bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3-02-03: Prognostic Value of Circulating Tumor Cells (CTC) in HER-2 Positive Metastatic Breast Cancer (MBC) Patients (pts) Treated with Anti-HER2-Based Therapy
Authors: Giuliano M , Giordano A , Handy BC , Ueno NT , Alvarez RH , Hortobagyi GN , Reuben JM , Valero V , Cristofanilli M.
Published: April 2011

Background. Detection of ≥5 CTC per 7.5 mL of blood before starting systemic treatment is a strong and independent predictor of worse progression-free survival (PFS) and overall survival (OS) in MBC. Trastuzumab (Herceptin®) was shown to be highly effective in reducing CTC number in all HER-2 amplified MBC cases after the first dose (De Giorgi et al., SABCS 2009). Furthermore, trastuzumab can target chemotherapy-resistant CTC in pts with breast cancer (Bozionellou et al. 2004). To date no significant analysis exploring the effect of another anti-HER2 therapy, lapatinib (Tykerb®) on CTC has been published.We hypothesized that anti-HER2-based therapy may modify the detection rate and prognostic value of CTC in pts with HER-2 amplified MBC. Patients and Methods. We retrospectively evaluated 91 HER-2 positive MBC pts, treated at the M. D. Anderson Cancer Center with trastuzumab-based (n=59), or lapatinib-based therapy (n=32). In all pts, CTC were enumerated before starting systemic treatments, using CellSearch™. Follow-up CTC counts were available for 41 (69%) pts treated with trastuzumab and 25 (78%) pts treated with lapatinib. PFS and OS were estimated by Kaplan-Meier product limit, and compared between groups according to baseline CTC count (<5 vs ≥5) by log-rank test. Results. Median follow-up was 27 months (range, 8-65) and 16 months (range, 3-33) for pts receiving trastuzumab and lapatinib, respectively. Treatments administered and CTC counts are reported in table 1. Two (5%) pts receiving trastuzumab and 4 (16%) pts treated with lapatinib had CTC ≥5 at the follow-up assessment. Estimated median PFS and OS according to baseline CTC value, for both treatments are shown in table 2. An elevated CTC count was associated with poor prognosis in pts treated with lapatinib (median OS: 25.8 vs 13.6 months in pts with CTC <5 and ≥5, respectively, P=.072). Surprisingly, in the trastuzumab group, PFS showed a non-statistically significant trend in favor of pts with CTC ≥5 compared to those with <5.

Conclusions. According to previously published data, our study confirms that trastuzumab-based therapies can drastically reduce the number of CTC and neutralize their negative prognostic value. Furthermore, we found that lapatinib had a limited effect on the detection rate and prognostic value of CTC, although the low number of pts and the high percentage of pre-treated women do not allow us to draw definitive conclusions.

Table 1

Table 2

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-03.
Abstract P3-02-04: Dendritic Cells from Breast Cancer Patients with ≥5 CTCs Are Functionally Impaired
Authors: Lee B-N , Gao H , Cohen EN , Tin S , Jackson SA , Valero V , Alvarez R , Cristofanilli M , Reuben JM.
Published: April 2011

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer patients and patients with ≥5 CTC per 7.5 mL of whole blood (WB) have a poor prognosis and overall survival than patients with fewer than 5 CTC. Dendritic cells (DCs), the most efficient antigen presenting cells (APC), are essential for a robust host immune response. Therefore, we investigated the relationship between CTCs and the immunophenotype and function of DCs in breast cancer (BC) patients.

Methods: This ongoing prospective study consists of 47 patients, 26 with metastatic breast cancer, 19 with inflammatory breast cancer; and 2 with locally advanced breast cancer, with a median age of 52 years (range: 34-71 years). Primary tumor characteristics of patients included 28 hormone receptor positive, 18 HER2 amplified, and 12 triple receptor negative. Fluorescence-activated cell sorting analysis was used to enumerate circulating DC subsets, myeloid (mDC) and plasmacytoid (pDC), that express costimulatory molecules (CD40, CD80, and CD86). Additionally, we assessed the ability of DCs to synthesize interleukin (IL)-10, IL-12, interferon-alpha (IFN-a) and tumor necrosis factor (TNF)-a constitutively and following activation with toll-like receptor (TLR) agonists. CTCs were enumerated in 7.5 mL of WB using CellSearch (Raritan, NJ). Results: CTCs were detected in 26 patients (range from 1 to 1009) including 12 patients with ≥5 CTCs. Compared with patients with < 5 CTCs, those with ≥5 CTCs have a significantly higher median number of mDC (P = 0.007) and higher median percentages (%) of TLR-activated mDC that expressed CD80 (P = 0.047) and CD86 (P = 0.063). Additionally, compared with patients who had < 5 CTCs, patients with ≥5 CTCs had a lower median % of TLR-activated mDC that synthesized IL-12 (P=0.028), TNF-α (P=0.006), or IL-10 (P = 0.031); and there was a higher median % of TLR-activated pDC that synthesized IL-10 (P = 0.000). Conclusions: Even though BC patients with ≥5 CTCs have more mDC, activated mDCs are less capable of synthesizing inflammatory factors (TNF-a and IL-12) responsible for adaptive immunity. Moreover, there was increased % of activated pDC that synthesized IL-10, a cytokine responsible for downregulating adaptive immune responses. These preliminary data suggest that activated DCs from BC patients with ≥5 CTCs are likely to have a suboptimal ability to sustain an efficient adaptive immune response compared with activated DCs of BC patients with < 5 CTCs. This is the first report linking CTCs, a proven poor prognostic indicator, with potential dysfunctional APC in BC patients.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-04.
Abstract P3-04-03: Combined Epigenetic Targeting Reverses Epithelial-Mesencymal Transition (EMT) and Reduces Invasiveness of Human Breast Cancer Cells
Authors: Joshi AD , Rao R , Fiskus W , Buckley KM , Nalluri S , Balusu R , Atadja P , Bhalla KN.
Published: April 2011

