bluuebunny/crossref_metadata_2025 huggingface dataset:
P1-01-10: Immune Suppression of Regulatory T Cells and M2 Macrophage in Breast Cancer Patients.
Authors: Imoto S , Sakemura N , Kamma H , Nakatsura T
Published: June 2012

Background: Recent multi-disciplinary treatments of breast cancer (BC) could decrease the mortality rate, but successful immune therapy remains uncertain. To explore new strategy of immune therapy of BC, we are investigating about host-tumor immune response in BC patients. Materials and methods: Tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were obtained from early and advanced BC patients. PBMC were also collected from healthy volunteers. Regulatory T (Treg) cells were examined by counting CD4+CD25highCD127low/−cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, and CD163. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/−, ++, +++). Results: HLA-ABC and DR were stained negative in 44% and 82% of 50 BC cases. When host-tumor immune response were compared between 38 early BC cases and 12 advanced BC cases, numbers of CD68-positive cells significantly increased in peripheral tumor tissues of advanced BC cases than in those of early BC cases (92% versus 53%). CD163-positive tumor cells were also detected more frequently in advanced BC cases than in early BC cases (75% versus 16%). There were no significant differences of distribution of CD4, CD8, CD56, and CD83-positive cells in early and advanced BC cases. Treg cells in PBMC significantly increased in percentage of the population in 37 BC patients than in 21 healthy volunteers (4.2% versus 2.5% of CD4-positive cells at mean value). Interestingly, Treg cells decreased in percentage of the population in 36 postoperative BC patients. Conclusions: Our results suggest that Treg cells render BC patients under immune suppression and M2 macrophage plays an important role of tumor progression. Targeted therapy against M2 macrophage may be a promising strategy of breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-10.
P1-01-12: Mesoporous Silicon Particles for the Presentation of Tumor Antigens and Adjuvant for Anti-Cancer Immunity.
Authors: Meraz IM , Melendez B , Gu J , Serda RE
Published: June 2012

Custom-made vaccines based on personalized tumor antigens are realistic options for secondary therapy, with the patient's excised tumor providing antigens. Overlap in the personalized repertoire of tumor antigens among patients also provides insight into targets for preventative cancer vaccines. Nanotechnology provides carriers for shielded delivery of antigens and presentation of immunostimulatory molecules. Rapid uptake of particles by phagocytic immune cells and migration to lymphatic tissue for antigen presentation provides opportunities to elicit tumor specific immune responses. Dendritic cells (DC) are the master antigen presenting cells (APC) for efficient processing and presentation of antigens. We have tested the ability of mesoporous silicon particles (pSi) to function as substrates for DC and to activate cells via engagement of surface toll-like receptors (TLR). pSi particles, surface labeled with lipopolysaccharide (LPS) or monophosphoryl lipid (MPL), were presented to bone marrow-derived DC. This resulted in rapid uptake of particles, with an enormous capacity for number of particles internalized per cell. Confocal microscopy studies supported higher uptake of LPS and MPL conjugated as compared to unlabeled pSi. pSi particle uptake into DC was also supported by electron microscopy imaging. TLR ligands induced morphological changes in GM-CSF stimulated cells consistent with maturation towards a DC phenotype. As expected, LPS conjugation to particles resulted in significant toxicity. MPL conjugated pSi showed little or no toxicity to DC and showed improved particle uptake into DC with morphological changes consistent with maturation towards a mature DC phenotype.The impact of cytokine cocktail on DC maturation and its impact on particle uptake were also examined. Flow cytometry analysis supported greater uptake of pSi by DC stimulated with GM-CSF and IL-4 compared to GM-CSF alone, with reduced uptake in the presence of TNF-alpha. Consistent with findings reported by others, addition of TNF-alpha to the cell media also resulted in higher levels of expression of costimulatory molecules by DC, compared to cells stimulated with GM-CSF alone. Thus immature DC are better able to internalize particle-based vaccines, with cytokine or TLR-driven maturation enhancing expression of costimulatory molecules for more effective antigen presentation.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-12.
P1-02-03: The Reciprocal Roles of E-Cadherin and ZEB1 Demonstrate the Mesenchymal-Epithelial Transition as a Primary Characteristic of Inflammatory Breast Cancer.
Authors: Chu K , Boley KM , Luo AZ , Ye Z , Wright MC , Freiter EM , Robertson FM
Published: June 2012

Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities.

Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used.

Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.
P1-02-05: Invasive Lobular Carcinoma – A Luminal Breast Cancer Histotype Enriched for Epithelial-to-Mesenchymal Transition Features.
Authors: Metzger O , Singhal SK , Michiels S , Ignatiadis M , Bertucci F , Galant C , Larsimont D , Salgado R , Veys I , Fumagalli D , Saini KS , Piccart M , Sotiriou C
Published: June 2012

Background: Invasive lobular carcinoma (ILC) represent the second most common breast cancer (BC) histotype, but little information is available about its molecular features. We sought to perform a comprehensive molecular classification of ILC and to investigate the molecular features that can differentiate ILC from invasive ductal carcinoma (IDC).

