bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3-02-02: Influence of Body Mass Index on Presence of Disseminated Tumor Cells in Clinical Stage I-III Breast Cancer Patients
Authors: Lodhi AK , Krishnamurthy S , Bhattacharyya A , Hall CS , Anderson AE , Singh B , Lucci A.
Published: April 2011

Background: Disseminated tumor cells (DTCs) are found in approximately one third of clinical stage I-III breast cancer (BC) patients, and published reports show that presence of DTCs is an independent predictor of outcome. While higher body mass index (BMI) is associated with increased risk of breast cancer recurrence and lower survival rates in BC patients, women with lower BMIs may have lower bone density and higher bone turnover. We hypothesized that increases in bone turnover may result in the release of bone growth and “homing” factors that facilitate BC metastasis to bone and provide a “pre-metastatic niche” for BC cells. The purpose of this study was to determine if a correlation existed between DTCs and BMI in early stage BC patients.

Methods: We obtained informed consent and collected bone marrow samples from 262 clinical stage I-III BC patients who were participants in an IRB-approved clinical study from 2/2005- 2/2010. All marrow samples were collected at the time of surgery for the primary tumor. DTCs were assessed using anti-pancytokeratin (CK) antibody cocktail (AE1/AE3, CAM5.2, MNF116, CK8 and 18) following cytospin. The presence of one or more CK positive cells meeting morphologic criteria for malignancy was considered a positive result for DTC. Patients with a BMI of (18.5 — 24.9) kg/m2 were considered “normal weight”, those with a BMI of (25 - 29.9) kg/m2 “overweight” and a BMI greater than 30 kg/m2 was used to designate them as “obese”. Information on clinicopathological factors including BMI (measured on initial presentation) was obtained from a prospective database. Statistical analyses used Chi-square and non-parametric tests for trend.

Results: Median follow-up was 19 months and mean age was 53 (25-80) years. Eighty-four patients (32%) were normal weight, 85 (32%) were overweight and 91 (35%) were obese. Seventy-eight (30%) patients had DTCs present at the time of assessment. Obese patients were significantly less likely to show presence of DTCs as compared to those who had a BMI < 30 kg/m2 (20/78; 26% vs. 71/184; 39%) {O.R. = 0.55, 95% C.I. = 0.29- 0.96, P = 0.03}. DTCs were also less likely to be found in patients with BMI ≥25 kg/m2 as compared to those with BMI < 25 kg/m2 (40/78; 51% vs. 136/184; 74%); {O.R. = 0.42, 95% C.I. = 0.04- 1.02, P = 0.03}. No statistically significant correlation was observed between primary tumor characteristics (ER, PR, HER2, lymph node status, tumor grade) and presence of DTCs. Finally, a non-parametric analysis demonstrated a trend in occurrence of DTCs across the ordered levels of patients’ BMI values (P= 0.013).

Conclusions: DTCs were much more common in patients with lower BMI. Further studies are needed to determine if patients with low BMI have unique microenvironmental factors within the bone that predisposes them to tumor cell dissemination.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-02.
Abstract P3-02-06: Correlation of BCL-2 and Apoptosis on Circulating Tumor Cells and Breast Cancer Tissue
Authors: Maranci V , Thomas DG , Brown M , Budd TG , Doyle G , Hayes DF , Griffith KA , Smeage J.
Published: April 2011

Background: Circulating Tumor Cell (CTC) levels are prognostic markers in metastatic breast cancer (MBC). Further phenotypic characterization of CTC may provide an opportunity for non-invasive evaluation of predictive and prognostic markers. Apoptosis is a common form of chemotherapy-induced cell death. Monoclonal antibody (MAb) M30 recognizes a neo-epitope on fragmented cytokeratin and is a marker of apoptosis. BCL-2 is an anti-apoptotic marker and may predict for resistance to anti-neoplastic therapy. We have previously reported the results of a pilot clinical trial to estimate M30 and BCL-2 expression in CTC from patients with MBC. The current study was performed to correlate the expression of both BCL-2 and M30 in CTC with breast cancer tissue samples. Methods: Of the 85 patients in the original pilot study, 52 (61%) had evaluable tissue available (39 primary and 12 metastatic lesions; one unknown) and were included in this analysis. CTC were collected at baseline and were isolated, enumerated, and phenotypically characterized for M30 and BCL-2 using the CellSearch® System. CTC phenotype is reported as percentage of cells with positive staining. Tissue Microarrays (TMA) were constructed and immunohistochemically stained for M30 (anti-Cytodeath™ M30, Roche) and Bcl-2 (M-0887, DAKO) and scored using the Allred method.