Background: Epithelial-Mesenchymal-transition (EMT) is a highly conserved biologic process that is involved in organ development, tissue regeneration, tumor invasion and metastasis. EMT is phenotypically associated with the loss E-cadherin and a gain of mesenchymal markers (e.g., vimentin). Both promoter methylation by DNMT1 and histone H3- K27 trimethylation by the histone methyl transferase EZH2 have been shown to cause E-cadherin expression. Several transcription factors, including ZEB1, Snail, Slug and Twist, directly bind to E-cadherin promoter, recruit histone deacetylase (HDAC) activity and repress E-cadherin. We have previously shown that the pan-HDAC inhibitor panobinostat (PS, Novartis Pharmaceuticals) depletes EZH2 and DNMT1 levels, and co-treatment with PS and DNMT1 inhibitor decitabine (DAC) synergistically depletes EZH2 and DNMT1.

Methods: Here, we determined the effects of PS (20 to 100 nM) and/or DAC (0.5 to 5.0 uM) on E-cadherin levels and other EMT-associated markers, as well as on invasiveness of human breast cancer MB-231 and SUM159PT cells, which have undetectable levels of E-cadherin. Results: Treatment with PS dose dependently inhibited the migration and invasion of the breast cancer cells (by Boyden chamber assay). This was associated with induction of mRNA and protein levels of E-cadherin and decline in vimentin levels. PS treatment inhibited HDAC activity and reduced DNMT1 and EZH2 levels. Additionally PS repressed ZEB1 mRNA and protein levels. Co-treatment with bortezomib partially restored ZEB1 levels, indicating that PS treatment promotes proteasomal degradation of ZEB1. Chromatin immunoprecipitation (ChIP) analyses demonstrated that PS treatment inhibited H3K27 chromatin repressive and increased H3K4 chromatin permissive mark associated with the E-cadherin promoter. PS treatment also diminished ZEB1 occupancy on the E-cadherin promoter. Pre-treatment of MB-231 and SUM 159PT cells with DAC or 5-azacytidine (AC, also a DNMT1 inhibitor) (0.5 to 5.0 uM) further enhanced PS-mediated induction of E-cadherin, which was associated with further decline in ZEB1 levels. Additionally, co-treatment with PS and DAC caused greater inhibition of in vitro invasion by the breast cancer cells. Discussion: Collectively, these findings indicate that combined epigenetic targeting with PS and DAC depletes ZEB1, induces E-cadherin and reverses EMT, thereby, attenuating invasiveness of human breast cancer cells.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-03.
Abstract P3-04-04: Novel Genome-Wide DNA Methylation Signatures of Primary Breast Tumor Subtypes
Authors: Lee MP , Kadota M , Yang HH , Clifford RJ , Dunn BK.
Published: April 2011

Background: Gene expression signatures can be used to classify breast tumor tissues into distinct categories: luminal, “basal”, and HER2 subtypes, which have distinct clinical prognoses. Since gene expression is largely determined at the level of epigenetic modification, we hypothesize that DNA methylation signatures may also be used to classify breast tumor subtypes and possibly to define new cancer subtypes; thus, such epigenetic signatures can complement gene expression signatures for a better understanding of breast tumorigenesis and in turn, may lead to improvement in clinical diagnosis.

Material and Methods: We used antibodies to 5-methylcytosine to immunoprecipitate DNA enriched for methylation by means of the MeDIP method on 66 breast primary tumors. Then we analyzed the MeDIP-derived DNA by means of the Affymetrix 500K SNP array. We normalized DNA methylation data from the 66 tumors using a modified quantile normalization procedure. The data were generated in two phases, phase 1 consisting of 42 tumors and phase 2 containing 24 tumors, data from the latter being reserved for validation purposes. We concentrated DNA methylation analysis on the SNPs that are located within promoter regions, defined here as +/− 2 kb flanking the transcription start site (TSS), and within CpG islands, since methylation in these regions is especially relevant for gene expression regulation. We searched for DNA methylation signatures that show distinct patterns among tumors with different clinical phenotypes such as histological grade and lymph node involvement. Results: We identified one DNA methylation signature consists of 78 genes that separated the tumors with high grade from those with low-mid grade; we found another DNA methylation signature consists of 42 genes that separated the tumors with lymph node positive status from those with lymph node negative status. We were able to validate these signatures using methylation data obtained from an independent set of 24 tumors. The performance of the signature for predicting lymph node status in the testing samples showed accuracy of 83%, sensitivity of 70%, and specificity of 90%.