Methods: A consecutive series of 174 ILC tumors from three different centers with mininum 5-year follow-up was selected. Gene expression data were generated using Affymetrix U133 Plus 2.0 arrays. Data were analyzed by using frozen robust multiarray analysis (Matthew et al. 2009). ILC tumors were classified into BC molecular subtypes using the PAM50 classifier. Invasive Disease Free Survival (IDFS) was defined as the interval between the date of surgery and the date of any invasive recurrence or death. Multivariate analyses for 10-y IDFS containing classical clinico-pathologic characteristics were performed for different gene signatures, which were scaled so that 2.5 and 97.5% percentiles equal −1 and 1. A cohort of 90 IDC tumor samples, normalised with the same method, was used to compare expression data between ILC and IDC.

Results: ILCs were mainly characterized as luminal A (LA: 76%, n=133) and luminal B (LB:; 20%, n=34) followed by a minority of HER2−positive (2%, n=3), basal (1%, n=2) and normal-like (1%, n=2) based on Perou's classification. LB had inferior 10-y IDFS than LA in univariate cox regression analysis (HR = 1.99, 95% CI, 1.08−3.67, p=0.026). The EMT gene module was significantly higher expressed in ILC as compared to IDC (p < 0.001). Adjusted hazard ratios (HR) for different gene modules investigated are detailed in the table below.

Conclusions: ILC is mainly composed of luminal tumors and a minority of HER2−positive tumors. Similarly to IDC, proliferation remains a statistically significant prognostic factor for ILC. ILC tumors were enriched with an EMT phenotype, which is not observed among IDC luminals. Of interest IGF signaling, which is known to be associated with poor prognosis in ER-positive BC, added prognostic value in this population which may open new therapeutics avenues for ILC.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-05.
P1-04-01: The Mechanism of Anti-Breast Cancer TICs Effect of Pyrvinium Pamoate Is through WNT/beta-Catenin Signaling.
Authors: Xu W , Debeb B , de Lacerda A , Li L , Larson R , Reuben J , Ueno N , Woodward W
Published: June 2012

We have previously shown that pyrvinium pamoate can decrease breast cancer TICs in vitro and shrink the tumor size in vivo. Although pyrvinium pamoate has been shown to target beta-catenin through activating CK-1alpha in a vitro model, the mechanism of its anti-breast cancer TICs effect is unknown. Herein, we use a constitutively active WNT/beta-catenin signaling construct EBETAP (ref) to determine if the anti-breast TIC effect of pyrvinium pamoate is through WNT/beta-catenin signaling. Using aldefluor expression and mammosphere formation efficiency as TIC surrogate assays, we found that TICs of SUM-159 transfected with EBETAP construct are resistant to pyrvinium pamoate treatment compared to control cells. Moreover, microarray analysis reveals a series of genes and signaling downstream of WNT-catenin were down-regulated in SUM-159 cells treated with pyrvinium pamoate. In summary, mechanism of anti-breast cancer TICs effect of pyrvinium pamoate is through WNT/beta-catenin signaling.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-01.
P1-04-04: Focal Adhesion Kinase Plays a Major Role in the Regulation of Human Ductal Carcinoma In Situ (DCIS) Stem Cell Activity.
Authors: Williams KE , Bundred NJ , Farnie G
Published: June 2012

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that has been implicated in the regulation of normal mammary stem cells and cancer stem cells (CSCs) within mouse models. FAK is frequently overexpressed in human breast cancers but its role in human CSCs is not known. We aimed to investigate the role of FAK in human ductal carcinoma in situ (DCIS) stem cells (SCs).

We have used PF573228 to inhibit FAK activity in SUM225 (HER2−overexpressing) and MCF10DCIS.com (HER2−normal) DCIS cell lines. PF573228 inhibits the autophosphorylation of FAK at tyrosine 397 causing inactivation. Western blot analysis confirmed PF573228 (0.1-5 μM) caused a dose dependent inhibition of FAK activity in both cell lines.

The in vitro mammosphere colony assay was used to measure CSC activity in both DCIS cell lines and primary DCIS samples (n=5) (LREC#01/012). Mammosphere-forming efficiency (MFE) was calculated as percentage colony formation (>60μm) in the presence or absence of PF573228 (0.1-5μM).

MFE was reduced in both the SUM225 cell line (p=0.02) and in the MCF10DCIS.com cell line (p=0.04) by PF573228 at a dose concentration of ≥0.1 μM (see Table 1). PF573228 (1 μM) also reduced mammosphere formation in human primary DCIS cells (n=5) by 18.1±5.75% when compared to control (p=0.03).

To measure the effect of PF573228 on DCIS SC self-renewal capacity we performed secondary generation mammosphere colony assays. Primary generation mammospheres that had been treated with PF573228 (0.1-5μM) were dissociated into a single cell suspension and reseeded out into mammosphere culture conditions with no additional PF573228. The mammosphere regeneration ratio (MRR) was calculated as the proportion of secondary mammospheres formed relative to the number of primary mammospheres.

MRR was reduced in both the SUM225 (p=0.005) and MCF10DCIS. com (p=0.001) cell lines after treatment with ≥0.5μM or ≥1.0μM PF573228 respectively (see Table 2). Preliminary data in human primary DCIS cells (n=1) also shows a reduction in MRR, from 0.56 to 0.14 with 0.5μM PF573228. These results suggest that CSC self-renewal capacity is reduced by FAK inhibition.