Results: Tissue staining for M30 and Bcl-2 were inversely correlated. Of the 52 patients with TMA available for investigation, 22 (42%) had > 5 CTC/7.5 ml whole blood. A positive, but non-significant, correlation was observed between increasing numbers of CTC levels and tissue BCL-2 Allred Score (Spearman r=0.36; p=0.1310). M30 staining was detected in ≥10% of CTC in 20 of the 22 (91%) patients with elevated CTC, while BCL-2 staining was detected in ≥10% of CTC in 19 of the 22 (86%) patients. Little if any correlation was observed between CTC M30 and tissue M30 expression, however CTC BCL-2 was positively correlated with tissue Bcl-2 expression (Spearman r=0.47; p=0.0440). Conclusions: BCL-2 and M30 can be characterized in both CTC and breast cancer tissue. The likelihood of having elevated CTC may be associated with higher BCL-2 expression, and CTC BCL-2 was associated with tissue BCL-2 expression. Although the clinical implications of these findings are unknown, they offer the opportunity for future trials to investigate whether CTC BCL-2 and M30 might be useful to identify patients with cancers that are resistant to standard therapies. Furthermore, CTC BCL-2 and M30 might be monitored in clinical trials of novel, apoptosis-inducing therapeutic agents.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-06.
Abstract P3-09-04: Exosome-Specific microRNA Signatures in Combination with Characteristic Surface Markers on the Circulating Exosomes Themselves Provide New Insights into the EMT
Authors: Paulaitis M , Guzman N , Agarwal K , Saji M.
Published: April 2011

Background: Recent discoveries have established that cancer tumors exhibit distinct microRNA (miR) expression profiles compared to normal tissues. Detection of miRs in the peripheral blood of cancer patients is possible due to their high stability. This stability is attributed to encapsulation of the miRs inside microvesicles (MVs) where they are protected from endogenous RNase activity in circulation. The tumor cell-secreted MVs of primary interest are circulating exosomes, a subpopulation of MVs distinguished by their relatively small size: 40 to 100 nm in diameter. The isolation of circulating tumor-derived exosomes from the other cell-secreted MVs in peripheral blood is the critical barrier to successful development of a robust assay for cancer-specific miR signatures. We describe the development of such an assay to: (1) capture/isolate circulating exosomes based on characteristic surface markers that correlate with different cell types, and (2) characterize exosome-specific miR signatures based on these surface markers.

Materials and Methods: We apply sequential ultracentrifugation to isolate cell-secreted MVs, followed by asymmetric flow field-flow fractionation to selectively separate exosomes based on their intrinsically small size and characteristic surface markers. Isolated exosomes are then screened for surface markers by selectively capturing them on microarrays printed with antibodies against a library of known cell-surface markers for breast cancer. For the assessment of exosome-specific miR signatures, we have devised an antibody microarray assay that also enables the in situ characterization of miR profiles of the captured exosomes locally by qRT-PCR analysis confined to subarrays of printed spots in 50-µl microwells of a custom-designed, multi-well microarray. Assay development has been carried out using two human cancer cell lines (MCF 7 and MDA MB 231) and a non-malignant cell line (MCF 10a) representing pre-malignant cells and cancer of the breast at different epithelial and mesenchymal states. Results: We have used light scattering and cryo-transmission electron microscopy to characterize the size, size distribution, and morphology of exosomes as a sub-population of the secreted MVs from each of the three cell lines. We have also screened for surface markers by selective capturing the exosome sub-populations derived from these cell lines on antibody microarrays, and correlated these surface markers with the miR content of MVs using qRT-PCR analysis.

Discussion: Our results demonstrate that correlating the miR signatures of circulating exosomes with characteristic surface markers on these exosomes leads to robust distinctions between the three cell lines that could not be achieved using either analysis or characterization alone.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-04.
Abstract P3-09-05: Let-7 microRNAs Regulate a Novel Variant of Estrogen Receptor Alpha, ER-≥36, Nongenomic Estrogen Signal Pathway, and Tamoxifen Resistance in Breast Cancer
Authors: Zhao Y , Deng C , Lu W , Xiao J , Recker RR , Guishan GG.
Published: April 2011

Background: MicroRNAs (miRNAs) have important regulatory functions in breast cancer tumorigenesis. We previously found that let-7 miRNAs were significantly downregulated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues.

Material and Methods: QRT-PCR was used to check let-7 family miRNAs in FFPE tissues and breast cancer cell lines as well as expression of estrogen receptor (ER)-≥36, a variant of ER-≥66, after let-7 miRNA transfecton. Immunoblot analysis was employed to check protein expression in FFPE tissue and breast cancer cell lines. Luciferase reporter assay was used to detect direct regulation of let-7 miRNA on ER-α expression. MTT assay was applied for cell proliferation after transfection of let-7 miRNAs. Results: We found that there was an inverse correlation between the expression of ER-≥36 and several members of let-7 family miRNAs in the FFPE tissue set. Let-7 miRNA sequences match sequence in the 3’ untranslated region (3’ UTR) of ER-≥36, indicating ER-≥36 may be a target of let-7. Cotransfection of let-7 mimics with ER-≥36 3’ UTR luciferase construct decreased the activity of reporter gene. Conversely, let-7 inhibitors enhanced the reporter gene activity. Transfection of let-7 mimics inhibited both the mRNA and protein levels of ER-≥36 and further inhibited the non-genomic estrogen pathway mediated by ER-≥36 in MDA-MB-231 cells. On the contrary, transfection of let-7 inhibitors enhanced the ER-≥36 expression at both mRNA and protein levels in 184A1 cells. The high expression of ER-≥36 in tamoxifen resistant MCF7 cells can be inhibited by transfection of let-7 mimics and sensitivity toward tamoxifen is enhanced.

Conclusion: Let-7 miRNAs regulate the expression of ER-≥36, which is involved in non-genomic estrogen pathway and tamoxifen resistance. Let-7 could be therapeutic target for breast cancer treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-05.
Abstract P3-10-03: The Implication of Androgen Receptor Expression in Breast Cancers
Authors: Park S , Koo JS , Kim MS , Park HS , Lee JS , Lee JS , Kim SI , Park B-W.
Published: April 2011

Purpose: To evaluate the implication of androgen receptor (AR) expression in patients with breast cancer.