Discussion: We have identified DNA methylation signatures that could classify breast tumor subtypes and correlate with clinical phenotypes. We are investigating whether such epigenetic signatures can complement gene expression signatures for a better understanding of breast tumorigenesis and for application in clinical diagnosis and treatment.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-04.
Abstract P3-04-07: Loss of Expression of the 14-3-3 Sigma by CpG Island Methylation and Relations with Prognostic Factor in Breast Cancer
Authors: Martínez-Galán J , Torres-Torres B , Ríos S , Del Moral R , Nuñez MI , González-Flores E , Conde V , Peñalver J , Delgado JR , Ruiz Almodóvar M.
Published: April 2011

Background: Expression of 14-3-3 σ is a tumor suppressor gene induced in response to DNA damage, and has been implicated in G2/M cell cycle arrest by p53. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. Objetive: To correlation methylation levels of promoter 14-3-3σ with association prognostic factors in breast cancer.Material and Methods:This is a prospective study we quantified methylation levels of promoter 14-3-3 σ gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women.Nodal involvement N0; 63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%,G2;37%,G3;30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047).The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (P<0.05),nodal positive status (P<0.05) and poor outcome. With a median follow up 6 years we saw more probability of developing distance metastasis in patients with methylation 14-3-3σ (P>0.05).Conclusions:Hypermethylation of the 14-3-3σ a promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3σ may be an important event in tumor progression and particularly in breast carcinogenesis.Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-07.
Abstract P3-04-08: Epigenetic Changes Due to DNA Methylation in CpG Islands during Breast Cancer Progression
Authors: Dimitrova N , Gorthi A , Prasada SK , Chakrabarty S , Keswarpu P , Banerjee N , Janevski A , Kiradi PH , Khandige S , Satyamoorthy K.
Published: April 2011

DNA methylation has been associated with several key events of gene regulation and to human cancer. It is not yet known how the epigenome of various populations is associated with clinical manifestations during the course of the disease. In this poster we describe a method for stratifying breast cancer patients from Indian origin using CpG island microarray from the University Healthcare Network (UHN) Toronto (human CpG island 12k microarray chip, HCGI12K). DNA samples were obtained from a prospective study cohort which consisted of 51 female primary breast cancers. All patients had been undergoing treatment in a tertiary care hospital and its associated centers in the southern part of India between 2007 and 2009. We have identified and classified the DNA methylation in CpG islands of patient samples using various clinical parameters such as age of disease onset, menopausal status, hormone receptor status and Her2 status. We present the methods to analyze the data from UHN CpG island arrays used in a high throughput methylation study in order to derive decision rules of stratifying the data into basic classes such as normal and benign conditions, and infiltrating ductal carcinoma. Results will be presented pertaining to differential methylation status of patients in different categories such as age of disease onset, menopausal status, hormone receptor status and Her2 status.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-08.
Abstract P3-06-02: Prevalence of TP53 p.R337H Mutation in Cases of Breast Phyllodes Tumours in Southern Brazil
Authors: Giacomazzi J , Koehler-Santos P , Alemar BB , Ashton-Prolla P , Graudenz MS.
Published: April 2011

Background: Previous studies in Brazilian patients diagnosed with the core-cancers of Li-Fraumeni syndrome (LFS) and its variants (LFL) indicate that a particular germline mutation, TP53 p.R337H, may be highly prevalent and strongly associated with pediatric adrenocortical tumors in Southern Brazil. However, other tumours types, including breast cancer, have been observed in carriers, raising the possibility that the TP53 p.R337H mutation may also contribute to breast tumorigenesis. Breast phyllodes tumour, a very rare and aggressive breast neoplasia that accounts for less than 1% of all breast cancer cases, may be associated with LFS, and has suggested by some to be particularly frequent in TP53 mutation carriers. Methods: Formalin fixed paraffin-embedded (FFPE) tissue from disease-free lymph nodes of an unselected consecutive series of women diagnosed with benign and malignant phyllodes tumours between 2000 and 2010, was obtained from 7 Pathology Laboratories in Southern Brazil. Genomic DNA was isolated from FFPE with the QIAamp DNA Tissue Kit. Genotyping was performed by qPCR using TaqMan technology and direct bi-directional sequencing for confirmation in mutation-positive cases. Results: Analysis of the first 48 cases included in the study (9 malignant, 3 borderline and 36 benign phyllodes tumors) resulted in identification of the TP53 p.R337H mutation in the germline of a 61 year-old patient with malignant phyllodes and a 38 year-old patient with a benign phyllodes tumor of the breast.