We conclude that FAK signalling is important in human DCIS SC activity measured by mammosphere growth and regeneration. Targeting FAK in the treatment of DCIS may reduce disease recurrence and improve patient outcome.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-04.
P1-04-08: Distribution of ALDH1 Positive Stem Cells in Benign Mammary Tissue from Women with and without Breast Cancer.
Authors: Isfoss BL , Holmqvist B , Alm P , Olsson H
Published: June 2012

Background and Aims. ALDH1 in female breast tissue has been linked to stem cells, but little is known about the benign cellular organisation in situ. We investigated the distibution of ALDH1 immunoreactive (ALDH1+) cells in histomorphologically benign breast tissue from 28 women with or without breast cancer.

Methods and Results. ALDH1+ cells were detected in benign tissue of women age 20 - 72, located most commonly within the luminal and intermediate ductular levels and in the stroma. ALDH1+ cell populations and Ki-67+ cell populations were present in separate ductules, both cell types rarely showing epithelial differentiation. ALDH1 + cells were non-reactive to Ki-67 and oestrogen receptor. Stromal round/oval ALDH1+ non-leukocyte cells in both age groups expressed contractile protein. There was a lower concentration of luminal and intermediate ductular ALDH1+ cells in post-menopausal women than pre-menopausal women, lower in cancer than non-cancer patients, and higher in women on exogenous hormones.

Conclusions. The study provides further evidence for the stem cell character of ALDH1+ cells, here in benign breast tissue of cancer and non-cancer women throughout non-lactating adult life, and contributes evidence of benign stromal ALDH1+ cells. The distribution of ductular ALDH1+ stem cells appears to be influenced by hormonal status.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-08.
P1-05-02: Epigenetic Regulation by Alcohol Reactivates Estrogen Receptor alpha in Estrogen Receptor alpha-Negative Cells.
Authors: Wong AW , Nunez N
Published: June 2012

Introduction: Alcohol consumption is an established risk factor for breast cancer development and contributes to mammary tumorigenesis through the regulation of estrogen receptor alpha (ERα) expression. Previously, we reported that alcohol consumption in the MMTV-neu ERα-negative mouse model of human breast cancer resulted in significantly increased ERα expression compared to non-alcohol consuming mice. Thus, our results indicated that alcohol consumption led to reexpression of ERα in an ERα-negative mouse model. Several lines of evidence suggest that ERα expression in ERα-negative cancer cells is inhibited through epigenetic mechanisms. In this study, we examine the role of alcohol on ERα reactivation through changes in histone modifications, DNA methylation, and recruitment of transcriptional regulation complexes on the ERα promoter, in vitro. Methods: ERα-negative MDA-MB-231 human breast cancer cells were treated with 0.1%, 0.2% and 0.5% v/v ethanol in culture media for 24 h. Chromatin immunoprecipitation (ChIP) assays were used to examine the enrichment of active and inactive markers of chromatin on the ERα promoter following alcohol treatment. DNA methylation at three ERα promoter CpG dinucleotide sites was assessed following alcohol treatment using methyl-sensitive restriction enzyme (MSRE)-qPCR. ChIP assays are used to determine the binding of the transcriptional regulation complexes, pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, to the ERa promoter following alcohol treatment. MCF-7 human breast cancer cells served as an ERα-positive control.

Results: We report that histone modifications were affected by alcohol treatment in a dose-dependent manner. Enrichment levels of all five chromatin markers tested, which includes markers of active and inactive transcription, were significantly altered as a result of alcohol treatment (p<0.05). We also found significantly decreased DNA methylation at three ERα promoter CpG sites as a result of alcohol treatment (p<0.05). Experiments testing the recruitment of the transcriptional regulation complexes, pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, are currently ongoing.

Discussion: ERα expression has been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, approximately one out of three breast cancers lack ERα expression at the time of diagnosis and many breast cancers lose ERα expression during the course of tumor progression. Thus, understanding the regulation of ERα expression in breast cancer cells is critical and results from this study will facilitate the development of novel therapeutic strategies and treatment options for hormone-resistant breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-02.
P1-06-16: BRCA2 Mutation Carriers Respond Poorly to Conventional Anthracylins/Taxanes-Based Neo-Adjuvant Chemotherapy.
Authors: Raphael J , Caron O , Rimareix F , Spielmann M , Al Ghuzlan A , Mathieu M-C , Delaloge S
Published: June 2012

Background: BRCA2 carriers typically develop luminal B-type breast cancers. Their exact sensitivity to conventional chemotherapy remains unclear. Promising drugs such as parp inhibitors are currently being developed in this indication. We attempted to evaluate pathological response rates of BRCA2 carriers in current the routine setting. Patients and methods: we screened our database of all patients seen at our genetic clinic over the past 15 years who were completely tested for BRCA1 and BRCA2 mutation and had received primary anthracyclines/taxanes-based chemotherapy (6-8 cycles) for invasive breast cancer as primary care. The pathological responses of pts who tested positive for BRCA2 were compared to those of BRCA1 and BRCA2-negative patients who had been diagnosed with luminal breast cancer (ER-positive, Her2-negative).