Methods: Immunohistochemically AR expression was investigated in 931 invasive breast cancers prepared in tissue microarray blocks that were collected in between November 1999 and August 2005. The clinicopathological features, disease-free (DFS) and overall survival (OS) were analyzed using chi-square test, Kaplan-Meier methods, and Cox's models. Tumors with ≥10% nuclear-stained cells were considered to be positive for AR, estrogen receptor (ER), and progesterone receptor (PR) expression and 3+ with immunohistochemical staing or FISH + were considered positive for HER2 expression.

Results: The positive expression of AR, ER, PR, and HER2 was 58.1%, 72.2%, 61.4%, and 24.7% of all patients, respectively. AR expression was significantly associated with an older age at diagnosis (p=0.007), smaller tumor size (P<0.001), well differentiation (P<0.001), higher hormone receptors expression (P<0.001), non-triple-negative breast cancers (non-TNBC) (P<0.001), and lower proliferation index (P<0.001). In ER-negative cancers, AR expression was associated with well differentiation, lower Ki-67 index, and distinctively HER2 overexpression. With median follow-up duration of 72.7 months, AR was significantly associated with better DFS (p=0.023) and OS (p=0.048) and the association was same in ER-positive cancers but was disappeared in ER-negative cancers. In Cox's models, AR expression was an independent prognostic factor for DFS (hazard ratio, 0.654; p=0.049), but it did not reach a statistical significance for OS (hazard ratio, 0.647; p=0.119) even in ER-positive tumors. Interestingly, tumors with molecular apocrine features (ER-negative, AR-positive, and HER2-positive) showed a trend of poorer outcome with AR expression, but there was no impact of AR expression on the survival in 156 patients with TNBC.

Conclusions: AR expression is significantly associated with favorable clinicopathological features and better outcomes in ER-positive cancers but tumors with molecular apocrine features showed a trend of poorer outcome with AR expression. These results suggest that AR expression could be an additional marker for endocrine responsiveness in ER-positive tumors and a target molecule for the molecular apocrine tumors.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-03.
Abstract P3-10-04: An Integration of Biological and Pathological Marker Panel in the TEAM Pathology Sub-Study: The Impact of Different Parameters on Risk Estimation of Relapse at Both 2.75 and 5 Years
Authors: Bartlett JMS , Stocken D , van de Velde CJH , Brookes CL , Robson T , Hasenburg A , Hille ETM , Kiebeck D , Markopoulos C , Mallon EA , Dirix L , Campbell FM , Seynaeve C , Rea DW.
Published: April 2011

Background: Recent evidence confirms the importance of both biological and pathological risk markers in predicting early relapse for breast cancer patients treated with endocrine therapy. Most studies use a two step process integrating biological markers into a “biological predictor (e.g. Oncotype Dx, “IHC4” etc) followed by assessment of the predictive value of such tests in the context of pathological markers (grade, nodal status etc). We have taken a one step process integrating both biological and pathological markers into a single model to assess key factors for predicting outcome at 2.75 years and 5 years of endocrine therapy; to inform choices between switching, upfront and extended adjuvant treatment with AIs. Patients & Methods: Pathology blocks from 4598 TEAM patients were collected and tissue microarrays constructed. Quantitative analysis ER, PgR, Ki67, HER1, HER2, and HER3 was performed centrally. A prognostic model, integrating data from biological and pathological markers was created to assess risk (disease-free survival) after 2.75 and 5 years of follow up in the TEAM trial.

Results: Of 4595 eligible cases samples received, 16 were excluded, and 3993 had complete biomarker data for all markers for the final biomarker analysis. In univariate analysis nodal status, grade, size, age at diagnosis, HER1, HER2, PgR, ER and Ki67 were all prognostic. At 2.75 years nodal status, age, PgR histoscore, size, grade, HER2, ER histoscore and HER1 positivity were significant prognostic variables (ranked by WaldX2 statistic), Ki67 and HER3 were not included in this model. At 5 years median follow up; age, nodal status, size, PgR histoscore, grade, Ki67, HER2, and HER1 positivity were significant prognostic variables (ranked by WaldX2 statistic), ER and HER3 were not included in this model. Conclusion: Combined biological and pathological marker panels are of significant value in predicting early relapse in breast cancer patients treated with endocrine therapy, however duration of follow-up may impact on the inclusion of variables in the model. This provides significant information relevant to the choice of different adjuvant endocrine therapies.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-04.
Abstract P3-10-05: Triple Negative Breast Cancer: Clinical Characteristics in the Different Histological Subtypes
Authors: Dreyer G , Vandorpe T , Forceville K , Neven P , Smeets A , Janssen H , Brouwers B , Deraedt K , Paridaens R , Wildiers H.
Published: April 2011

Purpose: To study clinical characteristics of different histological subtypes of triple negative breast cancer (TNC). TNC are often considered as one group, but consist of many different histological subtypes with different biology and behavior. We decided to analyze a large cohort of TNC in a single centre and focus on clinical behavior including age distribution, occurrence of lymph node invasion, and outcome in different histological subtypes.