Conclusion: Preliminary analysis of a case series of breast phyllodes tumors indicates that germline the founder mutation TP53 p.R337H may be particularly common in women with these tumors in Southern Brazil. Further analysis of the entire TP53 coding region and evaluation of the founder haplotype in the mutation-positive cases is ongoing. The identification of germline TP53 mutations in patients diagnosed with phyllodes tumors of the breast has important implications for disease management and in cancer risk counseling for these patients and families.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-06-02.
Abstract P3-08-03: Defects in BRCA1 Contribute to Global Differential Allele-Specific Expression
Authors: Chen X , Schaeffer A , Devarajan K , Liao C , Zhou Y , Slater CM , Vanderveer L , Conroy J , Godwin AK.
Published: April 2011

Differential allele-specific expression (DASE) has been shown to contribute to phenotypic variability in humans and more recently to the pathogenesis of cancer. DASE is associated with X-chromosome inactivation and genomic imprinting and is relatively common among non-imprinted autosomal genes. The DASE phenotype can also be transmitted by Mendelian inheritance. We have previously reported that nonsense-mediated mRNA decay (NMD) of mutant BRCA1 as well as other epigenetic mechanisms can lead to DASE of BRCA1 and enhanced susceptibility to breast cancer. BRCA1 has been implicated in many cellular processes including DNA repair, cell-cycle-checkpoint control, protein ubiquitination, and chromatin remodeling. Importantly, cells carrying a deleterious BRCA1 mutation exhibit increased genome instability, therefore, we hypothesize that defects in BRCA1 lead not only to DASE of itself, but increase genome-wide DASE and thus contribute to increased breast cancer susceptibility. To test this hypothesis, we employed a genome-wide ASE assay (Illumina Human Omni1-Quad BeadChip) using primary mammary epithelial cells [3 BRCA1 wild-type vs. 3 BRCA1 mutant carrying (2800delAA, 4154delA and R1751X)]. As shown in Table 1, cells carrying a BRCA1 mutation had significantly more DASE events as compared to wild-type cell (P<10-7). In addition, we identified 351 genes demonstrating DASE that were unique to the BRCA1 mutant cells. The cellular functions of these genes are wideranging, including DNA repair, cell cycle control, lipid metabolism and protein degradation. In summary, this study provides the first evidence that mutant BRCA1 can lead to global DASE, which in turn may contribute to the development of breast cancer in mutation carriers. This work was supported in part by the Eileen Stein-Jacoby Fund and a grant from the Congressionally Directed Medical Research Programs, Department of Defense, W81XWH-08-1-0361 (XC).

Table 1. BRCA1 mutations increase the global DASE in PMECs

t; A DASE event is defined as that the Log-ratio of allele-specific expression level of one gene is more than 1 or less than -1, i.e. the expression level from one allele is at least 50% less than the level of another allele

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-08-03.
Abstract P3-10-01: Quantitative Gene Expression Analysis in a Large Cohort of Estrogen-Receptor Positive Breast Cancers: Characterization of the Tumor Profiles in Younger Patients (≥40 yrs) and in Older Patients (≥70 yrs)
Authors: Shak S , Baehner FL , Stein M , Chen I , Yoshizawa C , Watson D , Swain SM.
Published: April 2011

Background: Previous studies have identified differences in the presentation, biology, and behavior of breast cancer as a function of patient age. To directly study breast cancer tumor profiles as a function of patient age, we examined the Oncotype DX Recurrence Score (RS) and the expression of ER, PR, and proliferation-related genes in a large cohort of invasive breast cancers.

Material and Methods: All tumor specimens successfully examined in the Genomic Health laboratory from June 2004 through March 2010 were included. Standard RS testing was performed. Quantitative expression for ER, PR, HER2, and the five proliferation genes, CCNB1, Ki-67, MYBL2, STK15, and Survivin, was measured by quantitative RT-PCR on a scale from 2 to 15 (relative to reference genes), where a one unit increment is associated with an approximate 2-fold increase in expression. The proliferation index (PI), a component of the RS, was calculated as the average of the expression of the five proliferation genes. ER negative tumors (ER<6.5) were excluded. Means and medians were calculated; with this very large sample size, all comparisons between groups were statistically significant (P<0.05).

Results: Among the 145,236 breast cancers, 5,794 (4.0%) were ≥40 yrs, 117,740 (81.1%) were 41 — 69 yrs, and 21,702 (14.9%) were ≥70 yrs. A wide range of RS (0 to >80) was observed in all age groups. The mean RS, median RS, and proportion of patients in each of the standard RS groups are shown in the Table. The average RS is slightly higher in the younger age group and lower in the older age group. Similarly, the proportion of high RS tumors is higher in the younger age group and lower in the older age group. However, many young patients are in the low RS group and many older patients are in the high RS group. Small differences in the mean expression of quantitative ER, quantitative PR, and the Proliferation Index were observed. Younger patients had lower mean ER, higher mean PR and higher proliferation.

Discussion: Among estrogen receptor positive breast cancers, the average RS is slightly higher in younger patients and is slightly lower in older patients. However, it is notable that a wide range of RS was observed in all age groups; many young patients are in the low RS group and many older patients are in the high RS group. These results demonstrate the importance of standardized quantitative measurement of gene expression on a continuous scale, and suggest that, for ER positive breast cancer, age per se does not predict individual tumor biology.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-01.
Abstract P3-10-16: Quantitative Subtractive Immunofluorescence To Develop a Surrogate for Mena Inv(asive) Isoform Is Associated with Poor Outcome in Breast Cancer
Authors: Agarwal S , Jones JG , Oktay M , Balsamo M , Condeelis J , Gertler F , Rimm DL
Published: April 2011

Introduction:

Previous work has shown that the inv isoform of Mena, an actin binding protein, is associated with invasion at the cellular level and metastasis in the context of the microenvironment in both animal models and humans (Robinson, B.D. et al. Clin Cancer Res 15, 2433-2441 (2009). However, the prognostic value for metastasis of MenaINV itself is unknown because there is no antibody that directly recognizes this isoform. Here we describe a method to assess a surrogate for MenaINV by measuring total Mena and subtracting the levels of the 11a (non-invasive) isoform. Method: Total Mena and Mena11a were measured in two independent retrospective breast cancer cohorts with 20 year follow-up using tissue microarray and quantitative immunofluorescence (AQUA) technology in a previously described multiplexed mode. AQUA scores for each marker were converted into z scores followed by subtraction of Mena 11a (noninvasive form of Mena) from total Mena (invasive and non-invasive) = Mena(inv) surrogate. This was calculated for each patient and correlated with clinical and pathological characteristics as well as disease-free survival in both cohorts.