Results: Among 155 BRCA2-carriers and 503 wild-type patients, 24 BRCA2 pts and 58 pts with luminal B-type breast cancer have received primary standard anthracyclin/taxane-based chemotherapy. Median age was 38 for both populations. Median tumor size was 45 and 40 mm respectively among carriers and WT pts. 60% of BRCA2 and 100% of WT luminal B pts had ER-positive disease. A pathological complete response occurred in 18% of BRCA2 carriers and of 39% of luminal B Wt-pts. 65% of BRCA2 and 51% of WT-pts pts remained node-positive.

Conclusion: Deleterious germline mutations of BRCA2 are associated with a low probability of complete pathological response and a high risk of axillary invasion after conventional primary chemotherapy. Alternative treatments are highly expected.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-16.
P1-06-21: Relationship between Body Mass Index and Preoperative Treatment Response to Aromatase Inhibitor Exemestane in Postmenopausal Patients with Primary Breast Cancer.
Authors: Takada M , Saji S , Masuda N , Kuroi K , Sato N , Takei H , Yamamoto Y , Ohno S , Yamashita H , Hisamatsu K , Aogi K , Iwata H , Ueno T , Sasano H , Toi M
Published: June 2012

Background: Some studies have shown that high body mass index (BMI) is associated with inferior outcome after adjuvant therapy with non-steroidal aromatase inhibitor, anastrozole in breast cancer patients. We aimed to investigate predictive effect of BMI on clinical response to neoadjuvant therapy with steroidal aromatase inhibitor, exemestane in postmenopausal patients with primary breast cancer.

Patients and methods: The study group consisted of 109 patients from the JFMC 34-0601 neoadjuvant endocrine therapy trial in which postmenopausal patients with estrogen receptor (ER)-positive primary breast cancer were administered exemestane (25 mg/day) for 24 weeks before surgery. Patients were categorized into three groups according to BMI: low (BMI < 22 kg/m2), intermediate (22 ≤ BMI < 25 kg/m2) and high (BMI ≥ 25 kg/m2). Statistical analyses were performed to explore the predictive effect of BMI on clinical response using a multivariable logistic regression model.

Results: Higher BMI correlated with positive progesterone receptor status (p < 0.01) and low Ki-67 index (p = 0.03). Objective response rates (ORR) were 21.7% in low BMI, 56.0% in intermediate BMI and 60.6% in high BMI, respectively (p = 0.01). In a multivariate analysis, only low BMI was an independent negative predictor of clinical response.

Conclusion: Low BMI was associated with a decreased ORR to neoadjuvant endocrine therapy with exemestane. Our results may suggest that the predictive effect of BMI varies according to the type of aromatase inhibitor and objective outcome.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-21.
P1-06-25: Changes in FDG PET SUV Correlates with Ki-67 Following 2 Weeks of Aromatase Inhibitor Therapy in ER+ Early Stage Breast Cancer, a Pilot Imaging Study.
Authors: Gadi VK , Kurland BF , Specht JM , Rodler E , Korde LA , Peterson LM , Schubert EK , Chai X , Mankoff DA , Linden HM
Published: June 2012

Background: In estrogen receptor positive (ER+) tumors, a low proliferative index (Ki-67) two weeks into endocrine therapy predicts response. FDG PET non-invasively measures tumor sites in vivo. The pre-operative window is an opportunity to assess impact of systemic therapies. We tested associations between FDG PET standardized uptake value (SUV) and Ki-67 after two weeks of aromatase inhibitor (AI) therapy in newly diagnosed, postmenopausal women.

Methods: Postmenopausal patients with clinical stage I-II ER+ HER2− primary tumors underwent a 9–35 day “run-in” of AI monotherapy prior to definitive surgery. FDG PET was performed before AI therapy, and 1–5 days before surgery. Ki-67 was measured in baseline core biopsy and surgical specimens.

Results: To date, 18 patients (median age 59) have been enrolled of whom 14 patients have undergone serial FDG PET imaging with 12 completed assessment of Ki-67 in surgical samples including one who had no residual invasive carcinoma. The majority harbored ductal carcinomas (n=16) with 10/18 having histologic grade ≥ 2. The median number of days exposed to AI was 18 (range, 9–35). Baseline SUV ranged from 1.8 to 10.9 (median 2.5), and post run-in SUV (7-34 days later) ranged from 1.0 to 10.7 (median 2.5). A median 14% decrease in SUV was observed between paired FDG PET studies (range, 44% decline to 13% increase). Five of 12 patients’ index lesion FDG SUV declined by 20% or more; all also had Ki-67 ≤5% at surgery. An additional 5 patients with Ki-67 ≤5% at surgery had percentage change in FDG PET SUV of 0% to 17% decline. Results will be updated as accrual is ongoing.

