast cancer angiogenesis using immunohistochemistry has been limited in generating detailed and reproducible data. To analyze the diversification of angiogenesis in the development of tumor growth and evaluate anti-vascular effects of Avastin, a new method of microvascular imaging was introduced in this paper. We used New X-ray microangiography and third-generation synchrotron radiation-based micro-computed tomography (SR micro-CT) to investigate the structures and density of microvessels at each stage of xenograft mouse models that were created by inoculation with MDA-MB-231HM cells (one breast cancer cell line with high metastasis potential). Barium Sulfate Nanoparticles, was used as a blood vessels contrast agent through left cardiac ventricle. Three dimensional structures of microvessels were displayed with a high spatial image resolution at around 20–30 μm. The density of microvessles was significantly reduced from the third week in mouse xenograft models. This observation with SR micro-CT was consistent with the results analyzed by immunohistochemical techniques. In addition, human breast cancer-bearing mice were treated site-specifically with humanized monoclonal antibody (Avastin) that targets all isoforms of VEGF-A. Tumor volume and the density of angiogenesis were significantly reduced in xenograft mouse models of breast cancer treated with avastin when compared with untreated mice as assessed by SR micro-CT. Specifically, the density of smaller vessels (di<50μm) was decreased significantly while the density of larger vessels was little changed in the Avastin-treated mice. Therefore, synchrotron radiation technique is a novel method for investigating breast cancer angiogenesis, and this might be considered as an additional complementary tool for more precise quantification of angiogenesis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-09-14.
P2-11-03: High Expression of CX3CL1 by Tumor Cells Correlates with a Good Prognosis and Increased Infiltrating CD8+ T Cells, Natural Killer Cells, and Dendritic Cells in Breast Carcinoma.
Authors: Lee JS , Park MH , Yoon JH
Published: June 2012

CX3CL1 is the only CX3C chemokine that can chemoattract CD8+ T cells, natural killer (NK) cells, and dendritic cells (DCs). Although studies have reported that CX3CL1 regulates the host immune response, the roles of the CX3CL1 in breast carcinoma remain unknown. We evaluated the expression CX3CL1 in 198 surgical specimens of breast carcinoma and analyzed any association with the clinicopathological factors, including CD8+ T cells, CD56+ NK cells, and CD1a+ DCs, while also evaluating the impact on the prognosis. High CX3CL1 expression was found in 136 of 198 (68.7%) breast carcinomas. The numbers of stromal CD8+ T cells, intratumoral CD1a+ DCs, and stromal CD56+ NK cells were significantly increased in the high CX3CL1 expression group (19.3 ± 15.2, 0.3 ± 0.9 and 1.4 ± 1.7 per field, respectively) compared with the low expression group (10.2 ± 13.1, 0.0 ± 0.3 and 0.7 ± 0.8 per field, respectively). Patients with high CX3CL1 expression had a significantly better prognosis for disease-free and overall survival than those with low expression (P = 0.002 and P = 0.000, respectively). In a multivariate analysis, the CX3CL1 expression was identified as one of the independent prognostic factors for overall survival (odds ratio, 2.29; P = 0.002). These data suggest that CX3CL1 expressed in the tumor cells appears to enhance the recruitment of CD8+ T cells, NK cells, and CD1a+ DCs, thereby bringing about a better prognosis in breast carcinoma. CX3CL1 is a new prognostic biomarker and be a novel candidate for development of a more effective therapeutic strategy for breast carcinoma.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-03.
P2-11-11: IGFBP Ratio Confers Resistance to IGF Targeting and Correlates with Increased Invasion and Poor Outcome in Breast Tumors.
Authors: Becker MA , Hou X , Harrington SC , Carboni JM , Gottardis MM , Haluska P
Published: June 2012

Purpose: To assess the role of insulin-like growth factor binding protein 5 (IGFBP-5) as a marker of relapse and survival in breast cancer tumors.

Experimental Design: Targeted regulation of IGFBP-5 was identified in MCF-7 cells resistant (MCF-7R4) to the IGF-1R/InsR inhibitor, BMS-536924 and examined by comparative microarray analysis, western and ELISA. Protein expression of IGFBP-5 was measured by immunohistochemistry in a cohort of 84 breast cancer patients to examine correlative associations with invasive tumor fraction and overall survival (OS). The expression ratio of IGFBP-5/IGFBP-4 (BPR) was determined in multiple breast tumor cohorts for univariate analysis.