Results: In the older Yale cohort, Kaplan Meier analysis dividing the Mena(inv) surrogate by quartiles suggested collapse of the top three quartiles followed by comparison to the fourth quartile (log rank p= 0.0003, n=501). The 4th quartile was also significant in node positive (log rank p=0.0047, n=267) and estrogen negative (ER) patient subgroups (log rank p=0.0003, n=234). Cox multivariate analysis showed Mena(inv) was independent of age, tumor size, nuclear grade, nodal status, ER, PR, Her2 (HR=0.636, 95% CI=0.47-0.86, p=0.0038, n=420). The newer Yale cohort showed similar results, but that cohort also had data on local vs. distant recurrence. The relative risk of distant recurrence in this cohort is 2.56 (p=0.011) for patients with high Mena (inv) compared to 1.96 (p=0.055) for any recurrence.

Conclusions: High Mena (inv) surrogate shows prognostic value for poor survival in two independent breast cancer cohorts with some suggestion of preferential prognostic value for distant recurrence.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-16.
Abstract P3-10-18: Early Detection of Recurrent Breast Cancer Using Metabolite Profiling
Authors: Asiago VM , Alverado LZ , Shanaiah N , Gowda NG , Owusu-Sarfo K , Ballas R , Raftery D
Published: April 2011

Background: The growing field of metabolite profiling (or metabolomics) focuses on the quantitative detection of multiple small molecules in biological systems that result from pathophysiological stimuli. As is the case for early detection of primary breast cancer, it is anticipated that early diagnosis of breast cancer recurrence will not only improve survival but also help clinicians determine the best therapeutic strategies for patients by avoiding under or over treatment. We report on the development of an early test for recurrent breast cancer using metabolite profiling methods. Methods: We applied a combination of nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (2D GC-MS) to analyze the metabolite profiles of 257 serial serum samples from breast cancer patients consisting 116 samples from breast cancer recurrence and 141 samples from breast cancer patients with no evidence of disease (NED). NMR and GC-MS data were analyzed by combining advanced univariate and multivariate statistical methods and comparison of individual spectral features between patients with and without recurrent breast cancer. Results: From multivariate analysis of hundreds of spectral features, ten metabolite markers (7 from NMR and 3 from GC-MS) were targeted to build logistic regression model, which yielded a prediction model with a high accuracy (AUROC >0.89 using 5 fold cross validation) with a sensitivity of 86% and specificity of 89%. When the model was tested using leave one patient cross validation it yielded a sensitivity of 75% and a specificity of 83% (AUROC >0.88). In addition, strikingly, over 55% of the patients could be correctly predicted to have recurrence as early as 13 months before the recurrence was actually diagnosed clinically, representing a large improvement over the current diagnostic assay CA 27.29. A second statistical approach using independent training and testing sets yielded very similar results. To the best of our knowledge, this is the first study to develop and validate a prediction model for early detection of recurrent breast cancer based on the metabolic profiling. The combination of complementary NMR and MS analytical methodologies is particularly useful for building accurate profiles. Conclusion: Metabolite profiling methods provide a powerful approach for the development of diagnostic tests for monitoring breast cancer patients.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-18.
Abstract P3-10-19: Breast Cancer Risk Prediction Model in Korean Women Using Five Polymorphisms Identified in Genome Wide Association Studies
Authors: Han W , Woo JH , Yu J-H , Ahn SK , Kim HS , Shin HC , Yom CK , Moon H-G , Noh D-Y
Published: April 2011

Background: Recently indentified genetic variants from genome-wide association studies of breast cancer have not been validated in Asian population except for Chinese. We sought to confirm the association in ethnically distinct Korean women and to make genetic risk assessment model using multi-gene markers

Materials and Methods: 3321 invasive breast cancer patients operated in Seoul National University Hospital and 3500 healthy control women from a population based cohort were genotyped for 5 single nucleotide polymorphisms (SNPs) using Taqman assay. The SNPs genotyped were rs2046210 (6q25.1), rs4973768 (3p), rs2981582 (FGFR2), rs3803662 (TNRC9), and rs889312 (MAP3K1).

Results: The five SNPs were significantly associated with the risk of breast cancer in dominant, recessive, and co-dominant model (p-values from 0.012 to 3.41E-08). Minor allele frequencies were between 0.2 and 0.5. Odds ratios were between 1.14 and 1.51. Multi-gene logistic regression risk model was made with this 5 SNPs. Women who have all protective variants showed odds ratio of 0.43, while women who are homozygous for risk variants in all 5 SNPs showed odds ratio of 2.36 compared with women with the most common genotype.