Conclusions: Substantial changes in FDG PET SUV in the breast tumor were appreciated early in AI therapy. SUV declined or was stable in all but one patient of 14, and all patients with ≥ 20% decrease in SUV had a low (≤5%) Ki-67 at surgery. Serial PET is a promising measure of early response to therapy.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-25.
P1-07-07: Assessing Two Methods of Meta-Analysis in Studies of Patients with Breast Cancer: Individual Patient Data-Based (IPD) Versus Literature Based Abstracted Data (AD) in 5 Meta-Analyses Including over 28,000 Patients. Are There Results Differences of Concern?
Authors: Gralla RJ , Bria E , Raftopoulos H , Sperduti I , Giannarelli D , Cognetti F
Published: June 2012

Background: Meta-analyses are widely used to provide high level evidence to resolve controversies. Differences of opinion continue concerning the two most frequently used methods (IPD and AD) and their roles in providing reliable recommendations for Evidence-Based Medicine. Some regard the IDP method as being more accurate, yet it is time-consuming, expensive, and can be troubled by getting data from all investigators. We tested whether overall conclusions would differ between the two methods when using the same studies for comparison. To overcome limitations related to simple correlation analysis, and: 1) to weigh the agreement between these 2 approaches (Bland-Altman [BA], Lancet 1986), and 2) to evaluate the eventual predictive role of the data coming from AD over IPD, an analysis with multiple methods was performed.

Methods: 5 meta-analyses in breast cancer examining 9 outcomes which had been subjected to IPD meta-analysis were selected. Literature-based AD meta-analyses were then conducted. Hazard Ratios (HR) and Risk Ratios (RRs) with 95% confidence intervals (CI) were extracted or derived from original meta-analyses. At least 90% of patients’ reports were required for the IDP analysis. Methods agreement was analyzed according to the BA method, as difference or ratios, and a comparison with the Variance-Ratio test (F-test), and the Concordance Correlation Coefficient (CCC) was done. Correlations between IPD/AD-ratios were estimated using a linear regression model with Pearson (r) and R2 coefficients (parametric) and Spearman's Rho/Kendall's Tau coefficient (non-parametric), to derive a predictive estimation on IPD when adopting AD. MedCalc® Software was used.

Results: 9 outcomes in 5 meta-analyses (28,358 patients) were analyzed, as seen in the table.

Graphical comparison between the deviation from the ‘line of identity’ identified with the B-A method did not show meaningful differences between AD and IPD (mean difference −0.01535, 95% CI −0.0315, 0.0008; mean ratio 0.9816, 95% CI 0.9625, 1.0008). No significant difference in the Variance (F-test 1.01, p=0.98), and a high concordance (CCC 0.9775, 95% CI 0.91, 0.99) were found. A highly significant correlation by both parametric and non-parametric tests allowed derivation of a linear regression equation able to predict estimation IPD results from AD data; R2 0.97, p<0.001.

Conclusions: When analyzing the results of studies (AD) or patient results from those same studies (IDP) we found: 1) No significant or meaningful differences between the 2 meta-analysis methods; and 2) Data from the AD analyses are able to predict those from IPD, without significant risk of overestimation.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-07.
P1-07-12: Assessment of Real World HER2 Status by Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) in Breast Cancers: Comparison with HERmark®, a Validated Quantitative Measure of HER2 Protein Expression.
Authors: Huang W , Paquet A , Sivaraman S , Pesano R , Goodman L , Sherwood T , Lie Y , Hickey J , Walworth C , Haddad M , Anderson S , Bates M , Weidler J
Published: June 2012

Background: Accurate assessment of the HER2 status is critical in determining appropriate therapy for patients with invasive breast cancer. ASCO/CAP HER2 testing guidelines caution that up to 20% of routine HER2 testing by IHC/FISH may be unreliable (Wolff et al. JCO 2007;25:118). The HERmark assay is a novel quantitative HER2 protein measurement for determining HER2 status in breast cancer. Central HER2 testing showed high concordance (96-98%) with HERmark for positive and negative categories when equivocal subsets were excluded (Huang et al. Am J Clin Pathol 2010;134:303; Joensuu et al, 2008 SABCS, abstract 2071). In this study, we examined concordance between HERmark and routine HER2 testing by IHC and FISH from “real world” formalin-fixed, paraffin-embedded (FFPE) breast cancers submitted commercially for HERmark testing.

Methods: 717 HERmark results on FFPE breast cancers tested from 2008 to 2010 and corresponding HER2 IHC/FISH results were reviewed. The IHC and FISH results, per pathology reports submitted at the time of HERmark testing, were compared to HERmark categorical (negative, equivocal, positive) results.

Results: 590 (419) samples had IHC (FISH) and HERmark results available. Of these cases, 92% (94%) were either negative or equivocal by IHC (FISH). The HERmark testing reported 33% HERmark negative, 33% HERmark equivocal and 34% HERmark positive. Comparisons of HER2 status by IHC and FISH vs. HERmark are detailed in Table 1.

19% of cases classified as IHC 0 or 1+ were positive by HERmark; 35% classified as FISH negative were HERmark positive. Of the 158 triple negative cases by IHC/FISH (ER, PR and HER2 negative, data not shown), 38 (24%) were reclassified as HER2 positive by HERmark.

Conclusions: Higher than expected discordance between HER2 IHC/FISH and the HERmark assay was observed in this study compared with central HER2 testing of prior study cohorts. Selection bias may have impacted these results as ≥ 92% of cases submitted for HERmark testing were either equivocal or negative by routine HER2 IHC/FISH.