Results: IGFBP-5 was markedly upregulated and highly localized to the membrane in MCF-7R4 resistant cells. When compared to pathologically normal reduction mammoplasty tissue, IGFBP-5 expression levels were upregulated in both invasive and histologically normal adjacent breast cancer tissue. In an independent cohort of breast cancer patients, IGFBP-5 protein levels correlated directly with invasion and OS. In univariate and multivariate modeling, metastasis-free survival, recurrence free survival (RFS) and OS were significantly associated with high IGFBP-5 expression. Prognostic power of IGFBP-5 was further increased with the addition of IGFBP-4 and tumors were ranked based upon IGFBP-5/IGFBP-4 expression ratio (BPR). Multiple breast cancer cohorts confirm that BPR (high vs. low) was a strong predictor of RFS and OS.

Conclusion: IGFBP-5 expression is a marker of poor outcome in breast cancer patients. An IGFBP-5/IGFBP-4 expression ratio may serve as a surrogate biomarker of IGF pathway activation and predict sensitivity to IGF-1R-targeted therapies.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-11.
P2-12-02: Correlation between BMI and Clinical Outcome of Patients with Early Stage HER2+ Breast Cancer from the N9831 Clinical Trial.
Authors: Crozier JA , Moreno-Aspitia A , Ballman KV , Martino S , Kutteh LA , Davidson NE , Kaufman PA , Perez EA
Published: June 2012

Background

Obesity, as defined by body mass index (BMI), has been associated with increased recurrence rate, shorter DFS and increased death rates due to breast cancer (BC). Most of the studies to date have examined the relationship of BMI and DFS in patients with hormone receptor positive disease. To our knowledge, BMI and its relationship with outcome in early stage HER2 positive breast cancer has not previously been examined. The N9831 is a large phase III trial testing the role of trastuzumab in the adjuvant setting of high risk patients with early stage HER2+ BC. We hypothesized that the occurrence of overweight and obesity may correlate with outcome.

Methods: This analysis presents BMI and its relation to tumor characteristics and DFS in patients (pts) enrolled in the N9831 clinical trial. Pts were categorized as normal weight, overweight or obese using the WHO BMI classification parameters of < 25%, 25–29% and ≥ 30% respectively. For patient characteristics, patients were grouped into non-obese (BMI< 30) and obese (≥ 30) cohorts. DFS was estimated by the Kaplan-Meier method. Comparisons between arms A (chemotherapy alone), B (chemotherapy plus sequential trastuzumab) and C (chemotherapy plus concurrent trastuzumab) were performed using the Cox proportional hazards model, stratified by BMI.

Results: Analysis was completed on 3,017 eligible pts. Obese pts were more likely to be older and postmenopausal (p<0.0001 for both). There was no significant association between BMI and ER/PR status (p=0.07) or histologic tumor grade (p=0.33). Obese pts were found to have significantly larger tumors ≥ 2 cm (p=0.002) and more positive lymph nodes (p=0.02). There was no significant difference in DFS within each intrinsic arm (A, B and C) between the obese and non-obese pts at 3, 5 or 7 yrs of follow up. However, pts in the non-obese group had significantly improved DFS in arm B and C compared to arm A (p=0.001 and p<0.0001 respectively). Also obese pts in arm C had significantly improved DFS compared to obese pts in arm A (p=0.008). There was a trend of improved DFS in the obese group in arm B compared to arm A, but this was not statistically significant (p=0.09). Pts in the normal weight and overweight groups did significantly better in arm B (p=0.02 for both) and arm C (p=0.01 and p=0.002 respectively) compared to arm A.

Conclusions: This analysis of data from the N9831 study confirms that obese pts with early stage HER2+ tumors have worse clinical outcome than pts with BMI < 30%. Adjuvant trastuzumab improved clinical outcome regardless of BMI. This study supports weight loss intervention for obese women with early stage HER2+ BC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-02.
P2-12-13: Topoisomerase 2 alpha (TOP2A) RNA Expression Provides Prognostic Information in Hormone Receptor Positive Breast Cancer That Is Complementary to a Simulated Algorithm for Recurrence Score.
Authors: Sparano JA , Goldstein LJ , Davidson NE , Sledge, Jr GW , Gray R
Published: June 2012

Background

We have previously reported that TOP2A RNA expression was prognostic in patients with operable estrogen receptor (ER) positive breast cancer treated with anthracycline containing adjuvant chemotherapy plus endocrine therapy in trial E2197, and provided complementary prognostic information in those who had a mid-range Recurrence Score (RS) (Sparano et al. Clin Cancer Res 2009; 15; 7693). The purpose of this analysis was to provide additional evidence regarding this observation.