Conclusion: We found that 5 SNPs from GWAS in mostly women of European ancestry were also significantly associated with breast cancer risk in Korean women. The multi-gene risk assessment model might be useful to classify women into relevant risk groups.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-19.
Abstract P3-10-22: Prognostic Significance of Aurora Kinase A Expression in Three Cohorts of Node-Negative Breast Cancer Patients
Authors: Schmidt M , Petry IB , Boehm D , Gebhard S , Lebrecht A , Koelbl H , Gehrmann M , Hengstler JG
Published: April 2011

Background: Aurora kinase A (AURKA) is important for cell cycle progression. Inhibitors that target AURKA are currently in clinical development. We examined the prognostic impact of AURKA in three cohorts of node-negative breast cancer patients without adjuvant systemic therapy (n=766).

Methods: AURKA (probe set ID 204092_s_at) was analysed utilizing microarray based gene-expression data of three independent and previously published cohorts of node-negative breast cancer patients (Mainz, Rotterdam, TRANSBIG). In addition to AURKA, we examined the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER) and HER2. Metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression.

Results: AURKA displayed a strong positive correlation with grade (P<0.001), tumor size (P<0.001), HER2 (P<0.001), and a negative correlation with ER (P<0.001), respectively. Patients with higher expression of IGKC showed shorter MFS in the Mainz cohort (HR 1.629, 95% CI 1.243-2.134, P<0.001), Rotterdam cohort (HR 1.567, 95% CI 1.271-1.933, P<0.001), and TRANSBIG cohort (HR 1.331, 95% CI 1.081-1.640, p=0.007), respectively. Accordingly, AURKA was significant in univariate analysis in the whole cohort of patients (HR 1.523, 95% CI 1.343-1.726, P<0.001). Using multivariate Cox regression, only tumor size retained its independent prognostic significance (HR 1.818, 95% CI 1.196-2.765, p=0.005). AURKA (HR 1.221, 95% CI 0.987-1.511, p=0.065) and histological grade (HR 1.551, 95% CI 0.955-2.520, p=0.076) showed only borderline significance for MFS.

Conclusion: AURKA has prognostic relevance in three independent cohorts of node-negative breast cancer patients making it a suitable target for therapy with Aurora kinase inhibitors.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-22.
Abstract P3-10-27: Caspase Variant as Prognostic Marker in Patients with Early Breast Cancer
Authors: Chae YS , Lee SJ , Jung JH , Park HY , Lee S-W , Kim WW , Lee SJ , Kang BW
Published: April 2011

Background: Since apoptosis induced by caspases may play a role in the prognosis of breast cancer, the present study analyzed the polymorphisms of caspase genes and their impact on the survival of 240 patients with early invasive ductal breast cancer.

Material and Methods: The genomic DNA was extracted from paraffin-embedded tumor-free tissue or blood, and 8 single nucleotide polymorphisms (SNPs) of 5 apoptosis-related genes in the apoptosis pathway determined using a Sequenom MassARRAY system. Results: During the median follow-up of 68.6 (range, 36.3-173.1) months, 43 relapses and 25 deaths occurred. Among the target polymorphisms, the caspase 9 gene polymorphism (CASP9 rs4645978) in a dominant model of the A allele was associated with survival in a log-rank test (P < 0.05). In a multivariate analysis, the GA+AA genotype of CASP9 rs4645978 was significantly correlated with a better disease-free survival (HR = 0.250; 95% CI = 0.075 - 0.840; P = 0.025), while marginally with distant disease-free or overall survival (HR = 0.360 and 0.337; P = 0.118 and 0.158, respectively). No clinicopathologic difference was observed according to the genotypes of this polymorphism. Discussion: The CASP9 rs4645978 was identified as a possible prognostic factor of survival in patients with operated invasive breast cancer.

Figures available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-27.
Abstract P3-10-28: Breast Cancer Patients with Neoadjuvant Chemotherapy Have a Different Clinical Significance of the Ki-67 Expression before and after the Treatment
Authors: Wada N , Asaga S , Yamauchi C , Fujii S
Published: April 2011

Background: Reliable predictive and prognostic markers for routine diagnostic purposes are needed for breast cancer patients treated with neoadjuvant chemotherapy (NAC). Ki-67 is a marker for proliferating cells and is overexpressed in many breast cancers. Aim of this analysis was to evaluate the potential role of Ki-67 as a biomarker for the breast cancer patients with NAC.

Patients and Methods: A retrospective search of a prospectively maintained clinical breast cancer database was performed to identify patients treated with NAC at the National Cancer Center Hospital East. The expression of Ki-67 was assessed using immunohistochemistry (MIB-1) in pretherapy core-needle biopsy and post-therapy surgical excision specimens. Levels of Ki-67 index (percentage of Ki-67 positive cancer nuclei) were dichotomized as high (over 10%) and low (less or equal 10%). Survival curve was calculated using Kaplan-Meier method. Log-Rank test was used to detect differences.