Patients and methods: We used data from the breast cancer database from UH Leuven, containing 479 newly diagnosed TNC from January 1999 till November 2009, of which 396 received upfront surgery, 64 neoadjuvant chemotherapy, and 18 with metastases at diagnosis. Univariate and multivariate relations were studied between age, lymph node status, histological subtype, treatment and outcome.

Results: Apocrine and invasive lobular TNC occur more often in older patients compared to IDC-NOS (resp. P = 0.0004, P = 0.0496), the latter being by far the most common subtype (80,2%).

Although TNC are considered as an aggressive subtype of breast cancer with a bad prognosis, only 35.1% have lymph nodal involvement. There were no significant differences in nodal invasion between subtypes, but most subtypes contained few patients. In contrast to previous reports, 7/14 (50%) of apocrine TNC had lymph node involvement.

DFS was different in different histological subtypes, but most of the histological subgroups contained insufficient numbers to be able to draw firm conclusions.

Within the histologically ‘homogeneous’ IDC-NOS group with primary surgery and outcome data (n=308), DFS with 3.5 year median FUP decreased with increasing age, but chemotherapy and also radiotherapy were much less frequently given with increasing age (in age <50, 50-69, 70+ no chemo in 2/135, 25/126, 39/44; no radiotherapy in 14/135, 22/126, 12/44 respectively). In multivariate analysis, increasing age (HR for 10 yr increase: 0.68 (0.52-0.89)), presence of lymph node involvement (HR 2.17 (1.24-3.77)), administration of chemotherapy (HR 0.28 (0.12 — 0.67)), and radiotherapy (HR 0.39 (0.20-0.73)) were significant predictors of relapse.

Conclusion: TNC is not a uniform disease. Different histological subtypes have different age distribution and outcome. The prognosis of the most common group of TNC, IDA-NOS is unexpectedly better in older patients, but this is counterbalanced by the significantly decreased use of chemotherapy and radiotherapy in this population.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-05.
Abstract P3-10-10: Progesterone Receptor-Negative Status Predicts Poorer Outcomes in Estrogen Receptor-Positive Patients Treated with Selective Estrogen Receptor Modulators
Authors: Chagpar AB , McMasters KM , Edwards MJ
Published: April 2011

Introduction: While estrogen receptor (ER) status has been used as the primary factor influencing the use of selective estrogen receptor modulators (SERMs), a number of ER-positive patients will relapse on this therapy. It has been postulated that SERM-resistance may be modulated by aberrant growth factor signaling, for which progesterone receptor (PR) status may be a surrogate. PR status, however, is frequently not considered as a strong prognostic or predictive marker in ER-positive patients. We sought to define the effect of PR-status on outcomes in such patients treated with SERMs.

Methods: The North American Fareston versus Tamoxifen Adjuvant (NAFTA) Trial is an investigator-initiated, multicenter, prospective trial that randomized patients with resected hormone-receptor positive breast cancers to receive either adjuvant tamoxifen (20 mg po daily) or toremifene (60 mg po daily) for five years. Between July 1998 and December 2002, 1692 ER-positive positive patients were randomized. PR was missing for 15 patients. The remaining 1677 (1424 PR-positive and 253 PR-negative) patients formed the cohort of interest for this analysis. The impact of PR status on overall (OS) and disease-free survival (DFS) was assessed using Kaplan Meier and Cox Proportional Hazards modeling. Results: The median patient age was 67 (range; 41-100) with a median tumor size of 1.2 cm (range; 0.01-14.00 cm). PR-positive status was correlated with node-negative disease (91.3% vs. 87.0%, p=0.035) and low grade tumors (34.9% vs. 27.7%, p=0.009), but was not associated with tumor size (p=0.533) or patient age (p=0.067). With a median follow-up of 59 months, the 5-year actuarial OS and DFS of PR-negative patients was significantly worse than that of PR-positive patients (88.5% vs. 94.3%, p=0.009 and 86.1% vs. 92.5%, p=0.003, respectively). Controlling for tumor size, grade and lymph node status, PR-negative status continued to predict worse DFS (OR=1.561; 95% CI: 1.013-2.406, p=0.043), although it lost significance in terms of OS (OR=1.619; 95% CI: 0.980-2.674, p=0.060).

Conclusion: PR-negative status is an independent predictor of worse DFS in ER-positive patients treated with SERMs. These data support the hypothesis that PR-negative status may signal a higher rate of SERM-resistance, and should be considered in adjuvant therapy decisionmaking.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-10.
Abstract P3-10-14: The Prognostic Significance of mib-1 (according to St Gallen Criteria) in Patients (pts) with Early Invasive Breast Cancer (EIBC) and Its Relationship with other Biologic Parameters in a Monoinstitutional Series
Authors: Ferro A , Caldara A , Eccher C , Triolo R , Aldovini D , Cuorvo LV , Frisinghelli M , Fasanella L , Berloffa F , Barbareschi M , Galligioni E
Published: April 2011

PURPOSE: To examine interactions between mib-1 and clinical-pathological markers and their impact on outcomes. PATIENTS AND METHODS: Mib-1 was identified by immunohistochemical staining in 3402 EIBC pts treated from 1995 to 2008. Mib-1 was defined as low (15%), intermediate (16%-30%) and high (>30%) value. Correlation between mib-1 and age, tumor size (T), nodal status (N), ER, PR, HER-2, grading (G) was performed by X2 test. Log-rank test and Cox regression model were performed to test mib-1 as prognostic factor for overall survival (OS) and Disease Free Survival (DFS) and its correlation with other known prognostic factors.