Methods: We evaluated TOP2A RNA expression in 4 independent data sets individually and in a pooled analysis including all data. We also used an algorithm to simulate the Oncotype DX Recurrence Score (SimRS). The analysis included 752 patients with early stage breast cancer that was ER-positive, HER2−negative defined by gene expression. Results are expressed as the hazard ratio (HR) for estimates of the effect of a one standard deviation increase in the value of the log gene expression (x + 1SD vs. x) as a continuous function. Five-year relapse free survival (RFS) rates were also evaluated for patients with a mid-range SimRS for high vs. low TOP2A expression (above vs. below the median, which was prognostic in E2197). The distribution of SimRS was categorized to be concordant with the distribution of actual RS in E2197 (47%, 34%, 19%, respectively for RS <18 - low, 18–30 - intermediate, >31 - high).

Results: The HR for models including TOP2A alone and joint models including TOP2A and SimRS are shown in the table.

TOP2A expression was significantly associated with recurrence in all 4 datasets when evaluated individually (HR 1.56, p<0.0001 in pooled analysis), and 2 of 4 datasets individually when adjusted for SimRS (HR 1.26, p=0.003 in pooled analysis). SimRS was significantly prognostic when adjusted for TOP2A individually in 3 of 4 datasets (HR 1.44, p<0.0001 in pooled analysis). For those with an intermediate SimRS, five year RFS rates were significantly higher in 2 datasets individually for high vs. low TOP2A expression (HR 1.82, p=0.007 in pooled analysis).

Conclusions: This analysis provides additional evidence that TOP2A expression provides prognostic information in patients with ER-positive, HER2−negative disease, a population known to have low incidence of TOP2A gene alterations. These findings also suggest that TOP2A expression provides information that is complementary to RS, and may be useful for identifying high risk subjects who have a mid-range RS. These findings require prospective validation in other prospective or prospective-retrospective trials using the actual rather than simulated RS.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-13.
P2-12-16: HER2 Expression Is the Major Risk Factor for Recurrence in pT1a-b,N0 Breast Cancer: A French Regional Population-Based Study of 671 Patients.
Authors: Rouanet P , Daures JP , Roger P , Mathieu A , Romieu G , Cretin J , Barneon G , Granier M , Maran-Gonzalez A , Thibault S , Boissiere F , Bibeau F
Published: June 2012

Background: To evaluate the prognostic impact of HER2 overexpression in patients with pT1a-b, node negative, breast cancers.

Patients and Methods: A population of1127 patients whose diseases were staged as pT 1a-b, pN0 and who were treated in the Languedoc-Roussillon (ONCO LR Southern French regional network) from 1999 to 2004, was identified. 95% of patients had conservative management, no patient received adjuvant trastuzumab, 5% received chemotherapy and 80% anti-estrogen endocrine therapy. HER 2 status was retrospectively assessed by immunohistochemistry in 671 patients (122 pT1a/549 pT1b). Kaplan-Meier method was used to estimate disease-free survival (DFS). Cox proportional hazard models were used to determine associations between HER2 status and disease-free survival adjusting on variables significantly linked to it.

Results: 9-year Overall survival was 95%. HER2 overexpression (3+) was observed in 5.2% of the patients (n=35).

HER2 3+ category was most frequently identified in the following sub groups: pT1a lesion (12.3% vs 3.6%; p: 0.0001), mastectomies (14% vs 4.4%; p:0.023), Grade 2–3 (91% vs 50%; p<0.0001), estrogen receptor (ER) negative (−) tumors (57% vs 30%; p<0.0001), progesterone receptor (PR) - tumors (74% vs 42%; p: 0.0002). HER2 3+ was less frequent with adjuvant hormonal treatment (43% vs 80%; p<0.0001). 33 relapse (5%) were observed with a median follow-up of 6.4 years (range, 0.3 to 9.9 years). The 5-year DFS rates were 78% and 95% in patients with HER2−positive and HER2−negative tumors, respectively (p:0.017).

According to the immunohistochemical phenotype DFS5 were 95%, 94%, 85%, 73.6% for ER+/PR+/HER2− (n:502/75%), ER-/PR-/ HER2− (n:134/20%), ER+/PR+/HER2 3+ (n:15/2%) and ER-/PR-/ HER2 3+ tumors (n:20/3%), respectively (p:0.02).