Central HER2 FISH retesting of these samples will be performed and compared with HERmark results to further evaluate the discordance observed in this analysis.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-12.
P1-07-18: Expanding an Online Tool for Genome-Wide Validation of Survival-Associated Biomarkers in Breast and Ovarian Cancer Using Microarray Data of 3,862 Patients.
Authors: Gyorffy B , Lanczky A , Szallasi Z
Published: June 2012

The pre-clinical validation of prognostic gene candidates in large independent patient cohorts is a pre-requisite for the development of robust biomarkers. We earlier implemented an online tool to assess the prognostic or predictive value of the expression levels of all microarray quantified genes in breast cancer patients. In present study, we further expanded our database, added additional analytical options and implemented the tool for ovarian cancer patients.

The database was set up using gene expression data and survival information of breast and ovarian cancer patients downloaded from GEO and TCGA (Affymetrix HGU133A, HGU133A 2.0 and HGU133+2 microarrays). After quality control and normalization only probes present on all three Affymetrix platforms were retained (n=22,277). Patients can be stratified into the various robust subtypes either by histology or by various gene expression profiling based methods. To analyze the prognostic value of the selected gene in the various cohorts the patients are divided into two groups according to the median expression of the gene. A Kaplan-Meier survival plot is generated and significance is computed.

All together 2,472 breast cancer patients and 1,390 ovarian cancer patients were entered into the database. These groups can be compared using relapse free survival (n=2,414 in breast cancer and 1,090 in ovarian cancer) or overall survival (n=463 and n=1,290). Follow-up threshold has been implemented to exclude long-term effects. The combination of several probe sets can be employed to assess the mean of their expression as a multigene predictor of survival and therapy efficiency.

In summary, we expanded our global online biomarker validation platform to mine all available microarray data to assess the prognostic power of 22,277 genes in 2,472 breast and 1,390 ovarian cancer patients. The tool can be accessed online at: www.kmplot.com/breast and www.kmplot.com/dev/ovar.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-18.
P1-07-20: Consistent High False Negative Rate of HER2 qRT-PCR of Oncotype DX® in Comparison to ASCO/CAP Recommended Combined IHC/FISH Method.
Authors: Dabbs DJ , Bhargava R
Published: June 2012

Background In our prior multi-institutional study of 843 cases we have shown a high false negative rate for HER2 testing by the oncotype DX® RT-PCR method, (ODX) compared to our combined FISH and IHC approach [J Clin Oncol 29: 2011 (suppl; abstr 603)]. This study is part of an ongoing quality assurance study, comparing HER2 test results by FISH/IHC and the ODX.

Methods From July 2010 till April, 2011, all patients who had either positive or equivocal HER2 results (on core biopsy) at Magee-Womens Hospital (MWH) by combined IHC/FISH, and who also had the ODX ordered (on resection specimen) by an oncologist, were reviewed for positive concordance of HER2 results. All tissues were fixed and FISH/IHC was reported according to ASCO/CAP HER2 guidelines. The core biopsy HER2 positive results were also confirmed positive on the same tissue block that was tested by ODX.

Results Of the total 234 cases, there were 21 cases that were either classified as positive (n=8) or equivocal (n=13) by combined IHC/FISH approach at MWH. Of the 8 MWH+ cases, 4 showed 3+ IHC score and these were also positive by ODX. The remainder 4 MWH+ cases were IHC 2+, and unequivocally amplified by FISH. These 4 cases were either equivocal (2 cases) or negative (2 cases) by ODX. Thirteen cases reported as equivocal by MWH FISH, were all reported as negative by ODX. Of the 213 MWH negative cases, 212 were also negative by ODX (percent negative agreement of >99%) and one case was called equivocal. If all equivocal cases are excluded, then percent positive agreement (PPA) between IHC/FISH and ODX is 67% (4/6). If “equivocal” cases are considered as “negative”, then PPA is 50% (4/8). If “equivocal” cases are considered as “positive”, then PPA is only 29% (6/21).

Conclusions (1) Similar to our prior study [J Clin Oncol 29: 2011 (suppl; abstr 603)], there is a consistent poor percent positive agreement for HER2 testing between FDA cleared FISH/IHC methods and ODX, and these results cast serious doubt on the robustness/reliability of ODX results for HER2. (2) There should be serious concern raised over the reliability of the other multivariate index platform results of ODX, and likely, the ODX recurrence score for an individual patient, as this sensitivity data suggests inadequate tissue microdissection, or issues with the ODX test platform. (3) Clinicians need to be aware that HER2 results of ODX are not reliable for patients who are equivocal or amplified by FDA approved methods, and only FDA approved methods should guide treatment decisions.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-20.
P1-07-22: A Venezuelan Study of Breast Cancer Estrogen Receptor, Progesterone Receptor and HER2 Receptor Expression by the Standard Method, Immunohistochemistry (IHC), Compared to a New Method, Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Authors: Marin C-EM , Ramirez AC , Baehner FL , Yoshizawa C , Acosta MM
Published: June 2012

Background: Assessment of ER, PR and HER2 is of significant importance in breast cancer diagnosis and treatment. We have performed the first large assessment of central IHC for ER, PR and HER2 by a South American (Caracas, Venezuela) Central Lab to Central Lab RT-PCR by Oncotype DX®.