RESULTS: Median mib-1 was 25%. Mib-1 was low in 1135, intermediate in 1094 and high in 1173 pts. Median age was 62 years. High mib-1 was significantly (P<0.001) associated with younger age, ductal type, greater size, positive N, poor G, absent or low ER and/or PR expression level, positive HER-2, triple negative subtypes, larger use of chemo±hormonotherapy. High mib-1 was a significant predictor of worse

DFS and OS (P<0.001). At median follow up of 42 months DFS and OS were 83.1 and 84.8% respectively in high, 88.9 and 89.3% in intermediate, 92.7 and 93.6% respectively in low Mib-1 group. There were 401 relapses (12%): 197 (6%) in high, 121 (3.5%) in intermediate and 83 (2,5%) in low mib-1 group. BC deaths were 368 (10.8 %): 178 (5.2%) in high, 117 (3.4%) intermediate and 73 (2.2%) in low mib-1 group. High mib1 was predictive of poorer prognosis in > 40 years pts (P<0.001), small tumors (T1) (P<0.001), N+ (P<0.001) and N0 (p=0.007), G1 (p=0.02), ER+ and/or PR+ (P<0.001), negative HER-2 (p=0.007), ductal type (P<0.001) respect to intermediate and low Mib1. At Cox regression analysis Mib1 showed independent prognostic value. According to the median value, high mib-1 (>25%) resulted a prognostic factor of worse outcome (P<0.001, HR=1.81, 95% CI=1.42-2.29), along with positive lymph nodes (P<0.001, HR=2.14, 95% CI=1.67-2.74), age<40 yrs (p=0.002, HR=1.93, 95% CI=1.30-2.86), greater tumors (P<0.001, HR=3.11, 95% CI=2.21-4.39), and ER/PR positive tumors (P<0.001, HR=1.742, 95% CI=1.305-2.327).

CONCLUSIONS: In our experience, Mib-1 confirms to be a significant prognostic biomarker for DFS and OS in EIBC, providing additional information besides other clinico-pathological parameters to help decision-making of treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-14.
Abstract P3-10-21: Audit of the Accuracy of Immunohistochemical (IHC) Testing of HER2 Status of Breast Cancer in the United Kingdom: An Interim Analysis
Authors: Starczynski JL , Campbell FM , Jones P , Gilbert J , Dowds JC , Miller K , Ibrahim M , Jasani B
Published: April 2011

Background: The analysis of the level and distribution of HER2 protein expressed by cancer cells (HER2 status) is of great clinical value in the management of breast cancer patients both for the determination of the prognosis of disease and for identification of those patients who are eligible for anti-HER2 therapy. Accurate assessment of the HER2 status is essential for identifying patients which will benefit from HER2 targeted therapy. HER2 status in the UK is established using a two tier strategy with IHC as the initial test and subsequent reflex of equivocal results to in situ hybridization (ISH). IHC staining of the HER2 protein is graded as 0- 3+ dependent upon the intensity of staining, cellular localisation and the percentage of cells positive in accordance with CAP/ASCO and UK guidelines. HER2 3+ cases are considered as positive, with HER2 2+ cases (equivocal) retested by ISH to ascertain the gene amplification status. Cases that are scored as 0 and 1+ by IHC have no additional testing and are classed as negative. The literature indicates that a subset of these IHC negative cases show HER2 gene amplification by FISH (range 1.1-10.7%). The aim of this audit is to evaluate the discordance rate of HER2 IHC negative, FISH positive breast cancer in the UK, with a secondary objective to resolve if this is related to the choice of antibody used. Materials and methods: This audit selected a total of 1000 sequential cases reported as HER2 negative on IHC, from three UK reference centres receiving cases from 29 different hospitals. The cases were given a unique identifying number and annonymised. Each of the three centres used a different IHC method for frontline HER2 testing with centre one using HercepTest™ (DAKO), centre two Pathway 4B5 (Roche), and centre three, Oracle (Leica Microsystems). HER2 gene amplification status was determined using dual colour FISH analysis, PathVysion (ABBOTT) fluorescence ISH (FISH) in a single centre to provide standardised methodology and assessment. HER2 was classed as amplified when the HER2/cep 17 ratio was two or greater in accordance with UK guidelines. All cases which showed discordance between IHC and FISH were retested with each of the HER2 IHC platforms to discover whether these are truly discordant results or if the discrepancy is a consequence of the choice of antibody.

Results: An interim analysis of 170 cases shows an overall IHC negative/FISH positive discordance rate of 2.37%. The discordance rate per antibody was 1.7% for HercepTest, 3.3% with 4B5 and 2.5% with Oracle. Of the FISH positive cases the HER2/cep 17 ratios ranged from 2.2 - 6.17. The overall discordance rate between IHC negative and FISH positive is in keeping with the literature and is comparable between the three IHC platforms.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-21.
Abstract P3-10-29: Health-Related Quality of Life (HRQoL) as Prognostic Factor for Time to Progression (TTP) and Survival in Women with Metastatic Breast Cancer
Authors: Svensson H , Einbeigi Z , Johansson H , Hatschek T , Brandberg Y
Published: April 2011

Background: Prognostic factors provide important information for women with breast cancer and they are used for information on the prognosis and choice of treatment. In early breast cancer, stage, malignancy grade and hormone receptor status are used regularly as prognostic factors, while age, performance status, sites of metastasis and disease-free interval (DFI) influence the outcome in metastatic disease.