In univariate analysis, HER2 positive tumors (p:0.017), phenotype classification (p:0.02) and adjuvant treatment (p:0.013) were significant prognostic factors. In multivariate analysis, only patients with HER2 3+ tumors had higher risks of recurrence (hazard ratio [HR], 2.41; 95% CI: [1.06−5.53]; p<0.05) than those with HER2 -tumors.

Discussion: Node-negative, pT1a-b, breast cancer patients overexpressing HER2 have a significant risk of recurrence at 6 years median follow-up. In our series of small breast tumors, HER2 status seems to be a better prognostic factor than ER status. In patients with hormone receptor-positive diseases, HER2 positivity is associated with a worse DFS despite an anti-estrogen treatment.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-16.
P2-12-32: Association between Progranulin (GP88) Expression and Recurrence Risk for Breast Cancer Patients with Estrogen Receptor Positive Invasive Ductal Carcinoma.
Authors: Serrero G , Hawkins DM , Ioffe O , Bejarano P , Phillips JT , Head JF , Elliott RL , Godwin AK , Weaver J , Yue B
Published: June 2012

Purpose: GP88 (progranulin) is a critical player of breast tumorigenesis for estrogen receptor positive (ER+) breast cancer. Pathological studies showed that GP88 was expressed in invasive ductal carcinoma (IDC), but not in normal mammary tissue, benign lesions or lobular carcinoma. The present study examines GP88 prognostic significance in association with recurrence risk for patients with ER+ IDC.

Patients and Methods: Two retrospective multi-site clinical studies examined GP88 expression by immunohistochemistry (IHC) analysis in paraffin-embedded tumor tissues in correlation with patients’ survival outcomes. The training study established a GP88 cut-off value associated with decreased disease-free (DFS) and overall (OS) survivals. The validation study verified the GP88 cut-off value and compared GP88 prognostic information with other prognostic factors in multivariate analysis.

Results: GP88 expression is associated with a statistically significant increase in recurrence risk for ER+ IDC patients. The training study established that GP88 3+ score by IHC analysis was associated with decreased DFS (p=0.0004) and OS (p=0.0036). The independent validation study verified that GP88 3+ score for the high risk group and demonstrated that GP88 3+ score was associated with a 5.9-fold higher hazard of disease recurrence and a 2.5-fold higher mortality hazard compared to patients with tumor GP88<3+. GP88 remained an independent risk predictor after considering age, nodal status, tumor size, tumor grade, progesterone receptor expression, treatment and disease stage.

Conclusion: Our training and validation studies demonstrate that the survival factor GP88 is a prognostic biomarker, predictive of recurrence risk and increased mortality for ER+ IDC patients, independent from other prognostic factors. These results provide support for measuring GP88 tissue expression for newly diagnosed early stage breast cancer patients.

This work was supported by grants R43CA124179, and U01CA113916 from the National Cancer Institute, grants 07-2007-064 and 02-2010-010 from the Avon Foundation for Women.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-32.
P2-13-04: Testing Women with Invasive Lobular Breast Cancer for BRCA Mutations.
Authors: Turco DL , Elsayegh N , Litton J , Hortobagyi GN , Arun B
Published: June 2012

Background: Infiltrating lobular carcinoma (ILC) is the second most common type of invasive breast cancer behind infiltrating ductal carcinoma (IDC). ILC makes up approximately eight percent of all invasive lesions. In addition, the mixed ductal and lobular carcinoma histology makes up approximately seven percent of all invasive diagnoses.

There are several biologic differences that are demonstrated by ILC compared to other common pathologies. They are more frequently bilateral and multicentric. They tend to be seen in women slightly older than the average age of diagnosis and are usually ER positive. It is also known that individuals with a genetic predisposition have an increased risk to develop breast cancers. Women with a mutation in their CDH1 gene have up to a 39% chance to develop ILC. The majority of hereditary breast cancer is caused by germ line mutations in the BRCA1 and BRCA2 genes. Women with a mutation in their BRCA1 or BRCA2 gene have up to an 87% risk to develop an invasive breast cancer, however, the presence of ILC in this population has not been well defined. Therefore, the aim of this study is to evaluate the rate of germline BRCA mutations in a cohort of patients both with pure ILC, as well as mixed ILC/IDC.