Design: Breast cancer specimens from the Fundación BADAN were evaluated by IHC for ER (clone 1D5), PR (clone 636) using 1% staining for positivity using ASCO/CAP guidelines and HER2 (clone A0485) using ASCO/CAP guidelines of 3+ in ≥30%. Standardized quantitative RT-PCR analysis for ER and PR used Oncotype DX with the pre-defined cutoffs of 6.5 units and 5.5 units for positivity, respectively. For HER2 the standard pre-defined cutoffs were used: positive ≥11.5 units, equivocal >10.7-<11.5 units, and negative ≥10.7 units. For HER2 concordance analysis the equivocal range was excluded from both assays according to ASCO/CAP Guidelines (Wolff et al, 2006). For all quantitative RT-PCR assessments the reference normalized expression measurements ranged from 0 to 15, where each 1-unit increase reflects about a 2-fold increase in RNA.

Result: Evaluable data was obtained in 96 pts for ER, 95 pts for PR (1 patient assessment unavailable) and 89 pts for HER2 (7 patient assessments unavailable). Two-by-two tables (below) compare IHC versus RT-PCR for ER, PR and HER2. The overall concordance for ER between IHC and RT-PCR was 94% (Kappa 0.467; 95% CI 0.107, 0.828); for PR between IHC and RT-PCR was 92% (Kappa 0.668; 95% CI 0.457, 0.878); and for HER2 between IHC and RT-PCR was 97% (Kappa = 0.649; 95% CI 0.280, 1.000). Three IHC 2+ cases and 4 equivocal cases by Oncotype DX were excluded from Concordance and Kappa statistics.

Conclusion: RT-PCR by Oncotype DX for ER, PR and HER2 status is useful for active monitoring of IHC assays as mandated by ASCO/CAP guidelines. There is a high degree of overall concordance between central IHC performed in South America (Caracas, Venezuela) and central RT-PCR for ER, PR and HER2 status.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-22.
P1-08-03: Huge Improvement in Relapse-Free Breast Cancer Survival over the Last 25 Years.
Authors: Geurts SME , van Dijck JAAM , de Vegt F , Paquay Y , Siesling S , Verbeek ALM , Tjan-Heijnen VCG
Published: June 2012

Introduction The purpose of this study was to evaluate the dwindling risk of relapse in breast cancer patients treated with curative intent since the 1970s.

Patients and methods 8570 women diagnosed with invasive breast cancer in 2003 or 2004 were compared with 307 consecutively diagnosed patients with invasive breast cancer between 1972 and 1986 in the Netherlands. Five-year overall survival and 5-year risk of relapse, i.e. second primary breast cancer, locoregional recurrence or distant metastasis, were calculated using the Kaplan-Meier method. Multivariable Cox-proportional hazards modelling was applied to correct the period-specific risk of relapse for tumour size, lymph node involvement and age at diagnosis.

Results Median (range) age at diagnosis was 52 years (27-82 years) in 1972–1986 and 58 years (20-96 years) in 2003–2004. Patients diagnosed in 1972–1986 had a larger tumour size and similar nodal involvement as compared to patients diagnosed in 2003–2004. In 2003–2004 more women were treated with breast conserving surgery, chemotherapy, and hormonal therapy as compared to 1972–1986. The 5-year overall survival rate increased from 71% (95% CI: 65% — 76%) in the period 1972–1986 to 85% (95% CI: 84% — 85%) in the years 2003–2004. Five-year risk of any relapse decreased from 37% to 16% across the calendar years (table 1). This decrease was observed for the risk of locoregional recurrence and the risk of distant metastasis (table 1). Risk of second primary breast cancer was similar for both periods of diagnosis (table 1). After adjustment for tumour size, nodal status and age at diagnosis the decrease in risk of relapse remained significant for patients diagnosed in 2003–2004 compared to 1972–1986 (HR= 0.5, 95% CI: 0.4 - 0.6).

Conclusion Over the last decades, the risk of breast cancer relapse has tremendously decreased, also after adjustment for tumour stage and age at diagnosis. The improved prognosis can be explained by the more often administered and intensified systemic treatment procedures and, further, by an earlier detection. The similar nodal stage probably resulted from stage migration caused by a more thoroughly performed lymph node staging in 2003–2004 compared to 1972–1986.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-03.
P1-08-04: Obesity, Adjuvant Therapy, and Survival Outcomes in Early-Stage Breast Cancer.
Authors: Jiralerspong S , Wang T , Rimawi MF , Nangia JR , Schiff R , Giordano SH , Pollak MN , Chenault CC , Osborne CK , Hilsenbeck SG
Published: June 2012

BACKGROUND: Obesity has risen to epidemic proportions and is associated with worse breast cancer (BC) prognosis in most studies. However, the effects of obesity according to adjuvant therapy choice are largely unknown. To address this issue, we examined the relationship between body mass index (BMI), adjuvant therapy, and survival outcomes in a large cohort of early-stage BC patients.