The aim of the present paper was to investigate the role of HRQoL (physical, role, emotional, cognitive, and social functioning, global quality of life, fatigue and pain) at randomization as independent prognostic factors to predict time to progression (TTP) and survival. Material and methods: In the TEX trial, 287 patients with documented locally advanced or distant metastatic breast cancer were randomized to either ET (epirubicin and paclitaxel) or TEX (epirubicin, paclitaxel and capecitabine). Treatment was repeated every 3 weeks. The EORTC QLQ-C30 questionnaire was used to assess baseline HRQoL data before randomization into the trial. Data on TTP and survival were collected from the study database.

Results

A total of 252 (88%) of the patients recruited within the trial completed questionnaires before randomization. A multivariate analysis shows that the HRQoL variables fatigue, pain and role functioning was related to prolonged survival (P<0. 01). Clinical variables included in the multivariate model were age, bone metastases, and treatment arm. Analyses of the relationship between HRQoL and TTP (time to progression) will be presented.

Conclusions: The present study shows that HRQoL variables, i.e. role functioning, fatigue and pain could contribute as important predictors of the outcome and may influence the choice of therapy in situations when two treatments are equally effective.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-29.
Abstract P3-10-33: Mammostrat® as an Immunohistochemical Multigene Assay for Prediction of Early Relapse Risk in Postmenopausal Early Breast Cancer: Preliminary Data of the TEAM Pathology Study
Authors: Bartlett JMS , Bloom KJ , Goldstein NS , van de Velde CJH , Ross DT , Seitz RS , Beck RA , Hasenburg A , Kieback D , Putter H , Markopoulos C , Dirix L , Robson T , Seynaeve C , Rea D.
Published: April 2011

Background: Postmenopausal early breast cancer patients, treated with endocrine therapy, have approximately 90% five year disease free survival (DFS). However, for patients at higher risk of relapse, additional adjuvant chemotherapy may be indicated. The challenge is to prospectively identify such patients. The Mammostrat test uses five immunohistochemical markers to stratify patients on tamoxifen (T) therapy into various risk groups potentially guiding treatment choices. We tested the efficacy of this panel in the TEAM trial (exemestane (E) versus T→E) to determine the relevance in patients treated with an AI.

Patients & Methods: Pathology blocks from 4598 TEAM patients were collected and tissue microarrays constructed. The cohort overall was 47% node positive, and 36% also received adjuvant chemotherapy. Samples were stained, using triplicate 0.6mm2 TMA cores, and positivity for p53, HTF9C, CEACAM5, NDRG1, SLC7A5 assessed. Each case was assigned a Mammostrat risk score and analysed for disease free survival (DFS) by marker positivity and risk score.

Results: Preliminary results on the UK TEAM cohort (1059 cases) showed 18.9% stained positive for p53 (184/972), 21.3% for NRDG1 (204/956), 26.4% for SLC7A (253/957), 21.9% for HTF9C (220/1004), 18.3% for CEACAM5 (185/1009). Complete data was available for 919 cases including patients treated with chemotherapy, with 447 (49%) designated low risk, 213 (23%) medium and 259 (28%) high risk. In univariate analysis, Mammostrat scores were prognostic (p=0.02), with 5 year DFS (see comment above) results being 86.9±1.7%, 80.1±3.0% and 80.8±2.6% for patients with low, medium and high Mammostrat scores respectively. Analyses on the entire TEAM pathology cohort are ongoing, and further data with sufficient power to evaluate the impact of Mammostrat in multivariate regression analyses will be presented. Conclusion: Preliminary analysis of the impact of the Mammostrat score in both tamoxifen and exemestane treated patients suggests it retains its prognostic value in this context. Further analysis with the power to evaluate the impact of Mammostrat in multivariate regression analyses will be presented.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-33.
Abstract P3-10-34: HER2 Testing by FISH: Discordance between HER2 Signals Per Cell and HER2:CEP 17 Ratio
Authors: Dintzis SM , Allison KH , Schmidt RA.
Published: April 2011

Background: HER2 gene amplification status may be tested by FISH using either HER2 signals per cell or HER2:CEP17 ratio. Established HER2 testing CAP/ASCO reporting guidelines allow for interpretation and reporting using either method. Both methods classify cases as amplified, not amplified or equivocal yet the two methods may yield different interpretations for the same case. We analyzed a large dataset to quantify concordance between these two testing and reporting methodologies. Materials and Methods:

We collected individual cell HER2 and CEP17 counts in 32,116 tumor cells from 1329 consecutive breast cancer cases. Data from Excel counting sheets were imported to a SQL Server database for analysis and statistics;

histograms and analyses were also performed in Excel. We specifically computed concordances between methods for HER2 signals per cell and HER2:CEP17 ratio reporting. Results:

Concordance between HER2 signals per cell and HER2:CEP17 ratio case averages was 89.8%. Most discordances involved cases classified as equivocal by either method. 7.8% of cases reported by HER2 signals per cell and 4.0% of cases reported by HER2:CEP17 ratio were classified as equivocal. 1% of cases were amplified by one method and not amplified by the other. Discussion:

This very large dataset shows that HER2 FISH reporting by established CAP/ASCO guidelines is generally concordant between the two testing methods. However, a substantial number of cases are discordant and patient treatment could vary depending on reporting criteria used. These results indicate the need for reporting by both methods to prevent missing patients who could potentially benefit from HER2 targeted therapy. Further study is necessary to determine the most clinically relevant reporting method and criteria.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-34.
Abstract P3-10-35: Using Automated Image Analysis To Validate Ki67 as a Clinical Prognostic Biomarker for Estrogen-Receptor Positive Breast Cancers
Authors: Magliocco AM , Kornaga EN , Klimowicz AC , Petrillo SK , Konno M , Yau A.
Published: April 2011

Background: Ki67, an indicator of proliferation, has been shown to be a useful prognostic and predictive marker for breast cancer. Ki67 can be used to identify two distinct estrogen-receptor positive subtypes: luminal A and luminal B. Luminal A breast cancers have been identified as having a lower proliferation and better outcome compared to luminal B. Furthermore, early clinical trials suggest that Ki67 may be useful in identifying a subset of patients that are sensitive to adjuvant docetaxel treatment. Currently, only estrogen-receptor (ER), progesterone-receptor (PR), and human epidermal growth-factor (HER2) are routinely performed. We have optimized and validated an immunohistochemical (IHC) Ki67 assay and automated computerized image analysis platform for routine clinical testing. Materials and Methods: Immunohistochemical staining was quantitatively assessed using the ACIS® III platform on a cohort (N=761) of tamoxifen treated patients who were diagnosed with breast cancer in Calgary between 1990 and 2001. Tissue microarrays were constructed using three 0.6 mm cores. Ki67 results were available for 510 patients, 461 of which are ER/PR positive and HER2 negative. Staining was performed using the DAKO FLEX ready-to-use system. The percent nuclear area positive was calculated using ACIS III and the maximum value was used in statistical analysis

Results: X-tile statistical software was used to identify an optimal Ki67 cut point to distinguish differential overall survival in node negative ER positive cancers of 18.75%. This cut point was then used to categorize the 461 ER/PR positive and HER2 negative breast cancers into luminal A (407; 88.3%) or luminal B (54; 11.7%) subtypes. The 8-year breast cancer specific survival was 85.2% (95% CI = 81.3% - 89.1%) for luminal A and 53.6% (95% CI = 39.3% - 67.9%) for luminal B (P<0.0001). Cox regression showed a hazard ratio of 1.63 (95% CI = 0.99 — 2.67, p=0.055), adjusting for age, tumor size, grade and lymph node status. Discussion: Quantification of Ki67 expression using automated image analysis can be used clinically to distinguish luminal A from luminal B in ER/PR positive and HER2 negative breast cancers. The purpose of this project was to develop a reliable Ki67 assay that can be easily adopted by other testing centers. Using a ready-to-use IHC system — such as DAKO FLEX — allows for consistent results between other clinical laboratories. Additionally, using an automated, quantitative imaging system — such as the ACIS® III — reduces inter-observer variation that can occur by human visual assessment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-35.
Abstract P3-11-09: Body Mass Index (BMI) and Survival: A Retrospective Review of Women with Triple Negative Breast Cancer (An Update)
Authors: Sudhindra A , Dammarich D , Ochoa R , Takita C , Mendiola MF , Hurley J , Gluck S , Welsh C , Slingerland J , Richman S , Gomez-Fernandez C , Timothee P , Diaz A , Silva OE.
Published: April 2011

Body Mass Index (BMI) has been reported as a risk factor for recurrence and decreased survival in women with breast cancer. At the Inter-American Breast Cancer Conference we presented data showing the effect BMI has on overall survival in women with triple negative breast cancer (TNBC). Here we present an update of this data which to our knowledge is the only study addressing the effect BMI has on outcomes in TNBC. We conducted a retrospective review of 170 patients with TNBC seen at the University of Miami Sylvester Comprehensive Cancer Center and Jackson Memorial Hospital between December of 1999 and April 2010. Charts were reviewed looking at BMI at the time of diagnosis and overall survival defined as date of last contact or date of death when known. 163 patients were included in the study. BMI was defined as normal (BMI 18.5-24.9) and overweight or obese (25 or greater). The average age at diagnosis was 50.96 years with a mean OS of 44.67 months. 21 patients (13%) had stage I disease, 61 (37.4%) had stage II disease, 81 (49.6%) had stage III disease. In patients with stage I disease 33% had a normal BMI with a mean OS of 51.7 months. 67% were overweight or obese with a mean OS of 60.2 months. In patients with stage II disease 33% had a normal BMI with a mean OS of 30.8 months. 67% were overweight or obese with a mean OS of 48.4 months. In patients with stage III disease 28% had a normal BMI with a mean OS of 37.2 months. 72% were overweight or obese with a mean OS of 43.1 months.

Survival in patients with TNBC does not appear to be negatively impacted by the patients BMI at diagnosis. While not statistically significant (p=0.274) patients with stage I TNBC that were overweight or obese tended to have an increased OS compared to those with a normal BMI. In patients with stage II disease there was a statistically significant (p=0.010) increase in OS in overweight or obese patients compared to those with a normal BMI. In the patients with stage III disease there was no significant difference in the OS (p=0.458) between patients with a normal BMI and those that were overweight or obese.