Results: Records were available for 296 patients who were received NAC (anthracycline and/or taxane based) between Nov 2001 and Nov 2009. High and low Ki-67 index in pre-therapy were 173 and 123 cases, respectively. 51 cases (17%) of all achieved a pathological complete response (pCR: no residual invasive disease in the breast). pCR rate (23%) in high Ki-67 was significantly different from that (9%) in low Ki-67. 5-year overall survival estimates were 69% and 75%, respectively. However, there was not difference between the groups in overall survival curve at a median follow-up time of 31 months. Of 245 cases with residual invasive, high and low Ki-67 index in post-therapy were 80 and 165 cases, respectively. 62 cases maintained high Ki-67 index before and after NAC, 94 cases maintained low Ki-67 index. There was significantly difference between high and low Ki-67 indexes of post-therapy in overall survival curve (P<0.01), 5-year overall survival estimates were 51% and 81%, respectively. Moreover, there was not significant different between 51 cases with pCR and low Ki-67 index in overall survival curve.

Conclusions: Although Ki-67 index in pre-therapy is a useful predictor for the therapeutic response to NAC, high Ki-67 index in pre-therapy dose not imply that the patients have a worse prognosis. Ki-67 index in post-therapy is useful to predict outcomes for patients with residual invasive disease.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-28.
Abstract P3-10-40: Expression and Prognostic Significance of Cancer Testis Antigens (CTA) in Primary Breast Cancer: Possible Diagnostic Markers and Therapeutical Targets
Authors: Balafoutas D , zur Hausen A , Jungbluth A , Denschlag D , Jäger M , Gitsch G , Stickeler E.
Published: April 2011

Backround: Cancer Testis Antigens (CTA) are a family of proteins normally expressed in the human germ cells such as in the testis. They also have been found in various types of malignant tumors including breast cancer. Because of their restricted expression pattern they are frequently able to elicit T-cell immune responses, and therefore they are considered as ideal targets for cancer immunotherapy. Breast differentiation antigen NY-BR-1 has also immunogenic properties. The aim of this study was to explore the CTA expression in breast cancer and detect possible clinical correlations.

Materials and Methods: The expression patterns of 6 CTAs (MAGE A1- MA454, MAGE A3-M3H67, MAGE A4-57B, NY-ESO-1-E978, GAGE, MAGE A-6C1) and NY-BR-1 were examined by immunohistochemistry in a series of 210 non-selected patients with primary invasive breast cancer using the tissue microarray technique. The intensity of the stain was scored semi-quantitatively in a scale 0 to+3. The expression of the antigens was correlated to established clinicopathological parameters as well as disease-free (DFS) and overall (OS) survival.

Results: CTA expression exhibited a predominantly cytoplasmic and occasionally nuclear localization. At least one CTA was identified in 37.2% of cases with expression of each antigen varying from 4.5 to 15%. NY-BR-1 was positive in 46.6% of tumors, with the well differentiated tumors showing more frequent expression. MAGE A4-57B (p=0.028 and 0.015) and MAGE A3-M3H67 (p=0.001 and 0.004, respectively) positivestaining was significantly correlated to shorter OS and DFS. MAGE A1-MA454 correlated significantly only with OS (p=0.028). GAGE and MAGE A-6C1 displayed a clear trend, but not statistically significant prognostic value concerning shorter DFS and OS. In multivariate analysis only MAGE A3-M3H67 (p=0.007 OS) and MAGE A4-57B (p=0.017 DFS, 0.052 OS) showed and independent prognostic relevance. Additionally, the mortality rate increased substantially if co-expression of any 3 or more CTAs was observed.

Discussion: Our findings suggest that CTAs could serve as potential prognostic markers in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define potential new targets for specific breast cancer therapies.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-40.
Abstract P3-10-41: Quantitative Immunohistochemical Analysis and Prognostic Significance of TRPS-1, a New GATA Transcription Factor Family Member, in Breast Cancer
Authors: Chen JQ , Litton J , Xiao L , Zhang H-Z , Warneke CL , Wu Y , Shen X , Sahin A , Katz R , Murray JL , Radvanyi L.
Published: April 2011

The Trichorhinophalangeal Syndrome-1 gene (TRPS-1) is a novel GATA transcription factor family member expressed in >90% of human breast cancers. In this study, we developed a new quantitative IHC (qIHC) method to study TRPS-1 as a possible indicator ofprognosis in BC. Using this method, a quantitative parameter for TRPS-1 expression called a Quick Score (QS) was derived from the measured Labeling Index (LI) and Mean Optical Density (MOD) after IHC and applied to a set of 152 Stage II/III BC patients from 1993-2006 who did not receive preoperative chemotherapy. Although highly prevalent in BC, a wide range of TRPS-1 QS was found among the sample set with higher TRPS-1 QS significantly associated with tumor ER-alpha (p= 0.023 for QS, and p= 0.028 for Allred score), PR (p= 0.009), GATA-3 (P<0.0001), and E-cadherin (p< 0.001). TRPS-1 QS was also positively associated with HER2 status (p=0.026). Analysis of different ductal structures revealed that TRPS-1 was expressed at low levels in normal ducts and in usual ductal hyperplasia, but showed a marked increase in DCIS and invasive carcinoma lesions. An analysis of TRPS-1 expression in association with overall survival revealed that TRPS-1 QS (≥4.0) was significantly associated with improved survival (p=0.0165). Patients with TRPS-1 QS <4 had a hazard ratio of 2 (p=0.019) after univariate Cox proportional hazards analysis. Further analysis of the patient samples revealed an ER-/lo, HER2+ (amplified) BC subset in which TRPS-1 expression above a critical quantitative threshold was predictive of greatly improved survival (median survival not reached after 10 years), while patients in this subset with TRPS-1 expression below this threshold having a median survival of less than 5 years. In summary, this new qIHC approach revealed critical differences in TRPS-1 expression in primary BC samples and found that this new GATA family member may be a promising positive prognostic marker that may regulate BC progression. In addition, TRPS-1 may also be a relevant new marker in a luminal ER-/low, HER2+ BC subset.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-41.
Abstract P3-11-10: Risk Factors for Pregnancy-Associated Breast Cancer: A Report from the Nigerian Breast Cancer Study
Authors: Huo D , Hou N , Ogundiran T , Olopade OI.
Published: April 2011