METHODS: We retrospectively studied patients from the Baylor Breast Center Tumor Bank treated from 1970–1995. Patients were divided into 3 BMI classes: normal/underweight (N, BMI<25), overweight (Ov, BMI 25–30), obese (Ob, BMI≥30); and 4 treatment groups: no adjuvant therapy, chemotherapy (mainly CMF), endocrine therapy (mainly tamoxifen), both chemo- and endocrine therapy. Time-to-recurrence (TTR), disease-free survival (DFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared among groups via the log-rank test. Multivariate analysis was conducted via Cox proportional hazards models.

RESULTS: There were 4,368 patients. Median age was 58. 74% were postmenopausal. 72% had stage I-II disease, 28% stage III. 76% were estrogen receptor (ER)-positive, 24% ER-negative. Patients distributed into BMI classes as follows: N 48%, Ov 30%, Ob 22%. Higher BMI was associated with postmenopausal status and increasing age, tumor size, positive lymph nodes, and stage, as well as a higher likelihood of receiving treatment. Median follow-up was 5 years. Kaplan-Meier analysis showed that TTR was significantly shorter in the Ov and Ob groups as compared to the N group (p=0.019), due to distant (p=0.001) rather than local (p=0.970) recurrences. DFS was also significantly worse in the Ov and Ob groups (p=0.002), as was OS (p=0.001). The Table shows the hazard ratios for the various survival outcomes after adjustment for age, tumor size, nodal status, and treatment groups. For all patients, TTR, DFS, and OS were significantly worse in the Ob vs. N groups. TTR and DFS were significantly worse in the chemo treated Ob vs. N groups. DFS and OS were significantly better in the endo treated Ov vs. N groups.

DISCUSSION: In this large cohort of BC patients, survival outcomes (TTR, DFS, OS) were significantly worse in the obese group. This remained true after adjustment for multiple factors. Obesity was associated with worse survival outcomes in the chemo treated (CMF) group. Overweight was associated with better survival outcomes in the endo treated (tamoxifen) group. These results confirm and extend the results of previous studies. Further studies to discover the reasons for these differences in outcomes are underway.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-04.
P1-08-06: Breast Cancer among Patients with Diabetes, Obesity and Abnormal Blood Lipids – A Population-Based Register Study in Sweden.
Authors: Olsson H , Attner B , Landin Olsson M , Lithman T , Noreen D
Published: June 2012

Objective: To study how the incidence of breast cancer is related to diabetes, obesity or abnormal blood lipids.

Methods: Diagnosis of diabetes, obesity or abnormal blood lipids was studied 0–10 years prior to the diagnosis of cancer in 2724 cases of cancer and in 20542 controls matched regarding age, sex and domicile in a population based material. Diagnoses were obtained by using out and inpatient population based registries. Also the use of glargine and metformin was studied in relation to cancer risk in diabetic patients using the national pharmacy prescription registry. Conditional logistic regression was used for the analyses.

Results: Diabetes was significantly more common prior to diagnosis in patients with breast cancer with diabetes diagnosed 0–4 years prior to the cancer diagnosis. The findings remained after adjusting for obesity and high blood lipids. Obesity was significantly more common in patients with breast cancer above the age of 60 years in those where obesity was diagnosed close to the diagnosis of cancer. High blood lipids were significantly less common in patients with breast cancer close to diagnosis.

Glargine use was associated with a doubled risk 2.88 (1.15−6.64) and metformin use with a lower risk of cancer in diabetic patients 0.92 (0.82−1.09).

Conclusions: Within 4 years of diagnosis diabetes, obesity after age 60 and low blood lipids are associated with breast cancer. Glargine use seems to increase overall cancer risk.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-06.
P1-08-10: Invasive Lobular Breast Cancer – No Increased Risk of Contralateral Disease.
Authors: Langlands F , Horgan K , Kearins O , Burns R , Dodwell D
Published: June 2012

Background: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer accounting for up to 14% of invasive breast cancers. Worldwide the incidence of breast cancer is increasing each year. Large population based studies using the Surveillance, Epidemiology, and End results (SEER) data have shown that the incidence of ILC has increased from 1977 to 1995. ILC has historically been thought to be associated with an increased risk of developing contralateral breast cancer and this belief may, in part, be responsible for the increasing trend towards contralateral prophylactic mastectomy.

Methods: All female patients diagnosed with breast cancer during the time period 1998–2003 were identified from two large cancer registries, Northern and Yorkshire Cancer Registry and Information Service (NYCRIS) and West Midlands Cancer Intelligence Unit (WMCIU). All patients diagnosed with either Infiltrating duct(adeno) carcinoma, microinvasive ductal carcinoma, infiltrating ductular carcinoma (all classified as ‘ductal') or lobular (adeno)carcinoma were included. Patients with a mixed type of breast cancer were excluded from the analysis. Follow-up was complete until October 2010. Data were compiled for diagnosed contralateral breast cancer of any histological type.

Results: Of the 32,735 patients with invasive ductal cancer 898 (2.74%) developed a contralateral breast cancer. In comparison 166 (3.1%) of 5397 patients with lobular cancer developed a contralateral breast cancer. The median time to first contralateral event was equivalent for both morphologies (ductal 40 months and lobular 39 months).

Conclusion: This study suggests that there is no increased risk for developing a contralateral breast cancer in patients diagnosed with an invasive lobular breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-10.