Our study shows that increased BMI at the time of diagnosis may have a positive impact on OS in patients with TNBC. Overweight and obese patients with stage I and II TNBC have an increased OS compared to those with a normal BMI. In those with stage II disease the difference was statistically significant. Although this study was limited by small sample size, if confirmed, it might suggest a relationship between obesity and survival in TNBC. Further studies are needed to evaluate these findings.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-11-09.
Abstract P3-12-06: Biomarkers of Malignant Potential in Breast Tissue in Relation to Short-Term Breast Cancer Risk
Authors: Hill D , Nibbe A , Lomo L , Royce M , Wallace AM , Bocklage T.
Published: April 2011

Background:

Biomarkers expressed in benign or normal breast tissue may be important indicators of subsequent risk of malignancy, but investigations identifying such have been few. Limited previous evidence suggests that loss of expression of several breast cancer tumor markers in benign tissue, relative to normal tissue, may contribute to malignant progression. We examined expression of markers implicated in tumorigenesis among women who developed breast cancer shortly after benign biopsy. Material and Methods:

We conducted a nested case-control study within a population-based cohort of 15,000 women who received benign breast biopsies between 1996 and 2005 in six New Mexico counties. We identified 95 women who developed breast cancer within 5 years of biopsy, and matched them to 3 similar controls who did not develop cancer. Benign breast as well as adjacent normal tissue was assayed using immunohistochemistry for epithelial and stromal markers plausibly related to risk of cancer development. Logistic regression models were employed to estimate odds ratios and 95% confidence intervals for breast cancer risk after adjustment for age, race/ethnicity, and risk category of benign biopsy. Results:

Women who expressed estrogen receptor alpha in their normal tissue (10% or greater staining) had a 2.9-fold increased breast cancer risk (95% confidence interval (CI) 1.0-8.3). Those who expressed Cytokeratin 5/6 (Odds ratio (OR) 3.3; 95% CI 1.2-8.8) or Her2/neu (30% or greater staining) (OR 8.2; 95% CI 0.9-70.6) in normal tissue also appeared to have an elevated breast cancer risk, relative to those with expression below the threshold. In contrast, the presence of these markers in benign tissue was unrelated to subsequent development of breast cancer. Estrogen receptor staining of 10% or greater in normal tissue, but absence of staining in benign tissue, was not a risk factor for progression to breast malignancy (OR 4.9; 95% CI 0.7-34.8) Discussion:

Our results suggest that biomarker expression in normal breast tissue may be an important component of risk assessment among women whoreceive benign breast biopsies. Findings such as these aim to identify women at highest risk for breast cancer development, allowing for the implementation of more frequent follow-up and new targeted interventions, while classifying most women as at low risk, providing reassurance and reducing the need for chemoprevention.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-12-06.
Abstract P3-12-08: Molecular Breast Cancer Subtypes in Women from Northeast China: Eight-Year Retrospective Study about Clinicopathologic Features and Survival
Authors: Nie G , Shi A-p , Song D , Yang M , Fu T , Li S-j , Han B , Zhao G , Wu D , Jia H-y , Fan Z-m.
Published: April 2011

Background and objectives: Analysis of gene expression has identified various molecular subtypes. These molecular subtypes defined by immunohistochemistry expression of estrogen receptor (ER) or progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) : luminal A, luminal B, basal cell-like and HER2-expresing. This study evaluates clinical and pathologic features and survival of the four molecular subtypes in premenopausal and postmenopausal women from northeast China.

Methods: A retrospective analysis of 1214 women diagnosed with breast cancer from 2000 to 2007 in Breast Surgery Department, First Hospital of Jilin University. Four molecular breast cancer subtypes defined as: luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-), and HER2-expresing, (ER-, PR-, and HER2+). We examined the prevalence of breast cancer subtypes and analyzed the associations with patient clinicopathologic features: age, menopausal status, tumor size, lymph node status, stage of cancer at diagnosis, histological characteristics, and breast cancer-specific survival.

Results: Among the all cases, the luminal A subtype was the most prevalent in our study sample (56.0%) compared with basal -like (18.5%), luminal B (13.7%), and HER2-expresing subtypes (11.8%). The four molecular subtypes differed significantly by menopausal status (P=0.011), age (P<0.001) and lymph node status (P<0.001). Luminal A subtype was more likely to with Stage II breast cancer (P<0.001) and most present with size of 2.1-5 cm (P<0.001). The luminal B and the HER2-expresing subtypes presented an increased association with more aggressive clinical course when compared with others subtypes. The estimated median follow-up period for all subjects was 54 months (range, 1 month to 120 months). The 8-year overall survival for all patients was 81.3% (95%CI, 79.4-83.7) and disease-free survival was 77.8% (95% CI, 75.4-79.9). The Kaplan-Meier curve showed breast cancer-specific survival differed significantly among the molecular subtypes (P < 0.03), with the luminal B and HER2-expresing subtypes having the poorest outcome.

Conclusion: Luminal A breast tumor appeared the better outcome than the others three subtypes and the shortest survival was HER2-expresing subtype. The poor clinical outcomes of luminal B subtype with women from northeast China were different from those of Western women could contribute to the poor prognosis of breast cancer observed in this cohort of patients and some of them couldn't accept molecular targeted therapy with trastuzumab by poor economic situations.

Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-12-08.