Background: It is known that pregnancy has a dual effect on breast cancer risk, with increased risk of breast cancer in short term and decreased risk in the long run. Pregnancy-associated breast cancer (PABC), occurring during pregnancy and several years postpartum, has poor prognosis and is challenging in clinical management. However, risk factors for PABC, except for pregnancy per se, are less understood. Other reproductive factors including age at menarche and age at first live birth may play important roles in the development of PABC.

Methods: The study is based on the Nigerian Breast Cancer Study, a case-control study of breast cancer conducted in Ibadan, Nigeria, between 1998 and 2009. In-person interviews were conducted using structured questionnaires to collect detailed epidemiologic data. Pre-menopausal women who were 50 years old or younger were eligible to this analysis. The breast cancer cases were divided in two groups: PABC (diagnosed during pregnancy or within 5 years postpartum) and non-PABC. Polytomous logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI), contrasting each group of cases versus controls. Age in 3-year category, ethnicity, education, family history of breast cancer, history of benign breast disease, hormonal contraceptive use, alcohol drinking, height, and body mass index were adjusted in the multivariate models.

Results: In total, 1440 women were eligible to this study, including 311 PABC cases, 357 non-PABC cases, and 772 healthy controls. In the multivariate analysis adjusting aforementioned potential confounders, age at menarche was inversely associated with non-PABC (p=0.002, Table 1) but only trend towards significance for PABC (p=0.12). Age at first live birth was associated with lower risk of non-PABC but was associated with elevated risk of PABC.

Table 1. Odds ratios and 95% confidence intervals for PABC and non-PABC

Conclusions: We found menarche at older age may be protective against all premenopausal breast cancer, but age at first live birth has different role in the development of pregnancy-associated breast cancer and other premenopausal breast cancer in Nigerian women. The findings suggest that the trend towards delayed child-bearing may increase in the occurrence of breast cancer complicated by pregnancy.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-11-10.
Abstract P3-11-11: A Comparison of Breast Cancer Treatment Rates in British Columbia, Scotland, and Western Australia, and a Comparison with Models of “Optimal” Therapy
Authors: Fong A , Shafiq J , Saunders C , Thompson A , Tyldesley S , Barton MB , Dewar JA , Ng W , Jacob S , Speers C , Olivotto I , Delaney GP.
Published: April 2011

Background: Evidence-based optimal utilization models provide estimates of optimal radiotherapy, chemotherapy and hormonal therapy utilization by stage and other clinically relevant patient sub-groups. We therefore compared predicted utilization with actual utilization of radiotherapy, hormone therapy and chemotherapy in 3 jurisdictions with population-based stage and treatment data: British Columbia, Canada; Dundee, Scotland; and Perth, Western Australia.

Methods: Previously published optimal radiotherapy, chemotherapy, and endocrine therapy treatment utilization trees for an Australian population were modified to incorporate epidemiological data from British Columbia, Dundee, and Perth, such that the optimal trees for each region reflected the casemix for each region. Frequency data on patient, tumour, and surgical factors were used to calculate optimal treatment rates for each region. Optimal rates were then compared with actual rates of surgery, radiotherapy, chemotherapy, and endocrine therapy use obtained from 2 population-based and 1 institution-based cancer registries for patients diagnosed with breast cancer between 2000 to 2004. Information on region-specific treatment guidelines was also collected.

Results: Region-specific optimal treatment utilization rates at diagnosis varied between 80% and 81% for radiotherapy (62 to 64% when patient preference is taken into account), 53% to 56% for chemotherapy, and 49% to 54% for endocrine therapy. The predicted ranges were due to local variations in demographics, and tumour stage. Actual radiotherapy utilization was 57%, 49%, and 52%; chemotherapy utilization was 32%, 24%, and 29%; and endocrine therapy utilization was 56%, 64%, and 52% for British Columbia, Dundee, and Perth, respectively. Conclusion: There are significant differences in actual treatment utilisation rates between the study populations. It is unlikely that all of this variation is due to differences in tumour characteristics alone. Actual utilization rates were lower than the calculated optimal rates for radiotherapy and chemotherapy, and higher for endocrine therapy. Differences between actual regional rates of treatment utilization were seen, and were associated with differences in mastectomy rates, and guideline recommendations for treatment use in that region. This methodology allows comparison of the treatment that occurs in a jurisdiction against what would be considered optimal based on evidence.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-11-11.