Methods: A retrospective chart review revealed one hundred and sixty nine women with ILC and mixed ILC/IDC who underwent genetic testing for mutations in the BRCA1 and BRCA2 genes through the Clinical Cancer Genetics Program at M. D. Anderson Cancer Center. Women are referred for genetic testing using referral guidelines based on the NCCN guidelines, this usually involves a personal history of early onset breast cancer and/or a family history of breast and/or ovarian cancer.

Results: Out of the 169 patients, 19 (11.24%) were found to have a germline mutation in their BRCA1 or BRCA2 gene. A significant majority (73.7%) of these patients were BRCA2 positive. Five women tested for a variant of uncertain significance. The average age of diagnosis for the cohort was 55.6 years (range 30–87); while the average age of diagnosis for a positive patient was 49.4 years (range 30–72). Of the 62 women with pure ILC, 5 (8.06%) were positive for a BRCA gene mutation. Historically, out of all the patients with breast cancer referred and tested through the Clinical Cancer Genetics Program, approximately 15% test positive, and research shows that in the general population, 7–10% of breast cancer patients will test positive.

Conclusions: while the positivity rates between the cohorts are not statistically significant, we have shown that patients with a BRCA mutation can develop ILC in addition to the more commonly seen IDC. We suggest that patients continue to be referred for genetic counseling according to the NCCN guidelines, regardless of the pathology of their tumor.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-04.
P2-13-11: Follow Up of BRCA1/2 Carriers: The Spectrum of Cancer Diagnoses in Healthy at Risk Individuals (HTR), and in Cancer Survivors (CS).
Authors: Clara AI , Costa CS , Silva J , Marques JC , Moura C , André S , Luís A , Bento S , Opinião A , Rodrigues P , Machado P , Fragoso S , Santos S , Vaz F
Published: June 2012

Introduction and Objective: Data from long term follow up of BRCA1/2 carriers is scarce and is mainly related to BRCA1 women. Our multidisciplinary program targets both women and men for counselling towards BRCA1/2 screening and inclusion in clinical follow up. In here we review all cancer diagnoses observed in our BRCA1/2 cohort during follow up.

Methods: Review of individual records of BRCA1/2 carriers registered from January 2000 to December 2010. Follow up was calculated since BRCA1/2 post-test counselling until the last visit to the Clinic. All new cancer diagnoses and preventive surgeries were registered.

Results: Two-hundred and fifty nine BRCA1/2 carriers (206 females and 53 males) were diagnosed with BRCA1/2 mutations (42 BRCA1 and 217 BRCA2). Medium follow up for all population is 25 months (1-98). At the date of initial BRCA1/2 diagnosis 99 women and 14 men were CS.

Female population: Eighty-eight female CS had been previously diagnosed with breast cancer (18 bilateral cases), 18 with ovarian cancer, and 1 with biliary tract cancer. Preventive surgeries in the CS female population were: bilateral adnexectomy (33 pts) and prophylactic contralateral mastectomy (10 pts). In this CS female population, new cancers, during follow up were: Contralateral breast cancer (4 cases), peritoneal cancer (2 cases in pts with previous prophylactic surgery) and skin non-melanoma cancers (2 cases). In female HTR, 23 preventive bilateral adnexectomies and 20 bilateral mastectomies were performed. Cancer diagnoses during follow up were: breast (11), peritoneum (1 in a pt with previous prophylactic surgery), gastric (1) and M3 leukemia (1). Global failure of prophylactic adnexectomy, so far (CS+HTR): 3/56 (5%).

Male population: The medium age for male CS is 73 yrs and for male HTR is 52 yrs. Male CS had mostly been previously diagnosed with BC (12; 4 bilateral) and prostate cancer (4). Other previous cancers: gastric (2), skin (2: 1Melanoma, 1 non-Melanoma), colorectal (1). One BRCA2 man with gynecomastia and prostate cancer was submitted to reduction mastectomy, as a preventive surgery. During follow up, we diagnosed second and third cancers in male CS: breast (2), prostate (6) and gastric (1). Only 1 male HTR was diagnosed with cancer: skin non-melanoma.

Conclusion: The proportion of second and third cancer diagnoses in the male BRCA2 CS population is higher than in the female BRCA1/2 CS population. Small numbers and the availability of preventive surgery for women influence this observation. No data from preventive mastectomy exists for males (we have one case). The low frequency of cancer in male HTR may be due to younger age and other unknown modifier factors. Longer follow is needed.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-11.